Impaired cerebellar plasticity hypersensitizes sensory reflexes in SCN2A-associated ASD

Abstract

Children diagnosed with autism spectrum disorder (ASD) commonly present with sensory hypersensitivity or abnormally strong reactions to sensory stimuli. Such hypersensitivity can be overwhelming, causing high levels of distress that contribute markedly to the negative aspects of the disorder. Here, we identify a mechanism that underlies hypersensitivity in a sensorimotor reflex found to be altered in humans and in mice with loss of function in the ASD risk-factor gene SCN2A. The cerebellum-dependent vestibulo-ocular reflex (VOR), which helps maintain one's gaze during movement, was hypersensitized due to deficits in cerebellar synaptic plasticity. Heterozygous loss of SCN2A-encoded Na_V1.2 sodium channels in granule cells impaired high-frequency transmission to Purkinje cells and long-term potentiation, a form of synaptic plasticity important for modulating VOR gain. VOR plasticity could be rescued in mice via a CRISPR-activator approach that increases Scn2a expression, demonstrating that evaluation of a simple reflex can be used to assess and quantify successful therapeutic intervention.

Keywords: autism spectrum disorder; cerebellum; gene therapy; human; mouse; neurodevelopmental disorder; sodium channel; vestibulo-ocular reflex.

Figure 1:. VOR gain is elevated in Scn2a haploinsufficiency conditions

A: Schematic of purpose-built eye tracking apparatus for VOR assessment in children. Head movements are measured from a device located at the center of the head while the right eye is imaged under infrared illumination (940 nm LED). Participants are seated in a swivel chair and oscillated ±5° at ~0.4 Hz to assess VOR gain. B: Head angle (purple) and contraversive eye angle for neurotypical (Nt, black) and SCN2A LoF conditions (cyan). Lines represent the average of a single cycle from all participants; shaded area is SEM. Dashed red lines indicate ±5° range. C: VOR was assessed in mice head-fixed at the center of a rotating table, imaged under IR illumination. D: VOR at 0.4 Hz rotation frequency, displayed as in panel B, for Scn2a^+/+ (left, black) and Scn2a^+/− mice (right, cyan). E: Baseline VOR gain in human and mouse. Circles are individuals; box plots are medians, quartiles and 90% tails. n: 11 Nt, 5 LoF humans; 13 +/+, 12 +/− mice. Mann Whitney test p-values shown. F: VOR gain across rotation frequencies in mouse. Lines connect repeated tests in single mice. Asterisks: p < 0.0001, Friedman test on overall distribution; Mann Whitney test on individual frequencies, Holm Šídák correction.

Figure 2:. Impaired VOR gain-down behavioral plasticity in Scn2a^+/− mice

A: Experimental design for VOR gain-down induction and simultaneous in vivo silicon probe recording in the cerebellar floccular complex. Inset, recording location in left hemisphere. Left: baseline VOR recording in the dark. Middle: VOR gain-down induction with virtual drum as visual stimulus. During gain-down induction, visual stimulus was moved in phase with table. Right: post-induction VOR recording in the dark. B: Head and eye angles in Scn2a^+/+ (black) and Scn2a^+/− (cyan) during baseline (dark colors) and post-induction (light colors) with table rotation (purple). Dashed red lines indicate ±5° range. C: Head angle (purple) and contraversive eye angle before (darker shade) and after (lighter shade) gain-down induction. Data presented as in Fig. 1D. D: VOR gain before and after gain-down induction. Data color coded as in B. Bars connect data from individual mice. * p < 0.001, Wilcoxon signed rank test, # p < 0.0001, Mann Whitney test. E: Putative Purkinje cell unit recordings before and after gain-down induction. Traces are aligned from table rotation onset. F: Average Purkinje cell simple spike firing frequency during sinusoidal head rotation, before and after gain-down induction, displayed as in D. Scn2a^+/+ (black, closed circles, n = 22 units, 6 mice), Scn2a^+/− mice (cyan, n = 13 units, 5 mice). * p < 0.05, all conditions, Mixed-effects modeling, # p < 0.001, Wilcoxon signed rank test. G: Normalized Purkinje cell simple spike firing frequency during induction protocol, normalized to firing rate in first minute per unit. Circles and bars are mean ± SEM, binned per minute. H: Normalized change in VOR gain vs. normalized change in simple spike rate (normalized per unit and averaged across units per animal). Circles are color-coded as in panel D and represent individual animals.

