Michael Anderson, PhD - US grants

The broad objective of the proposed research program is to investigate the role of inhibitory control mechanisms in causing both incidental and intentional forgetting in long-term memory. The importance of inhibitory processes in forgetting has been debated in psychology since the days of classical interference theory. Some investigators have attributed forgetting to passive processes such as response competition, contextual shifts, and decay. Others have emphasized active processes related to learning and attention, such as unlearning, response set suppression, and directed forgetting. In the past decade, I have developed a method that isolates the role of inhibition to forgetting. In the proposed studies, we will use this method to examine two issues: (1) the conditions under which memories might recover from suppression, and (2) the link between retrieval- inhibition and attentional control. It is suggested that, consistent with neuropsychological theories of prefrontal cortex, an attentional gating mechanism is recruited during retrieval to overcome distraction from interfering memories, and that this mechanism causes reversible suppression that underlies retrieval failures. Despite the proliferation of cognitive and neuropsychological theories asserting the importance of inhibition in achieving attentional control, the role of inhibition in intentional forgetting remains controversial. Work on directed forgetting and thought suppression offer conflicting conclusions about people's ability to exert mental control. As a third issue, an attempt will be made to relate cognitive work on inhibitory control in incidental forgetting to the strategic-use of such processes in achieving voluntary control over access to unwanted memories. A new laboratory model for studying voluntary forgetting has been developed and its relation to retrieval-based suppression will be studied. The proposed project offers an opportunity to bridge research on memory and attention with neurological theories of inhibitory control as well as with issues of clinical significance.

DESCRIPTION (provided by applicant): Melanosomes are a specialized organelle of melanocytes that exist in order to conduct a highly specialized task, producing and storing melanin pigment. Melanin acts as a protective agent against the damaging effects of UV radiation. Thus, the presence of melanin in human skin normally contributes to a key element promoting health. Unfortunately, melanosomes are also associated with disease causing potential. Of key concern, many of the intermediates of melanin synthesis are highly reactive quinones and capable of causing significant oxidative damage. Aberrant oxidative stress associated with pigment production has specifically been implicated in several skin diseases, including cutaneous autoimmune disease and malignant melanoma. Despite this importance, the biological processes and ultrastructural components contributing to the ability of melanosomes to contain or detoxify these compounds are largely unknown. Here, we present our preliminary experiments identifying a new melanosomal protein, GPNMB, which we hypothesize participates in the ability of melanosomes to contain or detoxify the potentially cytotoxic intermediates of melanin synthesis. We will test our hypothesis using a combination of genetic epistasis experiments that take advantage of existing Gpnmb mutant mouse strains and synchrotron based imaging techniques that utilize scanning transmission X-ray microscopy. Completion of these experiments will identify a function of the GPNMB protein in melanosomes of the skin, and begin to clarify the role of this protein in health and diseases of pigmentation. Melanosomes are a specialized organelle of melanocytes that in health produce and store melanin pigment and in disease contribute to a variety of skin diseases involving melanocytes. The goal of this research is to initiate functional tests of the newly identified melanosomal protein, GPNMB, utilizing a combination of genetic and innovative imaging approaches. Completion of these studies will yield important gains concerning the melanosomal functions of GPNMB and create needed resources that will facilitate growth of this area of research.

DESCRIPTION (provided by applicant): Pseudoexfoliation syndrome is a common age-related disease of worldwide significance and is the most commonly identified specific cause of open-angle glaucoma. The disease initiating mechanisms of pseudoexfoliation syndrome are almost completely unknown. Currently, all therapeutic strategies for pseudoexfoliative glaucoma aim to lower IOP and there are no specific therapies aimed at treating pseudoexfoliation syndrome itself. With increased knowledge of the initiating mechanisms, it should be possible to devise improved therapeutic strategies that specifically target pseudoexfoliation syndrome itself, promoting earlier interventions and improved medical outcomes. Our long-term goal is to contribute to the development of improved human glaucoma therapies by utilizing synergistic genetic approaches with mice and humans. Here, we take advantage of a phenotype-driven screening approach among mouse coat color variants that has identified a new mouse model of eye disease that strongly resembles aspects of pseudoexfoliation syndrome. Our objective in this proposal is to capitalize on this resource by initiating mechanistic studies and completing a phenotypic characterization of the strain. Using genetic approaches in mice, we are testing the hypothesis that susceptibility of the eye toward PEX syndrome is mediated via a mechanism influencing cellular morphology and oxidative stress associated with melanogenesis. Suspecting that the same mechanism likely underlies human PEX syndrome, we are simultaneously conducting human genetic association studies. Completion of these studies will not only identify PEX syndrome-related genetic pathways, but will also develop an animal model needed for development and testing of future therapeutic strategies. In the long-term, these experiments will contribute to a better understanding of glaucoma, and ultimately, to improved human therapies. Pseudoexfoliation syndrome is a common age-related disease of worldwide significance and is the most commonly identified specific cause of open-angle glaucoma. Here, we take advantage of a newly identified mouse model that strongly resembles aspects of pseudoexfoliation syndrome. Our objective in this proposal is to test the genetic pathways contributing to phenotypes of this mouse strain and test the significance of these genes among human pseudoexfoliation patients.

