A lifespan approach to neuroinflammatory and cognitive disorders: a critical role for glia

Abstract

Cognitive decline is a common problem of aging. Whereas multiple neural and glial mechanisms may account for these declines, microglial sensitization and/or dystrophy has emerged as a leading culprit in brain aging and dysfunction. However, glial activation is consistently observed in normal brain aging as well, independent of frank neuroinflammation or functional impairment. Such variability suggests the existence of additional vulnerability factors that can impact neuronal-glial interactions and thus overall brain and cognitive health. The goal of this review is to elucidate our working hypothesis that an individual's risk or resilience to neuroinflammatory disorders and poor cognitive aging may critically depend on their early life experience, which can change immune reactivity within the brain for the remainder of the lifespan. For instance, early-life infection in rats can profoundly disrupt memory function in young adulthood, as well as accelerate age-related cognitive decline, both of which are linked to enduring changes in glial function that occur in response to the initial infection. We discuss these findings within the context of the growing literature on the role of immune molecules and neuroimmune crosstalk in normal brain development. We highlight the intrinsic factors (e.g., chemokines, hormones) that regulate microglial development and their colonization of the embryonic and postnatal brain, and the capacity for disruption or "re-programming" of this crucial process by external events (e.g., stress, infection). An impact on glia, which in turn alters neural development, has the capacity to profoundly impact cognitive and mental health function at all stages of life.

Figure 1. Developing microglia participate in many processes of neural development

Timeline representation of microglial development (upper panel of timeline) juxtaposed with known processes of neurodevelopment (lower panel). Microglia are derived from primitive macrophages that originate within the yolk sac, enter the neuroectoderm during embryogenesis, and begin to colonize the parenchyma around embryonic day (E)12–14 (Ginhoux et al., 2010). During the early postnatal period (P4) several chemokines, cytokines, and receptors are several-fold higher than at P60 (e.g., Ccl12, Ccl3, Cxcl6); these may be involved in the ongoing recruitment of microglia into the neural tissue, among other functions (Schwartz and Bilbo, unpublished). In the adult (P60), the pattern of chemokine expression changes, with significant upregulation of different factors (e.g., C3, Cx3cl1) (Schwartz and Bilbo, unpublished). Cx3cl1 (fractalkine) is expressed by neurons and tonically inhibits microglia (Harrison et al., 1998); thus, its upregulation over the course of development may be important for shifting the morphology and function of microglia into their mature, ramified state in adulthood. In parallel with the colonization of microglia, progenitor cells in situ undergo proliferation and differentiate into neurons and glia, a process dependent upon local factors or intrinsic signals. Bone morphogenic protein (BMP)/TGFβ and interleukin (IL)-1, both released from developing microglia (Deverman and Patterson, 2009), are known mediators of these processes. As microglia continue to develop, they produce cytokines (including IL-1β, Tumor Necrosis Factor (TNF) α, and IL-11) that are important for ongoing neurogenesis within the developing brain; they also produce chemokines (Cxcl12 and its receptor Cxcr4) that guide the axons of new neurons via chemoattraction toward their new synaptic targets (Lu et al., 2002; Lieberam et al., 2005). At the level of the synapse, major histocompatability complex (MHC) I and TNFα are important for strengthening new synaptic connections within the brain (Corriveau et al., 1998; Santello et al., 2011). Finally, in the later stages of neurodevelopment, abundant or inappropriate synaptic connections are eliminated (synaptic pruning) and phagocytosed by microglia. Immune proteins such as complement component (C)1q and C3 tag synapses for elimination during this process (Stevens et al., 2007). These timelines are broadly representative of the pattern in both males and females, though there are sex differences in relative glial density at distinct stages (see Figure 3).

