Chronic Rapamycin administration via drinking water mitigates the pathological phenotype in a Krabbe disease mouse model through autophagy activation.

Krabbe disease (KD) is a rare disorder arising from the deficiency of the lysosomal enzyme galactosylceramidase (GALC), leading to the accumulation of the cytotoxic metabolite psychosine (PSY) in the nervous system. This accumulation triggers demyelination and neurodegeneration, and despite ongoing research, the underlying pathogenic mechanisms remain incompletely understood, with no cure currently available. Previous studies from our lab revealed the involvement of autophagy dysfunctions in KD pathogenesis, showcasing p62-tagged protein aggregates in the brains of KD mice and heightened p62 levels in the KD sciatic nerve. We also demonstrated that the autophagy inducer Rapamycin (RAPA) can partially reinstate the wild type (WT) phenotype in KD primary cells by decreasing the number of p62 aggregates. In this study, we tested RAPA in the Twitcher (TWI) mouse, a spontaneous KD mouse model. We administered the drug ad libitum via drinking water (15 mg/L) starting from post-natal day (PND) 21-23. We longitudinally monitored the mouse motor performance through grip strength and rotarod tests, and a set of biochemical parameters related to the KD pathogenesis (i.e. autophagy markers expression, PSY accumulation, astrogliosis and myelination). Our findings demonstrate that RAPA significantly enhances motor functions at specific treatment time points and reduces astrogliosis in TWI brain, spinal cord, and sciatic nerves. Utilizing western blot and immunohistochemistry, we observed a decrease in p62 aggregates in TWI nervous tissues, corroborating our earlier in-vitro results. Moreover, RAPA treatment partially removes PSY in the spinal cord. In conclusion, our results advocate for considering RAPA as a supportive therapy for KD. Notably, as RAPA is already available in pharmaceutical formulations for clinical use, its potential for KD treatment can be rapidly evaluated in clinical trials.

[Genetic analysis of a case with Adult-onset globoid cell leukodystrophy].

OBJECTIVE: To explore the clinical features and genetic etiology of a patient with Adult-onset globoid cell leukodystrophy/Krabbe disease (KD). METHODS: A patient who was admitted to the Tongji Hospital Affiliated to Tongji Medical College, Huazhong University of Science and Technology on February 15, 2022 due to exacerbation of right leg weakness for over 4 years was selected as the study subject. Clinical data and results of medical imaging and genetic analysis were analyzed. Candidate variants were verified by family analysis. RESULTS: The patient, a 36-year-old woman, had spasmodic gait as the primary presentation. Cranial magnetic resonance imaging (MRI) revealed symmetrical abnormalities in the bilateral corticospinal tracts, and the activity of ß-galactocerebrosidase (GALC) in her white blood cells was significantly decreased. The patient was found to harbor compound heterozygous variants of the GALC gene, namely c.461C>A (p.Pro154His) and c.1901T>C (p.Leu634Ser). Her mother, sister and nephew were heterozygous carriers of the c.461C>A (p.Pro154His) variant, whilst her father was heterozygous for the c.1901T>C (p.Leu634Ser) variant. CONCLUSION: The patient was ultimately diagnosed with adult-onset KD, for which the compound heterozygous variants of the GALC gene may be accountable.

Leukodystrophy Imaging: Insights for Diagnostic Dilemmas.

Leukodystrophies, a group of rare demyelinating disorders, mainly affect the CNS. Clinical presentation of different types of leukodystrophies can be nonspecific, and thus, imaging techniques like MRI can be used for a more definitive diagnosis. These diseases are characterized as cerebral lesions with characteristic demyelinating patterns which can be used as differentiating tools. In this review, we talk about these MRI study findings for each leukodystrophy, associated genetics, blood work that can help in differentiation, emerging diagnostics, and a follow-up imaging strategy. The leukodystrophies discussed in this paper include X-linked adrenoleukodystrophy, metachromatic leukodystrophy, Krabbe's disease, Pelizaeus-Merzbacher disease, Alexander's disease, Canavan disease, and Aicardi-Goutières Syndrome.

