Α-syn Preformed Fibrils Research Articles

Lewy Body Disease Primate Model with α-Synuclein Propagation from the Olfactory Bulb.

Lewy body diseases (LBDs), which are pathologically defined as the presence of intraneuronal α-synuclein (α-Syn) inclusions called Lewy bodies, encompass Parkinson's disease, Parkinson's disease with dementia, and dementia with Lewy bodies. Autopsy studies have shown that the olfactory bulb (OB) is one of the regions where Lewy pathology develops and initiates its spread in the brain. This study aims to clarify how Lewy pathology spreads from the OB and affects brain functions using nonhuman primates. We inoculated α-Syn preformed fibrils into the unilateral OBs of common marmosets (Callithrix jacchus) and performed pathological analyses, manganese-enhanced magnetic resonance imaging, and 18 F-fluoro-2-deoxy-d-glucose positron emission tomography up to 6months postinoculation. Severe α-Syn pathology was observed within the olfactory pathway and limbic system, while mild α-Syn pathology was seen in a wide range of brain regions, including the substantia nigra pars compacta, locus coeruleus, and even dorsal motor nucleus of the vagus nerve. The brain imaging analyses showed reduction in volume of the OB and progressive glucose hypometabolism in widespread brain regions, including the occipital lobe, and extended beyond the pathologically affected regions. We generated a novel nonhuman primate LBD model with α-Syn propagation from the OB. This model suggests that α-Syn propagation from the OB is related to OB atrophy and cerebral glucose hypometabolism in LBDs. © 2022 International Parkinson and Movement Disorder Society.

Rabphilin-3A as a novel target to reverse α-synuclein-induced synaptic loss in Parkinson’s disease

Toxic aggregates of α-synuclein (αsyn) are considered key drivers of Parkinson’s disease (PD) pathology. In early PD, αsyn induces synaptic dysfunction also modulating the glutamatergic neurotransmission. However, a more detailed understanding of the molecular mechanisms underlying αsyn-triggered synaptic failure is required to design novel therapeutic interventions. Here, we described the role of Rabphilin-3A (Rph3A) as novel target to counteract αsyn-induced synaptic loss in PD. Rph3A is a synaptic protein interacting with αsyn and involved in stabilizing dendritic spines and in promoting the synaptic retention of NMDA-type glutamate receptors. We found that in vivo intrastriatal injection of αsyn-preformed fibrils in mice induces the early loss of striatal synapses associated with decreased synaptic levels of Rph3A and impaired Rph3A/NMDA receptors interaction. Modulating Rph3A striatal expression or interfering with the Rph3A/αsyn complex with a small molecule prevented dendritic spine loss and rescued associated early motor defects in αsyn-injected mice. Notably, the same experimental approaches prevented αsyn-induced synaptic loss in vitro in primary hippocampal neurons. Overall, these findings indicate that approaches aimed at restoring Rph3A synaptic functions can slow down the early synaptic detrimental effects of αsyn aggregates in PD.

α-Synuclein fibril-specific nanobody reduces prion-like α-synuclein spreading in mice

Pathogenic α-synuclein (α-syn) is a prion-like protein that drives the pathogenesis of Lewy Body Dementia (LBD) and Parkinson’s Disease (PD). To target pathogenic α-syn preformed fibrils (PFF), here we designed extracellular disulfide bond-free synthetic nanobody libraries in yeast. Following selection, we identified a nanobody, PFFNB2, that can specifically recognize α-syn PFF over α-syn monomers. PFFNB2 cannot inhibit the aggregation of α-syn monomer, but can significantly dissociate α-syn fibrils. Furthermore, adeno-associated virus (AAV)-encoding EGFP fused to PFFNB2 (AAV-EGFP-PFFNB2) can inhibit PFF-induced α-syn serine 129 phosphorylation (pS129) in mouse primary cortical neurons, and prevent α-syn pathology spreading to the cortex in the transgenic mice expressing human wild type (WT) α-syn by intrastriatal-PFF injection. The pS129 immunoreactivity is negatively correlated with the expression of AAV-EGFP-PFFNB2. In conclusion, PFFNB2 holds a promise for mechanistic exploration and therapeutic development in α-syn-related pathogenesis.

