Neural connectivity predicts spreading of alpha-synuclein pathology in fibril-injected mouse models: Involvement of retrograde and anterograde axonal propagation.

In Parkinson's disease, some of the first alpha-synuclein aggregates appear in the olfactory system and the dorsal motor nucleus of the vagus nerve before spreading to connected brain regions. We previously demonstrated that injection of alpha-synuclein fibrils unilaterally into the olfactory bulb of wild type mice leads to widespread synucleinopathy in brain regions directly and indirectly connected to the injection site, consistently, over the course of periods longer than 6 months. Our previously reported observations support the idea that alpha-synuclein inclusions propagates between brain region through neuronal networks. In the present study, we further defined the pattern of propagation of alpha-synuclein inclusions and developed a mathematical model based on known mouse brain connectivity. Using this model, we first predicted the pattern of alpha-synuclein inclusions propagation following an injection of fibrils into the olfactory bulb. We then analyzed the fitting of these predictions to our published histological data. Our results demonstrate that the pattern of propagation we observed in vivo is consistent with axonal transport of alpha-synuclein aggregate seeds, followed by transsynaptic transmission. By contrast, simple diffusion of alpha-synuclein fits very poorly our in vivo data. We also found that the spread of alpha-synuclein inclusions appeared to primarily follow neural connections retrogradely until 9 months after injection into the olfactory bulb. Thereafter, the pattern of spreading was consistent with anterograde propagation mathematical models. Finally, we applied our mathematical model to a different, previously published, dataset involving alpha-synuclein fibril injections into the striatum, instead of the olfactory bulb. We found that the mathematical model accurately predicts the reported progressive increase in alpha-synuclein neuropathology also in that paradigm. In conclusion, our findings support that the progressive spread of alpha-synuclein inclusions after injection of protein fibrils follows neural networks in the mouse connectome

<i>C-elegans</i> model identifies genetic modifiers of alpha-synuclein inclusion formation during aging

Inclusions in the brain containing alpha-synuclein are the pathological hallmark of Parkinson's disease, but how these inclusions are formed and how this links to disease is poorly understood. We have developed a &lt;i&gt;C-elegans&lt;/i&gt; model that makes it possible to monitor, in living animals, the formation of alpha-synuclein inclusions. In worms of old age, inclusions contain aggregated alpha-synuclein, resembling a critical pathological feature. We used genome-wide RNA interference to identify processes involved in inclusion formation, and identified 80 genes that, when knocked down, resulted in a premature increase in the number of inclusions. Quality control and vesicle-trafficking genes expressed in the ER/Golgi complex and vesicular compartments were overrepresented, indicating a specific role for these processes in alpha-synuclein inclusion formation. Suppressors include aging-associated genes, such as sir-2.1/SIRT1 and lagr-1/LASS2. Altogether, our data suggest a link between alpha-synuclein inclusion formation and cellular aging, likely through an endomembrane-related mechanism. The processes and genes identified here present a framework for further study of the disease mechanism and provide candidate susceptibility genes and drug targets for Parkinson's disease and other alpha-synuclein related disorders

The synucleins.

PMC ID: PMC150459. The author can archive publisher's version/PDF.SUMMARY: Synucleins are small, soluble proteins expressed primarily in neural tissue and in certain tumors. The family includes three known proteins: alpha-synuclein, beta-synuclein, and gamma-synuclein. All synucleins have in common a highly conserved alpha-helical lipid-binding motif with similarity to the class-A2 lipid-binding domains of the exchangeable apolipoproteins. Synuclein family members are not found outside vertebrates, although they have some conserved structural similarity with plant 'late-embryo-abundant' proteins. The alpha- and beta-synuclein proteins are found primarily in brain tissue, where they are seen mainly in presynaptic terminals. The gamma-synuclein protein is found primarily in the peripheral nervous system and retina, but its expression in breast tumors is a marker for tumor progression. Normal cellular functions have not been determined for any of the synuclein proteins, although some data suggest a role in the regulation of membrane stability and/or turnover. Mutations in alpha-synuclein are associated with rare familial cases of early-onset Parkinson's disease, and the protein accumulates abnormally in Parkinson's disease, Alzheimer's disease, and several other neurodegenerative illnesses. The current challenge is to understand the normal cellular function of these proteins and how they might contribute to the development of human disease

