Huntington's disease (HD) is a neurodegenerative disorder caused by CAG repeat expansion in the huntingtin (HTT) gene, with longer repeats linked to earlier onset. Somatic CAG expansion, particularly in the striatum, contributes to disease progression and is influenced by HTT biology and genetic modifiers. Modulating somatic expansion is emerging as a promising approach to slow or prevent HD, and mouse models have been crucial for preclinical testing of different therapeutic strategies. The BAC-CAG model, developed on the FVB strain, has been used to study somatic expansion of human expanded HTT. However, comparisons with other key HD mouse models have been limited by differences in genetic background, as many other models are on the C57BL/6 strain. The BAC-CAG model has now been developed on a C57BL/6 background. To determine whether the C57BL/6 BAC-CAG model can be used to study and modulate somatic expansion, we compared CAG expansion in mice on C57BL/6 or FVB backgrounds, with and without intraventricular divalent small interfering RNAs (siRNA) targeting HD modifiers MutS homolog 3 (MSH3) and HTT. Both strains exhibited robust, comparable somatic expansion over two months, which was blocked by MSH3-, but not HTT-, targeted siRNA. RNA sequencing identified gene expression differences primarily in pseudogenes, with no differences in endogenous Htt , human HTT , or mismatch repair genes. These results demonstrate that BAC-CAG mice on a C57BL/6 background exhibit somatic CAG expansion comparable to the validated FVB strain, providing a model to study and preclinically test therapies targeting somatic expansion in HD.

Human and mouse genetics have established mismatch repair (MMR) as a central mediator of somatic repeat expansion, a key pathogenic process in Huntington's disease (HD) and related disorders. How individual MMR components function within the intact mammalian brain and interact with broader cellular networks remains poorly understood. We screened more than 500 chemically stabilized siRNAs targeting 10 MMR genes and used interventional RNAi in the Q111 HD mouse model to systematically dissect MMR function in vivo. MSH3 and PMS1 emerged as the most dose-sensitive regulators of somatic expansion but displayed markedly different effects on proteome stability. Quantitative proteomics generated an in vivo atlas of MMR component abundance and cross-regulation in the mammalian CNS, uncovering extensive connectivity between DNA repair, transcriptional regulation, chromatin remodeling, and mitochondrial biology. Together, these findings establish a systems-level framework linking MMR biology to neuronal function and offer mechanistic insight into selective neuronal vulnerability in HD.

Huntington's disease (HD) is marked by progressive neuronal loss and atrophy of grey matter structures, particularly the caudate and putamen. Brain imaging studies reveal that the white matter starts to decay in the brain of individuals bearing the HD mutation many years before symptomatic onset. However, the mechanism by which the HD mutation causes white matter loss remains to be further defined. In this study, we examined white matter pathology and explored the underlying mechanism in the HDQ140 knock-in mouse model of HD. Western blot analysis of proteins localized at different layers of the myelin sheath showed that myelin-associated glycoprotein (MAG), which is localized at the innermost myelin layer, was decreased earlier than proteins localized at outer layers of the myelin. The loss of MAG occurred at fully myelinated axons and was progressive with age. In postmortem symptomatic human HD brains, the level of MAG as well as other myelin proteins was also decreased. In HD mouse brains, MAG labeling was reduced at fiber bundles but accumulated in perinuclear structures of cells that expressed breast carcinoma amplified sequence 1, a marker for new oligodendrocytes. While their abundance was normal, new oligodendrocytes in HD brains were impeded in acquiring the expression of MAG. Compared with those in wild-type mouse brains, oligodendrocytes in HD mouse brains had a reduced frequency of MAG-bearing small vesicles and an increased abundance and enlargement of MAG-containing perinuclear structures. Further studies suggest that the MAG-accumulating perinuclear structures were derived from the late endosomal lysosomal compartment. Our study suggests that white matter decay in HD brains involves an early progressive loss of MAG in myelin membranes.

