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Understanding human lysosomal-autophagic dysfunction in CLN6 with IPSC-derived neural cells

Award Number: R21HD099551
ORGANIZATION: NATIONAL INSTITUTE OF CHILD HEALTH AND HUMAN DEVELOPMENT
OPDIV: NIH
AWARD CLASS: DISCRETIONARY
AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)
PERIOD OF PERFORMANCE START DATE: 03/01/2020
PERIOD OF PERFORMANCE END DATE: 02/28/2022

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Understanding human lysosomal-autophagic dysfunction in CLN6 with IPSC-derived neural cells - ABSTRACT 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.


Issue Date FY	Funding FY	Legal Entity Name	Legal Entity Address	Legal Entity City	Legal Entity State	Legal Entity Zip Code	Legal Entity COUNTY	Legal Entity COUNTRY	Assistance Listing	Award Code	Budget Year	Action Date	Action Type	Action Amount

Issue Date FY: 2023 ( Subtotal = -$4 )
2023	2021	CEDARS-SINAI MEDICAL CENTER	8700 BEVERLY BLVD	WEST HOLLYWOOD	CA	90048	LOS ANGELES	USA	Child Health and Human Development Extramural Research	000	2	5/15/2023	NON-COMPETING CONTINUATION	-$4
														Subtotal = -$4

Issue Date FY: 2022 ( Subtotal = -$3,254 )
2022	2021	CEDARS-SINAI MEDICAL CENTER	8700 BEVERLY BLVD	WEST HOLLYWOOD	CA	90048	LOS ANGELES	USA	Child Health and Human Development Extramural Research	000	2	9/23/2022	NON-COMPETING CONTINUATION	-$3,254
														Subtotal = -$3,254

Issue Date FY: 2021 ( Subtotal = $208,750 )
2021	2021	CEDARS-SINAI MEDICAL CENTER	8700 BEVERLY BLVD	LOS ANGELES	CA	90048	LOS ANGELES	USA	Child Health and Human Development Extramural Research	000	2	2/11/2021	NON-COMPETING CONTINUATION	$208,750
														Subtotal = $208,750

Issue Date FY: 2020 ( Subtotal = $250,500 )
2020	2020	CEDARS-SINAI MEDICAL CENTER	8700 BEVERLY BLVD	LOS ANGELES	CA	90048	LOS ANGELES	USA	Child Health and Human Development Extramural Research	000	1	2/17/2020	NEW	$250,500
														Subtotal = $250,500

Grand Total All Awards = $455,992

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Understanding human lysosomal-autophagic dysfunction in CLN6 with IPSC-derived neural cells

Award Number: R21HD099551

ORGANIZATION: NATIONAL INSTITUTE OF CHILD HEALTH AND HUMAN DEVELOPMENT

OPDIV: NIH

AWARD CLASS: DISCRETIONARY

AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)

PERIOD OF PERFORMANCE START DATE: 03/01/2020

PERIOD OF PERFORMANCE END DATE: 02/28/2022

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