36-008 Sigma-AldrichAnti-α-Synuclein Antibody, clone Syn211

Interneuronal propagation of α-synuclein has been demonstrated in a variety of experimental models and may be involved in disease progression during the course of human synucleinopathies. The aim of this study was to assess the role that neuronal injury or, vice versa, cell integrity could have in facilitating interneuronal α-synuclein transfer and consequent protein spreading in an in vivo animal model.Viral vectors carrying the DNA for human α-synuclein were injected into the rat vagus nerve to trigger protein overexpression in the medulla oblongata and consequent spreading of human α-synuclein toward pons, midbrain and forebrain. Two vector preparations sharing the same viral construct were manufactured using identical procedures with the exception of methods for their purification. They were also injected at concentrations that induced comparable levels of α-synuclein transduction/overexpression in the medulla oblongata. α-Synuclein load was associated with damage (at 6 weeks post injection) and death (at 12 weeks) of medullary neurons after treatment with only one of the two vector preparations. Of note, neuronal injury and degeneration was accompanied by a substantial reduction of caudo-rostral propagation of human α-synuclein.Interneuronal α-synuclein transfer, which underlies protein spreading from the medulla oblongata to more rostral brain regions in this rat model, is not a mere consequence of passive release from damaged or dead neurons. Neuronal injury and degeneration did not exacerbate α-synuclein propagation. In fact, data suggest that cell-to-cell passage of α-synuclein may be particularly efficient between intact, relatively healthy neurons.

α-Synuclein accumulation and pathology in Parkinson's disease typically display a caudo-rostral pattern of progression, involving neuronal nuclei in the medulla oblongata at the earliest stages. In this study, selective expression and accumulation of human α-synuclein within medullary neurons was achieved via retrograde transport of adeno-associated viral vectors unilaterally injected into the vagus nerve in the rat neck. The exogenous protein progressively spread toward more rostral brain regions where it could be detected within axonal projections. Propagation to the pons, midbrain and forebrain followed a stereotypical pattern of topographical distribution. It affected areas such as the coeruleus-subcoeruleus complex, dorsal raphae, hypothalamus and amygdala ipsilateral and, to a lesser extent, contralateral to the injection side. Spreading was accompanied by evidence of neuritic pathology in the form of axonal varicosities intensely immunoreactive for human α-synuclein and containing Thioflavin-S-positive fibrils. Thus, overexpression of human α-synuclein in the lower brainstem is sufficient to induce its long-distance caudo-rostral propagation, recapitulating features of Parkinson's disease and mechanisms of disease progression.

Intraneuronal inclusions containing alpha-synuclein (a-syn) constitute one of the pathological hallmarks of Parkinson's disease (PD) and are accompanied by severe neurodegeneration of A9 dopaminergic neurons located in the substantia nigra. Although to a lesser extent, A10 dopaminergic neurons are also affected. Neurodegeneration of other neuronal populations, such as the cholinergic, serotonergic and noradrenergic cell groups, has also been documented in PD patients. Studies in human post-mortem PD brains and in rodent models suggest that deficits in cholinergic and dopaminergic systems may be associated with the cognitive impairment seen in this disease. Here, we investigated the consequences of targeted overexpression of a-syn in the mesocorticolimbic dopaminergic and septohippocampal cholinergic pathways. Rats were injected with recombinant adeno-associated viral vectors encoding for either human wild-type a-syn or green fluorescent protein (GFP) in the ventral tegmental area and the medial septum/vertical limb of the diagonal band of Broca, two regions rich in dopaminergic and cholinergic neurons, respectively. Histopathological analysis showed widespread insoluble a-syn positive inclusions in all major projections areas of the targeted nuclei, including the hippocampus, neocortex, nucleus accumbens and anteromedial striatum. In addition, the rats overexpressing human a-syn displayed an abnormal locomotor response to apomorphine injection and exhibited spatial learning and memory deficits in the Morris water maze task, in the absence of obvious spontaneous locomotor impairment. As losses in dopaminergic and cholinergic immunoreactivity in both the GFP and a-syn expressing animals were mild-to-moderate and did not differ from each other, the behavioral impairments seen in the a-syn overexpressing animals appear to be determined by the long term persisting neuropathology in the surviving neurons rather than by neurodegeneration.

