Monomeric α-synuclein (αSN) species are abundant in nerve terminals where they are hypothesized to play a physiological role related to synaptic vesicle turn-over. In Parkinson's disease (PD) and dementia with Lewy body (DLB), αSN accumulates as aggregated soluble oligomers in terminals, axons and the somatodendritic compartment and insoluble filaments in Lewy inclusions and Lewy neurites. The autosomal dominant heritability associated to mutations in the αSN gene suggest a gain of function associated to aggregated αSN. We have conducted a proteomic screen to identify the αSN interactome in brain synaptosomes. Porcine brain synaptosomes were fractionated, solubilized in non-denaturing detergent and subjected to co-immunoprecipitation using purified recombinant human αSN monomers or oligomers as bait. The isolated αSN binding proteins were identified with LC-LTQ-orbitrap tandem mass spectrometry and quantified by peak area using Windows client application, Skyline Targeted Proteomic Environment. Data are available via ProteomeXchange with identifier PXD001462. To quantify the preferential binding an average fold increase was calculated by comparing binding to monomer and oligomer. We identified 10 proteins preferentially binding monomer, and 76 binding preferentially to oligomer and a group of 92 proteins not displaying any preferred conformation of αSN. The proteomic data were validated by immunoprecipitation in both human and porcine brain extracts using antibodies against monomer αSN interactors: Abl interactor 1, and myelin proteolipid protein, and oligomer interactors: glutamate decarboxylase 2, synapsin 1, glial fibrillary acidic protein, and VAMP-2. We demonstrate the existence of αSN conformation selective ligands and present lists of proteins, whose identity and functions will be useful for modeling normal and pathological αSN dependent processes.

Mechanisms underlying α-synuclein (αSyn) mediated neurodegeneration are poorly understood. Intramuscular (IM) injection of αSyn fibrils in human A53T transgenic M83+/- mice produce a rapid model of α-synucleinopathy with highly predictable onset of motor impairment. Using varying doses of αSyn seeds, we show that αSyn-induced phenotype is largely dose-independent. We utilized the synchrony of this IM model to explore the temporal sequence of αSyn pathology, neurodegeneration and neuroinflammation. Longitudinal tracking showed that while motor neuron death and αSyn pathology occur within 2 months post IM, astrogliosis appears at a later timepoint, implying neuroinflammation is a consequence, rather than a trigger, in this prionoid model of synucleinopathy. Initiating at 3 months post IM, immune activation dominates the pathologic landscape in terminal IM-seeded M83+/- mice, as revealed by unbiased transcriptomic analyses. Our findings provide insights into the role of neuroinflammation in αSyn mediated proteostasis and neurodegeneration, which will be key in designing potential therapies.

α-Synuclein (αS) is the major component of several types of brain inclusions including Lewy bodies, a hallmark of Parkinson's disease. Aberrant aggregation of αS also is associated with cellular demise in multiple neurologic disorders collectively referred to as synucleinopathies. Recent studies demonstrate the induction of αS pathology by a single intracerebral injection of exogenous amyloidogenic αS in adult non-transgenic and transgenic mice expressing human αS. To further investigate the mechanism of pathology induction and evaluate an experimental paradigm with potential for higher throughput, we performed similar studies in neonatal mice injected with αS.

Mutations in the gene encoding leucine-rich repeat kinase 2 (LRRK2) are the most frequent cause of familial Parkinson's disease (PD). The neuropathology of LRRK2-related PD is heterogeneous and can include aberrant tau phosphorylation or neurofibrillary tau pathology. Recently, LRRK2 has been shown to phosphorylate tau in vitro; however, the major epitopes phosphorylated by LRRK2 and the physiological or pathogenic consequences of these modifications in vivo are unknown. Using mass spectrometry, we identified multiple sites on recombinant tau that are phosphorylated by LRRK2 in vitro, including pT149 and pT153, which are phospho-epitopes that to date have been largely unexplored. Importantly, we demonstrate that expression of transgenic LRRK2 in a mouse model of tauopathy increased the aggregation of insoluble tau and its phosphorylation at T149, T153, T205, and S199/S202/T205 epitopes. These findings indicate that tau can be a LRRK2 substrate and that this interaction can enhance salient features of human disease.

