Christopher M. Norris - US grants

DESCRIPTION (provided by applicant): Our previous work indicates that protein phosphatase activity is increased in hippocampus of aged rats and contributes to age-related alterations in synaptic strength and cognition. The proposed studies investigate whether the protein phosphatase calcineurin interacts significantly with several other brain aging biomarkers, including increased Ca2+ channel function and altered gene expression. In each aim of this project, calcineurin activity in hippocampal cultures and in hippocampus of intact rats, is manipulated using recombinant viruses (adeno- and lentivirus). The first specific aim tests whether constitutively active calcineurin causes aging-like changes in voltage sensitive Ca2+ channels (VSCC) (i.e. an increase in L-type VSCC currents and a decrease in N-type VSCC mRNA levels). Molecular interactions of calcineurin with VSCCs also will be explored. The second aim tests whether increased calcineurin activity leads to aging-like changes in global gene expression, as assessed using gene microarrays. The specific role of the calcineurin/NF-AT transcriptional pathway will be explored using recombinant virus containing a potent NFAT inhibitor, VIVIT. The third aim uses recombinant viruses to test whether the calcineurin/NF-AT pathway contributes to age-related memory deficits on the Morris swim task. After completion of behavioral training, intact and partially dissociated hippocampal slices will be prepared from these rats to determine whether the calcineurin/NF-AT pathway also is responsible, in part, for age-related alterations in synaptic strength, plasticity (i.e. long-term potentiation and long-term depression), and L-type VSCC activity. Furthermore, individual neurons and glia will be harvested from zipper slices to examine single cell gene expression profiles using gene microarray technology.

Abstract Mounting evidence suggests that comorbid vascular contributions to cognitive impairment and dementia (VCID) may complicate the treatment of Alzheimer?s disease (AD)-related dementia. Our overarching hypothesis is that AD and VCID pathological sequelae converge at the level of activated astrocytes, providing a common druggable target for a wide range of dementias (i.e. AD alone, VCID alone, and mixed dementia). Central to this hypothesis is the Ca2+ -dependent phosphatase, calcineurin (CN), which appears at high levels in activated astrocytes and positively regulates multiple components of the activated astrocyte phenotype through direct interactions with NFAT transcription factors. We, and others have shown that hyperactivation of CN/NFAT occurs in astrocytes during early stages of cognitive decline in humans and mice with AD-like pathology and is linked to glutamate-dependent hyperexcitability. However, little is known about astrocytic CN/NFAT in VCID, presenting a critical knowledge gap in our understanding of mixed dementia. New preliminary data obtained from human cerebrovascular pathology cases and from an established diet-based VCID mouse model, suggest that hyperactive CN/NFAT signaling also arises with cerebrovascular pathology. Based on these observations, we predict that combined AD and VCID pathology will exacerbate aberrant CN/NFAT signaling leading to a ?neurotoxic? astrocyte phenotype, characterized by the loss of EAAT2/Glt-1 glutamate transporters and impaired glutamate uptake. We further predict that normalization of the CN/NFAT/Glt-1 axis, using cell-type specific AAV vectors and novel pharmacologic agents, will alleviate neuronal and cerebrovascular abnormalities in AD, VCID, and mixed AD/VCID models. Our overarching hypothesis and corollary predictions will be tested using human biospecimens and relevant mouse models including the hyperhomocysteinemia (HHcy) model of VCID and the 5xFAD model of A? pathology. Aim 1 will test the hypothesis that mixed AD and VCID pathologies in both humans and mice converge to exacerbate CN/NFAT hyperactivity in astrocytes, leading to a neurotoxic astrocyte molecular phenotype. Aim 2 will test the hypothesis that the astrocytic CN/NFAT/Glt-1 axis drives cerebrovascular dysfunction in AD, VCID, and mixed VCID/AD models. And Aim 3 will test the hypothesis that the astrocytic CN/NFAT/Glt-1 axis drives hyperexcitability, synapse dysfunction, and cognitive loss in AD, VCID, and mixed VCID/AD models. These aims will be pursued using novel reagents, and cutting-edge multidisciplinary approaches. This work is essential for assessing the role of astrocytic CN/NFATs in VCID and mixed pathology and may help stimulate the development of astrocyte-targeted approaches for treating a broad range of dementia cases.

7. Project Summary/Abstract Astrocytes are vital to brain metabolism and help to optimize synaptic function and promote neuronal health. Direct coupling of astrocytes to the extracellular milieu via unapposed hemichannels (uHCs)?made up of connexin 43 (Cx43) proteins?appears to be increased under a variety of pathological conditions. Elevated levels of amyloid-? (A?) peptides and pro-inflammatory cytokines, characteristic of Alzheimer?s disease (AD), lead to greater uHC permeability?which, in turn, impairs neuronal function and viability. In this project, we will test the novel hypothesis that A?-dependent changes in uHCs are mediated, in part, by aberrant activation of the protein phosphatase calcineurin (CN), shown by our lab and others to drive several critical components of the activated astrocyte phenotype. Aim 1 will use a combination of adenoviral vectors and pharmacological agents, in addition to Western blot, ethidium bromide uptake, patch-clamp electrophysiology, and a variety of biochemical assays, to determine whether CN and Cx43 are necessary and sufficient for the effects of A? on the functional status of HCs in primary astrocytes. In addition, this aim will determine the feasibility of using a novel peptide-based reagent, 43Gap52, to selectively disrupt CN/Cx43 interactions without inhibiting CN activity per se. This aim will establish whether aberrant CN/Cx43 interactions in astrocytes are a direct outcome of A? pathology. Aim 2 will use adeno-associated virus (AAV) bearing astrocyte-specific promoters and CN activators (active CN fragment) or CN inhibitors (CN-autoinhibitory peptide or 43Gap52) to selectively modulate astrocytic CN/Cx43 interactions in an intact mouse model of AD. The functional status of astrocytic uHCs will be investigated in acutely prepared brain slices using an EtBr uptake assay, as described for Aim 1. In addition, slice electrophysiology will be used to assess the impact of CN/Cx43 interactions on hippocampal synaptic strength and plasticity. This Aim will validate Aim 1 results in a commonly used animal model of AD, en route to determining the extent to which CN/Cx43 interactions in astrocytes disrupt synaptic function during the progression of A? pathology. These studies will shed light on the role of astrocytic HCs in AD pathophysiology and determine whether disruption of the CN/Cx43 interaction is a feasible strategy for ameliorating neurologic dysfunction due to AD and other types of neurodegenerative disease.