Eric Morrow - US grants

DESCRIPTION (provided by applicant): This application for a K23 Career Development Award is entitled "Genetic investigation of cognitive development in autistic spectrum disorders". The candidate has MD and PhD degrees. His PhD is in molecular neurodevelopment in genetic mouse models. His undergraduate training at MIT, and his MD in the Harvard-MIT Division of Health Sciences and Technology at Harvard Medical School involved studies in quantitative methods. The candidate has pursued clinical training through to Chief Residency in the MGH- McLean Psychiatry Residency. He is an Instructor in Psychiatry at Harvard Medical School with appointments at the MGH and Children's Hospital Boston, as well as a postdoctoral affiliation with the Broad Institute of MIT and Harvard. If successful, the career trajectory for this applicant is to lead a laboratory- based program in Molecular Psychiatry at MGH with a focus in autism and related disorders of cognitive development. Linked to this translational research lab, the candidate plans to direct a Clinical Research Program for Adults with Pervasive Development Disorders in MGH Psychiatry. The candidate's research proposal describes studies designed to identify genes in autism spectrum disorders (ASD). The candidate proposes to study two complementary patient populations: 1) a special founder population from the Arabic Middle East, Turkey and Pakistan, wherein parents of affected children are consanguineous;and 2) a large collection of North American families (>4000 samples) from AGRE and the Boston Autism Consortium, including a cohort of 200 families which the candidate proposes to characterized in MGH Pediatric Psychiatry. The consanguineous pedigrees will be enrolled as part of the Autism International Homozygosity Mapping Collaborative (for which the candidate serves as Associate Director) with the aim of discovering highly penetrant autosomal recessive genes. A principal tool for analysis will be the Affymetrix 500K SNP microarray, and studies will include genomic copy number, as well as deletion and homozygosity mapping. Genes thereby identified will be studied in the North American patients using large-scale resequencing, association studies and phenotype-genotype studies. Mentors include Chris A. Walsh, MD, PhD, Chair of Genetics at the Children's Hospital Boston and Director of the Boston Autism Consortium. Dr. Walsh's lab has expertise in international genetic collaborations and identifying recessive genes in neurodevelopmental and cognitive disorders. In addition, Mark J. Daly, PhD, a statistical geneticist at the Broad Institute will serve as co-mentor. The candidate is seeking training in human population and statistical genetics, and bioinformatics. He is also seeking patient- oriented research training in ASD which will be pursued through the Developmental Medicine Center at Children's Hospital, as well as with external autism research experts of national esteem.

PROJECT SUMMARY (See instructions): In this project, we will develop an understanding of the range of clinical symptoms and biological factors (genetics and brain morphometry) that correlate with obsessive-compulsive behavior in autism. Autism is a highly heterogeneous disorder. A significant number of patients do not improve substantially with current treatments. We refer to this group of patients as difficult-to-treat autism (DTT-Autism). Our preliminary data suggest that these patients exhibit obsessive-compulsive behavior (OCB). We will test the central hypothesis that participants with autism with high OCB represent a subtype of difficult-to-treat autism (DTT-Autism) who will have abnormal maturation of frontal-striatal circuitry and genetic susceptibilities analogous to those previously studied in obsessive-compulsive spectrum (OCS) conditions. Capitalizing on recent progress in neuroimaging and genetics in OC spectrum (OCS) disorders, this project will test the hypothesis that autism with OCB will share genetic and brain circuitry changes that have been demonstrated in OCS disorders. We will test the hypothesis that autism with OCB will correlate with abnormalities in frontal-striatal circuitry as is true for OCD and related OCS disorders. We will also look for associations between rare and common variation in OC-related genes and OCB symptoms in autism. Using a combination of approaches including clinical assessment, neuroimaging and genotyping, we will characterize the subtype of autism with OCB. This work is important as it studies a group of autism patients who are in greatest need of treatment development. If our hypotheses about the strong biologic relationship between OC spectrum disorders and autism with OCB are accurate, novel treatments for autism may be drawn from ongoing research in interventions in treatment-refractory OCD.

