Howard J Worman, M.D. - US grants

The long-term objective of the Physician-Scientist Award is for the principal investigator to prepare for a career as an independent researcher in the biomedical sciences through five years of basic science training. Phase I will consist of intensive training in molecular and cell biology which will be primarily accomplished through work on a research project in the Laboratory of Cell Biology at Rockefeller University. Specifically, the project will be concerned with characterization of protein components of the nuclear pore complex, an organelle involved in the transport of macromolecules between the nucleus and cytoplasm of eukaryotic cells. Pore complex proteins will be purified by monoclonal antibody affinity chromatography with antibodies found to inhibit macromolecule transport across the nuclear envelope of isolated nuclei. Polyclonal antibodies will be raised against these purified proteins and oligonucleotide probes will be synthesized complementary to the determined amino acid sequences of fragments if these proteins. The polyclonal antibodies and oligonucleotide probes will be used to screen cDNA libraries to islolate clones encoding pore complex proteins. The cDNA clones will be sequenced to obtain the entire amino acid sequences of the pore complex proteins. Such information should provide a better understanding of the role of the nuclear pore complex in the selective transport of macromolecules across the nuclear envelope. In Phase II of the Award, research will be conducted in the Department of Medicine at Cornell University Medical College. Methodology learned during Phase I will be used to characterize membrane bound proteins of hepatocytes or intestinal epithelial cells. Such proteins are involved in physiological functions ranging from billirubin metabolism to ion transport, and these proteins may be defective in various disease states. Characterization of membrane bound proteins of hepatic or intestinal cells at the molecular level should provide a better understanding of their functions in normal physiology and possible dysfunction in disease states.

This three-year award will support U.S.-France cooperative research in cell biology between Howard J. Worman of Columbia University and Jean-Claude Courvalin of the Institut Jacques Monod, University of Paris VI. The objective of their research is to study the mechanism of nuclear envelope or membrane reassembly that takes place at the end of cell division. They will focus on two nuclear- specific membrane proteins that have been identified in nuclear envelope reassembly. The U.S. investigator brings to this collaboration expertise in molecular biology. The engineering of the cDNA clones for the expression of the protein will be performed in his laboratory. This is complemented by the French investigator's expertise in cell biology. Most of the biochemical and morphological experiments on the nuclear envelope reassembly will be carried out in his laboratory. This joint effort will advance fundamental understanding of the cell nucleus and the role of these proteins in nuclear envelope reassembly.

DESCRIPTION: (adapted from the investigator's abstract) Proteins of the nuclear envelope are differentially expressed in human cancer and development. Variations in their expression may underlie the phenotypic differences in nuclear morphology, gene expression and mitotic potential that occur in cancer and cell differentiation. Nuclear envelope proteins have clinical utility as tumor rnarkers, especially in the diagnosis of leukemias and small cell lung carcinomas which do not express two proteins of the nuclear lamina called lamin A and lamin C. As nuclear envelope proteins play a prominent role in the disassembly and reassembly of the nucleus that occur during rnitosis, they could be potential targets for therapeutic agents designed to control the rate of cell division. Studies on the nuclear envelope are therefore significant to the pathobiology of cancer. For this reason, the present proposal is designed to examine the functions of human nuclear envelope proteins and the regulation of their genes. In the first specific aim, the interactions of LBR, an integral protein of the inner nuclear membrane, with lamin B 1 and chromatin will be studied to better understand the process of nuclear envelope reassembly at the end of mitosis. Alterations in these interactions, induced by mitosis-specific phosphorylation by p34cdc2 protein kinase and subsequent dephosphorylation, are likely responsible for nuclear envelope breakdown and reassembly. The affects of phosphorylation on these interactions will therefore be examined using cell-free chromatin and protein binding assays. The second specific aim of the proposal will further address the functions of LBR in cell division and nuclear morphology. Antisense methods will be used to inhibit LBR expression and determine how the lack of this protein affects nuclear structure and the potential of cells to divide. The results will provide insights into the function of this inner nuclear membrane protein in vivo. The experiments in the third specific aim will focus on the nuclear lamins, intermediate filament proteins of the nuclear lamina Because lamins A and C, which arise from the same gene by altemative splicing, are differentially expressed in cancer and development, an analysis of the factors that regulate the expression of this gene will be performed. The human gene for lamin B 1, a constitutively expressed lamin protein, will be investigated as a control. The regulatory regions of these genes will be analyzed using cell transfection assays in which the promoters are attached to reporter genes. By inhibiting expression using antisense methods, it will also be determined how lamin A and/or lamin C function in cell differentiation. The results of the proposed studies will provide basic information about the nuclear envelope relevant to the pathobiology of cancer. They will lay the foundation for the development of methods to inhibit cell division and for future studies on the role of nuclear envelope proteins in cell differentiation and cancer.

