Huntington’s disease (HD) is characterized by preferential loss of the medium spiny neurons in the striatum. Using CRISPR/Cas9 and somatic nuclear transfer technology, we established a knockin (KI) pig model of HD that endogenously expresses full-length mutant huntingtin (HTT). By breeding this HD pig model, we have successfully obtained F1 and F2 generation KI pigs. Characterization of founder and F1 KI pigs shows consistent movement, behavioral abnormalities, and early death, which are germline transmittable. More importantly, brains of HD KI pig display striking and selective degeneration of striatal medium spiny neurons. Thus, using a large animal model of HD, we demonstrate for the first time that overt and selective neurodegeneration seen in HD patients can be recapitulated by endogenously expressed mutant proteins in large mammals, a finding that also underscores the importance of using large mammals to investigate the pathogenesis of neurodegenerative diseases and their therapeutics.

Our previous studies showed that pigs overexpressing transgenic mutant HTT did not survive (), a fact that has prevented us from investigating neurodegeneration in adult animals. We used CRISPR/Cas9 to insert a large CAG repeat (150 CAGs) into the endogenous pig HTT gene in fibroblast cells and employed the SCNT to generate a HD knockin (KI) pig model that expresses full-length mutant HTT at the endogenous level. This KI model allowed us to explore whether misfolded proteins at the endogenous level can cause neurodegeneration in large mammals. We found germline transmittable neurological phenotypes in different generations of this KI pig model. More importantly, we provide convincing evidence that mutant HTT causes striking and selective neurodegeneration that recapitulates the typical neurodegeneration feature in HD. Our findings support the idea that species differences are critical for the nature of neuropathology and strengthen the rationale for using large mammals to investigate the pathogenesis of neurodegenerative diseases and to identify their therapeutics.

Pigs are genetically, anatomically, and physiologically closer to humans than are small mammals. More importantly, the existing genetic manipulation tools enable the generation of a variety of pig models of human diseases (). Somatic cell nuclear transfer (SCNT) in combination with CRISPR/Cas9 allows for genetic modifications of the endogenous pig genes (). The SCNT leads to non-chimeric animals in the first generation that may recapitulate endogenous genetic mutation-associated phenotypes. In addition, the fast breeding period (5–6 months for sexual maturation) and large litter size (average 7–8 piglets) of pigs hold obvious advantages over non-human primates when considering the time line of generating large animal models of human diseases.

The monogenic mutation feature of HD makes the disease ideal for investigating the pathogenesis of misfolded proteins common in neurodegenerative diseases. By expressing mutant HTT containing an expanded CAG repeat in different species, a variety of genetically modified animal models of HD have been established and characterized. Among these models, mouse models of HD have been widely used and provide valuable information regarding the pathogenesis and therapeutic development of HD. Although HD mouse models show age-dependent accumulation of mutant huntingtin and its associated neurological symptoms, the HD knockin mouse models, which express mutant HTT at the endogenous level, lack the overt and striking neurodegeneration, a typical pathological hallmark of HD (). Similar to HD mouse models, other genetically modified mouse models, including models for AD and PD, that express different types of misfolded proteins also show the absence of overt and selective neurodegeneration (). There are considerable differences between rodents and primates. For example, the striatum, which is the most affected region in HD, consists of the caudate nucleus and putamen in primate brains. However, the caudate nucleus and putamen are indistinguishable in rodents. The differences in neuropathology among rodent and human brains indicate that species differences determine the nature of neuropathology and also highlight the demand for investigation of larger mammals that are closer to humans.

A variety of neurodegenerative diseases, including Alzheimer’s disease (AD), Parkinson’s disease (PD), Huntington’s disease (HD), and amyotrophic lateral sclerosis (ALS), share the common features of age-dependent neurological symptoms and selective neuronal degeneration (). Current research indicates all these diseases show the accumulation of misfolded proteins in the brain (). However, the cause of the accumulation of misfolded proteins varies by disease, and the mechanisms underlying particular neurodegeneration phenomenon remain underinvestigated. While genetic mutations contribute to a small fraction of AD, PD, and ALS; HD results from a monogenetic mutation, which is a CAG repeat expansion in the exon1 of the gene for huntingtin (HTT) (). HTT is a multifaceted protein that is expressed ubiquitously and has numerous roles (). The CAG repeat expansion (>36 CAGs) in the HTT gene leads to a polyglutamine (polyQ) expansion that causes HTT to misfold and aggregate in the brain. Similarly, polyQ expansion causes at least 8 other neurodegenerative diseases, including various spinocerebellar ataxias and spinal-bulbar muscular atrophy ().

