Single-dose intranasal administration of α-syn PFFs induce lewy neurite-like pathology in olfactory bulbs.

INTRODUCTION: Pathological α-synuclein (α-Syn) propagation may cause Parkinson's disease progression. We aimed to verify whether single-dose intranasal administration of α-Syn preformed fibrils (PFFs) induces α-Syn pathology in the olfactory bulb (OB). METHODS: A single dose of α-Syn PFFs was administered to the left nasal cavity of wild-type mice. The untreated right side served as a control. The α-Syn pathology of the OBs was examined up to 12 months after the injection. RESULTS: Lewy neurite-like aggregates were observed in the OB 6 and 12 months after the treatment. CONCLUSIONS: These findings suggest that pathological α-Syn can propagate from the olfactory mucosa to the OB and reveal the potential dangers of α-Syn PFFs inhalation.

Striatal-Inoculation of α-Synuclein Preformed Fibrils Aggravated the Phenotypes of REM Sleep without Atonia in A53T BAC-SNCA Transgenic Mice.

Accumulation of α-synuclein (α-syn) is the pathological hallmark of α-synucleinopathy. Rapid eye movement (REM) sleep behavior disorder (RBD) is a pivotal manifestation of α-synucleinopathy including Parkinson's disease (PD). RBD is clinically confirmed by REM sleep without atonia (RWA) in polysomnography. To accurately characterize RWA preceding RBD and their underlying α-syn pathology, we inoculated α-syn preformed fibrils (PFFs) into the striatum of A53T human α-syn BAC transgenic (A53T BAC-SNCA Tg) mice which exhibit RBD-like phenotypes with RWA. RWA phenotypes were aggravated by PFFs-inoculation in A53T BAC-SNCA Tg mice at 1 month after inoculation, in which prominent α-syn pathology in the pedunculopontine nucleus (PPN) was observed. The intensity of RWA phenotype could be dependent on the severity of the underlying α-syn pathology.

Cellular analysis of SOD1 protein-aggregation propensity and toxicity: a case of ALS with slow progression harboring homozygous SOD1-D92G mutation.

Mutations within Superoxide dismutase 1 (SOD1) cause amyotrophic lateral sclerosis (ALS), accounting for approximately 20% of familial cases. The pathological feature is a loss of motor neurons with enhanced formation of intracellular misfolded SOD1. Homozygous SOD1-D90A in familial ALS has been reported to show slow disease progression. Here, we reported a rare case of a slowly progressive ALS patient harboring a novel SOD1 homozygous mutation D92G (homD92G). The neuronal cell line overexpressing SOD1-D92G showed a lower ratio of the insoluble/soluble fraction of SOD1 with fine aggregates of the misfolded SOD1 and lower cellular toxicity than those overexpressing SOD1-G93A, a mutation that generally causes rapid disease progression. Next, we analyzed spinal motor neurons derived from induced pluripotent stem cells (iPSC) of a healthy control subject and ALS patients carrying SOD1-homD92G or heterozygous SOD1-L144FVX mutation. Lower levels of misfolded SOD1 and cell loss were observed in the motor neurons differentiated from patient-derived iPSCs carrying SOD1-homD92G than in those carrying SOD1-L144FVX. Taken together, SOD1-homD92G has a lower propensity to aggregate and induce cellular toxicity than SOD1-G93A or SOD1-L144FVX, and these cellular phenotypes could be associated with the clinical course of slowly progressive ALS.

BCAS1-positive immature oligodendrocytes are affected by the α-synuclein-induced pathology of multiple system atrophy.