This study will determine if pts who have previiously failed Rx will have a better chance of achieving complete response after 24 wks. of Rx with interferon alpha-2b plus ribavirin than with interferon alpha-2b alone. It will also detemine if pts. will have a better chance of achieving a sustained response after 48 wks of treatment with ribavirin and interferon alpha-2b than after 24 weeks of treatment with ribavirin and 48 weeks of treatment with interferon alpha-2b.

DESCRIPTION (provided by applicant): Facioscapulohumeral muscular dystrophy (FSHD) is the third most common genetic disease of skeletal muscle, affecting approximately 1 in 20,000 persons. In contrast to most other muscular dystrophies, the molecular mechanisms responsible for FSHD remain unknown. More than 95% of cases of FSHD are associated with a partial deletion in an array of tandemly repeated 3.3 kilobase DNA units (D4Z4 repeat) near the telomeric region of chromosome 4q35; however, a specific genetic abnormality that causes the disease has not been identified. We have characterized a gene called DUX4 within each of the repeat elements at the FSHD locus and have obtained preliminary evidence that the encoded protein is expressed in muscle from affected subjects. The DUX4 protein appears to be localized to the nuclear envelope, the same location as emerin and lamins A and C, which are mutated in Emery-Dreifuss muscular dystrophy. This Exploratory/Developmental [R21] proposal is designed to further evaluate the expression of DUX4 in FSHD and to obtain pilot data on its function. Aim 1 will assess the expression of DUX4 in cells from patients with FSHD. Expression of the protein will be assessed by immuoblotting, immunofluorescence microscopy and mass spectroscopy using myoblasts from patients with FSHD and non-affected controls. In Aim 2, the targeting of the DUX4 protein to the nuclear envelope and its distribution throughout the cell cycle will be examined. Searches for DUX4 binding proteins, including the nuclear envelope proteins lamins and emerin, will be performed. Potential binding of DUX4 will also be investigated. As programmed cell death occurs in FSHD, the ability of DUX4 to induce apotosis will also be evaluated. The results obtained for DUX4 will be compared to DUX1, a non-pathological paralogue expressed in normal muscle cells. The results of this Exploratory/Developmental [R21] project will establish if abnormal expression of DUX4 occurs in FSHD and provide the prerequisite pilot data and direction for future studies on its role in disease pathogenesis. This work could lead to the development of new diagnostic methods as well as the identification of potential protein targets for the treatment of FSHD.

DESCRIPTION (provided by applicant): Mutations in the LMNA gene that encodes nuclear lamins A and C have been shown to cause Dunnigan-type partial lipodystrophy, an autosomal dominant inherited disease characterized by regional fat loss and insulin resistance. This finding implicates lamins A and C, intermediate filament proteins of the nuclear envelope, as part of a novel pathway involved in the control of body fat distribution. Our hypothesis is that dominantly acting mutations in lamins A and C interfere with this pathway that regulates fat cell differentiation or survival. The goal of this exploratory research project is to determine if mutant lamin A from patients with Dunnigan-type partial lipodystrophy blocks adipocyte differentiation or decreases adipocyte survival in vitro and in vitro. In Specific Aim 1, we will study 3T3-L1 pre-adipocyte cell lines that express wild-type lamin A with missense mutations found in patients with Dunnigan-type partial lipodystrophy. We will examine these cells and determine if the mutant lamin A blocks their in vitro differentiation into adipocytes or decreases their survival. In Specific Aim 2, we will create transgenic mouse lines that express wild-type and mutant lamin A under control of an Ap2 adipocyte-active promoter. Adipocyte cell distribution will be assessed in the transgenic mice, both at young and older ages as, in humans, fat loss in Dunnigan-type partial lipodystrophy occurs after the onset of puberty. The work in the exploratory project will prove or disprove the hypothesis that nuclear lamin A functions in fat cell development or survival. Confirmation of this hypothesis will have implications for the identification of new cellular targets to treat human obesity and diabetes mellitus.