Axonal degeneration is the early neuropathological event in old HD KI mice (). In the F1 KI (F1-15) pig brain, we found substantial axonal degeneration in the striatum and its projection area, globus pallidus. Compared to axons in the pig brain of WT control, those in the HD KI pig brain showed reduced myelination and contained degenerated organelles ( Figures 7 C and 7D). Altogether, these findings offer convincing evidence that HD KI pig brains display severe neurodegeneration with a similar pattern to that in HD patient brains ().

We then performed electron microscopy (EM) to explore the nature of neuronal loss in the striatum of the F1 KI pig (F1-15). EM revealed dark neurons in the cortex and striatum in the KI pig brain, which are characterized by electron-dense cytoplasm and the absence of organelles and a nuclear membrane ( Figure 7 A). These dark neurons were frequently seen in the cortex than in the striatum, perhaps because more neurons in the striatum have been lost ( Figure S7 A). In the striatum, degenerated neurons displayed the electron-lucent cytoplasm showing degenerated organelles or swollen mitochondria ( Figure 7 A). Different types of neurodegeneration in the HD KI pig cortex and striatum suggest that nature of neuronal degeneration depends on cell type. We also frequently observed reactive astrocytes and microglial cells near degenerated neurons. These reactive glial cells display increased density in chromatin on the nuclear membrane, highly clumped heterochromatin. Also, increased cytoplasmic region with variable-sized cytoplasmic vacuoles, and gaps in the nuclear membranes were evident ( Figures 7 B and S7 B).

(B) Different types of reactive microglia (middle photograph) and astrocytes (right photograph) in the KI pig brain show the enlarged cytoplasm with various sizes of vacuoles and degenerated organelles as compared to normal microglia (M) and astrocyte (AS) in a WT pig brain. Scale bars; (A), 2 μm; (B), 1 μm.

(A) Electron microscopy revealing a dark neuron that is adjacent to an astrocyte (AS) in the middle photograph. Two degenerated neurons in the middle and right photographs show different appearances (electronic cytoplasmic dense or lucent) without clear nuclear membrane and structure.

(C and D) Demyelinated axons and degenerated axons containing dark or disintegrated organelles are evident in the striatum (C) and globus pallidus (D) in the HD KI pig brain. In the globus pallidus, a reactive microglial cell (arrow) is among the demyelinated axons. An age-matched WT control served as a control.

(B) Reactive glial cells in the HD KI pig brain display dense chromatin on the nuclear membrane, variable-sized cytoplasmic vacuoles, and swollen mitochondria. Microglial cells are identified by having electron-dense and highly clumped heterochromatin, distinctly accumulated under the nuclear envelope. Astrocytes are characterized by their oval or round nuclei with an irregular ring beneath the nuclear envelope and evenly dispersed accumulations in the interior of the nucleus.

(A) Electron microscopy showed dark neurons in the cortex and striatum of the F1-15 KI pig brain. In the cortex, most degenerated neurons show electron-dense cytoplasm with no clear organelles and no identifiable nuclear membrane. In the striatum, degenerated neurons show an electron-lucent cytoplasmic region containing degenerated or swollen mitochondria and organelles and irregular and disintegrated nuclear membranes. Astrocytes (AS) and microglial (M) cells are in the vicinity of the neurons.

