Carboxyl truncation of α-synuclein occurs early and is influenced by human APOE genotype in transgenic mouse models of α-synuclein pathogenesis.

Post-translational modifications to the carboxyl (C) terminus domain of α-synuclein can play an important role in promoting the pathologic aggregation of α-synuclein. Various cleavages that diminish this highly charged, proline-rich region can result in exposure of hydrophobic, aggregation-prone regions, thereby accelerating the aggregation kinetics of α-synuclein into misfolded, pathologic forms. C-terminally truncated forms of α-synuclein are abundant in human diseased brains compared to controls, suggesting a role in disease pathogenesis. Factors that alter the homeostatic proteolytic processing of α-synuclein may ultimately tip the balance towards a progressive disease state. Apolipoprotein E (APOE) has been implicated in the acceleration of cognitive impairment in patients with Lewy body diseases. The APOE4 isoform has been found to cause dysregulation in the endosomal-lysosomal pathway, which could result in altered α-synuclein degradation as a potential mechanism for promoting its pathologic misfolding. Herein, we investigate the spatiotemporal accumulation of C-terminally truncated α-synuclein in a seeded and progressive mouse model of synucleinopathy. Furthermore, we study how this process is influenced in the context of mice that are altered to express either the human APOE3 or APOE4 isoforms. We found that specific C-terminal truncation of α-synuclein occurs at early stages of pathogenesis. We also found that proteolytic processing of this domain differs across various brain regions and is influenced by the presence of different human APOE isoforms. Our data demonstrate an early pathogenic role for C-terminally truncated α-synuclein, and highlight the influence of APOE isoforms in modulating its impact.

Cellular processing of α-synuclein fibrils results in distinct physiological C-terminal truncations with a major cleavage site at residue Glu 114.

α-synuclein (αS) is an abundant, neuronal protein that assembles into fibrillar pathological inclusions in a spectrum of neurodegenerative diseases that include Lewy body diseases (LBD) and Multiple System Atrophy (MSA). The cellular and regional distributions of pathological inclusions vary widely between different synucleinopathies contributing to the spectrum of clinical presentations. Extensive cleavage within the carboxy (C)-terminal region of αS is associated with inclusion formation, although the events leading to these modifications and the implications for pathobiology are of ongoing study. αS preformed fibrils can induce prion-like spread of αS pathology in both in vitro and animal models of disease. Using C truncation-specific antibodies, we demonstrated here that prion-like cellular uptake and processing of αS preformed fibrils resulted in two major cleavages at residues 103 and 114. A third cleavage product (122 αS) accumulated upon application of lysosomal protease inhibitors. In vitro, both 1-103 and 1-114 αS polymerized rapidly and extensively in isolation and in the presence of full-length αS. 1-103 αS also demonstrated more extensive aggregation when expressed in cultured cells. Furthermore, we used novel antibodies to αS cleaved at residue Glu114, to assess x-114 αS pathology in postmortem brain tissue from patients with LBD and MSA, as well as three different transgenic αS mouse models of prion-like induction. The distribution of x-114 αS pathology was distinct from that of overall αS pathology. These studies reveal the cellular formation and behavior of αS C-truncated at residues 114 and 103 as well as the disease dependent distribution of x-114 αS pathology.

Proteinopathies and Neurotrauma: Update on Degenerative Cascades.

Neurotrauma, especially repetitive neurotrauma, is associated with the development of progressive neurodegeneration leading to chronic traumatic encephalopathy (CTE). Exposure to neurotrauma regularly occurs during sports and military service, often not requiring medical care. However, exposure to severe and/or repeated sub-clinical neurotrauma has been shown cause physical and psychological disability, leading to reduce life expectancy. Misfolding of proteins, or proteinopathy, is a pathological hallmark of CTE, in which chronic injury leads to local and diffuse protein aggregates. These aggregates are an overlapping feature of many neurodegenerative diseases such as CTE, Alzheimer's Disease, Parkinsons disease. Neurotrauma is also a significant risk factor for the development of these diseases, however the mechanism's underlying this association are not well understood. While phosphorylated tau aggregates are the primary feature of CTE, amyloid-beta, Transactive response DNA-binding protein 43 (TDP-43), and alpha-synuclein (αSyn) are also well documented. Aberrant misfolding of these proteins has been shown to disrupt brain homeostasis leading to neurodegeneration in a disease dependent manor. In CTE, the interaction between proteinopathies and their associated neurodegeneration is a current area of study. Here we provide an update on current literature surrounding the prevalence, characteristics, and pathogenesis of proteinopathies in CTE.

