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Mechanisms of A-I RNA editing-mediated nuclear export of TDP-43

Award Number: R21NS130492
ORGANIZATION: NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE
OPDIV: NIH
AWARD CLASS: DISCRETIONARY
AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)
PERIOD OF PERFORMANCE START DATE: 09/22/2022
PERIOD OF PERFORMANCE END DATE: 08/31/2025

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Mechanisms of A-I RNA editing-mediated nuclear export of TDP-43 - TAR DNA binding protein – 43 (TDP-43) is a critical RNA binding protein that is intimately involved in many aspects of RNA metabolism. While primarily localized to the nucleus, TDP-43 shuttles between the nucleus and the cytoplasm performing its physiological functions. As an aggregation prone protein, TDP-43 is known to accumulate and from prion-like solid aggregates in the cytoplasm of cells leading to the sequestration of nuclear TDP-43. This behavior of TDP-43 has been well established as a pathological hallmark of a neurodegenerative disease spectrum encompassing amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD) and has been described in Alzheimer’s disease and related dementias. Pathological cytoplasmic TDP-43 inclusions have been hypothesized to contribute to disease pathogenesis through both a nuclear depletion and the cytoplasmic aggregation. Despite extensive research, mechanisms that initiate this pathology under disease conditions remain elusive. Recent studies in our laboratory showed that aberrant RNA A-I editing is present in multiple brain regions of C9orf72 ALS/FTD, where we detected bidirectional changes in A-I editing. Since then, we have generated preliminary data suggesting that TDP-43 nuclear export can be regulated via Adenosine Deaminase Acting on double stranded RNA (ADAR)-mediated A-I RNA editing. We show that enhancing RNA A-I editing through ADAR2 overexpression in mammalian cell lines induces TDP-43 translocation to the cytoplasm requiring functional RNA binding domains of TDP-43. In contrast, the overexpression of catalytically inactive ADAR2 does not alter the nuclear localization of TDP-43. These findings led us to hypothesize that aberrant increases in A-I editing induces TDP-43 cytoplasmic mislocalization through an RNA dependent mechanism. To determine if this editing induced TDP-43 nuclear export also occurs in a neuronal environment, we will expand on our preliminary data and examine human induced pluripotent stem cell (iPSC) differentiated into motor neurons for A-I editing-mediated TDP-43 nuclear export. We will validate A-I RNA editing mediated cytoplasmic accumulation of TDP-43 in iPSC-MNs expressing doxycycline inducible Tet-On ADAR2 constructs: wildtype ADAR2, a catalytically inactive ADAR2 (ADAR2E396A) and a catalytically hyperactive ADAR2 (ADAR2E488Q). To address the effects of RNA-editing induced TDP-43 mislocalization on TDP-43 function, we will examine TDP-43 inclusions for disease-relevant characteristics (Aim1). To determine the identity of mRNAs bound to TDP-43 and potentially being necessary for A-I RNA editing-mediated mislocalization, we will perform eCLIP-seq on iPSC-MNs genetically altered for hypo and hyper-editing as described in Aim1. In addition, we will perform eCLIP in C9orf72 iPSC-MNs to compare RNA-editing induced TDP-43 bound transcripts to those associated with endogenous disease (Aim 2). Finally, in Aim 3, we will perform exploratory studies towards the identification of molecular and cellular mechanisms involved in this new pathway of TDP-43 nuclear export using stably transduced SH-SY5Y cells expressing the doxycycline-induced Tet-On ADAR2 constructs described above.


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: 2026 ( Subtotal = $0 )
2026	2022	DIGNITY HEALTH	185 BERRY ST STE 200	SAN FRANCISCO	CA	94107	SAN FRANCISCO	USA	Extramural Research Programs in the Neurosciences and Neurological Disorders	000	1	4/22/2026	NEW	$0
														Subtotal = $0

Issue Date FY: 2024 ( Subtotal = $0 )
2024	2022	DIGNITY HEALTH	350 W THOMAS RD	PHOENIX	AZ	85013	MARICOPA	USA	Extramural Research Programs in the Neurosciences and Neurological Disorders	000	1	9/12/2024	NEW	$0
														Subtotal = $0

Issue Date FY: 2022 ( Subtotal = $345,566 )
2022	2022	DIGNITY HEALTH	185 BERRY ST STE 300	SAN FRANCISCO	CA	94107	SAN FRANCISCO	USA	Extramural Research Programs in the Neurosciences and Neurological Disorders	000	1	9/19/2022	NEW	$345,566
														Subtotal = $345,566

Grand Total All Awards = $345,566

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Mechanisms of A-I RNA editing-mediated nuclear export of TDP-43

Award Number: R21NS130492

ORGANIZATION: NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE

OPDIV: NIH

AWARD CLASS: DISCRETIONARY

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

PERIOD OF PERFORMANCE START DATE: 09/22/2022

PERIOD OF PERFORMANCE END DATE: 08/31/2025

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