Hallmark lesions of neurodegenerative synucleinopathies contain alpha-synuclein (alpha-syn) that is modified by nitration of tyrosine residues and possibly by dityrosine cross-linking to generated stable oligomers. Data gathered from in vitro experiments and from model systems of cells transfected with wild-type and mutant alpha-syn revealed that conditions resulting in alpha-syn nitration also induce formation of alpha-syn inclusions with similar biochemical characteristics to protein extracted from human lesions. The detection of tyrosine-nitrated alpha-syn signifies the formation of reactive nitrogen species capable of both radical and electrophilic attack on aromatic residues as well as nucleophilic additions and oxidations. The cellular sources and biochemical reactivity of reactive nitrogen species in the central nervous system remain largely unknown, but kinetically fast reactions of nitric oxide with superoxide to form peroxynitrite as well as enzymatic one-electron oxidation of nitrite are two important sources of reactive nitrogen species. Based on these findings a model is proposed where the process of fibrilization can be differentially affected by oxidants and nitrating species. Posttranslational modifications of alpha-syn by reactive nitrogen species inhibits fibril formation and results in urea- and SDS- insoluble, protease-resistant alpha-syn aggregates that maybe responsible for cellular toxicity.

Alpha-synuclein is regarded as a presynaptic protein, which may play an important role in neuronal plasticity. However, the actual physiological function of this protein is not completely clear. Abnormal accumulation of fibrillar alpha-synuclein in Lewy bodies, as well as mutations in the alpha-synuclein gene identified in the familial forms of Parkinson's disease, point to a central role of this protein in the pathophysiology of Lewy body-related disorders. In vivo and in vitro studies showed that overexpression of alpha-synuclein, its aggregation, and interaction with other proteins are the most critical factors affecting the survival of neurons. In Alzheimer's disease, the amount of alpha-synuclein is found to be elevated at synapses, whereas a peptide derived from alpha-synuclein is thought to represent an intrinsic component of amyloid plaques. It is likely that in this disorder alpha-synuclein plays a dual role by being involved not only in synaptic function but also in amyloid beta-fibrillogenesis.

Neuronal death in Parkinson's disease (PD), one of the most common neurodegenerative disorders in the adult and aging population is probably caused by misfolding of synaptic proteins such as alpha-synuclein. Although, some treatments are currently available to control some of the symptoms of PD, none of these approaches directly addresses the mechanisms of disease. With the advent of new experimental animal models for this disorder, the potential for development and discovery of new treatment has been significantly bolstered. Among them, overexpression of alpha-synuclein results in motor deficits. dopaminergic loss and formation of inclusion bodies. Co-expression of mutant amyloid precursor protein, accelerates alpha-synuclein aggregation and enhances the neurodegenerative pathology in these mice, providing a unique model where to investigate the interactions between Abeta1-42 and alpha-synuclein and to develop treatments for combined Alzheimer's disease and PD. Development of anti-parkinsonian treatments based on these models includes: (i) anti-aggregation or pro-degradation compounds, (ii) neuroprotective compounds, and (iii) neurotrophic agents. Among them, we characterized beta-synuclein, the non-amyloidogenic homologue of alpha-synuclein, as an inhibitor of aggregation of alpha-synuclein. Our results raise the intriguing possibility that beta-synuclein might be a natural negative regulator of alpha-synuclein aggregation, and that a similar class of endogenous factors might regulate the aggregation state of other molecules involved in neurodegeneration. Such an anti-amyloidogenic property of beta-synuclein might also provide a novel strategy for the treatment of neurodegenerative disorders.