Characterization of a Knock-In Mouse Model with a Huntingtin Exon 1 Deletion

Abstract

Background:

The Huntingtin (HTT) N-terminal domains encoded by Huntingtin’s (HTT) exon 1 consist of an N17 domain, the polyglutamine (polyQ) stretch and a proline-rich region (PRR). These domains are conserved in mammals and have been hypothesized to modulate HTT’s functions in the developing and adult CNS, including DNA damage repair and autophagy.

Objective:

This study longitudinally characterizes the in vivo consequences of deleting the murine Htt N-terminal domains encoded by Htt exon 1.

Methods:

Knock-in mice with a deletion of Htt exon 1 sequences (Htt^ΔE1) were generated and bred into the C57BL/6J congenic genetic background. Their behavior, DNA damage response, basal autophagy, and glutamatergic synapse numbers were evaluated.

Results:

Progeny from Htt^ΔE1/+ intercrosses are born at the expected Mendelian frequency but with a distorted male to female ratio in both the Htt^ΔE1/ΔE1 and Htt^+/+ offspring. Htt^ΔE1/ΔE1 adults exhibit a modest deficit in accelerating rotarod performance, and an earlier increase in cortical and striatal DNA damage with elevated neuronal pan-nuclear 53bp1 levels compared to Htt^+/+ mice. However, a normal response to induced DNA damage, normal levels of basal autophagy markers, and no significant differences in corticocortical, corticostriatal, thalamocortical, or thalamostriatal synapses numbers were observed compared to controls.

Conclusion:

Our results suggest that deletion of the Htt N-terminus encoded by the Htt exon 1 does not affect Htt’s critical role during embryogenesis, but instead, may have a modest effect on certain motor tasks, basal levels of DNA damage in the brain, and Htt function in the testis.

Keywords

Huntingtin huntingtin exon 1 DNA damage autophagy sex ratio

INTRODUCTION

The protein product of the Huntingtin (HTT) gene, Huntingtin (HTT), is a large 3144 amino acid protein (∼350 kD) containing a polyglutamine (polyQ) stretch encoded by a CAG repeat within HTT exon 1 that when expanded, causes Huntington’s disease (HD) [1]. The polyQ stretch is flanked by the first 17 amino acids of HTT (N17 domain), forming an amphipathic helix capable of interacting with membranes [2], and a proline-rich region (PRR), acting as a structural spacer with the potential to interact with SH3 and WW domain-containing proteins [3, 4]. The HTT N-terminal domains encoded by exon 1 appeared relatively late in evolution with the N17 domain highly conserved in all vertebrates, the polyQ stretch first appearing in sea urchins when protostomes diverged from deuterostomes, and the PRR evolving in mammals [5]. Based on the parallel evolution of the HTT N-terminus and the development of complex nervous systems with a telencephalon, it is hypothesized that the HTT N-terminus has evolved to play a critical role in HTT’s functions in vertebrate nervous system development, maturation, and maintenance [5].

The remainder of HTT contains multiple HEAT repeat domains (named after several proteins containing similar structural motifs: Huntingtin, Elongation factor 3, Protein phosphatase 2A, and yeast kinase TOR1 [6]) that can form an extended superhelical structure and interact with multiple proteins. X-ray crystallography of the HTT N-terminal domains has demonstrated that they can adopt many alternative structures [7]. More recently, cryo-electron microscopy of a 1:1 complex of HTT with HAP40 (an abundant cytoplasmic HTT-interacting protein involved in endosomal trafficking [8, 9]), revealed that HTT:HAP40 has a globular α-helical structure [10]. Importantly, no electron density was detected for the 90 aa encoded by HTT exon 1, an observation that suggests that this region of HTT is very flexible.

HTT is proposed to act as a multivalent protein-protein interaction scaffold [10 –12] based on its structure, the large number of HTT-interacting proteins that have been identified, and its many diverse functions (including neurogenesis [13, 14], synaptic development [15, 16], vesicular transport [17], selective macroautophagy [18, 19], and DNA damage repair [20 –22], among others). In mice, normal Huntingtin (Htt) expression is essential for embryonic development, and Htt knock-out mice die around embryonic day 7.5 to 8.5 [23 –25]. Conditional knockout of Huntingtin (Htt) in the central nervous system during embryonic or early postnatal development results in altered neurodevelopment and neurodegeneration [13 , 26], while loss of normal Htt expression in HD mouse models can affect pathogenesis [27]. Previous studies have also demonstrated a requirement of Htt for adult survival that correlates inversely with age, but the functional consequences of Htt inactivation varied between models [28, 29].

Mice with single- or double-domain deletions within Htt’s exon 1 are born at the expected Mendelian frequency, survive into adulthood, and are fertile, suggesting that Htt’s N-terminal domains are not required for its critical functions during embryogenesis or postnatal survival [28 , 30–33]. HTT has been proposed to participate in selective macroautophagy, and the N-terminal, middle, and C-terminal portions of HTT have sequence and structural similarity to the yeast autophagy proteins Atg23, Vac8, and Atg11 [18, 19]. Overexpressing a version of Htt with a CAG repeat deletion (Htt^ΔQ) in vitro leads to an increase in autophagosome formation, and the autophagosome marker microtubule-associated protein 1A/1B-light chain 3-II (LC3-II) is elevated in aged Htt^ΔQ/+ mice [34]. In primary neuronal cultures, however, neither Htt^ΔQ/ΔQ neurons nor neurons with both a polyQ and PRR deletion (Htt^ΔQP/ΔQP) exhibit changes in autophagic flux [31]. Atg23 and Atg11 coordinate autophagosome formation in yeast, and HTT aa1-568 interacts in vitro with an HTT C-terminal aa 2416–3144 fragment [18, 35]. However, HTT aa1–90 encoded by exon 1 are not required for this interaction [18]. Based on these findings, the extent to which the Htt N-terminus functions in basal neuronal autophagy is not clear.

In vitro studies of the HTT N-terminus have also demonstrated a role for the N17 domain in both the cell stress response and as sensor for oxidative stress [2 , 36]. Elevated reactive oxygen species (ROS) can oxidize methionine residues 1 and 8 within the N17 domain resulting in HTT’s translocation into the nucleus where it colocalizes with the ataxia-telangiectasia mutated protein (ATM) at sites of DNA damage [20]. Huntingtin is proposed to be involved in base excision repair [20, 37], transcription coupled DNA repair [22], and possibly also in double strand DNA break repair due to a functional interaction between HTT and ATM [20, 38], but how different HTT domains contribute to HTT’s involvement in DNA repair pathways is still unclear.

Aged mice homozygous for a deletion of the exon 1 sequences encoding the N17 domain (Htt^ΔN17) have reduced numbers of thalamostriatal glutamatergic synapses [31], although whether this is the result of a deficit in synaptic development or maintenance is not known. The phenotypic effects of deleting the Htt polyQ and PRR domains individually are not additive when both domains are deleted in mice [31 –34], suggesting that the function of the Htt N-terminus may not be fully understood by characterizing its domains in isolation. To address this, we have developed a mouse model with a deletion of all three functional domains of the Htt N-terminus (Htt^ΔE1), performed longitudinal behavioral characterization, and investigated several aging-related cellular processes in the brain including DNA damage repair, autophagy, and synaptic maintenance.

We have found that Htt^ΔE1/ΔE1 mice are born at the expected Mendelian frequency, can survive through 24 months of age, and are fertile. However, Htt^+/+ and Htt^ΔE1/ΔE1 progeny from Htt^ΔE1/+ intercrosses were born at a distorted male/female ratio. Htt^ΔE1/ΔE1 mice exhibit a modest deficit in accelerating rotarod performance, and an early increase in cortical and striatal DNA double-strand breaks (DSBs) at 3 months of age with elevated levels of pan-nuclear p53-binding protein 1 (53bp1, a protein functioning in double-strand DNA break repair [39]). However, no changes in the basal levels of autophagy markers or in glutamatergic corticocortical, corticostriatal, thalamocortical, or thalamostriatal synapse numbers were observed.

MATERIALS AND METHODS

Generation of the Htt^ΔE1 mouse allele

To generate mice lacking the 3 N-terminal domains of Htt (Htt^ΔE1), two synthetic oligomers 5^′-CTTCAGGGTCTGTCCCATCGGGCAGGAAGCCGTCATGCTGCCAGGTCCGGCAGAGGAACCGCTGCACCGACCGTGAGTCCGGTACCTCC-3^′ and 5^′-GGAGGTACCGGACTCACGGT-3^′, were annealed and filled in with the Klenow fragment of DNA polymerase I. The double strand DNA fragment was then digested with AlwN1 (restriction site bolded) and Kpn1 restriction enzymes (restriction site underlined) to generate a synthetic AlwN1-Kpn1 fragment that was used to replace the endogenous AlwN1-Kpn1 fragment in Htt exon 1. Two independent ES clones were used to generate germline transmitters as described [32]. Htt^ΔE1/+ males were back-crossed with C57BL/6J females for 11 generations to reach the C57BL/6J congenic background. No phenotypic differences were observed between mice derived from the two ES clones.

PCR genotyping

PCR was performed using the primers listed below: HdEpi-1:5^′-GCGTAGTGCCAGTAGGCTCCAAG-3^′ and 140 Reverse: 5^′-GAAGGCACTGGAGTCGTGAC-3^′ (Htt^ΔE1:233 bp, Htt⁺: no product due to high G/C-rich content in PRR). 140Forward: 5^′-CTGCACCGACCGTGAGTCC-3^′ and 140Reverse (see above) (Htt⁺: 236 bp, Htt^ΔE1:152 bp due to an intronic 84 bp deletion incorporated in the gene targeting construct).

