Highlights d 8,558 IgG1 + antigen-binding clonotypes were identified by high-throughput scRNA/VDJ-seq d 14 potent SARS-CoV-2 neutralizing antibodies were found from 60 convalescent patients d BD-368-2 showed high therapeutic and prophylactic efficacy in SARS-CoV-2-infected mice d Neutralizing antibodies can be directly selected based on predicted CDR3 H structures

α-synuclein (α-syn) is a main component of Lewy bodies (LB) that occur in many neurodegenerative diseases, includingParkinson's disease (PD), dementia with LB (DLB) and multi-system atrophy. α-syn mutations or amplifications are responsible for a subset of autosomal dominant familial PD cases, and overexpression causes neurodegeneration and motor disturbances in animals. To investigate mechanisms for α-syn accumulation and toxicity, we studied a mouse model of lysosomal enzyme cathepsin D (CD) deficiency, and found extensive accumulation of endogenous α-syn in neurons without overabundance of α-syn mRNA. In addition to impaired macroautophagy, CD deficiency reduced proteasome activity, suggesting an essential role for lysosomal CD function in regulating multiple proteolytic pathways that are important for α-syn metabolism. Conversely, CD overexpression reduces α-syn aggregation and is neuroprotective against α-syn overexpression-induced cell death in vitro. In a C. elegans model, CD deficiency exacerbates α-syn accumulation while its overexpression is protective against α-syn-induced dopaminergic neurodegeneration. Mutated CD with diminished enzymatic activity or overexpression of cathepsins B (CB) or L (CL) is not protective in the worm model, indicating a unique requirement for enzymatically active CD. Our data identify a conserved CD function in α-syn degradation and identify CD as a novel target for LB disease therapeutics.

Previous studies have argued that enhanced activity of the epidermal growth factor receptor (EGFR) and the mitogen-activated protein kinase (MAPK) pathway can promote tumor cell survival in response to cytotoxic insults. In this study, we examined the impact of MAPK signaling on the survival of primary hepatocytes exposed to low concentrations of deoxycholic acid (DCA, 50 microM). Treatment of hepatocytes with DCA caused MAPK activation, which was dependent upon ligand independent activation of EGFR, and downstream signaling through Ras and PI(3) kinase. Neither inhibition of MAPK signaling alone by MEK1/2 inhibitors, nor exposure to DCA alone, enhanced basal hepatocyte apoptosis, whereas inhibition of DCA-induced MAPK activation caused approximately 25% apoptosis within 6 h. Similar data were also obtained when either dominant negative EGFR-CD533 or dominant negative Ras N17 were used to block MAPK activation. DCA-induced apoptosis correlated with sequential cleavage of procaspase 8, BID, procaspase 9, and procaspase 3. Inhibition of MAPK potentiated bile acid-induced apoptosis in hepatocytes with mutant FAS-ligand, but did not enhance in hepatocytes that were null for FAS receptor expression. These data argues that DCA is causing ligand independent activation of the FAS receptor to stimulate an apoptotic response, which is counteracted by enhanced ligand-independent EGFR/MAPK signaling. In agreement with FAS-mediated cell killing, inhibition of caspase function with the use of dominant negative Fas-associated protein with death domain, a caspase 8 inhibitor (Ile-Glu-Thr-Asp-p-nitroanilide [IETD]) or dominant negative procaspase 8 blocked the potentiation of bile acid-induced apoptosis. Inhibition of bile acid-induced MAPK signaling enhanced the cleavage of BID and release of cytochrome c from mitochondria, which were all blocked by IETD. Despite activation of caspase 8, expression of dominant negative procaspase 9 blocked procaspase 3 cleavage and the potentiation of DCA-induced apoptosis. Treatment of hepatocytes with DCA transiently increased expression of the caspase 8 inhibitor proteins c-FLIP-(S) and c-FLIP-(L) that were reduced by inhibition of MAPK or PI(3) kinase. Constitutive overexpression of c-FLIP-(s) abolished the potentiation of bile acid-induced apoptosis. Collectively, our data argue that loss of DCA-induced EGFR/Ras/MAPK pathway function potentiates DCA-stimulated FAS-induced hepatocyte cell death via a reduction in the expression of c-FLIP isoforms.

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