Overexpression Of Human Α-synuclein Research Articles

Human α-synuclein overexpression upregulates SKOR1 in a rat model of simulated nigrostriatal ageing.

Parkinson's disease (PD) is characterised by progressive loss of dopaminergic (DA) neurons from the substantia nigra (SN) and α-synuclein (αSyn) accumulation. Age is the biggest risk factor for PD and may create a vulnerable pre-parkinsonian state, but the drivers of this association are unclear. It is known that ageing increases αSyn expression in DA neurons and that this may alter molecular processes that are central to maintaining nigrostriatal integrity. To model this, adult female Sprague-Dawley rats received a unilateral intranigral injection of adeno-associated viral (AAV) vector carrying wild-type human αSyn (AAV-αSyn) or control vector (AAV-Null). AAV-αSyn induced no detrimental effects on motor behaviour, but there was expression of human wild-type αSyn throughout the midbrain and ipsilateral striatum at 20 weeks post-surgery. Microarray analysis revealed that the gene most-upregulated in the ipsilateral SN of the AAV-αSyn group was the SKI Family Transcriptional Corepressor 1 (SKOR1). Bioenergetic state analysis of mitochondrial function found that SKOR1 overexpression reduced the maximum rate of cellular respiration in SH-SY5Y cells. Furthermore, experiments in SH-SY5Y cells revealed that SKOR1 overexpression impaired neurite growth to the same extent as αSyn, and inhibited BMP-SMAD-dependent transcription, a pathway that promotes DA neuronal survival and growth. Given the normal influence of ageing on DA neuron loss in human SN, the extent of αSyn-induced SKOR1 expression may influence whether an individual undergoes normal nigrostriatal ageing or reaches a threshold for prodromal PD. This provides new insight into mechanisms through which ageing-related increases in αSyn may influence molecular mechanisms important for the maintenance of neuronal integrity.

Striatal dopamine neurotransmission is altered in age- and region-specific manner in a Parkinson’s disease transgenic mouse

Dopamine (DA) plays a critical role in striatal motor control. The drop in DA level within the dorsal striatum is directly associated with the appearance of motor symptoms in Parkinson’s disease (PD). The progression of the disease and inherent disruption of the DA neurotransmission has been closely related to accumulation of the synaptic protein α-synuclein. However, it is still unclear how α-synuclein affects dopaminergic terminals in different areas of dorsal striatum. Here we demonstrate that the overexpression of human α-synuclein (h-α-syn) interferes with the striatal DA neurotransmission in an age‐dependent manner, preferentially in the dorsolateral striatum (DLS) of PDGF-h-α-syn mice. While 3-month-old mice showed an increase at the onset of h-α-syn accumulation in the DLS, 12-month-old mice revealed a decrease in electrically-evoked DA release. The enhanced DA release in 3-month-old mice coincided with better performance in a behavioural task. Notably, DA amplitude alterations were also accompanied by a delay in the DA clearance independently from the animal age. Structurally, dopamine transporter (DAT) was found to be redistributed in larger DAT-positive clumps only in the DLS of 3- and 12-month-old mice. Together, our data provide new insight into the vulnerability of DLS and suggest DAT-related dysfunctionalities from the very early stages of h-α-syn accumulation.

Hippocampal synaptic failure is an early event in experimental parkinsonism with subtle cognitive deficit.

