Age-dependent regulation of axoglial interactions and behavior by oligodendrocyte AnkyrinG

Abstract

The bipolar disorder (BD) risk gene ANK3 encodes the scaffolding protein AnkyrinG (AnkG). In neurons, AnkG regulates polarity and ion channel clustering at axon initial segments and nodes of Ranvier. Disruption of neuronal AnkG causes BD-like phenotypes in mice. During development, AnkG is also expressed at comparable levels in oligodendrocytes and facilitates the efficient assembly of paranodal junctions. However, the physiological roles of glial AnkG in the mature nervous system, and its contributions to BD-like phenotypes, remain unexplored. Here, we generated oligodendroglia-specific AnkG conditional knockout mice and observed the destabilization of axoglial interactions in aged but not young adult mice. In addition, these mice exhibited profound histological, electrophysiological, and behavioral pathophysiologies. Unbiased translatomic profiling revealed potential compensatory machineries. These results highlight the critical functions of glial AnkG in maintaining proper axoglial interactions throughout aging and suggests a previously unrecognized contribution of oligodendroglial AnkG to neuropsychiatric disorders.

Figure 1.. Loss of oligodendroglial AnkG leads to destabilization of paranodal junctions during aging.

a, Schematic of the Ank3 conditional allele with 2 loxp sites (triangles) flanking exons 23 and 24. Upon NG2^Cre-mediated recombination, exons 23 and 24 are excised, introducing premature stop codons in exons 25 and 26. b, Immunoblot of whole brain homogenates from 2-month-old control (Ank3^F/F), cHT (NG2^Cre+; Ank3^F/+), and cKO (NG2^Cre+; Ank3^F/F) animals. All three major isoforms of AnkG (480 kDa, 270 kDa, 190 kDa) are detected by guinea pig polyclonal AnkG antibody (SySy). β-actin serves as the loading control. c, Densitometry analysis of relative expression of AnkG (190 kDa isoform) in the brains of 2-month-old control, cHT, and cKO mice. Each data point represents an individual animal. One-way ANOVA with multiple comparison. *p=0.0436, **p=0.0031. d, Immunofluorescence staining of AnkG in the optic nerve of 2-month-old control and cKO mice. Arrowheads indicate paranodes. Scale bar, 10μm. e, Immunoblot of membrane-bound proteins from whole brain homogenates from 14-month-old control (Ank3^F/F) and cKO (NG2^Cre+; Ank3^F/F) animals. Known PNJ components are detected by antibodies against Caspr, Neurofascin, and Contactin. β-actin serves as the loading control. f-i, Immunofluorescence labeling of nodal (Yellow: Neurofascin), paranodal (Yellow: Neurofascin, Cyan: Caspr) and juxtaparanodal (Magenta: Kv1.2) proteins on small-diameter axons (f, g) in the optic nerve of 2-month-old control mice, or on large-diameter axons in the spinal cord (h, i) of 2-month-old control mice. (j-m) Immunofluorescence labeling of nodal (Yellow: Neurofascin186), paranodal (Cyan: Caspr) and juxtaparanodal (Magenta: Kv1.2) proteins on small-diameter axons in the optic nerve of 14-month-old control mice (j) and cKO (k) mice. In the spinal cord of 14-month-old cKO mice, severe paranodal unwinding is only observed in the large-diameter axons (l, m) but not in the small-diameter axons.

Figure 2.. Paranodal destabilization in AnkG cKO mice leads to wide-spread CNS pathology.

a, 14-month-old cKO mice show widespread histological changes in the entire CNS, including reduced myelin content in the optic nerve, increased reactive astrogliosis in the white matter of spinal cord, and Purkinje cell death (~50%) in the cerebellum, indicated by the arrowheads. These phenotypes are consistent with paranodal disruption. b-d, Quantifications of fluorescence intensity of MBP and GFAP, and density of Calbindin+ cells. Each data point represents an individual animal. (unpaired t-test. p=0.0236, p=0.0001, p<0.0001.) e, Immunoblot analyses of whole brain homogenates confirm the reduction of MBP from the membrane-bound portion, as well as increase in GFAP and decrease of Calbindin from the soluble portion.

