Beyond acute infection: mechanisms underlying post-acute sequelae of COVID-19 (PASC).

Immune dysregulation is a key aspect of post-acute sequelae of coronavirus disease 2019 (PASC), also known as long COVID, with sustained activation of immune cells, T cell exhaustion, skewed B cell profiles, and disrupted immune communication thereby resulting in autoimmune-related complications. The gut is emerging as a critical link between microbiota, metabolism and overall dysfunction, potentially sharing similarities with other chronic fatigue conditions and PASC. Immunothrombosis and neurological signalling dysfunction emphasise the complex interplay between the immune system, blood clotting, and the central nervous system in the context of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Clear research gaps in the design of PASC studies, especially in the context of longitudinal research, stand out as significant areas of concern.

Early existence and biochemical evolution characterise acutely synaptotoxic PrPSc.

Although considerable evidence supports that misfolded prion protein (PrPSc) is the principal component of "prions", underpinning both transmissibility and neurotoxicity, clear consensus around a number of fundamental aspects of pathogenesis has not been achieved, including the time of appearance of neurotoxic species during disease evolution. Utilizing a recently reported electrophysiology paradigm, we assessed the acute synaptotoxicity of ex vivo PrPSc prepared as crude homogenates from brains of M1000 infected wild-type mice (cM1000) harvested at time-points representing 30%, 50%, 70% and 100% of the terminal stage of disease (TSD). Acute synaptotoxicity was assessed by measuring the capacity of cM1000 to impair hippocampal CA1 region long-term potentiation (LTP) and post-tetanic potentiation (PTP) in explant slices. Of particular note, cM1000 from 30% of the TSD was able to cause significant impairment of LTP and PTP, with the induced failure of LTP increasing over subsequent time-points while the capacity of cM1000 to induce PTP failure appeared maximal even at this early stage of disease progression. Evidence that the synaptotoxicity directly related to PrP species was demonstrated by the significant rescue of LTP dysfunction at each time-point through immuno-depletion of >50% of total PrP species from cM1000 preparations. Moreover, similar to our previous observations at the terminal stage of M1000 prion disease, size fractionation chromatography revealed that capacity for acute synpatotoxicity correlated with predominance of oligomeric PrP species in infected brains across all time points, with the profile appearing maximised by 50% of the TSD. Using enhanced sensitivity western blotting, modestly proteinase K (PK)-resistant PrPSc was detectable at very low levels in cM1000 at 30% of the TSD, becoming robustly detectable by 70% of the TSD at which time substantial levels of highly PK-resistant PrPSc was also evident. Further illustrating the biochemical evolution of acutely synaptotoxic species the synaptotoxicity of cM1000 from 30%, 50% and 70% of the TSD, but not at 100% TSD, was abolished by digestion of immuno-captured PrP species with mild PK treatment (5µg/ml for an hour at 37°C), demonstrating that the predominant synaptotoxic PrPSc species up to and including 70% of the TSD were proteinase-sensitive. Overall, these findings in combination with our previous assessments of transmitting prions support that synaptotoxic and infectious M1000 PrPSc species co-exist from at least 30% of the TSD, simultaneously increasing thereafter, albeit with eventual plateauing of transmitting conformers.

PrPSc Oligomerization Appears Dynamic, Quickly Engendering Inherent M1000 Acute Synaptotoxicity.

