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Past Research Grants:

Tilly Hadlow Young Investigator Award:  Joseph Alaimo, Ph.D., Principal Investigator; Sarah Elsea, Ph.D., Co-Investigator/Mentor, Baylor College of Medicine, 2016

Delineating Therapeutic Targets using Global Metabolic Profiling in Pitt Hopkins syndrome: 

Defining the cellular defects due to alterations in TCF4 function is paramount in order to determine the proper molecular and biochemical targets for therapeutic intervention in Pitt-Hopkins syndrome. To identify and characterize the biochemical and molecular dysfunction due to altered TCF4 function, we plan to take a clinical and translational approach by recruiting a cohort of individuals with PTHS in collaboration with the Pitt-Hopkins Research Foundation and current PTHS clinics, phenotypically and molecularly characterizing the cohort, and employing state-of-the-art metabolomics screening to identify pharmacologically targetable molecular and biochemical pathways. Our unique approach will utilize a special type of biochemical genetic test called global metabolomics assisted pathway screening (Global MAPS). Global MAPS is currently the most comprehensive small molecule screen available in the clinical setting and is only available through Baylor College of Medicine’s Biochemical Genetics Diagnostic Laboratory. Global MAPS surveys greater than 1000 small molecules in human plasma, pinpointing defects in pathways unmeasurable by standard clinical testing methods, offering a comprehensive and in-depth analysis of patient samples and metabolic status. Our overall goal is to employ Global MAPS analysis in patients with PTHS to identify novel pathway alterations and to understand the basis of TCF4 function in cells. In addition, our proposal will serve as a functional confirmation of current molecular findings in PTHS research, including RNA-sequencing and gene expression profiling, thereby refining the molecular and biochemical targets that would benefit most from therapeutic intervention. Our novel but complementary approach will promote additional analysis to identify points of data convergence among other research groups thereby expediting the process toward targeted therapeutic intervention and clinical trials.

Benjamin D. Philpot, Ph.D., Principal Investigator; Alexander D. Kloth, Ph.D., Co-Principal Investigator; Courtney L. Thaxton, Ph.D., Co-Principal Investigator, The University of North Carolina at Chapel Hill, Awarded $75,000, PHRF, 2016

Characterization and Generation of PTHS Model Mice for Rational Therapeutic Discovery:  

Pitt-Hopkins syndrome (PTHS) is a rare neurodevelopmental disorder characterized by intellectual disability, absent speech, seizures, ataxia, and breathing anomalies. In support for future therapeutic development for PTHS, we will pursue two independent aims: (1) to uncover the neural impairments that are common across multiple PTHS mouse models, and (2) to develop new tools to analyze TCF4 expression in neuronal subtypes throughout development and adulthood. In the first aim, we will follow up on our finding that long-term changes in synaptic function related to experience are enhanced in multiple PTHS-related mouse models. We hypothesize that this deficit is related to altered function of a glutamate receptor, the NMDA receptor, and we will rigorously test this hypothesis using electrophysiology, biochemistry and pharmacological methods in multiple PTHS-related mouse models. In the second aim, we will develop a unique mouse model toward determining effective drug targets that affect TCF4 expression levels, as well as be able to alter TCF4 activity in a spatiotemporal manner. This novel binary “reporter-reinstatement” mouse will not only allow for a stream-lined and genetically precise approach to drug discovery for PTHS, but also will allow us to determine the most efficacious time in which to reinstate TCF4 function to alleviate the pathophysiologies associated with PTHS. In all, the proposed project pursues incisive approaches that will provide guidance to the development of PTHS therapeutics.

Kindal Kivisto Award for Promising Young Researchers: Andrew John Kennedy, Ph.D., Principal Investigator; J. David Sweatt, Ph.D., Co-Principal Investigator, Evelyn F. McKnight Brain Institute, The University of Alabama at Birmingham, Awarded $75,000, PHRF, 2016

Investigating Therapies for Pitt-Hopkins Syndrome:  