Figure 3.. Loss of oligodendroglial AnkG results in slower conduction velocity.

a, Representative compound action potential (CAP) traces recorded from freshly dissected 14-month-old optic nerves with suction electrodes ex vivo. b, The overall CAP velocity is calculated using the following formula: $V_{CAP}=\frac{\text{Length}\text{of}\text{optic}\text{nerve}}{T_{V_{max}}-T_{V_0}}$. The CAP velocity is significantly lower in AnkG cKO mice. Each data point represents an individual optic nerve. (Mann-Whitney test. p=0.0368.) c, Three Gaussian curves are fitted under each trace to differentiate fast-, medium- and slow-conducting axons. cKO mice show a slower conduction velocity in all three groups of axons. Each data point represents an individual optic nerve. (2-way ANOVA. p=0.0349, 0.0126, 0.0184 for subgroups 1, 2, and 3, respectively.)

Figure 4.. Loss of oligodendroglial AnkG is detrimental to survival and overall health.

a, cKO mice show postnatal lethality with incomplete penetrance. Chi-square test. b, representative image of a 14-month-old AnkG cKO with its littermate control mice.c, Quantification of body weight at different ages. Each data point represents an individual animal. (2-way ANOVA. 10 weeks: p=0.06; for all other ages ****p<0.0001.) d, Representative image of hind-foot prints for gait analysis. e-g, Quantification of stride (e), sway (f), and stance (g) show significant reduction in all categories. Each data point represents an individual animal. (unpaired t-test. ****p<0.0001.)

Figure 5.. Loss of oligodendroglial AnkG leads to reduced locomotion and motor learning.

a, Representative traces during 10-minute open field tests in 2 month-old control and AnkG cKO mice. b, Quantification of total distance traveled by each animal. Each data point represents an individual animal (unpaired t-test. p=0.4893). c, Quantification of time spent in the inner zone. Each data point represents an individual animal (Mann-Whitney test, p=0.0159). d, Representative traces during 10-minute open field tests in aged control and AnkG cKO mice (older than 6 months). e, Quantification of total distance traveled by each animal. Each data point represents an individual animal (unpaired t-test. p=0.0001). f, Quantification of time spent in the inner zone. Each data point represents an individual animal (Mann-Whitney test, p=0.7630). g-h, Accelerating rotarod test in 2 month-old control and AnkG cKO animals. Three trials per day were performed on three consecutive days. Data are presented as mean ± SEM. N=17 for control, N=13 for AnkG cKO. (2-way ANOVA. p=0.7794, 0.9780, 0.7277, 0.0123, 0.0728, 0.3681, 0.0281, 0.0358, 0.0063 for each consecutive trial, respectively).

Figure 6.. Loss of oligodendroglial AnkG leads to altered sociability, anxiety, and depression-like behaviors.

a, Representative trace during 10-minute elevated plus maze test. b, Quantification of the time spent in open arms for control and AnkG cKO mice during the elevated plus maze test. (Mann-Whitney U test. p<0.0001.) c, Illustration of three-chamber social tests in control and AnkG cKO mice. d, Sociability measured by time spent with mouse versus empty cage. (2-way ANOVA with multiple comparisons. p*=0.0109, p****<0.0001, p=0.7112, p^#=0.0174.) e, Social novelty measured by time spent with stranger versus familiar mice. (2-way ANOVA with multiple comparisons. p***=0.0007, p****<0.0001, p=0.4731, p=0.1713). f, Illustration of contextual fear conditioning test for learning and memory. g, Percentage of time spent freezing. (2-way ANOVA with multiple comparisons. p=0.9960, ***p=0.0007, p=0.9767.) h, Illustration of acoustic startle response and pre-pulse inhibition test. i, Amplitude of the startle response to 120 dB stimuli from AnkG cKO and littermate control mice. (unpaired t-test. p=0.8025.) j, %PPI for AnkG cKO and littermate control mice. (2way ANOVA with multiple comparisons. p>0.9999, p=0.9998, p=0.8671.) k, Illustration of tail suspension test for depression-like behavior in AnkG cKO and littermate control mice cKO mice. i, Quantification of immobility during the tail suspension test. (unpaired t-test. p<0.0001).

