Biology subjects

Tittle, T.

Publications and source records attributed to Tittle, T..

4 recordsLinked to original sources

bioRxiv2025

Alpha-synuclein phosphorylation is abundant in the non-synucleinopathy human brain

Phosphorylation of alpha-synuclein (syn) at serine 129 (PS129) marks aggregates in synucleinopathies but also occurs physiologically, potentially signaling protein interactions during neuronal activity. Technical barriers, including postmortem dephosphorylation, have hindered the study of physiological PS129 in the human brain. Using biotinylation by antibody recognition (BAR) on surgically resected temporal lobectomy tissues (without post-mortem interval), we mapped physiological PS129 and total syn interactomes. BAR identified 1,095 interactions with 513 syn-specific, 524 shared, and 58 PS129-specific, mostly associated with vesicles at presynaptic nerve terminals. PS129-specific interactions were uniquely associated with postsynaptic density proteins SHANK1/3, DLGAP1-4, DLGAP1-3, and DLG2-4, as well as nuclear-associated proteins HUWE1, HNRNPM, RBM14, ITCH, OGT, PHF24, and PPP2R5E. Fluorescent staining confirmed physiological PS129 proximal to dendrites and within the nucleus. Confirmation in healthy cynomolgus macaques (62% syn and 41% PS129 overlap) demonstrated that the interactomes were physiological rather than disease- or aggregate-associated. We conclude that physiological PS129 plays a unique and underappreciated role in postsynaptic neurons extending from the postsynaptic active zone to the nucleus. These interactomes benchmark normal syn biology, illuminating the transition to synucleinopathy pathology. Significance StatementDisease-associated syn phosphorylation (PS129) was recently identified in healthy mammalian brain and may signal syn-protein interactions during neuronal activity. Here, we surmounted technical hurdles and characterized syn and PS129 interactomes directly in the human brain. Results showed a unique significance for PS129 in post-synaptic active zones and nuclear compartments, which was confirmed in healthy non-human primates. These syn interactomes will be a valuable reference for understanding synucleinopathy mechanisms in the context of normal syn biology.

neuroscience↗

bioRxiv2024

Comparing alpha-synuclein-interactomes between multiple systems atrophy and Parkinsons disease reveals unique and shared pathological features.

IntroductionPrimary synucleinopathies, such as Parkinsons disease (PD), Dementia with Lewy bodies (DLB), and multiple system atrophy (MSA), are neurodegenerative disorders with some shared clinical and pathological features. Aggregates of alpha-synuclein (syn) phosphorylated at serine 129 (PSER129) are the hallmark of synucleinopathies, which for PD/DLB are found predominantly in neurons (Neuronal cytoplasmic inclusions "NCIs"), but for MSA, aggregates are primarily found in oligodendroglia (Glial cytoplasmic inclusions "GCIs"). It remains unclear if the distinct pathological presentation of PD/DLB and MSA are manifestations of distinct or shared pathological processes. We hypothesize that the distinct synucleinopathies MSA and PD/DLB share common molecular features. MethodsUsing the in-situ proximity labeling technique biotinylation by antibody recognition (BAR), we compare aggregated syn-interactomes (BAR-PSER129) and total syn-interactomes (BAR-MJFR1) between MSA (n=5) and PD/DLB (n=10) in forebrain and midbrain structures. ResultsFor BAR-PSER129 and BAR-MJFR1 captures, syn was the most significantly enriched protein in PD/DLB and MSA. In PD/DLB, BAR-PSER129 identified 194 syn-aggregate-interacting proteins, while BAR-MJFR1 identified 245 syn interacting proteins. In contrast, in the MSA brain, only 38 and 175 proteins were identified for each capture, respectively. When comparing MSA and PD/DLB, a high overlap (59.5%) was observed between BAR-MJFR1 captured proteins, whereas less overlap (14.4%) was observed for BAR-PSER129. Direct comparison between MSA and PD/DLB revealed 79 PD/DLB-associated proteins and only three MSA-associated proteins (CBR1, CRYAB, and GFAP). Pathway enrichment analysis revealed PD/DLB interactions were dominated by vesicle/SNARE-associated pathways, in contrast to MSA, which strongly enriched for metabolic/catabolic, iron, and cellular oxidant detoxification pathways. A subnetwork of cytosolic antioxidant enzymes called peroxiredoxins drove cellular detoxification pathways in MSA. A common network of 26 proteins, including neuronal-specific proteins (e.g., SNYGR3) with HSPA8 at the core, was shared between MSA and DLB/PD. Extracellular exosome pathways were universally enriched regardless of disease or BAR target protein. ConclusionSynucleinopathies have divergent and convergent syn-aggregate interactions, indicating unique and shared pathogenic mechanisms. MSA uniquely involves oxidant detoxification processes in glial cells, while vesicular processes in neurons dominate PD/DLB. Shared interactions, specifically SNYGR3 (i.e., a neuronal protein), between MSA and PD/DLB suggest neuronal axons origin for both diseases. In conclusion, we provide syn aggregates protein interaction maps for two distinct synucleinopathies.

neuroscience↗

bioRxiv2023

Aggregation inhibits alpha-synuclein dephosphorylation resulting in the observation of pathological enrichment

