Effect of Caloric Restriction on the in vivo Functional Properties of Aging Microglia

Abstract

Throughout the lifespan, microglia, the primary innate immune cells of the brain, fulfill a plethora of homeostatic as well as active immune defense functions, and their aginginduced dysfunctionality is now considered as a key trigger of aging-related brain disorders. Recent evidence suggests that both organism's sex and age critically impact the functional state of microglia but in vivo determinants of such state(s) remain unclear. Therefore, we analyzed in vivo the sex-specific functional states of microglia in young adult, middle aged and old wild type mice by means of multicolor two-photon imaging, using the microglial Ca 2+ signaling and directed process motility as main readouts. Our data revealed the sex-specific differences in microglial Ca 2+ signaling at all ages tested, beginning with young adults. Furthermore, for both sexes it showed that during the lifespan the functional state of microglia changes at least twice. Already at middle age the cells are found in the reactive or immune alerted state, characterized by heightened Ca 2+ signaling but normal process motility whereas old mice harbor senescent microglia with decreased Ca 2+ signaling, and faster but disorganized directed movement of microglial processes. The 6-12 months long caloric restriction (70% of ad libitum food intake) counteracted these aging-induced changes shifting many but not all functional properties of microglia toward a younger phenotype. The improvement of Ca 2+ signaling was more pronounced in males. Importantly, even short-term (6-week-long) caloric restriction beginning at old age strongly improved microglial process motility and induced a significant albeit weaker improvement of microglial Ca 2+ signaling. Together, these data provide first sex-specific in vivo characterization of functional properties of microglia along the lifespan and identify caloric restriction as a potent, cost-effective, and clinically relevant tool for rejuvenation of microglia.

Changing Functional Signatures of Microglia along the Axis of Brain Aging

“…The built-up of a spherical containment ( Figure 1 A) requires a high degree of coordination between the individual processes of different microglial cells. Indeed, it turned out that the movement speed of the individual process is directly proportional to its initial distance from the DAMP-containing (insult) area [ 39 , 40 ] ( Figure 1 C). In this way, processes located further away move faster and quickly join up with the processes located closer to the insult area.…”

“…Consistently, the functional in vivo studies from our group also document higher alertness of microglia in young adult male compared to female mice. Thus, under basal conditions the fraction of microglia showing spontaneous Ca 2+ transients was significantly higher in male compared to female mice [ 40 ]. To substantiate the differences between young adult male and female mice, we analyzed a previously published RNAseq data set [ 57 ].…”

“…In this data set, the genes associated with inflammation were significantly upregulated in young male compared with female mice. The Ca 2+ signaling-related pathways in general, as well as biological processes summarized under gene ontology annotations “release of sequestered calcium into cytosol,” “calcium-mediated signaling using intracellular calcium source,” or “positive regulation of cytosolic calcium ion concentration” were all upregulated in young adult male compared to female mice, but the difference observed did not reach the level of statistical significance [ 40 ]. These data are in good agreement with a study showing a higher transcriptional activation of NF-kB, a factor playing a key role in regulating the immune response, in male microglia [ 19 ].…”

“…Our in vivo data revealed a pronounced enhancement of spontaneous Ca 2+ signaling in middle-age microglia in terms of frequency, amplitude, duration, and integral of individual Ca 2+ transients [ 39 , 40 ], suggesting an activated, alerted functional state of middle-age microglia. This hyperactivity of Ca 2+ signaling in individual cells together with an aging-related increase in the fraction of spontaneously active microglia represents a functional hallmark of the middle-aged brain.…”

“…Importantly, we have also identified a severe impairment in the coordination of individual microglial processes from different cells, building up a spherical containment around the DAMP source ( Figure 1 D). Whereas in young adult mice, the initial distance of a process tip to the DAMP source is correlated with the mean velocity of extension ( Figure 1 B), this correlation decreases significantly in aged mice [ 39 , 40 ]. The final diameter of the containment formed by microglial processes, however, was similar in young adult, middle-aged, and old mice, suggesting that an equal number of processes contributed to the reaction in the different age groups [ 39 , 40 ].…”