Parkinson-related parkin reduces α-Synuclein phosphorylation in a gene transfer model

α-Synuclein aggregates in Lewy bodies and plays a central role in the pathogenesis of a group of neurodegenerative disorders, known as "Synucleinopathies", including Parkinson's disease. Parkin mutations result in loss of parkin E3-ubiquitin ligase activity and cause autosomal recessive early onset parkinsonism

Safety in Nonhuman Primates of Ocular AAV2-\u3cem\u3eRPE65\u3c/em\u3e, a Candidate Treatment for Blindness in Leber Congenital Amaurosis

Leber congenital amaurosis (LCA) is a molecularly heterogeneous disease group that leads to blindness. LCA caused by RPE65 mutations has been studied in animal models and vision has been restored by subretinal delivery of AAV- RPE65 vector. Human ocular gene transfer trials are being considered. Our safety studies of subretinal AAV-2/2. RPE65 in RPE65 -mutant dogs showed evidence of modest photoreceptor loss in the injection region in some animals at higher vector doses. We now test the hypothesis that there can be vector-related toxicity to the normal monkey, with its human-like retina. Good Laboratory Practice safety studies following single intraocular injections of AAV-2/2. RPE65 in normal cynomolgus monkeys were performed for 1-week and 3-month durations. Systemic toxicity was not identified. Ocular-specific studies included clinical examinations, electroretinography, and retinal histopathology. Signs of ocular inflammation postinjection had almost disappeared by 1 week. At 3 months, electroretinography in vector-injected eyes was no different than in vehicle-injected control eyes or compared with presurgical recordings. Healed sites of retinal perforation from subretinal injections were noted clinically and by histopathology. Foveal architecture in subretinally injected eyes, vector or vehicle, could be abnormal. Morphometry of central retina showed no photoreceptor layer thickness abnormalities occurring in a dose-dependent manner. Vector sequences were present in the injected retina, vitreous, and optic nerve at 1 week but not consistently in the brain. At 3 months, there were no vector sequences in optic nerve and brain. The results allow for consideration of an upper range for no observed adverse effect level in future human trials of subretinal AAV-2/2. RPE65. The potential value of foveal treatment for LCA and other retinal degenerations warrants further research into how to achieve gene transfer without retinal injury from surgical detachment of the retina

In vitro neuroprotective potential of four medicinal plants against rotenone-induced toxicity in SH-SY5Y neuroblastoma cells

BACKGROUND: Lannea schweinfurthii, Zanthoxylum capense, Scadoxus puniceus and Crinum bulbispermum are used traditionally to treat neurological disorders. The aim of this study was to evaluate the cytoprotective potential of the four plants, after induction of toxicity using rotenone, in SH-SY5Y neuroblastoma cells. METHODS: Cytotoxicity of the plant extracts and rotenone was assessed using the sulforhodamine B (SRB) assay. Fluorometry was used to measure intracellular redox state (reactive oxygen species (ROS) and intracellular glutathione content), mitochondrial membrane potential (MMP) and caspase-3 activity, as a marker of apoptotic cell death. RESULTS: Of the tested plants, the methanol extract of Z. capense was the least cytotoxic; LC(50) 121.3 ± 6.97 μg/ml, while S. puniceus methanol extract was the most cytotoxic; LC(50) 20.75 ± 1.47 μg/ml. Rotenone reduced intracellular ROS levels after 24 h exposure. Pre-treating cells with S. puniceus and C. bulbispermum extracts reversed the effects of rotenone on intracellular ROS levels. Rotenone exposure also decreased intracellular glutathione levels, which was counteracted by pre-treatment with any one of the extracts. MMP was reduced by rotenone, which was neutralized by pre-treatment with C. bulbispermum ethyl acetate extract. All extracts inhibited rotenone-induced activation of caspase-3. CONCLUSION: The studied plants demonstrated anti-apoptotic activity and restored intracellular glutathione content following rotenone treatment, suggesting that they may possess neuroprotective properties

Toll-Like Receptor 2 Signaling and Current Approaches for Therapeutic Modulation in Synucleinopathies