The central strategy of our research program consists of two goals: the near-term goal to identify FDA approved drugs as potential translatable therapies for Pitt-Hopkins Syndrome (PTHS) and the long-term goal to develop novel neuroepigenetic therapies that fundamentally reverse the effects of PTHS.  Over the past three years, we have characterized a genetically engineered heterozygous deletion mouse model of PTHS (Tcf4 +/-), validated the histone deacetylase enzyme Hdac2 as a target to treat the cognitive deficits associated with PTHS, and undertaken a drug screening program.  This grant will investigate the efficacy of Fingolimod (trade name Gilenya), as well as other FDA approved therapeutics that target Hdac2, to improve learning, problem solving, and associative memory in PTHS mice.  These experiments will focus on identifying a plausible drug candidate that can be translated to a clinical setting and effectively improve cognition in PTHS patients. Additionally, more advanced epigenetic therapies will be developed to address the genetic cause of PTHS.  Every person has two functioning copies of Tcf4 with the exception of individuals with PTHS, who have a mutation or deletion that yields only one functioning copy.  Epigenetic therapies, which alter the epigenetic states at specific genes within the genome, are being designed to allow PTHS models to use their one functioning copy of Tcf4 twice as much, hopefully restoring full Tcf4 function and reversing the cognitive deficits associated with Pitt-Hopkins.  Taken together, these approaches investigate already-available FDA approved drugs and cutting edge genetic techniques to identify potential therapies that improve cognition in the near-term and attempt to address and compensate for the underlying cause of Pitt-Hopkins Syndrome.

Brady Maher, Ph.D., Principal Investigator; Huei-Ying Chen Ph.D.; Stephanie Cerceo-Page, Ph.D.; Lieber Institute for Brain Development, Johns Hopkins School of Medicine, Awarded $50,000, UPenn, MDBR, 2015-2016

Exploring the impact of a TCF4 mutation on the physiology of inhibitory neurons of the prefrontal cortex:  

PTHS is a neurodevelopmental disorder due to mutation or deletion of one copy of the TCF4 gene. TCF4 is a transcription factor that can regulate the expression of many downstream genes and therefore regulates the genetic programs necessary for normal brain development. We measured the expression of TCF4 mRNA across the lifespan in humans and rodents and observed a peak in TCF4 expression occurs during the formation of the cerebral cortex, a region of the brain important to higher cognitive functions including learning and memory. Using a mouse model of PTHS that has a mutation in one copy of the TCF4 gene, we observed that TCF expression is blunted during the developmental peak in expression compared to control animals, and we believe this indicates a causal time period for the development of PTHS. Unfortunately, this critical period occurs in utero and prior to when diagnosis is currently made, thus complicating our ability design treatment strategies during this causal phase of the disorder. Therefore, our research group is focused on understanding the underlying pathophysiology that produces symptomatology in PTHS so that we can normalize this pathophysiology in children and adults. Using our animal models of PTHS, we have identified a sodium channel that is normally expressed in the peripheral nervous system, but is ectopically expressed in the central nervous system when TCF4 is mutated. Experiments are currently underway to determine if blocking this Na channel with drugs can lead to improvement on behavioral tests in our PTHS mouse model. In our current proposal, we would like to follow up a preliminary result that suggests inhibitory transmission onto excitatory neurons in the cortex is decreased in the PTHS mouse compared to control littermates. In addition, using RNA sequencing of the PTHS mouse model we observed that many genes that are specific to inhibitory neurons show decreased expression compared to control animals, and we identified a specific population of inhibitory neurons (cortistatin positive) that normally show high levels of TCF4 expression. These cortistatin positive interneurons are known to release a neuropeptide called cortistatin that has been shown to inhibit the generation of seizures and regulate sleep states. Given the prevalence of seizures and sleep disturbances in PTHS, we believe this population of inhibitory neurons may underlie clinical aspects of the disorder. Therefore, we propose to breed the PTHS mouse with another mouse that allows us to visualize cortistatin positive interneurons and we will use electrophysiology and microscopic imaging to determine if these cells are disrupted in the PTHS mouse model compared to control littermates. If deficits are observed in this population we will determine the cellular and molecular mechanism using pharmacological rescue and/or molecular phenocopy. Identified molecular mechanisms will then be deemed potential therapeutic targets and these targets will be tested for their ability to normalization of behavioral deficits in the PTHS mouse.

Tõnis Timmusk, Ph.D., Principal Investigator; Mari Sepp, Ph.D., Co-Investigator, Tallinn University of Technology, Estonia, Awarded $50,000, UPenn, MDBR, 2015-2016

Regulation of TCF4 transcriptional activity in neurons:

