Elucidating and Pharmacologically Targeting Secondary Injury Cascades following Neural Injury

3.4 million concussions occur each year in the United States. Recent evidence suggests that some of these individuals are susceptible to neurodegenerative disease development following traumatic brain injury. The unknown factor is how acute injury contributes to this degenerative process. A prominent neurotrauma related neurodegenerative disease is chronic traumatic encephalopathy (CTE). CTE is defined by neurofibrillary tau tangles with a perivascular distribution and mood disturbances. In order to elucidate the pathologic changes associated with CTE, it is imperative to utilize adequate preclinical models. We have strategically developed and tested a clinically relevant rodent blast model. The model reliably produces a CTE phenotype including tauopathy, cell death, impulsivity, and cognitive decline. Using this validated model, we investigated several important secondary injury cascades that link acute brain injury to chronic neurodegenerative changes. More importantly, we pharmacologically targeted these pathways and found improved pathologic and behavioral outcomes. In chapter 1, we discuss the potential mechanisms linking acute injury to CTE in athletes and soldiers. In chapter 2, we highlight the physics behind the compression wave produced by our model and how this wave produces injury. In chapter 3, data is presented regarding the CTE phenotype generated by our model following repetitive blast exposure in rodents. Chapter 4 focuses on the role of blood brain barrier disruption and how targeting protein kinase C activity with bryostatin reduces this disruption. In chapter 5, we look at the role endoplasmic reticulum stress plays in human pathologic specimens from patients diagnosed with CTE and in rodents following repeat blast. We found that docosahexaenoic acid successfully targeted endoplasmic reticulum stress, reduced tauopathy, and improved cognitive performance. In chapter 6, we looked at lipoic acid and its role in reducing NADPH oxidative stress following repetitive neurotrauma. We found that lipoic acid reduces impulsive-like behavior and decreased cell death. Finally, in chapter 7 we discuss important strategies for improving preclinical models going forward and what needs to be investigated to improve our understanding of CTE. In this dissertation, we highlight important secondary injury cascades including blood brain barrier disruption, protein kinase C activity, endoplasmic reticulum stress, and oxidative stress that warrant further investigation for the development of novel treatment approaches for CTE

Kinetic modeling of amyloid fibrillation and synaptic plasticity as memory loss and formation mechanisms

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, 2008.Includes bibliographical references (p. 141-150).The principles of biochemical kinetics and system engineering are applied to explain memory-related neuroscientific phenomena. Amyloid fibrillation and synaptic plasticity have been our focus of research due to their significance. The former is related to the pathology of many neurodegenerative diseases and the later is regarded as the principal mechanism underlying learning and memory. Claimed to be the number one cause of senile dementia, Alzheimer's disease (AD) is one of the disorders that involve misfolding of amyloid protein and formation of insoluble fibrils. Although a variety of time dependent fibrillation data in vitro are available, few mechanistic models have been developed. To bridge this gap we used chemical engineering concepts from polymer dynamics, particle mechanics and population balance models to develop a mathematical formulation of amyloid growth dynamics. A three-stage mechanism consisting of natural protein misfolding, nucleation, and fibril elongation phases was proposed to capture the features of homogeneous fibrillation responses. While our cooperative laboratory provided us with experimental findings, we guided them with experimental design based on modeling work. It was through the iterative process that the size of fibril nuclei and concentration profiles of soluble proteins were elucidated. The study also reveals further experiments for diagnosing the evolution of amyloid coagulation and probing desired properties of potential fibrillation inhibitors. Synaptic plasticity at various time ranges has been studied experimentally to elucidate memory formation mechanism. By comparison, the theoretical work is underdeveloped and insufficient to explain some experiments. To resolve the issue, we developed models for short-term, long-term, and spike timing dependent synaptic plasticity, respectively.(cont.) First, presynaptic vesicle trafficking that leads to the release of glutamate as neurotransmitter was taken into account to explain short-term plasticity data. Second, long-term plasticity data lasting for hours after tetanus stimuli has been matched by a calcium entrapment model we developed. Model differentiation was done to demonstrate the better performance of calcium entrapment model than an alternative bistable theory in fitting graded long-term potentiation responses. Finally, to decipher spike timing dependent plasticity (STDP), we developed a systematic model incorporating back propagation of action potential, dual requirement of NMDA receptors, and calcium dependent plasticity. This built model is supported by five different types of STDP experimental data. The accumulation of amyloid beta has been found to disrupt the sustainable modification of long-term synaptic plasticity which might explain the inability of AD patients to form new memory at early stage of the disease. Yet the linkage between the existence of amyloid beta species and failure of long-term plasticity was unclear. We suggest that the abnormality of calcium entrapment function caused by amyloid oligomers is the intermediate step that eventually leads to memory loss. Unsustainable calcium level and decreased postsynaptic activities result into the removal or internalization of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. The number of AMPA receptors as the indicators of synaptic strength may result into disconnection between neurons and even neuronal apoptosis. New experiments have been suggested to validate this hypothesis and to elucidate the pathology of Alzheimer's disease.by Chuang-Chung (Justin) Lee.Ph.D

