In the Howell lab, we apply genetics and genomics approaches to identify fundamental processes involved in the initiation and early propagation of age-related neurodegenerative diseases, focusing on Alzheimer's disease, non-Alzheimer's dementia and glaucoma. Understanding these processes provides the greatest opportunity of therapeutic intervention. We are particularly interested in the role of non-neuronal cells including astrocytes, monocyte-derived cells (such as microglia), endothelial cells and pericytes.
In previous work, I applied novel genomics and bioinformatics strategies to identify new molecular stages of glaucoma that preceded morphological changes. Genetic knockout and/or pharmaceutical approaches showed that targeting the Complement cascade and Endothelin system significantly lessened glaucomatous neurodegeneration in mice. Our work with glaucoma continues in collaboration with Dr. Simon John, and we are also now applying similar genetics and genomics strategies to understand initiating and early stages of Alzheimer's disease, Vascular Dementia and other dementias. A major aim is to combine knowledge from human genetic studies with the strengths of mouse genetics to develop new and improved mouse models for dementias and make them readily available to scientific community.
Identifying critical early events in Alzheimer's disease (AD)
Current mouse models develop AD-related phenotypes such as plaque deposition and glial activation, but the key early events in the pathogenesis of AD have not been studied. Identifying these early processes will provide novel targets to evaluate for human treatments. We are performing gene expression profiling combined with bioinformatics analyses and subsequent functional tests to determine early molecular processes of AD. I first applied this experimental design to glaucoma, where early pathological events were identified and their importance then tested using both gene knockouts and pharmacological intervention.
Bioinformatics approaches are being used to determine genes and biological processes that change early. Gene profiling datasets of this complexity do not currently exist for AD and will greatly facilitate our understanding of molecular changes that occur prior to its traditionally recognized hallmarks. This work is also likely to order processes that are currently known to be involved in AD but whose timing of activation is unclear. Our long-term goal is to determine the beneficial or damaging role(s) that key early events play using genetically engineered mice.
Figure 1: Hierarchical clustering identified molecularly defined stages of glaucoma.
Glaucoma-relevant genes were used to cluster samples into molecular stages based on expression profiles. Dendrogram (to the right) indicates the most similar eyes. Five new stages of disease were identified (ONH stages 1-5). Stages 1-3 contained eyes that could not be distinguished by conventional morphological assessment. For each gene, greens represent the lower normalized intensity values across all samples and reds the higher intensity values. Black horizontal lines indicate borders between stages. The red line indicates the cutoff level for inclusion in a stage.
Investigating cell specific roles of the complement cascade in dementia
The complement cascade has been implicated in human Alzheimer's disease, but the roles it and its constituent molecules play are not well understood. Members of the complement cascade are expressed in multiple cell types in animal models of Alzheimer's disease, and we are developing unique resources to perform a detailed characterization of the components:
The role of the C1 complex: The initiating complex of the classical pathway is expressed in neurons and monocyte-derived cells. The classical pathway has been implicated in synapse remodeling in development and disease, and monocytes are predicted to play both beneficial and damaging roles during the disease process. In collaboration with Beth Stevens (Harvard) we are using a conditional knockout approach to better understand the role of the C1 complex in all aspects of AD.
The role of membrane attack complex (MAC): We are testing the role of the MAC in AD on two genetic backgrounds (C57BL/6J (B6) and DBA/2J (D2)). Preliminary data from my collaborator, Dr. Bruce Lamb, suggests C5 (a controlling molecule of the MAC) modifies AD phenotypes. Also, network analysis shows biological links between the MAC and genes implicated in late-onset AD (Figure 2). D2 mice are naturally deficient for C5 and also likely more susceptible to neurodegeneration than B6 mice. We have generated resources including D2.C5B6 (C5 sufficient) and B6.C5D2 (C5 deficient) mice carrying AD mutations and a conditional knockout of C5 (also know as Hc) that are allowing us to comprehensively assess the role of the MAC in AD.
Figure 2: Network analysis predicts a role of the MAC in AD. Two biological networks, using late-onset AD genes as seeds (red), were constructed using Ingenuity Pathway Analysis (IPA). Network 1 includes genes from the MAC (green). CLU (Clusterin, also known as Apolipoprotein J or Complement cytolysis inhibitor) is known to regulate components of the MAC. APOE is known to modify plaque burden. (See also next section).
