Channelopathies in fragile X syndrome

Abstract

Fragile X syndrome (FXS) is the most common inherited form of intellectual disability and the leading monogenic cause of autism. The condition stems from loss of fragile X mental retardation protein (FMRP), which regulates a wide range of ion channels via translational control, protein-protein interactions, and second messenger pathways. Rapidly increasing evidence demonstrates that loss of FMRP leads to numerous ion channel dysfunctions, i.e., channelopathies, which in turn contribute significantly to FXS pathophysiology. Consistent with this, pharmacological or genetic interventions that target dysregulated ion channels effectively restore neuronal excitability, synaptic function, and behavioral phenotypes in FXS animal models. Recent studies further support a role for direct and rapid FMRP-channel interactions in regulating ion channel function. This review lays out the current state of knowledge in the field regarding channelopathies and the pathogenesis of FXS, including promising therapeutic implications.

Fragile X syndrome (FXS) is the leading monogenic cause of intellectual disability and autism. Affecting both males (~1/4000) and females (~1/5000-8000), FXS is typically associated with cognitive dysfunction (language delay, intellectual disabilities, learning deficits), social and behavioral problems (anxiety, autism spectrum disorders), neurological deficits (seizures, abnormal sleep patterns), and morphological abnormalities (dysmorphic faces and macroorchidism)¹. The syndrome is caused by loss of fragile X mental retardation protein (FMRP), which most commonly results from the expansion of an unstable triplet CGG-repeat motif (>200) located within the 5’-untranslated region of the FMR1 gene on the X chromosome. This expansion causes gene hypermethylation and transcriptional silencing. Rare mutations in FMR1 that result in ‘loss-of-function FMRP’ have also been linked to FXS².

Increasing evidence suggests that the symptoms of FXS stem from disruptions in neuronal activity³. Indeed, FMRP, with its multiple RNA-binding and protein-protein interaction domains (BOX 1), is highly expressed throughout the nervous system⁴. It is involved in a broad range of physiological functions, including genome stabilization, RNA-editing, pre-RNA splicing, regulation of cell differentiation, and modulation of ion channel function, neuronal excitability, and synaptic plasticity. Specific to ion channel function, FMRP works through multiple direct and/or indirect mechanisms to control their expression and activity (TABLE 1)^3,5,6. Indeed, many types of ion channels are dysfunctional in Fmr1 knockout (KO) models, including voltage-dependent ion channels (Na⁺ channels, K⁺ channels, Ca²⁺ channels, and HCN channels), voltage-independent ion channels (SK channels), and ligand-gated ion channels (ionotropic-glutamatergic and GABAergic receptors). While these abnormalities are not caused by genetic mutations in the ion channel genes themselves, we broadly refer here to these ion channel dysfunctions as channelopathies. At the functional level, these channelopathies contribute to neuronal and network hyperexcitability, cognitive dysfunction, and behavioral abnormalities, all of which are part of FXS pathophysiology (FIG. 1). In this review, we first summarize evidence of hyperexcitability at the behavioral, circuit, cellular, and synaptic level in FXS. We then present findings in support of channelopathies underlying these excitability-related defects, including key molecular mechanisms. Finally, we discuss implications for future therapeutic strategies targeting ion channels in FXS.

Box 1|. FMRP functional domains.

The multi-domain structure of FMRP enables it to modulate the functions of ion channels through multiple mechanisms including regulation of channel translation, surface expression, and/or gating dynamics, or by indirectly regulating channel activity through second-messenger signaling pathways. FMRP is a protein of 632 amino acids with a molecular weight of ~80 kDa. The N-terminal domain (aa1-202) contains two Tudor (Agenet) domains (AG1 aa3-49 and AG2 aa63-113, both of which are involved in protein-protein interactions), one nuclear localization signal (NLS, aa115-154) and one K Homology (KH) domain (KH0, aa126-202). The central domain (aa203-404) comprises the KH1 (aa216-280) and KH2 (aa281-404) motifs that mediate RNA binding. The C-terminal domain contains the RGG box (aa527-552), a high-affinity RNA-binding domain which interacts with G-quadruplex RNAs and is essential for FMRP’s association with polyribosomes. The C-terminus also harbors a low complexity domain (LCD, aa466-632), which can promote dynamic interactions with proteins and RNAs. The LCD is implicated in the formation of ribonucleoprotein particles. Schematic representation shows FMRP functional domains¹⁵⁹. Three major domains are color-coded. Numbers indicate position within the amino acid sequence.

Table 1 |.

Mechanisms of FMRP regulation of ion channels

Channel/modulation	Direction of modulation by FMRP	Refs
Translational control
Kv1.2	⇧ or ⇩	58, 65, 95
Kv3.1	⇩	67, 68
Kv4.2	⇧ or ⇩	95–97
Slack	⇄ or ⇩	72, 95
BK	⇧	158
Cav1.3	⇧ or ⇩	88, 99
Cav2.1	⇧	89, 95
Cav2.3	⇩	90, 95
HCN	⇧ or ⇩	56, 57, 95, 102
AMPAR	⇧ or ⇩	59, 83, 104, 105
NMDAR	⇧ or ⇩	59, 111–113, 116
GABAAR	⇩	64, 119–125
Ion channel activity via FMRP-channel interactions
Slack	⇧	72
BK	⇧	57, 62, 80
SK	⇧ or ⇩	51, 86
Kv1.2	⇧	65
Kv4	⇩	91
Cav3	⇩	91
Ion channel surface expression via FMRP-channel interactions
Kv1.2	⇧	65
Cav2.1	⇧	60, 89
Cav2.2	⇩	60
HCN	⇧ or ⇩	102
AMPAR	⇧ or ⇩	59, 108
Second messenger pathways
INaP	⇩	52
Unidentified
NKCC1	⇩	145, 146

⇧ or ⇩, FMRP increases or decreases channel function, respectively;

⇄, no significant change in channel function;

Figure 1 |. Overview of channelopathies in FXS.

a | Though FXS clinical manifestations are complex and vary considerably among individuals, most FXS patients exhibit cognitive dysfunction (intellectual disabilities, learning deficits, language delay), social and behavioral problems (anxiety, autism spectrum disorders), neurological deficits (seizures, abnormal sleep patterns), indicative of cellular and network hyperexcitability. b | Brain regions that are affected in FXS and are discussed in this review (somatosensory cortex, prefrontal cortex, hippocampus, entorhinal cortex, amygdala, cerebellum) are shown in a medial view of the brain (left panel, brain stem and cerebellum removed) and in a lateral view of the brain (right panel). c | A cartoon representation of the basic unitary neural circuit showing increased circuit excitability and excitation/inhibition imbalance, a key feature of the circuit changes observed in FXS. d | Hyperexcitability defects observed in FXS can be attributed to channelopathy-caused functional abnormalities in (1) AP initiation and firing; (2) neurotransmitter release; and/or (3) dendritic function, which may underlie various FXS phenotypes shown in a (multiple arrows). Ion channels contributing to these excitability defects are listed in corresponding inserts 1, 2 or 3. Insert 1, The FXS-related hyperexcitability is apparent in elevated neuronal firing frequency. These changes involve functional alterations in both potassium channels and sodium channels, including Kv1.2, Kv3.1, Slack, BK, SK, I_NaP and I_NaT. AP traces in light-green and green represent normal and FXS conditions, respectively, indicating that loss of FMPR increases AP firing frequency. Insert 2, The transduction of neuronal firing into neurotransmitter release at presynaptic terminals is mediated by Ca²⁺ influx through the voltage-gated calcium channels (VGCCs). Cav2.1 and Cav2.2 channels are the predominant VGCCs supporting neurotransmitter release in central neurons. FMRP can also indirectly regulate neurotransmitter release by controlling AP peak and duration, which in turn determine the amplitude and duration of presynaptic calcium influx through VGCCs. This mechanism is mediated by a subset of voltage-gated K⁺ channels, including Kv1.2 and BK channels. Insert 3, Dendritic excitability is governed, in large part, by ion channels, a number of which are dysregulated in the absence of FMRP, including HCN, Kv4, BK, Cav1, Cav2.3, Cav3, AMPAR, NMDAR, GABA_AR and NKCC1. EPSC waveforms in light-blue and blue denote normal and FXS conditions, respectively, indicating that loss of FMRP enhances EPSC integration and thus increases dendritic excitability.

