Spatacsin regulates directionality of lysosome trafficking by promoting the degradation of its partner AP5Z1.

The endoplasmic reticulum (ER) forms contacts with the lysosomal compartment, regulating lysosome positioning and motility. The movements of lysosomes are controlled by the attachment of molecular motors to their surface. However, the molecular mechanisms by which ER controls lysosome dynamics are still elusive. Here, using mouse brain extracts and mouse embryonic fibroblasts, we demonstrate that spatacsin is an ER-resident protein regulating the formation of tubular lysosomes, which are highly dynamic. Screening for spatacsin partners required for tubular lysosome formation showed spatacsin to act by regulating protein degradation. We demonstrate that spatacsin promotes the degradation of its partner AP5Z1, which regulates the relative amount of spastizin and AP5Z1 at lysosomes. Spastizin and AP5Z1 contribute to regulate tubular lysosome formation, as well as their trafficking by interacting with anterograde and retrograde motor proteins, kinesin KIF13A and dynein/dynactin subunit p150Glued, respectively. Ultimately, investigations in polarized mouse cortical neurons in culture demonstrated that spatacsin-regulated degradation of AP5Z1 controls the directionality of lysosomes trafficking. Collectively, our results identify spatacsin as a protein regulating the directionality of lysosome trafficking.

Update on the Genetics of Spastic Paraplegias.

PURPOSE OF REVIEW: Hereditary spastic paraplegias are a genetically heterogeneous group of neurological disorders. Patients present lower limb weakness and spasticity, complicated in complex forms by additional neurological signs. We review here the major steps toward understanding the molecular basis of these diseases made over the last 10 years. RECENT FINDINGS: Our perception of the intricate connections between clinical, genetic, and molecular aspects of neurodegenerative disorders has radically changed in recent years, thanks to improvements in genetic approaches. This is particularly true for hereditary spastic paraplegias, for which > 60 genes have been identified, highlighting (i) the considerable genetic heterogeneity of this group of clinically diverse disorders, (ii) the fuzzy border between recessive and dominant inheritance for several mutations, and (iii) the overlap of these mutations with other neurological conditions in terms of their clinical effects. Several hypotheses have been put forward concerning the pathophysiological mechanisms involved, based on the genes implicated and their known function and based on studies on patient samples and animal models. These mechanisms include mainly abnormal intracellular trafficking, changes to endoplasmic reticulum shaping and defects affecting lipid metabolism, lysosome physiology, autophagy, myelination, and development. Several causative genes affect multiple of these functions, which are, most of the time, interconnected. Recent major advances in our understanding of these diseases have revealed unifying pathogenic models that could be targeted in the much-needed development of new treatments.

Loss of spatacsin function alters lysosomal lipid clearance leading to upper and lower motor neuron degeneration.

Mutations in SPG11 account for the most common form of autosomal recessive hereditary spastic paraplegia (HSP), characterized by a gait disorder associated with various brain alterations. Mutations in the same gene are also responsible for rare forms of Charcot-Marie-Tooth (CMT) disease and progressive juvenile-onset amyotrophic lateral sclerosis (ALS). To elucidate the physiopathological mechanisms underlying these human pathologies, we disrupted the Spg11 gene in mice by inserting stop codons in exon 32, mimicking the most frequent mutations found in patients. The Spg11 knockout mouse developed early-onset motor impairment and cognitive deficits. These behavioral deficits were associated with progressive brain atrophy with the loss of neurons in the primary motor cortex, cerebellum and hippocampus, as well as with accumulation of dystrophic axons in the corticospinal tract. Spinal motor neurons also degenerated and this was accompanied by fragmentation of neuromuscular junctions and muscle atrophy. This new Spg11 knockout mouse therefore recapitulates the full range of symptoms associated with SPG11 mutations observed in HSP, ALS and CMT patients. Examination of the cellular alterations observed in this model suggests that the loss of spatacsin leads to the accumulation of lipids in lysosomes by perturbing their clearance from these organelles. Altogether, our results link lysosomal dysfunction and lipid metabolism to neurodegeneration and pinpoint a critical role of spatacsin in lipid turnover.

Loss of association of REEP2 with membranes leads to hereditary spastic paraplegia.

Hereditary spastic paraplegias (HSPs) are clinically and genetically heterogeneous neurological conditions. Their main pathogenic mechanisms are thought to involve alterations in endomembrane trafficking, mitochondrial function, and lipid metabolism. With a combination of whole-genome mapping and exome sequencing, we identified three mutations in REEP2 in two families with HSP: a missense variant (c.107T>A [p.Val36Glu]) that segregated in the heterozygous state in a family with autosomal-dominant inheritance and a missense change (c.215T>A [p.Phe72Tyr]) that segregated in trans with a splice site mutation (c.105+3G>T) in a family with autosomal-recessive transmission. REEP2 belongs to a family of proteins that shape the endoplasmic reticulum, an organelle that was altered in fibroblasts from an affected subject. In vitro, the p.Val36Glu variant in the autosomal-dominant family had a dominant-negative effect; it inhibited the normal binding of wild-type REEP2 to membranes. The missense substitution p.Phe72Tyr, in the recessive family, decreased the affinity of the mutant protein for membranes that, together with the splice site mutation, is expected to cause complete loss of REEP2 function. Our findings illustrate how dominant and recessive inheritance can be explained by the effects and nature of mutations in the same gene. They have also important implications for genetic diagnosis and counseling in clinical practice because of the association of various modes of inheritance to this new clinico-genetic entity.