Transcription factor TCF4 (alias ITF2, SEF2 or E2-2) is a broadly expressed protein involved in the development and functioning of many different cell types. Recent studies point to important roles for TCF4 in the nervous system. Specifically, human TCF4 gene is implicated in susceptibility to schizophrenia and mutations in TCF4 cause Pitt-Hopkins syndrome (PTHS), a rare developmental disorder characterized by severe motor and mental retardation, typical facial features and breathing anomalies. The mutation may be in different parts of the gene, but it appears in only one allele. Whereas in many other genes the other, unaffected allele would be able to compensate for the defect, this is not the case in TCF4. This indicates that the protein encoded by the TCF4 gene is essential for the development of the nervous system, and that human development depends significantly on the amount of this protein in the brain and body. Our previous data have suggested that synaptic activation of nerve cells, that is the basis of brain function, leads to activation and phosphorylation of TCF4 protein. Phosphorylation is the addition of a phosphate group to a protein or other organic molecule. Phosphorylation turns many proteins on and off, thereby altering their function and activity. The current project is aimed to find out how the activity and phosphorylation of TCF4 protein is regulated inside nerve cells of the brain, and to characterize the phosphorylation pattern of activated TCF4. Additionally, we want to determine which genes are targeted by TCF4 in nerve cells after synaptic activation. Since Pitt-Hopkins syndrome manifests itself at an early stage, there are better chances for its treatment due to the greater plasticity of children’s brains. Increasing the amount and/or activity of the functional TCF4 protein produced from the healthy allele is among possible approaches to develop drugs for Pitt-Hopkins syndrome treatment. We believe that our project could lead to the discovery of novel possibilities for increasing the activity of TCF4 in nerve cells that could be useful to develop treatments for therapeutic intervention of Pitt-Hopkins syndrome.

2014-2015:  The PHRF awarded four research grants and two additional research grants through UPenn Million Dollar Bike Ride.  A total of $430,000 awarded for Pitt Hopkins research this year.

 PHRF Awards:

Dr. Andrew Kennedy, University of Alabama Birmingham

$80,000; one year grant: Investigating therapies for Pitt Hopkins syndrome

Investigating Therapies for Pitt-Hopkins Syndrome: With the financial support of the Pitt-Hopkins Research Foundation, researchers in the Department of Neurobiology at the University of Alabama at Birmingham have started a research program to investigate the molecular and neural basis of Pitt-Hopkins Syndrome (PTHS). Their research bridges clinical and basic neuroscience by investigating the underlying neurobiology of PTHS and testing potential therapies for cognitive dysfunction using a PTHS mouse model. Specifically, Dr. Kennedy is working toward understanding how histone deacetylase (HDAC) inhibitors may serve to improve cognition in PTHS, and whether the endogenous HDAC inhibitor sphingosine 1-phosphate can serve as a blue print for a more comprehensive therapy. Using next-generation sequencing technology, the genetic and epigenetic consequences of PTHS that result in diminished cognition will be elucidated, which will significantly enhance our understand of the role Tcf4 plays in the learning and memory and help us identify critical downstream transcriptional targets. This grant also serves to broaden the scope of investigation to include potential therapies to improve myelination as well as alleviate the hyperventilation, apnea, and constipation associated with PTHS.

Dr. Stephen J. Haggarty, Harvard Medical School, Massachusetts General Hospital

$80,000; one year grant: Characterization of Pitt Hopkins Syndrome stem cell models & therapeutic screening (stem cells created from skin-derived fibroblasts of patients with Pitt Hopkins syndrome)

Developing therapeutics for neurodevelopmental disorders is one of the greatest medical needs of the 21st century. Of particular interest for defining underlying molecular and cellular mechanisms of these disorders is Pitt-Hopkins syndrome (PTHS), a neurodevelopmental disorder characterized by severe intellectual disability, seizures, as well as a other peripheral symptoms including failure to thrive, chronic constipation and recurrent facial dysmorphology. PTHS is tightly associated with mutations in the TCF4 gene encoding isoforms of the basic helix-loop-helix (bHLH) transcription factor 4 (TCF4), and recent genetic studies are beginning to connect other genes within the PTHS pathway, including patients with mutations in NRXN1 (neurexin 1) and CNTNAP2 (contactin-associated protein-like 2) that exhibit PTHS-like symptoms. The overall goal of our project is to advance a personalized medicine program for PTHS that seeks to identify novel targets and experimental therapeutics by testing approved drugs and novel synthetic compounds in patient-derived stem cell models in order to select compounds for testing in the animal model of PTHS, and eventually in PTHS patients given appropriate efficacy and safety properties. To this end, we have established a PTHS biorepository consisting of patient skin biopsy-derived fibroblasts and begun to use reprogramming technologies to create a panel of induced pluripotent stem cells (iPSC). The development of these powerful new in vitro culture systems allows: i) investigation of the step-by-step development of neuronal phenotypes due to TCF4 mutations; ii) determination of the role of TCF4 in neuronal gene expression; and iii) the opportunity to screen for experimental therapeutics that can enhance or restore TCF4 expression. Deep phenotyping of neurons derived from PTHS patient iPSCs is on-going using a variety of strategies ranging from imaging of their morphology and neurodevelopmental capacities using automated microscopy to genome-wide analysis of their transcriptome. With our preliminary findings, we have identified a set of novel pathways and targets for modulating TCF4 expression and are currently working to understand their precise mechanism of action as well as to identify compounds with improved efficacy and pharmacological properties. On-going efforts are also to expand the collection of PTHS iPSC models with a range of different genetic mutations and clinical symptoms as well as to generate isogenic controls of patient-derived iPSC models with the use of genome-editing methods to ‘correct’ mutations.

