Cell-specific Dyt1 ∆GAG knock-in to basal ganglia and cerebellum reveal differential effects on motor behavior and sensorimotor network function

Abstract

Dystonia is a neurological movement disorder characterized by repetitive, unintentional movements and disabling postures that result from sustained or intermittent muscle contractions. The basal ganglia and cerebellum have received substantial focus in studying DYT1 dystonia. It remains unclear how cell-specific ∆GAG mutation of torsinA within specific cells of the basal ganglia or cerebellum affects motor performance, somatosensory network connectivity, and microstructure. In order to achieve this goal, we generated two genetically modified mouse models: in model 1 we performed Dyt1 ∆GAG conditional knock-in (KI) in neurons that express dopamine-2 receptors (D2-KI), and in model 2 we performed Dyt1 ∆GAG conditional KI in Purkinje cells of the cerebellum (Pcp2-KI). In both of these models, we used functional magnetic resonance imaging (fMRI) to assess sensory-evoked brain activation and resting-state functional connectivity, and diffusion MRI to assess brain microstructure. We found that D2-KI mutant mice had motor deficits, abnormal sensory-evoked brain activation in the somatosensory cortex, as well as increased functional connectivity of the anterior medulla with cortex. In contrast, we found that Pcp2-KI mice had improved motor performance, reduced sensory-evoked brain activation in the striatum and midbrain, as well as reduced functional connectivity of the striatum with the anterior medulla. These findings suggest that (1) D2 cell-specific Dyt1 ∆GAG mediated torsinA dysfunction in the basal ganglia results in detrimental effects on the sensorimotor network and motor output, and (2) Purkinje cell-specific Dyt1 ∆GAG mediated torsinA dysfunction in the cerebellum results in compensatory changes in the sensorimotor network that protect against dystonia-like motor deficits.

Keywords: Dyt1; Purkinje cells; basal ganglia; cerebellum; diffusion MRI; dopamine-2 receptor; dystonia; fMRI; functional connectivity; sensorimotor; torsinA.

Figure 1.. Imaging setup.

Imaging was performed on an 11.1 Tesla MRI scanner with a Magnex Scientific 40 cm horizontal magnet (Bruker BioSpin, Billerica, MA). For imaging, animals were placed on a custom-designed mouse platform, which included anesthesia provided through a head stabilizer with a surface transmit/receive radiofrequency head coil. Core body temperature was maintained during imaging with a heated waterbed tube placed underneath the animal. A Medoc PATHWAY heating thermode was placed on the right hindlimb of the animal and controlled by an associated computer setup.

Figure 2.. Regions of Interest.

The top panel (A) shows diffusion MRI regions of interest overlaid on top of the fractional anisotropy template image: cerebellum (CER; orange), vermis (VER; yellow), posterior medulla (pMED; light green), anterior medulla (aMED; dark green), pons (PON; pink), midbrain (MID; turquoise), substantia nigra (SN; magenta), thalamus (THA; dark blue), globus pallidus (GP; orange), somatosensory cortex (SS; purple), primary motor cortex (M1; light blue), and striatum (STR; red).

Figure 3.. Motor performance in D2-KI and Pcp2-KI mice.

In the D2-KI cohort, mutant mice displayed a significant (*p < 0.05, reduced model) beam-walking deficit (A), but no difference in rotarod performance (B). In contrast, Pcp2-KI mutant mice showed no difference in beam-walking performance (C), but significantly improved performance (*p < 0.05 in the full model, p = 0.069 in the reduced model) on the rotarod (D). Data are shown as the mean number of slips for beam-walking and the mean latency to fall for rotarod (error bars: SEM).

Figure 4.. D2-KI sensory-evoked BOLD.

(A) The somatosensory cortex region of interest overlaid on top of mouse anatomical template image. (B) Blood oxygen level dependent (BOLD) percent signal change in the somatosensory cortex as a result of the sensory-evoked fMRI paradigm. There was a significant interaction between genotype and time-course, such that D2-KI mice (red) had blunted BOLD signal during the latter portion of sustained stimulus compared to controls (blue).

Figure 5.. Pcp2-KI sensory-evoked BOLD.

The top panel shows regions of interest for the striatum (A) and midbrain (B) regions of interest overlaid on top of the mouse anatomical template image. The bottom panel shows sensory-evoked blood oxygen level dependent (BOLD) percent signal change in the striatum (C) and midbrain (D), which showed a significant effect of genotype such that Pcp2-KI mice (red) had lower BOLD signal during 20–60 period after stimulus onset compared to controls (blue) in both brain regions.

Figure 6.. D2-KI resting-state functional connectivity.

Coronal slices representing functional connectivity from the anterior medulla seed (A). Group means are shown for D2-KI mutant mice (B) and control mice (C). Red colors show voxels with a high degree of functional connectivity and blue colors show voxels with low degree of functional connectivity. We observed significant differences (p_corrected < 0.05 ) in functional connectivity between D2-KI mutant and control mice, such that mutants had increased functional connectivity between the anterior medulla and a voxel cluster in the secondary somatosensory cortex (D).

Figure 7.. Pcp2-KI resting-state functional connectivity.

Coronal slices representing functional connectivity from the striatum seed (A). Group means are shown for Pcp2-KI mutant mice (B) and control mice (C). Red colors show voxels with a high degree of functional connectivity and blue colors show voxels with low degree of functional connectivity. We observed significant differences (p_corrected < 0.05 ) in functional connectivity between Pcp2-KI mutant and control mice, such that mutants had increased functional connectivity between the striatum and a voxel cluster in the medulla (D).