Time for T? Targeting language-associated gene Cntnap2 with a T-type calcium channel blocker corrects hyperexcitability driving sensory abnormalities, repetitive behaviors, and other ASD symptoms, but will it improve language? Will it also benefit Pitt Hopkins syndrome (PTHS) and broader autism?

 

  

 

There are at least 2 Natasas I can think of who will like this post.

Today’s post revisits the subject of calcium channels in autism.  Ion channel dysfunctions are a favourite area of mine because many should be treatable by repurposing safe, existing drugs. I do take note that many readers of this blog have reported success by targeting L-type calcium channels.

Many years ago, at the start of this blog, I recall reading about Timothy syndrome and a researcher at Stanford, Ricardo Dolmetsch, who was exploring treatment using a T-type calcium channel blocker.  It turned out that he had a son with severe autism, which was driving his interest at that time. He won all kinds of awards, but I always wondered why he did not treat his own son.

It is quite strange because Timothy syndrome is caused by a gain of function of an L-type channel. This mutation causes the Cav1.2 channel to fail to inactivate properly after opening. As a result, there is prolonged calcium influx into cells.

Instead of blocking Cav1.2, the researchers blocked the T-channels Cav3.2 and 3.3.

I did my homework on idiopathic autism a dozen years ago and concluded I needed to block Cav1.2. I went ahead and did it – it works like a charm.

It was a real drama back in those days, with self-injury and aggression, so Timothy syndrome and T channels remains stuck in my mind a decade later.

 

Language Genes

Even before parents worry about self-injurious behavior (SIB), they go through the phase of worrying about if their child will ever speak. Some do and some do not.  What really matters is communication, rather than speech.

 

FOXP2 - The language Gene

FOXP2 is a transcription factor involved in the development of neural circuits related to speech and language production, particularly in areas such as the basal ganglia and cerebellum. Mutations in FOXP2 can lead to speech and language deficits.

FOXP2 influences motor control and vocalization processes that are critical for speech, and it is thought to have evolved specifically in humans to support complex language abilities.

 

CNTNAP2 - The language-associated gene

CNTNAP2 (Contactin-associated protein-like 2) is a gene that encodes a cell adhesion protein. It plays a critical role in the development of neural connectivity and the formation of synapses in areas of the brain involved in language, such as the broca’s area and temporal lobes. CNTNAP2 is also involved in the regulation of neuronal excitability and is crucial for the development of white matter tracts that connect language-related brain regions.

Mutations in CNTNAP2 have been implicated in neurodevelopmental disorders such as specific language impairment (SLI), autism, and developmental language disorders.

 

FOXP2 and CNTNAP2 Interaction

FOXP2 and CNTNAP2 work together in the development of the neural circuits that are crucial for language and speech. They are both involved in the formation and maintenance of synaptic connections in key brain regions like the cortex, basal ganglia, and cerebellum, which are critical for motor control, vocalization, and language processing.

There is evidence to suggest that FOXP2 may regulate the expression of CNTNAP2 as part of a broader gene network that governs language development. FOXP2 may influence CNTNAP2 gene expression, which in turn impacts neural connectivity and synaptic function in brain regions responsible for speech and language.

 

CNTNAP2 sounds familiar?

We have come across this gene before.

At least one reader has a child with a mutation in this gene.

We also discovered that the Pitt Hopkins gene TCF4 regulates CNTNAP2 and that

“PTHS (Pitt Hopkins syndrome) is characterised by severe intellectual disability, absent or severely impaired speech, characteristic facial features and epilepsy. Many of these features are shared with patients carrying CNTNAP2 mutations, leading researchers to test patients with PTHS-like features for CNTNAP2 mutations”

Several readers have children with PTHS (Pitt Hopkins syndrome).

It is not inconceivable that what works for CNTNAP2 will also work for at least some PTHS (Pitt Hopkins syndrome).

The question is whether what works for CNTNAP2 will work much more broadly and could it even improve language development?

