Parkinson’s – Drug Discovery Opinion

Role for c-Abl in Parkinson’s Disease

The majority of Parkinson’s disease (PD) cases have no known cause, but have been associated with increased oxidative stress and mitochondrial dysfunction. Of the small proportion of hereditary cases, a number of defective genes have been identified including LRRK2 (PARK8), DJ-1 (PARK7), α-synuclein (SNCA) and parkin (PARK2). Mutations in parkin, which encodes an E3 ubiquitin ligase, are believed to interfere with the ability of parkin to clear the cell of its normal substrate proteins. Several substrates for parkin have been identified and shown to accumulate in the brain tissue of patients with hereditary PD.

Researchers at The University of Texas Health Science Center have now identified a link between the tyrosine kinase, c-Abl, and impaired parkin function. The scientists found that c-Abl was activated in cultured neuronal cells and the striatum of adult mice when subjected to oxidative and neuronal stress. They also identified parkin as a specific substrate for c-Abl and that the tyrosine-phosphorylated parkin lost its ubiquitin ligase activity.

The c-Abl inhibitor, imatinib (STI-571) was able to block the phosphorylation of parkin in vitro and in vivo, restoring ligase activity. Since there are several c-Abl inhibitors approved for the treatment of chronic myelogenous leukemia, tools are available to further explore the neuroprotective potential of c-Abl inhibition in sporadic PD.

The study is published in the Journal of Neuroscience.

Stressful ‘Lifestyle’ is Bad for Neurons

Parkinson’s disease is characterised by loss of dopaminergic neurons in the area of the midbrain known as the substantia nigra. Although mitochondrial stress – an accumulation of damaging superoxide and free radicals – is believed to be the cause of cell death, it is not understood why this subset of neurons is especially vulnerable.

Researchers at Northwestern University have now suggested a possible answer: these neurons have an inherently stressful ‘lifestyle’. The cells in the substantia nigra act as pacemakers, releasing rhythmic bursts of dopamine. This activity is accompanied by an influx of calcium ions which must then be pumped back out of the cell in an energy-demanding process. The inflow of calcium ions is not essential for pacemaking activity so, if the energy needed to pump calcium ions out of the cell is adding extra stress, blocking the influx of calcium should help to alleviate this. Using mice engineered to express a redox-sensitive fluorescent protein in their mitochondria, the team showed that the opening of L-type calcium channels during normal pacemaking activity created an oxidant stress that was specific to dopaminergic cells of the substantia nigra. The oxidative stress, in turn, caused a defensive mild mitochondrial depolarization or uncoupling.

Although most cases of Parkinson’s disease have no known genetic cause, loss-of-function DJ-1 (PARK7) mutations can cause early-onset Parkinson’s disease in humans and transgenic mice lacking DJ-1 also show damage to dopaminergic cells in the substantia nigra. Knocking out DJ-1 down-regulates expression of two uncoupling proteins and increases oxidation of mitochondrial matrix proteins in dopaminergic neurons of the substantia nigra. Treatment of the transgenic animals with the L-type calcium channel blocker, isradipine, was found to protect the dopaminergic cells of the substantia nigra from oxidative damage.

The study, which is published in the journal Nature, builds on previous studies linking calcium channel blockade with protective effects in Parkinson’s disease.

A clinical trial is currently underway to examine the safety, tolerability and efficacy of isradipine – which is already approved for the treatment of high blood pressure – in patients with Parkinson’s disease. The hope is that the drug will slow disease progression and allow a broader window for existing symptomatic treatments.

LRRK2 Inhibitor Protective in Parkinson’s Disease Model

"Moondance" photographed by Tena (Madmoiselle Lavender), a Parkinson's veteran

The main symptoms of Parkinson’s disease are tremor, rigidity and involuntary movement, caused by loss of dopaminergic neurons in the brain. Leucine-rich repeat protein kinase-2 (LRRK2) is mutated in a significant number of Parkinson’s disease cases, both familial and sporadic late-onset. A common mutation in which a glycine residue in the active site is altered to serine enhances catalytic activity of the kinase, suggesting that LRRK2 inhibitors might be useful for the treatment of Parkinson’s disease, although it is not entirely clear why enhanced LRRK2 activity causes loss of dopamine-producing neurons. Scientists led by a team at the Johns Hopkins University School of Medicine have now shown that inhibitors of the G2019S variant of LRRK2 can protect the nerve cells of mice genetically modified to produce the mutated kinase. Three weeks twice daily injections of GW5074 provided almost complete protection against loss of dopaminergic neurons compared with placebo treatment.

Although GW5074 is not an especially potent inhibitor of wild type or G2019S LRRK2 (IC₅₀ 0.2µM – 1.0µM depending on substrate) and is not selective (IC₅₀ vs cRaf ca 10nM), the study, which is published in Nature Medicine, provides encouragement that a more potent and selective inhibitor could lead to a new disease-modifying treatment for Parkinson’s disease. The John Hopkins team are collaborating with researchers at Southern Methodist University to design more selective inhibitors and many other groups in both industry and academia are engaged in the search for potent and selective LRRK2 inhibitors.

