Date of Award
Doctor of Philosophy (PhD)
Infantile neuronal ceroid lipofuscinosis (INCL, Infantile Batten) is typically an early onset, neurodegenerative lysosomal storage disorder. INCL is caused by mutations to the gene CLN1 which codes for the lysosomal enzyme palmitoyl-protein thioesterase-1 (PPT1). PPT1 is a soluble lysosomal enzyme that functions to cleave fatty acyl chains from proteins destined for degradation. Deficiency in PPT1 leads to the accumulation of autofluorescent storage material, a hallmark of the NCLs. The storage material has been implicated in progressive histopathological changes in the brain such as neuronal loss, astrocytosis, microgliosis, and immune cell infiltration. These histopathological changes result in a progression of clinical signs including vision loss, decline in motor function, cognitive deficits, seizures, and premature death. Currently, there are no cures or treatments for INCL. However, a murine model of INCL has been used in pre-clinical therapy studies. The PPT1-/- mouse has been shown to be a reliable model for the human INCL disease. Detailed temporal and spatial histopathological examinations of murine INCL in the brain have led to intracranial gene therapy studies. These pre-clinical studies have resulted in significant improvements in biochemical, histopathological, and functional deficits seen in the untreated PPT1-/- mouse. However, there have only been modest improvements in lifespan. Given the identification and development of improved gene therapy vectors, this was a surprising finding. Therefore, the first section of the dissertation, we pursued a more thorough characterization of the central nervous system to identify potential regions of disease not targeted by intracranial gene therapy. We identified the spinal cord as a significant site of disease that was not previously characterized or corrected. This allowed us to target both the brain and spinal cord with AAV-based gene therapy. We demonstrated that targeting the entirety of the central nervous system was necessary to treat INCL more effectively.
From these and historical studies, we identified a multitude of cell types that are involved with INCL pathogenesis. In the central nervous system, INCL has been shown to progress sequentially from astrocytosis, to neuronal loss, to microgliosis and immune cell infiltration. PPT1 is ubiquitously expressed; therefore, its deficiency in INCL could lead to pathology in every cell type. Currently, we are unable to model cellular and metabolic changes in specific cell types in INCL due to ‘cross-correction’. While ‘cross-correction’ is beneficial for the development of therapeutics, it interferes with our ability to understand the role of PPT1 in specific cell types. Therefore, in the second section of the dissertation, we sought to determine the cell-autonomous nature of PPT1. Because PPT1 is a soluble lysosomal hydrolase that can undergo ‘cross-correction’, we developed a chimeric enzyme whereby PPT1 is tethered to the lysosomal membrane. We demonstrated that tethered PPT1 retains its enzymatic function and does not ‘cross-correct’ in vitro and in vivo. We further demonstrated that near-ubiquitous expression of tethered PPT1 could prevent INCL. This lays the groundwork for future studies designed to determine the role of specific cell types in the pathogenesis of INCL.
Chair and Committee
Mark S. Sands
Daniel S. Ory, Steven J. Mennerick, David H. Gutmann, Jin-Moo Lee, John Cirrito
Shyng, Charles, "Infantile Batten Disease: Effective Therapy and Novel Model" (2016). Arts & Sciences Electronic Theses and Dissertations. 896.