Galactosylceramidase Activity Research Articles

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 (∼16weeks 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 41.2weeks 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).

GALC variants affect galactosylceramidase enzymatic activity and risk of Parkinson's disease.

The association between glucocerebrosidase, encoded by GBA, and Parkinson's disease (PD) highlights the role of the lysosome in PD pathogenesis. Genome-wide association studies in PD have revealed multiple associated loci, including the GALC locus on chromosome 14. GALC encodes the lysosomal enzyme galactosylceramidase, which plays a pivotal role in the glycosphingolipid metabolism pathway. It is still unclear whether GALC is the gene driving the association in the chromosome 14 locus and, if so, by which mechanism. We first aimed to examine whether variants in the GALC locus and across the genome are associated with galactosylceramidase activity. We performed a genome-wide association study in two independent cohorts from (i) Columbia University; and (ii) the Parkinson's Progression Markers Initiative study, followed by a meta-analysis with a total of 976 PD patients and 478 controls with available data on galactosylceramidase activity. We further analysed the effects of common GALC variants on expression and galactosylceramidase activity using genomic colocalization methods. Mendelian randomization was used to study whether galactosylceramidase activity may be causal in PD. To study the role of rare GALC variants, we analysed sequencing data from 5028 PD patients and 5422 controls. Additionally, we studied the functional impact of GALC knockout on alpha-synuclein accumulation and on glucocerebrosidase activity in neuronal cell models and performed in silico structural analysis of common GALC variants associated with altered galactosylceramidase activity. The top hit in PD genome-wide association study in the GALC locus, rs979812, is associated with increased galactosylceramidase activity (b = 1.2; SE = 0.06; P = 5.10 × 10-95). No other variants outside the GALC locus were associated with galactosylceramidase activity. Colocalization analysis demonstrated that rs979812 was also associated with increased galactosylceramidase expression. Mendelian randomization suggested that increased galactosylceramidase activity may be causally associated with PD (b = 0.025, SE = 0.007, P = 0.0008). We did not find an association between rare GALC variants and PD. GALC knockout using CRISPR-Cas9 did not lead to alpha-synuclein accumulation, further supporting that increased rather than reduced galactosylceramidase levels may be associated with PD. The structural analysis demonstrated that the common variant p.I562T may lead to improper maturation of galactosylceramidase affecting its activity. Our results nominate GALC as the gene associated with PD in this locus and suggest that the association of variants in the GALC locus may be driven by their effect of increasing galactosylceramidase expression and activity. Whether altering galactosylceramidase activity could be considered as a therapeutic target should be further studied.

Galactosylceramidase deficiency and pathological abnormalities in cerebral white matter of Krabbe disease

Krabbe Disease (KD) is an autosomal recessive disorder that results from loss-of-function mutations in the GALC gene, which encodes lysosomal enzyme galactosylceramidase (GALC). Functional deficiency of GALC is toxic to myelin-producing cells, which leads to progressive demyelination in both the central and peripheral nervous systems. It is hypothesized that accumulation of psychosine, which can only be degraded by GALC, is a primary initiator of pathologic cascades. Despite the central role of GALC in KD pathomechanism, investigations of GALC deficiency at a protein level are largely absent, due in part, to the lack of sensitive antibodies in the field. Leveraging two custom antibodies that can detect GALC at endogenous levels, we demonstrated that GALC protein is predominantly localized to oligodendrocytes in cerebral white matter of an infant brain, consistent with its functional role in myelination. Mature GALC could also be quantitatively detected as a 26 kDa band by western blotting and correlated to enzyme activity in brain tissues. The p.Ile562Thr polymorphic variant, which is over-represented in the KD population, was associated with reduced mature GALC protein and activity. In three infantile KD cases, homozygous null mutations in GALC lead to deficiency in total GALC protein and activity. Interestingly, although GALC activity was absent, normal levels of total GALC protein were detected by a sandwich ELISA using our custom antibodies in a later-onset KD brain, which suggests that the assay has the potential to differentiate infantile- and later-onset KD cases. Among the infantile KD cases, we quantified a 5-fold increase in psychosine levels, and observed increased levels of acid ceramidase, a key enzyme for psychosine production, and hyperglycosylated lysosomal-associated membrane protein 1, a marker for lysosomal activation, in periventricular white matter, a major pathological brain region, when compared with age-matched normal controls. While near complete demyelination was observed in these cases, we quantified that an early-infantile case (age of death at 10 months) had about 3-fold increases in both globoid cells, a pathological hallmark for KD, and CD8-positive T lymphocytes, a pathological marker for multiple sclerosis, in the white matter when compared with a slower progressing infantile case (age of death at 21 months), which suggests a positive correlation between clinical severity and neuropathology. Taken together, our findings have advanced the understanding of GALC protein biology in the context of normal and KD brain white matter. We also revealed new neuropathological changes that may provide insights to understand KD pathogenesis.

