Autistic-like behaviour and cerebellar dysfunction in Purkinje cell Tsc1 mutant mice

Abstract

Autism spectrum disorders (ASDs) are highly prevalent neurodevelopmental disorders, but the underlying pathogenesis remains poorly understood. Recent studies have implicated the cerebellum in these disorders, with post-mortem studies in ASD patients showing cerebellar Purkinje cell (PC) loss, and isolated cerebellar injury has been associated with a higher incidence of ASDs. However, the extent of cerebellar contribution to the pathogenesis of ASDs remains unclear. Tuberous sclerosis complex (TSC) is a genetic disorder with high rates of comorbid ASDs that result from mutation of either TSC1 or TSC2, whose protein products dimerize and negatively regulate mammalian target of rapamycin (mTOR) signalling. TSC is an intriguing model to investigate the cerebellar contribution to the underlying pathogenesis of ASDs, as recent studies in TSC patients demonstrate cerebellar pathology and correlate cerebellar pathology with increased ASD symptomatology. Functional imaging also shows that TSC patients with ASDs display hypermetabolism in deep cerebellar structures, compared to TSC patients without ASDs. However, the roles of Tsc1 and the sequelae of Tsc1 dysfunction in the cerebellum have not been investigated so far. Here we show that both heterozygous and homozygous loss of Tsc1 in mouse cerebellar PCs results in autistic-like behaviours, including abnormal social interaction, repetitive behaviour and vocalizations, in addition to decreased PC excitability. Treatment of mutant mice with the mTOR inhibitor, rapamycin, prevented the pathological and behavioural deficits. These findings demonstrate new roles for Tsc1 in PC function and define a molecular basis for a cerebellar contribution to cognitive disorders such as autism.

Figure 1. PC Tsc1 mutants display reduced PC numbers and abnormal PC morphology

A. Mutants displayed reduced Purkinje cell numbers by Calbindin staining. B. PC loss occurred by 2 months in mutants. No PC loss was observed in hets at 4 months. C. Increased cleaved caspase-3 (CC-3) staining in mutants. (for B-C, controls/mutants: n=3 mice; hets, n=2 mice; >500 cells/group) D., E. Mutant PCs displayed increased spine density. (control: n=10 cells, 3 mice; het: n=10 cells, 4 mice; mutant: n=11 cells, 3 mice). *** p < 0.001, two-way ANOVA, Bonferroni's post hoc analysis. Scale bars in A.100μm; D. 20μm (left), 5μm (middle), 2μm (right).

Figure 2. PC Tsc1 hets and mutants display autistic-like behaviors

A. In assays of social approach, unlike all control genotypes (WT, L7Cre, and Flox), hets and mutants (mut) demonstrated no significant preference for novel mouse over novel object by time spent in chamber (above) or time in close interaction (below) with novel mouse. B. Unlike controls, hets and mutants also failed to display preference for social novelty in chamber (above) and close interaction (below) times. n≥11 for each group. C. Mutants displayed normal acquisition of escape platform location but impairments on day 1 of reversal learning (RDay) 1 in a water T-maze. (total trials = 15), n≥13 for each group. D. Hets and mutants spent significantly more time self-grooming. n≥11 for each group. E. Hets (P10) and mutants (P7, 10) pups emitted significantly more ultrasonic vocalizations than controls. n≥8 for each time point and group. ns, p>0.05; *, p<0.05; ** p<0.01, *** p <0.001, two-way ANOVA, Bonferroni's post hoc analysis.

Figure 3. PC excitability is reduced in PC Tsc1 hets and mutants, but no significant difference in synaptic inputs to PCs is apparent

A. Schematic of the electrophysiological recording configuration and synaptic inputs onto cerebellar PCs. B. Electrical stimulation of climbing fibers (CF, top) and granule cell-mediated parallel fiber (PF, bottom). Whole cell mode voltage-clamp recordings showed no difference in the conductance (g) of single fiber CF inputs (control (con) n=20 cells, 5 mice; mutant (mut) n=16 cells, 5 mice, p=0.34), or the paired-pulse ratio (PPR) of either CF or PF inputs (CF PPR: con n=8, mut n=5, p=0.51). C. Electrical stimulation revealed no differences in the ratio of evoked synaptic excitation to inhibition (E/I ratio; con n=29, mut n=17, p=0.19). D. Extracelluar recording of spontaneous PC spiking (left) revealed a significantly lower spike rate in hets and mutants (right, con n=77 cells, 12 mice, het n=62 cells, 7 mice; n=42 cells, 9 mice). E. Whole cell mode current-clamp recordings showed reduced excitability in PCs from hets and mutants compared to control PCs. Left, current injections of 1 and 3 nA produced fewer spikes in PCs from hets and mutants. Right, average data showed a reduced spike output for mutant PCs (con n=77 cells, 12 mice, het n=48 cells, 7 mice; n=43 cells, 9 mice). ns, p>0.05, * p<0.05, ** p<0.01. two-way ANOVA, Bonferroni's post hoc analysis.

Figure 4. Rapamycin treatment prevents pathologic and behavior abnormalities in PC Tsc1 mutant mice

A. Treatment with rapamycin (Rapa) prevented cell loss seen in vehicle (Veh) treated PC Tsc1 mutants. Scale bar: 100 μm. Quantification of cell numbers on right (n > 500cells; 2 mice per group) B., C. Whereas vehicle treated mutants displayed behavioral deficits, rapamycin treated mutants displayed amelioration of these deficits in B. reversal learning in the water T maze (n≥9 in each group) and in C. social approach in the three chambered apparatus (n≥10). As behavioral phenotypes were not significantly different between the three control genotypes in untreated mice, control genotypes were pooled into a single group for these studies. ns, p>0.05; *, p<0.05; *** p <0.001, two-way ANOVA, Bonferroni's post hoc analysis.