Prion infection promotes extensive accumulation of α-synuclein in aged human α-synuclein transgenic mice

In neurodegenerative disorders of the aging population, misfolded proteins, such as PrP^Sc, α-synuclein, amyloid β protein and tau, can interact resulting in enhanced aggregation, cross seeding and accelerated disease progression. Previous reports have shown that in Creutzfeldt-Jakob disease and scrapie, α-synuclein accumulates near PrP^Sc deposits. However, it is unclear if pre-existing human α-synuclein aggregates modified prion disease pathogenesis, or if PrP^Sc exacerbates the α-synuclein pathology. Here, we inoculated infectious prions into aged α-synuclein transgenic (tg) and non-transgenic littermate control mice by the intracerebral route. Remarkably, inoculation of RML and mNS prions into α-synuclein tg mice resulted in more extensive and abundant intraneuronal and synaptic α-synuclein accumulation. In addition, infectious prions led to the formation of perineuronal α-synuclein deposits with a neuritic plaque-like appearance. Prion pathology was unmodified by the presence of α-synuclein. However, with the mNS prion strain there was a modest but significant acceleration in the time to terminal prion disease in mice having α-synuclein aggregates as compared with non-tg mice. Taken together, these studies support the notion that PrP^Sc directly or indirectly promotes α-synuclein pathology.     

β-III Tubulin Fragments Inhibit α-Synuclein Accumulation in Models of Multiple System Atrophy

Multiple system atrophy (MSA) is a neurodegenerative disease caused by α-synuclein aggregation in oligodendrocytes and neurons. Using a transgenic mouse model overexpressing human α-synuclein in oligodendrocytes, we previously demonstrated that oligodendrocytic α-synuclein inclusions induce neuronal α-synuclein accumulation and progressive neuronal degeneration. α-Synuclein binds to β-III tubulin, leading to the neuronal accumulation of insoluble α-synuclein in an MSA mouse model. The present study demonstrates that α-synuclein co-localizes with β-III tubulin in the brain tissue from patients with MSA and MSA model transgenic mice as well as neurons cultured from these mice. Accumulation of insoluble α-synuclein in MSA mouse neurons was blocked by the peptide fragment β-III tubulin (residues 235–282). We have determined the α-synuclein-binding domain of β-III tubulin and demonstrated that a short fragment containing this domain can suppress α-synuclein accumulation in the primary cultured cells. Administration of a short α-synuclein-binding fragment of β-III tubulin may be a novel therapeutic strategy for MSA.     

Fibrillar α-synuclein toxicity depends on functional lysosomes

Neurodegeneration in Parkinson''s disease (PD) can be recapitulated in animals by administration of α-synuclein preformed fibrils (PFFs) into the brain. However, the mechanism by which these PFFs induce toxicity is unknown. Iron is implicated in PD pathophysiology, so we investigated whether α-synuclein PFFs induce ferroptosis, an iron-dependent cell death pathway. A range of ferroptosis inhibitors were added to a striatal neuron-derived cell line (STHdhQ7/7 cells), a dopaminergic neuron–derived cell line (SN4741 cells), and WT primary cortical neurons, all of which had been intoxicated with α-synuclein PFFs. Viability was not recovered by these inhibitors except for liproxstatin-1, a best-in-class ferroptosis inhibitor, when used at high doses. High-dose liproxstatin-1 visibly enlarged the area of a cell that contained acidic vesicles and elevated the expression of several proteins associated with the autophagy-lysosomal pathway similarly to the known lysosomal inhibitors, chloroquine and bafilomycin A1. Consistent with high-dose liproxstatin-1 protecting via a lysosomal mechanism, we further de-monstrated that loss of viability induced by α-synuclein PFFs was attenuated by chloroquine and bafilomycin A1 as well as the lysosomal cysteine protease inhibitors, leupeptin, E-64D, and Ca-074-Me, but not other autophagy or lysosomal enzyme inhibitors. We confirmed using immunofluorescence microscopy that heparin prevented uptake of α-synuclein PFFs into cells but that chloroquine did not stop α-synuclein uptake into lysosomes despite impairing lysosomal function and inhibiting α-synuclein toxicity. Together, these data suggested that α-synuclein PFFs are toxic in functional lysosomes in vitro. Therapeutic strategies that prevent α-synuclein fibril uptake into lysosomes may be of benefit in PD.     

