Osteopontin Expression During the Acute Immune Response Mediates Reactive Synaptogenesis and Adaptive Outcome

Traumatic brain injury (TBI) is a worldwide epidemic as the number of victims living with the resulting cognitive and physical impairment continues to rise, principally due to limited treatment options which fail to address its multifaceted sequelae. By approaching TBI therapy from a molecular standpoint, we have the opportunity to develop a better understanding of the mechanisms which prevent effective recovery. With this information, we can move toward the identification of novel therapeutic treatments which target specific molecules to improve patient outcome following TBI. Here, we have focused on the therapeutic potential of osteopontin (OPN), an extracellular matrix (ECM) protein which is a substrate of several matrix metalloproteinases (MMPs), and capable of acting as both a cytokine and modulator of axonal outgrowth during synaptic recovery. The ECM and its components are of particular interest with respect to selecting novel TBI therapeutics since this network has been implicated in neuronal plasticity during both development and following central nervous system (CNS) insult. In this dissertation study, the temporal and spatial profile of OPN expression, its protein and transcript localization within reactive glia (IBA1 positive microglia or GFAP positive astroglia), and its interaction with the cytoarchitectural protein (microtubule associated protein 1B, MAP1B) after injury were each compared under conditions of deafferentation induced synaptogenesis. Two TBI models were employed: one exhibiting adaptive synaptic plasticity (unilateral entorhinal cortex lesion, UEC), and the other generating maladaptive synaptic plasticity (central fluid percussion injury followed by bilateral entorhinal cortex lesions, TBI+BEC), in each case targeting 1, 2, and 7d postinjury intervals. In addition, we examined the potential for converting the adaptive response to one of maladaptive plasticity by attenuating immune reactivity through acute administration of the tricyclic antibiotic minocycline, utilizing a dosing paradigm previously demonstrated to reduce inflammation. To more clearly confirm that OPN has a role in successful synaptic regeneration, we developed a colony of OPN knockout (KO) mice which were used to profile synaptic structure and functional outcome under conditions of UEC-induced synaptogenesis. In Chapter 2, we report that full length OPN responds robustly in the acute (1-2d postinjury) degenerative period following UEC and TBI+BEC. After UEC, time-dependent differences were observed for two alternative, MMP-processed OPN forms, including early increase in a RGD 45 kD, integrin binding fragment (1d), and delayed increase in a C-terminal 32 kD OPN peptide (7d). OPN transcript was also elevated acutely after UEC, a finding which was pronounced in enriched dentate molecular layer (ML) fractions. Parallel immunohistochemistry (IHC) and in situ hybridization localized OPN protein and transcript to reactive glia following UEC. This localization was concentrated within microglia which delineated the border between the intact and deafferented ML, a pattern which was less pronounced in maladaptive TBI+BEC animals. The timing of this glial movement suggests that OPN regulates microglial migration and, potentially, could act as an astrokine to recruit activated astrocytes for influencing subsequent synaptic regeneration. MAP1B staining confirmed dendritic loss during axonal degeneration and dendritic atrophy, with a reemergence during collateral axonal sprouting. However, OPN colocalization with MAP1B was minimal, suggesting a minor role for OPN in reorganization of dendritic/axonal cytoarchitecture in this model of deafferentation. Minocycline reduced acute OPN protein response 2d after UEC, and caused a more random OPN positive glial distribution, similar to that of the maladaptive TBI+BEC. The role of OPN in the inflammation-directed degeneration of terminals is supported by reduced MMP-9 activity, which is temporally correlated with the reduction of MMP-generated OPN lytic fragments (45 kD). Interestingly, this reduction of integrin-binding OPN peptide also matched the impaired removal of presynaptic terminals, evidenced by diminished synapsin 1 clearance in animals which received postinjury minocycline. In Chapter 3, we sought to more precisely evaluate the role of OPN following deafferentation, utilizing wild type (WT) C57BL/6 and OPN KO mice subjected to UEC, comparing the spatio-temporal injury response between WT and KO. To do this we profiled several outcome measures which assessed OPN role in different aspects of recovery: 1) expression of select proteins important in various stages of synaptic recovery, 2) glial response, 3) cognitive recovery, and 4) MMP enzymatic activity. Compared to WT mice, OPN KO mice did not show significant differences in the acute injury-induced alteration of proteins important to cytoarchitectural reorganization (MAP1B) or stabilization of the synaptic junction (N-cadherin). However, both Western blot and IHC analyses showed OPN KO mice had impaired presynaptic terminal clearance, supported by attenuated synapsin 1 breakdown, a result quite similar to that of the minocycline-treated rats with OPN reduction in Chapter 2. This impaired degeneration in OPN KO mice at 2d postinjury correlated with IHC evidence for altered microglial morphology, and hippocampal function assessed by the novel object recognition (NOR) task. Our NOR results confirmed cognitive dysfunction in OPN KO mice during the 4-21d period of synapse reorganization after UEC. In addition, OPN KO decreased MMP-9 activity, an effect associated with reduced MMP-9 bound lipocalin 2 (LCN2), a persistently activated form of that MMP. These latter findings further support the hypothesis that MMP processing of OPN contributes to effective regenerative response after injury. Collectively, the studies presented in the two chapters of this dissertation provide evidence that OPN is a critical element in the acute immune response following injury-induced CNS deafferentation. They suggest that the cytokine can be produced by reactive microglia, may mediate cell migration and acute degenerative clearance, potentially serves as an astrokine to recruit those glia to sites of synaptic repair, and that these processes are disrupted when OPN is either reduced or ablated. Interestingly, this OPN role in synaptogenesis appears to involve ECM interaction with MMP-9, possibly regulated by LCN2. Most importantly, OPN involvement seems to affect the time-dependent progression of synaptic repair, an effect which can be measured by efficacy of functional outcom

