LGALS3 (galectin 3) mediates an unconventional secretion of SNCA/α-synuclein in response to lysosomal membrane damage by the autophagic-lysosomal pathway in human midbrain dopamine neurons.

Numerous lines of evidence support the premise that the misfolding and subsequent accumulation of SNCA/α-synuclein (synuclein alpha) is responsible for the underlying neuronal pathology observed in Parkinson disease (PD) and other synucleinopathies. Moreover, the cell-to-cell transfer of these misfolded SNCA species is thought to be responsible for disease progression and the spread of cellular pathology throughout the brain. Previous work has shown that when exogenous, misfolded SNCA fibrils enter cells through endocytosis, they can damage and rupture the membranes of their endocytotic vesicles in which they are trafficked. Rupture of these vesicular membranes exposes intralumenal glycans leading to galectin protein binding, subsequent autophagic protein recruitment, and, ultimately, their introduction into the autophagic-lysosomal pathway. Increasing evidence indicates that both pathological and non-pathological SNCA species undergo autophagy-dependent unconventional secretion. While other proteins have also been shown to be secreted from cells by autophagy, what triggers this release process and how these specific proteins are recruited to a secretory autophagic pathway is largely unknown. Here, we use a human midbrain dopamine (mDA) neuronal culture model to provide evidence in support of a cellular mechanism that explains the cell-to-cell transfer of pathological forms of SNCA that are observed in PD. We demonstrate that LGALS3 (galectin 3) mediates the release of SNCA following vesicular damage. SNCA release is also dependent on TRIM16 (tripartite motif containing 16) and ATG16L1 (autophagy related 16 like 1), providing evidence that secretion of SNCA is mediated by an autophagic secretory pathway.

Pathological α-syn aggregation is mediated by glycosphingolipid chain length and the physiological state of α-syn in vivo.

GBA1 mutations that encode lysosomal ß-glucocerebrosidase (GCase) cause the lysosomal storage disorder Gaucher disease (GD) and are strong risk factors for synucleinopathies, including Parkinson's disease and Lewy body dementia. Only a subset of subjects with GBA1 mutations exhibit neurodegeneration, and the factors that influence neurological phenotypes are unknown. We find that α-synuclein (α-syn) neuropathology induced by GCase depletion depends on neuronal maturity, the physiological state of α-syn, and specific accumulation of long-chain glycosphingolipid (GSL) GCase substrates. Reduced GCase activity does not initiate α-syn aggregation in neonatal mice or immature human midbrain cultures; however, adult mice or mature midbrain cultures that express physiological α-syn oligomers are aggregation prone. Accumulation of long-chain GSLs (≥C22), but not short-chain species, induced α-syn pathology and neurological dysfunction. Selective reduction of long-chain GSLs ameliorated α-syn pathology through lysosomal cathepsins. We identify specific requirements that dictate synuclein pathology in GD models, providing possible explanations for the phenotypic variability in subjects with GCase deficiency.

Cargo and cell-specific differences in extracellular vesicle populations identified by multiplexed immunofluorescent analysis.

Extracellular vesicles (EVs) have been implicated in a wide variety of biological activities, have been implicated in the pathogenesis of numerous diseases, and have been proposed to serve as potential biomarkers of disease in human patients and animal models. However, characterization of EV populations is often performed using methods that do not account for the heterogeneity of EV populations and require comparatively large sample sizes to facilitate analysis. Here, we describe an imaging-based method that allows for the multiplexed characterization of EV populations at the single EV level following centrifugation of EV populations directly onto cover slips, allowing comprehensive analysis of EV populations with relatively small samples. We observe that canonical EV markers are present on subsets of EVs which differ substantially in a producer cell and cargo specific fashion, including differences in EVs containing different HIV-1 proteins previously reported to be incorporated into pathogenic EVs. We also describe a lectin binding assay to interrogate EVs based on their glycan content, which we observe to change in response to pharmacological modulation of secretory autophagy pathways. These studies collectively reveal that a multiplexed analysis of EV populations using fluorescent microscopy can reveal differences in specific EV populations that may be used to understand the biogenesis of specific EV populations and/or to interrogate small subsets of EVs of interest within larger EV populations in biological samples.