The basic understanding of Parkinson disease, the second most common neurodegenerative disorder after Alzheimer’s, is growing and with it comes new therapeutic targets and potential treatments. In just the last few weeks researchers have reported success with a pilot gene therapy trial and the development of a histone deacetylase inhibitor that may protect against α-synuclein toxicity, the cause of some inherited forms of the disease. New therapeutic leads have also come from the identification of a novel neurotrophic factor, and an antioxidant enzyme that is deactivated in response to MPTP, a chemical that induces Parkinson symptoms. These advances raise the hope of finding a treatment that tackles the underlying degenerative nature of the disease. A calcium channel that exacerbates neuronal toxicity in mouse models of the disease was also recently reported (see ARF related news story).

Dopaminergic Defense
In the July 5 Neuron, researchers led by David Park at the University of Ottawa, Ontario, report that the antioxidant peroxiredoxin 2 (Prx2), a neuronal peroxidase, is deactivated by cyclin dependent kinase 5 (Cdk5), which is itself activated by mitochondrial toxins, such as MPTP, that cause the death of dopaminergic neurons. The researchers suggest that this chain of events compromises the ability of neurons to eliminate reactive oxygen species (ROS), which are widely believed to play an important role in the pathology of PD and other neurodegenerative diseases including Alzheimer disease (see ARF related news story). Cdk5 has also been implicated in Alzheimer’s pathology through its effects on both amyloid-β and tau (see ARF related news story).

Park and colleagues had previously shown that activation of Cdk5 is a critical event in MPTP toxicity, but how the kinase contributes to neuronal death was unclear. To address this, first author Dianbo Qu and colleagues used the Cdk5 coactivator p35 as bait to fish out Cdk5 interacting proteins from mouse brain extracts. One of the proteins they isolated was Prx2. They subsequently determined that Prx2 and Cdk5 form a complex, that Cdk5 phosphorylates the peroxiredoxin, and that this modification reduces activity of the peroxidase by about two-thirds in vitro.

Qu and colleagues found that the Prx2 inactivation is a response to MPTP and other neuronal toxins, such as rotenone. In cultured cortical neurons treated with MPTP, Prx2 activity decreased by about one-third after 24 hours, just as phosphorylation at the Cdk5 site, tyrosine 89, increased. The loss in peroxidase activity was still apparent at 48 hours, when cell survival was about 50 percent. But when the researchers introduced a phosphorylation incompetent mutant of Prx2, (Prx2T89A), it partially protected the cells against MPTP toxicity.

The findings suggest that Prx2 plays an important role in protecting dopaminergic neurons from MPTP toxicity. The same seems true in vivo, because Qu and colleagues found that Prx2 in dopaminergic neurons of wild-type mice was phosphorylated in response to MPTP. Significantly, the phosphorylated peroxiredoxin was barely detected in MPTP-treated animals lacking p35, a coactivator of Cdk5. These results implicate the Cdk5-Prx2 pathway in MPTP toxicity in vivo. This idea is supported by the finding that the calpain inhibitor calpastatin blocks Prx2 phosphorylation in response to MPTP—activation of Cdk5 by calpain has been implicated in both PD and AD pathology (see ARF related news story).

The authors advise caution in drawing any direct comparison between the slow neurodegenerative process that underlies Parkinson’s and MPTP toxicity, which is acute. Nevertheless, there is reason to believe that the findings may be relevant to the human disease. For one, the authors found phosphorylated Prx2 in the soma of dopaminergic neurons in postmortem PD tissue samples, but not in control samples. Secondly, they found that expression of DJ-1, a gene that causes PD when mutated, prevented MPTP-driven phosphorylation of Prx2. Since it is unclear how DJ-1 protects against PD, this is a connection that warrants further investigation, suggest the authors.

