Mice lacking Astn2 have ASD-like behaviors and altered cerebellar circuit properties

Abstract

Astrotactin 2 (ASTN2) is a transmembrane neuronal protein highly expressed in the cerebellum that functions in receptor trafficking and modulates cerebellar Purkinje cell (PC) synaptic activity. Individuals with ASTN2 mutations exhibit neurodevelopmental disorders, including autism spectrum disorder (ASD), attention-deficit/hyperactivity disorder (ADHD), learning difficulties, and language delay. To provide a genetic model for the role of the cerebellum in ASD-related behaviors and study the role of ASTN2 in cerebellar circuit function, we generated global and PC-specific conditional Astn2 knockout (KO and cKO, respectively) mouse lines. Astn2 KO mice exhibit strong ASD-related behavioral phenotypes, including a marked decrease in separation-induced pup ultrasonic vocalization calls, hyperactivity, repetitive behaviors, altered behavior in the three-chamber test, and impaired cerebellar-dependent eyeblink conditioning. Hyperactivity and repetitive behaviors are also prominent in Astn2 cKO animals, but they do not show altered behavior in the three-chamber test. By Golgi staining, Astn2 KO PCs have region-specific changes in dendritic spine density and filopodia numbers. Proteomic analysis of Astn2 KO cerebellum reveals a marked upregulation of ASTN2 family member, ASTN1, a neuron-glial adhesion protein. Immunohistochemistry and electron microscopy demonstrate a significant increase in Bergmann glia volume in the molecular layer of Astn2 KO animals. Electrophysiological experiments indicate a reduced frequency of spontaneous excitatory postsynaptic currents (EPSCs), as well as increased amplitudes of both spontaneous EPSCs and inhibitory postsynaptic currents in the Astn2 KO animals, suggesting that pre- and postsynaptic components of synaptic transmission are altered. Thus, ASTN2 regulates ASD-like behaviors and cerebellar circuit properties.

Keywords: ASTN2; Purkinje cell; autism spectrum disorder; cerebellum; neurodevelopmental disorder.

Fig. 1.

Loss of Astn2 results in fewer and less dynamic USVs in the mouse pups. (A) Pups were isolated from their mothers at postnatal days (P)5–P14 and placed in a soundproof chamber fitted with an ultrasonic microphone. USVs were recorded for 5 min. (B) Sample spectrograms illustrating the differences in USVs from P7 WT (n = 20), heterozygous (HET) (n = 52), and Astn2 KO (n = 24) animals (frequency in kHz as a function of time). Loss of ASTN2 expression robustly influenced USV production and dynamics on a number of measures at P6–P10: (C) Number of USV calls during the 5-min recording session was reduced in Astn2 KOs. Heterozygous animals showed a significant reduction at P6–P8. (D) Bouts of calls were reduced in Astn2 KOs. (E) Call duration was reduced in Astn2 KOs. (F) Pitch range of all USV calls was reduced in Astn2 KOs. (G) Fraction of calls that are dynamic (contain a pitch jump) was reduced in Astn2 KOs. Heterozygous animals show a reduction in dynamic calls at P8. (H) Range of frequency pitch for flat calls was reduced in Astn2 KOs at P7–P10. Data are presented as beans ±SEM. Symbols represent statistically significant results between the genotypes as described in the legend within the figure. Statistical analysis can be found in SI Appendix, Table S1.

Fig. 2.

Astn2 KO mice show ASD- like behaviors. (A–C) Social behavior is altered in the Astn2 KO mice. (A) The three-chamber social test was performed on 8 to 12-week-old animals (n = 12 WT, 11 KO). The test consisted of three phases, starting with a 10-min habituation to the empty cages, followed by two 10-min testing phases. In phase 2, to test sociability, mice were exposed to a nonsocial (Lego block) and a social stimulus (stranger mouse). In phase 3, to test social novelty preference, the nonsocial stimulus was replaced by a novel mouse. The familiar mouse from phase 2 was left in their cage. The time spent interacting with the stimuli was manually scored by the investigator. (B) In the first phase of the experiment, WT mice show a strong preference for the social stimulus (S) rather than the Lego block (L) (P < 0.0001). Astn2 KO animals also show a preference for the social stimulus (P = 0.001), however, the absolute time they spend interacting with the social stimulus is significantly reduced as compared to the WT (P = 0.027). (C) In the third phase of the experiment, WT animals interact with the novel animals (N) significantly longer than with the familiar animals (F) (P = 0.0003). In contrast, the Astn2 KO animals do not show a preference for the novel animal and spend as much time with the familiar animal as with the novel animal (P = 0.35). Data are analyzed with two-tailed unpaired Student’s t test and presented as the mean + SEM. (D–K) Astn2 KO mice show hyperactivity and repetitive behaviors, but not anxiety, in the open-field test. (D) 8- to 12-week-old WT (n = 29), heterozygous (n = 17), and Astn2 KO (n = 23) animals were placed in an open-field arena for 1 h and allowed to explore freely. The center of the arena was assigned in the software. (E) The total distance traveled was increased significantly in the KO animals (P < 0.0001). (F) The number of vertical episodes (rearing) was significantly increased in the Astn2 KOs as compared to WTs and Hets (P = 0.005). (G) The number of revolutions (circling) was significantly increased in the Astn2 KOs (P < 0.0001). (H) Time spent in the center versus the periphery of the arena was measured. Astn2 KOs spend less time in the periphery of the arena (P = 0.026). (I) A subset of animals (n = 7 WT, 5 Het, and 8 KO) were tested in a light/dark open-field paradigm where a black box covers half of the arena. (J) All genotypes spent significantly more time in the light part of the arena (L) versus the dark part (D), and there were no differences in the amount to time spent in the light (L) versus the dark (D) between WT and KO animals (P = 0.9). Het animals spent significantly more time in the light compared to WT and KO animals (P < 0.0001). (K) The latency to enter the light compartment significantly decreased in the Astn2 KO (P < 0.0001). Data are presented as the mean + SEM. Data are analyzed with one-way ANOVA with Tukey Kramer post hoc test, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001, ns = not significant.

