Balraj Doray

In vertebrates, newly synthesized lysosomal enzymes traffic to lysosomes through the mannose-6-phosphate (M6P) pathway. The Golgi membrane protein LYSET was recently discovered to regulate lysosome biogenesis by controlling the level of GlcNAc-1-phosphotransferase (GNPT). However, its working mechanism remained unclear. In this study, we demonstrate that LYSET is a two-transmembrane protein essential for GNPT stability, cleavage by Site-1 Protease (S1P), and enzymatic activity. We reconcile conflicting models by showing that LYSET enhances GNPT cleavage and prevents its mislocalization to lysosomes for degradation. We further establish that LYSET achieves this by interacting with GOLPH3 and retromer complexes to anchor the LYSET-GNPT complex at the Golgi. Alanine mutagenesis identified an F ⁴ XXR ⁷ motif in LYSET’s N-tail for GOLPH3 binding. The retromer further promotes Golgi retention by binding to the C-terminal of LYSET and recycling it from endolysosomes. Together, our findings reveal LYSET’s multifaceted role in stabilizing GNPT, retaining it at the Golgi, and ensuring the fidelity of the M6P pathway, thereby providing insights into its molecular function.

Molecular genetic testing is increasingly used in clinical care to identify genetic variants and their impact on disease burden. However, variants of uncertain significance (VUS) hamper the utility of molecular diagnostic testing. In patients presenting with Hunter Syndrome and VUS in the IDS gene, clinical testing for iduronate-2-sulfatase enzyme activity has been the mainstay to determine whether a variant is likely damaging. However, enzyme assays alone fail to predict disease severity. In this study, we developed an image-based cellular assay using genome-engineered cells with IDS variants to determine whether a specific variant causes morphological changes that are associated with disease. Specifically, we generated twelve mutant cell lines and documented both IDS biochemical activity and reproducible phenotypic differences therein. Next, we examined patient-derived cell lines and found the same phenotypic differences compared to parental controls. The morphological changes were complex, but measured on a single scale, which we termed PathScore_LC. To determine whether the observed changes are specific to IDS, we reintroduced a recombinant human IDS enzyme (rhIDS) to rescue both the biochemical and phenotypic changes of these cells. We found a partial rescue in the presence of corrective levels of IDS enzyme. Finally, we examined the differences in gene expression and found that a recombinant enzyme was not sufficient to fully restore transcriptional changes in the mutant lines at the time points studied. This proof-of-concept study establishes preliminary validation of the method and sets the stage for future functional studies and broader IDS variant testing.

A biomarker in spinal fluid reflects brain disease in a rare genetic disorder, with implications for how we diagnose and monitor the disease.

The cellular etiology of seizures in CLN2 disease, a childhood-onset neurodegenerative lysosomal storage disorder caused by a deficiency of tripeptidyl peptidase 1 (TPP1), remains elusive. Given that Cln2 ^R207X/R207X mice display fatal spontaneous seizures and an early loss of several cortical GABAergic interneuron populations, we hypothesized that these 2 events might be causally related. To study the cell-autonomous effects of interneuron-specific TPP1 deficiency, we first generated transgenic mice expressing loxP -flanked lysosomal membrane–tethered TPP1 (TPP1LAMP1 mice) on the Cln2 ^R207X/R207X genetic background, and then crossed TPP1LAMP1 mice with Vgat-Cre mice. These Vgat-Cre; TPP1LAMP1 mice accumulated storage material in cortical and striatal interneurons. Vgat-Cre; TPP1LAMP1 mice also died more readily after pentylenetetrazole-induced seizures, indicating that interneuron-specific TPP1 deficiency renders these mice more susceptible to seizure-induced mortality. We also selectively activated interneurons using designer receptors exclusively activated by designer drugs (DREADDs) in Vgat-Cre; Cln2 ^R207X/R207X mice. Electroencephalogram monitoring revealed that DREADD-mediated activation of interneurons markedly accelerated the onset of spontaneous seizures and seizure-associated death in Vgat-Cre; Cln2 ^R207X/R207X mice, suggesting that modulating interneuron activity can exacerbate epileptiform abnormalities. Taken together, these results provide mechanistic insights into the underlying etiology of seizures and premature death that characterize CLN2 disease.

As with most enzyme-storage disorders, DNA testing at an early age is critical for identifying genetic variants and their impact on disease burden. Still, most variants in genes such as Iduronate-2-sulfatase, IDS, are Variants of Uncertain Significance (VUS). In patients presenting with Hunter Syndrome, clinical testing for IDS enzyme activity has been the mainstay to determine whether a variant is likely damaging. However, these enzyme assays are unable to predict disease severity or neuronal toxicity and may be missing many aspects of IDS pathology. In this study, we have developed an image-based assay using genome-engineered cells with IDS mutations to identify if a specific mutation causes lysosomal and membrane disruptions that characterize the disease. Specifically, we generate twelve mutant cell lines and document both the biochemical changes in IDS activity and the reproducible phenotypic differences therein. Next, we examine two patient-derived cell lines and find the same phenotypic differences in these lines compared to parental controls. The phenotypic changes are measured on a specific scale, which we term the PathScore _LC . To determine whether the observed changes are specific to IDS, we reintroduce a recombinant version of the IDS enzyme (rhIDS) to rescue both the biochemical and phenotypic changes of these cells. Interestingly, even after 120 hours, rescue is present, but not all the cells return to normal, which may also reflect the pathological state. Finally, we examine the gene expression differences and find that recombinant enzyme is not sufficient to induce transcriptional changes in the mutant lines at the time points studied. Overall, these cell-based lysosomal and membrane phenotypes may be key to quickly and accurately profiling clinical variants in the IDS gene.

LYSET is a recently identified Golgi transmembrane (TM) protein, and inactivating mutations in the LYSET gene phenocopy mucolipidosis II (MLII), the lysosomal storage disease caused by loss of function of GlcNAc-1-phosphotransferase αβ (GNPTαβ), which tags lysosomal hydrolases with the mannose 6-phosphate (M6P) tag for delivery to lysosomes. It is conceivable that LYSET facilitates integration of both hydrophilic TM helices (TMHs) of GNPTαβ and retain the latter in the Golgi, although this has only been directly demonstrated for the N-terminal TMH wherein a membrane-stabilized GNPTαβ variant restores lysosomal function in cells lacking LYSET. Here we show that the C-terminal TMH of GNPTαβ also contributes to LYSET-mediated Golgi retention. In addition, disease-causing patient mutations in the N-terminal TMH of GNPTαβ, which increase the hydrophilicity of the helix, are partly rescued by overexpression of LYSET. Finally, we show that a membrane-stabilized GNPTαβ variant, despite overcoming the requirement for LYSET, still requires COPI-mediated recycling via the N-terminal cytosolic domain (CD) for GNPTαβ retention and function in the Golgi.