Preclinical studies in Krabbe disease: A model for the investigation of novel combination therapies for lysosomal storage diseases

Krabbe disease (KD) is a lysosomal storage disease (LSD) caused by mutations in the galc gene. There are over 50 monogenetic LSDs, which largely impede the normal development of children and often lead to premature death. At present, there are no cures for LSDs and the available treatments are generally insufficient, short acting, and not without co-morbidities or long-term side effects. The last 30 years have seen significant advances in our understanding of LSD pathology as well as treatment options. Two gene therapy-based clinical trials, NCT04693598 and NCT04771416, for KD were recently started based on those advances. This review will discuss how our knowledge of KD got to where it is today, focusing on preclinical investigations, and how what was discovered may prove beneficial for the treatment of other LSDs

Axons mediate the distribution of arylsulfatase A within the mouse hippocampus upon gene delivery.

Axonal transport of the lysosomal enzyme arylsulfatase A (ARSA) may be an additional mechanism of enzyme distribution after in vivo brain gene transfer in an animal model of metachromatic leukodystrophy (MLD). Direct molecular demonstration of the movement of this lysosomal enzyme within axonal networks was missing. We generated lentiviral vectors carrying the ARSA cDNA tagged with hemagglutinin or the green fluorescent protein and examined the subcellular localization and anatomical distribution of the tagged enzymes within the MLD hippocampus after in vivo lentiviral gene transfer. The use of tagged ARSA allowed direct real-time observation and tracking of axon–dendritic transport of the enzyme after lentiviral gene therapy. Tagged ARSA was expressed in transduced pyramidal, granule, and hilar neurons within the lentiviral-injected side and was robustly contained in vesicles within ipsilateral axon–dendritic processes as well as in vesicles associated with contralateral axons and commissural axons of the ventral hippocampal commissure. Axonal transport of tagged ARSA led to the correction of hippocampal defects in long-term treated MLD mice, which was accompanied by enzyme uptake in nontransduced contralateral neurons, enzyme accumulation within the lysosomal compartment, and clearance of sulfatide storage deposits in this region of the MLD brain. These results contribute to the understanding of the mechanisms of distribution of lysosomal enzymes within the mammalian brain after direct gene therapy, demonstrating the use of neural processes for enzyme transport

P2x7 deficiency suppresses development of experimental autoimmune encephalomyelitis

<p>Abstract</p> <p>Background</p> <p>The purinergic receptor P2x7 is expressed on myeloid cells as well as on CNS glial cells, and P2x7 activation has been shown to increase both glial and T-cell activation. These properties suggest a role in the development of autoimmune disease including multiple sclerosis.</p> <p>Methods</p> <p>The animal model of MS, experimental autoimmune encephalomyelitis (EAE) using myelin oligodendrocyte glycoprotein (MOG) peptide residues 35–55 was induced in wildtype C57BL6 mice and in P2x7 deficient mice ('P2x7 mice') that were backcrossed to C57BL6 mice. Disease progression was monitored by appearance of clinical signs, immunocytochemical staining to assess brain inflammation and neuronal damage, and by measurement of Tcell cytokine production.</p> <p>Results</p> <p>The incidence of EAE disease in P2x7 mice was reduced 4-fold compared to the wildtype mice; however the P2x7 mice that became ill had similar days of onset and clinical scores as the wildtype mice. Splenic T-cells isolated from P2x7 null mice produced greater IFNγ and IL-17 (from 3 to 12 fold greater levels) than wildtype cells, however cytokine production from P2x7 derived cells was not increased by a selective P2x7 agonist as was cytokine production from wildtype cells. Although infiltrating cells were detected in brains of both the P2x7 and wildtype mice, astroglial activation and axonal damage was reduced versus wildtype mice, and the distribution of astroglial activation was markedly distinct in the two strains. In contrast, microglial activation was similar in the two strains.</p> <p>Conclusion</p> <p>P2x7 deficiency resulted in compensatory changes leading to increased T-cell cytokine production, and activated T-cells were detected in the brains of P2x7 null mice with no clinical signs. However, the greatly reduced incidence of disease suggests that an initiating event is absent in these mice, and points to a role for astroglial P2x7 in development of EAE disease.</p

Peripheral neuropathy in the Twitcher mouse involves the activation of axonal caspase 3