Investigating key factors underlying neurodegeneration linked to alpha-synuclein spread.

AimsIt has long been considered that accumulation of pathological alpha‐synuclein (aSyn) leads to synaptic/neuronal loss which then results in behavioural and cognitive dysfunction. To investigate this claim, we investigated effects downstream of aSyn preformed fibrils (PFFs) and 6‐hydroxydopamine (6‐OHDA), because aSyn PFFs induce spreading/accumulation of aSyn, and 6‐OHDA rapidly causes local neuronal loss.MethodsWe injected mouse aSyn PFFs into the medial forebrain bundle (MFB) of Sprague–Dawley rats. We investigated spread of pathological aSyn, phosphorylation of aSyn and tau, oxidative stress, synaptic/neuronal loss and cognitive dysfunction 60, 90 and 120 days after injection. Similarly, we injected 6‐OHDA into the MFB and examined the same parameters 1 and 3 weeks after injection.ResultsFollowing aSyn PFF injection, phosphorylated aSyn was found distant from the injection site in the hippocampus and frontal cortex. However, despite neuron loss being evident close to the site of injection in the substantia nigra at 120 days post injection, there were no other neurodegeneration‐associated features associated with aSyn including synaptic loss. In contrast, 6‐OHDA caused severe neuronal loss in the substantia nigra at 3 weeks post injection that was accompanied by phosphorylation of aSyn and tau, oxidative stress, loss of synaptic proteins, cognitive and motor dysfunction.ConclusionsOur results demonstrate that spread/replication and slow accumulation of pathological aSyn may not be sufficient to induce neurodegenerative changes. In contrast, oxidative stress responses in addition to aSyn accumulation were associated with other Parkinson's disease (PD)‐associated abnormalities and cognitive dysfunction. Our results may be important when considering why only some PD patients develop dementia.

The GBA1 D409V mutation exacerbates synuclein pathology to differing extents in two alpha-synuclein models.

ABSTRACTHeterozygous mutations in the GBA1 gene – encoding lysosomal glucocerebrosidase (GCase) – are the most common genetic risk factors for Parkinson's disease (PD). Experimental evidence suggests a correlation between decreased GCase activity and accumulation of alpha-synuclein (aSyn). To enable a better understanding of the relationship between aSyn and GCase activity, we developed and characterized two mouse models that investigate aSyn pathology in the context of reduced GCase activity. The first model used constitutive overexpression of wild-type human aSyn in the context of the homozygous GCase activity-reducing D409V mutant form of GBA1. Although increased aSyn pathology and grip strength reductions were observed in this model, the nigrostriatal system remained largely intact. The second model involved injection of aSyn preformed fibrils (PFFs) into the striatum of the homozygous GBA1 D409V knock-in mouse model. The GBA1 D409V mutation did not exacerbate the pathology induced by aSyn PFF injection. This study sheds light on the relationship between aSyn and GCase in mouse models, highlighting the impact of model design on the ability to model a relationship between these proteins in PD-related pathology.

Ulvan inhibits α-synuclein fibrillation and disrupts the mature fibrils: In vitro and in vivo studies

Misfolding and aggregation of α-synuclein (α-syn) play a key role in the pathogenesis of Parkinson's disease (PD). Herein, the inhibitory effect of ulvan on α-syn fibrillogenesis was studied using thioflavin T fluorescence and atomic force microscope assays. It is shown that ulvan could inhibit α-syn fibrillogenesis in a dose-dependent manner. Based on the circular dichroism results, it is found that ulvan delays greatly the conformational transition from its initial random coil to β-sheet rich structure. The protective effect of ulvan against celllular death induced by α-syn aggregates was investigated by MTT colorimetric and cellular staining methods. It is found that ulvan protects greatly PC12 cells from α-syn fibril-induced cytotoxicity. In addition, ulvan disaggregates preformed α-syn fibrils and reduces cytotoxicity in a dose-dependent manner. Thereafter, the inhibitory effects of ulvan against α-syn fibrillogenesis were probed using Caenorhabditis elegans model NL5901 expressing human α-syn. It is found that ulvan extends the lifespan of NL5901 and recovers the lipid deposition by reducing the accumulation of α-syn. Finally, the molecular interactions between ulvan and α-syn pentamer was also explored using molecular docking. These findings suggest that ulvan can be pursued as a novel candidate drug for treatment of PD.

