CRISPR-Cas9 Knock-In of T513M and G41S Mutations in the Murine ß-Galactosyl-Ceramidase Gene Re-capitulates Early-Onset and Adult-Onset Forms of Krabbe Disease.

Krabbe Disease (KD) is a lysosomal storage disorder characterized by the genetic deficiency of the lysosomal enzyme ß-galactosyl-ceramidase (GALC). Deficit or a reduction in the activity of the GALC enzyme has been correlated with the progressive accumulation of the sphingolipid metabolite psychosine, which leads to local disruption in lipid raft architecture, diffuse demyelination, astrogliosis, and globoid cell formation. The twitcher mouse, the most used animal model, has a nonsense mutation, which limits the study of how different mutations impact the processing and activity of GALC enzyme. To partially address this, we generated two new transgenic mouse models carrying point mutations frequently found in infantile and adult forms of KD. Using CRISPR-Cas9 gene editing, point mutations T513M (infantile) and G41S (adult) were introduced in the murine GALC gene and stable founders were generated. We show that GALC T513M/T513M mice are short lived, have the greatest decrease in GALC activity, have sharp increases of psychosine, and rapidly progress into a severe and lethal neurological phenotype. In contrast, GALC G41S/G41S mice have normal lifespan, modest decreases of GALC, and minimal psychosine accumulation, but develop adult mild inflammatory demyelination and slight declines in coordination, motor skills, and memory. These two novel transgenic lines offer the possibility to study the mechanisms by which two distinct GALC mutations affect the trafficking of mutated GALC and modify phenotypic manifestations in early- vs adult-onset KD.

Lipidomic analysis identifies age-disease-related changes and potential new biomarkers in brain-derived extracellular vesicles from metachromatic leukodystrophy mice.

BACKGROUND: Recent findings show that extracellular vesicle constituents can exert short- and long-range biological effects on neighboring cells in the brain, opening an exciting avenue for investigation in the field of neurodegenerative diseases. Although it is well documented that extracellular vesicles contain many lipids and are enriched in sphingomyelin, cholesterol, phosphatidylserines and phosphatidylinositols, no reports have addressed the lipidomic profile of brain derived EVs in the context of Metachromatic Leukodystrophy, a lysosomal storage disease with established metabolic alterations in sulfatides. METHODS: In this study, we isolated and characterized the lipid content of brain-derived EVs using the arylsulfatase A knockout mouse as a model of the human condition. RESULTS: Our results suggest that biogenesis of brain-derived EVs is a tightly regulated process in terms of size and protein concentration during postnatal life. Our lipidomic analysis demonstrated that sulfatides and their precursors (ceramides) as well as other lipids including fatty acids are altered in an age-dependent manner in EVs isolated from the brain of the knockout mouse. CONCLUSIONS: In addition to the possible involvement of EVs in the pathology of Metachromatic Leukodystrophy, our study underlines that measuring lipid signatures in EVs may be useful as biomarkers of disease, with potential application to other genetic lipidoses.

Synaptic Function and Dysfunction in Lysosomal Storage Diseases.

Lysosomal storage diseases (LSDs) with neurological involvement are inherited genetic diseases of the metabolism characterized by lysosomal dysfunction and the accumulation of undegraded substrates altering glial and neuronal function. Often, patients with neurological manifestations present with damage to the gray and white matter and irreversible neuronal decline. The use of animal models of LSDs has greatly facilitated studying and identifying potential mechanisms of neuronal dysfunction, including alterations in availability and function of synaptic proteins, modifications of membrane structure, deficits in docking, exocytosis, recycling of synaptic vesicles, and inflammation-mediated remodeling of synapses. Although some extrapolations from findings in adult-onset conditions such as Alzheimer's disease or Parkinson's disease have been reported, the pathogenetic mechanisms underpinning cognitive deficits in LSDs are still largely unclear. Without being fully inclusive, the goal of this mini-review is to present a discussion on possible mechanisms leading to synaptic dysfunction in LSDs.

Waning efficacy in a long-term AAV-mediated gene therapy study in the murine model of Krabbe disease.