Multiple system atrophy (MSA) is pathologically characterized by the presence of fibrillar α-synuclein-immunoreactive inclusions in oligodendrocytes. Although the myelinating process of oligodendrocytes can be observed in adult human brains, little is known regarding the presence of α-synuclein pathology in immature oligodendrocytes and how their maturation and myelination are affected in MSA brains. Recently, breast carcinoma amplified sequence 1 (BCAS1) has been found to be specifically expressed in immature oligodendrocytes undergoing maturation and myelination. Here, we analyzed the altered dynamics of oligodendroglial maturation in both MSA brains and primary oligodendroglial cell cultures which were incubated with α-synuclein pre-formed fibrils. The numbers of BCAS1-expressing oligodendrocytes that displayed a matured morphology negatively correlated with the density of pathological inclusions in MSA brains but not with that in Parkinson's disease and diffuse Lewy body disease. In addition, a portion of the BCAS1-expressing oligodendrocyte population showed cytoplasmic inclusions, which were labeled with antibodies against phosphorylated α-synuclein and cleaved caspase-9. Further in vitro examination indicated that the α-synuclein pre-formed fibrils induced cytoplasmic inclusions in the majority of BCAS1-expressing oligodendrocytes. In contrast, the majority of BCAS1-non-expressing mature oligodendrocytes did not develop inclusions on day 4 after maturation induction. Furthermore, exposure of α-synuclein pre-formed fibrils in the BCAS1-positive phase caused a reduction in oligodendroglial cell viability. Our results indicated that oligodendroglial maturation and myelination are impaired in the BCAS1-positive phase of MSA brains, which may lead to the insufficient replacement of defective oligodendrocytes. In vitro, the high susceptibility of BCAS1-expressing primary oligodendrocytes to the extracellular α-synuclein pre-formed fibrils suggests the involvement of insufficient oligodendroglial maturation in MSA disease progression and support the hypothesis that the BCAS1-positive oligodendrocyte lineage cells are prone to take up aggregated α-synuclein in vivo.

Differential roles of epigenetic regulators in the survival and differentiation of oligodendrocyte precursor cells.

During development or after brain injury, oligodendrocyte precursor cells (OPCs) differentiate into oligodendrocytes to supplement the number of oligodendrocytes. Although mechanisms of OPC differentiation have been extensively examined, the role of epigenetic regulators, such as histone deacetylases (HDACs) and DNA methyltransferase enzymes (DNMTs), in this process is still mostly unknown. Here, we report the differential roles of epigenetic regulators in OPC differentiation. We prepared primary OPC cultures from neonatal rat cortex. Our cultured OPCs expressed substantial amounts of mRNA for HDAC1, HDAC2, DNMT1, and DNMT3a. mRNA levels of HDAC1 and HDAC2 were both decreased by the time OPCs differentiated into myelin-basic-protein expressing oligodendrocytes. However, DNMT1 or DNMT3a mRNA level gradually decreased or increased during the differentiation step, respectively. We then knocked down those regulators in cultured OPCs with siRNA technique before starting OPC differentiation. While HDAC1 knockdown suppressed OPC differentiation, HDAC2 knockdown promoted OPC differentiation. DNMT1 knockdown also suppressed OPC differentiation, but unlike HDAC1/2, DNMT1-deficient cells showed cell damage during the later phase of OPC differentiation. On the other hand, when OPCs were transfected with siRNA for DNMT3a, the number of OPCs was decreased, indicating that DNMT3a may participate in OPC survival/proliferation. Taken together, these data demonstrate that each epigenetic regulator has different phase-specific roles in OPC survival and differentiation.

Zonisamide inhibits monoamine oxidase and enhances motor performance and social activity.

Zonisamide (ZNS) is an effective drug for not only motor symptoms but also non-motor symptoms in Parkinson's disease. However, the actions of ZNS as an anti-Parkinsonian drug are not well understood. To clarify the actions of ZNS in vivo, we administered ZNS to mice and examined the effects on neurotransmitter metabolism and behaviors, focusing on motor and non-motor symptoms. Administration of ZNS decreased dopamine (DA) turnover in various brain regions, including the striatum. In behavioral tests, ZNS enhanced locomotor activity and novelty seeking in the open field test, light-dark transition test, and the social interaction test. Consistent with these results of DA metabolism in ZNS-treated mice, monoamine oxidase activity was significantly inhibited by ZNS in primary neurons and astrocytes. Collectively, these data suggest that ZNS inhibits monoamine oxidase activity and decreases DA turnover, which increases locomotor activity and novelty seeking in mice. ZNS is potentially useful to improve not only motor symptoms but also neuropsychiatric non-motor symptoms such as apathy in PD.

Submembranous septins as relatively stable components of actin-based membrane skeleton.