Prion diseases are neurodegenerative disorders pathogenically linked to cellular prion protein (PrPC) misfolding into abnormal conformers (PrPSc), with PrPSc underpinning both transmission and synaptotoxicity. Although the biophysical features of PrPSc required to induce acute synaptic dysfunction remain incompletely defined, we recently reported that acutely synaptotoxic PrPSc appeared to be oligomeric. We herein provide further insights into the kinetic and requisite biophysical characteristics of acutely synaptotoxic ex vivo PrPSc derived from the brains of mice dying from M1000 prion disease. Pooled fractions of M1000 PrPSc located within the molecular weight range approximating monomeric PrP (mM1000) generated through size exclusion chromatography were found to harbor acute synaptotoxicity equivalent to preformed oligomeric fractions (oM1000). Subsequent investigation showed mM1000 corresponded to PrPSc rapidly concatenating in physiological buffer to exist as predominantly, closely associated, small oligomers. The oligomerization of PrP in mM1000 could be substantially mitigated by treatment with the antiaggregation compound epigallocatechin gallate, thereby maintaining the PrPSc as primarily nonoligomeric with completely abrogated acute synaptotoxicity; moreover, despite epigallocatechin gallate treatment, pooled oM1000 remained oligomeric and acutely synaptotoxic. A similar tendency to rapid formation of oligomers was observed for PrPC when monomeric fractions derived from size exclusion chromatography of normal brain homogenates (mNBH) were pooled, but neither mNBH nor preformed higher-order NBH complexes (oNBH) were acutely synaptotoxic. Oligomers formed from mNBH could be reduced to mainly monomers (<100 kDa) after enzymatic digestion of nucleic acids, whereas higher-order PrP assemblies derived from pooled mM1000, oM1000, and oNBH resisted such treatment. Collectively, these findings support that oligomerization of PrPSc into small multimeric assemblies appears to be a critical biophysical feature for engendering inherent acute synaptotoxicity, with preformed oligomers found in oM1000 appearing to be stable, tightly self-associated ensembles that coexist in dynamic equilibrium with mM1000, with the latter appearing capable of rapid aggregation, albeit initially forming smaller, weakly self-associated, acutely synaptotoxic oligomers.

The role of lipids in α-synuclein misfolding and neurotoxicity.

The misfolding and aggregation of α-synuclein (αsyn) in the central nervous system is associated with a group of neurodegenerative disorders referred to as the synucleinopathies. In addition to being a pathological hallmark of disease, it is now well-established that upon misfolding, αsyn acquires pathogenic properties, such as neurotoxicity, that can contribute to disease development. The mechanisms that produce αsyn misfolding and the molecular events underlying the neuronal damage caused by these misfolded species are not well-defined. A consistent observation that may be relevant to αsyn's pathogenicity is its ability to associate with lipids. This appears important not only to how αsyn aggregates, but also to the mechanism by which the misfolded protein causes intracellular damage. This review discusses the current literature reporting a role of lipids in αsyn misfolding and neurotoxicity in various synucleinopathy disorders and provides an overview of current methods to assess protein misfolding and pathogenicity both in vitro and in vivo.

Pathogenic mechanisms of prion protein, amyloid-ß and α-synuclein misfolding: the prion concept and neurotoxicity of protein oligomers.

Proteinopathies represent a group of diseases characterized by the unregulated misfolding and aggregation of proteins. Accumulation of misfolded protein in the central nervous system (CNS) is associated with neurodegenerative diseases, such as the transmissible spongiform encephalopathies (or prion diseases), Alzheimer's disease, and the synucleinopathies (the most common of which is Parkinson's disease). Of these, the pathogenic mechanisms of prion diseases are particularly striking where the transmissible, causative agent of disease is the prion, or proteinaceous infectious particle. Prions are composed almost exclusively of PrPSc ; a misfolded isoform of the normal cellular protein, PrPC , which is found accumulated in the CNS in disease. Today, mounting evidence suggests other aggregating proteins, such as amyloid-ß (Aß) and α-synuclein (α-syn), proteins associated with Alzheimer's disease and synucleinopathies, respectively, share similar biophysical and biochemical properties with PrPSc that influences how they misfold, aggregate, and propagate in disease. In this regard, the definition of a 'prion' may ultimately expand to include other pathogenic proteins. Unifying knowledge of folded proteins may also reveal common mechanisms associated with other features of disease that are less understood, such as neurotoxicity. This review discusses the common features Aß and α-syn share with PrP and neurotoxic mechanisms associated with these misfolded proteins. Several proteins are known to misfold and accumulate in the central nervous system causing a range of neurodegenerative diseases, such as Alzheimer's, Parkinson's, and the prion diseases. Prions are transmissible misfolded conformers of the prion protein, PrP, which seed further generation of infectious proteins. Similar effects have recently been observed in proteins associated with Alzheimer's disease and the synucleinopathies, leading to the proposition that the definition of a 'prion' may ultimately expand to include other pathogenic proteins. Unifying knowledge of misfolded proteins may also reveal common mechanisms associated with other features of disease that are less understood, such as neurotoxicity.