Human iPSC-derived astrocytes generated from donors with globoid cell leukodystrophy display phenotypes associated with disease.

Globoid cell leukodystrophy (Krabbe disease) is a fatal neurodegenerative, demyelinating disease caused by dysfunctional activity of galactosylceramidase (GALC), leading to the accumulation of glycosphingolipids including psychosine. While oligodendrocytes have been extensively studied due to their high levels of GALC, the contribution of astrocytes to disease pathogenesis remains to be fully elucidated. In the current study, we generated induced pluripotent stem cells (iPSCs) from two donors with infantile onset Krabbe disease and differentiated them into cultures of astrocytes. Krabbe astrocytes recapitulated many key findings observed in humans and rodent models of the disease, including the accumulation of psychosine and elevated expression of the pro-inflammatory cytokine IL-6. Unexpectedly, Krabbe astrocytes had higher levels of glucosylceramide and ceramide, and displayed compensatory changes in genes encoding glycosphingolipid biosynthetic enzymes, suggesting a shunting away from the galactosylceramide and psychosine pathway. In co-culture, Krabbe astrocytes negatively impacted the survival of iPSC-derived human neurons while enhancing survival of iPSC-derived human microglia. Substrate reduction approaches targeting either glucosylceramide synthase or serine palmitoyltransferase to reduce the sphingolipids elevated in Krabbe astrocytes failed to rescue their detrimental impact on neuron survival. Our results suggest that astrocytes may contribute to the progression of Krabbe disease and warrant further exploration into their role as therapeutic targets.

Krabbe Disease Associated With Mitochondrial Dysfunction in a Chinese Family.

Background: Krabbe disease is caused by biallelic mutations of GALC gene. NDUFAF1 gene mutations are related to mitochondrial encephalopathy. To date, there has been no report on the co-pathogenesis of these two gene mutations. There were three children in a family who presented with global developmental retardation. MRI showed lesions in the white matter and dentate nucleus of the cerebellum.Methods: Clinical data of the proband and her family members were gathered in a retrospective manner. Karyotype, FISH, whole exome sequencing was performed using genomic DNAs extracted from peripheral blood samples. Enzyme activities of galactosylceramidase (GALC) and mitochondria were determined to verify gene functions.Results: This study reported a pedigree of leukoencephalopathy, in which 3 of the 4 children showed phenotypes of developmental delay, hearing/visual impairment, and peripheral neuropathy. Mutations of NDUFAF1 (c.278A>G; p. His93Arg, c.247G> A; p. Asp83Asn) and GALC (c.599C>A; p.Ser200*) were identified in all three cases. The proband's parents carried these mutations as a heterozygous state. Clinical features, MRI changes, enzyme activity of GALC, and mitochondrial function analysis demonstrated that this pedigree was caused by GALC and NDUFAF1 gene mutations working together.Conclusion: We first report a pedigree of Krabbe disease with biallelic mitochondrial gene NDUFAF1 mutations. For multiple gene mutations found in genetic testing, clinical phenotypes, gene functions, and family history should be comprehensively analyzed. Gene panel examination may miss pathogenic mutations, and prenatal diagnosis of patients with polygenic inheritance needs careful consideration.

