Clinical and MRI findings in two unrelated cats with globoid cell leucodystrophy

Two unrelated male cats, both five months old, were referred for progressive neurological signs characterised by intentional tremors, paraplegia with absence of nociception in the pelvic limbs, weakness, dysmetria with reduced flexor reflex in the thoracic limbs, and bilaterally reduced menace response. MRI, performed by a 0.25 T Esaote Vet Grande, showed diffuse and irregular intramedullary T2 weighted hyperintensity between T3 and L3; these lesions were isointense on T1 weighted images without contrast uptake. In one patient MRI of the brain was obtained and mild cerebellar volume reduction was found. Histological examination of the brain and spinal cord demonstrated myelin loss and perivascular accumulation of periodic acid-Schiff-positive big macrophages. These findings were consistent with globoid cell leucodystrophy, as previously described in cats. Although not specific, in young cats with progressive spinal neurological signs, especially when associated with cerebellar signs, and irregular but diffuse T2 intramedullary hyperintensity and T1 isointensity without contrast uptake, globoid cell leucodystrophy should be suspected.

Impairment of Proteasome and Autophagy Underlying the Pathogenesis of Leukodystrophy

Impairment of the ubiquitin-proteasome-system (UPS) and autophagy causing cytoplasmic aggregation of ubiquitin andp62 have been implicated in the pathogenesis of most neurodegenerative disorders, yet, they have not been fully elucidated in leukodystrophies. The relationship among impairment of UPS, autophagy, and globoid cell leukodystrophy (GLD), one of the most common demyelinating leukodystrophies, is clarified in this study. We examined the ubiquitin and autophagy markers in the brains of twitcher mice, a murine model of infantile GLD, and in human oligodendrocytes incubated with psychosine. Immunohistochemical examinations showed spatiotemporal accumulation of ubiquitin- and p62-aggregates mainly in the white matter of brain and spinal cord at disease progression. Western blot analysis demonstrated a significant accumulation of ubiquitin, p62, and LC3-II in insoluble fraction in parallel with progressive demyelination and neuroinflammation in twitcher brains. In vitro study validated a dose- and time-dependent cytotoxicity of psychosine upon autophagy and UPS machinery. Inhibition of autophagy and UPS exacerbated the accumulation of insoluble ubiquitin, p62, and LC3-II proteins mediated by psychosine cytotoxicity as well as increased cytoplasmic deposition of ubiquitin- and p62-aggregates, and accumulation of autophagosomes and autolysosomes. Further, the subsequent accumulation of reactive oxygen species and reduction of mitochondrial respiration led to cell death. Our studies validate the impairment of proteasome and autophagy underlying the pathogenesis of GLD. These findings provide a novel insight into pathogenesis of GLD and suggest a specific pathomechanism as an ideal target for therapeutic approaches.

Human iPSC-based neurodevelopmental models of globoid cell leukodystrophy uncover patient- and cell type-specific disease phenotypes

SummaryGloboid Cell Leukodystrophy (GLD, or Krabbe disease) is a rare lysosomal storage disease caused by inherited deficiency of β-galactocerebrosidase (GALC). The build-up of galactosylsphingosine (psychosine) and other undegraded galactosylsphingolipids in the nervous system causes severe demyelination and neurodegeneration. The molecular mechanisms of GLD are poorly elucidated in neural cells and whether murine systems recapitulate critical aspects of the human disease is still to be defined.Here, we established a collection of GLD patient-specific induced pluripotent stem cell (iPSC) lines. We differentiated iPSCs from two patients (bearing different disease-causing mutations) into neural progenitors cells (NPCs) and their neuronal/glial progeny, assessing the impact of GALC deficiency and lentiviral vector-mediated GALC rescue/overexpression by means of phenotypic, biochemical, molecular, and lipidomic analysis. We show a progressive increase of psychosine during the differentiation of GLD NPCs to neurons and glia. We report an early and persistent impairment of oligodendroglial and neuronal differentiation in GLD cultures, with peculiar differences observed in the two GLD lines. GLD cells display a global unbalance of lipid composition during the iPSC to neural differentiation and early activation of cellular senescence, depending on the disease-causing mutation. Restoration of GALC activity normalizes the primary pathological hallmarks and partially rescues the differentiation program of GLD NPCs.Our results suggest that multiple mechanisms besides psychosine toxicity concur to CNS pathology in GLD and highlight the need of a timely regulated GALC expression for proper lineage commitment and differentiation of human NPCs. These findings have important implications for establishing tailored gene therapy strategies to enhance disease correction in GLD.