Although the striatum is the most affected brain region in HD, the neuronal loss in the striatum has a temporospatial distribution and occurs only with the medium spiny neurons while interneurons are primarily spared (). Examining 163 clinically diagnosed cases of HD revealed that the caudate nucleus was preferentially degenerated versus the putamen in early HD stages (). To investigate whether HD KI pig can recapitulate this pathological feature, we freshly isolated the brain of symptomatic HD F1 KI pigs (F1-14 and F1-15) at 5 months old and immediately fixed the brain with paraformaldehyde. Immunostaining of the striatum with an antibody to DAPRR-32, which is selectively expressed in the medium spiny neurons, clearly showed the greater reduction of DAPRR-32-positive cells in the caudate nucleus than the putamen ( Figure 6 A). We also used anti-calbindin D28k that specifically labels the medium spiny neurons as well and confirmed the reduced number of medium spiny neurons in the striatum ( Figures S6 C and S6D). There are different types of interneurons that express parvalbumin, neuropeptide Y (NPY), or choline acetyltransferase (ChAT) (). Despite the scarcity of these interneurons in the striatum, their density in the striatum is not different between WT and KI pig brains ( Figure 6 B). Quantitation of the relative numbers of DARRP-32 neurons and interneurons confirmed that only medium spiny neurons degenerated in the striatum as compared with different types of interneurons ( Figure 6 C).

(C) Quantitative analysis of the relative number of DARRP-32-positive medium spiny neurons and different types of interneurons in the striatum of the F1 KI and WT pigs.

(A) DARRP-32 staining shows fewer medium spiny neurons labeled in the caudate than in the putamen in the HD KI (F1-15) pig at 5 months old.

The selective loss of NeuN-positive cells was also confirmed by western blotting results, which showed a marked decrease in the NeuN level in the striatum as compared with the cortex and cerebellum ( Figure 5 C), and quantitative analysis of the relative level of NeuN (ratio to actin in Figure 5 D). Also, increases in GFAP level, which reflect an early neurodegenerative event, occurred in the striatum and cortex of the HD F1 KI pig brain ( Figure 5 D).

In HD F1 KI (FI-14) pig brains, we also saw the most severe loss of NeuN-positive cells in the striatum ( Figure 5 A). Because the neuronal loss in F1 pigs represents an important pathology that is germline transmissible, we performed unbiased stereology on 3 WT and 3 symptomatic F1 KI pigs at 4–5 months old to quantify the neuropathology in F1 pig brains. Compared with WT controls, the density of NeuN-positive cells is decreased to the greater extent in the caudate (WT 108,858 ± 7,449; KI 53,569 ± 4,908;p = 0.0034) and putamen in KI pigs (WT 111,398 ± 1,565, KI 76,936 ± 6,404,p = 0.0064) than the cortex (WT 143,727 ± 4,070, KI 113,704 ± 9,969,p = 0.0494). No significant difference is seen in the cerebellum in KI and WT pigs (WT 666,820 ± 8,989, KI 683,743 ± 3,633, p = 0.8699). We also quantified GFAP-positive cells and found there are a greater number of GFAP-positive cells in the caudate (WT 36,153 ± 2,121, KI 71,428 ± 3,735,p = 0.0012) and putamen (WT 34,235 ± 4,683, KI 72,045 ± 6,964,p = 0.0108) than the cortex (WT 31,284 ± 2,376, KI 52,183 ± 3,882,p = 0.0101), though the number of GFAP cells was similar in the WT (62,506 ± 3,219) and KI (62,862 ± 1,853) cerebellum. For reactive microglial cells, we quantified IBA1-positive cells. Increased IBA1 cells were also found in the cortex of F1 KI pig brains (WT 37,969 ± 6,252 KI, 63,715 ± 6,091p = 0.0136), putamen (WT 44,311 ± 2,362, KI 74,596 ± 4,364,p = 0.0036), caudate (WT 44,504 ± 3,809, KI 86,246 ± 3,690,p = 0.0014), but not in the cerebellum (WT 48,997 ± 2,299, KI 55,061 ± 3,808, p = 0.2445) ( Figure 5 B).

(D) Quantitation of the ratios of GFAP or NeuN to β-actin on the western blots (n = 6 fields/each brain section from 6 brain sections from 2 animals/per group). Western blot analysis was repeated independently at least three times.

(C) Western blotting of the brain tissues of WT and F1 KI founders with 1C2 for mutant HTT and antibodies against NeuN or GFAP. Actin served as a loading control.

(B) Stereology analysis of NeuN-, GFAP-, and IBA1-positive cells in the dorsal caudate nucleus, putamen, prefrontal cortex, and the anterior lobe of the cerebellum in WT and F1 KI pigs. Three F1 KI pigs and WT pigs at 4–5 months old were examined. ∗ p < 0.05; ∗∗ p < 0.01.