DESCRIPTION (provided by applicant): Emery-Dreifuss muscular dystrophy (EDMD) is characterized by region muscle contractures, slow progressive muscle wasting and cardiomyopathy with atrioventricular conduction block. Indistinguishable forms of EDMD are inherited in autosomal dominant and X-linked manners. Mutations in emerin, an integral protein of the nuclear envelope inner membrane, cause X-linked EDMD. Autosomal dominant EDMD is caused by mutations in the LMNA gene, which encodes the nuclear envelope intermediate filament proteins lamins A and C. It is not known how mutations in nuclear envelope proteins cause muscular dystrophy. We hypothesize that mutations in these chromatin-associated proteins cause changes in the expression of genes responsible for muscle cell differentiation or survival. Our goal is to test this hypothesis using a combination of studies in transfected cells, patients' cells and tissues and animals models. In the first specific aim, we will use fluorescence microscopy and photobleaching methods to investigate how lamin A and C mutants from patients with autosomal dominant EDMD influence the mobility of emerin in the inner nuclear membrane. We will determine if mutant lamins A and C cause emerin to "escape" from the inner nuclear membrane into the continuous endoplasmic reticulum. As patients with X-linked EDMD do not have emerin in the inner nuclear membrane, this finding would demonstrate a connection between the X-linked and autosomal dominant forms of the disease. In the second aim, we will use microarrays to compare gene expression in cells from patients with autosomal dominant EDMD to X-linked EDMD and Dunnigan-type partial lipodystrophy, a disease caused by mutations in different regions of lamins A and C. This will establish if emerin and lamin mutations responsible for EDMD alter expression of the same genes. We will also use microarrays to determine gene expression profiles in muscles from lamin A/C "knockout" mice that develop muscular dystrophy and compare the results to what is known about pathologic alterations in gene expression in Duchenne muscular dystrophy. The results will be confirmed in tissues from human subjects with EDMD. In Aim 3, we will generate transgenic mice expressing human lamin A mutants and determine if they develop pathological abnormalities of EDMD and similar gene expression changes. This work will help establish how abnormalities in the nuclear envelope cause muscular dystrophy.

DESCRIPTION (provided by applicant): Hutchinson-Gilford progeria syndrome (HGPS), a condition with features of premature aging, is caused by a dominant de novo mutation in LMNA, the gene that encodes lamins A and C, intermediate filament proteins associated with the nuclear envelope. The mutation in HGPS introduces an abnormal splice site that leads the expression of a lamin A mutant with 50 amino acids deleted near its carboxyl-terminal end. The mutant lamin A has been called progerin. Different mutations in lamins A and C cause cardiomyopathy and muscular dystrophy, partial lipodystrophy syndromes, a peripheral neuropathy and atypical Werner syndrome. Some of these disorders share clinical features with HGPS while others are quite different. It is not known how mutations in lamins A and C cause HGPS or other diseases. We hypothesize that different mutations in these proteins cause alterations in nuclear structure and chromatin organization that lead to abnormalities in gene expression. In HGPS, progerin expression, possibly in combination with decreased expression of normal lamins A and C, is responsible for this chain of events. Our goal is to test this hypothesis. In Aim 1, we will study the biochemistry of progerin and its effects on the cell nucleus. We will determine if progerin, like normal prelamin A, is farnesylated and processed by endoproteolysis. We will investigate the effects of progerin on nuclear and chromatin structure and on the dynamics of other nuclear envelope proteins using fluorescent photobleaching methods. In Aim 2, we will generate transgenic mice expressing progerin in epidermis and determine if they develop pathological and functional abnormalities similar to those in skin of human subjects HGPS and normal aging skin. We will also cross progerin transgenic mice to heterozygous Lmna "knockout" mice to determine if reduced wild type protein levels have additional effects. In Aim 3, we will determine if a farnesyltransferase inhibitor blocks prenylation of progerin and determine if blocking progerin prenylation reverses cellular alterations and tissue abnormalities in progerin-expressing transgenic mice, hence connecting the experimental work in Aims 1 and 2. This project will establish how mutations in nuclear lamins A and C cause HGPS, and if inhibition of protein farnesylation is a potential therapeutic intervention.

