Deficiency of galactosyl-ceramidase in adult oligodendrocytes worsens disease severity during chronic experimental allergic encephalomyelitis.

Galactosyl-ceramidase (GALC) is a ubiquitous lysosomal enzyme crucial for the correct myelination of the mammalian nervous system during early postnatal development. However, the physiological consequence of GALC deficiency in the adult brain remains unknown. In this study, we found that mice with conditional ablation of GALC activity in post-myelinating oligodendrocytes were lethally sensitized when challenged with chronic experimental allergic encephalomyelitis (EAE), in contrast with the non-lethal dysmyelination observed in Galc-ablated mice without the EAE challenge. Mechanistically, we found strong inflammatory demyelination without remyelination and an impaired fusion of lysosomes and autophagosomes with accumulation of myelin debris after a transcription factor EB-dependent increase in the lysosomal autophagosome flux. These results indicate that the physiological impact of GALC deficiency is highly influenced by the cell context (oligodendroglial vs. global expression), the presence of inflammation, and the developmental time when it happens (pre-myelination vs. post-myelination). We conclude that Galc expression in adult oligodendrocytes is crucial for the maintenance of adult central myelin and to decrease vulnerability to additional demyelinating insults.

Clinical feature, GALC variant spectrum, and genotype-phenotype correlation in Korean Krabbe disease patients: Multicenter experience over 13 years.

Krabbe disease (KD) is an autosomal recessive neurodegenerative disorder caused by deficiency of the galactocerebrosidase (GALC) due to variants in the GALC gene. Here, we provide the first and the largest comprehensive analysis of clinical and genetic characteristics, and genotype-phenotype correlations of KD in Korean in comparison with other ethnic groups. From June 2010 to June 2023, 10 patients were diagnosed with KD through sequencing of GALC. Clinical features, and results of GALC sequencing, biochemical test, neuroimaging, and neurophysiologic test were obtained from medical records. An additional nine previously reported Korean KD patients were included for review. In Korean KD patients, the median age of onset was 2 years (3 months-34 years) and the most common phenotype was adult-onset (33%, 6/18) KD, followed by infantile KD (28%, 5/18). The most frequent variants were c.683_694delinsCTC (23%) and c.1901T>C (23%), while the 30-kb deletion was absent. Having two heterozygous pathogenic missense variants was associated with later-onset phenotype. Clinical features were similar to those of other ethnic groups. In Korean KD patients, the most common phenotype was the adult-onset type and the GALC variant spectrum was different from that of the Caucasian population. This study would further our understanding of KD.

Splicing mutations of GALC in adult patient with adult-onset Krabbe disease: case report and review of literature.

Krabbe disease (KD) is classed as the lysosomal storage disease with mutations in the galactosylceramidase (GALC) gene, and commonly showed as autosomal recessive pattern with 30-kb deletion in infantile subtype. In this case, we report a 39-years adult-onset KD (AOKD) patient with multiple sclerosis-like symptoms and neuroimaging changes. She carries the heterozygous mutations in GALC included a missense mutation of c.1901T>C from her mother, and a splicing mutation of c.908+5G>A from her father. The splicing mutations in KD are reviewed and confirmed that c.908+5G>A is a novel splicing mutation in AOKD.

A novel compound heterozygous mutation in GALC associated with adult-onset Krabbe disease: case report and literature review.

Krabbe disease (KD) is a rare autosomal recessive lipid storage leukodystrophy. It is caused by deficient enzyme activity resulting from mutations of the ß-galactocerebrosidase (GALC) gene. KD is distinguished into subtypes based on the age of onset; these are early infantile, late infantile, juvenile, and adult-onset. We report a case of a 47-year-old Caucasian man with a 2-year history of muscle atrophy and weakness in both hands associated with pyramidal signs and mild spasticity in the lower limbs. An extensive work-up led this motor neuron disease-like disorder to be diagnosed as adult-onset KD. The patient was found to be compound heterozygous for two GALC mutations (p.G286D and p.Y490N). These two rare missense mutations have previously been reported with other heterozygous mutations. However, their co-occurrence in a KD patient is novel. From the perspective of this case, we review the current literature on compound heterozygous mutations in adult-onset KD and their phenotypic variability.