Prion infection impairs cholesterol metabolism in neuronal cells.

Conversion of prion protein (PrP(C)) into a pathological isoform (PrP(Sc)) during prion infection occurs in lipid rafts and is dependent on cholesterol. Here, we show that prion infection increases the abundance of cholesterol transporter, ATP-binding cassette transporter type A1 (ATP-binding cassette transporter type A1), but reduces cholesterol efflux from neuronal cells leading to the accumulation of cellular cholesterol. Increased abundance of ABCA1 in prion disease was confirmed in prion-infected mice. Mechanistically, conversion of PrP(C) to the pathological isoform led to PrP(Sc) accumulation in rafts, displacement of ABCA1 from rafts and the cell surface, and enhanced internalization of ABCA1. These effects were abolished with reversal of prion infection or by loading cells with cholesterol. Stimulation of ABCA1 expression with liver X receptor agonist or overexpression of heterologous ABCA1 reduced the conversion of prion protein into the pathological form upon infection. These findings demonstrate a reciprocal connection between prion infection and cellular cholesterol metabolism, which plays an important role in the pathogenesis of prion infection in neuronal cells.

Glycosaminoglycan sulfation determines the biochemical properties of prion protein aggregates.

Prion diseases are transmissible neurodegenerative disorders associated with the conversion of the cellular prion protein, PrP(C), to a misfolded isoform called PrP(Sc). Although PrP(Sc) is a necessary component of the infectious prion, additional factors, or cofactors, have been shown to contribute to the efficient formation of transmissible PrP(Sc). Glycosaminoglycans (GAGs) are attractive cofactor candidates as they can be found associated with PrP(Sc) deposits, have been shown to enhance PrP misfolding in vitro, are found in the same cellular compartments as PrP(C) and have been shown to be disease modifying in vivo. Here we investigated the effects of the sulfated GAGs, heparin and heparan sulfate (HS), on disease associated misfolding of full-length recombinant PrP. More specifically, the degree of sulfation of these molecules was investigated for its role in modulating the disease-associated characteristics of PrP. Both heparin and HS induced a ß-sheet conformation in recombinant PrP that was associated with the formation of aggregated species; however, the biochemical properties of the aggregates formed in the presence of heparin or HS varied in solubility and protease resistance. Furthermore, these properties could be modified by changes in GAG sulfation, indicating that subtle changes in the properties of prion disease cofactors could initiate disease associated misfolding.

Pathogenic mutations within the hydrophobic domain of the prion protein lead to the formation of protease-sensitive prion species with increased lethality.

