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Fall 2013:  Six Research Awards awarded, totaling $330,000

PTHSmouse1Dr. 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, 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