CONFERENCE COVERAGE SERIES

Society for Neuroscience Annual Meeting 2009

( 12 Articles Available, 0 Articles Pending )

Chicago, IL, U.S.A.

17 – 21 October 2009

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Chicago: Axonal Transport Not So Fast in Neurodegenerative Disease

03 Nov 2009

The long axons of neurons act as intracellular highways, with motor proteins shuttling their cargo up and down microtubule tracks. Block that traffic—by any number of ways—and the result is often feeble, dying neurons. The impairment of fast axonal transport (FAT) in a variety of neurodegenerative diseases was the theme at a mini-symposium held Sunday, 18 October 2009, at the Society for Neuroscience annual meeting in Chicago, Illinois (reviewed in Morfini et al., 2009).

“These diseases…share several common characteristics,” said Gerardo Morfini, who co-chaired the session with Gustavo Pigino. Both work at the University of Illinois in Chicago. A frequent pattern, Morfini said, is that defects in axonal transport and synapse function lead to a “dying back” axonal pathology, loss of connectivity between neurons, and, much later on, neuronal cell death.

Researchers recapped the impairment of FAT in models of Parkinson disease (see ARF related news story on Morfini et al., 2007), Alzheimer’s (see ARF related news story on Pigino et al., 2009), and hereditary spastic paraplegia (Edgar et al., 2004). They discussed new studies as well. For example, Daryl Bosco of the University of Massachusetts in Worcester presented data showing that two proteins associated with amyotrophic lateral sclerosis (ALS)—superoxide dismutase 1 (SOD1) and Fused in Sarcoma (FUS)—inhibit FAT. Skip Binder of the Northwestern University Medical School in Chicago shared results on a phosphorylation site that regulates tau’s interference in axonal trafficking. And, in a separate session on Huntington disease held October 20, Sarah Pollema of the University of Illinois at Chicago showed which part of polyglutamine-expanded huntingtin interferes with transport. (Hint: It’s not where you might think.)

For their experiments, the scientists depended on North Atlantic squid (Loligo pealii), netted off the coast of the Marine Biological Laboratory in Woods Hole, Massachusetts, so researchers could harvest their giant axons. “This animal seems to have been created by nature for neuroscientists,” quipped Morfini in a presentation last month at the André-Delambre Foundation Symposium on ALS in Québec City. Their giant axons are half a millimeter in diameter, and researchers can extrude the axoplasm “like a sausage,” Morfini said, onto a microscope slide. They can then watch molecular motors cart material up and down the microtubules, and perfuse proteins and drugs to see if they affect transport.

SOD1 and FUS: Each Blocks Transport in Its Own Way
Axonal transport has long been a topic of interest in ALS. Mutations in dynein cause motor neuron degeneration in mice (see ARF related news story on Hafezparast et al., 2003). And in a recent genomewide association study, researchers found an allele of kinesin-associated protein 3 (KIFAP3) that lengthened survival time among people with the disease (see ARF related news story on Landers et al., 2009).

Bosco, Morfini, and colleagues added SOD1 protein—mutations to the SOD1 gene are the most common cause of inherited ALS—to squid axoplasm. Wild-type protein had no effect, but G93A mutant SOD1 inhibited anterograde transport. Retrograde transport proceeded unimpeded. The same was true for other ALS-linked SOD1 mutants H46R, A4V, and G85R. To explore the mechanism by which SOD1 slowed transport, the researchers infused the squid axoplasm with various kinase inhibitors in addition to the mutant protein. They found that inhibiting p38 MAP kinase restored normal transport in the presence of mutant SOD1. To the authors, the data suggest that mutant SOD1 activates p38, which is known to phosphorylate kinesin, knocking the motor off the microtubules.

Mutant SOD1 is implicated in only 2 percent of ALS cases; other inherited mutations likely account for a further 8 percent, with the remaining instances currently thought to be sporadic. However, some scientists suspect wild-type SOD1 of involvement in motor neuron pathology in sporadic ALS, too, as mutations in the DNA sequence are not the only way to compromise a protein. Bosco suggested that the protein’s structure could be modified in various ways in disease. The protein normally functions as a dimer, with an intramolecular disulfide bond and zinc and copper cofactors—but any of those characteristics could change in disease, she said. Altered wild-type SOD1 might be just as bad for motor neurons as the mutant forms.

Bosco hypothesized that antibodies raised to mutant SOD1 (Urushitani et al., 2007) might also interact with wild-type protein in people with sporadic ALS. Among CNS tissue samples from 10 people who died of sporadic ALS, she found that four stained positive with the mutant SOD1 antibodies. Four did not and a further two had no evident motor neurons to examine. The researchers are currently using mass spectrometry to discover which SOD1 modifications are present in the immunoreactive samples.

That evidence led Bosco to wonder if modified, wild-type SOD1 would also impede axonal trafficking as the mutants did. Sure enough, purified protein from the immunoreactive patient samples did slow FAT in the squid axoplasm.

Earlier this year, researchers linked a new gene to familial ALS. FUS is involved in RNA transcription, splicing, and transport (see ARF related news story on Kwiatkowski et al., 2009 and Vance et al., 2009). When Bosco and colleagues added mutant FUS protein to squid axoplasm, they saw that both anterograde and retrograde transport slowed down. This contrasted with the effects of SOD1, which were solely on anterograde trafficking. The data suggest that FUS’s effects on axonal transport may be mediated by a different mechanism than SOD1’s.

Tau: Presenting PAD
It has been known for some time that tau filaments inhibit anterograde FAT. In previous work, Binder and colleagues discovered that deleting the amino terminus of tau—amino acids 2 through 18—prevented its interference with axonal transport (Lapointe et al., 2009). At the symposium, Binder reported on further research, led by former graduate student Nichole LaPointe, who is now at the University of California-Santa Barbara; Nick Kanaan, currently a post-doc in Binder’s lab; and Morfini. Kanaan wondered if the 2-18 region of tau required the rest of the protein, as well, to inhibit transport. Accordingly, he synthesized a peptide with only those amino acids—and found that this amino-terminal region alone impeded FAT.

Like SOD1, tau exerts its effects on FAT via phosphorylation of the motors. Previously, the researchers found that inhibitors of glycogen synthase kinase-3 (GSK3) and protein phosphatase 1 (PP1) prevent tau from slowing transport. PP1 dephosphorylates GSK3, activating it to dephosphorylate kinesin, detaching the motor from its cargo. The amino terminus of tau corresponds to a consensus sequence for PP1 binding, and the researchers christened amino acids 2 through 18 the Phosphatase Activation Domain (PAD). They do not yet know if this domain directly interacts with PP1 or activates it indirectly, perhaps through an enzymatic cascade.

The PAD contains a phosphorylation site at tyrosine 18, and Kanaan suspected the presence or absence of this phosphate would affect axonal transport. He engineered a mutant with glutamate at position 18 to mimic phosphorylation, and found that the pseudophosphorylated protein did not inhibit FAT. Nor did purified, phosphorylated wild-type tau filaments. Therefore, Kanaan concluded, the PAD’s effect on transport is mediated by phosphorylation at tyrosine 18, and the unphosphorylated form is the one that blocks FAT, presumably through some interaction with PP1.

Binder suspects that in a healthy brain, the PAD is tucked away inside the tau protein, unable to interfere with transport. But when tau is altered in disease, the PAD may stick out. “Anything that presents the PAD region to the cell should inhibit anterograde transport,” Binder said.

Huntingtin: It’s the Ps, Not the Qs
Morfini and colleagues previously showed that poly-glutamine expanded huntingtin, as well, interferes with anterograde transport: It activates cJun N-terminal kinase 3 (JNK3) to phosphorylate kinesin, uncoupling the motor from its tracks (see ARF related news story on Morfini et al., 2009). Pollema, a graduate student in Morfini’s and Brady’s labs, shared her work on which part of huntingtin mediates this effect.

Disease-causing huntingtin harbors an excess of glutamine repeats. Pollema showed that the first exon of the polyQ-expanded protein, containing those repeats, was sufficient to inhibit transport. Yet right next to those glutamines, and also in exon 1, lies a string of prolines. Further along the sequence is a second proline-rich domain, or PRD. To determine which part of the exon slowed axonal traffic, Pollema infused squid axoplasm with exon 1, along with antibodies to block either the glutamate or proline sequences. She found that only the proline antibody prevented the inhibition, indicating that the PRDs, not the polyglutamine repeats themselves, were the culprits. Further confirming the results, she showed that short polyproline peptides were sufficient to inhibit transport.

In conclusion, Morfini wrote in an e-mail to ARF that it might someday be possible to correct axonal transport defects with drugs that modify kinase activity. Several such pharmaceuticals are making their way through clinical trials for a variety of cancers. “Correcting fast axonal transport deficits in neurodegenerative disease by modulating kinase activities appears a promising avenue of research,” Morfini wrote.—Amber Dance.

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References

News Citations

Paper Citations

Morfini GA, Burns M, Binder LI, Kanaan NM, Lapointe N, Bosco DA, Brown RH, Brown H, Tiwari A, Hayward L, Edgar J, Nave KA, Garberrn J, Atagi Y, Song Y, Pigino G, Brady ST. Axonal transport defects in neurodegenerative diseases. J Neurosci. 2009 Oct 14;29(41):12776-86. PubMed.
Morfini G, Pigino G, Opalach K, Serulle Y, Moreira JE, Sugimori M, Llinás RR, Brady ST. 1-Methyl-4-phenylpyridinium affects fast axonal transport by activation of caspase and protein kinase C. Proc Natl Acad Sci U S A. 2007 Feb 13;104(7):2442-7. PubMed.
Pigino G, Morfini G, Atagi Y, Deshpande A, Yu C, Jungbauer L, Ladu M, Busciglio J, Brady S. Disruption of fast axonal transport is a pathogenic mechanism for intraneuronal amyloid beta. Proc Natl Acad Sci U S A. 2009 Apr 7;106(14):5907-12. PubMed.
Edgar JM, McLaughlin M, Yool D, Zhang SC, Fowler JH, Montague P, Barrie JA, McCulloch MC, Duncan ID, Garbern J, Nave KA, Griffiths IR. Oligodendroglial modulation of fast axonal transport in a mouse model of hereditary spastic paraplegia. J Cell Biol. 2004 Jul 5;166(1):121-31. PubMed.
Hafezparast M, Klocke R, Ruhrberg C, Marquardt A, Ahmad-Annuar A, Bowen S, Lalli G, Witherden AS, Hummerich H, Nicholson S, Morgan PJ, Oozageer R, Priestley JV, Averill S, King VR, Ball S, Peters J, Toda T, Yamamoto A, Hiraoka Y, Augustin M, Korthaus D, Wattler S, Wabnitz P, Dickneite C, Lampel S, Boehme F, Peraus G, Popp A, Rudelius M, Schlegel J, Fuchs H, Hrabe de Angelis M, Schiavo G, Shima DT, Russ AP, Stumm G, Martin JE, Fisher EM. Mutations in dynein link motor neuron degeneration to defects in retrograde transport. Science. 2003 May 2;300(5620):808-12. PubMed.
Landers JE, Melki J, Meininger V, Glass JD, van den Berg LH, van Es MA, Sapp PC, van Vught PW, McKenna-Yasek DM, Blauw HM, Cho TJ, Polak M, Shi L, Wills AM, Broom WJ, Ticozzi N, Silani V, Ozoguz A, Rodriguez-Leyva I, Veldink JH, Ivinson AJ, Saris CG, Hosler BA, Barnes-Nessa A, Couture N, Wokke JH, Kwiatkowski TJ, Ophoff RA, Cronin S, Hardiman O, Diekstra FP, Leigh PN, Shaw CE, Simpson CL, Hansen VK, Powell JF, Corcia P, Salachas F, Heath S, Galan P, Georges F, Horvitz HR, Lathrop M, Purcell S, Al-Chalabi A, Brown RH. Reduced expression of the Kinesin-Associated Protein 3 (KIFAP3) gene increases survival in sporadic amyotrophic lateral sclerosis. Proc Natl Acad Sci U S A. 2009 Jun 2;106(22):9004-9. PubMed.
Urushitani M, Ezzi SA, Julien JP. Therapeutic effects of immunization with mutant superoxide dismutase in mice models of amyotrophic lateral sclerosis. Proc Natl Acad Sci U S A. 2007 Feb 13;104(7):2495-500. PubMed.
Kwiatkowski TJ Jr, Bosco DA, Leclerc AL, Tamrazian E, Vanderburg CR, Russ C, Davis A, Gilchrist J, Kasarskis EJ, Munsat T, Valdmanis P, Rouleau GA, Hosler BA, Cortelli P, de Jong PJ, Yoshinaga Y, Haines JL, Pericak-Vance MA, Yan J, Ticozzi N, Siddique T, McKenna-Yasek D, Sapp PC, Horvitz HR, Landers JE, Brown RH Jr. Mutations in the FUS/TLS gene on chromosome 16 cause familial amyotrophic lateral sclerosis. Science. 2009 Feb 27;323(5918):1205-8. PubMed.
Vance C, Rogelj B, Hortobágyi T, De Vos KJ, Nishimura AL, Sreedharan J, Hu X, Smith B, Ruddy D, Wright P, Ganesalingam J, Williams KL, Tripathi V, Al-Saraj S, Al-Chalabi A, Leigh PN, Blair IP, Nicholson G, de Belleroche J, Gallo JM, Miller CC, Shaw CE. Mutations in FUS, an RNA processing protein, cause familial amyotrophic lateral sclerosis type 6. Science. 2009 Feb 27;323(5918):1208-11. PubMed.
Lapointe NE, Morfini G, Pigino G, Gaisina IN, Kozikowski AP, Binder LI, Brady ST. The amino terminus of tau inhibits kinesin-dependent axonal transport: implications for filament toxicity. J Neurosci Res. 2009 Feb;87(2):440-51. PubMed.
Morfini GA, You YM, Pollema SL, Kaminska A, Liu K, Yoshioka K, Björkblom B, Coffey ET, Bagnato C, Han D, Huang CF, Banker G, Pigino G, Brady ST. Pathogenic huntingtin inhibits fast axonal transport by activating JNK3 and phosphorylating kinesin. Nat Neurosci. 2009 Jul;12(7):864-71. PubMed.

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Chicago: Tau and α-Synuclein Oligomers Follow Aβ Footsteps

05 Nov 2009

In Alzheimer disease research, focus has recently shifted away from large plaques and toward small oligomers of amyloid as a potential cause of disease (e.g., ARF related news story on Pigino et al., 2009 and Moreno et al., 2009). At the Society for Neuroscience annual meeting held 17-21 October 2009 in Chicago, Illinois, dozens of presentations dealt with Aβ oligomers, but what stood out more as a budding trend were signs that research in the tau and α-synuclein fields may be going down the same road. Several presentations covered emerging tools to study oligomers of these two proteins, and they drew wide notice among attendees.

