A53T Α-synuclein Research Articles

Caffeic acid reduces A53T α-synuclein by activating JNK/Bcl-2-mediated autophagy in vitro and improves behaviour and protects dopaminergic neurons in a mouse model of Parkinson’s disease

The human A53T mutant of α-synuclein tends to aggregate and leads to neurotoxicity in familial Parkinson's disease (PD). The aggregation of α-synuclein is also found in sporadic PD. Thus, targeting α-synuclein clearance could be used as a drug-discovery strategy for PD treatment. Caffeic acid (CA) has shown neuroprotection in Alzheimer's disease or cerebral ischaemia; however, it is unclear whether CA confers neuroprotection in α-synuclein-induced PD models. Here we focus on whether and how A53T α-synuclein is affected by CA. We assessed the effect of CA on cell viability in SH-SY5Y cells overexpressing A53T α-synuclein. Pathway-related inhibitors were used to identify the autophagy mechanisms. Seven-month-old A53T α-synuclein transgenic mice (A53T Tg mice) received CA daily for eight consecutive weeks. Behaviour tests including the buried food pellet test, the pole test, the Rotarod test, open field analysis, and gait analysis were used to evaluate the neuroprotective effect of CA. Tyrosine hydroxylase and α-synuclein were assessed by immunohistochemistry or western blot in the substantia nigra (SN). We found that CA alleviated the cell damage induced by overexpressing A53T α-synuclein and that CA reduced A53T α-synuclein by activating the JNK/Bcl-2-mediated autophagy pathway. The efficacy of CA on A53T α-synuclein degradation was reversed by the autophagy inhibitor bafilomycin A1 and the JNK inhibitor SP600125. In A53T Tg mice, CA improved behavioural impairments, attenuated loss of dopaminergic neurons, enhanced autophagy and reduced α-synuclein in the SN. Thus, the results provide scientific evidence for the neuroprotective effect of CA in PD. Our work lays the foundation for CA clinical trials to treat PD in the future.

Synaptic vesicle mimics affect the aggregation of wild-type and A53T α-synuclein variants differently albeit similar membrane affinity.

α-Synuclein misfolding results in the accumulation of amyloid fibrils in Parkinson’s disease. Missense protein mutations (e.g. A53T) have been linked to early onset disease. Although α-synuclein interacts with synaptic vesicles in the brain, it is not clear what role they play in the protein aggregation process. Here, we compare the effect of small unilamellar vesicles (lipid composition similar to synaptic vesicles) on wild-type (WT) and A53T α-synuclein aggregation. Using biophysical techniques, we reveal that binding affinity to the vesicles is similar for the two proteins, and both interact with the helix long axis parallel to the membrane surface. Still, the vesicles affect the aggregation of the variants differently: effects on secondary processes such as fragmentation dominate for WT, whereas for A53T, fibril elongation is mostly affected. We speculate that vesicle interactions with aggregate intermediate species, in addition to monomer binding, vary between WT and A53T, resulting in different consequences for amyloid formation.

Ethanol extract from Gynostemma pentaphyllum ameliorates dopaminergic neuronal cell death in transgenic mice expressing mutant A53T human alpha-synuclein.

Gynostemma (G.) pentaphyllum (Cucurbitaceae) contains various bioactive gypenosides. Ethanol extract from G. pentaphyllum (GP-EX) has been shown to have ameliorative effects on the death of dopaminergic neurons in animal models of Parkinson’s disease (PD) induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine- and 6-hydroxydopamine. PD patients exhibit multiple symptoms, so PD-related research should combine neurotoxin models with genetic models. In the present study, we investigated the ameliorative effects of GP-EX, including gypenosides, on the cell death of dopaminergic neurons in the midbrain of A53T α-synuclein transgenic mouse models of PD (A53T). Both GP-EX and gypenosides at 50 mg/kg per day were orally administered to the A53T mice for 20 weeks. α-Synuclein-immunopositive cells and α-synuclein phosphorylation were increased in the midbrain of A53T mice, which was reduced following treatment with GP-EX. Treatment with GP-EX modulated the reduced phosphorylation of tyrosine hydroxylase, extracellular signal-regulated kinase (ERK1/2), Bcl-2-associated death promoter (Bad) at Ser112, and c-Jun N-terminal kinase (JNK1/2) due to α-synuclein overexpression. In the A53T group, GP-EX treatment prolonged the latency of the step-through passive avoidance test and shortened the transfer latency of the elevated plus maze test. Gypenosides treatment exhibited the effects and efficacy similar to those of GP-EX. Taken together, GP-EX, including gypenosides, has ameliorative effects on dopaminergic neuronal cell death due to the overexpression of α-synuclein by modulating ERK1/2, Bad at Ser112, and JNK1/2 signaling in the midbrain of A53T mouse model of PD. Further studies are needed to investigate GP-EX as a treatment for neurodegenerative synucleinopathies, including PD. This study was approved by the Animal Ethics Committee of Chungbuk National University (approval No. CBNUA-956-16-01) on September 21, 2016.

