Author ORCID Identifier

http://orcid.org/0000-0001-5450-937X

Year of Publication

2016

Degree Name

Doctor of Philosophy (PhD)

Document Type

Doctoral Dissertation

College

Medicine

Department

Pharmacology and Nutritional Sciences

First Advisor

Dr. Christopher M. Norris

Abstract

Calcineurin (CN) is a calcium (Ca2+)-sensitive serine/threonine protein phosphatase that plays a significant role in several cell signaling pathways, and has been implicated in many neurodegenerative diseases including Alzheimer’s disease (AD) and vascular cognitive impairment and dementia (VCID). Although normally found in neurons, CN also appears at high levels in activated astrocytes under conditions of injury and disease. To elucidate the role of astrocytic calcineurin signaling in neurodegenerative diseases, our lab has used primary rat astrocytes, transgenic and diet-induced mouse models of dementia, and human tissue biospecimens from confirmed AD and VCID cases.

To better understand mechanisms for aberrant activation of CN during injury and disease, we created a custom antibody that selectively identifies a proteolyzed, constitutively active CN fragment. Immunolabeling in human biospecimens was done to determine which cell types express high levels of CN proteolysis and hyperactivation. Our results revealed extensive proteolysis of CN in activated astrocytes near pathologic hallmarks of AD (Aβ plaques) and VCID (microinfarcts). When a similar activated CN fragment was expressed in hippocampal astrocytes of healthy adult rats using adeno-associated virus (AAV) vectors, we observed suppressed function of CA3-CA1 excitatory synapses, suggesting that proteolytic activation of CN in astrocytes is a key mechanism for driving neuronal dysfunction in neurodegenerative diseases.

To explore the interaction between astrocytic CN and a possible downstream signaling target, we studied the hemichannel-forming protein, connexin43 (Cx43). Previous work has shown that serine 368 near the C terminus of Cx43 is dephosphorylated by CN. Here, we found that dephosphorylation of ser368 was increased in human hippocampal specimens from subjects diagnosed with mild cognitive impairment (MCI). Dephospho-Cx43 levels were correlated, within subject, to elevated levels of CN proteolysis and signaling. Moreover, dephosphorylation of Cx43 could be mimicked in rat primary astrocyte cultures using both exogenous Ca2+ mobilizers (phorbol ester/ionomycin) and endogenous inflammatory mediators (IL-1b), found previously to activate CN in astrocytes. Finally, we created a custom peptide (43Gap52) that encompasses Ser368 and mimics a portion of the C-terminus of Cx43. 43Gap52 prevented IL-1b-mediated dephosphorylation of Cx43, but did not prevent CN-dependent activation of NFAT transcription factors, suggesting that 43Gap52 selectively disrupts CN/Cx43 interactions. Using 43Gap52 and the commercial CN inhibitor cyclosporin A (CsA), we found that blockade of CN/Cx43 interactions reduced hemichannel permeability in primary astrocytes following treatment with IL-1b, suggesting that aberrant CN activation in astrocytes may negatively affect neurons via interactions with Cx43-containing hemichannels.

To more clearly understand CN/NFAT signaling in VCID we used a diet-induced model of hyperhomocysteinemia (HHcy) that drives vascular pathology. Using AAV-mediated gene delivery, we injected mice with an astrocyte-targeted inhibitor of CN/NFAT binding (Gfa2-Egfp-VIVIT) or a control virus (Gfa2-Egfp). These mice were further split into two groups, one fed with the HHcy diet and one fed with control diet. Experiments are in progress to assess endpoint measurements including LTP and synaptic strength, RAWM behavior testing, as well as a panel of biochemical measurements.

Digital Object Identifier (DOI)

https://doi.org/10.13023/ETD.2016.470

Available for download on Wednesday, December 12, 2018

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