Glucosylceramide and galactosylceramide, small glycosphingolipids with significant impact on health and disease.

Numerous clinical observations and exploitation of cellular and animal models indicate that glucosylceramide (GlcCer) and galactosylceramide (GalCer) are involved in many physiological and pathological phenomena. In many cases, the biological importance of these monohexosylcermides has been shown indirectly as the result of studies on enzymes involved in their synthesis and degradation. Under physiological conditions, GalCer plays a key role in the maintenance of proper structure and stability of myelin and differentiation of oligodendrocytes. On the other hand, GlcCer is necessary for the proper functions of epidermis. Such an important lysosomal storage disease as Gaucher disease (GD) and a neurodegenerative disorder as Parkinson’s disease are characterized by mutations in the GBA1 gene, decreased activity of lysosomal GBA1 glucosylceramidase and accumulation of GlcCer. In contrast, another lysosomal disease, Krabbe disease, is associated with mutations in the GALC gene, resulting in deficiency or decreased activity of lysosomal galactosylceramidase and accumulation of GalCer and galactosylsphingosine. Little is known about the role of both monohexosylceramides in tumor progression; however, numerous studies indicate that GlcCer and GalCer play important roles in the development of multidrug-resistance by cancer cells. It was shown that GlcCer is able to provoke immune reaction and acts as a self-antigen in GD. On the other hand, GalCer was recognized as an important cellular receptor for HIV-1. Altogether, these two molecules are excellent examples of how slight differences in chemical composition and molecular conformation contribute to profound differences in their physicochemical properties and biological functions.

Reduction in miR-219 expression underlies cellular pathogenesis of oligodendrocytes in a mouse model of Krabbe disease.

Krabbe disease (KD), also known as globoid cell leukodystrophy, is an inherited demyelinating disease caused by the deficiency of lysosomal galactosylceramidase (GALC) activity. Most of the patients are characterized by early‐onset cerebral demyelination with apoptotic oligodendrocyte (OL) death and die before 2 years of age. However, the mechanisms of molecular pathogenesis in the developing OLs before death and the exact causes of white matter degeneration remain largely unknown. We have recently reported that OLs of twitcher mouse, an authentic mouse model of KD, exhibit developmental defects and endogenous accumulation of psychosine (galactosylsphingosine), a cytotoxic lyso‐derivative of galactosylceramide. Here, we show that attenuated expression of microRNA (miR)‐219, a critical regulator of OL differentiation and myelination, mediates cellular pathogenesis of KD OLs. Expression and functional activity of miR‐219 were repressed in developing twitcher mouse OLs. By using OL precursor cells (OPCs) isolated from the twitcher mouse brain, we show that exogenously supplemented miR‐219 effectively rescued their cell‐autonomous developmental defects and apoptotic death. miR‐219 also reduced endogenous accumulation of psychosine in twitcher OLs. Collectively, these results highlight the role of the reduced miR‐219 expression in KD pathogenesis and suggest that miR‐219 has therapeutic potential for treating KD OL pathologies.

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.

