Development of quantitative high-throughput screening assays to identify, validate, and optimize small-molecule stabilizers of misfolded ß-glucocerebrosidase with therapeutic potential for Gaucher disease and Parkinson's disease.

Glucocerebrosidase (GCase) is implicated in both a rare, monogenic disorder (Gaucher disease, GD) and a common, multifactorial condition (Parkinson's disease); hence, it is an urgent therapeutic target. To identify correctors of severe protein misfolding and trafficking obstruction manifested by the pathogenic L444P-variant of GCase, we developed a suite of quantitative, high-throughput, cell-based assays. First, we labeled GCase with a small pro-luminescent HiBiT peptide reporter tag, enabling quantitation of protein stabilization in cells while faithfully maintaining target biology. TALEN-based gene editing allowed for stable integration of a single HiBiT-GBA1 transgene into an intragenic safe-harbor locus in GBA1-knockout H4 (neuroglioma) cells. This GD cell model was amenable to lead discovery via titration-based quantitative high-throughput screening and lead optimization via structure-activity relationships. A primary screen of 10,779 compounds from the NCATS bioactive collections identified 140 stabilizers of HiBiT-GCase-L444P, including both pharmacological chaperones (ambroxol and non-inhibitory chaperone NCGC326) and proteostasis regulators (panobinostat, trans-ISRIB, and pladienolide B). Two complementary high-content imaging-based assays were deployed to triage hits: the fluorescence-quenched substrate LysoFix-GBA captured functional lysosomal GCase activity, while an immunofluorescence assay featuring antibody hGCase-1/23 provided direct visualization of GCase lysosomal translocation. NCGC326 was active in both secondary assays and completely reversed pathological glucosylsphingosine accumulation. Finally, we tested the concept of combination therapy, by demonstrating synergistic actions of NCGC326 with proteostasis regulators in enhancing GCase-L444P levels. Looking forward, these physiologically-relevant assays can facilitate the identification, pharmacological validation, and medicinal chemistry optimization of new chemical matter targeting GCase, ultimately leading to a viable therapeutic for two protein-misfolding diseases.

Characterization of Novel Human ß-glucocerebrosidase Antibodies for Parkinson's Disease Research.

BACKGROUND: Mutations in GBA1, which encodes the lysosome enzyme ß-glucocerebrosidase (also referred to as acid ß-glucosidase or GCase), are the most common genetic risk factor for Parkinson's disease (PD) and dementia with Lewy bodies (DLB). Evidence also suggests that loss of GCase activity is implicated in PD without GBA1 mutations. Consequently, therapies targeting GCase are actively being pursued as potential strategies to modify the progression of PD and related synucleinopathies. Despite this significant interest in GCase as a therapeutic target, the lack of well-characterized GCase antibodies continues to impede progress in the development of GCase-targeted therapies. OBJECTIVE: This study aims to independently evaluate human GCase (hGCase) antibodies to provide recommendations for western blot, immunofluorescence, immunoprecipitation, and AlphaLISA (Amplified Luminescent Proximity Homogeneous Assay) assays. METHODS: Two mouse monoclonal antibodies, hGCase-1/17 and hGCase-1/23, were raised against hGCase using imiglucerase, the recombinant enzyme developed to treat patients, as the antigen. These novel antibodies, alongside commonly used antibodies in the field, underwent evaluation in a variety of assays. RESULTS: The characterization of hGCase-1/17 and hGCase-1/23 using genetic models including GBA1 loss-of-function human neuroglioma H4 line and neurons differentiated from human embryonic stem cells revealed their remarkable specificity and potency in immunofluorescence and immunoprecipitation assays. Furthermore, a hGCase AlphaLISA assay with excellent sensitivity, a broad dynamic range, and suitability for high throughput applications was developed using hGCase-1/17 and hGCase-1/23, which enabled a sandwich assay configuration. CONCLUSIONS: The hGCase immunofluorescence, immunoprecipitation, and AlphaLISA assays utilizing hGCase-1/17 and hGCase-1/23 will not only facilitate improved investigations of hGCase biology, but can also serve as tools to assess the distribution and effectiveness of GCase-targeted therapies for PD and related synucleinopathies.