Progress in leukodystrophies with zebrafish.

Inherited leukodystrophies are genetic disorders characterized by abnormal white matter in the central nervous system. Although individually rare, there are more than 400 distinct types of leukodystrophies with a cumulative incidence of 1 in 4500 live births. The pathophysiology of most leukodystrophies is poorly understood, there are treatments for only a few, and there is significant morbidity and mortality, suggesting a critical need for improvements in this field. A variety of animal, cell, and induced pluripotent stem cell-derived models have been developed for leukodystrophies, but with significant limitations in all models. Many leukodystrophies lack animal models, and extant models often show no or mixed recapitulation of key phenotypes. Zebrafish (Danio rerio) have become increasingly used as disease models for studying leukodystrophies due to their early onset of disease phenotypes and conservation of molecular and neurobiological mechanisms. Here, we focus on reviewing new zebrafish disease models for leukodystrophy or models with recent progress. This includes discussion of leukodystrophy with vanishing white matter disease, X-linked adrenoleukodystrophy, Zellweger spectrum disorders and peroxisomal disorders, PSAP deficiency, metachromatic leukodystrophy, Krabbe disease, hypomyelinating leukodystrophy-8/4H leukodystrophy, Aicardi-Goutières syndrome, RNASET2-deficient cystic leukoencephalopathy, hereditary diffuse leukoencephalopathy with spheroids-1 (CSF1R-related leukoencephalopathy), and ultra-rare leukodystrophies. Zebrafish models offer important potentials for the leukodystrophy field, including testing of new variants in known genes; establishing causation of newly discovered genes; and early lead compound identification for therapies. There are also unrealized opportunities to use humanized zebrafish models which have been sparsely explored.

Combination HSCT and intravenous AAV-mediated gene therapy in a canine model proves pivotal for translation of Krabbe disease therapy.

Hematopoietic stem cell transplantation (HSCT) is the only approved treatment for presymptomatic infantile globoid cell leukodystrophy (GLD [Krabbe disease]). However, correction of disease is not complete, and outcomes remain poor. Herein we evaluated HSCT, intravenous (IV) adeno-associated virus rh10 vector (AAVrh10) gene therapy, and combination HSCT + IV AAVrh10 in the canine model of GLD. While HSCT alone resulted in no increase in survival as compared with untreated GLD dogs (∼16 weeks of age), combination HSCT + IV AAVrh10 at a dose of 4E13 genome copies (gc)/kg resulted in delayed disease progression and increased survival beyond 1 year of age. A 5-fold increase in AAVrh10 dose to 2E14 gc/kg, in combination with HSCT, normalized neurological dysfunction up to 2 years of age. IV AAVrh10 alone resulted in an average survival to 41.2 weeks of age. In the peripheral nervous system, IV AAVrh10 alone or in addition to HSCT normalized nerve conduction velocity, improved ultrastructure, and normalized GALC enzyme activity and psychosine concentration. In the central nervous system, only combination therapy at the highest dose was able to restore galactosylceramidase activity and psychosine concentrations to within the normal range. These data have now guided clinical translation of systemic AAV gene therapy as an addition to HSCT (NCT04693598, NCT05739643).

Nanomedicines to treat rare neurological disorders: The case of Krabbe disease.

The brain remains one of the most challenging therapeutic targets due to the low and selective permeability of the blood-brain barrier and complex architecture of the brain tissue. Nanomedicines, despite their relatively large size compared to small molecules and nucleic acids, are being heavily investigated as vehicles to delivery therapeutics into the brain. Here we elaborate on how nanomedicines may be used to treat rare neurodevelopmental disorders, using Krabbe disease (globoid cell leukodystrophy) to frame the discussion. As a monogenetic disorder and lysosomal storage disease affecting the nervous system, the lessons learned from examining nanoparticle delivery to the brain in the context of Krabbe disease can have a broader impact on the treatment of various other neurodevelopmental and neurodegenerative disorders. In this review, we introduce the epidemiology and genetic basis of Krabbe disease, discuss current in vitro and in vivo models of the disease, as well as current therapeutic approaches either approved or at different stage of clinical developments. We then elaborate on challenges in particle delivery to the brain, with a specific emphasis on methods to transport nanomedicines across the blood-brain barrier. We highlight nanoparticles for delivering therapeutics for the treatment of lysosomal storage diseases, classified by the therapeutic payload, including gene therapy, enzyme replacement therapy, and small molecule delivery. Finally, we provide some useful hints on the design of nanomedicines for the treatment of rare neurological disorders.