Neonatal AAV9-gene therapy of the lysosomal enzyme galactosylceramidase (GALC) significantly ameliorates central and peripheral neuropathology, prolongs survival, and largely normalizes motor deficits in Twitcher mice. Despite these therapeutic milestones, new observations identified the presence of multiple small focal demyelinating areas in the brain after 6-8 months. These lesions are in stark contrast to the diffuse, global demyelination that affects the brain of naive Twitcher mice. Late-onset lesions exhibited lysosomal alterations with reduced expression of GALC and increased psychosine levels. Furthermore, we found that lesions were closely associated with the extravasation of plasma fibrinogen and activation of the fibrinogen-BMP-SMAD-GFAP gliotic response. Extravasation of fibrinogen correlated with tight junction disruptions of the vasculature within the lesioned areas. The lesions were surrounded by normal appearing white matter. Our study shows that the dysregulation of therapeutic GALC was likely driven by the exhaustion of therapeutic AAV episomal DNA within the lesions, paralleling the presence of proliferating oligodendrocyte progenitors and glia. We believe that this is the first demonstration of diminishing expression in vivo from an AAV gene therapy vector with detrimental effects in the brain of a lysosomal storage disease animal model. The development of this phenotype linking localized loss of GALC activity with relapsing neuropathology in the adult brain of neonatally AAV-gene therapy-treated Twitcher mice identifies and alerts to possible late-onset reductions of AAV efficacy, with implications to other genetic leukodystrophies.

Biochemical Analysis of Lipid Rafts to Study Pathogenic Mechanisms of Neural Diseases.

The discovery of dynamic platforms in cell membranes, called lipid rafts or detergent resistant membrane domains, opened a new chapter on studies of membrane cell biology. Indeed, the analysis of lipid rafts enabled innovative ways to understand cellular and molecular mechanisms regulating normal and pathological processes. Lipid rafts have been studied in most cell types, where they work by providing transient and fluid architectural scaffolding platforms regulating a spectrum of important signaling pathways, including receptor activities, protein-protein interactions, posttranslational modifications of proteins and lipids and the function of ion channels. In this chapter, we will explain how to isolate these membrane domains from neural tissue samples and perform further analysis of proteins and lipids.

Standard-flow LC and thermal focusing ESI elucidates altered liver proteins in late stage Niemann-Pick, type C1 disease.

Aim: Mass spectrometry (MS)-based proteomics, particularly with the development of nano-ESI, have been invaluable to our understanding of altered proteins related to human disease. Niemann-Pick, type C1 (NPC1) disease is a fatal, autosomal recessive, neurodegenerative disorder. The resulting defects include unesterified cholesterol and sphingolipids accumulation in the late endosomal/lysosomal system resulting in organ dysfunction including liver disease. Materials & methods: First, we performed MS analysis of a complex mammalian proteome using both nano- and standard-flow ESI with the intent of developing a differential proteomics platform using standard-flow ESI. Next, we measured the differential liver proteome in the NPC1 mouse model via label-free quantitative MS using standard-flow ESI. Results: Using the standard-flow ESI approach, we found altered protein levels including, increased Limp2 and Rab7a in liver tissue of Npc1-/- compared to control mice. Conclusion: Standard-flow ESI can be a tool for quantitative proteomic studies when sample amount is not limited. Using this method, we have identified new protein markers of NPC1.

Extracellular vesicle fibrinogen induces encephalitogenic CD8+ T cells in a mouse model of multiple sclerosis.

Extracellular vesicles (EVs) are emerging as potent mediators of intercellular communication with roles in inflammation and disease. In this study, we examined the role of EVs from blood plasma (pEVs) in an experimental autoimmune encephalomyelitis mouse model of central nervous system demyelination. We determined that pEVs induced a spontaneous relapsing-remitting disease phenotype in MOG35-55-immunized C57BL/6 mice. This modified disease phenotype was found to be driven by CD8+ T cells and required fibrinogen in pEVs. Analysis of pEVs from relapsing-remitting multiple sclerosis patients also identified fibrinogen as a significant portion of pEV cargo. Together, these data suggest that fibrinogen in pEVs contributes to the perpetuation of neuroinflammation and relapses in disease.

Inhibition of the IGF-1-PI3K-Akt-mTORC2 pathway in lipid rafts increases neuronal vulnerability in a genetic lysosomal glycosphingolipidosis.