Parkinson Disease Mutant E46K Enhances α-Synuclein Phosphorylation in Mammalian Cell Lines,in Yeast,and in Vivo

Although α-synuclein (α-syn) phosphorylation has been considered as a hallmark of sporadic and familial Parkinson disease (PD), little is known about the effect of PD-linked mutations on α-syn phosphorylation. In this study, we investigated the effects of the A30P, E46K, and A53T PD-linked mutations on α-syn phosphorylation at residues Ser-87 and Ser-129. Although the A30P and A53T mutants slightly affected Ser(P)-129 levels compared with WT α-syn, the E46K mutation significantly enhanced Ser-129 phosphorylation in yeast and mammalian cell lines. This effect was not due to the E46K mutant being a better kinase substrate nor due to alterations in endogenous kinase levels, but was mostly linked with enhanced nuclear and endoplasmic reticulum accumulation. Importantly, lentivirus-mediated overexpression in mice also showed enhanced Ser-129 phosphorylation of the E46K mutant compared to WT α-syn, thus providing in vivo validation of our findings. Altogether, our findings suggest that the different PD-linked mutations may contribute to PD pathogenesis via different mechanisms.     

A Maladaptive Role for EP4 Receptors in Mouse Mesangial Cells

Roles of the prostaglandin E2 E-prostanoid 4 receptor (EP4) on extracellular matrix (ECM) accumulation induced by TGF-β1 in mouse glomerular mesangial cells (GMCs) remain unknown. Previously, we have identified that TGF-β1 stimulates the expression of FN and Col I in mouse GMCs. Here we asked whether stimulation of EP4 receptors would exacerbate renal fibrosis associated with enhanced glomerular ECM accumulation. We generated EP4^Flox/Flox and EP4^+/− mice, cultured primary WT, EP4^Flox/Flox and EP4^+/− GMCs, AD-EP4 transfected WT GMCs (EP4 overexpression) and AD-Cre transfected EP4^Flox/Flox GMCs (EP4 deleted). We found that TGF-β1-induced cAMP and PGE2 synthesis decreased in EP4 deleted GMCs and increased in EP4 overexpressed GMCs. Elevated EP4 expression in GMCs augmented the coupling of TGF-β1 to FN, Col I expression and COX2/PGE2 signaling, while TGF-β1 induced FN, Col I expression and COX2/PGE2 signaling were down-regulated in EP4 deficiency GMCs. 8 weeks after 5/6 nephrectomy (Nx), WT and EP4^+/− mice exhibited markedly increased accumulation of ECM compared with sham-operated controls. Albuminuria, blood urea nitrogen and creatinine (BUN and Cr) concentrations were significantly increased in WT mice as compared to those of EP4^+/− mice. Urine osmotic pressure was dramatically decreased after 5/6 Nx surgery in WT mice as compared to EP4^+/− mice. The pathological changes in kidney of EP4^+/− mice was markedly alleviated compared with WT mice. Immunohistochemical analysis showed significant reductions of Col I and FN in the kidney of EP4^+/− mice compared with WT mice. Collectively, this investigation established EP4 as a potent mediator of the pro-TGF-β1 activities elicited by COX2/PGE2 in mice GMCs. Our findings suggested that prostaglandin E2, acting via EP4 receptors contributed to accumulation of ECM in GMCs and promoted renal fibrosis.     

RECK-Mediated β1-Integrin Regulation by TGF-β1 Is Critical for Wound Contraction in Mice

