Michael S. Marshall

Neuronal inclusions of α-synuclein contribute to the pathogenesis of Krabbe disease.

Demyelination is a major contributor to the general decay of neural functions in children with Krabbe disease. However, recent reports have indicated a significant involvement of neurons and axons in the neuropathology of the disease. In this study, we have investigated the nature of cellular inclusions in the Krabbe brain. Brain samples from the twitcher mouse model for Krabbe disease and from patients affected with the infantile and late‐onset forms of the disease were examined for the presence of neuronal inclusions. Our experiments demonstrated the presence of cytoplasmic aggregates of thioflavin‐S‐reactive material in both human and murine mutant brains. Most of these inclusions were associated with neurons. A few inclusions were detected to be associated with microglia and none were associated with astrocytes or oligodendrocytes. Thioflavin‐S‐reactive inclusions increased in abundance, paralleling the development of neurological symptoms, and distributed throughout the twitcher brain in areas of major involvement in cognition and motor functions. Electron microscopy confirmed the presence of aggregates of stereotypic β‐sheet folded proteinaceous material. Immunochemical analyses identified the presence of aggregated forms of α‐synuclein and ubiquitin, proteins involved in the formation of Lewy bodies in Parkinsons disease and other neurodegenerative conditions. In vitro assays demonstrated that psychosine, the neurotoxic sphingolipid accumulated in Krabbe disease, accelerated the fibrillization of α‐synuclein. This study demonstrates the occurrence of neuronal deposits of fibrillized proteins including α‐synuclein, identifying Krabbe disease as a new α‐synucleinopathy. Copyright

Mechanism of Neuromuscular Dysfunction in Krabbe Disease

The atrophy of skeletal muscles in patients with Krabbe disease is a major debilitating manifestation that worsens their quality of life and limits the clinical efficacy of current therapies. The pathogenic mechanism triggering muscle wasting is unknown. This study examined structural, functional, and metabolic changes conducive to muscle degeneration in Krabbe disease using the murine (twitcher mouse) and canine [globoid cell leukodystrophy (GLD) dog] models. Muscle degeneration, denervation, neuromuscular [neuromuscular junction (NMJ)] abnormalities, and axonal death were investigated using the reporter transgenic twitcher–Thy1.1–yellow fluorescent protein mouse. We found that mutant muscles had significant numbers of smaller-sized muscle fibers, without signs of regeneration. Muscle growth was slow and weak in twitcher mice, with decreased maximum force. The NMJ had significant levels of activated caspase-3 but limited denervation. Mutant NMJ showed reduced surface areas and lower volumes of presynaptic terminals, with depressed nerve control, increased miniature endplate potential (MEPP) amplitude, decreased MEPP frequency, and increased rise and decay rate constants. Twitcher and GLD dog muscles had significant capacity to store psychosine, the neurotoxin that accumulates in Krabbe disease. Mechanistically, muscle defects involved the inactivation of the Akt pathway and activation of the proteasome pathway. Our work indicates that muscular dysfunction in Krabbe disease is compounded by a pathogenic mechanism involving at least the failure of NMJ function, activation of proteosome degradation, and a reduction of the Akt pathway. Akt, which is key for muscle function, may constitute a novel target to complement in therapies for Krabbe disease.

Beyond Krabbe's disease: The potential contribution of galactosylceramidase deficiency to neuronal vulnerability in late-onset synucleinopathies

New insights into the pathophysiological mechanisms behind late‐onset neurodegenerative diseases have come from unexpected sources in recent years. Specifically, the group of inherited metabolic disorders known as lysosomal storage diseases that most commonly affect infants has been found to have surprising similarities with adult neurodegenerative disorders. Most notable has been the identification of Gauchers disease as a comorbidity for Parkinsons disease. Prompted by the recent identification of neuronal aggregates of α‐synuclein in another lysosomal storage disease, Krabbes disease, we propose the idea that a similar connection exists between adult synucleinopathies and Krabbes. Similarities between the two diseases, including the pattern of α‐synuclein aggregation in the brain of the twitcher mouse (the authentic murine model of Krabbes disease), changes to lipid membrane dynamics, and possible dysfunction in synaptic function and macroautophagy, underscore a link between Krabbes disease and late‐onset synucleinopathies. Silent GALC mutations may even constitute a risk factor for the development of Parkinsons in certain patients. More research is required to identify definitively any link and the validity of this hypothesis, but such a connection would prove invaluable for developing novel therapeutic targets for Parkinsons based on our current understanding of Krabbes disease and for establishing new biomarkers for the identification of at‐risk patients.

