Internalized SNCA/α-synuclein fibrils become truncated and resist degradation in neurons while glial cells rapidly degrade SNCA fibrils

Parkinson disease (PD) and other α-synucleinopathies are characterized by the intracellular aggregates of SNCA/α-synuclein (synuclein, alpha) thought to spread via cell-to-cell transmission. To understand the contributions of various brain cells to the spreading of SNCA pathology, we examined the metabolism of SNCA aggregates in neuronal and glial cells. In neurons, while the full-length SNCA rapidly disappeared following SNCA pre-formed-fibril (PFF) uptake, truncated SNCA accumulated with a half-life of days rather than hours. Epitope mapping and fractionation studies indicate that SNCA fibrils internalized by neurons were truncated at the C-terminal region and remained insoluble. In contrast, microglia and astrocytes rapidly metabolized SNCA fibrils as the half-lives of SNCA fibrils in these glial cells were < 6 h. Differential uptake and processing of SNCA fibrils by neurons and glia was recapitulated in vivo where injection of fluorescently labeled SNCA fibrils initially accumulated in glial cells followed by rapid clearance while neurons stably accumulated SNCA fibrils at a slower rate. Immunolocalization and subcellular fractionation studies show that internalized SNCA PFF was initially localized to endosomes followed by lysosomes. The lysosome was largely responsible for the degradation of internalized SNCA PFF as the inhibition of lysosomal function led to the stabilization of SNCA in all cell types. Significantly, SNCA PFF causes lysosomal dysfunction in neurons. In summary, we show that neurons are inefficient in metabolizing internalized SNCA aggregates, partially because SNCA aggregates cause lysosomal dysfunction, potentially generating aggregation-prone truncated SNCA. In contrast, glial cells may protect neurons from SNCA aggregates by rapidly clearing these aggregates.<b>Abbreviations</b>: 3MA, 3-methyladenine; aa, amino acids; AF, Alexa Fluor; Baf A1, bafilomycin A1; DMEM, Dulbecco's modified Eagle's medium; DMSO, dimethyl sulfoxide; FL, full-length; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HMM, high molecular mass; Hs, human; kDa, kilodalton; MAP1LC3/LC3, microtubule-associated protein 1 light chain 3; ML, molecular layer; NAC domain, non-amyloidal component; PCN, primary cortical neuron; PD, Parkinson diseases; PFF, pre-formed-fibril; PFF-488, PFF Alexa Fluor-488; PMG, primary microglia; SNCA, synuclein, alpha; SNCA[∆], C-terminally truncated SNCA; SQSTM1/p62, sequestosome 1; TX-100, Triton X-100.