2013 IMSAloquium, Student Investigation Showcase

This year, we are proudly celebrating the twenty-fifth anniversary of IMSA’s Student Inquiry and Research (SIR) Program. Our first IMSAloquium, then called Presentation Day, was held in 1989 with only ten presentations; this year we are nearing two hundred.https://digitalcommons.imsa.edu/archives_sir/1005/thumbnail.jp

Low-intensity blast-induced mild traumatic brain injury : linking blast physics to biological outcomes

Blast-induced mild traumatic brain injury (mTBI) is of particular concern among military personnel due to exposure to blast energy during military training and combat. The impact of primary low-intensity blast (LIB) mediated pathophysiology upon later neurobehavioral disorders has been controversial. Our prior considerations of blast physics predicted ultrastructural injuries at nanoscale levels. Here, we provide quantitative data using a LIB injury murine model exposed to open-field detonation of 350 g of high-energy explosive C4. The use of an open-field experimental blast generated a primary blast wave with a peak overpressure of 6.76 pounds per square inch (PSI) (46.6 kPa) at a 3-meter (m) distance from the center of the explosion, with no apparent impact / acceleration in exposed animals. We first characterized neuropathological and behavioral changes. Using transmission electron microscopy (TEM), we further identified multifocal neuronal damages, myelin sheath defects, mitochondrial abnormalities, and synaptic dysregulation after LIB injury. Next, we used quantitative proteomics, bioinformatics analysis, biochemical investigations to seek insights into the molecular mechanisms underlying the ultrastructural pathology. Results illustrated the alterations of mitochondrial, axonal, synaptic proteins in related signaling pathways. These observations uncover unique ultrastructural brain abnormalities, biochemical correlates, and associated behavioral changes due to LIB injury. Insights on the early pathogenesis of LIB-induced brain damages provide a template for further characterization of its chronic effects, identification of potential biomarkers and targets for intervention.Includes bibliographical reference

DESIGN, SYNTHESIS, AND PHARMACOLOGICAL EVALUATION OF A SERIES OF NOVEL, GUANIDINE AND AMIDINE-CONTAINING NEONICOTINOID-LIKE ANALOGS OF NICOTINE: SUBTYPE-SELECTIVE INTERACTIONS AT NEURONAL NICOTINIC-ACETYLCHOLINE RECEPTOR.

The current project examined the ability of a novel series of guandine and amidine-containing nicotine analogs to interact with several native and recombinantlyexpressed mammalian neuronal nicotinic-acetylcholine receptor (nAChR) subtypes. Rational drug design methods and parallel organic synthesis was used to generate a library of guanidine-containing nicotine (NIC) analogs (AH compounds). A smaller series of amidine-containing nicotine analogs (JC compounds) were also synthesized. In total, \u3e150 compounds were examined. Compounds were first assayed for affinity in a high-throughput [3H]epibatidine radioligand-binding screen. Lead compounds were evaluated in subtype-selective binding experiments to probe for affinity at the α4β2* and α7* neuronal nAChRs. Several compounds were identified which possess affinity and selectivity for the α4β2* subtype [AH-132 (Ki=27nm) and JC-3-9 (Ki=11nM)]. Schild analysis of binding suggests a complex one-site binding interaction at the desensitized high-affinity nAChR. Whole-cell functional fluorescence (FLIPR) assays revealed mixed subtype pharmacology. AH-compounds were identified which act as activators and inhibitors at nAChR subtypes, while lead JC-compounds were found which possess full agonist activity at α4β2* and α3β4* subtypes. Compounds were identified as partial agonists, full agonists and inhibitors of multiple nAChR subtypes. Several SAR-based, ligand-receptor pharmacophore models were developed to guide future ligand design. Second-generation lead compounds were identified

Behavioral and histochemical characterization of a novel BACE Knockout x PDAPP mouse model of Alzheimer's Disease: examination of potential effects of BACE inhibition on Alzheimer's Disease and the role of APP, Aβ and BACE in normal and pathological memory function

