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Factors that Contribute to Huntington’s Disease Pathogenesis

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Huntington’s Disease Awareness Month

Huntington’s disease (HD) develops due to a rare genetic mutation in the huntingtin gene (Htt), leading to progressive neurodegeneration in the cortex and striatum (1). Normal Htt contain about 18 CAG repeats, whereas mutated ones have 40 or more (2). The length of CAG repeat predicts the age of HD onset, but the length of polyQ repeats—encoded by CAG and CAA—is not as important to the overall HD pathogenesis (3–5). Once expressed, mutated huntingtin aggregates and triggers other neuropathological changes, including striatal-selective degeneration of medium spiny neurons (MSN), astrocytosis, and microgliosis (6). As a result, patients with HD develop motor dysfunction, cognitive impairment, and psychiatric symptoms (1). It’s estimated that HD affects about 5 in every 100,000 people worldwide, and a cure for this disease is currently unavailable (7). 

Preclinical models play a critical role in the study of HD pathophysiology and the development of new therapies. They help elucidate the molecular mechanisms leading to disease onset and progression and make testing the efficacy of new drugs easier. However, available preclinical models fail to replicate all aspects of human HD at once (8,9). New mice models combining more core characteristics of human HD can advance the knowledge of HD pathophysiology and potentially contribute to developing new therapies.  

With this goal in mind, Gu et al. developed and described a new transgenic mouse model of HD that expresses full-length human mutant huntingtin protein (9). They generated the new model using bacterial artificial chromosome (BAC), encoding about 120 uninterrupted CAG repeats, hence the name BAC-CAG. Mice developed many pathophysiological changes replicating human HD, including progressive motor deficits, sleep disturbance, striatal-selective nuclear inclusion, synaptic loss, astrogliosis, and microgliosis. Other key attributes of the new preclinical model of HD included minimal weight gain, somatic CAG repeat instability, and striatum-selective transcriptional dysregulation. In addition, data from the Gu et al. study revealed that striatal-selective neuropathogenesis associates with the length of CAG repeat.  

Keep reading to learn more about the Gu et al. study, and check out other publication highlights on the blog. 

The Need for Novel Preclinical Models of Human HD  

Many preclinical models of HD are available. Each particular model develops some characteristics of human HD, but no single model presents with them all (Table 1) (8). For example, knockin models of HD express mutant huntingtin—either human or murine, full-length or truncated—under the endogenous Htt gene locus (8). They develop motor deficits but don’t show signs of sleep disturbances, which are also commonly observed in HD patients (8). Other mouse models of HD were generated expressing full-length human mutant huntingtin using either BAC or yeast artificial chromosome (YAC) (8). BACHD and YAC128 are examples of HD mouse models commonly used in research studies, but many others exist (8). Overexpression of mutant protein in these models associates with unintended excessive weight gain, which is not a characteristic of human HD (Table 1). In addition, they fail to replicate many aspects of human HD pathogenesis, such as uninterrupted long CAG repeats and striatal-selective transcriptional dysregulation (Table 1) (8). This leads to a partial understanding of HD pathophysiology and limited applicability to the development of new drugs. 

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Table 1. Huntington’s Disease Mouse Model Comparison

The new preclinical model developed by Gu et al.—BAC-CAG—is a BAC transgenic mouse model that expresses full-length human mutant huntingtin with about 120 uninterrupted CAG repeats. BAC-CAG expresses huntingtin protein at a lower level than other mouse models of HD. Thus, BAC-CAG mice didn’t gain excessive body weight throughout the course of the study.  

BAC-CAG Mice Replicates Behavioral Alterations Observed in Human HD 

Gu et al. assessed behavioral alterations in BAC-CAG mice at different ages and observed that performance in the accelerated rotarod started to decline at 6 months. In this behavioral test, mice need to balance and walk forward on an accelerated rotating rod to avoid falling off, and latency to fall is recorded as a measure of motor coordination (10). Impairment in grip strength, another measure of motor function, developed later, at 12 months, in BAC-CAG mice.  