DESCRIPTION (provided by applicant): A lack of validated preclinical models for exploring cellular mechanisms has limited treatment development in severe autism. In order to develop new preclinical models and therapeutics, rig- orous studies of cell phenotypes and cellular responses to potential agents in patient-derived neurons will be advantageous. The long-term goal of this research is to elucidate the molecular mechanisms underlying the pathology of severe autism that will serve as the basis to develop innovative treatments for these patients with few current therapeutic options. We recently identi- fied a novel mechanism in autism involving endosomal Na+/H+ exchangers (NHEs). We have now generated induced pluripotent stem cell lines (iPSCs) from patients with a range of NHE6 mutations. The objective for this application is to elucidate the role of NHE6 in human axon de- velopment and to test the response of patient-derived neurons to available, mechanism-based agents that may subsequently serve as treatments in patients. NHE6 regulates the efflux of pro- tons from endosomes. In our preliminary studies in human neurons, NHE6 deficiency leads to over-acidification of the endosomal lumen and defects in axon growth and branching. This mechanism is fortuitous because there are a number of well-known FDA-approved agents that target intra-endosomal pH. Our central hypothesis is that deficiency of NHE6 inhibits axon growth and branching due to diminished polarized membrane addition. The rationale for this re- search is that it constitutes the first critical steps toward the development of new treatments for individuals with severe autism and related disorders caused by reductions in NHE6 expression and/or by defects in axonal growth or arborization. We have published studies on NHE6 function in mouse models, yet the transition to patient-derived tissues is warranted at this time. Screening of potential human therapeutic agents is best performed on human (and patient-derived) tis- sues. Three specific aims are proposed: (1) Determine the role of NHE6 in regulating endosome lumen acidity and endosome recycling. (2) Determine the extent to which the function of RAB10-associated SVs in axon growth is perturbed by NHE6 mutation in patient-derived neu- rons. (3) Determine the reversibility of axon growth and branching defects in patient-derived neurons by exogenous growth factors. The proposed research is innovative because we are studying a novel cellular mechanism in autism, namely, regulation of intra-endosomal pH in ax- on growth. This research is significant because it will lead to the development of critically need- ed preclinical models in patient-derived neurons and potential treatments for severe autism and related disorders, an urgent public health problem.

? DESCRIPTION (provided by applicant): No effective therapeutic agents currently exist to improve outcomes in severe autism. We recently identified a new cellular mechanism in autism involving endosomal Na+/H+ exchanger 6 (NHE6). Studies of NHE6 offer a valuable opportunity for rapid development of innovative treatment strategies in severe autism and related dis- orders. Human mutations in endosomal NHE6 constitute a newly recognized, monogenic, autism-related disorder. Also, down-regulation of NHE6 gene expression is evident in postmortem brains in approximately 30% of patients with idiopathic autism. NHE6 is an integral membrane protein that regulates proton efflux out of endosomes. We have reported that loss of NHE6 in neurons leads to over-acidification of the endosome lumen, and diminished neuronal arborization and synapse development. We also have exciting, new data demonstrating that intra-endosomal pH in the mutant is normalized in vitro by FDA-approved medicines known to alka- linize endosomes. The objective for this research proposal is to elucidate the mechanisms by which NHE6 mu- tations lead to defects in circuit development, and to test the ability of alkalinizing agents to normalize these mutant phenotypes in mouse in vitro and in vivo. Our data support the following central hypothesis: Loss of NHE6 function leads to over-acidification of early endosomes, which enhances degradation of cargo. We also hypothesize that over-acidification of early endosomes drives trafficking to the degradative pathway (i.e., to late endosomes and lysosomes), and decreases endosome recycling and endosome signaling. Our data indicate that TrkB is cargo in NHE6-associated endosomes. We find attenuated TrkB signaling in NHE6 mutant neu- rons. The BDNF/TrkB pathway is well-known to govern circuit development via endosomal signaling, and has broad significance in neuropsychiatry. Our Aims include: 1) Determine the functional domains of NHE6 protein through study of patient mutations; 2) Determine the role of NHE6 and intra-endosomal pH regulation in TrkB trafficking and endosomal signaling; and 3) Determine the reversibility of defects in neuronal arborization and synapse development in NHE6 mutant neurons using alkalinizing medicines. These studies on the role of intra-endosomal proton concentration are innovative because they represent: 1) a new level of cellular analysis in endosome trafficking and neuronal differentiation; and 2) a novel, druggable cellular mechanism in autism and related disorders. This research is significant because it will lead to 1) elucidation of fundamental and disease- relevant mechanisms in endosome biology and circuit development; and 2) development of specific avenues for mechanism-based treatments for severe autism. This project is also a part of an integrated translational ap- proach in our research group that is coordinated with ongoing studies in patient-derived induced pluripotent stem cells (iPSCs) and with clinical studies in patients with NHE6 mutations. Finally, this application is strongly in line with the NIH Interagency Autism Coordinating Committee (IACC) Strategic Plan that calls for the identification of molecular targets amenable to interventions.