Untangling Tau
Oligomers form shapes distinct from either monomer or fibrillar aggregated forms. Accordingly, researchers can make antibodies specific for particular conformations. Rakez Kayed described how his group at the University of Texas Medical Branch in Galveston pursued a similar approach to generate first tau oligomers, then antibodies against them, that had borne fruit while he was a postdoc in Charlie Glabe’s laboratory at the University of California, Irvine (Kayed et al., 2003). In his Galveston lab, Kayed seeded recombinant full-length human tau with Aβ oligomers or with α-synuclein oligomers. “I think this way of making oligomers is relevant to what happens in vivo,” Kayed told ARF. The scientists used the resulting tau oligomers as an antigen and produced a series of anti-tau antibodies that they claim recognize trimers and higher-order oligomers, but not monomers or fibrils.

The lab gave several presentations in Chicago. In a slide talk, Kayed focused on tau oligomers with a distinct ring-like shape. The story starts with work on amyloid-β, which can form distinct pore-like structures called annular protofibrils. As postdoc, Kayed had raised an antibody, dubbed Officer, specific for these shapes. He and Glabe found that Officer also nabbed other pore-forming proteins, suggesting it is specific not for amyloid-β, but for the β-barrel structural motif of the pore (Kayed et al., 2009). In Chicago, Kayed described how when he used Officer to detect amyloid-β annular protofibrils in brain tissue from people who had had Alzheimer disease, he noticed that some of the labeled annular structures contained no amyloid-β and looked suspiciously like structures commonly formed by tau. Sure enough, double-immunostaining with tau antibodies and Officer suggested that the AD brain contains tau, as well as amyloid-β, annular protofibrils. The scientists found similar tau annular protofibrils in tissue from people who had dementia with Lewy bodies (DLB) and a tauopathy called progressive supranuclear palsy (see also ARF related news story). The researchers apparently were also able to make Officer-reactive tau oligomers in vitro; on a Western blot, these tau annular protofibrils ran as a smear with a molecular weight more than 50 kDa, Kayed reported.

Kayed suggested that tau annular protofibrils might form pores in the cell membrane, allowing ions to pass through and disrupting cellular homeostasis. This might be true especially in astrocytes and oligodendrocytes, which contain more annular tau structures than do neurons, Kayed said. Because the annular protofibrils are ubiquitinated, they might also impede the cell’s protein destruction pathway, he added, allowing other misfolded proteins to accumulate. For background on the pore hypothesis, see ARF Live Discussion.

On a poster, Cristian Lasagna-Reeves, a graduate student in Kayed’s laboratory, presented a similar approach with a different antibody that detects tau oligomers. Lasagna-Reeves noted that the presence of neurofibrillary tangles correlates less tightly with symptoms of AD than does the presence of pre-fibrillar tau. “We think [tau oligomers] are the real toxic species in Alzheimer disease,” Lasagna-Reeves said, adding that his work now gives researchers a method to detect those species.

Called T2286, his new antibody detects 109-kDa spherical oligomers, but not monomers or fibrils, nor other kinds of amyloid-forming protein. Using brain tissue extracts, Lasagna-Reeves reported that frontal cortex from people who had had AD showed immunoreactivity with T2286; control samples did not. Using CSF from some 20 people, he reported an increase in T2286 immunoreactivity in people with AD relative to normal controls, suggesting spherical tau could eventually become a biomarker for the disease as well as a potential therapeutic target. This early CSF data was not presented alongside established CSF tau assays, for example, the INNOTEST htau or phospho-tau ELISAs widely used in clinical AD research across the world, and no absolute concentrations of the tau oligomers in CSF were given on the poster. Therefore, a comparison of the proposed tau oligomers to prior data on CSF total tau or phospho-tau concentrations in AD and controls was not possible from this initial study. In the next few months, the Texas group is planning to measure tau oligomers in a large number of CSF and serum samples, Kayed noted.

In a conversation with ARF, Kayed said his lab did do a comparison both in CSF and brain extracts, that is, with various research antibodies against phosphorylated forms of tau. It showed that less than 30 percent of the tau oligomers his new antibody detected were phosphorylated. “This surprised us. We had expected it would be more,” Kayed said. Whether phosphorylated tau might act as a seed, or whether phosphorylation might not occur until after filaments have formed, remains debatable, Kayed noted. Beyond AD brain, the T2286 antibody detected tau oligomers in brain extracts of dementia with Lewy bodies, supranuclear palsy, and Parkinson disease (see ARF related news story). In addition, T2286 picked up tau oligomers in the P301L and rTg4510 tau mouse models, as well as an α-synuclein mouse model, the Tg2576, and an APP/PS1 line, Kayed said.

In the rTg4510 regulatable tau mouse, the presence of oligomers correlated with a behavioral phenotype Karen Ashe’s group at the University of Minnesota, Minneapolis, had demonstrated previously, whereby memory function recovers when the tau transgene gets switched off even as neurofibrillary tangles stay in place (Santacruz et al., 2005; Berger et al., 2007). “Our new contribution to that is showing specifically with our antibody that the oligomers are gone at this point of behavior improvement,” Kayed said. He added that other unpublished experiments indicate that these oligomers are toxic to cultured cells, and that scientists at University of Texas Medical Branch plan to begin humanizing this antibody toward a tau immunotherapy (see also Kayed and Jackson, 2009) and to elucidate the epitope recognized by this antibody.

Overall, other scientists were intrigued by this talk and poster. However, they cautioned that the prospect of a single antibody that specifically recognizes neurotoxic tau oligomers in Western blots, ELISA, on tissue sections, in CSF, and that can serve as a start for tau oligomer immunotherapy sounds almost too good to be true and will need careful experimental substantiation.

In another poster, Kristina Patterson, a graduate student in the laboratory of Skip Binder at the Northwestern University School of Medicine in Chicago, presented her work on tau oligomers. Patterson used chemical cross-linking to stabilize tau oligomers in vitro. Typical cross-linkers rely on tau’s two cysteines to hook peptides together, but that limits the linkable conformations to those with two cysteines in proximity. “We decided we wanted to give it more choices,” Patterson said. She used a benzophenone cross-linker that binds cysteine with one end, but any carbon-hydrogen bond with the other. The non-specific end is activated by ultraviolet light. In other words, Patterson allowed the cross-linker to bind the cysteines and then gave the tau molecules the opportunity to oligomerize before hitting them with UV light to stabilize whatever conformation they happened to be in.

Patterson rather expected to see a ladder in her gels, with step-like increases for each additional tau in the oligomer. Instead, she saw mostly a species that ran at 180 kDa. The researchers also discovered a 180-kDa tau oligomer in brain homogenates from four people who had AD; the oligomer was not present in control samples. The 180 kDa corresponds to a trimer, but Patterson noted that other tau combinations could run at that weight depending on their structure. Patterson is currently characterizing the exact makeup of the oligomers, but has not yet performed size exclusion chromatography to show definitively that the bands from the AD sample are oligomers. In general, the study of tau oligomers is in its infancy, Patterson said, opening up many new possibilities for research. “These results overall are similar to what we see,” Kayed told ARF at the conference.

Un-aggregating α-Synuclein
Test-tube cross-linkers are also contributing to α-synuclein research. Martin Ingelsson presented a talk on work he and Joakim Bergström are doing at Uppsala University in Sweden. They used reactive aldehydes; these are compounds formed in the body under conditions of oxidative stress, a likely contributor to α-synucleinopathies. The researchers compared α-synuclein oligomers cross-linked by either 4-hydroxy-2-nonenal (HNE) or 4-oxo-2-nonenal (ONE). Both of these reactive aldehydes converted α-synuclein monomers into stable, β-sheet rich structures of approximately 2,000 kDa, which Ingelsson estimates contain 40 to 50 monomers. However, atomic force microscopy revealed that the ONE-induced α-synuclein oligomers were amorphous and variable in size, while HNE-induced oligomers formed distinct donut-shaped structures that are similar to the annular protofibrils described for amyloid-β and tau. None of these oligomers aggregated further into fibrils such as seen in Lewy bodies. This supports a suggestion made previously by Glabe, Paul Muchowski, and others that oligomers can form in their own side pathways that dead-end with the oligomeric state and do not continue on to large fibrils.

Ingelsson further reported that both types of oligomer were taken up by cultured cells, where they proved cytotoxic (see also Vekrellis data in ARF related news story). “This is just another example of commonalities” in diseases based on amyloid-forming proteins, Ingelsson said. Based on this research, the Uppsala group is hoping to develop a future α-synuclein immunotherapy along the lines of an anti-Aβ oligomer antibody originally developed in their lab, which is in late preclinical development at present.

Last but not least, Karin Danzer, a postdoctoral researcher in the laboratory of Pam McLean at Massachusetts General Hospital in Charlestown, presented new data on how she detected α-synuclein oligomers secreted from living cells. The poster generated a crowd and persistent buzz among scientists. “This demonstration of α-synuclein oligomer secretion is very attractive, because it implies these species will be more accessible to therapeutic removal than previously thought,” commented Dennis Selkoe of Brigham and Women’s Hospital in Boston. Danzer characterized the transmission of oligomers through cell media. The group combined culturing neuroglioma cells in a microfluidic chamber with an assay they developed to determine if extracellular α-synuclein was monomeric or oligomeric. Danzer generated two α-synuclein fusions: one linking the protein to the amino terminus of luciferase, and one linking it to the carboxyl terminus. Alone, the fusion proteins produced no luminescence. Together in an oligomer, the luciferase domains worked together to release light, which Danzer measured to quantify oligomerization.

The researchers found that α-synuclein oligomers, ranging in size from 14 kDA to 40,000 kDa, were secreted by neuroglioma cells. In previous work, the same group had shown that the chaperone Hsp70 reduced α-synuclein aggregation (Klucken et al., 2004). In the new assay, they found that co-transfecting Hsp70 reduced the secreted oligomer signal by 24-fold, without decreasing the amount of α-synuclein present. The data suggest that Hsp70 blocks oligomerization without destroying the protein. This presentation was also summarized on PD Online).—Amber Dance and Gabrielle Strobel.

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References

News Citations

Webinar Citations

Paper Citations

Pigino G, Morfini G, Atagi Y, Deshpande A, Yu C, Jungbauer L, Ladu M, Busciglio J, Brady S. Disruption of fast axonal transport is a pathogenic mechanism for intraneuronal amyloid beta. Proc Natl Acad Sci U S A. 2009 Apr 7;106(14):5907-12. PubMed.
Moreno H, Yu E, Pigino G, Hernandez AI, Kim N, Moreira JE, Sugimori M, Llinás RR. Synaptic transmission block by presynaptic injection of oligomeric amyloid beta. Proc Natl Acad Sci U S A. 2009 Apr 7;106(14):5901-6. PubMed.
Kayed R, Head E, Thompson JL, McIntire TM, Milton SC, Cotman CW, Glabe CG. Common structure of soluble amyloid oligomers implies common mechanism of pathogenesis. Science. 2003 Apr 18;300(5618):486-9. PubMed.
Kayed R, Pensalfini A, Margol L, Sokolov Y, Sarsoza F, Head E, Hall J, Glabe C. Annular protofibrils are a structurally and functionally distinct type of amyloid oligomer. J Biol Chem. 2009 Feb 13;284(7):4230-7. PubMed.
Santacruz K, Lewis J, Spires T, Paulson J, Kotilinek L, Ingelsson M, Guimaraes A, DeTure M, Ramsden M, McGowan E, Forster C, Yue M, Orne J, Janus C, Mariash A, Kuskowski M, Hyman B, Hutton M, Ashe KH. Tau suppression in a neurodegenerative mouse model improves memory function. Science. 2005 Jul 15;309(5733):476-81. PubMed.
Berger Z, Roder H, Hanna A, Carlson A, Rangachari V, Yue M, Wszolek Z, Ashe K, Knight J, Dickson D, Andorfer C, Rosenberry TL, Lewis J, Hutton M, Janus C. Accumulation of pathological tau species and memory loss in a conditional model of tauopathy. J Neurosci. 2007 Apr 4;27(14):3650-62. PubMed.
Kayed R, Jackson GR. Prefilament tau species as potential targets for immunotherapy for Alzheimer disease and related disorders. Curr Opin Immunol. 2009 Jun;21(3):359-63. PubMed.
Klucken J, Shin Y, Masliah E, Hyman BT, McLean PJ. Hsp70 Reduces alpha-Synuclein Aggregation and Toxicity. J Biol Chem. 2004 Jun 11;279(24):25497-502. PubMed.

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Chicago: Nicotinic AChRs: α4β2 Iffy for AD, More Promise With α7?

05 Nov 2009

Days before the masses swarmed to Chicago for the Society for Neuroscience annual meeting, a more intimate assembly of 287 exchanged the latest buzz on nicotinic acetylcholine receptors (AChR) in a satellite symposium held 14-16 October in the northwest suburb of Lincolnshire. The 33 talks and 81 posters on the agenda [.pdf] ran the gamut from basic biology to drug development, and described therapeutic applications in smoking, addiction, and pain, as well as cognition. This story features clinical updates for a number of nicotinic AChR compounds in development for cognitive indications. Part 2 will describe newer approaches en route to the clinic, and Part 3 will give a glimpse into mechanistic advances driving future nAChR drug discovery.

Therapeutic approaches targeting nicotinic AChRs for cognition have focused primarily on two subtypes—high-affinity α4β2 and low-affinity α7 receptors. These neuronal membrane proteins adorn the cortex, hippocampus, and thalamus—the brain’s prime centers for learning and memory. Positron emission tomography (PET) studies show that expression of the α4β2 receptor drops in affected brain areas of patients with mild cognitive impairment (MCI) and AD, and α4β2 availability seems to correlate with severity of cognitive impairment (Sabri et al., 2008). The situation with α7 nAChRs has been more controversial (see ARF related news story), though in the clinic compounds targeting these receptors seem to have the upper hand on α4β2 agents, which have fared poorly thus far in trials for AD and schizophrenia.

