Immune proteins C1q and CD47 may contribute to aberrant microglia-mediated synapse loss in the aging monkey brain that is associated with cognitive impairment

Abstract

Cognitive impairment in learning, memory, and executive function occurs in normal aging even in the absence of Alzheimer's disease (AD). While neurons do not degenerate in humans or monkeys free of AD, there are structural changes including synapse loss and dendritic atrophy, especially in the dorsolateral prefrontal cortex (dlPFC), and these correlate with cognitive age-related impairment. Developmental studies revealed activity-dependent neuronal properties that lead to synapse remodeling by microglia. Microglia-mediated phagocytosis that may eliminate synapses is regulated by immune "eat me" and "don't eat me" signaling proteins in an activity-dependent manner, so that less active synapses are eliminated. Whether this process contributes to age-related synapse loss remains unknown. The present study used a rhesus monkey model of normal aging to investigate the balance between the "eat me" signal, complement component C1q, and the "don't eat me" signal, transmembrane glycoprotein CD47, relative to age-related synapse loss in dlPFC Area 46. Results showed an age-related elevation of C1q and reduction of CD47 at PSD95+ synapses that is associated with cognitive impairment. Additionally, reduced neuronal CD47 RNA expression was found, indicating that aged neurons were less able to produce the protective signal CD47. Interestingly, microglia do not show the hypertrophic morphology indicative of phagocytic activity. These findings suggest that in the aging brain, changes in the balance of immunologic proteins give microglia instructions favoring synapse elimination of less active synapses, but this may occur by a process other than classic phagocytosis such as trogocytosis.

Keywords: Aging; C1q; CD47; Cognitive decline; Microglia; Synapse.

Fig. 1

Summary of subjects and experimental parameters. a Table listing the 32 monkeys’ IDs, age, CII, and sex used for these experiments. Animals denoted with # were the 16 subjects used for the C1qA and CD47 RNAscope experiment. b Schematic summarizing behavioral tasks, cognitive characterization, and the dlPFC region of interest examined. c Linear regression of significant cognitive impairment, measured by CII score, with age for this cohort of subjects. Purple dashed line indicates the cognitive impairment cut off, where subjects above the line are severely impaired and those below are cognitively spared

Fig. 2

PSD95+ synapse % area with age and cognitive impairment. Example post synaptic marker PSD95, C1q, and CD47 immunohistochemical images in (a, c) young vs (b, c) old subjects. (d–i) Linear regression showing significant PSD95+ synapse loss with age across cortical layers 1–6. (j) Synapse loss correlates with cognitive impairment, measured by the cognitive impairment index (CII) in cortical layers 2–5. Scale bar represents 10 µm

Fig. 3

C1q and CD47 with age and cognitive impairment. (a) % area of C1q increases in cortical layers 1–6 with age. (b) CD47 % area decreases in all six cortical layers with age. (c) C1q increases with cognitive impairment in layers 1–6. (d) CD47 decreases are associated with cognitive impairment in layers 2–5

Fig. 4

C1q-PSD95 and CD47-PSD95 colocalization with age and cognitive impairment. (a) % area of colocalized C1q and PSD95 signal increases significantly with age and (c) associates with cognitive impairment in cortical layers 1–6. (b) % area of colocalized CD47 and PSD95 signal significantly decreases with age in cortical layers 2, 4–6 and approaches significance in layers 1 and 3. (d) Decreased CD47 and PSD95 colocalization significantly correlates in layer 6 and approaches significance in layers 3–5

Fig. 5

Classification and density of microglia morphology with age and related cognitive impairment. a Example images displaying ramified and hypertrophic morphology as well as C1q + or C1q − . b–e Ramified C1q − , ramified C1q + , hypertrophic C1q − , and hypertrophic C1q + microglia density did not change with age (p > 0.05). f–i Microglial morphology and C1q expression did not change with cognitive impairment (p > 0.05)

Fig. 6

C1qA expression in microglia and CD47 expression in neurons. a, b Staining of C1qA in Iba1 + microglia and CD47 in MAP2 + neurons in young and old subjects. c, d Quantification of CD47 in MAP2 + neurons with age and cognitive impairment, respectively. e, f C1qA puncta in Iba1 + microglia with age and cognitive impairment. Young < 10 years, old > 20 years; cognitively spared < 2.0, impaired > 2.0; N = 16, *p = 0.001