120 CAG R6/2 Mouse Model of Huntington’s Disease
Huntington’s disease (HD) is an incurable neurodegenerative disease characterized by abnormal motor movements, personality changes and early death. The disease typically starts in midlife and symptoms progress over the course of 15–20 years until death. The mutation responsible for HD is an unstable expansion of CAG repeats in the gene encoding huntingtin (Htt) protein giving rise to an abnormal number of glutamine repeats (polyQ) which will accumulate and form aggregates in the nucleus. These aggregates, called inclusion bodies (IBs), are the pathological hallmark of HD.
In collaboration with the CHDI Foundation, we employed molecular, behavioral, histological, and electrophysiological assessments of dysfunction in pre- and post-symptomatic R6/2 HD mouse models, expressing the N-terminal (exon 1) portion of huntingtin carrying around 120 CAG repeats. 120 CAG R6/2 mice remain the animal model of choice for preclinical testing, showing Huntington aggregation, neuronal loss, motor and cognition impairment, and deficits in synaptic transmission and plasticity, change in expression of molecular markers, and the kynurenine pathway.
Htt Aggregation and Brain Atrophy
Increased levels of aggregated Htt can be detected by mEM48 antibody at symptomatic ages in 120 CAG R6/2 mice. Aggregation of mutant huntingtin is accompanied by a decrease in the percentage of striatal DARPP-32-positive medium spiny neurons and a significant atrophy of both the dorsal striatum and the entire cerebral cortex.
Figure 1: Color composite depicting labeling of aggregated Htt by mEM48 (green), total Htt by LS-C204217 (red), and medium spiny neurons by DARPP-32 (blue) antibodies. Aggregates are concentrated in the striatal areas such as the caudate putamen (CPu) and in DARPP-32 positive neurons in deep cortical layers. Images above show the genotypic difference for the single labeling in more detail. Colored rectangles indicate the cut-out position as presented in the center. DAPI (white) was used to visualize nuclei.
(2B) Total Htt
Figure 2A – 2C: (A) mEM48-positive Htt aggregates in 120 CAG R6/2 (Left). Note the absence of Htt aggregates in WT mice (Right) in contrast the pathological nuclear accumulation of aggregates in medium spiny neurons are clearly present in 120 CAG R6/2 mouse. (B) Also, levels of total Htt protein increases in the 120 CAG R6/2 mice as compared to WT. (C) 120 CAG R6/2 shows a loss of medium spiny neurons as well as a general loss of DARPP-32-positive neuropil labeling. DARPP-32 positive neurons in deep cortical layers are widely absent in 120 CAG R6/2 mice.
Figure 3A-C: Histological quantification of (A) mEM48, (B) DARPP-32 and (C) atrophy in the dorsal striatum (Left) and the cerebral cortex (Right) of R6/2 versus WT. 120 CAG R6/2 mice at the age of 12 weeks show a strong increase of mEM48 positive aggregates, a loss of MSN as judged by reduction of DARPP-32 labeling. They also demonstrate a severe atrophy of the dorsal striatum and cerebral cortex.
Detection of Htt Aggregations by Filter Assay
Figure 4: Huntington filter aggregation assay in striatal tissue from 120 CAG R6/2 at different ages from 2 to 16 weeks. Huntington aggregation is observed in R6/2 in an age-dependent manner.
120 CAG R6/2 mice, the model of choice for preclinical testing, show a progressive decline with loss of motor function around 6 weeks of age, loss of body weight around 10 weeks of age, and premature death.
Figure 5: Kaplan-Meier curve recording death event from day of birth up to 25 weeks of age. No 120 CAG R6/2 mice survived past 23 weeks of age with a 50% survival at 16 weeks of age.
120 CAG R6/2 Mice Are Lighter Than WT Littermates
Figure 6: Body weight curve from 4 to 22 weeks age. 120 CAG R6/2 mice are significantly lighter than WT littermates from 10 weeks of age.
120 CAG R6/2 Mice Have Reduced Ambulatory Activity
Figure 7: Total traveled distance in open field chambers. Data was collected in 5 minutes bins during 30 minute sessions at 4, 6, 8, and 12 weeks of age. 120 CAG R6/2 mice displayed a lower total distance traveled compared to their WT littermates at each age timepoint.
