Protein Sorting in the Endomembrane System

EIPR1 (EARP–interacting protein 1, formerly TSSC1) is a WD40–domain protein (WD40 domains act as protein-interaction scaffolds) involved in delivering endosome-derived transmembrane cargos to the plasma membrane and the trans-Golgi network (TGN) and in the biogenesis of dense core vesicles. While in cultured cells and model organisms its role was known, its significance in humans was unclear. We identified five homozygous EIPR1 missense variants in eight individuals with a neurological disorder featuring neurodevelopmental delay, microcephaly, ataxia, and spasticity. These variants reduce EIPR1 protein levels and its interaction with EARP and GARP complexes (Golgi-assisted retrograde protein complexes), impairing endosomal recycling and dense-core vesicle biogenesis. Patient fibroblasts exhibit phenotypes consistent with EARP and GARP deficiencies. Knockout of eipr1 in zebrafish causes defects that are rescued by wild-type human EIPR1 mRNA but not by variant mRNAs, confirming impaired activity. These findings link EIPR1 to a neurodevelopmental disorder and highlight its essential role in endosomal recycling and vesicle biogenesis.

Hereditary spastic paraplegia type 21 (SPG21) is an inherited neurological disorder caused by biallelic mutations in the SPG21 gene, which encodes the protein maspardin. We found that the SPG21 protein localizes to endolysosomes via its interaction with the GTP–bound form of RAB7A (protein that regulates endolysosomal trafficking). Disease-associated SPG21 variants reduce SPG21 expression and disrupt its endolysosomal localization in both non-neuronal cells and neurons. Functional analysis connects SPG21 to endolysosomal and mTORC1 (a protein kinase complex that functions as a nutrient/energy/redox sensor and controls protein synthesis) signaling pathways. Biochemical studies show that SPG21 depletion does not affect the phosphorylation of canonical mTORC1 substrates such as ULK1, S6K1, and 4E-BP1, but reduces the phosphorylation of the non-canonical mTORC1 substrate TFEB, a central regulator of the autophagy/lysosomal-to-nucleus signaling pathway. This reduction enhances TFEB's nuclear localization and the expression of specific TFEB–target genes. Therefore, SPG21 acts as a RAB7A effector that promotes non-canonical mTORC1–mediated phosphorylation of TFEB, suppressing its nuclear localization and transcriptional activity. These findings link SPG21 dysfunction to altered endolysosomal signaling, providing new insights into SPG21 pathogenesis.

The BLOC-one–related complex (BORC) is a multiprotein complex involved in positioning of the lysosome within the cytoplasm, which is composed of eight subunits, named BORCS1 (also known as BLOC1S1) through BORCS8. BORC associates with the cytosolic surface of lysosomes, where it facilitates the recruitment of the small GTPase ARL8 and kinesin-1 and kinesin-3 microtubule motors. Such recruitment promotes the anterograde transport of lysosomes toward the peripheral cytoplasm in non-neuronal cells and toward distal axons in neurons. In previous work, we demonstrated that biallelic variants in BORCS8 cause an early-infantile neurodegenerative disorder. Over the past year, through two independent collaborations, one with Niccolò Mencacci and colleagues and another with Adeline Vanderver and colleagues, we identified additional patients with biallelic variants in two other BORC subunits: BORCS5 and BLOC1S1. These patients, all children, presented with a spectrum of neurodevelopmental abnormalities, including global developmental delay, severe to profound intellectual disability, hypotonia, limb spasticity, muscle wasting, dysmorphic facial features, optic atrophy, leuko-axonopathy with hypomyelination, and other neurodegenerative features predominantly affecting supratentorial brain regions. Cellular studies revealed that the pathogenic variants in BORCS5 and BLOC1S1 result in reduced protein expression, impaired assembly with other BORC subunits, and/or compromised ability to mediate lysosome translocation to the cell periphery. Together, these findings establish BORCS5 and BLOC1S1 as novel genetic loci for early-infantile neurodegenerative disorders and underscore the essential role of BORC and lysosomal dynamics in the development and maintenance of the central nervous system.

In collaboration with Yousuf Khan and other colleagues, we discovered a novel mechanism by which cells generate a proteoform of the lysosome-kinesin adaptor PLEKHM2 (also known as SKIP) through programmed ribosomal frameshifting. The process involves a subset of ribosomes that alter their reading frame on an mRNA. Although frameshifting is commonly utilized by viruses, there are very few known phylogenetically conserved examples in nuclear-encoded genes. We identified a +1 frameshifting event during the decoding of the human gene PLEKHM2, which encodes an essential adaptor for lysosomal trafficking, an event that grants access to a second, internally overlapping open reading frame. The resulting carboxyl-terminal domain of this frameshift protein forms an α-helix, releasing PLEKHM2 from autoinhibition and enabling its movement to the tips of cells independently of activation by the GTPase ARL8

ARL5 is a small GTPase of the ARF family that is recruited to the TGN by the ARF–family GTPase ARFRP1, in complex with the transmembrane protein SYS1. At the TGN, ARL5 mediates the recruitment of its known effector, the GARP–tethering complex, promoting SNARE–dependent fusion of endosome-derived retrograde transport carriers with the TGN. To explore additional functions of ARL5, we performed proximity biotinylation and protein-interaction assays to identify novel effectors. These analyses revealed that the armadillo-repeat protein (a family of proteins that contain several tandemly repeated copies composed of a pair of alpha helices that form a hairpin structure) ARMH3 (C10orf76) specifically binds to the active, GTP–bound form of ARL5 and is recruited to the TGN in a manner dependent on SYS1, ARFRP1, and ARL5. Unlike GARP, ARMH3 is not required for retrograde cargo transport. Instead, ARMH3 plays a distinct role by activating phosphatidylinositol 4-kinase III beta (PI4KB), which generates the major pool of phosphatidylinositol 4-phosphate (PI4P) at the TGN. This PI4P pool supports the recruitment of the oncoprotein GOLPH3 and facilitates glycan processing at the TGN. Collectively, these findings identify a novel SYS1–ARFRP1–ARL5–ARMH3 axis that regulates PI4KB–mediated PI4P production at the TGN, expanding our understanding of lipid signaling and membrane trafficking at this organelle.

In neurons, RNA granules are transported along the axon for local translation away from the soma. Recent studies indicate that some of this transport involves the hitchhiking of RNA granules on lysosome-related vesicles. To identify a subset of axonal mRNAs that depend on lysosome-related vesicles for transport, this past year we leveraged the ability to prevent transport of these vesicles into the axon by knocking out the lysosome-kinesin adaptor BLOC-one–related complex (BORC), which we had discovered in previous research. We found that BORC knockout causes depletion of a large group of axonal mRNAs that mainly encode ribosomal and mitochondrial/oxidative phosphorylation proteins. The depletion results in mitochondrial defects and eventually leads to axonal degeneration in human induced pluripotent stem cell (iPSC)–derived and mouse neurons. Pathway analyses of the depleted mRNAs revealed a mechanistic connection between BORC deficiency and common neurodegenerative disorders. The findings demonstrate that mRNA transport on lysosome-related vesicles is critical for the maintenance of axonal homeostasis and that its failure causes axonal degeneration.