Fibroblasts are critical for wound contraction; a pivotal step in wound healing. They produce and modify the extracellular matrix (ECM) required for the proper tissue remodeling. Reversion-inducing cysteine-rich protein with Kazal motifs (RECK) is a key regulator of ECM homeostasis and turnover. However, its role in wound contraction is presently unknown. Here we describe that Transforming growth factor type β1 (TGF-β1), one of the main pro-fibrotic wound-healing promoting factors, decreases RECK expression in fibroblasts through the Smad and JNK dependent pathways. This TGF-β1 dependent downregulation of RECK occurs with the concomitant increase of β1-integrin, which is required for fibroblasts adhesion and wound contraction through the activation of focal adhesion kinase (FAK). Loss and gain RECK expression experiments performed in different types of fibroblasts indicate that RECK downregulation mediates TGF-β1 dependent β1-integrin expression. Also, reduced levels of RECK potentiate TGF-β1 effects over fibroblasts FAK-dependent contraction, without affecting its cognate signaling. The above results were confirmed on fibroblasts derived from the Reck ^+/- mice compared to wild type-derived fibroblasts. We observed that Reck ^+/- mice heal dermal wounds more efficiently than wild type mice. Our results reveal a critical role for RECK in skin wound contraction as a key mediator in the axis: TGF-β1—RECK- β1-integrin.     

Kindlin-2 directly binds actin and regulates integrin outside-in signaling

Reduced levels of kindlin-2 (K2) in endothelial cells derived from K2^+/− mice or C2C12 myoblastoid cells treated with K2 siRNA showed disorganization of their actin cytoskeleton and decreased spreading. These marked changes led us to examine direct binding between K2 and actin. Purified K2 interacts with F-actin in cosedimentation and surface plasmon resonance analyses and induces actin aggregation. We further find that the F0 domain of K2 binds actin. A mutation, LK⁴⁷/AA, within a predicted actin binding site (ABS) of F0 diminishes its interaction with actin by approximately fivefold. Wild-type K2 and K2 bearing the LK⁴⁷/AA mutation were equivalent in their ability to coactivate integrin αIIbβ3 in a CHO cell system when coexpressed with talin. However, K2-LK⁴⁷/AA exhibited a diminished ability to support cell spreading and actin organization compared with wild-type K2. The presence of an ABS in F0 of K2 that influences outside-in signaling across integrins establishes a new foundation for considering how kindlins might regulate cellular responses.     

Regulation of Weibel-Palade Body Exocytosis by α-Synuclein in Endothelial Cells

α-Synuclein is a small presynaptic protein implicated in the pathogenesis of Parkinson disease. Nevertheless, its physiological roles and mechanisms remain incompletely understood. α-Synuclein is not only expressed in neurons but also in the vascular endothelium, which contains intracellular granules called Weibel-Palade bodies (WPBs) that contain a number of chemokines, adhesive molecules, and inflammatory cytokines. This study explored whether the exocytosis of WPB is regulated by α-synuclein. Phorbol 12-myristate 13-acetate-, thrombin-, or forskolin-induced von Willebrand factor release or translocation of P-selectin from endothelial cells were inhibited by α- and β-synuclein but not γ-synuclein. Three point mutants (A30P, A53T, and E46K) found in familial Parkinson disease also inhibited WPB exocytosis similar to that of wild-type α-synuclein. Furthermore, the negative regulation of WPB exocytosis required the N terminus or the nonamyloid β-component of Alzheimer disease amyloid region of α-synuclein, but not the C-terminal acidic tail, and α-synuclein affected WPB exocytosis through interference with RalA activation by enhancing the interaction of RalGDS-β-arrestin complexes. Immuno-EM analysis revealed that α-synuclein was localized close to WPBs. These findings imply that α-synuclein plays as a negative regulator in WPB exocytosis in endothelial cells.     

Anti-amyloid Compounds Inhibit α-Synuclein Aggregation Induced by Protein Misfolding Cyclic Amplification (PMCA)

Filaments made of α-synuclein form the characteristic Lewy pathology in Parkinson and other diseases. The formation of α-synuclein filaments can be reproduced in vitro by incubation of recombinant protein, but the filament growth is very slow and highly variable and so unsuitable for fast high throughput anti-aggregation drug screening. To overcome this obstacle we have investigated whether the protein misfolding cyclic amplification (PMCA) technique, used for fast amplification of prion protein aggregates, could be adapted for growing α-synuclein aggregates and thus suitable for screening of drugs to affect α-synuclein aggregation for the treatment of the yet incurable α-synucleinopathies. Circular dichroism, electron microscopy, and native and SDS-polyacrylamide gels were used to demonstrate α-synuclein aggregate formation by PMCA, and the strain imprint of the α-synuclein fibrils was studied by proteinase K digestion. We also demonstrated that α-synuclein fibrils are able to seed new α-synuclein PMCA reactions and to enter and aggregate in cells in culture. In particular, we have generated a line of “chronically infected” cells, which transmit α-synuclein aggregates even after multiple passages. To evaluate the sensitivity of the PMCA system as an α-synuclein anti-aggregating drug screening assay a panel of 10 drugs was tested. Anti-amyloid compounds proved efficient in inhibiting α-synuclein fibril formation induced by PMCA. Our results show that α-synuclein PMCA is a fast and reproducible system that could be used as a high throughput screening method for finding new α-synuclein anti-aggregating compounds.     