UNLABELLED: Prion diseases are a group of fatal and incurable neurodegenerative diseases affecting both humans and animals. The principal mechanism of these diseases involves the misfolding the host-encoded cellular prion protein, PrP(C), into the disease-associated isoform, PrP(Sc). Familial forms of human prion disease include those associated with the mutations G114V and A117V, which lie in the hydrophobic domain of PrP. Here we have studied the murine homologues (G113V and A116V) of these mutations using cell-based and animal models of prion infection. Under normal circumstances, the mutant forms of PrP(C) share similar processing, cellular localization, and physicochemical properties with wild-type mouse PrP (MoPrP). However, upon exposure of susceptible cell lines expressing these mutants to infectious prions, very low levels of protease-resistant aggregated PrP(Sc) are formed. Subsequent mouse bioassay revealed high levels of infectivity present in these cells. Thus, these mutations appear to limit the formation of aggregated PrP(Sc), giving rise to the accumulation of a relatively soluble, protease sensitive, prion species that is highly neurotoxic. Given that these mutations lie next to the glycine-rich region of PrP that can abrogate prion infection, these findings provide further support for small, protease-sensitive prion species having a significant role in the progression of prion disease and that the hydrophobic domain is an important determinant of PrP conversion. IMPORTANCE: Prion diseases are transmissible neurodegenerative diseases associated with an infectious agent called a prion. Prions are comprised of an abnormally folded form of the prion protein (PrP) that is normally resistant to enzymes called proteases. In humans, prion disease can occur in individuals who inherited mutations in the prion protein gene. Here we have studied the effects of two of these mutations and show that they influence the properties of the prions that can be formed. We show that the mutants make highly infectious prions that are more sensitive to protease treatment. This study highlights a certain region of the prion protein as being involved in this effect and demonstrates that prions are not always resistant to protease treatment.

The prion protein preference of sporadic Creutzfeldt-Jakob disease subtypes.

Sporadic Creutzfeldt-Jakob disease (CJD) is the most prevalent manifestation of the transmissible spongiform encephalopathies or prion diseases affecting humans. The disease encompasses a spectrum of clinical phenotypes that have been correlated with molecular subtypes that are characterized by the molecular mass of the protease-resistant fragment of the disease-related conformation of the prion protein and a polymorphism at codon 129 of the gene encoding the prion protein. A cell-free assay of prion protein misfolding was used to investigate the ability of these sporadic CJD molecular subtypes to propagate using brain-derived sources of the cellular prion protein (PrP(C)). This study confirmed the presence of three distinct sporadic CJD molecular subtypes with PrP(C) substrate requirements that reflected their codon 129 associations in vivo. However, the ability of a sporadic CJD molecular subtype to use a specific PrP(C) substrate was not determined solely by codon 129 as the efficiency of prion propagation was also influenced by the composition of the brain tissue from which the PrP(C) substrate was sourced, thus indicating that nuances in PrP(C) or additional factors may determine sporadic CJD subtype. The results of this study will aid in the design of diagnostic assays that can detect prion disease across the diversity of sporadic CJD subtypes.

Prion-infected cells regulate the release of exosomes with distinct ultrastructural features.

Exosomes are small membrane-bound vesicles released from cells and found in vivo in most biological fluids. Functions reported for exosomes include cell-cell communication, roles in modulating immune responses, and roles in the transfer of pathogens such as prions. Here we investigated the molecular characteristics of the structure of exosomes that harbor prion infectivity to determine the native structure of exosomes and whether infected exosomes have a distinct structure. Cryo-electron tomography revealed the previously unidentified ultrastructural detail of exosomes with high resolution. Exosomes were found to be naturally spherical in shape and to have a diverse population that varies in size and internal structure, such as differences in the number of membrane structures. Exosomes isolated from prion-infected cells contained a significantly different population of exosomes with distinct structural features compared to control vesicles from mock-infected cells. Exosomes are highly structured vesicles that can modify their structure on altering their protein cargo. This finding provides further insight into the role that the exosomal protein cargo plays on influencing the structure of the vesicles as well as highlighting the diversity of exosomes and their relationship to biological processes.

Conservation of a glycine-rich region in the prion protein is required for uptake of prion infectivity.

