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.