Glycosphingolipid (GSL) accumulation is implicated in the neuropathology of several lysosomal conditions, such as Krabbe disease, and may also contribute to neuronal and glial dysfunction in adult-onset conditions such as Parkinson's disease, Alzheimer's disease and multiple sclerosis. GSLs accumulate in cellular membranes and disrupt their structure; however, how membrane disruption leads to cellular dysfunction remains unknown. Using authentic cellular and animal models for Krabbe disease, we provide a mechanism explaining the inactivation of lipid raft (LR)-associated IGF-1-PI3K-Akt-mTORC2, a pathway of crucial importance for neuronal function and survival. We show that psychosine, the GSL that accumulates in Krabbe disease, leads to a dose-dependent LR-mediated inhibition of this pathway by uncoupling IGF-1 receptor phosphorylation from downstream Akt activation. This occurs by interfering with the recruitment of PI3K and mTORC2 to LRs. Akt inhibition can be reversed by sustained IGF-1 stimulation, but only during a time window before psychosine accumulation reaches a threshold level. Our study shows a previously unknown connection between LR-dependent regulation of mTORC2 activity at the cell surface and a genetic neurodegenerative disease. Our results show that LR disruption by psychosine desensitizes cells to extracellular growth factors by inhibiting signal transmission from the plasma membrane to intracellular compartments. This mechanism serves also as a mechanistic model to understand how alterations of the membrane architecture by the progressive accumulation of lipids undermines cell function, with potential implications in other genetic sphingolipidoses and adult neurodegenerative conditions. This article has an associated First Person interview with the first author of the paper.

microRNA-219 Reduces Viral Load and Pathologic Changes in Theiler's Virus-Induced Demyelinating Disease.

Analysis of microRNA (miR) expression in the central nervous system white matter of SJL mice infected with the BeAn strain of Theiler's murine encephalomyelitis virus (TMEV) revealed a significant reduction of miR-219, a critical regulator of myelin assembly and repair. Restoration of miR-219 expression by intranasal administration of a synthetic miR-219 mimic before disease onset ameliorates clinical disease, reduces neurogliosis, and partially recovers motor and sensorimotor function by negatively regulating proinflammatory cytokines and virus RNA replication. Moreover, RNA sequencing of host lesions showed that miR-219 significantly downregulated two genes essential for the biosynthetic cholesterol pathway, Cyp51 (lanosterol 14-α-demethylase) and Srebf1 (sterol regulatory element-binding protein-1), and reduced cholesterol biosynthesis in infected mice and rat CG-4 glial precursor cells in culture. The change in cholesterol biosynthesis had both anti-inflammatory and anti-viral effects. Because RNA viruses hijack endoplasmic reticulum double-layered membranes to provide a platform for RNA virus replication and are dependent on endogenous pools of cholesterol, miR-219 interference with cholesterol biosynthesis interfered virus RNA replication. These findings demonstrate that miR-219 inhibits TMEV-induced demyelinating disease through its anti-inflammatory and anti-viral properties.

Long-Term Improvement of Neurological Signs and Metabolic Dysfunction in a Mouse Model of Krabbe's Disease after Global Gene Therapy.

We report a global adeno-associated virus (AAV)9-based gene therapy protocol to deliver therapeutic galactosylceramidase (GALC), a lysosomal enzyme that is deficient in Krabbe's disease. When globally administered via intrathecal, intracranial, and intravenous injections to newborn mice affected with GALC deficiency (twitcher mice), this approach largely surpassed prior published benchmarks of survival and metabolic correction, showing long-term protection of demyelination, neuroinflammation, and motor function. Bone marrow transplantation, performed in this protocol without immunosuppressive preconditioning, added minimal benefits to the AAV9 gene therapy. Contrasting with other proposed pre-clinical therapies, these results demonstrate that achieving nearly complete correction of GALC's metabolic deficiencies across the entire nervous system via gene therapy can have a significant improvement to behavioral deficits, pathophysiological changes, and survival. These results are an important consideration for determining the safest and most effective manner for adapting gene therapy to treat this leukodystrophy in the clinic.

Psychosine enhances the shedding of membrane microvesicles: Implications in demyelination in Krabbe's disease.