Identifying alleles that can modify susceptibility to AD
Genetic interactions have a major impact on disease phenotypes and can be uncovered by varying the genetic background of mice. To date, AD research in mice has used primarily a single strain, C57BL/6J (B6), with a very limited number of other genetic backgrounds assessed. We are employing an unbiased forward genetics approach to identify new genetic modifiers of AD. These experiments will provide new mechanistic insight but also aim to develop improved mouse models for AD.
We are performing an extensive strain survey using mice carrying early-onset AD mutations. We are establishing AD mutations on eight genetically diverse mouse strains that are the founder strains for the Diversity Outcross (DO) and Collaborative Cross (CC) mouse resources. AD phenotypes (behavioral and histological) will be assessed with particular attention given to degrees of neuronal cell loss and behavioral deficits. Current AD mutations (e.g. in APP and PSEN) cause a dominant phenotype and we therefore plan to utilize the DO and CC strains for identification of genes that underlie dominant genetic modification of AD phenotypes. Given the lack of previous studies of AD mutations on multiple genetic backgrounds, this approach may result in the development of mouse models that better model human AD.
Investigating alleles that influence susceptibility to late-onset AD
Variations in at least ten genes potentially confer an increased risk for late-onset AD. These genes have been identified through linkage and genome-wide association studies, but their role in AD has not been proven. In particular, little is known about how combinations of these genetic variants may interact to cause AD. We are assessing whether mutations in these genes can cause AD-like phenotypes in mice. These genes are not likely to act in isolation to cause AD, so I will assess their combined effect on disease phenotypes. This work has the potential to provide the first animal model(s) for late-onset AD, the most common form of AD. Evidence from other disease research predicts that variations in these genes lead to a functional deficiency that increases risk for AD. To model this functional deficiency, we are first assessing AD phenotypes in mice that are haploinsufficient (one functional copy) for combinations of these AD genes. To determine the best combinations of genes to use in this study, we have performed bioinformatics analyses such as gene set enrichment and network analysis (using Ingenuity Pathway Analysis, IPA). Two networks contain seven genes associated with late-onset AD (Figure 2).
Perturbation of genes within one network or key genes in multiple networks may lead to AD. Therefore, we are assessing AD phenotypes in mice carrying mutations in multiple combinations of genes (such as APOE, CLU and CR1—all from network 1; BIN1 and PICALM Ð both from network 2; CLU, APOE and PICALM—from both networks). We are also incorporating late-onset genes not linked in networks (such as Epha1, Bst1 and Cd33). In addition, we are performing experiments with PSEN1 (an early-onset AD gene) as a sensitizer to assess whether mutations in these genes modify AD phenotypes. Some of these alleles are either already available at JAX or will be available as part of KOMP2. We are working closely with human geneticists (e.g. Julie Williams), to introduce human genes carrying variations that have been shown to confer an increased risk of late-onset AD into mice.
Determine cell-specific roles of the complement cascade in glaucoma
Glaucoma affects 70 million people and is a leading cause of blindness worldwide. It is characterized by the loss of retinal ganglion cells (RGCs, the output neurons of the retina). Age and intraocular pressure elevation are major risk factors for glaucoma. Complement expression increases in human glaucoma and in all animal models of glaucoma assessed to date. My work has extended previous studies by showing that induction of the complement cascade occurs very early during glaucoma (in both RGCs and monocyte-derived cells). We found that DBA/2J mice with a targeted null mutation in the gene for complement component 1qa (C1qa) are robustly protected from glaucoma. The robust protection highlights the importance of determining exactly how the complement cascade influences glaucoma. C1QA is expressed in at least two cell types (RGCs and microglia). Complement component C3 is also expressed in astrocytes. I will experimentally dissect the importance of C1Q and C3 from these three cell types using conditional knockouts. Determining the significance of complement biosynthesis by each of these cell types is important for designing therapeutic interventions that manipulate the complement system.
Principal Investigator: Gareth Howell, Ph.D.
Associate Research Scientist: Ileana Soto Reyes, Ph.D.
Postdoctoral Fellow: Kristen Onos, Ph.D.