Psychosine enhances the shedding of membrane microvesicles: Implications in demyelination in Krabbe's disease

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.

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 × 1011 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.

Analysis of age-related changes in psychosine metabolism in the human brain

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.

Long-Term Improvement of Neurological Signs and Metabolic Dysfunction in a Mouse Model of Krabbe’s Disease after Global Gene Therapy

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 Krabbes 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 GALCs 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.

α-Synuclein interacts directly but reversibly with psychosine: implications for α-synucleinopathies

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.

AAVrh10 Gene Therapy Ameliorates Central and Peripheral Nervous System Disease in Canine Globoid Cell Leukodystrophy (Krabbe Disease)

Globoid cell leukodystrophy (GLD), or Krabbe disease, is an inherited, neurologic disorder that results from deficiency of a lysosomal enzyme, galactosylceramidase. Most commonly, deficits of galactosylceramidase result in widespread central and peripheral nervous system demyelination and death in affected infants typically by 2 years of age. Hematopoietic stem-cell transplantation is the current standard of care in children diagnosed prior to symptom onset. However, disease correction is incomplete. Herein, the first adeno-associated virus (AAV) gene therapy experiments are presented in a naturally occurring canine model of GLD that closely recapitulates the clinical disease progression, neuropathological alterations, and biochemical abnormalities observed in human patients. Adapted from studies in twitcher mice, GLD dogs were treated by combination intravenous and intracerebroventricular injections of AAVrh10 to target both the peripheral and central nervous systems. Combination of intravenous and intracerebroventricular AAV gene therapy had a clear dose response and resulted in delayed onset of clinical signs, extended life-span, correction of biochemical defects, and attenuation of neuropathology. For the first time, therapeutic effect has been established in the canine model of GLD by targeting both peripheral and central nervous system impairments with potential clinical implications for GLD patients.

57. Intrathecal Administration of AAV/GALC Vectors in Juvenile Twitcher Mice Improves Survival and Is Enhanced by Bone Marrow Transplant

Globoid cell Leukodystrophy (GLD) or Krabbe disease is a rapidly progressing, neurodegenerative disease caused by the deficiency of the lysosomal enzyme Galactocerebrosidase (GALC). The pathological characteristics include presence of globoid cells and decreased myelin. In its severe infantile form, the symptoms appear within the first 6 months of life and complete loss of GALC function is fatal by 2-3 years old. The murine model of infantile GLD, the twitcher mouse, has been used to evaluate potential therapeutic approaches for GLD. Hematopoietic stem cell transplantation (HSCT) provides modest benefit in presymptomatic patients and mice indicating a slowdown in the progression of the disease, but no complete cure. Neonatal gene transfer experiments using viral vectors also has shown some limited success in extending the survival of the twitcher mice. The translatability of neonatal therapy in mice to human has met with difficulties as the stage of the disease in human and mice differ due to the difference in their gestation periods. So there is a need for testing out later stage interventions for GLD treatment. In the present study, we compare multiple vector designs along with a combination treatment of AAV plus bone marrow transplant (BMT) in juvenile twitcher mice. Initially, three single stranded (ss) AAV serotypes, two natural and one engineered (with oligodendrocyte tropism), were packaged with a codon-optimized murine GALC gene driven by the beta actin promoter. The vectors were delivered via a lumbar intrathecal route for global CNS distribution on post-natal day (PND) 10-11, at a dose of 2×1011 vg per mouse. The results show a significant extension of life span of the twitcher mice for all three serotypes (AAV9, AAVrh10, and AAV-Olig001) when compared to control cohorts. The treatment produced similar survival benefit regardless of which capsid was used. The rAAV gene transfer facilitates 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 and enzymatic activity over either treatment alone. Immunohistochemical analysis of the brain and spinal cord showed reduced inflammation and pathology. Additionally, we have also tested a novel self-complementary (sc) AAV vector with a minimal synthetic promoter, which would mediate a weaker overall level of GALC expression but express in more cells. Preliminary results indicate that this vector design provides a survival advantage over the ssAAV vector designs. In summary, we demonstrate that lumbar intrathecal delivery of rAAV/mGALCopt can significantly enhance the life span of twitcher mice treated at juvenile stage (PND10-11) and BMT synergizes with this treatment to further improve the survival. This effect is mediated by increased GALC activity in various parts of the nervous system as well as by the reduction in neuro-inflammation. Together, these studies detail a therapeutic approach for GLD in mice which is feasible and relevant for human translation.