Taub Institute: Genomics Core
AN NIA-FUNDED ALZHEIMER'S DISEASE RESEARCH CENTER
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Research

Cell Biology & Neuroscience


Michael L. Shelanski, M.D., Ph.D.: Director, Francis E. Delafield Professor and Chairman of the Department of Pathology and Cell Biology

Ottavio Arancio, M.D., Ph.D.: Assistant Professor of Pathology

Gilbert Di Paolo, Ph.D.: Assistant Professor of Pathology

Karen Duff, Ph.D.: Professor of Pathology

Lloyd Greene, Ph.D.: Professor of Pathology

Ulrich Hengst, Ph.D.: Assistant Professor of Pathology and Cell Biology

Tae-Wan Kim, Ph.D.: Associate Professor of Pathology

Ronald Liem, Ph.D.: Professor of Pathology and Cell Biology

Carol Troy, M.D., Ph.D.: Associate Professor of Clinical Pathology and Neurology


Introduction:



Michael L. Shelanski, M.D., Ph.D.: Director, Francis E. Delafield Professor and Chairman of the Department of Pathology and Cell Biology

Mechanism of Memory Disruption and Synaptic Dysfunction in Alzheimer's Disease
Research in my laboratory utilizes a combination of cell culture and transgenic animal approaches in an attempt to understand why the overexpression of the amyloid precursor protein (APP) or direct application of its active peptide, A-beta, inhibits intracellular signaling in neuronal cells and leads to alterations of electrical activity, dendritic spine morphology and behavior. These results are extended with analyses of neurons taken from post-mortem Alzheimer's disease brains. In the past two years our attention has been on the PKA-CREB signaling pathway and on the role of ubiquitin c-terminal hydrolase-L1 (Uch-L1) in regulating these events. We have used both drugs and protein transduction techniques to show that A-beta induced changes, both in culture and in the animal, can be reversed by restoring these pathways to their normal "balance". Other projects involve the induction of neurogenesis in neural stem cells by A-beta raising the possibility of an endogenous repair mechanism in AD, and the analysis of the action of the ginkgolides on neuronal function. The laboratory approaches these questions with a wide range of tools including biochemistry, cell biology, physiology and microscopy.

Ottavio Arancio, M.D., Ph.D.: Assistant Professor of Pathology

Mechanisms Underlying Changes of Synaptic Function Associated with Cognitive Impairment
Research in my laboratory stems from my life-long commitment to studying mechanisms of synaptic plasticity. I am interested in the cellular and molecular mechanisms that underlie long-lasting changes of synaptic function in both normal, healthy brains and in the brains of those affected by neurological disorders, in particular Alzheimer's disease (AD). Research in my laboratory has focused on the mechanisms by which amyloid-β (Aβ) peptides interfere with both memory formation and the regulation of hippocampal long-term potentiation (LTP), an activity-dependent model of synaptic plasticity that is thought to be related with learning and memory. I am interested in how regulation of gene activation and silencing, post-translational mechanisms, channel opening, intracellular calcium transients and changes in transmitter release machinery might participate in basal synaptic transmission and in synaptic plasticity. The research of my laboratory is answering the following questions:

1) How does Aβ elevation impair synaptic plasticity and memory? Experiments addressing this question examine Aβ-induced modifications in epigenetic and post-translational mechanisms.

Epigenetic mechanisms: We are exploring steps affected at the downstream level of CREB phosphorylation. CREB plays an important role together with CBP in gene transcription through histone acetylation leading to the loss of chromosomal repression and transcription of genes needed for synthesis of proteins underlying memory formation. Thus, we are investigating if reduced histone acetylation follows the reduction of CREB phosphorylation by Aβ elevation. Chromatin changes do not have to be necessarily limited to histone acetylation. As a mechanism which can "lock in" particular states of pathological gene expression in human cells, DNA methylation is an obvious candidate for contributing to the inexorably progression and irreversibility of AD in the middle to late stages of the disease. Additionally, DNA methylation may act early in AD, as some very recent work has shown that the proper regulation of gene expression in memory formation is not only controlled by the transcriptional machinery but also modulated by epigenetics. We are currently identifying genes that are differentially methylated following Aβ elevation.

Post-translational mechanisms: SUMOylation is a post-translational mechanism other than phosphorylation involving the covalent attachment of a small 11 kDA protein moiety, SUMO (Small Ubiquitin-like MOdifier), to substrate proteins. We are investigating if SUMOylation plays a role in learning and memory. We are also investigating whether it is modified following Aβ elevation and if by re-establishing normal SUMOylation one can revert synaptic and cognitive dysfunctions in AD mouse models.