Hyperphosphorylation and insolubility of alpha-synuclein in transgenic mouse oligodendrocytes

(Oligodendro)glial cytoplasmic inclusions composed of α-synuclein (αSYN) characterize multiple system atrophy (MSA). Mature oligodendrocytes (OLs) do not normally express αSYN, so MSA pathology may arise from aberrant expression of αSYN in OLs. To study pathological deposition of αSYN in OLs, transgenic mice were generated in which human wild-type αSYN was driven by a proteolipid protein promoter. Transgenic αSYN was detected in OLs but no other brain cell type. At the light microscopic level, the transgenic αSYN profiles resembled glial cytoplasmic inclusions. Strikingly, the diagnostic hyperphosphorylation at S129 of αSYN was reproduced in the transgenic mice. A significant proportion of the transgenic αSYN was detergent insoluble, as in MSA patients. The histological and biochemical abnormalities were specific for the disease-relevant αSYN because control green fluorescent protein was fully soluble and evenly distributed throughout OL cell bodies and processes. Thus, ectopic expression αSYN in OLs might initiate salient features of MSA pathology.     

The Nicotinic Receptor Alpha7 Impacts the Mouse Lung Response to LPS through Multiple Mechanisms

The nicotinic acetylcholine receptor alpha7 (α7) is expressed by neuronal and non-neuronal cells throughout the body. We examined the mechanisms of the lung inflammatory response to intranasal (i.n.) lipopolysaccharide (LPS) regulated by α7. This was done in mice using homologous recombination to introduce a point mutation in the α7 receptor that replaces the glutamate residue 260 that lines the pore with alanine (α7^E260A), which has been implicated in controlling the exceptional calcium ion conductance of this receptor. The α7^E260A mice exhibit normal inflammatory cell recruitment to the blood in response to i.n. LPS administration. This differs from the α7knock-out (α7^KO) in which upstream signaling to initiate the recruitment to the blood following i.n. LPS is significantly impaired. While hematopoietic cells are recruited to the bloodstream in the α7^E260A mouse, they fail to be recruited efficiently into both the interstitium and alveolar spaces of the lung. Bone marrow reconstitution experiments demonstrate that the responsiveness of both CD45⁺ and CD45^- cells of the α7^E260A mouse are impaired. The expression of several pro-inflammatory cytokine and chemokine RNAs including TNFα, IL-1α, Ccl2 and Cxcl10 are decreased in the α7^E260A mouse. However, there is a substantial increase in IL-13 expression by CD45^- lung interstitial cells in the α7^E260A mouse. Our results support the conclusion that α7 functional pleiotropy contributes to modulating the tissue response to an inflammatory insult through impacting upon a variety of mechanisms reflecting the individual cell composition of the lung.     

Spreading of pathological TDP-43 along corticospinal tract axons induces ALS-like phenotypes in Atg5+/- mice

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease, characterized by phosphorylated TDP-43 (pTDP-43)-positive inclusions in neurons and glial cells. However, the pathogenic mechanism that underlies ALS remains largely unknown. To investigate the effects of autophagy deficiency in the formation and spreading of pathological TDP-43 along corticospinal tract axons, TDP-43 preformed fibrils (PFFs) were prepared and unilaterally injected into the fifth layer of the left primary motor cortex (M1) or the left anterior horn of the seventh cervical spinal cord segment (C7) of Atg5^+/- mice. After the injection of TDP-43 PFFs, the elevated levels of pTDP-43 were present in several pyramidal tract-associated regions of Atg5^+/- mice. Additionally, the occurrence of spontaneous potentials detected by electromyography demonstrates evidence of lower motor neuron dysfunction in M1-TDP-43 PFFs-injected Atg5^+/- mice, and prolonged central motor conduction time detected by motor evoked potentials provides evidence of upper motor neuron dysfunction in C7-TDP-43 PFFs-injected Atg5^+/- mice. These results show that injection of TDP-43 PFFs into the M1 or C7 of Atg5^+/- mice induces the spreading of pathological TDP-43 along corticospinal tract axons in both an anterograde and retrograde manner. Importantly, TDP-43 PFFs-injected Atg5^+/- mice also display ALS-like motor dysfunction. Taken together, our findings provide direct evidence that TDP-43 PFFs-injected Atg5^+/- mice exhibited ALS-like neuropathology and motor phenotypes, suggesting that autophagy deficiency promotes the formation and spreading of pathological TDP-43 in vivo.     

