Vassilis Koliatsos, MD - US grants

In Project 3, we use a number of experimental approaches: to understand and possibly precipitate disease in Mo/Hu-APPswe transgenic (Tg) mice prior to the onset of amyloid deposition; and to influence the course and severity of amyloid deposition. In the first approach (Specific Aim 1 and 2), we hypothesize that mutant APP has adverse effects on the functional reserve (plasticity) of brain cholinergic, serotoninergic, and noradrenergic systems important for cognition and memory, and this effect explains the early deficits in cognitive performance of Mo/Hu-APPswe Tg mice (well before the onset of beta-amyloid protein [Abeta] deposits). To demonstrate deficits in plasticity, we challenge cholinergic and monoaminergic systems with surgical or neurotoxic injury or provoke them to respond to appropriate trophic factors. We predict that these systems in Mo/Hu-APPswe Tg mice will show reduced responsiveness to lesions and trophic stimulation that is characteristic of neurons in aged animals ("premature aging"). In the second approach (Specific Aims 3 and 4), our goal is to influence the course of amyloidogenesis in the brains of Mo/Hu-APPswe Tg mice by employing strategies to accelerate/enhance (i.e., via microglial stimulation with lipopolysaccharides or prevent (i.e., via facilitation of non-amyloidogenic APP processing by estrogens) Abeta deposition. Because inflammation and sex steroids are increasingly seen as major modifiers in amyloidogenesis related to AD, outcomes of research in Specific Aims 3 and 4 are likely to have significant clinical implications. In concert, Project 3 utilizes a number of interventions to uncover/precipitate/modify disease associated with Mo/Hu-APPswe mutation in vivo.

DESCRIPTION: (Verbatim from the Applicant's Abstract) Transsynaptic degeneration of corticocortical pathways is a central mechanism for the propagation of pathology an cell death in systems degenertions involving the cerebral cortex, including Alzheimer's disease. We propose that this type of degeneration may represent an appropriate and realistic target of pathogenic events in these disorders. In the present proposal, we focus on a very reliable in vivo model of transsynaptic cortical degeneration that we have recently developed, i.e., the apoptotic death of pyramidal neurons in the piriform cortex after their disconnection from the olfactory bulb. We investigate specific cellular/molecular events subsequent to bulbotomy such as excitotoxic-type alterations of distal dendrites of deafferented neurons, NOS/No signaling impacting on these neurons; and cell cycle and death genes such as Cyclin D1 and bax. To confirm data from anatomical/expression experiments, we use a number of pharmacological interventions, including: protein synthesis inhibitors that influence apoptosis but not excitotoxicity; small molecules that target glutamate synthesis, release and binding to block excitotoxicity and, in so doing, ameliorate of abolish apoptotic cortical cell death; NOS inhibitors/NO scavengers; cyclin-dependent kinase inhibitors to block cyclin signaling; and caspase inhibitors. We also use mice with genetic advantages that prevent apoptotic neuronal death (e.g., nNOS nulls, bax nulls, ICE-dominant negative Tgs). We expect that these carefully controlled investigations will shed light on critical intermediate mechanisms of cortical degenerations and will suggest clinically advantageous methods to treat these disorders.

DESCRIPTION (provided by applicant): Motor neuron diseases, including ALS and SMA, are featured by degeneration and death of lower motor neurons. Significant progress in genetics has allowed for the construction of spectacular transgenic models, but the basic pathogenic mechanisms of these illnesses remain unknown and there are no disease-modifying treatments. Cell death prevention strategies, including the use of trophic factors and small neuroprotective molecules, have had very limited clinical success. Perhaps the greatest promise lies in cell replacement strategies, based on our preliminary findings that exogenous neural stem cells (NSCs) can become avidly engrafted in the adult rat spinal cord and give rise to cells with clinically relevant phenotypes, i.e. neurons and ensheathing cells. Encouraged by these findings, we propose a stepwise approach to ensure that rodent and human NSCs differentiate into neurons and glia when transplanted at the sites of degenerated motor neurons in rat spinal cord after excitotoxic applications and in the ventral horn of transgenic animals which show the clinical features of ALS, i.e. SOD1 transgenic rodents. We are interested in the ability of NSC-derived neurons, suggested by our preliminary findings, to receive excitatory and inhibitory innervation and extend axons to ventral roots towards muscle targets. We expect that the restitution of the degenerating neuromuscular units will improve muscle strength in SOD1 transgenic animals, as assessed by behavioral testing on motorized devices. In concert, we propose to examine the essential preclinical parameters for the consideration of NSCs as therapeutic tools for motor neuron disease.