Gami-Chunggan Formula Prevents Motor Dysfunction in MPTP/p-Induced and A53T α-Synuclein Overexpressed Parkinson's Disease Mouse Model Though DJ-1 and BDNF Expression.

The Gami–Chunggan formula (GCF) is a modification of the Chunggan (CG) decoction, which has been used to treat movement disorders such as Parkinson’s disease (PD) in Traditional East Asian Medicine. To evaluate the neuroprotective effects of GCF in chronic PD animal models, we used either a 5-week treatment of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine with probenecid (MPTP/p) or the α-synuclein A53T overexpressed PD mouse model. C57BL/6 mice were treated with MPTP, in combination with probenecid, for 5 weeks. GCF was administered simultaneously with MPTP injection for 38 days. The A53T α-synuclein overexpressed mice were also fed with GCF for 60 days. Using behavioral readouts and western blot analyses, it was observed that GCF prevents motor dysfunction in the MPTP/p-induced and A53T α-synuclein overexpressed mice. Moreover, GCF inhibited the reduction of dopaminergic neurons in the substantia nigra (SN) and fibers in the striatum (ST) against MPTP/p challenge. The expression of DJ-1 was increased but that of α-synuclein was decreased in the SN of PD-like brains by GCF administration. In vitro experiments also showed that GCF inhibited 6-OHDA-induced neurotoxicity in SH-SY5Y neuroblastoma cell lines and that it did so to a greater degree than CG. Furthermore, GCF induced BDNF expression through phosphorylation of Akt, ERK, CREB, and AMPK in the SN of PD-like brains. Therefore, use of the herbal medicine GCF offers a potential remedy for neurodegenerative disorders, including Parkinson’s disease.

Deciphering the anti-Parkinson's activity of sulphated polysaccharides from Chlamydomonas reinhardtii on the α-Synuclein mutants A30P, A53T, E46K, E57K and E35K.

Parkinsonism-linked mutations in alanine and glutamic acid residues of the pre-synaptic protein α-Synuclein (α-Syn) affect specific tertiary interactions essential for stability of the native state and make it prone to more aggregation. Many of the currently available drugs used for the treatment of Parkinson's disease (PD) are not very effective and are associated with multiple side effects. Recently, marine algae have been reported to have sulphated polysaccharides which offers multiple pharmaceutical properties. With this background, we have isolated sulphated polysaccharides from Chlamydomonas reinhardtii (Cr-SPs) and investigated their effects on inhibition of fibrillation/aggregation of α-Syn mutants through a combination of spectroscopic and microscopic techniques. The kinetics of α-Syn fibrillation establishes that Cr-SPs are very effective in inhibiting fibrillation of α-Syn mutants. The morphological changes associated with the fibrillation/aggregation process have been monitored by transmission electron microscopy. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis gel image suggests that Cr-SPs increase the amount of soluble protein after completion of the fibrillation/aggregation process. The circular dichroism results showed that Cr-SPs efficiently delay the conversion of native protein into β-sheet-rich structures. Thus, the current work has considerable therapeutic implications towards deciphering the potential of Cr-SPs to act against PD and other protein aggregation-related disorders.

Serotonergic pathology and disease burden in the premotor and motor phase of A53T α-synuclein parkinsonism: a cross-sectional study