Unique seeding profiles and prion-like propagation of synucleinopathies are highly dependent on the host in human α-synuclein transgenic mice.

α-synuclein (αSyn) is an intrinsically disordered protein which can undergo structural transformations, resulting in the formation of stable, insoluble fibrils. αSyn amyloid-type nucleation can be induced by misfolded 'seeds' serving as a conformational template, tantamount to the prion-like mechanism. Accumulation of αSyn inclusions is a key feature of dementia with Lewy bodies (DLB) and multiple system atrophy (MSA), and are found as additional pathology in Alzheimer's disease (AD) such as AD with amygdala predominant Lewy bodies (AD/ALB). While these disorders accumulate the same pathological protein, they exhibit heterogeneity in clinical and histological features; however, the mechanism(s) underlying this variability remains elusive. Accruing data from human autopsy studies, animal inoculation modeling, and in vitro characterization experiments, have lent credence to the hypothesis that conformational polymorphism of the αSyn amyloid-type fibril structure results in distinct "strains" with categorical infectivity traits. Herein, we directly compare the seeding abilities and outcome of human brain lysates from these diseases, as well as recombinant preformed human αSyn fibrils by the intracerebral inoculation of transgenic mice overexpressing either human wild-type αSyn or human αSyn with the familial A53T mutation. Our study has revealed that the initiating inoculum heavily dictates the phenotypic and pathological course of disease. Interestingly, we have also established relevant host-dependent distinctions between propagation profiles, including burden and spread of inclusion pathology throughout the neuroaxis, as well as severity of neurological symptoms. These findings provide compelling evidence supporting the hypothesis that diverse prion-type conformers may explain the variability seen in synucleinopathies.

Targeted proteolytic products of τ and α-synuclein in neurodegeneration.

CNS pathological inclusions comprising τ or α-synuclein (αSyn) define a spectrum of neurodegenerative diseases, and these can often present concurrently in the same individuals. The aggregation of both proteins is clearly associated with neurodegeneration and the deleterious properties of each protein is further supported by mutations in each gene (MAPT and SNCA, respectively) resulting in disease. The initiating events in most sporadic neurodegenerative diseases are still unclear but growing evidence suggests that the aberrant proteolytic cleavage of τ and αSyn results in products that can be toxic and/or initiate aggregation that can further spread by a prion-like mechanism. The accumulation of some of these cleavage products can further potentiate the progression of protein aggregation transmission and lead to their accumulation in peripheral biofluids such as cerebrospinal fluid (CSF) and blood. The future development of new tools to detect specific τ and αSyn abnormal cleavage products in peripheral biofluids could be useful biomarkers and better understand of the role of unique proteolytic activities could yield therapeutic interventions.

Collusion of α-Synuclein and Aß aggravating co-morbidities in a novel prion-type mouse model.