(A) NeuN immunostaining reveals more extensive neuronal loss in the striatum than in the cortex of F1 KI (F1-14) pig as compared to the WT control at 5 months old. Scale bars, 20 μm.

It is known that reactive gliosis with increased GFAP, an astrocytic protein, is the early pathological event in HD KI mouse brains (). Immunohistochemical staining showed the increased staining in GFAP in the F0 KI pig brain ( Figure 4 C). Also, immunostaining with the antibody to IBA1, a microglial cell marker, showed a marked increase in its labeling in the KI pig brain, which is also more abundant in the striatum than the cortex ( Figure S6 A). Quantification of the number of different types of cells revealed that the KI striatum had the most severe loss of NeuN-positive cells and the highest increase in glial cell numbers ( Figures 4 D and S6 B).

(C) Immunostaining of the F1 KI pig striatum (caudate and putamen) with anti-calbindin-D28k showing a fewer numbers of calbindin-D28k-positive cells in the caudate than the putamen.

(B) Quantitative analysis of IBA1-positive microglial cells per image (20X) in WT and HD KI pig brains. The average numbers of IBA1-positive cells were obtained by examining at least 6 randomly selected images per brain section, 6 sections from 3 animals per group, and data are presented as mean ± SEM, ∗∗∗ p < 0.001 by Student’s-test.

(A) The brain striatum from WT and HD F1 KI pigs (F1-15) at 5 months of age were stained with antibodies to IBA1 for identifying microglial cells. Scale bars, 50 μm.

To provide substantial evidence for the neurodegeneration in HD KI pig brains, we performed immunohistochemical experiments. Immunostaining of the HD KI pig brains with mouse anti-HTT (mEM48) revealed the distribution of mutant HTT and small aggregates in the neurites and bodies of neuronal cells, and 1C2 immunostaining, which selectively labels expanded CAG repeats, also revealed mutant HTT aggregates in the nuclei ( Figure 4 A). It seems that mutant HTT aggregates in the pig brain have different conformations that are recognizable by different antibodies. We further performed immunostaining of the KI pig brains (F0-5 and F0-6) with antibody to NeuN, a neuronal marker protein, and found a drastic reduction in the number of NeuN-positive cells in the striatum ( Figure 4 B). Immunofluorescent labeling with nuclear staining allowed us to estimate the relative numbers of NeuN-positive cells over the total number of cells. This assay also showed the greater decrease of NeuN-positive cells in the striatum than the cortex. However, NeuN-positive cells in the KI cerebellum remained unaltered as compared to the WT control ( Figure S5 ).

(B) Quantitative analysis of NeuN-positive neuronal cells of the total cells per image (20X) in WT and HD KI pig brains. The average numbers of NeuN-positive cells were obtained by examining at least 8 randomly selected images per brain section, 6 sections from 3 animals per group, and data are presented as mean ± SEM, ∗ p < 0.05, ∗∗∗ p < 0.001 by Student’s-test. Scale bars, 100 μm.

(A) The brain cortex, striatum, and cerebellum from WT and HD F1 KI (F1-14) pigs at the age of 5 months were stained by immunofluorescent staining with anti-NeuN to label neuronal cells and DAPI to label the nuclei of cells. Note that the striatum of the HD KI pig brain shows the most significant reduction of NeuN-positive cells.

(D) Quantification of the numbers of NeuN-positive or GFAP-positive cells in WT and KI pigs (n = 6 fields/each brain section from 6 brain sections from three animals/per group).

(B) Anti-NeuN immunostaining of the cortex, striatum, and cerebellum of WT and HD KI pigs (F0-5 and F0-6) at 5 months old. NeuN immunostaining reveals more extensive neuronal loss in the striatum than in the cortex of the HD KI pig as compared to the WT control at 5 months old. Scale bars, 50 μm.

(A) EM48 and 1C2 immunocytochemistry of the brain striatum of wild-type (WT) and HD F1 KI pig (F1-14) at 5 months old. Neuropil aggregates (middle) and nuclear aggregates (right) were evident by immunostaining with mouse antibodies EM48 and 1C2, respectively. Scale bars, 20 μm.

