Abstract:
Alzheimer’s disease (AD) is the most common cause of dementia worldwide, however, there are only a few therapies available for this progressive and irreversible disorder. As the main immune cells of the brain, microglia protect against the spread of Aβ plaques in the brain parenchyma via phagocytosis and barrier formation. But they also release pro-inflammatory cytokines and seem to prune functional synapses thus facilitating synapse loss and potentiating disease progression. Caloric restriction (CR) is a cost-effective and easy-to-implement treatment, and previous data from our lab revealed that in WT midlife mice CR partially reverts the aging-associated changes in the ongoing microglial Ca2+ signaling. However, the effects of CR on the in vivo functional properties of microglia in mouse models of AD remain to be explored. Moreover, little is known about the sex specificity of microglial responses to AD and CR.
Here, we provided a detailed in vivo characterization of the sex-specific spatiotemporal Ca2+ signaling patterns in the frontal/motor cortex and investigated the effect of CR, using in vivo high-resolution Ca2+ imaging. To do so, 9-11 months old APPPS1 mice were put under 70% of initial ad libitum (AL) food intake for 6-8 months. Individual microglia were electroporated by a mixture of a Ca2+ indicator Oregon Green BAPTA-1 and a red fluorescent dye Alexa Fluor 594, and spontaneous microglial Ca2+ signaling (green channel), as well as cell morphology (red channel), were monitored simultaneously using ratiometric imaging in 4D (X/Y/Z/T). Besides, we analyzed the UDP-evoked somatic Ca2+ transients, process chemotaxis, and 3D morphology. By optimizing a new protocol for in vivo two-photon imaging and developing a precise 3D morphological reconstruction pipeline, we obtained extensive information about microglial somata and processes.
Our data revealed that compared with age-matched WT animals, the increases in in vivo spontaneous and UDP-evoked Ca2+ signaling, directed microglial process movement, soma sizes, spatial domains and process complexity were observed in AD microglia, indicating a pathologically elevated microglial activity. We next discovered the long-term CR had beneficial effects on AD microglia to some extent, as suggested by a reduction of the cerebral plaque load, partial normalization of some in vivo functional properties and the morphological alterations. However, CR enhanced the frequencies of in vivo spontaneous Ca2+ signaling in both somata and processes of hypertrophic AD microglia.
Moreover, in this work, we comprehensively investigated for the first time the sex-specific characteristics of WT and AD microglia. Our results revealed higher ramified processes in female WT microglia as well as hyper-ramified processes in plaque-distant male and female AD microglia, suggesting that female WT microglia were closer to the disease state than male WT microglia. Furthermore, some in vivo functional properties of plaque-associated microglia exhibited sex-specific heterogeneity in response to CR. Most of the CR-induced normalizing effects were pronounced in males. Additionally, alterations in the morphology of plaque-distant microglia also displayed sex specificity: the soma sizes and process complexity of male microglia responded to AD and CR far better than that of female microglia, while the spatial domains covered by processes of a single ramified microglia were better normalized by CR in females.
In conclusion, this research advances our knowledge of how CR affects the functional properties of AD microglia, including the in vivo calcium signaling, 3D morphology, and an in-depth understanding of sex-specific differences, thus paving the way for better disease-modifying treatments in the future.
Caloric restriction