Psychosis susceptibility is mediated in part by the dopaminergic neurotransmitter system. In humans, individual differences in vulnerability for psychosis are reflected in differential sensitivity for psychostimulants such as amphetamine. We hypothesize that the same genes and pathways underlying behavioral sensitization in mice are also involved in the vulnerability to psychosis.

Recently mutations in ubiquilin-2 were identified in patients with amyotrophic lateral sclerosis (ALS) and ALS/dementia providing direct evidence for the importance of this protein in neurodegenerative diseases. Histological studies have suggested that ubiquilin-1/-2 are associated with various pathological inclusions including Lewy bodies in Parkinson's disease, neurofibrillary tangles in Alzheimer's disease, polyQ inclusions in expansion repeat diseases and various proteinopathies associated with ALS and frontotemporal dementia. Using specific ubiquilin-2 antibodies and a series of transgenic mouse models of proteinopathies associated with neurodegenerative disease, we show that ubiquilin-2 preferentially associates with huntingtin polyQ expansion aggregates compared to α-synuclein, tau and several other types of protein inclusions. These results were confirmed by similar findings for ubiquilin-1 and -2 in human brain tissue sections, where accumulation was observed in huntingtin inclusions, but only infrequently in other types of protein inclusions. In cultured cells, ubiquilin-2 associates with huntingtin/polyQ aggregates, but this is not compromised by disease-causing mutations. Although ubiquilin proteins can function as chaperones to shuttle proteins for degradation, there is persistent co-localization between ubiquilin-2 and polyQ aggregated proteins during disease progression in the N586-82Q-C63 Huntington's disease mouse model. Thus, the co-localization of ubiquilin-2 with the huntingtin aggregates does not appear to facilitate aggregate removal.

Parkinson's disease (PD) is a progressive neurodegenerative disorder for which there is no existing therapeutic approach to delay or stop progression. Genetic, biochemical and pre-clinical studies have provided evidence that leucine-rich-repeat-kinase-2 (LRRK2) kinase is involved in the pathogenesis of PD, and small molecule LRRK2 inhibitors represent a novel potential therapeutic approach. However, potentially adverse target-related effects have been discovered in the lung and kidneys of LRRK2 knock-out (ko) mice and rats. It is unclear if the LRRK2 ko effect in the kidneys and lung is also induced by pharmacological inhibition of the LRRK2 kinase. Here, we show that treatment with the LRRK2 inhibitor PFE-360 in rats induces a morphological kidney phenotype resembling that of the LRRK2 ko rats, whereas no effects were observed in the lung. The PFE-360 treatment induced morphological changes characterised by darkened kidneys and progressive accumulation of hyaline droplets in the renal proximal tubular epithelium. However, no histopathological evidence of renal tubular injury or changes in the blood and urine parameters that would be indicative of kidney toxicity or impaired kidney function were observed after up to 12 weeks of treatment. Morphological changes were detected in the kidney after 2 weeks of treatment and were partially reversible within a 30 day treatment-free period. Our findings suggest that pharmacological LRRK2 inhibition may not have adverse consequences for kidney function.

Soluble aggregates of α-synuclein, so-called oligomers, are hypothesized to act as neurotoxic species in Parkinson's disease, Lewy body dementia and multiple systems atrophy, but specific tools to detect these aggregated species are only slowly appearing. We have developed an α-synuclein oligomer ELISA that allows us to detect and compare α-synuclein oligomer levels in different in vivo and in vitro experiments. The ELISA is based on commercially available antibodies and the epitope of the capture antibody MJF14-6-4-2 is folding- and aggregate-dependent and not present on monomers.