Learning and memory mainly rely on correct synaptic function in the hippocampus and other brain regions. In Parkinson's disease, subtle cognitive deficits may even precede motor signs early in the disease. Hence, we set out to unravel the earliest hippocampal synaptic alterations associated with human α-synuclein overexpression prior to and soon after the appearance of cognitive deficits in a parkinsonism model. We bilaterally injected adeno-associated viral vectors encoding A53T-mutated human α-synuclein into the substantia nigra of rats, and evaluated them 1, 2, 4 and 16 weeks post-inoculation by immunohistochemistry and immunofluorescence to study degeneration and distribution of α-synuclein in the midbrain and hippocampus. The object location test was used to evaluate hippocampal-dependent memory. Sequential window acquisition of all theoretical mass spectrometry-based proteomics and fluorescence analysis of single-synapse long-term potentiation were used to study alterations to protein composition and plasticity in isolated hippocampal synapses. The effect of L-DOPA and pramipexole on long-term potentiation was also tested. Human α-synuclein was found within dopaminergic and glutamatergic neurons of the ventral tegmental area, and in dopaminergic, glutamatergic and GABAergic axon terminals in the hippocampus from 1 week post-inoculation, concomitant with mild dopaminergic degeneration in the ventral tegmental area. In the hippocampus, differential expression of proteins involved in synaptic vesicle cycling, neurotransmitter release and receptor trafficking, together with impaired long-term potentiation were the first events observed (1 week post-inoculation), preceding cognitive deficits (4 weeks post-inoculation). Later on, at 16 weeks post-inoculation, there was a deregulation of proteins involved in synaptic function, particularly those involved in the regulation of membrane potential, ion balance and receptor signalling. Hippocampal long-term potentiation was impaired before and soon after the onset of cognitive deficits, at 1 and 4 weeks post-inoculation, respectively. L-DOPA recovered hippocampal long-term potentiation more efficiently at 4 weeks post-inoculation than pramipexole, which partially rescued it at both time points. Overall, we found impaired synaptic plasticity and proteome dysregulation at hippocampal terminals to be the first events that contribute to the development of cognitive deficits in experimental parkinsonism. Our results not only point to dopaminergic but also to glutamatergic and GABAergic dysfunction, highlighting the relevance of the three neurotransmitter systems in the ventral tegmental area-hippocampus interaction from the earliest stages of parkinsonism. The proteins identified in the current work may constitute potential biomarkers of early synaptic damage in the hippocampus and hence, therapies targeting these could potentially restore early synaptic malfunction and consequently, cognitive deficits in Parkinson's disease.

Cntnap4 partial deficiency exacerbates α-synuclein pathology through astrocyte–microglia C3-C3aR pathway

Parkinson’s disease (PD) is the most common progressive neurodegenerative movement disorder, which is characterized by dopaminergic (DA) neuron death and the aggregation of neurotoxic α-synuclein. Cntnap4, a risk gene of autism, has been implicated to participate in PD pathogenesis. Here we showed Cntnap4 lacking exacerbates α-synuclein pathology, nigrostriatal DA neuron degeneration and motor impairment, induced by injection of adeno-associated viral vector (AAV)-mediated human α-synuclein overexpression (AAV-hα-Syn). This scenario was further validated in A53T α-synuclein transgenic mice injected with AAV-Cntnap4 shRNA. Mechanistically, α-synuclein derived from damaged DA neuron stimulates astrocytes to release complement C3, activating microglial C3a receptor (C3aR), which in turn triggers microglia to secrete complement C1q and pro-inflammatory cytokines. Thus, the astrocyte–microglia crosstalk further drives DA neuron death and motor dysfunction in PD. Furthermore, we showed that in vivo depletion of microglia and microglial targeted delivery of a novel C3aR antagonist (SB290157) rescue the aggravated α-synuclein pathology resulting from Cntnap4 lacking. Together, our results indicate that Cntnap4 plays a key role in α-synuclein pathogenesis by regulating glial crosstalk and may be a potential target for PD treatment.

Systemic RNA Interference Defective (SID) genes modulate dopaminergic neurodegeneration in C. elegans.

