Mutant Α-synuclein Research Articles

Antagonizing pathological α-synuclein-mediated neurodegeneration by J24335 via the activation of immunoproteasome

The aggregation of misfolded proteins, such as α-synuclein in Parkinson's disease (PD), occurs intracellularly or extracellularly in the majority of neurodegenerative diseases. The immunoproteasome has more potent chymotrypsin-like activity than normal proteasome. Thus, degradation of α-synuclein aggregation via immunoproteasome is an attractive approach for PD drug development. Herein, we aimed to determine if novel compound, 11-Hydroxy-1-(8-methoxy-5-(trifluoromethyl)quinolin-2-yl)undecan-1-one oxime (named as J24335), is a promising candidate for disease-modifying therapy to prevent the pathological progression of neurodegenerative diseases, such as PD. The effects of J24335 on inducible PC12/A53T-α-syn cell viability and cytotoxicity were evaluated by MTT assay and LDH assay, respectively. Evaluation of various proteasome activities was done by measuring the luminescence of enzymatic activity after the addition of different amounts of aminoluciferin. Immunoblotting and real-time PCR were employed to detect the expression of various proteins and genes, respectively. We also used a transgenic mouse model for behavioral testing and immunochemical analysis, to assess the neuroprotective effects of J24335. J24335 inhibited wild-type and mutant α-synuclein aggregation without affecting the growth or death of neuronal cells. The inhibition of α-synuclein aggregation by J24335 was caused by activation of immunoproteasome, as mediated by upregulation of LMP7, and increased cellular chymotrypsin-like activity in 20S proteasome. J24335-enhanced immunoproteasome activity was mediated by PKA/Akt/mTOR pathway activation. Moreover, animal studies revealed that J24335 treatment markedly mitigated both the loss of tyrosine hydroxylase-positive (TH-) neurons and impaired motor skill development. This is the first report to use J24335 as an immunoproteasome enhancing agent to antagonize pathological α-synuclein-mediated neurodegeneration.

Dynamic reversibility of α-synuclein serine-129 phosphorylation is impaired in synucleinopathy models.

α-Synuclein phosphorylation at serine-129 (pS129) is a widely used surrogate marker of pathology in Parkinson's disease and other synucleinopathies. However, we recently demonstrated that phosphorylation of S129 is also a physiological activator of synaptic transmission. In a feed-forward fashion, neuronal activity triggers reversible pS129. Here, we show that Parkinson's disease-linked missense mutations in SNCA impact activity-dependent pS129. Under basal conditions, cytosol-enriched A30P, H50Q, and G51D mutant forms of α-synuclein exhibit reduced pS129 levels in rat primary cortical neurons. A53T pS129 levels are similar to wild-type, and E46K pS129 levels are higher. A30P and E46K mutants show impaired reversibility of pS129 after stimulation. For the engineered profoundly membrane-associated α-synuclein mutant "3K" (E35K + E46K + E61K), de-phosphorylation was virtually absent after blocking stimulation, implying that reversible pS129 is severely compromised. Importantly, pS129 excess resulting from proteasome inhibition is also associated with reduced reversibility by neuronal inhibition, kinase inhibition, or phosphatase activation. Our findings suggest that perturbed pS129 dynamics are probably a shared characteristic of pathology-associated α-synuclein, with possible implications for synucleinopathy treatment and diagnosis.

Transcriptional mutagenesis of α-synuclein caused by DNA oxidation in Parkinson’s disease pathogenesis

Oxidative stress plays an essential role in the development of Parkinson’s disease (PD). 8-oxo-7,8-dihydroguanine (8-oxodG, oxidized guanine) is the most abundant oxidative stress-mediated DNA lesion. However, its contributing role in underlying PD pathogenesis remains unknown. In this study, we hypothesized that 8-oxodG can generate novel α-synuclein (α-SYN) mutants with altered pathologic aggregation through a phenomenon called transcriptional mutagenesis (TM). We observed a significantly higher accumulation of 8-oxodG in the midbrain genomic DNA from PD patients compared to age-matched controls, both globally and region specifically to α-SYN. In-silico analysis predicted that forty-three amino acid positions can contribute to TM-derived α-SYN mutation. Here, we report a significantly higher load of TM-derived α-SYN mutants from the midbrain of PD patients compared to controls using a sensitive PCR-based technique. We found a novel Serine42Tyrosine (S42Y) α-SYN as the most frequently detected TM mutant, which incidentally had the highest predicted aggregation score amongst all TM variants. Immunohistochemistry of midbrain sections from PD patients using a newly characterized antibody for S42Y identified S42Y-laden Lewy bodies (LB). We further demonstrated that the S42Y TM variant significantly accelerates WT α-SYN aggregation by cell and recombinant protein-based assays. Cryo-electron tomography revealed that S42Y exhibits considerable conformational heterogeneity compared to WT fibrils. Moreover, S42Y exhibited higher neurotoxicity compared to WT α-SYN as shown in mouse primary cortical cultures and AAV-mediated overexpression in the substantia nigra of C57BL/6 J mice. To our knowledge, this is the first report describing the possible contribution of TM-generated mutations of α-SYN to LB formation and PD pathogenesis.

