Lee J. Martin, PhD - US grants

The Neuropathology Core has a central role in our Program Project. The approach of this Core will be multidisciplinary, using conventional neuropathological and state-of-the-art molecular neurobiological methods to evaluate mechanisms of ischemic brain injury. The staff of the Core will conduct quantitative neuropathological assessments of the regional/cellular localization and severity of brain damage in rat and mouse models of focal cerebral ischemia and in mouse and piglet models of cardiac arrest/CPR. In addition, the Core will function as a molecular neuropathology facility for centralized use of immunoblotting, northern blotting, in situ hybridization, histochemistry, and immunocytochemistry for the quantitative determination of ischemia/reperfusion-induced abnormalities in signal transduction and cell death pathways. The investigators supported by the Core have a long-standing history of productivity in clinical and experimental neuropathology, neuroanatomy, and/or molecular neuroscience; therefore, the core activities will support and coordinate the analysis of experimental material generated in the Program Project.

The mechanisms for motor neuron degeneration in amyotrophic lateral sclerosis (ALS) are not understood. Our preliminary studies indicate that motor neuron death in ALS may occur by programmed cell death (PCD). We have identified abnormalities in proapoptotic and antiapoptotic protein levels in individuals with ALS, leading to our hypothesis that neurodegeneration in ALS may occur by PCD, involving subcellular redistributions of cell death proteins. Our studies also suggest that neuronal apoptosis in ALS may be Fas-mediated. The proposed experiments are designed to further evaluate this mechanism for motor neuron death. We will examine, in postmortem central nervous system tissues from individuals with ALS and from age and disease matched controls, the possible role of Fas-mediated PCD as a mechanism for neuronal apoptosis in ALS, by measuring the expression of apoptosis- inducing cell surface receptors and their ligands as well as the subcellular expression of apoptosis regulatory proteins, specifically members of the Bcl-2 and caspase families and caspase target proteins. We have also found that axotomy and target deprivation of motor neurons (by sciatic nerve avulsion) in adult rodents causes apoptosis. Motor neuron apoptosis in this model is associated with mitochondrial accumulation within the cell body and oxidative stress. We will use this model of apoptosis to test the hypothesis that motor neurons undergoing apoptosis in adult mouse spinal cord develop mitochondrial abnormalities, oxidative stress, and release cytochrome c which corresponds termporally with activation of caspases. We will identify whether the mechanisms for motor neuron apoptosis are dependent on Bax, p53, or Fas/Fas ligand by evaluating the progression of avulsion-induced motor neuron apoptosis in mice deficient in these genes. We will then use interventions, including antioxidant, caspase inhibitor and immunosuppressant/mitochondrial premeability transition blocker therapies, to prevent or delay motor neuron apoptosis in mice. These experiments will identify the contributions of PCD mechanisms to the pathogenesis of ALS and will clarify the molecular pathways leading to motor neuron apoptosis in vivo. These studies should lead to a better understanding of motor neuron death and to the design of new therapeutic experiments critical for the future treatment of ALS.

