Tiscione et al. report a molecular mechanism that links lysosomal cholesterol export with the regulation of key intracellular Ca2+ signaling pathways to influence synaptic architecture in health and neurodegenerative NPC1 disease.

SUMMARY There is increasing evidence that the lysosome is involved in the pathogenesis of a variety of neurodegenerative disorders. Thus, mechanisms that link lysosome dysfunction to the disruption of neuronal homeostasis offer opportunities to understand the molecular underpinnings of neurodegeneration and potentially identify specific therapeutic targets. Here, using a monogenic neurodegenerative disorder, NPC1 disease, we demonstrate that reduced cholesterol efflux from lysosomes aberrantly modifies neuronal firing patterns. The molecular mechanism linking alterations in lysosomal cholesterol egress to intrinsic tuning of neuronal excitability is a transcriptionally mediated upregulation of the ABCA1 transporter, whose PtdIns(4,5) P 2 -floppase activity decreases plasma membrane PtdIns(4,5) P 2 . The consequence of reduced PtdIns(4,5) P 2 is a parallel decrease in a key regulator of neuronal excitability, the voltage-gated KCNQ2/3 potassium channel, which leads to hyperexcitability in NPC1 disease neurons. Thus, cholesterol efflux from lysosomes regulates PtdIns(4,5) P 2 to shape the electrical and functional identity of the plasma membrane of neurons in health and disease.

Ca2+ is the most ubiquitous second messenger in neurons whose spatial and temporal elevations are tightly controlled to initiate and orchestrate diverse intracellular signaling cascades. Numerous neuropathologies result from mutations or alterations in Ca2+ handling proteins; thus, elucidating molecular pathways that shape Ca2+ signaling is imperative. Here, we report that loss-of-function, knockout, or neurodegenerative disease–causing mutations in the lysosomal cholesterol transporter, Niemann-Pick Type C1 (NPC1), initiate a damaging signaling cascade that alters the expression and nanoscale distribution of IP3R type 1 (IP3R1) in endoplasmic reticulum membranes. These alterations detrimentally increase Gq-protein coupled receptor–stimulated Ca2+ release and spontaneous IP3R1 Ca2+ activity, leading to mitochondrial Ca2+ cytotoxicity. Mechanistically, we find that SREBP-dependent increases in Presenilin 1 (PS1) underlie functional and expressional changes in IP3R1. Accordingly, expression of PS1 mutants recapitulate, while PS1 knockout abrogates Ca2+ phenotypes. These data present a signaling axis that links the NPC1 lysosomal cholesterol transporter to the damaging redistribution and activity of IP3R1 that precipitates cell death in NPC1 disease and suggests that NPC1 is a nanostructural disease.

Cholesterol and phosphoinositides (PI) are two critically important lipids that are found in cellular membranes and dysregulated in many disorders. Therefore, uncovering molecular pathways connecting these essential lipids may offer new therapeutic insights. We report that loss of function of lysosomal Niemann‐Pick Type C1 (NPC1) cholesterol transporter, which leads to neurodegenerative NPC disease, initiates a signaling cascade that alters the cholesterol/phosphatidylinositol 4‐phosphate (PtdIns4P) countertransport cycle between Golgi‐endoplasmic reticulum (ER), as well as lysosome‐ER membrane contact sites (MCS). Central to these disruptions is increased recruitment of phosphatidylinositol 4‐kinases—PI4KIIα and PI4KIIIβ—which boosts PtdIns4P metabolism at Golgi and lysosomal membranes. Aberrantly increased PtdIns4P levels elevate constitutive anterograde secretion from the Golgi complex, and mTORC1 recruitment to lysosomes. NPC1 disease mutations phenocopy the transporter loss of function and can be rescued by inhibition or knockdown of either key phosphoinositide enzymes or their recruiting partners. In summary, we show that the lysosomal NPC1 cholesterol transporter tunes the molecular content of Golgi and lysosome MCS to regulate intracellular trafficking and growth signaling in health and disease.

ID 29773 Poster Board 262Persistent pollutants dichlorodiphenyltrichloroethane (DDT) and dichlorodiphenyldichloroethylene (DDE) have been associated with obesity across human and rodent studies. Current research suggests the impairment of thermogenesis in brown adipose tissue plays a significant role in the decreased energy expenditure and increased adiposity of exposed individuals. We hypothesized that mice perinatally exposed to DDT or DDE would exhibit impaired thermogenesis mediated by changes in DNA methylation. At 9 weeks, exposed offspring exhibited a lower rectal temperature than age-matched controls (DDE p< 0.05, DDT p<0.01), which continued into adulthood (DDT p<.001). Brown adipose tissue was isolated from mice at postnatal day 12 and 20 weeks for reduced representation bisulfite sequencing and RNA sequencing. 49 KEGG pathways were differentially methylated following DDT or DDE exposure (p<0.05). Of the 11 pathways enriched across more than one treatment, age, or nucleic acid; most notable were ovarian steroidogenesis, oxidative phosphorylation, mTOR signaling, and thermogenesis. Brown adipocytes were exposed to DDT or DDE over early differentiation and used to validate in vivo results. Preliminary results show increased expression of ribosomal subunit 18s in adipocytes exposed to DDE (p<0.05), suggesting a dysregulation of protein synthesis. Adipocytes exposed to DDE also exhibited a trend in decreased mitochondrial DNA copy number and increased expression of ATPase subunit g, Atp5mg, (p<0.05) consistent with the enrichment direction in the oxidative phosphorylation pathway for both DNA methylation and RNA expression. These preliminary data are consistent with published findings describing impaired mitochondrial function following DDE exposure and may provide insight into the mechanisms behind thermogenic impairment.