2) Does Aβ play a critical positive role in synaptic plasticity and memory? Recent research performed in my laboratory has shown that low levels of Aβ similar to those present in the brains of healthy individuals throughout life, enhance LTP and memory. We are continuing these studies to explore the role of endogenous Aβ in LTP and memory. We are addressing the following questions: can we visualize release of endogenous Aβ and follow its fate in normal physiological conditions? Are changes in APP processing by the secretases or other changes in APP metabolism responsible for the increase in Aβ levels during synaptic activity? Does release of Aβ from intracellular pools account for the increase in Aβ following activity in the presynaptic terminal? Most importantly, a fundamental question originating from the discovery of a positive function for Aβ is: how does it happen that a molecule performing a positive function gains a new and negative function?

All this work would be incomplete without the goal to move each project forward to the stage where it not only provides new biological insights but also, when appropriate, serve as the basis for future development of new therapeutic strategies. Such translational research is enhanced by collaborations with medicinal-chemists, biotech specialists, pathologists and clinicians. These studies should lead to the design of novel therapeutic approaches that might be effective in preventing or delaying the onset of AD and other neurodegenerative diseases characterized by cognitive disorders.

Gilbert Di Paolo, Ph.D.: Assistant Professor of Pathology

Endocytosis and Synaptic Vesicle Recycling in Mammalian CNS Neurons
Neurotransmission relies on the proper trafficking of synaptic vesicles (SVs) in nerve terminals. In the SV cycle, depolarization-induced calcium entry triggers the fusion of SVs with the plasma membrane. Subsequently, the SV membrane, along with essential membrane-bound SV proteins, must undergo recycling in order to replenish nerve terminals with new fusion-competent vesicles. This process is crucial to prevent plasma membrane expansion and SV depletion, both of which would impair neurotransmission. A major recycling pathway involves the retrieval of membrane through clathrin-mediated endocytosis, although other pathways (e.g. "kiss and run") have been described (Figure 1). Our laboratory studies the molecular mechanisms governing the recycling process in mammalian CNS synapses using molecular, cellular and genetic approaches. As a model system, we largely use primary cultures of cortical and hippocampal neurons prepared from rodent brain. Our research efforts focus on how protein-membrane interactions regulate the ability of vesicles to fuse, bud and travel within nerve terminals. Our interest can be subdivided into three themes.

The first theme concerns how modifications of the membrane environment affect the recruitment and assembly of endocytic components at the plasma membrane. In particular, phospholipids called phosphoinositides [e.g. PI(4,5)P2] are extensively studied in our laboratory, as they are major regulatory lipids involved in a myriad of cellular processes, such as organelle trafficking, actin dynamics and signal transduction (e.g. phospholipase C, PI 3-kinase pathways). Each of the seven known phosphoinositides appears to have a unique subcellular distribution in cell membranes. For instance, PI(4,5)P2 localizes to the plasma membrane, PI(4)P is enriched on the Golgi complex and on secretory organelles, and PI(3)P is concentrated on endosomes, as revealed by genetically-encoded fluorescent probes (Figure 2). Importantly, these lipids act as compartment-specific signals that regulate the cytosol-membrane interface. Our previous studies have shown that PI(4,5)P2 is a key factor involved in the recruitment of clathrin adaptors to the plasma membrane and thereby regulates the rate of endocytosis of synaptic vesicles. Nerve terminal proteins, such as synaptojanin [a PI(4,5)P2 phosphatase] and PIPK1g [a PI(4,5)P2-synthezing enzyme], are particularly essential to this process because they control the steady state levels of this regulatory lipid in nerve terminals. Therefore, we are interested in how these enzymes are regulated and interact with the exocytic/endocytic machineries. Furthermore, we have recently extended our analysis of lipids in nerve terminals by focusing on the role of phosphatidic acid, prompted by a growing number of studies implicating this major regulatory lipid to membrane trafficking in various systems. Our initial efforts have focused on the phospholipase D pathway, as the two main enzymes expressed in mammalian cells, PLD1 and PLD2, have been shown to play a key role in membrane budding, fusion and endocytosis in non-neuronal cells.

The second theme regards the mechanisms underlying membrane invagination during endocytosis. While many studies suggest that "coat" proteins, such as clathrin, are crucial to this process, recent evidence has indicated that a family of proteins can deform membranes (into buds or tubules) by inserting amphipathic helices into lipid bilayer and sense their curvature using domains, referred to as BAR. These domains have been recently crystallized and shown to consist of "crescents" which preferentially bind highly curved membranes, such as endocytic vesicles. Another interesting feature of these proteins is that they are generally coupled with other functional domains or activities. For instance, the BAR protein endophilin is the preferred interactor for synaptojanin, suggesting that it may control PI(4,5)P2 catabolism at sites of endocytosis in nerve terminals. More generally, our laboratory is currently testing whether a variety of BAR proteins are key factors for the recycling process in nerve terminals.