Dr. Hazel L. Sive, Whitehead Institute for Biomedical Research, Massachusetts Institute of Technology

$80,000; one year grant: Characterization and therapeutic screening of Pitt-Hopkins Syndrome using the Zebrafish

Our research has two goals: first, to understand how each variant form of TCF4 leads to specific symptoms of a PTHS patient, and second, to identify chemicals that change increase levels of TCF4, and that in the long term, may lead to therapies. We use the powerful zebrafish system where the transparent larva allows detailed analyses in single cells of the living animal. Fish can show many aspects of PTHS and can connect particular TCF4 mutations and particular symptoms. Zebrafish tcf4 and human TCF4 genes are similar and ‘humanized’ fish can be made where the human gene has been substituted for the fish gene, and so readily studied. Since zebrafish are small, they can be used in whole animal screens to test chemicals that may be potential medications. We have developed fish with reduced or no activity of the fish tcf4 gene, and will test these for analysis of brain development and function, face formation, digestive system formation and function, and muscle function, all of which can be affected in PTHS patients. We will work collaboratively with Dr. Stephen Haggarty (MGH) who uses patient-derived stem cells, making a powerful coordinated effort to understand and treat PTHS.

Dr. Brady Maher, Lieber Institute for Brain Development, Johns Hopkins School of Medicine

$80,000; one year grant: Exploring changes in neuronal translatomes in both cell- and non-cell autonomous animal models of Pitt Hopkins Syndrome

PTHS syndrome results from a mutation in one copy of the TCF4 gene that results in haploinsufficiency. TCF4 is a transcription factor that depending on the target gene can either produce repression or activation of target gene transcription. Therefore, PTHS results from dysregulation of target gene expression during fetal development. To begin to identify therapeutic targets for the treatment of this disorder we propose to identify target genes that are dysregulated downstream of TCF4 haploinsufficiency. We show that embryonic knockdown of TCF4 expression results in intrinsic excitability deficits in excitatory neurons of the rat frontal cortex. Using a novel combination of molecular approaches (iTRAP), we identify two ion channel genes (Kcnq1, Scn10a) whose expression is increased when TCF4 is suppressed and appear to be responsible for producing excitability defects observed with TCF4 knockdown. These proof-of-principle experiments suggest these methods are effective at identifying TCF4 target genes and therefore we propose to expand this technology to whole-genome RNA sequencing to identify more genes that are regulated by TCF4. To increase the validity of our results we propose to compare to independent animal models of PTHS. A cell autonomous model that uses shRNA constructs to knockdown the expression of TCF4 in a subset of neurons in the frontal cortex and a non-cell autonomous model (TCF4+/- mouse) in which the whole animal expresses only one copy of the TCF4 gene. In both animal models, we will use IUE to transfect a pure population of layer 2/3 neurons with the ribosomal protein L10a-EGFP and use antibodies against EGFP to affinity purify RNA at two distinct developmental time points. The first time point will be at the developmental peak of TCF4 expression and the second time point will be during rodent adolescents. We predict the early time point will identify target genes that are causal for PTHS and the adolescent time point will identify genes that maybe more suitable for treatment in the patient population.

UPenn Million Dollar Bike Ride awards:

Dr. Courtney Thaxton and Dr. Benjamin D. Philpot, University of North Carolina at Chapel Hill

$55,000; one year grant: Identification of genetic and molecular targets for Pitt-Hopkins Therapeutics

A major hurdle hindering the discovery of therapeutics to treat Pitt Hopkins Syndrome (PTHS) is the lack of knowledge of the genetic and molecular targets of TCF4 in the nervous system. Current research is utilizing a mouse model that lacks one complete copy of Tcf4, but to date there are no models phenocopying missense or nonsense mutations associated with PTHS. Both missense and nonsense mutations in TCF4 comprise the largest number of causative mutations for PTHS. Therefore, we generated a new mouse model mimicking the most prevalent mutation found in PTHS, as well as a unique mouse model lacking several common mutation sites linked to PTHS. We will use these two new mouse models, as well as a previously developed Tcf4-deletion mouse model, to uncover the genes and molecular pathways regulated by Tcf4. These mice will also be assessed for behavioral changes in order to identify potential genotype-phenotype characteristics. By discovering the genes or common pathways regulated by TCF4, we may be able to identify approved therapeutics that target these affected pathways or design new drugs to reverse the disregulation caused by the absence of TCF4.

