BTBR mice exhibited infancy-onset dystonic behavior and motor impairments
Movement disorders are widely reported in combination with autism in individuals. We found BTBR mice exhibited severe dystonic movements during tail suspension, when mice try to keep an upright body posture. The most prominent dystonic symptom was hyperflexion of one or both hyperkinetic hindlimbs during tail suspension. Hindlimb clasping or fore- and hindlimb clasping, hyperextension, and severe trunk twisting were also observed in conjunction with myodystonia (Fig.s1). The dystonia in BTBR mice onset began at postnatal day 10 (P10) (X2=3.902, P<0.05), and nearly 100% of BTBR mice exhibited significant dystonic behavior since P14 (X2=21.505, P<0.001). This behavior persisted to adulthood and remained stable (Fig.1 A and B). Physiological hypermyotonia was observed in WT mice between P7 and P30, but it disappeared after P30. In addition, BTBR mice also developed a weakened ability to hang from a wire grid since P21 (P<0.001) compared to the WT (Fig.1 C, F(1,258) = 343.614, P < 0.001) because of abnormal hindlimb clasping or twisting in some cases. Fine motor skill was assessed using regularly and irregularly spaced horizontal ladders at 8 weeks of age. Mice ran across the horizontal ladder in two patterns, as shown in Fig.1D. BTBR mice exhibited increased limb falls in both the regular (T20=2.377，P<0.05) and irregular (T20=2.812, P<0.05) patterns, which suggests a deficit in fine motor skill (Fig.1E). Interestingly, the time spent crossing the ladder was decreased in BTBR mice compared to WT controls (Pattern A: T20=4.381，P<0.001; Pattern B: T20=6.686，P<0.001), mainly because of the increased activity. BTBR mice were also significantly hyperactive in the open field, as indicated by the increased distance in all zones (T16=2.719, P<0.05) and the central zone (T16=2.504, P<0.05) (Fig.1 I, J). Motor skill learning was assessed by means of the consecutive rotarod learning test (Fig.1H). BTBR and WT mice learned the task, and the time on the rod gradually increased (WT: F(4,44) = 21.867, P < 0.001; BTBR: F(4,28) = 15.306, P < 0.001). However, learning was significantly slower in the BTBR mice (F(1,72) = 28.232, P < 0.001) (Fig. 1H). Notably, the BTBR mice showed abnormal behaviors, such as attention-deficit, when they were put on the rotarod, as shown in Fig.1G. Instead of concentrating on the motor learning, the BTBR mice explored and ignored the unstable rotating rod under their feet. It may be an important cause to the impaired learning process. In summary, BTBR mice exhibited infancy-onset dystonia accompanied by severe deficits in motor coordination and motor learning in addition to autistic behavior.
BTBR mice postnatally developed hyperplastic cerebella with increased foliation
In the adult BTBR mice, the overall structure of the cerebellum was abnormal, with an obviously larger area (T11=7.727, P<0.001) and more lobules (T11=6.826, P<0.001) (Fig. 2A, D). By dividing the area into three lamellas, the molecular layer (ML), granule cell layer (GCL) and white matter (WM), we found that the enlarged portion was mainly in the ML (T11=4.383, P<0.01) and GCL (T11=8.880, P<0.001) (Fig.2 B). The thickness of the ML was not altered in BTBR mice (data not shown), and its increased area may have resulted from an elongated perimeter. Therefore, the enlargement of the cerebella may be due to the extension of the GCL. Another noticeable change was considerably more foliation in BTBR cerebella than the WT controls (Fig.2 C). To determine when BTBR mice first exhibited enhanced foliation, paraffin sections of the cerebella with HE staining were observed sequentially during the first two postnatal weeks (Fig.2 F). The initial stages of cerebellar patterning, including cardinal fissure formation, were normal in BTBR mice until P3, but the average sagittal cerebellar area increased significantly (T9=2.447, P<0.05) compared to WT controls. The average sagittal cerebellar section perimeter was elongated concomitantly (T9=4.378, P<0.01), which indicates that the cerebellar surface area was increased. Thus, cortical expansion and increased cross-sectional area preceded supernumerary folia in BTBR mice.
BTBR mice first exhibited increased foliation at P7, with multiple lobules that were not present in controls (T10=20.125, P<0.001) (Fig. 2F and G). Additionally, the midsagittal area (T10=11.234, P<0.001) and perimeter (T10=20.698, P<0.001) increased more noticeably. At P14, when foliation patterns are established, BTBR were larger (T9=6.739, P<0.001) (Fig. 2H), had a longer perimeter (T9=10.639, P<0.001) (Fig. 2I), and were considerably more foliated than controls (T9=22.160, P<0.001) (Fig. 2F and G).
