The lysosomal ß-glucocerebrosidase strikes mitochondria: implications for Parkinson's therapeutics.

Parkinson's disease (PD) is a neurodegenerative disorder primarily known for typical motor features that arise due to the loss of dopaminergic neurons in the substantia nigra. However, the precise molecular etiology of the disease is still unclear. Several cellular pathways have been linked to PD, including the autophagy-lysosome pathway (ALP), α-synuclein (α-syn) aggregation, and mitochondrial function. Interestingly, the mechanistic link between GBA1, the gene that encodes for lysosomal ß-glucocerebrosidase (GCase), and PD lies in the interplay between GCase functions in the lysosome and mitochondria. GCase mutations alter mitochondria-lysosome contact sites. In the lysosome, reduced GCase activity leads to glycosphingolipid buildup, disrupting lysosomal function and autophagy, thereby triggering α-syn accumulation. Additionally, α-syn aggregates reduce GCase activity, creating a self-perpetuating cycle of lysosomal dysfunction and α-syn accumulation. GCase can also be imported into the mitochondria, where it promotes the integrity and function of mitochondrial complex I. Thus, GCase mutations that impair its normal function increase oxidative stress in mitochondria, the compartment where dopamine is oxidized. In turn, the accumulation of oxidized dopamine-adducts further impairs GCase activity, creating a second cycle of GCase dysfunction. The oxidative state triggered by GCase dysfunction can also induce mitochondrial DNA damage which, in turn, can cause dopaminergic cell death. In this review, we highlight the pivotal role of GCase in PD pathogenesis and discuss promising examples of GCase-based therapeutics such as gene and enzyme replacement therapies, small molecule chaperones, and substrate reduction therapies, among others, as potential therapeutic interventions.

Brain Disorders Due to Lysosomal Dysfunction.

Recent studies of autophagic and lysosomal pathways have significantly changed our understanding of lysosomes; once thought to be simple degradative and recycling centers, lysosomes are now known to be organelles capable of influencing signal transduction, via the mammalian target of rapamycin complex 1 (mTORC1), and regulating gene expression, via transcription factor EB (TFEB) and other transcription factors. These pathways are particularly relevant to maintaining brain homeostasis, as dysfunction of the endolysosomal and autophagic pathways has been associated with common neurodegenerative diseases, such as Alzheimer's, Parkinson's, and Huntington's, and lysosomal storage disorders, a group of inherited disorders characterized by the intralysosomal buildup of partially degraded metabolites. This review focuses on the cellular biology of lysosomes and discusses the possible mechanisms by which disruption of their function contributes to neurodegeneration. We also review and discuss how targeting TFEB and lysosomes may offer innovative therapeutic approaches for treating a wide range of neurological conditions.

A Mouse Systems Genetics Approach Reveals Common and Uncommon Genetic Modifiers of Hepatic Lysosomal Enzyme Activities and Glycosphingolipids.

Identification of genetic modulators of lysosomal enzyme activities and glycosphingolipids (GSLs) may facilitate the development of therapeutics for diseases in which they participate, including Lysosomal Storage Disorders (LSDs). To this end, we used a systems genetics approach: we measured 11 hepatic lysosomal enzymes and many of their natural substrates (GSLs), followed by modifier gene mapping by GWAS and transcriptomics associations in a panel of inbred strains. Unexpectedly, most GSLs showed no association between their levels and the enzyme activity that catabolizes them. Genomic mapping identified 30 shared predicted modifier genes between the enzymes and GSLs, which are clustered in three pathways and are associated with other diseases. Surprisingly, they are regulated by ten common transcription factors, and their majority by miRNA-340p. In conclusion, we have identified novel regulators of GSL metabolism, which may serve as therapeutic targets for LSDs and may suggest the involvement of GSL metabolism in other pathologies.

Lack of Annexin A6 Exacerbates Liver Dysfunction and Reduces Lifespan of Niemann-Pick Type C Protein-Deficient Mice.