Figure 3:. Impaired cerebellar plasticity in Scn2a^+/− conditions

A: Saggital section of mouse cerebellum in an animal with eGFP knocked into the Scn2a locus. Note GFP expression in granule cell layer and molecular layer and lack of expressing in Purkinje cell layer. B: High magnification single optical confocal section of region highlighted in yellow square in A. Area co-stained for eGFP, VGlut1, and DAPI. Top panels are single color. Bottom panels are overlays of 2 or 3 colors to show coexpression of eGFP and VGlut1. C: Left: Two EPSCs (20 Hz) before and after 166 Hz burst LTP induction in Scn2a^+/+ (black, n = 6 cells from 4 mice) and Scn2a^+/− (cyan, n = 6 cells from 4 mice) in the floccular region of the cerebellum. Inset, paired pulse ratio before and after LTP induction. *: p < 0.01, Wilcoxon signed-rank test. Right, first EPSC amplitude, normalized to pre-induction average. Data are binned each minute. Averaged baseline first EPSC amplitude (in pA) in flocculus: Scn2a^+/+: 451.54 ± 66.30; Scn2a^+/−: 417.41 ± 37.28. Circles and bars are mean ± SEM. D: Same as C, but for cells in vermis. Scn2a^+/+, black, n = 6 cells from 6 mice; Scn2a^+/−, cyan, n = 10 cells from 4 mice. Averaged baseline first EPSC amplitude (in pA): Scn2a^+/+: 359.50 ± 67.89, Scn2a^+/−: 383.42 ± 94.33

Figure 4:. Impaired cerebellar granule cell excitability in Scn2a^+/− conditions

A: APs generated by current injection (10–50 pA, 300 ms) in Scn2a^+/+ (black) and Scn2a^+/− (cyan). B: Top: APs (spikes) per 300 ms stimulation epoch for each current amplitude in the vermis (left) and flocculus (right) (lines and shadow are mean ± SEM of population, *: p < 0.05 of slope between 20–40 pA). Bottom: near-rheobase APs plotted as dV/dt vs voltage (phase-plane plot) from Scn2a^+/+ and Scn2a^+/−. C: AP threshold (top) and peak dV/dt (bottom) in Scn2a^+/+ (vermis: 13 cells from 3 mice; flocculus: 11 cells from 1 mouse) and Scn2a^+/− (vermis: 16 cells from 3 mice, flocculus: 15 cells from 2 mice). *: p < 0.05, Mann Whitney test. Circles are single cells, boxes are medians and quartiles with 90% tails. D: Spontaneous Purkinje cell AP train in Scn2a^+/+ and Scn2a^+/−. A single AP is highlight on the right. E: Left to right: single AP, phase-plane plot of all APs in train, and summary AP threshold and peak dV/dt in Scn2a^+/+ and Scn2a^+/−. F: Left, Parallel fiber volleys evoked in trains of 10 APs at 50 Hz or 20 APs at 166 Hz. Right, Fiber volley amplitude, normalized per volley to first event in train, then averaged across recordings. Lines and bars are mean ± SEM. 50 Hz (top) in Scn2a^+/+ (black, n = 5 from 2 mice) and Scn2a^+/− (cyan, n = 7 from 4 mice). 166 Hz (bottom) in Scn2a^+/+ (black, n = 9 from 3 mice) and Scn2a^+/− (cyan, n = 9 from 4 mice). G: Top, parallel fiber evoked EPSCs in Purkinje cells from train of 20 stimuli at 166 Hz. Bottom, charge transfer, normalized to transfer from EPSC1 in Scn2a^+/+ (black, n = 10 cells from 2 mice) and Scn2a^+/− (cyan, n = 9 cells from 2 mice) conditions.