Granule cell precursor (GCP) proliferation was increased in BTBR cerebella but migration did not change
The foliation pattern divided by fissures of different lengths is a representative morphology of cerebella. The formation is orchestrated by multicellular anchoring centers in which granule cells are the initiating factors and provide the driving physical force (32). In development, GCPs in the EGL proliferate and differentiate into granule cells, then gradually mature during migration through the ML to destinations in the IGL. BrdU was used to label the newborn GCPs in the EGL of P3 cerebella (Fig.3 A, B). Co-staining of nuclei with DAPI revealed that the EGL was much thicker in BTBR cerebella compared to WT (T9=3.218, P<0.05) (Fig.3 A2, B2, C). Simultaneously, the density of BrdU-positive GCPs in the EGL increased significantly (T9=3.910, P<0.05) (Fig.3 D). The total GCPs (T9=3.944, P<0.05) (Fig.3 E) and proportion (T9=4.899, P<0.01) (Fig.3 F) were also increased in BTBR mice. To confirm this result, another marker, Ki67, which is actively expressed during mitosis and degrades soon after caryomitosis, was used. Consistently, the Ki67-positive GCPs in the EGL were multiplied in BTBR mice compared to WT mice (T9=4.273, P<0.01) (Fig. G, H, I) at P3. Ki67 was further detected at P7, and it was still much greater in BTBR mice than WT mice (T10=3.747, P<0.01) (Fig.4 J, K, L). These results indicate increased granule cell precursor proliferation in the BTBR cerebella postnatally up to P7.
The granule neurons originate from the GCP in the EGL, then migrate radially through the scaffold-the Bergmann processes to reach their ultimate location in the IGL and maturation (17). To confirm whether the altered proliferation of GCPs affected this process, we investigated radial migration. We observed the distribution of mature granule neurons with NeuN staining at P7, which is the middle of the migration process. Almost all the mature granule neurons were in the IGL in WT mice, with invisible ectopic neurons in the EGL or ML (Fig.4 A). The results were similar in the BTBR, and no ectopic mature neurons were found (Fig.4 B). Notably, the EGL thickness was comparable in the two groups (T10=0.856, P=0.412) (Fig.4 C), which indicates that the overproduced granule neurons in BTBR mice were transferred efficiently. It was further confirmed that migrating granule neurons identified by slim nuclei in the ML (33) were increased in BTBR mice (T10=5.716, P<0.01) (Fig.s2 E), but the proportion or migrating rate was comparable between groups (T10=0.679, P=0.073) (Fig.s2 F). Bergmann glia play a vital role in granule neuron migration. Therefore, we also investigated this unique cell. Bergmann glia soma and fibers were clearly stained with s100b and GFAP in each group (Fig.4 E-H). No aberrations in Bergmann glia were found between groups, neither soma (T10=0.303, P=0.768) (Fig.4 I) nor fibers (T10=0.770, P=0.459) (Fig.4 J). The Bergmann glia in the WT and BTBR mice had the same morphology, with soma located in the Purkinje cell layer, diffuse distribution, and 3-4 cells surrounding a Purkinje neuron. Each soma extended approximately 1-3 processes upward, across the molecular layer, and ended at the pial surface. Furthermore, Nissl staining of adult 3-month cerebella for mature neurons revealed conspicuous gross morphological changes in BTBR mice, but the neuron density was similar in the ML between groups. The boundary of the IGL was well-defined with no stranded cells, which indicates that the migration of granule neurons was accomplished, in terms of results.