Fig. 3.

Astn2 KO mice show abnormal cerebellar-dependent associative learning but normal righting reflex and rotarod behavior. (A) Behavioral configuration for delay eyeblink conditioning. Mice (8 to 15 wk old, N = 9/genotype) were head-fixed and could run freely on a cylindrical treadmill while high-speed videography recorded eyelid position during stimuli presentation. (B) Selected average eyelid traces (100-trial average) over the course of training from an example WT mouse in response to a 250-ms LED CS coterminating with the delivery of a 30-ms aversive air puff US. (C, Top) binned (200-trial average) CR probability over training (last 600 trials P = 0.14.) (Middle) binned (200-trial average) CR amplitude fraction of eyelid closed (FEC) over training with CRs preserved in trial space (last 600 trials P = 0.22). (Bottom) CR probability plotted for individual WT (blue) and KO (maroon) mice. (D, Top) example eyelid traces showing various CR topographies observed during CS-US trials. CRs with three peaks comprised <1% of all observed CRs. (Middle) detected CR peaks from all CS-US trials of all WT mice (Inset, N = 7,128 trials) and proportional contribution of individual WT mice. (Bottom) same as Middle, for all CS-US trials of all KO mice (Inset, N = 4,943 trials) and proportional contribution of individual KO mice (P = 0.10). Data are analyzed with two-tailed unpaired Student’s t test and presented as mean ± SEM. E) P7 WT (n = 12) and Astn2 KO (n = 13) pups were evaluated using the righting reflex assay by placing pups in a supine position. Time to completely right themselves was recorded. No difference was found between WT and KO animals (P = 0.87). (F) Eight- to twelve-week-old WT (n = 8) and Astn2 KO (n = 10) animals were placed on an accelerating rotarod for five consecutive days (d). An average of three trials per day was recorded. Time to fall from the rotarod was measured. Astn2 KO animals had significantly higher latency to fall compared to WT animals [F(1,80) = 7.0205, P = 0.01]. Data are analyzed with the repeated measures ANOVA test and presented as the mean ± SEM.

Fig. 4.

Cerebellar lobule–specific changes in PC dendritic spine numbers and morphology in Astn2 KO mice. (A) Schematic of a mouse cerebellum. I marks the anterior vermis (lobule III), II marks the posterior vermis (lobule XI), and III marks Crus I. (B) Example images of sampled dendritic segments with dendritic spines in the anterior vermis, the posterior vermis, and Crus I, in WT (Top) and Astn2 KO (Bottom). (C–E) Analysis of PC dendritic spines in WT and Astn2 KO animals in C the anterior vermis, (D) the posterior vermis, and (E) Crus I. (i) The number of spines per 10 μm dendrite segment (C, P = 0.1; D, P = 0.00016; E, P = 0.15, Wilcoxon rank-sum test.) (ii) The distribution of spine lengths with all sampled dendritic segments pooled together (C, P = 0.078; D, P = 0.0038; E, P = 0.0047, two-sample Kolmogorov–Smirnov test.) (iii) Distribution of the fractions of filopodia on each sampled dendritic segment. (C, P = 0.65; D, P = 0.064; E, P = 0.013, two-sample Kolmogorov–Smirnov test.) (iv) Distribution of the total number of filopodia on each sampled dendritic segment (C, P = 0.54; D, P = 0.029; E, P = 0.019, two-sample Kolmogorov–Smirnov test.)

Fig. 5.