BACKGROUND: The misfolding of host-encoded proteins into pathological prion conformations is a defining characteristic of many neurodegenerative disorders, including Alzheimer's disease, Parkinson's disease, and Lewy body dementia. A current area of intense study is the way in which the pathological deposition of these proteins might influence each other, as various combinations of co-pathology between prion-capable proteins are associated with exacerbation of disease. A spectrum of pathological, genetic and biochemical evidence provides credence to the notion that amyloid ß (Aß) accumulation can induce and promote α-synuclein pathology, driving neurodegeneration. METHODS: To assess the interplay between α-synuclein and Aß on protein aggregation kinetics, we crossed mice expressing human α-synuclein (M20) with APPswe/PS1dE9 transgenic mice (L85) to generate M20/L85 mice. We then injected α-synuclein preformed fibrils (PFFs) unilaterally into the hippocampus of 6-month-old mice, harvesting 2 or 4 months later. RESULTS: Immunohistochemical analysis of M20/L85 mice revealed that pre-existing Aß plaques exacerbate the spread and deposition of induced α-synuclein pathology. This process was associated with increased neuroinflammation. Unexpectedly, the injection of α-synuclein PFFs in L85 mice enhanced the deposition of Aß; whereas the level of Aß deposition in M20/L85 bigenic mice, injected with α-synuclein PFFs, did not differ from that of mice injected with PBS. CONCLUSIONS: These studies reveal novel and unexpected interplays between α-synuclein pathology, Aß and neuroinflammation in mice that recapitulate the pathology of Alzheimer's disease and Lewy body dementia.

Disease-, region- and cell type specific diversity of α-synuclein carboxy terminal truncations in synucleinopathies.

Synucleinopathies, including Parkinson's disease (PD), Lewy body dementia (LBD), Alzheimer's disease with amygdala restricted Lewy bodies (AD/ALB), and multiple system atrophy (MSA) comprise a spectrum of neurodegenerative disorders characterized by the presence of distinct pathological α-synuclein (αSyn) inclusions. Experimental and pathological studies support the notion that αSyn aggregates contribute to cellular demise and dysfunction with disease progression associated with a prion-like spread of αSyn aggregates via conformational templating. The initiating event(s) and factors that contribute to diverse forms of synucleinopathies remain poorly understood. A major post-translational modification of αSyn associated with pathological inclusions is a diverse array of specific truncations within the carboxy terminal region. While these modifications have been shown experimentally to induce and promote αSyn aggregation, little is known about their disease-, region- and cell type specific distribution. To this end, we generated a series of monoclonal antibodies specific to neo-epitopes in αSyn truncated after residues 103, 115, 119, 122, 125, and 129. Immunocytochemical investigations using these new tools revealed striking differences in the αSyn truncation pattern between different synucleinopathies, brain regions and specific cellular populations. In LBD, neuronal inclusions in the substantia nigra and amygdala were positive for αSyn cleaved after residues 103, 119, 122, and 125, but not 115. In contrast, in the same patients' brain αSyn cleaved at residue 115, as well as 103, 119 and 122 were abundant in the dorsal motor nucleus of the vagus. In patients with AD/ALB, these modifications were only weakly or not detected in amygdala αSyn inclusions. αSyn truncated at residues 103, 115, 119, and 125 was readily present in MSA glial cytoplasmic inclusions, but 122 cleaved αSyn was only weakly or not present. Conversely, MSA neuronal pathology in the pontine nuclei was strongly reactive to the αSyn x-122 neo-epitope but did not display any reactivity for αSyn 103 cleavage. These studies demonstrate significant disease-, region- and cell type specific differences in carboxy terminal αSyn processing associated with pathological inclusions that likely contributes to their distinct strain-like prion properties and promotes the diversity displayed in the degrees of these insidious diseases.

Robust α-synuclein pathology in select brainstem neuronal populations is a potential instigator of multiple system atrophy.

Multiple system atrophy (MSA) is an insidious middle age-onset neurodegenerative disease that clinically presents with variable degrees of parkinsonism and cerebellar ataxia. The pathological hallmark of MSA is the progressive accumulation of glial cytoplasmic inclusions (GCIs) in oligodendrocytes that are comprised of α-synuclein (αSyn) aberrantly polymerized into fibrils. Experimentally, MSA brain samples display a high level of seeding activity to induce further αSyn aggregation by a prion-like conformational mechanism. Paradoxically, αSyn is predominantly a neuronal brain protein, with only marginal levels expressed in normal or diseased oligodendrocytes, and αSyn inclusions in other neurodegenerative diseases, including Parkinson's disease and Dementia with Lewy bodies, are primarily found in neurons. Although GCIs are the hallmark of MSA, using a series of new monoclonal antibodies targeting the carboxy-terminal region of αSyn, we demonstrate that neuronal αSyn pathology in MSA patient brains is remarkably abundant in the pontine nuclei and medullary inferior olivary nucleus. This neuronal αSyn pathology has distinct histological properties compared to GCIs, which allows it to remain concealed to many routine detection methods associated with altered biochemical properties of the carboxy-terminal domain of αSyn. We propose that these previously underappreciated sources of aberrant αSyn could serve as a pool of αSyn prion seeds that can initiate and continue to drive the pathogenesis of MSA.