Huntington's disease (HD) is a progressive neurodegenerative disorder with no approved therapies. Despite multiple clinical trials, huntingtin (HTT)-lowering strategies have yet to show meaningful clinical benefit. Both somatic expansion and toxic HTT species are key molecular drivers of HD, yet therapeutic strategies targeting these pathways have never been directly compared or evaluated in combination. Using therapeutic divalent siRNAs, we assessed the long-term impact of silencing MutS homolog 3 (MSH3), a critical regulator of somatic expansion, HTT, or both in Q111 HD mice (>110 CAGs), which develop robust expansion, mutant HTT inclusions, and transcriptional dysregulation by 12 months. Long-term MSH3 silencing blocked somatic expansion, reduced inclusions, and normalized gene expression. HTT silencing alone had a limited effect, whereas combined MSH3/HTT targeting synergistically eliminated inclusions and restored transcriptomic profiles. Parallel treatment in wild-type mice showed no toxicity, supporting the safety of long-term intervention. These findings position somatic expansion as a promising therapeutic target and demonstrate the potential of RNAi-based cosilencing of MSH3 and HTT as a disease-modifying strategy for HD.

Huntington's 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 (γ-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 γ-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 γ-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 γ-H2AX levels. Collectively, our findings identify WWOX as a contributor to DNA damage in HD, acting independently of the ATM pathway.

Background Huntington’s disease (HD) is a hereditary life-threatening disease marked by progressive neuronal loss and atrophy of grey matter structures, particularly the caudate putamen. Brain imaging studies have revealed that the degradation of the white matter occurs many years prior to symptomatic onset and neuronal loss, suggesting that the decay of brain white matter is an active contributor to the disease progression. However, the mechanisms by which the HD mutation triggers white matter loss is not well understood. Methods Western blot, immunohistochemistry, and electron microscopy were conducted to assess white matter pathology and explore the relevant mechanisms in CAG140 knock-in mice, which express the HD protein in the same way as patients suffering from HD and thus biologically replicate HD in human. Results Western blot analysis of proteins localized at different layers of the myelin coat revealed that the myelin-associated glycoprotein (MAG), which is localized at the innermost layer of the myelin coat and essential for maintaining the periaxonal space and the integrity of the myelin sheath, manifested as an early and progressive decline in HD mouse caudate putamen. The loss of MAG was detected at myelinated axons and in fiber bundles in HD mouse brains at an age when the abundance of myelinated axons was normal. Fluorescence immunohistochemical studies found that MAG labeling was concentrated in the soma of a subset of oligodendrocytes, which expressed breast carcinoma amplified sequence 1, a marker for new oligodendrocytes. While their abundance was normal, new oligodendrocytes in HD mouse caudate appeared to be impeded in acquiring the expression of MAG and in targeting MAG away from perinuclear punctate structures to processes, signs of impaired maturation. Compared with those in wildtype mouse brains, oligodendrocytes in HD mouse brains had a reduced abundance of small vesicles whereas an increased abundance of large punctate structures in the perinuclear region, implying defective generation of small vesicles transporting MAG from large punctate structures in the soma to processes. The MAG-containing perinuclear punctate structures were negative for proteins specifying trans-Golgi networks, early endosomes, or exosomes but had a minor portion labeled with lysosome-associated membrane protein 1, indicating that the structures where MAG accumulates in the soma are derived from the late endosomal lysosomal compartment. Conclusions Our study suggests that the decay of the brain white matter in Huntington’s disease involves a deficit in trafficking of myelin-associated glycoprotein, preventing its proper delivery from the soma of oligodendrocytes to myelin-forming processes. ### Competing Interest Statement The authors have declared no competing interest. Hereditary Disease Foundation, https://ror.org/02a3w7r50 The Lake Family Fund CHDI Foundation, https://ror.org/046eh8t80