Low-dose Cu exposure enhanced α-synuclein accumulation associates with mitochondrial impairments in mice model of Parkinson's disease

Parkinson's disease (PD) is a neurodegenerative disorder that mainly affects the elder population, and its etiology is enigmatic. Both environmental risks and genetics may influence the development of PD. Excess copper causes neurotoxicity and accelerates the progression of neurodegenerative diseases. However, the underlying mechanisms of copper-induced neurotoxicity remain controversial. In this study, A53T transgenic α-synuclein (A53T) mice and their matching wild-type (WT) mice were treated with a low dose of copper (0.13 ppm copper chlorinated drinking water, equivalent to the copper exposure of human daily copper intake dose) for 4 months, and copper poisoning was performed on human A53T mutant SHSY5Y cells overexpressed with α-synuclein (dose of 1/4 IC50), to test the effects of copper exposure on the body. The results of the open field test showed that the moto function of Cu-treated mice was impaired. Proteomics revealed changes in neurodevelopment, transport function, and mitochondrial membrane-related function in Cu-treated WT mice, which were associated with reduced expression of mitochondrial complex (NDUFA10, ATP5A), dopamine neurons (TH), and dopamine transporter (DAT). Mitochondrial function, nervous system development, synaptic function, and immune response were altered in Cu-treated A53T mice. These changes were associated with increased mitochondrial splitting protein (Drp1), decreased mitochondrial fusion protein (OPA1, Mfn1), abnormalities in mitochondrial autophagy protein (LC3BII/I, P62), decreased dopamine neuron (TH) expression, increased α-synuclein expression, inflammatory factors (IL-6, IL-1β, and TNF-α) release and microglia (Iba1) activation. In addition, we found that Cu2+ (30 μM) induced excessive ROS production and reduced mitochondrial ATP production in human A53T mutant α-synuclein overexpressing SHSY5Y cells by in vitro experiments. In conclusion, low-dose copper treatment altered critical proteins involved in mitochondrial, neurodevelopmental, and inflammatory responses and affected mitochondria's ROS and ATP production levels.

Membrane phospholipid peroxidation promotes loss of dopaminergic neurons in psychological stress-induced Parkinson's disease susceptibility.

Parkinson's disease (PD) is a neurodegenerative disorder associated with α-synuclein aggregation and dopaminergic neuron loss in the midbrain. There is evidence that psychological stress promotes PD progression by enhancing glucocorticoids-related oxidative damage, however, the mechanisms involved are unknown. The present study demonstrated that plasma membrane phospholipid peroxides, as determined by phospholipidomics, triggered ferroptosis in dopaminergic neurons, which in turn contributed to stress exacerbated PD-like motor disorder in mice overexpressing mutant human α-synuclein. Using hormonomics, we identified that stress stimulated corticosteroid release and promoted 15-lipoxygenase-1 (ALOX15)-mediated phospholipid peroxidation. ALOX15 was upregulated by α-synuclein overexpression and acted as a fundamental risk factor in the development of chronic stress-induced parkinsonism and neurodegeneration. Further, we demonstrated the mechanism by which corticosteroids activated the PKC pathway and induced phosphatidylethanolamine-binding protein-1 (PEBP1) to form a complex with ALOX15, thereby facilitating ALOX15 to locate on the plasma membrane phospholipids. A natural product isolated from herbs, leonurine, was screened with activities of inhibiting the ALOX15/PEBP1 interaction and thereby attenuating membrane phospholipid peroxidation. Collectively, our findings demonstrate that stress increases the susceptibility of PD by driving membrane lipid peroxidation of dopaminergic neurons and suggest the ALOX15/PEBP1 complex as a potential intervention target.

