Microglia are the resident immune cells of the central nervous system (CNS), derived from the myeloid lineage ^[1][24]. Under physiologic conditions, microglia exist in a homeostatic state, surveilling the brain for any potentially threatening signals, such as pathogens or evidence of neuronal death/neuroinflammation ^[2][25]. They regulate neural proliferation, neural differentiation, and some regenerative capabilities of the CNS, making them an ideal target for potential therapeutics ^[3][4][26,27]. In the presence of acute danger signals, microglia activate and take on an amoeboid morphology, leading to the production of pro-inflammatory cytokines through a variety of pathways and aiding in the clearance of foreign antigens ^[1][24]. Under pathological states or when the physiological functions of microglia are overwhelmed, they can take on aberrant phenotypes and enable disease progression ^[5][28]. Many factors are involved in microglial activation in neurodegenerative diseases, an example being mitochondrial and cellular metabolism dysregulation, which causes downstream inflammation from the build-up of reactive oxygen species (ROS), amino acids, iron, and eventual microglial activation in efforts to clear such cellular degradation products ^[6][7][8][9][21,29,30,31].

Microglia have also been repeatedly implicated in the pathogenesis of neurodegenerative diseases such as AD ^{[10][11][12][13]}[32,33,34,35], where they internalize and degrade amyloid-β (Aβ) plaques and become pro-inflammatory in nature through the secretion of cytokines and recruit other microglia around the excess extra-cellular protein. Microglial receptors in AD, such as Toll-like receptor 2 (TLR2), recognize α-syn, leading researchers to believe the same mechanism is involved with the progression of PD ^[14][15][36,37]. In fact, in brain PET studies of PD patients, pro-inflammatory microglia can be found dominating the substantia nigra. However, there is a duality to the role of microglia because their actions at the initial stages of synucleinopathies are posited to be more neuroprotective, while in later stages of synucleinopathies, they are postulated as more neurodegenerative ^[16][38]. This concept is commonly referred to as the “double-edged sword” of microglial functioning.

Toll-Like Receptors (TLRs) are proteins expressed on the cell membrane of microglia and other cells that play a key role in the immune system by recognizing pathogen-associated molecular patterns (PAMPs) ^[17][66]. They are most commonly associated with foreign pathogens but have been implicated in pathological protein accumulation as well ^[18][67]. Many studies have investigated the involvement of TLRs in microglial recognition of α-syn ^[18][19][20][67,68,69].

Specifically, microglial TLR4 has been studied with regard to full-length soluble, C-terminally truncated, fibrillar, and A53T mutant α-syn and is important for microglial activation and subsequent uptake of α-syn ^[21][70]. TLR4 has been shown to have high expression in patients with synucleinopathies and is crucial for α-syn uptake and downstream inflammatory activity, such as the production of ROS and cytokine release ^[21][70]. The removal of TLR4 cascades in vivo through the generation of a TLR4-specific knockout (KO) mouse has been shown to be protective against neuronal death in the striatum and to lead to decreased neuroinflammation ^[19][68]. Though this model was not a microglial-specific KO, it demonstrates that TLR4 is intimately involved in PD-related neuroinflammation.

To better understand the role of TLR4 specifically in microglia, primary microglia from TLR4 KO mice, as well as primary microglia from control mice, were cultured and treated with either full-length soluble, C-terminally truncated or fibrillar α-syn ^[21][70]. C-terminally truncated monomeric α-syn is used because it is very prone to form aggregates ^[22][71]. Measures of phagocytosis, pro-inflammatory cytokine release, and ROS production were taken for each of these conditions and revealed that TLR4 KO microglia experienced a reduction in all three categories. The primary microglia responded to each of these three forms of α-syn as well, but it was found that the C-terminally truncated monomeric α-syn induced the greatest microglial response. In terms of mutant A53T α-syn, the expression of TLR4 mRNA has been shown to be upregulated in primary microglia after incubation with A53T α-syn.

Apart from TLR4, TLR1 and TLR2 have also been shown to functionally interact with α-syn, with TLR2 having a greater described role ^[23][73]. It was found that the expression of TLR1 and TLR2 after exposure to mutant A53T α-syn was significantly upregulated in primary microglia ^[24][74]. Further studies on TLR2 have demonstrated that α-syn PFFs can activate both BV2 cells and primary microglia through TLR2 ^[20][69]. Primary microglia from TLR2 KO mice were also less effective with the uptake of extracellular monomeric α-syn. Additionally, the inhibition of TLR2 in PFF-seeded mice significantly reduced microglial activation in vivo. In humans, post-mortem PD brains demonstrate increased TLR2 expression across both microglia and neurons in comparison to matched controls ^[24][74]. It is thought that the monomeric, oligomeric, and fibrillar forms of α-syn interact with TLR2 ^[20][25][56,69].

Overall, the TLRs are heavily involved in both monomeric and aggregated α-syn uptake and the activation of microglia, with TLR2 and TLR4 being the major players. The activity of TLRs is implicated in microglial autophagy, increased the release of pro-inflammatory cytokines and increased α-syn clearance. Whether the interaction of microglial TLRs with α-syn protects or progresses the synucleinopathic state is complicated and depends on various factors, including the stage of disease and amount of α-syn build-up.