HTT1a was identified in human and mouse Huntington's disease brain as the pathogenic exon 1 mRNA generated from aberrant splicing between exon 1 and 2 of HTT that contributes to aggregate formation and neuronal dysfunction. 1 Detection of the huntingtin exon 1 protein (HTT1a) has been accomplished with fluorescence-based reporter assays (Meso Scale Discovery, Homogeneous Time Resolved Fluorescence) and immunoprecipitation assays in Huntington's disease knock-in mice but direct detection in homogenates by gel electrophoresis and western blot assay has been lacking. Subcellular fractions prepared from mouse and human Huntington's disease brain were separated by gel electrophoresis and probed by western blot with neo-epitope monoclonal antibodies 1B12 and 11G2 directed to the C-terminal eight residues of HTT1a. In caudate putamen of an allelic series of 6 month old Huntington's disease knock-in mice (Q50, Q80, Q111, Q140 and Q175) HTT1a migration was inversely correlated with CAG repeat length and appeared as a SDS soluble high molecular mass smear in Q111, Q140 and Q175 mice but weakly in Q80 and not in WT mice or Q50 indicating a CAG repeat size threshold for detecting HTT1a. HTT1a immunoreactivity diminished if 1B12 and 11G2 antibodies were preincubated with an eight amino acid peptide containing the C-terminus of HTT1a but not with unrelated peptide sequence. Migration of HTT1a and its high molecular mass smear changed with age in caudate putamen of Q111, Q175 and YAC128 mice. Treating Q111 mice with siRNA to MSH3 , a modifier of CAG repeat expansion, significantly reduced levels of the high molecular mass smear indicating that the effects of curbing CAG repeat expansion were quantifiable. A prominent 56-60 kDa doublet detected by 1B12 and 11G2 antibodies in lysates from human Huntington's disease brain was not blocked by preincubation with C-terminal HTT1a blocking peptide and also appeared in brains of Parkinson's disease patients. 1B12 and 11G2 antibodies did not immunoprecipitate HTT proteins from either Huntington's disease mouse or human brain lysates using conditions that pulled down full length HTT with anti-HTT antibody 2B7. Altogether these data show that 11G2 and 1B12 antibodies can be used in western blot assays to track and quantify immunoreactive HTT1a levels, solubility, and subcellular localization in Huntington's disease mouse brain. Abbreviated Summary:Sapp et al., report that pathogenic exon 1 protein HTT1a is detected in brain of mouse models of Huntington's disease by direct western blot assay using monoclonal antibodies 11G2 and 1B12. Lowering MSH3 mRNA in the caudate putamen to prevent CAG repeat expansion reduced levels of HTT1a.

Reducing the burden of mutant Huntingtin (mHTT) protein in brain cells is a strategy for treating Huntington's 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 cytosine-adenine-guanine (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 adeno-associated virus 9 (AAV9) bearing a cDNA for a zinc finger protein (ZFP), which preferentially targets mutant HTT (ZFP-HTT) to repress transcription. 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 index was assessed using capillary gel electrophoresis of PCR products and was shown to be impeded by ZFP-HTT. Lowering mHTT levels by 43% for 4 months prevented loss of total lipid content including the subclasses sphingomyelin, ceramide, phosphatidylethanolamine and others of caudate-putamen in zQ175DN mice. Moreover, LC-MS analysis of plasma demonstrated total lipid increases and lipid changes in monogalactosyl monoacylglyceride and certain phosphatidylcholine 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.

BackgroundRecent evidence suggests that accumulation of mutant exon 1 protein (HTT1a) may be critical to HD pathogenesis, but the relation of this to differential regional and cellular vulnerability in HD is unknown.ObjectiveWe assessed the contribution of the accumulation of the mutant huntingtin HTT1a to the regional and cellular variation in HD brain pathology by determining if more vulnerable regions and neuron types were relatively enriched.MethodsWe performed immunolabeling using the novel monoclonal antibodies 11G2 and 1B12 against the C-terminal proline 90 (P90) neoepitope of huntingtin HTT1a, which detect accumulation of monomeric, oligomeric and aggregated mutant HTT1a, on forebrain of Q175 and R6/2 mice and human HD cases.ResultsDiffuse nuclear and aggregate immunolabeling increased in abundance in Q175 with age, with striatal projection neurons showing immunolabeling earlier than cortical neurons, and only neuropil immunolabeling prominent in pallidal regions. Nonetheless, some regions less affected in HD, such as hippocampus, were rich in mutant HTT1a as well. In humans, striatal immunolabeling was sparser than in mouse, and mainly in the neuropil, but sparser in striatal target areas. In human HD cortex, the P90 antibodies detected predominantly neuropil aggregates, which appeared to, in part, localize to dendrites. Immunostaining in mouse and human could be blocked with HTT1a target peptide, demonstrating antibody specificity.ConclusionsOur results indicate that mutant HTT1a burden appears to partly account for overall differential forebrain regional vulnerability in HD, but additional factors may contribute to vulnerability differences among forebrain regions and between specific neuron types.