Research Assistant II: Harriet Williams, B.Sc.
Research Assistant I: Keating Pepper,B.A.
Laboratory Technician IV: Kelly Keezer
Predoctoral Associate: Leah Graham, B.S.
Research Administrative Assistant: Patricia Cherry
Cross SH, Macalinao DG, McKie L, Rose L, Kearney AL, Rainger J, Thaung C, Keighren M, Jadeja S, West K, Kneeland SC, Smith RS, Howell GR, Young F, Robertson M, van T' Hof R, John SW, Jackson IJ. A dominant-negative mutation of mouse Lmx1b causes glaucoma and is semi-lethal via LBD1-mediated dimerisation. PLoS Genet 2014 May 8; 10(5):e1004359. PMCID: PMC4014447
Howell GR, MacNicoll KH, Braine CE, Soto I, Macalinao DG, Sousa GL, John SWM. 2014. Combinatorial targeting of early pathways profoundly inhibits neurodegeneration in a mouse model of glaucoma. Neurobiol Dis. 71:44-52.
Soto I, Howell GR. 2014. The Complex Role of Neuroinflammation in Glaucoma. Cold Spring Harb Perspect Med 4(8)pii: a017269. doi: 10.1101/cshperspect.a017269.
Soto I, Howell GR, John CW, Kief JL, Libby RT, John SWM. 2014. DBA/2J Mice Are Susceptible to Diabetic Nephropathy and Diabetic Exacerbation of IOP Elevation. PLoS ONE 9(9):e107291 PMCID:PMC41602422013
Jackson HM, Soto I, Graham LC, Carter GW, Howell GR. 2013. Clustering of transcriptional profiles identifies changes to insulin signaling as an early event in a mouse model of Alzheimer's disease. BMC Genomic Nov 25;14(1):831. PMCID: PMC3907022
Howell GR, Libby RT. 2013. Adding metabolomics to the toolbox for studying retinal disease. Invest Ophthalmol Vis Sci 54(6):4260. Commentary
Howell GR, Soto I, Ryan M, Graham LC, Smith R, John SWM. 2013. Deficiency of complement component 5 ameliorates glaucoma in DBA/2J mice. J Neuroinflammation 10(1):76. PMCID: PMC3708765
Wiggs JL, Howell GR, Linkroum K, Abdrabou W, Hodges E, Braine CE, Pasquale LR, Hannon GJ, Haines JL, John SWM. 2013. Variations in COL15A1 influence age of onset of primary open angle glaucoma. Clin Genet 84(2):167-174. PMCID: PMC3771394
Williams PA, Howell GR, Barbay JM, Braine CE, Sousa GL, John SWM, Morgan JE. 2013. Retinal ganglion cell dendritic atrophy in DBA/2J glaucoma. PLoS One 8(8):e72282. PMCID: PMC3747092.
Howell GR, Soto I, Zhu X, Ryan M, Macalinao DG, Sousa GL, Caddle LB, Harmon K, Barbay JM, Porciatti V, Anderson MG, Smith RS, Clark AF, Libby RT, John SWM. 2012. Radiation treatment inhibits monocyte entry into the optic nerve head and prevents glaucoma in DBA/2J mice. J Clin Invest 122(4):1246-1261. PMCID: PMC3314470
Reinholdt LG*, Howell GR*, Czechanski AM, Macalinao DG, MacNicoll KH, Lin C-S, Donahue LR, John SWM. 2012. Generating embryonic stem cells from the inbred mouse strain DBA/2J, a model of glaucoma and other complex diseases. Plos One 7(11):e50081 PMCID: PMC3507949 *Authors contributed equally. PMCID: PMC3507949
Simon MM, Mallon AM, Howell GR, Reinholdt LG. 2012. High throughput sequencing approaches to mutation discovery in the mouse. Mamm Genome 23(9-10):499-513. PMCID: PMC3724459
Barabas P, Huang W, Chen H, Koehler CL, Howell G, John SWM, Tian N, Renter RC, Krista D. 2011. Missing opt motor head turning reflex in the DBA/2J mouse. IOVS 52(9):6766-6773. PMCID: PMC3175994