Abbreviations: AMPAR, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor; BK, large-conductance calcium-activated potassium channel; Cav1, voltage-gated calcium channel L-type; Cav2.1, voltage-gated calcium channel P/Q type; Cav2.2, voltage-gated calcium channel N type; Cav2.3, voltage-gated calcium channel R type; Cav3, voltage-gated calcium channel T type; GABA_AR, γ-aminobutyric acid type A receptor; HCN, hyperpolarization-activated, cyclic nucleotide-gated ion channel; I_NaP, persistent sodium current carried by voltage-gated sodium channel; I_NaT, transient sodium current carried by voltage-gated sodium channel; Kv1.2, voltage-gated potassium channel subfamily A member 2; Kv3, voltage-gated potassium channel subfamily C; Kv4, voltage-gated potassium channel subfamily D; NKCC1, Na⁺–K⁺–Cl⁻ cotransporter; NMDAR, N-methyl-D-aspartate receptor; NT, neurotransmitter; SK, small-conductance calcium-activated potassium channel; Slack, sodium-activated potassium channel.

Evidence of hyperexcitability in FXS

The clinical phenotypes of FXS are quite complex and vary considerably across individuals. However, most exhibit neurological symptoms indicative of network hyperexcitability, including seizures, hyperarousal, hyperactivity, and hypersensitivity to sensory stimuli⁷. Neuronal hyperexcitability is also consistently observed in FXS animal models, data from which indicate intrinsic hyperexcitability and/or excitation/inhibition (E/I) imbalance in neuronal circuits^3,8,9. We summarize the findings from both human patients and animal models below.

Behavioral abnormalities/symptoms

Epilepsy—an outward expression of circuit-level hyperexcitability—occurs in 10–20% of individuals with FXS. Seizure patterns on electroencephalography (EEG) typically present as benign focal epilepsy of childhood¹⁰, though many affected individuals continue to have seizures after the age of 20¹¹. FXS patients are also hyper-responsive to sensory stimulation and display tactile defensiveness^12,13. Sensory hyperreactivity can cause a hyperaroused state characterized by disruptions in circadian rhythms, including frequent awakenings from sleep^14,15. The seizures and hyperresponsivity are recapitulated in FXS models¹⁶; Fmr1 KO mice have a lower threshold for audiogenic seizures^17–23, hypersensitivity to sensory stimuli^24–26, and hyperactivity^27–29.

Circuit and network dysfunctions

Increased excitability at the network level has been measured in patients using various non-invasive techniques. For example, EEG abnormalities are commonly observed in FXS individuals^11,15,30, including centrotemporal spikes, slowed background rhythm, and multifocal epileptiform discharges. Sensory hypersensitivity is also apparent in the elevated amplitude of evoked potentials in response to auditory and/or visual stimuli³¹, including heightened event-related brain potentials within auditory cortex of children with FXS³². Looking into the mechanisms of cortical network hyperexcitability in FXS individuals, Morin-Parent et al.⁹ used transcranial magnetic stimulation to show that patients with FXS have significantly reduced short-interval intracortical inhibition (mediated by GABA_A receptors), increased long-interval intracortical inhibition (mediated by GABA_B receptors), and increased intracortical facilitation (mediated by glutamatergic receptors) compared to healthy controls. Overall, the data support that loss of FMRP leads to network hyperexcitability, hypersensitivity to sensory stimuli, and E/I imbalance.

Some of the specific neurological symptoms associated with FXS are also indicative of circuit dysfunction in particular brain regions. For example, tactile defensiveness, learning and memory deficits, fear and anxiety—all of which are common in FXS—can be linked to circuit/network abnormalities in the somatosensory cortex, hippocampus, or amygdala, respectively. Data from FXS animal models confirm the presence of circuit abnormalities and marked E/I imbalance in these regions. In the somatosensory cortex of Fmr1 KO mice, circuit hyperexcitability manifests as prolonged UP states^33–35. Studies also find elevated cortical network synchrony in layer 2/3 of barrel cortex³⁶, whereas network inhibition during the UP states is less synchronous³³. Also in this model, Domanski and colleagues observe diminished feed-forward inhibition (FFI) and increased E/I ratio during trains of activity in layer 4 barrel cortex neurons³⁷. Similar network E/I imbalance is also observed in primary visual cortex³⁸ and auditory brain stem^39–41 of FXS animal models. In the hippocampal circuits of Fmr1 KO mice, hyperexcitability manifests in elevated epileptiform activity^42,43 as well as abnormally high spiking probability and reduced spiking precision in hippocampal FFI circuits^44,45. These defects are associated with a marked shift in E/I circuit balance, primarily a result of reduced activity of FFI hippocampal interneurons. In the amygdala of Fmr1 KO mice, though the overall E/I balance appears to be maintained, the temporal window of peak excitation to peak inhibition is significantly narrower as a result of reduced tonic GABA_AR-mediated inhibition^46,47. This narrowing leads to circuit hyperexcitability and abnormal amygdala functions^46,48–50.

Collectively, these data support the presences of hyperexcitable circuits and networks in FXS individuals and animal models, some of which may underlie characteristic FXS symptoms.

Synaptic and cellular defects

Some of the behavioral and network-level hallmarks of FXS are driven by cellular and synaptic mechanisms. There is now extensive evidence of changes in synaptic transmission and plasticity, as well as dysregulated cellular excitability in FXS. For example, in animal models neurons across multiple brain regions exhibit an abnormally high action potential (AP) firing rate, including in hippocampus, somatosensory cortex, entorhinal cortex, and the auditory brainstem^{33,36,37,51–54}.

In terms of dendritic function and synaptic plasticity, Fmr1 KO mice have a higher threshold for spike-timing-dependent plasticity in the prefrontal cortex⁵⁵, and changes in the input resistance of dendritic compartments, which can significantly impact dendritic summation and thus cellular excitability^37,56–58. In line with elevated dendritic excitability, higher single spine glutamate currents and elevated dendritic gain (resulting in higher AP output) are observed in somatosensory cortex layer 4 stellate cells of Fmr1 KO mice⁵⁹. In addition, there are major FMRP-related abnormalities in neurotransmitter release across several types of neurons. For instance, glutamate release is elevated in the axon terminals of dorsal root ganglion neurons^60,61 and at the hippocampal CA3-CA1 synapse during spike trains^43,62,63.

Several studies have also observed synaptic deficits in inhibitory interneurons, including reduced GABA release in the hippocampus of Fmr1 KO mice^44,64, attributable in part to excessive presynaptic GABA_B receptor signaling in inhibitory presynaptic terminals⁴⁴. In contrast, Yang and colleagues report increased GABA release from basket cells in the cerebellum of Fmr1 KO mice, resulting in reduced firing frequency among postsynaptic Purkinje neurons⁶⁵. Though contradictory at first glance, Purkinje neurons are inhibitory, and thus the circuit-level effect of these changes are similar—both reduce circuit inhibition and promote hyperexcitability. Similarly, in the amygdala, loss of FMRP causes a marked reduction in the frequency and amplitude of inhibitory postsynaptic currents (IPSCs), reduced tonic inhibition, and decreased GABA availability⁴⁸. In the subiculum, loss of FMRP leads to selective reduction of tonic inhibition, but not phasic IPSCs, suggesting that specific subtypes of GABA_A receptors mediating tonic inhibitory currents are downregulated⁶⁶.