Niemann-Pick type C (NPC) disease is a lysosomal storage disorder characterized by cholesterol accumulation caused by loss-of-function mutations in the Npc1 gene. NPC disease primarily affects the brain, causing neuronal damage and affecting motor coordination. In addition, considerable liver malfunction in NPC disease is common. Recently, we found that the depletion of annexin A6 (ANXA6), which is most abundant in the liver and involved in cholesterol transport, ameliorated cholesterol accumulation in Npc1 mutant cells. To evaluate the potential contribution of ANXA6 in the progression of NPC disease, double-knockout mice (Npc1-/-/Anxa6-/-) were generated and examined for lifespan, neurologic and hepatic functions, as well as liver histology and ultrastructure. Interestingly, lack of ANXA6 in NPC1-deficient animals did not prevent the cerebellar degeneration phenotype, but further deteriorated their compromised hepatic functions and reduced their lifespan. Moreover, livers of Npc1-/-/Anxa6-/- mice contained a significantly elevated number of foam cells congesting the sinusoidal space, a feature commonly associated with inflammation. We hypothesize that ANXA6 deficiency in Npc1-/- mice not only does not reverse neurologic and motor dysfunction, but further worsens overall liver function, exacerbating hepatic failure in NPC disease.

Genetic Background Matters: Population-Based Studies in Model Organisms for Translational Research.

We are all similar but a bit different. These differences are partially due to variations in our genomes and are related to the heterogeneity of symptoms and responses to treatments that patients exhibit. Most animal studies are performed in one single strain with one manipulation. However, due to the lack of variability, therapies are not always reproducible when treatments are translated to humans. Panels of already sequenced organisms are valuable tools for mimicking human phenotypic heterogeneities and gene mapping. This review summarizes the current knowledge of mouse, fly, and yeast panels with insightful applications for translational research.

Complement Component C3 Participates in Early Stages of Niemann-Pick C Mouse Liver Damage.

Niemann-Pick type C (NPC), a lysosomal storage disorder, is mainly caused by mutations in the NPC1 gene. Niemann-Pick type C patients and mice show intracellular cholesterol accumulation leading to hepatic failure with increased inflammatory response. The complement cascade, which belongs to the innate immunity response, recognizes danger signals from injured tissues. We aimed to determine whether there is activation of the complement system in the liver of the NPC mouse and to assess the relationship between C3 activation, a final component of the pathway, and NPC liver pathology. Niemann-Pick type C mice showed high levels of C3 staining in the liver which unexpectedly decreased with aging. Using an inducible NPC1 hepatocyte rescue mouse model, we restored NPC1 expression for a short time in young mice. We found C3 positive cells only in non-rescued cells, suggesting that C3 activation in NPC cells is reversible. Then, we studied the effect of C3 ablation on NPC liver damage at two postnatal time points, P56 and P72. Deletion of C3 reduced the presence of hepatic CD68-positive cells at postnatal day 56 and prevented the increase of transaminase levels in the blood of NPC mice. These positive effects were abrogated at P72, indicating that the complement cascade participates only during the early stages of liver damage in NPC mice, and that its inhibition may serve as a new potential therapeutic strategy for the disease.

Is Parkinson's disease a lysosomal disorder?

Common forms of Parkinson's disease have long been described as idiopathic, with no single penetrant genetic factor capable of influencing disease aetiology. Recent genetic studies indicate a clear association of variants within several lysosomal genes as risk factors for idiopathic Parkinson's disease. The emergence of novel variants suggest that the aetiology of idiopathic Parkinson's disease may be explained by the interaction of several partially penetrant mutations that, while seemingly complex, all appear to converge on cellular clearance pathways. These newly evolving data are consistent with mechanistic studies linking α-synuclein toxicity to lysosomal abnormalities, and indicate that idiopathic Parkinson's disease resembles features of Mendelian lysosomal storage disorders at a genetic and biochemical level. These findings offer novel pathways to exploit for the development of disease-altering therapies for idiopathic Parkinson's disease that target specific components of the lysosomal system.

Gadolinium Chloride Rescues Niemann⁻Pick Type C Liver Damage.

Niemann⁻Pick type C (NPC) disease is a rare neurovisceral cholesterol storage disorder that arises from loss of function mutations in the NPC1 or NPC2 genes. Soon after birth, some patients present with an aggressive hepatosplenomegaly and cholestatic signs. Histopathologically, the liver presents with large numbers of foam cells; however, their role in disease pathogenesis has not been explored in depth. Here, we studied the consequences of gadolinium chloride (GdCl3) treatment, a well-known Kupffer/foam cell inhibitor, at late stages of NPC liver disease and compared it with NPC1 genetic rescue in hepatocytes in vivo. GdCl3 treatment successfully blocked the endocytic capacity of hepatic Kupffer/foam measured by India ink endocytosis, decreased the levels CD68-A marker of Kupffer cells in the liver-and normalized the transaminase levels in serum of NPC mice to a similar extent to those obtained by genetic Npc1 rescue of liver cells. Gadolinium salts are widely used as magnetic resonance imaging (MRI) contrasts. This study opens the possibility of targeting foam cells with gadolinium or by other means for improving NPC liver disease. Synopsis: Gadolinium chloride can effectively rescue some parameters of liver dysfunction in NPC mice and its potential use in patients should be carefully evaluated.