In addition to Prx2, scientists have uncovered another potential savior of dopaminergic neurons. Reporting in the July 5 Nature, researchers in Finland and Estonia led by Mart Saarma at the University of Helsinki, Finland, reveal a novel neurotrophic factor that they call conserved dopamine neurotrophic factor (CDNF). The trophin is a homolog of vertebrate and invertebrate mesencephalic-astrocyte-derived neurotrophic factors (MANFs). First author Païvi Lindholm and colleagues cloned human and mouse CDNF from brain tissue after a bioinformatics study identified MANF-homologous expressed sequence tags. In humans CDNF has been dubbed ARMET-like 1, where ARMET stands for “arginine-rich, mutated in early stage tumors.” But the authors point out that the arginine-rich region is found only in MANF from humans.

Lindholm and colleagues found CDNF mRNA expressed in several regions of the brain in postnatal pups, including the hippocampus, thalamus, the striatum and the substantia nigra, the major site of dopaminergic neuron loss seen in PD. They purified CDNF and after generating polyclonal antibodies discovered the protein in adult mouse heart, skeletal muscle, and testes. They found only low levels in whole brain extracts but immunohistochemical analysis detected CDNF in the CA1 and CA2 region of the hippocampus, the dentate gyrus, the striatum, and the substantia nigra. In the last, the trophin did not colocalize with tyrosine hydroxylase (TH), a marker for dopaminergic neurons.

Because MANF was earlier shown to promote survival of cultured dopaminergic cells, Lindholm and colleagues tested CDNF in a rat model of PD to see if it might be neuroprotective. When the authors injected CDNF into the brain prior to 6-hydroy DOPA, which induces ablation of dopaminergic neurons, they found that amphetamine-driven ipsilateral turning behavior, a symptom of dopamine neuron loss, was substantially reduced 2 weeks later, down from about 200 turns per hour to only about 40. Four weeks after the lesion, the number of TH-positive cells in the substantia nigra pars compacta of CDNF-treated animals was also significantly greater than in control rats. Protection against both the turning behavior and TH-positive cell loss was dose-dependent.

In addition to its protective effects, the researchers found that CDNF could help restore previously damaged TH neurons, as well. When they administered a single injection of the protein to rats treated with 6-hyrdroxy DOPA 4 weeks prior, they observed a partial recovery of injured neurons (58 percent) 8 weeks later. Again, amphetamine-driven turning behavior was significantly attenuated.

“Because CDNF is able to protect and restore the function of dopaminergic neurons in the rat Parkinson’s disease model, it has potential as a therapeutic protein or as a basis for the development of drugs for the treatment of Parkinson’s disease,” write the authors. In this respect, the trophin is reminiscent of glial-derived neurotrophic factor (GDNF). Clinical trials for GDNF therapy were stopped in 2004 because of lack of efficacy (see ARF related news story) despite indications of dopaminergic recovery in some patients (see ARF related news story). In fact, Lindholm and colleagues tested CDNF side-by-side with GDNF and found them to have almost identical efficacies. Whether CDNF might work for PD where GDNF failed is unclear at present, but the authors note that while GDNF promotes survival of embryonic and postnatal peripheral neurons, CDNF did not. “Our results suggest that CDNF is not a neurite outgrowth and survival promoting factor for neurons in the developing peripheral nervous system, but rather a neurotrophic factor for brain neurons,” write the authors.

Potential Treatments
New promising approaches to PD treatments include gene therapy and small molecules that might quell α-synuclein toxicity. In the June 23 Lancet, Matthew During, Weill Medical College of Cornell University, New York, and colleagues report results of a small pilot trial to test the safety and tolerability of a gene therapy that delivers glutamic acid decarboxylase (GAD) to the brains of Parkinson’s patients. GAD is an enzyme that is essential for production of γ-amino-butyric acid (GABA), a major inhibitory neurotransmitter. By delivering GAD to the subthalamic nucleus it is hoped that GABA production there will counter excitatory signals that contribute to motor problems. In Parkinson disease, loss of dopaminergic output from the substantia nigra results in hyperactivity of neurons in the subthalamic nucleus (STN), which in turn inhibits motor signals through the thalamus. Quelling the STN might relieve some of the motor symptoms of the disease.