Infantile Krabbe disease results in the accumulation of lipid-raft-associated galactosylsphingosine (psychosine), demyelination, neurodegeneration and premature death. Recently, axonopathy has been depicted as a contributing factor in the progression of neurodegeneration in the Twitcher mouse, a bona fide mouse model of Krabbe disease. Analysis of the temporal-expression profile of MBP (myelin basic protein) isoforms showed unexpected increases of the 14, 17 and 18.5 kDa isoforms in the sciatic nerve of 1-week-old Twitcher mice, suggesting an abnormal regulation of the myelination process during early postnatal life in this mutant. Our studies showed an elevated activation of the pro-apoptotic protease caspase 3 in sciatic nerves of 15- and 30-day-old Twitcher mice, in parallel with increasing demyelination. Interestingly, while active caspase 3 was clearly contained in peripheral axons at all ages, we found no evidence of caspase accumulation in the soma of corresponding mutant spinal cord motor neurons. Furthermore, active caspase 3 was found not only in unmyelinated axons, but also in myelinated axons of the mutant sciatic nerve. These results suggest that axonal caspase activation occurs before demyelination and following a dying-back pattern. Finally, we showed that psychosine was sufficient to activate caspase 3 in motor neuronal cells in vitro in the absence of myelinating glia. Taken together, these findings indicate that degenerating mechanisms actively and specifically mediate axonal dysfunction in Krabbe disease and support the idea that psychosine is a pathogenic sphingolipid sufficient to cause axonal defects independently of demyelination

Intrathecal administration of AAV/GALC vectors in 10-11-day-old twitcher mice improves survival and is enhanced by bone marrow transplant: Intrathecal AAV Combined With BMT To Treat Krabbe Disease

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

Astrocyte Support for Oligodendrocyte Differentiation can be Conveyed via Extracellular Vesicles but Diminishes with Age.

The aging brain is associated with significant changes in physiology that alter the tissue microenvironment of the central nervous system (CNS). In the aged CNS, increased demyelination has been associated with astrocyte hypertrophy and aging has been implicated as a basis for these pathological changes. Aging tissues accumulate chronic cellular stress, which can lead to the development of a pro-inflammatory phenotype that can be associated with cellular senescence. Herein, we provide evidence that astrocytes aged in culture develop a spontaneous pro-inflammatory and senescence-like phenotype. We found that extracellular vesicles (EVs) from young astrocyte were sufficient to convey support for oligodendrocyte differentiation while this support was lost by EVs from aged astrocytes. Importantly, the negative influence of culture age on astrocytes, and their cognate EVs, could be countered by treatment with rapamycin. Comparative proteomic analysis of EVs from young and aged astrocytes revealed peptide repertoires unique to each age. Taken together, these findings provide new information on the contribution of EVs as potent mediators by which astrocytes can extert changing influence in either the disease or aged brain

CRISPR-Cas9 knock-in of T513M and G41S mutations in the murine β-galactosyl-ceramidase gene re-capitulates early-onset and adult-onset forms of Krabbe Disease

Krabbe Disease (KD) is a lysosomal storage disorder characterized by the genetic deficiency of the lysosomal enzyme β-galactosyl-ceramidase (GALC). Deficit or a reduction in the activity of the GALC enzyme has been correlated with the progressive accumulation of the sphingolipid metabolite psychosine, which leads to local disruption in lipid raft architecture, diffuse demyelination, astrogliosis, and globoid cell formation. Th

microRNA-219 Reduces Viral Load and Pathologic Changes in Theiler's Virus-Induced Demyelinating Disease

Analysis of microRNA (miR) expression in the central nervous system white matter of SJL mice infected with the BeAn strain of Theiler's murine encephalomyelitis virus (TMEV) revealed a significant reduction of miR-219, a critical regulator of myelin assembly and repair. Restoration of miR-219 expression by intranasal administration of a synthetic miR-219 mimic before disease onset ameliorates clinical disease, reduces neurogliosis, and partially recovers motor and sensorimotor function by negatively regulating proinflammatory cytokines and virus RNA replication. Moreover, RNA sequencing of host lesions showed that miR-219 significantly downregulated two genes essential for the biosynthetic cholesterol pathway, Cyp51 (lanosterol 14-α-demethylase) and Srebf1 (sterol regulatory element-binding protein-1), and reduced cholesterol biosynthesis in infected mice and rat CG-4 glial precursor cells in culture. The change in cholesterol biosynthesis had both anti-inflammatory and anti-viral effects. Because RNA viruses hijack endoplasmic reticulum double-layered membranes to provide a platform for RNA virus replication and are dependent on endogenous pools of cholesterol, miR-219 interference with cholesterol biosynthesis interfered virus RNA replication. These findings demonstrate that miR-219 inhibits TMEV-induced demyelinating disease through its anti-inflammatory and anti-viral properties. MicroRNAs (miRs) are small noncoding RNAs that regulate a myriad of biological processes by controlling gene expression. In the latest issue of Molecular Therapy, Moyano et al. show that intranasal delivery of miR-219 in a mouse model of viral demyelination reduces neurological burden and improves life quality through anti-inflammatory and anti-viral mechanisms.Fil: Moyano, Ana Lis. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba; Argentina. Instituto Universitario de Ciencias Biomédicas de Córdoba; Argentina. University of Illinois; Estados UnidosFil: Steplowski, Jeffrey. University of Illinois; Estados UnidosFil: Wang, Haibo. Cincinnati Children's Hospital Medical Center; Estados UnidosFil: Son, Kyung No. University of Illinois; Estados UnidosFil: Rapolti, Diana I.. University of Illinois; Estados UnidosFil: Marshall, Jeffrey. University of Illinois; Estados UnidosFil: Elackattu, Vince. University of Illinois; Estados UnidosFil: Marshall, Michael S.. University of Illinois; Estados UnidosFil: Hebert, Amy K.. University of Illinois; Estados UnidosFil: Reiter, Cory R.. University of Illinois; Estados UnidosFil: Ulloa, Viviana. University of Illinois; Estados UnidosFil: Pituch, Katarzyna C.. University of Illinois; Estados UnidosFil: Givogri, Maria I.. University of Illinois; Estados UnidosFil: Lu, Q. Richard. Cincinnati Children's Hospital Medical Center; Estados UnidosFil: Lipton, Howard L.. University of Illinois; Estados UnidosFil: Bongarzone, Ernesto R.. University of Illinois; Estados Unidos. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Química Biológica; Argentin