Calcineurin stimulation by Cnb1p overproduction mitigates protein aggregation and α-synuclein toxicity in a yeast model of synucleinopathy

The calcium-responsive phosphatase, calcineurin, senses changes in Ca2+ concentrations in a calmodulin-dependent manner. Here we report that under non-stress conditions, inactivation of calcineurin signaling or deleting the calcineurin-dependent transcription factor CRZ1 triggered the formation of chaperone Hsp100p (Hsp104p)-associated protein aggregates in Saccharomyces cerevisiae. Furthermore, calcineurin inactivation aggravated α-Synuclein-related cytotoxicity. Conversely, elevated production of the calcineurin activator, Cnb1p, suppressed protein aggregation and cytotoxicity associated with the familial Parkinson’s disease-related mutant α-Synuclein A53T in a partly CRZ1-dependent manner. Activation of calcineurin boosted normal localization of both wild type and mutant α-synuclein to the plasma membrane, an intervention previously shown to mitigate α-synuclein toxicity in Parkinson’s disease models. The findings demonstrate that calcineurin signaling, and Ca2+ influx to the vacuole, limit protein quality control in non-stressed cells and may have implications for elucidating to which extent aberrant calcineurin signaling contributes to the progression of Parkinson’s disease(s) and other synucleinopathies.55zLzriqK_eYGg5W2i5yhiVideo

Mutant α-synuclein propagates via the lymphatic system of the brain in the monomeric state

Prion-like protein propagation is considered a common pathogenic mechanism in neurodegenerative diseases. Here we investigate the invivo propagation pattern and aggregation state of mutant α-synuclein by injecting adeno-associated viral (AAV)-α-synuclein-A53T-EGFP into the mouse olfactory cortex. Comparison of aggregation states in various brain regions at multiple time points after injection using western blot analyses shows that the monomeric state of the mutant/misfolded protein propagates to remote brain regions by 2weeks and that the propagated proteins aggregate in situ after being incorporated into neurons. Moreover, injection of Alexa 488-labeled α-synuclein-A53T confirms the monomeric propagation at 2weeks. Super-resolution microscopy shows that both α-synuclein-A53T proteins propagate via the lymphatic system, penetrate perineuronal nets, and reach the surface of neurons. Electron microscopy shows that the propagated mutant/misfolded monomer forms fibrils characteristic of Parkinson's disease after its incorporation into neurons. These findings suggest a mode of propagation different from that of aggregate-dependent propagation.

Navigating the cellular pathways: Chaperone-mediated autophagy as a targeted approach for management of parkinson\\\'s disease

Parkinson's disease (PD) is a common neurodegenerative condition marked by the degeneration of dopaminergic neurons and the amassing of α-synuclein protein in Lewy bodies. Chaperone-mediated autophagy (CMA), a selective form of autophagy, has been implicated in the development of PD. Mutant GBA1, α-synuclein, UCHL1, VPS35, and LRRK2 are affected proteins in PD that impair the CMA process. CMA Dysfunction cause accumulation of PD-associated proteins such as α-synuclein and many other, including DJ-1, MEF2D, PARK7,etc resulting in mitochondrial dysfunctioning and apoptosis. The impact of gene mutations associated with PD on CMA has been observed, along with dysregulation of miRNAs targeting CMA components. Toxicant-induced PD models demonstrate that impaired CMA increases α-synuclein aggregates and neurotoxicity. Understanding the molecular mechanisms of CMA has identified potential therapeutic targets, including increasing LAMP2A levels. Several compounds and substances have shown promise in enhancing CMA and reducing α- synuclein aggregates, such as 6-aminonicotinamide, geldanamycin, metformin, and natural compounds like trehalose and caffeine. Pharmacological modulation of CMA, such as through retinoic acid derivatives, has demonstrated positive effects on reducing protein aggregates in neurodegenerative diseases. However, the specific effects of inhibiting CMA on macroautophagy remain uncertain. Overcoming challenges in studying CMA, such as developing suitable models and monitoring methods, is crucial for advancing our understanding of CMA's role in neurodegenerative diseases and developing effective therapeutic strategies. Overall, CMA emerges as a key player in the pathogenesis of PD, and targeting this selective autophagy pathway holds promise for developing novel therapies to combat neurodegenerative disorders.

