Two scientists updated the audience on the continuing workup of patients in Elan’s ill-fated phase II trial of the AN-1792 vaccine. James Nicoll recounted what he got to see firsthand from his perch as a research and diagnostic pathologist at the University of Southampton’s General Hospital in England, when one of the eight English trial patients died a year after she’d fallen sick with probable meningoencephalitis (see ARF related news story). Since then, three more patients have died, and their brains are under investigation in Spain (Ferrer et al., 2004), California, and Cardiff, the latter two still unpublished. Initial results of these three additional cases show they had identical features to the Southampton case, Nicoll said. This suggests that Aβ immunotherapy can indeed remove some Aβ from the brain, but with complications that need resolving if this is ever to be a viable therapy.
In summary, the pathology profile that emerges from looks like this: Most strikingly, wide swaths of parietal and temporal cortex were devoid of plaques. Instead there were isolated patches of amyloid, and quantification showed an overall reduction. Aβ-reactive microglia were prominent, as well as amyloid around the blood vessels. This was all as predicted by prior animal models, as was the finding that dystrophic neurites straightened out, which has been described in mice, as well (Lombardo et al., 2003; for picture of neurites see ARF related news story. Yet there were unexpected findings, too. The pathologists were surprised to see that T lymphocytes and macrophages had infiltrated the brain, that there was evidence of white matter pathology, and that neurofibrillary tangles and neuropil threads remained stubbornly in place. On this last point, Isidro Ferrer’s study of the Spanish patient noted some indications that tau phosphorylation, and by implication further tangle formation, appeared to have slowed somewhat, which one could hope if indeed Aβ spurs tangle formation (Goetz et al., 2001).
What, then, caused this serious side effect? Nicoll suggested these possible explanations: First, microglial activation could have spun out of control in the heat of Aβ removal. Second, the solubilization of Aβ brought on by the antibody could have overburdened clearance mechanisms, effectively increasing Aβ flux, concentration, and then deposition in the perivascular pathway enough to cause the leucoencephalopathy. Third, T lymphocytes infiltrating across meningeal vessels could be the culprits. This last notion gets support from an independent study of seven people with CAA-related inflammation who had rapid cognitive decline and white matter disease, but got better when given immunosuppressive therapy, just like the Elan patients did (Eng et al., 2004). "These seven cases described by Steven Greenberg look remarkably similar to what we see," Nicoll said.
How about the immunized people who still live? The public has not yet seen an analysis of the patients from any location of this multicenter trial with the exception of the Swiss cohort, which is being followed by Roger Nitsch’s group at the University of Zurich.
Nitsch prefaced his lecture, titled “Can Antibodies Bring Back Memories?” by noting some of the prior data that convinced him of Aβ’s value as a therapeutic target. Standing out were Brian Bacskai’s and Brad Hyman’s multi-photon images showing the disappearance of plaques in mouse brain (Bacskai et al., 2002). “It was a shock to us who believe it takes 10 years for a plaque to develop to see it disappear within three days after a single dose of antibody,” Nitsch said.
This website has covered the Nitsch group’s initial report of the 30 Swiss patients extensively (Hock et al., 2003 ), as well as a prior update Nitsch offered at last year’s Society for Neuroscience conference in New Orleans (see ARF conference report ). Therefore this summary will simply add some details not previously mentioned. Nitsch noted that two years after the patients received their last vaccine injection, they still maintain high levels of IgG and IgM antibodies. These antibodies do not cross-react with APP or its derivatives, but specifically label amyloid deposits in brain tissue of AD and CAA patients. The antibodies are present in serum and in the cerebrospinal fluid. Nitsch said he suspected there are being transported across the blood-brain barrier by receptors.
Nitsch also offered an explanation for a data point that has drawn criticism. The “non-responder” patients who did not produce antibodies declined by six points on the MMSE scale in the first year of assessment, an unusually steep drop by some measures. However, the trial patients were in their 4th year of taking cholinesterase inhibitor drugs, and after the initial effect of these drugs wears off, patients are known to decline more quickly. Since the 19 “responders” all improved, they are unlikely to be outliers, Nitsch added.