The third theme concerns the potential involvement of our proteins/ genes of interest in disease-related processes. One project focuses on the role of synaptojanin 1 in Down syndrome, as the gene encoding this protein is located on chromosome 21 and is overexpressed due to the presence of three gene copies. Our lab has obtained evidence that PIP2 metabolism is altered in Down syndrome mouse models as a result of synaptojanin 1 overexpression, potentially interfering with normal synaptic vesicle trafficking and higher brain functions, such as learning and memory. Another project focuses on the role of the BAR protein oligophrenin 1 in endocytosis, as several X-linked cases of mental retardation were mapped to Ophn1, the gene encoding this protein. To test the implication of altered synaptic vesicle recycling in the animal, we now perform behavioral studies on various mouse models in collaboration with Dr. Ottavio Arancio in the same department. Finally, we are interested in investigating whether synaptic vesicle trafficking defects are occurring in major neurodegenerative disorders, such as Alzheimer's disease, using various mouse models. We extensively collaborate with the laboratory of Dr. Tae-Wan Kim on this topic.

Pilot projects in our laboratory also explore the role of phosphoinositides and protein-membrane interactions in the pathway of autophagy, which has been recently implicated in major human diseases, including neurodegenerative disorders and cancer. This pathway mediates the turnover of organelles (e.g. mitochondria) as well as long-lived proteins, including protein aggregates commonly accepted as major neurocytotoxic factors in neurodegenerative diseases (e.g. huntingtin in Huntington's disease, synuclein in Parkinson's disease). Our interest for this field has been stimulated by the recent implication of key endocytic factors, such as Vps34 [i.e. the main PI(3)P-synthesizing enzyme], in this pathway. Our laboratory now investigates the crosstalk between the endosomal pathway and that of autophagy (in part through a collaboration with the laboratory of Dr. Dave Sulzer in the Department of Pharmacology).
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Karen Duff, Ph.D.: Professor of Pathology


Understanding the Molecular Basis of Neurodegenerative and Neuropsychiatric Diseases

We are exploring what goes wrong in the brains of patients with diseases such as Alzheimer's, Parkinson's, and schizophrenia, and our aim is to test therapeutic approaches that may be beneficial for the treatment, or prevention of these diseases. Our main focus is on Alzheimer's disease and the contribution of tangles to disease, especially the role of aberrant phosphorylation of specific proteins such as tau, in disease progression. We also have new projects looking at pathogenic mechanisms in Parkinson's disease (especially the role of autophagy) and the identification and investigation of a new pathway implicated in schizophrenia. In addition, we are examining the impact of diabetes on tangle formation in Alzheimer's disease. We are using several transgenic mouse models which allow us to examine, and modulate, pathways of interest. We have examined the impact of pathways involved in, for example, aberrant phosphorylation of tau protein in tangles both genetically (through crosses to other transgenic mice, or through viral induction of genes) and also pharmacologically using drugs. In the case of our work on Lithium, data generated is of direct interest to clinicians looking for non-proprietary treatments for patients, and to support clinical trials. This sort of translational neuroscience has high clinical relevance, as it covers a wide range of techniques and approaches, and it is applicable to a number of different diseases.


Lloyd Greene, Ph.D.: Professor of Pathology


Neuronal Differentiation
The overall goal of research in this laboratory is to understand the mechanisms whereby neuronal precursors differentiate into mature functional neurons. To this end, we use the rat PC12 pheochromocytoma cell line developed in this laboratory as a model system to study the mechanism of action of nerve growth factor (NGF) and the steps that lead to neuronal differentiation. Among current projects in the laboratory are those addressing the following questions: 1) What is the essential property of the high-affinity NGF receptor that permits it to mediate the functional activities of NGF and how is this receptor different from the low-affinity, non-functional NGF receptor? 2) What is the transductional mechanism by which NGF receptor occupancy leads to subsequent response? 3) What are the steps beyond immediate transduction that lead to NGF mechanisms? 4) What is the mechanism by which NGF regulates growth cone motility? What are the molecules involved in this effect? 5) What genes are regulated by NGF? By what pathways are they regulated? 6) What are the mechanism by which NGF promotes the initiation and regeneration of neurites; specifically, what is the role of specific cytoskeleton proteins in this process? 7) How do NGF and other neuronotrophic substances maintain cell survival? 8) How do such agents regulate cell proliferation?