For brain pathology analysis, we examined 2 F0 and 3 F1 pigs ( Table S3 ). We isolated the brains of the F1 KI pigs (F1-14 and F1-15) at 5 months old and found that the brain size of these pigs was reduced when compared with the age-matched WT pigs. The thickness of the cortex and size of the striatum were smaller than those in the WT pig brain ( Figures 3 A and 3B ). MRI analysis of some symptomatic HD KI pigs at the age of 5 months also showed an enlarged lateral ventricle and the reduced size of the striatum when compared with the age-matched WT control ( Figure 3 C). Quantification of the volumes of the striatum and lateral ventricle in MRI confirmed the reduced volumes in HD KI pig brains as compared with WT pig brains ( Figure 3 D).

(D) The volume of the striatum and of lateral ventricle was decreased in HD KI pigs as observed by MRI.

(C) MRI analysis reveals T2-weighted coronal images of live WT (n = 3) and HD KI (n = 4) pigs at approximately 5 months old. The arrowheads indicate the enlargement of the frontal horns of the lateral ventricles in the HD KI pig brain compared with WT pig brain.

(B) Brain weight and ratios of the caudate and putamen to the total brain size in the WT (n = 4) and HD-KI (n = 4) pig brain at 5 months old. HD KI pigs showed reduced brain weight and decreased volume of the caudate and the putamen. ∗ p < 0.05.

(A) Gross morphology of the HD F1 KI (F1-14 and F1-15) pig brains at 5 months old showing the reduced thickness of their cortex (arrowheads) and decreased size of the caudate (c) and putamen (p) as compared with the age-matched WT control.

Analyzing CAG repeats in different pig generations showed that the CAG repeat is unstable in KI pigs ( Figure S4 C). We further analyzed the CAG repeat numbers in different tissues in F0 (F0-5, F0-6) and F1 (F1-14, F1-15) pigs. The results showed that there are different CAG repeats in different tissues ( Figures S4 D and S4E). We also compared the repeat numbers in the ear tissues of KI pigs of different generations and found that the CAG repeat is unstable during both male and female germline transmission ( Table S2 ). However, it remains to be determined how the repeat numbers change during germline transmission when more KI pigs are available. Although the instability of the CAG repeat has been reported previously in the rodents (), expansion of the CAG repeats appears to be more frequent in the pig genome than the mouse genome. Interestingly, a pig carrying 206 CAGs (F1-7) lived longer than other pigs carrying 113–138 CAGs ( Figure S4 C), suggesting that the CAG repeat length is not the sole determinant of the lifespan in these HD KI pigs and that environmental stress and other factors can also influence their lifespan. Indeed, we found that individually housing F1 KI pigs could reduce environmental stress and made them to live longer than F0 KI pigs that were kept in a large group. Also, the F1 KI pigs were generated by mating female Rongshui F0 pig with Bama male pigs so that F1 KI pigs carry mixed genetic backgrounds (Rongshui/Bama), which could also influence the age of onset. It also seems that the breathing difficulty is unique to the pig KI model since it is not found in mice and other animal models of HD.

For F1 KI pigs (F1-14 and F1-15), western blotting also verified the expression of full-length mutant HTT and its fragments in the cortex, striatum, and cerebellum ( Figure 2 F), with a similar pattern to that in the F0 KI brains. Further, we also observed less body weight gain and the early death of F1 KI pigs ( Figure 2 G) and similar difficulties in body movement and breathing ( Movies S3 and S4 ), indicating that F1 KI pigs shared the similar phenotypes as F0 KI pigs ( Table S1 ). For the dead KI founders (F0-3), gross examination revealed lung edema with hemorrhaging and extensively dilated alveolar lumen ( Figures S4 A and S4B), further supporting the idea that respiratory failure was likely the cause of animal death.

(C) The death and the age at death as well as the CAG repeat numbers of HD KI pigs in different generations.

(B) Quantification of the area and length of alveolar lumen indicates the dilation of the alveolar lumen in the HD KI lung. ∗∗∗ p < 0.001 by Student’s t test.

(A) The pathology of the HD KI pig (F0-3) lung is evident by edema with hemorrhaging (arrows, upper panel) and extensively dilated alveolar lumen (red arrow) that is revealed by hematoxylin-eosin staining (lower panel). Scale bars, 50 μm.