Because of the highly penetrant gene mutation and clinical features consistent with idiopathic Parkinson's disease, carriers of the autosomal dominant Ala53Thr (A53T; 209G→A) point mutation in the α-synuclein (SNCA) gene are an ideal population to study the premotor phase and evolution of Parkinson's pathology. Given the known neurochemical changes in the serotonergic system and their association with symptoms of Parkinson's disease, we hypothesised that carriers of the A53T SNCA mutation might show abnormalities in the serotonergic neurotransmitter system before the diagnosis of Parkinson's disease, and that this pathology might be associated with measures of Parkinson's burden. In this cross-sectional study, we recruited carriers of the A53T SNCA mutation from specialist Movement Disorders clinics in Athens, Greece, and Salerno, Italy, and a cohort of healthy controls with no personal or family history of neurological or psychiatric disorders from London, UK (recruited via public advertisement) who were age matched to the A53T SNCA carriers. We also recruited one cohort of patients with idiopathic Parkinson's disease (cohort 1) from Movement Disorders clinics in London, UK, and retrieved data on a second cohort of such patients (cohort 2; n=40) who had been scanned with a different scanner. 7-day continuous recording of motor function was used to determine the Parkinson's disease status of the A53T carriers. To assess whether serotonergic abnormalities were present, we used [11C]DASB PET non-displaceable binding to quantify serotonin transporter density. We constructed brain topographic maps reflecting Braak stages 1-6 and used these as seed maps to calculate [11C]DASB non-displaceable binding potential in our cohort of A53T SNCA carriers. Additionally, all participants underwent a battery of clinical assessments to determine motor and non-motor symptoms and cognitive status, and [123I]FP-CIT single-photon emission CT (SPECT) to assess striatal dopamine transporter binding and MRI for volumetric analyses to assess whether pathology is associated with measures of Parkinson's disease burden. Between Sept 1, 2016, and Sept 30, 2018, we recruited 14 A53T SNCA carriers, 25 healthy controls, and 25 patients with idiopathic Parkinson's disease. Seven (50%) of 14 A53T SCNA carriers were confirmed to have motor symptoms and confirmed to have Parkinson's disease, and the absence of motor symptoms was confirmed in seven (50%) A53T SCNA carriers (ie, premotor), in whom [123I]FP-CIT SPECT confirmed the absence of striatal dopaminergic deficits. Compared with healthy controls, premotor A53T SNCA carriers showed loss of [11C]DASB non-displaceable binding potential in the ventral (p<0·0001) and dorsal (p=0·0002) raphe nuclei, caudate (p=0·00015), putamen (p=0·036), thalamus (p=0·00074), hypothalamus (p<0·0001), amygdala (p=0·0041), and brainstem (p=0·046); and in A53T SNCA carriers with Parkinson's disease this loss was extended to the hippocampus (p=0·0051), anterior (p=0·022) and posterior cingulate (p=0·036), insula (p=0·0051), frontal (p=0·0016), parietal (p=0·019), temporal (p<0·0001), and occipital (p=0·0053) cortices. A53T SNCA carriers with Parkinson's disease showed a loss of striatal [123I]FP-CIT-specific binding ratio compared with healthy controls (p<0·0001). Premotor A53T SNCA carriers had loss of [11C]DASB non-displaceable binding potential in brain areas corresponding to Braak stages 1-3, whereas [11C]DASB non-displaceable binding potential was largely preserved in areas corresponding to Braak stages 4-6. Except for one participant who was diagnosed with Parkinson's disease in the past year, all A53T SNCA carriers with Parkinson's disease had decreases in [11C]DASB non-displaceable binding potential in brain areas corresponding to Braak stages 1-6. Decreases in [11C]DASB non-displaceable binding potential in the brainstem were associated with increased Movement Disorder Score-Unified Parkinson's Disease Rating Scale total scores in all A53T SNCA carriers (r -0·66, 95% CI -0·88 to -0·20; p=0·0099), idiopathic Parkinson's disease cohort 1 (r -0·66, -0·84 to -0·36; p=0·00031), and idiopathic Parkinson's disease cohort 2 (r -0·71, -0·84 to -0·52; p<0·0001). The presence of serotonergic pathology in premotor A53T SNCA carriers preceded development of dopaminergic pathology and motor symptoms and was associated with disease burden, highlighting the potential early role of serotonergic pathology in the progression of Parkinson's disease. Our findings provide evidence that molecular imaging of serotonin transporters could be used to visualise premotor pathology of Parkinson's disease in vivo. Future work might establish whether serotonin transporter imaging is suitable as an adjunctive tool for screening and monitoring progression for individuals at risk or patients with Parkinson's disease to complement dopaminergic imaging, or as a marker of Parkinson's burden in clinical trials. Lily Safra Hope Foundation and National Institute for Health Research (NIHR) Biomedical Research Centre at King's College London.