Precision therapeutic targets for COVID-19.

Beginning in late 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged as a novel pathogen that causes coronavirus disease 2019 (COVID-19). SARS-CoV-2 has infected more than 111 million people worldwide and caused over 2.47 million deaths. Individuals infected with SARS-CoV-2 show symptoms of fever, cough, dyspnea, and fatigue with severe cases that can develop into pneumonia, myocarditis, acute respiratory distress syndrome, hypercoagulability, and even multi-organ failure. Current clinical management consists largely of supportive care as commonly administered treatments, including convalescent plasma, remdesivir, and high-dose glucocorticoids. These have demonstrated modest benefits in a small subset of hospitalized patients, with only dexamethasone showing demonstrable efficacy in reducing mortality and length of hospitalization. At this time, no SARS-CoV-2-specific antiviral drugs are available, although several vaccines have been approved for use in recent months. In this review, we will evaluate the efficacy of preclinical and clinical drugs that precisely target three different, essential steps of the SARS-CoV-2 replication cycle: the spike protein during entry, main protease (MPro) during proteolytic activation, and RNA-dependent RNA polymerase (RdRp) during transcription. We will assess the advantages and limitations of drugs that precisely target evolutionarily well-conserved domains, which are less likely to mutate, and therefore less likely to escape the effects of these drugs. We propose that a multi-drug cocktail targeting precise proteins, critical to the viral replication cycle, such as spike protein, MPro, and RdRp, will be the most effective strategy of inhibiting SARS-CoV-2 replication and limiting its spread in the general population.

Novel monoclonal antibodies targeting the RRM2 domain of human TDP-43 protein.

Transactive response DNA-binding protein of 43 kilodaltons (TDP-43) is a 414 amino acid protein that under physiologic conditions localizes to the nucleus and participates in the regulation of RNA metabolism through two RNA recognition motifs (RRM1 and RRM2). In neurodegenerative diseases, TDP-43 may become hyperphosphorylated, ubiquitinated, and aggregate into cytoplasmic inclusions. TDP-43 is now well-characterized as a pathologic protein of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with TDP-43 proteinopathy (FTLD-TDP). Additionally, a common TDP-43 proteinopathy arising outside of the context of ALS and FTLD-TDP has been recently described, termed "limbic predominant age-related TDP-43 encephalopathy (LATE)." In the current study, two novel mouse-derived monoclonal antibodies, 2G11 and 2H1, raised against an epitope within the RRM2 domain of TDP-43 (residues 198-216), were characterized for specificity and immunohistochemical application in human brain from cases of Alzheimer's disease (AD), Lewy Body Disease (LBD), amyotrophic lateral sclerosis (ALS), and frontotemporal lobe degeneration with TDP-43 inclusions (FTLD-TDP). Immunoblot analysis of these antibodies in HEK293T cells revealed efficient detection of intact human TDP-43 protein, and in N2A cells showed no reactivity for mouse TDP-43. Immunohistochemically applied to formalin-fixed paraffin-embedded tissues, 2G11 and 2H1 robustly identified the classic inclusions of ALS and FTLD-TDP, and efficaciously provided a diagnosis of LATE in cases of AD and LBD. These novel antibodies label aberrant intracytoplasmic protein inclusions without relying on hyperphosphorylated epitopes, and provide elegant discrimination between TDP-43 and tau neurofibrillary tangles within neurodegenerative comorbidity.