In prior studies, our laboratory showed that psychosine accumulates and disrupts lipid rafts in brain membranes of Krabbe's disease. A model of lipid raft disruption helped explaining psychosine's effects on several signaling pathways important for oligodendrocyte survival and differentiation but provided more limited insight in how this sphingolipid caused demyelination. Here, we have studied how this cationic inverted coned lipid affects the fluidity, stability and structure of myelin and plasma membranes. Using a combination of cutting-edge imaging techniques in non-myelinating (red blood cell), and myelinating (oligodendrocyte) cell models, we show that psychosine is sufficient to disrupt sphingomyelin-enriched domains, increases the rigidity of localized areas in the plasma membrane, and promotes the shedding of membranous microvesicles. The same physicochemical and structural changes were measured in myelin membranes purified from the mutant mouse Twitcher, a model for Krabbe's disease. Areas of higher rigidity were measured in Twitcher myelin and correlated with higher levels of psychosine and of myelin microvesiculation. These results expand our previous analyses and support, for the first time a pathogenic mechanism where psychosine's toxicity in Krabbe disease involves deregulation of cell signaling not only by disruption of membrane rafts, but also by direct local destabilization and fragmentation of the membrane through microvesiculation. This model of membrane disruption may be fundamental to introduce focal weak points in the myelin sheath, and consequent diffuse demyelination in this leukodystrophy, with possible commonality to other demyelinating disorders.

Port-to-port delivery: Mobilization of toxic sphingolipids via extracellular vesicles.

The discovery that most cells produce extracellular vesicles (EVs) and release them in the extracellular milieu has spurred the idea that these membranous cargoes spread pathogenic mechanisms. In the brain, EVs may have multifold and important physiological functions, from deregulating synaptic activity to promoting demyelination to changes in microglial activity. The finding that small EVs (exosomes) contain α-synuclein and ß-amyloid, among other pathogenic proteins, is an example of this notion, underscoring their potential role in the brains of patients with Parkinson's and Alzheimer's diseases. Given that they are membranous vesicles, we speculate that EVs also have an intrinsic capacity to incorporate sphingolipids. In conditions under which these lipids are elevated to toxic levels, such as in Krabbe's disease and metachromatic leukodystrophy, EVs may contribute to spread disease from sick to healthy cells. In this essay, we discuss a working hypothesis that brain cells in sphingolipidoses clear some of the accumulated lipid material to attempt restoring cell homeostasis via EV secretion. We hypothesize that secreted sphingolipid-loaded EVs shuttle pathogenic lipids to cells that are not intrinsically affected, contributing to establishing non-cell-autonomous defects. © 2016 Wiley Periodicals, Inc.

Sulfatides in extracellular vesicles isolated from plasma of multiple sclerosis patients.

Extracellular vesicles (EVs) are membrane nanovesicles of diverse sizes secreted by different cell types and are involved in intercellular communication. EVs shuttle proteins, nucleic acids, and lipids that reflect their cellular origin and could mediate their biological function in recipient cells. EVs circulate in biological fluids and are considered as potential biomarkers that could be used to analyze and characterize disease development, course and response to treatment. EVs exhibit specific distribution of glycolipids and membrane organization, but little is known about the biological significance of this distribution or how it could contribute to pathological conditions such as multiple sclerosis (MS). We provide the first description of sulfatide composition in plasma-derived EVs by ultra-high-performance liquid chromatography tandem mass spectrometry. We found that EVs of different sizes showed C16:0 sulfatide but no detectable levels of C18:0, C24:0, or C24:1 sulfatide species. Small EVs isolated at 100,000 × g-enriched in exosomes-from plasma of patients with MS showed a significant increase of C16:0 sulfatide compared with healthy controls. Nanoparticle tracking analysis showed that the particle size distribution in MS plasma was significantly different compared with healthy controls. Characterization of small EVs isolated from MS plasma showed similar protein content and similar levels of exosomal markers (Alix, Rab-5B) and vesicular marker MHC class I (major histocompatibility complex class I) compared with healthy controls. Our findings indicate that C16:0 sulfatide associated with small EVs is a candidate biomarker for MS that could potentially reflect pathological changes associated with this disease and/or the effects of its treatment. © 2016 Wiley Periodicals, Inc.

Generation of a LacZ reporter transgenic mouse line for the stereological analysis of oligodendrocyte loss in galactosylceramidase deficiency.