Temporal changes in neuroinflammation and brain glucose metabolism in a rat model of viral vector-induced α-synucleinopathy

Rat models based on viral vector-mediated overexpression of α-synuclein are regarded as highly valuable models that closely mimic cardinal features of human Parkinson's disease (PD) such as L-DOPA-dependent motor impairment, dopaminergic neurodegeneration and α-synuclein inclusions. To date, the downstream effects of dopaminergic cell loss on brain glucose metabolism, including the neuroinflammation component, have not been phenotyped in detail for this model. Cerebral glucose metabolism was monitored throughout different stages of the disease using in vivo 2-[18F]-fluoro-2-deoxy-d-glucose ([18F]FDG) positron emission tomography (PET) and was combined with in vitro [18F]DPA-714 autoradiography to assess concomitant inflammation. Rats were unilaterally injected with recombinant adeno-associated viral vector serotype 2/7 (rAAV2/7) encoding either A53T α-synuclein or eGFP. Brain [18F]FDG microPET was performed at baseline, 1, 2, 3, 4, 6, and 9 weeks post-surgery, in combination with behavioral tests. As a second experiment, [18F]DPA-714 autoradiography was executed across the same timeline. Voxel-based analysis of relative [18F]FDG uptake showed a dynamic pattern of PD-related metabolic changes throughout the disease progression (weeks 2–9). Glucose hypermetabolism covering a large bilateral area reaching from the insular, motor- and somatosensory cortex to the striatum was observed at week 2. At week 4, hypermetabolism presented in a cluster covering the ipsilateral nigra-thalamic region, whereas hypometabolism was noted in the ipsilateral striatum at week 6. Elevated [18F]FDG uptake was seen in a cluster extending across the contralateral striatum, motor- and somatosensory cortex at week 9. Increased [18F]FDG in the region of the substantia nigra was associated with increased [18F]DPA-714 binding, and correlated significantly with motor symptoms. These findings point to disease-associated metabolic and neuroinflammatory changes taking place in the primary area of dopaminergic neurodegeneration but also closely interconnected motor and somatosensory brain regions.

Gene co-expression analysis of the human substantia nigra identifies BMP2 as a neurotrophic factor that can promote neurite growth in cells overexpressing wild-type or A53T α-synuclein

Introductionα-synuclein-induced degeneration of dopaminergic neurons has been proposed to be central to the early progression of Parkinson's disease. This highlights the need to identify factors that are neuroprotective or neuroregenerative against α-synuclein-induced degeneration. Due to their potent neurotrophic effects on nigrostriatal dopaminergic neurons, we hypothesized that members of the bone morphogenetic protein (BMP) family have potential to protect these cells against α-synuclein. MethodsTo identify the most relevant BMP ligands, we used unbiased gene co-expression analysis to identify all BMP family members having a significant positive correlation with five markers of dopaminergic neurons in the human substantia nigra (SN). We then tested the ability of lead BMPs to promote neurite growth in SH-SY5Y cells and in primary cultures of ventral mesencephalon (VM) dopaminergic neurons, treated with either 6-OHDA or MPP+, or overexpressing wild-type or A53T α-synuclein. ResultsOnly the expression of BMP2 was found to be significantly correlated with multiple dopaminergic markers in the SN. We found that BMP2 treatment promoted neurite growth in SH-SY5Y cells and in dopaminergic neurons. Moreover, BMP2 treatment promoted neurite growth in both SH-SY5Y cells and VM neurons, treated with the neurotoxins 6-OHDA or MPP+. Furthermore, BMP2 promoted neurite growth in cells overexpressing wild-type or A53T-α-synuclein. ConclusionThese findings are important given that clinical trials of two neurotrophic factors, GDNF and neurturin, have failed to meet their primary endpoints. Our findings are a key first step in rationalising the further study of BMP2 as a potential neurotrophic factor in α-synuclein-based translational models of Parkinson's disease.

Layer-specific axonal degeneration of serotonergic fibers in the prefrontal cortex of aged A53T α-synuclein–expressing mice

Axonal pathology precedes dopaminergic cell loss in Parkinson's disease (PD), indicating a dying back axonopathy of nigrostriatal projections. Although most attention focused on the dopaminergic system, increasing evidence implies a compromised serotonergic system in PD as well. By combining immunohistological and biochemical approaches, a profound layer-specific reduction of the serotonergic input to the prefrontal cortex (PFC) layers II and V/VI in aged mutant A53T α-synuclein–expressing mice (A53T mice) was detected. In addition, the altered fiber network was characterized by swollen axons and enlarged axonal varicosities within all PFC layers, but most pronounced in PFC layer I. Although prefrontal serotonin levels and synaptic protein expression were preserved, aged A53T mice showed increased levels of kinesin family member 1a and vesicular monoamine transporter 2. Together with increased tryptophan hydroxylase 2 mRNA levels in the raphe nuclei and an elevated serotonin receptor 1b expression in the PFC, these findings point to compensatory mechanisms within the serotonergic system to overcome the reduced neuritic input to the PFC in this transgenic animal model for PD.

