Is Alpha-Synuclein Loss-of-Function a Contributor to Parkinsonian Pathology? Evidence from Non-human Primates

Accumulation of alpha-synuclein (α-syn) in Lewy bodies and neurites of midbrain dopamine neurons is diagnostic for Parkinson’s disease (PD), leading to the proposal that PD is a toxic gain-of-function synucleinopathy. Here we discuss the alternative viewpoint that α-syn displacement from synapses by misfolding and aggregation results in a toxic loss-of-function. In support of this hypothesis we provide evidence from our pilot study demonstrating that knockdown of endogenous α-syn in dopamine neurons of nonhuman primates reproduces the pattern of nigrostriatal degeneration characteristic of PD

Loss of α-Synuclein Does Not Affect Mitochondrial Bioenergetics in Rodent Neurons.

Increased α-synuclein (αsyn) and mitochondrial dysfunction play central roles in the pathogenesis of Parkinson's disease (PD), and lowering αsyn is under intensive investigation as a therapeutic strategy for PD. Increased αsyn levels disrupt mitochondria and impair respiration, while reduced αsyn protects against mitochondrial toxins, suggesting that interactions between αsyn and mitochondria influences the pathologic and physiologic functions of αsyn. However, we do not know if αsyn affects normal mitochondrial function or if lowering αsyn levels impacts bioenergetic function, especially at the nerve terminal where αsyn is enriched. To determine if αsyn is required for normal mitochondrial function in neurons, we comprehensively evaluated how lowering αsyn affects mitochondrial function. We found that αsyn knockout (KO) does not affect the respiration of cultured hippocampal neurons or cortical and dopaminergic synaptosomes, and that neither loss of αsyn nor all three (α, β and γ) syn isoforms decreased mitochondria-derived ATP levels at the synapse. Similarly, neither αsyn KO nor knockdown altered the capacity of synaptic mitochondria to meet the energy requirements of synaptic vesicle cycling or influenced the localization of mitochondria to dopamine (DA) synapses in vivo. Finally, αsyn KO did not affect overall energy metabolism in mice assessed with a Comprehensive Lab Animal Monitoring System. These studies suggest either that αsyn has little or no significant physiological effect on mitochondrial bioenergetic function, or that any such functions are fully compensated for when lost. These results implicate that αsyn levels can be reduced in neurons without impairing (or improving) mitochondrial bioenergetics or distribution

Targeted gene delivery to the enteric nervous system using AAV: a comparison across serotypes and capsid mutants

Recombinant adeno-associated virus (AAV) vectors are one of the most widely used gene transfer systems in research and clinical trials. AAV can transduce a wide range of biological tissues, however to date, there has been no investigation on targeted AAV transduction of the enteric nervous system (ENS). Here, we examined the efficiency, tropism, spread, and immunogenicity of AAV transduction in the ENS. Rats received direct injections of various AAV serotypes expressing green fluorescent protein (GFP) into the descending colon. AAV serotypes tested included; AAV 1, 2, 5, 6, 8, or 9 and the AAV2 and AAV8 capsid mutants, AAV2-Y444F, AAV2-tripleY-F, AAV2-tripleY-F+T-V, AAV8-Y733F, and AAV8-doubeY-F+T-V. Transduction, as determined by GFP-positive cells, occurred in neurons and enteric glia within the myenteric and submucosal plexuses of the ENS. AAV6 and AAV9 showed the highest levels of transduction within the ENS. Transduction efficiency scaled with titer and time, was translated to the murine ENS, and produced no vector-related immune response. A single injection of AAV into the colon covered an area of ~47 mm(2). AAV9 primarily transduced neurons, while AAV6 transduced enteric glia and neurons. This is the first report on targeted AAV transduction of neurons and glia in the ENS

Loss of functional alpha-synuclein: A toxic event in Parkinson\u27s disease?

