Immune protein interactions with microglia: contributors to both synapse loss and myelin damage in the aging rhesus monkey brain
OA Version
Citation
Abstract
Normal aging is characterized by cognitive decline in learning, memory, and executive function even in the absence of neurodegenerative diseases such as Alzheimer’s disease (AD). Although originally believed to be a result of neuron loss, the source of cognitive decline during normal aging is now understood to reflect damage sustained by neurons, particularly disruption of the insulative myelin sheath around the axon and disruption of synaptic connections. As a result of myelin pathology, white matter volume is lost, and axon conduction is disrupted leading to loss of cortical connectivity. Concurrently, gray matter volume loss occurs due to the combined loss of myelin and synapses and atrophy of dendrites, resulting in decreased synaptic transmission between neurons and ultimately cortical disconnection. Although the underlying cause of this damage is unknown, increasing neuroinflammation is likely involved as it renders microglia reactive, disrupting proper maintenance of myelin and synaptic homeostasis. With age, the ability of microglia to clear myelin debris declines and it has been shown that myelin debris impedes remyelination by oligodendrocytes. Additionally, secretion of proinflammatory molecules increases, making microglia more sensitive to inflammatory stimuli, resulting in an exaggerated inflammatory response. This exaggerated response may lead to aberrant engulfment and phagocytosis of both myelin and synapses during normal aging, ultimately leading to cognitive decline. Microglia engulfment is regulated through innate immune signals that either promote or inhibit phagocytosis. Two key components in this regulation are the initiatory signal of the classical complement cascade, C1q, that signals “eat me” to initiate engulfment of targets and the inhibitory neuroimmune regulatory protein CD47 that signals “don’t eat me” to prevent engulfment. The overall goal of this dissertation study was to investigate regional differences in innate immune signals C1q and CD47 in the aging brain, where microglial homeostatic function deteriorates under neuroinflammatory conditions. To do this, immunofluorescence (IF) was used to examine changes in C1q and CD47 in relation to synapse loss in the dorsolateral prefrontal cortex (dlPFC) gray matter, myelin damage in the cingulum bundle white matter, and cognitive decline in the normal aging rhesus monkey. Modeling human aging with the rhesus monkey is a highly translatable model as monkeys have extended lifespans, a gray to white matter ratio comparable to humans, and a similar pattern of cognitive impairment in learning and memory tasks during aging. First, using IF on archived brain sections collected from a large cohort of behaviorally tested monkeys, we found that synapses identified by PSD95 decreased with age, and existing PSD95+ synapses had increased colocalization of C1q along with decreased CD47 colocalization in dlPFC gray matter. However, gray matter microglia did not show age-related changes in morphology or expression of C1q, likely indicating a process other than conventional phagocytosis by which microglia engulf synapses. Next, these immune signals were investigated in cingulum bundle white matter, where IF showed colocalization of C1q with myelin increased with age. In contrast CD47 showed decreased colocalization with myelin from young to middle age but then increased in the oldest subjects. In aging white matter, IF revealed an increase in the number of microglia with hypertrophic morphology along with in the phagocytic activation marker Gal-3, changes that may reflect microglial reactivity. Further, RNAscope in situ hybridization revealed C1qA mRNA expression increased in white matter microglia with age, while CD47 mRNA expression decreased in oligodendrocytes, likely making them more vulnerable to attack by microglia. Importantly, these changes in C1q, CD47, and microglia reactivity are associated with age-related cognitive decline. Finally, microglia engulfment of myelin and synapses was investigated in relation to changes in C1q, CD47, and downstream complement components. IF and spectral confocal reflectance (SCoRe) microscopy, which enables visualization of myelin, revealed increased microglial engulfment of myelin in aging white matter and increased synaptic engulfment in aging gray matter. Electron microscopy confirmed that C1q and CD47 both colocalize to synaptic structures and to myelin. At the molecular level, quantitative polymerase chain reaction showed differences in upregulation of downstream complement molecules, where aging white matter had upregulated expression of C1q and CR1 genes that mediate phagocytosis. In contrast, aging gray matter showed increased expression of C4b which has been implicated in excess synapse loss. Together, these results provide strong evidence that C1q and CD47 immune signaling are dysregulated during aging and likely contribute to synapse loss, myelin damage, and microglia reactivity, and thus presents a novel target for therapies aimed at slowing cognitive decline.
Description
2024