Quantitative Characterization of α-Synuclein Aggregation in Living Cells through Automated Microfluidics Feedback Control

SummaryAggregation of α-synuclein and formation of inclusions are hallmarks of Parkinson’s disease (PD). Aggregate formation is affected by cellular environment, but it has been studied almost exclusively in cell-free systems. We quantitatively analyzed α-synuclein inclusion formation and clearance in a yeast cell model of PD expressing either wild-type (WT) α-synuclein or the disease-associated A53T mutant from the galactose (Gal)-inducible promoter. A computer-controlled microfluidics device regulated α-synuclein in cells by means of closed-loop feedback control. We demonstrated that inclusion formation is strictly concentration dependent and that the aggregation threshold of the A53T mutant is about half of the WT α-synuclein (56%). We chemically modulated the proteasomal and autophagic pathways and demonstrated that autophagy is the main determinant of A53T α-synuclein inclusions’ clearance. In addition to proposing a technology to overcome current limitations in dynamically regulating protein expression levels, our results contribute to the biology of PD and have relevance for therapeutic applications.

Beneficial Effects of Trehalose on Striatal Dopaminergic Deficits in Rodent and Primate Models of Synucleinopathy in Parkinson's Disease.

Disease modification in Parkinson's disease (PD) is an unmet medical need. In the current study, we evaluated trehalose, a safe and well-tolerated disaccharide that has previously demonstrated efficacy in rodent models of neurodegenerative diseases, including PD. In a rat model of PD, based on delivery of adeno-associated virus serotype 1/2 containing the mutated human A53T α-synuclein gene (AAV1/2-hourA53T-aSyn) to the substantia nigra (SN), we showed that rats administered trehalose (2.67 g/kg per day, by mouth) for 6 weeks had less forelimb asymmetry (93% reduction) and higher striatal dopamine (54% increase) compared with rats receiving vehicle. In a pharmacokinetic study, we determined that efficacy was associated with plasma C max of 8900 ng/ml and area under the curve from time 0 to infinity (AUC0-inf) of 11,136 hour⋅ng/ml. We then showed, in macaques, that oral administration of trehalose (2.67 g/kg per day) produced plasma exposures of similar magnitude, with plasma C max of 10,918 ng/ml and AUC0-inf of 27,445 hour⋅ng/ml. In a macaque model of PD, also based on delivery of AAV1/2-hourA53T-aSyn to the SN, trehalose (2.67 g/kg per day, by mouth), administered for 142 days, produced higher striatal dopamine (by 39%) and dopamine transporter levels (by 50%), compared with macaques receiving vehicle. In neither model did trehalose treatment prevent loss of tyrosine hydroxylase (TH) positive (TH+ve) cells in the SN or alter α-synuclein levels in the striatum. These studies demonstrated that trehalose reduces striatal dopaminergic deficits in a rodent and macaque model of synucleinopathy in PD. Furthermore, we have determined the pharmacokinetic parameters associated with efficacy, and thus defined exposures to target in future clinical trials.

Impact of Dopamine Oxidation on Dopaminergic Neurodegeneration.

Parkinson's disease (PD) is the second most common neurodegenerative disease. The characteristic feature of PD is the progressive degeneration of the dopaminergic (DAergic) neurons in the substantia nigra (SN). DAergic neurons in the SN accumulate black and insoluble membrane structures known as neuromelanin during aging. The oxidation of dopamine (DA) to form neuromelanin generates many o-quinones, including DA o-quinones, aminochrome, and 5,6-indolequinone. The focus of this review is to discuss the role of DA oxidation in association with PD. The oxidation of DA produces oxidative products, inducing mitochondrial dysfunction, impaired protein degradation, α-synuclein aggregation into neurotoxic oligomers, and oxidative stress, in vitro. Recent studies have demonstrated that the DA content is critical for both DJ-1 knockout and A53T α-synuclein transgenic mice to develop PD pathological features, providing evidence for DA action in PD pathogenesis in vivo. The effects of L-DOPA, as the most effective anti-PD drug, are also briefly discussed.