Ashwagandha- Withania somnifera (L.) Dunal as a multipotent neuroprotective remedy for genetically induced motor dysfunction and cellular toxicity in human neurodegenerative disease models of Drosophila

Ethnopharmacological relevanceAshwagandha-Withania somnifera (L.) Dunal, well known for its multipotent therapeutic properties has been used in Ayurveda for 3000 years. The plant with more than 50 active phytoconstituents is recognised for its anti-cancerous, anti-diabetic, anti-inflammatory, anti-microbial, and neurotherapeutic properties demonstrated in in vitro studies and chemically induced rodent models. Genetically targeted Parkinson’s, Alzheimer’s and other neurodegenerative disease models have been created in Drosophila and have been used to get mechanistic insight into the in vivo cellular events, and genetic pathways that underlie respective neurodegenerative condition. But hitherto, there aren’t enough attempts made to capitalize the genetic potential of these disease models to validate the therapeutic efficacy of different reagents used in traditional medicine, in the context of specific disease-causing genetic mutations. Aim of the studyDrugs discovered using in vitro platforms might fail in several instances of clinical trials because of the genetic heterogeneity and variability in the physiological context found among the patients. Drosophila by virtue of its genetically regulated experimental potential forms an ideal in vivo model to validate the candidate reagents discovered in in vitro screens for their efficacy under specific genetic situations. Here we have used genetically induced α-synucleinopathy and tauopathy transgenic fly models to study the efficacy of Ashwagandha treatment, assessing cellular and behavioural parameters. MethodsWe have expressed the disease-causing human gene mutations in specific cell types of Drosophila using GAL4/UAS targeted expression system to create disease models. Human α-synuclein mutant (A30P) was expressed in dopaminergic neurons using Ddc-GAL4 driver strain to induce dopaminergic neurodegeneration and assayed for motor dysfunction. Human TauE14, mutant protein was expressed in photoreceptor neurons using GMR-GAL4 driver to induce photoreceptor degeneration. Microtubular destability and mitotic arrest in the dividing photoreceptor precursor cells were studied using αPH3 antibody. Lysosomal dysregulation caused necrotic black spots were induced by TauE14 with GMR-GAL4 driver, in a white mutant background. These flies mimicking neurodegenerative conditions were supplemented with different concentrations of Ashwagandha aqueous root extract mixed with regular fly food. The treated flies were analysed for cellular and behaviour parameters. ResultsLifespan assay shows that, Ashwagandha-root extract imparts an extended lifespan in male Drosophila flies which are intrinsically less stress resistant. Motor dysfunction caused due to human α-synuclein mutant protein expressed in dopaminergic neurons is greatly brought down. Further, Ashwagandha extract treatment significantly reduces TauE14 induced microtubular destability, mitotic arrest and neuronal death in photoreceptor neurons. Our experiment with tauopathy model in white mutant background exemplify that, Ashwagandha-root extract treatment can bring down lysosomal dysregulation induced necrosis of photoreceptor neurons. ConclusionWe have carried out a multifaceted study which elucidates that Ashwagandha can serve as a comprehensive, phytotherapeutic formulation to combat neurodegeneration, targeting multiple causative genetically defective conditions.

Potent New Targets for Autophagy Enhancement to Delay Neuronal Ageing.

Autophagy is a lysosomal-dependent degradation process of eukaryotic cells responsible for breaking down unnecessary and damaged intracellular components. Autophagic activity gradually declines with age due to genetic control, and this change contributes to the accumulation of cellular damage at advanced ages, thereby causing cells to lose their functionality and viability. This could be particularly problematic in post-mitotic cells including neurons, the mass destruction of which leads to various neurodegenerative diseases. Here, we aim to uncover new regulatory points where autophagy could be specifically activated and test these potential drug targets in neurodegenerative disease models of Drosophila melanogaster. One possible way to activate autophagy is by enhancing autophagosome-lysosome fusion that creates the autolysosome in which the enzymatic degradation happens. The HOPS (homotypic fusion and protein sorting) and SNARE (Snap receptor) protein complexes regulate the fusion process. The HOPS complex forms a bridge between the lysosome and autophagosome with the assistance of small GTPase proteins. Thus, small GTPases are essential for autolysosome maturation, and among these proteins, Rab2 (Ras-associated binding 2), Rab7, and Arl8 (Arf-like 8) are required to degrade the autophagic cargo. For our experiments, we used Drosophila melanogaster as a model organism. Nerve-specific small GTPases were silenced and overexpressed. We examined the effects of these genetic interventions on lifespan, climbing ability, and autophagy. Finally, we also studied the activation of small GTPases in a Parkinson's disease model. Our results revealed that GTP-locked, constitutively active Rab2 (Rab2-CA) and Arl8 (Arl8-CA) expression reduces the levels of the autophagic substrate p62/Ref(2)P in neurons, extends lifespan, and improves the climbing ability of animals during ageing. However, Rab7-CA expression dramatically shortens lifespan and inhibits autophagy. Rab2-CA expression also increases lifespan in a Parkinson's disease model fly strain overexpressing human mutant (A53T) α-synuclein protein. Data provided by this study suggests that Rab2 and Arl8 serve as potential targets for autophagy enhancement in the Drosophila nervous system. In the future, it might be interesting to assess the effect of Rab2 and Arl8 coactivation on autophagy, and it would also be worthwhile to validate these findings in a mammalian model and human cell lines. Molecules that specifically inhibit Rab2 or Arl8 serve as potent drug candidates to modulate the activity of the autophagic process in treating neurodegenerative pathologies. In the future, it would be reasonable to investigate which GAP enzyme can inhibit Rab2 or Arl8 specifically, but not affect Rab7, with similar medical purposes.