Krabbe's disease is a leukodystrophy resulting from deficiency of galactosylceramidase and the accumulation of galactosylsphingosine (psychosine) in the nervous system. Psychosine is believed to cause central demyelination by killing oligodendrocytes. Quantitative analysis of this process is lacking. To address this, we generated a new transgenic reporter twitcher line in which myelinating oligodendrocytes are genetically marked by the expression of LacZ under control of the myelin basic protein (MBP) promoter. MBP-LacZ-twitcher transgenic mice were used for unbiased stereological quantification of ß-galactosidase+ oligodendrocytes in the spinal cord. As expected, we found decreased numbers of these cells in mutant cords, paralleling the severity of clinical disease. The decrease of oligodendrocytes does not correlate well with the increase of psychosine. The new MBP-LacZ-twitcher line will be a useful genetic tool for measuring changes in oligodendrocyte numbers in different regions of the mutant CNS and in preclinical trials of therapies to prevent demyelination. © 2016 Wiley Periodicals, Inc.

Differential effects on glial activation by a direct versus an indirect thrombin inhibitor.

Thrombin is a potent regulator of brain function in health and disease, modulating glial activation and brain inflammation. Thrombin inhibitors, several of which are in clinical use as anti-coagulants, can reduce thrombin-dependent neuroinflammation in pathological conditions. However, their effects in a healthy CNS are largely unknown. In adult healthy mice, we compared the effects of treatment by the direct thrombin inhibitor dabigatran etexilate (DE), to those of warfarin, which acts by preventing vitamin K recycling essential for coagulation. After 4weeks, warfarin increased both astrocyte GFAP and microglia Iba-1 staining throughout the CNS; whereas DE reduced expression of both markers. Warfarin, but not DE, reduced sulfatide levels; and warfarin showed longer lasting changes in cerebellar gene expression. DE also reduced glial activation in a mouse model of Alzheimer's disease, although no changes in amyloid plaque burden were observed. These results suggest that treatment with direct thrombin inhibitors may be preferable to those agents which reduce vitamin K levels and have the potential to increase glial activation.

Dysfunction of platelet-derived growth factor receptor α (PDGFRα) represses the production of oligodendrocytes from arylsulfatase A-deficient multipotential neural precursor cells.

The membrane-bound receptor for platelet-derived growth factor A (PDGFRα) is crucial for controlling the production of oligodendrocytes (OLs) for myelination, but regulation of its activity during OL differentiation is largely unknown. We have examined the effect of increased sulfated content of galactosylceramides (sulfatides) on the regulation of PDGFRα in multipotential neural precursors (NPs) that are deficient in arylsulfatase A (ASA) activity. This enzyme is responsible for the lysosomal hydrolysis of sulfatides. We show that sulfatide accumulation significantly impacts the formation of OLs via deregulation of PDGFRα function. PDGFRα is less associated with detergent-resistant membranes in ASA-deficient cells and showed a significant decrease in AKT phosphorylation. Rescue experiments with ASA showed a normalization of the ratio of long versus short sulfatides, restored PDGFRα levels, corrected its localization to detergent-resistant membranes, increased AKT phosphorylation, and normalized the production of OLs in ASA-deficient NPs. Moreover, our studies identified a novel mechanism that regulates the secretion of PDGFRα in NPs, in glial cells, and in the brain cortex via exosomal shedding. Our study provides a first step in understanding the role of sulfatides in regulating PDGFRα levels in OLs and its impact in myelination.

Neuronal inclusions of α-synuclein contribute to the pathogenesis of Krabbe disease.

Demyelination is a major contributor to the general decay of neural functions in children with Krabbe disease. However, recent reports have indicated a significant involvement of neurons and axons in the neuropathology of the disease. In this study, we have investigated the nature of cellular inclusions in the Krabbe brain. Brain samples from the twitcher mouse model for Krabbe disease and from patients affected with the infantile and late-onset forms of the disease were examined for the presence of neuronal inclusions. Our experiments demonstrated the presence of cytoplasmic aggregates of thioflavin-S-reactive material in both human and murine mutant brains. Most of these inclusions were associated with neurons. A few inclusions were detected to be associated with microglia and none were associated with astrocytes or oligodendrocytes. Thioflavin-S-reactive inclusions increased in abundance, paralleling the development of neurological symptoms, and distributed throughout the twitcher brain in areas of major involvement in cognition and motor functions. Electron microscopy confirmed the presence of aggregates of stereotypic ß-sheet folded proteinaceous material. Immunochemical analyses identified the presence of aggregated forms of α-synuclein and ubiquitin, proteins involved in the formation of Lewy bodies in Parkinson's disease and other neurodegenerative conditions. In vitro assays demonstrated that psychosine, the neurotoxic sphingolipid accumulated in Krabbe disease, accelerated the fibrillization of α-synuclein. This study demonstrates the occurrence of neuronal deposits of fibrillized proteins including α-synuclein, identifying Krabbe disease as a new α-synucleinopathy.