Electron microscopy analysis of alpha-synuclein and LRRK2 transgenic C. elegans

Thesis (M.A.)--Boston UniversityMutations in alpha-synuclein and leucine-rich repeat kinase 2 (LRRK2) have been implicated in the cause of Parkinson’s disease (PD). These two proteins have been the targets of a great deal of recent research that has transformed our understanding of this disorder. Recent research using C. elegans as a model species has shown that alpha- synuclein expression and the LRRK2-G2019S mutation potentiate neurodegeneration similar to that seen in cases PD. Further exploration revealed that defects in autophagy of dopaminergic neurons may be the cause for the observed pathology. In the current study, the confirmation of autophagy as a possible cause of pathology due to the expression of alpha-synuclein and the LRRK2-G2019S mutation is completed through the use of electron microscopy. We observed that large vacuoles had formed in the cephalic dopaminergic neurons of alpha-synuclein + LRRK2 transgenic samples not seen in wild-type samples. Further, large morphological changes in the nerve ring area of the transgenic nematodes were also observed that may implicate that alpha- synuclein expression in conjunction with the LRRK2-G2019S mutation may have a widespread effect on many neurons that was not previously expected

The potential role of dietary polyphenols in Parkinson’s disease

Cumulative evidence now suggests that the abnormal aggregation of the neuronal protein alpha-synuclein is critically involved in the pathogenesis of synucleinopathies, of which Parkinson’s disease (PD) is the most prevalent. Development of neuropathology appears to be linked to events that accelerate the rate of aggregation of alpha-synuclein from monomers, via soluble oligomeric intermediates, into amyloid fibrils. Although increasing data suggest that oligomeric aggregates, not amyloid fibrils, disrupt or permeabilise cellular membranes, the nature of the neurotoxic species and its precise molecular mechanism still remain largely unknown, hampering the development of an effective treatment for the disease. Currently, there is no approved therapeutic agent directed toward preventing alpha-synuclein aggregation and only symptomatic therapies are available with a limited time-frame of utility. Numerous studies have demonstrated the protective effects of dietary polyphenols against neuronal damage in PD. The aim of this review is to look at what research has been done so far to show that dietary polyphenolic compounds can effectively interfere with alpha-synuclein oligomerisation. Evidence in the role and mechanisms of diet-derived phenolic products may guide the design of novel therapeutic drugs that can block early stages of amyloid self-assembly in PD and related synucleinopathies.peer-reviewe

The HSP70 Molecular Chaperone Is Not Beneficial in a Mouse Model of α-synucleinopathy

BACKGROUND: Aggregation and misfolded alpha-synuclein is thought to be central in the pathogenesis of Parkinson's disease (PD). Heat-shock proteins (HSPs) that are involved in refolding and degradation processes could lower the aggregate load of alpha-synuclein and thus be beneficial in alpha-synucleinopathies. METHODOLOGY/PRINCIPAL FINDINGS: We co-overexpressed human A53T point-mutated alpha-synuclein and human HSP70 in mice, both under the control of Thy1 regulatory sequences. Behavior read-outs showed no beneficial effect of HSP70 expression in mice. In contrast, motor coordination, grip strength and weight were even worse in the alpha-synucleinopathy model in the presence of HSP70 overexpression. Biochemical analyses revealed no differences in alpha-synuclein oligomers/aggregates, truncations and phosphorylation levels and alpha-synuclein localization was unchanged in immunostainings. CONCLUSION/SIGNIFICANCE: Overexpressing HSP70 in a mouse model of alpha-synucleinopathy did not lower the toxic load of alpha-synuclein species and had no beneficial effect on alpha-synuclein-related motor deficits

Combined Active Humoral and Cellular Immunization Approaches for the Treatment of Synucleonopathies

Objectives: Parkinson’s Disease (PD), Dementia with Lewy bodies (DLB), and Multiple System Atrophy (MSA) are neurodegenerative disorders of the aging population characterized by the progressive accumulation of alpha-synuclein. Jointly these disorders have been denominated synucleinopathies and currently no disease modifying treatments are available. Previous in vivo studies in transgenic (tg) mice have shown that active and passive immunization targeting alpha-synuclein ameliorates to some extent deficits and synuclein accumulation, however it’s unknown if combining humoral and cellular immunization might synergize and also reduce inflammation and improve microglial cell mediated synuclein clearance. Methods: PDGF- alpha-synuclein tg mice and control non-tg mice were immunized with: 1) Glucan Particle (GP) adjuvant alone, 2) GP human (hu)- alpha-synuclein (active immunization), 3) GP plus rapamycin and 4) GP plus rapamycin and hu-alpha-synuclein (combined active and humoral) and analyzed by neuropathological and biochemical markers. Results: Compared to tg mice treated with adjuvant alone, mice immunized with GP hu-alpha-synuclein displayed a 30% reduction in alpha-synuclein accumulation. Combined immunotherapy with GP plus rapamycin and hu-alpha-synuclein resulted in 50% reduction in alpha-synuclein accumulation which was accompanied by reduced neuro-inflammation (Iba-1, GFAP, IL6, TNFalpha), phospho and insoluble alpha-synuclein, microglia and astroglia cell numbers, and retention of CD25, FoxP3 and CD4 positive cells. Levels of TGFb1 were also increased. Serological studies showed that active immunization resulted in higher levels of total IgG, IgG1 and IgG2 titers, levels were slightly higher in the combined group. Conclusions: In vivo studies targeting alpha-synuclein support the hypothesis that cellular immunization might enhance the effects of active immunotherapy for the treatment of synucleionopathies