First author Michael Kaplitt and colleagues tested the safety and tolerability of an adeno-associated virus harboring the GAD gene. Three different doses of the virus were injected unilaterally into the subthalamus in a total of 12 Parkinson disease patients. The subjects were examined at baseline and again 1, 3, 6, and 12 months after treatment.

The researchers reported no complication with the procedure and no related adverse effects with the therapy. Clinically, the patients seemed to improve. Though there was no significant change 1 month after surgery, over the next 11 months there was, on average, a significant improvement in motor function of up to 28 percent on the unified Parkinson disease rating scale. Brain imaging scans showed reduced glucose utilization in the thalamus on the treated side of the brain 12 months after surgery.

Though the trial was not designed to test efficacy, the results seem promising. However, A Jon Stoessl, University of British Columbia, notes some caveats in an accompanying Lancet commentary. “Because the placebo effect in Parkinson’s disease can be large, especially after surgical intervention, no firm conclusions can be drawn about efficacy, despite objective improvement in motor function and functional imaging,” he writes. Nevertheless, he suggests that further double-blind and sham-surgery controlled trials are now warranted.

An alternative type of treatment might grow from a small molecule enzyme inhibitor reported in the June 21 Science online. Aleksey Kazantsev and colleagues at Massachusetts General Hospital, Boston, report that inhibitors of SIRT2, a histone deacetylase, can prevent α-synuclein toxicity in cells and fruit flies. Kazantsev and colleagues had previously identified a small molecule that promoted formation of large α-synuclein inclusions in neurons, protecting them against synuclein-associated toxicity (see ARF related news story). Now, first author Tiago Fleming Outeiro and colleagues report that that molecule, dubbed B2, inhibits SIRT2. Armed with this knowledge, the researchers have screened a library of B2 analogs to find more potent and specific SIRT2 inhibitors. Outeiro shows that these more potent molecules protect human neuroblastoma cells against overexpression of α-synuclein, and dopaminergic cells against mutant α-synuclein that causes Parkinson’s. The compounds also protect fruit flies expressing the mutant protein. At the highest doses, the SIRT2 inhibitors limited dopaminergic neuronal loss to only 10 percent of that seen in untreated animals.

Though it is not clear how inhibiting this histone deacetylase promotes recruitment of α-synuclein into inclusion bodies, the finding supports the idea that these protein aggregates, a hallmark of Parkinson’s and other neurodegenerative diseases, protect cells by sequestering toxic proteins. “Thus, SIRT2 targeting may be therapeutically beneficial in other diseases where aggregation of misfolded proteins is central to disease pathogenesis,” write the authors.—Tom Fagan.

References:
Lindholm P, Voutilainen MH, Luren J, Peranen J, Leppanen V-M, Andressoo J-O, Lindhal M, Janhunen S, Kalkkinen N, Timmusk T, Tuominen RK, Saarma M. Novel neurotrophic factor CDNF protects and rescues midbrain dopamine neurons in vivo. Nature. 2007, July 5;448:73-77. Abstract

Qu D, Rashidian J, Mount MP, Aleyasin H, Parsanejad M, Lira A, Haque E, Zhang Y, Callaghan S, Daigle M, Rousseaux MWC, Slack RS, Albert PR, Vincent I, Woulfe JM, Park DS. Role of Cdk5-mediated phosphorylation of Prx2 in MPTP toxicity and Parkinson’s disease. Neuron. 2007, July 5;55:37-52. Abstract

Kaplitt MG, Feigin A, Tang C, Fitzsimons HL, Mattis P, Lawlor PA, Bland RJ, Young D, Strybing K, Eidelberg D, During MJ. Safety and tolerability of gene therapy with an adeno-associated virus (AAV) borne GAD gene for Parkinson’s disease: an open label, phase I trial. Lancet. 2007, June 23;369:2097-2105. Abstract