Aβ4β2—Slow Going
In a talk by Ed Johnson of AstraZeneca in Wilmington, Delaware, the clinical outlook for the selective α4β2 agonist AZD3480 (TC-1734) looked cloudy at best. Discovered by partner company Targacept in Winston-Salem, North Carolina, this compound appeared promising in rodent models of episodic memory, working memory, and spatial memory, and has been safe and well tolerated in numerous clinical studies. However, efficacy signals have yet to appear reliably. Earlier this year, the company reported, and Johnson reiterated in Lincolnshire, that a three-month Phase 2b trial of the α4β2 agonist in mild to moderate AD patients was inconclusive based on primary outcome. Not only did the treatment group fail to show meaningful change in ADAS-Cog scores at week 12 compared to baseline, but even participants taking donepezil (i.e., symptomatic drugs among the AD standard of care) did not show an uptick at 12 weeks, relative to those on placebo. A company news release attributes these results in part to unexpected improvement in the placebo group, a much-debated feature of recent AD trials (see ARF related news story). On safety and tolerability, the α4β2 agonist was similar to placebo and had fewer gastrointestinal-related adverse events than did donepezil. In a separate Phase 2b trial, AZD3480 did not improve cognitive deficits in schizophrenia patients who were taking an antipsychotic drug, Johnson said.

A ray of hope appeared in a more recent Phase 2 trial of adults with attention deficit hyperactivity disorder (ADHD). In that study, people on the higher dose (50 mg) showed improvement on the primary outcome measure (total symptom score on the Conners Adult ADHD Rating Scale-Investigator Rating), and the compound was not associated with any serious adverse events. AstraZeneca plans to continue development of AZD3480 for ADHD, and is recruiting for a Phase 1 study of another selective α4β2 agonist, AZD1446 (TC-6683), for AD.

Jeffrey Baker of Abbott Laboratories in Abbott Park, Illinois, updated the audience on ABT-089, an α4β2 partial agonist that improves working memory and selective attention in monkeys. The compound has also been safe and well tolerated in six Phase 1 studies totaling 198 healthy adults. However, earlier this year the company terminated a Phase 2 trial testing ABT-089 as an adjunct therapy in mild to moderate AD patients already taking cholinesterase inhibitors. This 12-week study enrolled nearly 400 people at 39 U.S. sites. It used a new trial design called response-adaptive randomization, whereby primary efficacy data are evaluated every two weeks in a sponsor-blinded fashion. This allows participants to be shifted into the most informative treatment arms as the study proceeds. Another benefit of the adaptive trial design is the pre-specification of futility criteria that warrant termination before the study proceeds to its bitter end. In other words, things fail faster, allowing sponsors to redirect the saved time and money toward other projects. Indeed, Abbott did terminate the AD trial of its α4β2 compound after an interim analysis revealed that none of the six treatment arms was separating from placebo on the study’s primary outcome measure, the ADAS-Cog. Like the AstraZeneca α4β2 compound, ABT-089 looked promising in a four-week Phase 2 study of adults with ADHD; it improved some ADHD symptoms, relative to placebo, but that trial was not powered to assess cognition. Abbott has since decided to discontinue ABT-089 studies in ADHD, a company representative confirmed by e-mail.

Lucky Seven?
In an overview of nAChRs in drug discovery, Steve Arneric of Eli Lilly and Company in Indianapolis, Indiana, speculated whether AD drug developers have been barking up the wrong tree in their attempts to target α4β2 receptors. He noted several lines of research hinting that α7 nAChRs are the way to go. In a study published several months ago, treatment with an α7 agonist (S 24795) brought functional recovery to cortical synaptosomes from AD patients by disrupting interactions between Aβ peptides and α7 receptors (Wang et al., 2009 and ARF related news story). Earlier this year, scientists reported that lack of α7 protected AD mice from Aβ-induced synaptic loss, restored long-term potentiation, and improved cognition (Dziewczapolski et al., 2009 and ARF related news story). In addition, researchers recently reported a new α7 nAChR subtype, the α7β2 receptor, which may not only outnumber homomeric α7 receptors in the basal forebrain, but also seems more sensitive to blockage by Aβ oligomers (Liu et al., 2009 and ARF related news story). Finally, a recent review proposes that α7 receptors may link a number of apparently disparate mechanisms thought to underlie AD (Bencherif and Lippiello, 2009).

Judging by talks and posters at the nAChR symposium, α7 compounds are outpacing α4β2 compounds in the clinic, and the field’s hot new pursuit—positive allosteric modulators (PAMs)—could be poised to dethrone the more tried-and-true agonists (see Part 2). But it’s still early days with the PAMs, which differ from agonists in that they target sites away from the substrate binding action. Several α7 PAMs look decent in preclinical studies, but only one has gotten the go-ahead for Phase 1 trials. At the meeting, many scientists were unable, or unwilling, to confidently say whether modulators would ultimately beat out agonists in the clinic.

In the meantime, several α7 agonists have undergone early Phase 2 studies and, more importantly, survived them to stay in the running for future trials. Tanya Wallace of Roche in Palo Alto, California, briefed attendees with clinical data on the company’s α7 partial agonist R3487 (MEM3454), developed in collaboration with Memory Pharmaceuticals, which Roche acquired last year. In a Phase 2a proof-of-concept monotherapy study, mild to moderate AD patients receiving the lowest dose (5 mg) showed improvement in accuracy and speed of memory in the study’s primary endpoint, the CDR test battery. “As we increased the dose (to 15 or 50 mg), the pro-cognitive improvement was reduced,” Wallace said, noting this trend had also appeared in preclinical analyses. She said the dosing effects could stem from one of the α7 nAChR’s key features—its tendency to rapidly desensitize upon prolonged agonist exposure. Earlier this spring, the company began recruitment for a Phase 2b study that aims to enroll 420 mild to moderate AD patients at 62 worldwide sites. This is a six-month trial testing a lower dose range (1, 5, or 15 mg) as an add-on to donepezil treatment, with ADAS-Cog as the primary endpoint, Wallace said. A poster by Wallace and other researchers at Roche and Memory Pharmaceuticals showed that the compound improves attention and working memory in non-human primates with properties in line with clinical data.

Gerhard Koenig spoke of another α7 agonist with promising Phase 2 signals—EnVivo Pharmaceuticals’ EVP-6124. This compound is “very selective” and has “truly superior brain penetration,” which would mean limited systemic exposure, Koenig noted in an e-mail to ARF. “We believe that could be a key unique feature of our compound,” he wrote. In a poster presentation at this year’s International Conference on Alzheimer’s Disease (ICAD) in Vienna, Koenig’s team reported cognitive benefit (measured as improvement on some parts of the CogState or NTB batteries) in mild to moderate AD patients who received EVP-6124 for four weeks as an adjunct therapy with acetylcholinesterase (AChE) inhibitors (see ARF related conference story). That trial was small (48 patients). A longer Phase 2b AD study involving more than 200 participants is set to begin in the first half of 2010, Koenig said. He noted in an e-mail that primary endpoints for this trial are still being intensely discussed and cannot at this point be disclosed publicly. EnVivo is also wrapping up data analysis of a Phase 1b/2a monotherapy trial of its α7 agonist in AD patients who did not take AChE inhibitors, Koenig said. Meanwhile, the Watertown, Massachusetts-based biopharmaceutical company plans to move ahead with a three-month Phase 2b trial of its α7 agonist in schizophrenia patients by late 2009, based on promising biomarker Phase 1b data (see ARF related conference story). The schizophrenia trial will test EnVivo’s compound on top of existing antipsychotics, using parts of the CogState and NTB batteries as primary endpoints and functional measures from a new interview-based assessment of cognition, the Schizophrenia Cognition Rating Scale (SCoRS), as secondary endpoints.

Targacept, Inc. of Winston-Salem, North Carolina, also has an α7 agonist in the running—at this point for treatment of cognitive dysfunction in schizophrenia. The compound (TC-5619) is a full agonist that is selective for α7 and, unlike many other α7 compounds, does not hit 5-HT serotonin receptors. In rat models of social interaction and novel object recognition, the compound worked best at lower doses, and its pharmacokinetics and safety profiles have looked good in Phase 1 studies. The take-home message from Pat Lippiello’s talk at the nAChR symposium was that α7 compounds have potential not only to help with cognitive dysfunction, but also with other symptoms such as withdrawal and flattened affect in schizophrenia. The company plans to begin recruitment for a Phase 2 proof-of-concept trial of its α7 compound in schizophrenia later this year, and will consider clinical development of the agonist for AD and ADHD going forward, Lippiello said.

Partnering with Siena Biotech in Siena, Italy, Wyeth also has an α7 agonist (WYE-103914/SEN34625) in the works. This compound is a full agonist that steers clear of other nAChR subtypes and 5-HT receptors. It uses a flex-bridge chemical platform thus far unique to α7 compounds in in-vitro assays. This agonist seems to hit a different pathway; it increases glutamate transmission, whereas other α7 agonists have only elicited increases in dopamine and acetylcholine signaling. Whether these features are advantageous or disadvantageous remains to be seen, said Wyeth chemist Simon Haydar, a coauthor on two posters showing preclinical data on the compound. WYE-103914/SEN34625 has shown pro-cognitive and potential neuroprotective activities in rodent studies. In addition, the agonist seems able to improve cognition without affecting the activity of an antipsychotic drug offered in combination. This property would be critical for future drugs treating cognitive dysfunction associated with schizophrenia. For coverage of newer nAChR approaches, see Part 2. For more detail, see Neuroscience 2009 abstracts.—Esther Landhuis.

This is Part 1 of a three-part series. See also Parts 2 and 3.

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References

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Paper Citations

Sabri O, Kendziorra K, Wolf H, Gertz HJ, Brust P. Acetylcholine receptors in dementia and mild cognitive impairment. Eur J Nucl Med Mol Imaging. 2008 Mar;35 Suppl 1:S30-45. PubMed.
Wang HY, Stucky A, Liu J, Shen C, Trocme-Thibierge C, Morain P. Dissociating beta-amyloid from alpha 7 nicotinic acetylcholine receptor by a novel therapeutic agent, S 24795, normalizes alpha 7 nicotinic acetylcholine and NMDA receptor function in Alzheimer's disease brain. J Neurosci. 2009 Sep 2;29(35):10961-73. PubMed.
Dziewczapolski G, Glogowski CM, Masliah E, Heinemann SF. Deletion of the alpha 7 nicotinic acetylcholine receptor gene improves cognitive deficits and synaptic pathology in a mouse model of Alzheimer's disease. J Neurosci. 2009 Jul 8;29(27):8805-15. PubMed.
Liu Q, Huang Y, Xue F, Simard A, Dechon J, Li G, Zhang J, Lucero L, Wang M, Sierks M, Hu G, Chang Y, Lukas RJ, Wu J. A novel nicotinic acetylcholine receptor subtype in basal forebrain cholinergic neurons with high sensitivity to amyloid peptides. J Neurosci. 2009 Jan 28;29(4):918-29. PubMed.
Bencherif M, Lippiello PM. Alpha7 neuronal nicotinic receptors: the missing link to understanding Alzheimer's etiopathology?. Med Hypotheses. 2010 Feb;74(2):281-5. PubMed.

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Chicago: Move Over, Agonists; Make Way for Modulators

06 Nov 2009

At a satellite symposium, “Nicotinic Acetylcholine Receptors (nAChRs) as Therapeutic Targets,” held days before the Society of Neuroscience’s annual meeting in Chicago last month, clinical discussion of cognitive treatments focused largely on nAChR agonists. Clearly, though, the arena of potential compounds has expanded. Mirroring a trend seen in muscarinic AChR drug development (see ARF related news story), the newest nAChR compounds are positive allosteric modulators (PAMs), which avoid the neurotransmitter site and instead bind alternate regions on the nicotinic AChR.

Though PAMs have yet to strut their stuff in the clinic, many in the field suspect they have an edge over agonists. Nicotinic AChRs desensitize with repeated stimulation at the neurotransmitter binding site, so a nagging problem with agonists has been that they induce tolerance and thereby lose effectiveness over time.

In an overview of advances in α7 PAMs, John Dunlop of Wyeth Research in Princeton, New Jersey, noted that in preclinical studies, the efficacy profile for PAMs parallels that of agonists. α7 PAMs can correct deficits in sensory gating (see, e.g., Hurst et al., 2005 and Ng et al., 2007) and prepulse inhibition (Dunlop et al., 2009)—two noted endophenotypes that serve as electrophysiological biomarkers in schizophrenics. Modulators have also shown cognitive enhancement in vivo, for example, in rats subjected to water-maze learning tasks (Timmermann et al., 2007).

However, one perceived disadvantage of PAMs is their reliance on cholinergic transmission, which may already be sagging in Alzheimer disease and other conditions. This could also be viewed as an advantage, some scientists say, because it means PAMs do not “create” additional signaling but rather amplify normal nAChR signaling—a feature that may be favorable for long-term use. Furthermore, some PAMs, namely those classified as Type 1, may do little for the tolerance problem that plagues agonists, because only Type 2 PAMs slow receptor desensitization, said Vince Simmon, CEO of Xytis, a private biotech company in Irvine, California. But Type 2 PAMs are a double-edged sword in another way. In order to reduce desensitization rates, they gate large amounts of Ca2+, which could have neurotoxic effects, as was the case with Pfizer’s PNU-120596 and Eli Lilly’s ampakine drug LY-503430, Simmon said. Type 1 PAMs only induce a moderate, that is, two- to threefold, increase in Ca2+ influx and maintain the quick receptor desensitization, which implies they may be less potent as agonists but possibly better because they do not create extra signaling. All told, scientists said it is too early to predict whether the ideal α7 compound would be an agonist or a PAM.

Thus far, one α7 PAM has gained clearance from the U.S. Food and Drug Administration to enter clinical development. It is compound XY4083, made by Xytis. The company has published preclinical data on this Type 1 PAM (Ng et al., 2007) but did not present new findings at the recent nAChR symposium. Nor has it begun Phase 1 trials of the compound, despite having gained FDA approval for such studies in November 2008. “We are looking for financing and/or partnership,” Simmon told ARF in a phone interview. The decision about which disease(s) to target would be made with a partner, but at this point the company is leaning toward standard-of-care therapy in schizophrenia as a first indication. “With schizophrenia, there are short-term tests that can be done for cognition, for example, sensory gating. Whereas in AD, you have to do pretty long-term studies to see effects,” Simmon said. At the nAChR symposium, posters described α7 PAMs in preclinical development at Johnson & Johnson (JNJ-1930942), Abbott (A-716096), Roche (dimethylcyclopropyl-benzamides), and GlaxoSmithKline (PheTQS).

Puff, Puff
A presentation by Paul Newhouse of the University of Vermont, Burlington, provided a respite from the flood of αnumeric compound names. Rather than boosting cognitive function by tickling nAChRs with agonists or modulators, his team recruited people with mild cognitive impairment (MCI) for a pilot trial of a physiological nAChR substrate—nicotine itself.