Aegeline, a natural product from the plant Aegle marmelos, mimics the yeast SNARE protein Sec22p in suppressing α-synuclein and Bax toxicity in yeast

Herein, we have identified yeast Sec22p (ySec22p), a SNARE protein essential for endoplasmic reticulum to Golgi trafficking, as a suppressor of Bax-induced yeast apoptosis and corroborated published observations that ySec22p suppresses α-synuclein’s toxicity in yeast. It has been suggested that compounds which enhance expression, in neurons, of human homologues of ySec22p (Sec22Bp/Sec22p/Sec22A) would prevent synucleinopathies, such as Parkinson’s disease. With the aim of finding a small molecule that would mimic ySec22p, a library of natural products consisting of 394-compounds was screened using yeast cells that express either human α-synuclein or human Bax. The antioxidant aegeline, an alkaloid-amide occurring in the leaves of the plant Aegle marmelos Correa, was the only molecule that overcame apoptosis induced by both α-synuclein and Bax in yeast. Besides, aegeline also prevented growth block in cells expressing the more toxic A53T α-synuclein mutant. Restoration of cell growth occurred through inhibition of increased ROS levels, mitochondrial membrane potential loss and nuclear DNA fragmentation, characteristics of apoptosis manifested in α-synuclein or Bax-expressing cells. These results highlight the importance of yeast systems to identify rapidly molecules that may prevent the onset of apoptosis that occurs in Parkinson’s disease.

Cerebral inoculation of human A53T α-synuclein reduces spatial memory decline and amyloid-β aggregation in APP/PS1 transgenic mice of Alzheimer’s disease

Amyloid-β (Aβ) peptide and α-synuclein (α-syn) are major components of senile plaques in Alzheimer’s disease (AD) and Lewy bodies in Parkinson’s disease (PD), respectively. Co-occurrence of Aβ and α-syn in the senile brains of AD and LB diseases suggests interactions between the two proteins. However, the significance of the overlapping deposition, especially the effects of α-syn on the Aβ aggregation, still remains to be clarified. In the present study, we investigated the effects of α-syn pre-formed fibrils (PFFs) injection on the cognitive behaviors and Aβ deposition in the brain of APP/PS1 transgenic AD mice by using Morris water maze (MWM) test, immunohistochemistry and western blot techniques. We found that APP/PS1 transgenic mice exhibited an obvious elevation in the α-syn load, as well as Aβ deposition in the brain compared with wild type of C57 BL littermates. 5 months after cerebral injection of exogenous α-syn, MWM tests showed an alleviation in cognitive impairments in APP/PS1 mice; western blot and immunohistochemistry experiments also exhibited a significant reduction in Aβ level in the brain of APP/PS1 mice injected with α-syn. These results suggest that α-syn aggregated in the brain of AD may act as a protective factor and defend the brain tissue from early Aβ deposition and cognitive deficits.

A53T Mutant Alpha-Synuclein Induces Tau-Dependent Postsynaptic Impairment Independently of Neurodegenerative Changes

Abnormalities in α-synuclein are implicated in the pathogenesis of Parkinson's disease (PD). Because α-synuclein is highly concentrated within presynaptic terminals, presynaptic dysfunction has been proposed as a potential pathogenic mechanism. Here, we report novel, tau-dependent, postsynaptic deficits caused by A53T mutant α-synuclein, which is linked to familial PD. We analyzed synaptic activity in hippocampal slices and cultured hippocampal neurons from transgenic mice of either sex expressing human WT, A53T, and A30P α-synuclein. Increased α-synuclein expression leads to decreased spontaneous synaptic vesicle release regardless of genotype. However, only those neurons expressing A53T α-synuclein exhibit postsynaptic dysfunction, including decreased miniature postsynaptic current amplitude and decreased AMPA to NMDA receptor current ratio. We also found that long-term potentiation and spatial learning were impaired by A53T α-synuclein expression. Mechanistically, postsynaptic dysfunction requires glycogen synthase kinase 3β-mediated tau phosphorylation, tau mislocalization to dendritic spines, and calcineurin-dependent AMPA receptor internalization. Previous studies reveal that human A53T α-synuclein has a high aggregation potential, which may explain the mutation's unique capacity to induce postsynaptic deficits. However, patients with sporadic PD with severe tau pathology are also more likely to have early onset cognitive decline. Our results here show a novel, functional role for tau: mediating the effects of α-synuclein on postsynaptic signaling. Therefore, the unraveled tau-mediated signaling cascade may contribute to the pathogenesis of dementia in A53T α-synuclein-linked familial PD cases, as well as some subgroups of PD cases with extensive tau pathology.SIGNIFICANCE STATEMENT Here, we report mutation-specific postsynaptic deficits that are caused by A53T mutant α-synuclein, which is linked to familial Parkinson's disease (PD). The overexpression of WT, A53T, or A30P human α-synuclein leads to decreased spontaneous synaptic vesicle release. However, only those neurons expressing A53T α-synuclein exhibit tau phosphorylation-dependent postsynaptic dysfunction, which is characterized by decreased miniature postsynaptic current amplitude and decreased AMPA to NMDA receptor current ratio. The mutation-specific postsynaptic effects caused by human A53T α-synuclein will help us better understand the neurobiological basis of this specific form of familial PD. The differential effects of exogenous human WT, A53T, A30P, and E46K α-synuclein on glutamatergic synaptic responses will help to explain the clinical heterogeneity of sporadic and familial PD.