ARL6IP5 Ameliorates α-Synuclein Burden by Inducing Autophagy via Preventing Ubiquitination and Degradation of ATG12.

Recent advanced studies in neurodegenerative diseases have revealed several links connecting autophagy and neurodegeneration. Autophagy is the major cellular degradation process for the removal of toxic protein aggregates responsible for neurodegenerative diseases. More than 30 autophagy-related proteins have been identified as directly participating in the autophagy process. Proteins regulating the process of autophagy are much more numerous and unknown. To address this, in our present study, we identified a novel regulator (ARL6IP5) of neuronal autophagy and showed that the level of ARL6IP5 decreases in the brain with age and in Parkinson's disease in mice and humans. Moreover, a cellular model of PD (Wild type and A53T mutant α-synuclein overexpression) has also shown decreased levels of ARL6IP5. ARL6IP5 overexpression reduces α-synuclein aggregate burden and improves cell survival in an A53T model of Parkinson's disease. Interestingly, detailed mechanistic studies revealed that ARL6IP5 is an autophagy inducer. ARL6IP5 enhances Rab1-dependent autophagosome initiation and elongation by stabilizing free ATG12. We report for the first time that α-synuclein downregulates ARL6IP5 to inhibit autophagy-dependent clearance of toxic aggregates that exacerbate neurodegeneration.

Presence of Extracellular Alpha-Synuclein Aggregates Trigger Astrocytic Degeneration Through Enhanced Membrane Rigidity and Deregulation of Store-Operated Calcium Entry (SOCE) into the Endoplasmic Reticulum.

α-Synuclein has a critical role in Parkinson's disease, but the mechanism of how extracellular α-synuclein aggregates lead to astrocytic degeneration remains unknown. Our recent study in astrocytes highlighted that α-synuclein aggregates undergo lower endocytosis than the monomeric-form, even while displaying a higher impact on glutathione-machinery and glutamate-metabolism under sublethal conditions. As optimal intracellular calcium levels are essential for these functions, we aimed to study the effect of extracellular α-synuclein aggregates on ER calcium entry. We assessed the association of extracellular aggregated-α-synuclein (WT and A30P/A53T double-mutant) with the astrocytic membrane (lipid rafts) and studied its effects on membrane fluidity, ER stress, and ER calcium refilling in three systems-purified rat primary midbrain astrocyte culture, human iPSC-derived astrocytes, and U87 cells. The corresponding timeline effect on mitochondrial membrane potential was also evaluated. Post-24h exposure to extracellular WT and mutant α-synuclein aggregates, fluorescence-based studies showed a significant increase in astrocyte membrane rigidity over control, with membrane association being significantly higher for the double mutant aggregates. α-Synuclein aggregates also showed preferentially higher association with lipid rafts of astrocytic membrane. A simultaneous increase in ER stress markers (phosphorylated PERK and CHOP) with significantly higher SOCE was also observed in aggregate-treated astrocytes, with higher levels for double mutant variant. These observations correlate with increased expression of SOCE markers, especially Orai3, on plasma membrane. Alterations in mitochondrial membrane potential were only noted post-48h of exposure to α-synuclein aggregates. We therefore suggest that in astrocytes, α-synuclein-aggregates preferentially associate with lipid rafts of membrane, altering membrane fluidity and consequently inducing ER stress mediated by interaction with membrane SOCE proteins, resulting in higher Ca2+ entry. A distinct cascade of events of sequential impairment of ER followed by mitochondrial alteration is observed. The study provides novel evidence elucidating relationships between extracellular α-synuclein aggregates and organellar stress in astrocytes and indicates the therapeutic potential in targeting the association of α-synuclein aggregates with astrocytic membrane.