Motivation for this trial came from earlier work by Newhouse and others showing that cholinergic mechanisms help mediate age-related shifts in the way our brains handle cognitive tasks. These studies tested predictions extending from the Resource Reduction Hypothesis, which presumes that reduced efficiency in lower-level core processes (e.g., attention, working memory, speed of memory) leads to higher-level impairments (e.g., decision making, language, problem solving). Researchers have found that people compensate for age-induced declines in core processes by increasingly shifting cognitive processing forward in the brain. As such, elders performing at the same level as younger people show more activity in frontal brain structures. Blockage of nicotinic or muscarinic AChRs with antagonists can reproduce this effect in young people, suggesting that the cholinergic system is involved in this caudal to frontal shift (aka the PASA effect).

On these grounds, Newhouse and colleagues simply asked whether nicotine, in this case offered through skin patches, would provide any measurable cognitive boost to MCI patients whose nAChR function is presumably better preserved than that of people with outright AD. Their study, which was funded by the National Institute on Aging (NIA), enrolled 74 non-smokers with amnestic MCI at three sites for a six-month double-blinded study, followed by a six-month open-label extension. During the double-blinded phase, the treatment group showed improvement on various measures including delayed word recall accuracy, choice reaction time, and speed of memory (see Newhouse et al. SfN poster abstract). “If we saw cognitive improvement, we did not lose that effect over the relatively lengthy trial,” Newhouse said. The study had no major drug-related adverse events, though the nicotine-treated group did end up with lower blood pressure. Curiously, the treatment effects were more prominent in ApoE4 homozygotes compared to people with the ApoE3 allele or just one copy of E4 (see Wilkins et al. SfN poster abstract). Thirty of 70 participants in that MCI trial had at least one E4 allele. At the Cognitive Neuroscience Society’s annual meeting held in San Francisco earlier this spring, UK researchers (Marchant et al.) also reported E4 preferential benefit to young people (ages 18-30) treated with a nicotine nasal spray. In a separate trial presented by Newhouse at the nAChR symposium, nicotine improved several core cognitive deficits in non-smoking adolescents with attention deficit hyperactivity disorder (ADHD). And recently, Pfizer has moved varenicline (a nicotinic receptor partial agonist sold under trade name Chantix for smoking addiction) into a Phase 2 trial of mild to moderate AD patients. For more on mechanisms behind cognitive enhancement, see Part 3. For details, see Neuroscience 2009 abstracts.—Esther Landhuis.

This is Part 2 of a three-part series. See also Parts 1 and 3.

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References

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Hurst RS, Hajós M, Raggenbass M, Wall TM, Higdon NR, Lawson JA, Rutherford-Root KL, Berkenpas MB, Hoffmann WE, Piotrowski DW, Groppi VE, Allaman G, Ogier R, Bertrand S, Bertrand D, Arneric SP. A novel positive allosteric modulator of the alpha7 neuronal nicotinic acetylcholine receptor: in vitro and in vivo characterization. J Neurosci. 2005 Apr 27;25(17):4396-405. PubMed.
Ng HJ, Whittemore ER, Tran MB, Hogenkamp DJ, Broide RS, Johnstone TB, Zheng L, Stevens KE, Gee KW. Nootropic alpha7 nicotinic receptor allosteric modulator derived from GABAA receptor modulators. Proc Natl Acad Sci U S A. 2007 May 8;104(19):8059-64. PubMed.
Timmermann DB, Grønlien JH, Kohlhaas KL, Nielsen EØ, Dam E, Jørgensen TD, Ahring PK, Peters D, Holst D, Christensen JK, Chrsitensen JK, Malysz J, Briggs CA, Gopalakrishnan M, Olsen GM. An allosteric modulator of the alpha7 nicotinic acetylcholine receptor possessing cognition-enhancing properties in vivo. J Pharmacol Exp Ther. 2007 Oct;323(1):294-307. PubMed.

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Chicago: Nicotinic AChRs—Mechanistic Basis for New Drug Discovery?

07 Nov 2009

All too often, basic science findings that initially seem promising never translate into viable pharmaceutical approaches, or when they do, fail miserably in clinical trials. Amid such tales of disappointment, it’s refreshing to hear about studies showing clear convergence between biology, drug action, and behavior, all in the space of a few seconds, no less. At a satellite symposium on therapeutic approaches targeting nicotinic acetylcholine receptors (nAChRs), which took place days before the Society of Neuroscience’s annual meeting in Chicago last month, Martin Sarter of the University of Michigan, Ann Arbor, had such a story to tell. His lab developed a way to record neurotransmitter release at sub-second resolution in freely moving animals. Using this technology, the researchers identified an electrophysiological readout for key attention tasks in rodents. Better yet, treatment with nicotine or nAChR agonists can tweak this readout and, correspondingly, affect task performance. “The neuroscience and the pharmacology come together,” Sarter told ARF. “That’s something you don’t see very often in this business.” The findings could have implications for screening new nAChR-targeting drugs.

Noting its role in regulating sleep/wake cycles and other types of arousal setting, scientists had presumed that the cholinergic system operates on the level of minutes, even tens of minutes. But once Sarter and colleagues pioneered their technology for listening to neurotransmitter release in real time, they made a surprising discovery. By recording acetylcholine release from presynaptic terminals in the prefrontal cortex of rats engaged in a cue detection task, the researchers saw characteristic “transients”—bursts of cholinergic activity—that mediated task performance on a timescale not of minutes, but of seconds (Parikh et al., 2007). “That’s really, really short,” Sarter said. “It was a whole new story.”

The rats in these studies wore surgically implanted, choline-sensitive microelectrodes, and were trained to perform a sustained attention task involving repeated rounds of pressing one lever if a cue light went on and pressing another lever if the light remained off. The researchers measured the accuracy of cue detection as well as response speed and other parameters. When they treated the rats with mecamylamine to block nACh receptors, the cue detection rate dropped considerably; this indicates that the nAChR system is required for this attention task and for generating the underlying electrophysiological signature, that is, the rapid transients. Quicker detection rates were found to be associated with larger amplitudes and faster decay rates of cholinergic transients, Sarter said.

For the next phase of analysis, the researchers made the attention task harder by introducing a distractor—for example, house lights flashing on and off. This lowered the baseline task performance and made the system more useful for determining how well various treatments influence the transients and, in turn, improve behavior. With the lower baseline, behavior was measured in terms of how long it took the rats to recover normal (i.e., distractor-free) levels of performance. The researchers found that nicotine helped the rats detect cues faster; importantly, this corresponded with larger amplitudes on their transients compared to vehicle treatment. Treatment with Abbott’s α4β2 agonist ABT-089 improved task performance even more, and further sharpened the transients—that is, gave them higher amplitude and faster decay rate. However, treatment with an α7 agonist (ABT-107) did not help attention, and increased the duration of acetylcholine release. The readout for this was cholinergic transients with a longer “tail.”

Astute readers will recall from Part 1 of this series that Abbott has discontinued development of ABT-089 for AD and ADHD, in part because the α4β2 agonist failed to show efficacy in a recent Phase 2 AD trial. Sarter does not necessarily find these clinical disappointments inconsistent with his rat studies, where the compound has shown promise. “It is difficult to see how such a compound would work in AD, given that the prefrontal ‘cue detection network’ including the glutamatergic-cholinergic interactions and the prefrontal output neurons that are required for mediation of the performance effects (in the rat cue detection task) are quite disrupted,” he wrote in an e-mail to ARF. “To use a simple analogy, it is difficult to enhance the workings of a component of a circuit if the circuit is in a state of advanced disintegration.” As for the α7 agonist (ABT-107), the fact it did not improve task performance in the rat studies does not imply these compounds would be ineffective in other cognitive domains, such as memory, he said.

Despite these nuances, Sarter believes his animal set-up could be useful in screening for nAChR-stimulating cognition enhancers. “The rats do the detection task. You see the transients. You give the drug. You see what it does to transients and performance at the same time,” he said. “You look for drugs that make (transients) bigger and sharper.” When it comes to targeting the cholinergic system for therapeutics, it is too simplistic to think in terms of having too much or too little neurotransmitter, he said. Compounds should work if they allow proper orchestration of the transients.

Sarter’s team has preliminary data suggesting that the transients could have relevance in a disease context. In a rat neurodevelopmental model for schizophrenia that performs poorly in attention tasks, nicotine treatment failed to generate cholinergic transients (see Wescott et al. SfN poster abstract). Thus far, standard antipsychotics also have been unable to bring back these transients. Any compound that could restore the cholinergic bursts could thus be interesting to pursue, Sarter said.—Esther Landhuis.

This story concludes a three-part series. See also Parts 1 and 2.

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Chicago: AD and Epilepsy—Joined at the Synapse?

13 Nov 2009

One disease presents as electrical jolts that sporadically seize the brain, the other as forgetfulness and disorientation that progressively worsen. “At first pass, you’d think they are different universes,” said Helen Scharfman, Nathan Kline Institute for Psychiatric Research, Orangeburg, New York, in a symposium on epilepsy and Alzheimer disease at the Society for Neuroscience annual meeting held 17-21 October 2009 in Chicago. In the past two years, however, a closer look at these two conditions has revealed some fundamental similarities. “In both diseases, neuronal activity has gone awry in timing and synchronization,” Scharfman told the assembly of epilepsy and AD researchers. The symposium’s four presentations, as well as several posters on the topic, gave attendees plenty to chew on—highlighting links in electrophysiology, epidemiology, and animal models of the two diseases. “We believe there is some compelling clinical and experimental evidence suggesting that there could be an overlap between epilepsy and dementia, and that there could be a fruitful ground for collaboration for investigators in these areas,” said symposium organizer Lennart Mucke of the Gladstone Institute of Neurological Disease in San Francisco, California.

Mucke and colleagues provided the spark for this interface when they teamed up with epileptologist Jeffrey Noebels of Baylor College of Medicine in Houston, Texas, to reveal epileptiform activity in an AD mouse model (Palop et al., 2007 and ARF related news story). These abnormal discharges escape casual observation but readily showed up in electroencephalography (EEG) recordings done on freely moving J20 mice. “This confirmed our suspicion that, against what people expected, Aβ wasn’t shutting the network down,” Mucke told the SfN audience. Instead, Aβ was inducing peaks of overexcitation, which then trigger suppressive mechanisms, he suggested. “The system flips and flops between these states, resulting in an imbalance between overexcitation and inhibitory pathways in memory centers that we predict are key components of AD pathophysiology,” Mucke said.

His lab then asked whether this network instability affects adult neurogenesis. Using retrovirus to label newborn neurons in the dentate gyrus of J20 mice, the researchers followed the cells’ morphological and functional development from birth. The newborn granule cells seemed to develop on a fast track in the early stages, but later their maturation slowed down as compared to wild-type granule cells, Mucke reported. His team was able to restore normal neurogenesis by either blocking GABAergic signaling early on or inhibiting calcineurin at later stages. These findings are in press in Cell Stem Cell, Mucke said.

In addition to demonstrating that the network instability in AD mice has functional consequences, Mucke noted that his lab has found sodium channel abnormalities in J20 inhibitory interneurons. These findings, currently under review, could help reconcile the conundrum that Aβ, which is thought to dampen synaptic activity (Kamenetz et al., 2003), can in fact make networks hyperactive. If inhibitory interneurons are impaired, the end result is network disinhibition, Mucke told ARF in a post-symposium interview. Work published earlier this year by Dennis Selkoe, Brigham and Women’s Hospital, Boston, and colleagues suggests that Aβ-induced neuronal overstimulation can disrupt uptake of extracellular glutamate, making neurons more prone to long-term depression (Li et al., 2009 and ARF related news story).

A recent in-vivo calcium imaging study led by Arthur Konnerth at Technical University Munich in Germany also lent credence to the notion that Aβ can make some neurons hyperactive (Busche et al., 2008 and ARF related news story). In that analysis, researchers imaged cortical neurons from APP23xPS45 mice and showed that while a third of the neurons were less active, about a fifth of them actually became more active.

At SfN, Konnerth presented new data from his lab showing that these changes in activity status seem to have functional consequences. His team analyzed neurons from the primary visual cortex of wild-type and APP23xPS45 mice. They chose the visual system because the relationship between physical input (i.e., shape and orientations of images) and how the neurons typically respond to it is well described. Plus, the researchers had found amyloid plaques and impaired spontaneous neuronal activity in the mice’s visual cortex in their earlier study. In the new analysis, some cells in the APP23xPS45 visual cortex were hypoactive and some were hyperactive, as expected. But more than that, the silent neurons in the AD mice showed no response to sensory stimuli. This was in contrast to wild-type mice, where a proportion of the silent cells were still responsive. More serious problems showed up in the APP23xPS45 hyperactive neurons. These cells essentially lost their ability to respond precisely to specific orientations, compared with hyperactive cells in the wild-type visual cortex, Konnerth reported.

The Mucke lab presented several SfN posters with mechanistic data on the relationship between synaptic dysfunction, network hyperexcitability, and behavioral deficits. Julie Harris and colleagues tried to get a handle on where Aβ first acts within the entorhinal-hippocampal network to wreak havoc on networks and behavior. To that end, the scientists analyzed transgenic mice expressing human mutant APP primarily in layer 2/3 pyramidal cells of the medial entorhinal cortex. These neurons signal to granule cells of the dentate gyrus, which had no detectable APP expression. By the time they were six months old, the transgenic mice had molecular changes in the dentate gyrus, as well as defects in several behavioral assays, but with still hardly any Aβ deposition in the dentate gyrus. From these findings, they determined that Aβ acts trans-synaptically to induce molecular and functional impairments (see Harris et al. SfN abstract). In a separate poster (Palop et al. SfN abstract), Jorge Palop and colleagues showed that exacerbating Aβ-induced epileptiform activity in J20 mice further intensified the remodeling of hippocampal circuits and other molecular abnormalities they had characterized in their previous study (Palop et al., 2007). The data suggest to the authors that these changes are indeed a downstream consequence of the neuronal overexcitability.

Scientists in Finland have recently extended the observations of hyperactivity to another AD transgenic line, PSAPP (Minkeviciene et al., 2009 and ARF related news story). In an SfN poster (Leiser et al. SfN abstact), researchers at Pfizer Global Research and Development, Princeton, New Jersey, report epileptiform activity in that same strain as well as another, Tg2576. Steve Leiser—who has since moved to AstraZeneca in Wilmington, Delaware—and colleagues found hyperexcitability in PSAPP mice at 21, 34, and 47 weeks, and in Tg2576 mice at 27 and 34 weeks. Like the J20 mice, the Tg2576 and PSAPP animals showed freezing behavior but had no tremors, convulsions, or other visible signs of overt seizures. Both strains showed epileptiform activity only after Aβ deposition had begun but before measurable cognitive decline, Leiser wrote in an e-mail to ARF. The EEG recordings also revealed a shift from delta (slow-wave) toward theta activity in the transgenic mice, suggesting their brains are in a hyperexcited state. Leiser noted that this pattern parallels that of AD patients, who show an increase in theta power early in the disease, and later shift back to a high delta state. “These EEG features could indicate a hyperexcitable state predictive of seizures, and might serve as a biomarker for preclinical AD,” he wrote. For epidemiological and clinical data supporting the AD-epilepsy connection, see also Part 2.—Esther Landhuis.