Modeling diseases in multiple mouse strains for precision medicine studies.

The genetic basis of the phenotypic variability observed in patients can be studied in mice by generating disease models through genetic or chemical interventions in many genetic backgrounds where the clinical phenotypes can be assessed and used for genome-wide association studies (GWAS). This is particularly relevant for rare disorders, where patients sharing identical mutations can present with a wide variety of symptoms, but there are not enough number of patients to ensure statistical power of GWAS. Inbred strains are homozygous for each loci, and their single nucleotide polymorphisms catalogs are known and freely available, facilitating the bioinformatics and reducing the costs of the study, since it is not required to genotype every mouse. This kind of approach can be applied to pharmacogenomics studies as well.

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.

Neuronal and epithelial cell rescue resolves chronic systemic inflammation in the lipid storage disorder Niemann-Pick C.

Chronic systemic inflammation is thought to be a major contributor to metabolic and neurodegenerative diseases. Since inflammatory components are shared among different disorders, targeting inflammation is an attractive option for mitigating disease. To test the significance of inflammation in the lipid storage disorder (LSD) Niemann-Pick C (NPC), we deleted the macrophage inflammatory gene Mip1a/Ccl3 from NPC diseased mice. Deletion of Ccl3 had been reported to delay neuronal loss in Sandhoff LSD mice by inhibiting macrophage infiltration. For NPC mice, in contrast, deleting Ccl3 did not retard neurodegeneration and worsened the clinical outcome. Depletion of visceral tissue macrophages also did not alter central nervous system (CNS) pathology and instead increased liver injury, suggesting a limited macrophage infiltration response into the CNS and a beneficial role of macrophage activity in visceral tissue. Prevention of neuron loss or liver injury, even at late stages in the disease, was achieved through specific rescue of NPC disease in neurons or in liver epithelial cells, respectively. Local epithelial cell correction was also sufficient to reduce the macrophage-associated pathology in lung tissue. These results demonstrate that elevated inflammation and macrophage activity does not necessarily contribute to neurodegeneration and tissue injury, and LSD defects in immune cells may not preclude an appropriate inflammatory response. We conclude that inflammation remains secondary to neuronal and epithelial cell dysfunction and does not irreversibly contribute to the pathogenic cascade in NPC disease. Without further exploration of possible beneficial roles of inflammatory mediators, targeting inflammation may not be therapeutically effective at ameliorating disease severity.

The unique case of the Niemann-Pick type C cholesterol storage disorder.

Niemann-Pick type C disease (NPC) is a neurovisceral lysosomal cholesterol storage disorder that arises from loss-of-f unction mutations in either the NPCI or NPC2 genes. Both genes code for proteins involved in lysosomal cholesterol efflux. NPC is often diagnosed in early childhood, with patients typically displaying cerebellar ataxia, difficulties in Unfortunately, to date, there is no curative treatment for this devastating and fatal disorder, although several symptomatic manifestations of NPC are treatable. In this review, we discuss the cell biology of the disease, clinical aspects, diagnostic approaches, and current and potential therapeutic strategies against NPC.

Lysosomal storage disorders: old diseases, present and future challenges.

Lysosomal storage diseases (LSDs), which are inborn errors of metabolism, encompass around 50 different inherited syndromes. Together, they have an incidence of 1/7000 newborns. LSDs are caused by deficiencies in lysosomal enzymes or transporters, resulting in intra-lysosomal buildup of under graded metabolites. Common features of LSDs include bone disease, organomegaly and central and peripheral nervous system degeneration. These diseases were first described in the 1880s. Despite more than an hundred years of study of the genetic and molecular bases of LSDs, little is known about the events that lead from intra-lysosomal accumulation to the distinctive cell dysfunction and pathology that is characteristic of each disease. This review focuses on the main historical discoveries in LSD biology, from the original descriptions of their phenotypes, to animal models, including therapeutic strategies and challenges to treat this family of devastating diseases.

Understanding the phenotypic variability in Niemann-Pick disease type C (NPC): a need for precision medicine.