Purkinje neurons in BTBR cerebella was hypotrophy with abnormal dendritic spine formation
Purkinje cells are the sole efferent neurons in the cerebella and play a key role in motor function, the development of which is regulated by multiple factors and cells. We investigated whether the Purkinje neurons of BTBR mice were affected during the critical time when the dystonia behavior reached the fastigium at P14. The Purkinje neurons were labeled with the specific marker calbindin (CB) (Fig.5 A-D). The distribution of Purkinje neurons and the development process were not different between WT and BTBR mice. Purkinje neurons were arranged in a monolayer, and the bushy dendrites grew into the ML. Purkinje neuron density was comparable in each lobe between groups (Fig.5 E) However, Purkinje neurons in BTBR cerebella exhibited significant cell soma hypotrophy compared to WT, especially the posterolateral lobes, from lobe IV to X (lobe IV/V: T10=3.217, P<0.01; love VI/VII: T10=5.470, P<0.001; lobe VIII: T10=4.142, P<0.01; lobe IX: T10=2.972, P<0.05; lobe X: T10=2.649, P<0.05) (Fig.5 F). Western blotting was used to confirm this result (Fig.5 G), and calbindin protein expression was decreased in BTBR cerebella (T10=2.416, P<0.05) (Fig.5 H), in accordance with the decrease in soma size. The cerebellar lobular development occurs from posterior to anterior, and lobes IX and X originally develop. Therefore, we speculated this that phenotype in BTBR was progressing. We observed the Purkinje cells in adulthood and found a sparse cell distribution with significant cell loss in the posterior lobes in BTBR mice, including lobes VIII (T6=3.019, P<0.05), IX (T6=4.971, P<0.001) and X (T6=2.662, P<0.05) (Fig.s3). These results indicate that the hypotrophy in Purkinje neurons may be a foreboding of cell death.
We investigated synaptic formation of the Purkinje dendrites. Golgi staining was used to examine the dendrites (Fig.6 A-B). Representative images showed no differences in gross morphology of Purkinje neurons between groups. There were no abnormalities in extended areas of dendrites (T6=2.315, P=0.060) (Fig.6 C), primary dendrite length (T6=0.951, P=0.378) (Fig.6 D), or the complexity of the dendrite arborization as assessed by Sholl analysis (F(1,204) = 0.105, P =0.757) (Fig.6 E, F) of BTBR mice. Finally, the synaptic structure was detected. Spine density in BTBR cerebella was significantly increased (T6=5.793, P<0.01) compared to WT, with a close array in dendritic branches (Fig.6 A1’-B2’ and H). Spines exhibited a transformed morphology during their development and maturation, which reflects different synaptic function at different stages (34). After dividing the spines into three subtypes, thin, stubby and mushroom (Fig.6 G), the maturation of the spines was generally assessed. The proportion of the immature long, thin subtype was significantly increased (T6=6.147, P<0.01) in BTBR mice compared to WT mice, and the transitional stubby subtype (T6=2.617, P<0.05) and mature wide-headed mushroom spines (T6=7.738, P<0.001) were decreased (Fig.6 A1’-B2’ and I). These results demonstrate that the formation of the spines was strongly promoted but the maturation was suffocated by some factor in BTBR mice.
Dysregulated TRPC genes impaired cerebellar development in BTBR mice
To examine the underlying mechanism of the abnormal cerebellar development and connection formation in BTBR mice, we performed RNA-Seq in whole cerebella tissue of WT and BTBR mice at postnatal day 14. Previous studies were performed in other brain regions of BTBR mice vs WT mice (35-37), but few studies were done in cerebella. We identified 3992 differentially expressed genes (P<0.05), with 1858 upregulated and 2134 downregulated (Fig.6 A) genes in BTBR mice compared to the WT. For functional annotation, the GO term enrichment in biological processes of differentially expressed genes was analyzed. The greatest differences primarily were involved in central nervous system development, neurogenesis, differentiation, cell development and morphogenesis (Fig.6 B), which are highly consistent with the abnormal development of the cerebella in BTBR mice. The negative regulation of nervous system development was noticeable, in the top and the coverage of cerebellar development. After further screening using protein-protein interaction (PPI) networks analysis (Fig.s4), the critical genes were identified, and significantly increased TRPC6 was a highly suspicious candidate in BTBR mice (Fig.6 C). TRPC6 is especially expressed during cerebellar development (38), and it regulates neurogenesis and synaptic formation (39, 40). PPI networks of the DEGs in this pathway (Fig.s4) indicated the direct interaction of TRPC6 to the changed allele, Itpr3, of BTBR mice, which further supports its core status. The expression was verified using RT-PCR (Fig.6 D). We unexpectedly detected CAMK IV gene expression, which acts downstream of TRPC6, and found that it was upregulated as expected (Fig.6 E). TRPC3 and 4 were also detected and exhibited decreased and increased expression, respectively, consistent with the RNA-seq results (Fig.6 E). Therefore, the RNA-seq suggests that TRPC protein, with TRPC6 as the core factor, mistakenly regulated the development of the cerebellum and resulted in more serious disorders over time.