The Roles of Channelopathies in FXS

Overview

Most of the pathological changes in excitability associated with FXS can be attributed to ion channel dysfunction that occurs as a result of the loss of FMRP, which regulates multiple types of channels across various neuronal subcompartments (FIG 1. and TABLE 1). The specific functional consequences of FMRP loss include changes in AP initiation and firing pattern (involving various K⁺, and Na⁺ channels), neurotransmitter release (involving Ca²⁺ and K⁺ channels), and dendritic function (involving K⁺, Ca²⁺, and HCN channels, as well as AMPAR, NMDAR and GABA_AR) (BOX 2, TABLE 2). In turn, these functional effects are likely part of the mechanisms underlying cognitive and behavioral hallmarks of FXS. In the following sections we discuss recent advances in our understanding of channelopathy-based mechanisms of FXS pathophysiology, and potential new therapeutic strategies stemming from our growing knowledge of FXS-related ion channel defects. Where applicable, we also review efforts to investigate potential therapeutic interventions that target particular channelopathies (TABLE 3).

Box 2 |. Mechanisms underlying excitability defects in FXS models.

Loss of FMRP dysregulates a large number of ion channels. This in turn affects multiple aspects of neuronal excitability, including changes in AP initiation and firing rate, presynaptic vesicle release, dendritic properties, and synaptic plasticity (TABLE 2).

• The combined activity of voltage-gated Na⁺ and K⁺ channels determine AP shape, firing rate and pattern. For example, channels that active around threshold potentials, such as SK channels and Na⁺ channels carrying I_NaP, contribute to AP initiation. Na⁺ channels carrying I_NaT determine AP rising speed and amplitude. Several families of voltage-gated K⁺ channels act in concert to determine AP repolarization dynamics, and thus AP duration.

• AP firing patterns are transduced into synaptic vesicle release at synaptic terminals via activation of VGCCs, among which P/Q- and N-type Ca²⁺ channels are the major source of Ca²⁺ for synaptic vesicle release in central neurons.

• Multiple channels act together to control dendritic excitability, input resistance, and thus spatial and temporal summation, including K⁺ channels, VGCCs, and HCN channels. Many of these channels are also involved in the initiation and maintenance of various forms of synaptic plasticity. Ligand-gated ion channels/ionotropic receptors (AMPA, NMDA and GABA_A receptors) function to directly evoke excitatory or inhibitory synaptic currents.

• Many channels are expressed in multiple cellular compartments and be activated by different factors, in cell-type and brain-region specific manners.

Table 2 |.

Mechanisms underlying excitability defects in FXS models

Channel	Changes	Functional abnormality	Refs
AP initiation and firing pattern
Kv3.1	↑	↑AP repolarization speed and firing rate	53, 68
Slack	↓	↑ AP duration and firing rate	73
BK	↓	↑ AP duration, ↓ fAHP	62
Kv1.2	↓	↑ AP duration	65
SK	↓ ↑	↓ AP threshold and mAHP, ↑ AP firing rate ↑ mAHP, ↓AP firing rate	51 86
INaP	↑	↓ AP threshold, ↑ AP firing rate	52
INaT	↑	↑ AP rising speed and amplitude ↑ AP firing rate, ↓ AP duration	54
Neurotransmitter release
Cav2.1	↓	May be a compensation for Cav2.2	89
Cav2.2	↑	↑ Ca2+ influx and vesicle release	60, 61
BK	↓	↑ Ca2+ influx and vesicle release	62
Kv1.2	↓	↑ Ca2+ influx and vesicle release	65
Dendritic function
Kv4	↑	↑ threshold for LTP, Lack of TBS-induced LTP	91, 97
Cav3	↑	↑ Ca2+ influx	88
BK	↓	↑ dendritic excitability, ↑ sensory sensitivity, ↑ dendritic spines	57, 77
Cav1	↓	↑ threshold for tLTP	99,55
	↑	↑ progenitor differentiation to glutamate-responsive cells	88
Cav2.3	↑	↑ Ca2+ influx	90
HCN	↓	↑ input resistance and summation	56, 102
	↑	↓ input resistance and summation	57,59,102
AMPAR	↓	↓ LTP and ↑ LTD	104, 105
	↑	↓ LTP, ↑ AMPAR current ↑ silent dendritic spines	59, 83
NMDAR	↓	↓ NMDAR currents	113, 116
	↑	↑ NMDAR mEPSC frequency	59
GABAAR	↓	↓ inhibition	64,120,121
NKCC1	↑	delayed GABAAR polarity switch delayed maturation of glutamatergic synapses	145, 146

↑, increase; ↓, decrease; AP, action potential; fAHP, fast afterhyperpolarization; LTD, long-term depression; LTP, long-term potentiation; mEPSC, miniature excitatory postsynaptic current; MF, mossy fiber; RMP, resting membrane potential; TBS, theta burst stimulation; tLTP, spike-timing-dependent long-term potentiation.

Table 3 |.

Manipulations that rescue cellular, circuit and behavioral phenotypes in FXS models

Target	Rescue strategy	Behavioral phenotypes normalized	Refs
Kv1.2	Oral administration of Kv1.2 agonist DHA	Acoustic startle reflex and social interaction	65
Kv3.1	Kv3.1 positive modulator AUT2	MNTB neurons firing rate Auditory brain response	53
Kv4	Kv4 blocker heteropodatoxin	TBS-induced LTP in hippocampus	97
Slack	FMRP1-298	Slack open probability.	72, 73
BK	Intracellular application of FMRP1-298 Genetic upregulation of BK activity by ablation of BKβ4 subunit	AP duration, BK open probability, glutamate release Short-term plasticity Epileptiform activity	43, 62, 76
	Intraperitoneal injection of BK channel opener BMS-204352	Acoustic startle reflex, locomotor activity, nest building, grooming, social interactions, anxiety and spatial memory	57, 77, 84
SK	Intracellular administration of FMRP1-298, Genetic reintroduction of FMRP1-234 or SK channel opener 1-EBIO or NS309	AP threshold, mAHP and AP firing rate   MF-CA3 input-output transmission.	51
NMDAR	Bath application of NMDAR coagnoists glycine or D-serine	NMDAR-dependent LTP in dentate gyrus	113
GABAAR	Intraperitoneal injection of GABAAR agonist ganaxolone, acamprosate or metadoxine.	UP states, locomotor activity, anxiety, marble burying behavior and sensory hyperresponsiveness.	127,131
NKCC1	Intraperitoneal injection of NKCC1 inhibitor bumetanide	GABAAR reversal potential of L4 somatosensory neurons Excitatory synapse development LTP in somatosensory cortex Whisker-evoked responses in the cortex	146
FMRP loss	Intravenous injection of FMRP297-tat	Cav3 and Kv4 currents Cerebellar MF-granule cell LTP Protein levels (APP, αCaMKII and PSD95), locomotor activity	91

αCaMKII, alpha-Ca²⁺/calmodulin-dependent protein kinase II; AUT2, ((4-(⁵³oxy)-2-(1-methylethyl) benzonitrile; CP, critical period; DHA, docosahexaenoic acid; mAHP, medium afterhyperpolarization; MF, mossy fiber; NS309, 3-Oxime-6,7-dichloro-1H-indole-2,3-dione; MNTB, medial nucleus of the trapezoid body; PSD, postsynaptic density.

