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

Superwarfarins were developed following the emergence of warfarin resistance in rodents. Superwarfarins have much longer half-lives and stronger affinity to vitamin K epoxide reductase versus warfarin, and therefore can cause death in warfarin-resistant rodents. By the mid-1970s, the superwarfarins brodifacoum (BDF) and difenacoum (DiF) were the most widely used rodenticides throughout the world. Unfortunately, increased use was accompanied by a rise in accidental poisonings, reaching >16,000 per year in the United States. Risk of exposure has become a concern since large quantities, up to hundreds of kilograms of rodent bait, are applied by aerial dispersion over regions with rodent infestations. Reports of intentional use of superwarfarins in civilian and military scenarios raises the specter of larger incidents or mass casualties. Unlike warfarin overdose, for which 1–2 days of treatment with vitamin K is effective, treatment of superwarfarin poisoning with vitamin K is limited by extremely high cost and can require daily treatment for a year or longer. Furthermore, superwarfarins have actions that are independent of their anticoagulant effects, including both vitamin K–dependent and –independent effects, which are not mitigated by vitamin K therapy. In this review, we will summarize superwarfarin development, biology and pathophysiology, their threat as weapons, and possible therapeutic approaches.

Aggregation of α-synuclein, the hallmark of α-synucleinopathies such as Parkinson’s disease, occurs in various glycosphingolipidoses. Although α-synuclein aggregation correlates with deficiencies in the lysosomal degradation of glycosphingolipids (GSL), the mechanism(s) involved in this aggregation remains unclear. We previously described the aggregation of α-synuclein in Krabbe’s disease (KD), a neurodegenerative glycosphingolipidosis caused by lysosomal deficiency of galactosyl-ceramidase (GALC) and the accumulation of the GSL psychosine. Here, we used a multi-pronged approach including genetic, biophysical and biochemical techniques to determine the pathogenic contribution, reversibility, and molecular mechanism of aggregation of α-synuclein in KD. While genetic knock-out of α-synuclein reduces, but does not completely prevent, neurological signs in a mouse model of KD, genetic correction of GALC deficiency completely prevents α-synuclein aggregation. We show that psychosine forms hydrophilic clusters and binds the C-terminus of α-synuclein through its amino group and sugar moiety, suggesting that psychosine promotes an open/aggregation-prone conformation of α-synuclein. Dopamine and carbidopa reverse the structural changes of psychosine by mediating a closed/aggregation-resistant conformation of α-synuclein. Our results underscore the therapeutic potential of lysosomal correction and small molecules to reduce neuronal burden in α-synucleinopathies, and provide a mechanistic understanding of α-synuclein aggregation in glycosphingolipidoses.

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

α-Synuclein aggregation has been linked to Gaucher’s disease (GD) and Krabbe’s disease (KD), lysosomal conditions affecting glycosphingolipid metabolism. α-Synuclein pathology has been directly attributed to the dysregulation of glycosphingolipids in both conditions, specifically to increased galactosylsphingosine (psychosine) content in the context of KD. Furthermore, the gene (GALC) coding for the psychosine degrading enzyme galactosylceramidase (GALC), has recently been identified as a risk loci for Parkinson’s disease. However, it is unknown if changes in psychosine metabolism and GALC activity in the context of the aging human brain correlate with Parkinson’s disease. We investigated psychosine accumulation and GALC activity in the aging brain using fresh frozen post-mortem tissue from Parkinson’s (PD, n = 10), Alzheimer’s (AD, n = 10), and healthy control patients (n = 9), along with tissue from neuropsychiatric patients (schizophrenia, bipolar disorder and depression, n = 15 each). An expanded mutational analysis of PD (n = 20), AD (n = 10), and healthy controls (n = 30) examined if PD was correlated with carriers for severe GALC mutations. Psychosine content within the cerebral cortex of PD patients was elevated above control patients. Within all patients, psychosine displayed a significant (p<0.05) and robust regional distribution in the brain with higher levels in the white matter and substantia nigra. A mutational analysis revealed an increase in the incidence of severe GALC mutations within the PD patient population compared to the cohorts of Alzheimer’s patients and healthy controls tested. In addition to α-synuclein pathology identified in the KD brain, control patients identified as GALC mutational carriers or possessing a GALC pathogenic variant had evidence of α-synuclein pathology, indicating a possible correlation between α-synuclein pathology and dysregulation of psychosine metabolism in the adult brain. Carrier status for GALC mutations and prolonged exposure to increased psychosine could contribute to α-synuclein pathology, supporting psychosine metabolism by galactosylceramidase as a risk factor for Parkinson’s disease.