Reversal of Pathologic Lipid Accumulation in NPC1-Deficient Neurons by Drug-Promoted Release of LAMP1-Coated Lamellar Inclusions

Abstract

Aging and pathologic conditions cause intracellular aggregation of macromolecules and the dysfunction and degeneration of neurons, but the mechanisms are largely unknown. Prime examples are lysosomal storage disorders such as Niemann-Pick type C (NPC) disease, where defects in the endosomal-lysosomal protein NPC1 or NPC2 cause intracellular accumulation of unesterified cholesterol and other lipids leading to neurodegeneration and fatal neurovisceral symptoms. Here, we investigated the impact of NPC1 deficiency on rodent neurons using pharmacologic and genetic models of the disease. Improved ultrastructural detection of lipids and correlative light and electron microscopy identified lamellar inclusions as the subcellular site of cholesterol accumulation in neurons with impaired NPC1 activity. Immunogold labeling combined with transmission electron microscopy revealed the presence of CD63 on internal lamellae and of LAMP1 on the membrane surrounding the inclusions, indicating their origins from intraluminal vesicles of late endosomes and of a lysosomal compartment, respectively. Lamellar inclusions contained cell-intrinsic cholesterol and surface-labeled GM1, indicating the incorporation of plasma membrane components. Scanning electron microscopy revealed that the therapeutic drug candidate ␤-cyclodextrin induces the subplasmalemmal location of lamellar inclusions and their subsequent release to the extracellular space. In parallel, ␤-cyclodextrin mediated the NPC1-independent redistribution of cholesterol within neurons and thereby abolished a deleterious cycle of enhanced cholesterol synthesis and its intracellular accumulation, which was indicated by neuron-specific transcript analysis. Our study provides new mechanistic insight into the pathologic aggregation of macromolecules in neurons and suggests exocytosis as cellular target for its therapeutic reversal.

Synthetic high-density lipoprotein nanoparticles for the treatment of Niemann–Pick diseases

“…2f). These data contrast the observation that the cholesterol reducing agent 2-hydroxypropyl-beta cyclodextrin (cyclodextrin) preloaded with cholesterol remains effective at reducing unesterified cholesterol in Niemann–Pick C cells [37–39], and raise that possibility that sHDL and cyclodextrin have distinct mechanisms of action.…”

“…Unesterified cholesterol that accumulates within Niemann–Pick C cells resides in LAMP1-positive LE/Lys [39]. To determine if 5A-SM traffics to this compartment, we conducted a time course and analyzed co-localization of 5A-SM-DiD with LAMP1 and filipin.…”

Pre-clinical Mouse Models of Neurodegenerative Lysosomal Storage Diseases

“…Pre-clinical trials evaluating receptor antagonism (RA), AMPA-RA (Kovacs and Pearce, 2008) and NMDA-RA (Finn et al, 2013) have improved the neurobehavioral phenotypes associated with LSDs. Furthermore, a number of pharmacological agents have been explored eliciting various effects from modulating membrane fluidity (Schultz et al, 2018), modulating Ca 2+ (Chang et al, 2007) or cholesterol levels (Erickson et al, 2000;Pelled et al, 2003;Kim et al, 2007;Repa et al, 2007;Abi-Mosleh et al, 2009;Liu et al, 2010;Taylor et al, 2012;Hovakimyan et al, 2013;Nusca et al, 2014;Tanaka et al, 2014;Soga et al, 2015;Demais et al, 2016;Liou et al, 2016), and enhancing enzyme activity (Arroyo et al, 2014) via the use of neurosteroids (NS) (Griffin et al, 2004;Liao et al, 2009). There is also interest in facilitating sphingolipid degradation via the upregulation of heat shock proteins (HSP) (Chung et al, 2016;Kirkegaard et al, 2016).…”

“…--SRT (Platt et al, 1997) (Sango et al, 1995) + + AAV (Sargeant et al, 2011;Cachon-Gonzalez et al, 2014), BMT (Norflus et al, 1998;Wada et al, 2000), CM (Pelled et al, 2003), Diet (Denny et al, 2010), HSP (Kirkegaard et al, 2016), MIP-1 (Wu and Proia, 2004), SRT (Jeyakumar et al, 1999) HexB ( Npc1 (−/−) (Morris et al, 1977) + -AAV (Chandler et al, 2017b;Xie et al, 2017;Hughes et al, 2018), AI (Alvarez et al, 2008), AO (Fu et al, 2013), CM (Erickson et al, 2000;Repa et al, 2007;Abi-Mosleh et al, 2009;Liu et al, 2010;Taylor et al, 2012;Hovakimyan et al, 2013;Nusca et al, 2014;Tanaka et al, 2014;Soga et al, 2015;Demais et al, 2016), Diet (Jelinek et al, 2015;Soga et al, 2015), HSP (Chung et al, 2016;Kirkegaard et al, 2016), NS (Griffin et al, 2004;Liao et al, 2009), Transplant (Veyron et al, 1996) Npc1 (Otterbach and Stoffel, 1995) + +…”

Loss of Mevalonate/Cholesterol Homeostasis in the Brain: A Focus on Autism Spectrum Disorder and Rett Syndrome

“…At present, there is no curative treatment for this disease, and reduction of glycolipids by N -butyl-deoxynojirimycin (Miglustat) is the only therapeutic option in the EU [39]. Recent data suggest a new therapeutic drug candidate, β-cyclodextrin [40], which may resolubilize cholesterol within cells and induce the release of cholesterol-rich membrane inclusions [41].…”