Accelerated accumulation of retinal α-synuclein (pSer129) and tau, neuroinflammation, and autophagic dysregulation in a seeded mouse model of Parkinson's disease

Parkinson's disease (PD) is a neurodegenerative disorder characterized by accumulation of misfolded α-synuclein within the central nervous system (CNS). Visual problems in PD patients are common, although retinal pathology associated with PD is not well understood. The purpose of this study was to investigate retinal pathology in a transgenic mouse model (TgM83) expressing the human A53T α-synuclein mutation and assess the effect of α-synuclein “seeding” on the development of retinal pathology. Two-month-old TgM83 mice were intracerebrally inoculated with brain homogenate from old (12–18 months) TgM83 mice. Retinas were then analyzed at 5 months of age. We analyzed retinas from 5-month-old and 8-month-old uninoculated healthy TgM83 mice, and old (12–18 months) mice that were euthanized following the development of clinical signs. Retinas of B6C3H mice (genetic background of the TgM83 mouse) served as control. We used immunohistochemistry and western blot analysis to detect accumulation of α-synuclein, pTauThr231, inflammation, changes in macroautophagy, and cell death. Raman spectroscopy was used to test the potential to differentiate between retinal tissues of healthy mice and diseased mice. This work demonstrates retinal changes associated with the A53T mutation. Retinas of non-inoculated TgM83 mice had accumulation of α-synuclein, “pre-tangle” tau, activation of retinal glial cells, and photoreceptor cell loss by 8 months of age. The development of these changes is accelerated by inoculation with brain homogenate from clinically ill TgM83 mice. Compared to non-inoculated 5-month-old TgM83 mice, retinas of inoculated 5-month-old mice had increased accumulation of α-synuclein (pSer129) and pTauThr231 proteins, upregulated microglial activation, and dysregulated macroautophagy. Raman spectroscopic analysis was able to discriminate between healthy and diseased mice. This study describes retinal pathology resulting from the A53T mutation. We show that seeding with brain homogenates from old TgM83 mice accelerates retinal pathology. We demonstrate that Raman spectroscopy can be used to accurately identify a diseased retina based on its biochemical profile, and that α-synuclein accumulation may contribute to accumulation of pTauThr231 proteins, neuroinflammation, metabolic dysregulation, and photoreceptor cell death. Our work provides insight into retinal changes associated with Parkinson's disease, and may contribute to a better understanding of visual symptoms experienced by patients.

Lipid metabolism alterations in the neuronal response to A53T α-synuclein and Fe-induced injury

Pathological α-synuclein (α-syn) overexpression and iron (Fe)-induced oxidative stress (OS) are involved in the death of dopaminergic neurons in Parkinson's disease (PD). We have previously characterized the role of triacylglycerol (TAG) formation in the neuronal response to Fe-induced OS. In this work we characterize the role of the α-syn variant A53T during Fe-induced injury and investigate whether lipid metabolism has implications for neuronal fate. To this end, we used the N27 dopaminergic neuronal cell line either untransfected (UT) or stably transfected with pcDNA3 vector (as a transfection control) or pcDNA-A53T-α-syn (A53T α-syn). The overexpression of A53T α-syn triggered an increase in TAG content mainly due to the activation of Acyl-CoA synthetase. Since fatty acid (FA) β-oxidation and phospholipid content did not change in A53T α-syn cells, the unique consequence of the increase in FA-CoA derivatives was their acylation in TAG moieties.Control cells exposed to Fe-induced injury displayed increased OS markers and TAG content. Intriguingly, Fe exposure in A53T α-syn cells promoted a decrease in OS markers accompanied by α-syn aggregation and elevated TAG content.We report here new evidence of a differential role played by A53T α-syn in neuronal lipid metabolism as related to the neuronal response to OS.