Dr. Daniel Marenda, Drexel University, and Dr. Wenhui Hu, Temple University School of Medicine

$55,000; one year grant: Understanding TCF4 function in post-mitotic neuron synaptic plasticity

In the study of human disease, animal models often act as surrogates for people when (as is often the case) testing on humans is unethical or not feasible. The common thread of evolution among species allows for discoveries that are made in nonhuman species to be applied across the diversity of life forms, from single-celled bacteria and yeast to large, complex, many-celled human beings. These nonhuman surrogates are collectively known as “model organisms”. They bring with them an enormous battery of sophisticated experimental tools that allow for the study of human diseases and offer clues about the underlying factors that contribute to these diseases.

In collaboration with Dr. Wenhui Hu’s laboratory at Temple University, Dr. Daniel Marenda at Drexel University is using two of these model organisms (the fruit fly and the common mouse) to further understand the function of the TCF4 gene. TCF4 is well known to function during development, to help form proper muscle cells, nerve cells, and other tissues in the body. After these cells develop, they form mature cells (that is, cells that do a job). Dr. Marenda and Dr. Hu have discovered that while TCF4 does function to help cells develop, TCF4 also functions in mature cells as well (particularly mature nerve cells). This is exciting, as there previously has been no described function of TCF4 in mature nerve cells. Further, this opens the possibility of therapeutic intervention for Pitt-Hopkins outside of embryonic development. The hope is that once we better understand what TCF4 is doing in mature nerves, we will be better able to intervene with drugs to fix TCF4 function in those mature cells when TCF4 function is lost due to mutation.

2013-2014:  Six Research Awards awarded, totaling $330,000

Dr. David Sweat, University of Alabama Birmingham

$80,000, one year grant

New Research Into the Neurobiological Basis of PTHS

The underlying genetic basis of Pitt-Hopkins Syndrome (PTHS) has recently been discovered – PTHS is caused by heterozygous null mutation or deletion of the TCF4 gene on human chromosome 18. The identification of the dysfunctional TCF4 transcription factor gene as the genetic basis of the disorder is a critical step forward in beginning to understand the diagnosis, etiology and molecular biology of PTHS. This project encompasses a set of studies to investigate the cognitive dysfunction associated with PTHS, focusing on mechanistic studies to understand the role of the TCF4 transcription factor in central nervous system function, using genetically engineered mouse models for PTHS. The project is focused on investigating: 1. Cognitive dysfunction in the domain of learning and memory; 2. Alterations in CNS neuronal function and the regulation of long-term gene readout in the CNS, and; 3. Testing one specific hypothesis concerning a potential new drug treatment for PTHS. The longer-term priorities for the proposed project will be two-fold: rapid drug development of a new PTHS treatment and understanding the molecular biology underlying PTHS intellectual disability.

Dr. Stephen J. Haggarty, Harvard Medical School, Massachusetts General Hospital

$80,000 one year grant

Generation and Characterization of Pitt-Hopkins Syndrome Stem Cell Models

Developing therapeutics for neurodevelopmental and intellectual disability disorders presents significant challenges and is one of the greatest areas of unmet medical need in the 21st century. Of particular interest for gaining insight into the underlying molecular and cellular mechanisms of neuroplasticity and cognition is Pitt- Hopkins syndrome, an autism-spectrum disorder, known to be caused by mutations in the TCF4 gene on Chromosome 18q21. The TCF4 gene is known to encode multiple isoforms of the basic helix-loop-helix (bHLH) transcription factor 4 (TCF4). Gaining a more complete understanding of the regulation and function of TCF4 in the human nervous system may provide critically needed insight into the pathophysiology of multiple human disorders sharing the features of intellectual disability and developmental delay.

With the overarching goal of advancing a Personalized Medicine Program for Pitt-Hopkins syndrome that seeks to identify novel targets and experimental therapeutics for testing in pre-clinical animal models and eventually in patients, we have established a Pitt-Hopkins syndrome biorepository and are actively collecting skin-derived fibroblasts from patients from which we have created multiple patient-specific, induced pluripotent stem cell (iPSC) models. The development of these powerful new in vitro culture systems now allow: i) investigation of the step-by-step development of neuronal phenotypes due to TCF4 mutations; and ii) determination of the role of TCF4 in neuronal gene expression. In addition to generating cellular tools to study TCF4 function, we are also developing and validated molecular probes to quantify the expression of multiple TCF4 mRNA transcripts in response to experimental therapeutics along with molecular tools for silencing and nucleotide-specific genome editing at the TCF4 locus to create isogenic TCF4 mutant and corrected iPSCs.

