Feeling sleepy? The amino acid behind the dreaded drowsiness that sets in after a big meal makes headlines in today’s Cell, where researchers report that tweaking a tryptophan degradation pathway with an oral compound relieved behavioral deficits and synaptic loss in Alzheimer’s and Huntington’s mouse models. Three additional studies made for a hodgepodge of recent news on therapeutic approaches being pursued across the field. A paper posted online May 25 in the Journal of Alzheimer’s Disease describes a cysteine protease inhibitor that lowers brain Aβ and relieves cognitive deficits in AD mice. In the May 6 Journal of Biological Chemistry, a study suggests that a histone deacetylase inhibitor approved for cancer treatment may help in certain cases of familial frontotemporal dementia. And, some food for thought for researchers preferring approaches au natural: A company developing an oral zinc-based therapy announced that it plans to conduct future trials with this compound in older AD patients while selling it at the same time.

JM6
For the Cell paper, researchers led by senior author Paul Muchowski, Gladstone Institute of Neurological Disease, San Francisco, and Robert Schwarcz, University of Maryland School of Medicine, Baltimore, fed AD and HD transgenic mice a compound that manipulates the kynurenine pathway for tryptophan breakdown. By inhibiting the enzyme kynurenine 3-monooxygenase (KMO), the compound (JM6) diverts the pathway toward a side reaction that produces a neuroprotective metabolite—kynurenate. AD and HD patients have reduced brain levels of kynurenate (Gulaj et al., 2010; Beal et al., 1992), and accumulate two neurotoxic kynurenine metabolites in their blood and brains (Guidetti et al., 2004; Heyes et al., 1992). The researchers decided to synthesize JM6, a “slow-release” version of a KMO inhibitor widely used in animal studies, because Muchowski’s group had shown that KMO-deficient yeast could resist the toxic effects of polyglutamine-expanded huntingtin protein (ARF related news story on Giorgini et al., 2005).

First author Daniel Zwilling and colleagues tested JM6 in pre-symptomatic AD mice (J20 strain) and found that the KMO inhibitor prevented spatial memory and anxiety deficits, and preserved synapses in the cortex and hippocampus. When given to R6/2 HD mice starting at early stages of disease, the compound helped the animals live longer, and prevented synaptic loss and microglial activation.

The authors hypothesize that when they give mice JM6, the KMO substrate kynurenine accumulates in the blood as peripheral monocytes break down tryptophan. Then, kynurenine gets pumped into the brain where astrocytes convert it to kynurenate. “The take-home message is that regulating the activity of KMO in the blood is sufficient to regulate brain levels of kynurenate,” Muchowski said in an interview with ARF. Underscoring the importance of the kynurenine pathway in neurodegeneration, a team led by Muchowski’s former postdoc Flaviano Giorgini showed that manipulating the pathway in various ways to increase kynurenate levels protected against disease in an HD fly model. Giorgini is now at the University of Leicester in the U.K., and reported the findings in a Current Biology paper published online June 2.

The possibility of alleviating both AD and HD with a single strategy is appealing, suggested Peter Reinhart, Proteostasis Therapeutics, Inc., Cambridge, Massachusetts, and Jeffrey Kelly, Scripps Research Institute, La Jolla, California, in a commentary accompanying the Cell paper. Reinhart and Kelly note, however, that future studies would need to address whether the approach works in animals with disease already well underway. Meanwhile, Muchowski said his lab is in the process of initiating safety and toxicology studies on their KMO inhibitor.

E64d
Another oral inhibitor—this one targeting cysteine proteases—looked promising in animal studies reported in the JAD paper. Greg Hook of American Life Science Pharmaceuticals, a small biotech company in San Diego, California, and colleagues report that the compound E64d reduced brain Aβ and memory deficits when fed to plaque-free, young AD transgenic mice or older animals with strong amyloid deposition. E64d also lowered brain Aβ levels in guinea pigs, whose amyloid precursor protein (APP) has the same β-secretase cleavage site as wild-type human APP.