Levels of plasma sulfatides C18 : 0 and C24 : 1 correlate with disease status in relapsing-remitting multiple sclerosis.

Multiple sclerosis (MS) is considered an autoimmune demyelinating disease of the CNS and myelin-derived glycolipids are one of the targets of this autoimmune attack. In this study, we examined for the first time the plasma distribution of sulfatide isoforms. Sulfatides with long-chain (C24 : 0 or C24 : 1) and short-chain (C16 : 0 or C18 : 0) fatty acids were quantified in plasma of relapsing­remitting MS patients by ultra-high-performance liquid chromatography tandem mass spectrometry. We found that C18 : 0 and C24 : 1 sulfatide plasma levels positively correlated with the Expanded Disability Status Scale. C16/C18 : 0 and C16/C24 : 0 ratios also correlated with the age and the time since last relapse. Healthy women showed higher levels of C16 : 0 sulfatide than healthy men; however, this gender difference disappeared in MS patients. Our data underline the potential use of sulfatides as biomarkers in relapsing­remitting MS and points to a possible association with the higher susceptibility of women to develop MS.Sulfatides are glycolipids highly enriched in myelin that have been associated with multiple sclerosis (MS). In this study, we have found a positive correlation between levels of specific sulfatides in plasma and increased disability in patients with relapsing-remitting MS. These findings underline the potential use of these molecules as biomarkers for MS.

The sphingolipid psychosine inhibits fast axonal transport in Krabbe disease by activation of GSK3ß and deregulation of molecular motors.

Loss of function of galactosylceramidase lysosomal activity causes demyelination and vulnerability of various neuronal populations in Krabbe disease. Psychosine, a lipid-raft-associated sphingolipid that accumulates in this disease, is thought to trigger these abnormalities. Myelin-free in vitro analyses showed that psychosine inhibited fast axonal transport through the activation of axonal PP1 and GSK3ß in the axon. Abnormal levels of activated GSK3ß and abnormally phosphorylated kinesin light chains were found in nerve samples from a mouse model of Krabbe disease. Administration of GSK3ß inhibitors significantly ameliorated transport defects in vitro and in vivo in peripheral axons of the mutant mouse. This study identifies psychosine as a pathogenic sphingolipid able to block fast axonal transport and is the first to provide a molecular mechanism underlying dying-back degeneration in this genetic leukodystrophy.

Psychosine induces the dephosphorylation of neurofilaments by deregulation of PP1 and PP2A phosphatases.

Patients with Krabbe disease, a genetic demyelinating syndrome caused by deficiency of galactosyl-ceramidase and the resulting accumulation of galactosyl-sphingolipids, develop signs of a dying-back axonopathy compounded by a deficiency of large-caliber axons. Here, we show that axonal caliber in Twitcher mice, an animal model for Krabbe disease, is impaired in peripheral axons and is accompanied by a progressive reduction in the abundance and phosphorylation of the three neurofilament (NF) subunits. These changes correlate with an increase in the density of NFs per cross-sectional area in numerous mutant peripheral axons and abnormal increases in the activity of two serine/threonine phosphatases (PP1 and PP2A) in mutant tissue. Similarly, acutely isolated mutant cortical neurons show abnormal phosphorylation of NFs. Psychosine, the neurotoxin accumulated in Krabbe disease, was sufficient to induce abnormal dephosphorylation of NF subunits in a normal motor neuron cell line as well as in acutely isolated normal cortical neurons. This in vitro effect was mediated by PP1 and PP2A, which specifically dephosphorylated NFs. These results demonstrate that the reduced caliber observed in some axons in Krabbe disease involves abnormal dephosphorylation of NFs. We propose that a psychosine-driven pathogenic mechanism through deregulated phosphotransferase activities may be involved in this process.