SUMMARY: Glaucoma is a leading cause of irreversible blindness and visual disability that has a major impact on the quality of life and productivity of millions of Americans. With no new pharmaceutical classes for treating glaucoma introduced into clinical practice since the 1990s, there remains a continuing need for improved regimes that treat glaucoma more effectively. Our long-term goal is to contribute to the development of these improved therapies by utilizing synergistic genetic approaches with mice and humans. Here, we focus on a sub-type of glaucoma, pigmentary glaucoma, and its major risk factor, pigment dispersion syndrome. Pigment dispersion is an alarmingly common condition characterized by aberrant release and collection of pigment throughout the anterior chamber of the eye. In most people, pigment dispersion causes no significant problems. However, in others, pigment dispersion leads to elevated intraocular pressure and pigmentary glaucoma. The factors initiating pigment dispersion and determining these very different potential outcomes are largely unknown. Our central hypothesis is that dispersed pigment elicits active, modifiable, physiological responses by the trabecular meshwork that are shaped by genetics and that dictate whether or not the insult progresses to secondary glaucoma. Using human genetics, we are studying families affected by pigment dispersion to identify genetic factors causing initiation of pigment dispersion. Using approaches with mice, we have developed an inducible mouse model for studying physiological responses to pigment dispersion and identified genetic suppressors of pigmentary glaucoma for studying potential treatments. Our objective in this proposal is to utilize and build on these resources to study molecular events contributing to pigment dispersion and its conversion to pigmentary glaucoma. To accomplish this, we propose: (SA1) to identify genes linked with pigmentary glaucoma using human genetics, (SA2) to define predictors of ocular responses to pigment dispersion using inducible mouse models, and (SA3) to identify suppressors of pigmentary glaucoma using mouse models.

Abstract Leber congenital amaurosis (LCA) is a group of inherited retinal degenerative diseases characterized by nystagmus and blindness that typically manifest in the first year of life. As with several retinal degenerative diseases, many forms of LCA involve dysfunction of photoreceptor cilia. Mutations in the CEP290 gene are the most common cause of LCA, implicating CEP290 as a major contributor to the disease. CEP290 encodes a large protein proposed to regulate protein transport through the photoreceptor connecting cilium spanning the inner and outer segments. Using a mouse model of CEP290-mediated LCA, the rd16 mouse, we have found that the relative severity of Cep290 phenotypes in mice is highly sensitive to genetic background. Here, we propose experiments using mice that take advantage of this background sensitivity to identify genetic modifiers of Cep290-mediated retinal degeneration. Identification of these modifiers has both basic, and clinical, significance. From a basic biology perspective, studies of genetic modifiers can uncover basic biological functions of CEP290, photoreceptor cilia, and their gestalt contributions to retinal disease in a physiologic context. From a clinical perspective, identification of genetic modifiers offers an opportunity to identify therapeutic surrogates. The premise for our current proposal, its feasibility, and evidence of our ability to conduct quantitative modifier studies of retinal degeneration all stem from a relatively large body of recent work. We have performed large genetic crosses with mice and identified quantitative trait loci modifying retinal disease severity of mice homozygous for the Cep290rd16 mutation. Among regions of the genome identified as particularly important, our current proposal focuses on the Modifier of retinal degeneration quantitative trait locus 1 (Mrdq1) located on mouse chromosome 12. A unique feature of this modifier that has aided our ability to identify its molecular basis is that it shows imprinting?its influence varies according to parent-of-origin. Using physical mapping in combination with a study of retinal expressed genes that are imprinted, we have identified an overt mutation within a previously unstudied microRNA that is highly likely to be the causative mutation. Experiments of this proposal describe the work to stringently confirm that we have identified the precise mutation underlying the Mrdq1 modifier (Specific Aim 1), and begin to study its mechanisms of action through identification of downstream targets of the microRNA present in photoreceptors. At completion, we expect this work to have impact on CEP290-mediated LCA, as well as promote a better understanding of how two understudied phenomena, gene regulation via microRNAs and imprinting, influence retinal disease.