HD KI pigs did not show obvious symptoms before the age of 4 months. By continuously monitoring the growth and body weights of HD KI founders, we saw that F0 KI pigs gained less body weight than the age- and sex-matched WT pigs ( Figure 2 B). The old HD KI pigs often displayed wrinkled and sagging skin on their bodies ( Figures 1 G, S2 A, and S2B), which is similar to transgenic TDP-43 pigs we generated previously (). Moreover, some of F0 KI pigs died earlier between the ages of 5 to 10 months ( Figure 2 B). F0 KI pigs often showed walking abnormalities ( Figure 2 C; Movie S1 ). Foot-print analysis demonstrated the abnormal walking pattern with asymmetric steps and reduced distance between front and hind paw of the HD KI pigs as compared with WT pigs that showed normal alternate steps while walking ( Figures 2 D and 2E). Importantly, HD KI pigs displayed respiratory difficulty or irregular breathing patterns and abnormal movement ( Movie S1 ). The breath difficulty suggests that respiratory failure could contribute to the death of animals and is consistent with the observation that pulmonary dysfunction and aspiration pneumonia/suffocation are the major cause of the death in HD patients (). We also examined F0 KI pigs for their motor function using treadmill, as we did previously with our transgenic ALS pigs (). Video recording of a symptomatic HD KI pig at 5 months old (F0-5) revealed that this KI pig was unable to run as compared with its age- and sex-matched control ( Movie S2 ). Other F0 KI pigs (F0-6 and F0-7) showed similar running difficulties ( Figure S2 A). In addition, this HD KI pig died 2 days after the treadmill test, suggesting that the HD KI pig was susceptible to exercise stress. This post-treadmill death did not allow us to use the treadmill to continuously test more KI pigs for their limb movement impairment.

Given that the expanded CAG repeats are transmitted via germline cells, we wanted to know whether the HD KI pigs develop age-dependent neurological symptoms and whether these symptoms are transmittable via germline cells. First, we needed to verify the expression of full-length mutant HTT in the HD KI pigs. Thus, we isolated brain tissues from some F0 KI founders (F0-2, F0-3, and F0-6) at 5 months old and performed western blotting with 1C2 antibody that specifically reacts with the expanded polyQ repeat. Western blotting clearly showed the expression of full-length mutant HTT (arrow above 245 kDa in Figure 2 A) and multiple fragmented HTT products, which are absent in the brain tissues of WT pigs. The expression profiling of the intact full-length mutant HTT and its degraded products is very similar to that in HTT KI mouse brains (). As with mutant HTT in rodent brains, full-length mutant HTT in the pig brains also undergoes a proteolytic process to generate multiple N-terminal HTT fragments that carry 1C2-labeled polyQ repeats. Western blotting also suggests that mutant HTT is more abundant in the cortex than the striatum and cerebellum where the lowest level of HTT is seen. Such brain region-dependent expression levels were not found in the HTT KI mouse brains (). Thus, different expression levels of mutant HTT in distinct brain regions are likely species dependent.

(F) 1C2 western blots of the brain tissues from HD F1 KI pigs (F1-14, F1-15) at 5 months old. For western blots in (A) and (F), the full-length mutant HTT is indicated by an arrow. Arrowheads indicate non-specific bands. Western blot analysis was repeated independently at least three times.