Mouse husbandry and behavioral analyses

Mice were housed in a temperature-and humidity-controlled facility on a 12-hour light-dark cycle with unrestricted access to food and water. Whole body γ-irradiation was administered with a J.L. Shepherd Mark 1 Model 68A Cesium-137 irradiator located in the vivarium. KBrO₃ was added in the drinking water at the concentration of 2 g/liter for 7 days. All experiments using mice were approved by the University of Virginia Animal Care and Use Committee. Both male and female mice in the C57BL/6J congenic background were used for all experiments (18 male Htt^+/+, 30 female Htt^+/+, 28 male Htt^ΔE/ΔE1, and 19 female Htt^ΔE/ΔE1 mice) except for longitudinal behavioral testing. An additional 20 male mice for each genotype were used for behavioral testing to eliminate the potential effects of the estrus cycle on behavior and tissues from these mice were collected for analysis at 24 months of age. Experimenters were blinded to genotype during data collection and analysis when appropriate.

Behavioral analyses were performed as described [31], except all testing were performed between 8 AM and 2 PM (light phase). Mice were examined, weighed, and then subjected to accelerating rotarod, open field, and grip strength testing at 3, 6, 12, and 18 months of age, along with Morris water maze testing at 14 and 19 months of age. Open field testing was performed with the VersaMax Animal Activity Monitoring System and analysis software (AccuScan Instruments). Accelerating rotarod testing using an initial speed of 2 rpm with 0.1 rpm/s acceleration was performed with an Economex accelerating rotarod (Columbus Instruments). Forelimb grip strength was assessed using a grip strength meter (San Diego Instruments). Each mouse was tested on one day per experimental time point, and the average force required to break the mouse’s grip over 3 trials was recorded. Morris water maze testing was performed over 8 consecutive days, including the acquisition phase (4 days), the probe phase (1 day), the reversal phase (2 days), and the visible platform control phase (1 day). Data was collected and analyzed using EthoVision XT 13 software (Noldus Information Technology).

Brain tissue lysate preparation and subcellular fractionation

Brain lysates from 3- 6- or 24-month-old mice were separated into enriched nuclear, microsomal, and cytosolic fractions as described [31], with the following modifications. The 800 xg supernatant was centrifuged at 100 k xg for 60 min at 4°C to obtain a microsomal (pellet) and a cytosolic (supernatant) fraction. The 800 xg pellet was resuspended in 1.3 M sucrose buffer, layered on a cushion of the same buffer, and centrifuged for 45 min at 3 k xg to obtain a nuclear fraction.

For detection of protein carbonyls, cortical and striatal tissues were dissected and homogenized in 25 mM Tris (pH 7.9), 500 mM NaCl, 1 mM EDTA, 1%NP40, 0.1%SDS, 1%sodium deoxycholate, 1 mM DTT, 5 mM NaF, 1 mM NaVO4, and Halt protease inhibitor (Thermo Scientific 78425). Samples were centrifuged at 15 k xg for 15 min at 4°C and the supernatant was collected.

For detection of 53bp1, cortical tissue was dissected and homogenized in 25 mM Tris (pH 7.9), 500 mM NaCl, 1 mM EDTA, 1 mM DTT, 5 mM NaF, 1 mM NaVO4, 1%TritonX-100 and Halt protease inhibitor. The homogenate was sonicated for 3 min (10 s on/20 s off) at 4°C (Fisher Scientific Sonic Dismembrator, Fisherbrand Model 505), centrifuged at 15 k xg for 15 min at 4°C, and the supernatant was collected.

For cortical and striatal cytosolic and microsomal fractions, tissues were dissected and homogenized in the same buffer used in the whole brain fractionation (see above), except the supernatant from the 800 xg spin was centrifugated at 15 k xg to obtain the microsomal fraction (pellet) and cytosolic fraction (supernatant). Protein levels were estimated using a Thermo Fisher Pierce BCA Protein Assay kit (PI23227).

Western blotting

Western blotting was performed as described [31], with the following modifications. 60μg protein was fractionated on Mini-PROTEAN TGX 4–15%gradient gels (Bio-Rad) for synaptic marker detection, on 7%acrylamide gels for p62 and LAMP2A detection, or on 15%acrylamide gels for LC3 detection, and then transferred onto 0.2μm nitrocellulose membranes (Bio-Rad, 1704158) using a Bio-Rad Transblot Turbo transfer system.

To detect Htt, proteins from cytosolic, nuclear, and microsomal fractions were separated on 4.4%acrylamide gels and transferred electrophoretically onto Immun-Blot Low Fluorescence PVDF membranes (Bio-Rad 162-0263) at 30 V overnight using a mini TransBlot system (BioRad) at 4°C.

To quantify cortical 53bp1 levels, 60μg of total protein were fractionated on 2-step 4.4%/8.8%acrylamide gels and transferred electrophoretically onto Immun-Blot Low Fluorescence PVDF membranes at 100 V for 2.5 h at 4°C.

To examine protein carbonyl levels, 15μg of total cortical and striatal protein were fractionated on 7%acrylamide gels and transferred onto Immun-Blot Low Fluorescence PVDF membranes using the Transblot Turbo transfer system. Derivatization of carbonyl groups with 2,4-dinitrophenylhydrazine (DNPH) and immunoblotting with a DNP antibody were performed using the OxiSelect Protein Carbonyl Kit (Cell BioLabs STA-308) and signals were detected with Thermo Scientific SuperSignal WestDura substrate (PI34076). Blots were then re-probed with β-actin antibody.

Primary antibodies used were: Huntingtin (aa181-810) (Millipore MAB 2166, 1:3000), Huntingtin (aa1-17) (Sigma-Aldrich H7540, 1:1500), Huntingtin (D7F7, amino acids surrounding P1220) (Cell Signaling 5656, 1:1000), 53bp1 (Abcam 175933, 1:5000), LC3 (Novus NB100-2220, 1:1000), p62 (American Research Products 03-GP62-C; 1:2000), LAMP2A (Thermo Fisher 51-2200, 1:3000), β-actin (Cell Signaling 3700; 1:1000), Vglut1 (Millipore AB5905 1:10,000), PSD95 (Cell Signaling 2507; 1:1000), Vglut2 (Millipore AB2251; 1:10,000), and DNP (Cell BioLabs 230801). IRDye 800CW and IRDye 680RD conjugated secondary antibodies were purchased from Li-Cor (926–32212, 926–68073, 925–68070, 925–32211) and Alexa Fluor 680 and 790 donkey anti-guinea pig conjugated secondary antibodies were purchased from Jackson ImmunoResearch (706-625-148, 706-655-148). Blots were imaged on a Li-Cor Odyssey Fc system and analyzed using Li-Cor ImageStudio software.

Immunohistochemistry

For immunohistochemistry, 2-, 3-, 4-, 12-, or 18-month-old mice were anesthetized and perfused with 10%sucrose in PBS for 1 min (5 ml/min) using a peristaltic pump (Cole-Parmer Master flex L/S pump equipped with a model 77200-60 pump head), followed by 4%paraformaldehyde-10%sucrose in 0.1 M phosphate buffer for 5 min. Brains were removed and post-fixed for 2 h, rinsed in 10%sucrose in PBS, incubated for 1 h in 0.125 M glycine-10%sucrose in PBS, and washed 3 times in 10%sucrose in PBS (including one overnight wash, all steps were performed at 4°C). Tissue was then frozen in O.C.T. compound (Tissue-Tek 4583). Frozen forebrains were sectioned coronally at 14 or 20μm, while cerebella were sectioned sagittally at 14μm, and processed as described [31]. For samples to be labeled with primary antibodies generated in mice, an additional blocking step with a monovalent FAB anti-mouse antibody (Jackson ImmunoResearch, 715-007-003) at 1:100 was performed for 1 h at RT.

Primary antibodies used: γH2AX (Abcam 26350, 1:8000), 53bp1 (Abcam 175933, 1:5000), p62 (American Research Products 03-GP62-C; 1:900), LC3 (Cell Signaling 12741; 1:900), Vglut1 (Millipore AB5905 1:3750), Vglut2 (Millipore AB2251;1:5000), PSD95 (Cell Signaling 3409; 1:800), NeuN (Millipore ABN90P, 1:500), calbindin (Novus NBP2-50028, 1:4000). Secondary antibodies were purchased from Jackson ImmunoResearch Laboratories (715-165-151, 711-165-152, 706-165-148, 715-545-151, 711-545-152, 706-545-148, 703-175-155). DAPI (Sigma-Aldrich, D9542) was used to label nuclei.

Images of brain sections were obtained using an Olympus FV1200 confocal system with a 60x objective. Coronal sections were collected for synapse quantification: from bregma –0.555 mm to 1.245 mm (dorsal striatum) and bregma –1.155 mm to 1.245 mm (motor cortex layers I and II) (20μm sections at 200μm intervals, 10–13 sections/mouse), for autophagy marker quantification: from bregma –0.18 mm to 1.42 mm (20μm sections at 200μm intervals, 9 sections/mouse), and for DNA damage marker quantification: from bregma –0.26 mm to 1.42 mm (14μm sections at 210μm intervals, 8–9 sections/mouse). Images of sagittal cerebellar sections were collected from 1.10 mm to 2.78 mm lateral to the midline (14μm sections at 210μm intervals, 9 sections/mouse, 3 images/section).

For Purkinje cell density quantification, images of sagittal cerebellar sections were obtained using a Keyence BZ-X800E fluorescence microscope with a 20x objective. Images were acquired from 1.10 mm to 2.99 mm lateral to the midline (14μm sections at 630μm intervals, 4 sections/mouse) and stitched into a single image per section.