ABSTRACT Glaucoma is the leading cause of irreversible blindness worldwide. Pigmentary glaucoma is caused by a release of iris pigment known as pigment dispersion syndrome. Pigment dispersion syndrome is a common disorder that affects about 1 in 40 (2.5%) Americans. As many as 10 to 20% of those with pigment dispersion syndrome develop high intraocular pressure (ocular hypertension) and a secondary glaucoma - pigmentary glaucoma. Young to middle-aged people are affected by pigmentary glaucoma and consequently the visual disability it causes may lead to decades more loss of function and productivity than late-onset forms of glaucoma. Thus, research to determine the cause of pigmentary glaucoma is an important public concern. Strong evidence indicates that pigmentary glaucoma has a genetic basis. DBA/2J mice are an inbred strain of mice that develop pigmentary glaucoma that is a clearly inherited trait. Large human pedigrees have also been reported, in which pigmentary glaucoma is dominantly inherited. Cases of pigmentary glaucoma are also frequently clustered within families and ethnic groups. These data provide strong evidence for the existence of genes that cause or predispose pigmentary glaucoma. However, no genes that cause pigmentary glaucoma have been identified to date. The largely unknown genetic basis of pigmentary glaucoma has been an obstacle to discovery of the mechanisms that cause this disease and has also hindered development of new more effective therapies for this potentially blinding disease. As a result, there is critical need to identify and study the genes that cause pigmentary glaucoma. Aberrant release of iris pigment is a defining feature of pigmentary glaucoma. Moreover, mutations in genes involved in melanin synthesis and melanosomes (Gpnmb and Typr1) are known to cause pigmentary glaucoma in DBA/2J mice. Based on these observations, we hypothesize that some cases of pigmentary glaucoma are caused by mutations in genes involved in melanin synthesis. We will evaluate our hypothesis by studying whole exome data collected from 5 pigmentary glaucoma pedigrees and a cohort of pigmentary glaucoma patients (n=214) and matched controls (n=354) with the aims described below: AIM 1 PEDIGREE-BASED exome analysis of several large pigmentary glaucoma pedigrees. AIM 2 POPULATION-BASED exome analysis of a cohort of pigmentary glaucoma patients (n=214) and matched controls (n=254) We will validate our human studies with analyses of pigmentary glaucoma in mice using 1) publicly available strains of mice from The Jackson Laboratory and 2) our inducible pigmentary glaucoma mouse model. Identifying the first human pigmentary glaucoma genes will provide new insights into the pathogenesis of this important disease and may advance development of sight-saving therapies.

