Background
Fragile X syndrome (FXS) is a heritable developmental disorder resulting from abnormal trinucleotide CGG repeat expansion and transcriptional silencing of the X-linked
FMR1 gene that encodes fragile X messenger ribonucleoprotein 1 (FMRP) [
1,
2]. FXS is the most prevalent known genetic cause of autism spectrum disorder (ASD) and intellectual disability (ID), with approximately 2–5% of all ASD cases linked to FXS, and 85% of males and 25% of females with FXS exhibiting ID [
3,
4]. Clinical presentation of FXS is complex, ranging from normal functioning (mainly in mosaic females) to severe ID and ASD, and is associated with additional psychiatric conditions, including anxiety, social avoidance, self-injurious behavior and hyperactivity/impulsivity, which are generally more severe in males [
1,
2,
5]. Overall, more than half of males and ~ 20% of females with FXS meet the
Diagnostic and Statistical Manual of Mental Disorders (5th edition, DSM-V) criteria for ASD diagnosis based on impairments in communication and social interaction, and presentation of restrictive, repetitive behaviors [
3,
4]. Even though much research has been devoted to the biology and cellular function of FMRP, the mechanism of how reduction or loss of FMRP leads to ASD symptoms has not been clearly elucidated.
FMRP is an mRNA binding protein that can regulate mRNA translation, stability and transport, and was shown to bind > 800 mRNAs encoding cellular and synaptic proteins in the mouse brain, many of which have been identified as ASD susceptibility genes [
6‐
10]. FMRP typically functions as a repressor of translation in cells and its loss results in global elevation of protein synthesis in neurons and leads to aberrant synaptic structure, function, and plasticity [
11,
12]. Many of the behavioral features associated with FXS, such as impairments in social interaction and repetitive behaviors, can be modeled in
Fmr1 knockout mice (
Fmr1−/y mouse) [
13]. Furthermore, the pharmacological and genetic manipulation of several proteins involved in translational regulation was shown to rescue molecular, synaptic, and behavioral deficits observed in
Fmr1−/y mouse, including restoration of elevated protein synthesis [
12,
14‐
16].
The link between aberrant protein synthesis and autistic-like behavioral deficits has been demonstrated in mice by studies utilizing genetic manipulation of proteins involved in cap-dependent translation, and recently it has been demonstrated that cell type-specific deletion of the translational repressor eukaryotic translation initiation factor 4E-binding protein 2 (4E-BP2) specifically in γ-aminobutyric acid (GABA)ergic inhibitory neurons results in autistic-like behavioral alterations in mice [
17‐
19]. There is a large diversity of GABAergic neuron subtypes in the neocortex that vary in morphology, electrophysiological properties, connectivity patterns and expression of specific molecular markers. Ca
2+-binding protein parvalbumin (PV) and the neuropeptide somatostatin (SOM)-expressing inhibitory neurons are the major inhibitory neuron subtypes in CNS, accounting for 40% and 30% of all GABAergic neurons, respectively [
20]. PV-positive inhibitory neurons are fast-spiking basket and chandelier cells, targeting soma and axon initial segment of neurons where they provide strong inhibition of synaptic firing, whereas SOM-expressing Martinotti cells target distal dendrites to gate inputs to pyramidal neurons [
21]. Recent studies indicate that these major inhibitory neuron subtypes play very distinct roles in neural circuits and perform different functions in behaving animals. For example, in medial prefrontal cortex (mPFC), SOM, but not PV, neuron activity is critical for discrimination of affective states during social interaction, whereas PV neuron activity is necessary for social investigation behavior [
22]. In the amygdala, during threat conditioning, PV and SOM neurons are activated during distinct behavioral phases to exert bidirectional control on acquisition of fear [
23]. Cell type-specific function of FMRP and its role in the performance of behavioral tasks, as well as its contribution to FXS-associated behavioral deficits has not been examined in detail.
Here, we used Cre-lox recombinase technology to assess whether deletion of Fmr1 in either PV or SOM-expressing neurons leads to behavioral deficits in mice. We found that cell type-specific deletion of Fmr1 in PV, but not SOM-expressing neurons, results in behavioral alterations, including anxiety-like behavior and deficits in social interaction. Furthermore, FMRP loss from PV-positive neurons was associated with brain region-specific dysregulation of de novo protein synthesis, whereas its absence from SOM-positive neurons did not impact global protein synthesis in mouse cortex or hippocampus. Our findings uncover a distinct cell type-specific role for FMRP in two major inhibitory neuron populations in mediating specific cellular and behavioral deficits associated with FXS.
