Cellular Modeling of CLN6 with IPSC-derived Neurons and Glia

Otero, M. G.; Kim, J.; Kumar, Y.; Rajewski, A.; Nonis, F. D.; Santiskulvong, C.; Bannykh, S.; Oza, H.; Farooqi, H. M. U.; Babros, M.; Freeman, C.; Dupuis, L.; Andrews, S.; Mendoza, R.; Bresee, C.; Adams, D.; Tifft, C.; Toro, C.; Khanlou, N.; Gahl, W. A.; Salamon, N.; Pierson, T. M.

doi:10.1101/2024.01.29.577876

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bioRxiv · 10.1101/2024.01.29.577876

Cellular Modeling of CLN6 with IPSC-derived Neurons and Glia

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Abstract

Neuronal ceroid lipofuscinosis (NCL), type 6 (CLN6) is a neurodegenerative disorder associated with progressive neurodegeneration leading to dementia, seizures, and retinopathy. CLN6 encodes a resident-ER protein involved in trafficking lysosomal proteins to the Golgi. CLN6p deficiency results in lysosomal dysfunction and deposition of storage material comprised of Nile Red+ lipids/proteolipids that include subunit C of the mitochondrial ATP synthase (SUBC). White matter involvement has been recently noted in several CLN6 animal models and several CLN6 subjects had neuroimaging was consistent with leukodystrophy. CLN6 patient-derived induced pluripotent stem cells (IPSCs) were generated from several of these subjects. IPSCs were differentiated into oligodendroglia or neurons using well-established small-molecule protocols. A doxycycline-inducible transgenic system expressing neurogenin-2 (the I3N-system) was also used to generate clonal IPSC-lines (I3N-IPSCs) that could be rapidly differentiated into neurons (I3N-neurons). All CLN6 IPSC-derived neural cell lines developed significant storage material, CLN6-I3N-neuron lines revealed significant Nile Red+ and SUBC+ storage within three and seven days of neuronal induction, respectively. CLN6-I3N-neurons had decreased tripeptidyl peptidase-1 activity, increased Golgi area, along with increased LAMP1+ in cell bodies and neurites. SUBC+ signal co-localized with LAMP1+ signal. Bulk-transcriptomic evaluation of control- and CLN6-I3N-neurons identified >1300 differentially-expressed genes (DEGs) with Gene Ontogeny (GO) Enrichment and Canonical Pathway Analyses having significant changes in lysosomal, axonal, synaptic, and neuronal-apoptotic gene pathways. These findings indicate that CLN6-IPSCs and CLN6-I3N-IPSCs are appropriate cellular models for this disorder. These I3N-neuron models may be particularly valuable for developing therapeutic interventions with high-throughput drug screening assays and/or gene therapy.

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Otero, M. G., Kim, J., Kumar, Y., Rajewski, A., Nonis, F. D., Santiskulvong, C., Bannykh, S., Oza, H., Farooqi, H. M. U., Babros, M., Freeman, C., Dupuis, L., Andrews, S., Mendoza, R., Bresee, C., Adams, D., Tifft, C., Toro, C., Khanlou, N., Gahl, W. A., Salamon, N., Pierson, T. M.. 2024-01-30. Cellular Modeling of CLN6 with IPSC-derived Neurons and Glia. https://doi.org/10.1101/2024.01.29.577876

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