Previously, the lab of Greg Hook’s wife and collaborator Vivian Hook at UCSD had shown that inhibitors of cathepsin B (CatB), a lysosomal cysteine protease, reduced brain amyloid in mice expressing the London APP mutant, which has the wild-type β-secretase site. The inhibitors did not work in mice expressing APP with the Swedish mutation, which is at the β-secretase site (Hook et al., 2008). (There is controversy in the field about cathepsin B being a bona fide β-secretase; see below). Those compounds were injected into the mouse brain. The current study tested an oral inhibitor—an approach more amenable to human studies.

“We got a big effect feeding E64d in chow to severely demented mice,” Hook told ARF. Furthermore, the compound seems safe, he said. It was developed in Japan in the 1980s to treat muscular dystrophy (Satoyoshi, 1992), failing Phase 3 because it lacked efficacy for that disease, the authors wrote. “We know its pharmacokinetics and its oral bioavailability,” Hook said. “It has overcome two of the biggest hurdles—druggability and toxic side effects—and is efficacious in AD animal models.”

The data are “intriguing,” noted Li Gan, at the Gladstone Institute for Neurological Disease. However, the mechanism by which E64d lowers Aβ is unclear because the compound inhibits many cysteine proteases besides cathepsin B, including the ubiquitously expressed calpain. To complicate matters, Gan and colleagues showed that cathepsin B-deficient AD mice had more plaques than AD mice with wild-type CatB (ARF related news story on Mueller-Steiner et al., 2006). This is the opposite of what one would expect if cathepsin B inhibition lowers Aβ, as suggested in the current study. Hook’s findings also run counter to recent work by Ralph Nixon’s group at the Nathan Kline Institute for Psychiatric Research, Orangeburg, New York. Nixon and colleagues reported reduced Aβ and plaques in AD mice with enhanced cathepsin activity owing to deletion of an endogenous inhibitor, cystatin B (Yang et al., 2011). In another recent paper by the Nixon lab (Lee et al., 2011), inhibitors of cathepsins, including E64, disrupted transport and caused dystrophic swelling in axons, urging caution for the E64d inhibition approach, Gan noted (see full comment below).

SAHA
In the Journal of Biological Chemistry paper, researchers at the University of Texas Southwestern Medical Center, Dallas, describe a potential therapeutic strategy for frontotemporal dementia that, like the E64d approach, uses a compound with a prior safety record. Led by senior author Joachim Herz and lead author Basar Cenik, the team showed that the cancer drug suberoylanilide hydroxamic acid (SAHA), a histone deacetylase (HDAC) inhibitor, restored normal progranulin expression in cells from patients with frontotemporal dementia (FTD) caused by progranulin haploinsufficiency. In light of recent evidence that progranulin may tone down inflammation by blocking tumor necrosis factor (Tang et al., 2011), compounds that upregulate progranulin expression would be expected to be neuroprotective “not only in FTD but in all neurodegenerative syndromes in which neuroinflammation is a significant factor,” Herz suggested in an e-mail to ARF. Several companies are working on developing more brain-penetrant and more specific HDAC inhibitors than SAHA.

Zinc Cysteine
Finally, some approaches are trying to harness the power of basic nutrients, like zinc. People with Alzheimer’s or Parkinson’s have low blood levels of zinc (Brewer et al., 2010), and mice deficient in synaptic zinc develop an AD-like syndrome (ARF related news story on Adlard et al., 2010). Earlier this spring at the American Academy of Neurology (AAN) annual meeting in Honolulu, Hawaii, Adeona Pharmaceuticals of Ann Arbor, Michigan, presented Phase 2 trial data on its zinc cysteine tablet (reaZin), given daily to 57 AD and mild cognitive impairment (MCI) patients for six months. The treatment was safe and well tolerated, and achieved the trial’s primary outcomes of reducing serum levels of copper and increasing serum zinc. Six-month changes on three cognitive batteries (ADAS-Cog, CDR-SOB, MMSE) were reported to “favor the treatment group,” the poster authors wrote. Based on post-hoc analysis of age-related subgroups, Adeona recently announced plans to evaluate reaZin in a larger 12-month study of AD patients ages 70 and up. According to its press release, the company intends to develop the zinc tablet as a drug and, in parallel, market it as a prescription medical food.