α-Synuclein Impacts on Intrinsic Neuronal Network Activity Through Reduced Levels of Cyclic AMP and Diminished Numbers of Active Presynaptic Terminals.

α-synuclein (α-Syn) is intimately linked to synucleinopathies like Parkinson’s disease and dementia with Lewy bodies. However, the pathophysiological mechanisms that are triggered by this protein are still largely enigmatic. α-Syn overabundance may cause neurodegeneration through protein accumulation and mitochondrial deterioration but may also result in pathomechanisms independent from neuronal cell death. One such proposed pathological mechanism is the influence of α-Syn on non-stimulated, intrinsic brain activity. This activity is responsible for more than 90% of the brain’s energyconsumption, and is thus thought to play an eminent role in basic brain functionality. Here we report that α-Syn substantially disrupts intrinsic neuronal network burst activity in a long-term neuronal cell culture model. Mechanistically, the impairment of network activity originates from reduced levels of cyclic AMP and cyclic AMP-mediated signaling as well as from diminished numbers of active presynaptic terminals. The profound reduction of network activity due to α-Syn was mediated only by intracellularly expressed α-Syn, but not by α-Syn that is naturally released by neurons. Conversely, extracellular pre-formed fibrils of α-Syn mimicked the effect of intracellular α-Syn, suggesting that they trigger an off-target mechanism that is not activated by naturally released α-Syn. A simulation-based model of the network activity in our cultures demonstrated that even subtle effect sizes in reducing outbound connectivity, i.e., loss of active synapses, can cause substantial global reductions in non-stimulated network activity. These results suggest that even low-level loss of synaptic output capabilities caused by α-Syn may result in significant functional impairments in terms of intrinsic neuronal network activity. Provided that our model holds true for the human brain, then α-Syn may cause significant functional lesions independent from neurodegeneration.

Spreading of Aggregated α-Synuclein in Sagittal Organotypic Mouse Brain Slices.

The accumulation of α-synuclein (α-syn) in the brain plays a role in synucleinopathies and it is hypothesized to spread in a prion-like fashion between connected brain regions. In the present study, we aim to investigate this spreading in well-characterized sagittal organotypic whole brain slices taken from postnatal wild type (WT) and transgenic mice overexpressing human α-syn under the promoter of proteolipid protein (PLP). Collagen hydrogels were loaded with monomers of human α-syn, as well as human and mouse pre-formed fibrils (PFFs), to allow local application and slow release. The spreading of α-syn was evaluated in different brain regions by immunohistochemistry for total α-syn and α-syn phosphorylated at the serine129 position (α-syn-P). The application of human and mouse PFFs of α-syn caused the aggregation and spreading of α-syn-P in the brain slices, which was pronounced the most at the region of hydrogel application and surrounding striatum, as well as along the median forebrain bundle. The organotypic slices from transgenic mice showed significantly more α-syn pathology than those from WT mice. The present study demonstrates that seeding with α-syn PFFs but not monomers induced intracellular α-syn pathology, which was significantly more prominent in brain slices with α-syn overexpression. This is consistent with the prion-like spreading theory of α-syn aggregates. The sagittal whole brain slices characterized in this study carry the potential to be used as a novel model to study α-syn pathology.