Characterization of Novel Human ß-glucocerebrosidase Antibodies for Parkinson Disease Research.

BACKGROUND: Mutations in GBA1, which encodes the lysosome enzyme ß-glucocerebrosidase (also referred to as acid ß-glucosidase or GCase), are the most common genetic risk factor for Parkinson disease (PD) and dementia with Lewy bodies (DLB). Evidence also suggests that loss of GCase activity is implicated in PD without GBA1 mutations. Consequently, therapies targeting GCase are actively being pursued as potential strategies to modify the progression of PD and related synucleinopathies. Despite this significant interest in GCase as a therapeutic target, the lack of well-characterized GCase antibodies continues to impede progress in the development of GCase-targeted therapies. OBJECTIVE: This study aims to independently evaluate human GCase (hGCase) antibodies to provide recommendations for western blot, immunofluorescence, immunoprecipitation, and AlphaLISA (Amplified Luminescent Proximity Homogeneous Assay) assays. METHODS: Two mouse monoclonal antibodies, hGCase-1/17 and hGCase-1/23, were raised against hGCase using imiglucerase, the recombinant enzyme used to treat patients, as the antigen. These novel antibodies, alongside commonly used antibodies in the field, underwent evaluation in a variety of assays. RESULTS: The characterization of hGCase-1/17 and hGCase-1/23 using genetic models including GBA1 loss-of-function human neuroglioma H4 line and neurons differentiated from human embryonic stem cells (hESCs) revealed their remarkable specificity and potency in immunofluorescence and immunoprecipitation assays. Furthermore, a hGCase AlphaLISA assay with excellent sensitivity, a broad dynamic range, and suitability for high throughput applications was developed using hGCase-1/17 and hGCase-1/23, which enabled a sandwich assay configuration. CONCLUSIONS: The hGCase immunofluorescence, immunoprecipitation, and AlphaLISA assays utilizing hGCase-1/17 and hGCase-1/23 will not only facilitate improved investigations of hGCase biology, but can also serve as tools to assess the distribution and effectiveness of GCase-targeted therapies for PD and related synucleinopathies.

C2-lacking isoform of Nedd4-2 regulates excitatory synaptic strength through GluA1 ubiquitination-independent mechanisms.

Neural precursor cell expressed developmentally downregulated gene 4-like (Nedd4-2) is an epilepsy-associated gene, which encodes a ubiquitin E3 ligase that is highly expressed in the brain. Nedd4-2's substrates include many ion channels and receptors because its N-terminal C2 domain guides Nedd4-2 to the cell membrane. We previously found that Nedd4-2 ubiquitinates the glutamate receptor subunit 1 (GluA1) subunit of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor, which leads to reduction of neuronal excitability and seizures in mice. However, despite awareness of a Nedd4-2 isoform with no C2 domain, the functions of this isoform remain elusive. In this study, we showed that the C2-lacking Nedd4-2 has reduced membrane distribution and exhibits reduced affinity toward ubiquitinating GluA1. However, when expressed in primary cortical neurons, we found that the C2-lacking Nedd4-2 exhibits a similar activity toward reducing excitatory synaptic strength as does the C2-containing Nedd4-2. In an attempt to identify novel Nedd4-2 substrates that could mediate excitatory synaptic strength, we used unbiased proteomic screening and found multiple synaptic regulators that were up-regulated in the brain of conditional Nedd4-2 knockout mice, including protein phosphatase 3 catalytic subunit-α (PPP3CA; alternately called calcineurin A-α). We confirmed PPP3CA as a substrate of the C2-lacking Nedd4-2 and showed that all three epilepsy-associated missense mutations of Nedd4-2 disrupted PPP3CA ubiquitination. Altogether, our results revealed novel potential Nedd4-2 substrates and suggest that the C2-lacking Nedd4-2 represses excitatory synaptic strength most likely through GluA1 ubiquitination-independent mechanisms. These findings provide novel information to further our knowledge about Nedd4-2-dependent neuronal excitability homeostasis and pathological hyperexcitability when Nedd4-2 is compromised.