Morphological characteristics and corresponding functional properties of homeostatic human microglia

Microglia, the resident immune cells of the central nervous system, exhibit a wide array of functional states, even in their so-called “homeostatic” condition, when they are not actively responding to overt pathological stimuli. These functional states can be visualized using a combination of multi-omics techniques (e.g., gene and protein expression, posttranslational modifications, mRNA profiling, and metabolomics), and, in the case of homeostatic microglia, are largely defined by the global (e.g., genetic variations, organism’s age, sex, circadian rhythms, and gut microbiota) as well as local (specific area of the brain, immediate microglial surrounding, neuron-glia interactions and synaptic density/activity) signals (Paolicelli et al., 2022). While phenomics (i.e., ultrastructural microglial morphology and motility) is also one of the key microglial state-defining parameters, it is known that cells with similar morphology can belong to different functional states.

Heterogeneity of human microglial morphotypes: Based on the plethora of rodent data, homeostatic microglia are usually considered ramified, although the detailed appearance of such ramified microglia can vary dramatically between different studies. In the case of human microglia, its morphology under homeostatic conditions is much more heterogeneous (Figure 1). Postmortem immune-labeling studies of middle-aged subjects suggested a substantial regional variability by showing that primed (with approximately twice as large cell bodies compared to ramified microglia and fewer higher-order branches, 34% of all cells) and reactive (with large cell bodies and only a few processes, 32% of all cells) microglia predominate in the gray matter of, for example, the dorsal anterior cingulate cortex. Interestingly, only 16% of microglia in this preparation were ramified. In contrast, ramified microglia (43%) were most common in the white matter of the same cortical region, followed by primed (27%), amoeboid (18%), and reactive (12%) morphotypes (Torres-Platas et al., 2014). Of note, with approximately 20%, amoeboid microglia constituted a substantial fraction of the population both in the white and grey matter. A similar distribution of cell morphotypes was observed in human microglia residing in the parietal, temporal, or frontal cortical tissue, which was freshly resected, sliced, and cultured in human cerebrospinal fluid for 7–9 days (Nevelchuk et al., 2024). In this preparation, 47% of microglia belonged to ramified, 31% to hypertrophic (reactive), and 22% to amoeboid morphotypes (Figure 1).

Figure 1.

Morphological diversity of human microglia under homeostatic conditions.

The figure illustrates six microglial morphotypes – ramified, hypertrophic, amoeboid, dystrophic, rod-shaped, and dark microglia – highlighting their distinct structural characteristics, Ca²⁺ signaling profiles, and functional roles. The images of ramified, hypertrophic, and amoeboid microglia are adapted from Nevelchuk et al. (2024), the rod-shaped and dark microglia from Savage et al. (2019) and St-Pierre et al. (2022), respectively, all licensed under CC BY 4.0. The image of dystrophic microglia is modified, with permission from Neumann et al. (2023). The brain shape included in the figure was created using a template from BioRender.com.

Aging leads to further diversification in the morphology of human microglia. For instance, dystrophic microglia (Figure 1), are characterized by deramification, spheroidal swellings, shortened and gnarled processes, and cytoplasmic fragmentation. These alterations suggest a shift towards cellular senescence and a reduction in functional capacity, contrasting with the more reactive phenotypes typically observed in younger brains. Although dystrophic microglia are already present at midlife, they become significantly more abundant in the aging human brain (Neumann et al., 2023). Aging also brings a rise in rod-shaped microglia (Figure 1), identified by elongated cell bodies and polarized processes, which play a role in synaptic stripping (Savage et al., 2019). Similarly, dark microglia, another intriguing phenotype, are rare under homeostatic conditions in young brains but become more abundant in the course of aging. Characterized by hyperramified processes and electron-dense nature, dark microglia frequently interact with blood vessels and synapses, likely contributing to synaptic remodeling and vascular maintenance (Paolicelli et al., 2022). Thus, in stark contrast to the rodent brain, in which the great majority (≥ 90%) of homeostatic microglia belong to the ramified morphotype (Torres-Platas et al., 2014), human microglia present with much higher structural diversity. Consistently, recent single-cell transcriptome analyses revealed that human microglia show greater heterogeneity than the other mammalian species including mice also at the molecular level (Paolicelli et al., 2022). This poses the question about the functional properties of different microglial morphotypes.

Role of intracellular Ca²⁺ signaling for microglial function: Intracellular Ca²⁺ signaling is essential for microglial function, as changes in the intracellular free Ca²⁺ concentration ([Ca²⁺]_i) within these immune cells often serve as a connecting link between their sensory inputs and effector responses. Ca²⁺-mediated signaling pathways regulate microglial morphology, process movement, gene expression, nuclear factor κB-mediated cytokine production, and release, as well as the production of reactive oxygen species or brain-derived neurotrophic factor, vital for neuroprotection and synaptic function. Moreover, intracellular Ca²⁺ signaling influences fundamental microglial behaviors, including proliferation, differentiation, migration, phagocytosis, as well as transitions between different vigilance states (Lushchak et al., 2021; Pan and Garaschuk, 2023; Ma et al., 2024). Therefore, understanding the specific Ca²⁺ signaling patterns across different microglial morphotypes shall provide valuable functional insights into their distinct roles in maintaining central nervous system homeostasis and/or responding to subclinical pathologies.