Rapid Induction of Dopaminergic Neuron Loss Accompanied by Lewy Body-Like Inclusions in A53T BAC-SNCA Transgenic Mice

Parkinson's disease (PD), the most common neurodegenerative movement disorder, is characterized by dopaminergic neuron loss in the substantia nigra pars compacta (SNpc) and intraneuronal α-synuclein (α-syn) inclusions. It is highly needed to establish a rodent model that recapitulates the clinicopathological features of PD within a short period to efficiently investigate the pathological mechanisms and test disease-modifying therapies. To this end, we analyzed three mouse lines, i.e., wild-type mice, wild-type human α-syn bacterial artificial chromosome (BAC) transgenic (BAC-SNCA Tg) mice, and A53T human α-syn BAC transgenic (A53T BAC-SNCA Tg) mice, receiving dorsal striatum injections of human and mouse α-syn preformed fibrils (hPFFs and mPFFs, respectively). mPFF injections induced more severe α-syn pathology in most brain regions, including the ipsilateral SNpc, than hPFF injections in all genotypes at 1-month post-injection. Although these Tg mouse lines expressed a comparable amount of α-syn in the brains, the mPFF-injected A53T BAC-SNCA Tg mice exhibited the most severe α-syn pathology as early as 0.5-month post-injection. The mPFF-injected A53T BAC-SNCA Tg mice showed a 38% reduction in tyrosine hydroxylase (TH)-positive neurons in the ipsilateral SNpc, apomorphine-induced rotational behavior, and motor dysfunction at 2 months post-injection. These data indicate that the extent of α-syn pathology induced by α-syn PFF injection depends on the types of α-syn PFFs and exogenously expressed α-syn in Tg mice. The mPFF-injected A53T BAC-SNCA Tg mice recapitulate the key features of PD more rapidly than previously reported mouse models, suggesting their usefulness for testing disease-modifying therapies as well as analyzing the pathological mechanisms.

Alpha-synuclein from patient Lewy bodies exhibits distinct pathological activity that can be propagated in vitro

Lewy bodies (LBs) are complex, intracellular inclusions that are common pathological features of many neurodegenerative diseases. They consist largely of aggregated forms of the protein alpha-Synuclein (α-Syn), which misfolds to give rise to beta-sheet rich amyloid fibrils. The aggregation of monomers into fibrils occurs readily in vitro and pre-formed fibrils (PFFs) generated from recombinant α-Syn monomers are the basis of many models of LB diseases. These α-Syn PFFs recapitulate many pathological phenotypes in both cultured cells and animal models including the formation of α-Syn rich, insoluble aggregates, neuron loss, and motor deficits. However, it is not clear how closely α-Syn PFFs recapitulate the biological behavior of LB aggregates isolated directly from patients. Direct interrogation of the cellular response to LB-derived α-Syn has thus far been limited. Here we demonstrate that α-Syn aggregates derived from LB disease patients induce pathology characterized by a prevalence of large somatic inclusions that is distinct from the primarily neuritic pathology induced by α-Syn PFFs in our cultured neuron model. Moreover, these LB-derived aggregates can be amplified in vitro using recombinant α-Syn to generate aggregates that maintain the unique, somatic pathological phenotype of the original material. Amplified LB aggregates also showed greater uptake in cultured neurons and greater pathological burden and more rapid pathological spread in injected mouse brains, compared to α-Syn PFFs. Our work indicates that LB-derived α-Syn from diseased brains represents a distinct conformation species with unique biological activities that has not been previously observed in fully recombinant α-Syn aggregates and demonstrate a new strategy for improving upon α-Syn PFF models of synucleinopathies using amplified LBs.

Alpha-synuclein alters the faecal viromes of rats in a gut-initiated model of Parkinson\u2019s disease

Parkinson’s disease (PD) is a chronic neurological disorder associated with the misfolding of alpha-synuclein (α-syn) into aggregates within nerve cells that contribute to their neurodegeneration. Recent evidence suggests α-syn aggregation may begin in the gut and travel to the brain along the vagus nerve, with microbes potentially a trigger initiating α-syn misfolding. However, the effects α-syn alterations on the gut virome have not been investigated. In this study, we show longitudinal faecal virome changes in rats administered either monomeric or preformed fibrils (PFF) of α-syn directly into their enteric nervous system. Differential changes in rat viromes were observed when comparing monomeric and PFF α-syn, with alterations compounded by the addition of LPS. Changes in rat faecal viromes were observed after one month and did not resolve within the study’s five-month observational period. These results suggest that virome alterations may be reactive to host α-syn changes that are associated with PD development.

