We are committed to advancing genetic discoveries in stroke towards novel biology and advanced therapeutics.  A new genetic association in the COL4A2 gene has been discovered in association with ICH, but the way that this genetic region changes cellular biology is not clear.  Yet we know this gene is very important, because rare diseases associated with mutations in COL4A2 result in ICH at a very young age.  By applying bioinformatic techniques to genome sequencing data, exploration of regulatory gene regions using high-throughput reporter assays, and genome editing using CRISPR-Cas9 methodologies, we are interrogating the COL4A2 locus to help understand how genetic variants in the region raise the risk of ICH.

We are actively working with collaborators, both internally and from other institutions around the world, to conduct genetic studies to better define the molecular basis of the NCL disorders. Although the majority of NCL genes are now identified, enabling a molecular diagnosis in >90% of cases, some patients do not harbor mutations in the known NCL genes, and interpretation of genetic data even in known genes remains a challenge. We are also using our genetic model systems to screen for and test candidate disease modifiers in preclinical studies using our genetic disease models.

The most recent genetic advances in the field have highlighted an increasingly complex genetic and clinicopathologic picture of the NCL disorders and one that shows significant overlap with other neurodegenerative diseases. We are interested in better understanding these complex genotype-phenotype relationships and the pathway crosstalk across neurodegenerative diseases, and we are using our disease models and other genetic tools to accomplish this.

We study the predictors of depression and the cognitive, social, and biological pathways through which experience can shape risk for depression.

Physiological and functional characterization of variants in the SLC16A11 gene

We are leading an international clinical trial that is performing ex vivo lentiviral gene therapy in childhood cerebral adrenoleukodystrophy (NCT01896102). We were also the first to show the ability of an adeno-associated virus to target the intracellular peroxisomal membrane and lower very long chain fatty acids. This was critical proof of concept to justify developing an in vivo gene therapy for adrenomyeloneuropathy, the most common metabolic hereditary spastic paraplegia.

Using both imaging tools and post mortem pathological analysis, our group has contributed to the understanding of disease progression and the pathophysiology of adrenoleukodystrophy. We have defined microglial dysfunction in the disease as distinct from that of other demyelinating disorders. More recently, the lab has discovered that the disease-causing gene ABCD1 has a specific role in maintaining barrier function in brain microvascular endothelial cells.

Understanding the role of lysosomal channel TRPML1 in iron homeostasis and oligodendrocyte function.

Understanding the pro-inflammatory signaling in the TRPML1 deficiency and modulation of neuroinflammation to treat mucolipidosis IV.

Role of Neuroepigenetic Mechanisms in Cognitive & Mood Disorders. A major focus of the Haggarty Laboratory has been to advance understanding of the critical function of epigenetic mechanisms in the regulation of neuroplasticity and neurocircuits involved in memory and mood regulation. Using a combination of chemical genomics, biochemistry, functional genomics, and in vivo mouse models we helped elucidated a key role for Class I histone deacetylases (HDACs), in particular HDAC2 complexes, in the regulation of learning and memory and the dysregulation of these epigenetic mechanisms in the context of Alzheimer’s disease (Kilgore et al. Neuropsychopharmacology 2009; Guan et al. Nature, 2009; Fass et al. ACS Medicinal Chemistry Letters, 2012; Fass et al. Neuropharmacology, 2013; Graff et al. Nature, 2014; Wagner et al. Chemical Science, 2015; Gosh et al. Bioorganic & Medicinal Chemistry Letters, 2016). We also demonstrated, for the first time, that selective pharmacological inhibition of Class I HDAC1/2 complexes is sufficient to have anti-manic-like and anti-depressant-like activities in pre-clinical rodent models of depression and bipolar disorder (Covington et al. Journal of Neuroscience, 2009; Schroeder et al. PloS ONE, 2013). Most recently, with our colleague Dr. Ralph Mazitschek at MGH we co-developed an innovative technology of “optoepigenetic” probes that allow the precise spatial and temporal control of the epigenome and transcription with light (Reis et al. Nature Chemical Biology, 2016). We are applying these and the other neuroepigenetic probes we have developed to target rare neurogenetic disorders with cognitive deficits and dysregulated neuroplasticity.