The cases of subacute aseptic meningoencephalitis that arose in 18 of the 298 patients vaccinated worldwide are clearly related to the vaccine, Nitsch confirmed. Yet antibodies probably had little to do with it, as their levels did not correlate with it, and some of the people who developed it but recovered are among the highest responders clinically.
On the mechanism of Aβ clearance, Nitsch noted that Nicoll’s observation of activated microglia and amyloid-free patches of brain hinted that Fc-mediated clearance was at work. To try to test the peripheral sink hypothesis (see, e.g., DeMattos et al., 2002), Zurich researchers Christoph Hock and Uwe Konietzko analyzed blood samples collected from the patients every month, but they were unable to find changes of Aβ42 levels in plasma. Neither did they see significant changes in CSF. Future directions being explored for better immunotherapy approaches include passive vaccination, more careful epitope selection, and anti-inflammatory strategies, Nitsch concluded.
Privately, several scientists at the conference voiced concern that all patients of the halted Elan trial may not be followed as carefully as the Zurich cohort, raising concerns that a valuable learning opportunity might be lost to the field.
Microglia: Good or Bad? Conditional K.O. Offers an Answer
Quite a separate question in immunotherapy, and more broadly in neurodegeneration and neuroinflammation, revolves around the role of microglia. To this day, these ‘macrophages of the brain’ remain shrouded in mystery, and scientists trying to lift this shroud variously come across contradictory results. In general, research finds that normally quiescent microglia become activated in most brain diseases, and that they begin spewing oxygen radicals, nitric oxide, cytokines and chemokines in the process. Yet is this activation beneficial (as in removing amyloid) or detrimental (as in killing neurons)? At what point does one tilt over into the other? In short, precise mechanisms of how microglia function in the pathogenic process remain unclear. To probe the role of microglia in vivo, Frank Heppner at Zurich’s Institute for Neuropathology created a clever transgenic mouse line that essentially allows him to selectively silence microglia. Working with Aguzzi and others, Heppner started out with a promoter specific to monocytes/macrophage (microglia are of hematopoietic origin.) Behind it he spliced a suicide gene that makes the otherwise inactive pro-drug ganciclovir toxic to proliferating cells. Heppner’s twist to this widely used system lies in transplanting wild-type bone marrow into these transgenic so that the chimeras had normal, ganciclovir-insensitive hematopoietic cells in the periphery but transgenic, ganciclovir-sensitive microglia. “Ganciclovir only hits the brain macrophages,” Heppner said.
To confirm that the transgene worked as expected, Heppner cut the facial nerve of these mice, an operation that activates microglia without affecting the blood-brain-barrier, in other words avoids confounding infiltration of peripheral macrophages. Unlike control microglia, which proliferated, the transgenic microglia did not respond to the severed nerve at all, Heppner said.
Next, Heppner used his model to define the role of microglia in multiple sclerosis, a demyelinating neuroinflammatory and, in its later stages, neurodegenerative disease known to involve both T cell and microglial activation. The question was who is doing what in the pathogenesis, and are the two responses independent of each other? When the scientists injected the MOG peptide antigen to induced an established mouse version of multiple sclerosis called EAE, they noticed that the ensuing T cell response occurred in the transgenic mice as expected, showing that microglia are not necessary for the peripheral immune system to mount its autoimmune response. However, the mice with the impotent microglia barely got sick. They had a much milder disease with later onset and they recovered, unlike normal mice given the same injection. Inflammatory infiltration of the spinal cord and cerebellum were also muted in the transgenic mice. This suggests that taking microglia out of commission represses the clinical course of multiple sclerosis, and establishes these cells as valid drug targets, Heppner said. Precisely what did the trick remains to be shown, however; candidate mechanisms include that the microglia were unable to present antigens (see ARF related news story) or the absence of the noxious substances that activated microglia usually release.
On the broader front of mouse genetics, Klaus Rajewsky, Harvard Medical School, introduced the audience to new techniques of generating mutant models. In collaboration with Rudolph Jaenisch at MIT, Rajewski is working out methods that could do away with the cross-breeding of single-mutant strains by which so-called compound mutant strains are currently being generated. Cross-breeding is laborious, expensive, and introduces confounds stemming from strain backgrounds. Instead, the scientists are devising methods to grow mice from embryonic stem cells that already contain all the desired mutations.