Comet assay and nuclear immunostaining

Cortex, striatum, thalamus, and cerebellum were dissected from mice at 3 or 13–16 months of age and homogenized in 250 mM sucrose, 20 mM HEPES (pH 7.3), 1 mM MgCl₂, 1%DMSO, and Halt protease inhibitor. Homogenates were centrifuged at 800 xg for 4 min at 4°C. Pellets were gently resuspended in 250 mM sucrose, 20 mM HEPES (pH7.3), 1 mM MgCl₂, 25 mM KCl, 1%DMSO, and Halt protease inhibitor. Iodixanol solution (Optiprep, Sigma-Aldrich D1556) was added to 25%, and the samples were layered on top of an 29%–35%iodixanol cushion and centrifuged at 3 k xg for 30 min at 4°C. The nuclear-enriched band between the 25%and 29%iodixanol layers was removed and added to 3x volume of PBS and centrifuged at 1 k xg for 5 min at 4°C. Excess PBS was removed and the nuclei were resuspended in the remaining PBS and counted using a hemocytometer.

For comet assays, ∼5,500 nuclei in 10μl PBS were mixed with 100μl of 1%low-melting agarose, plated onto agarose-coated slides, covered with a 22 mm×50 mm coverslip, and then allowed to solidify for 30 min at 4°C. Coverslips were removed and slides were incubated in a lysis buffer (20 mM Tris, 50 mM EDTA, 1.2 M NaCl, 1%sarkosyl, and 2%DMSO) at either neutral (pH 8) or alkaline (pH 10) pH for 30 min at 4°C to lyse the nuclei. The slides were then electrophoresed at 23 V for 20 min in either a neutral (TBE pH 7.4) or an alkaline buffer (50 mM NaOH, 1 mM EDTA). Slides were washed twice in water (alkaline treated slides were neutralized in TBE for 5 min before washing), fixed in 70%ethanol for 10 min, and dried. Slides were rehydrated in water, incubated in 2.5μg/ml propidium iodide for 15 min to stain DNA, and washed twice in water for 10 min each, then mounted with Vectashield mounting media (Vector Lab H-1000) before imaging. Images were obtained using a Nikon TE2000 inverted microscope with a 20x oil objective and a Nikon DS-Qi2 camera, and quantified using the public domain CaspLab software (casplab.com) [40]. The DNA in the comet tail, as a percentage of the total DNA in the comet, was quantified. This value represents the amount of total DNA damage and is not affected by the length of the tail which may be influenced by slight changes in buffer composition, electrophoresis conditions, and DNA fragment size.

To quantify 8-OHdG levels, ∼22,500 nuclei in 45μl 0.9%low-melt agarose were plated onto agarose-coated slides, covered with a 22 mm×22 mm coverslip, and then allowed to solidify for 30 min at 4°C. Coverslips were removed and the slides were then lysed in an alkaline (pH 10) buffer, neutralized, fixed, and dried as described above. Slides were then rehydrated in water and immunostaining with anti-8-OHdG antibody (Millipore AB5830, 1:200) following standard procedures. Secondary antibody was obtained from Jackson ImmunoResearch Laboratories (705-165-147), and DNA was stained with DAPI. Images of isolated nuclei were obtained using a Nikon TE2000 inverted microscope with a 20x oil objective and a Nikon DS-Qi2 camera.

Image and statistical analyses

Analysis of immunohistochemistry and nuclear immunostaining images was performed with Nikon NIS-Elements AR Analysis software (5.02.01). For γH2AX, 53bp1, LC3, p62, and PSD95 quantification, puncta under 2 pixels were excluded. For Vglut2 and Vglut1 quantification, puncta under 3 pixels were excluded. For DAPI quantification in immunohistochemistry, nuclei under 15 pixels were excluded. To determine the calbindin⁺ cell density, the number of calbindin⁺ cells along the entire length of the Purkinje cell layer were counted and divided by the total length of the layer. Numerical data were analyzed using GraphPad Prism 9. Statistical significance in the χ² test, unpaired t-test, paired t-test, 1-way ANOVA, or 2-way ANOVA was set at p < 0.05. Tukey’s multiple comparison test was used for post-hoc analysis of ANOVA tests.

RESULTS

Generation of the Htt^ΔE1 mouse allele

The Htt^ΔE1 knock-in mice were generated as described [31] by replacing the wild type Htt exon 1 sequence with a synthetic sequence lacking amino acids (aa) 2–17 of the N17 domain, the polyQ, and the PRR domains of Htt (Fig. 1). To confirm expression of ΔE1-Htt, cytosolic, microsomal, and nuclear protein fractions from 6-month-old Htt^+/+, Htt^ΔE1/+, and Htt^ΔE1/ΔE1 mouse brains were analyzed by western blotting with the MAB2166 antibody, which recognizes an epitope present in both WT-Htt and ΔE1-Htt (aa181-810), and the H7540 antibody, recognizing the first 17 aa of Htt that are deleted in ΔE1-Htt. ΔE1-Htt migrates faster through the gel than WT-Htt, resulting in a doublet in the Htt^ΔE1/+ sample detected by the MAB2166 antibody and a single band corresponding to WT-Htt detected by the H7540 antibody (Fig. 2A and data not shown).

Fig. 1

Diagram of wild-type (Htt⁺) and N-terminal deletion (Htt^ΔE1) versions of Htt’s exon 1. The amino acid sequence of the Htt N-terminus includes the N17 domain (bolded), the polyQ domain (italicized), and PRR domain (underlined).

Fig. 2

Deletion of the 3 N-terminal domains of Htt does not affect its subcellular distribution. A) Western blot of whole brain total microsomal fractions (60μg) from Htt^+/+ (+/+), Htt^ΔE1/+ (ΔE1/+), and Htt^ΔE1/ΔE1 (ΔE1/ΔE1) mice probed with the MAB2166 antibody (Millipore), which recognizes an epitope present in both wild type (WT-Htt, white arrow) and N-terminal deleted Htt (ΔE1-Htt, black arrow), and the H7540 antibody (Sigma-Aldrich), which recognizes the first 17 amino acids of Htt. B) Western blots of whole brain cytosolic (C, 30μg), nuclear (N, 90μg), and microsomal (M, 30μg) fractions from Htt^ΔE1/+ mice were probed with MAB2166 (Millipore) and D7F7 (Cell Signaling). White arrows: WT-Htt, black arrows: ΔE1-Htt. C) Quantification of the relative fluorescence of ΔE1-Htt to WT-Htt in each fraction. No differences were observed in the ΔE1-Htt to WT-Htt ratio between fractions. Mean±SEM, 1-way ANOVA, n = 3 mice. 2166: C: 0.76±0.04, N: 0.67±0.02, M: 0.72±0.05, p = 0.3460 F = 1.273, D_F = 2; D7F7: C: 0.78±0.04, N: 0.71±0.05, M: 0.81±0.04, p = 0.3659 F = 1.194, D_F = 2.

The Htt N-terminus encoded by exon 1 is not essential for embryonic survival

Htt knockout mice die early during embryogenesis [23 –25]. To determine if the Htt N-terminus is required for embryonic development, progeny from Htt^ΔE1/+ intercrosses (124 litters, average litter size 5.94) were sexed and genotyped, and the ratio of Htt^+/+, Htt^ΔE1/+, and Htt^ΔE1/ΔE1 pups was compared to the expected Mendelian ratio (Table 1). Female and male pups were born at 1:1 ratio, and the ratio of total (male + female) Htt^+/+, Htt^ΔE1/+, and Htt^ΔE1/ΔE1 progeny was not significantly different from the Mendelian ratio, suggesting that the Htt exon 1-encoded N-terminal domains are not required for its critical functions during early embryogenesis. When separated by sex, however, the percentage of male Htt^ΔE1/ΔE1 progeny (30.7%) was increased and Htt^+/+ progeny (18.6%) was decreased (Table 1, χ² = 10.677, p = 0.0048) compared to the Mendelian ratio. Conversely, the percentage of female Htt^+/+ progeny (29.4%) was increased, while the Htt^ΔE1/ΔE1 progeny (19.7%) was decreased (χ² = 7.119, p = 0.0285). Both male and female Htt^ΔE1/ΔE1 mice are fertile and the litter sizes obtained from Htt^ΔE1/ΔE1 intercrosses are within normal range (8 litters, average litter size 5.11) without a significant change in the male to female ratio of their progeny (males: 41.3%, females: 58.7%, χ² = 1.391, p = 0.2382).

Table 1

The number (percentage) of Htt^+/+, Htt^ΔE1/+, and Htt^ΔE1/ΔE1 progeny from Htt^ΔE1/+ intercrosses compared to the expected Mendelian ratio

	Htt^+/+ (%)	Htt^ΔE1/+ (%)	Htt^ΔE1/ΔE1 (%)	Total	χ²	p
Total	177 (24.1%)	374 (50.8%)	185 (25.1%)	736	0.370	0.8313
Male	68 (18.6%)	185 (50.7%)	112 (30.7%)	365	10.677	0.0048
Female	109 (29.4%)	189 (50.9%)	73 (19.7%)	371	7.119	0.0285

To identify which Htt N-terminal domain deletions may be responsible for this phenotype, we performed a retrospective analysis of the numbers of male and female progeny obtained from Htt^ΔN17/+, Htt^ΔP/+ or Htt^ΔQ/+ intercrosses in a mixed C57BL/6J and 129/Sv genetic background and we did not identify any significant deviation from the Mendelian ratio (Supplementary Table 1, [31 –33] and data not shown). However, in a retrospective analysis of 261 progeny obtained from Htt^ΔQP/+ intercrosses [31], we observed a trend towards an increase in the percentage of male Htt^ΔQP/ΔQP pups (32%) and a decrease in the percentage of male Htt^+/+ pups (22.7%) (Supplementary Table 1, χ2 = 3.375, p = 0.185), while the percentage of female Htt^+/+ pups was significantly increased (33.8%) compared to the Mendelian ratio (χ2 = 6.226, p = 0.0445), suggesting that deletion of more than one Htt N-terminal domain may elicit a sex distortion phenotype in the progeny from heterozygous intercrosses.