Selective targeting of the TLR2/MyD88/NF-\u03baB pathway reduces \u03b1-synuclein spreading in vitro and in vivo

Pathways to control the spreading of α-synuclein (α-syn) and associated neuropathology in Parkinson’s disease (PD), multiple system atrophy (MSA) and dementia with Lewy bodies (DLB) are unclear. Here, we show that preformed α-syn fibrils (PFF) increase the association between TLR2 and MyD88, resulting in microglial activation. The TLR2-interaction domain of MyD88 (wtTIDM) peptide-mediated selective inhibition of TLR2 reduces PFF-induced microglial inflammation in vitro. In PFF-seeded A53T mice, the nasal administration of the wtTIDM peptide, NEMO-binding domain (wtNBD) peptide, or genetic deletion of TLR2 reduces glial inflammation, decreases α-syn spreading, and protects dopaminergic neurons by inhibiting NF-κB. In summary, α-syn spreading depends on the TLR2/MyD88/NF-κB pathway and it can be reduced by nasal delivery of wtTIDM and wtNBD peptides.

DNAJB6 suppresses alpha-synuclein induced pathology in an animal model of Parkinson's disease

Backgroundα-synuclein (α-syn) aggregation can lead to degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNpc) as invariably observed in patients with Parkinson's Disease (PD). The co-chaperone DNAJB6 has previously been found to be expressed at higher levels in PD patients than in control subjects and was also found in Lewy bodies. Our previous experiments showed that knock out of DNAJB6 induced α-syn aggregation in cellular level. However, effects of overexpression of DNAJB6 against α-syn aggregation remains to be investigated. MethodsWe used a α-syn CFP/YFP HEK293 FRET cell line to investigate the effects of overexpression of DNAJB6 in cellular level. α-syn aggregation was induced by transfection α-syn preformed fibrils (PPF), then was measured FRET analysis. We proceeded to investigate if DNAJB6b can impair α-syn aggregation and toxicity in an animal model and used adeno associated vira (AAV6) designed to overexpress of human wt α-syn, GFP-DNAJB6 or GFP in rats. These vectors were injected into the SNpc of the rats, unilaterally. Rats injected with vira to express α-syn along with GFP in the SNpc where compared to rats expressing α-syn and GFP-DNAJB6. We evaluated motor functions, dopaminergic cell death, and axonal degeneration in striatum. ResultsWe show that DNAJB6 prevent α-syn aggregation induced by α-syn PFF's, in a cell culture model. In addition, we observed α-syn overexpression caused dopaminergic cell death and that this was strongly reduced by co-expression of DNAJB6b. The lesion caused by α-syn overexpression resulted in behavior deficits, which increased over time as seen in stepping test, which was rescued by co-expression of DNAJB6b. ConclusionWe here demonstrate for the first time that DNAJB6 is a strong suppressor of α-syn aggregation in cells and in animals and that this results in a suppression of dopaminergic cell death and PD related motor deficits in an animal model of PD.

Brain iron enrichment attenuates α-synuclein spreading after injection of preformed fibrils.

