Biology subjects

Kegel-Gleason, K. B.

Publications and source records attributed to Kegel-Gleason, K. B..

3 recordsLinked to original sources

bioRxiv2026

WWOX contributes to DNA damage, but not somatic instability in Huntington s disease

Huntingtons disease (HD), caused by a CAG repeat expansion in the huntingtin (HTT) gene, is characterized by progressive neurodegeneration and accumulation of DNA damage with multiple disease-modifier genes involved in DNA repair pathways. Previous studies have implicated ataxia telangiectasia mutated (ATM) signaling in the regulation of genomic stability and DNA damage repair (DDR) pathways in HD. ATM has also been linked to the WW domain-containing oxidoreductase (WWOX), a protein involved in DNA repair and maintenance of genomic stability, through the E3 ubiquitin ligase ITCH. However, whether this signaling pathway contributes to HD pathogenesis remains unknown. Here, we investigated the role of ATM-ITCH-WWOX signaling in HD. Our results revealed no significant alterations in total ATM, phosphorylated ATM (pATM-S1981), or ITCH in HD post-mortem prefrontal cortex (PFC) compared to controls. Although treatment of human neuroblastoma SH-SY5Y cells with HD PFC lysates did not alter pATM-S1981 levels, it increased histone H2AX phosphorylation at S139 ({gamma}-H2AX), a marker of DNA double-strand breaks. This finding suggested the presence of persistent DNA damage signaling independent of canonical ATM activation. Conversely, WWOX levels were increased in both HD PFC and HD embryonic stem cell-derived cortical neurons. Additionally, treatment of SH-SY5Y cells with recombinant human WWOX protein or WWOX overexpression increased {gamma}-H2AX levels, supporting a role for WWOX in promoting DNA damage. To determine whether WWOX contributed to DNA damage in HD, SH-SY5Y cells were treated with HD PFC lysates that were depleted of WWOX. Immuno-depletion of WWOX reduced the ability of HD PFC lysates to increase {gamma}-H2AX, suggesting that WWOX contributes to DNA damage in HD. Finally, overexpression of WWOX in RPE1-AAVS1-CAG115 cells did not affect somatic CAG repeat instability, despite persistent increases in {gamma}-H2AX levels. Collectively, our findings identify WWOX as a contributor to DNA damage in HD, acting independently of the ATM pathway.

neuroscience↗

bioRxiv2025

Intrastriatal delivery of a zinc finger protein targeting the mutant HTT gene obviates lipid phenotypes in zQ175DN HD mice

Reducing the burden of mutant Huntingtin (mHTT) protein in brain cells is a strategy for treating Huntingtons disease (HD). However, it is still unclear what pathological changes can be reproducibly reversed by mHTT lowering and whether these changes can be measured in peripheral biofluids. We previously found that lipid changes that occur in brain with HD progression could be prevented by attenuating HTT transcription of the mutant allele in a genetic mouse model (LacQ140) with inducible whole body lowering. Here, we tested whether intrastriatal injection of a therapeutic capable of repressing the mutant HTT allele with expanded CAG can provide similar protection against lipid changes in HD mice with a deletion of neo cassette (zQ175DN). Wild-type or zQ175DN mice were injected with AAV9 bearing a cDNA for a zinc finger protein (ZFP) which preferentially targets mutant HTT (ZFP-HTT) to repress transcription (1). Proteins from brain tissues were analyzed using western blot, capillary electrophoresis, and nitrocellulose filtration methods. Lipid analyses of brain tissue and plasma collected from the same mice were conducted by liquid chromatography and mass spectrometry (LC-MS). Somatic instability (SI) index was assessed using capillary gel electrophoresis of PCR products and was shown to be impeded by HTT-ZFP. Lowering mHTT levels by 43% for 4 months prevented loss of total lipid content including subclasses sphingomyelin (SM), ceramide, phosphatidylethanolamine (PE) and others of caudate-putamen in zQ175DN mice. Moreover, LC-MS analysis of plasma demonstrated total lipid increases and lipid changes in monogalactosyl monoacylglyceride (MGMG) and certain phosphatidylcholine (PC) species were reversed with the therapy. In summary, our data demonstrate that analyzing lipid signatures of brain tissue and peripheral biofluids are valuable approaches for evaluating potential therapies in a preclinical model of HD. FundingCHDI Foundation, Dake Family Fund Disclosure statementThe authors have nothing to disclose. Author contributionsAI and KS extracted lipids and performed computational and statistical analysis; AB and CS collected plasma and brain tissues; SL and ES performed protein chemistry; KC maintained mouse colonies, RM performed stereotaxic injections, RB subcloned ZFP cDNAs and prepared virus, MSE, NA, MD, and KKG planned experiments and wrote manuscript.

neuroscience↗

bioRxiv2023

Early whole-body mutant huntingtin lowering preserves proteins and lipids important for synapse function and white matter maintenance in the LacQ140 mouse model

BackgroundExpansion of a triplet repeat tract in exon1 of the HTT gene causes Huntingtons disease (HD). The mutant HTT protein (mHTT) has numerous aberrant interactions with diverse, pleiomorphic effects. No disease modifying treatments exist but lowering mutant huntingtin (mHTT) by gene therapy is a promising approach to treat Huntingtons disease (HD). It is not clear when lowering should be initiated, how much lowering is necessary and for what duration lowering should occur to achieve benefits. Furthermore, the effects of mHTT lowering on brain lipids have not been assessed. MethodsUsing a mHtt-inducible mouse model we analyzed whole body mHtt lowering initiated at different ages and sustained for different time-periods. Subcellular fractionation (density gradient ultracentrifugation), protein chemistry (gel filtration, western blot, and capillary electrophoresis immunoassay), liquid chromatography and mass spectrometry of lipids, and bioinformatic approaches were used to test effects of mHTT transcriptional lowering. ResultsmHTT protein in cytoplasmic and synaptic compartments of the caudate putamen, which is most affected in HD, was reduced 38-52%. Little or no lowering of mHTT occurred in nuclear and perinuclear regions where aggregates formed at 12 months of age. mHtt transcript repression partially or fully preserved select striatal proteins (SCN4B, PDE10A). Total lipids in striatum were reduced in LacQ140 mice at 9 months and preserved by early partial mHtt lowering. The reduction in total lipids was due in part to reductions in subclasses of ceramide (Cer), sphingomyelin (SM), and monogalactosyldiacylglycerol (MGDG), which are known to be important for white matter structure and function. Lipid subclasses phosphatidylinositol (PI), phosphatidylserine (PS), and bismethyl phosphatidic acid (BisMePA) were also changed in LacQ140 mice. Levels of all subclasses other than ceramide were preserved by early mHtt lowering. Pathway enrichment analysis of RNAseq data imply a transcriptional mechanism is responsible in part for changes in myelin lipids, and some but not all changes can be rescued by mHTT lowering. ConclusionsOur findings suggest that early and sustained reduction in mHtt can prevent changes in levels of select striatal proteins and most lipids but a misfolded, degradation-resistant form of mHTT hampers some benefits in the long term.

neuroscience↗