“The results are interesting, but since the numbers are small and the trial only lasted six months, it is difficult to interpret. It is good that they are planning a bigger trial, and hopefully they will include biomarkers related to Aβ,” commented Colin Masters of the University of Melbourne, Australia.

Paul Aisen noted that, while the study addressed the product’s safety and bioavailability, “there is no meaningful information on efficacy.” Furthermore, the concept of medical foods is problematic, “particular for the AD field,” wrote Aisen, who directs the Alzheimer’s Disease Cooperative Study headquartered at the University of California, San Diego. “A product can be marketed as a medical food, even using phrases such as “FDA approved,” in a way that suggests there is evidence of efficacy, when in fact there is no such evidence.” (See also ARF series on medical foods.) John Breitner of McGill University, Montreal, Canada, agrees that the cognitive claims are not statistically founded. “I cannot agree with the authors' conclusion that these results provide a "strong trend toward cognitive benefit favoring the treatment group," he wrote in an e-mail to ARF (see full comment below).—Esther Landhuis

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Comments on News and Primary Papers

  1. In the Cell paper by Zwilling et al. on oral kynurenine 3-monooxygenase (KMO) inhibition in Huntington's and Alzheimer's transgenic mouse models, a striking effect was the prevention of pre-synaptic protein loss in the transgenic models of AD (APP-Tg mice) and HD (R6/2 mice). In these models, systemic (oral) administration using the small-molecule KMO inhibitor JM6 increased brain levels of kynurenic acid, prevented behavioral deficits in APP-Tg mice, and increased survival and reduced CNS microglial activation in R6/2 mice. Importantly, in both models, JM6 prevented the loss of the pre-synaptic protein synaptophysin. Recent reports (e.g., Chung et al., 2009) have highlighted synaptic changes as an early pre-degenerative event in neurodegenerative diseases, and such changes are a target for pre-symptomatic neuroprotective interventions. The striking effect of JM6 on synaptic changes and other degenerative alterations in these two in vivo models are encouraging for future protective therapies for several neurodegenerative diseases.

    References:

    . Dynamic changes in presynaptic and axonal transport proteins combined with striatal neuroinflammation precede dopaminergic neuronal loss in a rat model of AAV alpha-synucleinopathy. J Neurosci. 2009 Mar 18;29(11):3365-73. PubMed.

    View all comments by Penny Hallett
  2. Aβ-lowering effects of E64d are very intriguing. However, since E64d inhibits numerous cathepsins, the mechanism by which E64d lowers Aβ remains unclear. Moreover, given the recent finding from Randy Nixon's group that inhibitors of cathepsins, including E64, disrupt axonal transport and cause axonal dystrophic swelling (Lee et al., 2011), one needs to be very cautious about this approach.

    The Hook lab has suggested in the past that cathepsin B (CatB) only acts as a β-secretase with wild-type human APP (see Hook comment on Mueller-Steiner et al., 2006). We have deleted and overexpressed CatB in hAPP-I63 mice, which overexpress wild-type human APP. In our analyses of these mice, which will be presented this fall at the Society for Neuroscience meeting in Washington, DC, deletion or overexpression of CatB in neurons had no effect on β-CTF derived from wild-type hAPP. In contrast, deleting CatB increased Aβ while overexpressing CatB reduced it. Consistent with these findings, deleting cystatin C (an endogenous inhibitor of cathepsin B) in hAPP-I63 mice lowered Aβ levels.

    Furthermore, consistent with our findings on cystatin C deletion, Randy Nixon's group reported that deletion of cystatin B (another endogenous inhibitor of cathepsins, including CatB) led to reduction of Aβ and plaques in TgCRND8 AD transgenic mice (Yang et al., 2011). These findings contrast with the effects of E64d, which has been shown to inhibit numerous lysosomal proteases (Barrett et al., 1982).

    References:

    . Lysosomal proteolysis inhibition selectively disrupts axonal transport of degradative organelles and causes an Alzheimer's-like axonal dystrophy. J Neurosci. 2011 May 25;31(21):7817-30. PubMed.

    . Antiamyloidogenic and neuroprotective functions of cathepsin B: implications for Alzheimer's disease. Neuron. 2006 Sep 21;51(6):703-14. PubMed.