Adult-onset Krabbe disease presenting as isolated sensorimotor demyelinating polyneuropathy: A case report.

Krabbe disease is a rare autosomal recessive neurodegenerative disease, caused by mutations in the GALC gene, which encodes for the lysosomal enzyme galactocerebrosidase. Typical clinical manifestations of Krabbe include psychomotor deterioration, visual loss, seizures, and spasticity, that result from central nervous system demyelination. We report a case of a 35-year-old male with Krabbe who presented in adulthood with isolated severe, upper extremity predominant demyelinating sensorimotor polyneuropathy and did not develop other distinguishing clinical or radiological features of Krabbe until the later stages of the disease. The patient's diagnostic odyssey lasted 13 years from presentation to diagnosis, which was ultimately determined with the use of whole exome sequencing (WES) at the age of 48 years. The expanding phenotypic spectrum of adult-onset Krabbe Disease (AOKD) presents a diagnostic challenge that can lead to diagnostic delays and potentially affect treatment options. Our patient's case underscores the importance of pursuing WES in those with undiagnosed progressive neuromuscular disorders.

A novel GALC gene mutation associated with adult-onset Krabbe disease: a case report.

To analyze the clinical, imaging, and genetic characteristics of a patient diagnosed with adult-onset Krabbe disease (KD). Clinical and imaging features of the patient were retrospectively reviewed. The patient, a 40-year-old female, presented adult-onset spastic paraplegia. Brain magnetic resonance imaging (MRI) showed white matter hyperintensities along bilateral optic radiations. Colorimetry of galactocerebrosidase enzyme activity showed low enzyme levels. A heterozygous missense mutation: c.1658G>A (p.G553E) and c.1901T>C (p.L634S) was identified in the GALC gene by whole exome sequencing, and was verified by Sanger sequencing. KD should be considered when patients presented adult-onset spastic paraplegia with classical MRI imaging features. Mutation c.1658G>A (p.G553E) was novel in GALC gene and broaden the mutation spectrum.

ß-Galactosylceramidase Deficiency Causes Upregulation of Long Pentraxin-3 in the Central Nervous System of Krabbe Patients and Twitcher Mice.

Globoid cell leukodystrophy (GLD), or Krabbe disease, is a neurodegenerative sphingolipidosis caused by genetic deficiency of lysosomal ß-galactosylceramidase (GALC), characterized by neuroinflammation and demyelination of the central (CNS) and peripheral nervous system. The acute phase protein long pentraxin-3 (PTX3) is a soluble pattern recognition receptor and a regulator of innate immunity. Growing evidence points to the involvement of PTX3 in neurodegeneration. However, the expression and role of PTX3 in the neurodegenerative/neuroinflammatory processes that characterize GLD remain unexplored. Here, immunohistochemical analysis of brain samples from Krabbe patients showed that macrophages and globoid cells are intensely immunoreactive for PTX3. Accordingly, Ptx3 expression increases throughout the course of the disease in the cerebrum, cerebellum, and spinal cord of GALC-deficient twitcher (Galctwi/twi) mice, an authentic animal model of GLD. This was paralleled by the upregulation of proinflammatory genes and M1-polarized macrophage/microglia markers and of the levels of PTX3 protein in CNS and plasma of twitcher animals. Crossing of Galctwi/twi mice with transgenic PTX3 overexpressing animals (hPTX3 mice) demonstrated that constitutive PTX3 overexpression reduced the severity of clinical signs and the upregulation of proinflammatory genes in the spinal cord of P35 hPTX3/Galctwi/twi mice when compared to Galctwi/twi littermates, leading to a limited increase of their life span. However, this occurred in the absence of a significant impact on the histopathological findings and on the accumulation of the neurotoxic metabolite psychosine when evaluated at this late time point of the disease. In conclusion, our results provide the first evidence that PTX3 is produced in the CNS of GALC-deficient Krabbe patients and twitcher mice. PTX3 may exert a protective role by reducing the neuroinflammatory response that occurs in the spinal cord of GALC-deficient animals.

A new compound heterozygous mutation in adult-onset Krabbe disease.