AP Initiation and Firing Patterns

The FXS-related hyperexcitability noted at the behavioral and circuit level is apparent in lowered AP threshold and elevated firing frequency characteristic of Fmr1 KO animal models. These changes involve functional alterations to both potassium (K⁺) channels and sodium (Na⁺) channels.

The role of K⁺ channels

Potassium channels crucially contribute to establishing resting membrane potential, and setting AP dynamics and firing patterns. FMRP regulates the translation of a number of K⁺ channels, as well as their activity and surface expression (FIG. 2).

Figure 2 |. FMRP controls ion channel functions through multiple mechanisms.

There are four major mechanisms by which FMRP regulates channel functions. a | FMRP regulates ion channel’s gating. By interacting directly with the pore-forming subunit (1) and/or with the regulatory subunit (2) of ion channels, FMRP modulates the function of the pore-forming subunit and thus regulates neuronal excitability and neurotransmitter release. Ion channels regulated by FMRP through this mechanism include Slack, BK, SK, Kv1.2, Kv4.3 and Cav3. b | By binding to mRNA of ion channels, FMRP regulates ion channel translation and thus modulates neural excitability. Ion channels regulated by FMRP through this mechanism include Slack, BK, Kv1.2, Kv3.1, Kv4.2, Cav1.3, Cav2.1, Cav2.3, HCN, AMPAR, NMDAR and GABA_AR. c | By binding to ion channels, FMRP modulates ion channel trafficking and surface expression. Ion channels regulated by FMRP through this mechanism include Kv1.2, Cav2.1, Cav2.2, HCN and AMPAR. d | FMRP controls ion channel activity via second messenger pathways. By modulating mGluR5-PKC-PLC pathway, FMRP modulates persistent sodium current (I_NaP) and thus regulates AP threshold and excitability. Note that the same channel can be modulated via multiple mechanisms. Also, the direction of changes, extent and the mechanism of channel modulation by FMRP is often brain-region, cell-type and even subcellular-compartment specific

Abbreviations: AMPAR, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor; BK, large-conductance calcium-activated potassium channel; Cav1.3, voltage-gated calcium channel with α1D subunit L-type; Cav2.1, voltage-gated calcium channel P/Q type; Cav2.2, voltage-gated calcium channel N type; Cav2.3, voltage-gated calcium channel R type; Cav3, voltage-gated calcium channel T type; GABA_AR, γ-aminobutyric acid type A receptor; HCN, hyperpolarization-activated, cyclic nucleotide-gated ion channel; I_NaP, persistent sodium current carried by voltage-gated sodium channel; Kv1.2, voltage-gated potassium channel subfamily A member 2; Kv3.1, voltage-gated potassium channel subfamily C member 1; Kv4.2, voltage-gated potassium channel subfamily D member 2; Kv4.3, voltage-gated potassium channel subfamily D member 3; mGluR5, metabotropic glutamate receptor subtype 5; NMDAR, N-methyl-D-aspartate receptor; PKC, protein kinase C; PLC, phosphoinositide phospholipase C; SK, small-conductance calcium-activated potassium channel; Slack, sodium-activated potassium channel.

Kv3.1 channels.

FMRP directly regulates the expression of delayed rectifier K⁺ channel (Kv3.1) mRNA^67,68. The rapid voltage-dependent activation/deactivation characteristics of Kv3.1 facilitate high firing rates in cells that express these channels. For example, there is normally a gradient of Kv3.1 expression within the sound localization circuitry of the brainstem (the medial nucleus of the trapezoid body, MNTB). However, when FMRP is absent, the normal gradient distribution of Kv3.1 is flattened and the levels of Kv3.1b protein are higher overall⁶⁹. Consequently, the high-threshold (Kv3.1-dependent) current is increased in the MNTB of Fmr1 KO mice, leading to faster AP repolarization and higher firing rates (i.e., hyperexcitability)⁵³.

Therapeutic implications.

Since increased Kv3.1 channel expression contributes to impaired encoding and processing of auditory information in Fmr1 KO mice⁶⁸, these channels are promising therapeutic targets. Indeed, the Kv3.1 channel modulator AUT2 normalizes AP firing rate in auditory brainstem neurons and restores the auditory brainstem response in vivo in Fmr1 KO mice⁵³. Targeting Kv3.1 channels may have an even broader therapeutic potential to reduce sensory hypersensitivity across multiple modalities in FXS, as Kv3.1 channels are widely expressed in rapidly firing neurons throughout the nervous system⁷⁰. However, it remains to be determined if loss of FMRP also increases excitability among these rapidly firing neurons in various brain regions in a Kv3.1-dependent manner.

Slack channels.

Slack channels are widely expressed in the brain, including in brainstem, cerebellum, prefrontal cortex, and hippocampus. As a major component of the delayed outward current, Slack channels are critical in regulating AP firing patterns and temporal precision⁷¹. FMRP directly modulates Slack channel activity⁷². In a seminal study, Kaczmarek and colleagues show that the N-terminal domain of FMRP interacts with the Slack channel C-terminus to increase the frequency of the channel opening (i.e. gating) in a rapid and reversible manner^72,73. Through this protein-protein interaction, FMRP dynamically and precisely tunes Slack channel activity and thus neuronal firing. In the absence of FMRP, Slack currents are significantly reduced, thus increasing neuronal excitability and reducing temporal precision of spiking, as observed in the auditory brainstem of Fmr1 KO mice⁷². Together with the aforementioned effects on Kv3.1 channels, the FMRP-related decrease in Slack currents likely contributes to auditory hypersensitivity in FXS.

Interestingly, an additional “nonconducting” function for Slack channels in regulating neuronal firing has been reported in Aplysia bag cell neurons in which activation of Slack channels is linked to protein synthesis-dependent, prolonged changes in neuronal firing patterns⁷³. This function raises the possibility that Slack-FMRP interactions may link changes in neuronal firing to changes in protein translation, known to be disrupted in FXS.

BK channels.

Large conductance voltage-and Ca²⁺-activated K⁺ (BK) channels are widely expressed throughout the brain, and in nearly every cellular compartment of neurons⁷⁵. Being both voltage- and calcium-activated, these channels play critical roles in regulating neuronal excitability, AP duration, firing, and neurotransmitter release⁷⁵. FMRP targets BK channels, regulating translation and function^{43,57,62,76–80}. Specifically, the N-terminal domain of FMRP interacts with the channel’s regulatory β4 subunit to increase BK channel’s Ca²⁺ sensitivity and thus open probability^43,62. FMRP can also directly bind to the BK channel’s pore-forming α subunit^76,80 to modulate channel gating and increase BK currents⁸⁰. Indeed, studies of both Fmr1 KO mice and stem cell-derived neurons from FXS patients report that the loss of FMRP reduces BK channel activity, which in turn leads to a wide range of functional consequences. For example, in hippocampal and cortical neurons these effects include increased firing, excessive AP broadening, elevated presynaptic Ca²⁺ influx, and increased glutamate release^43,62,76,81.

More recent studies support the idea that the loss of FMRP-BK channel interactions contribute to FXS pathophysiology. Particularly interesting is the discovery that a rare FMRP missense mutation (R138Q), which is associated with a partial clinical FXS phenotype (intellectual disability and seizures) in at least one human patient, impairs the protein’s translation-independent presynaptic functions, including its ability to interact with BK channels to regulate AP duration and control glutamate release (i.e., excitability)⁷⁶. In drosophila, this mutation impairs the presynaptic-specific function of FMRP to control axonal elaboration in the neuromuscular junction⁷⁶. And in mouse cortical neurons, while the R138Q mutation preserves FMRP’s canonical mRNA binding and translational regulation functions—generally associated with (but not limited to) postsynaptic compartments—it impairs interactions with BK channels that ultimately control glutamate release⁷⁶. These results suggest a model in which the effects of R138Q mutation are caused by disruption of FMRP’s protein-protein interactions, including FMRP-BK channel interactions, which are important in regulating neuronal excitability and glutamate release, while retaining translational regulation functions of FMRP.