DESCRIPTION (Adapted from the applicant's abstract): The mechanisms for neuronal degeneration in adult-onset central nervous system (CNS) diseases, including Alzheimer's disease (AD) and amyotrophic lateral sclerosis (ALS), are not understood. Recent studies suggest that neurodegeneration in AD and ALS is apoptosis, occurring by programmed cell death (PCD). The investigator has developed an animal model to study neuronal apoptosis. Occipital cortex ablation in adult rat and mice, a model of axotomy and target deprivation, causes progressive retrograde neuronal degeneration in thalamus that is structurally apoptosis. This apoptosis is associated with accumulation of active mitochondria within the neuronal cell body and oxidative damage to DNA. The investigator proposes to evaluate the mechanisms for neuronal apoptosis in vivo. The investigator will test the hypothesis that apoptosis in neurons is signaled by subcellular translocation of Bcl-2 and Bax and release of cytochrome C from mitochondria, which correspond temporally with activation of caspases and DNA fragmentation factors. The participation Bcl-2 and Bax to the mechanisms for neuronal apoptosis will be determined by evaluating whether neuronal loss is reduced in lesioned transgenic mice overexpressing Bcl-2 and in mice deficient in Bax. The participation of mitochondrial permeability transition and cytochrome C release will be determined by post-injury treatment with the permeability transition blocker cyclosporin A. In addition, the investigator proposes that a signal for PCD in these neurons is oxidative stress. The investigator will test the hypothesis that retrograde neuronal death after axotomy is nuclear DNA damage-induced, p53-dependent apoptosis. The investigator will evaluate whether dying neurons sustain oxidative damage to DNA and proteins during the transition between chromatolysis and early apoptosis. The participation of oxidative stress as a mechanism for the induction of neuronal apoptosis in vivo after axotomy/target deprivation will be further examined by determining whether oxidative injury and apoptosis are attenuated in transgenic mice that are deficient in neuronal or inducible nitric oxide synthase and in mice that overexpress human wild-type superoxide dismutase 1. The dependence of this neuronal apoptosis on p53 will be evaluated in lesioned p53-deficient mice. The investigator will then use antioxidant therapies (Trolox and uric acid) to prevent or delay neuronal apoptosis. These studies will identify possible molecular mechanisms of neuronal apoptosis in vivo and could lead to the design of new therapeutic neuroprotection experiments critical for the future treatment of AD and ALS.

DESCRIPTION (provided by applicant): Motor neurons (MN) in patients with amyotrophic lateral sclerosis (ALS) degenerate through unclear mechanisms. Apoptosis could be a mechanism for this neuronal loss. Previous work on MN death mechanisms in ALS has lacked cellular resolution for MN-specific events. We propose to study the levels of selected cell death molecules in MN from humans with ALS and transgenic ALS mice using laser capture microdissection and proteomic analyses with ProteinChip arrays. The profiles of human and mouse ALS MN will be compared to MN induced to degenerate (by sciatic nerve avulsion) through a process that is unequivocally apoptosis and is p53- and Bax-dependent and also involves mitochondrial accumulation, oxidative stress, DNA damage, and caspase-3 activation. We hypothesize that MN degeneration in human and mouse ALS is structurally a nonclassical form of apoptosis that is DNA damage-induced and mediated by p53, or its homologue p73, and caspases. The upstream mechanisms for MN death that we will study in human and mouse ALS and in axotomy will be the formation of DNA lesions (abasic sites and single- and double-strand breaks) and signaling pathways leading to accumulation of p53/p73. We will measure, in human and mouse ALS, different types of damage to chromosomal and mitochondrial DNA. We will use an innovative method (the comet assay) to quantify different forms of DNA lesions in single MN and to determine if known and potentially new pharmacotherapies (creatine and selenomethionine) for mouse ALS attenuate the formation of DNA lesions. Our preliminary data also implicate DNA damage-responsive kinases in the mechanisms of MN death in human and mouse ALS and in axotomy. We will measure the activation of selected DNA damage-responsive protein kinases (ATM and c- Abl) in MN in human and mouse ALS and in avulsion. We will use mouse ALS and avulsion models of MN degeneration to directly identify upstream mechanisms leading to MN death. We will test the hypothesis that MN degeneration is stimulated by accumulation of DNA single-strand breaks and mediated by ATM and/or c- Abl activation and p53/p73 activation. We will determine if avulsion-induced MN apoptosis is dependent on ATM. Pharmacological inhibition of c-Abl with STI571 and inhibition of p53/p73 with pifithrin-V will be used to modify the degeneration of MN in ALS mice. We will also measure DNA repair enzymes in human and mouse ALS MN because failed or defective DNA repair in MN could lead to the accumulation of DNA lesions and cellular degeneration. This work is essential for the further understanding of the biological substrates and molecular mechanisms of MN death and the pathogenesis of ALS and is critical for the identification of novel molecular targets and new drug therapies for the treatment of ALS.