The fine-tuning of gene expression is critical for all cellular processes; aberrations in this activity can lead to pathology, and conversely, resilience. As their role in coordinating organismal responses to both internal and external factors have increasingly come into focus, small non-coding RNAs have emerged as an essential component to disease etiology. Using Systemic RNA interference Defective (SID) mutants of the nematode Caenorhabditis elegans, deficient in gene silencing, we examined the potential consequences of dysfunctional epigenomic regulation in the context of Parkinson’s disease (PD). Specifically, the loss of either the sid-1 or sid-3 genes, which encode a dsRNA transporter and an endocytic regulatory non-receptor tyrosine kinase, respectively, conferred neuroprotection to dopaminergic (DA) neurons in an established transgenic C. elegans strain wherein overexpression of human α-synuclein (α-syn) from a chromosomally integrated multicopy transgene causes neurodegeneration. We further show that knockout of a specific microRNA, mir-2, attenuates α-syn neurotoxicity; suggesting that the native targets of mir-2-dependent gene silencing represent putative neuroprotective modulators. In support of this, we demonstrated that RNAi knockdown of multiple mir-2 targets enhanced α-syn-induced DA neurodegeneration. Moreover, we demonstrate that mir-2 overexpression originating in the intestine can induce neurodegeneration of DA neurons, an effect that was reversed by pharmacological inhibition of SID-3 activity. Interestingly, sid-1 mutants retained mir-2-induced enhancement of neurodegeneration. Transcriptomic analysis of α-syn animals with and without a sid-1 mutation revealed 27 differentially expressed genes with human orthologs related to a variety of diseases, including PD. Among these was pgp-8, encoding a P-glycoprotein-related ABC transporter. Notably, sid-1; pgp-8 double mutants abolished the neurodegeneration resulting from intestinal mir-2 overexpression. This research positions known regulators of small RNA-dependent gene silencing within a framework that facilitates mechanistic evaluation of epigenetic responses to exogenous and endogenous factors influencing DA neurodegeneration, revealing a path toward new targets for therapeutic intervention of PD.

Time-Course of Alterations in the Endocannabinoid System after Viral-Mediated Overexpression of α-Synuclein in the Rat Brain.

Since the discovery of α-synuclein as the major component in Lewy bodies, research into this protein in the context of Parkinson’s disease pathology has been exponential. Cannabinoids are being investigated as potential therapies for Parkinson’s disease from numerous aspects, but still little is known about the links between the cannabinoid system and the pathogenic α-synuclein protein; understanding these links will be necessary if cannabinoid therapies are to reach the clinic in the future. Therefore, the aim of this study was to investigate the time-course of alterations in components of the endocannabinoid system after viral-mediated α-synuclein overexpression in the rat brain. Rats were given unilateral intranigral injections of AAV-GFP or AAV-α-synuclein and sacrificed 4, 8 and 12 weeks later for qRT-PCR and liquid chromatography–mass spectrometry analyses of the endocannabinoid system, in addition to histological visualization of α-synuclein expression along the nigrostriatal pathway. As anticipated, intranigral delivery of AAV-α-synuclein induced widespread overexpression of human α-synuclein in the nigrostriatal pathway, both at the mRNA level and the protein level. However, despite this profound α-synuclein overexpression, we detected no differences in CB1 or CB2 receptor expression in the nigrostriatal pathway; however, interestingly, there was a reduction in the expression of neuroinflammatory markers. Furthermore, there was a reduction in the levels of the endocannabinoid 2-AG and the related lipid immune mediator OEA at week 12 post-surgery, indicating that α-synuclein overexpression triggers dysregulation of the endocannabinoid system. Although this research does show that the endocannabinoid system is impacted by α-synuclein, further research is necessary to more comprehensively understand the link between the cannabinoid system and the α-synuclein aspect of Parkinson’s disease pathology in order for cannabinoid-based therapies to be feasible for the treatment of this disease in the coming years.

The Small Molecule Alpha-Synuclein Aggregator, FN075, Enhances Alpha-Synuclein Pathology in Subclinical AAV Rat Models.

Animal models of Parkinson’s disease, in which the human α-synuclein transgene is overexpressed in the nigrostriatal pathway using viral vectors, are widely considered to be the most relevant models of the human condition. However, although highly valid, these models have major limitations related to reliability and variability, with many animals exhibiting pronounced α-synuclein expression failing to demonstrate nigrostriatal neurodegeneration or motor dysfunction. Therefore, the aim of this study was to determine if sequential intra-nigral administration of AAV-α-synuclein followed by the small α-synuclein aggregating molecule, FN075, would enhance or precipitate the associated α-synucleinopathy, nigrostriatal pathology and motor dysfunction in subclinical models. Rats were given unilateral intra-nigral injections of AAV-α-synuclein (either wild-type or A53T mutant) followed four weeks later by a unilateral intra-nigral injection of FN075, after which they underwent behavioral testing for lateralized motor functionality until they were sacrificed for immunohistological assessment at 20 weeks after AAV administration. In line with expectations, both of the AAV vectors induced widespread overexpression of human α-synuclein in the substantia nigra and striatum. Sequential administration of FN075 significantly enhanced the α-synuclein pathology with increased density and accumulation of the pathological form of the protein phosphorylated at serine 129 (pS129-α-synuclein). However, despite this enhanced α-synuclein pathology, FN075 did not precipitate nigrostriatal degeneration or motor dysfunction in these subclinical AAV models. In conclusion, FN075 holds significant promise as an approach to enhancing the α-synuclein pathology in viral overexpression models, but further studies are required to determine if alternative administration regimes for this molecule could improve the reliability and variability in these models.