The discovery that alpha-synuclein (α-syn) is the primary component of the neuropathological hallmarks of Parkinson\u27s disease (PD) and the identification of α-syn mutations in numerous inherited forms of PD has positioned α-syn at the top of the list of important factors in the pathogenesis of PD. Based on the pathological accumulation of α-syn in the brains of patients, the field is currently focused on therapeutic strategies that aim to reduce or eliminate α-syn. However, recent evidence suggests α-syn is a critical protein in neuron (i.e. dopamine neurons) survival and that maintaining a certain level of biologically functional α-syn is an important consideration in targeting α-syn for therapies. Despite the widespread interest in α-syn, the normal biological functions remain elusive, but a large body of work is focused on addressing this issue. In this review, we will discuss the current evidence related to α-syn function, α-syn folding and aggregation, and α-syn\u27s role in disease. Finally, we will propose a relatively novel hypothesis on the pathogenesis of PD that hinges upon the premises that functional α-syn is critical to cell survival and that a reduction in biologically functional α-syn, whether through aggregation or reduced expression, may lead to the neurodegeneration in PD. © 2012 - IOS Press and the authors. All rights reserved

Intraparenchymal stereotaxic delivery of rAAV and special considerations in vector handling

Stereotaxic surgery enables precise and consistent microinjections to discrete neural nuclei. Using stereotaxic surgery to deliver viral vectors is a powerful tool that provides the ability to manipulate gene expression in specific regions, or even specific cell types in the brain. Here, we describe the proper handling and stereotaxic delivery of recombinant adeno-associated virus to various neuroanatomical structures of the rodent brain

The development of flexible lentiviral vectors for gene transfer in the CNS

The use of recombinant lentiviral vectors (rLV) is emerging as a viable candidate for clinical gene therapy of the central nervous system. New generation vectors are being produced while addressing viral safety concerns as well as production capabilities. Furthermore, the ability to combine envelope proteins targeting specific cell types with specific promoters guiding the expression of the genetic payload will allow researchers and clinicians to precisely guide transgene expression to anatomically and phenotypically distinct populations of cells. In a recent issue of Experimental Neurology, Cannon and colleagues demonstrate the ability to transduce specific populations of cells in the rat midbrain by using differently pseudotyped lentiviral vectors which results in significant differences in transgene spread throughout the nigrostriatal tract. These results highlight the potential utility of rLV in clinical applications as well as in research involving neurodegenerative disease. © 2011 Elsevier Inc

Development of gene therapy for neurological disorders

Given improvements in viral vector design, production and efficiency of transduction in the central nervous system (CNS), as well as increased knowledge of neuropathological mechanisms in neurological disorders, success in treating a CNS disorder with gene transfer seems inevitable. Several different vector systems have been studied extensively and the adeno-associated viral vector system has been utilized in most early stage clinical trials in neurological disorders. Other vector systems, such as lentivirus, adenovirus, and herpes simplex virus are also viable vector platforms that should fill significant clinical niches based on their specific characteristics. In addition to the choice of the appropriate vector, the proper choice of transgene for the appropriate strategy to treat a neurological disorder is also critical. The example of glial cell line-derived neurotrophic factor ligands to treat Parkinson\u27s disease is used to illustrate the importance of the interface between interpretation of pre-clinical data and consideration of the natural history of the disorder. This interface dictates the proper design of clinical trials that are capable of testing whether the treatment is actually successful

Induction of Alpha-Synuclein Pathology in the ENS of the Rat and Non-Human Primates Results in Gastointestinal Dymotility and Transient CNS Pathology