Deletion of Htt’s N-terminal domains has no apparent effect on ΔE1-Htt’s subcellular localization

In vitro studies have identified HTT N17 as both a membrane association and a nuclear export domain, suggesting that HTT’s N-terminus may regulate its subcellular localization [2 , 42]. To examine if the combined deletion of the three N-terminal domains of Htt affects its subcellular localization, we first quantified Htt levels in whole brain cytosolic, microsomal, and nuclear protein fractions obtained from 6-month-old Htt^+/+, Htt^ΔE1/+ and Htt^ΔE1/ΔE1 mice by western blotting. We did not observe a significant change in Htt subcellular localization between genotypes (Fig. 2A and data not shown). To circumvent the confounding factor of comparing Htt levels between different animals, we then prepared whole brain lysates from 5-month-old Htt^ΔE1/+ mice and separated them into cytosolic, nuclear, and microsomal fractions. WT-Htt and ΔE1-Htt levels in each fraction were analyzed by western blotting using the anti-Huntingtin antibodies MAB2166 and D7F7 (which recognizes an epitope surrounding P1220) (Fig. 2B). There were no significant differences in the ratio of the fluorescence intensity of the ΔE1-Htt to WT-Htt bands between any of the fractions (Fig. 2C), indicating that ΔE1-Htt subcellular localization is not significantly altered under homeostatic conditions in vivo.

Htt^ΔE1/ΔE1 mice exhibit a modest rotarod deficit

For phenotypic characterization, a cohort of 20 male mice of each genotype was observed for 24 months. Body weight, accelerating rotarod performance, forelimb grip strength, and activity levels were evaluated at 3, 6, 12, and 18 months of age. There were no differences in body weight between Htt^+/+ and Htt^ΔE1/ΔE1 mice at 3, 6, or 18 months of age (Supplementary Figure 1A). At 12 months of age, Htt^ΔE1/ΔE1 mice exhibit a slight but significantly higher body weight compared to Htt^+/+ mice (Supplementary Figure 1A, Htt^+/+: 30.94±0.65 g, Htt^ΔE1/ΔE1: 34.25±1.50 g, p = 0.0458, mean±SEM, unpaired t-test, n = 19–20/genotype). There is no difference in body weight between limited numbers of Htt^ΔE1/ΔE1 and Htt^+/+ female mice examined between 3–12 months of age (data not shown). The percentage of surviving mice at 24 months of age was not significantly different between Htt^ΔE1/ΔE1 and Htt^+/+ controls (Htt^+/+: 70%, Htt^ΔE1/ΔE1: 80%, χ² = 0.952, p = 0.3291).

We observed a modest but significant deficit in accelerating rotarod performance in the Htt^ΔE1/ΔE1 mice compared to the Htt^+/+ controls (Fig. 3). At 3 months of age, the Htt^ΔE1/ΔE1 mice were able to stay on an accelerating rod for ∼12%less time than the control mice, and this trend was maintained through 18 months of age (6 months: ∼10%less time, 12 months: ∼16%less time, 18 months: ∼19%less time). Although the difference in performance between the Htt^ΔE1/ΔE1 and Htt^+/+ mice did not reach significance at 6 months of age, there was a trend toward a deficit (Htt^+/+: 212.2±5.5 s, Htt^ΔE1/ΔE1: 191.4±4.6 s, p = 0.0627, F = 3.677, D_F = 1, mean±SEM, 2-way ANOVA, 20 mice/genotype). The accelerating rotarod performance of Htt^ΔE1/+ mice was not significantly different from either Htt^+/+ or Htt^ΔE1/ΔE1 mice at any experimental time point (data not shown). There were no differences observed in forelimb grip strength or in activity levels between Htt^+/+ and Htt^ΔE1/ΔE1 mice (Supplementary Figure 1B-D).

Fig. 3

Htt^ΔE1/ΔE1 mice exhibit a rotarod deficit. Latency to fall from the accelerating rotarod was evaluated in Htt^+/+ (+/+) and Htt^ΔE1/ΔE1 (ΔE1/ΔE1) mice on 3 consecutive days at 3, 6, 12, and 18 months of age. ^*p < 0.05, mean±SEM, 2-way ANOVA, 18–20 mice/genotype. The exact number of mice tested at each time point is listed in Supplementary Table 2. 3 months: Htt^+/+: 218.5±6.9 s, Htt^ΔE1/ΔE1: 192.8±6.4 s, p = 0.0455, F = 4.276, D_F = 1; 6 months: Htt^+/+: 212.2±5.5 s, Htt^ΔE1/ΔE1: 191.4±4.6 s, p = 0.0627, F = 3.677, D_F = 1, 12 months: Htt^+/+: 197.2±6.2 s, Htt^ΔE1/ΔE1: 166.5±5.6 s, p = 0.0231, F = 5.614, D_F = 1; 18 months: Htt^+/+: 174.8±7.4 s, Htt^ΔE1/ΔE1: 142.2±5.7 sec, p = 0.0327, F = 4.945, D_F = 1.

To assess if Htt^ΔE1/ΔE1 mice have a deficit in spatial learning and memory, Morris water maze testing was performed at 14 and 19 months of age. No differences between Htt^ΔE1/ΔE1 and Htt^+/+ mice were observed in the latency to the platform during the acquisition trials, reversal trials, or visible platform trials. There was also no difference between genotypes in the time the mice spent in the platform quadrant or in their swim velocity during the probe trial (Supplementary Figure 2).

These data suggest that the three N-terminal domains of Htt are not required for survival in adulthood or for normal spatial learning and memory, but deletion of the N-terminal domains has a modest impact on accelerating rotarod performance.

Double-strand DNA breaks are increased in the cortex and striatum of 3-month-old Htt^ΔE1/ΔE1 mice

In vitro studies have shown that HTT localizes to sites of DNA damage upon oxidative stress or following UV laser micro-irradiation [20]. To examine if the deletion of Htt’s N-terminal domains results in any change in the DNA damage response, we first used the single-nucleus gel electrophoresis comet assay [43] to quantify DNA breaks in young (3-month-old) and older (13- to 16-month-old) Htt^ΔE1/ΔE1 and Htt^+/+ mice. Nuclei from dissected cortex, striatum, thalamus, and cerebellum were isolated using an iodixanol gradient, embedded in low-melting point agarose, and then lysed and electrophoresed at alkaline pH to detect both DNA single strand breaks (SSBs) and double strand breaks (DSBs) or under neutral pH to detect only DSBs. A small but significant increase in the percentage of DNA detected in the tail of the comet was observed in the cortex and striatum of 3-month-old Htt^ΔE1/ΔE1 mice compared to controls under neutral pH conditions (Fig. 4A, B, cortex: Htt^+/+: 15.00±1.43%, Htt^ΔE1/ΔE1: 18.00±1.35%, p = 0.0053; striatum: Htt^+/+: 17.33±1.57%, Htt^ΔE1/ΔE1: 22.62±1.78%, p = 0.0289, mean±SEM, paired t-test, 104–202 nuclei/mouse, 6 mice/genotype), indicating that there is an elevated level of DSBs in the Htt^ΔE1/ΔE1 cortex and striatum at 3 months of age. However, no difference in the levels of DSBs were observed in the thalamus or cerebellum between 3-month-old Htt^ΔE1/ΔE1 and control mice (data not shown), suggesting that the increased DSBs in the Htt^ΔE1/ΔE1 brain is region-specific. In contrast, no differences in the level of SSBs were observed in the cortex, striatum, thalamus, and cerebellum in a comparison between Htt^ΔE1/ΔE1 and controls at 3 months of age (Fig. 4A and data not shown), and no differences in either DNA SSBs or DSBs were observed in these brain regions obtained from 13–16-month-old Htt^ΔE1/ΔE1 and Htt^+/+ mice (Fig. 4A and data not shown).

Fig. 4

Double strand DNA breaks are increased in 3-month-old Htt^ΔE1/ΔE1 mice. A) Quantification of neutral and alkaline comet assays from the cortex and striatum of 3- and 13–16-month-old mice. The percentage of DNA in the comet tail under neutral conditions was significantly increased in the cortex and striatum of Htt^ΔE1/ΔE1 (ΔE1/ΔE1) mice compared to Htt^+/+ (+/+) mice at 3 months of age. ^*p < 0.05, ^**p < 0.01, mean±SEM, paired t-test, 104–202 nuclei/mouse, 5–6 mice/genotype. B) Neutral comet assay images from the cortex and striatum of 3-month-old Htt^+/+ and Htt^ΔE1/ΔE1 mice. Examples of CaspLab comet quantification analyses are shown in white boxes: comet head circumference (white circle), comet head center (black cross), comet tail area (purple), and end of comet tail (vertical white line) are indicated. Neutral assay: 3 months: cortex: Htt^+/+: 15.00±1.43%, Htt^ΔE1/ΔE1: 18.00±1.35%, p = 0.0053; striatum: Htt^+/+: 17.33±1.57%, Htt^ΔE1/ΔE1: 22.62±1.78%, p = 0.0289; 13–16 months: cortex: Htt^+/+: 13.58±2.37%, Htt^ΔE1/ΔE1: 15.17±2.55%, p = 0.2315; striatum: Htt^+/+: 18.50±2.95%, Htt^ΔE1/ΔE1: 19.20±1.92%, p = 0.7558. Alkaline assay: 3 months: cortex: Htt^+/+: 13.28±1.73%, Htt^ΔE1/ΔE1: 11.29±2.78%, p = 0.3987; striatum: Htt^+/+: 23.22±3.33%, Htt^ΔE1/ΔE1: 23.01±4.72%, p = 0.9382; 13–16 months: cortex: Htt^+/+: 18.68±6.83%, Htt^ΔE1/ΔE1: 13.65±4.41%, p = 0.1914; striatum: Htt^+/+: 23.07±5.84%, Htt^ΔE1/ΔE1: 18.25±4.77%, p = 0.1288.