Anders Nykjaer presented his data, recently published in Nature, on nerve growth factor signaling (Nykjaer et al., 2004). Working with Claus Petersen at Aarhus University in Denmark, Nykjaer analyzed how a new receptor family called sortilins might influence the signaling of NGF. Part of the reason why NGF signaling is complicated lies in its ability to promote both cell survival and cell death, and to do so via different receptor types. Simply put, NGF generally signals survival through TrkA receptors, while its immature form, pro-NGF, signals cell death via the receptor P75NTR. Both functions have an important role in shaping the developing nervous system; in neurodegenerative diseases, NGF could potentially be a treatment were it not for the difficulties of delivering it to needy neurons in a targeted way. Earlier basic research had indicated that additional receptors likely participate in transmitting NGF’s death signal. Nykjaer and colleagues suspected that sortilin, which had been described a few years ago but had few functions assigned to it yet, might be the one. In St. Moritz, Nykjaer reviewed data showing that sortilin binds pro-NGF tightly in its pro-domain whereas, in the same complex, p75NTR binds to the ligand’s mature NGF domain. This appears to fit with new crystallographic images of NGF-p75NTR binding (see ARF related news story). Sortilin antagonists can protect against cell death induced by pro-NGF. By contrast, sortilin does not interact with TrkA, Taken together, this suggests that sortilin essentially functions as a switch that can “sort” life from death.
All told, this conference drew on some of the liveliest neurodegeneration science, particularly from Europe. As with every good meeting, this Alzforum summary cannot cover nearly all of the 43 talks and 51 posters but instead highlights selected presentations and common motifs. As always, the writer invites conference participants to fill the gaps, and indeed all readers to comment on any of the points raised.—Gabrielle Strobel.
- Trials and Tribulations—Autopsy Reveals Pros and Cons of AD Vaccine
- New Orleans: Immunotherapy—The Game Is Still in Town
- New Orleans: New Approaches to Lift Microglia Mysteries
- NGF—From Crystal Structure to Human Trials
- Ferrer I, Boada Rovira M, Sánchez Guerra ML, Rey MJ, Costa-Jussá F. Neuropathology and pathogenesis of encephalitis following amyloid-beta immunization in Alzheimer's disease. Brain Pathol. 2004 Jan;14(1):11-20. PubMed.
- Götz J, Chen F, van Dorpe J, Nitsch RM. Formation of neurofibrillary tangles in P301l tau transgenic mice induced by Abeta 42 fibrils. Science. 2001 Aug 24;293(5534):1491-5. PubMed.
- Eng JA, Frosch MP, Choi K, Rebeck GW, Greenberg SM. Clinical manifestations of cerebral amyloid angiopathy-related inflammation. Ann Neurol. 2004 Feb;55(2):250-6. PubMed.
- Bacskai BJ, Kajdasz ST, McLellan ME, Games D, Seubert P, Schenk D, Hyman BT. Non-Fc-mediated mechanisms are involved in clearance of amyloid-beta in vivo by immunotherapy. J Neurosci. 2002 Sep 15;22(18):7873-8. PubMed.
- Hock C, Konietzko U, Streffer JR, Tracy J, Signorell A, Müller-Tillmanns B, Lemke U, Henke K, Moritz E, Garcia E, Wollmer MA, Umbricht D, de Quervain DJ, Hofmann M, Maddalena A, Papassotiropoulos A, Nitsch RM. Antibodies against beta-amyloid slow cognitive decline in Alzheimer's disease. Neuron. 2003 May 22;38(4):547-54. PubMed.
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- Nykjaer A, Lee R, Teng KK, Jansen P, Madsen P, Nielsen MS, Jacobsen C, Kliemannel M, Schwarz E, Willnow TE, Hempstead BL, Petersen CM. Sortilin is essential for proNGF-induced neuronal cell death. Nature. 2004 Feb 26;427(6977):843-8. PubMed.
- Heppner FL, Greter M, Marino D, Falsig J, Raivich G, Hövelmeyer N, Waisman A, Rülicke T, Prinz M, Priller J, Becher B, Aguzzi A. Experimental autoimmune encephalomyelitis repressed by microglial paralysis. Nat Med. 2005 Feb;11(2):146-52. PubMed.