This is Part 1 of a two-part series. See also Part 2.

References

News Citations

Paper Citations

Palop JJ, Chin J, Roberson ED, Wang J, Thwin MT, Bien-Ly N, Yoo J, Ho KO, Yu GQ, Kreitzer A, Finkbeiner S, Noebels JL, Mucke L. Aberrant excitatory neuronal activity and compensatory remodeling of inhibitory hippocampal circuits in mouse models of Alzheimer's disease. Neuron. 2007 Sep 6;55(5):697-711. PubMed.
Kamenetz F, Tomita T, Hsieh H, Seabrook G, Borchelt D, Iwatsubo T, Sisodia S, Malinow R. APP processing and synaptic function. Neuron. 2003 Mar 27;37(6):925-37. PubMed.
Li S, Hong S, Shepardson NE, Walsh DM, Shankar GM, Selkoe D. Soluble oligomers of amyloid Beta protein facilitate hippocampal long-term depression by disrupting neuronal glutamate uptake. Neuron. 2009 Jun 25;62(6):788-801. PubMed.
Busche MA, Eichhoff G, Adelsberger H, Abramowski D, Wiederhold KH, Haass C, Staufenbiel M, Konnerth A, Garaschuk O. Clusters of hyperactive neurons near amyloid plaques in a mouse model of Alzheimer's disease. Science. 2008 Sep 19;321(5896):1686-9. PubMed.
Minkeviciene R, Rheims S, Dobszay MB, Zilberter M, Hartikainen J, Fülöp L, Penke B, Zilberter Y, Harkany T, Pitkänen A, Tanila H. Amyloid beta-induced neuronal hyperexcitability triggers progressive epilepsy. J Neurosci. 2009 Mar 18;29(11):3453-62. PubMed.

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Chicago: AD and Epilepsy—Lessons from the Clinic, Animals

13 Nov 2009

The Society for Neuroscience annual meeting, which took place 17-21 October 2009 in Chicago, featured a rare convergence of epilepsy and Alzheimer disease researchers at a symposium focused on shared features of these two disorders. There is accumulating animal data for such a connection: At least three strains of AD transgenic mice show epileptiform activity on electroencephalography (EEG) recordings, and the data hint that the EEG changes could be a signature for impending cognitive decline (see Part 1). There is also growing suspicion that the same may be true for AD patients. In a slide talk, Jeffrey Noebels, an epilepsy researcher at Baylor College of Medicine in Houston, Texas, reviewed clinical and pathological evidence for overlap between AD and temporal lobe epilepsy (TLE). He proposed that seizures may be a common feature of AD that escapes detection by conventional EEG. Physicians spend a lot of time figuring out whether patients have AD or temporal lobe epilepsy (TLE), Noebels said, noting, “It’s actually possible to have both.”

Epidemiological data points in this direction. Epilepsy is generally a childhood disease but seems to occur more frequently in seniors with dementia (Cloyd et al., 2006). More than half of the rare early onset AD patients with APP duplications have seizures (Cabrejo et al., 2006), and for those with very early onset of dementia (i.e., under 40 years of age), the incidence of epilepsy rises to 83 percent (Snider et al., 2005).

When made to hyperventilate, people who carry an ApoE4 allele, which puts them at increased AD risk, show epileptiform activity on EEG (Ponomareva et al., 2008). An SfN poster by Jesse Hunter, Eli Lilly and Co., Indianapolis, Indiana, and colleagues showed mouse data that seem to jibe with this. The scientists analyzed E2, E3, and E4 targeted replacement mice (i.e., transgenic mice that express a given human ApoE allele in the endogenous locus) and report that old E4 females developed seizures whereas E2 and E3 mice did not (Hunter et al. SfN poster abstract).

Meanwhile, the overall proportion of people with AD and mild cognitive impairment patients who have non-convulsive seizures remains unknown—in large part because probing the right brain regions is difficult. It is much easier to pick up epileptiform activity in mice, where the cortex is just a millimeter thick and the hippocampus is disproportionately large, Noebels said. In the human brain, surface EEG readily misses abnormal activity in the temporal lobe, the main site of early AD pathology, because this brain structure is small and deep—buried within a comparatively thick cortex. Noebels described a case where nothing showed up on a surface EEG recording, but implanted depth electrodes revealed a full-blown seizure. Mindful of these challenges, scientists at the University of California, San Francisco, launched a study earlier this year to determine the incidence of epileptiform activity in dementia patients by doing 36-hour EEG and magnetoencephalography (MEG) on study volunteers with AD and MCI, said symposium chair Lennart Mucke of the Gladstone Institute of Neurological Disease in San Francisco, California.

The notion of an interface between AD and TLE appears to be gaining traction among AD researchers. “People I talked with were fascinated by the overlap, which seems wider than is realized by the experimental and clinical evidence that has accumulated in a fairly short period of time,” Mucke told ARF. Steve Barger of the University of Arkansas, Little Rock, said the overlap with TLE might explain the vacillating clinical behavior of some AD patients, who can seem completely disoriented for a time and then snap back into an almost-normal state. “Maybe you don’t really get cognitive deficits until you get something that approaches epileptiform activity,” he said.

Helen Scharfman of the Nathan Kline Institute for Psychiatric Research, Orangeburg, New York, proposed several potential shared mechanisms between AD and TLE. The first was inflammation. Clinical data suggest that infection is a risk factor for TLE, and animal studies have shown that cyclooxygenase-2 inhibitors can help restore long-term potentiation in an AD model (Kotilinek et al., 2008) and decrease seizures in a TLE model (Jung et al., 2006). Another common mechanism could be neurogenesis, Scharfman said. In rodents, the dentate gyrus makes new neurons after seizures (Bengzon et al., 1997), and the neurogenesis seems to contribute to hippocampal network reorganization (Parent et al., 1997). Neurogenesis is also increased early—by three to four months of age—in Tg2576 AD mice (Lopez-Toledano and Shelanski, 2007).

Some TLE models might actually be unique tools for studying AD because they model features that amyloidosis models don’t capture, Scharfman suggested. For example, the loss of basal forebrain cholinergic neurons is a common feature in AD patients, yet is hardly detectable in mouse models based on APP mutations and overexpression. However, cholinergic deficits do appear in the kindling model of TLE. (Kindling refers to repeated stimulation that leaves neurons oversensitized and prone to seizures.) The kindling model also shares a number of common dentate gyrus features with the APP/PS1 mouse, including reduced dentate hilar cells, interneuron loss, survival of granule cells, mossy fiber sprouting, and increased expression of neuropeptide Y.

The experimental and clinical support for an AD-TLE overlap begs the obvious question of whether anti-epileptic drugs can stem the network dysfunction that presumably leads to dementia. So far, this hasn’t been the case. When Mucke and colleagues treated J20 mice with the anti-epileptic drug phenytoin, a sodium channel blocker, the animals had even more seizure activity. The findings were perplexing, he said, but perhaps not so unreasonable given that some epilepsy patients with loss-of-function mutations in voltage-gated sodium channels also respond poorly to phenytoin. That observation led his team to look for sodium channel abnormalities in the cortex of their J20 mice (see Part 1).

Thus far, AD patients treated with epilepsy drugs have not fared any better. Phenytoin (sold under the trade name Dilantin) is often given to stop seizures in AD, but it tends to worsen cognition, Mucke told ARF. And a recent Phase 3 trial of the anticonvulsant valproate failed to improve neuropsychiatric symptoms in AD patients. Principal investigator Pierre Tariot of Banner Alzheimer’s Institute, Phoenix, Arizona, presented the trial results at this year’s International Conference on Alzheimer’s Disease (ICAD) in Vienna (see Tariot et al. ICAD abstract).

Much work remains to contrast and compare the mechanisms at work in epilepsy and network dysfunction in AD, Mucke said. “But at least people felt there was good reason to pay attention to that interface.”—Esther Landhuis.

This concludes a two-part series. See also Part 1.

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References

News Citations

Paper Citations

Cloyd J, Hauser W, Towne A, Ramsay R, Mattson R, Gilliam F, Walczak T. Epidemiological and medical aspects of epilepsy in the elderly. Epilepsy Res. 2006 Jan;68 Suppl 1:S39-48. PubMed.
Cabrejo L, Guyant-Maréchal L, Laquerrière A, Vercelletto M, De la Fournière F, Thomas-Antérion C, Verny C, Letournel F, Pasquier F, Vital A, Checler F, Frebourg T, Campion D, Hannequin D. Phenotype associated with APP duplication in five families. Brain. 2006 Nov;129(Pt 11):2966-76. PubMed.
Snider BJ, Norton J, Coats MA, Chakraverty S, Hou CE, Jervis R, Lendon CL, Goate AM, McKeel DW Jr, Morris JC. Novel presenilin 1 mutation (S170F) causing Alzheimer disease with Lewy bodies in the third decade of life. Arch Neurol. 2005 Dec;62(12):1821-30. PubMed.
Ponomareva NV, Korovaitseva GI, Rogaev EI. EEG alterations in non-demented individuals related to apolipoprotein E genotype and to risk of Alzheimer disease. Neurobiol Aging. 2008 Jun;29(6):819-27. PubMed.
Kotilinek LA, Westerman MA, Wang Q, Panizzon K, Lim GP, Simonyi A, Lesne S, Falinska A, Younkin LH, Younkin SG, Rowan M, Cleary J, Wallis RA, Sun GY, Cole G, Frautschy S, Anwyl R, Ashe KH. Cyclooxygenase-2 inhibition improves amyloid-beta-mediated suppression of memory and synaptic plasticity. Brain. 2008 Mar;131(Pt 3):651-64. PubMed.
Jung KH, Chu K, Lee ST, Kim J, Sinn DI, Kim JM, Park DK, Lee JJ, Kim SU, Kim M, Lee SK, Roh JK. Cyclooxygenase-2 inhibitor, celecoxib, inhibits the altered hippocampal neurogenesis with attenuation of spontaneous recurrent seizures following pilocarpine-induced status epilepticus. Neurobiol Dis. 2006 Aug;23(2):237-46. PubMed.
Bengzon J, Kokaia Z, Elmér E, Nanobashvili A, Kokaia M, Lindvall O. Apoptosis and proliferation of dentate gyrus neurons after single and intermittent limbic seizures. Proc Natl Acad Sci U S A. 1997 Sep 16;94(19):10432-7. PubMed.
Parent JM, Yu TW, Leibowitz RT, Geschwind DH, Sloviter RS, Lowenstein DH. Dentate granule cell neurogenesis is increased by seizures and contributes to aberrant network reorganization in the adult rat hippocampus. J Neurosci. 1997 May 15;17(10):3727-38. PubMed.
López-Toledano MA, Shelanski ML. Increased neurogenesis in young transgenic mice overexpressing human APP(Sw, Ind). J Alzheimers Dis. 2007 Nov;12(3):229-40. PubMed.

External Citations

Chicago: New Technologies Help Drugs Cross Blood-Brain Barrier

15 Nov 2009

It’s hard enough to make a drug that does the right thing. Designing compounds to fight neurodegenerative disease comes with the additional challenge of making sure they actually reach the brain. At the Society for Neuroscience annual meeting held 17-21 October 2009 in Chicago, a handful of groups tackled this “problem behind the problem” of drug development by presenting different tactics for overcoming the blood-brain barrier.

Thanks to an intricate, coordinated system of tight endothelial junctions and active transport enzymes, the brain enjoys remarkable protection from pathogens and most blood-borne substances greater than 400 daltons. Unfortunately, this physiological barrier also keeps out the vast majority of compounds that could help sustain and restore the critical organ when it succumbs to Alzheimer disease or any number of central nervous system scourges.

AngioChem Inc., a biotech company in Montreal, Canada, has addressed this problem by harnessing proteins naturally expressed at high levels on brain capillaries. Low-density lipoprotein receptor-related proteins (LRPs) are endocytic and signaling receptors; they help shuttle into the brain large, essential molecules, such as glucose and insulin, which would ordinarily be excluded because of size.

Taking advantage of this transport pathway, researchers at AngioChem have designed a set of peptides that can be attached to candidate drugs, enabling them to hitch a ride with LRP across the blood-brain barrier. These peptides, cleverly called Angiopeps, bind to LRPs and can range in size from six to more than 30 amino acids. In the case of small molecules, the peptides are hooked to the compound of interest using linkers that enable them to be cleaved off once the drug crosses the blood-brain barrier. However, larger biologics—such as enzymes, nucleic acids, and monoclonal antibodies—do not require this release step.

The system is adaptable. “We can have different types of linkers, different length peptides, different numbers of peptides incorporated. There are numerous possibilities,” Betty Lawrence, vice president of development, told ARF. “It’s something that needs to be examined for each project.” The technology is called the Engineered Peptide Compound (EPiC) platform.

Thus far, AngioChem has applied the EPiC system with small anticancer drugs, larger peptides, as well as with small interfering RNAs (siRNAs) and monoclonal antibodies. At SfN, the company presented data showing brain uptake of various EPiC compounds and early clinical trials in brain cancer patients of its lead candidate, ANG1005 (paclitaxel, a mitotic inhibitor used in cancer chemotherapy). With the EPiC platform, three anticancer drugs (paclitaxel, doxorubicin, and etoposide) given intravenously were able to reach the brain five to 10 times faster than they would have without EPiC.

The researchers showed similar enhancement with monoclonal antibodies and siRNAs—large molecules that are far worse at crossing the blood-brain barrier. Ordinarily, these biologics enter the brain >100 times more slowly than glucose, i.e., their transport rates are less than 1 percent of glucose’s. With EPiC, they were getting into the brain at rates ranging from five to 10 percent of glucose’s, CEO Jean-Paul Castaigne told ARF. In Alzheimer disease immunotherapy, how much antibody actually crosses the BBB remains a big, unsolved question even as expensive large-scale trials are under way.

In a Phase 1 trial of 55 people with malignant gliomas, the EPiC-paclitaxel drug had no CNS toxicity and did not trigger an immune reaction when given to patients as two intravenous infusions three weeks apart. The drug was able to reach the brain tumors at therapeutic concentrations (see Drappatz et al. SfN poster abstract). And in a separate Phase 1b/2 study of patients with more advanced disease (i.e., advanced solid tumors and brain metastases), the drug reduced tumor sizes in multiple organs in 15 of 21 participants (see Kurzrock et al. SfN poster abstract).