CRISPR/Cas9 can break double-stranded DNAs to facilitate homologous recombination, a process that is required for genetic replacement of the targeted gene or genomic KI (). We designed two guide RNAs (gRNAs) to target the pig HTT intron after exon 1 to promote homologous recombination by replacing the pig HTT exon 1 with the human exon 1 containing a 150-CAG repeat. We transfected fetal pig fibroblast cells from a female Rongshui pig with the gRNAs and Cas9, as well as a donor vector that carries human HTT exon1 with 150-CAGs repeat flanked by two pig HTT DNA fragments (1 kb for each left and right arm) for homologous recombination ( Figure 1 A). We screened 2,430 fetal pig fibroblast cells by PCR and identified 9 positive cell clones that contained the heterozygous expanded human HTT exon1 in the right locus of the pig HTT gene. We selected a cell clone for SCNT and obtained 2,880 embryos, which were transferred into 16 surrogate pigs. Of these surrogate animals, 10 became pregnant, yielding some miscarried fetuses and 7 naturally delivered piglets ( Figure 1 B). Genotyping by PCR analysis identified that 6 piglets carried the human HTT exon1 with expanded CAG repeats ( Figure 1 C). The female founder (F0) pigs (Rongshui) were used to mate with wild-type (WT) male Bama miniature pigs () to generate F1 pigs (Rongshui/Bama), and the male F1 KI pigs were crossed with WT female Bama pigs to yield F2 generation pigs. This breeding over the last two years gave rise to 15 F1 pigs and 10 F2 pigs, which were all positive for the mutant HTT ( Figures 1 C–1E). PCR and DNA sequencing ( Figure S1 ) verified the human exon1 sequences and large CAG repeats in the targeted pig HTT allele in F1 and F2 KI pigs. Interestingly, genotyping revealed that the CAG repeats were unstable, ranging from 130 to 150 CAGs in the F0 founders, 113 to 206 CAGs in F1, and 118 to 230 CAGs in F2 generation ( Figure 1 F). The oldest F0 KI animal is 30 months old, and F1 KI pigs are 12 months old, whereas F2 KI pigs are newborn piglets ( Figures 1 G and S2 ). Whole-genome sequencing revealed no off-targets in the F1 KI pig brain cortex ( Figure S3 ). Thus, our characterization of the HD KI pigs was focused on F0 and F1 KI pigs to investigate their behavioral and pathological changes.

Whole genome sequencing analysis shows no off-target mutations in the cortex of F1 KI pig. Genomic DNAs from the cortical tissues of F1 KI (F0-6, F1-14, F1-15) pig were subjected to whole genome sequencing. Relative sequencing depth for most likely off-target loci by HTT gRNA-1 and gRNA-2 was calculated by normalizing the number of mapped reads in those loci to the genome-wide average of mapped reads. The on-target rate is not shown, as Cas9/sgRNA, generated nicks followed by homologous recombination that resulted in the same sequences as WT HTT for the on-target. Mismatched nucleotides are indicated in red.

(B) Photos show HD F1 KI pigs at the age of 5 months. Arrows indicate symptomatic HD KI (F1-11 and F1-13) pigs. The age-matched WT control pig is next to the HD KI pig (right panel).

(A) Photos of HD KI (F0-7) founder (arrow) at the age of 15 months (left panel) and HD KI (F0-6) and WT pigs at the age of 5 months during treadmill running test (right panel).

(A) PCR analysis of targeted allele in blood cells of the HD F0, F1, and F2 KI pigs. The PCR was performed using primers that could amplify the right arm of homologous DNA targeted in the endogenous locus of the pig HTT gene.

(F) The number of KI pigs in each generation and the range of the CAG repeat numbers in the HD KI pigs.

(C–E) PCR analysis of targeted allele containing the expanded CAG repeats in the ear tissues of the HD KI founders (F0) (C), F1 (D), and F2 (E) generation pigs. Note that the sizes of PCR products vary because of different CAG repeat numbers.

(A) Schematic diagram of the strategy to generate HD KI pigs via homologous recombination. Two gRNAs were used to target the pig HTT intron after exon 1 to promote DNA breaks and homologous recombination. The donor DNA consisting of human exon 1 HTT with 150 CAGs and homologous pig HTT sequences (left arm and right arm) was used to replace the endogenous pig exon 1 HTT in cultured pig fibroblast cells. Cells containing the knockin (KI) allele were identified via PCR and selected for somatic nuclear transfer technology.

Discussion

By establishing a HD KI pig model, our findings demonstrate for the first time that large mammals can recapitulate overt and selective neurodegeneration and the severe symptoms caused by the mutant protein expressed at the endogenous level. These findings also raise some important issues regarding the differences in the pathology and phenotypes between small and large mammalian animal models of neurodegenerative diseases.