The Nrf2-NLRP3-caspase-1 axis mediates the neuroprotective effects of Celastrol in Parkinson's disease

Parkinson's disease (PD) is a chronic neurodegenerative disorder that is characterized by motor symptoms as a result of a loss of dopaminergic neurons in the substantia nigra pars compacta (SNc), accompanied by chronic neuroinflammation, oxidative stress, formation of α-synuclein aggregates. Celastrol, a potent anti-inflammatory and anti-oxidative pentacyclic triterpene, has emerged as a neuroprotective agent. However, the mechanisms by which celastrol is neuroprotective in PD remain elusive. Here we show that celastrol protects against dopamine neuron loss, mitigates neuroinflammation, and relieves motor deficits in MPTP-induced PD mouse model and AAV-mediated human α-synuclein overexpression PD model. Whole-genome deep sequencing analysis revealed that Nrf2, NLRP3 and caspase-1 in SNc may be associated with the neuroprotective actions of celastrol in PD. By using multiple genetically modified mice (Nrf2-KO, NLRP3-KO and Caspase-1-KO), we identified that celastrol inhibits NLRP3 inflammasome activation, relieves motor deficits and nigrostriatal dopaminergic degeneration through Nrf2-NLRP3-caspase-1 pathway. Taken together, these findings suggest that Nrf2-NLRP3-caspase-1 axis may serve as a key target of celastrol in PD treatment, and highlight the favorable properties of celastrol for neuroprotection, making celastrol as a promising disease-modifying agent for PD.

α-Synuclein E46K Mutation and Involvement of Oxidative Stress in a Drosophila Model of Parkinson's Disease.

Parkinson's disease (PD) is an age-associated neurodegenerative condition in which some genetic variants are known to increase disease susceptibility on interaction with environmental factors inducing oxidative stress. Different mutations in the SNCA gene are reported as the major genetic contributors to PD. E46K mutation pathogenicity has not been investigated as intensive as other SNCA gene mutations including A30P and A53T. In this study, based on the GAL4-UAS binary genetic tool, transgenic Drosophila melanogaster flies expressing wild-type and E46K-mutated copies of the human SNCA gene were constructed. Western blotting, immunohistochemical analysis, and light and confocal microscopy of flies' brains were undertaken along with the survival rate measurement, locomotor function assay, and ethanol and paraquat (PQ) tolerance to study α-synuclein neurotoxicity. Biochemical bioassays were carried out to investigate the activity of antioxidant enzymes and alterations in levels of oxidative markers following damages induced by human α-synuclein to the neurons of the transgenic flies. Overexpression of human α-synuclein in the central nervous system of these transgenic flies led to disorganized ommatidia structures and loss of dopaminergic neurons. E46K α-synuclein caused remarkable climbing defects, reduced survivorship, higher ethanol sensitivity, and increased PQ-mediated mortality. A noticeable decline in activity of catalase and superoxide dismutase enzymes besides considerable increase in the levels of lipid peroxidation and reactive oxygen species was observed in head capsule homogenates of α-synuclein-expressing flies, which indicates obvious involvement of oxidative stress as a causal factor in SNCAE46K neurotoxicity. In all the investigations, E46K copy of the SNCA gene was found to impose more severe defects when compared to wild-type SNCA. It can be concluded that the constructed Drosophila models developed PD-like symptoms that facilitate comparative studies of molecular and cellular pathways implicated in the pathogenicity of different α-synuclein mutations.

Overexpression of α-Synuclein Reorganises Growth Factor Profile of Human Astrocytes.

