Dominant Mutations in the Autoimmune Regulator AIRE Are Associated with Common Organ-Specific Autoimmune Diseases.

The autoimmune regulator (AIRE) gene is crucial for establishing central immunological tolerance and preventing autoimmunity. Mutations in AIRE cause a rare autosomal-recessive disease, autoimmune polyendocrine syndrome type 1 (APS-1), distinguished by multi-organ autoimmunity. We have identified multiple cases and families with mono-allelic mutations in the first plant homeodomain (PHD1) zinc finger of AIRE that followed dominant inheritance, typically characterized by later onset, milder phenotypes, and reduced penetrance compared to classical APS-1. These missense PHD1 mutations suppressed gene expression driven by wild-type AIRE in a dominant-negative manner, unlike CARD or truncated AIRE mutants that lacked such dominant capacity. Exome array analysis revealed that the PHD1 dominant mutants were found with relatively high frequency (>0.0008) in mixed populations. Our results provide insight into the molecular action of AIRE and demonstrate that disease-causing mutations in the AIRE locus are more common than previously appreciated and cause more variable autoimmune phenotypes.

Substrate reduction therapy using Genz-667161 reduces levels of pathogenic components in a mouse model of neuronopathic forms of Gaucher disease.

Most lysosomal storage diseases (LSDs) have a significant neurological component, including types 2 and 3 Gaucher disease (neuronal forms of Gaucher disease; nGD). No therapies are currently available for nGD since the recombinant enzymes used in the systemic form of Gaucher disease do not cross the blood-brain barrier (BBB). However, a number of promising approaches are currently being tested, including substrate reduction therapy (SRT), in which partial inhibition of the synthesis of the glycosphingolipids (GSLs) that accumulate in nGD lowers their accumulation. We now induce nGD in mice by injection with conduritol B-epoxide (CBE), an irreversible inhibitor of acid beta-glucosidase (GCase), the enzyme defective in nGD, with or without co-injection with Genz-667161, a prototype for SRT which crosses the BBB. Significant neuropathology, and a reduction in lifespan, was observed upon CBE injection, and this was largely reversed by co-injection with Genz-667161, along with a reduction in glucosylceramide and glucosylsphingosine levels. Analysis of gene expression by RNAseq revealed that Genz-667161 largely reversed the changes in genes and pathways that were differentially expressed upon CBE injection, specifically pathways of GSL metabolism, lipoproteins and other lipid metabolic pathways, lipid droplets, astrocyte activation, neuronal function, and to some extent, neuroinflammation. Together, this demonstrates the efficacy of SRT to reverse the effects of substrate accumulation on pathological components and pathways in nGD brain.

Fingolimod phosphate inhibits astrocyte inflammatory activity in mucolipidosis IV.

Mucolipidosis IV (MLIV) is an orphan neurodevelopmental disease that causes severe neurologic dysfunction and loss of vision. Currently there is no therapy for MLIV. It is caused by loss of function of the lysosomal channel mucolipin-1, also known as TRPML1. Knockout of the Mcoln1 gene in a mouse model mirrors clinical and neuropathologic signs in humans. Using this model, we previously observed robust activation of microglia and astrocytes in early symptomatic stages of disease. Here we investigate the consequence of mucolipin-1 loss on astrocyte inflammatory activation in vivo and in vitro and apply a pharmacologic approach to restore Mcoln1-/- astrocyte homeostasis using a clinically approved immunomodulator, fingolimod. We found that Mcoln1-/- mice over-express numerous pro-inflammatory cytokines, some of which were also over-expressed in astrocyte cultures. Changes in the cytokine profile in Mcoln1-/- astrocytes are concomitant with changes in phospho-protein signaling, including activation of PI3K/Akt and MAPK pathways. Fingolimod promotes cytokine homeostasis, down-regulates signaling within the PI3K/Akt and MAPK pathways and restores the lysosomal compartment in Mcoln1-/- astrocytes. These data suggest that fingolimod is a promising candidate for preclinical evaluation in our MLIV mouse model, which, in case of success, can be rapidly translated into clinical trial.

Mice defective in interferon signaling help distinguish between primary and secondary pathological pathways in a mouse model of neuronal forms of Gaucher disease.