Aggregation of α-synuclein is a hallmark of Parkinson's disease and dementia with Lewy bodies. We here investigate the relationship between cytosolic Ca2+ and α-synuclein aggregation. Analyses of cell lines and primary culture models of α-synuclein cytopathology reveal an early phase with reduced cytosolic Ca2+ levels followed by a later Ca2+ increase. Aggregated but not monomeric α-synuclein binds to and activates SERCA in vitro, and proximity ligation assays confirm this interaction in cells. The SERCA inhibitor cyclopiazonic acid (CPA) normalises both the initial reduction and the later increase in cytosolic Ca2+ CPA protects the cells against α-synuclein-aggregate stress and improves viability in cell models and in Caenorhabditis elegans in vivo Proximity ligation assays also reveal an increased interaction between α-synuclein aggregates and SERCA in human brains affected by dementia with Lewy bodies. We conclude that α-synuclein aggregates bind SERCA and stimulate its activity. Reducing SERCA activity is neuroprotective, indicating that SERCA and down-stream processes may be therapeutic targets for treating α-synucleinopathies.

In vivo functional and structural brain imaging of synucleinopathies in humans have provided a rich new understanding of the affected networks across the cortex and subcortex. Despite this progress, the temporal relationship between α-synuclein (α-syn) pathology and the functional and structural changes occurring in the brain is not well understood. Here, we examine the temporal relationship between locomotor ability, brain microstructure, functional brain activity, and α-syn pathology by longitudinally conducting rotarod, diffusion magnetic resonance imaging (MRI), resting-state functional MRI (fMRI), and sensory-evoked fMRI on 20 mice injected with α-syn fibrils and 20 PBS-injected mice at three timepoints (10 males and 10 females per group). Intramuscular injection of α-syn fibrils in the hindlimb of M83+/- mice leads to progressive α-syn pathology along the spinal cord, brainstem, and midbrain by 16 weeks post-injection. Our results suggest that peripheral injection of α-syn has acute systemic effects on the central nervous system such that structural and resting-state functional activity changes occur in the brain by four weeks post-injection, well before α-syn pathology reaches the brain. At 12 weeks post-injection, a separate and distinct pattern of structural and sensory-evoked functional brain activity changes was observed that are co-localized with previously reported regions of α-syn pathology and immune activation. Microstructural changes in the pons at 12 weeks post-injection were found to predict survival time and preceded measurable locomotor deficits. This study provides preliminary evidence for diffusion and fMRI markers linked to the progression of synuclein pathology and has translational importance for understanding synucleinopathies in humans.SIGNIFICANCE STATEMENT α-Synuclein (α-syn) pathology plays a critical role in neurodegenerative diseases such as Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy. The longitudinal effects of α-syn pathology on locomotion, brain microstructure, and functional brain activity are not well understood. Using high field imaging, we show preliminary evidence that peripheral injection of α-syn fibrils induces unique patterns of functional and structural changes that occur at different temporal stages of α-syn pathology progression. Our results challenge existing assumptions that α-syn pathology must precede changes in brain structure and function. Additionally, we show preliminary evidence that diffusion and functional magnetic resonance imaging (fMRI) are capable of resolving such changes and thus should be explored further as markers of disease progression.

The conversion of endogenous alpha-synuclein (asyn) to pathological asyn-enriched aggregates is a hallmark of Parkinson's disease (PD). These inclusions can be detected in the central and enteric nervous system (ENS). Moreover, gastrointestinal symptoms can appear up to 20 years before the diagnosis of PD. The dual-hit hypothesis posits that pathological asyn aggregation starts in the ENS, and retrogradely spreads to the brain. In this study, we tested this hypothesis by directly injecting preformed asyn fibrils into the duodenum wall of wild-type rats and transgenic rats with excess levels of human asyn. We provide a meticulous characterization of the bacterial artificial chromosome (BAC) transgenic rat model with respect to initial propagation of pathological asyn along the parasympathetic and sympathetic pathways to the brainstem, by performing immunohistochemistry at early time points post-injection. Induced pathology was observed in all key structures along the sympathetic and parasympathetic pathways (ENS, autonomic ganglia, intermediolateral nucleus of the spinal cord (IML), heart, dorsal motor nucleus of the vagus, and locus coeruleus (LC)) and persisted for at least 4 months post-injection. In contrast, asyn propagation was not detected in wild-type rats, nor in vehicle-injected BAC rats. The presence of pathology in the IML, LC, and heart indicate trans-synaptic spread of the pathology. Additionally, the observed asyn inclusions in the stomach and heart may indicate secondary anterograde propagation after initial retrograde spreading. In summary, trans-synaptic propagation of asyn in the BAC rat model is fully compatible with the "body-first hypothesis" of PD etiopathogenesis. To our knowledge, this is the first animal model evidence of asyn propagation to the heart, and the first indication of bidirectional asyn propagation via the vagus nerve, i.e., duodenum-to-brainstem-to-stomach. The BAC rat model could be very valuable for detailed mechanistic studies of the dual-hit hypothesis, and for studies of disease modifying therapies targeting early pathology in the gastrointestinal tract.

