A point mutation in the dynein heavy chain gene leads to striatal atrophy and compromises neurite outgrowth of striatal neurons

Abstract

The molecular motor dynein and its associated regulatory subunit dynactin have been implicated in several neurodegenerative conditions of the basal ganglia, such as Huntington's disease (HD) and Perry syndrome, an atypical Parkinson-like disease. This pathogenic role has been largely postulated from the existence of mutations in the dynactin subunit p150(Glued). However, dynactin is also able to act independently of dynein, and there is currently no direct evidence linking dynein to basal ganglia degeneration. To provide such evidence, we used here a mouse strain carrying a point mutation in the dynein heavy chain gene that impairs retrograde axonal transport. These mice exhibited motor and behavioural abnormalities including hindlimb clasping, early muscle weakness, incoordination and hyperactivity. In vivo brain imaging using magnetic resonance imaging showed striatal atrophy and lateral ventricle enlargement. In the striatum, altered dopamine signalling, decreased dopamine D1 and D2 receptor binding in positron emission tomography SCAN and prominent astrocytosis were observed, although there was no neuronal loss either in the striatum or substantia nigra. In vitro, dynein mutant striatal neurons displayed strongly impaired neuritic morphology. Altogether, these findings provide a direct genetic evidence for the requirement of dynein for the morphology and function of striatal neurons. Our study supports a role for dynein dysfunction in the pathogenesis of neurodegenerative disorders of the basal ganglia, such as Perry syndrome and HD.

Figure 1.

Locomotor and behavioural abnormalities in Cra/+ mice. (A) Grip muscle strength of forelimbs (left panel) and all limbs (right panel) in wild-type mice (+/+) and heterozygous Cra/+ mice at 3 and 12 months of age. ***P < 0.001 versus corresponding wild-type (n = 12 mice per group). (B) Latency to fall in an accelerating rotarod test in wild-type mice (+/+) and heterozygous Cra/+ mice at 3 and 12 months of age. ***P < 0.001 versus corresponding wild-type, ^#P < 0.05 versus 3-month-old Cra/+ mice (n = 12 mice per group). (C) Track length (left panel) and average velocity (right panel) in an open field test in wild-type mice (+/+) and heterozygous Cra/+ mice at 3 and 12 months of age. ***P < 0.001 versus corresponding wild-type (n = 12 mice per group). (D) Time spent in closed arms (upper panel) and open arms (lower panel) in an elevated plus maze test (during a 300 s session) by wild-type mice (+/+) and heterozygous Cra/+ mice at 3 and 12 months of age. Non-significant differences (n = 12 mice per group). (E) Time spent (upper panels) and distance swum (lower panel) to reach the hidden platform in a Morris water maze test by wild-type mice (+/+) and heterozygous Cra/+ mice at 3 (left panel) and 12 (right panel) months of age. The platform was in position A during the first 3 days of the test, and then moved to position C for the last 2 days. Six trials were performed per day, and each point is the mean of two consecutive trials. *P < 0.05 versus corresponding wild-type in time spent and non-significant differences in distance swum (n = 12 mice per group).

Figure 2.

Striatal atrophy in Cra/+ mice. (A) Wet weight of hindbrain (left) and forebrain (right) of wild-type mice (+/+, empty columns) and heterozygous Cra/+ mice (black columns) at 12 months of age. *P < 0.05 versus corresponding wild-type (n = 4 mice per group). (B) Low magnification photomicrographs of haematoxylin and eosin staining of wild-type (+/+) and heterozygous Cra/+ brains at 18 months of age. CC, corpus callosum. Scale bar = 100 µm. (C) Higher magnification of B showing the aspect of the six layers of the cortex. Scale bar = 50µm. (D) Representative horizontal T2-weighted MRI slices of a wild-type (+/+) and Cra/+ mouse at 5 and 10 months of age. Note the enlargement of the lateral ventricles (white arrows) of the Cra/+ mouse. (E) Striatal volume of wild-type (+/+, empty columns) and heterozygous Cra/+ mice (black columns) at 5 (left) and 10 (right) months of age. ***P < 0.001 versus corresponding wild-type (n = 20 mice per group). (F) Lateral ventricle volume of wild-type (+/+, empty columns) and heterozygous Cra/+ mice (black columns) at 5 (left) and 10 (right) months of age. ***P < 0.001 versus corresponding wild-type (n = 20 mice per group).