Untargeted Lipidomic Approach for Studying Different Nervous System Tissues of the Murine Model of Krabbe Disease.

Krabbe disease is a rare neurodegenerative disease with an autosomal recessive character caused by a mutation in the GALC gene. The mutation leads to an accumulation of psychosine and a subsequent degeneration of oligodendrocytes and Schwann cells. Psychosine is the main biomarker of the disease. The Twitcher mouse is the most commonly used animal model to study Krabbe disease. Although there are many references to this model in the literature, the lipidomic study of nervous system tissues in the Twitcher model has received little attention. This study focuses on the comparison of the lipid profiles of four nervous system tissues (brain, cerebellum, spinal cord, and sciatic nerve) in the Twitcher mouse compared to the wild-type mouse. Altogether, approximately 230 molecular species belonging to 19 lipid classes were annotated and quantified. A comparison at the levels of class, molecular species, and lipid building blocks showed significant differences between the two groups, particularly in the sciatic nerve. The in-depth study of the lipid phenotype made it possible to hypothesize the genes and enzymes involved in the changes. The integration of metabolic data with genetic data may be useful from a systems biology perspective to gain a better understanding of the molecular basis of the disease.

The development of a broad-spectrum retaining ß-exo-galactosidase activity-based probe.

Acid ß-galactosidase (GLB1) and galactocerebrosidase (GALC) are retaining exo-ß-galactosidases involved in lysosomal glycoconjugate metabolism. Deficiency of GLB1 may result in the lysosomal storage disorders GM1 gangliosidosis, Morquio B syndrome, and galactosialidosis, and deficiency of GALC may result in Krabbe disease. Activity-based protein profiling (ABPP) is a powerful technique to assess the activity of retaining glycosidases in relation to health and disease. This work describes the use of fluorescent and biotin-carrying activity-based probes (ABPs) to assess the activity of both GLB1 and GALC in cell lysates, culture media, and tissue extracts. The reported ABPs, which complement the growing list of retaining glycosidase ABPs based on configurational isomers of cyclophellitol, should assist in fundamental and clinical research on various ß-galactosidases, whose inherited deficiencies cause debilitating lysosomal storage disorders.

Brain Targeted AAV1-GALC Gene Therapy Reduces Psychosine and Extends Lifespan in a Mouse Model of Krabbe Disease.

Krabbe disease (KD) is a progressive and devasting neurological disorder that leads to the toxic accumulation of psychosine in the white matter of the central nervous system (CNS). The condition is inherited via biallelic, loss-of-function mutations in the galactosylceramidase (GALC) gene. To rescue GALC gene function in the CNS of the twitcher mouse model of KD, an adeno-associated virus serotype 1 vector expressing murine GALC under control of a chicken ß-actin promoter (AAV1-GALC) was administered to newborn mice by unilateral intracerebroventricular injection. AAV1-GALC treatment significantly improved body weight gain and survival of the twitcher mice (n = 8) when compared with untreated controls (n = 5). The maximum weight gain after postnatal day 10 was significantly increased from 81% to 217%. The median lifespan was extended from 43 days to 78 days (range: 74-88 days) in the AAV1-GALC-treated group. Widespread expression of GALC protein and alleviation of KD neuropathology were detected in the CNS of the treated mice when examined at the moribund stage. Functionally, elevated levels of psychosine were completely normalized in the forebrain region of the treated mice. In the posterior region, which includes the mid- and the hindbrain, psychosine was reduced by an average of 77% (range: 53-93%) compared to the controls. Notably, psychosine levels in this region were inversely correlated with body weight and lifespan of AAV1-GALC-treated mice, suggesting that the degree of viral transduction of posterior brain regions following ventricular injection determined treatment efficacy on growth and survivability, respectively. Overall, our results suggest that viral vector delivery via the cerebroventricular system can partially correct psychosine accumulation in brain that leads to slower disease progression in KD.