AngioChem is collaborating with two different companies to develop EPiC compounds for neurodegenerative disease—one a monoclonal antibody (ANG3101), the other a siRNA (ANG3201). Castaigne said he cannot at this point disclose which companies and therapeutics are involved. However, one can make educated guesses. At an AngioChem-sponsored SfN breakfast symposium, Karoly Nikolich, chairman of AngioChem’s scientific advisory board and a consulting professor at Stanford University, Palo Alto, California, specifically mentioned Alzheimer and Parkinson diseases in a talk on how EPiC can be used to deliver substances besides paclitaxel. The next speaker at the symposium was Dinah Sah of Alnylam Pharmaceuticals in Cambridge, Massachusetts. Alnylam, which specializes in therapeutics based on RNA interference technology, has a grant from the Michael J. Fox Foundation to develop novel PD therapies (see company news release).

The monoclonal antibody and siRNA programs are in the early preclinical stage, with only animal data. The company has successfully incorporated the Angiopep backbone onto the target molecules, and has shown that the new products efficiently cross the blood-brain barrier, reach the brain parenchyma, and get to their target cells in mice, Castaigne told ARF. “This is really early research. We have signals that it is going in the right direction,” he said. “We are now improving and optimizing.”

Meanwhile, other researchers are taking a different tack to help therapeutics reach the brain. These methods use high-frequency sound waves to open the blood-brain barrier in a temporary, localized manner. In a SfN slide talk, James Choi (a Ph.D. student in the lab of Elisa Konofagou at Columbia University, New York) described his latest work using focused ultrasound to deliver BBB-impermeable molecules into specific brain regions. After demonstrating they could use the technique to open the blood-brain barrier in the hippocampus of wild-type mice (Choi et al., 2007), Choi and colleagues showed they could do this in an AD mouse model (APP/PS1) without affecting the timing of BBB opening and molecular delivery (Choi et al., 2008). For the new study, presented at SfN and published last week in the journal Ultrasound in Medicine & Biology (Choi et al., 2009), the focus was safety and efficacy (i.e., how large the compounds can be, and how precisely they are delivered). First, the researchers injected into the blood microbubbles—gas-filled contrast agents used in medical sonography—which interact with the focused ultrasound (into the left hippocampus, in this case) and serve as a contrast agent to enhance imaging. Once the BBB is open (it usually remains so for a day), they intravenously injected fluorescent-tagged dextrans of 3, 70, and 2,000 kDa. The 3- and 70-kDa dextrans made it into the brain—specifically into the left hippocampus and not the right. But there was a cutoff; the 2,000-kDa compound did not go through. Concluding his talk, Choi said, “We’ve delivered into the brain pharmacologically sized agents that were localized to the target region using a noninvasive technique.” The scientists are now optimizing the ultrasound parameters for uniform BBB opening; specifically, they are trying to identify exactly how and when it opens, and to target specific brain regions associated with AD and PD (such as the hippocampus and substantia nigra), Konofagou wrote in an e-mail to ARF.

Jessica Jordao is a Ph.D. student working with Isabelle Aubert and Kullervo Hynynen at Sunnybrook Health Sciences Center of the University of Toronto, Ontario, Canada. She used a similar focused ultrasound method for passive immunotherapy in an AD mouse model (TgCRND8). The researchers injected microbubbles, along with a magnetic resonance imaging (MRI) contrast agent and anti-Aβ antibodies into the tail veins of four-month-old mice. The TgCRND8 strain develops plaques by three months. They then applied focused ultrasound to four cortical spots in the right hemisphere of the brain while the left side served as the negative control, and sacrificed the mice at four hours, two days, and four days after treatment. Using immunoprecipitation and Western blot analysis, they confirmed that the Aβ antibodies only found their way into the right side of the brain. By four days, the treatment had brought a 12 percent reduction in number of plaques and mean plaque size, as well as a 23 percent decrease in Aβ surface area, Jordao said.

One difference between the two ultrasound protocols is that the Columbia method does not require MRI for targeting, and is hence less expensive, Konofagou said.—Esther Landhuis.

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References

Paper Citations

Choi JJ, Pernot M, Small SA, Konofagou EE. Noninvasive, transcranial and localized opening of the blood-brain barrier using focused ultrasound in mice. Ultrasound Med Biol. 2007 Jan;33(1):95-104. PubMed.
Choi JJ, Wang S, Brown TR, Small SA, Duff KE, Konofagou EE. Noninvasive and transient blood-brain barrier opening in the hippocampus of Alzheimer's double transgenic mice using focused ultrasound. Ultrason Imaging. 2008 Jul;30(3):189-200. PubMed.
Choi JJ, Wang S, Tung YS, Morrison B, Konofagou EE. Molecules of various pharmacologically-relevant sizes can cross the ultrasound-induced blood-brain barrier opening in vivo. Ultrasound Med Biol. 2010 Jan;36(1):58-67. PubMed.

External Citations

Chicago: The Vampire Principle—Young Blood Rejuvenates Aging Brain?

20 Nov 2009

Bram Stoker would have loved it. One hundred twelve years after the Irish author penned Dracula, his bone-chilling masterpiece, scientists at last month’s Society for Neuroscience conference in Chicago confirmed the vampire principle, whereby young blood keeps an aging organism vigorous. Of course, the qualifications that must follow such a breezy analogy are manifold. To begin with, the research involved not fair maidens and an ancient count but young and old mice, none of them evil. But in essence, scientists did report that admixing young blood to old rejuvenated the aging brain’s otherwise flagging output of newly generated neurons. The scientists identified some of the molecular factors, to boot. Presented October 19 in a talk by Saul Villeda, a Ph.D. student in the laboratory of Tony Wyss-Coray at Stanford University, the research features a combination of the lab’s parabiosis (i.e., blood-mixing) experiments with proteomics and follow-up studies in vitro and in vivo. The data presented to date are about aging, not Alzheimer disease, but the scientists are actively studying what their findings could mean for this neurodegenerative disease.

Aging remains the biggest risk factor for AD, and it can be viewed as a shift over time in the body’s balance of regenerative capacity versus degeneration, Villeda said. The immune system, in particular, changes with aging. Indeed, many of the signaling proteins active in aging, in stressed neurons, and in activated glial cells are proteins that were originally named by immunologists but function in the brain, as well. “It has been shown that as we age, and in AD, inflammatory proteins in blood correlate with degenerative brain disease in certain ways. The drive behind my work is that I am interested particularly in the regenerative aspect of blood-derived factors, and I view neural stem cells as a readout of that,” Villeda said.

The importance of adult neurogenesis is not fully established, but scientists increasingly believe the new neurons are functionally relevant to olfaction and perhaps learning (e.g., Kokovay et al., 2008). Adult neurogenesis in young and old animals also responds to external stimuli such as exercise, which increases blood supply to the brain and benefits learning and memory.

Wyss-Coray’s group has found, as have published studies before, that adult neurogenesis in the hippocampal subgranular zone of mice dwindles as they age, to where it hovers near zero by the time they are two years old. To pinpoint peripheral factors that might correlate with the age-related decline in neurogenesis, the Wyss-Coray lab conducted a proteomics study of blood from healthy humans at different ages and then tested if the protein signature that came up in this experiment was able to predict age in a similar proteomics run in mice. (As in an earlier proteomics study [Ray et al., 2007; Britschgi et al., 2009], the Stanford scientists restricted their comparison to proteins involved in what they call the “communicome,” i.e., the several hundred secreted proteins known to be involved in signaling among cells of the immune system. This narrows by about a factor of 100 to 1,000, respectively, the challenge of drawing biological meaning out of changes in the entire transcriptome or proteome.) This new proteomics experiment generated a short list of some 12 plasma proteins, of which eotaxin and MCP-1 looked most intriguing, Villeda said.

To take these correlative clues to a functional level, the researchers availed themselves of a technique called parabiosis, where a scientist opens the peritoneum of two mice and sutures them together. This is not vascular surgery; rather, the capillary beds of both mice fuse as their tissue heals. Jian Luo in the Wyss-Coray lab in this way conjoined several dozen pairs of one two-month old and one 18-month old mice. The pairs then lived as unequal Siamese twins of sorts (one small and fluffy, one large and a bit scruffy). They did that for two months while old blood mixed with young. Parabiosis is not new in science (e.g., Mildner et al., 2007), and indeed has inspired artists over the years (e.g., The Two Fridas [.pdf]; Parabiosis). The new aspect here is the combination of young, old, and a neurogenesis readout.

When the mice were four and 20 months old, respectively, Villeda assessed several readouts in the brain. The scientists were startled to find that the young mice exposed to old blood had a drop in neurogenesis as measured by doublecortin-positive cells in the hippocampus, whereas the old mice exposed to young blood enjoyed a threefold boost in their neurogenesis. The newly differentiated neurons in these “Dracula” mice grew longer neurites, too. Also in the hippocampus, the 20-month-old mice were spared the increase in activated microglia that is typically seen in aging mice, whereas the four-month-old mice did show that increase.

Next, the scientists probed whether the candidate factors eotaxin and MCP-1 might play a role in this phenomenon. The concentration of both proteins went up in the brain of young mice connected to old blood, as indeed it does in normally aging mice. These proteins also increase with age in human plasma and CSF, Villeda noted. When tested in vitro, eotaxin slowed stem cell proliferation and neurosphere growth. Similar results came out of in-vivo imaging using a transgenic mouse that expresses the luciferase reporter gene under the control of the doublecortin promoter. In this model, differentiation of neural progenitor cells lights up in living animals placed under a bioluminescence camera (Couillard-Despres et al., 2008). This readout dimmed within days after these mice were injected with eotaxin. The mice were made in the lab of Ludwig Aigner of Paracelsus Medical University in Salzburg, Austria, a coauthor of this study.

“We think these proteins are bad guys in aging,” Villeda said. Eotaxin has its own literature, though hardly any published work in brain (but see Xia et al., 1998; Choi et al., 2008). At the conference, the Stanford scientists presented no experimental data on MCP-1, which is receiving growing attention in AD research (see e.g., Galimberti et al., 2006; Galimberti et al., 2006). In an interview, Villeda noted that experiments with MCP-1 are ongoing, as well as studies with AD models and repeat parabiosis experiments in other mouse strains.

Villeda emphasized that he believes eotaxin and other such signals enter the brain from the periphery. In fact, the neurogenic niche—a specialized microenvironment in the subventricular zone and the dentate gyrus—appears particularly well positioned to receive molecular cues from the blood. These niches occupy a physical space in which the blood-brain barrier appears to be altered. Several recent studies on the microanatomy of the neurogenic niche have concluded that not only do neural stem cells in the niche make intimate contact with the vascular surface, but they also react to soluble factors released from vascular endothelial cells. Stem cells in vivo were reported to proliferate right next to blood vessels. By comparison, the rest of the brain is more solidly walled off from blood by the classic blood-brain barrier comprising pericytes and astrocytes endfeet, both of which cover the outside of the vascular endothelium (Tavazoie et al., 2008; Shen et al., 2008; Kazanis et al., StemBook).

Besides neurogenesis, what else did the scientists observe about the parabiotic mice? The lab did not conduct a comprehensive examination, but Villeda did notice one curious change. By the end of the blood-sharing period, the old mice, which had gone into it with quite a bit of gray hair, had regrown the dark fur of the C57 black 6 strain used in the experiment. They also had lower mortality than their naturally aging fellow mice and looked a little less—well—old. “There is a rejuvenating ability in young blood onto an aged brain,” said Villeda. Then he quipped, “Maybe Dracula was right: Suck young blood and live forever.”—Gabrielle Strobel.

References

Paper Citations

Kokovay E, Shen Q, Temple S. The incredible elastic brain: how neural stem cells expand our minds. Neuron. 2008 Nov 6;60(3):420-9. PubMed.
Ray S, Britschgi M, Herbert C, Takeda-Uchimura Y, Boxer A, Blennow K, Friedman LF, Galasko DR, Jutel M, Karydas A, Kaye JA, Leszek J, Miller BL, Minthon L, Quinn JF, Rabinovici GD, Robinson WH, Sabbagh MN, So YT, Sparks DL, Tabaton M, Tinklenberg J, Yesavage JA, Tibshirani R, Wyss-Coray T. Classification and prediction of clinical Alzheimer's diagnosis based on plasma signaling proteins. Nat Med. 2007 Nov;13(11):1359-62. PubMed.
Britschgi M, Wyss-Coray T. Blood protein signature for the early diagnosis of Alzheimer disease. Arch Neurol. 2009 Feb;66(2):161-5. PubMed.
Mildner A, Schmidt H, Nitsche M, Merkler D, Hanisch UK, Mack M, Heikenwalder M, Brück W, Priller J, Prinz M. Microglia in the adult brain arise from Ly-6ChiCCR2+ monocytes only under defined host conditions. Nat Neurosci. 2007 Dec;10(12):1544-53. PubMed.
Couillard-Despres S, Finkl R, Winner B, Ploetz S, Wiedermann D, Aigner R, Bogdahn U, Winkler J, Hoehn M, Aigner L. In vivo optical imaging of neurogenesis: watching new neurons in the intact brain. Mol Imaging. 2008 Jan-Feb;7(1):28-34. PubMed.
Xia MQ, Qin SX, Wu LJ, Mackay CR, Hyman BT. Immunohistochemical study of the beta-chemokine receptors CCR3 and CCR5 and their ligands in normal and Alzheimer's disease brains. Am J Pathol. 1998 Jul;153(1):31-7. PubMed.
Choi C, Jeong JH, Jang JS, Choi K, Lee J, Kwon J, Choi KG, Lee JS, Kang SW. Multiplex analysis of cytokines in the serum and cerebrospinal fluid of patients with Alzheimer's disease by color-coded bead technology. J Clin Neurol. 2008 Jun;4(2):84-8. Epub 2008 Jun 20 PubMed.
Galimberti D, Schoonenboom N, Scheltens P, Fenoglio C, Bouwman F, Venturelli E, Guidi I, Blankenstein MA, Bresolin N, Scarpini E. Intrathecal chemokine synthesis in mild cognitive impairment and Alzheimer disease. Arch Neurol. 2006 Apr;63(4):538-43. PubMed.
Galimberti D, Fenoglio C, Lovati C, Venturelli E, Guidi I, Corrà B, Scalabrini D, Clerici F, Mariani C, Bresolin N, Scarpini E. Serum MCP-1 levels are increased in mild cognitive impairment and mild Alzheimer's disease. Neurobiol Aging. 2006 Dec;27(12):1763-8. Epub 2005 Nov 22 PubMed.
Tavazoie M, Van der Veken L, Silva-Vargas V, Louissaint M, Colonna L, Zaidi B, Garcia-Verdugo JM, Doetsch F. A specialized vascular niche for adult neural stem cells. Cell Stem Cell. 2008 Sep 11;3(3):279-88. PubMed.
Shen Q, Wang Y, Kokovay E, Lin G, Chuang SM, Goderie SK, Roysam B, Temple S. Adult SVZ stem cells lie in a vascular niche: a quantitative analysis of niche cell-cell interactions. Cell Stem Cell. 2008 Sep 11;3(3):289-300. PubMed.