Although the comet assay can provide a direct assessment of the level of DNA breaks in a single cell, slight differences during nuclear isolation, lysis, and electrophoresis made it difficult to compare results between different sets of experiments. Therefore, we next examined the expression of DSB markers by immunohistochemistry in the cortex and striatum of 3- and 12-month-old Htt^+/+ and Htt^ΔE1/ΔE1 mice. The histone H2A variant H2AX is phosphorylated by ATM at the sites of DSBs to form γH2AX [44], which then helps to recruit DNA repair proteins into the DNA repair complex. The presence of nuclear γH2AX puncta has been widely used to visualize DSBs in vitro and in vivo [45]. A greater percentage of nuclei with γH2AX⁺ puncta was observed in both the cortex and striatum of Htt^ΔE1/ΔE1 mice in comparison to Htt^+/+ controls at 3 months of age (Fig. 5A, B, cortex: Htt^+/+: 17.66±3.18%, Htt^ΔE1/ΔE1: 49.50±8.76%, p = 0.0142; striatum: Htt^+/+: 6.47±1.79%, Htt^ΔE1/ΔE1: 31.35±9.12%, p = 0.0368; mean±SEM, unpaired t-test, 8–9 images/mouse, 4 mice/genotype). In contrast, no difference between genotypes was detected at 12 months of age (Fig. 5A, B).

Fig. 5

The percentage of nuclei with γH2AX puncta is increased in 3-month-old Htt^ΔE1/ΔE1 mice. A) Cortex and striatum from 3- and 12-month-old Htt^+/+ (+/+) and Htt^ΔE1/ΔE1 (ΔE1/ΔE1) mice without γ-irradiation and 3-month-old Htt^+/+ and Htt^ΔE1/ΔE1 mice 3 or 24 h after exposure to 7Gy of γ-irradiation immunostained with a γH2AX antibody (green). Nuclei were labeled with DAPI (blue), scale bar = 10μm. White dashed boxes indicate the locations of inset images, scale bar = 2μm. B) Quantification of the percentage of nuclei containing γH2AX⁺ puncta in 3- and 12-month-old untreated mice. ^*p < 0.05, mean±SEM, unpaired t-test, 8–9 images/mouse, 3–4 mice/genotype. Cortex: 3 months: Htt^+/+: 17.66±3.18%, Htt^ΔE1/ΔE1: 49.50±8.76%, p = 0.0142; 12 months: Htt^+/+: 45.77±14.80%, Htt^ΔE1/ΔE1: 40.76±12.81%, p = 0.8108; striatum: 3 months: Htt^+/+: 6.47±1.79%, Htt^ΔE1/ΔE1: 31.35±9.12%, p = 0.0368; 12 months: Htt^+/+: 27.14±12.18%, Htt^ΔE1/ΔE1: 25.24±6.26%, p = 0.8964. C) Quantification of the percentage of nuclei containing γH2AX⁺ puncta before and after γ-irradiation. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001, mean±SEM, unpaired t-test, 8–9 images/mouse, 3–5 mice/genotype. Cortex: Htt^+/+: 3 h post-irradiation: 59.81±4.67%, p = 0.0002 compared to control; 24 h post-irradiation: 29.54±3.67%, p = 0.0583 compared to control, p = 0.0043 compared to 3 h post-irradiation; Htt^ΔE1/ΔE1: 3 h post-irradiation: 64.52±3.86%, p = 0.1334 compared to control, p = 0.4590 compared to Htt^+/+ 3 h post-irradiation; 24 h post-irradiation: 24.91±4.66%, p = 0.0771 compared to control, p = 0.0007 compared to 3h post-irradiation, p = 0.4788 compared to Htt^+/+ 24 h post-irradiation. Striatum: Htt^+/+: 3 h post-irradiation: 48.96±4.57%, p = 0.0001 compared to control; 24 h post-irradiation: 26.35±2.84%, p = 0.0015 compared to control, p = 0.0126 compared to 3 h post-irradiation; Htt^ΔE1/ΔE1: 3 h post-irradiation: 55.83±3.52%, p = 0.0292 compared to control, p = 0.2678 compared to Htt^+/+ 3 h post-irradiation; 24 h post-irradiation: 33.08±4.74%, p = 0.8862 compared to control, p = 0.0080 compared to 3 h post-irradiation, p = 0.2902 compared to Htt^+/+ 24 h post-irradiation.

The increased basal levels of DSBs observed in the Htt^ΔE1/ΔE1 cortex and striatum at 3 months of age could result from either an increase in DSB formation or from a deficit in DSB repair. To determine whether DNA repair is altered in the Htt^ΔE1/ΔE1 cortex and striatum, we induced DSB DNA damage with γ-irradiation and examined if DNA repair occurs in the Htt^ΔE1/ΔE1 mice at a comparable level to the Htt^+/+ controls. 3-month-old Htt^+/+ and Htt^ΔE1/ΔE1 mice were exposed to a single 7Gy dose of γ-irradiation, and their brains were collected at 3- or 24-h following irradiation. At 3 h after irradiation, there was a further increase in the percentage of nuclei with γH2AX⁺ puncta in the Htt^ΔE1/ΔE1 striatum (and a trend toward an increase in the cortex) (Fig. 5A, C), which then decreased by 24 h after irradiation (Fig. 5A, C). A similar response to γ-irradiation was observed in Htt^+/+ controls (Fig. 5A, C), and there was no significant difference in the percentage of nuclei with γH2AX⁺ puncta in a comparison between genotypes at either 3 or 24 h after γ-irradiation (Fig. 5A, C, 3 h post-irradiation cortex: Htt^+/+: 59.81±4.67%, Htt^ΔE1/ΔE1: 64.52±3.86%, p = 0.4590; striatum: Htt^+/+: 48.96±4.57%, Htt^ΔE1/ΔE1: 55.83±3.52%, p = 0.2678; 24 h post-irradiation cortex: Htt^+/+: 29.54±3.67%, Htt^ΔE1/ΔE1: 24.91±4.66%, p = 0.4788; striatum: Htt^+/+: 26.35±2.84%, Htt^ΔE1/ΔE1: 33.08±4.74%, p = 0.2902; mean±SEM, unpaired t-test, 8–9 images/mouse, 3–5 mice/genotype). These results suggest that DNA DSB repair following irradiation is not significantly impaired in the Htt^ΔE1/ΔE1 mice compared to the controls.

Pan-nuclear 53bp1 levels are elevated in the cortex and striatum of 3-month-old Htt^ΔE1/ΔE1 mice

We next examined the expression of 53bp1, a protein involved in the non-homologous end-joining (NHEJ) repair pathway for DNA DSBs, by immunohistochemistry (for review of 53bp1 functions, see [39]). We expected to observe an increase in 53bp1⁺ puncta at the sites of DNA DSBs in the Htt^ΔE1/ΔE1 brains where elevated γH2AX⁺ puncta were present, but instead, we detected pan-nuclear 53bp1 staining in both the cortex and striatum of Htt^ΔE1/ΔE1 mice at 3 months of age that is absent in the Htt^+/+ mice (Fig. 6A). By 12 months of age, pan-nuclear 53bp1 can be detected in the cortex and striatum of both the Htt^+/+ and Htt^ΔE1/ΔE1 mice (Fig. 6A).