Misfolding and accumulation of aberrant α-synuclein in the brain is associated with the distinct class of neurodegenerative diseases known as α-synucleinopathies, which include Parkinson's disease, dementia with Lewy bodies and multiple system atrophy. Pathological changes in astrocytes contribute to all neurological disorders, and astrocytes are reported to possess α-synuclein inclusions in the context of α-synucleinopathies. Astrocytes are known to express and secrete numerous growth factors, which are fundamental for neuroprotection, synaptic connectivity and brain metabolism; changes in growth factor secretion may contribute to pathobiology of neurological disorders. Here we analysed the effect of α-synuclein overexpression in cultured human astrocytes on growth factor expression and release. For this purpose, the intracellular and secreted levels of 33 growth factors (GFs) and 8 growth factor receptors (GFRs) were analysed in cultured human astrocytes by chemiluminescence-based western/dot blot. Overexpression of human α-synuclein in cultured foetal human astrocytes significantly changes the profile of GF production and secretion. We found that human astrocytes express and secrete FGF2, FGF6, EGF, IGF1, AREG, IGFBP2, IGFBP4, VEGFD, PDGFs, KITLG, PGF, TGFB3 and NTF4. Overexpression of human α-synuclein significantly modified the profile of GF production and secretion, with particularly strong changes in EGF, PDGF, VEGF and their receptors as well as in IGF-related proteins. Bioinformatics analysis revealed possible interactions between α-synuclein and EGFR and GDNF, as well as with three GF receptors, EGFR, CSF1R and PDGFRB.

Anti-α-synuclein ASO delivered to monoamine neurons prevents α-synuclein accumulation in a Parkinson's disease-like mouse model and in monkeys.

BackgroundProgressive neuronal death in monoaminergic nuclei and widespread accumulation of α-synuclein are neuropathological hallmarks of Parkinson's disease (PD). Given that α-synuclein may be an early mediator of the pathological cascade that ultimately leads to neurodegeneration, decreased α-synuclein synthesis will abate neurotoxicity if delivered to the key affected neurons.MethodsWe used a non-viral gene therapy based on a new indatraline-conjugated antisense oligonucleotide (IND-ASO) to disrupt the α-synuclein mRNA transcription selectively in monoamine neurons of a PD-like mouse model and elderly nonhuman primates. Molecular, cell biology, histological, neurochemical and behavioral assays were performed.FindingsIntracerebroventricular and intranasal IND-ASO administration for four weeks in a mouse model with AAV-mediated wild-type human α-synuclein overexpression in dopamine neurons prevented the synthesis and accumulation of α-synuclein in the connected brain regions, improving dopamine neurotransmission. Likewise, the four-week IND-ASO treatment led to decreased levels of endogenous α-synuclein protein in the midbrain monoamine nuclei of nonhuman primates, which are affected early in PD.Conclusions: The inhibition of α-synuclein production in dopamine neurons and its accumulation in cortical/striatal projection areas may alleviate the early deficits of dopamine function, showing the high translational value of antisense oligonucleotides as a disease modifying therapy for PD and related synucleinopathies.FundingGrants SAF2016-75797-R, RTC-2014-2812-1 and RTC-2015-3309-1, Ministry of Economy and Competitiveness (MINECO) and European Regional Development Fund (ERDF), UE; Grant ID 9238, Michael J. Fox Foundation; and Centres for Networked Biomedical Research on Mental Health (CIBERSAM), and on Neurodegenerative Diseases (CIBERNED).

Impairment of Macroautophagy in Dopamine Neurons Has Opposing Effects on Parkinsonian Pathology and Behavior

SummaryParkinson’s disease (PD) is characterized by the death of dopamine neurons in the substantia nigra pars compacta (SNc) and accumulation of α-synuclein. Impaired autophagy has been implicated and activation of autophagy proposed as a treatment strategy. We generate a human α-synuclein-expressing mouse model of PD with macroautophagic failure in dopamine neurons to understand the interaction between impaired macroautophagy and α-synuclein. We find that impaired macroautophagy generates p62-positive inclusions and progressive neuron loss in the SNc. Despite this parkinsonian pathology, motor phenotypes accompanying human α-synuclein overexpression actually improve with impaired macroautophagy. Real-time fast-scan cyclic voltammetry reveals that macroautophagy impairment in dopamine neurons increases evoked extracellular concentrations of dopamine, reduces dopamine uptake, and relieves paired-stimulus depression. Our findings show that impaired macroautophagy paradoxically enhances dopamine neurotransmission, improving movement while worsening pathology, suggesting that changes to dopamine synapse function compensate for and conceal the underlying PD pathogenesis, with implications for therapies that target autophagy.