Discussion
Our data indicate that ablating FMRP expression in PV-positive neurons recapitulates some of the behavioral deficits associated with FXS, including impaired social interaction and elevated anxiety, but does not result in impairments in learning and memory. Furthermore, we found that
Fmr1 deletion in PV-positive neurons resulted in brain region-specific dysregulation of de novo protein synthesis in PV-positive cells whereas deletion of
Fmr1 in SOM-expressing inhibitory neurons did not result in any behavioral deficits and no effect on global de novo protein synthesis was observed in these cells. Our findings suggest a differential role for FMRP expression in two major inhibitory neuron populations and increase our understanding of the cell type-specific mechanisms underlying FXS phenotypes. As FMRP is ubiquitously expressed in cells and present in most brain regions [
30‐
33], selective deletion in specific cell types likely will result in a particular pattern of FXS-like phenotypes. The contribution of FMRP expression in different CNS cell types to FXS pathophysiology is now beginning to be elucidated, but the effects of
Fmr1 deletion in GABAergic neurons have not been previously described. Selective deletion of
Fmr1 from forebrain excitatory neurons induces cellular, electrophysiological, and behavioral phenotypes in mice including increased mTOR/Akt phosphorylation and enhanced locomotor activity, but not anxiety-like behavior [
34]. In addition, mice with deletion of
Fmr1 in forebrain excitatory neurons exhibit deficits in PV-positive neuron density and perineuronal net formation. FMRP ablation in astroglial cells results in cellular, synaptic, and behavioral deficits, including elevated protein synthesis, increased neuronal spine density, enhanced phosphorylation of rpS6, and impaired learning of a motor task [
35,
36].
Fmr1−/y mice exhibit several autistic-like behaviors and cognitive impairments, although specific findings vary between different labs, which has been attributed to differences in the mouse genetic background [
37].
Fmr1−/y-PV mice recapitulate some of the behavioral deficits previously described in
Fmr1−/y. Hyperactivity has been reported in
Fmr1−/y mice [
38,
39], although we did not observe hyperactivity in
Fmr1−/y-PV mice (data not shown). No changes in anxiety-like behavior or reduced anxiety have both been reported in
Fmr1−/y mice [
26,
39], which contrasts with elevated anxiety we observed in
Fmr1−/y-PV mice (Fig.
2B). Furthermore,
Fmr1−/y mice exhibit stereotypic and repetitive behaviors in the marble burying task and increased self-grooming [
13,
26]. We did not observe these behaviors in
Fmr1−/y-PV mice (Fig.
2C, D). In addition,
Fmr1−/y mice exhibit perseverative behaviors during water Y maze reversal [
15] whereas we observed deficit during training but not reversal portion of the Y maze in
Fmr1−/y-PV mice (Fig.
2E). Impairments in communication (reduced number of ultrasonic vocalizations) and deficits in social interaction have been reported in
Fmr1−/y mice [
13,
40]. Social vocalizations were not tested in
Fmr1−/y-PV mice. We observed impaired social novelty behavior in
Fmr1−/y-PV (Fig.
2G) which is in line with findings in
Fmr1−/y [
13]. Deficits in learning and memory have also been reported in
Fmr1−/y mice. During MWM, fewer platform crossings in MWM on probe trial day, slight deficit in training or no deficits have been reported in
Fmr1−/y mice [
26,
41,
42]. We did not observe significant deficits in
Fmr1−/y-PV mice during MWM (Fig.
2C, D and E). Furthermore, no deficits in threat conditioning or impaired cue and context freezing were reported in
Fmr1−/y mice [
26,
41,
43]. We did not observe deficits in threat conditioning in
Fmr1−/y-PV mice, but we observed enhanced freezing during auditory tone presentation (Fig.
3G–J). Thus, cell-type specific deletion of
Fmr1 in PV + neurons recapitulates some, but not all, of the behavioral deficits described in global
Fmr1−/y mice.
Elevated or dysregulated neuronal protein synthesis has been postulated as one of the key mechanisms underlying development of ASD and it has been demonstrated that manipulating expression of proteins controlling cap-dependent translation initiation, such as 4E-BP and eIF4E, results in aberrant, autistic-like behavioral deficits in mice [
17,
18,
44]. Cell type-specific conditional knockout of the translational repressor 4E-BP2 in GABAergic inhibitory neurons is sufficient to induce autistic-like phenotypes, although analysis of protein synthesis was not performed [
19]. Here, we show that cell-type specific deletion of
Fmr1 in PV inhibitory neurons results in increased de novo protein synthesis in these cells in the mPFC. In addition, we found impaired social novelty behavior in
Fmr1−/y-PV mice. PV neuron activity in mPFC has been implicated in mediating social behavior in mice [
22,
45,
46]; therefore, it is not surprising that exaggerated protein synthesis in these cells may be associated with deficits in social behavior, as in our studies. On the other hand, de novo protein synthesis in hippocampal PV neurons was decreased rather than elevated upon cell type-specific deletion of
Fmr1, which is inconsistent with the cellular function of FMRP as a translational repressor. In addition, this contrasted with elevated de novo protein synthesis observed in PV-positive neurons in
Fmr1−/y mouse hippocampus (Fig.