? DESCRIPTION (provided by applicant): Christianson syndrome (CS) is a recently discovered X-linked neurodevelopmental disorder caused by deleterious mutations in SLC9A6 which encodes the sodium-hydrogen exchanger known as NHE6. This protein, along with 8 other NHE proteins, are members of the family of solute carriers found localized to different membranes in cells where they are thought to influence the pH of luminal areas. NHE6 is associated with early endosomes and recycling endosomes and recent studies of a knockout mouse model have shown defects in growth factor signaling, neurotransmitter receptor cycling as well as in lysosomal function. Patients with CS are clinically recognized by features of intellectual disability, ataxia, epilepsy, minimal verbal status, postnatal microcephaly, and cerebellar degeneration. Autistic features have also been ascribed to some patients. Prior to identification of the CS gene, the presence of a `happy demeanor' typically led to a diagnosis of Angelman syndrome, and thus was referred to as X-linked Angelman-like syndrome. Importantly, males with CS described to date have epilepsy, with seizure types including: infantile spasms, tonic seizures, tonic-clonic seizures, myoclonic seizures, drop seizures, and episodes described as staring spells. Clinical involvement in female carriers of CS has been less closely analyzed to date. While CS is a rare condition, Morrow and colleagues have identified 21 affected families and 26 individuals, which include families and patients from the United States, Canada, and Europe. A Christianson Syndrome Association (CSA) was formed in 2011 by a family in Houston, Texas, and a similar organization has now been established in Canada. The CSA held an initial meeting at Brown University in 2013 (hosted by Dr. Eric Morrow) that brought together families with interested clinicians and scientists. The CSA strongly supports the inclusion of an international scientific conference in conjunction with its 2015 family meeting and the purpose of this proposal is to secure funding to facilitate this event. Planned for this meeting are presentations from a diverse array of well-known speakers and CS experts - national and international, including leaders in the field of neurodevelopmental and seizure disorders. We also anticipate the creation of impactful opportunities for junior investigators, including women and minorities, to participate in scientific exchange and to meet CS patients and their families. Key outcomes expected from this meeting include: (i) networking and establishment of collaborative research and clinical outreach programs; (ii) generation of new ideas on the pathogenesis and possible treatment of CS, including new avenues of research and collaborative grants; (iii) expansion of the CS research and clinical community, including the introduction of junior scientists and clinicians to the importance of studying CS and other related rare diseases; and (iv) establishment of an international CS research and clinical network to foster fully collaborative, multi-laboratory basic research and to encourage initiation of a patien registry and natural history study in order to advance patient care and treatment.