Using these novel cellular and molecular tools, we aim to address whether the loss of function of TCF4 leads to deficits in the regulation of genes important for synaptic functions that are known to play an important role in cognition. We will also be able to compare the phenotypes of our Pitt-Hopkins syndrome patient-derived neurons to those of other neurodevelopmental disorders, such as Fragile X syndrome and Rett syndrome, that have been developed in our lab in order to identify potentially distinct, as well as shared, aspects of each disorder.

Overall, we anticipate that our research, in combination with studies using animal models, will help lay the foundation for the discovery of therapeutic agents targeting early steps of the pathophysiological mechanisms associated with Pitt-Hopkins syndrome. In doing so, these efforts are likely to advance our understanding of the molecular and cellular neurobiology of neurodevelopmental disorders that share common mechanisms of aberrant chromatin-mediated neuroplasticity.

Dr.Hazel L. Sive, Massachusetts Institute of Technology

$50,000, one year grant

Characterization and Therapeutic Screening of Pitt-Hopkins Syndrome using the Zebrafish

We propose to develop the zebrafish as a tool to understand the biology of and to assess potential therapeutic treatments for Pitt-Hopkins Syndrome (PTHS), a rare but devastating disorder associated with the TCF4 gene. While there are common symptoms associated with PTHS including intellectual disability, locomotion deficits, epilepsy and chronic constipation, the severity of these can vary between patients. The connection between the multiple mutations observed in the TCF4 gene in individual patients and the symptoms of each patient is not understood. This study will contribute to a “personalized” approach in categorizing PTHS TCF4 genes and patient symptoms. The study will also use a whole animal screening approach to identify chemicals that may lead to treatments for PTHS.

Dr. Courtney Thaxton and Dr. Benjamin D. Philpot and Dr. Mark Zylka, University of North Carolina at Chapel Hill

$50,000, one year grant

Identification of genetic and molecular targets for Pitt-Hopkins Therapeutics

Pitt-Hopkins syndrome (PTHS) is a rare neurodevelopmental disorder on the autism spectrum. Since the discovery of TCF4 as the causative gene for Pitt-Hopkins syndrome (PTHS), little progress has been made towards uncovering the genetic and molecular pathways regulated by TCF4 and their involvement in brain functions. Progress has been further limited due to the embryonic lethality observed in mouse models that entirely lack expression of the TCF4 gene. Here, we will uncover the genes and molecular pathways regulated by TCF4 in primary brain neurons, a principal cell type affected in PTHS, with the expectation that this information will provide the most rational path towards designing therapeutic strategies. We will use whole- genome analyses to identify every gene affected by different genetic models of PTHS. To mimic large deletions of TCF4, we will determine the genetic and molecular consequences of TCF4 haploinsufficiency. To mimic PTHS-causing mutations, we will establish the consequence of common and recurring TCF4 point mutations on gene expression. By comparing the genes affected by these diverse manipulations associated with PTHS, we will better pinpoint the relevant genetic and molecular pathways to be targeted for future therapeutics.

Dr. Joseph D. Buxbaum, Icahn School of Medicine at Mount Sinai

$20,000, one year grant, to develop new antibodies and testing those antibodies with the specific goal of identifying a ChIP-grade antibody. Once ChIP grade antibodies are identified, funding to complete aims 1 and 2 could be negotiated, at 30-50K to continue project.

Functional analysis of TCF4 in neurons

Pitt Hopkins syndrome is a debilitating childhood disorder caused by deficiency of a protein called TCF4. To date, how TCF4 participates in the development of the brain and the connection between the brain cells (neurons) is not known. Here, we propose to identify the genes regulated by TCF4 in human and mouse, and to define if these target genes are turned on or turned off. In addition, we propose to study the functions of TCF4 at synapses, which provide for communication between adjacent neurons. We will study if depleting TCF4 in a mouse model alters the biochemical composition, the architecture, and the function of the synapses. Our study will help in the understanding of what are the specific defects caused by deficiency of TCF4, increasing the current knowledge on the pathological processes occurring in patients and laying the groundwork for the development of novel therapeutic strategies.

Aim 1. Identify the target genes of TCF4 in purified neuronal nuclei from human brain.
Aim 2. Identify the target genes of TCF4 in purified neuronal nuclei from mouse brain and correlate with RNA expression in Tcf4+/- mice and littermate controls.
Aim 3. Dissection of Tcf4 function in mouse.

