Neuraminidase 1 regulates the cellular state of microglia by modulating the sialylation of Trem2.

Neuraminidase 1 (Neu1) cleaves terminal sialic acids from sialoglycoproteins in endolysosomes and at the plasma membrane. As such, Neu1 regulates immune cells, primarily those of the monocytic lineage. Here we examined how Neu1 influences microglia by modulating the sialylation of full-length Trem2 (Trem2-FL), a multifunctional receptor that regulates microglial survival, phagocytosis, and cytokine production. When Neu1 was deficient/downregulated, Trem2-FL remained sialylated, accumulated intracellularly, and was excessively cleaved into a C-terminal fragment (Trem2-CTF) and an extracellular soluble domain (sTrem2), enhancing their signaling capacities. Sialylated Trem2-FL (Sia-Trem2-FL) did not hinder Trem2-FL-DAP12-Syk complex assembly but impaired signal transduction through Syk, ultimately abolishing Trem2-dependent phagocytosis. Concurrently, Trem2-CTF-DAP12 complexes dampened NFκB signaling, while sTrem2 propagated Akt-dependent cell survival and NFAT1-mediated production of TNFα and CCL3. Because Neu1 and Trem2 are implicated in neurodegenerative/neuroinflammatory diseases, including Alzheimer disease (AD) and sialidosis, modulating Neu1 activity represents a therapeutic approach to broadly regulate microglia-mediated neuroinflammation.

Glycosphingolipids within membrane contact sites influence their function as signaling hubs in neurodegenerative diseases.

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.

Preclinical Enzyme Replacement Therapy with a Recombinant ß-Galactosidase-Lectin Fusion for CNS Delivery and Treatment of GM1-Gangliosidosis.

GM1-gangliosidosis is a catastrophic, neurodegenerative lysosomal storage disease caused by a deficiency of lysosomal ß-galactosidase (ß-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 ß-Gal fused to the plant lectin subunit B of ricin (mß-Gal:RTB). We show that long-term, bi-weekly systemic injection of mß-Gal:RTB in the ß-Gal-/- mouse model resulted in widespread internalization of the enzyme by cells of visceral organs, with consequent restoration of enzyme activity. Most importantly, ß-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 ß-Gal ERT, with the potential to translate to a clinical setting for the treatment of GM1-gangliosidosis.

Isolation of Mitochondria-Associated ER Membranes (MAMs), Synaptic MAMs, and Glycosphingolipid Enriched Microdomains (GEMs) from Brain Tissues and Neuronal Cells.

Subcellular fractionation is a valuable procedure in cell biology to separate and purify various subcellular constituents from one another, i.e., nucleus, cytosol, membranes/organelles, and cytoskeleton. The procedure relies on the use of differential centrifugation of cell and tissue homogenates. Fractionated subcellular organelles may be subjected to additional purification steps that enable the isolation of specific cellular sub-compartments, including interorganellar membrane contact sites. Here we outline a protocol tailored to the isolation of mitochondria, mitochondria-associated ER membranes (MAMs), and glycosphingolipid enriched microdomains (GEMs) from the adult mouse brain, primary neurospheres, and murine embryonic fibroblasts (MEFs). We also provide a detailed protocol for the purification of synaptosomes and their corresponding MAMs .