Despite the compelling alignment of both clinical and preclinical data linking these specific FMRP functions to a specific subset of FXS phenotypes, the story is likely more complicated. Two other individuals with FXS who harbor the same mutation present with different clinical phenotypes: one (male) presents with a more extensive set of FXS features, including intellectual disability, ADHD, autism, seizures, craniofacial changes, and hyperextensibility²; the other (unrelated female) presents with mild intellectual disability and attention deficits⁸². Data from a rodent model of FXS specific to the R138Q mutation indicate that at least some postsynaptic functions of FMRP are also impacted by this mutation; surface expression of AMPA receptors is increased, as is dendritic spine density, hippocampal LTP is impaired, and there are socio-cognitive deficits at the behavioral level⁸³. These data suggest that the R138Q mutation may disrupt FMRP interactions with other channels as well, resulting in both pre- and postsynaptic defects.

Therapeutic implications.

Upregulating BK channel activity may be a useful approach to normalize a range of excitability-associated phenotypes in FXS. Indeed, genetically upregulating BK channel activity normalizes multiple synaptic and circuit defects in an FXS mouse model, including AP broadening, elevated glutamate release, and epileptiform activity in the hippocampal circuit⁴³. Moreover, the BK channel opener BMS-204352 corrects a number of behavioral phenotypes in Fmr1 KO mice, including hyperactivity, hypersensitivity to sensory stimuli, social defects, anxiety, deficient spatial memory, impaired nest building, and excessive grooming^57,77,84. Together, these studies suggest that targeting BK channels may have therapeutic potential in alleviating a number of excitability-associated FXS phenotypes.

Kv1.2 channels.

Kv1.2 channels are widely distributed across various cellular compartments of neurons, and play important roles in regulating intrinsic excitability and AP initiation⁸⁵. In Fmr1 KO mice, the Kv1-mediated current is downregulated in one population of layer 5 pyramidal cells (PCs) of the medial prefrontal cortex (called long tract-projecting neurons), leading to increased excitability of these neurons⁵⁸. Interestingly, this defect appears to be cell-type specific, as it is not observed in the neighboring intratelencephalic projecting PCs of the same brain region. Mechanistically, FMRP binds Kv1.2 channel mRNA, as well as interacts directly with the channel through protein-protein interactions to regulate channel activity and surface expression. Specifically, recent studies indicate that the N-terminus of FMRP directly binds to a phosphorylated serine motif on the C-terminus of Kv1.2 to form a dimer-dimer configuration in which two C-terminal strands of the same Kv1.2 channel bind to one FMRP dimer⁶⁵. This interaction enhances axonal trafficking, membrane expression, and gating dynamics of Kv1.2 in axonal terminals of cerebellar basket cells, ultimately impacting presynaptic excitability and Ca²⁺ influx. Consequently, loss of FMRP-Kv1.2 interaction in Fmr1 KO basket cells increases the duration and amplitude of APs, leading to enhanced presynaptic Ca²⁺ influx and excessive GABA release onto Purkinje neurons⁶⁵. Since Purkinje neurons are inhibitory, the overall effect of these changes is to promote circuit hyperexcitability.

Therapeutic implications.

As Purkinje neurons are the sole output cells from cerebellar cortex, the Kv1.2 channelopathy is likely to lead to abnormal cerebellar functions. Targeting Kv1.2 may thus help normalize cerebellar circuit hyperexcitability and cerebellar-related abnormalities in FXS. Indeed, the therapeutic potential of one Kv1.2 agonist, docosahexaenoic acid, was recently tested in an FXS mouse model, where it was found to rectify the dysregulated cerebellar inhibition in vitro and to rescue acoustic startle reflex and social interaction phenotypes in vivo⁶⁵.

SK channels.

Voltage-independent K⁺ channels also play key roles in regulating neuronal excitability by modulating the resting membrane potential, AP threshold, and afterpotentials⁸⁵. Within the context of Fmr1 KO mice, there is evidence of hypofunction of small conductance Ca²⁺-activated K⁺ (SK) channel, which is linked to neuronal hyperexcitability (lower AP threshold, smaller medium AHP, increased firing rate, and exaggerated synaptic gain) in CA3 PCs⁵¹. The SK-current defect stems from the loss of FMRP interactions with the SK2 channel isoform, though channel expression is unaffected. This idea is further supported by the finding that all observed excitability defects in CA3 PCs of Fmr1 KO mice are normalized by acute intracellular reintroduction of the N-terminal FMRP fragment (aa1-298), or the genetic reintroduction of an even shorter N-terminal FMRP fragment (aa1-234), both of which lack the KH2 domain and are thus incapable of translational regulation⁵¹. Interestingly, similar to other FXS-related channelopathies, the SK defects are cell-type specific. Within a single Fmr1 KO animal, changes to the SK current in CA1 PCs mirror those apparent in CA3: SK current goes up, as does spike rate variability and medium AHP, but AP rate goes down⁸⁶. Similar cell-type specific changes are also reported for Kv1⁵⁸, and HCN channels^56–58 (TABLE 1).

The role of Na⁺ channels

In most neurons, AP initiation and rising phase are governed by voltage-gated Na⁺ channel availability and activation/inactivation dynamics. Na⁺ channels can produce two distinct currents: the fast transient Na⁺ current (I_NaT) and the non-inactivating persistent Na⁺ current (I_NaP). I_NaP is active at subthreshold voltages and thus is critical for AP initiation, while I_NaT underlies the AP rising phase⁸⁵. Thus, disruption to either current is likely to impact neuronal excitability.

I_NaP within the context of FXS.

In Fmr1 KO mice, I_NaP is abnormally increased in the entorhinal cortex layer 3 PCs, i.e., neurons that project directly to CA1 region. This upregulation of I_NaP leads to profound excitability defects, including lower AP threshold and increased AP firing⁵². Unlike many K⁺ channel defects, which are mediated by FMRP-channel interactions within individual neurons, I_NaP-mediated defects are not apparent in neurons isolated from their circuit. This indicates that the defects are not cell-autonomous. Indeed, the enhanced I_NaP is caused by exaggerated activation of mGluR5 signaling, which in turn acts on Na⁺ channels through phospholipase C (PLC) and protein kinase C (PKC)⁵². This signaling mechanism requires circuit activity and is distinct from the well-established mGluR5 signaling cascade affecting local translation in FXS animal models. Thus, Na⁺ channel dysregulation is likely a major contributor to neuronal hyperexcitability in the FXS mouse model, at least in this subpopulation of cortical neurons.

I_NaT within the context of FXS.

While the above study did not include direct measures of I_NaT, observation of increased AP rising speed, an I_NaT-dependent parameter, suggests that I_NaT is also increased in Fmr1 KO neurons⁵². Indeed, a direct evidence of increased I_NaT resulting in larger and faster rising APs and a higher gain of somatic excitability is reported in layer 2/3 prefrontal cortex neurons of Fmr1 KO mice⁵⁴. This study also finds that the observed excitability changes are accompanied by a large depolarizing shift in the activation of A-type K⁺ channels. This shift is expected to decrease AP threshold, as is the case when I_NaP is higher. However, no such changes were observed⁵⁴, indicating that some form of compensatory changes may be involved.