Novel genetic variant associated with globoid cell leukodystrophy in a family of mixed breed dogs.

BACKGROUND: Globoid cell leukodystrophy (GCL) is a fatal autosomal recessive disease caused by variants in the galactosylceramidase (GALC) gene. Two dog breed-specific variants are reported. OBJECTIVES: Characterize the putatively causative GALC variant for GCL in a family of dogs and determine population allele frequency. ANIMALS: Four related mixed-breed puppies with signs of neurologic disease were evaluated. Subsequently, 33 related dogs were tested for genetic markers for parentage and the identified GALC variant. Additional GALC genotyping was performed on 278 banked samples from various breeds. METHODS: The 4 affected puppies had neurological exams and necropsies. DNA was isolated from blood samples. Variants in GALC were identified via Sanger sequencing. Parentage testing was performed using short tandem repeat markers. Prevalence of the GALC variant of interest was investigated in other breeds. RESULTS: GCL was confirmed histopathologically. A novel missense variant in GALC (NC_006590.4:g.58893972G>A) was homozygous in all affected animals (n = 4). A recessive mode of inheritance was confirmed by parentage testing as was variant linkage with the phenotype (LOD = 3.36). Among the related dogs (n = 33), 3 dogs were homozygous and 7 heterozygous. The variant allele was not detected in screening 278 dogs from 5 breeds. The novel variant is either unique to this family or has an extremely low allele frequency in the general population. CONCLUSIONS AND CLINICAL IMPORTANCE: A novel GALC variant was identified that likely explains GCL in this cohort. The identification of multiple causal variants for GCL in dogs is consistent with findings in humans.

Genetic ablation of Saposin-D in Krabbe disease eliminates psychosine accumulation but does not significantly improve demyelination.

Krabbe disease is an inherited demyelinating disease caused by a genetic deficiency of the lysosomal enzyme galactosylceramide (GalCer) ß-galactosidase (GALC). The Twitcher (Twi) mouse is a naturally occurring, genetically and enzymatically authentic mouse model that mimics infantile-onset Krabbe disease. The major substrate for GALC is the myelin lipid GalCer. However, the pathogenesis of Krabbe disease has long been explained by the accumulation of psychosine, a lyso-derivative of GalCer. Two metabolic pathways have been proposed for the accumulation of psychosine: a synthetic pathway in which galactose is transferred to sphingosine and a degradation pathway in which GalCer is deacylated by acid ceramidase (ACDase). Saposin-D (Sap-D) is essential for the degradation of ceramide by ACDase in lysosome. In this study, we generated Twi mice with a Sap-D deficiency (Twi/Sap-D KO), which are genetically deficient in both GALC and Sap-D and found that very little psychosine accumulated in the CNS or PNS of the mouse. As expected, demyelination with the infiltration of multinucleated macrophages (globoid cells) characteristic of Krabbe disease was milder in Twi/Sap-D KO mice than in Twi mice both in the CNS and PNS during the early disease stage. However, at the later disease stage, qualitatively and quantitatively comparable demyelination occurred in Twi/Sap-D KO mice, particularly in the PNS, and the lifespans of Twi/Sap-D KO mice were even shorter than that of Twi mice. Bone marrow-derived macrophages from both Twi and Twi/Sap-D KO mice produced significant amounts of TNF-α upon exposure to GalCer and were transformed into globoid cells. These results indicate that psychosine in Krabbe disease is mainly produced via the deacylation of GalCer by ACDase. The demyelination observed in Twi/Sap-D KO mice may be mediated by a psychosine-independent, Sap-D-dependent mechanism. GalCer-induced activation of Sap-D-deficient macrophages/microglia may play an important role in the neuroinflammation and demyelination in Twi/Sap-D KO mice.