Here is the recent research from Stanford:

 

Reticular Thalamic Hyperexcitability Drives Autism Spectrum Disorder Behaviors in the Cntnap2 Model of Autism

Autism spectrum disorders (ASDs) are a group of neurodevelopmental disorders characterized by social communication deficits, repetitive behaviors, and comorbidities such as sensory abnormalities, sleep disturbances, and seizures. Dysregulation of thalamocortical circuits has been implicated in these comorbid features, yet their precise roles in ASD pathophysiology remain elusive. This study focuses on the reticular thalamic nucleus (RT), a key regulator of thalamocortical interactions, to elucidate its contribution to ASD-related behavioral deficits using a Cntnap2 knockout (KO) mouse model. Our behavioral and EEG analyses comparing Cntnap2^+/+ and Cntnap2^-/- mice demonstrated that Cntnap2 knockout heightened seizure susceptibility, elevated locomotor activity, and produced hallmark ASD phenotypes, including social deficits, and repetitive behaviors. Electrophysiological recordings from thalamic brain slices revealed increased spontaneous and evoked network oscillations with increased RT excitability due to enhanced T-type calcium currents and burst firing. We observed behavior related heightened RT population activity in vivo with fiber photometry. Notably, suppressing RT activity via Z944, a T-type calcium channel blocker, and via C21 and the inhibitory DREADD hM4Di, improved ASD-related behavioral deficits. These findings identify RT hyperexcitability as a mechanistic driver of ASD behaviors and underscore RT as a potential therapeutic target for modulating thalamocortical circuit dysfunction in ASD.

Teaser RT hyperexcitability drives ASD behaviors in Cntnap2-/- mice, highlighting RT as a therapeutic target for circuit dysfunction.

 

Overall, this study identifies elevated RT burst firing and aberrant thalamic oscillatory dynamics in Cntnap2^−/− mice as a key driver of ASD-related behavioral deficits. If this is a common mechanism of ASD-circuit pathology arising from a variety of genetic causes, then compounds such as Z944, or subtype specific T-type calcium channel antagonists that would target the Cav3.2 and Cav3.3 expressed in RT neurons, might be an effective therapeutic strategy. Furthermore, future research should focus on elucidating RT’s roles in sensory, emotional, and sleep regulation to optimize therapeutic strategies in the context of ASD.

 

Existing T-type calcium channel blockers for humans

Mibefradil is one of the most well-known T-type calcium channel blockers. It was initially developed for hypertension and angina because of its ability to block T-type channels. However, mibefradil was withdrawn from the market in 1998 due to serious drug interactions with other medications, particularly those that inhibit liver enzymes involved in drug metabolism, like statins.

Despite its withdrawal, mibefradil has been studied for other potential uses, including in epilepsy and chronic pain, due to its effects on neuronal excitability.

Zonisamide is an anticonvulsant medication that has some T-type calcium channel blocking properties. It is approved for epilepsy and partial seizures, but it is not typically used specifically for Timothy syndrome or conditions involving T-type channel dysfunction.

Zonisamide is also used to treat seizures in pet dogs and cats.  

Zonisamide: chemistry, mechanism of action, and pharmacokinetics

Zonisamide is a novel antiepileptic drug (AED) that was developed in search of a less toxic, more effective anticonvulsant. The drug has been used in Japan since 1989, and is effective for simple and complex partial seizures, generalized tonic-clonic seizures, myoclonic epilepsies, Lennox–Gastaut syndrome, and infantile spasms. In Japan, zonisamide is currently indicated for monotherapy and adjunctive therapy for partial onset and generalized onset seizures in adults and children. In the United States, zonisamide was approved by the Food and Drug Administration (FDA) in 2000 as an adjunctive treatment for partial seizures.

The drug’s broad spectrum of activity and favorable pharmacokinetic profile offer certain advantages in the epilepsy treatment armamentarium. Chemically distinct from other AEDs, zonisamide has been shown to be effective in patients whose seizures are resistant to other AEDs. Zonisamide’s long plasma elimination half-life has allowed it to be used in a once-daily or twice-daily treatment regimen in Japan.