Up-regulation of Translation Eukaryotic Initiation Factor 4E in Nucleophosmin 1 Haploinsufficient Cells Results in Changes in CCAAT Enhancer-binding Protein α Activity: IMPLICATIONS IN MYELODYSPLASTIC SYNDROME AND ACUTE MYELOID LEUKEMIA*

NPM1 is a ubiquitously expressed nucleolar phosphoprotein, the gene for which maps to chromosome 5q35 in close proximity to a commonly deleted region associated with (del)5q, a type of myelodysplastic syndrome (MDS). This region is also a frequent target of deletions in de novo and therapy-related MDS/acute myeloid leukemia. Previous studies have shown that Npm1^+/− mice develop an MDS-like disease that transforms to acute myeloid leukemia over time. To better understand the mechanism by which NPM1 haploinsufficiency causes an MDS phenotype, we generated factor-dependent myeloid cell lines from the bone marrow of Npm1^+/+ and Npm1^+/− mice and demonstrated compromised neutrophil-specific gene expression in the MNPM1^+/− cells. We attribute these observations to increased levels of the shorter, dominant negative leukemogenic isoform (p30) of CCAAT enhancer-binding protein α (C/EBPα). We show that this increase is caused, in part, by elevated levels of the activated translation initiation factor eIF4E, overexpression of which also increases translation of C/EBPαp30 in HEK293 cells. In a positive feedback loop, eIF4E expression is further elevated both at the mRNA and protein levels by C/EBPαp30 but not by the full-length C/EBPαp42. Re-expression of C/EBPαp42 or NPM1 but not C/EBPαp30 in MNPM1^+/− cells partially rescues the myeloid phenotype. Our observations suggest that the aberrant feed-forward pathway that keeps eIF4E and C/EBPαp30 elevated in NPM1^+/− cells contributes to the MDS phenotype associated with NPM1 deficiency.     

The H50Q Mutation Induces a 10-fold Decrease in the Solubility of α-Synuclein

The conversion of α-synuclein from its intrinsically disordered monomeric state into the fibrillar cross-β aggregates characteristically present in Lewy bodies is largely unknown. The investigation of α-synuclein variants causative of familial forms of Parkinson disease can provide unique insights into the conditions that promote or inhibit aggregate formation. It has been shown recently that a newly identified pathogenic mutation of α-synuclein, H50Q, aggregates faster than the wild-type. We investigate here its aggregation propensity by using a sequence-based prediction algorithm, NMR chemical shift analysis of secondary structure populations in the monomeric state, and determination of thermodynamic stability of the fibrils. Our data show that the H50Q mutation induces only a small increment in polyproline II structure around the site of the mutation and a slight increase in the overall aggregation propensity. We also find, however, that the H50Q mutation strongly stabilizes α-synuclein fibrils by 5.0 ± 1.0 kJ mol⁻¹, thus increasing the supersaturation of monomeric α-synuclein within the cell, and strongly favors its aggregation process. We further show that wild-type α-synuclein can decelerate the aggregation kinetics of the H50Q variant in a dose-dependent manner when coaggregating with it. These last findings suggest that the precise balance of α-synuclein synthesized from the wild-type and mutant alleles may influence the natural history and heterogeneous clinical phenotype of Parkinson disease.     