Alpha galactosidase A activity in Parkinson's disease

Glucocerebrosidase (GCase, deficient in Gaucher disease) enzymatic activity measured in dried blood spots of Parkinson's Disease (PD) cases is within healthy range but reduced compared to controls. It is not known whether activities of additional lysosomal enzymes are reduced in dried blood spots in PD. To test whether reduction in lysosomal enzymatic activity in PD is specific to GCase, we measured GCase, acid sphingomyelinase (deficient in Niemann–Pick disease types A and B), alpha galactosidase A (deficient in Fabry), acid alpha-glucosidase (deficient in Pompe) and galactosylceramidase (deficient in Krabbe) enzymatic activities in dried blood spots of PD patients (n = 648) and controls (n = 317) recruited from Columbia University. Full sequencing of glucocerebrosidase (GBA) and the LRRK2 G2019S mutation was performed. Enzymatic activities were compared between PD cases and controls using t-test and regression models adjusted for age, gender, and GBA and LRRK2 G2019S mutation status. Alpha galactosidase A activity was lower in PD cases compared to controls both when only non-carriers were included (excluding all GBA and LRRK2 G2019S carriers and PD cases with age-at-onset below 40) [2.85 μmol/l/h versus 3.12 μmol/l/h, p = 0.018; after controlling for batch effect, p = 0.006 (468 PD cases and 296 controls)], and when including the entire cohort (2.89 μmol/l/h versus 3.10 μmol/l/h, p = 0.040; after controlling for batch effect, p = 0.011). Because the alpha galactosidase A gene is X-linked, we stratified the analyses by sex. Among women who were non-carriers of GBA and LRRK2 G2019S mutations (PD, n = 155; control, n = 194), alpha galactosidase A activity was lower in PD compared to controls (2.77 μmol/l/h versus 3.10 μmol/l/h, p = 0.044; after controlling for a batch effect, p = 0.001). The enzymatic activity of acid sphingomyelinase, acid alpha-glucosidase and galactosylceramidase was not significantly different between PD and controls. In non-carriers, most lysosomal enzyme activities were correlated, with the strongest association in GCase, acid alpha-glucosidase, and alpha galactosidase A (Pearson correlation coefficient between 0.382 and 0.532). In a regression model with all five enzymes among non-carriers (adjusted for sex and age), higher alpha galactosidase A activity was associated with lower odds of PD status (OR = 0.54; 95% CI:0.31–0.95; p = 0.032). When LRRK2 G2019S PD carriers (n = 37) were compared to non-carriers with PD, carriers had higher GCase, acid sphingomyelinase and alpha galactosidase A activity. We conclude that alpha galactosidase A may have a potential independent role in PD, in addition to GCase.

Metabolic profiling reveals biochemical pathways and potential biomarkers associated with the pathogenesis of Krabbe disease.

Krabbe disease (KD) is caused by mutations in the galactosylceramidase (GALC) gene, which encodes a lysosomal enzyme that degrades galactolipids, including galactosylceramide and galactosylsphingosine (psychosine). GALC deficiency results in progressive intracellular accumulation of psychosine, which is believed to be the main cause for the demyelinating neurodegeneration in KD pathology. Umbilical cord blood transplantation slows disease progression when performed presymptomatically but carries a significant risk of morbidity and mortality. Accurate presymptomatic diagnosis is therefore critical to facilitate the efficacy of existing transplant approaches and to avoid unnecessary treatment of children who will not develop KD. Unfortunately, current diagnostic criteria, including GALC activity, genetic analysis, and psychosine measurement, are insufficient for secure presymptomatic diagnosis. This study performs a global metabolomic analysis to identify pathogenetic metabolic pathways and novel biomarkers implicated in the authentic mouse model of KD known as twitcher. At a time point before onset of signs of disease, twitcher hindbrains had metabolic profiles similar to WT, with the exception of a decrease in metabolites related to glucose energy metabolism. Many metabolic pathways were altered after early signs of disease in the twitcher, including decreased phospholipid turnover, restricted mitochondrial metabolism of branched-chain amino acids, increased inflammation, and changes in neurotransmitter metabolism and osmolytes. Hypoxanthine, a purine derivative, is increased before signs of disease appear, suggesting its potential as a biomarker for early diagnosis of KD. Additionally, given the early changes in glucose metabolism in the pathogenesis of KD, diagnostic modalities that report metabolic function, such as positron emission tomography, may be useful in KD. © 2016 Wiley Periodicals, Inc.

Mesenchymal Stem Cells Yield Transient Improvements in Motor Function in an Infant Rhesus Macaque with Severe Early-Onset Krabbe Disease.