Block of A1 astrocyte conversion by microglia is neuroprotective in models of Parkinson's disease.

Activation of microglia by classical inflammatory mediators can convert astrocytes to a neurotoxic A1 phenotype in a variety of neurological diseases1,2. Development of agents that could inhibit the formation of A1 reactive astrocytes could be used to treat these diseases for which there are no disease modifying therapies. Glucagon-like peptide-1 receptor (GLP-1R) agonists have been touted as potential neuroprotective agents for neurologic disorders such as Alzheimer’s disease (AD) and Parkinson’s disease (PD)3-13. The mechanisms by which GLP-1R agonists are neuroprotective are not known. Here we show that a potent, brain penetrant long acting GLP-1R agonist NLY01 protects against the loss of dopamine neurons and behavioral deficits in the α-synuclein preformed fibril (α-syn PFF) model of sporadic PD14,15. NLY01 also prolongs the life and reduces the behavioral deficits and neuropathological abnormalities in the human A53T α-synuclein (hA53T) transgenic (Tg) model of α-synucleinopathy induced neurodegeneration16. We found that NLY01 is a potent GLP-1R agonist with favorable properties that is neuroprotective via the direct prevention of microglial mediated conversion of astrocytes to an A1 neurotoxic phenotype. In light of NLY01 favorable properties it should be evaluated in the treatment of PD and related neurologic disorders characterized by microglial activation.

Impaired Photic Entrainment of Spontaneous Locomotor Activity in Mice Overexpressing Human Mutant α-Synuclein.

Parkinson’s disease (PD) is characterized by distinct motor and non-motor symptoms. Sleep disorders are the most frequent and challenging non-motor symptoms in PD patients, and there is growing evidence that they are a consequence of disruptions within the circadian system. PD is characterized by a progressive degeneration of the dorsal vagal nucleus and midbrain dopaminergic neurons together with an imbalance of many other neurotransmitters. Mutations in α-synuclein (SNCA), a protein modulating SNARE complex-dependent neurotransmission, trigger dominantly inherited PD variants and sporadic cases of PD. The A53T SNCA missense mutation is associated with an autosomal dominant early-onset familial PD. To test whether this missense mutation affects the circadian system, we analyzed the spontaneous locomotor behavior of non-transgenic wildtype mice and transgenic mice overexpressing mutant human A53T α-synuclein (A53T). The mice were subjected to entrained- and free-running conditions as well as to experimental jet lag. Furthermore, the vesicular glutamate transporter 2 (VGLUT2) in the suprachiasmatic nucleus (SCN) was analyzed by immunohistochemistry. Free-running circadian rhythm and, thus, circadian rhythm generation, were not affected in A53T mice. A53T mice entrained to the light–dark cycle, however, with an advanced phase angle of 2.65 ± 0.5 h before lights off. Moreover, re-entrainment after experimental jet lag was impaired in A53T mice. Finally, VGLUT2 immunoreaction was reduced in the SCN of A53T mice. These data suggest an impaired light entrainment of the circadian system in A53T mice.

Phosphorylation of Parkin at serine 131 by p38 MAPK promotes mitochondrial dysfunction and neuronal death in mutant A53T \u03b1-synuclein model of Parkinson\u2019s disease

α-synuclein abnormal accumulation and mitochondria dysfunction are involved in the pathogenesis of Parkinson’s disease. Selective autophagy of mitochondria (mitophagy) is a crucial component of the network controlling the mitochondrial homeostasis. However, the underlying mechanism that mutant α-synuclein induces mitochondrial abnormality through mitophagy impairment is not fully understood. Here, we showed that mutant A53T α-synuclein accumulation impaired mitochondrial function and Parkin-mediated mitophgy in α-synucleinA53T model. α-synucleinA53T overexpression caused p38 MAPK activation, then p38 MAPK directly phosphorylated Parkin at serine 131 to disrupt the Parkin’s protective function. The p38 MAPK inhibition significantly reduced cellular apoptosis, restored mitochondrial membrane potential as well as increased synaptic density both in SN4741 cells and primary midbrain neurons. These findings show that the p38 MAPK-Parkin signaling pathway regulates mitochondrial homeostasis and neuronal degeneration, which may be a potential therapeutic strategy of PD via enhancing mitochondrial turn-over and maintenance.