Seeding of protein aggregation causes cognitive impairment in rat model of cortical synucleinopathy

Cortical α-synuclein pathology plays a role in the development of cognitive dysfunction in both Parkinson's disease and dementia with Lewy bodies, although the causative cellular lesions have remained unclear. We aimed to address causal links between α-synuclein-driven pathology in the cerebral cortex and the development of cognitive impairments using new experimental models. Neuronal overexpression of human α-synuclein was induced in the rat medial prefrontal cortex using viral vectors. This was combined with inoculations of preformed fibrils of human α-synuclein in some animals. Rats were evaluated with tests probing prefrontal cognitive functions (delayed matching/nonmatching to position and 5-choice serial reaction time task). Patterns of neuropathology were characterized immunohistochemically. Neither α-synuclein overexpression nor the fibril seeds alone yielded any behavioral phenotype. In contrast, combining the 2 approaches produced significant impairments in working memory, attention, and inhibitory control. All animals injected with α-synuclein vectors exhibited high immunoreactivity for human α-synuclein in the medial prefrontal cortex and its primary projection targets. However, only when this overexpression was combined with fibril inoculations did animals exhibit large, proteinase K-resistant and Ser129 -phosphorylated α-synuclein intraneuronal inclusions in the medial prefrontal cortex and its closely interconnected brain regions. The inclusions were associated with distorted dendritic morphologies and partial neuronal loss in the targeted cortical areas. Cortical overexpression of human α-synuclein is not sufficient to produce cognitive dysfunction, whereas combining this overexpression with fibril seeds yields both cognitive and histopathological phenotypes that are relevant to human Lewy body disease. © 2019 International Parkinson and Movement Disorder Society.

Mitochondrial Dysregulation and Impaired Autophagy in iPSC-Derived Dopaminergic Neurons of Multiple System Atrophy.

SummaryMultiple system atrophy (MSA) is a progressive neurodegenerative disease that affects several areas of the CNS, whose pathogenesis is still widely unclear and for which an effective treatment is lacking. We have generated induced pluripotent stem cell-derived dopaminergic neurons from four MSA patients and four healthy controls and from two monozygotic twins discordant for the disease. In this model, we have demonstrated an aberrant autophagic flow and a mitochondrial dysregulation involving respiratory chain activity, mitochondrial content, and CoQ10 biosynthesis. These defective mechanisms may contribute to the onset of the disease, representing potential therapeutic targets.

Motor learning and metaplasticity in striatal neurons: relevance for Parkinson's disease.

Nigro-striatal dopamine transmission is central to a wide range of neuronal functions, including skill learning, which is disrupted in several pathologies such as Parkinson's disease. The synaptic plasticity mechanisms, by which initial motor learning is stored for long time periods in striatal neurons, to then be gradually optimized upon subsequent training, remain unexplored. Addressing this issue is crucial to identify the synaptic and molecular mechanisms involved in striatal-dependent learning impairment in Parkinson's disease. In this study, we took advantage of interindividual differences between outbred rodents in reaching plateau performance in the rotarod incremental motor learning protocol, to study striatal synaptic plasticity ex vivo. We then assessed how this process is modulated by dopamine receptors and the dopamine active transporter, and whether it is impaired by overexpression of human α-synuclein in the mesencephalon; the latter is a progressive animal model of Parkinson's disease. We found that the initial acquisition of motor learning induced a dopamine active transporter and D1 receptors mediated long-term potentiation, under a protocol of long-term depression in striatal medium spiny neurons. This effect disappeared in animals reaching performance plateau. Overexpression of human α-synuclein reduced striatal dopamine active transporter levels, impaired motor learning, and prevented the learning-induced long-term potentiation, before the appearance of dopamine neuronal loss. Our findings provide evidence of a reorganization of cellular plasticity within the dorsolateral striatum that is mediated by dopamine receptors and dopamine active transporter during the acquisition of a skill. This newly identified mechanism of cellular memory is a form of metaplasticity that is disrupted in the early stage of synucleinopathies, such as Parkinson's disease, and that might be relevant for other striatal pathologies, such as drug abuse.