Prion diseases are associated with the misfolding of the endogenously expressed prion protein (designated PrP(C)) into an abnormal isoform (PrP(Sc)) that has infectious properties. The hydrophobic domain of PrP(C) is highly conserved and contains a series of glycine residues that show perfect conservation among all species, strongly suggesting it has functional and evolutionary significance. These glycine residues appear to form repeats of the GXXXG protein-protein interaction motif (two glycines separated by any three residues); the retention of these residues is significant and presumably relates to the functionality of PrP(C). Mutagenesis studies demonstrate that minor alterations to this highly conserved region of PrP(C) drastically affect the ability of cells to uptake and replicate prion infection in both cell and animal bioassay. The localization and processing of mutant PrP(C) are not affected, although in vitro and in vivo studies demonstrate that this region is not essential for interaction with PrP(Sc), suggesting these residues provide conformational flexibility. These data suggest that this region of PrP(C) is critical in the misfolding process and could serve as a novel, species-independent target for prion disease therapeutics.

Residues surrounding the glycosylphosphatidylinositol anchor attachment site of PrP modulate prion infection: insight from the resistance of rabbits to prion disease.

Prion diseases are a group of transmissible, invariably fatal neurodegenerative diseases that affect both humans and animals. According to the protein-only hypothesis, the infectious agent is a prion (proteinaceous infectious particle) that is composed primarily of PrP(Sc), the disease-associated isoform of the cellular prion protein, PrP. PrP(Sc) arises from the conformational change of the normal, glycosylphosphatidylinositol (GPI)-anchored protein, PrP(C). The mechanism by which this process occurs, however, remains enigmatic. Rabbits are one of a small number of mammalian species reported to be resistant to prion infection. Sequence analysis of rabbit PrP revealed that its C-terminal amino acids differ from those of PrP from other mammals and may affect the anchoring of rabbit PrP through its GPI anchor. Using a cell culture model, this study investigated the effect of the rabbit PrP-specific C-terminal amino acids on the addition of the GPI anchor to PrP(C), PrP(C) localization, and PrP(Sc) formation. The incorporation of rabbit-specific C-terminal PrP residues into mouse PrP did not affect the addition of a GPI anchor or the localization of PrP. However, these residues did inhibit PrP(Sc) formation, suggesting that these rabbit-specific residues interfere with a C-terminal PrP(Sc) interaction site.

The brain to gut pathway: a possible route of prion transmission.

OBJECTIVE: The intestine is recognised to play a key role in the transmission of prion diseases. These diseases are associated with pathological isoforms (PrP(Sc)) of the normal cellular prion protein (PrP(C)) and can be transmitted between individuals or arise spontaneously. The brain, as the primary site of prion replication, could provide infectious prions to peripheral tissues. Here, we examine whether the brain is a source of intestinal prion accumulation. METHODS: Following intracerebral inoculation with human origin prions the ileums of BalbC mice with clinical prion disease were assessed by Western immunoblot and immunohistochemical analysis for the presence of PrP(Sc) and the survival of enteric glial cells (EGCs) and specific neuronal subpopulations in the myenteric and submucosal plexus. RESULTS: PrP(Sc) was detected in the ileum of 13/13 mice following intracerebral inoculation with prions and 0/4 saline-inoculated mice. PrP(Sc) was localised at detectable levels in the Peyer's patches of infected mice. Investigation of neuronal subpopulations revealed a significant decrease in neurofilament reactive neurons (11±8%, p<0.05, n=5) compared with saline-inoculated mice (23±5%, n=3). Neuronal nitric oxide synthase (nNOS) and tyrosine hydroxylase reactive neurons were decreased in some (2 of 4 and 1 of 3, respectively) but not all prion-infected mice, whereas calretinin and vasoactive intestinal peptide reactive neurons were unaffected. EGCs were highly distorted in circumscribed ganglia of the myenteric plexus. In areas of glial derangement, the neurons showed undefined outlines and faint cytoplasmic immunoreactivity for the pan-neuronal marker Hu and loss of nNOS reactivity. CONCLUSIONS: The present work shows that PrP(Sc) can be transmitted from the brain to the intestine. This causes pathological changes in enteric glia and neurons. We conclude that PrP(Sc) of brain origin finds a substrate in the naturally occurring PrP(C) of EGCs and neurons. This results in a reservoir of PrP(Sc) in the intestine, which may represent a source of prion disease transmission through surgical procedures and environmental contamination.

