Farber disease is a fatal inherited disorder of lipid metabolism. It is characterized by a deficiency of the lysosomal enzyme acid ceramidase (AC), resulting in the buildup of the bio-effector molecule ceramide. A previous attempt to generate a Farber mouse model by Asah1 knockout was unsuccessful owing to embryonic lethality in homozygous embryos. Here, we successfully generated viable homozygotes that developed Farber disease by introducing a single-nucleotide human patient mutation into a conserved region of murine Asah1 (P361R). Indeed, homozygotes manifested Farber disease symptoms and died within 7-13 weeks. Furthermore, treatment of neonatal pups with intravenous injections of AC lentiviral vectors (LVs) yielded homozygotes with intermediate Farber phenotypes highlighted by reduced symptoms and increased longevity. Therefore, this first model of Farber disease can facilitate the advancement of experimental therapies and offer mechanistic insights into the integral roles of acid ceramidase, ceramide, and related sphingolipids in cell signaling, growth, and oncogenesis.

Farber disease is a fatal inherited disorder of lipid metabolism. It is characterized by a deficiency of the lysosomal enzyme acid ceramidase (AC), resulting in the buildup of the bio-effector molecule ceramide. A previous attempt to generate a Farber mouse model by Asah1 knockout was unsuccessful owing to embryonic lethality in homozygous embryos. Here, we successfully generated viable homozygotes that developed Farber disease by introducing a single-nucleotide human patient mutation into a conserved region of murine Asah1 (P361R). Indeed, homozygotes manifested Farber disease symptoms and died within 7-13 weeks. Furthermore, treatment of neonatal pups with intravenous injections of AC lentiviral vectors (LVs) yielded homozygotes with intermediate Farber phenotypes highlighted by reduced symptoms and increased longevity. Therefore, this first model of Farber disease can facilitate the advancement of experimental therapies and offer mechanistic insights into the integral roles of acid ceramidase, ceramide, and related sphingolipids in cell signaling, growth, and oncogenesis.

Sphingolipids modulate multiple cellular functions, including steroid hormone biosynthesis. Sphingosine is an antagonist ligand for the nuclear receptor steroidogenic factor 1 (SF-1), which is the primary transcriptional regulator of most steroidogenic genes. Furthermore, sphingosine-dependent repression of SF-1 function is dependent on the expression of acid ceramidase (ASAH1), an enzyme that forms sphingosine. Based on these data, I hypothesized that ACTH/cAMP signaling regulates ASAH1 function at both transcriptional and post-transcriptional levels. In addition, because SF-1 is predominantly a nuclear protein, I postulated that ASAH1 modulates SF-1 function and, therefore, steroidogenic gene expression by controlling the nuclear concentrations of SPH. To test these hypotheses, I first examined the effect of chronic ACTH/cAMP signaling on the transcription of the ASAH1 gene. Next, the functional significance of ASAH1 expression in adrenocortical cells was probed by generating an ASAH1-knockdown cell line. I subsequently characterized the role of ASAH1 as a transcriptional nuclear receptor coregulator. Finally, I defined the role of sphingosine-1-phosphate, a bi-product of ASAH1 activity, in the acute phase of cortisol biosynthesis. Using a variety of experimental approaches, I identified cAMP response element binding protein as an essential transcriptional activator of the ASAH1 gene. Analysis of adrenocortical cells lacking ASAH1 revealed that ASAH1 is a global regulator of steroidogenic capacity. Furthermore, I identified ASAH1 as a nuclear protein and defined the molecular determinants of the interaction between ASAH1 and SF-1. Collectively, this body of work establishes the integral role of ASAH1 in the regulation of ACTH-dependent adrenocortical cortisol biosynthesis.

Spinal Muscular Atrophy with Progressive Myoclonic Epilepsy (SMA-PME), Farber Lipogranulomatosis (FL), and a rare variant form of Neonatal Progeroid Syndrome (NPS) are three monogenetic rare disorders caused by pathogenic variation in genes encoding lipid modifying proteins. FL and SMA-PME are caused by loss of function mutations in ASAH1, encoding the acid ceramidase (aCDase) enzyme. It is not, however, known how aCDase deficiency can produce either the isolated neurological symptoms of SMA-PME or the predominantly systemic symptoms of FL. Further, a recently identified variant form of NPS has been attributed to variants in ANO6, encoding a dual function calcium-activated chloride channel and glycerophosphoserine (GPS) scramblase. Here, it is not known how ANO6 mutation causes the premature aging phenotype that defines NPS. To address these questions, I sought to elucidate pathogenic changes in lipid metabolism that associate clinical phenotype. I show here that the different patient mutations in ANO6 cause a non-physiological gain of channel function and either a loss or gain of scramblase function depending on the variant expressed. Both variants, however, alter GPS metabolic homeostasis suggesting a common mechanism of action. To provide in vivo insight, I characterized a novel mouse model based on our NPS patient genetics, showing extremely low penetrance of disease symptoms in terms of live births yet confirming that affected animals show impaired GPS metabolism in affected organs. Next, I characterized the clinical presentation of six new patients with SMA-PME and identified distinct sphingolipid metabolic fingerprints in FL and SMA-PME cells. I show that FL is defined by a hypometabolic sphingolipid phenotype with cellular and molecular features of a classic lysosomal storage disorder. By contrast, SMA-PME has a hypermetabolic sphingolipid phenotype with features of non-classic lysosomal trafficking disorders. To provide clinical insight, I assessed the potential of enzyme replacement therapy, demonstrating a rescue of sphingolipid metabolism in SMA-PME patient cells. Together, this thesis identified changes in the cellular and tissue lipid profiles of patients with ANO6-NPS, SMA-PME, or FL, elucidating some of the lipid-centric pathomechanisms of these diseases.