Huntington’s disease (HD) is a progressive neurodegenerative disorder with no approved therapies. Two major molecular drivers—somatic expansion of inherited CAG repeats and toxic mutant HTT (mHTT) variants—lead to neuronal dysfunction. Despite multiple trials, HTT-lowering strategies have not shown meaningful clinical benefit. Using therapeutic divalent siRNAs, we assessed the long-term impact of silencing MSH3 (a key regulator of somatic expansion), HTT, or both. In Q111 HD mice (>110 CAGs), which exhibit robust expansion, mHTT inclusions, and transcriptional dysregulation by 12 months, long-term MSH3 silencing blocked expansion, reduced inclusions, and reversed gene expression changes. HTT silencing alone had limited effect, but combined MSH3/HTT targeting synergistically eliminated inclusions and restored transcriptomic profiles. Parallel treatment in wild-type mice showed no toxicity, supporting the safety of long-term intervention. These findings position somatic expansion as a promising therapeutic target and demonstrate the potential of RNAi-based co-silencing of MSH3 and HTT as a disease-modifying strategy for HD.

Abstract: Reducing the burden of mutant Huntingtin (mHTT) protein in brain cells is a strategy for treating Huntington's disease (HD). However, it is still unclear what pathological changes can be reproducibly reversed by mHTT lowering. We previously found that lipid changes that occur 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). Methods: 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 (Zeitler et al., 2019). Proteins were analyzed using western blot, capillary electrophoresis, and nitrocellulose filtration methods. Lipid analyses were conducted by liquid chromatography and mass spectrometry (LC-MS). Somatic expansion index was assessed using capillary gel electrophoresis of PCR products. Conclusions: Lowering mHTT levels by 43% for 4 months prevented numerous changes in lipids of caudate-putamen in zQ175DN mice. Our data support the idea that mHTT lowering can provide meaningful benefits and support brain health. Furthermore, our data demonstrate that analyzing lipid signatures is a valuable method for evaluating potential therapies in a preclinical model of HD. Key words: AAV9, striatum, Huntington's disease, transcription, metabolomics, gene therapy ### Competing Interest Statement The authors have declared no competing interest.

AbstractHTT1ahas been identified in human and mouse HD brain as the pathogenic exon 1 mRNA generated from aberrant splicing between exon 1 and 2 that contributes to aggregate formation and neuronal dysfunction (Sathasivam et al., 2013). Detection of the HTT exon 1 protein (HTTex1p) has been accomplished with surrogate antibodies in fluorescence-based reporter assays (MSD, HTRF), and immunoprecipitation assays, in HD postmortem cerebellum and knock-in mice but direct detection by SDS-PAGE and western blot assay has been lacking. Here proteins in subcellular fractions prepared from human and mouse HD brain were separated by SDS-PAGE and probed by western blot with neo-epitope monoclonal antibodies (P90-1B12 and 11G2) directed to the C-terminal 8 residues of HTTex1p. In human HD putamen and cortex, HTTex1p migrated at 56-60 kD and at higher molecular masses (HMM) consistent with the presence of CAG repeat expansion inHTT1a. HTTex1p in control brain was low or undetectable. Immunofluorescence labeling of human HD cortex using P90-11G2 revealed small aggregates that sparsely populated the neuropil in layers 3 and 5. In caudate putamen of 6 month old HD knock-in mice (Q50, Q80, Q111, Q140 and Q175) HTTex1p migration was inversely correlated with CAG repeat length and appeared as a SDS soluble high molecular mass (HMM) smear in HD Q111, Q140 and Q175 mice but not in Q50 and Q80 mice indicating a CAG repeat size threshold for detecting HTTex1p aggregation. Migration of HTTex1p and HMM smear changed with age in caudate putamen of Q111, Q175 and YAC128 mice. Treating HD Q111 mice with siRNA to MSH3, a modifier of CAG repeat expansion, significantly reduced levels of the HMM smear indicating that the effects of curbing CAG repeat expansion was quantifiable. These results show that P90 antibodies can be used in western blot assays and immunostaining to track and quantify HTTex1p levels, subcellular localization, and solubility.