External Citations

Chicago: NFATs, Calcineurin—Mediators of AD, PD Pathogenesis?

02 Dec 2009

At the Society for Neuroscience annual meeting held 17-21 October 2009 in Chicago, several posters beefed up the concept that signaling via calcineurin and nuclear factors of activated T cells (NFATs) may play a central role in neurodegenerative disease pathogenesis. One study puts forth calcineurin activation as a critical step linking soluble Aβ to downstream spine morphological changes in neurons. Other analyses reveal elevated levels of activated calcineurin in people with mild cognitive impairment (MCI), suggesting that this pathway starts to malfunction early in the disease course. And work from a third research group indicates that calcineurin may function similarly in Parkinson disease, mediating the neurotoxic effects of α-synuclein oligomers.

Using multiphoton imaging, Brad Hyman and colleagues at Massachusetts General Hospital, Charlestown, have reported dramatic changes in dendrites and dendritic spines in the vicinity of amyloid plaques in Tg2576 AD transgenic mice (Spires et al., 2005). Paul Greengard’s lab at Rockefeller University, New York, showed that interfering with extracellular signal-regulated kinase (ERK) or calcineurin pathways was sufficient to block Aβ-induced spine changes in vitro (Snyder et al., 2005). And more recent work led by Massachusetts General colleague Brian Bacskai demonstrated that Aβ plaques have even more far-reaching effects—namely, chronic elevations of resting calcium levels in surrounding astrocytes that spread as calcium waves across large distances (Kuchibhotla et al., 2009 and ARF related news story). Taken together, these findings “led to the plausible hypothesis that one of the things calcium does is activate calcineurin,” Hyman told ARF. “That would be a nice way to link Greengard’s observations in vitro with our observations in vivo.”

In their SfN poster, first author Haiyan Wu and colleagues showed that Aβ exposure led to dendritic spine changes, as well as activation and nuclear translocation of NFATc4, in cultured cortical neurons. They were able to block all these effects using calcineurin or NFAT inhibitors. “That suggests that calcineurin activation was a critical signaling mechanism that converts soluble Aβ to neuronal abnormality,” Hyman said. Furthermore, his team was able to phenocopy the Aβ-induced changes in wild-type neurons by transfecting them with a constitutively active form of calcineurin, showing that calcineurin is not only necessary but sufficient to mediate the Aβ effects.

Hyman and colleagues have some evidence to suggest these findings are relevant to AD. They found accumulation of NFATc4 and an active form of calcineurin in nuclear extracts from AD compared to control brain tissue.

This jibes with new data from Chris Norris’s lab at the University of Kentucky in Lexington. In a study published last month, first author Hafiz Abdul and colleagues found higher amounts of several activated NFATs in nuclear fractions of postmortem brain tissue from MCI and AD patients, relative to healthy seniors (Abdul et al., 2009 and ARF related news story).

At SfN, Abdul and colleagues presented a poster showing a corresponding increase in activation of calcineurin—the phosphatase that regulates NFAT activity—in the same MCI and AD samples. Previous work had demonstrated increases in calpain-mediated proteolysis and activation of calcineurin in severe AD (Liu et al., 2005), and the new data from Norris’s group suggest that these abnormalities begin in earlier stages of disease. Specifically, Abdul and colleagues found that MCI cytosolic fractions had higher levels of calpain-1 and the 45 kDa activated calcineurin-Aα fragment. Treatment with oligomeric Aβ was able to induce proteolysis of calcineurin to the 45 kDa fragment in mixed hippocampal cultures, and the calpain inhibitor calpeptin tempered this activity.

On another poster from the Norris lab, Jennifer Furman and colleagues reported looking at production of inflammatory cytokines, the downstream effect of NFAT-mediated signal transduction. They found that GM-CSF, TNFα, and IL-1β are upregulated in AD, and to some extent in brain samples from MCI and milder “preclinical” patients, too. (Patients were classified as control, MCI, or preclinical based on pathology and cognitive status as determined by the Mini-Mental State Examination. The control and preclinical groups had MMSE scores of 28-29, and MCI participants had scores around 24.) Levels of the three upregulated cytokines seemed to correlate with nuclear accumulation of NFAT1, but not NFATs 2 or 3. As reported on the poster, these findings suggest that “at least some components of neuroinflammation are increased in the very early stages of AD and are due, in part, to elevated NFAT1 activation.”

Last but not least, work by Giulio Taglialatela and colleagues at University of Texas Medical Branch, Galveston, suggests that the calcineurin/NFAT pathway may play a central role in Parkinson disease, too. Earlier, his group showed that oligomeric Aβ induces calcineurin activity and triggers downstream neurotoxic events in Tg2576 neurons (Reese et al., 2008). In their SfN poster, Taglialatela and colleagues show that oligomers of α-synuclein that are structurally similar to Aβ oligomers mediate similar calcineurin-dependent events in human neuroblastoma cells and mice. Furthermore, they report increased calcineurin activity in the brains of transgenic mice overexpressing mutant α-synuclein and in brain tissue from people with dementia with Lewy bodies (DLB), a dementia spectrum disorder that combines elements of AD and PD.—Esther Landhuis.

References

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Spires TL, Meyer-Luehmann M, Stern EA, McLean PJ, Skoch J, Nguyen PT, Bacskai BJ, Hyman BT. Dendritic spine abnormalities in amyloid precursor protein transgenic mice demonstrated by gene transfer and intravital multiphoton microscopy. J Neurosci. 2005 Aug 3;25(31):7278-87. PubMed.
Snyder EM, Nong Y, Almeida CG, Paul S, Moran T, Choi EY, Nairn AC, Salter MW, Lombroso PJ, Gouras GK, Greengard P. Regulation of NMDA receptor trafficking by amyloid-beta. Nat Neurosci. 2005 Aug;8(8):1051-8. PubMed.
Kuchibhotla KV, Lattarulo CR, Hyman BT, Bacskai BJ. Synchronous hyperactivity and intercellular calcium waves in astrocytes in Alzheimer mice. Science. 2009 Feb 27;323(5918):1211-5. PubMed.
Abdul HM, Sama MA, Furman JL, Mathis DM, Beckett TL, Weidner AM, Patel ES, Baig I, Murphy MP, Levine H, Kraner SD, Norris CM. Cognitive decline in Alzheimer's disease is associated with selective changes in calcineurin/NFAT signaling. J Neurosci. 2009 Oct 14;29(41):12957-69. PubMed.
Liu F, Grundke-Iqbal I, Iqbal K, Oda Y, Tomizawa K, Gong CX. Truncation and activation of calcineurin A by calpain I in Alzheimer disease brain. J Biol Chem. 2005 Nov 11;280(45):37755-62. PubMed.
Reese LC, Zhang W, Dineley KT, Kayed R, Taglialatela G. Selective induction of calcineurin activity and signaling by oligomeric amyloid beta. Aging Cell. 2008 Dec;7(6):824-35. PubMed.

Chicago: Interest in PyroGluAβ Flares Up in Academia

09 Dec 2009

Pyroglutamate Aβ has been making a return to center stage in AD research after languishing in relative obscurity for a decade following its initial discovery. Researchers from the German biotech company Probiodrug AG in Halle rekindled interest in this post-translational modification of the peptide implicated in Alzheimer disease. Outsiders to the field of AD research, these researchers claimed at conferences that a two-step process of truncation of Aβ’s N-terminus, followed by enzymatic cyclization of its new end, generates a particularly incendiary form of Aβ that makes for a better drug target than does the full-length version itself. Meeting sessions sponsored by the company featured mostly its own scientists, leaving the field at large skeptical at first. But pyroGlu Aβ’s flame is spreading. At recent conferences and increasingly in the literature, a growing number of academic groups have begun presenting their independent studies. These academic groups are confirming some of the company’s ideas, questioning others, and pushing the topic forward in the process. See below for a summary of the main points thus far. (For previous Alzforum stories on pyroGluAβ, see ARF related AD/PD 2007 Salzburg story; ARF related Keystone 2008 story; ARF related AD/PD 2009 Prague story.)

At the ICAD conference held last July in Vienna, Austria, academic research presentations began with a talk by Cynthia Lemere of Brigham and Women’s Hospital in Boston. In order to establish how common this form of Aβ truly is in brain, Lemere obtained monoclonal antibodies the Probiodrug scientists had raised against Aβ cyclized at the 3 position, i.e., pyrogluAβ3-42, and tested the antibody’s performance across a wide swath of AD-relevant material including human, two different species of non-human primate, and at least six different lines of transgenic mouse. This work recapitulated and extended earlier work by Takaomi Saido at RIKEN in Saitama, Japan, who had used a polyclonal antibody in his early work that put pyroGluAβ on the map (Saido et al., 1995).

“These new monoclonals are extremely specific, and they confirm Takaomi’s work,” Lemere told Alzforum. Comparing the new pyroGluAβ antibodies to a standard Aβ antibody, Lemere first showed that it stained all plaques in the brains of 12 of 12 AD brains examined to date. The pyroGluAβ antibody also stained all plaques in all Down syndrome brains examined so far, as well as small amounts of diffuse plaque found in seven of 10 aged controls. PyroGluAβ was apparent in human AD cortex and hippocampus. “In humans, just about every amyloid plaque was positive for pyroglutamate Aβ,” Lemere said.

Lemere next examined the brains of vervet monkeys from a colony kept on the eastern Caribbean island of St. Kitts. Starting at age 15, these animals develop cerebral Aβ plaques in the parenchyma and blood vessels; these deposits, too, were heavily labeled with the new pyroGluAβ antibodies. A different primate model showed similar results. Cottontop tamarins are a small native species living in Colombia’s rain forests. They are endangered and not sacrificed for research, but some archival tissue is on hand for study at the New England Primate Research Center in Southborough, Massachusetts, Lemere said. Sections from most of the 18 brains available in this way had Aβ plaques and some CAA starting at around 12 years of age; about half had pyroGluAβ.

At the Society for Neuroscience Conference last October in Chicago, Rebecca Rosen of Atlanta’s Emory University told this reporter that she has obtained similar results as Lemere. Using a commercial anti-pyroGluAβ antibody from IBL Japan, Rosen first confirmed with Western blots that the antibody did not recognize Aβ1-40 or 1-42, and then tested it on human and non-human primate brain. PyroGluAβ staining came up intensely on Aβ plaques and vascular amyloid in cortical section from AD brain, from aged chimpanzees, rhesus macaques, and squirrel monkeys. In 2006, Rosen presented a poster at SfN in Atlanta, Georgia, reporting that she and her colleagues had detected pyroGluAβ3-42 in cortical tissue extracts of both human and monkey temporal and occipital cortex using MALDI-TOF mass spectrometry.

A separate, international collaboration of scientists found much the same. In the October 13 Journal of Neural Transmission, the groups of Thomas Bayer at Germany’s University of Goettingen, Lars Lannfelt and Martin Ingelsson of the Sweden’s Uppsala University; Gerd Multhaup at Free University of Berlin; Paul Lucasson at University of Amsterdam; and David Brody of Washington University, St. Louis, Missouri, reported results of their own two new monoclonal antibodies against pyroGluAβ. These antibodies heavily stained amyloid plaques in all of 14 samples of sporadic AD, as well as sections of familial AD caused by the Arctic and by the Swedish APP mutations and by a PS1 mutation (Wirths et al., 2009). PyroGluAβ staining is abundant in brains of people from a large Colombian pedigree with a different PS1 mutation, as well, Lemere told this reporter. When asked at recent conferences, other scientists said they harbored little doubt that this modified form of Aβ constitutes a significant component of deposited amyloid in AD. On this point, the new data confirm published studies.

Both the Lemere lab and the international collaboration noticed pyroGluAβ-positive plaques in a fraction of cognitively normal human controls, though fewer than in AD brains. This suggests, as has other research before (e.g., Vanderstichele, 2005), that pyroGluAβ appears in early stages of Alzheimer disease, but it also differs from observations in a recent publication (Schilling et al., 2008). Active discussion also arises from the question of whether pyroGluAβ acts as the seed for plaque formation or not. Many scientists agree with the conclusion that pyroGluAβ aggregates more readily and is more stable and toxic than full-length Aβ. But whether it precipitates amyloid deposition in the development of human disease is still unclear. Based on data emerging this year, academic groups tend to argue that the pyroGlu form is unlikely to be the initial seed. Rather, they say, deposition might start with full-length Aβ; perhaps even harmlessly enough for a while, at least in some people. Later on, possibly fanned by neuroinflammation that upregulates pyroGlu’s generating enzyme glutaminyl cyclase in local brain areas, cyclization could occur on existing plaques and render them more toxic, Lemere speculated. This hypothesis is difficult to test directly in humans. One way to look at it indirectly would be to see if people who age cognitively intact have predominantly full-length Aβ, if any, in their brains.

The question of what comes first can be addressed in mouse models by staining brain sections at different time points across the mice’s lifespan. At ICAD, the Lemere lab reported initial data of exactly such a study, and added further data at SfN. In early summer, Lemere approached six colleagues to request sections of their respective transgenic mouse lines. “All responded promptly, showing not just generosity but also growing interest in pyroGluAβ in the research community,” Lemere said. So far, data from 10 widely used transgenic strains are in, including, for example, the mThy-1-hAPP751 (Rockenstein et al., 2001), TgSwDI: APP (Davis et al., 2004), PSAPP (Holcomb et al., 1998), 5XFAD-APP/PS1 (Oakley et al., 2006), 3XTg-AD (Oddo et al., 2003), and J20APP (Mucke et al., 2000; Chin et al., 2005; Palop et al., 2005; Aucoin et al., 2005; Patel et al., 2005; Chin et al., 2004; Moolman et al., 2004; Seabrook et al., 2004; Palop et al., 2003). As expected, results varied somewhat along with the known variation of amyloid deposition between and even within a given transgenic model; however, all had in common that as the mice aged, full-length Aβ deposition showed up first, followed by pyroGluAβ, in a subset of amyloid plaques. Whether it was at two months, three months, or six months of age that Aβ plaques first appeared, whether in parenchyma or blood vessels, they were always full-length Aβ plaques. As the mice grew older, some of these plaques became positive for pyroGluAβ, as well. It was never the other way around, Lemere said. In those mouse models that feature neuronal loss, this loss generally occurred around the time pyroGluAβ became abundant, Lemere noted.

