Mouse models of the GM2 gangliosidoses [Tay-Sachs, late onset Tay-Sachs (LOTS), Sandhoff] and GM1 gangliosidosis have been studied to determine whether there is a common neuro-inflammatory component to these disorders. During the disease course, we have: (i) examined the expression of a number of inflammatory markers in the CNS, including MHC class II, CD68, CD11b (CR3), 7/4, F4/80, nitrotyrosine, CD4 and CD8; (ii) profiled cytokine production [tumour necrosis factor alpha (TNF alpha), transforming growth factor (TGF beta 1) and interleukin 1 beta (IL1 beta)]; and (iii) studied blood-brain barrier (BBB) integrity. The kinetics of apoptosis and the expression of Fas and TNF-R1 were also assessed. In all symptomatic mouse models, a progressive increase in local microglial activation/expansion and infiltration of inflammatory cells was noted. Altered BBB permeability was evident in Sandhoff and GM1 mice, but absent in LOTS mice. Progressive CNS inflammation coincided with the onset of clinical signs in these mouse models. Substrate reduction therapy in the Sandhoff mouse model slowed the rate of accumulation of glycosphingolipids in the CNS, thus delaying the onset of the inflammatory process and disease pathogenesis. These data suggest that inflammation may play an important role in the pathogenesis of the gangliosidoses.

Summary Mitochondria-associated ER membranes or MAMs define the sites of endoplasmic reticulum/mitochondria juxtaposition that control Ca2+ flux between these organelles. We found that in a mouse model of the human lysosomal storage disease GM1-gangliosidosis, GM1-ganglioside accumulates in the glycosphingolipid-enriched microdomain (GEM) fractions of MAMs, where it interacts with the phosphorylated form of IP3 receptor-1, influencing the activity of this channel. Ca2+ depleted from the ER is then taken up by the mitochondria, leading to Ca2+ overload in this organelle. The latter induces mitochondrial membrane permeabilization (MMP), opening of the permeability transition pore and activation of the mitochondrial apoptotic pathway. This study identifies the GEMs as the sites of Ca2+ diffusion between the ER and the mitochondria. We propose a new mechanism of Ca2+-mediated apoptotic signalling, whereby GM1 accumulation at the GEMs alters Ca2+ dynamics and acts as a molecular effector of both ER stress–induced and mitochondria-mediated apoptosis of neuronal cells.

Neuraminidases (sialidases) have an essential role in the removal of terminal sialic acid residues from sialoglycoconjugates and are distributed widely in nature. The human lysosomal enzyme occurs in complex with 13-galactosidase and protective protein/cathepsin A (PPCA), and is deficient in two genetic disorders: sialidosis, caused by a structural defect in the neuraminidase gene, and galactosialidosis, in which the loss of neuraminidase activity is secondary to a deficiency of PPCA. We identified a full-length cDNA clone in the dbEST data base, of which the predicted amino acid sequence has extensive homology to other mammalian and bacterial neuraminidases, including the F(Y)RIP domain and "Asp-boxes." In situ hybridization localized the human neuraminidase gene to chromosome band 6p21, a region known to contain the HLA locus. Transient expression of the cDNA in deficient human fibroblasts showed that the enzyme is compartmentalized in lysosomes and restored neuraminidase activity in a PPCA-dependent manner. The authenticity of the cDNA was verified by the identification of three independent mutations in the open reading frame of the mRNA from clinically distinct sialidosis patients. Coexpression of the mutant cDNAs with PPCA failed to generate neuraminidase activity, confirming the inactivating effect of the mutations. These results establish the molecular basis of sialidosis in these patients, and clearly identify the cDNA-encoded protein as lysosomal neuraminidase.

GM1-ganglioside (GM1) is a major sialoglycolipid of neuronal membranes that, among other functions, modulates calcium homeostasis. Excessive accumulation of GM1 due to deficiency of lysosomal beta-galactosidase (beta-gal) characterizes the neurodegenerative disease GM1-gangliosidosis, but whether the accumulation of GM1 is directly responsible for CNS pathogenesis was unknown. Here we demonstrate that activation of an unfolded protein response (UPR) associated with the upregulation of BiP and CHOP and the activation of JNK2 and caspase-12 leads to neuronal apoptosis in the mouse model of GM1-gangliosidosis. GM1 loading of wild-type neurospheres recapitulated the phenotype of beta-gal-/- cells and activated this pathway by depleting ER calcium stores, which ultimately culminated in apoptosis. Activation of UPR pathways did not occur in mice double deficient for beta-gal and ganglioside synthase, beta-gal-/-/GalNAcT-/-, which do not accumulate GM1. These findings suggest that the UPR can be induced by accumulation of the sialoglycolipid GM1 and this causes a novel mechanism of neuronal apoptosis.

In normal human fibroblasts, an enzymically active 85,000-dalton precursor form of P-galactosidase is processed, via a number of intermediates, into a mature 64,000-dalton form. In addition there is an enzymically inactive 32,000-dalton component and its 54,000-dalton precursor. In fibroblasts from patients with a combined deficiency of P-galactosidase and neuraminidase these last two components are absent and hardly any mature /3-galactosidase can be demonstrated. Nevertheless, in the mutant fibroblasts, precursor f3-galactosidase is synthesized and processed normally. The excessive intralysosomal degradation that is responsible for the deficiency of mature P-galactosidase can be partially corrected by addition of the protease inhibitor leupeptin, which results in the accumulation of 85,000-dalton precursor P-galactosidase and of a partially processed 66,000-dalton form. When mutant cells were grown in the presence of a "corrective factor" purified from the medium of NH4CI-stimulated cell cultures, both (-galactosidase and neuraminidase activities were restored to low control levels. The immunoprecipitation pattern was completely normal after addition ofthe corrective factor, and mature 64,000-dalton (3-galactosidase accumulated in the mutant fibroblasts. We propose that the combined P-galactosidase/neuraminidase deficiency is caused by a defective 32,000-dalton glycoprotein which is normally required to protect /3-galactosidase and neuraminidase against excessive intralysosomal degradation and to give these enzymes their full hydrolytic activity.