Figure 3.

Progressive striatal astrocytosis in Cra/+ mice. (A) Representative microphotographs showing haematoxylin/eosin staining (left panels) and GFAP immunoreactivity (right panels) in the striatum from wild-type mice (+/+, upper panels) and heterozygous Cra/+ mice (lower panels) at 18 months of age. Scale bar = 25 µm. (B) Quantification of the surface occupied by GFAP positive cells in the striatum from wild-type mice (+/+) and heterozygous Cra/+ mice at 8 and 18 months of age. Data are expressed as percentage of the total surface in the picture. *P < 0.05 versus indicated condition (n = 5 mice per group). (C) mRNA levels of GFAP in the striatum from wild-type mice (+/+) and heterozygous Cra/+ mice at 4 and 8 months of age. *P < 0.05 versus wild-type (n = 5–7 mice per group).

Figure 4.

No significant neuronal loss in the striatum of Cra/+ mice. (A and B) Representative photographs of striatal sections processed for DARPP-32 immunohistochemistry from wild-type mice (+/+) at 6 months of age as well as heterozygous Cra/+ mice at 6 and 18 months of age. (C) Stereological estimations of the total number of DARPP-32 positive neurons in the unilateral striatum did not reveal any statistically significant differences between the groups (n = 4–6 mice per group).

Figure 5.

Altered dopamine signalling and binding in the striatum of Cra/+ mice. (A) mRNA levels of the dopamine D1 and D2 receptor, substance P and pre-proenkephalin in the striatum of wild-type mice (+/+) and heterozygous Cra/+ mice at 4 and 8 months of age. *P < 0.05 versus wild-type (n = 5–7). (B) Representative [¹¹C] SCH-23390 images (all frames averaged together) through the striatum of a wild-type mouse brain (+/+, upper panels) and a heterozygous mouse brain (Cra/+, lower panels) are shown. (C) Binding potentials (BP_ND) of [¹¹C] SCH-23390 (n = 5) and [¹⁸F] Fallypride (n = 10) calculated using SRTM. *P < 0.05 versus wild-type.

Figure 6.

Dramatic defect in dendritic morphology of cultured dynein mutant striatal neurons. (A and B) Cell survival of primary striata neuronal culture from wild-type embryo (+/+), heterozygous Cra/+ embryo and homozygous Cra/Cra embryo after 7 days in culture as assessed using MTT reduction assay (A) or direct counting of nuclei (B). (C and D) DNA (C) and RNA (D) content of primary striata neuronal culture from wild-type embryo (+/+), heterozygous Cra/+ embryo and homozygous Cra/Cra embryo after 7 days in culture. (E) mRNA levels of neurofilament heavy (NF-H) or medium (NF-M) subunits and of DARPP-32 in primary striata neuronal culture from wild-type embryo (+/+), heterozygous Cra/+ embryo and homozygous Cra/Cra embryo after 7 days in culture. (F) Representative microphotographs of MAP2 and β3-tubulin immunostaining of primary striata neuronal culture from wild-type embryo (+/+), heterozygous Cra/+ embryo and homozygous Cra/Cra embryo. (G) Length of longest neurite of individual striatal cells stained by MAP2 antibody from wild-type embryo (+/+), heterozygous Cra/+ embryo and homozygous Cra/Cra embryo. Total number of cells analysed: +/+108 cells, Cra/+162 cells and Cra/Cra 81 cells, **P < 0.01 versus wild-type (ANOVA followed by Newman–Keuls post hoc test). (H) Quantification of the number of MAP2 immunopositive primary, secondary and tertiary neurites from wild-type embryo (+/+), heterozygous Cra/+ embryo and homozygous Cra/Cra embryo. Total number of cells analysed: +/+68 cells, Cra/+126 cells and Cra/Cra 61 cells. **P < 0.01 versus wild-type, ***P < 0.001 versus wild-type (ANOVA followed by Newman–Keuls post hoc test).