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et al. A transgenic minipig model of Huntington’s disease. The most important finding in our study is the presence of robust and selective neurodegeneration in the HD KI pig brains, which mimics the severe and preferential neurodegeneration of the medium spiny neurons in HD patients. Mutant HTT preferentially targets the striatum in humans and mice (). Although mutant HTT can affect multiple types of cells and impaired cell-cell interactions contribute to HD pathology (), the medium spiny neurons are more vulnerable in the patient brains at the early stages of HD (). The severe neurodegeneration of medium spiny neurons in HD KI pig brains is evident by the loss of DARPP32- or calbindin-D28k-positive neurons, which are the majority of neurons (>90%) in the striatum. In contrast, the numbers of interneurons, which specifically express parvalbumin, NPY, or ChAT and are spared in HD, are not altered in HD KI pig brains compared with the WT control. Thus, this striatal degeneration in the KI pig brain remarkably recapitulates the selective degeneration of medium spiny neurons in HD. Also, the increased reactive gliosis, including increased GFAP and IBA1 staining, is evident for the glial response to neuronal damage. The transgenic pig model, which expresses the first 548 amino acids (aa) of HTT with 124 glutamines under the control of human HTT promoter, did not show distinct movement phenotypes and neurodegeneration (). The differences between HD KI and transgenic pigs suggest that the context and expression level of HTT are essential for neurodegeneration. Because the preferential loss of striatal medium spiny neurons in the HD KI pigs mimics the critical pathological feature in HD patients, this finding also points out the possibility of pig models for replicating selective neurodegeneration in other neurodegenerative diseases.

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Brooks S.P. Light and electron microscopic characterization of the evolution of cellular pathology in the Hdh(CAG)150 Huntington’s disease knock-in mouse. Ultrastructural alterations also confirm the severe loss of striatal neurons in HD pig brains revealed by EM, which identified dark neurons that were previously found in transgenic HD mice expressing small N-terminal HTT fragments (). Also, HD KI pig brains show degenerated axons and demyelination, as reported in HD KI mice (). However, these pathological changes were often found in old (>21 months) HD KI mice and these changes in HD KI mice were milder than those in HD KI pigs (). Considering the lifespan differences between rodent (average 2 years) and pig (average 15 years), neurodegeneration caused by large polyQ repeats in the HD KI pigs apparently occurs much earlier and to a more severe extent. It should be pointed out that the expression of a large polyQ repeat (150Q) in the KI pig brains is more likely to elicit the neuropathology resembling juvenile HD. However, stereological analysis showed that loss of medium spiny neurons in the sacrificed F1 KI pigs is not as robust as that in postmortem brains of HD patients. It is possible that the extent of neurodegeneration would become more severe if these F1 KI pigs were kept to live longer. The robust neurodegeneration in the HD KI pigs is undoubtedly an advantage of using the KI pig model to explore the mechanism underlying the selective neurodegeneration in HD and to develop effective therapeutics.

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et al. Full-length human mutant huntingtin with a stable polyglutamine repeat can elicit progressive and selective neuropathogenesis in BACHD mice. Why can HD KI pigs more faithfully recapitulate the phenotypes and neurodegeneration seen in HD patients? While addressing this issue would require substantial experiments to test a variety of hypotheses, several possible explanations exist. First, the species-dependent differences in lifespan, genomics, anatomy, and physiology play essential roles in determining the severity of neurodegeneration in different species. Indeed, the lack of distinguishable caudate nucleus and putamen structures in the rodent striatum accounts for the inability to mimic the preferential caudate degeneration in HD. Second, the development of the central nervous system is remarkably different in various species. The rapid development and maturation of the rodent brain may render neuronal cells resistant to toxic proteins. On the other hand, the toxic effect of misfolded proteins during the lengthy early brain development in large mammals may be required for the more severe neuropathology in adult brains after the differentiation and maturation of neuronal cells. In support of this idea, some studies show that toxic effects of misfolded proteins in the embryonic or postnatal stage can affect the development of neuropathology in the adult brains (). Also, HTT in the brains of small and large mammals may associate with different partners and function differentially. The pig HTT exhibited 96% peptide sequence homology to human huntingtin (). Since transgenic yeast artificial chromosome (YAC) and bacterial artificial chromosome (BAC) mice expressing human HTT with a large repeat show more severe neurodegenerative phenotypes than HD KI mice (), it is also possible that the large glutamine repeat in pig HTT can lead to the molecular changes that are more similar to those caused by mutant human HTT. Addressing the above possibilities would help in understanding the pathogenesis of neurodegenerative diseases.