DESCRIPTION (provided by applicant): Emery-Dreifuss muscular dystrophy (EDMD) results from mutations in two genes. Autosomal dominant EDMD, and infrequent autosomal recessive cases, result from mutations in LMNA. LMNA encodes A-type nuclear lamins, which are intermediate filament proteins associated with the inner nuclear membrane. Xlinked EDMD results from mutations in EMD, which encodes an integral protein of the nuclear envelope inner membrane called emerin. A commonly observed phenomenon in EDMD is defects in nuclear structure;however, little is known about how these structural defects relate to abnormal function. An emerging body of evidence demonstrates that the inside of the nucleus is connected to the cytoskeleton by a complex of interacting proteins termed the LING complex. These proteins include lamins, SUNs, which are transmembrane proteins of the inner nuclear membrane, and nesprins, some of which are transmembrane proteins localized to the outer nuclear membrane. Nesprins in turn can interact with cytoskeletal components such as actin. We hypothesize that mutations in the genes encoding A-type lamins and emerin cause a disruption of the LINC complex, which leads to abnormal nucleocytoskeletal connections and related defects in nuclear positioning and migration. Using a model system of fibroblasts undergoing polarization, we have obtained preliminary data showing that expression of lamin A mutants found in EDMD lead to nuclear movement defects. In Aim 1 of this project, we will carefully examine nuclear migration and centrosome positioning in cells expressing A-type lamins with amino acid substitutions that cause EDMD and related muscle disorders, cells lacking A-type lamins and cells expressing A-type lamins with amino acid substitutions that cause different diseases. In Aim 2, we will use fluorescence photobleaching techniques to determine the effects of mutations in A-type lamins on the dynamics of proteins of the LINC complex. In Aim 3, to link the pathogenic processes in autosomal EDMD to X-linked EDMD, we will examine the effects of emerin on nuclear movement and LINC complex protein dynamics. As nuclear positioning and nuclear migration are of critical importance in muscle fiber organization and differentiation, these studies will establish if defects in nucleocytoplasmic interactions lead to functional anomalies that can underlie the pathogenesis of autosomal and X-linked EDMD.

DESCRIPTION (provided by applicant): Hutchinson-Gilford progeria syndrome (HGPS) is a rare genetic disorder that resembles physiological aging but presents in early childhood. Understanding the mechanism by which HGPS triggers aging may reveal novel aspects of normal aging. HGPS is caused by mutations in the LMNA gene, which encodes the A-type nuclear lamins, intermediate filament proteins associated with the inner nuclear membrane. The mutations increase use of a cryptic splice site and the deletion of a proteolytic processing site resulting in the abnormal accumulation of farnesylated prelamin A variant called progerin in the nuclear lamina. Mutations in the ZMPSTE24 gene encoding the protease that processes prelamin A result in a similar accumulation of farnesylated prelamin A and cause restrictive dermopathy (RD), a related but more severe progeriod disease. Low levels of progerin have also been detected in normal cells where they may contribute to aged phenotypes. Although morphological alterations in the nuclear envelope have been described in HGPS, RD and normal aging, the mechanism by which progerin leads to abnormal cellular function and disease is unknown. It is likely that progerin expression alters the nuclear lamina and that this affects other structures in the cell. An emerging body of evidence has suggested that the nucleus is connected to the cytoskeleton via a complex of interacting proteins known as the LINC complex. These proteins include the lamins, Suns and nesprin proteins. Suns are transmembrane proteins in the inner nuclear membrane that interact directly with lamins and nesprins. Nesprins are in the outer nuclear membrane and associate with Suns and cytoskeletal elements such as actin. We hypothesize that progerin expression leads to a disruption in the LINC complex, resulting in defective nucleocytoskeletal connections and consequently alterations in nuclear movement and centrosome positioning. Using a model system of fibroblasts undergoing polarization, we have obtained preliminary data showing that expression of progerin alters localization of nesprin2G in the nucleus and interferes with nuclear movement and centrosome positioning. In Aim1 of the proposed experiments, we will test whether farnesylated progerin and prelamin A interfere with LINC complex formation and function and identify where in the pathways for nuclear movement and centrosome positioning progerin acts. In Aim 2, we will determine how progerin affects the biophysical behavior of LINC complex components by examining their mobility in the nuclear membranes and their interactions with each other. In Aim 3, we will test whether progerin is an important component of normal aging by examining whether normal aged cells exhibit defects in the LINC complex and whether the defects can be ameliorated by interventions that reduce progerin expression or farnesylation. These studies will establish whether defects in nucleocytoplasmic interactions contribute to cellular dysfunction in progeria and in normal aging.