Figure 5:. Scn2a heterozygosity in granule cells alone impairs VOR gain-down plasticity

A: Experimental design for VOR gain-down induction as in Fig. 2, but for Scn2a^+/fl mice crossed to the alpha6-Cre driver line (α6-Cre), which restricts Cre expression largely to cerebellar granule cells. B: Head angle (purple) and contraversive eye angle before (darker shade) and after (lighter shade) gain-down induction for α6-Cre not crossed to Scn2a^+/fl animals (black, WT-equivalent), or α6-Cre::Scn2a^+/fl mice (green). C: VOR gain before and after gain-down induction. Data color coded as in B. Bars connect data from individual mice. *: p < 0.001, Wilcoxon signed rank test, # p < 0.0001, Mann Whitney test. D: Putative Purkinje cell unit recordings before and after gain-down induction. Traces aligned to table rotation onset. E: Average Purkinje cell simple spike firing frequency during sinusoidal head rotation, before and after gain-down induction, displayed as in C. α6-Cre WT (gray, open circles, n = 24 units, 5 mice) and α6-Cre::Scn2a^+/fl mice (green, n = 28 units, 5 mice). *: p < 0.005, all conditions, Mixed-effects modeling, #: p = 0.01, Mann Whitney test. Note that baseline firing rates are not different than those observed in constitutive Scn2a^+/− mice: Scn2a^+/− baseline firing: 45.5 ± 7.5 Hz, n = 13 units; a6-Cre::Scn2a+/fl: 52.5 ± 5.2 Hz, n = 28 units; p = 0.34, Wilcoxon signed rank test. F: Average Purkinje cell simple spike firing frequency during induction protocol, normalized to firing rate in first minute per cell. Circles and bars are mean ± SEM, binned per minute. G: Normalized change in VOR gain vs. normalized change in simple spike rate (normalized per unit and averaged across units per animal). Circles are color-coded as in C and represent individual animals. Data from Scn2a^+/+ (gray) and Scn2a^+/− (light cyan) are in background for comparison.

Figure 6:. Rescue of VOR gain-down behavioral plasticity with CRISPR activation

A: Two AAV-PHP vectors are injected together for systemic infection via the retro-orbital sinus. AAV1 contains a guide RNA and mCherry. AAV2 contains dCas9 and the transcriptional regulator VP64. B: Cerebellar coronal section from animals injected at P30 and P3 animal. Note broad, but incomplete, infection across cerebellum, as signaled by mCherry fluorescence. A region of cerebellum containing the left floccular complex was dissected immediately after harvesting brain for quantitative PCR and is missing from section. C. Quantitative PCR of Scn2a mRNA mouse cerebellum from CRISPRa-injected mice. In 5/6 animals injected at P30, marked upregulation was noted (closed circles). In 1 animal, no upregulation was noted (open circle). At P3, all animals exhibited robust upregulation. Data are plotted vs. the normalized change in VOR gain (e.g., slope per animal in E). Dashed line is a linear regression. D: Example VOR before and after gain-down induction in CRISPRa-treated animals (P30 and P3). E. VOR gain before and after gain-down induction in Scn2a^+/− mice with Scn2a CRISPRa at P30 (red, 6 mice), P3 (purple, 6 mice) and empty vector (blue, 4 mice), depicted as in Fig 2D. WT and Scn2a^+/− data replotted from 2D for comparison. *: p < 0.05 Wilcoxon-signed rank test; all data included. F: Average Purkinje cell simple spike firing frequency during sinusoidal head rotation, before and after gain-down induction in all RO-injected mice. Circles are color-coded as in D and represent individual units. *: p < 0.005, Mixed-effects modeling. G: Normalized Purkinje cell simple spike firing frequency during gain-down induction for all RO-injected mice. Circles and bars are mean ± SEM, binned per minute. H: Normalized change in VOR gain vs. normalized change in simple spike firing frequency (normalized per unit and averaged across units per animal). Circles are color-coded as in D and represent individual units. Data from Scn2a^+/+ (gray) and Scn2a^+/− (light cyan) are in background for comparison. Note overlap of CRISPRa population with Scn2a^+/+ population (except open circle) and empty vector with Scn2a^+/− population.