PURPOSE: The purpose of this study is to determine whether alpha-synuclein (a-syn) pathology can be initiated in the enteric nervous system (ENS) and spread to the central nervous system (CNS) over time. A-syn is a major protein involved in familial and sporadic Parkinson’s disease (PD); PD is a progressive neurodegenerative disorder, the cause of which remains unknown. The ENS is important to study for two reasons: first, the most significant clinical presentations of PD is gastric dysfunction, and second, accumulation of Lewy bodies (primarily a-syn aggregates) can be found in ENS myenteric and submucosal neurons. CHALLENGE: Although aggregated a-syn has been shown to cause conversion of functional a-syn to insoluble proteins, how a-syn becomes toxic to the neurons and relates to the pathophysiology of PD is unknown. EXPERIENCE: I had worked independently on several assigned tasks such as brain dissection and its sample collection and staining, as well as working as part of the research team. OUTCOME: The outcome of this project implied that the spread of a-syn pathology from enteric neurons to the CNS is limited and not adequate to sustain pathological spread of a-syn throughout the CNS on its own. IMPACT: I had gained many technical and professional skills during my one-year internship. It also helped me better understand a research lab dynamic. The challenges I had while performing certain task or repeating protocols helped me to adapt to become a better scientist. I was hired as a research technician to continue the research after my internship

Gene Silencing of Striatal Cav 1.3 Channels can Prevent Levodopa-Induced Dyskinesia in Parkinsonian Rats

PURPOSE: Parkinson’s Disease (PD) is a debilitating neurodegenerative disorder that affects dopaminergic neurons in the brain. Levodopa, the gold standard medication for treating PD, can lead to several side effects, with Levodopa-Induced Dyskinesia (LID) as most severe. LID are abnormal and involuntary movements of the limbs and/or trunk that arise from the loss of dendritic spines of medium spiny neurons associated with the over-activity of striatal Cav 1.3 channels. Gene knockdown of Cav 1.3 channels prior to inducing parkinsonism and administration of levodopa in rats has been reported to prevent LID. The purpose of this study was to determine the amount of Cav 1.3 protein knockdown after gene silencing. CHALLENGE: Cav 1.3 protein is present in very low amounts in the brain and is therefore notoriously difficult to quantify. EXPERIENCE: Rats were given unilateral injections of either control or sh-RNA to silence Cav 1.3 channels. This was followed by induction of parkinsonism via 6-OHDA and administration of levodopa prior to LID monitoring (blinded). Rat brain tissues were sectioned and stained using fluorescent dye and Cav 1.3 protein expression was analyzed using a confocal microscope. OUTCOME: The amount of Cav 1.3 protein knockdown is an important next step after analysis of gene knockdown. Targeted gene silencing of Cav 1.3 channels can be used as therapeutic approach for preventing/potentially reversing LID in PD patients. IMPACT: This study allowed learning and development of several new skills such as tissue sectioning and staining, animal handling, and confocal microscopy

Gene therapy for neurological disorders: Challenges and future prospects for the use of growth factors for the treatment of Parkinson\u27s disease

Glial cell line-derived neurotrophic factor (GDNF) family of ligands (GDFLs) as well as other trophic factors have, in animal models of Parkinson\u27s disease (PD), demonstrated the potential for excellent ameliorative properties. Clinical trials that have mechanically injected GDNF intracerebrally, while demonstrating relative safety, have been clinically disappointing to date. Likewise, recombinant adeno-associated virus (rAAV) delivered neurturin (cere-120) has also been demonstrated to be safe in humans, however clinical results have been negative. The failure of the major clinical trials has cast some doubt in the field about trophic factor delivery for the treatment of PD. In this review, we make the case that GDFLs are likely to function only when there are remaining dopamine neurons in the nigrostriatal pathway as opposed to other candidate modes of action. Thus, it is our view that utilizing earlier stage PD patients who have significant nigrostriatal dopamine innervation remaining would be more ideal to demonstrate the efficacy of GDFLs. This is particularly true when considering a novel delivery method such as gene transfer. However, if earlier stage patients are to be enrolled in GDFL gene transfer trials, then a much better safety profile must be demonstrated by preclinical experiments. One important safety advance might be the use of an external regulation system to control the expression level of the transgene. However, gene regulation systems pose unique safety issues and we will discuss these in detail. It is our view that GDFLs still remain as a promising therapeutic approach for PD. © 2009 Bentham Science Publishers Ltd