Regional iron accumulation and α-synuclein (α-syn) spreading pathology within the central nervous system are common pathological findings in Parkinson's disease (PD). Whereas iron is known to bind to α-syn, facilitating its aggregation and regulating α-syn expression, it remains unclear if and how iron also modulates α-syn spreading. To elucidate the influence of iron on the propagation of α-syn pathology, we investigated α-syn spreading after stereotactic injection of α-syn preformed fibrils (PFFs) into the striatum of mouse brains after neonatal brain iron enrichment. C57Bl/6J mouse pups received oral gavage with 60, 120, or 240mg/kg carbonyl iron or vehicle between postnatal days 10 and 17. At 12weeks of age, intrastriatal injections of 5-µg PFFs were performed to induce seeding of α-syn aggregates. At 90days post-injection, PFFs-injected mice displayed long-term memory deficits, without affection of motor behavior. Interestingly, quantification of α-syn phosphorylated at S129 showed reduced α-syn pathology and attenuated spreading to connectome-specific brain regions after brain iron enrichment. Furthermore, PFFs injection caused intrastriatal microglia accumulation, which was alleviated by iron in a dose-dependent way. In primary cortical neurons in a microfluidic chamber model in vitro, iron application did not alter trans-synaptic α-syn propagation, possibly indicating an involvement of non-neuronal cells in this process. Our study suggests that α-syn PFFs may induce cognitive deficits in mice independent of iron. However, a redistribution of α-syn aggregate pathology and reduction of striatal microglia accumulation in the mouse brain may be mediated via iron-induced alterations of the brain connectome.

Lovastatin Alleviates α-Synuclein Aggregation and Phosphorylation in Cellular Models of Synucleinopathy.

Parkinson’s disease (PD) is one of the most common neurodegenerative diseases. Pathologically, it is characterized by the aberrant aggregation of α-synuclein (α-syn) in neurons. Clinical evidence shows that patients with hypercholesterolemia are more likely to get PD, while lovastatin users have a lower risk of suffering from it. In this study, we investigated the effects of lovastatin on the aggregation and phosphorylation of α-syn in vitro. Our results demonstrate that α-syn preformed fibrils induce the phosphorylation and aggregation of α-syn in HEK293 cells stably transfected with α-syn-GFP and SH-SY5Y cells as well, which could be attenuated by in a concentration-dependent manner. Besides, lovastatin inhibited oxidative stress, histone acetylation, and the activation of casein kinase 2 (CK2). Collectively, lovastatin alleviates α-syn aggregation and phosphorylation in cellular models of synucleinopathy, indicating its potential value of being adopted in the management of PD.

PKC Delta Activation Promotes Endoplasmic Reticulum Stress (ERS) and NLR Family Pyrin Domain-Containing 3 (NLRP3) Inflammasome Activation Subsequent to Asynuclein-Induced Microglial Activation: Involvement of Thioredoxin-Interacting Protein (TXNIP)/Thioredoxin (Trx) Redoxisome Pathway.

A classical hallmark of Parkinson’s disease (PD) pathogenesis is the accumulation of misfolded alpha-synuclein (αSyn) within Lewy bodies and Lewy neurites, although its role in microglial dysfunction and resultant dopaminergic (DAergic) neurotoxicity is still elusive. Previously, we identified that protein kinase C delta (PKCδ) is activated in post mortem PD brains and experimental Parkinsonism and that it participates in reactive microgliosis; however, the relationship between PKCδ activation, endoplasmic reticulum stress (ERS) and the reactive microglial activation state in the context of α-synucleinopathy is largely unknown. Herein, we show that oxidative stress, mitochondrial dysfunction, NLR family pyrin domain containing 3 (NLRP3) inflammasome activation, and PKCδ activation increased concomitantly with ERS markers, including the activating transcription factor 4 (ATF-4), serine/threonine-protein kinase/endoribonuclease inositol-requiring enzyme 1α (p-IRE1α), p-eukaryotic initiation factor 2 (eIF2α) as well as increased generation of neurotoxic cytokines, including IL-1β in aggregated αSynagg-stimulated primary microglia. Importantly, in mouse primary microglia-treated with αSynagg we observed increased expression of Thioredoxin-interacting protein (TXNIP), an endogenous inhibitor of the thioredoxin (Trx) pathway, a major antioxidant protein system. Additionally, αSynagg promoted interaction between NLRP3 and TXNIP in these cells. In vitro knockdown of PKCδ using siRNA reduced ERS and led to reduced expression of TXNIP and the NLRP3 activation response in αSynagg-stimulated mouse microglial cells (MMCs). Additionally, attenuation of mitochondrial reactive oxygen species (mitoROS) via mito-apocynin and amelioration of ERS via the eIF2α inhibitor salubrinal (SAL) reduced the induction of the ERS/TXNIP/NLRP3 signaling axis, suggesting that mitochondrial dysfunction and ERS may act in concert to promote the αSynagg-induced microglial activation response. Likewise, knockdown of TXNIP by siRNA attenuated the αSynagg-induced NLRP3 inflammasome activation response. Finally, unilateral injection of αSyn preformed fibrils (αSynPFF) into the striatum of wild-type mice induced a significant increase in the expression of nigral p-PKCδ, ERS markers, and upregulation of the TXNIP/NLRP3 inflammasome signaling axis prior to delayed loss of TH+ neurons. Together, our results suggest that inhibition of ERS and its downstream signaling mediators TXNIP and NLRP3 might represent novel therapeutic avenues for ameliorating microglia-mediated neuroinflammation in PD and other synucleinopathies.

