Broadly, the goal of this application is to understand how ER-endosome contact site dysfunction underlies the pathomechanisms of hereditary spastic paraplegia (HSP). Membrane contact sites (MCS) form between the endoplasmic reticulum (ER) and endosomes/lysosomes to facilitate processes such as fission, receptor dephosphorylation, and lipid exchange. Contact sites specifically with late endosomes (LE) have been observed to govern vesicular positioning and cholesterol transfer to the ER additionally. Mammalian cells have been reported to form contact sites with the ER and 99% of LEs. Most LEs will undergo fusion with the lysosome, completing their endocytic life-cycle. However, a mobile subset traffic directionally to the PM in response to protrusion demand. Recent efforts have focused on understanding how the formation of MCS with LEs is initiated and how observed changes in vesicular mobility occur. To date, how MCS mediate the increasing mobility of LEs in response to protrusion demand remains poorly understood. Recent investigation has uncovered novel regulatory functions of ER/endosome MCS during neurodevelopment, including protrusion and neurite outgrowth.
I aim to understand how mutations in proteins responsible for mediating MCS impair neurite outgrowth. Specifically, I seek to characterize the transitional forces of the ER-resident protein Protrudin using single-molecule force spectroscopy. Several Protrudin effectors and interacting partners may alter MCS organization in mammalian cells, including the well-known VAP homologs. Protrudin has been implicated in the initiation of late endosome mobility required for protrusion. However, this process is poorly understood. By measuring the forces necessary for MCS formation and endosome release, I will gain insight into how neurons select endosomes and subsequently direct vesicular trafficking during development. I also aim to use optogenetic imaging to artificially generate ER/endosome MCS during neurite outgrowth to delineating mechanisms responsible for vesicular mobility. HSPs are characterized by spastic progressive paralysis of the lower limbs through length dependent distal axonopathy of the cortical spinal tract, consistent with neurite outgrowth deficits. A missense mutation (p.G191V) in the membrane anchoring hairpin domain of Protrudin causes a dominant form of hereditary spastic paraplegia (HSP). This project emphasizes the generation of genome edited spinal motor neurons to study HSP and model the effects of ER-endosome MCS dysfunction during neurodevelopment. Using these motor neurons edited to express the mutant Protrudin at endogenous levels, I will examine the sub- cellular morphology using FIB-SEM to determine the impact on ER/endosome MCS. My project aims to build a detailed understanding of how Protrudin increases vesicular mobility. This represents an essential step in our understanding of the link between ER-endosome/lysosome MCS regulation and neurodegeneration. This award will help me become increasingly independent and establish an area of focus in the membrane trafficking field from which to operate in the future.

Public Health Relevance

ER-endosome/lysosome membrane contact sites are abundant in mammalian cells and regulate a multitude of the physiological process including late endosome mobility. Trafficking of late endosomes through coordination with ER contact sites is essential for neurite outgrowth and protrusion during neurodevelopment. This project aims to delineate how the ER-resident protein Protrudin coordinates late endosome mobility with other membrane contact site regulators and how Protrudin dysfunction underlies the movement disorder hereditary spastic paraplegia.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Postdoctoral Individual National Research Service Award (F32)
Project #
5F32NS108448-02
Application #
9743634
Study Section
NST-2 Subcommittee (NST)
Program Officer
Gubitz, Amelie
Project Start
2018-08-01
Project End
2021-01-31
Budget Start
2019-08-01
Budget End
2020-07-31
Support Year
2
Fiscal Year
2019
Total Cost
Indirect Cost
Name
Yale University
Department
Neurosciences
Type
Schools of Medicine
DUNS #
043207562
City
New Haven
State
CT
Country
United States
Zip Code
06520