Ulrich Hengst, Ph.D.: Assistant Professor of Pathology and Cell Biology


Local protein synthesis in developing and degenerating neurons
Neurons are arguable the cells with the most extreme morphological polarization, with distances between the periphery and the neuronal cell bodies ranging from millimeters to several feet. This extreme architectural polarization is mirrored in the existence of functionally distinct subcellular compartments, chiefly dendrites, axon, and soma. Spatially restricted protein expression is crucial for the establishment and maintenance of polarized neuronal morphology and function. Indeed, it has become apparent that alterations of polarized protein expression can cause or contribute to the pathogenesis of a wide variety of disorders. Our laboratory studies the physiological role of intra-axonal translation during development as well as the possible role of local protein synthesis during neurodegenerative disorders, especially Alzheimer's disease. We seek to understand how changes in local protein synthesis can either attenuate or ameliorate neuronal integrity in AD brain.


Tae-Wan Kim, Ph.D.: Associate Professor of Pathology


Molecular Mechanisms and Translational Research in Alzheimer's Disease
The major goal of our laboratory is to understand the molecular basis of Alzheimer's disease (AD) using a multidisciplinary approach based on molecular, cellular and chemical biology. We are also conducting translation research aimed at discovery and pre-clinical development of novel therapies for AD. Several cellular disease models are being used, including mouse embryonic stem (ES) cell-derived neurons as an alternative to primary cortical neurons for small molecule screening and functional genetic analyses. Relevant mouse models are also being utilized.

The first theme of our research is to understand the fundamental biochemical and cellular defects associated with the familial forms of AD, which occur in a small, but significant proportion of AD cases. Although familial AD (FAD) accounts for a small percentage of all AD cases, at the neuropathological level it is phenotypically indistinguishable from the more common (sporadic) form of AD. Thus, understanding the genotype-to-phenotype transition in presenilin-dependent FAD is likely to shed light on the pathogenesis of the more common, non-familial AD. Mutations in the genes encoding the presenilins (PS1 and PS2) are the most common cause of early-onset FAD and give rise to multiple cellular deficits. It has been shown that PS1 or PS2 serve as catalytic components of the γ-secretase complex that is essential for regulated intramembrane proteolysis (RIP) of select transmembrane receptor-like substrates, including APP. At the same time, the presenilins are well-accepted as regulators of calcium and several ion channels via a γ-secretase-independent mechanism. We investigate the molecular basis for the multi-functional nature of the presenilins as regulators of both intracellular ion homeostasis and intramembrane proteolysis. Both γ-secretase-independent and dependent pathogenic mechanisms have been studied in the lab.

The second subject of our research is to identify molecular factors controlling biogenesis and synaptic action of amyloid β-peptide (A β), a pathogenic agent in AD. Our recent studies reveal that alterations in phosphatidyl-4,5-bisphosphate [known as PI(4,5)P2], a phosphoinositide lipid that controls several essential neural functions, contributes to the biochemical and cellular defects associated with AD. Specific emphasis has been given to the role of PI(4,5)P2 in amyloid β-peptide (A β)-induced impairments in synaptic plasticity (synaptic dysfunction). Synaptic dysfunction caused by A β has been linked to cognitive decline associated with AD. Molecular, physiological and mouse genetic approaches are currently being used to investigate the hypothesis that A β-induced PI(4,5)P2 breakdown is an early and critical event that precedes other A β-associated morphological and functional synaptic changes such as loss of dendritic spines and suppression of long-term potentiation (LTP). We are also addressing whether the PI(4,5)P2 pathway can be targeted for novel AD therapeutics. This project is being conducted in close collaboration with the laboratory of Dr. Gilbert Di Paolo.

The goal of third project is to understand how BACE1, one of the key enzymes responsible for A β biogenesis, is regulated in neural cells. BACE1 mediates the proteolytic cleavage of β-amyloid precursor protein (APP) and the activity/levels of BACE1 are elevated in brains of AD mouse models as well as in postmortem AD brain tissue. We have conducted a high throughput cell-based assay and identified small molecules that can modulate BACE1 function via either a direct or indirect mechanism. Using these chemical probes, our laboratory is trying to understand the mechanism of BACE1 regulation by identifying cellular target(s) of these novel chemical modulators of BACE1. Furthermore, some of the small molecule hits are being developed as therapeutic candidates for the treatment of AD. Complementary to the chemical biology approach, biochemical experiments to isolate the BACE1-haboring molecular complex have been conducted. Several BACE1-associated proteins, including members of the sorting nexin and sortilin families of protein trafficking modulators, have been identified. The function and pathological relevance of these proteins are being investigated.