Parkinson's disease (PD) is a progressive neurodegenerative disorder associated with impaired motor function and several non-motor symptoms, with no available disease modifying treatment. Intracellular accumulation of pathological α-synuclein inclusions is a hallmark of idiopathic PD, whereas, dominant mutations in leucine-rich repeat kinase 2 (LRRK2) are associated with familial PD that is clinically indistinguishable from idiopathic PD. Recent evidence supports the hypothesis that an increase in LRRK2 kinase activity is associated with the development of not only familial LRRK2 PD, but also idiopathic PD. Previous reports have shown preclinical effects of LRRK2 modulation on α-synuclein-induced neuropathology. Increased subthalamic nucleus (STN) burst firing in preclinical neurotoxin models and PD patients is hypothesized to be causally involved in the development of the motor deficit in PD. To study a potential pathophysiological relationship between α-synuclein pathology and LRRK2 kinase activity in PD, we investigated the effect of chronic LRRK2 inhibition in an AAV-α-synuclein overexpression rat model. In this study, we report that chronic LRRK2 inhibition using PFE-360 only induced a marginal effect on motor function. In addition, the aberrant STN burst firing and associated neurodegenerative processes induced by α-synuclein overexpression model remained unaffected by chronic LRRK2 inhibition. Our findings do not strongly support LRRK2 inhibition for the treatment of PD. Therefore, the reported beneficial effects of LRRK2 inhibition in similar α-synuclein overexpression rodent models must be considered with prudence and additional studies are warranted in alternative α-synuclein-based models.

Apolipoprotein (APOE) is a major risk factor of Alzheimer's disease (AD), with the E2, E3 and E4 isoforms differentially regulating the burden of AD-associated neuropathologies, such as amyloid β and tau. In AD, pathological tau is thought to spread along neuroanatomic connections following a prion-like mechanism. To provide insights into whether APOE isoforms differentially regulate the prion properties of tau and determine trans-synaptic transmission of tauopathy, we have generated human P301S mutant tau transgenic mice (PS19) that carry human APOE (APOE2, APOE3 or APOE4) or mouse Apoe allele. Mice received intrahippocamal injections of preformed aggregates of K18-tau at young ages, which were analyzed 5 months post-inoculation. Compared to the parental PS19 mice with mouse Apoe alleles, PS19 mice expressing human APOE alleles generally responded to K18-tau seeding with more intense AT8 immunoreactive phosphorylated tau athology. APOE3 homozygous mice accumulated higher levels of AT8-reactive ptau and microgliosis relative to APOE2 or APOE4 homozygotes (E3 > E4~2). PS19 mice that were heterozygous for APOE3 showed similar results, albeit to a lesser degree. In the timeframe of our investigation, we did not observe significant induction of argentophilic or MC1-reactive neurofibrillary tau tangle in PS19 mice homozygous for human APOE. To our knowledge, this is the first comprehensive study in rodent models that provides neuropathological insights into the dose-dependent effect of APOE isoforms on phosphorylated tau pathology induced by recombinant tau prions.