    . Reversal of autophagy dysfunction in the TgCRND8 mouse model of Alzheimer's disease ameliorates amyloid pathologies and memory deficits. Brain. 2011 Jan;134(Pt 1):258-77. PubMed.

    . L-trans-Epoxysuccinyl-leucylamido(4-guanidino)butane (E-64) and its analogues as inhibitors of cysteine proteinases including cathepsins B, H and L. Biochem J. 1982 Jan 1;201(1):189-98. PubMed.

    View all comments by Li Gan
  3. The primary outcome data from the reaZin study appear to be consistent with the proposed action of the intervention. The three cognitive and functional measures used for the series of secondary outcomes are appropriate, but the small size of the sample means that the study was underpowered with respect to any clinical outcome measures. The small sample size was probably responsible also for the lack of balance in baseline measures across the randomized groups. Whether one should see the preliminary clinical outcome results as encouraging is a matter of judgment. The poster presentation does not make it clear whether the composite outcome was specified a priori. If not, the meaning of the p-value of 0.15 is hard to discern. In any event, I cannot agree with the authors' conclusion that these results provide a "strong trend toward cognitive benefit favoring the treatment group."

  4. This is an interesting study that examined the neuroprotective efficacy of JM6, a small molecule inhibitor of kynurenine 3-monooxygenase (KMO). The authors demonstrate that oral administration of JM6 inhibits KMO in the blood, resulting in an increase in kynurenic acid levels. Interestingly, they demonstrate that JM6 does not cross the blood-brain barrier, and does not inhibit KMO in the brain, yet there are neuroprotective effects in transgenic mouse models of AD and of HD. The inhibition of KMO in the blood results in an increase in kynurenine, which then leads to an increase in kynurenic acid concentrations both in blood as well as in the brain. In the brain, the increase in kynurenic acid is thought to exert neuroprotective effects by blocking both AMPA and NMDA excitatory amino acid receptors, and by blocking the pre-synaptic α7 nicotinic acetylcholine receptors, resulting in a reduction in glutamate release and excitotoxicity.

    In APP transgenic mice, JM6-increased brain kynurenic acid levels prevent synapse loss and spatial memory loss in the Morris water maze. The authors also carried out studies in the R6/2 transgenic mouse model of HD, using both a high or a low dose of JM6 administered orally. These studies demonstrated that there was a significant increase in survival, with either dose level of JM6, as well as protection against loss of synaptophysin immunoreactivity in the striatum. There was a significant reduction in the number of neurons immunoreactive for the calcium regulated immediate early gene c-Fos, and a reduction in IBA1-positive activated microglia, but no alterations in huntingtin (Htt) inclusions. This is consistent with a number of other studies, which have shown that Htt inclusions do not correlate with neuroprotection or improved survival.

    Overall, these are novel and interesting findings, which show that manipulation of the kynurenine pathway in the periphery, leading to increased kynurenine and kynurenic acid levels in the brain, produces neuroprotective and behavioral improvement in transgenic mouse models of AD and HD. In the HD mice, the improvement in survival was several weeks, which is consistent with other compounds that block excitatory amino acid receptors, such as remacemide and memantine. These results are also consistent with our studies in which we administered kynurenine in combination with probenecid, an inhibitor of organic acid transport, which increases kynurenic acid concentrations in the brain and exerts protective effects against pentylentetrazol and NMDA-induced seizures (Vecsei et al., 1992; Miller et al., 1992). Other studies showed that administration of L-kynurenine and probenecid, produced dose-dependent attenuation of quinolinic acid striatal lesions (Harris et al., 1998). We also demonstrated that administration of L-kynurenine alone, or in combination with probenecid, produced dose-dependent neuroprotection against hypoxia-ischemia and NMDA lesions in neonatal rats (Nozak and Beal, 1992).