The cell cortex is organized by the dynamic interplay between the plasma membrane, membrane proteins, and the cytoskeleton. Despite the cortical localization of septin heteropolymers in vivo and their direct interaction with phospholipid membranes in vitro, their behavior and roles remain elusive. This study characterizes the major cortical septin assembly found in mammalian tissue culture cells by fluorescence recovery after photobleaching analysis. GFP-tagged septin subunits, which colocalized with cortical actin, exhibited slower turnover than some other cortical proteins that were analyzed (e.g., actin, syntaxin-1A and a glutamate aspartate transporter [GLAST]). Perturbation of actin turnover by cytochalasin D or jasplakinolide retarded the cortical septin turnover, while septin depletion by RNAi did not recognizably affect cortical actin turnover. These phenomena are compatibly interpreted by septins' selective association with a subset of actin-based membrane skeleton, as revealed by rapid-freeze deep-etch immuno-replica electron microscopy. We applied the assay system to test septins' presumptive scaffold function on their physiological binding partners. Septin filament destabilization by RNAi-mediated subunit depletion facilitated the turnover of GLAST, depending on the carboxyl-terminal 29 residues, while a septin filament-stabilizing drug forchlorfenuron restrained more GLAST in the unexchangeable fraction. These data indicate that cortical septin heteropolymers are components of the actin-based membrane skeleton providing scaffolds for their interacting partners probably by impeding their lateral diffusion. We predict that diverse submembranous septin clusters found in vivo may serve as scaffolds or reserve pools for specific membrane-bound proteins.

The endoplasmic reticulum stress sensor, ATF6α, protects against neurotoxin-induced dopaminergic neuronal death.

Oxidative stress and endoplasmic reticulum (ER) stress are thought to contribute to the pathogenesis of various neurodegenerative diseases including Parkinson disease (PD), however, the relationship between these stresses remains unclear. ATF6α is an ER-membrane-bound transcription factor that is activated by protein misfolding in the ER and functions as a critical regulator of ER quality control proteins in mammalian cells. The goal of this study was to explore the cause-effect relationship between oxidative stress and ER stress in the pathogenesis of neurotoxin-induced model of PD. 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a dopaminergic neurotoxin known to produce oxidative stress, activated ATF6α and increased ER chaperones and ER-associated degradation (ERAD) component in dopaminergic neurons. Importantly, MPTP induced formation of ubiquitin- immunopositive inclusions and loss of dopaminergic neurons more prominently in mice deficient in ATF6α than in wild-type mice. Cultured cell experiments revealed that 1-methyl-4-phenylpyridinium (MPP(+))-induced oxidative stress not only promoted phosphorylation of p38 mitogen-activated protein kinase (p38MAPK) but also enhanced interaction between phosphorylated p38MAPK and ATF6α, leading to increment in transcriptional activator activity of ATF6α. Thus, our results revealed a link between oxidative stress and ER stress by showing the importance of ATF6α in the protection of the dopaminergic neurons from MPTP that occurs through oxidative stress-induced activation of ATF6α and p38MAPK-mediated enhancement of ATF6α transcriptional activity.

Sept4, a component of presynaptic scaffold and Lewy bodies, is required for the suppression of alpha-synuclein neurotoxicity.

In Parkinson disease (PD), alpha-synuclein aggregates called Lewy bodies often involve and sequester Septin4 (Sept4), a polymerizing scaffold protein. However, the pathophysiological significance of this phenomenon is unclear. Here, we show the physiological association of Sept4 with alpha-synuclein, the dopamine transporter, and other presynaptic proteins in dopaminergic neurons; mice lacking Sept4 exhibit diminished dopaminergic neurotransmission due to scarcity of these presynaptic proteins. These data demonstrate an important role for septin scaffolds in the brain. In transgenic mice that express human alpha-synuclein(A53T) (a mutant protein responsible for familial PD), loss of Sept4 significantly enhances neuropathology and locomotor deterioration. In this PD model, insoluble deposits of Ser129-phosphorylated alpha-synuclein(A53T) are negatively correlated with the dosage of Sept4. In vitro, direct association with Sept4 protects alpha-synuclein against self-aggregation and Ser129 phosphorylation. Taken together, these data show that Sept4 may be involved in PD as a dual susceptibility factor, as its insufficiency can diminish dopaminergic neurotransmission and enhance alpha-synuclein neurotoxicity.