This dissertation describes the phenotypic characterization of a BACE knockout (KO) x PDAPP transgenic mouse line, utilizing behavioral, histochemical, and pharmacologic methods. Overproduction and accumulation of the amyloid-|3 (A|3) peptide in the brain has been implicated as one of the causal factors in the development of Alzheimer's Disease (AD). Based on this concept, several transgenic mouse models have been created that overexpress human mutant Amyloid Precursor Protein (hAPP) that reproduces many of the cognitive and histopathological features of AD. Recently, the (3-site cleaving enzyme (BACE) responsible for the first proteolytic cleavage of APP has been characterized, and subsequent research has led to the propagation of BACE inhibition as a prime experimental strategy for AD therapy.Currently, there are many academic and pharmaceutical company laboratories actively engaged in developing therapeutic inhibitors of BACE for AD. However, the theoretical repercussions of BACE activity reduction have not yet been fully addressed in an in vivo model. Indeed, although overproduction of A|3 leads to neuroanatomical and cognitive pathology in human patients and animal models, lack ofA|3 may also result in deleterious cognitive effects. Examining the behavioral and histological phenotypes of BACE KO animals on normal and hAPP overexpressing backgrounds is an effective way to assess whether the inhibition of BACE is a reasonable strategy for the treatment of AD.To examine this issue a series of behavioral studies were conducted using homozygous and hemizygous BACE KO mice, PDAPP mice, and BACE KO; PDAPP lines together with relevant controls. The studies included various protocols in a cued and spatial watermaze task and detailed analysis of the occurrence of epileptiform seizures. Objective methods were used to analyse the changes in learning ability and the frequency of seizures.The results from the characterization of the BACE KO x PDAPP mouse line indicate that the absolute loss of BACE and A|3 caused profound spatial memory deficits, sometimes greater even than that of hAPP mice alone. In addition, absolute BACE KO was associated with spontaneous seizures as well as greater seizure activity in drug-induced seizure experiments. However, the partial hemizygous deletion of the BACE gene on a hAPP background appeared to improve spatial memory performance on certain measures and protect against drug-induced seizure responses relative to hAPP mice. The research described in this dissertation is consistent with the notion that, under certain circumstances, therapeutic inhibition of BACE may prove to be a valuable strategy for treatment of AD. In addition, these studies also support an important role for the [3-amyloid processing pathway in "normal" learning and memory processes, possibly by regulating neuronal activity levels

Understanding the brain through its spatial structure

The spatial location of cells in neural tissue can be easily extracted from many imaging modalities, but the information contained in spatial relationships between cells is seldom utilized. This is because of a lack of recognition of the importance of spatial relationships to some aspects of brain function, and the reflection in spatial statistics of other types of information. The mathematical tools necessary to describe spatial relationships are also unknown to many neuroscientists, and biologists in general. We analyze two cases, and show that spatial relationships can be used to understand the role of a particular type of cell, the astrocyte, in Alzheimer's disease, and that the geometry of axons in the brain's white matter sheds light on the process of establishing connectivity between areas of the brain. Astrocytes provide nutrients for neuronal metabolism, and regulate the chemical environment of the brain, activities that require manipulation of spatial distributions (of neurotransmitters, for example). We first show, through the use of a correlation function, that inter-astrocyte forces determine the size of independent regulatory domains in the cortex. By examining the spatial distribution of astrocytes in a mouse model of Alzheimer's Disease, we determine that astrocytes are not actively transported to fight the disease, as was previously thought. The paths axons take through the white matter determine which parts of the brain are connected, and how quickly signals are transmitted. The rules that determine these paths (i.e. shortest distance) are currently unknown. By measurement of axon orientation distributions using three-point correlation functions and the statistics of axon turning and branching, we reveal that axons are restricted to growth in three directions, like a taxicab traversing city blocks, albeit in three-dimensions. We show how geometric restrictions at the small scale are related to large-scale trajectories. Finally we discuss the implications of this finding for experimental and theoretical connectomics

Finding the pathology of major depression through effects on gene interaction networks

The disease signature of major depressive disorder is distributed across multiple physical scales and investigative specialties, including genes, cells and brain regions. No single mechanism or pathway currently implicated in depression can reproduce its diverse clinical presentation, which compounds the difficulty in finding consistently disrupted molecular functions. We confront these key roadblocks to depression research - multi-scale and multi-factor pathology - by conducting parallel investigations at the levels of genes, neurons and brain regions, using transcriptome networks to identify collective patterns of dysfunction. Our findings highlight how the collusion of multi-system deficits can form a broad-based, yet variable pathology behind the depressed phenotype. For instance, in a variant of the classic lethality-centrality relationship, we show that in neuropsychiatric disorders including major depression, differentially expressed genes are pushed out to the periphery of gene networks. At the level of cellular function, we develop a molecular signature of depression based on cross-species analysis of human and mouse microarrays from depression-affected areas, and show that these genes form a tight module related to oligodendrocyte function and neuronal growth/structure. At the level of brain-region communication, we find a set of genes and hormones associated with the loss of feedback between the amygdala and anterior cingulate cortex, based on a novel assay of interregional expression synchronization termed "gene coordination". These results indicate that in the absence of a single pathology, depression may be created by dysynergistic effects among genes, cell-types and brain regions, in what we term the "floodgate" model of depression. Beyond our specific biological findings, these studies indicate that gene interaction networks are a coherent framework in which to understand the faint expression changes found in depression and complex neuropsychiatric disorders