Taken together, the data discussed here reflect a complex interplay of celltype-specific Na⁺ and K⁺ channelopathies caused by loss of FMRP. In nearly all cases, the deficits contribute to overall neuronal and circuit hyperexcitability. To elucidate further mechanistic detail will require careful consideration of celltype-specific effects, as well as the impact of compensatory changes.

Neurotransmitter Release

The transduction of neuronal firing into neurotransmitter release at presynaptic terminals is mediated by Ca²⁺ influx through the voltage-gated calcium channels (VGCC)⁸⁷. Interestingly, in FXS models all five types of mammalian VGCCs [P/Q (Cav2.1), N (Cav2.2), R (Cav2.3), L (Cav1), and T (Cav3)] are affected (FIG. 2, TABLE 2)^{6,60,61,88–91}. N- and P/Q-type channels are the predominant VGCCs supporting neurotransmitter release at most central synapses and are discussed here; those VGCC types that contribute to dendritic excitability changes in FXS are discussed in the next section.

N-type and P/Q-type VGCCs

The N-type VGCC (Cav2.2) plays a prominent role in neurotransmitter release at presynaptic terminals. It was also the first Ca²⁺ channel found to be directly modulated by FMRP through protein-protein interactions⁶⁰. The mechanism of this interaction is distinct from the known FMRP interactions with K⁺ channels; in the case of N-type VGCC, it is FMRP’s C-terminal domain that interacts with N-type channel’s synaptic targeting domain^60,61 to suppress the channels’ presynaptic localization and surface expression. Consequently, loss of FMRP increases N-type VGCC surface expression and glutamate release at presynaptic terminals of sensory neurons in dorsal root ganglia^60,61.

The P/Q-type (Cav2.1) channel is also critical for AP-evoked neurotransmitter release in many types of neurons. It is involved in various forms of synaptic plasticity as well as spatial learning and memory in mice⁹². In cortical neurons, overall levels of P/Q-type channels are unaltered by FMRP loss. However, we know that FMRP can bind to these channels to increase their surface expression^60,89; this is opposite of the effect of FMRP interactions with N-type channels. Thus, loss of FMRP results in reduced surface expression of P/Q-type channels, while surface expression of N-type channels is simultaneously increased⁸⁹. Interestingly, it has been suggested that N- and P/Q-type channels have channel-type preferred slots in hippocampal synapses, which can be occupied by the competing channel type upon certain disease conditions⁹³. Thus, the loss of FMRP may contribute to synaptic transmission deficits by creating an imbalance between the surface levels of N- and P/Q-type channels, which differentially contribute to the efficiency of neurotransmitter release and synaptic plasticity⁹³.

Indirect modulation of calcium influx

In addition to controlling presynaptic surface expression of VGCCs, FMRP can also indirectly regulate neurotransmitter release by controlling AP duration (which in turn determines the duration of presynaptic calcium influx through VGCCs). This mechanism is mediated by a subset of voltage-gated K⁺ channels that regulate AP duration, and has major implications for glutamate and GABA release, both of which are significantly impacted in Fmr1 KO mice. As discussed above, reduced BK channel activity in hippocampal and cortical excitatory neurons of Fmr1 KO mice leads to extensive AP broadening. This in turn elevates presynaptic calcium influx and glutamate release⁶². Similarly, the increased AP duration apparent in cerebellar basket cells (secondary to reduced Kv1.2 channel activity) leads to excessive GABA release⁶⁵. VGCC-K⁺ channel activity interdependence is often reciprocal; for example, VGCCs are the main source of Ca²⁺ to activate BK channels, whereas BK channels can physically couple to pore-forming α-subunits of L, P/Q, and N-type VCGGs⁹⁴. This reciprocal interdependence is present to various extents across different types of central neurons. Thus, it likely contributes to neuronal hyperexcitability within the context of FXS.

Dendritic function

There are numerous reports of disrupted dendritic function in FXS models, including changes in excitability, gain, integration, and multiple forms of long-term synaptic plasticity localized to the dendritic compartment^37,55–59. These functions are governed, in large part, by ion channels, a number of which are dysregulated in the absence of FMRP (BOX 2, TABLE 2).

K⁺ channels

Kv4 channels.

The Kv4.2 channel regulates hippocampal and neocortical dendritic excitability and synaptic plasticity. FMRP can bind Kv4.2 mRNA and regulate its translation^95–97. FMRP can also directly interact with and regulate Kv4 activity. Specifically, reduced A-type K⁺ currents (carried by Kv4 channels) in hippocampal PCs of Fmr1 KO mice are associated with hyperpolarizing shifts in channel’s activation and inactivation kinetics⁹⁸, suggesting changes to channel gating properties. Moreover, FMRP constitutively binds to the complex of Kv4 and Cav3 channels in cerebellar granule cells to modulate activity of both channels⁹¹. In both cases, FMRP decreases channel currents primarily through a strong hyperpolarizing shift in inactivation kinetics, while channel density is unchanged⁹¹. The Cav3-Kv4-FMRP interaction has important functional consequences within the context of FXS. In Fmr1 KO mice, the theta burst-induced form of long-term potentiation (LTP) is absent in mossy fiber-granule cell synapses of cerebellar cortex, a change that is attributed to increased Kv4 current⁹¹. Significantly, acute intracellular administration of the N-terminal FMRP fragment (aa1–297) is sufficient to restore LTP defects; it directly modulates Cav3-Kv4 channel complex activity in the absence of translational changes⁹¹.

Therapeutic implications.

The fact that FMRP directly modulates Cav3-Kv4 complex activity raises the possibility that this interaction can be targeted as a therapeutic strategy. When the N-terminal fragment of FMRP is modified to facilitate blood-brain barrier crossing (conjugation to tat; FMRP₂₉₇-tat) and infused into Fmr1 KO mice, it rapidly improves multiple functions at the cellular and behavioral levels, including restoration of Cav3-Kv4 complex activity, rescue of cerebellar mossy fiber LTP, and normalization of hyperactivity in the open field test⁹¹. Interestingly, FMRP₂₉₇-tat also restores levels of several selected proteins (PSD95, CamKII) for at least 24 hours. It remains unclear whether the observed changes in synaptic protein levels are a cause or a consequence of normalized LTP, since the FMRP₂₉₇ fragment lacks the ability to associate with polyribosomes and regulate translation. Additionally, there are still no data directly linking improvements in hyperactivity in vivo with restored Cav3-Kv4 complex function or the normalization of mossy fiber LTP. Understanding the mechanisms underlying FMRP₂₉₇-tat treatment is important because FMRP₂₉₇-tat is likely taken up by a wide range of excitatory and inhibitory neurons throughout the brain, including cerebellum but also cortex, hippocampus, and other brain areas⁹¹. However, regardless of the precise mechanisms, a tat-conjugate approach is a promising strategy through which to reintroduce a combination of different FMRP segments containing various functional domains, thus wielding broad power to address FXS channelopathies.

BK channels.

In additional to their prominent presynaptic roles in controlling AP duration and glutamate release, BK channels are also localized to dendrites and regulate dendritic excitability. In Fmr1 KO mice, BK channel activity is reduced in dendrites of neocortical neurons, leading to exaggerated Ca²⁺ influx accompanying backpropagating APs and reduced dendritic spike threshold⁵⁷. The BK channel opener BMS-204352 rescues a wide range of behavioral phenotypes in these mice, which likely reflects the effect of normalizing both pre- and post-synaptic functions^57,77,84.

Calcium channels

L-type VGCCs.

