The neuronal ceroid lipofuscinoses (NCLs) are the most common type of inherited pediatric neurodegenerative diseases. They are a group of heterogenous disorders with a progressive clinical course that includes seizures, blindness, dementia, brain atrophy and death. CLN6 is an NCL disorder due to mutations in the CLN6 gene that encodes a resident endoplasmic reticulum (ER) transmembrane protein (CLN6p). CLN6p?s function is poorly understood; however its dysfunction leads to the intracellular accumulation of storage material whose prominent component is the Subunit C (SUBC) of the mitochondrial ATP synthase protein. Storage material is thought to be due to disruption of the autophagic-lysosomal system, but the pathologic mechanism has remained unclear. Currently, CLN6 has no cure and therapeutic intervention is limited by inadequate knowledge of its mechanism. Animal models of CLN6 recapitulate the human disease with progressive neurological decline and deposition of SUBC+ storage material within the brain. Investigations with primary neural cultures from mice and sheep have provided some insight into the disorder, with common changes including SUBC+ accumulation, decreased acidification of lysosomes, and autophagic dysfunction. Unfortunately, primary neurons can vary from one preparation to another, as well as being difficult to manipulate genetically. Furthermore, although primary animal neurons can be similar to human neurons, they still may not adequately recapitulate human genetic disease. Currently, there are no renewable sources of human cell models available to investigate human CLN6 pathology. Non-neuronal patient-derived cells (e.g. fibroblasts) do not accumulate significant storage material. Therefore, we generated patient-derived induced pluripotent stem cells (IPSCs) from CLN6-affected subjects, that exhibited significant SUBC storage in mixed neural cultures after conventional differentiation protocols. Unfortunately, the mixed-type populations present in these cultures (e.g. progenitors, neurons) and the length of time required to generate cells with storage has limited these methods? ability to be applied effectively. Therefore, we performed preliminary work using an inducible neurogenin-2 expression (I3N) system. This I3N system was engineered into CLN6- and CTL-IPSC lines each and produced homogeneous and synchronous cortical neuron cultures within days of doxycycline induction. I3N-CLN6-neurons had increased SUBC+ storage material, confirming the system?s value for modeling CLN6. Our current project seeks to increase the number of CLN6- and CTL-I3N- IPSC lines, integrate additional inducible-trangenes for regulated expression of the CLN6p, and investigate how CLN6p dysfunction affects the morphology and localization of organelles along the ER to lysosomal-autophagic systems. Activation of ER stress and lysosomal stress markers will also be evaluated to determine potential targets for future activation or depletion. These preliminary studies will evaluate how CLN6 affects organellar function including ER stress, lysosomal stress, and autophagic function. This work seeks to establish adequate and renewable models for mechanistic studies and the potential identification of therapeutic targets.
The most common inherited pediatric neurodegenerative disorders are the neuronal ceroid lipofuscinoses (CLN1-CLN14). Late infantile-onset CLN6 is a rare autosomal recessive disorder that causes progressive visual failure, seizures, and dementia; similar to other CLN disorders, CLN6 has no treatment and causes premature death. We have generated induced pluripotent stem cells (IPSCs) from several CLN6 patients to generate neural progenitor cells and neurons to determine the mechanisms underlying dysfunction, and to be a potential platform for therapeutic screening.