    We and others previously showed that there were significant reductions in kynurenic acid concentrations in HD postmortem brain tissue, which may increase the vulnerability of neurons to excitotoxic cell death (Beal et al., 1992; Jauch et al., 1995). The present approach is a novel one in that they blocked KMO activity in the periphery, which results in increased kynurenine, which is transported across the blood-brain barrier and increases kynurenic acid levels in the central nervous system, reducing release of extracellular glutamate as assessed using microdialysis. Administration of probenecid to a transgenic mouse model of HD increased cortical kynurenic acid levels fourfold and increased survival by 35 percent (Vamos et al., 2009). Manipulations of the kynurenine pathway to increase kynurenic acid levels are also protective in a Drosophila model of Huntington’s disease (Campesan et al., 2011). This is a particularly attractive approach, since one is manipulating an endogenous pathway to exert neuroprotective effects, which may be well tolerated without the behavioral toxicity associated with other excitatory amino acid receptor antagonists.

    References:

    . Kynurenic acid concentrations are reduced in Huntington's disease cerebral cortex. J Neurol Sci. 1992 Mar;108(1):80-7. PubMed.

    . The kynurenine pathway modulates neurodegeneration in a Drosophila model of Huntington's disease. Curr Biol. 2011 Jun 7;21(11):961-6. PubMed.

    . Modulation of striatal quinolinate neurotoxicity by elevation of endogenous brain kynurenic acid. Br J Pharmacol. 1998 May;124(2):391-9. PubMed.

    . Dysfunction of brain kynurenic acid metabolism in Huntington's disease: focus on kynurenine aminotransferases. J Neurol Sci. 1995 May;130(1):39-47. PubMed.

    . The effect of peripheral loading with kynurenine and probenecid on extracellular striatal kynurenic acid concentrations. Neurosci Lett. 1992 Oct 26;146(1):115-8. PubMed.

    . Neuroprotective effects of L-kynurenine on hypoxia-ischemia and NMDA lesions in neonatal rats. J Cereb Blood Flow Metab. 1992 May;12(3):400-7. PubMed.

    . Neuroprotective effects of probenecid in a transgenic animal model of Huntington's disease. J Neural Transm. 2009 Sep;116(9):1079-86. PubMed.

    . Kynurenine and probenecid inhibit pentylenetetrazol- and NMDLA-induced seizures and increase kynurenic acid concentrations in the brain. Brain Res Bull. 1992 Feb;28(2):233-8. PubMed.

    . Kynurenine 3-monooxygenase inhibition in blood ameliorates neurodegeneration. Cell. 2011 Jun 10;145(6):863-74. PubMed.

    View all comments by M Flint Beal
  5. Dr. Gan’s comments do not address the main point of our paper, which is that oral E64d improves memory in an AD mouse model that represents the majority of AD patients. Feeding E64d-containing chow to transgenic APPL on AD mice improved memory and reduced both Aβ and amyloid plaques in brain. Furthermore, guinea pigs that express human wild-type APP show reduced brain Aβ after oral administration of E64d. Because E64d has been shown to be safe in patients at the effective dose used in the animal experiments, this compound can be tested in the clinic for AD.

    Dr. Gan makes an excellent comment about E64d inhibition of cathepsins. We, too, discuss the multiple beneficial effects of E64d on inhibition of several cysteine cathepsins as well as calpain. These properties result in reduction of toxic Aβ and neuroprotection.

    Dr. Gan’s point on potential mechanisms of drug action is impossible to evaluate. She cites unpublished data from her lab, but we cannot evaluate data not shown. Therapeutic agents can improve the human disease condition without knowledge of their mechanism. Many drugs are used for disease treatments for which mechanisms of action are unknown or controversial, for example, lithium for bipolar disorder.

    The key finding about E64d is that it is efficacious at improving memory in animal models expressing APP with the wild-type β-secretase site that is expressed in the majority of AD patients.

    View all comments by Greg Hook

References

News Citations

  1. Huntington Disease: Three Ways to Tackle Triplet Disorder
  2. Role Reversal—AD Mouse Desperately Seeks CatB
  3. Think Zinc—Mice Missing Key Ion Transporter Develop AD-like Problems
  4. Medical Foods—Fallback Option for Elusive AD Drug Status?