Krabbe disease, or globoid cell leukodystrophy, is a rare disorder caused by deficient galactosylceramidase activity and loss of myelin‐forming oligodendrocytes, resulting in progressive demyelination and severely impaired motor function. Disease symptoms in humans appear within 3–6 months of age (early infantile) and manifest as marked irritability, spasticity, and seizures. The disease is often fatal by the second year of life, with few effective treatment options. Herein we evaluated the therapeutic potential of mesenchymal stem cells (MSCs) administered intracranially to a 1‐month‐old rhesus macaque diagnosed with severe early‐onset Krabbe disease that displayed neurologic and behavioral symptoms similar to those of human patients. The infant was subjected to physical and neurological behavior examinations and nerve conduction velocity tests to assess efficacy, and outcomes were compared with age‐matched normal infants and Krabbe‐affected rhesus monkeys with late‐onset disease. Changes in major blood lymphocyte populations were also monitored to assess host immune cell responses. MSC administration resulted in transient improvements in coordination, ambulation, cognition, and large motor skills, which correlated with increased peripheral nerve conduction velocities and decreased latencies. Improvements also corresponded to transient increases in peripheral blood lymphocyte counts, but secondary challenge failed to elicit allo‐antibody production. Nevertheless, white cell and neutrophil counts showed dramatic increases, and CD20+ B cell counts underwent a precipitous decline at late stages of disease progression. Correlative data linking MSC administration to transient improvements in motor function suggest that MSCs should be evaluated further as an experimental therapy for rare neurodegenerative diseases. Stem Cells Translational Medicine 2017;6:99–109

56. Intracerebroventricular and Intravenous AAV Gene Therapy in Canine Globoid Cell Leukodystrophy

Globoid cell leukodystrophy (GLD), or Krabbe disease, results from a deficiency in the hydrolytic enzyme galactosylceramidase (GALC), which is responsible for degrading the central and peripheral nervous system (CNS, PNS) myelin lipids galactosylceramide and galactosylsphingosine (psychosine). Symptoms in infants include irritability and stiffness with disease progressing rapidly to a vegetative state and death typically resulting by 2 years of age. GLD is naturally occurring in dogs and signs include ataxia, tremors, pelvic limb paralysis, loss of hearing, and blindness, with disease progression warranting euthanasia at 15.9 ± 4.6 weeks. In GLD dogs, GALC concentrations are decreased in leukocytes, CSF, and tissues and psychosine concentrations are elevated in serum and CSF. Conduction velocities in peripheral nerves of GLD dogs are < 50% of normal and G-ratios of the sciatic nerve are significantly higher than normal. Brain stem auditory evoked response testing exhibits a loss of waveform integrity and increased central conduction time consistent with auditory system demyelination. MRI of the brain shows T2-weighted hyperintensity of the white matter, widened sulci, and enlarged ventricles and MRS identified decreases in N-acetylaspartate and increases in choline. Diffusion tensor imaging established decreased fractional anisotropy and increased radial diffusivity in multiple white matter tracts including the corpus callosum and posterior internal capsule. Histologically, GLD dogs revealed severe loss of myelin, a decrease in oligodendrocytes, astrogliosis, and microgliosis. Combination gene therapy has been evaluated in GLD affected dogs using AAVrh10 encoding canine GALC (AAVrh10-cGALC). Two GLD dogs received a low dose of AAVrh10-cGALC, 1.2E12, by combination intravenous (IV) and intracerebroventricular (ICV) injection routes and displayed a modest increase in survival to 17.9 and 22.1 weeks of age. Two additional GLD dogs were treated with an increased dose of AAVrh10-cGALC, 1.9E13, and survival was further increased to 30.3 and 43.1 weeks of age. Both low and high dose combination IV and ICV therapy delayed the onset of neurological signs and prevented the onset of tremors, one of the debilitating neurologic signs in untreated GLD dogs. High dose AAVrh10-cGALC, but not low dose, resulted in complete normalization of pelvic and thoracic limb NCV and near normal sensory NCV. Combination therapy of either dose had negligible effect on the auditory system, as treated animals showed little to no improvement in distance between waveform peaks or hearing threshold over untreated animals. After high dose treatment with AAVrh10-cGALC, GALC activity reached near normal levels in the cerebellum and sciatic nerve, with levels decreasing in more distal brain regions. GALC levels in the liver and heart were intermediate between affected untreated GLD and normal control dogs. Interestingly, the highest GALC activity was found in the quadriceps muscle. GLD dogs treated with high dose AAV had CSF psychosine concentrations lower than untreated and low dose AAV-treated GLD dogs, despite being substantially older. Efficacy of hematopoietic stem cell transplant (HSCT) alone and in combination with IV AAVrh10-cGALC is currently being evaluated in GLD dogs. Ongoing studies suggest that addition of IV infusion of AAVrh10-cGALC substantially increases survival as compared to HSCT alone.