 

Dr. Tonis Timmusk, Tallinn University of Technology, Estonia

$50,000; one year grant

Signaling pathways and compounds regulating transcriptional activity and phosphorylation of TCF4 protein in neurons

Pitt-Hopkins syndrome is a cognitive functional disorder (a form of mental retardation) diagnosed in less than 200 people in the world. Polymorphism or variation in the DNA sequence of the same gene has been linked to schizophrenia. Pitt-Hopkins syndrome is caused by a so-called de novo genetic mutation – one that is non-hereditary. The mutation may be in any part of the gene, but it appears in only one allele. Whereas in many other genes the other, unaffected allele would be able to compensate for the defect, this is not the case in TCF4. This indicates that the protein encoded by the TCF4 gene is essential for the development of the nervous system, and that human development depends significantly on the amount of this protein in the brain and body.

Our previous data have suggested that synaptic activation of nerve cells, that is the basis of brain function, leads to phosphorylation of TCF4 at specific sites of the protein. Phosphorylation is the addition of a phosphate group to a protein or other organic molecule. Phosphorylation turns many proteins on and off, thereby altering their function and activity. The current project is aimed at identification of signalling pathways inside nerve cells of the brain that regulate the phosphorylation and activity of TCF4 protein, characterization of the phosphorylation pattern of activated TCF4 and assessment of the efficiency of chemical compounds targeted at different components of the identified signalling pathways to increase TCF4 activity and correct PTHS deficiencies in neurons.

Since Pitt-Hopkins syndrome manifests itself at an early stage, there are better chances for its treatment due to the greater plasticity of children’s brains. If previously it was believed that mental retardation cannot be treated, then the results of recent tests on mice give hope that it can. In addition, the identification of treatment could be simpler for disorders caused by one gene than for disorders caused by the malfunction of hundreds of genes. Therefore, increasing the amount and/or activity of the functional TCF4 protein produced from the healthy allele is among possible approaches to develop drugs for Pitt-Hopkins syndrome treatment. We believe that current project could lead to the discovery of novel possibilities for increasing the activity of TCF4 in nerve cells that could be useful to develop treatments for therapeutic intervention of Pitt-Hopkins syndrome.

 

Two new grants awarded, Spring 2013:

David Sweatt, PhD,  Evelyn F. McKnight Chair, Dept of Neurobiology; Director, McKnight Brain Institute; University of Alabama at Birmingham

The identification of the dysfunctional TCF4 transcription factor gene as the genetic basis of Pitt-Hopkins Syndrome is a critical step forward in beginning to understand the diagnosis, etiology and molecular biology of PTHS. This project encompasses a set of studies to investigate the cognitive dysfunction associated with PTHS, focusing on mechanistic studies to understand the role of the TCF4 transcription factor in central nervous system function. For this project we are using genetically engineered mice in which the TCF4 gene has been manipulated in order to mimic human PTHS. This particular project is focused on investigating whether there is aberrant regulation of epigenetic molecular mechanisms, and altered transcriptional regulation of genes and small non-coding gene products in the PTHS model mice. For these studies we are particularly interested in learning and memory function as it relates to these molecular biological mechanisms in the CNS. Toward that end we are using next-generation high-throughput DNA sequencing methodologies coupled with epigenomics and bio-informatics approaches.  Grant amount:  $63,920.

Stephen J. Haggarty, PhD,  Associate Professor of Neurology | Harvard Medical School and Massachusetts General Hospital

The additional funds will be used to further support a post-doctoral research scientist in the Haggarty laboratory who is developing assays with human patient specific, stem-cell derived neuronal to measure TCF4 expression at the mRNA and protein level. Additionally, methods for mapping TCF4 target genes using state-of-the-art techniques for chromatin immunoprecipitation coupled with high-throughput DNA sequencing (ChIP-seq) are being piloted. These studies are anticipated to provide important new insight into how the loss of TCF4 function may lead to changes in pathways important for neuroplasticity.  Grant amount:  $50,000.

Grants given by the Pitt Hopkins Syndrome Fund, March-September 2012:

  • Molecular and Neural Basis of PTHS in TCF4 Mouse Models, with Dr. David Sweatt at the University of Alabama at Birmingham (a mutation or deletion in the TCF4 gene causes PTHS), $85,000
  • Determining if TCF4 is necessary for normal cognitive function in a developed mature central nervous system, using mouse models, with Dr. David Sweatt at the University of Alabama at Birmingham, $75,000
  • Generation and characterization of isogenic PTHS Stem cell models with Dr. Stephen Haggarty at Harvard Medical School, $85,000
  • Creating an accurate description of the physical and behavioral consequences of an abnormality in the TCF4 gene with Dr. Jannine Cody at the Chromosome 18 Clinical Research Center at the University of Texas at San Antonio, partnering with Dr. Stephen Haggarty at the Harvard Stem Cell Institute, using skin cells to create stem cells.  For this grant, Dr. Cody budgeted: $5,719 for year one (given in April 2012 by the PTHS Fund).  

