Title: In vivo functional imaging reveals impaired neuronal and astrocytic calcium regulation in Alzheimer's disease
Description: Alzheimer's disease (AD) is characterized by the deposition of senile plaques, neurofibrillary tangles and progressive dementia. Despite decades of research, the direct molecular mechanisms that couple plaque deposition to neural system failure remain unclear. I hypothesized that plaque deposition disrupts the function of cortical networks by destabilizing calcium homeostasis. To test this, I adapted existing in vivo multiphoton imaging techniques in novel ways to measure the impact of plaque deposition on calcium regulation on populations of astrocytes and neurons with subcellular resolution. I first measured neuronal calcium homeostasis in individual neurites and spines of mice that express mutations associated with AD. I utili7ed a FRET-based, ratiometric and genetically-encoded calcium indicator (GECI): Yellow-Cameleon 3.6. Intravital gene transfer of YC3.6 permitted me to measure resting calcium levels across four different transgenic mouse strains. This revealed calcium overload in ∼20% of neurites in APP mice with cortical plaques, compared to less than 5% in wildtype mice, PSI-mutant mice, or young APP mice (animals without cortical plaques). Calcium overload depended on the existence and proximity to plaques and led to the activation of the phosphatase calcineurin. These data demonstrated that senile plaques disrupted the structure and function of neuronal networks by impairing neuronal calcium homeostasis.

I next posited that plaque deposition might also impact the function of astrocyte networks. Using multiphoton fluorescence lifetime imaging microscopy in vivo, I quantitatively imaged astrocytic calcium homeostasis in a mouse model of Alzheimer's disease. Resting calcium was globally elevated in the astrocytic network, but was independent of proximity to individual plaques. Time lapse imaging revealed that calcium transients in astrocytes were more frequent, were synchronously coordinated across long distances, and were uncoupled from neuronal activity. Furthermore, rare intercellular calcium waves were observed, but only in mice with amyloid-13 plaques, originating near plaques and spreading radially at least 200 μm. Thus, while neurotoxicity is observed near amyloid-B deposits, there exists a more general astrocyte-based network response to focal pathology. Taken together, it appears that plaque deposition does not lead to the deterministic degeneration, or dysfunction, of all cells in the cortical network: certain dendrites are vulnerable to calcium overload whereas others are less so. Moreover, Aβ appears to globally impact astrocytes whereas plaques locally impair neuronal function. This complex and multidimensional impact of Aβ argues that a novel, combinatorial approach to therapeutic intervention might prove effective.