**2. Harmful and beneficial effects of microglia**

Microglia play the role of phagocytes in the CNS, thus, maintaining the homeostasis of the brain [21]. In aging brains, microglia will cause synaptic clearance leading to forgetting *via* complement pathway [22]. In AD pathology, microglia also prove to be phagocytose synapses [23, 24]. Nevertheless, with CSF1R blockade to remove microglia in Aβ models, increased Aβ is detected [25]. Despite negative outcomes of microglia, synapse loss and behavior deficits can be avoided [26, 27]. The production of neurotoxic inflammatory cytokines and reactive oxygen species are found to be related to chronic activation of microglia [28]. However, it still remains unclear whether microglia play positive or negative roles in the process of neurodegenerative diseases.

In recent research, microglia in patients with AD show specific characteristics such as aging and upregulation of apolipoprotein E (APOE) [29]. The fat droplets appearing in microglia of aged mice suggest that the main manifestations of aging are the accumulation of fat droplets and excessive secretion of pro-inflammatory factors [30], which may be a new biological hallmark of AD. Additionally, it is not difficult to find that the branching of microglia has been reduced in aged brains, thus cutting the size of microglia's area for surveillance and leading to the harm of homeostatic functions [31–34]. One important function of microglia in AD is the phagocytosis of Aβ amyloid. For instance, microglia can mediate clearance of Aβ *via* receptors including β1 integrin in neurodegenerative diseases [35]. The acute inflammatory response can also promote phagocytosis of impaired neurons and neuronal toxic accumulation [36]. Despite the protection of microglia, prolonged inflammatory reaction will exacerbate neuronal degeneration [37]. The TAM receptor tyrosine kinases (RTKs) are a distinct family of three protein tyrosine kinases, namely Tyro3, Axl, and Mer3, which play an important role in phagocytosis and phagocytic clearance of apoptotic cells and cell membranes in the adult tissues [38, 39]. Axl and Mer play pivotal roles in macrophages like phagocytosis of apoptotic cells and negative feedback inhibition of toll-like receptor and cytokine receptor signaling. In AD mice with double knockout of Axl or Mer, the ability of microglia to phagocytize the plaque is weakened, suggesting the inhibition of TAM signal promotes plaque formation [40]. A cluster of differentiation-22 (CD22), a canonical B-cell receptor and a negative regulator of phagocytosis, is found highly expressed in microglia of aged brains, and rarely in young brains [41]. The finding suggests that the inhibition of CD22 can delay aging-related dysfunction and

neurodegenerative diseases. The pellino-1 (peli1) is a ubiquitin E3 ligase, expressed in many kinds of nerve cells in the mouse brain, and with the highest expression level of microglia [42]. Similarly, Peli1 negatively regulates the ability of phagocytosis of microglia to Aβ, resulting in the inability of clearance of deposition, leading to the deterioration of AD [43].

Perineuronal nets (PNNs), with their structure remaining unknown in detail, surround the cell bodies and dendrites, and spare free space for synaptic contact [44]. In the AD mice model and human cortical tissue, PNNs are largely lost in proportion to plaque burden and depletion of microglia. Loss is prevented regardless of plaque persistence and suggests that microglia can enhance the loss of PNNs in the AD brain [45]. Besides, CD163-positive amyloid-responsive microglia are depleted in TREM2 and APOE variants in AD like TREM2 R47H and APOE4 [46].

Microglia may be detrimental to neurons in the pathological process. Recently, interleukin 3 (IL-3) from astrocytes was found to re-encode microglia, thus improving the situation of Aβ pathology [47]. Injection of IL-3 enables microglia to focus on clearing amyloid deposition and neurofibrillary tangles instead of causing extensive neuroinflammation [47]. This signaling pathway is expected to provide ideas for new drug research and development in the future and bring new drugs for the treatment of AD. A study suggested that some damaging characteristics of microglia behavior may be reversible by short-term treatment with CSF1R inhibitors [48–50]. In the mice model, removal of microglia did not improve the cognitive ability in a traumatic brain injury (TBI) [51]. Interestingly, repopulating microglia can reverse the decrease of nerve regeneration caused by brain injury and improve cognitive dysfunction in mice in an IL-6-dependant manner [51]. This study opens up a new understanding of the role of microglia in the brain injury. Remarkably, the ubiquitin ligase COP1 (also called RFWD2) is shown to dampen the neuroinflammation through inhibiting the expression of the transcription factor CCAAT/ enhancer-binding protein beta (c/EBPβ), which regulates the pro-inflammatory gene of microglia [52], marking a new target for suppressing neuroinflammation in AD patients.

Disease-associated microglia (DAM), which was identified in AD patients by single-cell RNA sequencing (RNA-seq) [53], has recently become a hot topic, characterized by molecules including Iba1, Cst3, and Hexb, typically expressed in microglia. DAM also experiences downregulation of physiologically expressed genes such as P2ry12, P2ry13, Cx3cr1, CD33, and Tmem119 [9]. It is remarkable to find that DAM is identified in areas that are affected by diseases such as cortical tissue [53] and postmortem human AD brain [54]. The evidence suggests that DAM is specifically expressed in CNS pathological process, serving as an important pathological diagnostic standard. However, in the late set of neurodegenerative diseases, its role still remains unclear, which needs further investigation.