It is believed that zonisamide’s effect on the propagation of seizure discharges involves blocking the repetitive firing of voltage-sensitive sodium channels, and reducing voltage-sensitive T-type calcium currents without affecting L-type calcium currents. These mechanisms stabilize neuronal membranes and suppress neuronal hypersynchronization, leading to the suppression of partial seizures and generalized tonic–clonic seizures in humans.

Zonisamide possesses mechanisms of action that are similar to those of sodium valproate, e.g., suppression of epileptogenic activity and depression of neuronal responses. These mechanisms are thought to contribute to the suppression of absence and myoclonic seizures.

  

Conclusion

It would seem that zonisamide should be trialed in:

·        CNTNAP2-related neurodevelopmental disorder

·        Pitt Hopkins syndrome (PTHS)

·        Timothy syndrome

·        Idiopathic/polygenic autism

(But, don’t hold your breath!)

Due to the nature of CNTNAP2 disorder and PTHS, I think the greatest impact will be if given from a very young age. However, we do see improvements with many autism interventions regardless of age.

It is certainly conceivable that even mild autism can benefit from damping down reticular thalamic (RT) hyperexcitability.

If shown effective, zonisamide would join the long list of anti-epileptic drugs (AEDs) “repurposable” to treat certain subtypes of autism.

Low-dose clonazepam for autism - SCN2A deficient, SCN1A deficient, BTBR polygenic autism and Maternal Immune Activation (MIA) all respond to the same cheap treatment

 

Restoring the excitation/inhibition balance in neurons is a good way to treat both epilepsy and autism. Professor Catterall performed the groundwork in one model of epilepsy and one model of autism more than a decade ago, using low-dose clonazepam.

At that point I did endeavour to translate that science from mouse to human, in part because Professor Catterall made clear he was not going to.

A number of readers of this blog, biased towards doctor parents, did use this therapy for several years.

In 2021 the Chinese showed low-dose clonazepam effective in the Maternal Immune Activation (MIA) model of autism.

Roll forward to 2025 and Chinese researchers looking into another single gene autism (SCN2A) have found the same therapy to be effective.  Fancy that !!

It is actually the cheapest therapy I ever investigated, costing a few dollars/euros/pounds a year.

At a tiny dose, clonazepam, which acts as a positive allosteric modulator of GABA receptors, enhances the activity of receptors containing α2 and α3 subunits and restores the excitation/inhibition balance.

The effective dose in us humans is about 0.0006 mg/kg per day. Due to its long half-life, you need to take the dose for 3 days before the level in the body rises to the therapeutic level. As suggested by Catterall, there is a narrow therapeutic window; too high a dose, or too low a dose, will not be effective.

I used tablets, but it is much easier to use the liquid version of clonazepam, since you need to produce a very dilute version and then measure the dose with a syringe. Some people used a compounding pharmacy to do the hard work.

Not everyone responds and I think not everyone finds their therapeutic dosage window. I think people may need to adjust the dosage over the years.

There are many posts in this blog that refer to this therapy.

 

https://www.epiphanyasd.com/search/label/Clonazepam

 

What are the effects in children?

The effects are improved cognition, ability to learn new skills and a reduction in broad symptoms of autism.

In kids already taking bumetanide, and are responsive to that therapy, there is an additional benefit. In our case the incremental effect was less than bumetanide, but welcome nonetheless.

Some people use it as an alternative to bumetanide, when diuresis is problematic.

The dose is so low, the usual risks of benzodiazepines are not present. It might be better described as a micro-dose.

 

 The science, for those who are interested:

 

The recent paper from 2025:

  

Restoration of excitation/inhibition balance enhances neuronal signal-to-noise ratio and rescues social deficits in autism-associated Scn2a-deficiency