Molecular Ageing of Alpha- and Beta-Synucleins: Protein Damage and Repair Mechanisms

Abnormal α-synuclein aggregates are hallmarks of a number of neurodegenerative diseases. Alpha synuclein and β-synucleins are susceptible to post-translational modification as isoaspartate protein damage, which is regulated in vivo by the action of the repair enzyme protein L-isoaspartyl O-methyltransferase (PIMT). We aged in vitro native α-synuclein, the α-synuclein familial mutants A30P and A53T that give rise to Parkinsonian phenotypes, and β-synuclein, at physiological pH and temperature for a time course of up to 20 days. Resolution of native α-synuclein and β-synuclein by two dimensional techniques showed the accumulation of a number of post-translationally modified forms of both proteins. The levels of isoaspartate formed over the 20 day time course were quantified by exogenous methylation with PIMT using S-Adenosyl-L-[³H-methyl]methionine as a methyl donor, and liquid scintillation counting of liberated ³H-methanol. All α-synuclein proteins accumulated isoaspartate at ∼1% of molecules/day, ∼20 times faster than for β-synuclein. This disparity between rates of isoaspartate was confirmed by exogenous methylation of synucleins by PIMT, protein resolution by one-dimensional denaturing gel electrophoresis, and visualisation of ³H-methyl esters by autoradiography. Protein silver staining and autoradiography also revealed that α-synucleins accumulated stable oligomers that were resistant to denaturing conditions, and which also contained isoaspartate. Co-incubation of approximately equimolar β-synuclein with α-synuclein resulted in a significant reduction of isoaspartate formed in all α-synucleins after 20 days of ageing. Co-incubated α- and β-synucleins, or α, or β synucleins alone, were resolved by non-denaturing size exclusion chromatography and all formed oligomers of ∼57.5 kDa; consistent with tetramerization. Direct association of α-synuclein with β-synuclein in column fractions or from in vitro ageing co-incubations was demonstrated by their co-immunoprecipitation. These results provide an insight into the molecular differences between α- and β-synucleins during ageing, and highlight the susceptibility of α-synuclein to protein damage, and the potential protective role of β-synuclein.     

Bi-Allelic TCRα or β Recombination Enhances T Cell Development but Is Dispensable for Antigen Responses and Experimental Autoimmune Encephalomyelitis

Dual TCRα-expressing T cells outnumber dual TCRβ-expressing cells by ~10:1. As a result, efforts to understand how dual TCR T cells impact immunity have focused on dual TCRα expression; dual TCRβ expression remains understudied. We recently demonstrated, however, that dual TCRβ expression accelerated disease in a TCR transgenic model of autoimmune arthritis through enhanced positive selection efficiency, indicating that dual TCRβ expression, though rare, can impact thymic selection. Here we generated mice hemizygous for TCRα, TCRβ, or both on the C57BL/6 background to investigate the impact bi-allelic TCR chain recombination has on T cell development, repertoire diversity, and autoimmunity. Lack of bi-allelic TCRα or TCRβ recombination reduced αβ thymocyte development efficiency, and the absence of bi-allelic TCRβ recombination promoted γδ T cell development. However, we observed no differences in the numbers of naïve and expanded antigen-specific T cells between TCRα^+/-β^+/- and wildtype mice, and TCR repertoire analysis revealed only subtle differences in Vβ gene usage. Finally, the absence of dual TCR T cells did not impact induced experimental autoimmune encephalomyelitis pathogenesis. Thus, despite more stringent allelic exclusion of TCRβ relative to TCRα, bi-allelic TCRβ expression can measurably impact thymocyte development and is necessary for maintaining normal αβ/γδ T cell proportions.     

Parkin Deficiency Delays Motor Decline and Disease Manifestation in a Mouse Model of Synucleinopathy