Transgenic Mice Expressing Human α-Synuclein in Noradrenergic Neurons Develop Locus Ceruleus Pathology and Nonmotor Features of Parkinson's Disease.

Degeneration of locus ceruleus (LC) neurons and dysregulation of noradrenergic signaling are ubiquitous features of Parkinson's disease (PD). The LC is among the first brain regions affected by α-synuclein (asyn) pathology, yet how asyn affects these neurons remains unclear. LC-derived norepinephrine (NE) can stimulate neuroprotective mechanisms and modulate immune cells, while dysregulation of NE neurotransmission may exacerbate disease progression, particularly nonmotor symptoms, and contribute to the chronic neuroinflammation associated with PD pathology. Although transgenic mice overexpressing asyn have previously been developed, transgene expression is usually driven by pan-neuronal promoters and thus has not been selectively targeted to LC neurons. Here we report a novel transgenic mouse expressing human wild-type asyn under control of the noradrenergic-specific dopamine ß-hydroxylase promoter (DBH-hSNCA). These mice developed oligomeric and conformation-specific asyn in LC neurons, alterations in hippocampal and LC microglial abundance, upregulated GFAP expression, degeneration of LC fibers, decreased striatal DA metabolism, and age-dependent behaviors reminiscent of nonmotor symptoms of PD that were rescued by adrenergic receptor antagonists. These mice provide novel insights into how asyn pathology affects LC neurons and how central noradrenergic dysfunction may contribute to early PD pathophysiology.SIGNIFICANCE STATEMENT ɑ-Synuclein (asyn) pathology and loss of neurons in the locus ceruleus (LC) are two of the most ubiquitous neuropathologic features of Parkinson's disease (PD). Dysregulated norepinephrine (NE) neurotransmission is associated with the nonmotor symptoms of PD, including sleep disturbances, emotional changes such as anxiety and depression, and cognitive decline. Importantly, the loss of central NE may contribute to the chronic inflammation in, and progression of, PD. We have generated a novel transgenic mouse expressing human asyn in LC neurons to investigate how increased asyn expression affects the function of the central noradrenergic transmission and associated behaviors. We report cytotoxic effects of oligomeric and conformation-specific asyn, astrogliosis, LC fiber degeneration, disruptions in striatal dopamine metabolism, and age-dependent alterations in nonmotor behaviors without inclusions.

Prominent amyloid plaque pathology and cerebral amyloid angiopathy in APP V717I (London) carrier - phenotypic variability in autosomal dominant Alzheimer's disease.

The discovery of mutations associated with familial forms of Alzheimer's disease (AD), has brought imperative insights into basic mechanisms of disease pathogenesis and progression and has allowed researchers to create animal models that assist in the elucidation of the molecular pathways and development of therapeutic interventions. Position 717 in the amyloid precursor protein (APP) is a hotspot for mutations associated with autosomal dominant AD (ADAD) and the valine to isoleucine amino acid substitution (V717I) at this position was among the first ADAD mutations identified, spearheading the formulation of the amyloid cascade hypothesis of AD pathogenesis. While this mutation is well described in multiple kindreds and has served as the basis for the generation of widely used animal models of disease, neuropathologic data on patients carrying this mutation are scarce. Here we present the detailed clinical and neuropathologic characterization of an APP V717I carrier, which reveals important novel insights into the phenotypic variability of ADAD cases. While age at onset, clinical presentation and widespread parenchymal beta-amyloid (Aß) deposition are in line with previous reports, our case also shows widespread and severe cerebral amyloid angiopathy (CAA). This patient also presented with TDP-43 pathology in the hippocampus and amygdala, consistent with limbic predominant age-related TDP-43 proteinopathy (LATE). The APOE ε2/ε3 genotype may have been a major driver of the prominent vascular pathology seen in our case. These findings highlight the importance of neuropathologic examinations of genetically determined AD cases and demonstrate striking phenotypic variability in ADAD cases.