CD8+ T cell depletion prevents neuropathology in a mouse model of globoid cell leukodystrophy.

Globoid cell leukodystrophy (GLD) or Krabbe's disease is a fatal genetic demyelinating disease of the central nervous system caused by loss-of-function mutations in the galactosylceramidase (galc) gene. While the metabolic basis for disease is known, the understanding of how this results in neuropathology is not well understood. Herein, we report that the rapid and protracted elevation of CD8+ cytotoxic T lymphocytes occurs coincident with clinical disease in a mouse model of GLD. Administration of a function-blocking antibody against CD8α effectively prevented disease onset, reduced morbidity and mortality, and prevented CNS demyelination in mice. These data indicate that subsequent to the genetic cause of disease, neuropathology is driven by pathogenic CD8+ T cells, thus offering novel therapeutic potential for treatment of GLD.

Identifying altered developmental pathways in human globoid cell leukodystrophy iPSCs-derived NSCs using transcriptome profiling.

BACKGROUND: Globoid cell leukodystrophy (GLD) is a devastating neurodegenerative disease characterized by widespread demyelination caused by galactocerebrosidase defects. Changes in GLD pathogenesis occurring at the molecular level have been poorly studied in human-derived neural cells. Patient-derived induced pluripotent stem cells (iPSCs) are a novel disease model for studying disease mechanisms and allow the generation of patient-derived neuronal cells in a dish. RESULTS: In this study, we identified gene-expression changes in iPSCs and iPSC-derived neural stem cells (NSCs) from a patient with GLD (K-iPSCs/NSCs) and normal control (AF-iPSCs/NSCs), in order to investigate the potential mechanism underlying GLD pathogenesis. We identified 194 (K-iPSCs vs. AF-iPSCs) and 702 (K-NSCs vs. AF-NSCs) significantly dysregulated mRNAs when comparing the indicated groups. We also identified dozens of Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway terms that were enriched for the differentially expressed genes. Among them, 25 differentially expressed genes identified by RNA-sequencing analysis were validated using real-time quantitative polymerase chain reaction analysis. Dozens of pathways involved in neuroactive ligand-receptor interactions, synaptic vesicle cycle signaling, serotonergic synapse signaling, phosphatidylinositol-protein kinase B signaling, and cyclic AMP signaling were identified as potential contributors to GLD pathogenesis. CONCLUSIONS: Our results correspond to the fact that mutations in the galactosylceramidase gene may disrupt the identified signaling pathways during neural development, suggesting that alterations in signaling pathways contribute to GLD pathogenesis. At the same time, our results demonstrates that the model based on K-iPSCs is a novel tool that can be used to study the underlying molecular basis of GLD.

Favorable outcome of hematopoietic stem cell transplantation in late-onset Krabbe disease.

BACKGROUND: Late-onset Krabbe disease is a disorder with autosomal recessive inheritance caused by a deficiency in galactocerebrosidase (GALC) activity. Its late-onset form usually shows slow disease progression with atypical symptoms including spastic paresis. The efficacy of hematopoietic stem cell transplantation (HSCT) in late-onset Krabbe disease has not been fully established. CASE REPORT: We describe the case of a patient with late-onset Krabbe disease showing progressive spastic paraparesis. At the age of 18, one and a half years after the development of symptoms, the patient underwent HSCT. After HSCT, the patient's GALC activity returned to a normal level and the lesions in the brain and spinal cord became faint on images. Over two and a half years after the HSCT, the patient's gait remained spastic, however, an improvement in gait speed and modified Rankin Scale score was observed. No severe adverse events occurred during this period. CONCLUSION: Our experience reported herein provides additional evidence for a favorable course in HSCT conducted in the early course of late-onset Krabbe disease.

Rapamycin Alleviates Protein Aggregates, Reduces Neuroinflammation, and Rescues Demyelination in Globoid Cell Leukodystrophy.

