Hereditary spastic paraplegia‐linked REEP1 modulates endoplasmic reticulum/mitochondria contacts

Abstract

Objective Mutations in receptor expression enhancing protein 1 (REEP1) are associated with hereditary spastic paraplegias (HSPs). Although axonal degeneration is thought to be a predominant feature in HSP, the role of REEP1 mutations in degeneration is largely unknown. Previous studies have implicated a role for REEP1 in the ER, whereas others localized REEP1 with mitochondria. We sought to resolve the cellular localization of REEP1 and to further elucidate the pathobiology underlying REEP1 mutations in patients. Methods A combination of cellular imaging and biochemical approaches was used to refine the cellular localization of REEP1. Next, Reep1 mutations associated with HSP were functionally tested in neuritic growth and degeneration assays using mouse cortical culture. Finally, a novel assay was developed and used with wild type and mutant Reep1s to measure the interactions between the ER and mitochondria. Results We found that REEP1 is present at the ER-mitochondria interface, and it contains subdomains for mitochondrial as well as ER localization. Knockdown of Reep1 and the expression of pathological Reep1 mutations resulted in neuritic growth defects and degeneration. Finally, using our novel split-RLuc8 assay, we show REEP1 facilitates ER-mitochondria interactions, a function diminished by disease-associated mutations. Interpretation Our data potentially reconcile the current conflicting reports regarding REEP1 being either an ER or a mitochondrial protein. Furthermore, our results connect, for the first time, the disrupted ER-mitochondria interactions to a failure in maintaining health of long axons in HSPs. Finally, the split-RLuc8 assay offers a new tool to identify potential drugs for multiple neurodegenerative diseases with ER-mitochondria interaction defects.

Ascorbate peroxidase proximity labeling coupled with biochemical fractionation identifies promoters of endoplasmic reticulum–mitochondrial contacts

“…For the split Renilla luciferase complementation assay (22), HEK293T cells (4 ϫ 10 5 /well) were plated in a 12-well culture plate the day before transfection. Expression constructs pcDNA3-Mit-NRluc91 and pcDNA3-CRluc92-ER were cotransfected with expression constructs encoding RTN1A, RTN2B, or RTN3B using PEI (1:4 DNA/PEI ratio, Polysciences).…”

“…For example, overexpression of familial Alzheimer's disease-associated presenilin 2 mutations significantly increases EMCs and causes an imbalance of intracellular Ca 2ϩ homeostasis (20,21). Overexpression of hereditary spastic paraplegia-associated REEP1 mutations impairs the level of ER-mitochondrial contacts and results in neuritic degeneration in cortical neurons (22). Missense mutation (P56S) in the integral ER membrane protein vesicle-associated membrane protein-associated protein B (VAPB) has been linked to atypical amyotrophic lateral sclerosis (ALS8).…”

Mitochondrial junctions with cellular organelles: Ca2+ signalling perspective

“…The ability of reticulons (specifically of RTN1A, RTN2B, and RTN3B) to increase ER-mitochondria interaction was determined by split luciferase assay [23]. Similar technique was used to ascertain the role of receptor expression-enhancing protein 1 (REEP1) in potentiating ER-mitochondria interaction [100]. Another regulator of ER-mitochondrial junctions is the endoplasmic-reticulum-associated E3 ubiquitin ligase Gp78, which is particularly important for rough ER-mitochondria contacts [168].…”

From dysfunctional endoplasmic reticulum-mitochondria coupling to neurodegeneration

“…Measuring the colocalization of ER and mitochondrial markers is not the optimal approach for the assessment of organelle proximity, because of the limited resolution of confocal light microscopy (200 nm). Alternative fluorescent-based methods, such as Fluorescence Resonance Energy Transfer (FRET, (Csordas et al, 2010)), Proximity Ligation Assay (PLA, (Bernard-Marissal et al, 2015;De Vos et al, 2012;Hedskog et al, 2013;Stoica et al, 2016)) or dimerization-dependent fluorescence/luminescence (Alford et al, 2012;Lim et al, 2015;Naon et al, 2016), bypass this limitation by detecting specific interactions between ER and OM membranelocalized proteins. Super-resolution imaging approaches allow the assessment of changes in individual ER-mitochondria contacts, and are, so far, the most adapted method to investigate this subcellular compartment.…”

“…wt or disease-related substitutions (M337V, Q331K, A382T, G384C) in cell lines and in motor neurons of mouse model (Stoica et al, 2014) • No effect of down-regulation • ↓ juxtaposiZon, ↓ Ca 2+ transfers, ↓ ATP production when overexpr. wt or disease-related substitutions (R521C, R518K) in cell lines and in motor neurons of mouse model (Stoica et al, 2016) Hereditary Spastic Paraplegia (HSP) axonopathy of corticospinal motor neurons Receptor expression enhancing protein 1 (Reep1) mouse brain Expression of wt, but not of disease-related mutant forms, increases ERmitochondria proximity in cell lines (Lim et al, 2015) Charcot Marie Tooth ( • ↓ juxtaposition, ER dilatation, perturbed mt connectivity, motility and distribution when down-regulated in cell lines; ↑ juxtaposiZon, altered mt distribution when overexpr (Pla-Martin et al, 2013) • ↓ levels of triglycerides in fibroblasts and serum from a mutaZon M A N U S C R I P T…”