Postsynaptic synucleins mediate endocannabinoid signaling

Endocannabinoids are among the most powerful modulators of synaptic transmission throughout the nervous system, and yet little is understood about the release of endocannabinoids from postsynaptic compartments. Here we report an unexpected finding that endocannabinoid release requires synucleins, key contributors to Parkinson’s disease. We show that endocannabinoids are released postsynaptically by a synuclein-dependent and SNARE-dependent mechanism. Specifically, we found that synuclein deletion blocks endocannabinoid-dependent synaptic plasticity; this block is reversed by postsynaptic expression of wild-type but not of mutant α-synuclein. Whole-cell recordings and direct optical monitoring of endocannabinoid signaling suggest that the synuclein deletion specifically blocks endocannabinoid release. Given the presynaptic role of synucleins in regulating vesicle lifecycle, we hypothesize that endocannabinoids are released via a membrane interaction mechanism. Consistent with this hypothesis, postsynaptic expression of tetanus toxin light chain, which cleaves synaptobrevin SNAREs, also blocks endocannabinoid-dependent signaling. The unexpected finding that endocannabinoids are released via a synuclein-dependent mechanism is consistent with a general function of synucleins in membrane trafficking and adds a piece to the longstanding puzzle of how neurons release endocannabinoids to induce synaptic plasticity.

Decreased levels of alpha synuclein in families with autism spectrum disorder and relationship between the disease severity

Autism spectrum disorder (ASD) is a heterogeneous neurodevelopmental disorders that begin in early childhood. Mutations in α-synuclein (SNCA) gene have been shown to result in the accumulation of α-synuclein, which occurs in many neurodegenerative diseases. Our aim was to determine the changes in the expression profile and protein level of this gene by comparing the autistic children with their healthy siblings, their mothers and healthy controls in order to elucidate the possible contribution of the SNCA gene to the etiology of ASD. 50 autistic patients, their mothers, siblings and 25 healthy controls and their mothers were enrolled to determine SNCA gene expression and serum α-synuclein levels. It was determined that α-synuclein serum levels decreased in the autistic patients. Similarly, it was found that SNCA gene expression and serum α-synuclein levels were significantly decreased in the mothers of the patients. Significant negative correlation was observed between the SNCA gene and protein expression amounts in the 6–8 age of the patients. This family-based study is the first in the literature, with both gene expression and serum levels of α-synuclein. The relationship between ASD severity and α-synuclein level needs to be confirmed in larger-scale studies.

In vivo18F-DOPA PET imaging identifies a dopaminergic deficit in a rat model with a G51D α-synuclein mutation.

Parkinson's disease (PD) is a neurodegenerative condition with several major hallmarks, including loss of substantia nigra neurons, reduction in striatal dopaminergic function, and formation of α-synuclein-rich Lewy bodies. Mutations in SNCA, encoding for α-synuclein, are a known cause of familial PD, and the G51D mutation causes a particularly aggressive form of the condition. CRISPR/Cas9 technology was used to introduce the G51D mutation into the endogenous rat SNCA gene. SNCAG51D/+ and SNCAG51D/G51D rats were born in Mendelian ratios and did not exhibit any severe behavourial defects. L-3,4-dihydroxy-6-18F-fluorophenylalanine (18F-DOPA) positron emission tomography (PET) imaging was used to investigate this novel rat model. Wild-type (WT), SNCAG51D/+ and SNCAG51D/G51D rats were characterized over the course of ageing (5, 11, and 16 months old) using 18F-DOPA PET imaging and kinetic modelling. We measured the influx rate constant (Ki) and effective distribution volume ratio (EDVR) of 18F-DOPA in the striatum relative to the cerebellum in WT, SNCAG51D/+ and SNCAG51D/G51D rats. A significant reduction in EDVR was observed in SNCAG51D/G51D rats at 16 months of age indicative of increased dopamine turnover. Furthermore, we observed a significant asymmetry in EDVR between the left and right striatum in aged SNCAG51D/G51D rats. The increased and asymmetric dopamine turnover observed in the striatum of aged SNCAG51D/G51D rats reflects one aspect of prodromal PD, and suggests the presence of compensatory mechanisms. SNCAG51D rats represent a novel genetic model of PD, and kinetic modelling of 18F-DOPA PET data has identified a highly relevant early disease phenotype.