Astn2 KO mice have increased levels of ASTN1 and an increase in Bergmann glia. (A) Proteomic analysis of Astn2 KO animals at P22. A volcano plot depicting differentially expressed proteins in whole cerebellar lysates of Astn2 KO animals and WT littermates (n = 8 WT, 8 KO). ASTN2 is down-regulated and ASTN1 and TRIM32 are up-regulated in Astn2 KO animals. (B) Western blot showing the upregulation of ASTN1 and TRIM32 in P22 cerebellar samples. (C) Western blot for GFAP in P22 cerebellar tissue of Astn2 KO mice. There is an increase in the amount of GFAP protein in Astn2 KO mice. (D) Immunohistochemistry with antibodies against GFAP (a Bergmann glia marker, green), calbindin (a PC marker, red), and Hoechst (blue) in P22 Astn2 KO mice (Bottom) and WT littermates (Top Left). An increase in GFAP signal as well as disorganization of BG fibers is observed in Astn2 KO mice (Right). (E) Astn2 KO mice have a higher mean fluorescent intensity of GFAP staining indicating an increase in Bergmann glia (P = 0.003). (F) Volcano plot depicting differentially expressed genes (P-adj < 0.05, indicated with red) in P22 Astn2 KO cerebellum, compared with WT littermates, identified using DESeq2. Astn1 gene is not up-regulated suggesting that ASTN1 protein overexpression is posttranscriptionally regulated in Astn2 KO animals. (G) Electron microscopy imaging of WT (Top) Astn2 KO (Bottom) cerebellar molecular layer at P22. An example image of 2,900× direct magnification EM image pseudocolored revealing a PC dendrite (green) and Bergmann glia fibers (red). H) Quantification of the area covered by glia in EM images in WT and Astn2 KO animals. There is a significant increase in Bergmann glia fibers in the Astn2 KO (P = 0.0007). Three mice per genotype for all datasets. Data are presented as the mean + SEM. Data are analyzed with Student’s t test, *P < 0.05, **P < 0.01, and ***P < 0.001

Fig. 6.

Astn2 KO mice show differences in spontaneous and evoked synaptic currents. (A, Top) sEPSCs in whole-cell recordings (V_m = −70 mV) of PCs. (Bottom Left) cumulative distribution of sEPSC amplitudes. P = 6.6e-148, Wilcoxon rank-sum test. (Bottom Right) same as Left, for IEI. P = 3.3e-14, Wilcoxon rank-sum test. (B, Top) sIPSCs in whole-cell recordings of PCs (V_m = 0 mV). (Bottom Left) cumulative distribution of sIPSC amplitudes. P = 3.5e-123, Wilcoxon rank-sum test. (Bottom Right) same as Left, for IEI, P = 0.99, Wilcoxon rank-sum test. (C, Left) example of mean evoked EPSC in whole-cell recordings of PCs (V_m = −70 mV). (Right) summary of EPSC amplitudes across cells. P = 0.02, two-tailed unpaired Student’s t test. (D) Summary of EPSC decay kinetics (τ) across cells. P = 0.91, two-tailed Student’s t test. (E, Left) example of mean evoked IPSC in whole-cell recordings of PCs (V_m = 0 mV). (Right) summary of IPSC amplitudes across cells. P = 0.33, two-tailed unpaired Student’s t test. (F) Summary of IPSC τ across cells. P = 0.94, two-tailed unpaired Student’s t test. (G, Left) example of mean evoked EPSC and IPSC from an individual WT and KO PC. KO traces scaled to the WT EPSC. (Right) summary of the ratios of excitation to inhibition (E/I ratio) across cells. P = 0.46, two-tailed unpaired Student’s t test. (H, Left) example of mean evoked EPSCs from an individual WT and KO PC. KO traces scaled to the first WT EPSC. (Right) summary of paired-pulse ratios (PPR) across cells. P = 0.09, two-tailed unpaired Student’s t test. (I, Left) example of mean extracellular recordings of PC spiking activity from an individual WT and KO PC. Middle, summary of spike rates across cells. (Right) summary of coefficients of variation (CV) across cells. P = 0.10, two-tailed unpaired Student’s t test. (J, Left) cumulative distribution of sEPSC amplitudes of PCs in the posterior vermis. P = 2.0e-106, Wilcoxon rank-sum. (Right) same as (left) for IEIs. P = 1.2e-17, Wilcoxon rank-sum test. (K, Left) cumulative distribution of sIPSC amplitudes of PCs in the posterior vermis. P < 0.0001, Wilcoxon rank-sum test. (Right) same as Left, for IEIs. P = 0.004, Wilcoxon rank-sum test. (L) Summary of EPSC amplitudes across PCs in the posterior vermis. P = 0.02, two-tailed unpaired Student’s t test. (M) Summary of IPSC amplitudes across PCs in the posterior vermis. P = 0.19, two-tailed unpaired Student’s t test. Error bars ± SEM.