Stoessl AJ. Gene therapy for Parkinson’s disease: early data. Lancet. 2007, June 23;369:2056-2057. Abstract

Outeiro TF, Kontopoulos E, Altman S, Kufareva I, Strathearn KE, Amore AM, Volk CB, Maxwell MM, Rochet J-C, McLean PJ, Young AB, Abagyan R, Feany MB, Hyman BT, Kazantsev A. Sirtuin 2 inhibitors rescue alpha-synuclein–mediated toxicity in models of Parkinson’s disease. Science. 2007, June 21 online. Abstract

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References

News Citations

  1. Teaching Old Neurons Young Tricks—“Rejuvenation” Protects from PD
  2. Aβ Production Linked to Oxidative Stress
  3. Tangles, Neurodegeneration, Plaques—p25 Does it All
  4. The Calpain Connection
  5. End of the Road for GDNF Parkinson Therapy—or Just a Detour?
  6. GDNF Powers Neuron Sprouting in Human Brain
  7. Inclusions: Part of the Problem, or the Solution?

Paper Citations

  1. . Novel neurotrophic factor CDNF protects and rescues midbrain dopamine neurons in vivo. Nature. 2007 Jul 5;448(7149):73-7. PubMed.
  2. . Role of Cdk5-mediated phosphorylation of Prx2 in MPTP toxicity and Parkinson's disease. Neuron. 2007 Jul 5;55(1):37-52. PubMed.
  3. . Safety and tolerability of gene therapy with an adeno-associated virus (AAV) borne GAD gene for Parkinson's disease: an open label, phase I trial. Lancet. 2007 Jun 23;369(9579):2097-105. PubMed.
  4. . Gene therapy for Parkinson's disease: early data. Lancet. 2007 Jun 23;369(9579):2056-8. PubMed.
  5. . Sirtuin 2 inhibitors rescue alpha-synuclein-mediated toxicity in models of Parkinson's disease. Science. 2007 Jul 27;317(5837):516-9. PubMed.

Further Reading

Papers

  1. . Role of Cdk5-mediated phosphorylation of Prx2 in MPTP toxicity and Parkinson's disease. Neuron. 2007 Jul 5;55(1):37-52. PubMed.
  2. . Safety and tolerability of gene therapy with an adeno-associated virus (AAV) borne GAD gene for Parkinson's disease: an open label, phase I trial. Lancet. 2007 Jun 23;369(9579):2097-105. PubMed.
  3. . Gene therapy for Parkinson's disease: early data. Lancet. 2007 Jun 23;369(9579):2056-8. PubMed.
  4. . Sirtuin 2 inhibitors rescue alpha-synuclein-mediated toxicity in models of Parkinson's disease. Science. 2007 Jul 27;317(5837):516-9. PubMed.
  5. . Novel neurotrophic factor CDNF protects and rescues midbrain dopamine neurons in vivo. Nature. 2007 Jul 5;448(7149):73-7. PubMed.

Primary Papers

  1. . Role of Cdk5-mediated phosphorylation of Prx2 in MPTP toxicity and Parkinson's disease. Neuron. 2007 Jul 5;55(1):37-52. PubMed.
  2. . Safety and tolerability of gene therapy with an adeno-associated virus (AAV) borne GAD gene for Parkinson's disease: an open label, phase I trial. Lancet. 2007 Jun 23;369(9579):2097-105. PubMed.
  3. . Gene therapy for Parkinson's disease: early data. Lancet. 2007 Jun 23;369(9579):2056-8. PubMed.
  4. . Sirtuin 2 inhibitors rescue alpha-synuclein-mediated toxicity in models of Parkinson's disease. Science. 2007 Jul 27;317(5837):516-9. PubMed.
  5. . Novel neurotrophic factor CDNF protects and rescues midbrain dopamine neurons in vivo. Nature. 2007 Jul 5;448(7149):73-7. PubMed.