Mechanisms Mediating Adaptive Presynaptic Muting Induction

Neurons are responsible for information processing within the nervous system, so strong perturbations of neuronal function have far-reaching consequences within the neural network. Damage in response to excess excitation, as occurs during stroke or seizure, is known as excitotoxicity. One method utilized by neurons for reducing excitotoxicity within an overly activated neuronal network is to arrest excitatory neurotransmitter release from presynaptic terminals. The mechanisms responsible for inducing this presynaptic silencing: or muting ), however, have been elusive. In order to elucidate the signals responsible, I used molecular techniques in defined networks of cultured neurons from the mammalian hippocampus, a well-studied brain region known to be important for learning and memory but susceptible to excitotoxic damage. Calcium serves as a signal transducer during excitotoxicity and many forms of synaptic plasticity, but the signaling cascades in presynaptic silencing were previously unknown. In neurons individually depolarized via heterologous ion channel activation, I showed that calcium influx led to cell death while channel expression led to synaptic depression, although muting was not confirmed. Calcium, however, was not necessary for presynaptic muting after strong depolarization. Instead, inhibitory G-protein signaling induced silencing through cyclic adenosine monophosphate: cAMP) reduction but surprisingly not via activation of likely candidate receptors. This cAMP reduction contributed to loss of proteins important for vesicle fusion at the presynaptic terminal. I also found that astrocytes, support cells in the nervous system that have garnered attention recently for their ability to modulate neuronal function, were required for the proper development of presynaptic muting in hippocampal neurons. Soluble factors released by astrocytes were permissive, but not instructive, for silencing induction. Thrombospondins were identified as the astrocyte-derived factors responsible for muting competence in neurons, and they act through binding to the a2d-1 subunit of voltage-gated calcium channels. cAMP-activated protein kinase A exhibited dysfunctional behavior in the absence of thrombospondins, potentially explaining the presynaptic muting deficit in an astrocyte-deficient environment. Together these results clarify the molecular mechanisms responsible for an underappreciated form of neuroprotective synaptic plasticity and provide potential therapeutic targets for a number of disorders expressing excitotoxic damage