Selective α-Synuclein Knockdown in Monoamine Neurons by Intranasal Oligonucleotide Delivery: Potential Therapy for Parkinson’s Disease

Progressive neuronal death in brainstem nuclei and widespread accumulation of α-synuclein are neuropathological hallmarks of Parkinson's disease (PD). Reduction of α-synuclein levels is therefore a potential therapy for PD. However, because α-synuclein is essential for neuronal development and function, α-synuclein elimination would dramatically impact brain function. We previously developed conjugated small interfering RNA (siRNA) sequences that selectively target serotonin (5-HT) or norepinephrine (NE) neurons after intranasal administration. Here, we used this strategy to conjugate inhibitory oligonucleotides, siRNA and antisense oligonucleotide (ASO), with the triple monoamine reuptake inhibitor indatraline (IND), to selectively reduce α-synuclein expression in the brainstem monoamine nuclei of mice after intranasal delivery. Following internalization of the conjugated oligonucleotides in monoamine neurons, reduced levels of endogenous α-synuclein mRNA and protein were found in substantia nigra pars compacta (SNc), ventral tegmental area (VTA), dorsal raphe nucleus (DR), and locus coeruleus (LC). α-Synuclein knockdown by ∼20%-40% did not cause monoaminergic neurodegeneration and enhanced forebrain dopamine (DA) and 5-HT release. Conversely, a modest human α-synuclein overexpression in DA neurons markedly reduced striatal DA release. These results indicate that α-synuclein negatively regulates monoamine neurotransmission and set the stage for the testing of non-viral inhibitory oligonucleotides as disease-modifying agents in α-synuclein models of PD.

Brain-to-stomach transfer of α-synuclein via vagal preganglionic projections.

Detection of α-synuclein lesions in peripheral tissues is a feature of human synucleinopathies of likely pathogenetic relevance and bearing important clinical implications. Experiments were carried out to elucidate the relationship between α-synuclein accumulation in the brain and in peripheral organs, and to identify potential pathways involved in long-distance protein transfer. Results of this in vivo study revealed a route-specific transmission of α-synuclein from the rat brain to the stomach. Following targeted midbrain overexpression of human α-synuclein, the exogenous protein was capable of reaching the gastric wall where it was accumulated into preganglionic vagal terminals. This brain-to-stomach connection likely involved intra- and inter-neuronal transfer of non-fibrillar α-synuclein that first reached the medulla oblongata, then gained access into cholinergic neurons of the dorsal motor nucleus of the vagus nerve and finally traveled via efferent fibers of these neurons contained within the vagus nerve. Data also showed a particular propensity of vagal motor neurons and efferents to accrue α-synuclein and deliver it to peripheral tissues; indeed, following its midbrain overexpression, human α-synuclein was detected within gastric nerve endings of visceromotor but not viscerosensory vagal projections. Thus, the dorsal motor nucleus of the vagus nerve represents a key relay center for central-to-peripheral α-synuclein transmission, and efferent vagal fibers may act as unique conduits for protein transfer. The presence of α-synuclein in peripheral tissues could reflect, at least in some synucleinopathy patients, an ongoing pathological process that originates within the brain and, from there, reaches distant organs innervated by motor vagal projections.

Photobiomodulation Suppresses Alpha-Synuclein-Induced Toxicity in an AAV-Based Rat Genetic Model of Parkinson's Disease.