Alpha-synuclein (syn) is an intrinsically disordered protein that aggregates in the brain in several neurodegenerative diseases collectively called synucleinopathies. Phosphorylation of syn at serine 129 (PSER129) was considered rare in the healthy human brain but is enriched in pathological syn aggregates and is used as a specific marker for disease inclusions. However, recent observations challenge this assumption by demonstrating that PSER129 results from neuronal activity and can be readily detected in the non-diseased mammalian brain. Here, we investigated experimental conditions under which two distinct PSER129 pools, namely endogenous-PSER129 and aggregated-PSER129, could be detected and differentiated in the mammalian brain. Results showed that in the wild-type (WT) mouse brain, perfusion fixation conditions greatly influenced the detection of endogenous-PSER129, with endogenous-PSER129 being nearly undetectable after delayed perfusion fixation (30-minute and 1-hour postmortem interval). Exposure to anesthetics (e.g., Ketamine or xylazine) before perfusion did not significantly influence endogenous-PSER129 detection or levels. In situ, non-specific phosphatase calf alkaline phosphatase (CIAP) selectively dephosphorylated endogenous-PSER129 while syn preformed fibril (PFF)-seeded aggregates and genuine disease aggregates (Lewy pathology and Papp-Lantos bodies in Parkinsons disease and multiple systems atrophy brain, respectively) were resistant to CIAP-mediated dephosphorylation. The phosphatase resistance of aggregates was abolished by sample denaturation, and CIAP-resistant PSER129 was closely associated with proteinase K (PK)-resistant syn (i.e., a marker of aggregation). CIAP pretreatment allowed for highly specific detection of seeded syn aggregates in a mouse model that accumulates non-aggregated-PSER129. We conclude that syn aggregates are impervious to phosphatases, and CIAP pretreatment increases detection specificity for aggregated-PSER129, particularly in well-preserved biological samples (e.g., perfusion fixed or flash-frozen mammalian tissues) where there is a high probability of interference from endogenous-PSER129. Our findings have important implications for the mechanism of PSER129-accumulation in the synucleinopathy brain and provide a simple experimental method to differentiate endogenous-from aggregated PSER129. Significance StatementPhosphorylated alpha-synuclein (PSER129) was widely regarded as a sensitive, specific marker for pathological aggregates in synucleinopathies until recent data demonstrated that PSER129 is abundant in the healthy mammalian nervous system and results from normal neuronal activity. Differentiating pathological (i.e., aggregated PSER129) and biological (non-aggregated PSER129) has thus become of critical importance to the field. Here, we describe our discovery that aggregated-PSER129 is impervious to enzymatic dephosphorylation. We leverage this discovery to develop a technique (CIAP-PSER129) to detect normal or pathological PSER129 selectively. Our technique allowed us to unambiguously differentiate pathological inclusions in brain regions and mouse models where excessive non-aggregated PSER129 severely limits the sensitivity of aggregate detection. CIAP-PSER129 is nondestructive and compatible with most downstream assays, including mass spectrometry-based peptide identification. These findings have important implications and utility for the synucleinopathy field and may have applicability to other neuropathological proteins (e.g., tau).

neuroscience↗

bioRxiv2023

Neither alpha-synuclein-preformed fibrils derived from patients with GBA1 mutations nor the host murine genotype significantly influence seeding efficacy in the mouse olfactory bulb.

Parkinsons disease (PD) is a neurodegenerative disease characterized by progressive motor symptoms and alpha-synuclein (syn) aggregation in the nervous system. For unclear reasons, PD patients with certain GBA mutations (GBA-PD) have a more aggressive clinical progression. Two testable hypotheses that can potentially account for this phenomenon are that GBA1 mutations promote syn spread or drive the generation of highly pathogenic syn polymorphs (i.e., strains). We tested these hypotheses by treating homozygous GBA1 D409V knockin (KI) mice with human -syn-preformed fibrils (PFFs) and treating wild-type mice (WT) with several syn-PFF polymorphs amplified from brain autopsy samples collected from patients with idiopathic PD and GBA-PD patients with either homozygous or heterozygous GBA1 mutations. Robust phosphorylated-syn (PSER129) positive pathology was observed at the injection site (i.e., the olfactory bulb granular layer) and throughout the brain six months following PFF injection. The PFF seeding efficiency and degree of spread were similar regardless of the mouse genotype or PFF polymorphs. We found that PFFs amplified from the human brain, regardless of patient genotype, were generally more effective seeders than wholly synthetic PFFs (i.e., non-amplified); however, PFF concentration differed between these two studies, and this might also account for the observed differences. To investigate whether the molecular composition of pathology differed between different seeding conditions, we permed Biotinylation by Antibody Recognition on PSER129 (BAR-PSER129). We found that for BAR-PSER129, the endogenous PSER129 pool dominated identified interactions, and thus, very few potential interactions were explicitly identified for seeded pathology. However, we found Dctn2 interaction was shared across all PFF conditions, and Nckap1 and Ap3b2 were unique to PFFs amplified from GBA-PD brains of heterozygous mutation carriers. In conclusion, both the genotype and syn strain had little effect on overall seeding efficacy and global PSER129-interactions.

neuroscience↗