Stephen W. Scheff, PhD - US grants

Following trauma to the CNS a regenerative process known as axon sprouting or reactive synaptogenesis begins. In this way the circuitry of the damaged area is reordered, resulting in functional changes. The aged CNS has a diminished injury-induced growth response when compared to young adults. The proposed experiments will determine the mechanism(s) behind the aged brain's response to damage. In the first set of experiments we will assess the ability of the aged animal to support reactive growth in two different areas of the CNS, the hippocampus and septum. Both areas are denervated by a transection of the fimbria-fornix. This will explore the extent of the growth reponse in the aged brain. The second set of experiments will determine if experimental alteration in the neuropil milieu can directly influence the altered lesion-induced growth response in the aged brain. Following injury in the young adult, a timed accumulation of a trophic substance, neuronotrophic factor (NTF), is produced which supports cell survival and promotes process growth. By administering a purified form of NTF we will test its ability to promote lesion-induced growth in the aged brain. In the third series of investigations, fetal tissue will be implanted into the aged host to determine if the alteration of plasticity in the senescent brain is the loss of the aged neuron's ability to innervate a target area or the loss of the target area to support reinnervation. By providing a detaileds qualitative and quantitative analysis of the restoration process at various times post surgical intervention, it will be possible to gain information on the rate and magnitude of the reactive growth response. Quantitative electron microscopic and biochemical indices of lesion-induced growth will be employed.

DESCRIPTION: In Alzheimers disease (AD) there is a progressive cognitive deterioration which is far greater than that observed in normal aging. This decline is paralleled by neuropathological and neurochemical alterations and has been closely linked to a continuous decrement of large neurons and extensive synapse loss in the neocortex. Synaptic loss may well occur before the neuronal loss. In the young adult CNS, growth quiescent neurons manifest a reactive repair process in response to damage, leading to a replacement of lost synaptic contacts. Because the final common pathway for neurotransmission involves synaptic integrity, it is important to assess the extent of this compensatory response throughout aging. It is also important to understand the effect of the aging factor on synaptic loss in AD as wells its relationship to any age-related cell loss or shrinkage. The present studies will utilize ultrastructural techniques to evaluate the synaptic density in the neocortex and hippocampus throughout the adult human life span (20-95 yrs). These studies will test the hypothesis that there is an age-related decline in synaptic density in human neocortex and hippocampal formation which is beyond the normal compensatory mechanisms of the aging brain. This age-related decline in synaptic numbers is closely related to the age-dependent decline in larger teurons~either within the cortical area itself or in areas which have a direct projection to the region which manifests synapse loss. Specific aim #1 will quantify and correlate the synaptic density and neuronal density in frontal and temporal neocortex throughout the human life span in cognitively normal subjects. Specific aim #2 will quantify and correlate the synaptic density in the molecular layer of the hippocampal dentate gyrus and neuronal density in the entorhinal cortex throughout the life span in cognitively normal subjects. These studies will definitively answer whether there is an age-related decline in synapses and define the relationship to neuronal loss.

Maintenance of calcium (Ca2+) homeostasis is extremely important not only for normal cellular function but also cell survival. The mitochondrial cycling of Ca2+ during excitotoxic insults, such as that occurring after traumatic brain injury (TBI), is key to this Ca2+ homeostasis and hence cellular homeostasis. Excessive sequestering of Ca2+ by mitochondria uncouples electron transport from ATP synthesis leading to the increased production of free radicals, and opening of the mitochondrial permeability transition pore (MPTP). The MPTP is an important component contributing to the cell death cascade. Opening of the MPTP abolishes the mitochondrial transmembrane potential (deltapsi) resulting in excessive amounts of Ca2+ and free radicals in the cytosol. Maintenance of the deltapsi is critical for synthesis of ATP, the primary energy source for the cell, which is in great demand following injury. Without an adequate source of ATP the cell has a problem maintaining Ca2+ homeostasis. The central hypothesis of this proposal is that the cycling of Ca2+ by the mitochondria is a key element in excitotoxic neuronal damage. Cyclosporin A (CsA), a widely used immunosuppressant, inhibits the opening of the MPTP and maintains mitochondrial homeostasis. We have strong evidence that systemic injections of CsA significantly reduces neuronal death in an animal model of TBI. The specific aims of this proposal examine the following hypotheses: 1) that stabilizing mitochondrial homeostasis will stabilize cellular homeostasis and protect cortical neurons following TBI, 2) that inhibiting opening of the MPTP after TBI enhances basic metabolic functions of synapses and mitochondria, and 3) that mitochondrial homeostasis promotes synaptic plasticity following TBI. These studies will significantly enhance our understanding of the mechanisms following TBI and hopefully lead to therapies resulting in increased recovery.

