Julie C. Lauterborn - US grants

[unreadable] DESCRIPTION (provided by applicant): Fragile X syndrome (fraX), resulting from the loss of fragile X mental retardation protein (FMRP), is the most common cause of inheritable mental retardation. In addition to cognitive impairment, fraX is characterized by abnormal "stress-related" behaviors, and children with fraX have greater basal and stress-induced salivary levels of the adrenal hormone cortisol, as compared to unaffected siblings. These data suggest that hypothalamic-pituitary-adrenal (HPA) axis function is altered in fraX. A murine model of fraX has been developed that exhibits several features of this syndrome and holds promise for identifying the cellular and behavioral consequences of Fmr1 deletion. Work by the investigator has demonstrated that fragile X mental retardation 1 gene knockout (Fmr1-K0) mice also have greater responses to stress including elevated gene expression and glucocorticoid levels than do wild-type mice. These data indicate that Fmr1-KOs are exhibiting a hyper-stress response, similar to the phenotype in human fraX, yet more information is needed as to the extent the HPA axis is altered, the cellular basis of this dysfunction, and potential therapeutic targets. The goal of the proposed research is to obtain such information and lay the groundwork for understanding the contribution of an exaggerated stress response to cognitive impairment in fraX. Three aims are proposed. Specific Aim 1 will test the hypothesis that there is a generalized increase in HPA tone in fragile X mutants (Aims 1A & 1B), and that the disparity among genotypes is enhanced by chronic stress (Aim 1A). In Aim 1A, adrenocorticotropic hormone (ACTH) release and corticotropin-releasing factor receptor 1 (CRH-R1) mRNA levels will be analyzed at three ages (3 mo, 12 mo, 24 mo) in handled (unstressed) and stressed Fmr1-KO and WT mice. Aim 1B will examine basal, diurnal corticosterone fluctuations in Fmr1-KOs and WTs to determine if levels are altered as in fraX humans. Specific Aim 2 will test the hypothesis that immobilization stress will alter the subcellular compartmentalization of the glucocorticoid receptor in fraX mutants as compared to WT mice (Aim 2A) at the light microscopic level and, in particular, that there are greater levels of glucocorticoid receptor in nuclear fractions of cortical cells from Fmr1-KOs as compared to WTs under basal conditions and following stress (2B) using Western blot analysis. Specific Aim 3 will test the hypothesis that fraX mutant mice have an exaggerated stress-induced hyperthermic response, and that this is attenuated by antagonism of group I metabotropic glutamate receptor type 5 (mGluR5) function. The proposed research will build upon initial findings in the adult male Fmr1-KO to test the general hypothesis that in fraX there is a dysregulated HPA axis that leads to a heightened stress response, and that antagonism of the group I mGluR5 will reduce stress-related anxiety seen in this syndrome. Given that stress results in cognitive impairment and has been reported by some to increase dendritic spine densities, two attributes of fraX, studies on stress and the HPA axis in fraX may give valuable insight into the cause of mental retardation in this syndrome. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): Fragile X syndrome (fraX), resulting from the loss of fragile X mental retardation protein (FMRP), is the most common cause of inheritable mental retardation. In addition to cognitive impairment, fraX is characterized by abnormal "stress-related" behaviors, and children with fraX have greater basal and stress-induced salivary levels of the adrenal hormone cortisol, as compared to unaffected siblings. These data suggest that hypothalamic-pituitary-adrenal (HPA) axis function is altered in fraX. A murine model of fraX has been developed that exhibits several features of this syndrome and holds promise for identifying the cellular and behavioral consequences of Fmr1 deletion. Work by the investigator has demonstrated that fragile X mental retardation 1 gene knockout (Fmr1-K0) mice also have greater responses to stress including elevated gene expression and glucocorticoid levels than do wild-type mice. These data indicate that Fmr1-KOs are exhibiting a hyper-stress response, similar to the phenotype in human fraX, yet more information is needed as to the extent the HPA axis is altered, the cellular basis of this dysfunction, and potential therapeutic targets. The goal of the proposed research is to obtain such information and lay the groundwork for understanding the contribution of an exaggerated stress response to cognitive impairment in fraX. Three aims are proposed. Specific Aim 1 will test the hypothesis that there is a generalized increase in HPA tone in fragile X mutants (Aims 1A & 1B), and that the disparity among genotypes is enhanced by chronic stress (Aim 1A). In Aim 1A, adrenocorticotropic hormone (ACTH) release and corticotropin-releasing factor receptor 1 (CRH-R1) mRNA levels will be analyzed at three ages (3 mo, 12 mo, 24 mo) in handled (unstressed) and stressed Fmr1-KO and WT mice. Aim 1B will examine basal, diurnal corticosterone fluctuations in Fmr1-KOs and WTs to determine if levels are altered as in fraX humans. Specific Aim 2 will test the hypothesis that immobilization stress will alter the subcellular compartmentalization of the glucocorticoid receptor in fraX mutants as compared to WT mice (Aim 2A) at the light microscopic level and, in particular, that there are greater levels of glucocorticoid receptor in nuclear fractions of cortical cells from Fmr1-KOs as compared to WTs under basal conditions and following stress (2B) using Western blot analysis. Specific Aim 3 will test the hypothesis that fraX mutant mice have an exaggerated stress-induced hyperthermic response, and that this is attenuated by antagonism of group I metabotropic glutamate receptor type 5 (mGluR5) function. The proposed research will build upon initial findings in the adult male Fmr1-KO to test the general hypothesis that in fraX there is a dysregulated HPA axis that leads to a heightened stress response, and that antagonism of the group I mGluR5 will reduce stress-related anxiety seen in this syndrome. Given that stress results in cognitive impairment and has been reported by some to increase dendritic spine densities, two attributes of fraX, studies on stress and the HPA axis in fraX may give valuable insight into the cause of mental retardation in this syndrome. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Efforts to identify causes of memory and cognition deficits in Fragile X Syndrome (FXS) led to the discovery of synaptic plasticity impairments in cortex of a knock-out mouse model (Fmr1-KO) of the disorder. The applicants have extended these results by showing that hippocampal long-term potentiation (LTP), induced by learning-related patterns of afferent activity, fails to stabilize in Fmr1-KO mice. Analysis of cytoskeletal changes required for lasting LTP pointed to the hypothesis that a critical FXS-defect involves failed stabilization of new actin filaments during the first few minutes after LTP induction. Preliminary results showing abnormal expression of several actin-associated proteins in the knockouts (KOs) support this argument. Objectives of the proposed studies are to (1) identify causes for the failure in filamentous (F) actin stabilization and LTP in Fmr1-KO mice, and (2) develop treatments for normalizing cytoskeletal changes and stable LTP. Pilot studies have shown that treatment with the mGluR5 antagonist MPEP, or with a positive AMPA receptor modulator (ampakine), can restore stable LTP to Fmr1-KO hippocampus. Further results indicate that both drugs also reverse measures of aberrant spine morphology in the KOs. The proposed research will build on these findings in 4 specific aims. Aim 1 will test the hypothesis that MPEP can normalize stabilization of spine F-actin and LTP in hippocampal slices from adult Fmr1-KO mice. Further studies will test if LTP impairments are offset by translation inhibitors and linked to aberrant signaling by integrin-associated tyrosine kinases. Aim 2 will test if abnormal basal levels of actin regulatory proteins in Fmr1-KO dendritic spines lead to aberrations in TBS- induced signaling to the actin cytoskeleton. Studies will employ deconvolution immunofluorescent techniques to test effects of theta burst afferent stimulation on levels of target proteins in spines of KO and WT mice. Aim 3 will use acute slices to test if MPEP and ampakine treatments have additive or synergistic effects in the rescue of hippocampal LTP in Fmr1-KO mice (3A). Follow on acute slice experiments will test if the treatments that rescue LTP also normalize (3B) pyramidal cell spine measures and (3C) levels and activity-induced changes in spine actin-regulatory proteins in hippocampal field CA1. Studies in Aim 4 complement those in Aim 3 to test if drugs that rescue hippocampal LTP also restore stable potentiation (4A) and spine measures (4B) in slices from somatosensory neocortex of Fmr1-KO mice. Aim 4C will then test if in vivo treatments with an ampakine, MPEP, or both, normalize spine measures in somatosensory cortex and hippocampal field CA1. Aims 3 and 4 will use Fmr1-KO and WT mice that constitutively express yellow fluorescent protein (YFP) in scattered pyramidal cells to provide a bright label of dendritic spines. These studies are expected to produce a specific explanation for why spine plasticity and structure are disturbed by the Fragile X mutation, and to generate potential therapies for correcting the defects. PUBLIC HEALTH RELEVANCE: Efforts to identify causes of mental retardation associated with Fragile X Syndrome led to the discovery of synaptic plasticity impairments in a mouse model of the disorder. The present studies will test the hypothesis that impairments are due to abnormal levels of actin regulatory proteins, which are critical for changes in synaptic function during learning. Studies will also test potential therapeutics for correcting these synaptic defects that might improve learning in this syndrome and other autism spectrum disorders.