Purpose: Krabbe disease (KD) or globoid cell leukodystrophy is an autosomal recessive lysosomal disorder caused by a lack of the lysosomal enzyme galactocerebrosidase (GALC) because of mutations in GALC. Patients with KD exhibit a wide spectrum of clinical symptoms; therefore, their diagnosis can be challenging. We report the clinical features and gene mutations in a 48-year-oldpatient with adult-onset KD.Methods: We collected and analyzed clinical data of the patientwith a diagnosis of KD. Gene mutations were identified by whole exome sequencing.Results: We describe a case of adult-onset KD caused by a novel compound heterozygous mutation; a missense mutation, c. 1901 T > C (p. L634S); and a novel nonsense mutation, c.1005C > G (p. Y335X), in GALC. The disease onset started when the patient was 40 years old, and manifested as typical paralytic paraplegia. Magnetic resonance imaging indicated demyelination of the white matter, which is consistent with the typical symptoms of adult-onset KD. Biochemical analysis revealed GALC activity to be 1.5 nmol/17 h/mg protein, confirming its deficiency and KD diagnosis.Conclusions: Our findings provide evidence of a novel mutation, providing additional information toward to the GALC mutation database.

GALC mutations in Chinese patients with late-onset Krabbe disease: a case report.

BACKGROUND: Krabbe disease (also known as globoid cell leukodystrophy) cause by a deficiency of the enzyme ß-galactocerebrosidase (galactosylceramidase, GALC). The deficiency of GALC leads to accumulation of galactosylceramide and psychosine, the latter GALC substrate having a potential role in triggering demyelination. Typically, the disease has an infantile onset, with rapid deterioration in the first few months, leading to death before the age of 2 years. The late onset forms (late-infantile, juvenile, and adult forms) are rare with variable clinical outcomes, presenting spastic paraplegia as the main symptom. CASE PRESENTATION: We recruited a family with two affected individuals. The proband (Patient 1), a 25-year-old male, was presented with slow progressive symptoms, including spastic gait disturbance and vision loss since the 5th year of life. His elder sister (Patient 2), became wheelchair-bound and demented at the age of 22 years. Brain magnetic resonance imaging (MRI) showed increased signal intensity in the white matter along with the involvement of the bilateral corticospinal tracts. GALC deficiency was confirmed by biochemical analysis. DNA sequencing revealed two mutations (c.865G > C: p. G289R and c.136G > T: p. D46Y) in GALC. The clinical characteristics, brain MRI, biochemical and molecular findings led to the diagnosis of Krabbe disease. CONCLUSION: Clinical and neuroimaged signs, positive enzymatic analysis and molecular data converged to definite diagnosis in this neurodegenerative disease.

A novel homozygous GALC variant has been associated with Krabbe disease in a consanguineous family.

Krabbe disease (KD) or globoid cell leukodystrophy is an autosomal recessive lysosomal storage disorder involving the white matter of the peripheral and the central nervous systems. It is caused by a deficiency of galactocerebrosidase enzyme activity. The most common manifestation is the classical early onset KD that leads to patient's loss before the age of 2. Herein, we report the evaluation of a consanguineous family with three affected children manifesting severe neurological findings that ended with death before the age of 2, in an attempt to provide genetic diagnosis to the family. One of the children underwent detailed physical and neurological examinations, including brain magnetic resonance imaging (MRI) and scalp electroencephalography (EEG) evaluations. GALC genetic testing on this child enabled identification of a novel homozygous variant (NM_000153.3: c.1394C>T; p.(Thr465Ile)), which confirmed diagnosis as KD. Familial segregation of this variant was performed by PCR amplification and Sanger sequencing that revealed the parents as heterozygous carriers. We believe this novel GALC variant will not only help in genetic counseling to this family but will also aid in identification of future KD cases.

In Silico Analysis of Missense Mutations as a First Step in Functional Studies: Examples from Two Sphingolipidoses.