Human metabolic adaptations and prolonged expensive neurodevelopment: A review

1.	After weaning, human hunter-gatherer juveniles receive substantial (≈3.5-7 MJ day^-1^), extended (≈15 years) and reliable (kin and nonkin food pooling) energy provision.
2.	The childhood (pediatric) and the adult human brain takes a very high share of both basal metabolic rate (BMR) (child: 50-70%; adult: ≈20%) and total energy expenditure (TEE) (child: 30-50%; adult: ≈10%).
3.	The pediatric brain for an extended period (≈4-9 years-of-age) consumes roughly 50% more energy than the adult one, and after this, continues during adolescence, at a high but declining rate. Within the brain, childhood cerebral gray matter has an even higher 1.9 to 2.2-fold increased energy consumption. 
4.	This metabolic expensiveness is due to (i) the high cost of synapse activation (74% of brain energy expenditure in humans), combined with (ii), a prolonged period of exuberance in synapse numbers (up to double the number present in adults). Cognitive development during this period associates with volumetric changes in gray matter (expansion and contraction due to metabolic related size alterations in glial cells and capillary vascularization), and in white matter (expansion due to myelination). 
5.	Amongst mammals, anatomically modern humans show an unique pattern in which very slow musculoskeletal body growth is followed by a marked adolescent size/stature spurt. This pattern of growth contrasts with nonhuman primates that have a sustained fast juvenile growth with only a minor period of puberty acceleration. The existence of slow childhood growth in humans has been shown to date back to 160,000 BP. 
6.	Human children physiologically have a limited capacity to protect the brain from plasma glucose fluctuations and other metabolic disruptions. These can arise in adults, during prolonged strenuous exercise when skeletal muscle depletes plasma glucose, and produces other metabolic disruptions upon the brain (hypoxia, hyperthermia, dehydration and hyperammonemia). These are proportional to muscle mass.
7.	Children show specific adaptations to minimize such metabolic disturbances. (i) Due to slow body growth and resulting small body size, they have limited skeletal muscle mass. (ii) They show other adaptations such as an exercise specific preference for free fatty acid metabolism. (iii) While children are generally more active than adolescents and adults, they avoid physically prolonged intense exertion. 
8.	Childhood has a close relationship to high levels of energy provision and metabolic adaptations that support prolonged synaptic neurodevelopment. 
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The Tenets of Teneurin: Conserved Mechanisms Regulate Diverse Developmental Processes in the Drosophila Nervous System

To successfully integrate a neuron into a circuit, a myriad of developmental events must occur correctly and in the correct order. Neurons must be born and grow out toward a destination, responding to guidance cues to direct their path. Once arrived, each neuron must segregate to the correct sub-region before sorting through a milieu of incorrect partners to identify the correct partner with which they can connect. Finally, the neuron must make a synaptic connection with their correct partner; a connection that needs to be broadly maintained throughout the life of the animal while remaining responsive to modes of plasticity and pruning. Though many intricate molecular mechanisms have been discovered to regulate each step, recent work showed that a single family of proteins, the Teneurins, regulates a host of these developmental steps in Drosophila – an example of biological adaptive reuse. Teneurins first influence axon guidance during early development. Once neurons arrive in their target regions, Teneurins enable partner matching and synapse formation in both the central and peripheral nervous systems. Despite these diverse processes and systems, the Teneurins use conserved mechanisms to achieve these goals, as defined by three tenets: (1) transsynaptic interactions with each other, (2) membrane stabilization via an interaction with and regulation of the cytoskeleton, and (3) a role for presynaptic Ten-a in regulating synaptic function. These processes are further distinguished by (1) the nature of the transsynaptic interaction – homophilic interactions (between the same Teneurins) to engage partner matching and heterophilic interactions (between different Teneurins) to enable synaptic connectivity and the proper apposition of pre- and postsynaptic sites and (2) the location of cytoskeletal regulation (presynaptic cytoskeletal regulation in the CNS and postsynaptic regulation of the cytoskeleton at the NMJ). Thus, both the roles and the mechanisms governing them are conserved across processes and synapses. Here, we will highlight the contributions of Drosophila synaptic biology to our understanding of the Teneurins, discuss the mechanistic conservation that allows the Teneurins to achieve common neurodevelopmental goals, and present new data in support of these points. Finally, we will posit the next steps for understanding how this remarkably versatile family of proteins functions to control multiple distinct events in the creation of a nervous system

