The glycosylation of nucleocytoplasmic proteins with O-linked N-acetylglucosamine residues (O-GlcNAc) is conserved among metazoans and is particularly abundant within brain. O-GlcNAc is involved in diverse cellular processes ranging from the regulation of gene expression to stress response. Moreover, O-GlcNAc is implicated in various diseases including cancers, diabetes, cardiac dysfunction, and neurodegenerative diseases. Pharmacological inhibition of O-GlcNAcase (OGA), the sole enzyme that removes O-GlcNAc, reproducibly slows neurodegeneration in various Alzheimer's disease (AD) mouse models manifesting either tau or amyloid pathology. These data have stimulated interest in the possibility of using OGA-selective inhibitors as pharmaceuticals to alter the progression of AD. The mechanisms mediating the neuroprotective effects of OGA inhibitors, however, remain poorly understood. Here we show, using a range of methods in neuroblastoma N2a cells, in primary rat neurons, and in mouse brain, that selective OGA inhibitors stimulate autophagy through an mTOR-independent pathway without obvious toxicity. Additionally, OGA inhibition significantly decreased the levels of toxic protein species associated with AD pathogenesis in the JNPL3 tauopathy mouse model as well as the 3×Tg-AD mouse model. These results strongly suggest that OGA inhibitors act within brain through a mechanism involving enhancement of autophagy, which aids the brain in combatting the accumulation of toxic protein species. Our study supports OGA inhibition being a feasible therapeutic strategy for hindering the progression of AD and other neurodegenerative diseases. Moreover, these data suggest more targeted strategies to stimulate autophagy in an mTOR-independent manner may be found within the O-GlcNAc pathway. These findings should aid the advancement of OGA inhibitors within the clinic.

Loss of activity of the lysosomal glycosidase β-glucocerebrosidase (GCase) causes the lysosomal storage disease Gaucher disease (GD) and has emerged as the greatest genetic risk factor for the development of both Parkinson disease (PD) and dementia with Lewy bodies. There is significant interest into how GCase dysfunction contributes to these diseases, however, progress toward a full understanding is complicated by presence of endogenous cellular factors that influence lysosomal GCase activity. Indeed, such factors are thought to contribute to the high degree of variable penetrance of GBA mutations among patients. Robust methods to quantitatively measure GCase activity within lysosomes are therefore needed to advance research in this area, as well as to develop clinical assays to monitor disease progression and assess GCase-directed therapeutics. Here, we report a selective fluorescence-quenched substrate, LysoFQ-GBA, which enables measuring endogenous levels of lysosomal GCase activity within living cells. LysoFQ-GBA is a sensitive tool for studying chemical or genetic perturbations of GCase activity using either fluorescence microscopy or flow cytometry. We validate the quantitative nature of measurements made with LysoFQ-GBA using various cell types and demonstrate that it accurately reports on both target engagement by GCase inhibitors and the GBA allele status of cells. Furthermore, through comparisons of GD, PD, and control patient-derived tissues, we show there is a close correlation in the lysosomal GCase activity within monocytes, neuronal progenitor cells, and neurons. Accordingly, analysis of clinical blood samples using LysoFQ-GBA may provide a surrogate marker of lysosomal GCase activity in neuronal tissue.

Glycosyltransferases carry out important cellular functions in species ranging from bacteria to humans.Despite their essential roles in biology,simple and robust activity assays that can be easily applied to high-throughput screening for inhibitors of these enzymes have been challenging to develop. Herein, we report abead-based strategy to measure the grouptransfer activity of glycosyltransferases sensitively using simple fluorescence measurements,w ithout the need for coupled enzymes or secondary reactions.W ev alidate the performance and accuracy of the assayusing O-GlcNAc transferase (OGT) as am odel system through detailed Michaelis-Menten kinetic analysis of various substrates and inhibitors.O ptimization of this assay and application to high-throughput screening enabled screening for inhibitors of OGT,l eading to an ovel inhibitory scaffold. We believe this assay will prove valuable not only for the study of OGT,b ut also more widely as ag eneral approach for the screening of glycosyltransferases and other group-transfer enzymes.Supportinginformation and the ORCID identification number(s) for the author(s) of this article can be found under: https://doi.

