A haploinsufficiency restoration strategy corrects neurobehavioral deficits in Nf1+/– mice
Su Jung Park, … , Steven P. Angus, D. Wade Clapp
Su Jung Park, … , Steven P. Angus, D. Wade Clapp
Published July 1, 2025
Citation Information: J Clin Invest. 2025;135(13):e188932. https://doi.org/10.1172/JCI188932.
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Research Article Genetics Neuroscience

A haploinsufficiency restoration strategy corrects neurobehavioral deficits in Nf1+/– mice

Abstract

Neurofibromatosis type 1 (NF1) is a genetic disorder caused by mutations of the NF1 tumor suppressor gene resulting in the loss of function of neurofibromin, a GTPase-activating protein (GAP) for Ras. While the malignant manifestations of NF1 are associated with loss of heterozygosity of the residual WT allele, the nonmalignant neurodevelopmental sequelae, including autism spectrum disorder (ASD) and/or attention deficit hyperactivity disorder (ADHD) are prevalent morbidities that occur in the setting of neurofibromin haploinsufficiency. We reasoned that augmenting endogenous levels of WT neurofibromin could serve as a potential therapeutic strategy to correct the neurodevelopmental manifestations of NF1. Here, we used a combination of genetic screening and genetically engineered murine models to identify a role for the F-box protein FBXW11 as a regulator of neurofibromin degradation. Disruption of Fbxw11, through germline mutation or targeted genetic manipulation in the nucleus accumbens, increased neurofibromin levels, suppressed Ras-dependent ERK phosphorylation, and corrected social learning deficits and impulsive behaviors in male Nf1+/– mice. Our results demonstrate that preventing the degradation of neurofibromin is a feasible and effective approach to ameliorate the neurodevelopmental phenotypes in a haploinsufficient disease model.

Authors

Su Jung Park, Jodi L. Lukkes, Ka-Kui Chan, Hayley P. Drozd, Callie B. Burgin, Shaomin Qian, Morgan McKenzie Sullivan, Cesar Gabriel Guevara, Nolen Cunningham, Stephanie Arenas, Makenna A. Collins, Jacob Zucker, JinHee Won, Abbi Smith, Li Jiang, Dana K. Mitchell, Steven D. Rhodes, Steven P. Angus, D. Wade Clapp

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Figure 3

FBXW11 preferentially interacts with and polyubiquitinates the GRD of neurofibromin isoform 1.

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FBXW11 preferentially interacts with and polyubiquitinates the GRD of ne...
(A) Schematic map of the neurofibromin (NF1) interaction domains used to test interactions with FBXW11. Full-length NF1 was divided into the 6 indicated domains and subcloned into GFP-expressing plasmids. Flag-tagged FBXW11 and the various GFP-tagged NF1 subdomains were transfected into HEK293T cells. Six hours prior to harvesting, the cells were treated with 15 μM MG-132. Anti-Flag IP followed by immunoblotting was used to detect GFP-tagged NF1 co-IP with FBXW11. Relative binding activity as determined by immunoblotting is indicated. Image created in BioRender. Angus, S. (2025) https://BioRender.com/n28p603 (B) HEK293T cells were treated as in A to confirm the specific interaction of FBXW11 with the domain 3 (D3) fragment of the NF1 peptide, which encompasses the GRD of NF1 isoform 1 (GRD1), but not GRD2. (C) A NanoBiT complementation assay was performed after cotransfection of HEK293T cells with LgBiT-FBXW11 and SmBiT-GRD1 or GRD2 fusion proteins. Forty-five hours after transfection, the interaction was detected by luminescence. The negative control (neg. cont.) refers to luminescence values obtained from wells containing cells with LgbiT-FBXW11 and SmBiT-empty. Vehicle or the proteasome inhibitor bortezomib (1 mM) was included to enhance the interaction due to GRD1 accumulation. *P < 0.05 and ***P < 0.001, by unpaired 2-tailed t test. Data indicate the mean ± SEM. Image created in BioRender. Angus, S. (2025) https://BioRender.com/e61l197 (D) The SCF complex (SKP1, CUL1, RBX1, and Flag-FBXW11) was purified after cotransfection and subsequent Flag-IP (and Flag-peptide elution) from HEK293T cell lysates. Isolated GFP-GRD1 was incubated at 37°C for 60 minutes in the presence of E1 (100 nM), E2 (2 mM), Mg-ATP (5 mM), and ubiquitin (Ub) in the presence (+) or absence (–) of SCFFBXW11. Samples were then subjected to immunoblotting, and GRD1 ubiquitination was detected by GFP antibody and the smearing due to higher-molecular-weight species. (E) HEK293T cells were transfected with control or FBXW11-targeting siRNAs for 72 hours. A TUBEs assay was performed using agarose-TUBE2 to pull down total ubiquitinated protein. Immunoblotting was used to detect ubiquitin and NF1.