In order to delineate a better approach to functional studies, we have selected 23 missense mutations distributed in different domains of two lysosomal enzymes, to be studied by in silico analysis. In silico analysis of mutations relies on computational modeling to predict their effects. Various computational platforms are currently available to check the probable causality of mutations encountered in patients at the protein and at the RNA levels. In this work we used four different platforms freely available online (Protein Variation Effect Analyzer- PROVEAN, PolyPhen-2, Swiss-model Expert Protein Analysis System-ExPASy, and SNAP2) to check amino acid substitutions and their effect at the protein level. The existence of functional studies, regarding the amino acid substitutions, led to the selection of the distinct protein mutants. Functional data were used to compare the results obtained with different bioinformatics tools. With the advent of next-generation sequencing, it is not feasible to carry out functional tests in all the variants detected. In silico analysis seems to be useful for the delineation of which mutants are worth studying through functional studies. Therefore, prediction of the mutation impact at the protein level, applying computational analysis, confers the means to rapidly provide a prognosis value to genotyping results, making it potentially valuable for patient care as well as research purposes. The present work points to the need to carry out functional studies in mutations that might look neutral. Moreover, it should be noted that single nucleotide polymorphisms (SNPs), occurring in coding and non-coding regions, may lead to RNA alterations and should be systematically verified. Functional studies can gain from a preliminary multi-step approach, such as the one proposed here.

Mutations in GALC cause late-onset Krabbe disease with predominant cerebellar ataxia.

Mutations in GALC cause Krabbe disease. This autosomal recessive leukodystrophy generally presents in early infancy as a severe disorder, but sometimes manifests as a milder adult-onset disease with spastic paraplegia as the main symptom. We recruited a family with five affected individuals presenting with adult-onset predominant cerebellar ataxia with mild spasticity. Whole exome sequencing (WES) revealed one novel and one previously reported compound heterozygous variants in GALC. Magnetic resonance imaging (MRI) confirmed the presence of typical Krabbe features. Our findings expand the phenotypic spectrum of adult-onset Krabbe disease and demonstrate the usefulness of combining WES and pattern-specific MRI for the diagnosis of neurodegenerative diseases.

Krabbe disease: involvement of connexin43 in the apoptotic effects of sphingolipid psychosine on mouse oligodendrocyte precursors.

Krabbe disease is a genetic demyelinating syndrome characterized by deficiency of the enzyme ß-galactosylceramidase, lysosomal psychosine accumulation, and loss of myelin-forming cells. In this study, some apoptotic markers such as apoptotic index (AI), DNA fragmentation, caspase-3, PTEN, Bad, and PI3K were determined in oligodendrocyte precursors from wild type or twitcher mice untreated or treated with psychosine. Twitcher is a natural mouse model of Krabbe disease containing a premature stop codon (W339X) in the ß-galactosylceramidase gene. Moreover, a possible involvement of connexin (Cx)43 in cell death of oligodendrocyte precursors induced by psychosine was investigated with the final aim to provide a contribution to the knowledge of the molecular mechanisms and pathophysiological events that occur in Krabbe disease. Connexins are a multigene family of structurally related trans-membrane proteins able to modulate essential cellular processes such as proliferation, differentiation and migration. Among these, Cx43 is the predominant isoform in many cell types, including neural progenitor cells. Our results showed an increase of AI, DNA fragmentation, caspase-3, PTEN, Bad, and Cx43 associated to a decrease of PI3K, pAKT and pBad. Taken together, these findings suggest an involvement of Cx43 in the psychosine-mediated apoptosis of primary oligodendrocyte progenitors from wild type or twitcher mice, used for the first time as cell models in comparison. It could open unexplored perspective also for other demyelinating diseases.

Newborn screening for Krabbe's disease.

Live newborn screening for Krabbe's disease (KD) was initiated in New York on August 7, 2006, and started in Missouri in August, 2012. As of August 7, 2015, nearly 2.5 million infants had been screened, and 443 (0.018%) infants had been referred for followup clinical evaluation; only five infants had been determined to have KD. As of August, 2015, the combined incidence of infantile KD in New York and Missouri is ∼1 per 500,000; however, patients who develop later-onset forms of KD may still emerge. This Review provides an overview of the processes used to develop the screening and followup algorithms. It also includes updated results from screening and discussion of observations, lessons learned, and suggested areas for improvement that will reduce referral rates and the number of infants defined as at risk for later-onset forms of KD. Although current treatment options for infants with early-infantile Krabbe's disease are not curative, over time treatment options should improve; in the meantime, it is essential to evaluate the lessons learned and to ensure that screening is completed in the best possible manner until these improvements can be realized. © 2016 Wiley Periodicals, Inc.