Fig. 6

Pan-nuclear 53bp1 is elevated in the cortex and striatum of 3-month-old Htt^ΔE1/ΔE1 mice. A) Cortex and striatum from 2-, 3-, 4-, and 12-month-old Htt^+/+ (+/+) and Htt^ΔE1/ΔE1 (ΔE1/ΔE1) mice immunostained with a 53bp1 antibody (red). Nuclei were labeled with DAPI (blue), scale bar = 10μm. B) Quantification of the percentage of the nuclear area with 53bp1. ^*p < 0.05, ^**p < 0.01, mean±SEM, unpaired t-test, 9 images/mouse, 3–4 mice/genotype. Cortex: 2 months: Htt^+/+: 11.56±6.22%, Htt^ΔE1/ΔE1: 23.04±4.25%, p = 0.2018; 3 months: Htt^+/+: 3.73±2.61%, Htt^ΔE1/ΔE1: 44.29±9.69%, p = 0.0068; 4 months: Htt^+/+: 51.22±6.6%, Htt^ΔE1/ΔE1: 33.05±3.36%, p = 0.0702; 12 months: Htt^+/+: 45.70±11.875, Htt^ΔE1/ΔE1: 61.30±9.59%, p = 0.3790. Striatum: 2 months: Htt^+/+: 8.08±5.37%, Htt^ΔE1/ΔE1: 27.77±3.175, p = 0.0344; 3 months: Htt^+/+: 0.26±0.16%, Htt^ΔE1/ΔE1: 23.02±4.14%, p = 0.0015; 4 months: Htt^+/+: 27.72±6.51%, Htt^ΔE1/ΔE1: 18.49±4.80%, p = 0.3174; 12 months: Htt^+/+: 33.13±11.08%, Htt^ΔE1/ΔE1: 55.81±16.35%, p = 0.2849. C) Images of the cortex and striatum of 3-month-old Htt^ΔE1/ΔE1 mice immunostained with NeuN (green) and 53bp1 antibodies (red). Nuclei were labeled with DAPI (blue), scale bar = 10μm. D) Quantification of the percentage of total cells that are NeuN⁺ per image. Mean±SEM, unpaired t-test, 9 images/mouse, 4 mice/genotype. Cortex: Htt^+/+: 61.53±1.16%, Htt^ΔE1/ΔE1: 62.35±1.31%, p = 0.6559; striatum: Htt^+/+: 58.57±1.94%, Htt^ΔE1/ΔE1: 61.33±0.99%, p = 0.2521. E) Quantification of the percentage of NeuN⁺ cells with pan-nuclear 53bp1. ^***p < 0.001, ^****p < 0.0001, mean±SEM, unpaired t-test, 9 images/mouse, 4 mice/genotype. Cortex: Htt^+/+: 21.58±11.62%, Htt^ΔE1/ΔE1: 93.90±3.44%, p = 0.0010; striatum: Htt^+/+: 0.77±0.45%, Htt^ΔE1/ΔE1: 83.90±8.25%, p < 0.0001. F) Quantification of the percentage of NeuN^– cells with pan-nuclear 53bp1, ^**p < 0.01, mean±SEM, unpaired t-test, 9 images/mouse, 4 mice/genotype. Cortex: Htt^+/+: 0.89±0.52%, Htt^ΔE1/ΔE1: 20.04±5.01%, p = 0.0090; striatum: Htt^+/+: 0.07±0.07%, Htt^ΔE1/ΔE1: 4.23±2.14%, p = 0.0996.

To determine the age at which pan-nuclear 53bp1 is first elevated, additional brains from Htt^+/+ and Htt^ΔE1/ΔE1 mice at 2 and 4 months of age were collected and examined. In both cortex and striatum of Htt^+/+ mice, pan-nuclear 53bp1 levels are low at 2 and 3 months of age but are increased to a level comparable to that observed in Htt^ΔE1/ΔE1 mice at 4 months of age (Fig. 6A). At 4 months of age, there is also no difference between genotypes in the percentage of nuclei with γH2AX staining (cortex: Htt^+/+: 45.72±3.79%, Htt^ΔE1/ΔE1: 30.95±3.97%, p = 0.0545; striatum: Htt^+/+: 26.40±2.88%, Htt^ΔE1/ΔE1: 17.69±2.43%, p = 0.0821; mean±SEM, unpaired t-test, 9 images/mouse, 3 mice/genotype). Higher levels of pan-nuclear 53bp1 staining can be detected in the Htt^ΔE1/ΔE1 striatum at 2 and 3 months of age in comparison to the controls (Fig. 6A, B, 2 months: Htt^+/+: 8.08±5.37%, Htt^ΔE1/ΔE1: 27.77±3.17%, p = 0.0344; 3 months: Htt^+/+: 0.26±0.16%, Htt^ΔE1/ΔE1: 23.02±4.14%, p = 0.0015. mean±SEM, unpaired t-test, 9 images/mouse, 3–4 mice/genotype). An increase in the pan-nuclear 53bp1 staining can also be detected in the Htt^ΔE1/ΔE1 cortex at 3 months of age (Fig. 6A, B. Htt^+/+: 3.73±2.61%, Htt^ΔE1/ΔE1: 44.29±9.69%, p = 0.0068. mean±SEM, unpaired t-test, 9 images/mouse, 4 mice/genotype). To confirm these observations, 53bp1 levels in cortical tissue from 3-month-old Htt^ΔE1/ΔE1 and Htt^+/+mice were analyzed by western blotting. A higher level of 53bp1 was detected in Htt^ΔE1/ΔE1 cortical protein lysates in comparison to the controls (Supplementary Figure 3A, BHtt^+/+: 0.14±0.01, Htt^ΔE1/ΔE1: 0.17±0.01; p = 0.0257. mean±SEM, unpaired t-test, 3 mice/genotype).

To investigate if the pan-nuclear 53bp1 is present in neurons or glia, brain sections from 3-month-old Htt^+/+ (2 males and 2 females) and Htt^ΔE1/ΔE1 mice (1 male and 3 females) were co-immunostained with 53bp1 and NeuN (a protein present in many but not all neurons, for review, see [46]) antibodies (Fig. 6C). The percentage of NeuN⁺ cells was not significantly different between genotypes (Fig. 6D), but the percentage of NeuN⁺ cells with elevated 53bp1 levels was ∼72%higher in the Htt^ΔE1/ΔE1 cortex and ∼ 83%higher in the Htt^ΔE1/ΔE1 striatum compared to the controls. (Fig. 6E, cortex: Htt^+/+: 21.58±11.62%, Htt^ΔE1/ΔE1: 93.90±3.44%, p = 0.0010; striatum: Htt^+/+: 0.77±0.45%, Htt^ΔE1/ΔE1: 83.90±8.25%, p < 0.0001. mean±SEM, unpaired t-test, 9 images/mouse, 4 mice/genotype). A higher percentage of NeuN cells in the Htt^ΔE1/ΔE1 cortex also exhibited elevated nuclear 53bp1 levels in comparison to the controls (Fig. 6F, Htt^+/+: 0.89±0.52%, Htt^ΔE1/ΔE1: 20.04±5.01%, p = 0.0090, mean±SEM, unpaired t-test, 9 images/mouse, 4 mice/genotype). These NeuN^–53bp1⁺ cells (∼8%of the total cells) could be either NeuN^– neurons or glial cells. In contrast, very few NeuN^– cells in the striatum exhibited elevated levels of 53bp1 in both genotypes (Fig. 6F). These results suggest that the elevated levels of pan-nuclear 53bp1 occur predominantly in neurons.

Purkinje cell density and DNA damage marker levels are not altered in 3-month-old Htt^ΔE1/ΔE1 mice

Purkinje cell loss affects motor function [47, 48], and has been reported in mouse models of HD [49] and in postmortem brains of HD patients with primarily motor symptoms [50]. To determine if Purkinje cell loss could contribute to the rotarod deficit observed in the Htt^ΔE1/ΔE1 mice, Purkinje cell density was examined in 3-month-old Htt^+/+ and Htt^ΔE1/ΔE1 mice by immunohistochemistry using a calbindin antibody. There were no differences in Purkinje cell density between genotypes (Fig. 7A, B, Htt^+/+: 24.40±1.05 cells/mm, Htt^ΔE1/ΔE1: 25.12±0.62 cells, p = 0.5747. mean±SEM, unpaired t-test, 4 images/mouse, 4 mice/genotype).

Fig. 7

Purkinje cell density and DNA damage marker levels are not altered in 3-month-old Htt^ΔE1/ΔE1 mice. A) Cerebellum from 3-month-old Htt^+/+ (+/+) and Htt^ΔE1/ΔE1 (ΔE1/ΔE1) mice immunostained with a calbindin antibody (green). Nuclei were labeled with DAPI (blue), scale bar = 50μm. B) Quantification of the number of calbindin⁺ cells per mm. Mean±SEM, unpaired t-test, 4 images/mouse, 4 mice/genotype. Htt^+/+: 24.40±1.05 cells, Htt^ΔE1/ΔE1: 25.12±0.62 cells, p = 0.5747. C) Cerebellum from 3-month-old Htt^+/+ (+/+) and Htt^ΔE1/ΔE1 (ΔE1/ΔE1) mice immunostained with 53bp1 (red) and calbindin (green) antibodies. Nuclei were labeled with DAPI (blue), scale bar = 10μm. D) Quantification of the percentage of calbindin⁺ cells with pan-nuclear 53bp1. Mean±SEM, unpaired t-test, 157–224 cells/mouse, 4 mice/genotype. Htt^+/+: 89.73±4.48%, Htt^ΔE1/ΔE1: 97.56±1.01%, p = 0.1390. E) Quantification of the percentage of calbindin⁺ cells with γH2AX puncta. Mean±SEM, unpaired t-test, 157–224 cells/mouse, 4 mice/genotype. Htt^+/+: 3.69±1.78%, Htt^ΔE1/ΔE1: 10.37±2.74%, p = 0.0870.

We did not observe a difference between genotypes in either DNA SSB or DSB damage in the cerebellum by comet assay (data not shown); however, Purkinje cells represent a small percentage of the total neurons in the mouse cerebellum (∼1 Purkinje cell per 175 granule cells, [51]). Therefore, we also examined γH2AX and 53bp1 levels in Purkinje cells by immunohistochemistry. Interestingly, in contrast to what we observed in the cortex and striatum, pan-nuclear 53bp1 staining was present in the majority of Purkinje cells in the Htt^+/+ mice at 3 months of age at a level similar to that observed in the Htt^ΔE1/ΔE1 cerebellum (Fig. 7C, D, Htt^+/+: 89.73±4.48%, Htt^ΔE1/ΔE1: 97.56±1.01%, p = 0.1390. Mean±SEM, unpaired t-test, 157–224 cells/mouse, 4 mice/genotype). No differences in the percentage of Purkinje cells with γH2AX puncta were observed between genotypes (Fig. 7E). These data suggest that loss of the Htt N-terminus does not affect cell density or the levels of DNA damage repair markers in Purkinje cells.