Howell GR, Macalinao DR, Sousa G, Walden M, Soto I, Kneeland S, Barbay J, King BL, Marchant JK, Hibbs M, Stevens B, Barres BA, Clark AF, Libby RT, John SWM. 2011. Molecular clustering identifies complement and endothelin induction as early events in a mouse model of glaucoma. 121(4):1429-44. J Clin Invest 121(4):1429-1444. PMCID: PMC3069778
Howell GR, Walton D, King BJ, Libby RT, John SWM. 2011. Datgan: a reusable software system for visualizing transcriptome profiling data. BMC Genomics 14:429. PMCID: PMC3171729
Lachke SA, Alkuraya FS, Kneeland SC, Ohn T, Aboukhalil A, Howell GR, Saadi I, Cavallesco R, Yue Y, Tsai A, Nai S, Cosma MI, Smith RS, Hodges E, Alfadhli SM, Al-Hajeri A, Shamseldin H, Behbehani A, Hannon GJ, Bulyk ML, Drack AV, Anderson P, John SWM, Maas RL. 2011. Mutations in the RNA granule component TDRD7 cause cataract, glaucoma and male sterility. Science 331(6024):1571-1576. PMCID: PMC3279122
Nair KS, Hmani-Aifa M, Ali Z, Kearney A, Salem SB, Macalinao DG, Cosma IM, Bouassida W, Hakim B, Benzina Z, Soto I, Söderkvist P, Howell GR, Smith RS, Ayadi H, John SWM. 2011. Alteration of the serine protease PRSS56 causes angle-closure glaucoma in mice and posterior microphthalmia in humans and mice. Nat Genet 43(6):579-584. PMCID: Letter
Howell GR, Libby RT, John SW. 2008. Mouse genetic models: an ideal system for understanding glaucomatous neurodegeneration and neuroprotection. Prog Brain Res 173:303-321.
Howell GR, Libby RT, Jakobs TC, Smith RS, Phalan FC, Barter JW, Barbay JM, Marchant JK, Mahesh N, Porciatti V, Whitmore AV, Masland RH, John SW. 2007. Axons of retinal ganglion cells are insulted in the optic nerve early in DBA/2J glaucoma. J Cell Biol. 179:1523-37. PMCID: PMC2373494
Howell GR, Libby RT, Marchant JK, Wilson LA, Cosma IM, Smith RS, Anderson MG, John SW. 2007. Absence of glaucoma in DBA/2J mice homozygous for wild-type versions of Gpnmb and Tyrp1. BMC Genet 3:45. PMCID: PMC1937007
Howell GR, Shindo M, Murray S, Gridley T, Wilson LA, Schimenti JC. 2007. Mutation of a ubiquitously expressed mouse transmembrane protein (Tapt1) causes specific skeletal homeotic transformations. Genetics 175(2):699-707. PMCID: PMC1800629
Libby RT, Howell GR, Pang IH, Savinova OV, Mehalow AK, Barter JW, Smith RS, Clark AF, John SW. 2007. Inducible nitric oxide synthase, Nos2, does not mediate optic neuropathy and retinopathy in the DBA/2J glaucoma model. BMC Neurosci 8:108. PMCID: PMC2211487
Stevens B, Allen NJ, Vazquez LE, Howell GR, Christopherson KS, Nouri N, Micheva KD, Mehalow A, Huberman AD, Stafford B, Sher A, Litke AM, Lambris JD, Smith SJ, John SWM, Barres BA. 2007. The classical complement cascade mediated CNS synapse elimination. Cell 131:1164-1178.
From 1997 to 2006
Das RM, Van Hateren NJ, Howell GR, Farrell ER et al. 2006. A robust system for RNA interference in the chicken using a modified microRNA operon. Dev Biol 294:554-563.
Howell GR, Bergstrom R, Munroe R, Masse J and Schimenti JC. 2005. Identification of a cryptic lethal mutation in the mouse tw73 haplotype. Genet Res 84(3):153-59.
Howell GR, Munroe RJ and Schimenti JC. 2005. Transgenic rescue of the mouse t complex haplolethal locus Thl1. Mamm Genome 16(11):838-46.
Ross MT, Grafham DV, Scherer S, Coffey J, McLay K, Muzny D, Platzer M, Howell GR, Burrows C et al. 2005. The DNA sequence of the human X chromosome. Nature 434(7031):325-37.