Figure 2. Colonization of the developing brain by microglia

On the day of birth (first column), microglia first appear within the brain around the hippocampus (top row), cortex (second row), and amygdala (third row). By P4 (second column), microglia have begun to enter hypothalamic nuclei such as the paraventricular nucleus (fourth row), and density begins to peak in hippocampus. At P0 and P4, microglial morphology is either amoeboid or round with short, stout processes (best seen above in the cortex at P0 and P4). By P30 (third column) and P60 (fourth column) microglia reside in most brain regions (including striatum and others not shown here). At P30, many microglia still have a relatively immature morphology. Specifically, microglia at this time have thicker, longer processes than microglia at P60. By P60, microglia have matured and are maintained in the healthy brain in a ramified state characterized by thin, long processes. This timeline is broadly representative of the pattern in both males and females, though there are sex differences in relative glial density at distinct stages (see Figure 3). (Scale bar = 500 µm and applies to all)

Figure 3. The density and morphology of microglia within cognitive regions of the brain are significantly affected by sex and age

At embryonic day 17 (Prenatal; first column from left), males (top row of representative coronal sections) and females (bottom row) have a similar number of microglia within the hippocampus (HP), cortex (CX), and the amygdala (AMY.) At birth (Postnatal day 0), females (bottom series of representative coronal sections) have slightly more microglia than males (top series of representative coronal sections) within the CA3 region of HP and AMY, though not within the CX. As the brain develops and microglia continue to enter the brain, this sex difference reverses. By Postnatal day 4, males have significantly more microglia within the CX, all subregions of the HP, and AMY than females. Beginning around adolescence (postnatal day 30) and into adulthood (postnatal day 60) the sex difference reverses again. At this age, microglia generally appear within all brain regions in a ramified state, represented by thin, long processes. However, at postnatal day 30 and 60, females have a significantly greater population of microglia with thicker, longer processes than males in all three regions, suggestive of a differential function between the sexes at this age. One representative light micrograph is displayed below each diagram, which schematize cell density. Scale bars (in the first column from left): dotted line = 100 µm (for representative pictures at 4× for all ages), solid line = 50 µm (for inset pictures at 20× for all ages). One schematized coronal section is equivalent to 10 sections analyzed for cell counts and morphology at all ages. 1 cell shown above at postnatal day 0 and postnatal day 4 represents approximately 10 counted cells. 1 cell shown above at postnatal day 30 – 60 represents approximately 200 counted cells.

Figure 4. Early-life bacterial infection leads to LPS-induced memory impairment in adult males but not females

Male and female rat pups injected with PBS or E. coli on postnatal day 4 were tested for memory as adults using a contextual fear-conditioning paradigm. Half of the rats in each neonatal group were injected with LPS immediately after learning. (A) Freezing to the context 48 h later was significantly reduced in adult males injected with LPS after learning, but only if they had also received E. coli as neonates (from Bilbo et al., 2006; *p<0.05). (B) In contrast to males, LPS had no effect on memory in females from either neonatal treatment.

Figure 5. Bacterial infection in neonatal male rats alters long-term microglial morphology, surface antigen expression, and function

(A) Male rats treated on P4 with PBS or live E. coli were injected systemically as adults with saline (SAL) or 25 µg/kg lipopolysaccharide (LPS), and microglia were rapidly isolated from whole HP 24 h later following cold saline perfusion to eliminate infiltrating cells as described in detail previously (Frank et al.; Henry et al., 2009). Isolated microglia were stained for APC-conjugated CD11b. Cell size (forward light scatter) and CD11b+ expression were assessed using a FACSCanto™ II flow cytometer (Becton, Dickinson and Co.) and FlowJo™ software (Treestar, Inc.). The gating strategy and representative contour plot are shown. (B) There is a greater proportion of large, CD11b^bright microglia in adult rats infected neonatally with E. coli. Mean fluorescence of CD11b+ staining in gated cells was also greater on a per cell basis in rats infected neonatally with E. coli. *Overall effect of E. coli for each, p<0.01. (C) Sensitized/primed microglia in neonatally-infected rats produce more IL-1β in response to LPS in adulthood compared to controls (Bilbo et al., 2005a; ; Bilbo et al., submitted data).