DNA and protein oxidation products are not altered in 3-month-old Htt^ΔE1/ΔE1 brains

In a cell culture model, the N17 domain was shown to act as a sensor for oxidative stress [36], suggesting that without exon 1-encoded domains, oxidative damage could be altered in the Htt^ΔE1/ΔE1 mice. To determine whether DNA oxidation levels are altered in the cortex and striatum of 3-month-old Htt^ΔE1/ΔE1 mice in comparison to controls, isolated nuclei were embedded in agarose and lysed under alkaline conditions to expose 8-hydroxy-2'-deoxyguanosine (8-OHdG), a common product of DNA oxidation [52]. Alkaline treatment denatures DNA and increases nuclear volume that together helps to expose the 8-OHdG epitopes, but the treatment also results in a more diffuse DAPI staining (Fig. 8A). No differences in 8-OHdG levels between genotypes were observed in either the cortical or the striatal nuclei, but the amount of 8-OHdG detected in both Htt^+/+ and Htt^ΔE1/ΔE1 nuclei was low (Fig. 8A, B). To determine whether increased oxidative stress would differentially affect 8-OHdG levels in the Htt^ΔE1/ΔE1 and Htt^+/+ cortex and striatum, KBrO₃ was administered to 3-month-old Htt^+/+ and Htt^ΔE1/ΔE1 mice in their drinking water at 2 g/L for 7 days to induce oxidative DNA damage. Administration of KBrO₃, which is a renal carcinogen that preferentially produces 8-OHdG lesions [53], also elevates reactive oxygen species in the brain [54]. Although 8-OHdG levels were elevated in cortical and striatal nuclei isolated from both Htt^+/+ and Htt^ΔE1/ΔE1 mice after KBrO₃ treatment, no significant differences between genotypes were observed (Fig. 8A, B) and no sex-specific differences were observed (data not shown).

Fig. 8

DNA and protein oxidation markers are not significantly altered in Htt^ΔE1/ΔE1 mice at 3 months of age. A) Nuclei isolated from the cortex and striatum of 3-month-old Htt^ΔE1/ΔE1 (ΔE1/ΔE1) and Htt^+/+ (+/+) mice with or without KBrO₃ treatment and immunostained with an 8-OHdG antibody (red). Nuclei were labeled with DAPI (blue), scale bar = 10μm. B) Quantification of the percentage of DAPI⁺ pixels that are 8-OHdG⁺. Mean±SEM, unpaired t-test, 19–20 images/mouse, 3 mice/genotype. Cortex: without KBrO₃: Htt^+/+: 2.40±1.04%, Htt^ΔE1/ΔE1: 1.17±0.29%, p = 0.3229; with KBrO₃: Htt^+/+: 15.64±2.20%, Htt^ΔE1/ΔE1: 12.51±1.72%, p = 0.3247; striatum: without KBrO₃: Htt^+/+: 1.57±0.51%, Htt^ΔE1/ΔE1: 1.81±0.59%, p = 0.7717; with KBrO₃: Htt^+/+: 24.90±8.72%, Htt^ΔE1/ΔE1: 16.27±4.51%, p = 0.4285. C) Western blots of cortical and striatal lysates probed with a DNP antibody to evaluate protein carbonyl levels. D) Protein carbonyl levels were quantified and normalized to β-actin. Mean±SEM, unpaired t-test, 3 mice/genotype. Cortex: Htt^+/+: 2.43×10⁷±1.39×10⁶, Htt^ΔE1/ΔE1: 2.47×10⁷±1.52×10⁶, p = 0.8317; striatum: Htt^+/+: 2.38×10⁷±1.14×10⁶, Htt^ΔE1/ΔE1: 2.68×10⁷±1.29×10⁶, p = 0.1566.

To examine protein oxidation levels, total proteins from the cortex and striatum of 3-month-old Htt^+/+ and Htt^ΔE1/ΔE1 mice were used to assess the level of protein carbonyls, a product of protein oxidation [55], by western blotting. No differences in the levels of protein carbonyls were observed between genotypes (Fig. 8C, D). The absence of altered DNA and protein oxidation product levels in the Htt^ΔE1/ΔE1 cortex and striatum in comparison to Htt^+/+ controls suggests that deletion of the Htt N-terminal domains does not significantly affect the levels of oxidative damage.

Autophagy markers are not altered under basal conditions in Htt^ΔE1/ΔE1 mice

To determine whether steady-state autophagy is affected by the ΔE1-Htt N-terminal deletion, we evaluated the levels of selective autophagy markers LC3 and p62 in the microsomal protein fractions obtained from dissected cortex and striatum of 3-month-old Htt^+/+ and Htt^ΔE1/ΔE1 mice. During autophagosome assembly, phosphatidylethanolamine is conjugated to cytosolic LC3 (LC3-I), that is then incorporated into the autophagosome membrane as LC3-II (56–58). LC3-II is subsequently linked to ubiquitinated cargo though the adaptor protein p62 [56, 59]. We found no significant differences in the levels of LC3-II or p62 between genotypes (Supplementary Figure 4A-C).

We also evaluated levels of the chaperone-mediated autophagy (CMA) marker Lamp2A in the microsomal protein fractions obtained from dissected cortex of 3-month-old Htt^+/+ and Htt^ΔE1/ΔE1 mice. Lamp2A is a CMA-specific lysosomal membrane receptor, and CMA rates are correlated with Lamp2A levels [60]. We observed no significant differences in the levels of Lamp2A between genotypes (Supplementary Figure 4D, E).

To ascertain that autophagy is not affected in aged animals, we performed LC3 western blotting using whole brain microsomal fractions from 24-month-old Htt^+/+ and Htt^ΔE1/ΔE1 mice, and no difference in the LC3-II to LC3-I ratio was detected between genotypes (Supplementary Figure 4F, G). To confirm that the levels of autophagosomes in Htt^ΔE1/ΔE1 mice were not altered, we examined the co-localization of LC3 and p62 in the cortex and striatum of 3-month-old Htt^+/+ and Htt^ΔE1/ΔE1 mice by immunohistochemistry, where we did not observe a difference between genotypes (Supplementary Figure 4H, I). These data suggest that basal levels of selective autophagy and CMA are not affected in Htt^ΔE1/ΔE1 mouse brains in vivo.

Glutamatergic corticocortical, corticostriatal, thalamocortical, and thalamostriatal synapse numbers are not altered in Htt^ΔE1/ΔE1 mice at 18 months of age

Mice with a conditional knockout of cortical Htt exhibit altered numbers of glutamatergic synapses in the cortex and striatum, and mice with a conditional knockout of Htt in striatal projection neurons exhibit altered numbers of inhibitory synapses in the external globus pallidus [15, 16]. Additionally, Htt^ΔN17/ΔN17 mice exhibit a reduction in glutamatergic thalamostriatal synapses at 24 months of age. To determine whether Htt^ΔE1/ΔE1 mice exhibit a phenotype similar to the Htt^ΔN17/ΔN17 mice, colocalization of the presynaptic markers Vglut1 (expressed in cortical projection neurons) or Vglut2 (expressed in thalamic projection neurons) with the post-synaptic marker PSD95 was quantified in the cortex and striatum of 18-month-old Htt^ΔE1/ΔE1 and Htt^+/+ mice. There were no significant differences in the numbers of glutamatergic corticocortical, corticostriatal, thalamocortical, or thalamostriatal synapses between genotypes (Supplementary Figure 5A-D). In addition, no significant differences in the levels of Vglut1, Vglut2, and PSD95 expression were detected in whole brain microsomal fractions from 24-month-old Htt^ΔE1/ΔE1 and Htt^+/+ mice (Supplementary Figure 5E-H).

DISCUSSION

Htt^ΔE1/ΔE1 progeny from Htt^ΔE1/+ x Htt^ΔE1/+ crosses were born at the expected Mendelian frequency and survived up to 24 months of age, suggesting that the domains encoded by exon 1 are not required for Htt’s critical role during embryogenesis or for survival in adulthood. This is consistent with prior work showing that transgenic expression of an N-terminal truncated fragment of HTT lacking the first 169 aa can support survival in adult mice that lack normal Htt expression [28].

We do not know the cause of the sex-specific non-Mendelian genotype frequency observed in the progeny from Htt^ΔE1/+ intercrosses. Two independent Htt^ΔE1 knock-in lines were included in this study, and therefore off-target effects are not likely to contribute to our observation. This difference could be explained by an increase in Htt^ΔE1 Y and Htt⁺ X sperm with a similar decrease in Htt⁺ Y and Htt^ΔE1 X sperm, indicating that non-random meiotic chromosomal segregation could be a possible cause of this effect. Although no meiotic deficits were noted in mice with a conditional knockout of Htt in the testes [61], a small change in the segregation frequency might not be detected. Alternatively, a change in sperm swim rate or ability to fertilize an egg could skew the ratio of the expected progeny.

A retrospective analysis of progeny obtained from Htt^ΔQP/+ intercrosses in comparison to Htt^ΔN17/+, Htt^ΔQ/+, and Htt^ΔP/+ intercrosses suggests that a combined deletion of the HTT polyQ stretch and PRR may elicit a sex distortion phenotype. The difference in the sex distortion phenotype between the Htt^ΔQP/+ and Htt^ΔE1/+ intercrosses could be due to a contribution from the N17 domain deletion, smaller numbers of progeny analyzed from the Htt^ΔQP/+ intercrosses, or differences in genetic background (the single domain and combined polyQ and PRR deletion mice were in a mixed C57BL/6J and 129/Sv genetic background, while the Htt^ΔE1/+ mice are congenic in the C57BL/6J genetic background). A thorough characterization of gametes from Htt^ΔE1/+ mice and Htt^ΔQP/+ mice will be required to understand the sex-specific non-Mendelian transmission we observed. Other than the sex distortion in the progeny from the Htt^ΔE1/+ intercrosses, we did not detect any obvious differences between sexes in all analyses performed with both male and female mice.