In synucleinopathies, including Parkinson''s disease, partially ubiquitylated α-synuclein species phosphorylated on serine 129 (P^S129-α-synuclein) accumulate abnormally. Parkin, an ubiquitin-protein ligase that is dysfunctional in autosomal recessive parkinsonism, protects against α-synuclein-mediated toxicity in various models.We analyzed the effects of Parkin deficiency in a mouse model of synucleinopathy to explore the possibility that Parkin and α-synuclein act in the same biochemical pathway. Whether or not Parkin was present, these mice developed an age-dependent neurodegenerative disorder preceded by a progressive decline in performance in tasks predictive of sensorimotor dysfunction. The symptoms were accompanied by the deposition of P^S129-α-synuclein but not P^S87-α-synuclein in neuronal cell bodies and neuritic processes throughout the brainstem and the spinal cord; activation of caspase 9 was observed in 5% of the P^S129-α-synuclein-positive neurons. As in Lewy bodies, ubiquitin-immunoreactivity, albeit less abundant, was invariably co-localized with P^S129-α-synuclein. During late disease stages, the disease-specific neuropathological features revealed by ubiquitin- and P^S129-α-synuclein-specific antibodies were similar in mice with or without Parkin. However, the proportion of P^S129-α-synuclein-immunoreactive neuronal cell bodies and neurites co-stained for ubiquitin was lower in the absence than in the presence of Parkin, suggesting less advanced synucleinopathy. Moreover, sensorimotor impairment and manifestation of the neurodegenerative phenotype due to overproduction of human α-synuclein were significantly delayed in Parkin-deficient mice.These findings raise the possibility that effective compensatory mechanisms modulate the phenotypic expression of disease in parkin-related parkinsonism.     

Changes in the miRNA-mRNA Regulatory Network Precede Motor Symptoms in a Mouse Model of Multiple System Atrophy: Clinical Implications

Multiple system atrophy (MSA) is a fatal rapidly progressive α-synucleinopathy, characterized by α-synuclein accumulation in oligodendrocytes. It is accepted that the pathological α-synuclein accumulation in the brain of MSA patients plays a leading role in the disease process, but little is known about the events in the early stages of the disease. In this study we aimed to define potential roles of the miRNA-mRNA regulatory network in the early pre-motor stages of the disease, i.e., downstream of α-synuclein accumulation in oligodendroglia, as assessed in a transgenic mouse model of MSA. We investigated the expression patterns of miRNAs and their mRNA targets in substantia nigra (SN) and striatum, two brain regions that undergo neurodegeneration at a later stage in the MSA model, by microarray and RNA-seq analysis, respectively. Analysis was performed at a time point when α-synuclein accumulation was already present in oligodendrocytes at neuropathological examination, but no neuronal loss nor deficits of motor function had yet occurred. Our data provide a first evidence for the leading role of gene dysregulation associated with deficits in immune and inflammatory responses in the very early, non-symptomatic disease stages of MSA. While dysfunctional homeostasis and oxidative stress were prominent in SN in the early stages of MSA, in striatum differential gene expression in the non-symptomatic phase was linked to oligodendroglial dysfunction, disturbed protein handling, lipid metabolism, transmembrane transport and altered cell death control, respectively. A large number of putative miRNA-mRNAs interaction partners were identified in relation to the control of these processes in the MSA model. Our results support the role of early changes in the miRNA-mRNA regulatory network in the pathogenesis of MSA preceding the clinical onset of the disease. The findings thus contribute to understanding the disease process and are likely to pave the way towards identifying disease biomarkers for early diagnosis of MSA.     

α-Synuclein Disrupts Vesicle Fusion by Two Mutant-Specific Mechanisms

Synaptic accumulation of α-synuclein (α-Syn) oligomers and their interactions with VAMP2 have been reported to be the basis of synaptic dysfunction in Parkinson’s disease (PD). α-Syn mutants associated with familial PD have also been known to be capable of interacting with VAMP2, but the exact mechanisms resulting from those interactions to eventual synaptic dysfunction are still unclear. Here, we investigate the effect of α-Syn mutant oligomers comprising A30P, E46K, and A53T on VAMP2-embedded vesicles. Specifically, A30P and A53T oligomers cluster vesicles in the presence of VAMP2, which is a shared mechanism with wild type α-Syn oligomers induced by dopamine. On the other hand, E46K oligomers reduce the membrane mobility of the planar bilayers, as revealed by single-particle tracking, and permeabilize the membranes in the presence of VAMP2. In the absence of VAMP2 interactions, E46K oligomers enlarge vesicles by fusing with one another. Our results clearly demonstrate that α-Syn mutant oligomers have aberrant effects on VAMP2-embedded vesicles and the disruption types are distinct depending on the mutant types. This work may provide one of the possible clues to explain the α-Syn mutant-type dependent pathological heterogeneity of familial PD.