Niemann-Pick type C (NPC) disease is a lysosomal storage disease (LSD) characterized by the buildup of endo-lysosomal cholesterol and glycosphingolipids due to loss of function mutations in the NPC1 and NPC2 genes. NPC patients can present with a broad phenotypic spectrum, with differences at the age of onset, rate of progression, severity, organs involved, effects on the central nervous system, and even response to pharmacological treatments. This article reviews the phenotypic variation of NPC and discusses its possible causes, such as the remaining function of the defective protein, modifier genes, sex, environmental cues, and splicing factors, among others. We propose that these factors should be considered when designing or repurposing treatments for this disease. Despite its seeming complexity, this proposition is not far-fetched, considering the expanding interest in precision medicine and easier access to multi-omics technologies.

Anatomically defined neuron-based rescue of neurodegenerative Niemann-Pick type C disorder.

Niemann-Pick type C disease is a fatal lysosomal storage disorder caused by loss of NPC1 function. The disorder severely affects multiple body systems, particularly the nervous system. To test whether rescue of NPC1 activity in neurons, astrocytes, or other cell types can correct the neurological defects, a Tet-inducible Npc1-YFP transgene was introduced into Npc1(-/-) mice for the cell type-specific rescue of NPC1 loss. NPC1-YFP produced in neurons prevented neuron degeneration, slowed reactive glial activity, and ameliorated the disease. NPC1-YFP produced in astrocytes or in cells of visceral tissue did not. These results suggest that loss of NPC1 activity from neurons is the primary cause of the neuropathology and that rescue of NPC1 function in neurons is sufficient to mitigate the disease. The ability of neurons to survive and function in a cell-autonomous fashion allowed the use of this newly engineered rescue system to further define the brain regions or neuron populations required to ameliorate a neurological symptom. NPC1-YFP produced specifically in cerebellar Purkinje neurons reduced ataxia, increased weight, and prolonged life, but it did not prevent the eventual decline and premature death of Npc1(-/-) mice. Significant increase in lifespan correlated with sustained reduction of inflammation in the thalamus. Neuron rescue of other forebrain areas provided little benefit. Future work targeting increasingly discrete neuronal networks should reveal which CNS areas are critical for survival. This work may have broad implications for understanding the anatomical and cellular basis of neurological signs and symptoms of other neurodegenerative and lysosomal disorders.

Complement is dispensable for neurodegeneration in Niemann-Pick disease type C.

BACKGROUND: The immune system has been implicated in neurodegeneration during development and disease. In various studies, the absence of complement (that is, C1q deficiency) impeded the elimination of apoptotic neurons, allowing survival. In the genetic lysosomal storage disease Niemann-Pick C (NPC), caused by loss of NPC1 function, the expression of complement system components, C1q especially, is elevated in degenerating brain regions of Npc1-/- mice. Here we test whether complement is mediating neurodegeneration in NPC disease. FINDINGS: In normal mature mice, C1q mRNA was found in neurons, particularly cerebellar Purkinje neurons (PNs). In Npc1-/- mice, C1q mRNA was additionally found in activated microglia, which accumulate during disease progression and PN loss. Interestingly, C1q was not enriched on or near degenerating neurons. Instead, C1q was concentrated in other brain regions, where it partially co-localized with a potential C1q inhibitor, chondroitin sulfate proteoglycan (CSPG). Genetic deletion of C1q, or of the downstream complement pathway component C3, did not significantly alter patterned neuron loss or disease progression. Deletion of other immune response factors, a Toll-like receptor, a matrix metalloprotease, or the apoptosis facilitator BIM, also failed to alter neuron loss. CONCLUSION: We conclude that complement is not involved in the death and clearance of neurons in NPC disease. This study supports a view of neuroinflammation as a secondary response with non-causal relationship to neuron injury in the disease. This disease model may prove useful for understanding the conditions in which complement and immunity do contribute to neurodegeneration in other disorders.

Npc1 deficiency in the C57BL/6J genetic background enhances Niemann-Pick disease type C spleen pathology.