The innate immune response in the central nervous system (CNS) is implicated as both beneficial and detrimental to health. Integral to this process are microglia, the resident immune cells of the CNS. Microglia express a wide variety of pattern-recognition receptors, such as Toll-like receptors, that detect changes in the neural environment. The activation of microglia and the subsequent proinflammatory response has become increasingly relevant to synucleinopathies, including Parkinson's disease the second most prevalent neurodegenerative disease. Within these diseases there is evidence of the accumulation of endogenous α-synuclein that stimulates an inflammatory response from microglia via the Toll-like receptors. There have been recent developments in both new and old pharmacological agents designed to target microglia and curtail the inflammatory environment. This review will aim to delineate the process of microglia-mediated inflammation and new therapeutic avenues to manage the response

Synuclein and microglial activation in Parkinson's disease

Thesis (Ph.D.)--University of Rochester. School of Medicine and Dentistry. Dept. of Microbiology and Immunology, 2007.Parkinson’s disease (PD) is an age-related slowly progressive neurodegenerative disorder with preferential loss of nigrostriatal dopamine neurons. The pathological hallmark of PD is the presence of intracytoplasmic proteinaceous synuclein positive inclusions, so-called Lewy bodies, in the remaining dopaminergic neurons. It is well documented that over-expression of wild-type (WT) α-synuclein (SYN) or mutant forms, A30P and A53T, play an important role in the pathogenesis of PD. In addition, a growing body of evidence suggests an inflammatory process in the substantia nigra (SN) and striatum (STR), characterized by activation of resident microglial cells in PD patients. Based on this information, we hypothesize that overexpression of WT and mutant SYN will induce microglial-mediated inflammation. Furthermore, we posit that activation of microglia will exacerbate nigrostriatal pathology. To test this hypothesis, we used two transgenic mouse models as well as primary microglial-enriched cell cultures. The first model utilizes human WT SYN overexpression exclusively in tyrosine hydroxylase (TH) containing cells (SYNWT+/+) while the second model employs a similar strategy for overexpression of doublymutated human SYN (A30P and A53T, SYNDM+/+). Utilizing these transgenic models, we determined microglial activation at various ages compared with nontransgenic animals (NTG) and characterized upregulated inflammatory molecules. Furthermore, to examine the direct effect of SYN on microglial activation, we utilized primary microglia-enriched cultures from non-transgenic mice treated with WT and DM SYN. Finally, primary microglia-enriched cell cultures from CD36-deficient vii mice (CD36-/-) were used to determine the necessity of this scavenger receptor for SYN phagocytosis. Together, the results of these studies demonstrated that an early microglia-mediated inflammation occurred in both SYNWT+/+ and SYNDM+/+ transgenic mice, and the degree of inflammation in SYNDM+/+ was much greater than in SYNWT+/+. Furthermore, the early and transient microglial activation in SYNDM+/+ mice was associated with delayed degeneration of striatal nerve terminals within 12 months. In vitro studies showed that both WT and DM SYN were able to activate microglia directly and induce a cascade of inflammatory events, which processes are partially mediated by CD36. To conclude, these studies reveal the pathological effect of SYN overexpression or mutation in triggering microglial activaton as well as the neurotoxic role of microglial activation in dopaminergic neuron dysfunction in the SYN-related transgenic models of Parkinson’s disease

Lentiviral vectors expressing chimeric NEDD4 ubiquitin ligases: An innovative approach for interfering with alpha-synuclein accumulation

One of the main pathological features of Parkinson’s disease (PD) is a diffuse accumulation of alpha-synuclein (aS) aggregates in neurons. The NEDD4 E3 Ub ligase promotes aS degradation by the endosomal–lysosomal route. Interestingly, NEDD4, as well as being a small molecule able to trigger its functions, is protective against human aS toxicity in evolutionary distant models. While pharmacological activation of E3 enzymes is not easy to achieve, their flexibility and the lack of “consensus” motifs for Ub-conjugation allow the development of engineered Ub-ligases, able to target proteins of interest. We developed lentiviral vectors, encoding well-characterized anti-human aS scFvs fused in frame to the NEDD4 catalytic domain (ubiquibodies), in order to target ubiquitinate aS. We demonstrate that, while all generated ubiquibodies bind to and ubiquitinate aS, the one directed against the non-amyloid component (NAC) of aS (Nac32HECT) affects aS’s intracellular levels. Furthermore, Nac32HECT expression partially rescues aS’s overexpression or mutation toxicity in neural stem cells. Overall, our data suggest that ubiquibodies, and Nac32HECT in particular, represent a valid platform for interfering with the effects of aS’s accumulation and aggregation in neurons