Ronald Liem, Ph.D.: Professor of Pathology and Cell Biology


The Neuronal Cytoskeleton in Neurodegenerative Diseases
Charcot-Marie-Tooth disease (CMT) is the most commonly inherited neurological disorder with a reported prevalence of 1 in 2500 people world-wide. It is found in all races and ethnic groups. CMT is slowly progressive and CMT patients suffer from degeneration of the peripheral nerves that control sensory information of the foot/leg and hand/arm. The nerve degeneration causes the subsequent degeneration of the muscles in the extremities. CMT is divided in two major types, CMT1 and CMT2. CMT1 is a demyelinating neuropathy, and due to mutations in genes important in myelin formation, whereas CMT2 is axonal. Mutations in the neuronal intermediate filament gene, NEFL have been shown to be the primary cause of CMT2. NEFL encodes the neurofilament light (NFL) protein that we have previously shown to be a necessary component for the assembly of neuronal intermediate filaments. Neuronal intermediate filaments form the intermediate filament network in neurons and are the predominant cytoskeletal structure in the axon.

Neurofilamentous aggregates both in the neuronal cell bodies and axons are seen in patients with mutations in NEFL, as well as in other neurodegenerative diseases. We have studied the NEFL mutations in transfected cells and found that in both neuronal and non-neuronal cells, mutant NFL resulted in misassembly of the intermediate filament network. This effect was dominant in agreement with the dominant nature of the NEFL mutations associated with CMT2E. In neuronal cells, we found that the mutant proteins caused defects in axonal transport leading to degeneration of neurites. Our studies showed a perfect correlation between pathogenic mutant NFL and the misassembly of filaments in the cultured cells. We are characterizing these mutations in more detail in transgenic animals. We hypothesize that inhibitors of neurofilament misassembly will lead to therapies for CMT. Our cell and animal models will be useful for screening of potential therapeutic agents against the disease.

We are also studying a family of cytoskeletal linker proteins called plakins. One of these plakins, BPAG1 was originally described as a component of the hemidesmosome in the epithelia, where it links the intermediate filaments to the extracellular matrix. Interestingly, the mutant mouse dystonia musculorum (dt), which suffers from a severe hereditary sensory neuropathy is due to mutations in the BPAG1 gene. Focal axonal swellings filled with neurofilaments, mitochondria and membrane bound dense bodies are hallmarks of the pathology of these mice and we are studying the neuronal form of this molecule. A closely related plakin called MACF1 (Microtubule actin crosslinking factor) is also highly expressed in the nervous system. MACF1-/- mice are embryonic lethal, and we have generated a neuron-specific knock-out of MACF1, which shows defects in neuronal migration. We are further characterizing this neuron-specific knock-out and also determining whether MACF1 and BPAG1 have related functions in various tissues.


Carol Troy, M.D., Ph.D.: Associate Professor of Clinical Pathology and Neurology


The Study of the Molecular Mechanisms of Neuronal Dysfunction and Death with an Emphasis on the Regulation of Caspase Activity
Neuronal loss is an outstanding feature of neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease and ischemic stroke. We employ model systems of neuronal death to define the death pathways. We are particularly interested in the regulation of the caspases, the multi-membered family of death proteases that are central to the execution of death. Current death paradigms under study include β-amyloid toxicity, peroxynitrite mediated death, neurotrophin-withdrawal-induced death and in vivo models of ischemia. We have developed specific molecular tools for knocking down individual members of the death pathways in post-mitotic (neuronal) cells and for blocking caspase activity/activation in cultured neurons and in vivo.

We have shown that the specificity of the death pathway is determined by the stimulus inducing death but also that there is flexibility in the pathways chosen for executing death. The dominant pathway depends on the relative concentrations of anti- and pro-apoptotic proteins. This illustrates that the maintenance of life and execution of death of a neuron is a delicate balance of the pro- and anti-apoptotic molecules in the cell, a balance that can be altered in disease.

Our studies of oxidative stress mediated death show that cytokines can induce an autocrine mediated death. Down-regulation of superoxide dismutase 1 leads to activation of caspase-1 which releases the cytokine interleukin-1b and the cells undergo a peroxynitrite-dependent death. Thus, although caspase-1 has been defined as a non-apoptotic caspase with a role in inflammation, in response to specific death stimuli caspase-1 can activate a death pathway. It is important to understand the interaction that can occur between the cytokine signaling pathway and the death pathway to determine the appropriate intervention that will result in increased neuronal survival.

 

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