Converging lines of evidence indicate that near-infrared light treatment, also known as photobiomodulation (PBM), may exert beneficial effects and protect against cellular toxicity and degeneration in several animal models of human pathologies, including neurodegenerative disorders. In the present study, we report that chronic PMB treatment mitigates dopaminergic loss induced by unilateral overexpression of human α-synuclein (α-syn) in the substantia nigra of an AAV-based rat genetic model of Parkinson’s disease (PD). In this model, daily exposure of both sides of the rat’s head to 808-nm near-infrared light for 28 consecutive days alleviated α-syn-induced motor impairment, as assessed using the cylinder test. This treatment also significantly reduced dopaminergic neuronal loss in the injected substantia nigra and preserved dopaminergic fibers in the ipsilateral striatum. These beneficial effects were sustained for at least 6 weeks after discontinuing the treatment. Together, our data point to PBM as a possible therapeutic strategy for the treatment of PD and other related synucleinopathies.

Depressive-like phenotype induced by AAV-mediated overexpression of human α-synuclein in midbrain dopaminergic neurons

Parkinson's disease (PD) is a neurodegenerative disorder characterized by a progressive loss of nigral dopaminergic neurons and by the presence of aggregates containing α-synuclein called Lewy bodies. Viral vector-induced overexpression of α-synuclein in dopaminergic neurons represents a model of PD which recapitulates disease progression better than commonly used neurotoxin models. Previous studies using this model have reported motor and cognitive impairments, whereas depression, mood and anxiety phenotypes are less described. To investigate these psychiatric phenotypes, Sprague–Dawley rats received bilateral injections of a recombinant adeno-associated virus (AAV) vector expressing human α-synuclein or GFP into the substantia nigra pars compacta. Behavior was assessed at two timepoints: 3 and 8weeks post-injection. We report that nigral α-synuclein overexpression led to a pronounced nigral dopaminergic cell loss accompanied by a smaller cell loss in the ventral tegmental area, and to a decreased striatal density of dopaminergic fibers. The AAV-α-synuclein group exhibited modest, but significant motor impairments 8weeks after vector administration. The AAV-α-synuclein group displayed depressive-like behavior in the forced swim test after 3weeks, and reduced sucrose preference at week 8. At both timepoints, overexpression of α-synuclein was linked to a hyperactive hypothalamic–pituitary–adrenal (HPA) axis regulation of corticosterone. The depressive-like phenotype was also correlated with decreased nigral brain-derived neurotrophic factor and spinophilin levels, and with decreased striatal levels of the activity-regulated cytoskeleton-associated protein. This study demonstrates that AAV-mediated α-synuclein overexpression in dopamine neurons is not only useful to model motor impairments of PD, but also depression. This study also provides evidence that depression in experimental Parkinsonism is correlated to dysregulation of the HPA axis and to alterations in proteins involved in synaptic plasticity.

Emotional memory impairments induced by AAV-mediated overexpression of human α-synuclein in dopaminergic neurons of the ventral tegmental area

Parkinson’s disease (PD) is associated with extensive degeneration of dopaminergic neurons originating in the substantia nigra pars compacta, but neuronal loss is also found in the ventral tegmental area (VTA). The VTA projects to areas involved in cognitive and emotional processes, including hippocampus, amygdala, nucleus accumbens and prefrontal cortex, and has thus been proposed to play a role in emotional memory impairments in PD. Since the formation of α-synuclein inclusions throughout the central nervous system is a pathological hallmark of PD, we studied the progressive effects of α-synuclein overexpression in the VTA on motor functions, emotional behaviour and emotional memory. Adeno-associated viral (AAV) vectors encoding either human α-synuclein or green fluorescent protein (GFP) were injected stereotactically into the VTA, and behaviour was monitored 3 and 8 weeks following AAV injection. At week 8, there was a 22% reduction of TH+ neurons in the VTA. We demonstrate that α-synuclein overexpression in dopaminergic neurons of the VTA induced mild motor deficits that appeared 3 weeks following AAV-α-synuclein injection and were aggravated at week 8. No depressive- or anxiety-like behaviours were found. To address emotional memory, we used the passive avoidance test, a one-trial associative learning paradigm based on contextual conditioning which requires minimal training. Interestingly, emotional memory impairments were found in α-synuclein overexpressing animals at week 8. These findings indicate that α-synuclein overexpression induces progressive memory impairments likely caused by a loss of function of mesolimbic dopaminergic projections.