BACKGROUND: The type 1 interferon (IFN) response is part of the innate immune response and best known for its role in viral and bacterial infection. However, this pathway is also induced in sterile inflammation such as that which occurs in a number of neurodegenerative diseases, including neuronopathic Gaucher disease (nGD), a lysosomal storage disorder (LSD) caused by mutations in GBA. METHODS: Mice were injected with conduritol B-epoxide, an irreversible inhibitor of acid beta-glucosidase, the enzyme defective in nGD. MyTrMaSt null mice, where four adaptors of pathogen recognition receptors (PRRs) are deficient, were used to determine the role of the IFN pathway in nGD pathology. Activation of inflammatory and other pathways was analyzed by a variety of methods including RNAseq. RESULTS: Elevation in the expression of PRRs associated with the IFN response was observed in CBE-injected mice. Ablation of upstream pathways leading to IFN production had no therapeutic benefit on the lifespan of nGD mice but attenuated neuroinflammation. Primary and secondary pathological pathways (i.e., those associated or not with mouse survival) were distinguished, and a set of ~210 genes including those related to sphingolipid, cholesterol, and lipoprotein metabolism, along with a number of inflammatory pathways related to chemokines, TNF, TGF, complement, IL6, and damage-associated microglia were classified as primary pathological pathways, along with some lysosomal and neuronal genes. CONCLUSIONS: Although IFN signaling is the top elevated pathway in nGD, we demonstrate that this pathway is not related to mouse viability and is consequently defined as a secondary pathology pathway. By elimination, we defined a number of critical pathways that are directly related to brain pathology in nGD, which in addition to its usefulness in understanding pathophysiological mechanisms, may also pave the way for the development of novel therapeutic paradigms by targeting such pathways.

Absence of infiltrating peripheral myeloid cells in the brains of mouse models of lysosomal storage disorders.

Approximately 70 lysosomal storage diseases are currently known, resulting from mutations in genes encoding lysosomal enzymes and membrane proteins. Defects in lysosomal enzymes that hydrolyze sphingolipids have been relatively well studied. Gaucher disease is caused by the loss of activity of glucocerebrosidase, leading to accumulation of glucosylceramide. Gaucher disease exhibits a number of subtypes, with types 2 and 3 showing significant neuropathology. Sandhoff disease results from the defective activity of ß-hexosaminidase, leading to accumulation of ganglioside GM2. Niemann-Pick type C disease is primarily caused by the loss of activity of the lysosomal membrane protein, NPC1, leading to storage of cholesterol and sphingosine. All three disorders display significant neuropathology, accompanied by neuroinflammation. It is commonly assumed that neuroinflammation is the result of infiltration of monocyte-derived macrophages into the brain; for instance, cells resembling lipid-engorged macrophages ('Gaucher cells') have been observed in the brain of Gaucher disease patients. We now review the evidence that inflammatory macrophages are recruited into the brain in these diseases and then go on to provide some experimental data that, at least in the three mouse models tested, monocyte-derived macrophages do not appear to infiltrate the brain. Resident microglia, which are phenotypically distinct from infiltrating macrophages, are the only myeloid population present in significant numbers within the brain parenchyma in these authentic mouse models, even during the late symptomatic stages of disease when there is substantial neuroinflammation. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/. This article is part of the Special Issue "Lysosomal Storage Disorders".

Ablation of the pro-inflammatory master regulator miR-155 does not mitigate neuroinflammation or neurodegeneration in a vertebrate model of Gaucher's disease.

Bi-allelic mutations in the glucocerebrosidase gene (GBA1) cause Gaucher's disease, the most common human lysosomal storage disease. We previously reported a marked increase in miR-155 transcript levels and early microglial activation in a zebrafish model of Gaucher's disease (gba1-/-). miR-155 is a master regulator of inflammation and has been implicated in a wide range of different neurodegenerative disorders. The observed miR-155 upregulation preceded the subsequent development of widespread pathology with marked neuroinflammation, closely resembling human Gaucher's disease pathology. We now report similar increases of miR-155 expression in mammalian models of GD, confirming that miR-155 upregulation is a shared feature in glucocerebrosidase (GCase) deficiency across different species. Using CRISPR/Cas9 mutagenesis we then generated a miR-155 mutant zebrafish line (miR-155-/-) with completely abolished miR-155 expression. Unexpectedly, loss of miR-155 did not mitigate either the reduced lifespan or the robust inflammatory phenotypes of gba1-/- mutant zebrafish. Our data demonstrate that neither neuroinflammation nor disease progression in GCase deficiency are dependent on miR-155 and suggest that miR-155 inhibition would not be a promising therapeutic target in Gaucher's disease.