Figure 7.. Loss of AnkG from adult mice.

a, Illustration of inducible loss of AnkG from oligodendrocytes in NG2^CreERTM; Ank3 ^F/F mice (AnkG icKO). b-c, Immunofluorescence staining of AnkG in the optic nerve of 3-month-old AnkG icKO mice (b) with littermate control (c). d-e, Immunofluorescence staining of the node, paranode, and juxtaparanode in the optic nerve of 3-month-old AnkG icKO mice (d) with littermate control (e) (Yellow: AnkG, Cyan: Caspr, Magenta: Kv1.2). f, Body weight of AnkG icKO vs control mice. (unpaired t-test, p=0.6588) g-I, Gait analyses of AnkG icKO vs control mice. (unpaired t-test for stride, p=0.7416; Mann-Whitney test for sway, p=0.3963; unpaired t-test for stance, p=0.1112.) j-k, Open field test of AnkG icKO vs control mice. (unpaired t-test for distance, p=0.7450; Mann-Whitney test for time in inner zone, p=0.3033.) l, Latency to fall from the accelerating rotarod for AnkG icKO vs control mice. (2-way ANOVA with multiple comparisons, p= 0.0576, 0.0935, 0.5004, 0.2071, 0.1259, 0.0141, 0.5468, 0.5935, 0.3180 for each trial, respectively). m, Time spent in open arms during the elevated plus maze test for AnkG icKO vs control mice. (Mann-Whitney test, p=0.3769.) n, Immobility during the suspension test for AnkG icKO vs control mice. (unpaired t-test, p=0.0417.) o, Three-chamber test of sociability for AnkG icKO vs control mice. (2-way ANOVA with multiple comparisons, p=0.3414, p****<0.0001, p^####<0.0001.) p, Three-chamber test of social novelty test for AnkG icKO vs control mice. (2-way ANOVA with multiple comparisons, p=0.8263 for familiar, p=0.2136 for stranger, p**=0.0024, p*=0.0336.)

Figure 8.. Translatomic profiling of AnkG-deficient oligodendrocytes reveals compensatory cytoskeletal mechanisms.

a, Illustration of the experimental design for translatomic profiling in NG2⁺ oligodendroglial-lineage cells. b, Relative enrichment of major cell-type markers (oligodendrocytes: Olig2, astrocytes: Aldh1l1, neurons: NeuN) and major AnkG isoforms (190 kDa, 270 kDa, 480 kDa) by HA-IP. c, Volcano plot of differentially expressed genes between AnkG icKO IP sample and control IP sample. Magenta dots represent significantly upregulated genes, blue dots represent significantly downregulated genes, and gray dots represent genes that were not statistically differentially expressed (cutoffs: p < 0.05, |log₂^FC| > 0.6). The names of representative genes-of-interest are labeled in black. d Immunoblot of AnkB and spectrins to validate translatomic data. e, Bar plots for downregulated cellular component (CC, solid bar) and molecular functions (MF, empty bar) identified by Gene Ontology (GO) analyses. Enrichment scores are calculated by −log10(Pvalue). f, Bar plots for upregulated cellular component (CC, solid bar) and molecular functions (MF, empty bar) identified by Gene Ontology (GO) analyses. Enrichment scores are calculated by −log10(Pvalue).