[unreadable] DESCRIPTION (provided by applicant): Abundant clinical data indicate increased morbidity and mortality following trauma to the aging nervous system. Little is currently known about the cellular substrates underlying this amplified adverse response. The proposed studies lead to testing of the hypothesis, that age-related changes associated with traumatic brain injury (TBI) can be negated by enhancing the ability of neurons to produce ATP. This idea is based on the theory that age-related changes in brain mitochondria lead to a disruption of ATP necessary to meet the energy demands of neurons following trauma. Mitochondria are the major source of ATP required for neuronal function. Age-related defects in mitochondrial oxidative phosphorylation result in deceased energy production, impaired cellular calcium buffering, activation of proteases and phospholipases, and the generation of increased free radicals. All of these pathways can lead to enhanced cell death depending on the severity of the insult. Preliminary data from our laboratory demonstrate age-related differences following experimental TBI, supporting its usefulness as an aging animal model. We also present data that a diet supplemented by creatine provides a neuroprotective intervention for TBI. Because of a lack of sufficient information on aged animal models of TBI, this study will first characterize age-related changes. Specific aim #1 will characterize age-related decline in morphologic and behavioral changes following TBI employing an animal model of controlled cortical contusion. Specific aim #2 will characterize age-related changes in synaptic and non-synaptic mitochondria following TBI. Specific aim #3 will explore age-related changes in the generation of lactic acid and free fatty acids, sensitive markers of secondary injury, and changes in isoprostanes and neuroprostanes, markers of lipid peroxidation following TBI. Specific aim #4 will use a 'creatine-supplemented' dietary intervention that enhances cytosolic phosphocreatine and increases the ability of neurons to produce ATP, an intervention to reverse the age-related response to TBI. [unreadable] [unreadable]

The Animal Surgery &TBI/SCI Model Core will be available to all members of the center and their students and technicians. Services provided by this core will include: assistance in performing 1) traumatic brain injury using one of the standard closed head injury models (controlled cortical impact, fluid percussion, Marmarou diffuse impact-acceleration);2) spinal cord injury (NYU device, IH 0400 device OSU device). The facility will be designed to handle both rats and mice with the stipulation that only one species will be present at any given surgery appointment. The core facility will also provided assistance in post-injury animal welfare and care including bladder expression following SCI.

[unreadable] DESCRIPTION (provided by applicant): Mounting evidence suggests that individuals with mild cognitive impairment (MCI) have an increased likelihood to develop Alzheimer's disease (AD). It is unclear what early pathophysiological changes may underlie this transition. The brains of individuals with definite AD manifest several pathological changes including a substantial loss of synapses in association areas of the neocortex. Recent work has now demonstrated that synaptic loss provides an excellent correlation with cognitive ability and provides a strong correlate of dementia. The relationship between synapse loss, early cognitive decline, such as that observed in MCI, is poorly understood. There is increasing evidence that amyloid beta peptide (Abeta) and oxidative damage may be fundamentally involved in the pathogenesis of AD and contribute to MCI. The interaction between Abeta, oxidative damage and synapse loss may provide important keys to the mechanisms that lead to MCI. This proposal will examine the hypothesis that synapse loss is associated with cognitive deficits observed in the early phase of the disease process, and is responsible for amnestic memory problems associated with MCI. Studies will be carried out on short postmortem interval brains from individuals characterized as no cognitive impairment (NCI), MCI, and early AD, and evaluation of total synapses will be obtained by coupling unbiased stereology with transmission electron microscopy. Since Abeta is considered by many researchers to play an important role in progression of AD, we will study the relationship of soluble Abeta1-42 with synaptic loss and pre/post synaptic proteins. The specific aims will also test the hypothesis that oxidative damage is an early indicator of MCI and is associated with changes in total synaptic numbers in neocortical association areas known to be affected early in AD. Finally, we will study possible changes in mitochondrial bioenergetics that occur in MCI and early AD since mitochondria can be affected by both Ap and oxidative stress and are important for synapse function. Successful completion of the proposed studies will lead to new insights into the mechanisms underlying MCI and early AD and contribute to the development of effective pharmacologic therapies for AD. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Traumatic brain injury (TBI) is a global health problem that is financially crippling for many families because of both specialized care and lost financial income. TBI involves at least two separate injury cascades that lead to neuronal and cognitive dysfunction. The secondary injury cascade may be amenable to pharmacologic intervention but the mechanisms are complicated requiring a multi-faceted approach. Oxidative stress, loss of mitochondrial function, and neuroinflammation appear to play major roles. Naturally occurring flavonoids are unique in possessing not only tremendous free radical scavenging properties but also the ability to modulate cellular homeostasis leading to a reduction in inflammation and cell toxicity. The proposed studies will investigate the idea that Pycnogenol(r) (PYC), a combination of bioflavonoids, can significantly reduce the secondary injury cascade following TBI. This is an extremely novel approach to TBI therapy since it utilizes a family of very closely related biologically active polyphenols derived from tree bark. The first series of studies will explore the best possible dosing of PYC to maximally reduce oxidative stress and neuroinflammation following experimental TBI in rodents. The second set of studies will explore the protective qualities of PYC on mitochondrial bioenergetics and cell survival in the hippocampus following experimental TBI. Successful completion of the studies will pave the way for using PYC in more involved experimental TBI studies and contribute to the development of a rational therapy leading to a more favorable outcome. PUBLIC HEALTH RELEVANCE: The proposed studies investigate a specialized compound, called a bioflavonoid, to determine if it can help protect cells in the brain following injury. Using a well characterized animal model of traumatic brain injury, subjects are treated with the compound, Pycnogenol(r), and tested for a variety of outcome measures. Bioflavonoids are known to manifest multiple different helpful properties such as reducing oxidative stress. If the studies work, then more complex and longer term studies can be proposed, hopefully leading to a more rational therapy for brain injury.