Exfoliation syndrome is common and may affect 5-10% of Americans and up to 50% in some populations (i.e. Icelanders). As many as 10 to 50% of those with exfoliation syndrome develop high intraocular pressure and a secondary glaucoma - exfoliative glaucoma, which causes blindness and visual disability in millions worldwide. Exfoliative syndrome has a strong genetic basis, but complex inheritance. Genome-wide association studies (GWAS) of exfoliation syndrome have successfully identified 7 genetic factors that increase risk for disease: LOXL1, CACNA1A, POMP, TMEM136, AGPAT1, RBMS3, and SEMA6A. However, an animal model has not yet been built based upon these gene discoveries. There is a critical need for these human GWAS discoveries to be translated into animal models. We previously reported mice, B6-Lystbg-j, with a mutation in the Lyst gene, that have some features of exfoliation syndrome, but produce only tiny amounts of exfoliation material and do not develop glaucoma. We hypothesize that inactivating the 7 exfoliation syndrome risk factor genes using CRISPR/Cas9 methods will generate animal models of exfoliation syndrome and glaucoma. We further hypothesize that introducing these mutations on the B6-Lystbg-j background strain will take advantage of its subtle exfoliation syndrome features and facilitate developing a new line with pronounced features, high pressure and glaucoma. We have created CRISPR reagents to inactivate the seven exfoliation syndrome risk factors and we have begun injecting mouse embryos. We are injecting pools of CRISPR reagents for our target genes in order to get founder mice with one or more inactivated risk factor genes. All mutations are made in B6-Lystbg-j mice. AIM 1A: ASSESS FOUNDER MICE. We will assess the eyes of founder mice (in vivo) that have one or more inactivated exfoliation syndrome risk factor genes for exfoliation material. Those with exfoliative material in their eyes will be aged to 1 year, assessed for high intraocular pressure, and glaucomatous optic neuropathy. AIM 1B: GENERATE MICE WITH MULTIPLE INACTIVATED RISK FACTORS. We will conduct crosses to produce mice with multiple inactivated exfoliation syndrome risk factor genes on a B6-Lystbg-j background: 1) To produce mice with 2 heterozygously inactivated risk factor genes (with extreme individual phenotypes). 2) To produce mice with 2 risk factors (Loxl1, Cacn1a1) heterozygously inactivated (top GWAS hits). AIM 2A DETERMINE EXPRESSION PATTERNS OF RISK FACTOR GENES IN B6-Lystbg-j MICE. We will assess expression of the 7 risk factor genes at the mRNA and protein level in ocular tissues of B6-Lystbg-j mice relevant to exfoliation syndrome pathophysiology (lens, iris, and iridocorneal angle). AIM 2B DETERMINE EXPRESSION PATTERNS OF RISK FACTOR GENES IN OUR CRISPR/Cas9 MICE. INNOVATION. Pooling CRISPR guide RNAs to make mice with inactivated risk factor genes (alone and in combination). Creating the 1st animal model of exfoliation syndrome from GWAS hits as a step towards a cure.

Abstract Glaucoma is a group of age-related neurodegenerative diseases characterized by loss of retinal ganglion cells and their axons. Because RGCs are post-mitotic neurons, their loss is permanent and causes a gradual decline leading toward irreversible blindness. Unfortunately, glaucoma manifests in a way that damages US society more broadly?it disproportionately affects certain racial groups. Glaucoma is a worse problem amongst African Americans than Caucasians. Though socioeconomic factors often contribute to this disparity, a large part is biological and driven by genomic differences between racial groups. A recent breakthrough points to APBB2 as central to glaucoma in African Americans. A large genome-wide association study (GWAS) has found a single nucleotide polymorphism in APBB2 (rs59892895) with genome-wide significance (P=2x10-8; Odds ratio=1.33), and successfully replicated the finding in additional cohorts. identified a variant in the APBB2 gene as an important cause of glaucoma in people of African descent. ABPP2 encodes a cytoplasmic adaptor protein with multiple protein-protein interaction domains. Remarkably, the high-risk allele is present in approximately 21% of African Americans and apparently absent from Caucasians. Our team has contributed to this discovery of APBB2 as a gene of importance to glaucoma in African Americans, and as additionally shown in our Preliminary Data, have developed a hypothesis that overexpression of APBB2 is the disease-causing mechanism. To test this new hypothesis, we recently generated and now have in hand, a mouse model on a pure C57BL/6J genetic background that is overexpressing Apbb2. The strain was created by the University of Iowa Genome Editing Facility and features a full-length mouse Apbb2 (transcript variant 1) under control of a ubiquitous promotor (CAG) and includes a 6His/3XFlag attached to the N-terminus (abbreviated as B6- Tg(Apbb2). The experiments of this proposal utilize this new strain as a central resource to complete the important, but somewhat high risk, test via manipulation that over-expression of Apbb2 promotes glaucoma. In Specific Aim 1, we propose to test the anatomical and physiological consequences over time of Apbb2 overexpression in mice. This Aim will also complete a thorough characterization of the B6-Tg(Apbb2) strain. Specific Aim 2 will study APBB2 from a molecular perspective, identifying retinal APBB2 binding partners that will guide future mechanistic and candidate-driven genetic experiments.