Differential seeding and propagating efficiency of \u03b1-synuclein strains generated in different conditions

BackgroundAccumulation of alpha-synuclein (α-syn) is a main pathological hallmark of Parkinson’s and related diseases, which are collectively known as synucleinopathies. Growing evidence has supported that the same protein can induce remarkably distinct pathological progresses and disease phenotypes, suggesting the existence of strain difference among α-syn fibrils. Previous studies have shown that α-syn pathology can propagate from the peripheral nervous system (PNS) to the central nervous system (CNS) in a “prion-like” manner. However, the difference of the propagation potency from the periphery to CNS among different α-syn strains remains unknown and the effect of different generation processes of these strains on the potency of seeding and propagation remains to be revealed in more detail.MethodsThree strains of preformed α-syn fibrils (PFFs) were generated in different buffer conditions which varied in pH and ionic concentrations. The α-syn PFFs were intramuscularly (IM) injected into a novel bacterial artificial chromosome (BAC) transgenic mouse line that expresses wild-type human α-syn, and the efficiency of seeding and propagation of these PFFs from the PNS to the CNS was evaluated.ResultsThe three strains of α-syn PFFs triggered distinct propagation patterns. The fibrils generated in mildly acidic buffer led to the most severe α-syn pathology, degeneration of motor neurons and microgliosis in the spinal cord.ConclusionsThe different α-syn conformers generated in different conditions exhibited strain-specific pathology and propagation patterns from the periphery to the CNS, which further supports the view that α-syn strains may be responsible for the heterogeneity of pathological features and disease progresses among synucleinopathies.

Effects of Alpha-Synuclein Targeted Antisense Oligonucleotides on Lewy Body-Like Pathology and Behavioral Disturbances Induced by Injections of Pre-Formed Fibrils in the Mouse Motor Cortex.

Background:Alpha-synuclein (αsyn) characterizes neurodegenerative diseases known as synucleinopathies. The phosphorylated form (psyn) is the primary component of protein aggregates known as Lewy bodies (LBs), which are the hallmark of diseases such as Parkinson’s disease (PD). Synucleinopathies might spread in a prion-like fashion, leading to a progressive emergence of symptoms over time. αsyn pre-formed fibrils (PFFs) induce LB-like pathology in wild-type (WT) mice, but questions remain about their progressive spread and their associated effects on behavioral performance.Objective:To characterize the behavioral, cognitive, and pathological long-term effects of LB-like pathology induced after bilateral motor cortex PFF injection in WT mice and to assess the ability of mouse αsyn-targeted antisense oligonucleotides (ASOs) to ameliorate those effects.Methods:We induced LB-like pathology in the motor cortex and connected brain regions of male WT mice using PFFs. Three months post-PFF injection (mpi), we assessed behavioral and cognitive performance. We then delivered a targeted ASO via the ventricle and assessed behavioral and cognitive performance 5 weeks later, followed by pathological analysis.Results:At 3 and 6 mpi, PFF-injected mice showed mild, progressive behavioral deficits. The ASO reduced total αsyn and psyn protein levels, and LB-like pathology, but was also associated with some deleterious off-target effects not involving lowering of αsyn, such as a decline in body weight and impairments in motor function.Conclusions:These results increase understanding of the progressive nature of the PFF model and support the therapeutic potential of ASOs, though more investigation into effects of ASO-mediated reduction in αsyn on brain function is needed.

