Beyond Activation: Characterizing Microglial Functional Phenotypes

Classically, the following three morphological states of microglia have been defined: ramified, amoeboid and phagocytic. While ramified cells were long regarded as “resting”, amoeboid and phagocytic microglia were viewed as “activated”. In aged human brains, a fourth, morphologically novel state has been described, i.e. dystrophic microglia, which are thought to be senescent cells. Since microglia are not replenished by blood-borne mononuclear cells under physiological circumstances, they seem to have an “expiration date” limiting their capacity to phagocytose and support neurons. Identifying factors that drive microglial aging may thus be helpful to delay the onset of neurodegenerative diseases, such as Alzheimer’s disease (AD). Recent progress in single-cell deep sequencing methods allowed for more refined differentiation and revealed regional-, age- and sex-dependent differences of the microglial population, and a growing number of studies demonstrate various expression profiles defining microglial subpopulations. Given the heterogeneity of pathologic states in the central nervous system, the need for accurately describing microglial morphology and expression patterns becomes increasingly important. Here, we review commonly used microglial markers and their fluctuations in expression in health and disease, with a focus on IBA1 low/negative microglia, which can be found in individuals with liver disease.

Dystrophic microglia are a disease associated microglia morphology in the human brain

Microglia activation—typically described in terms of hypertrophic appearance—is a well-established feature of aging. Recent studies have suggested that microglia dystrophy, not activation, may increase the propagation of progressive neurodegenerative diseases such as Alzheimer’s disease (AD). Yet, a clear understanding of cause and consequences of dystrophic microglia is lacking. Although frequently observed in diseased brains, the appearance of dystrophic microglia in the hippocampus of individuals free of cognitive impairment suggests that microglia may be undergoing senescence with age, leading to dystrophy. Therefore, we hypothesized that chronological age could be a significant contributor to the presence of dystrophic microglia. To investigate this relationship, we employed stereological counts of total microglia, hypertrophic, and dystrophic microglia across the decades of the human lifespan. The microglia counts were performed in the frontal cortex gray and white matter and the CA1 subregion of the hippocampus in individuals without known neurodegenerative disease. There was age-associated increase in the number of dystrophic microglia in the CA1 region and the frontal cortex gray matter related to age. However, the increase in dystrophic microglia was proportional to the age-related increase in the total number of microglia, suggesting that aging alone was not sufficient to explain the presence of dystrophic microglia. We next tested if dystrophic microglia could be a disease-associated microglia phenotype. Compared to controls without neuropathology, the number of dystrophic microglia was significantly greater in aged-matched cases with either AD, dementia with Lewy bodies, or LATE-NC. These results provide evidence that healthy aging is only associated with a modest increase in dystrophic microglia and suggest that dystrophic microglia may be a disease-associated microglia phenotype. Finally, we found strong evidence for iron homeostasis changes, with increases in ferritin light chain, in dystrophic microglia compared to ramified or hypertrophic microglia. Based on our findings, microglia dystrophy, and not hypertrophic microglia, are the disease-associated microglia morphology. Future work is required to understand the links between the increase in dystrophic microglia and neurodegenerative disorders.

Model Senescent Microglia Induce Disease Related Changes in α-Synuclein Expression and Activity

Aging is the most prominent risk factor for most neurodegenerative diseases. However, incorporating aging-related changes into models of neurodegeneration rarely occurs. One of the significant changes that occurs in the brain as we age is the shift in phenotype of the resident microglia population to one less able to respond to deleterious changes in the brain. These microglia are termed dystrophic microglia. In order to better model neurodegenerative diseases, we have developed a method to convert microglia into a senescent phenotype in vitro. Mouse microglia grown in high iron concentrations showed many characteristics of dystrophic microglia including, increased iron storage, increased expression of proteins, such as ferritin and the potassium channel, Kv1.3, increased reactive oxygen species production and cytokine release. We have applied this new model to the study of α-synuclein, a protein that is closely associated with a number of neurodegenerative diseases. We have shown that conditioned medium from our model dystrophic microglia increases α-synuclein transcription and expression via tumor necrosis factor alpha (TNFα) and mediated through nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). The conditioned medium also decreases the formation of α-synuclein tetramers, associated ferrireductase activity, and increases aggregates of α-synuclein. The results suggest that we have developed an interesting new model of aged microglia and that factors, including TNFα released from dystrophic microglia could have a significant influence on the pathogenesis of α-synuclein related diseases.