Abstract Body

Generation of Alpha-Synuclein Aggregates in Human Olfactory Epithelia Includes Mucus-Producing Glands

The majority of patients with Parkinson disease features hyposmia. At brain autopsy, they show accumulation of alpha-synuclein aggregates in their olfactory bulbs. We hypothesized that alpha-synuclein misfolding may occur in response to environmental triggers, including within the nasal cavity. We tested this first in intact nasal cavity tissues collected at autopsy from 45 adults, including typical Lewy body disorders, tauopathies, fatal COVID19, and controls. There, we detected diffuse alpha-synuclein reactivity in the cytoplasm of olfactory sensory neurons in all subjects, as expected, and to a lesser extent in sustentacular cells, but not in basal stem cells. Using a panel of monoclonal antibodies against misfolded alpha-synuclein, we found that select regions and strata of epithelia revealed different types of aggregates, including 1-2 mM-sized punctae found in the cytoplasm of olfactory neurons, within apical dendrites and in epithelial cells of submucosal glands and their ducts. We also identified larger aggregates within extracellular deposits above the lamina propria following injury. The aggregates of alpha-synuclein within epithelial cells of mucin-producing Bowman’s glands often were proteinase K-resistant and co-labelled with anti-LAMP2. These alpha-synuclein aggregates were seen at a rate of ~40% in Lewy body diseases, in >80% of fatal COVID19 cases and in ~50% of controls. Further, using organoids of nasal cavity epithelia from wild-type mice, we detected an increased release rate of alpha-synuclein and intraellular aggregate formation following inoculation with vesicular stomatitis virus. We conclude that the neuroepithelium of the nasal cavity, including lysosomal ocompartments of mucin-producing cells, can physiologically generate alpha-synuclein aggregates. Our findings support the concept that alpha-synuclein aggregation may be involved in host defence against microbes within the nasal cavity.

Aims

Objective: To apply new biological definition and Integrated Staging System of Neuronal Alpha-Synuclein Disease (NSD-ISS) to the PPMI dataset

Background: The NSD-ISS Working Group developed a data-driven approach to: 1) determine a biologic definition for disease; 2) establish a framework for a disease staging platform.

Methods

We applied data driven staging criteria to the PPMI cohort.

Results

NSD is defined by the presence of pathologic n-asyn (S) assessed by a validated in vivo biomarker and ultimate presence of dopaminergic neuronal dysfunction (D). This biologic definition is independent of the presence of clinical features, or if present, of the specific clinical syndrome. NSD-ISS integrates these biological anchors (S and D) and degree of functional impairment caused by motor, cognitive or other non-motor signs (Table 1). Individuals in stage 0 and 1 are free of clinical signs and can be diagnosed either by presence of fully penetrant pathogenic variants (G) in SNCA gene (Stage 0), n-asyn alone (Stage 1A) or in combination with dopaminergic neuronal dysfunction (Stage 1B). Stage 2 is characterized by subtle clinical signs but no functional impairment, with either n-asyn (S) alone (Stage 2A) or in combination with dopaminergic neuronal dysfunction (Stage 2B). Stages 3-6 require the presence of both biomarkers and stage-specific increase in severity of functional impairment. Table 2 presents staging of the PPMI PD cohor

Conclusions

The NSD-ISS provides a comprehensive biologically-based framework essential to advance biologically-targeted therapeutic development. The NSD-ISS is expected to evolve as additional biomarkers emerge. The NSD-ISS will inform our understanding of early disease and accelerate development of disease-targeted therapies. We present the first data-driven application of the NSD-ISS, highlighting the heterogeneity of the biological stages in a phenotypically homogeneous cohort.

Aims

Lewy body (LB) disorders have a heterogeneous clinical presentation, possibly due to variations in the underlying trajectories of LB accumulation. Here, we aimed to identify and characterize distinct data-driven spatial-temporal progression patterns of LB pathology using post-mortem data.

Methods

We applied the Ordinal Subtype and Stage Inference (SuStaIn) model to post-mortem Lewy-type α-synuclein density scores measured in 10 regions (extracted from the olfactory bulb and tract [OBT], limbic system, brainstem, and neocortex) of 814 subjects from the Sun Health Research Institute Brain Donation Program (Table-1). Each subject was assigned to a subtype and a subtype-specific disease stage derived using SuStaIn. In 781 subjects with detectable LB pathology and confident subtype assignment, regression models adjusted for SuStaIn stage, age, and sex compared subtypes based on (i) clinicopathological diagnosis, (ii) demographics, (iii) Alzheimer’s disease (AD) pathology, and (iv) cross-sectional and longitudinal clinical symptoms (additionally adjusted for education). Nonlinear trajectories of peripheral LB pathology measured in 7 additional non-brain regions were modelled.