DESCRIPTION (provided by applicant): Neuronal cell death induced by DNA damage has been implicated in the pathogenesis of many acute and chronic neurological disorders in humans, and DNA damage-induced cell death is a fundamental principle driving therapy for many types of cancer. The major goal of the experiments in this grant proposal is to determine if DNA damage is a cause or consequence of neurodegeneration. Cultured embryonic mouse cortical neurons will be used as a model to study cause-effect links between specific types of DNA lesions and cell death and the mechanisms of DNA damage-induced apoptosis in young and mature neurons. In Specific Aim 1 we will identify lethal levels of DNA damage in neurons and the relationships between DNA damage level, DNA lesion type, and cell death pattern (apoptosis, necrosis, or hybrid) and will test the hypothesis that toxicity of DNA damaging agents on neurons is maturation-related. We will study if DNA damage occurs preferentially at specific sites within the neuronal genome using comet-FISH analysis. We will identify the major DNA repair and DNA degradation pathways in cortical neurons and will determine whether DNA repair is different in neurons at different stages of maturation. We will test the hypothesis that the dependence of DNA damage-triggered death on caspases and MARK signaling differs in neurons of different maturational states. In Specific Aim 2, we will evaluate upstream mechanisms that link DNA damage to downstream apoptosis mechanisms in cortical neurons. We will test the hypothesis that DNA damage-induced apoptosis in young and mature neurons is mediated by the ATM-p53 or cAbl-p53 signaling networks. In Specific Aim 3, we will determine if enforced expression of DNA repair enzymes that function in base excision repair can be neuroprotective. These experiments will provide important information on the toxicity of DNA damage in neurons, the specific types of DNA damage that can occur in neurons, the effects of DNA damage on neurons of different ages, and the molecular mechanisms that transduce DNA damage signals to apoptotic pathways in neurons. This work is relevant to human disease because it will contribute to the understanding of the pathobiology of DNA damage-induced neurodegeneration and to the identification of possible molecular targets for reducing the toxic and neurologic side-effects of cancer therapy.

DESCRIPTION (provided by applicant): Amyotrophic lateral sclerosis (ALS) is the 3rd most common human neurodegenerative disease with an adult onset. It is a fatal paralytic disease of motor neurons (MNs) without any effective treatments. Novel mechanism-based targets need to be identified for drug discovery relevant to diseased MNs. Some forms of ALS are inherited and are caused by mutations in the superoxide dismutase-1 (SOD1) gene, thus providing a clue about MN vulnerability. Many different SOD1 mutations occur, but the mechanisms of human SOD1 (hSOD1) toxicity to MNs are unresolved. Importantly, the autonomy of the MN degeneration in ALS is an important unresolved problem. We hypothesize that skeletal muscle is a primary site of pathogenesis in ALS that triggers MN degeneration. We have created new transgenic (tg) mice with skeletal muscle-specific expression of hSOD1 gene variants. These hSOD1mus tg mice develop neurologic and pathologic phenotypes consistent with ALS. Using these novel mice we propose to study skeletal muscle as a disease-causing entity in ALS. In Aim 1 we will analyze the age-related neurologic and pathologic phenotypes of hSOD1mus tg mice. We hypothesize that the mechanisms of MN degeneration in our hSOD1mus tg mice are consistent with distal axonopathy and target deprivation-induced apoptosis. In Aim 2, we will analyze the involvement of oxidative stress and activation of the mitochondrial permeability pore in skeletal muscle as mediators of muscle pathology in hSOD1mus tg mice. In Aim 3 we will use cultured cells to examine if hSOD1 expression in skeletal muscle cells alters their intracellular redox state, Ca2+ handling, and ion channel function and disrupts the neuromuscular junction, thus provoking MN degeneration. The work can lead to new concepts about the non-autonomous death of MNs in ALS pathogenesis. The discovery of instigating toxicities or disease progression determinants within skeletal muscle would be very valuable for development of new effective therapies in the treatment and cure of ALS.