Mechanisms underlying morphotype-specific intracellular Ca²⁺ signaling: In the intact mouse brain, different microglial morphotypes present with highly specific patterns of intracellular Ca²⁺ signals. The basal levels of [Ca²⁺]i are low in ramified in vivo microglia but rise significantly under conditions of brain damage, promoting the appearance of hypertrophic and amoeboid morphotypes (Lushchak et al., 2021; Pan and Garaschuk, 2023). Ongoing somatic Ca²⁺ signals are rare in ramified homeostatic microglia but abundant in hypertrophic and amoeboid cells, which in these species are found in vivo only under pathological conditions (Brawek et al., 2014). Instead, homeostatic mouse microglia present with a reach repertoire of the ongoing Ca²⁺ signals compartmentalized in microglial processes (Umpierre et al., 2020).

Mechanistically, these Ca²⁺ signals more often rely on Ca²⁺ release from the intracellular Ca²⁺ stores than on transmembrane Ca²⁺ entry (Figure 1 in Umpierre et al., 2020), and are most likely mediated by the activation of ATP and glutamate receptors or by the store-operated Orai channels (Pan and Garaschuk, 2023). Physiological levels of these abundant neuro- and gliotransmitters likely define the “healthy” microenvironment of the homeostatic microglia. Consistently, the bi-directional shifts in the activity of the surrounding neuroglial network (e.g., kainate-induced seizures or anesthesia-induced network hypoactivity) notably enhance Ca²⁺ signaling in microglial processes, often accompanied by process extension and outgrowth (Umpierre et al., 2020). Importantly, such activity-dependent regulation of the process of Ca²⁺ signaling takes place in microglia not only in the case of (sub)clinical pathology but also when switching the vigilance states (Ma et al., 2024). Moreover, in mouse microglia, the interactions between the process of Ca²⁺ signaling and the sleep-wake cycle are reciprocal, as experimentally-induced potentiation of such Ca²⁺ signals was recently shown to decrease wakefulness while increasing slow-wave sleep (Ma et al., 2024).

Metabotropic receptor-mediated Ca²⁺ release from the intracellular Ca²⁺ stores likely also underlies the ongoing Ca²⁺ signals in the hypertrophic/ameboid mouse microglia. For example, in cells located in the vicinity of amyloid plaques in a mouse model of Alzheimer’s disease (Brawek et al., 2014), the ongoing Ca²⁺ signals were blocked by a non-selective antagonist of purinergic P2 receptors pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS) as well as by depletion of the intracellular Ca²⁺ stores with the help of a blocker of sarcoplasmic/endoplasmic reticulum Ca²⁺ ATPases cyclopiazonic acid. Around 80% of plaque-associated hypertrophic/amoeboid microglia exhibited ongoing somatic Ca²⁺ transients, marking a significant increase compared to the low (4%–20%) number of cells with somatic Ca²⁺ signaling among homeostatic wild-type microglia or plaque-distant ramified microglia in amyloid-depositing mice. Interestingly, plaque-distant microglia in amyloid-depositing mice displayed larger ongoing Ca²⁺ transients than controls, though their frequency remains unchanged. In turn, the plaque-associated hypertrophic/amoeboid microglia showed diminished amplitudes of ongoing Ca²⁺ signals and impaired directed process motility, failing to effectively protect the brain (Brawek et al., 2014).

After the acute kainate-induced status epilepticus mouse microglia quickly (within hours) increased the frequency of ongoing process Ca²⁺ transients and slowly (within days) changed their morphology from ramified to hypertrophic/amoeboid (Umpierre et al., 2020; Umpierre et al., 2024). In parallel to changes in morphology, somatic Ca²⁺ transients become more and more frequent, engaging approximately 60% of all cells at days 1–7 post kainate injection. The observed changes in Ca²⁺ signaling primarily reflect the enhanced P2Y₆ receptor activation, paced by transient ongoing releases of extracellular UDP during epileptogenesis, and are largely absent in P2Y₆ knock-out mice (Umpierre et al., 2024). In the kainate-induced model of epilepsy, the heightened Ca²⁺ signaling described above supports essential microglial functions such as lysosome biogenesis, process chemotaxis, and engulfment/phagocytosis of neuronal somata, as knocking out P2Y₆ receptors or experimentally inhibiting P2Y₆ receptor-mediated Ca²⁺ signaling impaired these critical functions (Umpierre et al., 2024). Interestingly, the development of kainate-induced status epilepticus also promoted the appearance of rod-shaped microglia (Umpierre et al., 2024) but the functional properties of these cells were not studied in detail.