Results

SuStaIn identified three LB subtypes, which we termed “OBT-early/Limbic-early” (n=475;60.8%), “OBT-early/Brainstem-early” (n=165;21.1%), and “OBT-late/Brainstem-early” (n=141;18.1%) based on the initial regions to become abnormal, with 82% of individuals having the OBT as the first affected region. (Figure-1). Compared to both other subtypes, the OBT-early/Limbic-early subtype showed a higher frequency of AD and lower frequency of LB disorder clinicopathological diagnosis; more APOE-e4 carriers; higher post-mortemamyloid and tau burden; lower MMSE (Table-1, Figure-2) and a delayed onset of peripheral LB pathology (Figure-3). The OBT-early/Brainstem-early subtype showed slower global cognitive and motor decline over time (Figure-2H-I).

Conclusions

In this large post-mortem study, we identified three LB subtypes, possibly reflecting different epicenters and spreading trajectories of misfolded α-synuclein. The OBT-early/Limbic-early subtype was strongly associated with AD-pathology.

Aims

Missense mutations in the VPS35 gene are a rare cause of familial Parkinson’s disease (PD) and VPS35 protein is a subunit of the retromer complex . Prior studies reported thet retromer complex controls accumulation of α-Synuclein (αSyn) aggregates.

Our fisrt aim has been the validation of a pharmacological approach using a retromer stabilizer (1,3 phenyl bis guanylhydrazone, 2a) in protecting against αSyn pathology, dopaminergic neuronal degeneration and striatal fiber loss in a PD mouse model. Subsequently we assessed the effect of 2a oncontrolling the main αSyn degradation pathways in PD mouse model and in dopaminergic-like cell line

Methods

Here, we validate the efficacy of 2a in protecting against αSynuclein pathology and dopaminergic neuronal degeneration in a PD mouse model generated by unilateral injection of AAV-A53T-αSynuclein in the substantia nigra.

Results

Daily intraperitoneal administration of 2a at 10 mg/Kg for 100 days led to robust protection against behavioral deficits, dopaminergic neuronal loss and loss of striatal dopaminergic fibers and striatal monoamines. Treatment with 2a activated αSynuclein degradation pathways in the SN and led to significant reductions in aggregates and pathological αSynuclein. We analyzed the effect of 2a on αSynuclein degradation pathways in dopaminergic-like cell line (Sh-SY5Y cells)

Conclusions

The data presented here in a pathophysiologically relevant αSyn-based PD mouse model highlight the neuroprotective potential of increasing retromer levels to block the accumulation of pathological αSyn and to protect DA neurons against neurodegeneration in PD. We tested 2a, a patented VPS35-targeting small molecule (pharmacological chaperone) that may itself be a promising drug candidate. These data support the potential for future clinical testing of 2a or other VPS35-targeted therapies for disease-modifying effects in PD.

Aims

Parkinson’s Disease (PD) and dementia with Lewy bodies are among the most common neurodegenerative disorders. A 140 aa protein alpha-synuclein (αSyn) is the major component of intraneuronal and glial aggregates that comprise the hallmark pathology for synucleinopathies, including PD and the AD-related dementia (ADRD) dementia with Lewy bodies (DLB). GWAS and rare familial mutations in SNCA gene (encoding αSyn) linked this protein directly to disease etiology. Cellular pathophysiology of αSyn, such as vesicular trafficking and lipid homeostasis disruption, is mostly attributed to its association with cellular membranes. Apart from αSyn’s residence on the membranes, the cytosolic function of αSyn and its contribution to disease etiology is unknown.

Methods

We used multiple interdisciplinary approaches ranging from high-throughput protein aggregation assay, RNA turnover assays, high-content imaging and novel genetic analysis of GWAS data. We used yeast, flies, patient derived human cortical neurons and human brain samples.

Results

Using a yeast-based RBP perturbation platform combined with proteomics and fly genetics, we discovered a cytosolic function of αSyn in regulating Processing bodies(P-bodies). P-bodies are conserved in all eukaryotic cells and are membraneless organeless of post-transcriptional regulation of mRNAs, especially mRNA decay. αSyn interacts strongly with decapping machinery. Membrane binding and decapping module binding use the same protein surface on αSyn, rendering these interactions mutually exclusive. In pathophysiological levels of αSyn (either in patient derived neurons and post-mortem brains), P-body numbers and integrity is compromised, leading to aberrant mRNA stabilization globally. Human genetic analysis shows accumulated mutational burden in PD patients compared to controls.

Conclusions

P-bodies represent a new molecular pathway in the pathophsiology of αSyn. We propose that αSyn is a messenger between RNA world and lipid world, with important therapeutic implications.

Aims

The aim of my lab’s research is to understand how structural (amino-acid sequence, post-translational modifications) and cellular (lipid environment, stressors, pathways, activity...) factors affect the dynamic structure of alpha-synuclein in the cellular content. We seek to and find strategies to strategically alter dynamic alpha-synuclein folding, molecular interactions, post-translational modifications, and half-life in favorable ways.