We have shown in vivo and in vitro previously that psychosine causes dysfunction of autophagy and the ubiquitin-proteasome system underlying the pathogenesis of globoid cell leukodystrophy (GLD), a devastating lysosomal storage disease complicated by global demyelination. Here, we investigated the therapeutic efficacy of the mTOR inhibitor rapamycin in twitcher mice, a murine model of infantile GLD, in biochemical, histochemical, and clinical aspects. Administration of rapamycin to twitcher mice inhibited mTOR signaling in the brains, and significantly reduced the accumulation of insoluble ubiquitinated protein and the formation of ubiquitin aggregates. The astrocytes and microglia reactivity were attenuated in that reactive astrocytes, ameboid microglia, and globoid cells were reduced in the brains of rapamycin-treated twitcher mice. Furthermore, rapamycin improved the cortical myelination, neurite density, and rescued the network complexity in the cortex of twitcher mice. The therapeutic action of rapamycin on the pathology of the twitcher mice's brains prolonged the longevity of treated twitcher mice. Overall, these findings validate the therapeutic efficacy of rapamycin and highlight enhancing degradation of aggregates as a therapeutic strategy to modulate neuroinflammation, demyelination, and disease progression of GLD and other leukodystrophies associated with intracellular aggregates.

Impaired Autophagy in Krabbe Disease: The Role of BCL2 and Beclin-1 Phosphorylation.

Autophagic impairment was identified in many lysosomal storage diseases and adult neurodegenerative diseases. It seems that this defect could be directly related to the appearance of a neurodegenerative phenotype and could contribute to worsen metabolite accumulation and lysosomal distress. Thus, autophagy is becoming a promising target for supportive therapies. Autophagy alterations were recently identified also in Krabbe disease. Krabbe disease is characterized by extensive demyelination and dysmyelination and it is due to the genetic loss of function of the lysosomal enzyme galactocerebrosidase (GALC). This enzyme leads to the accumulation of galactosylceramide, psychosine, and secondary substrates such as lactosylceramide. In this paper, we induced autophagy through starvation and examined the cellular response occurring in fibroblasts isolated from patients. We demonstrated that the inhibitory AKT-mediated phosphorylation of beclin-1 and the BCL2-beclin-1 complex concur to reduce autophagosomes formation in response to starvation. These events were not dependent on the accumulation of psychosine, which was previously identified as a possible player in autophagic impairment in Krabbe disease. We believe that these data could better elucidate the capability of response to autophagic stimuli in Krabbe disease, in order to identify possible molecules able to stimulate the process.

Expanding the Spectrum of NUBPL-Related Leukodystrophy.

Mitochondrial leukodystrophies constitute a group of different conditions presenting with a wide range of clinical presentation but with some shared neuroradiological features. Genetic defects in NUBPL have been recognized as cause of a pediatric onset mitochondrial leukodystrophy characterized by onset at the end of the first year of life with motor delay or regression and cerebellar signs, followed by progressive spasticity. Early magnetic resonance imagings (MRIs) show white matter abnormalities with predominant involvement of frontoparietal regions and corpus callosum. A striking cerebellar involvement is usually observed. Later MRIs show spontaneous improvement of white matter abnormalities but worsening of the cerebellar involvement evolving to global atrophy and progressive involvement of brainstem. After the 7 cases initially described, 11 more subjects were reported. Some of them were similar to patients from the original series while few others broadened the phenotypic spectrum. We performed a literature review and report on a new patient who further expand the spectrum of NUBPL-related leukodystrophy. With our study we confirm that the association of cerebral white matter and cerebellar cortex abnormalities is a feature commonly observed in early stages of the disease but beside the original and so far prevalent presentation, there are also uncommon phenotypes: clinical onset can be earlier and more severe than previously thought and signs of extraneurological involvement can be observed. Brain white matter can be diffusely abnormal without anteroposterior gradient, can progressively worsen, and cystic degeneration can be present. Thalami can be involved. Basal ganglia can also become involved during disease evolution.