The following is a detailed description of grants that have been funded by the Pitt Hopkins Research Foundation, as of September 2012:

Dr. David Sweatt, Department of Neurobiology, the University of Alabama at Birmingham

PTHS Foundation Funding Enables New Research Into the Neurobiological Basis of PTHS

With the help and financial support of the PTHS Foundation Dr. J. David Sweatt, Professor and Chairman of the Department of Neurobiology at the University of Alabama at Birmingham, has started a new project to investigate the molecular and neural basis of Pitt-Hopkins Syndrome (PTHS). Dr. Sweatt is a senior scientist in the field of learning and memory, with an track record of prior discovery related to intellectual disability.

The research in the Sweatt laboratory capitalizes on the recent discovery that PTHS is a neurodevelopmental disorder, the underlying genetic basis of which is mutation/deletion of the TCF4 gene. In PTHS patients the altered gene product is present throughout development but is also present in the fully developed CNS. PTHS is an orphan disease, and at present there is a profound lack of information concerning the molecular neurobiology underlying PTHS. Thus, the Sweatt laboratory will begin to investigate the molecular and cellular basis of nervous system dysfunction in PTHS, to lay a cornerstone of research that will allow the hope of the development of a treatment for these patients in the future.

In their study the Sweatt laboratory will undertake comprehensive behavioral testing of genetically engineered PTHS model mice (TCF4 heterozygous deficiency mice) focusing on learning and memory, and investigating whether there is altered function in the neurons of PTHS model mice. In addition the Sweatt lab will testing whether a specific category of drugs, histone deacetylase (HDAC) inhibitors, might ameliorate any behavioral and neurophysiologic deficits observed in the mice. The proposed studies will yield valuable information concerning the Intellectual Disabilities aspect of PTHS, and whether HDAC inhibitors might be a candidate treatment for this aspect of PTHS.

A gift of $85,000 was given to Dr. David Sweatt to begin this project by the PTHS Fund in June 2012.

Partnering with the Simons Foundation Autism Research Initiative

The PTHS research in the Sweatt laboratory is also being supported by a brand-new Simons Foundation Autism Research Initiative (SFARI) Explorer Award. In addition to manifesting intellectual disabilities, PTHS is an Autism Spectrum Disorder (ASD) and the autistic characteristics of PTHS are a crucial aspect of the syndrome. Thus, given their focus on autism the Simons Foundation has generously now begun funding research into PTHS. For their research project, in addition to studying the memory and neuronal characteristics of the PTHS model mice, the Sweatt laboratory will study the PTHS model mice in the domain of autistic behaviors as well. This important aspect of the research project will undertake behavioral studies to assess whether the TCF4 heterozygous deficiency mice manifest autistic-like behaviors, and also address whether HDAC inhibitors might ameliorate autism-like phenotypes in the mice. Studies of the PTHS model mice in terms of ASD-like behaviors are important not only because of their direct relevance to PTHS patients, but also because studies of the TCF4-deficient animals may give fundamental insights into the molecular neurobiology underlying the broader spectrum of autistic disorders. Furthermore, any potential drug treatments that ameliorate autism-like behaviors in PTHS mice might also be useful to consider for possible use with other ASD’s as well.

Additional grant funded in September 2012:
Dr. Sweatt’s group has just recently received a second grant to determine if TCF4 is necessary for normal cognitive function in a developed mature central nervous system. This work will lay the ground work for determining if PTHS is a developmental or chemical disorder in learning and memory or both.
Grant amount: $75,000, Fall 2012

Dr. Stephen Haggarty, Harvard Medical School:

Grant funded in September 2012:

The Chromosome 18 Clinical Research Center at the University of Texas San Antonio is partnering with Dr. Stephen Haggarty, at the Harvard Stem Cell Institute and Assistant Professor of Neurology at Harvard Medical School, to work on drug development. They will be using skin cells (fibroblasts) from individuals with Pitt Hopkins to create stem cells and then differentiate them into neurons (or other important cell types). They can then use these cell lines to screen a large variety of possible drugs relatively rapidly. This project is very significant, both for its importance for Pitt Hopkins and as a “proof of principle” that we can apply to other conditions involving the central nervous system.

In addition to his collaborative work with Dr. Cody at the University of Texas, San Antonio, Dr. Haggarty’s group is working on identifying common molecular pathways and networks dysregulated due to TCF4 haploinsufficiency, using specially developed isogenic PTHS stem cell models or neurons. This work will set the stage for the discovery of therapeutic targets aimed at improving cognition.
Grant amount: $85,000, Fall 2012