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.Fil: Abdelkarim, Hazem. University of Illinois; Estados UnidosFil: Marshall, Michael S.. University of Illinois; Estados UnidosFil: Scesa, Giuseppe. University of Illinois; Estados UnidosFil: Smith, Rachael A.. University of Illinois; Estados UnidosFil: Rue, Emily. University of Illinois; Estados UnidosFil: Marshall, Jeffrey. University of Illinois; Estados UnidosFil: Elackattu, Vince. University Of Illinois Chicago; Estados UnidosFil: Stoskute, Monika. University Of Illinois Chicago; Estados UnidosFil: Issa, Yazan. University Of Illinois Chicago; Estados UnidosFil: Santos, Marta. University Of Illinois Chicago; Estados UnidosFil: Nguyen, Duc. University Of Illinois Chicago; Estados UnidosFil: Hauck, Zane. University Of Illinois Chicago; Estados UnidosFil: Van Breemen, Richard B.. University Of Illinois Chicago; Estados UnidosFil: Celej, Maria Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; ArgentinaFil: Gaponenko, Vadim. University Of Illinois Chicago; Estados UnidosFil: Bongarzone, Ernesto R.. University Of Illinois Chicago; Estados Unido

Hematopoietic Stem cell transplantation and lentiviral vector-based gene therapy for Krabbe's disease: Present convictions and future prospects: BMT-Lentiviral Vectors Therapy for Krabbe's Disease

Currently, presymtomatic hematopoietic stem and progenitor cell transplantation (HSPCT) is the only therapeutic modality that alleviates Krabbe's disease (KD)‐induced central nervous system damage. However, all HSPCT‐treated patients exhibit severe deterioration in peripheral nervous system function characterized by major motor and expressive language pathologies. We hypothesize that a combination of several mechanisms contribute to this phenomenon, including 1) nonoptimal conditioning protocols with consequent inefficient engraftment and biodistribution of donor‐derived cells and 2) insufficient uptake of donor cell‐secreted galactocerebrosidease (GALC) secondary to a naturally low expression level of the cation‐independent mannose 6‐phosphate‐receptor (CI‐MPR). We have characterized the effects of a busulfan (Bu) based conditioning regimen on the efficacy of HSPCT in prolonging twi mouse average life span. There was no correlation between the efficiency of bone marrow engraftment of donor cells and twi mouse average life span. HSPCT prolonged the average life span of twi mice, which directly correlated with the aggressiveness of the Bu‐mediated conditioning protocols. HSPC transduced with lentiviral vectors carrying the GALC cDNA under control of cell‐specific promoters were efficiently engrafted in twi mouse bone marrow. To facilitate HSPCT‐mediated correction of GALC deficiency in target cells expressing low levels of CI‐MPR, a novel GALC fusion protein including the ApoE1 receptor was developed. Efficient cellular uptake of the novel fusion protein was mediated by a mannose‐6‐phosphate‐independent mechanism. The novel findings described here elucidate some of the cellular mechanisms that impede the cure of KD patients by HSPCT and concomitantly open new directions to enhance the therapeutic efficacy of HSPCT protocols for KD. © 2016 The Authors. Journal of Neuroscience Research Published by Wiley Periodicals, Inc