1D). These discrepancies could be driven by altered activity in another brain region in
Fmr1−/y-PV mice, as global
Fmr1 deletion may have different circuit-level effects compared to cell type-specific
Fmr1 ablation. Furthermore, we observed elevated PV expression in
Fmr1−/y-PV hippocampus but not in the
Fmr1−/y mouse hippocampus, which may also be driven by differences in the activity in PV-positive cells in these models as it known that parvalbumin expression can be regulated by activity [
47,
48].
Signaling by group 1 mGluRs stimulates protein synthesis in response to synaptic activity and in
Fmr1−/y mice protein synthesis is exaggerated and not responsive to further induction by group 1 mGluR activation [
49,
50]. Several receptor-mediated signal transduction pathways that regulate neuronal protein synthesis, including muscarinic acetylcholine receptors, dopamine D1/5 receptors and tyrosine kinase receptor B, were also found to be dysregulated in
Fmr1−/y mice [
51‐
53]. Thus, it’s possible that in PV-positive neurons in
Fmr1−/y-PV mice, mTORC1 signaling is decoupled from protein synthesis. In the hippocampus, we observed decreased protein synthesis despite elevated rpS6 phosphorylation which may indicate that mTORC1 activation was unable to elicit an increase in protein synthesis. On the other hand, in mPFC we observed elevated protein synthesis whereas rpS6 phosphorylation was unaltered, which may also suggest that protein synthesis is not responsive to mTORC1 signaling in PV-positive neurons in
Fmr1−/y-PV mice.
In the context of global
Fmr1 deletion in
Fmr1−/y mice, several studies reported abnormal PV inhibitory neuron development and function. In
Fmr1−/y mouse somatosensory cortex, PV neurons showed altered expression and morphology, delayed maturation, and deficits in local excitation [
54‐
57]. In addition, impaired visual discrimination in
Fmr1−/y mice, was shown to be correlated with decreased PV inhibitory neuron activity in the primary visual cortex and can be rescued by normalizing PV neuron activity [
58]. Deficits in SOM neurons have not been described in
Fmr1−/y mice. Our data indicate that de novo protein synthesis was elevated in SOM and PV inhibitory neurons in the
Fmr1−/y mouse hippocampus. Furthermore, we found that cell type-specific deletion of
Fmr1 in PV, but not SOM, neurons was associated with dysregulated protein synthesis and can recapitulate some FXS-like behavioral deficits. How might dysregulated protein synthesis in PV inhibitory neurons lead to behavioral deficits? PV inhibitory neurons regulate pyramidal neuron activity and play an important role in gating cortical excitation/inhibition (E/I) balance, which is critical for proper cortical function as E/I imbalance is associated with multiple psychiatric conditions [
59,
60]. FMRP has been shown to regulate the expression of Kv4.2 potassium channels and surface expression of Cav2.2 calcium channels [
61,
62]. FMRP also interacts with Slack and BK potassium channels to modulate their activity [
63,
64]. In addition, mRNAs of numerous ion channel subunits, including N-type, L-type, and R-type and T-type calcium channel, among others, have been identified as putative FMRP targets [
9,
65]. Altered expression or function of these proteins in the absence of FMRP could potentially lead to altered intrinsic cell excitability [
66]. Previous studies in
Fmr1−/y mice suggest neuronal and circuit hyperexcitability in several brain regions, which is in line with clinical features of sensory hypersensitivity observed in FXS individuals [
66]. Furthermore, PV neurons appear to be especially sensitive to insults, including disruption of ASD-associated genes as deficits in PV neurons were observed in mouse models harboring deletion of other ASD-associated genes including
Cntnap2,
Mecp2,
Nlgn3 and
Shank3 and in human postmortem neocortical tissue from subjects with autism [
67‐
70]. Thus, PV inhibitory neurons may contribute to mediating ASD pathophysiology and represent a potential therapeutic target in ASD and other psychiatric diseases.
We found that following threat conditioning,
Fmr1−/y-PV mice spend significantly more time freezing in response to the auditory tone. In the basolateral amygdala, PV neuron activity during presentation of the CS was shown to correlate with enhanced learning [
23]. Thus, our findings implicate FMRP in the molecular mechanisms underlying acquisition and retrieval of auditory threat memory and suggest that PV neuronal activity may be altered following
Fmr1 deletion as inhibition of PV neurons enhances US-induced freezing [
23]. Because GABAergic inhibitory neuron activity is linked to anxiety, more studies are needed to uncover a potential role of FMRP in amygdala in anxiety-like behavior observed in FXS [
71,
72].
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