Mutant induced neurons and humanized mice enable identification of Niemann-Pick type C1 proteostatic therapies.

Therapeutics that rescue folding, trafficking, and function of disease-causing missense mutants are sought for a host of human diseases, but efforts to leverage model systems to test emerging strategies have met with limited success. Such is the case for Niemann-Pick type C1 disease, a lysosomal disorder characterized by impaired intracellular cholesterol trafficking, progressive neurodegeneration, and early death. NPC1, a multipass transmembrane glycoprotein, is synthesized in the endoplasmic reticulum and traffics to late endosomes/lysosomes, but this process is often disrupted in disease. We sought to identify small molecules that promote folding and enable lysosomal localization and functional recovery of mutant NPC1. We leveraged a panel of isogenic human induced neurons expressing distinct NPC1 missense mutations. We used this panel to rescreen compounds that were reported previously to correct NPC1 folding and trafficking. We established mo56-hydroxycholesterol (mo56Hc) as a potent pharmacological chaperone for several NPC1 mutants. Furthermore, we generated mice expressing human I1061T NPC1, a common mutation in patients. We demonstrated that this model exhibited disease phenotypes and recapitulated the protein trafficking defects, lipid storage, and response to mo56Hc exhibited by human cells expressing I1061T NPC1. These tools established a paradigm for testing and validation of proteostatic therapeutics as an important step toward the development of disease-modifying therapies.

Endogenous Protein-Protein Interaction Network of the NPC Cholesterol Transporter 1 in the Cerebral Cortex.

NPC intracellular cholesterol transporter 1 (NPC1) is a multipass, transmembrane glycoprotein mostly recognized for its key role in facilitating cholesterol efflux. Mutations in the NPC1 gene result in Niemann-Pick disease, type C (NPC), a fatal, lysosomal storage disease. Due to the progressively expanding implications of NPC1-related disorders, we investigated endogenous NPC1 protein-protein interactions in the mouse cortex and human-derived iPSCs neuronal models of the disease through coimmunoprecipitation-coupled with LC-MS based proteomics. The current study investigated protein-protein interactions specific to the wild-type and the most prevalent NPC1 mutation (NPC1I1061T) while filtering out any protein interactor identified in the Npc1-/- mouse model. Additionally, the results were matched across the two species to map the parallel interactome of wild-type and mutant NPC1I1061T. Most of the identified wild-type NPC1 interactors were related to cytoskeleton organization, synaptic vesicle activity, and translation. We found many putative NPC1 interactors not previously reported, including two SCAR/WAVE complex proteins that regulate ARP 2/3 complex actin nucleation and multiple membrane proteins important for neuronal activity at synapse. Moreover, we identified proteins important in trafficking specific to wild-type and mutant NPC1I1061T. Together, the findings are essential for a comprehensive understanding of NPC1 biological functions in addition to its classical role in sterol efflux.

Species-specific differences in NPC1 protein trafficking govern therapeutic response in Niemann-Pick type C disease.

The folding and trafficking of transmembrane glycoproteins are essential for cellular homeostasis and are compromised in many diseases. In Niemann-Pick type C disease, a lysosomal disorder characterized by impaired intracellular cholesterol trafficking, the transmembrane glycoprotein NPC1 misfolds due to disease-causing missense mutations. While mutant NPC1 has emerged as a robust target for proteostasis modulators, drug development efforts have been unsuccessful in mouse models. Here, we demonstrated unexpected differences in trafficking through the medial Golgi between mouse and human I1061T-NPC1, a common disease-causing mutant. We established that these distinctions are governed by differences in the NPC1 protein sequence rather than by variations in the endoplasmic reticulum-folding environment. Moreover, we demonstrated direct effects of mutant protein trafficking on the response to small molecules that modulate the endoplasmic reticulum-folding environment by affecting Ca++ concentration. Finally, we developed a panel of isogenic human NPC1 iNeurons expressing WT, I1061T-, and R934L-NPC1 and demonstrated their utility in testing these candidate therapeutics. Our findings identify important rules governing mutant NPC1's response to proteostatic modulators and highlight the importance of species- and mutation-specific responses for therapy development.

Loss of POGZ alters neural differentiation of human embryonic stem cells.

POGZ is a pogo transposable element derived protein with multiple zinc finger domains. Many de novo loss-of-function (LoF) variants of the POGZ gene are associated with autism and other neurodevelopmental disorders. However, the role of POGZ in human cortical development remains poorly understood. Here we generated multiple POGZ LoF lines in H9 human embryonic stem cells (hESCs) using CRISPR/CAS9 genome editing. These lines were then differentiated into neural structures, similar to those found in early to mid-fetal human brain, a critical developmental stage for studying disease mechanisms of neurodevelopmental disorders. We found that the loss of POGZ reduced neural stem cell proliferation in excitatory cortex-patterned neural rosettes, structures analogous to the cortical ventricular zone in human fetal brain. As a result, fewer intermediate progenitor cells and early born neurons were generated. In addition, neuronal migration from the apical center to the basal surface of neural rosettes was perturbed due to the loss of POGZ. Furthermore, cortical-like excitatory neurons derived from multiple POGZ homozygous knockout lines exhibited a more simplified dendritic architecture compared to wild type lines. Our findings demonstrate how POGZ regulates early neurodevelopment in the context of human cells, and provide further understanding of the cellular pathogenesis of neurodevelopmental disorders associated with POGZ variants.