Modeling Pathogenesis & Treatment of Neuropsychiatric & Neurodegenerative Disorders Using Patient-Specific Stem Cells. To advance our understanding of human disease biology and a platform for discovery of novel therapeutic targets, the Haggarty Laboratory has been a pioneer in the application of reprogramming technology to the generation and characterization of patient-specific iPSCs models from individuals with a range of single gene, Mendelian neuropsychiatric disorders, including Fragile X syndrome due to FMR1 mutations (Sheridan et al. PloS One, 2011), Rett syndrome due to MECP2 mutations (manuscript under review), Pitt-Hopkins syndrome (manuscript under review), Frontotemporal dementia (FTD) due to MAPT/tau mutations (Silva et al. Stem Cell Reports, 2016) and GRN/progranulin mutations (manuscript under review), as well as complex, polygenic disorders, including bipolar disorder and schizophrenia (Rueckert et al. Molecular Psychiatry, 2013; Madison et al. Molecular Psychiatry, 2015; Bavamian et al. Molecular Psychiatry, 2015). As part of these efforts, we generated the first ever iPSC models of MGH patients within both the Departments of Psychiatry (bipolar disorder subjects) and Neurology (FTD subjects). Collectively, these studies have begun to reveal fundamental insight into the underlying mechanisms of human disease neurobiology. Furthermore, through our advancement of robust strategies for large-scale generation of highly expandable neural progenitors cells (NPCs) with the ability for rapid directed neural differentiation to disease relevant subtypes these studies have created new avenues for the discovery of novel targets and lead compounds using high-throughput drug screening, functional genomics (shRNA/CRISPR-Cas9), and quantitative proteomics (Zhao et al. Journal of Biomolecular Screening, 2012; Silva et al. Stem Cell Reports, 2016; Cheng et al. Current Protocols, 2016; Silva et al. Current Medicinal Chemistry, 2016), which we are applying to systematically discover next-generation neurotherapeutics.

Disease signatures for schizophrenia and bipolar disorder using patient iPSC-derived neurons

Modulating synaptogenesis with HDAC6 inhibitors through acetylation of non-histone proteins

Gene expression-based screen to identify novel therapeutic leads for Parkinson’s Disease

We are leading an international clinical trial that is testing the Sur1 inhibitor glyburide for the prevention of brain edema after large hemispheric stroke. Following the successful completion of phase 2a and 2b studies, we currently co-lead the CHARM study, a phase 3 randomized, placebo-controlled, double-blind trial testing the clinical efficacy of intravenous glyburide for the prevention of brain edema after large hemispheric stroke (gov Identifier: NCT02864953).

Our group develops and evaluates neuroimaging and proteomic biomarkers that guide prognosis and treatment decisions after acute brain injury. A major focus is to characterize which patients are prone to edema formation and its relationship to subsequent neurological recovery. Having established tools to non-invasively characterize brain edema in patients, we identify novel pathways that are markers for and contribute to brain edema formation. Our analyses suggest that the sterile, innate inflammatory response to ischemia drives edema formation and contributes to worse outcome.

Systems genetics: Huntington’s disease allelic series

Computational and experimental genomic strategies to define the function of all mitochondrial proteins. We built a comprehensive catalog of mitochondrial proteins (A) and developed genomic algorithms to predict function of unstudied genes using shared evolutionary history (www.gene-clime.org) (B) and shared co-expression across many conditions (www.gene-clic.org) (C).

Using both post mortem pathological analysis and in-vitro modeling of the human blood brain barrier, our group has contributed to the understanding of disease progression and the pathophysiology of adrenoleukodystrophy. We have discovered that the disease-causing geneABCD1 has a specific role in maintaining barrier function in brain microvascular endothelial cells.

We have been pursuing the molecular mechanisms by which genetic variation in atrial natriuretic peptide and the natriuretic peptide receptor influences blood pressure and risk of hypertension through cellular models.

We are developing methods for modeling microglia-mediated synaptic pruning in vitro, and are using them to understand pruning abnormalities in schizophrenia and to identify novel treatments.

We are developing new tools for visualizing risk in clinical populations, allowing clinicians to make better use of emerging clinical and genomic predictors.