? DESCRIPTION (provided by applicant): Emery-Dreifuss muscular dystrophy (EDMD) is caused by mutations in genes encoding proteins of the nuclear envelope. Autosomal EDMD results from mutations in LMNA, which encodes A-type lamins, and X-linked EDMD from mutations in EMD, which encodes emerin. Mutations in genes encoding nesprins and SUNs are also associated with the EDMD phenotype. Nesprins and SUNs comprise the linker of the nucleoskeleton and cytoskeleton (LINC) complex that spans the nuclear membranes. The LINC complex connects the nuclear lamina, which binds to SUNs, to cytoskeletal components including actin, which bind to nesprins. Emerin also associates with lamins and nesprins and modifies LINC complex function. Alterations in expression or primary structure of the nuclear envelope proteins implicated in EDMD prevent the proper movement and positioning of nuclei in migrating cells. In parallel, there is an activation in signaling by the MAP kinase ERK1/2, which itself blocks nuclear movement. This has lead us to hypothesize that all of the nuclear envelope EDMD proteins contribute to a common cellular pathway that controls nuclear positioning, which is essential for proper skeletal muscle structure and directed migration of myogenic progenitors. We propose to test this hypothesis by examining links between nuclear movement, ERK1/2 activity, nuclear positioning in skeletal muscle, muscle progenitor cell migration and EDMD pathogenesis in three specific aims. Aim 1 will involve a series of cell biological experiments designed to uncover how hyperactivated ERK1/2 prevents nuclear movement, including investigation of a previously uncharacterized hypothetical brake. In Aim 2, we will determine how nuclear movement affects ERK1/2 activation by dissecting its relationship to ERK1/2 activity during physiological activation of the kinase and then testing if moving the nucleus is necessary and sufficient to regulate ERK1/2 signaling. In Aim 3, we will first determine if EDMD-associated protein alterations that block nuclear movement interfere with myoblast fusion and differentiation in vitro. We will then use mouse models of EDMD to investigate the role A-type lamins and emerin on nuclear movement in regenerating skeletal muscle and to determine if alterations in these proteins block migration of myogenic progenitors into injured muscle. We will also determine if reducing ERK1/2 activity, which is elevated in skeletal muscle in EDMD, has effects on these processes. Overall, the proposed research will provide novel insights into the cellular pathology of EDMD, a poorly understood muscular dystrophy, and simultaneously uncover new information about nuclear movement, a process of broad significance to basic cell biology.

PROJECT SUMMARY The most important determinant of cardiovascular health is a person's age, as the risk of cardiovascular disease (CVD) increases significantly as we grow old. In this project, we will generate a new mutant Lmna mouse, as a model to probe the role of permanently farnesylated prelamin A in driving the CVD of physiological aging. LMNA encodes prelamin A, the precursor of the nuclear scaffold protein lamin A. Normally, prelamin A undergoes farnesylation and subsequent proteolytic cleavage by the protease ZMPTE24 that removes the farnesylated C-terminal portion of the protein. In the premature aging disorder Hutchinson- Gilford Progeria Syndrome (HGPS), an internally deleted (?50aa) form of prelamin A called progerin that remains permanently farnesylated causes disease phenotypes, including CVD. However, HGPS mouse models may not effectively model the CVD of physiological aging in unaffected individuals, because progerin is produced in vanishingly small amounts and the ?50aa deletion may impart novel binding properties to this prelamin A variant. On the other hand, compelling recent studies provide evidence that prelamin A, normally a transiently expressed farnesylated precursor that is rapidly converted to mature lamin A, accumulates in vascular smooth muscle cells of aged, but not young, individuals and in atherosclerotic lesions. Although the observed accumulation of farnesylated prelamin A in the vasculature of old human subjects is intriguing, there is no mouse model to directly assess experimentally the role of this protein in the development of CVD. The existing Zmpste24-/- mouse is not ideal for such studies because this enzyme has another critical cellular function besides prelamin A processing, which may confound analysis. Instead, to test the hypothesis that permanently farnesylated prelamin A promotes accelerated CVD, we propose to generate mice with a LmnaL648R mutation. The L648R amino acid substitution abolishes the ZMPSTE24 cleavage recognition site, leading to the accumulation of permanently farnesylated full-length prelamin A, essentially the same species seen in aging vessels. In Aim 1, we will generate knock-in LmnaL648R mice and analyze them for multiple organismal and cellular phenotypes associated with progeria and physiological aging. In Aim 2, we will characterize the development of vascular pathology in heterozygous and homozygous LmnaL648R mice by performing a longitudinal study of vascular stiffness using non-invasive pulse-wave velocity testing over their lifetimes. We will also perform interventional and ex vivo assessments of vascular mechanics, vasoreactivity, vasomechanics and vascular pathology. This R21 proposal involves development ? generation of a novel mouse model to study the potential role of prelamin A in CVD of aging ? and exploration ? studying these mice for the progression of CVD as they age. The potential for reward is huge, as these studies could pave the way for a paradigm-shifting understanding of CVD, the most common cause of morbidity and mortality for old Americans.

