Role of GALC in Adult Brain Myelination

Krabbe Disease (KD) is a demyelinating leukodystrophy instigated by various mutations in the GALC gene. KD has multiple subtypes, but the most common and most aggressive variant is lethal within the first years of life without treatment. The GALC enzyme is responsible for catabolism of galactosphingolipids, particularly galactosylceramide and psychosine. Psychosine is cytotoxic and its accumulation leads to widespread pathology, particularly demyelination. KD is studied through animal models, particularly the Twitcher (TWI, GALC -/-) mouse model that features a nonsense mutation leading to no functional GALC enzyme. A previous study from this lab treated TWI with an adeno-associated virus (AAV) with a GALC transgene (TWI-AAV). This therapy increased survival, reduced signs of disease, increased GALC activity to supraphysiologic levels, and reduced pathology. However, the protective effect of the AAV waned over time such that GALC activity decreased and psychosine increased. Chapter II examines the aged TWI-AAV as compared to WT, untreated TWI, and younger TWI-AAV. The aged TWI-AAV have multi-focal demyelination, specifically in the brain, as compared to the widespread demyelination of untreated TWI. The demyelinated lesions mostly resembled untreated TWI with no GALC mRNA, no GALC enzyme, and microgliosis. However, the demyelinated lesions also had novel pathology, previously not documented in untreated TWI, including blood brain barrier disruptions and fibrinogen extravasation. This pathology has however been documented in Multiple Sclerosis (MS), for which GALC is a known risk factor. Furthermore, certain proteins related to MS pathology were also elevated in the aged TWI-AAV brain. Chapter III investigates the episome dilution hypothesis as the cause for why the AAV treatment efficacy declined over time. Upon transducing cells, the DNA within AAV is known to primarily exist as non-replicating extrachromosomal episomes. The demyelinated lesions of aged TWI-AAV were shown to have actively replicating oligodendrocyte precursor cells (OPC). Furthermore, the OPCs specifically within the lesions did not express GALC in contrast to OPCs in other regions. In vitro and in vivo experiments provided evidence in support of the episome dilution hypothesis. AAV DNA and GALC transgene were shown to decrease over time in TWI neuroglial cells, inversely proportional to total cell count. GFP fluorescence, from AAV encoded GFP, diminished over time in WT mouse brains. Together, this data shows it is highly likely the AAV treatment efficacy declined over time due to episomal dilution and eventual exhaustion. Chapter IV explores the role of GALC in adulthood, particularly its role in myelination, through the tamoxifen (TMX) inducible GALC knockout GFPC model. Adult GALC loss within oligodendrocytes (OG) lead to mild to moderate dysmyelination and locomotor impairment, particularly in complex tasks requiring balance and coordination. The mice were then challenged with an additional demyelinating insult in the form of experimental autoimmune encephalitis (EAE), a model of MS, to examine if the GALC deficiency made them more vulnerable to secondary demyelination. The GALC deficiency and EAE were additive in worsening signs of disease, significantly decreasing median survival, associated demyelination, gliosis, microgliosis, decreasing GALC activity, and increasing psychosine. GALC deletion and EAE demonstrated an additive effect showing that GALC loss sensitizes myelin to further demyelinating insults. Lastly, Chapter V summarizes the findings of this thesis. The main findings of this thesis being that episomal AAV DNA is lost through continuous cellular replication as defined in the episomal dilution hypothesis and GALC is required for myelin maintenance throughout life. The gradual dilution of episomal AAV DNA leads to focal loss of therapeutic episome with reversion to an untreated state over time in regions with suspected higher basal rates of cellular replication. GALC being required for myelin maintenance throughout life is a novel finding, this highlights how a successful therapy for KD will need to continuously supply GALC throughout life for it to be successful. Lastly, the conclusion discusses the limitations of the experiments in this thesis and ideas for future experimentation to address unanswered questions.