In general, increased somatic Ca²⁺ signaling in hypertrophic/amoeboid microglia promotes the Ca²⁺-dependent generation and release of pro-inflammatory molecules such as reactive oxygen species, interleukine 1β or tumor necrosis factor α, exacerbating neuronal hyperactivity, synaptic loss, and local inflammation, and thereby contributing to a vicious cycle accelerating disease progression (Lushchak et al., 2021; Pan and Garaschuk, 2023). As discussed in detail below, in situ human microglia closely recapitulate the morphotype-specific spatiotemporal patterns of ongoing intracellular Ca²⁺ signaling found in mice (Nevelchuk et al., 2024). The molecular mechanisms underlying the ongoing Ca²⁺ signals in human microglia remain, however, unclear and subject to further studies.

Impact of aging on microglial Ca²⁺ signaling: Normal aging modifies the ongoing Ca²⁺ signaling in mouse microglia. Of note, significant increases in the frequency and duration of somatic Ca²⁺ transients are observed already at midlife, in 9–11 months old mice (Olmedillas del Moral et al., 2020), roughly corresponding to 40–45 years old humans. This increase likely signals an accumulation of pro-inflammatory self-derived “wear-and-tear” molecules in the microglial microenvironment. Upon further aging both frequency and duration of somatic Ca²⁺ transients decrease, in line with a significant downregulation of the “homeostatic” P2Y receptors (Paolicelli et al., 2022, Pan and Garaschuk, 2023). This likely reflects the microglial adaptation to the pro-inflammatory environment or its transition towards cellular senescence. Although the morphology of aging mouse microglia still looks ramified, its detailed ultrastructure remains to be deciphered.

Translational insights into Ca²⁺ signaling across microglial morphotypes: Is the knowledge about morphotype-specific Ca²⁺ signaling obtained in mouse microglia also applicable to humans? Or, in other words, can it be used for predicting the morphotype-specific Ca²⁺ signaling in homeostatic human microglia? Surprisingly, it can. In marked accordance with the mouse data, the basal levels of [Ca²⁺]i in decades-old human cells in organotypic slices briefly cultured in human cerebrospinal fluid progressively increased from ramified to hypertrophic and ameboid microglia. Moreover, a transition from ramified to amoeboid morphotypes was accompanied by an increased amplitude and incidence of ongoing somatic and a decreased amplitude and incidence of process Ca²⁺ transients. The ongoing Ca²⁺ signals also exhibited a striking shift in spatial compartmentalization, transitioning from a predominance in individual microglial processes in ramified to a wider and soma-centric localization in hypertrophic/ameboid microglia (Nevelchuk et al., 2024). While the fraction of active cells, characterized by exhibiting at least one Ca²⁺ transient during a 15-minute recording period, was similar between ramified and hypertrophic microglia, a significant decline in this fraction was noted for ameboid microglia.

These findings suggest that the dynamics of ongoing Ca²⁺ signaling is linked to microglial morphology both across species (humans versus mice) and conditions (homeostatic in humans versus disease models in mice). Ramified microglia likely use compartmentalized Ca²⁺ signaling in fine processes to detect and respond to activity changes in the surrounding neural network and to interact with synapses, glia, or vasculature. In contrast, hypertrophic and amoeboid microglia require more distributed and soma-centered intracellular Ca²⁺ signaling to support lysosome biogenesis, engulfment/phagocytosis of neuronal (sub)compartments, or cytokine production.

If so, there are several predictions to be tested: (i) as aged ramified mouse microglia differ in their Ca²⁺ signaling from their young counterparts (Olmedillas del Moral et al., 2020), their fine morphology also might be different; (ii) hypertrophic and amoeboid human microglia might belong to activated rather than homeostatic state and (iii) their early accumulation in the nonsymptomatic human brain might reflect weaker immune defense abilities of human microglia. Therefore, future studies are warranted to explore the exact time courses, preferred hormonal, metabolic, or environmental conditions as well as brain areas in which hypertrophic and amoeboid human microglia first appear. Advancements in new technologies, such as live imaging of human organoids xenotransplanted in animal brains, longitudinal in vivo tracing of individual microglial cells, and artificial intelligence/machine learning-driven analyses of morphology and function, hold great promise for addressing current knowledge gaps in human microglial research. These tools will allow for real-time visualization and tracking of microglial behaviors, investigation of cellular interactions in complex tissue environments, and precise, large-scale analysis of the relationship between microglial morphology and function.

This work was supported by Deutsche Forschungsgemeinschaft, German Research Foundation grant GA 654/13-2 to OG.