Leonard Kaczmarek, PhD—Yale University School of Medicine
Potassium channel modulators for the treatment of Fragile X

Leonard Kaczmarek, PhD, Principal Investigator (2006 to Present)
Lynda EL-Hassar , PhD, Postdoctoral Fellow (2012 to Present)
Jack Kronengold, PhD, FRAXA Postdoctoral Fellow (2011)
Vali Gazula, PhD, FRAXA Postdoctoral Fellow (2009)

FRAXA Awards:

$67,000 in 2013
$67,000 in 2012
$45,000 in 2011
$61,670 in 2010
$60,000 in 2009
$60,000 in 2008
$40,000 in 2007
$40,000 in 2006


Processing sensory input is dependent on a precise complement of ion channels that shape neuronal action potentials and determine their firing patterns and frequency of firing. Specifically, Na+ - activated potassium channels (termed KNa channels) are critical for this function. Dr. Kaczmarek's team has found that the Slack KNa channel directly binds the Fragile X Mental Retardation Protein (FMRP), resulting in a several-fold activation of Slack channel activity. The association of Slack channels with FMRP suggests that pharmacological manipulations of channel activity could prove to be a novel therapy for Fragile X syndrome. This team is developing a range of compounds that can activate Slack channels.
2013 Project description - channels as treatment targets

by Dr. Kaczmarek, 5/13/2013

Loss of the messenger RNA-binding protein FMRP, which causes Fragile X Syndrome, is associated with hypersensitivity to a variety of sensory stimuli, particularly sound. This sensitivity occurs because the excitability of groups of neurons in the brain is increased. Such over-excitability may also contribute to other features of Fragile X syndrome, such as the increased incidence of seizures in childhood and an inability to concentrate on or learn very fine motor tasks.

A variety of work has demonstrated that increased excitability occurs because FMRP normally controls a class of proteins called potassium channels. Potassium channels are key controllers of how intensely a neuron responds to stimulation and how accurately a neuron can follow different patterns of stimulation. The Kaczmarek group has been investigating pharmacological agents that modify these potassium channels and that are likely to prove to be therapeutic in Fragile X syndrome.

The first type of potassium channel implicated in Fragile X syndrome is called the Kv3.1 channel. This protein is normally required for neurons to generate electrical impulses at very high rates (many hundreds of times a second, as is required for normal hearing). When FMRP is absent, levels of Kv3.1 increase in the brain, and this allows neurons to fire at rates that are inappropriately high for the stimulation they are receiving.

In collaboration with a company, Autifony, the Kaczmarek group is studying drugs that directly counteract the effects of the increased Kv3.1 protein levels.

A second potassium channel, termed Slack, becomes reduced when FMRP is absent. A key role that Slack plays in the brain is to enhance the accuracy with which a neuron responds to its inputs, such as those coming from sensory organs. Slack normally binds directly to FMRP and this mutual interaction is required to maintain the activity of Slack. Thus neurons with low Slack activity are likely to make mistakes in timing when responding to inputs, and genetic changes in Slack channels themselves have devastating effects on psychomotor development.

At present there are no known pharmaceutical compounds that act on Slack channels, and so the Kaczmarek group is using novel high-throughput drug screening techniques to discover new compounds that act to correct the abnormal firing patterns resulting from low Slack activity.

The Slack Potassium Ion channel as a Therapeutic Target for Fragile X Syndrome

A paper on this work has been published in Journal of Neuroscience on 2010 August 4: Fragile X mental retardation protein is required for rapid experience-dependent regulation of the potassium channel Kv3.1b

by Leonard Kaczmarek and Jack Kronengold, 6/8/2011

Our laboratory has investigated how the excitability of neurons becomes modified in the absence of the FMRP protein. We have found that the levels of two potassium channels, termed Slack and Kv3.1 are altered in mice that lack this protein. We have made significant progress in identifying novel pharmacological activators of the Slack potassium channel for potential therapeutic intervention in FXS individuals.

The Slack potassium channel is widely expressed in the brain. Using neurons of the central auditory system, our laboratory has demonstrated that Slack is required for accurate timing of action potentials in response to synaptic stimuli. This channel is activated by the FMRP protein through a direct association of FMRP with the cytoplasmic domain of the channel itself. We have shown that levels of the Slack potassium current are reduced by about 50% in neurons of FMR1-/- mice. Treatment of neurons with pharmacological activators of Slack would be expected to restore levels of this current back to levels found in wild type animals. Using a combination of electrophysiological and biochemical assays, we have now identified five compounds that are effective activators of Slack channels and are now testing their specificity for this class of channels.

In addition, we have recently published work (see below) that has identified another potassium channel, Kv3.1, that is altered by loss of FMRP. The physiological role of this channel is to allow specific types of neurons to fire at very high rates. We have found that Kv3.1 mRNA is a binding partner for FMRP, and that in FMR1-/- mice, levels of this channel are elevated. Moreover, in the central auditory system Kv3.1 is normally expressed in an orderly tonotopic gradient, such that neurons responding to the highest frequency sounds have the highest levels of Kv3.1 channel. This orderly gradient is abolished in the absence of FMRP, causing all these neurons to have uniformly high levels of this channel. This finding suggests that auditory neurons are likely to be hyperexcitable in FXS individuals. In collaboration with a pharmaceutical company, we are therefore now investigating the actions of activators and inhibitors of the Kv3.1 potassium channel.