Increased Percentage of 120 CAG R6/2 Mice Displaying Full Clasping With Age
Figure 8: Percentage of 120 CAG R6/2mice displaying a four limb clasp from 5 to 12 weeks of age. Only 120 CAG R6/2 mice display full clasping with age as this neurological response is absent in age-matched WT littermates.
120 CAG R6/2 Mice Have Poor Performance in Rotarod
Figure 9: Latency to fall collected from 3 consecutive trials at 4, 6, 8 and 12 weeks of age. 120 CAG R6/2 mice demonstrated a loss of motor coordination as early as 6 weeks of age, with a faster latency to fall when compared to their WT littermates.
Progressive Downregulation of Striatal mRNA Transcripts
The 120 CAG R6/2 mouse model showed progressive neurodegeneration in the striatum, recapitulating what is observed in Huntington’s disease (HD). Transcripts for medium spiny neuron markers- Drd1, DARPP32, DrD2, pde10A and cnr1- are downregulated in R6/2 in an age-dependent manner.
Figure 10A – E: Quantitative PCR (qPCR) analysis of striatal tissue from 120 CAG R6/2 at different ages (WT in gray; R6/2 in red). Relative level of target genes (A: Drd1=D1 receptor, B: Darpp32, C: Drd2=D2 receptor, D: Pde10a=cGMP phosphodiesterase 10A, and E: Cnr1=cannabinoid receptor 1) were normalized by the geometric mean of housekeeping genes (Gapdh, ATP5b, Elf4a2) and then normalized to the 2 week old WT average (n=24 6-week old mice; n=12-24 per age for ages 2-12 weeks).
Hyperexcitability in Dorsal Striatal Medium Spiny Neurons (MSNs) in 120 CAG R6/2 Mice
Medium spiny neuron (MSN) within the striatum, have been the most extensively studied cell type in HD mouse models. Ex vivo electrophysiological recordings of MSNs from acutely prepared slices using whole-cell patch clamp technique, show an age dependent hyperexcitability phenotype and some deficits in synaptic transmission. Somatic recordings indicate that cells become modestly depolarized, exhibit reduced rheobase in response to current injection, and exhibit significantly higher membrane resistance around resting state in figure below (Right Panel), implying the loss of hyperpolarizing conductance that maintain MSN quiescence. Additionally, the frequency of miniature excitatory synaptic potentials recorded in MSNs is significantly reduced, suggesting weakening or loss of cortical input (Left Panel).
Figure 11: Striatal MSNs from 8-12 week old R6/2 120 CAG mice exhibit elevated input resistance (Rm) (Left Panel) and reduced frequency of spontaneous excitatory post-synaptic currents (mEPSCs) (Right Panel). The amplitude of mEPSCs was unaffected (not shown). These readouts are consistent with both intrinsic hyperexcitability and the loss of excitatory synapses. Animal numbers are indicated in parentheses.
Alterations of Kynurenine Pathway Metabolites in the Striatum of 12-13 Weeks Old 120 CAG R6/2 Mice
Comparison of striatum levels of kynurenine metabolites in 12-13 weeks old 120 CAG R6/2 vs. wild type mice demonstrated a significant increase in the neurotoxic metabolite 3-hydroxykynurenine in R6/2 mice and this neurotoxin is believed to play a causative role in HD. Such changes in kynurenine metabolism are associated with neuroinflammation and have been demonstrated to be involved in numerous CNS conditions (HD, Parkinson’s, Alzheimer, depression, Schizophrenia and ALS). In particular, increased levels of quinolinic acid have been associated with suicidality in humans, so it is important to measure changes in kynurenine metabolites to monitor disease progression.
Figure 12: LC/MS/MS results for measures of Kynurenine and related analytes in striatal tissue of the 120 CAG R6/2 vs. wild type mice. Neuroactive metabolites of the kynurenine pathway (KP) of tryptophan degradation have been implicated in the pathophysiology of neurodegenerative disorders, including HD. Similar to HD patients, significant impact is observed on these important neurochemicals in the 120 CAG R6/2 mice. Same measures are available in microdialysis studies, and other matrices, all with the option to measure drug level.
Bioanalysis & Microdialysis