Pervasive supply of therapeutic lysosomal enzymes in the CNS of normal and Krabbe-affected non-human primates by intracerebral lentiviral gene therapy.

Metachromatic leukodystrophy (MLD) and globoid cell leukodystrophy (GLD or Krabbe disease) are severe neurodegenerative lysosomal storage diseases (LSD) caused by arylsulfatase A (ARSA) and galactosylceramidase (GALC) deficiency, respectively. Our previous studies established lentiviral gene therapy (GT) as a rapid and effective intervention to provide pervasive supply of therapeutic lysosomal enzymes in CNS tissues of MLD and GLD mice. Here, we investigated whether this strategy is similarly effective in juvenile non‐human primates (NHP). To provide proof of principle for tolerability and biological efficacy of the strategy, we established a comprehensive study in normal NHP delivering a clinically relevant lentiviral vector encoding for the human ARSA transgene. Then, we injected a lentiviral vector coding for the human GALC transgene in Krabbe‐affected rhesus macaques, evaluating for the first time the therapeutic potential of lentiviral GT in this unique LSD model. We showed favorable safety profile and consistent pattern of LV transduction and enzyme biodistribution in the two models, supporting the robustness of the proposed GT platform. We documented moderate inflammation at the injection sites, mild immune response to vector particles in few treated animals, no indication of immune response against transgenic products, and no molecular evidence of insertional genotoxicity. Efficient gene transfer in neurons, astrocytes, and oligodendrocytes close to the injection sites resulted in robust production and extensive spreading of transgenic enzymes in the whole CNS and in CSF, leading to supraphysiological ARSA activity in normal NHP and close to physiological GALC activity in the Krabbe NHP, in which biological efficacy was associated with preliminary indication of therapeutic benefit. These results support the rationale for the clinical translation of intracerebral lentiviral GT to address CNS pathology in MLD, GLD, and other neurodegenerative LSD.

Altered Trafficking and Processing of GALC Mutants Correlates with Globoid Cell Leukodystrophy Severity.

Globoid cell leukodystrophy (GLD, Krabbe disease) is diagnosed by measuring galactosylceramidase (GALC) activity and DNA analysis. However, genotype and phenotype often do not correlate due to considerable clinical variability, even for the same mutation, for unknown reasons. We find that altered trafficking to the lysosome and processing of GALC correlates with GLD severity and is modulated by cis-polymorphisms. Current diagnosis of GLD is based on GALC activity of total cell lysates from blood, which does not discriminate whether the activity comes from the lysosome or other subcellular organelles. Measurement of GALC activity in lysosomes may predict which infants are at high risk for the infantile phenotype while distinguishing other children who will develop later-onset phenotypes without onset of symptoms for years.

Region- and Age-Dependent Alterations of Glial-Neuronal Metabolic Interactions Correlate with CNS Pathology in a Mouse Model of Globoid Cell Leukodystrophy