Maternal Nutrient Restriction in Pregnant Guinea Pigs and the Impact on Fetal Growth and Brain Development

Maternal nutrient restriction (MNR) in guinea pigs results in placental structural abnormalities that reduce nutrient transport contributing to fetal growth restriction (FGR). However, whether brain weights are similarly reduced, or preserved by “brain sparing” mechanisms, and whether energy levels are depleted leading to membrane failure and overt injury remains unknown. Guinea pig sows were fed ad libitum (Controls) or 70% of the control diet pre-pregnant switching to 90% at mid-pregnancy (MNR). Animals were necropsied near term for fetal growth measures and fetal brains were assessed for markers of necrotic cell injury, apoptotic cell injury, endoplasmic reticulum stress, and altered development proteins. MNR resulted in FGR with brains that are large relative to body weight and livers that are small relative to body weight, which suggests a degree of blood flow redistribution. These fetuses have reduced brain weights, but with substantial brain sparing, and with no increased necrotic cell injury and no changes in synaptic development, indicating that the threshold for membrane failure or aberrant development with energy depletion has likely not been reached. However, apoptotic indices were increased in FGR-MNR cohort compared to appropriate for gestational age (AGA)-control cohort and more so in males than females. Changes in apoptosis were primarily in hippocampal regions and were not accompanied by significant changes of protein levels of investigated pro-apoptotic factors

Development of variable and robust brain wiring patterns in the fly visual system

Precise generation of synapse-specific neuronal connections are crucial for establishing a robust and functional brain. Neuronal wiring patterns emerge from proper spatiotemporal regulation of axon branching and synapse formation during development. Several neuropsychiatric and neurodevelopmental disorders exhibit defects in neuronal wiring owing to synapse loss and/or dys-regulated axon branching. Despite decades of research, how the two inter-dependent cellular processes: axon branching and synaptogenesis are coupled locally in the presynaptic arborizations is still unclear. In my doctoral work, I investigated the possible role of EGF receptor (EGFR) activity in coregulating axon branching and synapse formation in a spatiotemporally restricted fashion, locally in the medulla innervating Dorsal Cluster Neuron (M- DCN)/LC14 axon terminals. In this work I have explored how genetically encoded EGFR randomly recycles in the axon branch terminals, thus creating an asymmetric, non-deterministic distribution pattern. Asymmetric EGFR activity in the branches acts as a permissive signal for axon branch pruning. I observed that the M-DCN branches which stochastically becomes EGFR ‘+’ during development are synaptogenic, which means they can recruit synaptic machineries like Syd1 and Bruchpilot (Brp). My work showed that EGFR activity has a dual role in establishing proper M-DCN wiring; first in regulating primary branch consolidation possibly via actin regulation prior to synaptogenesis. Later in maintaining/protecting the levels of late Active Zone (AZ) protein Brp in the presynaptic branches by suppressing basal autophagy level during synaptogenesis. When M-DCNs lack optimal EGFR activity, the basal autophagy level increases resulting in loss of Brp marked synapses which is causal to increased exploratory branches and post-synaptic target loss. Lack of EGFR activity affects the M-DCN wiring pattern that makes adult flies more active and behave like obsessive compulsive in object fixation assay. In the second part of my doctoral work, I have asked how non-genetic factors like developmental temperature affects adult brain wiring. To test that, I increased or decreased rearing temperature which is known to inversely affect pupal developmental rate. We asked if all the noisy cellular processes of neuronal assembly: filopodial dynamics, axon branching, synapse formation and postsynaptic connections scale up or down accordingly. I observed that indeed all the cellular processes slow down at lower developmental temperature and vice versa, which changes the DCN wiring pattern accordingly. Interestingly, behavior of flies adapts to their developmental temperature, performing best at the temperature they have been raised at. This shows that optimal brain function is an adaptation of robust brain wiring patterns which are specified by noisy developmental processes. In conclusion, my doctoral work helps us better understand the developmental regulation of axon branching and synapse formation for establishing precise brain wiring pattern. We need all the cell intrinsic developmental processes to be highly regulated in space and time. It is infact a combinatorial effect of such stochastic processes and external factors that contribute to the final outcome, a functional and robust adult brain