PROJECT SUMMARY No effective therapeutic agents currently exist to improve outcomes in autism and related neurodevelopmental disorders. Genetic research has led to the discovery of a growing list of highly penetrant mutations that contribute to disease pathophysiology. This recent progress provides an important opportunity to define the molecular mechanisms underlying these disorders, as well as to identify targets for new treatment strategies. However, given the large number of emergent loci, a major challenge for the current phase of autism research is to establish convergent cellular mechanisms that group apparently distinct genetic etiologies. Here we focus on the following disease-associated proteins: the E3 ubiquitin ligase UBE3A/E6-AP and Na+/H+ exchangers (NHEs), with an emphasis on NHE6 (also known as SLC9A6). Genetic mutations affecting these proteins are associated with overlapping clinical syndromes. These shared clinical spectra suggest that UBE3A and NHEs may function in a convergent cellular pathway. Exciting preliminary data support a functional interaction between UBE3A and NHE6 in the regulation of intra-organellar pH. Discovery of a shared pathway for UBE3A and NHEs involving intra-organellar pH would be fortuitous because FDA-approved drugs known to target this cellular process are currently available. Therefore, success in this research would lay the foundation for future drug screening in available models of disease, including both patient-derived iPSCs and animal models. Our central hypothesis is that UBE3A controls trafficking and degradation of NHEs, and that perturbations in UBE3A activity (through loss-of-function or gain-of-function mutations) cause abnormalities in the regulation of intra-organellar pH as a result of mislocalization and aberrant accumulation of NHEs. Such abnormalities in intra-organellar pH will disturb organellar functions, including protein processing, trafficking, and signaling, and ultimately disrupt circuit development. We will test this hypothesis and build a path for future research through these Specific Aims: (1) Determine the extent to which UBE3A governs the trafficking and degradation of NHE proteins; and (2) Investigate perturbations in regulation of intra-organellar pH mediated by UBE3A and NHE6. We are conducting innovative, live-cell imaging of intra-organellar pH in neurons, from both patient-derived iPSCs and also animal models, by targeting ratiometric pHluorin to specific organellar compartments. The research is significant because: (1) The discovery of a functional linkage between autism-associated genes serves the important goal of developing more unified pathogenic mechanisms yielding treatment targets; and (2) UBE3A has been a widely studied protein; however, a role in modulation of intra-organellar pH represents a new mechanism with a potential for targeting by therapeutic drugs.

PROJECT SUMMARY Human genetics offers a powerful approach to dissect cellular mechanisms in neurodegenerative disease. We are studying new genetic conditions with neurodegeneration caused by mutations in the X-linked endosomal Na+/H+ exchanger 6 (NHE6, also known as SLC9A6). Dysfunction of the endolysosomal system is a common feature in many neurodegenerative disorders. Loss-of-function mutations in NHE6 in males cause Christianson syndrome (CS), which displays mixed neurodevelopmental and neurodegenerative pathology. My research group has recently discovered adult-onset, neurodegenerative disease in female NHE6 mutation carriers. Data in NHE6-related disease support pathology, including axonal degeneration, cerebellar degeneration, and diffuse tau-related disease. The objective of the research in this R01 proposal is to define the cellular mechanisms that cause NHE6-related neurodegeneration, as well as to develop mechanistic linkages to other related neurodegenerative disorders, including Alzheimer?s disease (AD) and AD-related dementias (ADRD). Our central hypothesis is that loss of NHE6 leads to abnormal maturation of late endosomes, thereby causing aberrant retrograde axonal transport and lysosomal dysfunction. My research group, with our collaborators, is in an excellent position to study NHE6-related neurologic disease, both in males and females, as we have developed unique resources including: an international patient registry with patient phenotypic information; the mouse Nhe6 conditional mutant; a panel of patient-derived iPSC cells with robust controls; and an Nhe6-null rat model. We capitalize on the relative strengths of each experimental model to address our scientific questions. We will pursue the following Specific Aims: 1) Demonstrate that neuronal, cell-autonomous loss of NHE6 function in the mature brain causes neurodegeneration; 2) Determine the mechanism by which loss of NHE6 leads to aberrant endosome maturation, lysosomal function, and retrograde axonal transport; and 3) Determine the extent to which impairments in neuronal connectivity in NHE6-null neurons are mediated by tau- related mechanisms. In these Aims, we study mechanisms in CS, as well as neurodegenerative mechanisms in the female-specific NHE6-related syndrome. This research will have a sustained impact on both fundamental neuronal cell biology and on translational neuroscience. These studies will define the neurodegenerative mechanisms in new genetic diseases in males and females, and will establish linkages with more common neurodegenerative disorders, potentially identifying new therapeutic targets. Additionally, our research uses a powerful integrated translational approach, bridging patient-oriented studies to experimental models. Finally, we are establishing valuable experimental resources for these studies, which we will share broadly in order to maximize their utility for the research community.