Globoid cell leukodystrophy (GLD) or Krabbe disease is a lysosomal storage disorder caused by genetic defects in the expression and activity of galactosylceramidase, a key enzyme in the catabolism of myelin-enriched sphingolipids. While there are several histologic, biochemical, and functional studies on GLD, correlations between morphologic and biochemical alterations in central nervous system (CNS) tissues during disease progression are lacking. Here, we combined immunohistochemistry and metabolic analysis using (1)H and (13)C magnetic resonance (MR) spectra of spinal cord, cerebellum, and forebrain to investigate glial-neuronal metabolic interactions and dysfunction in a GLD murine model that recapitulates the human pathology. In order to assess the temporal- and region-dependent disease progression and the potential metabolic correlates, we investigated CNS tissues at mildly symptomatic and fully symptomatic stages of the disease. When compared with age-matched controls, GLD mice showed glucose hypometabolism, alterations in neurotransmitter content, N-acetylaspartate, N-acetylaspartylglutamate, and osmolytes levels. Notably, age- and region-dependent patterns of metabolic disturbances were in close agreement with the progression of astrogliosis, microglia activation, apoptosis, and neurodegeneration. We suggest that MR spectroscopy could be used in vivo to monitor disease progression, as well as ex vivo and in vivo to provide criteria for the outcome of experimental therapies.

The galactocerebrosidase enzyme contributes to maintain a functional neurogenic niche during early post-natal CNS development

We report a novel role for the lysosomal galactosylceramidase (GALC), which is defective in globoid cell leukodystrophy (GLD), in maintaining a functional post-natal subventricular zone (SVZ) neurogenic niche. We show that proliferation/self-renewal of neural stem cells (NSCs) and survival of their neuronal and oligodendroglial progeny are impaired in GALC-deficient mice. Using drugs to modulate inflammation and gene transfer to rescue GALC expression and activity, we show that lipid accumulation resulting from GALC deficiency acts as a cell-autonomous pathogenic stimulus in enzyme-deficient NSCs and progeny before upregulation of inflammatory markers, which later sustain a non-cell-autonomous dysfunction. Importantly, we provide evidence that supply of functional GALC provided by neonatal intracerebral transplantation of NSCs ameliorates the functional impairment in endogenous SVZ cells. Insights into the mechanism/s underlying GALC-mediated regulation of early post-natal neurogenic niches improve our understanding of the multi-component pathology of GLD. The occurrence of a restricted period of SVZ neurogenesis in infancy supports the implications of our study for the development of therapeutic strategies to treat this severe pediatric neurodegenerative disorder.

Bone Marrow Transplantation Augments the Effect of Brain- and Spinal Cord-Directed Adeno-Associated Virus 2/5 Gene Therapy by Altering Inflammation in the Murine Model of Globoid-Cell Leukodystrophy

Globoid-cell leukodystrophy (GLD) is an inherited demyelinating disease caused by the deficiency of the lysosomal enzyme galactosylceramidase (GALC). A previous study in the murine model of GLD (twitcher) demonstrated a dramatic synergy between CNS-directed adeno-associated virus 2/5 (AAV2/5) gene therapy and myeloreductive bone marrow transplantation (BMT). However, the mechanism by which these two disparate therapeutic approaches synergize is not clear. In addition, the therapeutic efficacy may have been limited since the CNS-directed gene therapy was restricted to the forebrain and thalamus. In the current study, intrathecal and intracerebellar injections were added to the therapeutic regimen and the mechanism of synergy between BMT and gene therapy was determined. Although AAV2/5 alone provided supraphysiological levels of GALC activity and reduced psychosine levels in both the brain and spinal cord, it significantly increased CNS inflammation. Bone marrow transplantation alone provided essentially no GALC activity to the CNS and did not reduce psychosine levels. When AAV2/5 is combined with BMT, there are sustained improvements in motor function and the median life span is increased to 123 d (range, 92-282 d) compared with 41 d in the untreated twitcher mice. Interestingly, addition of BMT virtually eliminates both the disease and AAV2/5-associated inflammatory response. These data suggest that the efficacy of AAV2/5-mediated gene therapy is limited by the associated inflammatory response and BMT synergizes with AAV2/5 by modulating inflammation.