Under basal conditions, we did not observe an alteration in the subcellular localization of ΔE1-Htt compared to WT-Htt. This result is consistent with prior observations showing that deletion of the Htt N17 domain does not affect the subcellular localization of ΔN17-Htt in vivo [31]. A second HTT membrane-association domain (aa172-372) is retained in ΔE1-Htt, as well as a nuclear export signal in the C-terminus [62, 63]. Our data does not exclude the possibility that differences in Htt localization exist in different brain regions or cell types that cannot be detected in whole brain lysate, or that there may be subtle differences in Htt localization that cannot be detected by western blotting. A brain region-specific study with a more sensitive detection method will be required to reveal subtle differences in Htt localization in vivo.

Previous evaluations of the role of Htt’s N-terminal domains in motor performance and spatial learning and memory have reported differential effects from the deletion of individual N-terminal Htt domains. Htt^ΔQ/ΔQ and Htt^ΔQP/ΔQP mice exhibited improved rotarod performance while rotarod performance was mostly unaffected in Htt^ΔN17/ΔN17 and Htt^ΔP/ΔP mice [31 –33]. In contrast, a modest deficit in rotarod performance was observed in the Htt^ΔE1/ΔE1 mice. At 18–19 months of age, Htt^ΔP/ΔP mice exhibited impaired Morris water maze performance, while no differences were observed in Htt^ΔN17/ΔN17, Htt^ΔQP/ΔQP, or Htt^ΔE1/ΔE1 mice compared to controls [31, 33]. However, a direct comparison of behavioral phenotypes between the Htt^ΔE1/ΔE1 mice and the previously characterized domain-deletion mice is difficult, as the Htt^ΔE1/ΔE1 mice are congenic in the C57BL/6J background while the Htt^ΔN17/ΔN17, Htt^ΔQ/ΔQ, Htt^ΔP/ΔP, and Htt^ΔQP/ΔQP mice were in a mixed C57BL/6J and 129/Sv genetic background. Nonetheless, the differential motor and cognitive performances observed between Htt^ΔN17/ΔN17, Htt^ΔQ/ΔQ, Htt^ΔP/ΔP, Htt^ΔQP/ΔQP, and Htt^ΔE1/ΔE1 mice suggest that the N17, polyQ, and PRR domains do not function independently of each other, consistent with the hypothesis that polyQ domain flexibility allows for interactions between the N17 and PRR domains [64]. Further experiments will be needed to address if there are any female-specific changes in behavior, as only male mice were used in our longitudinal behavioral analyses.

Elevated pan-nuclear 53bp1 levels and increased neuronal γH2AX puncta have been reported in postmortem Alzheimer’s disease brain tissue and from individuals with mild cognitive impairment in comparison to age-matched control tissue [65]. Elevated pan-nuclear 53bp1 has also been reported in fibroblasts collected from centenarians (aged 95–105) compared to fibroblasts from younger subjects (aged 65–80) [66]. The centenarian fibroblasts were able to resolve induced oxidative DNA damage more quickly than the fibroblasts obtained from younger subjects (aged 65–80) [66], suggesting that increased pan-nuclear 53bp1 levels may be a compensatory response to genotoxic stress. The increase in pan-nuclear 53bp1 levels observed in the Htt^ΔE1/ΔE1 cortex and striatum may therefore be a compensatory response to the elevated DSBs caused by either an early elevation in genotoxic stress or a subtle deficit in DSB repair. A similar elevation in pan-nuclear 53bp1 levels that occurs slightly later in the cortex and striatum of wild type mice could be due to normal aging. The brain regional differences in the timing of 53bp1 elevation that we observed may correlate with potential brain-regional susceptibilities to DNA damage.

ROS is a possible source of genotoxic stress, and the Htt N-terminus is able to respond to the presence of ROS in vitro [36]. DNA oxidation, however, results in the formation of multiple forms of DNA damage, including SSBs and DSBs [67]. The lack of elevated 8-OHdG in nuclei isolated from Htt^ΔE1/ΔE1 cortex and striatum is consistent with the lack of elevated SSBs detected by the comet assay in 3-month-old Htt^ΔE1/ΔE1 mice in comparison to Htt^+/+ mice and suggests that SSB repair is functioning normally in the Htt^ΔE1/ΔE1 mice. However, we cannot rule out the possibility that nuclei with relatively high levels of DNA damage were preferentially lost during the isolation process and were therefore not included in the comet assay or 8-OHdG immunostaining analyses. In addition to oxidative damage, other mechanisms could also cause increased DSBs in the brain, including neuronal activity [68]. Additionally, DNA repair in the cortex and striatum following γ-irradiation appeared to function normally in Htt^ΔE1/ΔE1 mice at 3 months of age, suggesting that DNA damage caused by γ-irradiation may recruit compensatory repair pathways that do not require participation of Htt’s N-terminal domains. Based on our data, we cannot differentiate between the possibilities that a small increase in genotoxicity, a subtle deficit in DNA repair, or a combination of both contribute to the elevated DSBs observed in the Htt^ΔE1/ΔE1 mice at 3 months of age.

Our results are consistent with prior studies demonstrating that deletion of the N17 domain or the polyQ and PRR domains together do not affect basal autophagy levels [31]. Htt^ΔN17/ΔN17 mice exhibit a slight reduction in glutamatergic thalamostriatal synapses at 24 months of age [31]; however, the numbers of glutamatergic corticocortical, corticostriatal, thalamocortical, and thalamostriatal synapses in 18-month-old Htt^ΔE1/ΔE1 mice are not significantly different from the numbers in Htt^+/+ mice. Both age and genetic background differences in the mice we examined may contribute to this discrepancy.

Similar to the phenotypes exhibited by Htt^ΔN17/ΔN17, Htt^ΔQ/ΔQ, Htt^ΔP/ΔP, and Htt^ΔQP/ΔQP mouse models, the behavioral and cellular phenotypes exhibited by adult Htt^ΔE1/ΔE1 mice are subtle and suggest that deletion of all three domains encoded by Htt exon 1 only modestly impact the efficiency of normal Htt functions in mice. Future experiments with conditional Htt domain deletions will be necessary to distinguish between a role for the Htt N-terminus in normal Htt functions during development and in adult tissues.

Footnotes

ACKNOWLEDGMENTS

This work was supported by an NIH training grant 4T32GM008328 (E. Braatz), F31NS083289 (E. André), and NIH/NINDS NS077926 and NS090914 (E. Braatz, J.-P. Liu, and S. Zeitlin).

CONFLICT OF INTEREST

The authors have no conflict of interest to report.

The supplementary material is available in the electronic version of this article: .

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Characterization of a Knock-In Mouse Model with a Huntingtin Exon 1 Deletion

Abstract

Background:

Objective:

Methods:

Results:

Conclusion:

Keywords

INTRODUCTION

MATERIALS AND METHODS

Generation of the HttΔE1 mouse allele

PCR genotyping

Mouse husbandry and behavioral analyses

Brain tissue lysate preparation and subcellular fractionation

Western blotting

Immunohistochemistry

Comet assay and nuclear immunostaining

Image and statistical analyses

RESULTS

Generation of the HttΔE1 mouse allele

The Htt N-terminus encoded by exon 1 is not essential for embryonic survival

Deletion of Htt’s N-terminal domains has no apparent effect on ΔE1-Htt’s subcellular localization

HttΔE1/ΔE1 mice exhibit a modest rotarod deficit

Double-strand DNA breaks are increased in the cortex and striatum of 3-month-old HttΔE1/ΔE1 mice

Pan-nuclear 53bp1 levels are elevated in the cortex and striatum of 3-month-old HttΔE1/ΔE1 mice

Purkinje cell density and DNA damage marker levels are not altered in 3-month-old HttΔE1/ΔE1 mice

DNA and protein oxidation products are not altered in 3-month-old HttΔE1/ΔE1 brains

Autophagy markers are not altered under basal conditions in HttΔE1/ΔE1 mice

Glutamatergic corticocortical, corticostriatal, thalamocortical, and thalamostriatal synapse numbers are not altered in HttΔE1/ΔE1 mice at 18 months of age

DISCUSSION

Footnotes

ACKNOWLEDGMENTS

CONFLICT OF INTEREST

References

Generation of the Htt^ΔE1 mouse allele

Generation of the Htt^ΔE1 mouse allele

Htt^ΔE1/ΔE1 mice exhibit a modest rotarod deficit

Double-strand DNA breaks are increased in the cortex and striatum of 3-month-old Htt^ΔE1/ΔE1 mice

Pan-nuclear 53bp1 levels are elevated in the cortex and striatum of 3-month-old Htt^ΔE1/ΔE1 mice

Purkinje cell density and DNA damage marker levels are not altered in 3-month-old Htt^ΔE1/ΔE1 mice

DNA and protein oxidation products are not altered in 3-month-old Htt^ΔE1/ΔE1 brains

Autophagy markers are not altered under basal conditions in Htt^ΔE1/ΔE1 mice

Glutamatergic corticocortical, corticostriatal, thalamocortical, and thalamostriatal synapse numbers are not altered in Htt^ΔE1/ΔE1 mice at 18 months of age