Intrathecal administration of AAV/GALC vectors in 10-11-day-old twitcher mice improves survival and is enhanced by bone marrow transplant.

Globoid cell leukodystrophy (GLD), or Krabbe disease, is an autosomal recessive neurodegenerative disease caused by the deficiency of the lysosomal enzyme galactocerebrosidase (GALC). Hematopoietic stem cell transplantation (HSCT) provides modest benefit in presymptomatic patients but is well short of a cure. Gene transfer experiments using viral vectors have shown some success in extending the survival in the mouse model of GLD, twitcher mice. The present study compares three single-stranded (ss) AAV serotypes, two natural and one engineered (with oligodendrocyte tropism), and a self-complementary (sc) AAV vector, all packaged with a codon-optimized murine GALC gene. The vectors were delivered via a lumbar intrathecal route for global CNS distribution on PND10-11 at a dose of 2 × 10(11) vector genomes (vg) per mouse. The results showed a similar significant extension of life span of the twitcher mice for all three serotypes (AAV9, AAVrh10, and AAV-Olig001) as well as the scAAV9 vector, compared to control cohorts. The rAAV gene transfer facilitated GALC biodistribution and detectable enzymatic activity throughout the CNS as well as in sciatic nerve and liver. When combined with BMT from syngeneic wild-type mice, there was significant improvement in survival for ssAAV9. Histopathological analysis of brain, spinal cord, and sciatic nerve showed significant improvement in preservation of myelin, with ssAAV9 providing the greatest benefit. In summary, we demonstrate that lumbar intrathecal delivery of rAAV/mGALCopt can significantly enhance the life span of twitcher mice treated at PND10-11 and that BMT synergizes with this treatment to improve the survival further. © 2016 Wiley Periodicals, Inc.

Cellular transplant therapies for globoid cell leukodystrophy: Preclinical and clinical observations.

Globoid cell leukodystrophy (GLD) is a progressive neurodegenerative disorder caused by the deficiency of galactocerebrosidase (GALC), resulting in accumulation of toxic metabolites in neural tissues. Clinically variable based on age of onset, infantile GLD is generally a rapidly fatal syndrome of progressive neurologic and cognitive decline, whereas later-onset GLD has a more indolent, protracted clinical course. Animal models, particularly the twitcher mouse, have allowed investigation of both the pathophysiology of and the potential treatment modalities for GLD. Cellular therapy for GLD, notably hematopoietic cell transplantation (HCT; transplantation of bone marrow, peripheral blood stem cells, or umbilical cord blood cells) from a normal related or unrelated allogeneic donor provides a self-renewing source of GALC in donor-derived cells. The only currently available treatment option in human GLD, allogeneic HCT, can slow the progression of the disease and improve survival, especially when performed in presymptomatic infants. Because persistent neurologic dysfunction still occurs after HCT in GLD, preclinical studies are evaluating combinations of HCT with other treatment modalities. © 2016 Wiley Periodicals, Inc.

Immunological considerations for treating globoid cell leukodystrophy.

Globoid cell leukodystrophy (GLD, or Krabbe's disease) is a severe inherited neurodegenerative disease caused by the lack of a lysosomal enzyme, GALC. The disease has been characterized in humans as well as three naturally occurring animal models, murine, canine, and nonhuman primate. Multiple treatment strategies have been explored for GLD, including enzyme replacement therapy, small-molecule pharmacological approaches, gene therapy, and bone marrow transplant. No single therapeutic approach has proved to be entirely effective, and the reason for this is not well understood. It is unclear whether initiation of a neuroinflammatory cascade in GLD precedes demyelination, a hallmark of the disease, but it does precede overt symptoms. This Review explores what is known about the role of inflammation and the immune response in the progression of GLD as well as how various treatment strategies might interplay with innate and adaptive immune responses involved in GLD. The focus of this Review is on GLD, but these concepts may have relevance for other, related diseases. © 2016 Wiley Periodicals, Inc.

Expression of individual mutations and haplotypes in the galactocerebrosidase gene identified by the newborn screening program in New York State and in confirmed cases of Krabbe's disease.