The liver is a major site of lipid metabolism. Abnormalities of hepatic lipid metabolism cause nonalcoholic fatty liver disease (NAFLD), a burgeoning public health epidemic estimated to affect approximately 25% of the U.S. population. NAFLD encompasses a spectrum of liver pathologies, beginning with lipid droplet accumulation in hepatocytes, called simple steatosis, which may progress to nonalcoholic steatohepatitis (NASH). Individuals with NASH are at increased risk for subsequent fibrosis, cirrhosis and hepatocellular carcinoma. Although there is appropriate focus on the progression of simple steatosis to NASH and fibrosis, there is also an urgent need to advance understanding of the mechanisms governing the development of simple steatosis. This proposal addresses this need, identifying novel molecular components that are critical to the normal regulation of hepatic lipid metabolism and, when impaired, cause profound steatosis. Our extensive preliminary studies in vivo and in vitro newly identify the torsinA/lamina-associated polypeptide 1 (LAP1) complex of the inner membrane of the nuclear envelope as a novel regulator of intrahepatic lipid metabolism, and a potent driver of steatosis. Conditional hepatocyte-specific depletion of either torsinA (A-CKO) or LAP1 (L-CKO) causes significant steatosis, which can progress to NASH in mice fed a regular chow diet. These mice demonstrate significant reductions in the assembly and secretion of very low density lipoprotein (VLDL) triglycerides (TG) and apolipoprotein B (apoB). The hepatic steatosis in these mice is far more severe, however, than observed in existing murine models of reduced VLDL secretion or in humans with mutations in genes that affect VLDL secretion, indicating additional effects of loss of the torsinA/LAP1 complex. These effects are cell autonomous as the genetic deletion is hepatocyte-specific, and these mice exhibit normal body mass and no evidence of insulin resistance. Based on these and additional preliminary results, we hypothesize that disruption of the torsinA/LAP1 complex at inner nuclear membrane causes steatosis by impairing the transfer of newly synthesized lipids to downstream processes. In Aim 1, we will conduct in vivo, ex vivo, and in vitro studies to test the hypothesis that loss of the torsinA/LAP1 complex impairs VLDL assembly and secretion by preventing the transfer of TG to nascent apoB. In Aim 2, we will examine hepatic lipid metabolism in detail, including studies of nuclear lipid droplets (LD) present in L-CKO mice and lipid accumulated in the ER in A-CKO mice. Studies of fatty acid and TG synthesis, fatty acid oxidation, phospholipid synthesis, and lipid droplet formation and turnover, as well as examination of key proteins in LD biogenesis, will allow for identification of critical pathways involved in hepatic lipid accumulation. In Aim 3, we will define the epistatic relationship between torsinA and LAP1 in steatosis by testing if we can prevent it in L-CKO mice by overexpressing torsinA. Successful completion of these Aims will implicate new intrahepatic targets to control VLDL secretion and prevent hepatic steatosis. It will also advance understanding of the role of the nuclear envelope as a critical node of liver lipid metabolism.