In addition to the Cav3 (T-type) VGCC discussed above, a large proportion of depolarization-induced Ca²⁺influx in dendrites is mediated by L-type (Cav1) VGCCs. The L-type channels regulate dendritic excitability, gene expression, synaptic plasticity, and neural differentiation and migration⁸⁷. Cav1.3 mRNA is FMRP target; channel expression is downregulated in the frontal cortex and cerebellum of Fmr1 KO mice⁹⁹. In the dendritic spines of prefrontal cortex layer 2/3 neurons from Fmr1 KO mice, the reduced expression of L-type channels is linked with defects in spike timing-dependent plasticity⁵⁵. Interestingly, in both FXS human patients and mouse models, Ca²⁺ influx through L-type VGCCs is increased in neural progenitors derived from stem cells⁸⁸. Specifically, the ratio of L-type to T-type channels increases, leading to enhanced progenitor differentiation to glutamate-responsive cells. These findings suggest that there are significant implications of upregulated L-type channel function on differentiation, migration, and fate determination in neural progenitor cells within the context of FXS⁸⁸.

R-type VGCCs.

The R-type calcium current is carried by Cav2.3, whose mRNA is also targeted by FMRP⁹⁵. This channel regulates burst-firing and afterdepolarization in dendrites, and its inhibition disrupts a form of mGluR-dependent long-term depression (LTD)⁸⁷. Consistent with profound alterations in dendritic excitability and calcium spiking in neurons lacking FMRP, Cav2.3 expression and R-type Ca²⁺ current are increased in brain synaptosomes and hippocampal cultured neurons from Fmr1 KO mice⁹⁰. Moreover, loss of FMRP occludes mGluR-dependent Cav2.3 upregulation⁹⁰. We do not yet completely understand how these defects contribute to altered dendritic excitability in FXS models, as other factors are also involved in modulating excitability following mGluR stimulation, including the downregulation of several K⁺ channels¹⁰⁰.

HCN channels

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are active at rest and therefore play a crucial role in controlling the resting membrane potential, input resistance, and thus dendritic excitability¹⁰¹. They are prominently expressed in the soma and dendrites of excitatory neurons, following an expression gradient that increases toward more distal regions to regulate the integration of synaptic events occurring at different dendritic locations. Numerous cell type-specific alterations in HCN channels are observed in FXS models. For example, in CA1 PCs of Fmr1 KO mice, HCN1 subunit expression and dendritic HCN currents are elevated. This in turn decreases input resistance, reduces temporal summation, and increases membrane resonance frequency⁵⁶. In contrast, in both layer 5 PCs of the somatosensory cortex and layer 4 stellate cells in these mice, HCN1 subunit expression and HCN current are decreased, leading to opposite changes in excitability, i.e., increased dendritic gain^57,59. Mechanistically, FMRP bidirectionally regulates HCN channels via a protein-protein interaction mechanism in dendrites of hippocampal CA1 PCs and prefrontal cortex neurons¹⁰². FMRP associates with the HCN-TRIP8b complex to down-regulate (in hippocampus) or up-regulate (in prefrontal cortex) the number of functional dendritic HCN channels. This previously unrecognized mechanism expands the repertoire of modulatory mechanisms by which FMRP regulates ion channel functions and excitability (FIG. 2), in opposite directions depending upon the cellular milieu. If this type of regulation is observed more broadly in future studies, it may explain many cell-type specific changes in channel expression/activity in neurons lacking FMRP.

Ligand-gated ion channels

The ligand-gated ion channels are a group of postsynaptic neurotransmitter receptors, also known as ionotropic receptors. There are three major ionotropic receptors involved in FXS: AMPA-, NMDA-, and GABA_A receptors (Rs). The AMPARs and NMDARs are nonselective cation channels, while GABA_AR is a Cl⁻ channel. In addition to these channels, we discuss the impact of FMRP loss on the chloride transporters that maintain intracellular Cl⁻ ion homeostasis, as these transporters determine the GABA_AR polarity switch early in development involved in synaptogenesis and synapse maturation.

AMPARs.

AMPARs mediate fast excitatory synaptic transmission in the brain. Altering the number of postsynaptic AMPARs is a fundamental mechanism of activity-dependent plasticity at excitatory synapses, including some forms of LTP and LTD. Local protein synthesis in dendrites is required for stable expression of LTP and LTD, and there is now evidence that these newly expressed proteins are regulated by specific RNA-binding proteins, including FMRP^103–108. Thus, FMRP may indirectly regulate synaptic plasticity through AMPAR-dependent functions^{59,83,103–108}. In Fmr1 KO mice, expression of the AMPAR subunit GluA1 is decreased in cortex (but not in the hippocampus or cerebellum¹⁰⁴), as is LTP^104,105. AMPARs also show brain region/cell-type specific defects in FXS models. Specifically, in the hippocampus, FMRP promotes GluA1 surface expression in adult-born granule cells of dentate gyrus, but has no effect on total GluA1 protein levels^107,108. Consequently, loss of FMRP decreases surface expression of GluA1, reduces GluA1-mediated AMPA currents, and delays dendritic maturation. In contrast, layer 4 stellate cells of primary somatosensory cortex in Fmr1 KO mice show elevated single-spine excitation with normal spine morphology, in part due to increased AMPAR currents⁵⁹. Consistent with this, studies of a new FXS mouse model (point missense mutation R138Q in FMRP) reveal an increase in both total and surface expression of AMPAR subunits GluA1 and GluA2, decreased thickness of postsynaptic density, and reduced LTP⁸³.

Therapeutic implications.

The evidence presented above raise the possibility that positive allosteric modulators of AMPARs could be useful for restoring some of the synaptic deficits associated with FXS. However, a double-blind clinical trial of one such compound, CX516, did not show significant improvements in cognition, language, or attention/executive function in individuals with FXS¹⁰⁹. AMPARs also show brain region-/cell type-specific defects in FXS models. Thus, globally targeting AMPARs is unlikely to be effective.

NMDARs.

NMDARs are involved in synaptic transmission, plasticity, and experience-dependent synaptic refinement during development. FMRP regulates transport and stabilization of NMDAR subunits GluN1, GluN2B¹¹⁰, and GluN2A¹¹¹. In the dentate gyrus of Fmr1 KO mice, NMDAR currents are decreased and LTP is reduced¹¹². The reduction in NMDAR-dependent synaptic plasticity in this region is accompanied by lower levels of GluN1, GluN2A, and GluN2B subunits. Indeed, the NMDAR co-agonists glycine and D-serine can restore NMDAR-dependent LTP in these mice to WT levels^113,114. At the behavioral level, NMDAR hypofunction is also linked to impaired context discrimination in adult Fmr1 KO mice¹¹⁵. Most recently, Yau and colleagues found that lower NMDAR currents in the dentate gyrus of Fmr1 KO mice is associated with a significant decrease in dendritic complexity (i.e., reduced dendritic length and number of intersections)¹¹⁶. Yet again, evidence points to cell-type specific changes in NMDAR levels and functions as a result of FMRP loss. NMDAR signaling is elevated in stellate cells of somatosensory cortex layer 4, and dendritic morphology is normal⁵⁹. In these cells, the increase in dendritic gain and enhanced summation appear to be caused by a 3-fold increase in the number of poly-synaptic spines and an increase in intrinsic excitability. Interestingly, while the loss of FMRP causes abnormal synaptogenesis, there is little correlation between spine structure and function, at least at the age of animals used in this study⁵⁹.

GABA_ARs.