Methods

My lab uses a wide variety of molecular biology (mutagenesis), protein biochemistry (Western blot, crosslinking, sequential protein extraction…), biophysics (NMR), cell-biology (immuno-cytochemistry, EM, live-cell imaging), electrophysiology (cell culture, brain slice recordings) and in-vivo methods. Models range from purified recombinant protein, neuroblastoma cells, iPSC neurons, primary rodent neurons to live mice and rats.

Results

We have identified defined amino-acid substitutions that interfere with the proteins normal rapid switching between cytosol and vesicle membranes. Both membrane-only and cytosol-only alpha-synuclein variants are toxic in our assays, and we have gained insight into differences and similarities between the respective pathways involved. We understand better and better how fine-tuning alpha-synuclein/membrane interactions pharmacologically can overcome key aspects of alpha-synuclein pathology. We provide evidence that phospho-serine 129 plays different roles in physiological vs. pathological settings. Moreover, we are characterizing new cellular pathways to strategically reduce alpha-synuclein half-life in the cell.

Conclusions

We have gained new insight on how to strategically alter alpha-synuclein dynamics in the cellular context, including membrane interactions, post-translational modifications, and degradation pathways.

Aims

α-Synuclein (α-syn) phosphorylation at the Serine-129 (Ser129P) residue is a recognized pathologic hallmark of PD. Though only ~4% of α-syn is phosphorylated at this site under normal conditions, the tight endogenous regulation of α-syn Ser129P suggests a physiologic role. Nevertheless, almost all studies to date have focused on α-syn Ser129 in the context of pathology, and no firm physiologic role has been assigned. Accordingly, I propose that α-syn Ser129P has a role in normal α-syn function, and that this modification is relevant to the pathophysiology of PD and related disorders.

Methods

We have used immunocytochemistry and immunohistochemistry experiments to localize α-syn Ser-129P, optical pHluorin assays to report SV-recycling and trafficking, multielectrode arrays to examine neurotransmitter release, immunoprecipitation and GST Pulldown assays to evaluate protein-protein interactions, and AlphaFold2-driven modeling to visualize conformational changes in α-syn Ser-129P.

Results

We found that unlike native (total) α-syn that is widely expressed throughout the brain, the overall pattern of α-syn Ser129P is restricted, suggesting intrinsic regulation and putative physiologic roles. Surprisingly, preventing phosphorylation at the Ser-129 site blocked the ability of α-syn to attenuate activity-dependent synaptic vesicle (SV) recycling – widely thought to reflect its normal function. Exploring mechanisms, we found that neuronal activity augments α-syn Ser-129P, and this phosphorylation is required for α-syn binding to VAMP2 and synapsin – two functional binding partners that are necessary for α-syn function. AlphaFold2-driven modeling suggests a scenario where Ser129P induces conformational changes in the C-terminus that stabilize this region and facilitate protein-protein interactions.

Conclusions

Our data from cellular, in vivo, cell-free systems, and molecular simulations strongly suggest that the pathology- associated Ser129 modification has a physiologic role at the synapse, which has broad implications for α-syn pathophysiology and drug development.

Aims

High levels of the protein alpha-synuclein (aSyn) are strongly associated with increased aSyn aggregation and neurodegeneration in Parkinson’s disease (PD). Genetic evidence of pathogenic aSyn gene (SNCA) multiplication reinforces these connections. In this study, we explored the specific dose effect of aSyn on the impairment of neuronal integrity.

Methods

We differentiated human-induced pluripotent stem cells (hiPSCs) into midbrain dopaminergic neurons (mDAns) i) from a PD patient with an SNCA duplication (SNCA^Dupl); ii) with isogentically corrected SNCA dosage (SNCA^Corr) using CRISPR/Cas9; and iii) from healthy controls (HC). We performed parallel RNAseq and proteome analyses on mDAns to uncover global molecular changes driven specifically by respective SNCA dosage, followed by mechanistic validation of candidate proteins.

Results

SNCA^Dupl-mDAns exhibited enhanced aSyn aggregation compared to SNCA^corr- and HC-mDAns. Furthermore, SNCA^Dupl-mDAns displayed significant changes in neuronal morphology and neuritogenesis, rescueable by reducing SNCA dosage. Both gene expression and proteome analyses of SNCA^Dupl mDAns revealed remarkable shifts in the expression of mRNAs and proteins involved in cytoskeletal organization with a reduction in b-tubulin-III and an increase in vimentin compared to SNCA^corr- and HC-mDAns. Mechanistic studies revealed preferential interaction of aggregated aSyn with b-tubulin-III as indicated by direct binding and an increased co-distribution within disassembled microtubules. Finally, a distinct truncated form of vimentin was significantly elevated in SNCA^Dupl-mDAns which was corrected by lowering aSyn genetically through SNCA^corr or treatment with the intermediate filament-interfering compound okadaic acid.

Conclusions

Our data highlight a specific SNCA dose effect on the interplay between aSyn and its aggregation with cytoskeletal organization, such as the microtubule and intermediate filament networks. aSyn-mediated dysregulation of cytoskeletal networks contributes to the impairment of neuronal integrity in PD, and was rescued by lowering SNCA dose.