Newborn screening (NBS) for Krabbe's disease (KD) has been instituted in several states, and New York State has had the longest experience. After an initial screening of dried blood spots, samples from individuals with galactocerebrosidase (GALC) values below a given cutoff level were subjected to additional testing, including sequencing of the GALC gene. This resulted in the identification of mutations that had previously been found in confirmed KD patients and of variants that had never previously been reported. Some individuals had variants considered to be polymorphisms, alone or on the same allele as another mutation. To help with counseling of families on the risk for a newborn to develop KD, expression studies were conducted with these variants identified by NBS. GALC activity was measured in COS1 cells for 140 constructs and compared with mutations that had previously been seen in confirmed cases of KD. When a polymorphism was present on the same allele as the variant, expressed activity was measured with and without the polymorphism. In some cases the presence of the polymorphism greatly lowered the measured GALC activity, possibly making it disease causing. Although it is not possible to predict conclusively whether a variant is severe and will result in infantile KD if two such variants are present or whether a variant is mild and will result in late-onset disease, some variants clearly are not disease causing. This is the largest expression study of GALC variants/mutations found in NBS and confirmed KD cases. This work will be helpful for counseling families of screen-positive newborns found to have low GALC activity. © 2016 Wiley Periodicals, Inc.

New therapeutic approaches for Krabbe disease: The potential of pharmacological chaperones.

Missense mutations in the lysosomal hydrolase ß-galactocerebrosidase (GALC) account for at least 40% of known cases of Krabbe disease (KD). Most of these missense mutations are predicted to disrupt the fold of the enzyme, preventing GALC in sufficient amounts from reaching its site of action in the lysosome. The predominant central nervous system (CNS) pathology and the absence of accumulated primary substrate within the lysosome mean that strategies used to treat other lysosomal storage disorders (LSDs) are insufficient in KD, highlighting the still unmet clinical requirement for successful KD therapeutics. Pharmacological chaperone therapy (PCT) is one strategy being explored to overcome defects in GALC caused by missense mutations. In recent studies, several small-molecule inhibitors have been identified as promising chaperone candidates for GALC. This Review discusses new insights gained from these studies and highlights the importance of characterizing both the chaperone interaction and the underlying mutation to define properly a responsive population and to improve the translation of existing lead molecules into successful KD therapeutics. We also highlight the importance of using multiple complementary methods to monitor PCT effectiveness. Finally, we explore the exciting potential of using combination therapy to ameliorate disease through the use of PCT with existing therapies or with more generalized therapeutics, such as proteasomal inhibition, that have been shown to have synergistic effects in other LSDs. This, alongside advances in CNS delivery of recombinant enzyme and targeted rational drug design, provides a promising outlook for the development of KD therapeutics. © 2016 The Authors. Journal of Neuroscience Research Published by Wiley Periodicals, Inc.

Quantum dots and potential therapy for Krabbe's disease.

Enzyme replacement therapy and substrate reduction therapy have proved useful in reversing many pathological consequences of many nonneural lysosomal storage diseases but have not yet reversed pathology or influenced disease outcome in Krabbe's disease (KD). This Review discusses the relative merits of stem cell therapy, molecular chaperone therapy, gene therapy, substrate reduction therapy, enzyme replacement therapy, and combination therapy. Given the limitations of these approaches, this Review introduces the idea of using tiny, 6-nm, intensely fluorescent quantum dots (QDs) to deliver a cell-penetrating peptide and 6 histidine residue-tagged ß-D-galactocerebrosidase across the blood-brain barrier. We can therefore follow the fate of injected material and ensure that all targets are reached and that accumulated material is degraded. Uptake of lysosomal hydrolases is a complex process, and the cell-penetrating peptide JB577 is uniquely able to promote endosomal egress of the QD cargo. This Review further shows that uptake may depend on the charge of the coating of the QD, specifically, that negative charge directs the cargo to neurons. Because KD involves primarily glia, specifically oligodendroglia, we experiment with many coatings and discover a coating (polyethylene glycol 600 amino) that has a positive charge and targets oligodendrocytes. A similar effect is achieved by treating with chondroitinase ABC to degrade the extracellular matrix, indicating that enzyme replacement has several hurdles to overcome before it can become a routine CNS therapy. © 2016 Wiley Periodicals, Inc.