Patients with HD also develop sleep disturbances, and symptoms start to appear during the early stages of the disease (11). Likewise, BAC-CAG mice slept less and had a more fragmented sleep than wild-type mice. In addition, BAC-CAG mice showed reduced nighttime activity over 10 days—mice are nocturnal animals—compared with wild-type controls.   

Neuropathological Changes in HD Mice are Similar to Those Observed in Patients with HD 

Next, Gu et al. assessed whether BAC-CAG mice replicate various aspects of striatal-specific HD pathophysiology, including MSN loss, astrogliosis, microgliosis, presence of nuclear inclusions, and aggregation of mutant huntingtin (6). Using brain sections immunostained for Actn2, an MSN postsynaptic marker, Gu et al. observed that fluorescence intensity in the striata was lower in BAC-CAG than in wild-type mice at 12 months. Researchers also quantified MSN spine density using MORF3/Camk2a-CreER mice, which provides spatially spare fluorescent labeling of neuronal cells (12). In agreement with Actn2 immunofluorescence data, 12-month-old BAC-CAG mice displayed reduced spine densities in striata compared with wild-type mice.  

BAC-CAG mice showed morphological evidence of astrocytosis and microgliosis, other key hallmarks of human HD pathophysiology. Gu et al. performed immunostaining for GFAP, an astrocyte marker, in brain sections from 12-month-old BAC-CAG and wild-type mice. Quantification of immunofluorescence intensity revealed that BAC-CAG showed higher expression of GFAP in the striata and corpus callosum than wild-type mice. In addition, glial cells in BAC-CAG mice had a hypertrophic morphology. Together, both changes indicate that astrogliosis is part of the neuropathology of HD in the BAC-CAG mouse model. Researchers also quantified the number of Iba1+ and Gal3+ microglia cells in striata from 12-month-old BAC-CAG and wild-type mice. After immunohistochemistry and staining with Vector® SG Substrate Kit, Peroxidase (HRP), cell quantification revealed that BAC-CAG mice had more reactive microglia than wild-type mice.  

At 12 months, BAC-CAG mice exhibited striatum-selective nuclear inclusions containing mutant huntingtin, a feature of human HD present in knockin models but absent from other BAC and YAC transgenic mice. In addition, the striatum-selective nuclear inclusions further increased at 18 months, affecting about 98% of striatal neurons. BAC-CAG mice also showed the presence of aggregated mutant huntingtin in the striatum, which increased from 12 to 18 months. At 18 months, BAC-CAG mice also started to display aggregated mutant huntingtin in the cortex and cerebellum. However, researchers didn’t observe changes in soluble mutant huntingtin at any age.  

Transcriptional Dysregulation in BAC-CAG Mice Correlate with CAG Repeat Length 

BAC-CAG mice also displayed age-dependent striatum-selective transcriptional dysregulation. RNA-seq analysis at 2, 6, and 12 months showed that BAC-CAG mice exhibited dysregulation of gene expression in the striatum but not in the cortex. Mice exhibited transcriptional dysregulation as early as 6 months, but the number of differentially expressed genes increased with age. In addition, changes in gene transcription observed in BAC-CAG partially overlapped with those observed in the brains of patients with HD.  

Transcriptomic changes in the BAC-CAG model also shed light on a novel finding related to HD pathophysiology. Transcriptional dysregulation strongly correlated with the length of CAG repeats but not the length of polyQ. In knockin mice models of HD, the length of CAG and Q repeats matches. But because Q is encoded by CAG and CAA, human genomic models can have long Q repeats with short CAG repeats. Data from Gu et al. showed that CAG repeat length is a critical driving factor of HD transcriptionopathy. 