Aggregated α-synuclein (α-syn) accumulates in the neuronal Lewy body (LB) inclusions in Parkinson's disease (PD) and LB dementia. Yet, under nonpathological conditions, monomeric α-syn is hypothesized to exist in an equilibrium between disordered cytosolic- and partially α-helical lipid-bound states: a feature presumably important in synaptic vesicle release machinery. The exact underlying role of α-syn in these processes, and the mechanisms regulating membrane-binding of α-syn remains poorly understood. Herein we demonstrate that Protein kinase R (PKR) can phosphorylate α-syn at several Ser/Thr residues located in the membrane-binding region that is essential for α-syn's vesicle-interactions. α-Syn phosphorylated by PKR or α-syn isolated from PKR overexpressing cells, exhibit decreased binding to lipid membranes. Phosphorylation of Thr64 and Thr72 appears as the major contributor to this effect, as the phosphomimetic Thr64Glu/Thr72Glu-α-syn mutant displays reduced overall attachment to brain vesicles due to a decrease in vesicle-affinity of the last two thirds of α-syn's membrane binding region. This allows enhancement of the "double-anchor" vesicle-binding mechanism that tethers two vesicles and thus promote the clustering of presynaptic vesicles in vitro. Furthermore, phosphomimetic Thr64Glu/Thr72Glu-α-syn inhibits α-syn oligomerization and completely abolishes nucleation, elongation, and seeding of α-syn fibrillation in vitro and in cells, and prevents trans-synaptic spreading of aggregated α-syn pathology in organotypic hippocampal slice cultures. Overall, our findings demonstrate that normal and abnormal functions of α-syn, like membrane-binding, synaptic vesicle clustering and aggregation can be regulated by phosphorylation, e.g., via PKR. Mechanisms that could potentially be modulated for the benefit of patients suffering from α-syn aggregate-related diseases.

The accumulation of α-synuclein (α-syn) in intracellular formations known as Lewy bodies (LBs) is associated with several neurodegenerative diseases including Parkinson's disease and Lewy Body Dementia. There is still limited understanding of how α-syn and LB formation is associated with cellular dysfunction and degeneration in these diseases. To examine the clearance and production dynamics of α-syn we transduced organotypic murine brain slice cultures (BSCs) with recombinant adeno-associated viruses (rAAVs) to express Dendra2-tagged human wild-type (WT) and mutant A53T α-syn, with and without the addition of exogenous α-syn fibrillar seeds and tracked them over several weeks in culture using optical pulse labeling. We found that neurons expressing WT or mutant A53T human α-syn show similar rates of α-syn turnover even when insoluble, phosphorylated Ser129 α-syn has accumulated. Taken together, this data reveals α-syn aggregation and overexpression, pSer129 α-syn, nor the A53T mutation affect α-syn dynamics in this system. Prion-type seeding with exogenous α-syn fibrils significantly slows α-syn turnover, in the absence of toxicity but is associated with the accumulation of anti-p62 immunoreactivity and Thiazin Red positivity. Prion-type induction of α-syn aggregation points towards a potential protein clearance deficit in the presence of fibrillar seeds and the ease of this system to explore precise mechanisms underlying these processes. This system facilitates the exploration of α-syn protein dynamics over long-term culture periods. This platform can further be exploited to provide mechanistic insight on what drives this slowing of α-syn turnover and how therapeutics, other genes or different α-syn mutations may affect α-syn protein dynamics.

Parkinson's disease (PD) is a progressive neurodegenerative disease that is typically diagnosed late in its progression. There is a need for biomarkers suitable for monitoring the disease progression at earlier stages to guide the development of novel neuroprotective therapies. One potential biomarker, α-synuclein, has been found in both the familial cases of PD, as well as the sporadic cases and is considered a key feature of PD. α-synuclein is naturally present in the retina, and it has been suggested that early symptoms of the visual system may be used as a biomarker for PD. Here, we use a viral vector to induce a unilateral expression of human wild-type α-synuclein in rats as a mechanistic model of protein aggregation in PD. We employed functional magnetic resonance imaging (fMRI) to investigate whether adeno-associated virus (AAV) mediated expression of human wild-type α-synuclein alter functional activity in the visual system. A total of 16 rats were injected with either AAV-α-synuclein (n = 7) or AAV-null (n = 9) in the substantia nigra pars compacta (SNc) of the left hemisphere. The expression of α-synuclein was validated by a motor assay and postmortem immunohistochemistry. Five months after the introduction of the AAV-vector, fMRI showed robust blood oxygen level-dependent (BOLD) responses to light stimulation in the visual systems of both control and AAV-α-synuclein animals. However, our results demonstrate that the expression of AAV-α-synuclein does not affect functional activation of the visual system. This negative finding suggests that fMRI-based read-outs of visual responses may not be a sensitive biomarker for PD.