Identification of a feedback loop involving ß-glucosidase 2 and its product sphingosine sheds light on the molecular mechanisms in Gaucher disease.

The lysosomal acid ß-glucosidase GBA1 and the non-lysosomal ß-glucosidase GBA2 degrade glucosylceramide (GlcCer) to glucose and ceramide in different cellular compartments. Loss of GBA2 activity and the resulting accumulation of GlcCer results in male infertility, whereas mutations in the GBA1 gene and loss of GBA1 activity cause the lipid-storage disorder Gaucher disease. However, the role of GBA2 in Gaucher disease pathology and its relationship to GBA1 is not well understood. Here, we report a GBA1-dependent down-regulation of GBA2 activity in patients with Gaucher disease. Using an experimental approach combining cell biology, biochemistry, and mass spectrometry, we show that sphingosine, the cytotoxic metabolite accumulating in Gaucher cells through the action of GBA2, directly binds to GBA2 and inhibits its activity. We propose a negative feedback loop, in which sphingosine inhibits GBA2 activity in Gaucher cells, preventing further sphingosine accumulation and, thereby, cytotoxicity. Our findings add a new chapter to the understanding of the complex molecular mechanism underlying Gaucher disease and the regulation of ß-glucosidase activity in general.

Delineating pathological pathways in a chemically induced mouse model of Gaucher disease.

Great interest has been shown in understanding the pathology of Gaucher disease (GD) due to the recently discovered genetic relationship with Parkinson's disease. For such studies, suitable animal models of GD are required. Chemical induction of GD by inhibition of acid ß-glucosidase (GCase) using the irreversible inhibitor conduritol B-epoxide (CBE) is particularly attractive, although few systematic studies examining the effect of CBE on the development of symptoms associated with neurological forms of GD have been performed. We now demonstrate a correlation between the amount of CBE injected into mice and levels of accumulation of the GD substrates, glucosylceramide and glucosylsphingosine, and show that disease pathology, indicated by altered levels of pathological markers, depends on both the levels of accumulated lipids and the time at which their accumulation begins. Gene array analysis shows a remarkable similarity in the gene expression profiles of CBE-treated mice and a genetic GD mouse model, the Gba(flox/flox) ;nestin-Cre mouse, with 120 of the 144 genes up-regulated in CBE-treated mice also up-regulated in Gba(flox/flox) ;nestin-Cre mice. We also demonstrate that various aspects of neuropathology and some behavioural abnormalities can be arrested upon cessation of CBE treatment during a specific time window. Together, our data demonstrate that injection of mice with CBE provides a rapid and relatively easy way to induce symptoms typical of neuronal forms of GD. This is particularly useful when examining the role of specific biochemical pathways in GD pathology, since CBE can be injected into mice defective in components of putative pathological pathways, alleviating the need for time-consuming crossing of mice. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Proteomics analysis of a human brain sample from a mucolipidosis type IV patient reveals pathophysiological pathways.

BACKGROUND: Mucolipidosis type IV (MLIV), an ultra-rare neurodevelopmental and neurodegenerative disorder, is caused by mutations in the MCOLN1 gene, which encodes the late endosomal/lysosomal transient receptor potential channel TRPML1 (mucolipin 1). The precise pathophysiogical pathways that cause neurological disease in MLIV are poorly understood. Recently, the first post-mortem brain sample became available from a single MLIV patient, and in the current study we performed mass spectrometry (MS)-based proteomics on this tissue with a view to delineating pathological pathways, and to compare with previously-published data on MLIV, including studies using the Mcoln1-/- mouse. RESULTS: A number of pathways were altered in two brain regions from the MLIV patient, including those related to the lysosome, lipid metabolism, myelination, cellular trafficking and autophagy, mTOR and calmodulin, the complement system and interferon signaling. Of these, levels of some proteins not known previously to be associated with MLIV were altered, including APOD, PLIN4, ATG and proteins related to interferon signaling. Moreover, when proteins detected by proteomics in the human brain were compared with their orthologs detected in the Mcoln1-/- mouse by RNAseq, the results were remarkably similar. Finally, analysis of proteins in human and mouse CSF suggest that calbindin 1 and calbindin 2 might be useful as biomarkers to help chart the course of disease development. CONCLUSIONS: Despite the sample size limitations, our findings are consistent with the relatively general changes in lysosomal function previously reported in MLIV, and shed light on new pathways of disease pathophysiology, which is required in order to understand the course of disease development and to determine the efficacy of therapies when they become available for this devastating disease.