CORE B. Animal Modeling Core Plans: This core, previously named the Animal Surgery & TBI/SCI Model Core will be renamed Animal Modeling Core to reflect the expansion of the capabilities of this widely used facility beyond its previous use as an animal surgery and neurotrauma modeling resource. While these functions will continue to constitute the central component of the core which will be the Surgery and In Vivo Models Component, we request funding to add physiological (blood pressure, heart rate, arterial oxygenation, body temperature and electrophysiological (e.g. somatosensory evoked potentials, magnetically-evoled inter-enlargement recordings) monitoring capability during the acute recovery period after experimental neurotrauma. In addition, we request support to establish a Viral Vectors Component and a Genetically-Modified Mice Component. These latter two capabilities currently exist within individual SCoBIRC laboratories (Dr. George Smith's lab in the former instance and Dr. Geddes' in the latter case). However, the increasing need for the application of these across multiple laboratories has made it sensible to now expand and incorporate them under the umbrella of our Animal Modeling Core in order to improve access to them and to insure technical standardization across user laboratories.

DESCRIPTION (provided by applicant): Alzheimer's disease (AD) manifests severe pathological changes in the CNS including increased levels of amyloid, hyperphosphorylated tau, and synaptic loss. Synaptic dysfunction is a hallmark of the disease that associates with the cognitive ability and level of dementia during the progression of AD. It is unclear why synaptic numbers are reduced in the early stages of AD and how it is linked to other features of the pathology. We believe that oxidative damage and microtubule/actin changes are early events in the progression of AD and underlie synaptic dysfunction. Increasing evidence suggests that the medial temporal lobe (MTL) is the earliest regions of the brain affected and may provide important clues to the progression of the disease. Our hypothesis is that multiple different cellular changes occur in the MTL initiating the loss of synaptic plasticity resulting in a declinein cognition and the onset of clinical AD. The proposed experiments will evaluate changes in this brain region in regards to synaptic proteins, oxidative stress, and structural proteins. Studies ar carried out on short post mortem samples from longitudinally followed individuals with detailed cognitive testing. Individuals with amnestic mild cognitive impairment (aMCI) will be compared to individuals that clinically show no cognitive impairment (NCI). The NCI group is further classified as individuals with very low pathology (LP- NCI) or high (AD levels) of histopathology (HP-NCI). Current literature suggests that HP-NCI represents individuals with preclinical AD. Aim one assess the direct relationship between different key synaptic proteins and oxidative stress in the MTL. Aim two probes whether or not NADPH-oxidase (NOX) activity and its subunits change during the disease progression and how it associates with changes in synaptic proteins and soluble A beta. The NOX enzyme is normally expressed throughout the central nervous system and is a key non-mitochondrial source of free radicals. The third aim explores whether or not key cytoskeletal proteins, such as the actin binding protein cofilin and tau, increase in the MTL and alters different levels of key synaptic proteins. Successful completion of the proposed studies will reveal new insights into the mechanisms underlying the very early stages in the progression of AD and contribute to the development of rational therapies.