Clinical signs and neuropathologic abnormalities in working Australian Kelpies with globoid cell leukodystrophy (Krabbe disease)

To characterize the clinical signs of globoid cell leukodystrophy (GLD) in Australian Kelpies from a working line (AWKs) and determine whether an association existed between these signs and degrees of demyelination and inflammatory responses in affected brains. Case-control study. 4 AWKs with GLD (cases) and 7 unaffected young adult dogs of mixed breeding (controls). Clinical records were reviewed for information on signalment, and samples of neurologic tissues underwent histological processing, immunohistochemical staining, and image analysis. Findings were compared between case and control dogs. The 4 affected AWKs had progressive ataxia, tremors, and paresis and low leukocyte activity of galactosylceramidase, the lysosomal enzyme deficient in GLD. Image analysis of neurologic tissue revealed globoid cells characteristic of GLD and substantial demyelination in the peripheral and central nervous systems, relative to that in neurologic tissue from control dogs. This was accompanied by microglial activation, reactive astrocyto-sis, and axonal spheroid formation. The demyelination, inflammatory responses, and axo-nal spheroids evident in the AWKs were consistent with the clinical signs of peripheral nerve, spinal cord, and cerebellar dysfunction. Because GLD is an autosomal recessive inherited disease, with considerable overlap in galactosylceramidase activity existing among heterozygotes and noncarriers, development of a molecular test is important for preventing the perpetuation of this disease in the Australian Kelpie breed.

Combined hematopoietic and lentiviral gene‐transfer therapies in newborn Twitcher mice reveal contemporaneous neurodegeneration and demyelination in Krabbe disease

This study characterized the therapeutic benefits of combining hematogenous cell replacement with lentiviral-mediated gene transfer of galactosylceramidase (GALC) in Twitcher mice, a bona fide model for Krabbe disease. Bone marrow cells and GALC-lentiviral vectors were administered intravenously without any preconditioning to newborn Twitcher pups before postnatal day 2. Treated Twitchers survived up to 4 months of age. GALC activity remained less than 5% of normal values in the nervous system for the first 2 months after treatment and reached approximately 30% in long-term-surviving mice. Long-term reconstitution of GALC activity in the nervous system was provided primarily by infiltrating macrophages and to a lesser extent by direct lentiviral transduction of neural cells. Treated Twitchers had significant preservation of myelin, with a G-ratio (ratio of the axon diameter to the diameter of the myelinated fiber) in sciatic nerve myelin of 0.75 +/- 0.08 compared with 0.85 +/- 0.10 in untreated mutants. Although treated mutants had improved locomotor activities during their long-term survival, they died with symptoms of progressive neurological degeneration, indistinguishable from those seen in untreated Twitchers. Examination of long-lived Twitchers showed that treated mutants were not protected from developing degeneration of axons throughout the neuroaxis. These results suggest that GALC deficiency not only affects myelinating glia but also leads to neuronal dysfunction. The contemporaneous neuropathology might help to explain the limited efficacy of current gene and cell therapies.

Central Nervous System-directed AAV2/5-Mediated Gene Therapy Synergizes with Bone Marrow Transplantation in the Murine Model of Globoid-cell Leukodystrophy

Globoid-cell leukodystrophy (GLD) is a rapidly progressing inherited neurodegenerative disorder caused by a deficiency in galactosylceramidase activity. Previous studies in the murine model of GLD (Twitcher mouse) have shown that both bone marrow transplantation (BMT) and central nervous system (CNS)-directed gene therapy can be moderately effective at ameliorating certain aspects of GLD. As BMT and CNS-directed gene therapy target fundamentally different tissues, we tested the hypothesis that combining these disparate therapies would be more efficacious than either therapy alone. Mice receiving myeloreductive conditioning at birth followed by syngeneic BMT had approximately 25-35% donor chimerism. Untreated Twitcher mice, Twitcher mice treated with BMT alone, AAV2/5 alone, or a combination of BMT and AAV2/5 had mean lifespans of 39, 44, 49, and 104 days, respectively. Twitcher mice treated with a combination of BMT and AAV2/5 also had significantly improved performance in several behavioral tests and greater reduction in demyelination, astrocytosis, and macrophage infiltration compared to untreated Twitcher mice or mice that received either therapy alone. These data suggest that CNS-directed gene therapy synergizes with BMT. The combination of these disparate therapeutic approaches may form the basis for more effective treatment of this inherited neurodegenerative disorder.