Distribution of microRNA profiles in pre-clinical and clinical forms of murine and human prion disease.

Prion diseases are distinguished by long pre-clinical incubation periods during which prions actively propagate in the brain and cause neurodegeneration. In the pre-clinical stage, we hypothesize that upon prion infection, transcriptional changes occur that can lead to early neurodegeneration. A longitudinal analysis of miRNAs in pre-clinical and clinical forms of murine prion disease demonstrated dynamic expression changes during disease progression in the affected thalamus region and serum. Serum samples at each timepoint were collected whereby extracellular vesicles (EVs) were isolated and used to identify blood-based biomarkers reflective of pathology in the brain. Differentially expressed EV miRNAs were validated in human clinical samples from patients with human sporadic Creutzfeldt-Jakob disease (sCJD), with the molecular subtype at codon 129 either methionine-methionine (MM, n = 14) or valine-valine (VV, n = 12) compared to controls (n = 20). EV miRNA biomarkers associated with prion infection predicted sCJD with an AUC of 0.800 (85% sensitivity and 66.7% specificity) in a second independent validation cohort (n = 26) of sCJD and control patients with MM or VV subtype. This study discovered clinically relevant miRNAs that benefit diagnostic development to detect prion-related diseases and therapeutic development to inhibit prion infectivity.

ATH434 Reverses Colorectal Dysfunction in the A53T Mouse Model of Parkinson's Disease.

BACKGROUND: Gastrointestinal (GI) complications, that severely impact patient quality of life, are a common occurrence in patients with Parkinson's disease (PD). Damage to enteric neurons and the accumulation of alpha-synuclein in the enteric nervous system (ENS) are thought to contribute to this phenotype. Copper or iron chelators, that bind excess or labile metal ions, can prevent aggregation of alpha-synuclein in the brain and alleviate motor-symptoms in preclinical models of PD. OBJECTIVE: We investigated the effect of ATH434 (formally PBT434), a small molecule, orally bioavailable, moderate-affinity iron chelator, on colonic propulsion and whole gut transit in A53T alpha-synuclein transgenic mice. METHODS: Mice were fed ATH434 (30 mg/kg/day) for either 4 months (beginning at ∼15 months of age), after the onset of slowed propulsion ("treatment group"), or for 3 months (beginning at ∼12 months of age), prior to slowed propulsion ("prevention group"). RESULTS: ATH434, given after dysfunction was established, resulted in a reversal of slowed colonic propulsion and gut transit deficits in A53T mice to WT levels. In addition, ATH434 administered from 12 months prevented the slowed bead expulsion at 15 months but did not alter deficits in gut transit time when compared to vehicle-treated A53T mice. The proportion of neurons with nuclear Hu+ translocation, an indicator of neuronal stress in the ENS, was significantly greater in A53T than WT mice, and was reduced in both groups when ATH434 was administered. CONCLUSION: ATH434 can reverse some of the GI deficits and enteric neuropathy that occur in a mouse model of PD, and thus may have potential clinical benefit in alleviating the GI dysfunctions associated with PD.

Strain variation in treatment and prevention of human prion diseases.