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Glycosyltransferases carry out important cellular functions in species ranging from bacteria to humans. Despite their essential roles in biology, simple and robust activity assays that can be easily applied to high‐throughput screening for inhibitors of these enzymes have been challenging to develop. Herein, we report a bead‐based strategy to measure the group‐transfer activity of glycosyltransferases sensitively using simple fluorescence measurements, without the need for coupled enzymes or secondary reactions. We validate the performance and accuracy of the assay using O‐GlcNAc transferase (OGT) as a model system through detailed Michaelis–Menten kinetic analysis of various substrates and inhibitors. Optimization of this assay and application to high‐throughput screening enabled screening for inhibitors of OGT, leading to a novel inhibitory scaffold. We believe this assay will prove valuable not only for the study of OGT, but also more widely as a general approach for the screening of glycosyltransferases and other group‐transfer enzymes.

Within mammals, there are often several functionally related glycoside hydrolases, which makes monitoring their activities problematic. This problem is particularly acute for the enzyme β-glucocerebrosidase (GCase), the malfunction of which is a key driver of Gaucher's disease (GD) and a major risk factor for Parkinson's disease (PD). Humans harbor two other functionally related β-glucosidases known as GBA2 and GBA3, and the currently used fluorogenic substrates are not selective, which has driven the use of complicated subtractive assays involving the use of detergents and inhibitors. Here we describe the preparation of fluorogenic substrates based on the widely used nonselective substrate resorufin β-D-glucopyranoside. Using recombinant enzymes, we show that these substrates are highly selective for GCase. We also demonstrate their value through the analysis of GCase activity in brain tissue homogenates from transgenic mice expressing mutant human GCase and patient fibroblasts expressing mutant GCase. This approach simplifies the analysis of cell and tissue homogenates and should facilitate the analysis of clinical and laboratory tissues and samples.

Biallelic mutations in GBA1 that lead to reduced β-glucocerebrosidase (GCase) activity result in the monogenic lysosomal storage disease Gaucher disease (GD). Variants in one GBA1 allele are the most common genetic risk factor for multiple synucleinopathies including Parkinson’s disease (PD). Therapies to increase GCase activity in the brain hold great promise for the treatment of these diseases. To this end, we have developed blood-brain barrier penetrant therapeutic molecules by fusing antibody moieties that bind the transferrin receptor (TfR) to murine or human GCase (referred to as mGCase-mBS or hGCase-hBS, respectively). We demonstrate that these fusion proteins maintain full enzymatic activity and, while their total cellular uptake is only marginally increased compared to the enzyme alone, they have up to 100-fold better lysosomal uptake and function. Uptake and efficacy of GCase-BS relies primarily on binding to the TfR, rather than to mannose phosphate receptors (M6PRs) as conventional enzyme replacement therapy. In a GD cellular model, GCase-BS rapidly rescues the lysosomal proteome and lipid accumulations beyond known GCase substrates. Intravenous injection of mGCase-mBS leads to significant reduction of brain lysosomal membrane lipids in a GD mouse model which is sustained for four weeks. Monthly dosing over six months shows sustained efficacy and reduces neurofilament-light chain (NFL) plasma levels. Collectively, these findings demonstrate the great potential of TfR-targeted GCase for treating GBA1-associated neurodegeneration, provide insight into candidate biomarkers of GD lysosomal dysfunction, and ultimately may open a new treatment paradigm for lysosomal storage diseases (LSDs) extending beyond the central nervous system (CNS).

Gaucher disease is a lysosomal storage disorder caused by mutations which destabilize the native folded form of GCase, triggering degradation and ultimately resulting in low enzyme activity. Pharmacological chaperones (PCs) which stabilize mutant GCase have been used to increase lysosomal activity through improving trafficking efficiency. By engineering their inherent basicity, we have synthesized PCs that change conformation between the ER and the lysosomal environment, thus weakening binding to GCase after its successful trafficking to the lysosome. NMR studies confirmed the conformational change while X‐ray data reveal bound conformations and binding modes. These results were further corroborated by cell studies showing increases in GCase activity when using the pH‐switchable probe at low dosing. Preliminary in vivo assays with humanized mouse models of Gaucher showed enhanced GCase activity levels in relevant tissues, including the brain, further supporting their potential.