Arf1-PI4KIIIß positive vesicles regulate PI(3)P signaling to facilitate lysosomal tubule fission.

Formation and fission of tubules from autolysosomes, endolysosomes, or phagolysosomes are required for lysosome reformation. However, the mechanisms governing these processes in these different lysosomal organelles are poorly understood. Thus, the role of phosphatidylinositol-4-phosphate (PI(4)P) is unclear as it was shown to promote the formation of tubules from phagolysosomes but was proposed to inhibit tubule formation on autolysosomes because the loss of PI4KIIIß causes extensive lysosomal tubulation. Using super-resolution live-cell imaging, we show that Arf1-PI4KIIIß positive vesicles are recruited to tubule fission sites from autolysosomes, endolysosomes, and phagolysosomes. Moreover, we show that PI(4)P is required to form autolysosomal tubules and that increased lysosomal tubulation caused by loss of PI4KIIIß represents impaired tubule fission. At the site of fission, we propose that Arf1-PI4KIIIß positive vesicles mediate a PI(3)P signal on lysosomes in a process requiring the lipid transfer protein SEC14L2. Our findings indicate that Arf1-PI4KIIIß positive vesicles and their regulation of PI(3)P are critical components of the lysosomal tubule fission machinery.

ORP1L mediated PI(4)P signaling at ER-lysosome-mitochondrion three-way contact contributes to mitochondrial division.

Mitochondrial division is not an autonomous event but involves multiple organelles, including the endoplasmic reticulum (ER) and lysosomes. Whereas the ER drives the constriction of mitochondrial membranes, the role of lysosomes in mitochondrial division is not known. Here, using super-resolution live-cell imaging, we investigate the recruitment of lysosomes to the site of mitochondrial division. We find that the ER recruits lysosomes to the site of division through the interaction of VAMP-associated proteins (VAPs) with the lysosomal lipid transfer protein ORP1L to induce a three-way contact between the ER, lysosome, and the mitochondrion. We also show that ORP1L might transport phosphatidylinositol-4-phosphate (PI(4)P) from lysosomes to mitochondria, as inhibiting its transfer or depleting PI(4)P at the mitochondrial division site impairs fission, demonstrating a direct role for PI(4)P in the division process. Our findings support a model where the ER recruits lysosomes to act in concert at the fission site for the efficient division of mitochondria.

Loss of spatacsin impairs cholesterol trafficking and calcium homeostasis.

Mutations in SPG11, leading to loss of spatacsin function, impair the formation of membrane tubules in lysosomes and cause lysosomal lipid accumulation. However, the full nature of lipids accumulating in lysosomes and the physiological consequences of such accumulation are unknown. Here we show that loss of spatacsin inhibits the formation of tubules on lysosomes and prevents the clearance of cholesterol from this subcellular compartment. Accumulation of cholesterol in lysosomes decreases cholesterol levels in the plasma membrane, enhancing the entry of extracellular calcium by store-operated calcium entry and increasing resting cytosolic calcium levels. Higher cytosolic calcium levels promote the nuclear translocation of the master regulator of lysosomes TFEB, preventing the formation of tubules and the clearance of cholesterol from lysosomes. Our work reveals a homeostatic balance between cholesterol trafficking and cytosolic calcium levels and shows that loss of spatacsin impairs this homeostatic equilibrium.

Inhibition of Lysosome Membrane Recycling Causes Accumulation of Gangliosides that Contribute to Neurodegeneration.

Lysosome membrane recycling occurs at the end of the autophagic pathway and requires proteins that are mostly encoded by genes mutated in neurodegenerative diseases. However, its implication in neuronal death is still unclear. Here, we show that spatacsin, which is required for lysosome recycling and whose loss of function leads to hereditary spastic paraplegia 11 (SPG11), promotes clearance of gangliosides from lysosomes in mouse and human SPG11 models. We demonstrate that spatacsin acts downstream of clathrin and recruits dynamin to allow lysosome membrane recycling and clearance of gangliosides from lysosomes. Gangliosides contributed to the accumulation of autophagy markers in lysosomes and to neuronal death. In contrast, decreasing ganglioside synthesis prevented neurodegeneration and improved motor phenotype in a SPG11 zebrafish model. Our work reveals how inhibition of lysosome membrane recycling leads to the deleterious accumulation of gangliosides, linking lysosome recycling to neurodegeneration.