In the past six months, Jeffrey Frost in the lab performed extensive single and double immunofluorescence labeling comparing full-length and pyroGluAβ species. “Using this method, we see much more pyroGluAβ in the mouse models, even at younger ages. However, it is not apparent in every plaque or amyloid-bearing blood vessel. Instead, in the mice, it tends to be associated with compacted, thioflavin S-positive fibrillar plaques and vessels. In humans, it is observed in both compacted and diffuse Aβ deposits. We have experiments underway to help determine if pyroGluAβ is, in fact, necessary for plaque deposition,” Lemere wrote to ARF.

The international group led by Bayer further extended this finding with one particular mouse line. Using an APP/PS1 knock-in mouse initially made by Laurent Pradier at Aventis (Casas et al., 2004; ARF related news story), these scientists found that as the mice aged, the number of pyroGluAβ plaques kept increasing over time, whereas that of full-length Aβ plaques even decreased somewhat. The interpretation here would be that, as disease progresses, the N-terminus of Aβ in plaques gradually becomes chewed off and the exposed glutamate cyclized. This would generate more and more of the stable pyroglutamate form at the expense of the full-length form, implying that deposited amyloid undergoes continuous rearrangement over the course of years. A recent paper correlating amyloid pathology and dementia drew attention for showing that this link weakens in the oldest old; however, the study assessed plaques only with full-length Aβ antibodies, not anti-pyroGluAβ antibodies (Savva et al., 2009).

Last but not least, here’s one question where the pyroGluAβ field is quite unsettled: Does pyroGluAβ play an important role inside neurons? Academic groups have no broadly overlapping data yet to suggest as much. The Lemere lab, in surveying a range of different mouse models, found no significant intraneuronal pyroGluAβ in any of them, including the 5XFAD-APP/PS1 line that demonstrably accumulates Aβ42 inside neurons (Oakley et al., 2006; ARF related SfN story). On the other hand, Bayer noted evidence linking intraneuronal aggregation of pyroGluAβ to neuron loss in the APP/PS1KI model (Breyhan et al., 2009), as well as in a separate model that expresses only transgenic Aβ3-42 (not APP) in neurons (Wirths et al., 2009; see comment below). This issue generated discussion at ICAD, but no emerging consensus as yet.—Gabrielle Strobel.

This is Part 1 of a two-part series. See also Part 2.

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Wirths O, Bethge T, Marcello A, Harmeier A, Jawhar S, Lucassen PJ, Multhaup G, Brody DL, Esparza T, Ingelsson M, Kalimo H, Lannfelt L, Bayer TA. Pyroglutamate Abeta pathology in APP/PS1KI mice, sporadic and familial Alzheimer's disease cases. J Neural Transm. 2010 Jan;117(1):85-96. PubMed.
Vanderstichele H, De Meyer G, Andreasen N, Kostanjevecki V, Wallin A, Olsson A, Blennow K, Vanmechelen E. Amino-truncated beta-amyloid42 peptides in cerebrospinal fluid and prediction of progression of mild cognitive impairment. Clin Chem. 2005 Sep;51(9):1650-60. Epub 2005 Jul 14 PubMed.
Schilling S, Zeitschel U, Hoffmann T, Heiser U, Francke M, Kehlen A, Holzer M, Hutter-Paier B, Prokesch M, Windisch M, Jagla W, Schlenzig D, Lindner C, Rudolph T, Reuter G, Cynis H, Montag D, Demuth HU, Rossner S. Glutaminyl cyclase inhibition attenuates pyroglutamate Abeta and Alzheimer's disease-like pathology. Nat Med. 2008 Oct;14(10):1106-11. Epub 2008 Sep 28 PubMed.
Rockenstein E, Mallory M, Mante M, Sisk A, Masliaha E. Early formation of mature amyloid-beta protein deposits in a mutant APP transgenic model depends on levels of Abeta(1-42). J Neurosci Res. 2001 Nov 15;66(4):573-82. PubMed.
Davis J, Xu F, Deane R, Romanov G, Previti ML, Zeigler K, Zlokovic BV, Van Nostrand WE. Early-onset and robust cerebral microvascular accumulation of amyloid beta-protein in transgenic mice expressing low levels of a vasculotropic Dutch/Iowa mutant form of amyloid beta-protein precursor. J Biol Chem. 2004 May 7;279(19):20296-306. Epub 2004 Feb 25 PubMed.
Holcomb L, Gordon MN, McGowan E, Yu X, Benkovic S, Jantzen P, Wright K, Saad I, Mueller R, Morgan D, Sanders S, Zehr C, O'Campo K, Hardy J, Prada CM, Eckman C, Younkin S, Hsiao K, Duff K. Accelerated Alzheimer-type phenotype in transgenic mice carrying both mutant amyloid precursor protein and presenilin 1 transgenes. Nat Med. 1998 Jan;4(1):97-100. PubMed.
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Oddo S, Caccamo A, Kitazawa M, Tseng BP, Laferla FM. Amyloid deposition precedes tangle formation in a triple transgenic model of Alzheimer's disease. Neurobiol Aging. 2003 Dec;24(8):1063-70. PubMed.
Mucke L, Masliah E, Yu GQ, Mallory M, Rockenstein EM, Tatsuno G, Hu K, Kholodenko D, Johnson-Wood K, McConlogue L. High-level neuronal expression of abeta 1-42 in wild-type human amyloid protein precursor transgenic mice: synaptotoxicity without plaque formation. J Neurosci. 2000 Jun 1;20(11):4050-8. PubMed.
Chin J, Palop JJ, Puoliväli J, Massaro C, Bien-Ly N, Gerstein H, Scearce-Levie K, Masliah E, Mucke L. Fyn kinase induces synaptic and cognitive impairments in a transgenic mouse model of Alzheimer's disease. J Neurosci. 2005 Oct 19;25(42):9694-703. PubMed.
Palop JJ, Chin J, Bien-Ly N, Massaro C, Yeung BZ, Yu GQ, Mucke L. Vulnerability of dentate granule cells to disruption of arc expression in human amyloid precursor protein transgenic mice. J Neurosci. 2005 Oct 19;25(42):9686-93. PubMed.
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Chicago: Fanning the Flames of PyroGluAβ in Academia

09 Dec 2009

Exactly which form the amino end of Aβ takes in the brains of people with Alzheimer disease is a question that increasingly crops up in talks about immunotherapy and even other dementias, as well. For example, last October at the Society for Neuroscience meeting in Chicago, David Cribbs of the University of California at Irvine presented new data from his lab in collaboration with that of Goar Gevorkian at Universidad Nacional Autonoma de Mexico in Mexico City. Cribbs is thinking about how to exploit pyroGluAβ aggregates as a target for immunotherapy. Probiodrug researchers have begun probing this potential new target by way of small-molecule inhibitors of glutaminyl cyclase. For his part, Cribbs noted that immunotherapy research, too, stands to gain by directing new therapeutic antibodies specifically toward the modified N-terminus of Aβ.

Many antibodies in preclinical and clinical research recognize the EFRH sequence at residues 3-6 of full-length Aβ’s N-terminus, and many immunogens studied for use as active vaccines tend to induce antibodies against this epitope. The problem with that, Cribbs said in Chicago, is that this epitope may be absent from plaques in AD brain because it gets lost during in-situ modification of full-length Aβ to the pyroGlu3 form. Furthermore, the EFRH epitope is immuno-dominant in mice but not necessarily in rabbits or humans. “If you produce anti-Aβ antibodies in mice, then humanize them and give them to people, you may be targeting a part of the Aβ peptide that is actually gone from many plaques in human brain,” Cribbs said. To circumvent this problem, the Californian-Mexican colleagues generated rabbit polyclonal antibodies specifically against Aβ pyroglutamated on the 3 position, and used them to characterize the requisite immuno-dominant epitope. “This approach may offer alternatives in AD immunotherapy,” Cribbs concluded. The work appeared online last summer (Acero et al., 2009).

Chris Dealwis at Case Western Reserve University in Cleveland, Ohio, and collaborators elsewhere, are pursuing a similar vein of investigation by way of structural biology. In 2007, these scientists solved the crystal structure of their anti-Aβ antibody PF1 complexed with its quarry, treating the field at large to its first glimpse of an Aβ-antibody interaction in atom-by-atom resolution (see ARF related news story and Q&A). Earlier this year, they followed up with a second paper noting that the PF1 antibody binds pyroGlu3Aβ with 77-fold loss in affinity compared to full-length Aβ. PF1 recognizes the EFRH motif. In their latest study, Dealwis and colleagues dissect exactly why the antibody binds pyroGlu3Aβ so weakly. The scientists present new crystal structures of the antibody complexed with pyroGlu3Aβ, describing how the pyro-glutaminyl ring at residue 3 alters the hydrogen bonds that can form between the antibody and its prey. The goal of this work, as well, is to pave the way for high-affinity antibodies specifically directed against the pyroGlu3 form of Aβ for use in future immunotherapies (Gardberg et al., 2009).

Biomarker research, too, has taken note of pyroGluAβ. Last July, researchers led by Tony Wyss-Coray at Stanford University noted in a broad-based study of circulating natural antibodies that pyroGluAβ forms (there are several different kinds) were the target of surprisingly many of those presumably protective antibodies, and that their concentration in plasma tends to go down with age (Britschgi et al., 2009; ARF related news story). At ICAD in Vienna, and soon after in Neurobiology of Aging, Andrea Marcello and colleagues working with Thomas Bayer at the University of Goettingen, Germany, and Lars Lannfelt at Uppsala University in Sweden, followed on with their own, smaller study of 75 people. Similarly, these investigators found that people with AD tended to have fewer anti-pyroGluAβ IgM autoantibodies in their blood than did people with MCI who, in turn, had lower levels than normal controls. These scientists reported a correlation of plasma anti-pyroGluAβ IgMs and the MMSE in their MCI group of 15 people, though not in the AD group (Marcello et al., 2009).

Finally, a last hint toward therapy development came from Yasushi Tomidokoro at University of Tsukuba in Japan, working with Jorge Ghiso’s group at New York University School of Medicine in New York City. Four years ago, while studying the rare disease Familial Danish Dementia, these authors had noted that a pyroGlu-modified form of the pathogenic protein at play in this illness was a major constituent of its amyloid deposits but was absent from the soluble protein that floats in plasma of patients with this disease (Tomidokoro et al., 2005). At SfN in Chicago, Tomidokoro and colleagues examined the pyroGlu aspect of a similar disease, called Familial British Dementia. FBD is an autosomal-dominant dementia that comes with both central and peripheral amyloidoses. Importantly, the disease serves as a model for AD featuring even neurofibrillary tangles, and yet the original offending peptide has nothing to do with Aβ. The protein comes into being thanks to a stop-to-arginine mutation in the BRI2 gene, which generates a de-novo amyloidogenic peptide that does not exist in normal people.

FBD is a central and peripheral disease, making it more accessible for direct study than AD. In Chicago, the scientists presented a poster showing that soluble ABri peptides circulating in the blood of mutation carriers all are 34 amino acids long, with a glutamine at the N-terminus. In contrast, ABri extracted from FBD brains was in large part pyroglutamated, suggesting that this modification occurs locally where the protein deposits, not throughout the body. To examine this more closely, the scientists extracted ABri from muscle, pancreas, and myocardium and subjected the samples to biochemical analysis to see where in the process of amyloid deposition pyroglutamate formation occurs. More broadly, however, the point was to show that the ability to take amyloid tissue biopsies in FBD might open up opportunities for testing therapeutic strategies directed against pyroGlu formation in FBD, with likely significance for AD.

All told, scientists across the field are taking pyroGluAβ increasingly seriously, Christian Czech of F. Hoffmann-La Roche in Basel, Switzerland, told this reporter. Beyond that, they are hoping to see validation of its clinical potential soon, said Barry Greenberg at University Health Network, Toronto.—Gabrielle Strobel.

This is Part 2 of a two-part series. See also Part 1.

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References

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Acero G, Manoutcharian K, Vasilevko V, Munguia ME, Govezensky T, Coronas G, Luz-Madrigal A, Cribbs DH, Gevorkian G. Immunodominant epitope and properties of pyroglutamate-modified Abeta-specific antibodies produced in rabbits. J Neuroimmunol. 2009 Aug 18;213(1-2):39-46. PubMed.
Gardberg A, Dice L, Pridgen K, Ko J, Patterson P, Ou S, Wetzel R, Dealwis C. Structures of Abeta-related peptide--monoclonal antibody complexes. Biochemistry. 2009 Jun 16;48(23):5210-7. PubMed.
Britschgi M, Olin CE, Johns HT, Takeda-Uchimura Y, Lemieux MC, Rufibach K, Rajadas J, Zhang H, Tomooka B, Robinson WH, Clark CM, Fagan AM, Galasko DR, Holtzman DM, Jutel M, Kaye JA, Lemere CA, Leszek J, Li G, Peskind ER, Quinn JF, Yesavage JA, Ghiso JA, Wyss-Coray T. Neuroprotective natural antibodies to assemblies of amyloidogenic peptides decrease with normal aging and advancing Alzheimer's disease. Proc Natl Acad Sci U S A. 2009 Jul 21;106(29):12145-50. PubMed.
Marcello A, Wirths O, Schneider-Axmann T, Degerman-Gunnarsson M, Lannfelt L, Bayer TA. Reduced levels of IgM autoantibodies against N-truncated pyroglutamate Aβ in plasma of patients with Alzheimer's disease. Neurobiol Aging. 2011 Aug;32(8):1379-87. PubMed.
Tomidokoro Y, Lashley T, Rostagno A, Neubert TA, Bojsen-Møller M, Braendgaard H, Plant G, Holton J, Frangione B, Révész T, Ghiso J. Familial Danish dementia: co-existence of Danish and Alzheimer amyloid subunits (ADan AND A{beta}) in the absence of compact plaques. J Biol Chem. 2005 Nov 4;280(44):36883-94. PubMed.

Chicago: Axonal Transport Not So Fast in Neurodegenerative Disease

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Chicago: Tau and α-Synuclein Oligomers Follow Aβ Footsteps

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Chicago: Nicotinic AChRs: α4β2 Iffy for AD, More Promise With α7?

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Chicago: Move Over, Agonists; Make Way for Modulators

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Chicago: Nicotinic AChRs—Mechanistic Basis for New Drug Discovery?

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Chicago: AD and Epilepsy—Joined at the Synapse?

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Chicago: AD and Epilepsy—Lessons from the Clinic, Animals

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Chicago: New Technologies Help Drugs Cross Blood-Brain Barrier

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Chicago: The Vampire Principle—Young Blood Rejuvenates Aging Brain?

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