Wilson L, Ching Y-C, Farias M, Hartford S, Howell GR, Shoa H, Bucan M and Schimenti JC. 2005. A region-specific ENU mutagenesis screen on mouse chromosome 5. Genome Res 15(8):1095-105.
Woodward KJ, Cundall M, Sperle K, Sistermans EA, Ross M, Howell GR, et al. 2005. Heterogeneous duplications in patients with Pelizaeus-Merzbacher disease suggest a mechanism of coupled homologous and nonhomologous recombination. Am J Hum Genet 77(6):966-87.
Mungall AJ et al. 2003. The DNA sequence and analysis of human chromosome 6. Nature 425:805-811.
McMullen TW, Crolla JA, Gregory SG, Carter NP, Cooper RA, Howell GR, Robinson DO. 2002. A candidate gene for congenital bilateral isolated ptosis identified by molecular analysis of a de novo balanced translocation. Human Genetics 110(3):244-250.
Stephan D, Howell GR, Teslovich TM, Coffey AJ, et al. 2002. Physical and transcript map of the hereditary prostate cancer region at Xq27. Genomics 79(1):41-50.
Bentley DB, et al. 2001. The physical maps for sequencing human chromosomes 1, 6, 9, 10, 13, 20 and X. Nature 409(6822):942-3.
Blanco P, Sargent C, Boucher C, Howell G, Ross M, Affara N . 2001. (2001). A novel poly(A)-binding protein gene (PABPC5) maps to an X-specific subinterval in the Xq21.3/YP11.2 homology block of the human sex chromosomes. Genomics 74:1-11.
Braybrook C, Warry G, Howell G, Arnason A, Bjornsson A, Moore G, Ross M, Stanier P. 2001. Identification and characterization of KLHL4, a novel human homologue of the Drosophila Kelch gene that maps within the X-linked cleft palate and Ankyloglossia (CPX) critical region. Genomics 72:128-136.
Carrie A, Jun L, Bienvenu T, Vinet MC, McDonell N, Couvert P, Zemni R, Cardona A, Van Buggenhout G, Frints S, Hamel B, Moraine C, Ropers HH, Strom T, Howell GR, et al. 1999. A new member of the IL-1 receptor family highly expressed in hippocampus and involved in X-linked mental retardation. Nat Genet 23(1):25-31.
Steingruber HE, Dunham A, Coffey AJ, Clegg SM, Howell GR, Maslen GL, Scott CE, Gwilliam R, Hunt PJ, Sotheran EC, Huckle EJ, Hunt SE, Dhami P, Soderlund C, Leversha MA, Bentley DR, Ross MT. 1999. High-resolution landmark framework for the sequence-ready mapping of Xq23-q26.1. Genome Res 9(8):751-762. PMCID: PMC310799
Coffey AJ, Brooksbank RA, Brandau O, Oohashi T, Howell GR, et al. 1998. Host responses to EBV infection in X-linked lymphoproliferative results from mutations in an SH2-domain encoding gene. Nat Genet 20(2):129-35.
Gregory SG, Howell GR, Bentley DR. 1997. Genome mapping by fluorescent fingerprinting. Genome Res 79(12):1162-68.
Walpole SM, Nicolaou A, Howell GR, Bentley DR, Ross MT, Yates JR, Trump D. 1997. High-resolution physical map of the X-linked retinoschisis interval in Xp22. Genomics 44(3):300-08.
Books Chapters and Reviews:
Howell GR, Soto I, Libby RT, John SWM. 2012. Intrinsic axonal degeneration pathways are critical for glaucomatous damage. Exp NeurolNickells RW, Howell GR, Soto I, John SWM. 2012. Under pressure: cellular and molecular responses in glaucoma. Annual Reviews of Neuroscience 35:153-179
Howell GR, John SWM. 2010. Genetic and genomic approaches for understanding retinal diseases. Animal models of retinal diseases. Clark AF and Pang I-H (Eds). Springer Protocols.
Howell GR, Marchant JK and John SWM. 2008. Mouse Models: A Key system for revolutionizing understanding of glaucoma. Eye, Retina and Visual System of the Mouse. Chalupa LM and Williams RW (Eds). MIT Press.