α-Synuclein A53T Binds to Transcriptional Adapter 2-Alpha and Blocks Histone H3 Acetylation.

α-Synuclein (α-syn) is a hallmark amyloidogenic protein component of Lewy bodies in dopaminergic neurons affected by Parkinson’s disease (PD). Despite the multi-faceted gene regulation of α-syn in the nucleus, the mechanism underlying α-syn crosstalk in chromatin remodeling in PD pathogenesis remains elusive. Here, we identified transcriptional adapter 2-alpha (TADA2a) as a novel binding partner of α-syn using the BioID system. TADA2a is a component of the p300/CBP-associated factor and is related to histone H3/H4 acetylation. We found that α-syn A53T was more preferentially localized in the nucleus than the α-syn wild-type (WT), leading to a stronger disturbance of TADA2a. Indeed, α-syn A53T significantly reduced the level of histone H3 acetylation in SH-SY5Y cells; its reduction was also evident in the striatum (STR) and substantia nigra (SN) of mice that were stereotaxically injected with α-syn preformed fibrils (PFFs). Interestingly, α-syn PFF injection resulted in a decrease in TADA2a in the STR and SN of α-syn PFF-injected mice. Furthermore, the levels of TADA2a and acetylated histone H3 were significantly decreased in the SN of patients with PD. Therefore, histone modification through α-syn A53T-TADA2a interaction may be associated with α-syn-mediated neurotoxicity in PD pathology.

Multiple system atrophy-associated oligodendroglial protein p25\u03b1 stimulates formation of novel \u03b1-synuclein strain with enhanced neurodegenerative potential

Pathology consisting of intracellular aggregates of alpha-Synuclein (α-Syn) spread through the nervous system in a variety of neurodegenerative disorders including Parkinson’s disease, dementia with Lewy bodies, and multiple system atrophy. The discovery of structurally distinct α-Syn polymorphs, so-called strains, supports a hypothesis where strain-specific structures are templated into aggregates formed by native α-Syn. These distinct strains are hypothesised to dictate the spreading of pathology in the tissue and the cellular impact of the aggregates, thereby contributing to the variety of clinical phenotypes. Here, we present evidence of a novel α-Syn strain induced by the multiple system atrophy-associated oligodendroglial protein p25α. Using an array of biophysical, biochemical, cellular, and in vivo analyses, we demonstrate that compared to α-Syn alone, a substoichiometric concentration of p25α redirects α-Syn aggregation into a unique α-Syn/p25α strain with a different structure and enhanced in vivo prodegenerative properties. The α-Syn/p25α strain induced larger inclusions in human dopaminergic neurons. In vivo, intramuscular injection of preformed fibrils (PFF) of the α-Syn/p25α strain compared to α-Syn PFF resulted in a shortened life span and a distinct anatomical distribution of inclusion pathology in the brain of a human A53T transgenic (line M83) mouse. Investigation of α-Syn aggregates in brain stem extracts of end-stage mice demonstrated that the more aggressive phenotype of the α-Syn/p25α strain was associated with an increased load of α-Syn aggregates based on a Förster resonance energy transfer immunoassay and a reduced α-Syn aggregate seeding activity based on a protein misfolding cyclic amplification assay. When injected unilaterally into the striata of wild-type mice, the α-Syn/p25α strain resulted in a more-pronounced motoric phenotype than α-Syn PFF and exhibited a “tropism” for nigro-striatal neurons compared to α-Syn PFF. Overall, our data support a hypothesis whereby oligodendroglial p25α is responsible for generating a highly prodegenerative α-Syn strain in multiple system atrophy.