Neuroprotective Small Molecule TrkB Agonist

Roles have been suggested for brain-derived neurotrophic factor (BDNF) – which helps to support neurons and also stimulates and controls neurogenesis – in preventing or treating degenerative diseases such amyotrophic lateral sclerosis, Parkinson’s disease, and Alzheimer’s disease. The use of BDNF itself in therapy is limited by a poor pharmokinetic profile including rapid metabolism and poor CNS penetration. BDNF elicits at least some of its effects through binding to the high affinity tyrosine kinase receptor B, TrkB, and investigators at Emory University School of Medicine have now identified a small, high-affinity molecule that can also activate signalling through TrkB.

7,8-dihydroxy flavone structure — 7,8-dihydroxyflavone

7,8-Dihydroxyflavone was shown to protect wild-type, but not TrkB-deficient, neurons from apoptosis. Following intraperitoneal administration, the compound was also found to activate TrkB in the brain and to be protective in animal models of seizure, stroke and Parkinson’s disease. The compound was also found to have low toxicity on chronic dosing. Although favonoids such as 7,8-dihydroxyflavone occur in a wide range of foodstuffs, levels obtained from a normal diet are believed to be insufficient for a sustained effect.

The study is published in the online early edition of PNAS.

Statins Protect Mice from Symptoms of Parkinson’s Disease

Parkinsons Disease Tulip Statins lower cholesterol by inhibiting HMG-CoA reductase, the rate-limiting step in cholesterol biosynthesis, and new indications have continued to emerge for these medicines since their introduction in the late 1980s. The association between cholesterol levels, statin use, and Parkinson’s disease has been much debated in recent years, with some studies suggesting that low cholesterol levels (although not statin use) are linked to the disease, others suggesting that statin use is associated with a reduced incidence of disease, and yet others finding no effect of statin use on disease incidence.

New research by scientists at Rush University Medical Center and the University of Nebraska Medical Center now adds evidence that statins are protective by demonstrating that simvastatin can reverse the toxic effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in mice. MPTP causes selective dopaminergic neurotoxicity in cells of the substantia nigra, causing parkinsonism in humans and some laboratory animals. MPTP itself is not neurotoxic, but is metabolised by monoamine oxidase-B into the toxic cation, methyl-4-phenylpyridinium (MPP⁺). The team found that MPP⁺ induced activation of p21^ras and nuclear factor-_κB (NF-_κB) in mouse microglial cells and that this effect was attenuated by simvastatin. p21^ras was also found to be rapidly activated in vivo in the substantia nigra pars compacta of mice treated with MPTP. Oral administration of simvastatin reduced nigral activation of p21^ras and NF-_κB, inhibited expression of proinflammatory molecules, and suppressed activation of glial cells. These changes were associated with protection of dopaminergic neurones, normalized striatal neurotransmitters, and improved motor function. Pravastatin was also shown to protect dopaminergic neurons from the toxic effects of MPTP, but to a lesser extent than simvastatin. Both statins were still able to protect dopaminergic neurons when administered 2 days after treatment with MPTP, suggesting that statins may provide benefit to Parkinson’s disease patients. The use of statins would be particularly attractive because of their proven safety profile in very large patient populations.

The study is published in the current issue of the Journal of Neurosciences.

Phosphorylation Modulates α-Synuclein Neurotoxicity

Immunohistochemistry for α-synuclein showing positive staining (brown) of an intraneural Lewy-body in the Substantia nigra in Parkinson disease

Image: Wikimedia - Marvin 101

α-Synuclein, expressed primarily in neural tissue, is normally an unstructured, soluble, protein. Under some circumstances, however, it has the ability to aggregate and form insoluble fibrils. A missense mutation in α-synuclein, A53T, was the first defined genetic lesion in familial Parkinson’s disease. Subsequently, α-synuclein was identified as a major component of Lewy bodies, even in the more common later-onset variant of Parkinson’s, which in most cases does not involve a clear family history of the disease. Lewy bodies are also seen in a group of related disorders, synucleinopathies, which may share important pathways of pathogenesis with Parkinson’s disease.

A new study from scientists at Brigham and Women’s Hospital and Harvard Medical School, Università di Padova and Massachusetts General Hospital has now established a role for phosphorylation in the control of α-synuclein neurotoxicity. The group had previously identified Ser¹²⁹ phosphorylation as a key event in α-synuclein neurotoxicity in a Drosophila model. Ser¹²⁹ phosphorylation conferred toxicity to α-synuclein without a substantial increase in the number of fibrillar deposits, suggesting that nonfibrillar species of α-synuclein may be neurotoxic.

The latest work shows that α-synuclein is also phosphorylated at Tyr¹²⁵ in transgenic Drosophila expressing wild-type human α-synuclein and that this tyrosine phosphorylation protects from α-synuclein neurotoxicity in a Drosophila model of Parkinson’s disease. Further, the team found that there was an age-related decrease in Tyr¹²⁵ phosphorylation in humans (as well as in the transgenic Drosophila) and that this phosphorylation was undetectable in the brains of patients who had dementia with Lewy bodies.