Bi-allelic inactivating mutations in the NF2 tumor suppressor gene leads to CNS tumors such as schwannomas and meningiomas in NF2 patients and in a majority of sporadic cases. Our studies employing NF2/sporadic patient-derived tumor cells established that the NF2 protein (NF2/merlin) plays a role in mTOR signaling (James et al., Mol. Cell. Biol. 2009; James et al., Mol. Can. Res. 2012; Beauchamp et al., Oncotarget 2015). The NF2 protein negatively regulates various signaling pathways including mTORC1 and mTORC2, and meningiomas and schwannomas with NF2 loss display abnormal activation of these pathways. Rapamycin is a selective inhibitor of mTORC1, and AZD2014 is a dual inhibitor that blocks mTORC1 and mTORC2 (Fig. 1).

Our current projects focus on the mechanistic aspects of mTOR signaling activation upon NF2 loss, identification of other kinases and pathway targets in NF2-deficent human cells and addressing the crosstalk among various pathways dysregulated in NF2-associated tumor cells.

Tuberous sclerosis complex (TSC) is a multisystem genetic syndrome caused by mutations in TSC1 or TSC2, with increased prevalence of epileptic seizures, autism spectrum disorders (ASD) and intellectual disability (ID). Analyses of brain pathophysiology in TSC can lead to a better understanding of this neurodevelopmental disorder and provide clues for the shared molecular mechanisms between TSC and related ASD and ID. Although successful studies in animal models and cellular models of TSC have led to clinical trials with rapamycin analogs targeting mTORC1, progress toward fully comprehending TSC pathophysiology, along with development of more effective disease-modifying therapeutics, has been hampered by challenges in establishing disease-relevant human cell models to support cell-dependent studies and drug screens. To overcome these limitations, we take advantage of recent advances in reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) and in genome editing technologies that provide a powerful strategy to establish isogenic, TSC patient-specific iPSCs that differ exclusively at the disease-causing genetic alteration. Such isogenic iPSCs can be differentiated into CNS neural progenitor cells (NPCs) and neurons (Fig. 4) as well as other non-CNS cell types, thereby providing novel avenues to investigate the pathological basis of TSC and discover new treatment targets.

Charting a course from gene discovery to translation: type 2 diabetes-associated melatonin receptor variation. We collaborate with leading chronobiologists and nutritionists on genotype-targeted physiologic studies, in-vitro human tissue studies, and gene x environment studies in shift-workers (SHIFT) and late-night eaters (ONTIME) to investigate mechanisms linking food timing, circadian rhythms and melatonin signaling to glucose metabolism and to evaluate possible interventions.

Schematic model of TS polygenic risk where disease arises from a cumulative burden of hundreds of small effect-size genetic risk variants (identified by genome-wide association studies (GWAS)), and symptoms manifest when a threshold of risk is surpassed.  Under this hypothesis, the same genetic risk factors may contribute to each of the developmental tic disorders – transient (provisional) tic disorder, chronic tic disorder, and TS, with a higher burden of risk causing more severe and/or persistent disease.  Our lab uses computational biology methods to capture aggregated TS polygenic risk and to test how this genomic risk may be used to predict disease severity, risk of co-occurring neuropsychiatric disorders and outcome in adulthood.  In parallel, these methods provide us with a “TS genomic fingerprint” which can be used to probe gene expression (transcriptomic) and gene regulation (epigenomic) datasets to identify the cell types, brain regions and neurodevelopmental time points where TS risk genes act to cause disease.

Our overarching goal is to understand the impact of polyQ expansion on full-length huntingtin and to beneficially modify mutant huntingtin’s structure and functional activities. As summarized below for current projects, we have investigated the pathogenic mechanism of HD guided by human genetic study by developing biochemical cellular assays and reagents. Through the achievement and experience of HD research, our expertise has contributed to other genetic diseases through closed collaborations in CGM colleagues.

Mucolipidosis Type IV is caused by mutation of the mucolipin-1 lysosomal membrane channel. This is a devastating disease that causes severe psychomotor and mental retardation in infants, and progressive retinal disease leads to blindness by the time patients are in their early teens.  Our goal is to develop gene therapy for both the retinal and CNS dysfunction.

Mitral valve prolapse (MVP) is a common cardiac valve disease that affects nearly 1 in 40 individuals. It can manifest as mitral regurgitation and is the leading indication for mitral valve surgery. Despite a clear heritable component, the genetic etiology leading to non-syndromic MVP has remained elusive. We used a large multigenerational family segregating non-syndromic MVP to identify a novel missense mutation in the DCHS1 gene, the human homologue of the Drosophila cell polarity gene dachsous (ds)

Brain genomics: the neural and genetic architecture of mental illness

In order to study the response to the antidiabetic drug metformin we use the power of gene discovery in C. elegans to illuminate genetic pathways necessary for the drug to act. We then bring discoveries to mammalian models of metformin action. We are collaborating with human geneticists to interface genetic pathways with understanding of the human genetic determinants of metformin action. We recently discovered an ancient pathway conserved from worm to human by which metformin extends lifespan and blocks cancer growth (Wu et al., Cell 2016, In Press.)