DESCRIPTION (provided by applicant): Mechanisms of antidepressant drug actions are not clearly understood. In the present application, we propose that antidepressant drugs work via a classical trophic mechanism, i.e. proliferation of terminal branches (sprouting) of central monoamine axons in cortex and the limbic system. We also propose that the molecular signals mediating these structural effects are neurotrophins, e.g. for the serotonin system, the critical signal is brain-derived neurotrophic factor (BDNF). Our experimental design takes a stepwise approach using cellular and molecular methodologies, including transgenic mice. We first evaluate the ability of serotonin (5-HT)- and norepinephrine (NE)-promoting compounds to cause 5-HT and NE sprouting in selected areas of neocortex, hippocampus and the subcortical limbic system. In parallel, we evaluate the role of antidepressants in enhancing neurotrophin expression and transduction in the same areas, focusing on BDNF and 5-HT reuptake inhibitors/enhancersx We subsequently test whether strategies that abolish BDNF expression/binding (such as blocking BDNF with antibodies and scavenging peptides or partially eliminating BDNF with transgenic approaches) inhibit the effects of antidepressants on 5-HT fiber sprouting. Finally, we assess the significance of trophic mechanisms for the mediation of antidepressant effects using BDNF +/- mice in laboratory models of depression. In concert, our proposal pursues a novel hypothesis for the effects of antidepressant drugs which has implications for an understanding of mood disorders as disorders of blunted structural plasticity of central monoamine systems.

DESCRIPTION (provided by applicant): Schizophrenic patients show high-level olfactory deficits that may be caused, at least in part, by structural alterations in olfactory cortex and its connections. Our group has studied animal models relevant to the structural plasticity of the rodent primary olfactory (piriform) cortex for a decade and has established lesion and genetic models to study piriform cortex remodeling. The central hypothesis of this proposal is that BACE1- neuregulin 1 (NRG1)-ErbB4 signaling is critical for the ongoing remodeling of piriform cortex, primarily by affecting the induction and differentiation of basal forebrain neurogenic niches and that perturbations of these processes may cause some of the clinical manifestations of schizophrenia. The principal mechanism explored here is the activation and/or induced migration of GABAergic interneurons of layer I, especially evident after olfactory lesions. These interneurons then lay the groundwork for reconstitution of the pyramidal layer of piriform cortex. In Specific Aim 1, we establish the role of BACE1- NRG1 in the development and lesion- induced plasticity (remodeling) of piriform cortex. In Aim 2, we explore specific cellular and molecular mechanisms of piriform remodeling involving GABAergic interneurons and neurogenic niches in the basal forebrain. Together, experiments proposed here link NRG1 signaling pathways with GABAergic cortical interneurons in animal models relevant to schizophrenia. PUBLIC HEALTH RELEVANCE: Schizophrenia is a debilitating mental illness whose causes and mechanisms have been very difficult to decipher. A major problem in the past was the absence of animal models in which to alter the expression of genes or make lesions and then follow behaviors and study biological links (mechanisms) between interventions and outcomes. This significant obstacle is now starting to yield, with the discovery of susceptibility genes that contribute to the disease cause and the availability of transgenic animals with altered expression of these genes or their downstream molecular targets. In this application, we combine our traditional strengths in neural plasticity of the olfactory limbic system and in transgenic animal models and propose that at least a portion of psychotic symptoms may be modeled on the basis of a stalled plasticity of the olfactory cortex and its connections because of deficient signals in GABAergic cortical interneurons.