Niemann-Pick type C (NPC) disease is an autosomal recessive neurovisceral lipid storage disorder. The affected genes are NPC1 and NPC2. Mutations in either gene lead to intracellular cholesterol accumulation. There are three forms of the disease, which are categorized based on the onset and severity of the disease: the infantile form, in which the liver and spleen are severely affected, the juvenile form, in which the liver and brain are affected, and the adult form, which affects the brain. In mice, a spontaneous mutation in the Npc1 gene originated in the BALB/c inbred strain mimics the juvenile form of the disease. To study the influence of genetic background on the expression of NPC disease in mice, we transferred the Npc1 mutation from the BALB/c to C57BL/6J inbred background. We found that C57BL/6J-Npc1(-/-) mice present with a much more aggressive form of the disease, including a shorter lifespan than BALB/c-Npc1(-/-) mice. Surprisingly, there was no difference in the amount of cholesterol in the brains of Npc1(-/-) mice of either mouse strain. However, Npc1(-/-) mice with the C57BL/6J genetic background showed striking spleen damage with a marked buildup of cholesterol and phospholipids at an early age, which correlated with large foamy cell clusters. In addition, C57BL/6J Npc1(-/-) mice presented red cell abnormalities and abundant ghost erythrocytes that correlated with a lower hemoglobin concentration. We also found abnormalities in white cells, such as cytoplasmic granulation and neutrophil hypersegmentation that included lymphopenia and atypias. In conclusion, Npc1 deficiency in the C57BL6/J background is associated with spleen, erythrocyte, and immune system abnormalities that lead to a reduced lifespan.

NPC1 as a Modulator of Disease Severity and Viral Entry of SARSCoV- 2.

The COVID-19 plague is hitting mankind. Several viruses, including SARS-CoV-1, MERS-CoV, EBOV, and SARS-CoV-2, use the endocytic machinery to enter the cell. Genomic variants in NPC1, which encodes for the endo-lysosomal Niemann-Pick type C1 protein, restricts the host-range of viruses in bats and susceptibility to infections in humans. Lack of NPC1 and its pharmacological suppression inhibits many viral infections including SARS-CoV-1 and Type I Feline Coronavirus Infection. Antiviral effects of NPC1-inhibiting drugs for COVID-19 treatment should be explored.

Identification of genetic modifiers of murine hepatic ß-glucocerebrosidase activity.

The acid ß-glucocerebrosidase (GCase) enzyme cleaves glucosylceramide into glucose and ceramide. Loss of function variants in the gene encoding for GCase can lead to Gaucher disease and Parkinson's disease. Therapeutic strategies aimed at increasing GCase activity by targeting a modulating factor are attractive and poorly explored. To identify genetic modifiers, we measured hepatic GCase activity in 27 inbred mouse strains. A genome-wide association study (GWAS) using GCase activity as a trait identified several candidate modifier genes, including Dmrtc2 and Arhgef1 (p=2.1x10-7), and Grik5 (p=2.1x10-7). Bayesian integration of the gene mapping with transcriptomics was used to build integrative networks. The analysis uncovered additional candidate GCase regulators, highlighting modules of the acute phase response (p=1.01x10-8), acute inflammatory response (p=1.01x10-8), fatty acid beta-oxidation (p=7.43x10-5), among others. Our study revealed previously unknown candidate modulators of GCase activity, which may facilitate the design of therapies for diseases with GCase dysfunction.

Proteomic Analysis of Niemann-Pick Type C Hepatocytes Reveals Potential Therapeutic Targets for Liver Damage.

Niemann-Pick type C disease (NPCD) is a lysosomal storage disorder caused by mutations in the NPC1 gene. The most affected tissues are the central nervous system and liver, and while significant efforts have been made to understand its neurological component, the pathophysiology of the liver damage remains unclear. In this study, hepatocytes derived from wild type and Npc1-/- mice were analyzed by mass spectrometry (MS)-based proteomics in conjunction with bioinformatic analysis. We identified 3832 proteins: 416 proteins had a p-value smaller than 0.05, of which 37% (n = 155) were considered differentially expressed proteins (DEPs), 149 of them were considered upregulated, and 6 were considered downregulated. We focused the analysis on pathways related to NPC pathogenic mechanisms, finding that the most significant changes in expression levels occur in proteins that function in the pathways of liver damage, lipid metabolism, and inflammation. Moreover, in the group of DEPs, 30% (n = 47) were identified as lysosomal proteins and 7% (n = 10) were identified as mitochondrial proteins. Importantly, we found that lysosomal DEPs, including CTSB/D/Z, LIPA, DPP7 and GLMP, and mitocondrial DEPs, AKR1B10, and VAT1 had been connected with liver fibrosis, damage, and steatosis in previous studies, validiting our dataset. Our study found potential therapeutic targets for the treatment of liver damage in NPCD.