2020 Michael, Marcia, and Christa Parseghian Scientific Conference for Niemann-Pick Type C Research

Basic, translational, and clinical research towards a greater understanding and a cure for Niemann-Pick Type C disease.

This past year, the annual in-person conference in Tucson, AZ was cancelled due to the COVID-19 / novel Coronavirus pandemic. This conference has always been a phenomenal opportunity to gather families, scientists, and clinicians to share advances and exchange ideas related to curing NPC disease, which is why we are deeply disappointed that we were not able to meet together this year.

Because sharing research progress is essential, we developed a new format in place of the Tucson conference. We hosted a series of virtual symposia throughout the month of May and June to provide key research updates from APMRF-funded researchers and invited speakers, clinical trial updates, and other select research topics.

The symposia was hosted on a password protected Zoom platform and included talks and poster presentations.  Details are below.

These symposia were held from 11am to 2pm EST on:

• Monday, May 11
• Monday, May 18
• Tuesday, May 26
• Monday, June 1

Session #1, May 11^th: Niemann Pick Type C disease is a disease of the lysosome, and the first session brought together leaders in the field who are studying the consequences of NPC mutations on lysosome biology.  Our keynote speaker, Dr. Andrea Ballabio from the Telethon Institute of Genetics and Medicine and Federico II University, Naples, Italy, discovered a master transcriptional activation pathway that regulates the expression of a large set of lysosomal proteins.  Controlled by a transcription factor called TFEB, this pathway responds to cellular nutrient status sensed by the mTOR kinase.  Ballabio reported that phosphorylation of TFEB, which dictates its localization and activity, is regulated in a manner that differs from other mTORC1 substrates: it is highly sensitive to amino acid dependent activation of Rag GTPases (but not serum starvation), and requires Folliculin-dependent activation of RagC/D.  This mechanism plays a crucial role in Birt–Hogg–Dubé syndrome, a disorder that is caused by mutations in folliculin.  Roberto Zoncu has discovered that mTORC1 can sense the level of cholesterol in lysosomes and regulate downstream metabolic events.  Moreover, NPC1 protein is a negative regulator of mTORC1 activity and he presented an update of his current research that seeks to understand the consequences of dysregulated cholesterol-mTORC1 signaling on lysosome and mitochondrial function.

Cholesterol egress from lysosomes is tightly linked to lipid synthesis and storage at the endoplasmic reticulum (ER).  Bob Farese presented elegant new structures of dimeric human DGAT enzyme, with and without bound Acyl CoA substrates, that reveal important clues to the mechanism of triglyceride biosynthesis in the ER.  Mike Henne described his discovery of the association of the yeast cholesterol pathway synthesis enzyme, Hmg1, with the nuclear-vacuole junction created by the Nvj1 protein.  This unexpected spatial regulation of mevalonate synthesis suggests that ER-lysosome contacts spatially compartmentalize sterol synthesis.  Bob Keenan described his exciting discovery of a protein complex that assists in the membrane integration of newly synthesized, multi-pass membrane proteins such as NPC1.  Finally, Bill Pavan described their recent progress testing highly CNS-penetrant adeno-associated viruses to achieve NPC1 gene therapy in mice. He also described the hydroxypropyl-beta cyclodextrin-triggered down-regulation of GPNMB, an Alzheimer’s disease associated marker expressed in a subset of activated microglia.

Session #2, May 18^th: The session featured several outstanding speakers discussing the molecular mechanisms of cholesterol transport and signaling, with emphasis on the biochemical and structural characterization of NPC1 and the downstream routes for lysosomal cholesterol egress.