PROJECT SUMMARY Traumatic brain injury (TBI) is a very common problem with over 2 million new cases requiring medical attention annually in the US. A neuropathology that is widely shared among various types of TBI and across spectrum of severity is diffuse or traumatic axonal injury (TAI), a form of ultra-rapid deformation and disruption of axons caused by rotational acceleration and shearing of brain tissue. In this project we use a visual model of TAI and explore the hypothesis that the DLK-JNK signaling cascade, known to be activated in simple axotomy lesions like nerve crush, triggers downstream degenerative effects of TAI in neurons projecting in the optic nerve, i.e. retinal ganglion cells (RGCs). We ask three questions, in logical order: is DLK-JNK pathway activated in visual TAI? If so, does DLK-JNK activation trigger retrograde degenerative effects in RGCs as in simple axotomy models? And does blocking of the DLK-JNK pathway prevent or treat RGC degeneration and related system impairments such as disconnection with CNS targets and loss of vision? To inflict TBI, we use the impact acceleration model of diffuse TAI with which we have consistently produced primary optic nerve injury in our laboratories. Our experimental subjects are conditional knockout mice for key members of the dual leucine zipper kinase (DLK-JNK) cascade. We also employ CRISPR strategies to disrupt the initiating kinases of the DLK-JNK cascade, DLK and LZK, and also treat wild-type injured mice with the DLK inhibitor sunitinib. Our experimental readouts include kinase activation, cell death, axonal and terminal degeneration using conventional and high-resolution (CLARITY) neuroanatomical methods, and behavioral measures of visual acuity. Besides exploring the role of key members of the DLK-JNK signaling pathway in perikaryal and axonal degeneration in visual TAI, the proposed experiments provide proof of concept for the protective or therapeutic effect of DLK/LZK blockade in TAI. The project leverages the complementary strengths of our laboratories and the design and experimental methods are supported by extensive preliminary studies. In Specific Aim 1 that serves as the foundation of the proposal, we will determine whether TAI in the visual system is associated with activation of the DLK-JNK signaling cascade in RGCs and we will identify key signaling molecules. In Specific Aim 2, we will explore whether blocking JNK and DLK/LZK signaling with knockout/genome editing strategies or pharmacological inhibition prevents or aborts RGC perikaryal and axonal degeneration in visual TAI. In Specific Aim 3, we will determine whether blocking DLK/LZK activation with knockout/genome editing strategies or pharmacological approaches prevents or aborts neural system impairments (visual disconnection and dysfunction). Taken together, these experiments examine the role of the DLK-JNK pathway in neuronal degeneration, disconnection, and dysfunction following TAI in a model CNS system and explore novel protective and therapeutic strategies for diffuse TBI.

PROJECT SUMMARY In this application we propose that traumatic axonal injury (TAI) leads to an active axonopathy with the molecular features of Wallerian degeneration and that targeting some of the relevant signals may protect axons as well as brain systems. This hypothesis is based on recent findings in our laboratory showing that axonal breakdown after diffuse TAI depends on the activation of Sterile Alpha and TIR Motif Containing 1 (SARM1) signaling and that molecular interventions to block SARM1 lead to significant gains in preserving axons and rescuing functions/behaviors that rely on axonal integrity. The case of TAI is unique in the sense that that many axons are only partially injured and are potentially salvageable, therefore blocking Wallerian-type self-destruction may afford long-term neuroprotection and change the prognosis of traumatic brain injury. Our proposal is organized in three specific aims. In Aim 1 we establish that TAI in an index CNS tract, i.e. the corticospinal system, leads to progressive axonopathy with the molecular signatures of Wallerian degeneration, i.e. activation of SARM1. In Aim 2 we ask whether axonal protection by genetic or pharmacological blockade of SARM1 signaling in the injured corticospinal tract translate into protection at the systems level, i.e. prevention of retrograde atrophy of corticospinal neurons, preservation of corticospinal connectivity and rescue of CST-dependent motor skills. In Aim 3 we explore the synergistic role of the mitogen-activated protein kinase (MAPK) pathway, specifically signaling by the dual leucine zipper kinase (DLK) and related leucine zipper kinase (LZK), in corticospinal axonal degeneration following TAI. The MAPK pathway signals general neuronal responses to injury and there is evidence that specific members of the pathway cooperate with SARM1-related signals in triggering or affecting the outcome of Wallerian degeneration. To achieve the previous aims, we use a complement of molecular genetic tools including knockout mice, dominant negative strategies and genome editing with CRISPR-Cas9, metabolomic assessments, CLARITY-based high-resolution neuropathology, structural and functional connectivity markers, behavioral testing, and small molecules as probes for molecular targets and also as therapeutic agents (the NAMPT inhibitor FK866 that serves as indirect inhibitor of SARM1 and the pan-Aurora inhibitor tozasertib that blocks DLK/LZK signaling). In summary, here we explore specific molecular mechanisms related to Wallerian degeneration and, in the course of doing this, we establish molecular targets for potential pharmacological interventions in traumatic brain injury.