DESCRIPTION (provided by applicant): Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease with worldwide incidence and no racial, ethnic, or socioeconomic boundaries. Most ALS cases are sporadic with no known inherited component. Epidemiological studies implicate some environmental and acquired factors in its pathogenesis; however, previous genetic studies have been unable to uncover the genes involved in elucidating the molecular mechanisms of sporadic ALS. We hypothesize the aberrant epigenetic regulation of DNA contributes to the neurodegeneration in ALS. Specifically, we propose that aberrant silencing of neuronal or skeletal muscle synapse maintenance genes or cell survival genes by DNA methylation could be a critical factor in the cause of neurodegeneration in ALS. Our preliminary studies of human genomic DNA obtained from ALS patients and age-matched controls reveal differential patterns of hyper- and hypomethylation. CpG island microarrays have identified the neuroligin-1 gene promoter as being hypermethylated in human ALS nervous tissue. We also find increased levels of DNA methyltransferase isoforms (Dnmt1 and 3A) and 5- methylcytosine in human ALS nervous tissue. In cell culture and mouse models, we find that enforced expression and activity of Dnmts induce neuronal degeneration. We propose to conduct the following studies. In Specific Aim 1 we will perform additional CpG microarray analyses of human ALS and age-matched control nervous tissue and skeletal muscle to determine the specific genes that are hypermethylated or hypomethylated and to validate the patterns of gene methylation using alternative methods such as methylation- sensitive-restriction-fingerprinting and also demonstrate cell-type specificities in aberrant Dnmt expression and DNA methylation. In Specific Aim 2 we will test the hypothesis that neuroligin 1 silencing participates in the mechanisms of ALS through interneuronopathy and failure of synaptic inhibition. By studying the DNA methylation and gene silencing in ALS, we could identify novel cell-based, genome-based, and physiology-based mechanisms of ALS pathogenesis that could lead to novel strategies for treating ALS.

Neonatal hypoxic-ischemic encephalopathy (HIE) from birth asphyxia causes persistent and severe neurologic disabilities, even in patients who receive therapeutic hypothermia. We found in clinical studies that white matter injury on MRI persists after hypothermic treatment. Thus, hypothermia is not fully protective. White matter injury is a prominent yet understudied component of the neurologic disabilities observed in neonates who receive hypothermia for HIE. Therapeutic adjuncts that protect the white matter might reduce the risk of permanent neurologic injury in HIE. Hypoxia-ischemia (HI) in neonatal pig, which has human-like white matter tracts, produces brain damage similar to that of full-term human newborns with HIE, including the white matter injuries observed in clinical studies. Our model includes clinically relevant whole-body hypothermia, rewarming at 0.5°C/h, sedation, continuous hemodynamic monitoring, ventilator support, and correction of blood gas and electrolyte abnormalities to mimic clinical neonatal intensive care. Preliminary data suggest that insufficient proteasome function mediates persistent white matter injury after HI and hypothermia. We postulate that white matter proteasome insufficiency causes a failure to clear oxidatively damaged proteins, causing oligodendrocyte apoptosis, potential disruption of oligodendrocyte precursor maturation, myelin and axonal injury, and white matter volume loss after HI and hypothermia. We will elucidate the proteasome?s role in white matter injury after whole-body HI and overnight hypothermia in neonatal swine. White matter injury and oligodendrocyte biology will be studied with neuropathology (including oligodendrocyte precursor maturation, stereology, cell death, and electron microscopy) and biochemistry (including protein post-translational modification and proteasome composition and activity) through 1 month recovery after HI. T- maze neurocognitive behavior testing with neuropathology correlation will provide a functional outcome. We developed new methods to genetically modulate proteasome activity in distinct, targeted regions of white matter in neonatal pig forebrain using virus-mediated enforced expression of a proteasome activator subunit or proteasome inhibition with short hairpin small interfering RNA. We will also use a small molecule proteasome inhibitor to determine whether proteasome inhibition aggravates white matter injury. Moreover, we will test the potential of the drug oleuropein to protect white matter. Oleuropein is a readily bioavailable compound with proteasome activating properties and few clinical side effects. An intravenous oleuropein dosing regimen will be used that protects oligodendrocytes and myelin, increases proteasome expression, and promotes clearance of oxidized proteins after HI and hypothermia. We will identify whether oleuropein acts on the standard proteasome or the immunoproteasome. Cultured human oligodendrocyte experiments will validate the proteasome as a therapeutic target and oleuropein?s actions after oxygen glucose deprivation. This project will advance the neonatal HI and cell biology fields by investigating novel mechanisms by which proteasome insufficiency mediates hypothermia-resistant injury in white matter. We will discover whether proteasome activation is a relevant therapeutic adjunct to hypothermia to protect white matter and improve neurologic outcomes in HIE.