Hypothalamic-pituitary-adrenal (HPA) axis dysfunction contributes to numerous human diseases and disorders. We developed a high-affinity monoclonal antibody, CTRND05, targeting corticotropin-releasing factor (CRF). In mice, CTRND05 blocks stress-induced corticosterone increases, counteracts effects of chronic variable stress, and induces other phenotypes consistent with suppression of the HPA axis. CTRND05 induces skeletal muscle hypertrophy and increases lean body mass, effects not previously reported with small-molecule HPA-targeting pharmacologic agents. Multiorgan transcriptomics demonstrates broad HPA axis target engagement through altering levels of known HPA-responsive transcripts such as Fkbp5 and Myostatin and reveals novel HPA-responsive pathways such as the Apelin-Apelin receptor system. These studies demonstrate the therapeutic potential of CTRND05 as a suppressor of the HPA axis and serve as an exemplar of a potentially broader approach to target neuropeptides with immunotherapies, as both pharmacologic tools and novel therapeutics.

Enhancing production of protein cargoes delivered by gene therapies can improve efficacy by reducing the amount of vector or simply increasing transgene expression levels. We explored the utility of a 126-amino acid collagen domain (CD) derived from the C1qTNF3 protein as a fusion partner to chaperone secreted proteins, extracellular "decoy receptor" domains, and single-chain variable fragments (scFvs). Fusions to the CD domain result in multimerization and enhanced levels of secretion of numerous fusion proteins while maintaining functionality. Efficient creation of bifunctional proteins using the CD domain is also demonstrated. Recombinant adeno-associated viral vector delivery of the CD with a signal peptide resulted in high-level expression with minimal biological impact as assessed by whole-brain transcriptomics. As a proof-of-concept in vivo study, we evaluated three different anti-amyloid Aβ scFvs (anti-Aβ scFvs), alone or expressed as CD fusions, following viral delivery to neonatal CRND8 mice. The CD fusion increased half-life, expression levels, and improved efficacy for amyloid lowering of a weaker binding anti-Aβ scFv. These studies validate the potential utility of this small CD as a fusion partner for secretory cargoes delivered by gene therapy and demonstrate that it is feasible to use this CD fusion to create biotherapeutic molecules with enhanced avidity or bifunctionality.

The relative polymerization of specific tau protein cores that define Alzheimer's disease, Pick's disease and corticobasal degeneration were investigated using amyloid fluorometry and electron microscopy. In addition, the relative prion-like activities of polymers comprised of these respective tau protein segments were investigated in a cell-based assay. It is demonstrated that the seeding activities of specific tau core fibrils are affected by the presence of pathogenic tau missense mutations and the microtubule binding domain composition of tau. The unique impact of tau phosphorylation on seeding propensity was also investigated by altering stretches of phospho-mimetic and phospho-null residues in the presence of Alzheimer's disease tau core fibrils. These results have important mechanistic implications for mutation and isoform-specific driven pathogenesis.

It has been challenging to produce ex vivo models of the inclusion pathologies that are hallmark pathologies of many neurodegenerative diseases. Using three-dimensional mouse brain slice cultures (BSCs), we have developed a paradigm that rapidly and robustly recapitulates mature neurofibrillary inclusion and Lewy body formation found in Alzheimer's and Parkinson's disease, respectively. This was achieved by transducing the BSCs with recombinant adeno-associated viruses (rAAVs) that express α-synuclein or variants of tau. Notably, the tauopathy BSC model enables screening of small molecule therapeutics and tracking of neurodegeneration. More generally, the rAAV BSC "toolkit" enables efficient transduction and transgene expression from neurons, microglia, astrocytes, and oligodendrocytes, alone or in combination, with transgene expression lasting for many months. These rAAV-based BSC models provide a cost-effective and facile alternative to in vivo studies, and in the future can become a widely adopted methodology to explore physiological and pathological mechanisms related to brain function and dysfunction.