Fast inhibitory synaptic neurotransmission in the brain is mostly mediated by GABA and its ionotropic target, GABA_ARs¹¹⁷. GABA_AR is a pentameric ion channel that is selectively permeable to Cl⁻ ions. Animal models of FXS show abnormalities in GABA_AR expression and function, as do human FXS patients (for review see ref ¹¹⁸). Kooy and colleagues initially found decreased expression of GABA_AR δ subunit mRNA in Fmr1 KO mice¹¹⁹. Further studies from multiple groups found that the protein levels of more than half of GABA_AR subunits (including α1–α4, β1-β3, γ1, γ2 and δ) are lower in FXS mouse models^{48,64,120–125}, and throughout the brain of FXS patients¹²⁶. In addition, the amount of GABA itself is also lower across multiple brain regions in FXS mouse models^127,128. Such hypofunction of the GABAergic system is likely to cause profound circuit hyperexcitability. Indeed, Morin-Parent and colleagues demonstrate that reduced GABA_AR function causes impaired short-interval intracortical inhibition, which contributes to cortical hyperexcitability in individuals with FXS⁹.

Therapeutic implications.

Given the critical role of the GABAergic system in network excitability, GABA_AR agonists have the potential to ameliorate excitability-associated deficits in FXS^129,130. The GABA_AR agonists acamprosate, ganaxolone, and metadoxine can improve multiple defects in Fmr1 KO models, including neuromuscular junction overgrowth and locomotor defects¹³¹, prolonged cortical UP state and anxiety¹³², and marble burying behavior and sensory hyperresponsiveness¹²⁷. However, when these compounds were tested in clinical trials of individuals with FXS, there were no significant improvements in outcome measures^133–136. Another strategy is to target metabotropic GABA_BR receptors to alleviate FXS deficits by normalizing the GABAergic system. However, again, although the GABA_BR agonist baclofen restores cognitive and behavioral deficits in animal models^137–140, it did not impact primary outcome measures in clinical trials^141,142. These results highlight the limitations of targeting a single aspect of excitability defects in FXS. Ultimately, the most effective treatment strategies are likely to be multifactorial.

Chloride transporters

In addition to wielding fine control over circuit excitability, GABAergic transmission plays a critical trophic role in cortical development through its early depolarizing action. During the first few postnatal weeks (critical period) in rodents, activating GABA_AR depolarizes neurons, increasing their excitability and promoting Ca²⁺ entry¹⁴³. This in turn regulates cell migration, proliferation, and synaptogenesis. The early depolarizing effect of GABA results from a transient high intracellular Cl⁻ concentration that is only present early in development and is determined by the expression pattern of two Cl⁻ cotransporters: Na⁺–K⁺–Cl⁻ cotransporter (NKCC1) and K⁺–Cl⁻ cotransporter (KCC2). The NKCC1, which transports Cl⁻ into neurons, is expressed at high levels early after birth, while the levels of KCC2, which extrudes Cl⁻ from neurons, gradually increases over the course of development¹⁴⁴. In the FXS mouse model, NKCC1 expression stays high through the end of the critical period, thus delaying the GABA_AR polarity switch and disrupting the normal trophic function of GABA^145,146,147.

Therapeutic implications.

Systemic administration of bumetanide to inhibit NKCC1 during the critical period rectifies the intracellular Cl⁻ imbalance in layer 4 somatosensory cortex neurons and corrects the development of thalamocortical excitatory synapses in Fmr1 KO mice¹⁴⁶. Inhibiting NKCC1 during early development also leads to broad remodeling of the proteome in the barrel cortex, and restores adult tactile response maps in Fmr1 KO mice¹⁴⁶. These findings provide rationale for considering NKCC1 as a novel therapeutic target in FXS.

Caveats of targeting channelopathies

As we have presented above, channelopathies play wide and varied roles in FXS pathophysiology. This makes ion channel-targeting therapies of particular interest. Indeed, multiple ion channel modulators show promise based on their ability to normalize cellular-, circuit-, and behavior-level abnormalities in FXS animal models (TABLE 3). We note above many cases in which specific ion channel-targeting therapies have already been tested in both animals and human patients. However, in most cases clinical trials fail to show efficacy. In fact, recent studies emphasize that loss of FMRP can have diverse effects on the same ion channel depending on the brain region, cell-type, or even subcellular compartment^56,58,98. This will make targeting any given ion channel for therapeutic intervention in FXS particularly challenging. Recent modeling studies also indicate that many cellular and synaptic pathologies in Fmr1 KO mice are antagonistic on the circuit level, and hence may be compensatory to the primary pathology³⁷. Thus, while there has been major progress towards identifying the molecular dysfunctions at synaptic and cellular levels, we need to better understand the overall effects of various channelopathies on circuit functions to design more effective therapeutic strategies. Moreover, because no single channelopathy can account for all, or even most of defects in FXS, a combinatory approach is almost certainly necessary. Alternatively, a strategy that targets the primary causes of ion channel dysfunctions (such as loss of interactions with, or translational regulation by, FMRP) might prove more efficient. For example, a tat-conjugate approach to reintroduce N-terminal FMRP fragment via intravenous injection restores ion channel and synaptic functions in at least part of the brain, as well as locomotion activity at the behavioral level in Frm1 KO mice⁹¹. Finally, a role for astrocytes in modulating neuronal excitability and plasticity in FXS is beginning to emerge^148–150. This may represent another potential target through which to normalize excitability deficits in FXS.

Implications for ASD

FXS is one of the many causes of autism spectrum disorders (ASD) and it has comorbidities with other genetic causes and also with idiopathic ASD. FXS and FMRP-loss-independent ASD, while genetically distinct, share significant clinical phenotypes. Indeed, their similarities suggest some level of common pathophysiology, including similar ion channel-based deficits¹⁵¹. There are multiple types of ion channels and transporters that are affected, in similar ways, in both FXS and FMRP-loss-independent ASD¹⁵², including Kv4.2^153,154, BK channels¹⁵⁵, Na⁺ channels¹⁵⁶, Ca²⁺ (L, R and T-type) channels, HCN1¹⁵⁷, GABA_AR, and NKCC1¹⁵². Moreover, channelopathies responsible for hyperexcitability of somatosensory neurons are part of the core developmental pathology in ASD models, leading to region-specific brain abnormalities during the critical period¹⁵⁷. Peripherally-restricted GABA_AR agonists, which reduce hyperexcitability within peripheral sensory circuits, improve a number of ASD-associated behaviors in mouse models, including tactile over-reactivity, anxiety-like behaviors, and certain social impairments¹⁵⁷. Together, these findings indicate that what we learn about the role of channelopathies in FXS pathogenesis is likely to have important implications for ASD stemming from other causes.

Concluding remarks

We now understand that FMRP wields control over neuronal and circuit excitability by regulating ion channels (FIG. 1, TABLES 1,2). Indeed, FMRP modulates the physiological functions of many different types of ion channels, each of which contributes uniquely to controlling intrinsic excitability, AP properties, firing patterns, synaptic transmission, synaptic plasticity, and dendritic integration. FMRP can regulate ion channel function via protein-protein interactions with pore-forming or regulatory subunits, or channel expression, and some channels are regulated via both mechanisms (FIG. 2). FMRP can also indirectly modulate channel activity through second messenger pathways. The various mechanisms through which FMRP regulates channel function may be brain region-, cell type-, or even subcellular compartment-specific. Given that direct FMRP-channel interactions are rapid and reversible, this mechanism means that ion channels can be precisely and dynamically tuned, which is critical for normal excitability, synaptic plasticity, and information processing, and, ultimately, normal behavioral and cognitive functions. Consequently, loss of FMRP, as occurs in FXS, triggers a series of channelopathies that contribute to circuit hyperexcitability-related abnormalities which in turn underlie core symptoms of FXS. Focusing on the role of channelopathies in such neuronal and circuit-level defects will be crucial in understanding the mechanistic basis of FXS pathophysiology and in designing novel therapeutic strategies.