Hongyuan Yang presented ongoing work on the coupling between cholesterol transport and phosphoinositide transport by the Oxysterol Binding Protein RelatedProteins (ORP) family proteins, which reside at membrane contact sites. In particular, he discussed how ORP2, which localizes to endosome-plasma membrane contacts, utilizes a gradient of PI(4,5)P2 to transfer cholesterol in the endosome-to-PM direction. Dr. Yang also showed that ORP1L, which localizes to contacts between late endosome/lysosome and ER, may also exhibit a phosphoinositide-dependent cholesterol transport toward the ER, although in this case PI(4,5)P2 and PI(3,4)P2 may function as allosteric regulators rather than substrates for counter-transport. Nieng Yan  presented exciting structural work that reconstitutes the interaction between NPC1 and NPC2 in enabling cholesterol transport out of the lysosome. In particular, the near-atomic reconstruction of the NPC1-NPC2 complex by cryo-EM revealed a continuous tunnel connecting the N-terminal and Sterol-sensing domains of NPC1, through which cholesterol moves down its concentration gradient, eventually ending up on the outer (cytoplasmic) leaflet of the lysosomal membrane. The NPC1-NPC2 structure also provided mechanistic insight into how several disease-associated mutations in NPC1 disrupt cholesterol transport.  Xiaochun Li also presented a cryo-EM structure of human NPC1 but in complex with an inihbitor, itraconazole. The itraconazole binding site within the NPC1 molecule occurs within the hydrophobic tunnel that, based on the studies of Hongyuan Yang and Bjorn Pedersen (see below), provides the path for cholesterol transport from the lumen to the lysosomal limiting membrane. Thus, these three studies nicely complement each other on clarifying the exit route and the mechanism of sterol transport by NPC1. Bjorn Pedersen described their recently published cryo-EM structure of the yeast NPC1 homologue, NCR1, in complex with NPC2. Their structure delineates a path for cholesterol transfer that connects the hydrophobic pocket of NPC2 to the luminal domain of NCR1, continuing through the transmembrane portion of the latter protein. The arrangement of charged residues within this sterol-transfer tunnel reveals a proton-dependent translocation mechanism, explaining the requirement for lysosomal acidification in cholesterol egress. Arun Radhakrishnan discussed the use of bacterial cholesterol-binding proteins as probes to monitor cholesterol at the plasma membrane and the mechanisms that control its organization and trafficking. In particular, these studies identify three distinct pools of plasma membrane cholesterol, one of which is in rapid equilibrium with the ER and actively participates in homeostatic regulation of cholesterol synthesis. The last speaker of the session, Suzanne Pfeffer, performed a detailed analysis of cholesterol translocation across the NPC1 protein using inducible disulfide bonds to probe the requirement for large conformational changes within the protein. Moreover, she presented the results of a genome-wide Crispr-Cas9 screen for genetic modifiers of cholesterol accumulation in a cellular model of NPC. This screen led to the identification of new candidate regulators of sterol transport, including Sortin Nexin 13 (SNX13), a protein found at membrane contacts between the ER and late endosomes-lysosomes. Depletion of SNX13 rescued cholesterol accumulation caused by NPC1 loss, suggesting the existence of an additional pathway for cholesterol transport that acts in parallel to NPC1 and which could represent a therapeutic target in Niemann-Pick type C.  The session was concluded by 5 short poster presentations.

Session #3, May 26^th: This session focused on diagnostics, treatments, and animal models. The first speaker, Dan Heller, described the use of novel lipid-sensitive nanoparticles that can report changes in lipid storage in endolysosomal organelles, including the cholesterol storage organelles in NPC cells in vivo. Changes in infrared fluorescence from these particles can be sensed in deep tissues and can report changes in endolysosomal lipid storage. Viviana Gradinaru presented exciting new work on engineered gene therapy vectors with enhanced  potential for selective cell type gene expression in the central nervous system. Charles Vite showed that in some NPC model cats an optimized NPC1 gene therapy vector could restore NPC1 expression in many Purkinje neurons and ameliorate cerebellar ataxia. Eamonn Dickson discussed ways in which altered calcium signaling in NPC neurons is associated with changes in neuronal plasticity. John Pluvinage showed that dysfunction in microglia, the main innate immune system cells in the brain, can influence neuronal behavior, and that restoration of microglial homeostasis can extend the survival of NPC1 null mice. Mark Schultz investigated the differences between mouse and human forms of the NPC1 protein, which have a high degree of homology. It was found that the same mutations affect the two forms of the protein differently – arguing that therapies should be tested in human cells with mutant forms of the human NPC1gene. Kevin Vaughan discussed the role that the loss of tubule formation in lysosomes plays in NPC mutant cells and the role of a sterol-binding protein, STARD9, in the formation of these tubules. Possible strategies to restore lysosome tubulation were discussed. Paul Helquist and Olaf Wiest discussed their synthetic chemistry program to provide tools for the study of cholesterol transport in cells and possible new strategies for treating Niemann Pick C disease. There were seven posters that were presented briefly and discussed in more depth in breakout sessions.

Session #4, June 1: The final session highlighted advances in translational and clinical studies. Results of the arimoclomol trial conducted by Orphazyme were presented by Marc Patterson, who reported that the drug had a significant reduction over placebo in the annualized disease progression. Fred Maxfield provided an update on targeting endogenous chaperones to upregulate NPC1 protein trafficking, demonstrating that either inhibition of Hsp90 or increased expression of Hsp70 could improve NPC1 protein trafficking and reduce lysosomal cholesterol storage. Using the response of NPC1 protein to proteostasis regulators, Bill Balch shared machine and deep learning approaches to understanding the effect of individual variants on protein function and disease. Liz Berry-Kravis reported on the long-term benefits (up to 5 years) of VTS-270/adrabetadex to reduce disease progression in multiple cohorts, including the Phase 1/2, Phase 2/3 and expanded access patients. Beth Solomon presented findings showing that miglustat stabilized swallowing outcomes in NPC1 patients from the NPC1 Observational Study at NIH. An update on NPC Newborn Screening Initiative was provided by Allison Rosen, followed Melissa Wasserstein, who shared plans to include NPC1 newborn screening in the Screening Plus initiative in New York State. The final presentation in the session and conference was delivered by Paulina Ordonez who is examining the potential benefits of mitoprotective compounds in NPC1 disease models.

Thank you to all of the presenters and attendees for working with us as we adapted to this new format. In total we had over 275 attendees throughout the symposium and were able to facilitate some great discussions throughout the oral and poster presentations.  Next year’s conference will be May 22-25, 2021.  We look forward to seeing you back in Tucson, AZ!