The study, published in the Journal of Clinical Investigation, suggests that the loss of protective tyrosine phosphorylation may predispose to clinically relevant α-synuclein neurotoxicity in human disease.

Yeast Cells: Drug Discovery Factories

Whitehead Institute researchers have described a new drug discovery technique which uses yeast cells to both synthesise and screen novel compounds. Writing in the journal Nature Chemical Biology, the team have demonstrated that they can negate the toxic effects of α-synuclein in a yeast model that mimics much of the cellular pathology of Parkinson’s disease. α-Synuclein accumulates in vulnerable brain cells in patients with Parkinson’s disease, and the team had previously shown that yeast cells engineered to express large amounts of α-synuclein do not survive. In the new study, yeast cells were engineered to produce cyclic peptides – which target protein-protein interactions – and the α-synuclein was then switched on. The cells that produced cyclic peptides that protect against α-synuclein toxicity survived: the rest died. Out of a library of millions of cyclic peptides, only two were found to rescue yeast cells from α-synuclein toxicity. Although it is not yet clear how the cyclic peptides protect the cells – they were shown not to affect vesicle trafficking – both peptides share a structural motif with thioredoxins, proteins that act as antioxidants; metal transport proteins and proteins that regulate gene activity. The team are now working to determine the precise mechanism of action and to develop new analogues of the peptides.

In a follow-on study carried out by researchers at the University of Alabama, these two cyclic peptides were also found to protect dopaminergic neurones in a C. elegans model of Parkinson’s disease.

The new technique is rapid and inexpensive compared with other methods of lead discovery, and should be applicable to other diseases where key aspects of the pathology can be modelled in yeast or mammalian cells.

E3 Ubiquitin Ligase CHIP Regulates LRRK2 Stability

Parkinson's Tulip logo Although mutations in the gene for leucine-rich repeat kinase 2 (LRRK2) have been linked to both familial and sporadic cases of Parkinson’s disease, the exact cellular function of LRRK2 remains unclear. The mechanisms by which mutated LRRK2 variants contribute to Parkinson’s disease are also unclear but it is believed that they lead, directly or indirectly, to increased kinase activity and promote inclusion formation leading to neurotoxicity. Researchers at the University of Texas Southwestern Medical Center have now identified a strong interaction between LRRK2 and CHIP (C-terminus of Hsp70-Interacting Protein), an E3 ubiquitin ligase which is crucial for the ubiquitination of several heat shock protein (Hsp70/Hsp90) client proteins involved in neurodegenerative disease. The screen also confirmed a robust interaction between LRRK2 and Hsp90, which had previously been identified as a LRRK2 binding protein. CHIP was further found to be able to significantly reduce cellular levels of LLRK2 in a dose-dependent fashion, most likely by ubiquitination and subsequent proteasome-dependent degradation. CHIP-mediated degradation of both wild type and mutant LRRK2 was found to be similar.

CHIP was found to be able to bind to LRRK2 in at least two different ways: directly or indirectly, but independent of Hsp90, to the ROC (Ras of complex) domain of LRRK2 via CHIP’s charged domain; indirectly, via Hsp90, to the N-terminal domain of LRRK2 via the TPR (tetratricopeptide repeat) domain of CHIP. Hsp90 was shown to block CHIP-mediated degradation of LRRK2, which could be overcome using the Hsp90 inhibitor, geldanamycin.

The authors hope that the discovery of cellular mechanisms that regulate LRRK2 will provide new therapeutic targets for the treatment of familial and sporadic Parkinson’s disease.

The study is published in the journal PloS ONE.

New Brain Delivery Strategy for Parkinson’s Disease

The blood-brain barrier (BBB) fulfills an essential role by restricting the entry of potentially neurotoxic chemicals into brain tissue. The downside of this protective function is that entry of therapeutic molecules into the brain may also be severely restricted; delivering adequate amounts of drugs is one of the biggest challenges in treating many brain diseases.

L-Dopa, used to treat Parkinson’s Disease, is transported into the brain using a carrier system (LAT 1) which normally transports large neutral amino acids. L-Dopa is close enough in structure to one of the endogenous substrates, phenylalanine, to gain entry using this transporter, but the constraints in terms of size and shape on the transported molecule mean that opportunities for such carrier-mediated transport are very limited.

Now Armagen Technologies has announced funding by the Michael J. Fox Foundation for Parkinson’s Research to develop a receptor-mediated system to deliver a neurotrophin into the brain. Receptor-mediated transport mechanisms involve attaching the drug molecule to a protein recognized by cell surface receptors and triggering an energy-dependant transcytosis. In this case, the neurotrophin, which protects the part of the brain that degenerates in Parkinson’s Disease, is fused to a monoclonal antibody which is able to cross the blood brain barrier and so deliver the neurotrophin into the brain tissue.

Receptor-mediated transport mechanisms offer greater flexibility in terms of the size and shape of drug molecules that can be transported, and are likely to be more widely applicable than carrier-mediated systems.