A de novo mutation in the transcription factor Nucleus accumbens associated protein 1 (NACC1) gene (c.892C > T p.R298W) causes a rare, severe neurodevelopmental disorder which manifests postnatally. Genome editing was used to generate human isogenic ESCs (control, mutant heterozygote and homozygote lines) which were differentiated to cortical neurons. Mutant neurons expressed higher levels of NACC1 protein by western blot. RNAseq, GO term and SynGO analysis revealed altered expression of transcripts involved with pre- and postsynaptic signaling, neurotransmission, extracellular matrix, and adhesion. Western blot revealed increased expression of the presynaptic proteins SNAP25 and VAMP2 and the postsynaptic protein SYNGAP1. A functional assay showed increased adhesion of neural stem cells to collagen 1 and 4. The mutation also changed levels of transcripts measured by qPCR involved with dorsal ventral patterning to favor a ventral signature. These results suggest that the NACC1 R298W mutation causes molecular changes in an embryonic cell model that may impact postnatal development of cortical neurons.

HTT1a was identified in human and mouse Huntington's disease brain as the pathogenic exon 1 mRNA generated from aberrant splicing between exon 1 and 2 of HTT that contributes to aggregate formation and neuronal dysfunction. Detection of the huntingtin exon 1 protein (HTT1a) has been accomplished with Meso Scale Discovery, Homogeneous Time Resolved Fluorescence and immunoprecipitation assays in Huntington's disease knock-in mice, but direct detection in homogenates by gel electrophoresis and western blot assay has been lacking. Subcellular fractions prepared from mouse and human Huntington's disease brain were separated by gel electrophoresis and probed by western blot with neoepitope monoclonal antibodies 1B12 and 11G2 directed to the C-terminal eight residues of HTT1a. In caudate putamen of an allelic series of 6-month-old Huntington's disease knock-in mice (Q50, Q80, Q111, Q140 and Q175), HTT1a migration was inversely correlated with CAG repeat length and appeared as a sodium dodecyl sulphate soluble high molecular mass smear in Q111, Q140 and Q175 mice but weakly in Q80 and not in wild-type mice or Q50 indicating a CAG repeat size threshold for detecting HTT1a. HTT1a immunoreactivity diminished if 1B12 and 11G2 antibodies were preincubated with an eight amino acid peptide containing the C-terminus of HTT1a but not with an unrelated peptide sequence. Migration of HTT1a and its high molecular mass smear changed with age in caudate putamen of Q111, Q175 and YAC128 mice. Reducing levels of MutS Homolog 3 (MSH3) protein >84% in Q111 mice caudate putamen with small interfering RNA to MSH3, a modifier of CAG repeat expansion, significantly reduced levels of the high molecular mass smear suggesting that the effects of curbing CAG repeat expansion on HTT1a were quantifiable. A prominent 56-60 kDa doublet detected by 1B12 and 11G2 antibodies in lysates from human Huntington's disease brain was not blocked by preincubation with C-terminal HTT1a blocking peptide and also appeared in brains of Parkinson's disease patients. 1B12 and 11G2 antibodies did not immunoprecipitate huntingtin (HTT) proteins from either Huntington's disease mouse or human brain lysates using conditions that pulled down full-length HTT with anti-HTT antibody 2B7. Altogether, these data show that 11G2 and 1B12 antibodies can be used in western blot assays to track and quantify immunoreactive HTT1a levels, solubility and subcellular localization in Huntington's disease mouse brain.