Development of pharmacological activators for FMRP-regulated potassium channels

by Leonard Kaczmarek and Jack Kronengold, 4/2/2010

The Slack Potassium Ion channel as a Therapeutic Target for Fragile X Syndrome

Individuals with Fragile X syndrome have a range of perceptual processing deficits related to auditory stimuli. They commonly report an increased awareness of environmental sounds, abnormal loudness perception, and difficulty in filtering or "hearing out" the important auditory information in background noise. This manifests itself as paying too much attention to stimuli that most people ignore, or not responding to the most important messages that are confronting the person. These symptoms interfere with attention, learning, language development and social interactions and are likely due to changes in synaptic connections in sensory circuits; the breakdown of the normal flow of information along pathways in the brain.

The integrity of neuronal encoding and processing of sensory input is dependent on a precise complement of ion channels that shape neuronal action potentials and determine their firing patterns and frequency of firing. Specifically, Na+ - activated potassium channels (termed KNa channels) are critical for this function. We have found that the Slack KNa channel directly binds the Fragile X Mental Retardation Protein (FMRP), resulting in a several-fold activation of Slack channel activity. This is an entirely novel finding because previous work has focused on the role of FMRP in binding messenger RNAs.

The association of Slack channels with FMRP suggests that pharmacological manipulations of channel activity could prove to be a novel therapy for Fragile X syndrome. In particular, FMRP knockout animals (fmr1-/-) lack a physiological activator of Slack channel (FMRP itself) and expression of a C-terminal Slack isoform is completely abolished in fmrl-/- animals, leading to significantly reduced Slack currents. Thus some of the neuronal abnormalities associated with Fragile X may result from the effect of altered K currents on neuronal excitability and timing. Moreover our findings suggest that the interaction of Slack channels with FMRP could regulate rates of translation of mRNAs bound to FMRP.

Our laboratory is developing a range of compounds that can activate Slack channels. We plan to determine whether these compounds enhance Slack channel activity and restore normal firing patterns in neurons from fmrl-/- mice. If we find potent and selective compounds, this will provide a strong impetus for the evaluation of Slack activators as novel therapeutic agents that could act either independently or in concert with agents that act at group1 metabotropic glutamate receptors, which have been found to regulate Slack channels.

Paper published in Nature Neurosci. 2010 May 30. Fragile X mental retardation protein controls gating of the sodium-activated potassium channel Slack


 
Electrophysiological, Biochemical And Immunohistochemical Characterization Of Kv3.1 In Auditory Brainstem Nuclei In The Fragile X Knockout Mouse

by Leonard Kaczmarek and Jack Kronengold, 4/2/2007

Individuals with Fragile X Syndrome exhibit extreme sensitivity to auditory stimuli. The debilitating behavioral symptoms associated with "sensory overload" interfere with attention, learning, language development and social interactions and are likely due to changes in synaptic connections in the auditory circuitry.

Studies have shown that the Fragile X knockout mouse also exhibits abnormal sensitivity to auditory stimuli including hyper reactivity and the induction of audiogenic seizures by acoustic stimulation. While audiogenic seizures have not been reported in Fragile X patients, the onset and manifestation of autistic behavior in these individuals has been directly correlated with auditory hypersensitivity. The source of the audiogenic seizures is believed to be due to increased excitation in auditory nuclei and not to an overall increase in brain excitability.

A paper in Cell by Darnell et al. (2001) has identified the mRNA for the voltage gated potassium (K+) channel, Kv3.1, as a candidate "binding target" for the Fragile X Mental Retardation Protein (FMRP). The absence of FMRP in the Fragile X knockout mouse would be expected to result in the altered regulation of Kv3.1 which is critical for normal synaptic function. The goals of this project are to determine the changes in expression of Kv3.1 in auditory neurons of the Fragile X knockout mouse. The Kv3.1 channel, which belongs to the Kv3 family of voltage-dependent K+ channels, is found at particularly high levels in neurons of auditory nuclei. The biophysical properties of the Kv3.1 channel impart a "fast spiking" (FS) phenotype to neurons that need to fire repetitively at high frequencies, in response to high frequency stimuli. Moreover, the response to all sound frequencies is dependent on a precise tonotopic expression of Kv3.1 in neurons of the auditory nuclei. Disruption of this auditory space code, or map, by altered regulation of Kv3.1, would be expected to interfere with auditory processing in auditory nuclei of the brain stem and in the auditory cortex.

We have identified a new FMRP-interacting protein which is widely expressed in neurons in many parts of the brain.We are currently examining its function in the auditory brainstem and how this is likely to be misregulated in Fragile X and Autism Spectrum Disorder individuals.

Our project now includes not only studies of the Kv3.1 channel but of the newly discovered protein as well. Because of the exciting discoveries centered around the mGluR theory of Fragile X, we have added studies involving the inferior colliculus, a brain region which expresses high levels of mGluR5. Fortuitously, we can study all these components simultaneously in neurons of the auditory brainstem.