Inhibition of p38α MAPK restores neuronal p38γ MAPK and ameliorates synaptic degeneration in a mouse model of DLB/PD.

Alterations in the p38 mitogen-activated protein kinases (MAPKs) play an important role in the pathogenesis of dementia with Lewy bodies (DLB) and Parkinson's disease (PD). Activation of the p38α MAPK isoform and mislocalization of the p38γ MAPK isoform are associated with neuroinflammation and synaptic degeneration in DLB and PD. Therefore, we hypothesized that p38α might be associated with neuronal p38γ distribution and synaptic dysfunction in these diseases. To test this hypothesis, we treated in vitro cellular and in vivo mouse models of DLB/PD with SKF-86002, a compound that attenuates inflammation by inhibiting p38α/β, and then investigated the effects of this compound on p38γ and neurodegenerative pathology. We found that inhibition of p38α reduced neuroinflammation and ameliorated synaptic, neurodegenerative, and motor behavioral deficits in transgenic mice overexpressing human α-synuclein. Moreover, treatment with SKF-86002 promoted the redistribution of p38γ to synapses and reduced the accumulation of α-synuclein in mice overexpressing human α-synuclein. Supporting the potential value of targeting p38 in DLB/PD, we found that SKF-86002 promoted the redistribution of p38γ in neurons differentiated from iPS cells derived from patients with familial PD (carrying the A53T α-synuclein mutation) and healthy controls. Treatment with SKF-86002 ameliorated α-synuclein-induced neurodegeneration in these neurons only when microglia were pretreated with this compound. However, direct treatment of neurons with SKF-86002 did not affect α-synuclein-induced neurotoxicity, suggesting that SKF-86002 treatment inhibits α-synuclein-induced neurotoxicity mediated by microglia. These findings provide a mechanistic connection between p38α and p38γ as well as a rationale for targeting this pathway in DLB/PD.

Sporadic SNCA mutations A18T and A29S exhibit variable effects on protein aggregation, cell viability and oxidative stress.

α-synuclein aggregation is the hallmark feature of Parkinson's disease. Both familial and sporadic forms of the disease exhibit this feature. Several mutations have been identified in patients and are associated with the disease pathology. We have used site-directed mutagenesis to generate α-synuclein mutant variants tagged with GFP. Fluorescence microscopy, flow cytometry, western blotting, cell viability and oxidative stress analysis were performed to investigate the effect of two less studied α-synuclein variants. In this study we characterized two less studied α-synuclein mutations, A18T and A29S, in the well-established yeast model. Our data shows variable expression, distribution and toxicity of the protein in the mutant variants A18T, A29S, A53T and WT. The cells expressing the double mutant variant A18T/A53T showed the most increase in the aggregation phenotype and also depicted reduced viability suggesting a more substantial effect of this variant. The outcome of our study highlights the variable localization, aggregation phenotype and toxicity of the studied α-synuclein variants. This underscores the importance of in-depth analysis of every disease-associated mutation which may result in variable cellular phenotype.

Mutations in α-synuclein, TDP-43 and tau prolong protein half-life through diminished degradation by lysosomal proteases

