Dysregulation of dopamine receptor D2 as a sensitive measure for Huntington disease pathology in model mice

Abstract

The ability to quantitatively evaluate the impact of a potential therapeutic intervention for Huntington disease (HD) in animal models for the disease is a critical step in the pathway to development of an effective therapy for this devastating neurodegenerative disorder. We report here an approach that combines a cell-based assay's quantitative accuracy and direct relationship to molecular processes with the ability to directly monitor effects in HD model mouse neurons. To accomplish this goal, we have developed an accurate quantitative reporter assay for a transcript known to be down-regulated as an early consequence of mutant huntingtin expression. This system uses mouse strains carrying a GFP reporter for the expression of the dopamine receptor D2, expressed in the medium spiny neurons of the basal ganglion. This receptor consistently demonstrates reduced expression in patients and murine models, and the FACS-based assay gives a highly accurate and quantitative readout of this pathology in mouse neurons expressing mutant huntingtin. For four genetic models and one viral model, a highly reproducible time course of loss of reporter expression is observed. This quantitative measure of HD pathology can be used to measure the effects of HD therapeutics in small cohorts with high confidence. We further demonstrate that the introduction of an shRNA against the huntingtin transgene by virus can improve this pathological status in medium spiny neurons transduced with the construct. We believe this system can be of great utility in the validation of effective therapeutic interventions for HD.neurodegeneration | polyglutamine | viral vectors | gene expression | neuroprotection I n the search for effective therapeutic interventions for neurodegenerative diseases such as ALS, Parkinson disease, Alzheimer's disease, and Huntington disease (HD), cell and animal models for all of these conditions have been developed. Whereas cell models allow higher throughput and lower cost, any therapeutic intervention initially evaluated in a cell-culture model must subsequently be assessed in an appropriate animal model system before clinical trials in humans can be considered. However, the inherent variance in quantitation of the behavioral or pathological readouts in animals has practical consequences, requiring large cohorts to detect all but the most overt benefits, which limits experimental throughput. We sought to develop and implement an approach to this problem that combines the quantitative accuracy and direct relationship to molecular processes of a cell-based assay with the medical relevance of evaluating endogenous neurons in the animal brain.In the present study, we have focused on HD. The uniform genetic etiology of HD, caused by a poly(CAG) expansion within exon 1 of huntingtin (HTT) (1), has provided an excellent starting point for the derivation of animal models for the disease. A number of similar animal models have been developed and characterized by behavioral and/or morphometric assessments of pathology (reviewed in...

Synthetic zinc finger repressors reduce mutant huntingtin expression in the brain of R6/2 mice

“…RNA fold-repression hATN1 (16,22) hATXN1 (12,29) hATXN2 (13,23) hATXN3 (7,14) hATXN7 (10) hCACNA1A (13) hTBP (18,38) hHTT (mut 45/wt 21) titer) into the other brain hemisphere. ZFP striatal expression was confirmed by Western blot (Fig.…”

“…The rAAV pseudotype 2/1 (AAV2/1), containing AAV2 terminal repeats and AAV1 capsid, efficiently transduces neurons (and most glial and some ependymal cells) and appears to be the most efficient pseudotype for transducing large volumes of the striatum (37). R6/2 mice are one of the most widely used models of HD because they show highly reproducible early-onset symptoms, allowing the use of fewer animals (38). Although R6/2 mice have very long CAG repeats (range: 115-160 repeats), which are rarely found in patients (typically in the range of 42-45 repeats), they are nonetheless ideal to assay acute phenotypic reversal at the molecular, histological, and early-symptom levels (39).…”

“…CAG-WPRE constructs were injected into the striatum of one brain hemisphere, with AAV2/1-GFP control injections (equal STHdH hATN1 (16,22) hATXN1 (12,29) hATXN2 (13,23) hATXN3 (7,14) hATXN7 (10) hCACNA1A (13) hTBP (18,38) hHTT (21,23) mATN1 (3,10) mATXN1 (2) mATXN2 (6,10) mATXN3 (5,6) mRgs12 (N//A) mATXN7 (5,7) mCACNA1A (2,3) mTBP (3,13) mGRK4 (N/A) STHdH:…”

Profilin reduces aggregation and phase separation of huntingtin N-terminal fragments by preferentially binding to soluble monomers and oligomers

“…The age of onset as well as severity of HD are inversely correlated with the length of the polyglutamine (polyQ) tract within the N-terminal region of huntingtin (Htt) (32). The disease is characterized by deposition of insoluble intranuclear deposits of Htt-NTFs with expanded polyQ tracts in afflicted neurons (33,34). Htt-NTFs consist of a 17-residue N-terminal stretch (MATLEKLMKAFESLKSF) designated as N17, a polyQ tract of length n (Q n ), and a 38-residue C-terminal stretch (C38) that includes two polyproline (polyP) modules P 11 and P 10 connected by a 17-residue linker denoted as L17 (QLPQPPPQAQPLLPQPQ) (Figure 1a).…”

TAK-063, a Novel Phosphodiesterase 10A Inhibitor, Protects from Striatal Neurodegeneration and Ameliorates Behavioral Deficits in the R6/2 Mouse Model of Huntington’s Disease

“…Those phenotypes might reflect the higher vulnerability of indirect pathway MSNs than direct pathway MSNs at earlier phases, although the direct evidence is limited. Interestingly, green fluorescent protein, selectively expressed in indirect pathway MSNs under Drd2 promoter control, was reduced from early stages of disease progression in R6/2, R6/1, CAG140, and HdhQ111 mice (Crook and Housman, 2012). Those HD model mice might not completely replicate the HD pathology; however, these mice would be useful for preclinical evaluation of potential therapeutics for the treatment of HD.…”