SUMMARY The cytoskeleton and its connections to the nucleus play fundamental critical roles in establishing cellular morphology, polarity, migration and substrate adhesion. We have discovered a fundamental cell polarity defect that occurs in physiological aging and in children with the accelerated aging disorder Hutchinson-Gilford progeria syndrome. This defect results from imbalanced connections between the nuclear lamina on the inner aspect of the inner nuclear membrane and two major cytoskeletal protein systems: actin/microfilaments and microtubules. These connections are mediated by the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex composed of inner nuclear membrane SUN and outer nuclear membrane KASH proteins. In aging, there is a preferential interaction of increased SUN1 with microtubules versus SUN2 with actin/microfilaments. The parent funded grant (R01 AG064944) for this supplemental application is designed to test the hypothesis that altered nucleocytoskeletal connections mediated by the LINC complex cause an intrinsic cell polarity defect in aging. In this supplement, we will expand the parent project to examine nucleocytoskeletal connections in Alzheimer disease (AD) and related neurodegenerative dementias. The neuronal microtubule-associated protein tau accumulates in a hyperphosphorylated form in neurons in these disorders and amyloid-beta (A?) oligomers accumulate extracellularly and induce hyperphosphorylation of tau. We will therefore test the hypothesis that tau and oligomeric A? alter microtubule association with LINC complexes and interfere with the generation of cell polarity in a fibroblast model system and in primary neurons. In Aim 1, we will determine if tau and A? influence the interactions of microtubules with LINC complexes and alter the generation of cell polarity. To do so, we will use a robust fibroblast model system as proposed in the funded parent award. In Aim 2, we will determine if tau hyperphosphorylation induced by oligomeric A? oligomers increases microtubule interactions with the nucleus and perform experiments to establish if these altered interactions underlie concurrent pathological changes in primary neurons. Finally, we will also assess the effects of SUN1 overexpression, which occurs with aging, on primary neurons. This research will implicate tau/A?-induced dysfunction of the LINC complex in the pathogenesis of AD and related dementia, and potentially identify targets for the development of therapeutic treatments.

Project Summary Dilated cardiomyopathy caused by mutations in the lamin A/C gene (LMNA) encoding A-type nuclear lamins is a life-threatening disease with no definitive cure. The pathogenic mechanisms responsible for cardiomyopathy in this inherited disease are poorly understood. In particular, it is not known how alterations in proteins expressed in nuclei of virtually all terminally differentiated cells selectively cause heart disease. Our hypothesis is that alterations in A-type lamins predispose cells to oxidative stress-induced remodeling of ryanodine receptors (RyRs), creating a sarcoplasmic reticulum (SR) Ca2+ ?leak.? Oxidative stress and increased cytosolic Ca2+ also contribute to hyper-activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2), which occurs in cardiomyopathy caused by LMNA mutations. The increased cytosolic Ca2+ and ERK1/2 activity generates various defects, including mitochondrial dysfunction, that cause cardiomyopathy. A corollary of our hypothesis is that blocking the SR Ca2+ ?leak? will have beneficial effects in cardiomyopathy caused by LMNA mutations. Using mouse models of the disease and human tissue, we will test our hypothesis and its corollary. In Aim 1, we will determine if alterations in A-type lamins that cause cardiomyopathy lead to enhanced cardiac muscle oxidative stress, resultant RyR2 remodeling and a SR Ca2+ ?leak.? We will also determine if the SR Ca2+ ?leak? stimulates ERK1/2 activity, causes mitochondrial dysfunction and damages DNA. In addition to heart, we will similarly examine skeletal muscle, which is often simultaneously affected in human patients with cardiomyopathy caused by LMNA mutations as well as in model mice. We will further assess these processes in cultured cells that stably express a cardiomyopathy-causing lamin A variant or lack A-type lamins. In Aim 2, we will utilize the three-dimensional structure of RyR to determine how specific oxidative modifications that occur in striated muscle of Lmna mutant mice affect its structure and make it ?leaky? to Ca2+. In Aim 3, we will perform experiments to determine if a Rycal, drugs that stabilize remodeled RyRs and block the SR Ca2+ ?leak,? improves heart function and prolongs survival in Lmna mutant mice and if it blocks the ?leak? in hearts from human subjects with cardiomyopathy caused by LMNA mutations. We will further determine if a Rycal has synergistically beneficial effects when combined with an inhibitor of ERK1/2 activity, which has previously been shown to partially improve heart function in Lmna mutant mice with cardiomyopathy. These studies will reveal basic information about the pathogenesis of cardiomyopathy caused by LMNA mutations and connect an intranuclear protein defect with a tangible mechanism of cardiac dysfunction. They will also determine if drugs already in clinical development can be translated to trials in patients with this lethal heart disease.