BAC-CAG mice also showed the presence of CAG repeat instability in somatic tissues, another pathological characteristic of human HD. Gu et al. observed the presence of PCR products amplified from human mutated huntingtin in the striatum, cortex, cerebellum, liver, heart, testis, and tail from 2-month-old BAC-CAG mice. In addition, at 12 months, somatic CAG instability increased in the striatum and liver but not in other tissue types from BAC-CAG mice.   

The presence of repeat-associated non-AUG (RAN) proteins translated from either CUG or CAG transcripts promotes cellular toxicity. Gu et al. used immunohistochemistry targeting the C-terminal region of polySer to detect the expression of RAN proteins on brain sections of 12, 18, and 22-month-old BAC-CAG mice. After incubation with primary and secondary antibodies, researchers used VECTASTAIN® Elite® ABC-HRP Kit, Peroxidase (Standard) and ImmPACT NovaRED® Substrate Kit, Peroxidase (HRP) to stain targeted cells. BAC-CAG but not wild-type mice expressed polySer RAN-positive cells in the striatum and cortex at 18 and 22 months. Researchers detected no changes in RAN protein levels in 12-month-old BAC-CAG mice. Most behavioral, pathological, and molecular changes start to develop in BAC-CAG mice at 12 months or earlier. Thus, the accumulation of RAN proteins is unlikely to contribute to disease onset but could contribute to disease progression.  

The new BAC-CAG mouse model of HD replicates more characteristics of human HD than any of the previous models. In addition, data acquired using BAC-CAG mice provided new insight into HD pathophysiology and revealed that CAG repeat length correlates with transcriptional dysregulation. The molecular, pathological, and behavioral characteristics of this new mouse model make it a promising candidate for testing therapies targeting gene expression levels and CAG repeat-induced toxicity. In addition, future studies using the BAC-CAG model might help to unravel new mechanisms underlying the pathophysiology of human HD. Altogether, these outcomes could potentially impact the lives of patients with this disease.   

Check out other resources in the blog to learn about other scientific advances and tips & tricks to improve your experiments.  

References 

  1. Ross CA, et al. 2014. Huntington Disease: Natural History, Biomarkers and Prospects for Therapeutics. Nature Reviews Neurology.
  2. Warby SC, et al. 2009. CAG Expansion in the Huntington Disease Gene is Associated With a Specific and Targetable Predisposing Haplogroup. The American Journal of Human Genetics.
  3. Genetic Modifiers of Huntington’s Disease  (GeM-HD) Consortium. 2019. CAG Repeat Not Polyglutamine Length Determines Timing of Huntington’s Disease Onset. Cell.
  4. Orr HT, et al. 2007. Trinucleotide Repeat Disorders. Annual Review of Neuroscience.
  5. Wright GEB, et al. 2019. Length of Uninterrupted CAG, Independent of Polyglutamine Size, Results in Increased Somatic Instability, Hastening Onset of Huntington Disease. The American Journal of Human Genetics.
  6. Vonsattel JPG, et al. 1998. Huntington Disease. Journal of Neuropathology & Experimental Neurology.
  7. Medina A, et al. 2022. Prevalence and Incidence of Huntington’s Disease: An Updated Systematic Review and Meta-Analysis. Movement Disorders.
  8. Ferrante RJ. 2009. Mouse Models of Huntington’s Disease and Methodological Considerations for Therapeutic Trials. Biochimica et Biophysica Acta (BBA) – Molecular Basis of Disease.
  9. Gu X, et al. 2022. Uninterrupted CAG Repeat Drives Striatum-Selective Transcriptionopathy and Nuclear Pathogenesis in Human Huntingtin BAC Mice. Neuron.
  10. Deacon RMJ. 2013. Measuring Motor Coordination in Mice. Journal of Visualized Experiments.
  11. Morton AJ. 2013. Circadian and Sleep Disorder in Huntington’s Disease. Experimental Neurology.
  12. Veldman MB, et al. 2020. Brainwide Genetic Sparse Cell Labeling to Illuminate the Morphology of Neurons and Glia with Cre-Dependent MORF Mice. Neuron.
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Camila Suhett, PhD