DESCRIPTION (provided by applicant): Spine disturbances are a common feature of intellectual developmental disorders (IDD) such as Down Syndrome (DS), which occurs in ~ 1 in 700 live births and is associated with an average IQ of 50. In DS, cortical dendritic spines are often described as appearing immature (long, thin and tortuous) and spine densities are reduced. The cellular mechanisms underlying these morphological disturbances are not known but likely involve dysregulation of the spine actin cytoskeleton that largely determines spine shape and is critical for plasticity underlying memory encoding. Our previous work has shown that function of one Rho-family GTPase pathway involved in regulating spine filamentous (F-) actin, the Rac cascade, is markedly disturbed in a mouse model of another IDD, Fragile X syndrome; this aligns with observations that several IDDs exhibit abnormalities in Rho GTPase pathways. However, it is not known the degree to which specific defects in spine Rho GTPase signaling are shared across IDDs or converge on the same down-stream proteins that directly regulate spine F-actin. In preliminary studies using fluorescence deconvolution microscopy, we evaluated two Rac pathway proteins, p21-activated kinase 3 (PAK3) and Arp2, in middle-aged human DS parietal cortex. The results demonstrate that levels of both proteins are reduced at excitatory, PSD95-immunopositive (+) synapses suggesting that the actin regulatory machinery is indeed disturbed in DS. The results also suggest the possibility that, like Fragile X syndrome, DS exhibits abnormalities specific to the Rac cascade that regulates the branching and stabilization of the spine actin network. The proposed studies will expand upon these findings and test the hypothesis that trisomy giving rise to DS leads to disturbances in the dendritic spine Rac GTPase cascade while leaving elements in the RhoA cascade relatively normal. Aim 1 studies will further test if abnormalities in actin regulatory proteins are present in DS individuas across a broad age range or preferentially at later ages, and if these perturbations are greatest in DS individuals with Alzheimer's Disease (AD) tau pathology. Findings will provide insight as to whether the actin signaling disturbances are core features of DS, or secondary to emergent AD pathology. Aim 2 will then test if abnormalities in Rac pathway proteins are present in the Ts65Dn mouse model of DS that exhibits both spine and synaptic plasticity abnormalities. Confirmation of this point is essential for the use of the Ts65Dn model in preclinical studies aimed at devising therapies to offset spine defects, and facilitate learning, in DS. Pertinent to this, we have shown that manipulation of signaling through several synaptic modulatory receptors can dramatically alter local actin regulatory signaling and, in some cases, restore normal actin remodeling, synaptic plasticity, and cognitive function in otherwise impaired animals. Thus, the proposed studies will determine if actin regulatory deficits are present in DS spines that might be similarly responsive to actin based strategies for cognitive enhancement.

PROJECT SUMMARY Initial stages of Alzheimer?s disease (AD) appear to be correlated with elevated electrical activity and synaptic abnormalities in brain regions first affected by pathology. This pathologically shifting towards excitation suggests that there are alterations in the synaptic excitation and inhibition balance (E/I ratio) within these areas (e.g., entorhinal cortex), which in turn may accelerate activity-dependent AD pathology. However, there are no quantitative, regional measurements of the E/I ratio in the human brain, and alterations in this measure in AD are unknown. In preliminary work leading to this proposal, we have found evidence of inhibitory signaling disturbances at early stages of AD. Levels of Gephyrin expression, an inhibitory postsynaptic synaptic density (iPSD) protein, are reduced in entorhinal cortex neuronal cell bodies of postmortem brain from donors diagnosed with mild cognitive impairment (MCI), a prodromal stage of AD. In addition, by microtransplanting receptors from temporal cortices of human AD donors into Xenopus oocytes, we discovered electrophysiological abnormalities of GABA receptors (GABAARs) suggesting that inhibitory tone is reduced in AD. Importantly though, it is not known whether these collective alterations also occur at the level of synapses in AD or if they are emergent in MCI. Given these preliminary findings, we hypothesize that 1) MCI is characterized by abnormally large E/I ratios in brain regions particularly affected early on in AD and 2) E/I ratio imbalance is driven by impairment in the clustering of synaptic excitatory or inhibitory receptors, or by alteration of the electrophysiological properties of major synaptic glutamate and GABA receptors (GluRs and GABAARs). This general hypothesis will be evaluated in two Specific Aims. Aim 1 will test whether there are specific pro- excitatory alterations in the ratio of excitatory to inhibitory postsynaptic density (ePSD/iPSD) proteins in MCI and AD versus controls. Studies will use Fluorescence Deconvolution Tomography (FDT), developed by part of our research group, whereby immunolabeling of PSD markers are measured within the size constraints of synapses from 3D reconstructions; FDT analysis will determine if pro-excitatory E/I ratios based on counts, volume, and intensity of synaptic markers characterize and differentiate MCI from control and AD cases. Complementing the anatomical work, Aim 2 will test whether electrophysiological alterations of synaptic receptors contribute to larger E/I ratio in MCI and AD using the Microtransplantation of Synaptic Membranes (MSM), a novel technique that allows for electrophysiological studies of GluRs and GABAARs from postmortem human brain tissue. Understanding the degree to which abnormal synaptic E/I ratios are present in MCI and/or AD, and which PSD receptors or proteins are affected, would greatly facilitate targeted pharmacological interventions aimed at restoring E/I balance and may provide substantial benefit to patients showing early signs of cognitive decline by delaying or stopping the progression to AD which currently affects ~5.3 million Americans.