Social behavior is critical for survival and adaptation, which is profoundly disrupted in autism spectrum disorders (ASD). Social withdrawal due to information overload was often described in ASD, and it was suspected that increased basal noise, i.e., excessive background neuronal activities in the brain could be a disease mechanism. However, experimental test of this hypothesis is limited. Loss-of-function mutations (deficiency) in SCN2A, which encodes the voltage-gated sodium channel Na_V1.2, have been revealed as a leading monogenic cause of profound ASD. Here, we revealed that Scn2a deficiency results in robust and multifaceted social impairments in mice. Scn2a-deficient neurons displayed an increased excitation-inhibition (E/I) ratio, contributing to elevated basal neuronal noise and diminished signal-to-noise ratio (SNR) during social interactions. Notably, the restoration of Scn2a expression in adulthood is able to rescue both SNR and social deficits. By balancing the E/I ratio and reducing basal neuronal firing, an FDA-approved GABA_A receptor-positive allosteric modulator improves sociability in Scn2a-deficient mice and normalizes neuronal activities in translationally relevant human brain organoids carrying autism-associated SCN2A nonsense mutation. Collectively, our findings revealed a critical role of the Na_V1.2 channel in the regulation of social behaviors, and identified molecular, cellular, and circuitry mechanisms underlying SCN2A-associated disorders.

HIGHLIGHTS

1.     Na_V1.2 deficiency leads to pronounced social deficits in mice.

2.     Na_V1.2 deficiency results in an overall enhanced E/I ratio, elevated basal neuronal activity, and impaired signal-to-noise ratio.

3.     Both the enhanced E/I ratio and impaired sociability are reversible through the restoration of Na_V1.2 expression in adulthood.

4.     Targeted restoration of Na_V1.2 in striatum-projecting neurons rescues social impairments.

5.     GABA transmission is reduced in both mouse and human organoid models of SCN2A deficiency, and acute systemic administration of GABA_A receptor-positive allosteric modulators restores sociability.

 

 

 

Because reduced GABAergic signaling can enhance the E/I ratio and contribute to in vivo neuronal hyperexcitability, we examined whether potentiating GABA_A receptor activity using a positive allosteric modulator (PAM) could normalize neuronal firing. Notably, clonazepam, an FDA-approved benzodiazepine has been shown to rescue social deficits in both a Scn1a knockout model of Dravet syndrome37 and the BTBR model of idiopathic autism38. In WT mice, baseline recordings from putative MSNs showed low firing rates that remained unchanged following acute systemic administration of a low dose of clonazepam (Clz, 0.05 mg/kg, i.p.) (Figure 5C, D). In contrast, clonazepam markedly suppressed the abnormally high firing rates in HOM mice (Figure 5E, F).

 

In summary, our findings reveal that severe Na_V1.2 deficiency produces profound and reversible social deficits, underpinned by disproportionate reductions in excitatory and inhibitory synaptic transmission. We demonstrate a direct, dose-dependent relationship between Scn2a expression and sociability, whereby a ∼70% reduction in Na_V1.2 leads to an elevated E/I ratio, increased noisy basal activity, and impaired neuronal coding, while restoration of Scn2a or pharmacological enhancement of GABA_A receptor function reverses these deficits. Collectively, our work provides a comprehensive exploration, from molecular and cellular mechanisms to neural circuits, of the pathophysiology underlying social impairments in SCN2A-associated disorders. These insights lay a robust foundation for the development of targeted therapeutic interventions aimed at normalizing synaptic function to ameliorate social impairments.

 

The original papers from Professor Catterall in a model of polygenic autism and in Dravet syndrome:

 

Enhancement of Inhibitory Neurotransmission by GABA_A Receptors Having α_2,3-Subunits Ameliorates Behavioral Deficits in a Mouse Model of Autism

Autism spectrum disorder (ASD) may arise from increased ratio of excitatory to inhibitory neurotransmission in the brain. Many pharmacological treatments have been tested in ASD, but only limited success has been achieved. Here we report that BTBR T⁺ Itpr3^tf/J (BTBR) mice, a model of idiopathic autism, have reduced spontaneous GABAergic neurotransmission. Treatment with low non-sedating/non-anxiolytic doses of benzodiazepines, which increase inhibitory neurotransmission through positive allosteric modulation of postsynaptic GABA_A receptors, improved deficits in social interaction, repetitive behavior, and spatial learning. Moreover, negative allosteric modulation of GABA_A receptors impaired social behavior in C57BL/6J and 129SvJ wild-type mice, suggesting reduced inhibitory neurotransmission may contribute to social and cognitive deficits. The dramatic behavioral improvement after low-dose benzodiazepine treatment was subunit-specific—the α_2,3-subunit-selective positive allosteric modulator L-838,417 was effective, but the α₁-subunit-selective drug zolpidem exacerbated social deficits. Impaired GABAergic neurotransmission may contribute to ASD, and α_2,3-subunit-selective positive GABA_A receptor modulation may be an effective treatment.