Transmissible spongiform encephalopathies or prion diseases describe a number of different human disorders that differ in their clinical phenotypes, which are nonetheless united by their transmissible nature and common pathology. Clinical variation in the absence of a conventional infectious agent is believed to be encoded by different conformations of the misfolded prion protein. This misfolded protein is the target of methods designed to prevent disease transmission in a surgical setting and reduction of the misfolded seed or preventing its continued propagation have been the focus of therapeutic strategies. It is therefore possible that strain variation may influence the efficacy of prevention and treatment approaches. Historically, an understanding of prion disease transmission and pathogenesis has been focused on research tools developed using agriculturally relevant strains of prion disease. However, an increased understanding of the molecular biology of human prion disorders has highlighted differences not only between different forms of the disease affecting humans and animals but also within diseases such as Creutzfeldt-Jakob Disease (CJD), which is represented by several sporadic CJD specific conformations and an additional conformation associated with variant CJD. In this chapter we will discuss whether prion strain variation can affect the efficacy of methods used to decontaminate prions and whether strain variation in pre-clinical models of prion disease can be used to identify therapeutic strategies that have the best possible chance of success in the clinic.

Markers of A1 astrocytes stratify to molecular sub-types in sporadic Creutzfeldt-Jakob disease brain.

Astrocytes are glial cells of the central nervous system that become reactive under conditions of stress. The functional properties of reactive astrocytes depend on their stimulus that induces the upregulation of specific genes. Reactive astrocytes are a neuropathological feature of prion disorders; however, their role in the disease pathogenesis is not well understood. Here, we describe our studies of one polarization state of reactive astrocytes, termed A1 astrocytes, in the frontal cortex region of 35 human sporadic Creutzfeldt-Jakob disease brains encompassing a range of molecular sub-types. Examination of two mRNA markers of A1 astrocytes, C3 and GBP2, revealed a strong linear correlation between the two following their log-normalization (P = 0.0011). Both markers were found upregulated in the sporadic Creutzfeldt-Jakob disease brain compared with age-matched control tissues (P = 0.0029 and 0.0002, for C3log and GBP2log, respectively), and stratifying samples based on codon 129 genotype revealed that C3log is highest in homozygous methionine and lowest in homozygous valine patients, which followed a linear trend (P = 0.027). Upon assessing other disease parameters, a significant positive correlation was found between GBP2log and disease duration (P = 0.031). These findings provide evidence for a divergence in the astrocytic environment amongst patients with sporadic Creutzfeldt-Jakob disease based on molecular sub-type parameters of disease. While more research will be needed to determine the global changes in the genomic profiles and resulting functional properties of reactive astrocytes in disease, considering the evidence demonstrating that A1 astrocytes harbour neurotoxic properties, the changes seen in C3log and GBP2log in the current study may reflect differences in pathogenic mechanisms amongst the sporadic Creutzfeldt-Jakob disease sub-types associated with the A1 polarization state.

Misfolded α-synuclein causes hyperactive respiration without functional deficit in live neuroblastoma cells.

The misfolding and aggregation of the largely disordered protein, α-synuclein, is a central pathogenic event that occurs in the synucleinopathies, a group of neurodegenerative disorders that includes Parkinson's disease. While there is a clear link between protein misfolding and neuronal vulnerability, the precise pathogenic mechanisms employed by disease-associated α-synuclein are unresolved. Here, we studied the pathogenicity of misfolded α-synuclein produced using the protein misfolding cyclic amplification (PMCA) assay. To do this, previous published methods were adapted to allow PMCA-induced protein fibrillization to occur under non-toxic conditions. Insight into potential intracellular targets of misfolded α-synuclein was obtained using an unbiased lipid screen of 15 biologically relevant lipids that identified cardiolipin (CA) as a potential binding partner for PMCA-generated misfolded α-synuclein. To investigate whether such an interaction can impact the properties of α-synuclein misfolding, protein fibrillization was carried out in the presence of the lipid. We show that CA both accelerates the rate of α-synuclein fibrillization and produces species that harbour enhanced resistance to proteolysis. Because CA is virtually exclusively expressed in the inner mitochondrial membrane, we then assessed the ability of these misfolded species to alter mitochondrial respiration in live non-transgenic SH-SY5Y neuroblastoma cells. Extensive analysis revealed that misfolded α-synuclein causes hyperactive mitochondrial respiration without causing any functional deficit. These data give strong support for the mitochondrion as a target for misfolded α-synuclein and reveal persistent, hyperactive respiration as a potential upstream pathogenic event associated with the synucleinopathies.This article has an associated First Person interview with the first author of the paper.