A variety of experiments in the laboratory have demonstrated how loss of the TLE genes leads to hematopoietic cell proliferation and cooperates with oncogenes to cause leukemia.

Conditional knockout mice for Tle1 and Tle4 were created to better understand the role of these genes in normal developmental and cancer.

Neural and Non-Neural Contributions to Spinal Muscular Atrophy: A Human Tissue Study. Dr. Swoboda’s Lab Research Flow Chart. This illustration was created using templates from Servier Medical Art (https://smart.servier.com) licensed under a Creative Commons Attribution 3.0 Unported License

Functional genomic modeling of reciprocal genomic disorders

We have conducted a genetic screen using a Drosophila model of NF1 to look for dominant modifiers of the dNf1 pupal size defect. Initially we screened deficiencies on the 1st and 2nd chromosome that uncover 80% of annotated genes. We subsequently identified responsible genes in crosses with mutant alleles or by tissue-specific RNA interference (RNAi) knockdown.

We are using two complementary mass-spectrometry-based approaches to define (i) the proteome and phosphoproteome and (ii) the activated kinome signature of NF1-deficient Schwann cells (SCs) derived from plexiform neurofibromas from patients with NF1. Together these data sets will provide a detailed view of the cellular signaling networks and the molecular targets controlled by neurofibromin in a cell-type relevant to the disease. Small molecule inhibitors to both known and novel signaling pathways will be used to examine the adaptive kinome in treated cell lines.

Patient mutations causing NF1 have been identified throughout NF1­ gene – not just within the segment known to control Ras. We hypothesize that investigating these mutations may give a clue to the diverse clinical symptoms and the variable nature of NF1. We are using CRISPR/Cas9 gene editing to generate selected mutations in human induced pluripotent stem cells (iPSCs). These iPSCs will be differentiated into neurons to examine neuronal morphology, proliferation, signaling and electrophysiological properties to look for correlates with specific mutations in different regions of NF1.

Effective disease-modifying therapies for HD will require an understanding of the earliest cellular and molecular changes that occur as a result of expression of the mutant huntingtin gene, as these are most likely to drive, rather than be a response to pathogenesis. To uncover these alterations we are using precise genetic knock-in mouse models of HD that provide a window into early, pre-manifest HD. This forms the foundation for uncovering underlying mechanisms using genetic modifier approaches as well as defining endpoints for pre-clinical testing.

The HD CAG repeat is highly unstable both in the germline and in somatic tissues where it progressively lengthens over time. We are using a variety of complementary methodologies to uncover the mechanisms underlying CAG instability, including genetic and pharmacological approaches in HD mice and in human iPSC-based models.

Improving the targeting range and activities of CRISPR genome editing technologies. A few major limitations of genome editing enzymes include: 1) the inability to target sequences of interest, or 2) suboptimal/inadequate activity. We have engineered naturally occurring CRISPR nucleases to enable them to target more frequently and to access previously untargetable sequences in the genome. These engineered enzymes have implications for various genome editing applications including gene knock-out and knock-in, epigenome editing, base editing, and allele specific discrimination.

Defining and minimizing the off-target effects of CRISPR nucleases. Genome editing technologies can exhibit the propensity to target DNA sequences that resemble the intended site. The recognition and cleavage of ‘off-target’ sites can lead to undesirable consequences for research applications, and can have major implications for the therapeutic use of genome editing technologies. To prevent off-target cutting, we have ongoing efforts to develop nucleases with vastly improved genome-wide specificities.

Characterizing and developing alternative technologies with enhanced properties. Beyond the prototypical commonly used SpCas9 nuclease, there is a tremendous variety of enzymes that have distinctive properties. The diverse characteristics of these alternative platforms may therefore be advantageous for biomedical research or potentially for the treatment of disease.

Identify new pathways or biomarkers for a given disease, through molecular profiling of asymptomatic people at the extremes of a polygenic score distribution