Paper Citations

  1. . Kynurenine and its metabolites in Alzheimer's disease patients. Adv Med Sci. 2010 Dec 30;55(2):204-11. PubMed.
  2. . Kynurenic acid concentrations are reduced in Huntington's disease cerebral cortex. J Neurol Sci. 1992 Mar;108(1):80-7. PubMed.
  3. . Neostriatal and cortical quinolinate levels are increased in early grade Huntington's disease. Neurobiol Dis. 2004 Dec;17(3):455-61. PubMed.
  4. . Quinolinic acid and kynurenine pathway metabolism in inflammatory and non-inflammatory neurological disease. Brain. 1992 Oct;115 ( Pt 5):1249-73. PubMed.
  5. . A genomic screen in yeast implicates kynurenine 3-monooxygenase as a therapeutic target for Huntington disease. Nat Genet. 2005 May;37(5):526-31. PubMed.
  6. . Inhibitors of cathepsin B improve memory and reduce beta-amyloid in transgenic Alzheimer disease mice expressing the wild-type, but not the Swedish mutant, beta-secretase site of the amyloid precursor protein. J Biol Chem. 2008 Mar 21;283(12):7745-53. PubMed.
  7. . Therapeutic trials on progressive muscular dystrophy. Intern Med. 1992 Jul;31(7):841-6. PubMed.
  8. . Antiamyloidogenic and neuroprotective functions of cathepsin B: implications for Alzheimer's disease. Neuron. 2006 Sep 21;51(6):703-14. PubMed.
  9. . Reversal of autophagy dysfunction in the TgCRND8 mouse model of Alzheimer's disease ameliorates amyloid pathologies and memory deficits. Brain. 2011 Jan;134(Pt 1):258-77. PubMed.
  10. . Lysosomal proteolysis inhibition selectively disrupts axonal transport of degradative organelles and causes an Alzheimer's-like axonal dystrophy. J Neurosci. 2011 May 25;31(21):7817-30. PubMed.
  11. . The growth factor progranulin binds to TNF receptors and is therapeutic against inflammatory arthritis in mice. Science. 2011 Apr 22;332(6028):478-84. PubMed.
  12. . Subclinical zinc deficiency in Alzheimer's disease and Parkinson's disease. Am J Alzheimers Dis Other Demen. 2010 Nov;25(7):572-5. PubMed.
  13. . Cognitive loss in zinc transporter-3 knock-out mice: a phenocopy for the synaptic and memory deficits of Alzheimer's disease?. J Neurosci. 2010 Feb 3;30(5):1631-6. PubMed.

Other Citations

  1. J20 strain

External Citations

  1. Phase 2 trial data
  2. announced plans

Further Reading

Papers

  1. . A genomic screen in yeast implicates kynurenine 3-monooxygenase as a therapeutic target for Huntington disease. Nat Genet. 2005 May;37(5):526-31. PubMed.
  2. . Inhibitors of cathepsin B improve memory and reduce beta-amyloid in transgenic Alzheimer disease mice expressing the wild-type, but not the Swedish mutant, beta-secretase site of the amyloid precursor protein. J Biol Chem. 2008 Mar 21;283(12):7745-53. PubMed.
  3. . Antiamyloidogenic and neuroprotective functions of cathepsin B: implications for Alzheimer's disease. Neuron. 2006 Sep 21;51(6):703-14. PubMed.
  4. . Cognitive loss in zinc transporter-3 knock-out mice: a phenocopy for the synaptic and memory deficits of Alzheimer's disease?. J Neurosci. 2010 Feb 3;30(5):1631-6. PubMed.

Primary Papers

  1. . Kynurenine 3-monooxygenase inhibition in blood ameliorates neurodegeneration. Cell. 2011 Jun 10;145(6):863-74. PubMed.
  2. . Suberoylanilide hydroxamic acid (vorinostat) up-regulates progranulin transcription: rational therapeutic approach to frontotemporal dementia. J Biol Chem. 2011 May 6;286(18):16101-8. PubMed.
  3. . The cysteine protease inhibitor, E64d, reduces brain amyloid-β and improves memory deficits in Alzheimer's disease animal models by inhibiting cathepsin B, but not BACE1, β-secretase activity. J Alzheimers Dis. 2011;26(2):387-408. PubMed.
  4. . The kynurenine pathway modulates neurodegeneration in a Drosophila model of Huntington's disease. Curr Biol. 2011 Jun 7;21(11):961-6. PubMed.
  5. . Treating the periphery to ameliorate neurodegenerative diseases. Cell. 2011 Jun 10;145(6):813-4. PubMed.