An intact membrane is essential for small extracellular vesicle-induced modulation of α-synuclein fibrillization.

The misfolding and fibrillization of the protein, α-synuclein (αsyn), is associated with neurodegenerative disorders referred to as the synucleinopathies. Understanding the mechanisms of αsyn misfolding is an important area of interest given that αsyn misfolding contributes to disease pathogenesis. While many studies report the ability of synthetic lipid membranes to modulate αsyn folding, there is little data pertaining to the mechanism(s) of this interaction. αSyn has previously been shown to associate with small lipid vesicles released by cells called extracellular vesicles (EVs) and it is postulated these interactions may assist in the spreading of pathological forms of this protein. Together, this presents the need for robust characterisation studies on αsyn fibrillization using biologically-derived vesicles. In this study, we comprehensively characterised the ability of lipid-rich small extracellular vesicles (sEVs) to alter the misfolding of αsyn induced using the Protein Misfolding Cyclic Amplification (PMCA) assay. The biochemical and biophysical properties of misfolded αsyn were examined using a range of techniques including: Thioflavin T fluorescence, transmission electron microscopy, analytical centrifugation and western immunoblot coupled with protease resistance assays and soluble/insoluble fractionation. We show that sEVs cause an acceleration in αsyn fibrillization and provide comprehensive evidence that this results in an increase in the abundance of mature insoluble fibrillar species. In order to elucidate the relevance of the lipid membrane to this interaction, sEV lipid membranes were modified by treatment with methanol, or a combination of methanol and sarkosyl. These treatments altered the ultrastructure of the sEVs without changing the protein cargo. Critically, these modified sEVs had a reduced ability to influence αsyn fibrillization compared to untreated counterparts. This study reports the first comprehensive examination of αsyn:EV interactions and demonstrates that sEVs are powerful modulators of αsyn fibrillization, which is mediated by the sEV membrane. In doing so, this work provides strong evidence for a role of sEVs in contributing directly to αsyn misfolding in the synucleinopathy disorders.

Chronic isolation stress is associated with increased colonic and motor symptoms in the A53T mouse model of Parkinson's disease.

BACKGROUND: Chronic stress exacerbates motor deficits and increases dopaminergic cell loss in several rodent models of Parkinson's disease (PD). However, little is known about effects of stress on gastrointestinal (GI) dysfunction, a common non-motor symptom of PD. We aimed to determine whether chronic stress exacerbates GI dysfunction in the A53T mouse model of PD and whether this relates to changes in α-synuclein distribution. METHODS: Chronic isolation stress was induced by single-housing WT and homozygote A53T mice between 5 and 15 months of age. GI and motor function were compared with mice that had been group-housed. KEY RESULTS: Chronic isolation stress increased plasma corticosterone and exacerbated deficits in colonic propulsion and whole-gut transit in A53T mice and also increased motor deficits. However, our results indicated that the novel environment-induced defecation response, a common method used to evaluate colorectal function, was not a useful test to measure exacerbation of GI dysfunction, most likely because of the reported reduced level of anxiety in A53T mice. A53T mice had lower corticosterone levels than WT mice under both housing conditions, but single-housing increased levels for both genotypes. Enteric neuropathy was observed in aging A53T mice and A53T mice had a greater accumulation of alpha-synuclein (αsyn) in myenteric ganglia under both housing conditions. CONCLUSIONS & INFERENCES: Chronic isolation stress exacerbates PD-associated GI dysfunction, in addition to increasing motor deficits. However, these changes in GI symptoms are not directly related to corticosterone levels, worsened enteric neuropathy, or enteric αsyn accumulation.