Phosphorylation does not prompt, nor prevent, the formation of α-synuclein toxic species in a rat model of Parkinson's disease

Phosphorylation is involved in numerous neurodegenerative diseases. In particular, alpha-synuclein is extensively phosphorylated in aggregates in patients suffering from synucleinopathies. However, the share of this modification in the events that lead to the conversion of alpha-synuclein to aggregated toxic species needed to be clarified. The rat model that we developed through rAAV2/6-mediated expression of alpha-synuclein demonstrates a correlation between neurodegeneration and formation of small filamentous alpha-synuclein aggregates. A mutation preventing phosphorylation (S129A) significantly increases alpha-synuclein toxicity and leads to enhanced formation of beta-sheet-rich, proteinase K-resistant aggregates, increased affinity for intracellular membranes, a disarrayed network of neurofilaments and enhanced alpha-synuclein nuclear localization. The expression of a mutation mimicking phosphorylation (S129D) does not lead to dopaminergic cell loss. Nevertheless, fewer but larger aggregates are formed, and signals of apoptosis are also activated in rats expressing the phosphorylation-mimicking form of alpha-synuclein. These observations strongly suggest that phosphorylation does not play an active role in the accumulation of cytotoxic pre-inclusion aggregates. Unexpectedly, the study also demonstrates that constitutive expression of phosphorylation-mimicking forms of alpha-synuclein does not protect from neurodegeneration. The role of phosphorylation at Serine 129 in the early phase of Parkinson's disease is examined, which brings new perspective to therapeutic approaches focusing on the modulation of kinases/phosphatases activity to control alpha-synuclein toxicit

Nitrated α-Synuclein Induces the Loss of Dopaminergic Neurons in the Substantia Nigra of Rats

BACKGROUND: The pathology of Parkinson's disease (PD) is characterized by the degeneration of the nigrostriatal dopaminergic pathway, as well as the formation of intraneuronal inclusions known as Lewy bodies and Lewy neurites in the substantia nigra. Accumulations of nitrated alpha-synuclein are demonstrated in the signature inclusions of Parkinson's disease. However, whether the nitration of alpha-synuclein is relevant to the pathogenesis of PD is unknown. METHODOLOGY/PRINCIPAL FINDINGS: In this study, effect of nitrated alpha-synuclein to dopaminergic (DA) neurons was determined by delivering nitrated recombinant TAT-alpha-synuclein intracellular. We provide evidence to show that the nitrated alpha-synuclein was toxic to cultured dopaminergic SHSY-5Y neurons and primary mesencephalic DA neurons to a much greater degree than unnitrated alpha-synuclein. Moreover, we show that administration of nitrated alpha-synuclein to the substantia nigra pars compacta of rats caused severe reductions in the number of DA neurons therein, and led to the down-regulation of D(2)R in the striatum in vivo. Furthermore, when administered to the substantia nigra of rats, nitrated alpha-synuclein caused PD-like motor dysfunctions, such as reduced locomotion and motor asymmetry, however unmodified alpha-synuclein had significantly less severe behavioral effects. CONCLUSIONS/SIGNIFICANCE: Our results provide evidence that alpha-synuclein, principally in its nitrated form, induce DA neuron death and may be a major factor in the etiology of PD

Insight Into Parkinson\u27s Disease Using Yeast as a Model to Evaluate the Role of Autophagy Genes in alpha-Synuclein Toxicity

Parkinson’s disease is a fatal and incurable human neurodegenerative disorder that destroys midbrain neurons. The misfolding, accumulation, and aggregation of the protein alpha-synuclein is thought to kill these cells. Enhancing alpha-synuclein degradation may help prevent its accumulation and aggregation, while protecting cells against toxicity. For this thesis, we used the model organism budding yeast to evaluate the hypothesis that alpha-synuclein is degraded by the cellular organelle lysosome via a specific route: the MVB/endocytosis pathway. Specifically, we evaluated whether three disease-related properties of alpha-synuclein (aggregation, accumulation, and toxicity) worsened in yeast strains that were individually deleted for genes coding for proteins required for the MVB/endocytosis pathway. In support of our hypothesis, each gene deletion altered one or more alpha-synuclein properties. While our data indicates that the MVB pathway is a route for alpha-synuclein degradation, the specificity and extent of alpha-synuclein involvement with proteins within the ESCRT complexes appears unexpectedly complex