Novel insights into the coupling of osteoclasts and resorption to bone formation.

Bone remodeling consists of resorption by osteoclasts (OCs) and formation by osteoblasts (OBs). Precise coordination of these activities is required for the resorbed bone to be replaced with an equal amount of new bone in order to maintain skeletal mass throughout the lifespan. This coordination of remodeling processes is referred to as the "coupling" of resorption to bone formation. In this review, we discuss the essential role for OCs in coupling resorption to bone formation, mechanisms for this coupling, and how coupling becomes less efficient or disrupted in conditions of bone loss. Lastly, we provide perspectives on targeting coupling to treat human bone disease.

Adipocyte-Specific Deletion of Lamin A/C Largely Models Human Familial Partial Lipodystrophy Type 2.

Mechanisms by which autosomal recessive mutations in Lmna cause familial partial lipodystrophy type 2 (FPLD2) are poorly understood. To investigate the function of lamin A/C in adipose tissue, we created mice with an adipocyte-specific loss of Lmna (Lmna ADKO). Although Lmna ADKO mice develop and maintain adipose tissues in early postnatal life, they show a striking and progressive loss of white and brown adipose tissues as they approach sexual maturity. Lmna ADKO mice exhibit surprisingly mild metabolic dysfunction on a chow diet, but on a high-fat diet they share many characteristics of FPLD2 including hyperglycemia, hepatic steatosis, hyperinsulinemia, and almost undetectable circulating adiponectin and leptin. Whereas Lmna ADKO mice have reduced regulated and constitutive bone marrow adipose tissue with a concomitant increase in cortical bone, FPLD2 patients have reduced bone mass and bone mineral density compared with controls. In cell culture models of Lmna deficiency, mesenchymal precursors undergo adipogenesis without impairment, whereas fully differentiated adipocytes have increased lipolytic responses to adrenergic stimuli. Lmna ADKO mice faithfully reproduce many characteristics of FPLD2 and thus provide a unique animal model to investigate mechanisms underlying Lmna-dependent loss of adipose tissues.

Enrichment of NPC1-deficient cells with the lipid LBPA stimulates autophagy, improves lysosomal function, and reduces cholesterol storage.

Niemann-Pick C (NPC) is an autosomal recessive disorder characterized by mutations in the NPC1 or NPC2 genes encoding endolysosomal lipid transport proteins, leading to cholesterol accumulation and autophagy dysfunction. We have previously shown that enrichment of NPC1-deficient cells with the anionic lipid lysobisphosphatidic acid (LBPA; also called bis(monoacylglycerol)phosphate) via treatment with its precursor phosphatidylglycerol (PG) results in a dramatic decrease in cholesterol storage. However, the mechanisms underlying this reduction are unknown. In the present study, we showed using biochemical and imaging approaches in both NPC1-deficient cellular models and an NPC1 mouse model that PG incubation/LBPA enrichment significantly improved the compromised autophagic flux associated with NPC1 disease, providing a route for NPC1-independent endolysosomal cholesterol mobilization. PG/LBPA enrichment specifically enhanced the late stages of autophagy, and effects were mediated by activation of the lysosomal enzyme acid sphingomyelinase. PG incubation also led to robust and specific increases in LBPA species with polyunsaturated acyl chains, potentially increasing the propensity for membrane fusion events, which are critical for late-stage autophagy progression. Finally, we demonstrated that PG/LBPA treatment efficiently cleared cholesterol and toxic protein aggregates in Purkinje neurons of the NPC1I1061T mouse model. Collectively, these findings provide a mechanistic basis supporting cellular LBPA as a potential new target for therapeutic intervention in NPC disease.

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

BACKGROUND: Niemann-Pick disease type C is a fatal and progressive neurodegenerative disorder characterized by the accumulation of unesterified cholesterol in late endosomes and lysosomes. We sought to develop new therapeutics for this disorder by harnessing the body's endogenous cholesterol scavenging particle, high-density lipoprotein (HDL). METHODS: Here we design, optimize, and define the mechanism of action of synthetic HDL (sHDL) nanoparticles. RESULTS: We demonstrate a dose-dependent rescue of cholesterol storage that is sensitive to sHDL lipid and peptide composition, enabling the identification of compounds with a range of therapeutic potency. Peripheral administration of sHDL to Npc1 I1061T homozygous mice mobilizes cholesterol, reduces serum bilirubin, reduces liver macrophage size, and corrects body weight deficits. Additionally, a single intraventricular injection into adult Npc1 I1061T brains significantly reduces cholesterol storage in Purkinje neurons. Since endogenous HDL is also a carrier of sphingomyelin, we tested the same sHDL formulation in the sphingomyelin storage disease Niemann-Pick type A. Utilizing stimulated Raman scattering microscopy to detect endogenous unlabeled lipids, we show significant rescue of Niemann-Pick type A lipid storage. CONCLUSIONS: Together, our data establish that sHDL nanoparticles are a potential new therapeutic avenue for Niemann-Pick diseases.