BackgroundAutosomal dominant mutations in α-synuclein, TDP-43 and tau are thought to predispose to neurodegeneration by enhancing protein aggregation. While a subset of α-synuclein, TDP-43 and tau mutations has been shown to increase the structural propensity of these proteins toward self-association, rates of aggregation are also highly dependent on protein steady state concentrations, which are in large part regulated by their rates of lysosomal degradation. Previous studies have shown that lysosomal proteases operate precisely and not indiscriminately, cleaving their substrates at very specific linear amino acid sequences. With this knowledge, we hypothesized that certain coding mutations in α-synuclein, TDP-43 and tau may lead to increased protein steady state concentrations and eventual aggregation by an alternative mechanism, that is, through disrupting lysosomal protease cleavage recognition motifs and subsequently conferring protease resistance to these proteins.ResultsTo test this possibility, we first generated comprehensive proteolysis maps containing all of the potential lysosomal protease cleavage sites for α-synuclein, TDP-43 and tau. In silico analyses of these maps indicated that certain mutations would diminish cathepsin cleavage, a prediction we confirmed utilizing in vitro protease assays. We then validated these findings in cell models and induced neurons, demonstrating that mutant forms of α-synuclein, TDP-43 and tau are degraded less efficiently than wild type despite being imported into lysosomes at similar rates.ConclusionsTogether, this study provides evidence that pathogenic mutations in the N-terminal domain of α-synuclein (G51D, A53T), low complexity domain of TDP-43 (A315T, Q331K, M337V) and R1 and R2 domains of tau (K257T, N279K, S305N) directly impair their own lysosomal degradation, altering protein homeostasis and increasing cellular protein concentrations by extending the degradation half-lives of these proteins. These results also point to novel, shared, alternative mechanism by which different forms of neurodegeneration, including synucleinopathies, TDP-43 proteinopathies and tauopathies, may arise. Importantly, they also provide a roadmap for how the upregulation of particular lysosomal proteases could be targeted as potential therapeutics for human neurodegenerative disease.

Disrupting the α-synuclein-ESCRT interaction with a peptide inhibitor mitigates neurodegeneration in preclinical models of Parkinson’s disease

Accumulation of α-synuclein into toxic oligomers or fibrils is implicated in dopaminergic neurodegeneration in Parkinson’s disease. Here we performed a high-throughput, proteome-wide peptide screen to identify protein-protein interaction inhibitors that reduce α-synuclein oligomer levels and their associated cytotoxicity. We find that the most potent peptide inhibitor disrupts the direct interaction between the C-terminal region of α-synuclein and CHarged Multivesicular body Protein 2B (CHMP2B), a component of the Endosomal Sorting Complex Required for Transport-III (ESCRT-III). We show that α-synuclein impedes endolysosomal activity via this interaction, thereby inhibiting its own degradation. Conversely, the peptide inhibitor restores endolysosomal function and thereby decreases α-synuclein levels in multiple models, including female and male human cells harboring disease-causing α-synuclein mutations. Furthermore, the peptide inhibitor protects dopaminergic neurons from α-synuclein-mediated degeneration in hermaphroditic C. elegans and preclinical Parkinson’s disease models using female rats. Thus, the α-synuclein-CHMP2B interaction is a potential therapeutic target for neurodegenerative disorders.

Calcium/calmodulin-dependent serine protein kinase exacerbates mitochondrial calcium uniporter-related mitochondrial calcium overload by phosphorylating α-synuclein in Parkinson's disease.

α-Synuclein phosphorylation and mitochondrial calcium homeostasis are important mechanisms underlying mitochondrial dysfunction in Parkinson's disease, but the network regulating these mechanisms remains unclear. We identified the role of key phosphokinases and the pathological effects of α-synuclein phosphorylation on mitochondrial calcium influx and mitochondrial function in Parkinson's disease. The function of the key phosphokinase, calcium/calmodulin-dependent serine protein kinase, was investigated through loss- and gain-of-function experiments using a cell model of Parkinson's disease. The regulation of mitochondrial calcium uniporter-mediated mitochondrial calcium influx by calcium/calmodulin-dependent serine protein kinase was explored using a cellular model of Parkinson's disease. Coimmunoprecipitation experiments and α-synuclein mutation were used to explore the mechanism through which calcium/calmodulin-dependent serine protein kinase regulates mitochondrial calcium uniporter-mediated mitochondrial calcium influx and exacerbates mitochondrial damage in Parkinson's disease. Here, we show the pathogenic role of calcium/calmodulin-dependent serine protein kinase in Parkinson's disease progression. Calcium/calmodulin-dependent serine protein kinase phosphorylated α-synuclein to activate mitochondrial calcium uniporter and thus increase mitochondrial calcium influx, and these effects were blocked by α-synuclein S129A mutant expression. Furthermore, the calcium/calmodulin-dependent serine protein kinase inhibitor CASK-IN-1 exerted neuroprotective effects in Parkinson's disease. Collectively, our results suggest that calcium/calmodulin-dependent serine protein kinase phosphorylates α-synuclein to activate the mitochondrial calcium uniporter and thereby causes mitochondrial calcium overload and mitochondrial damage in Parkinson's disease. We elucidated a new role of calcium/calmodulin-dependent serine protein kinase in Parkinson's disease and revealed the potential therapeutic value of targeting calcium/calmodulin-dependent serine protein kinase in Parkinson's disease treatment.