Loss-of-function mutations of the gene encoding the trafficking protein particle complex subunit 9 (Trappc9) cause autosomal recessive intellectual disability and obesity by unknown mechanisms. Genome-wide analysis links Trappc9 to nonalcoholic fatty liver disease (NAFLD). Trappc9-deficient mice have been shown to appear overweight shortly after weaning. Here, we analyzed serum biochemistry and histology of adipose and liver tissues to determine the incidence of obesity and NAFLD in Trappc9-deficient mice and combined transcriptomic and proteomic analyses, pharmacological studies, and biochemical and histological examinations of postmortem mouse brains to unveil mechanisms involved. We found that Trappc9-deficient mice presented with systemic glucose homeostatic disturbance, obesity, and NAFLD, which were relieved upon chronic treatment combining dopamine receptor D2 (DRD2) agonist quinpirole and DRD1 antagonist SCH23390. Blood glucose homeostasis in Trappc9-deficient mice was restored upon administering quinpirole alone. RNA-sequencing analysis of DRD2-containing neurons and proteomic study of brain synaptosomes revealed signs of impaired neurotransmitter secretion in Trappc9-deficient mice. Biochemical and histological studies of mouse brains showed that Trappc9-deficient mice synthesized dopamine normally, but their dopamine-secreting neurons had a lower abundance of structures for releasing dopamine in the striatum. Our study suggests that Trappc9 loss of function causes obesity and NAFLD by constraining dopamine synapse formation.

A missense mutation in the transcription repressor Nucleus accumbens-associated 1 (NACC1) gene at c.892C>T (p.Arg298Trp) on chromosome 19 causes severe neurodevelopmental delay ( Schoch et al., 2017). To model this disorder, we engineered the first mouse model with the homologous mutation (Nacc1+/R284W) and examined mice from E17.5 to 8 months. Both genders had delayed weight gain, epileptiform discharges and altered power spectral distribution in cortical electroencephalogram, behavioral seizures, and marked hindlimb clasping; females displayed thigmotaxis in an open field. In the cortex, NACC1 long isoform, which harbors the mutation, increased from 3 to 6 months, whereas the short isoform, which is not present in humans and lacks aaR284 in mice, rose steadily from postnatal day (P) 7. Nuclear NACC1 immunoreactivity increased in cortical pyramidal neurons and parvalbumin containing interneurons but not in nuclei of astrocytes or oligodendroglia. Glial fibrillary acidic protein staining in astrocytic processes was diminished. RNA-seq of P14 mutant mice cortex revealed over 1,000 differentially expressed genes (DEGs). Glial transcripts were downregulated and synaptic genes upregulated. Top gene ontology terms from upregulated DEGs relate to postsynapse and ion channel function, while downregulated DEGs enriched for terms relating to metabolic function, mitochondria, and ribosomes. Levels of synaptic proteins were changed, but number and length of synaptic contacts were unaltered at 3 months. Homozygosity worsened some phenotypes including postnatal survival, weight gain delay, and increase in nuclear NACC1. This mouse model simulates a rare form of autism and will be indispensable for assessing pathophysiology and targets for therapeutic intervention.

Oligonucleotide therapeutics (ASOs and siRNAs) have been explored for modulation of gene expression in the central nervous system (CNS), with several drugs approved and many in clinical evaluation. Administration of highly concentrated oligonucleotides to the CNS can induce acute neurotoxicity. We demonstrate that delivery of concentrated oligonucleotides to the CSF in awake mice induces acute toxicity, observable within seconds of injection. Electroencephalography and electromyography in awake mice demonstrated seizures. Using ion chromatography, we show that siRNAs can tightly bind Ca2+ and Mg2+ up to molar equivalents of the phosphodiester/phosphorothioate bonds independently of the structure or phosphorothioate content. Optimization of the formulation by adding high concentrations (above biological levels) of divalent cations (Ca2+ alone, Mg2+ alone, or Ca2+ and Mg2+) prevents seizures with no impact on the distribution or efficacy of the oligonucleotide. The data here establish the importance of adding Ca2+ and Mg2+ to the formulation for the safety of CNS administration of therapeutic oligonucleotides.