Diantha van de Vlekkert

High-energy-demanding tissues, such as skeletal muscle, rely on mitochondrial proteostasis for proper function. Two key quality-control mechanisms -the ubiquitin proteasome system (UPS) and the release of mitochondria-derived vesicles- help maintain mitochondrial proteostasis, but whether these processes interact remains unclear. Here, we show that CRL5^Ozz and its substrate, Alix, localize to mitochondria and together regulate the levels and distribution of the mitochondrial solute carrier Slc25A4, which is essential for ATP production. In Ozz^−/− or Alix^−/− mice, skeletal muscle mitochondria exhibit similar morphological abnormalities, including swelling and dysmorphism, along with partially overlapping metabolomic alterations. We demonstrate that CRL5^Ozz ubiquitinates Slc25A4, targeting it for proteasomal degradation, while Alix facilitates Slc25A4 loading into exosomes for lysosomal degradation. Loss of Ozz or Alix in vivo disrupts the steady-state levels of Slc25A4, impairing mitochondrial metabolism and triggering a switch in muscle fiber composition from oxidative, mitochondria-rich slow to glycolytic fast fibers.

Sialidosis is a glycoprotein storage disease caused by deficiency of the lysosomal sialidase NEU1, which leads to pathogenic accumulation of sialylated glycoproteins and oligosaccharides in tissues and body fluids. The disease belongs to the group of orphan disorders with no therapy currently available. Here, we have tested the therapeutic potential of AAV-mediated gene therapy for the treatment of sialidosis in a mouse model of the disease. One-month-old Neu1 ^−/− mice were co-injected with two scAAV2/8 vectors, expressing NEU1 and its chaperone PPCA, and sacrificed at 3 months post-injection. Treated mice were phenotypically indistinguishable from their WT controls. Histopathologically, they showed diminished or absent vacuolization in cells of visceral organs, including the kidney, as well as the choroid plexus and other areas of the brain. This was accompanied by restoration of NEU1 activity in most tissues, reversal of sialyl-oligosacchariduria, and normalization of lysosomal exocytosis in the CSF and serum of treated mice. AAV injection prevented the occurrence of generalized fibrosis, which is a prominent contributor of disease pathogenesis in Neu1 ^−/− mice and likely in patients. Overall, this therapeutic strategy holds promise for the treatment of sialidosis and may be applicable to adult forms of human idiopathic fibrosis with low NEU1 expression.

Summary: Endoplasmic reticulum–plasma membrane (ER-PM) junctions mediate Ca ²⁺ flux across neuronal membranes. The properties of these membrane contact sites are defined by their lipid content, but little attention has been given to glycosphingolipids (GSLs). Here, we show that GM1-ganglioside, an abundant GSL in neuronal membranes, is integral to ER-PM junctions; it interacts with synaptic proteins/receptors and regulates Ca ²⁺ signaling. In a model of the neurodegenerative lysosomal storage disease, GM1-gangliosidosis, pathogenic accumulation of GM1 at ER-PM junctions due to β-galactosidase deficiency drastically alters neuronal Ca ²⁺ homeostasis. Mechanistically, we show that GM1 interacts with the phosphorylated NMDAR Ca ²⁺ channel, thereby increasing Ca ²⁺ flux, activating ERK signaling, and increasing the number of synaptic spines without increasing synaptic connectivity. Thus, GM1 clustering at ER-PM junctions alters synaptic plasticity and exacerbates the generalized neuronal cell death characteristic of GM1-gangliosidosis.

Intracellular organelles carry out many of their functions by engaging in extensive interorganellar communication through specialized membrane contact sites (MCSs) formed where two organelles tether to each other or to the plasma membrane (PM) without fusing. In recent years, these ubiquitous membrane structures have emerged as central signaling hubs that control a multitude of cellular pathways, ranging from lipid metabolism/transport to the exchange of metabolites and ions (i.e., Ca²⁺), and general organellar biogenesis. The functional crosstalk between juxtaposed membranes at MCSs relies on a defined composite of proteins and lipids that populate these microdomains in a dynamic fashion. This is particularly important in the nervous system, where alterations in the composition of MCSs have been shown to affect their functions and have been implicated in the pathogenesis of neurodegenerative diseases. In this review, we focus on the MCSs that are formed by the tethering of the endoplasmic reticulum (ER) to the mitochondria, the ER to the endo‐lysosomes and the mitochondria to the lysosomes. We highlight how glycosphingolipids that are aberrantly processed/degraded and accumulate ectopically in intracellular membranes and the PM change the topology of MCSs, disrupting signaling pathways that lead to neuronal demise and neurodegeneration. In particular, we focus on neurodegenerative lysosomal storage diseases linked to altered glycosphingolipid catabolism.

Rhabdomyosarcoma, the most common pediatric sarcoma, has no effective treatment for the pleomorphic subtype. Still, what triggers transformation into this aggressive phenotype remains poorly understood. Here we used Ptch1^+/−/ETV7^TG/+/− mice with enhanced incidence of rhabdomyosarcoma to generate a model of pleomorphic rhabdomyosarcoma driven by haploinsufficiency of the lysosomal sialidase neuraminidase 1. These tumors share mostly features of embryonal and some of alveolar rhabdomyosarcoma. Mechanistically, we show that the transforming pathway is increased lysosomal exocytosis downstream of reduced neuraminidase 1, exemplified by the redistribution of the lysosomal associated membrane protein 1 at the plasma membrane of tumor and stromal cells. Here we exploit this unique feature for single cell analysis and define heterogeneous populations of exocytic, only partially differentiated cells that force tumors to pleomorphism and promote a fibrotic microenvironment. These data together with the identification of an adipogenic signature shared by human rhabdomyosarcoma, and likely fueling the tumor’s metabolism, make this model of pleomorphic rhabdomyosarcoma ideal for diagnostic and therapeutic studies.

GM1-gangliosidosis is a catastrophic, neurodegenerative lysosomal storage disease caused by a deficiency of lysosomal β-galactosidase (β-Gal). The primary substrate of the enzyme is GM1-ganglioside (GM1), a sialylated glycosphingolipid abundant in nervous tissue. Patients with GM1-gangliosidosis present with massive and progressive accumulation of GM1 in the central nervous system (CNS), which leads to mental and motor decline, progressive neurodegeneration, and early death. No therapy is currently available for this lysosomal storage disease. Here, we describe a proof-of-concept preclinical study toward the development of enzyme replacement therapy (ERT) for GM1-gangliosidosis using a recombinant murine β-Gal fused to the plant lectin subunit B of ricin (mβ-Gal:RTB). We show that long-term, bi-weekly systemic injection of mβ-Gal:RTB in the β-Gal^−/− mouse model resulted in widespread internalization of the enzyme by cells of visceral organs, with consequent restoration of enzyme activity. Most importantly, β-Gal activity was detected in several brain regions. This was accompanied by a reduction of accumulated GM1, reversal of neuroinflammation, and decrease in the apoptotic marker caspase 3. These results indicate that the RTB lectin delivery module enhances both the CNS-biodistribution pattern and the therapeutic efficacy of the β-Gal ERT, with the potential to translate to a clinical setting for the treatment of GM1-gangliosidosis.