Brain pathology and cerebellar purkinje cell loss in a mouse model of chronic neuronopathic Gaucher disease.

Gaucher disease (GD) is currently the focus of considerable attention due primarily to the association between the gene that causes GD (GBA) and Parkinson's disease. Mouse models exist for the systemic (type 1) and for the acute neuronopathic forms (type 2) of GD. Here we report the generation of a mouse that phenotypically models chronic neuronopathic type 3 GD. Gba-/-;Gbatg mice, which contain a Gba transgene regulated by doxycycline, accumulate moderate levels of the offending substrate in GD, glucosylceramide, and live for up to 10 months, i.e. significantly longer than mice which model type 2 GD. Gba-/-;Gbatg mice display behavioral abnormalities at ∼4 months, which deteriorate with age, along with significant neuropathology including loss of Purkinje neurons. Gene expression is altered in the brain and in isolated microglia, although the changes in gene expression are less extensive than in mice modeling type 2 disease. Finally, bone deformities are consistent with the Gba-/-;Gbatg mice being a genuine type 3 GD model. Together, the Gba-/-;Gbatg mice share pathological pathways with acute neuronopathic GD mice but also display differences that might help understand the distinct disease course and progression of type 2 and 3 patients.

In vivo inactivation of glycosidases by conduritol B epoxide and cyclophellitol as revealed by activity-based protein profiling.

Glucocerebrosidase (GBA) is a lysosomal ß-glucosidase-degrading glucosylceramide. Its deficiency causes Gaucher disease (GD), a common lysosomal storage disorder. Carrying a genetic abnormality in GBA constitutes at present the largest genetic risk factor for Parkinson's disease (PD). Conduritol B epoxide (CBE), a mechanism-based irreversible inhibitor of GBA, is used to generate cell and animal models for investigations on GD and PD. However, CBE may have additional glycosidase targets besides GBA. Here, we present the first in vivo target engagement study for CBE, employing a suite of activity-based probes to visualize catalytic pocket occupancy of candidate off-target glycosidases. Only at significantly higher CBE concentrations, nonlysosomal glucosylceramidase (GBA2) and lysosomal α-glucosidase were identified as major off-targets in cells and zebrafish larvae. A tight, but acceptable window for selective inhibition of GBA in the brain of mice was observed. On the other hand, cyclophellitol, a closer glucose mimic, was found to inactivate with equal affinity GBA and GBA2 and therefore is not suitable to generate genuine GD-like models. ENZYMES: Glucocerebrosidase (EC 3.2.1.45), nonlysosomal ß-glucocerebrosidase (EC 3.2.1.45); cytosolic ß-glucosidase (EC 3.2.1.21); α-glucosidases (EC 3.2.1.20); ß-glucuronidase (EC 3.2.1.31).

Emerging therapeutic targets for Gaucher disease.

INTRODUCTION: Gaucher disease (GD) is an inherited metabolic disorder caused by mutations in the glucocerebrosidase (GBA1) gene. Although infusions of recombinant GBA ameliorate the systemic effects of GD, this therapy has no effect on the neurological manifestations. Patients with the neuronopathic forms of GD (nGD) are often severely disabled and die prematurely. The search for innovative drugs is thus urgent for the neuronopathic forms. AREAS COVERED: Here we briefly summarize the available treatments for GD. We then review recent studies of the molecular pathogenesis of GD, which suggest new avenues for therapeutic development. EXPERT OPINION: Existing treatments for GD are designed to target the primary consequence of the inborn defects of sphingolipid metabolism, that is, lysosomal accumulation of glucosylceramide (GlcCer). Here we suggest that targeting other pathways, such as those that are activated as a consequence of GlcCer accumulation, may also have salutary clinical effects irrespective of whether excess substrate persists. These pathways include those implicated in neuroinflammation, and specifically, receptor-interacting protein kinase-3 (RIP3) and related components of this pathway, which appear to play a vital role in the pathogenesis of nGD. Once available, inhibitors to components of the RIP kinase pathway will hopefully offer new therapeutic opportunities in GD.