Calcineurin/NFAT Signaling in Activated Astrocytes Drives Network Hyperexcitability in Aß-Bearing Mice.

Hyperexcitable neuronal networks are mechanistically linked to the pathologic and clinical features of Alzheimer's disease (AD). Astrocytes are a primary defense against hyperexcitability, but their functional phenotype during AD is poorly understood. Here, we found that activated astrocytes in the 5xFAD mouse model were strongly associated with proteolysis of the protein phosphatase calcineurin (CN) and the elevated expression of the CN-dependent transcription factor nuclear factor of activated T cells 4 (NFAT4). Intrahippocampal injections of adeno-associated virus vectors containing the astrocyte-specific promoter Gfa2 and the NFAT inhibitory peptide VIVIT reduced signs of glutamate-mediated hyperexcitability in 5xFAD mice, measured in vivo with microelectrode arrays and ex vivo brain slices, using whole-cell voltage clamp. VIVIT treatment in 5xFAD mice led to increased expression of the astrocytic glutamate transporter GLT-1 and to attenuated changes in dendrite morphology, synaptic strength, and NMDAR-dependent responses. The results reveal astrocytic CN/NFAT4 as a key pathologic mechanism for driving glutamate dysregulation and neuronal hyperactivity during AD.SIGNIFICANCE STATEMENT Neuronal hyperexcitability and excitotoxicity are increasingly recognized as important mechanisms for neurodegeneration and dementia associated with Alzheimer's disease (AD). Astrocytes are profoundly activated during AD and may lose their capacity to regulate excitotoxic glutamate levels. Here, we show that a highly active calcineurin (CN) phosphatase fragment and its substrate transcription factor, nuclear factor of activated T cells (NFAT4), appear in astrocytes in direct proportion to the extent of astrocyte activation. The blockade of astrocytic CN/NFAT signaling in a common mouse model of AD, using adeno-associated virus vectors normalized glutamate signaling dynamics, increased astrocytic glutamate transporter levels and alleviated multiple signs of neuronal hyperexcitability. The results suggest that astrocyte activation drives hyperexcitability during AD through a mechanism involving aberrant CN/NFAT signaling and impaired glutamate transport.

Biomimetic bone mechanotransduction modeling in neonatal rat femur organ cultures: structural verification of proof of concept.

The goal of this work was to develop and validate a whole bone organ culture model to be utilized in biomimetic mechanotransduction research. Femurs harvested from 2-day-old neonatal rat pups were maintained in culture for 1 week post-harvest and assessed for growth and viability. For stimulation studies, femurs were physiologically stimulated for 350 cycles 24 h post-harvest then maintained in culture for 1 week at which time structural tests were conducted. Comparing 1 and 8 days in culture, bones grew significantly in size over the 7-day culture period. In addition, histology supported adequate diffusion and organ viability at 2 weeks in culture. For stimulation studies, 350 cycles of physiologic loading 24 h post-harvest resulted in increased bone strength over the 7-day culture period. In this work, structural proof of concept was established for the use of whole bone organ cultures as mechanotransduction models. Specifically, this work established that these cultures grow and remain viable in culture, are adequately nourished via diffusion and are capable of responding to a brief bout of mechanical stimulation with an increase in strength.

The alpha1D-adrenergic receptor is expressed intracellularly and coupled to increases in intracellular calcium and reactive oxygen species in human aortic smooth muscle cells.

BACKGROUND: The cellular localization of the alpha1D-adrenergic receptor (alpha1D-AR) is controversial. Studies in heterologous cell systems have shown that this receptor is expressed in intracellular compartments. Other studies show that dimerization with other ARs promotes the cell surface expression of the alpha1D-AR. To assess the cellular localization in vascular smooth muscle cells, we developed an adenoviral vector for the efficient expression of a GFP labeled alpha1D-AR. We also measured cellular localization with immunocytochemistry. Intracellular calcium levels, measurement of reactive oxygen species and contraction of the rat aorta were used as measures of functional activity. RESULTS: The adenovirally expressed alpha1D-AR was expressed in intracellular compartments in human aortic smooth muscle cells. The intracellular localization of the alpha1D-AR was also demonstrated with immunocytochemistry using an alpha1D-AR specific antibody. RT-PCR analysis detected mRNA transcripts corresponding to the alpha1A-alpha1B- and alpha1D-ARs in these aortic smooth muscle cells. Therefore, the presence of the other alpha1-ARs, and the potential for dimerization with these receptors, does not alter the intracellular expression of the alpha1D-AR. Despite the predominant intracellular localization in vascular smooth muscle cells, the alpha1D-AR remained signaling competent and mediated the phenylephrine-induced increases in intracellular calcium. The alpha1D-AR also was coupled to the generation of reactive oxygen species in smooth muscle cells. There is evidence from heterologous systems that the alpha1D-AR heterodimerizes with the beta2-AR and that desensitization of the beta2-AR results in alpha1D-AR desensitization. In the rat aorta, desensitization of the beta2-AR had no effect on contractile responses mediated by the alpha1D-AR. CONCLUSION: Our results suggest that the dimerization of the alpha1D-AR with other ARs does not alter the cellular expression or functional response characteristics of the alpha1D-AR.