 

 

Autistic behavior in Scn1a^+/− mice and rescue by enhanced GABAergic transmission

Haploinsufficiency of the SCN1A gene encoding voltage-gated sodium channel Na_V1.1 causes Dravet Syndrome (DS), a childhood neuropsychiatric disorder including recurrent intractable seizures, cognitive deficit, and autism-spectrum behaviors. The neural mechanisms responsible for cognitive deficit and autism-spectrum behaviors in DS are poorly understood. Here we show that mice with Scn1a haploinsufficiency display hyperactivity, stereotyped behaviors, social interaction deficits, and impaired context-dependent spatial memory. Olfactory sensitivity is retained, but novel food odors and social odors are aversive to Scn1a^+/− mice. GABAergic neurotransmission is specifically impaired by this mutation, and selective deletion of Na_V1.1 channels in forebrain interneurons is sufficient to cause these behavioral and cognitive impairments. Remarkably, treatment with low-dose clonazepam, a positive allosteric modulator of GABA_A receptors, completely rescued the abnormal social behaviors and deficits in fear memory in DS mice, demonstrating that they are caused by impaired GABAergic neurotransmission and not by neuronal damage from recurrent seizures. These results demonstrate a critical role for Na_V1.1 channels in neuropsychiatric functions and provide a potential therapeutic strategy for cognitive deficit and autism-spectrum behaviors in DS.

 

The 2021 paper from China using the maternal immune activation model of autism:

  

Clonazepam attenuates neurobehavioral abnormalities in offspring exposed to maternal immune activation by enhancing GABAergic neurotransmission

Ample evidence indicates that maternal immune activation (MIA) during gestation is linked to an increased risk for neurodevelopmental and psychiatric disorders, such as autism spectrum disorder (ASD), anxiety and depression, in offspring. However, the underlying mechanism for such a link remains largely elusive. Here, we performed RNA sequencing (RNA-seq) to examine the transcriptional profiles changes in mice in response to MIA and identified that the expression of Scn1a gene, encoding the pore-forming α-subunit of the brain voltage-gated sodium channel type-1 (Na_V1.1) primarily in fast-spiking inhibitory interneurons, was significantly decreased in the medial prefrontal cortex (mPFC) of juvenile offspring after MIA. Moreover, diminished excitatory drive onto interneurons causes reduction of spontaneous gamma-aminobutyric acid (GABA)ergic neurotransmission in the mPFC of MIA offspring, leading to hyperactivity in this brain region. Remarkably, treatment with low-dose benzodiazepines clonazepam, an agonist of GABA_A receptors, completely prevented the behavioral abnormalities, including stereotypies, social deficits, anxiety- and depression-like behavior, via increasing inhibitory neurotransmission as well as decreasing neural activity in the mPFC of MIA offspring. Our results demonstrate that decreased expression of Na_V1.1 in the mPFC leads to abnormalities in maternal inflammation-related behaviors and provides a potential therapeutic strategy for the abnormal behavioral phenotypes observed in the offspring exposed to MIA.

 

Conclusion

On the one hand, it is great that you can use this published research to treat your own child, but it is rather sad that this research is never going to be applied widely to children with severe autism.

Professor Catterall did not want to take on the massive task of “commercializing” his discovery. It would be a huge and expensive job, with no financial return, since clonazepam is already a widely available cheap generic drug.

This is the same problem faced by bumetanide, leucovorin and other generic drugs that can be repurposed for autism.

You have to adjust to the imperfect world we live in, rather than assume everything is being done on your child’s behalf, by those thousands of autism researchers. They want to get published and get paid — that is their success. For me, what matters is getting results, and I did. Hopefully, so will you.