Lymphocyte activation gene 3 (Lag3) is a receptor that is a member of the immunoglobulin superfamily, which binds with pathologic α-syn fibrils ^[26][75] and the C-terminus of α-syn ^[27][76]. The depletion of Lag3 can reduce the neuronal uptake of α-syn fibrils and the subsequent neuron-to-neuron transmission of pathogenic α-syn. In an in vivo experiment, KO of Lag3 in mice injected with α-syn PFF led to reduced dopaminergic neuron loss and reduced neurodegenerative phenotypes compared to WT mice injected with α-syn PFF. Such findings were also seen when using Lag3 antibodies ^[26][27][75,76]. Similar results of neuroprotection were seen when using murine models overexpressing hA53T α-syn driven by the mouse prion protein promoter ^[28][77]. These mice were bred with Lag3 KO mice, and the Lag3 KO mice demonstrated reduced α-syn pathology and microglial activation, along with improvements in behavioral tests. In follow-up studies using Lag3 KO mice and a Lag3 reporter mouse line, Lag3 expression has been proven not only in neurons ^[27][76] but also in microglia ^[29][78]. In the gene expression profiles of purified microglia isolated at autopsy of individuals without neurodegenerative disease, microglial Lag3 was expressed at levels similar to known microglial marker ITGAM (CD11B), with confirmation at the protein level using DAB staining and immunofluorescence ^[30][79]. This is increasingly relevant in the setting of synucleinopathy, where Lag3 levels and microglial activation, in general, are known to be markedly elevated ^[31][80]. Functionally, it is interesting to note that microglial Lag3 is being studied as a target for the treatment of depression ^[29][78] and that α-syn is more highly expressed in patients with major depressive disorder ^[32][81], as many patients with synucleinopathies experience depressive symptoms ^[33][34][35][82,83,84]. Lag3 could therefore be a possible link between synucleinopathies and depression. Overall, given its expression level in microglia and known functions, Lag3 should be studied as a potentially important microglial receptor for α-syn ^{[26][27][28][29][30][36][37][38]}[75,76,77,78,79,85,86,87].

Triggering Receptor Expressed on Myeloid Cells 2 (TREM2) is a transmembrane receptor found in several myeloid cells, including microglia, that binds to a host of extracellular ligands, leading to a downstream signaling cascade that promotes survival, proliferation, and inflammation regulation ^[39][88]. Some of these ligands that are especially disease relevant include ApoE, Aβ, and the most recently discovered interactor, TDP-43, which is a protein that can become pathologically accumulated in neurodegenerative diseases, such as frontotemporal dementia (FTD) and ALS ^[40][89]. Each of these ligands is involved in neurodegenerative disease; however, there remains no documentation as to whether α-syn is a ligand for TREM2. This is an important question to address, as TREM2 mutations have been identified as risk factors for PD ^[41][90], and a functional interaction between TREM2 and α-syn has been demonstrated ^[42][91]. This functional interaction revealed that TREM2 deficiency leads to increased α-syn induced neurodegeneration and neuroinflammation in vitro and in vivo.

The importance of interrogating whether a TREM2–α-syn interaction exists is further highlighted by the function of TREM2 mutations in AD. The R47H mutation in TREM2, which has a significant correlation to FTD, AD, and PD, is known to decrease the binding of TREM2 to Aβ, decreasing microglial activation and subsequent clearance of Aβ ^[43][44][92,93]. This finding is further bolstered and shown to be cell autonomous by work conducted using TREM2-KO iPSC-derived microglia monocultures, showing that they exhibit disease phenotypes, including reduced survival, altered phagocytic ability, and impaired chemotaxis ^[45][94]. TREM2-deficient microglia become locked in a homeostatic state, indicating the necessity of TREM2 to react to a neurodegeneration-associated stimulus. However, it was also recently published that TREM2 activation over time has the potential to worsen Aβ-induced tau pathology ^[46][95], further contributing to the theme of the double-edged sword of microglia and the potential temporal aspect of their function.

A general consensus across various data implicates TREM2 function as neuroprotective given that mutations in TREM2 confer PD risk. Since TREM2 is known to bind to other pathologically accumulated proteins in neurodegenerative disease, the relationship between TREM2 and α-syn should be further considered.

The group 1 metabotropic glutamate receptor 5 (mGluR5) is a G-protein coupled receptor (GPCR) expressed throughout the brain ^[47][96]. Though its expression is highest in neurons, microglia also express mGluR5 ^[48][97]. Microglial mGluR5 has been an attractive target due to its involvement in neuroinflammation since the activation of mGluR5 in microglia significantly inhibits their inflammatory response ^[49][98]. Researchers built upon this previous knowledge by reporting a pathway that demonstrates mGluR5 involvement in α-syn mediated microglial activation ^[48][97]. Using BV2 cells and primary microglia, the group showed that monomeric α-syn physically interacts with mGluR5 and that the activation of mGluR5 dampens microglial activation by monomeric α-syn and protects neurons from toxicity. The group went on to show that the overexpression of monomeric α-syn leads to the degradation of mGluR5 in the lysosome, increasing the potential of α-syn to induce an inflammatory response. The use of a specific mGluR5 agonist prevents the α-syn mGluR5 interaction, thereby preventing the degradation of mGluR5 and presenting a potential therapeutic for synucleinopathies. The group also generated an AAV–α-syn mouse model of PD by using an intrastriatal injection, which is known to form α-syn aggregates. They demonstrated the colocalization and direct interaction of the aggregated form of α-syn with the mGluR5 receptor and demonstrated the capacity of mGluR5 to reduce inflammation in an in vivo model of PD.

Additionally, aside from α-syn, mGluR5 is also involved in a complex with Aβ with an interestingly sex-dependent interaction, with the cortical and hippocampal mGluR5 binding Aβ in males but not females ^[50][99]. A sex-dependent relationship may be an important consideration when investigating the role of mGluR5 with α-syn. Overall, mGluR5 aids microglia in the maintenance of a homeostatic state and decreases neuronal toxicity in the synucleinopathic brain.

There are several other receptors that have been studied regarding α-syn interaction, and some have been shown to have some relevance for microglia with limited work. The N-methyl-D-aspartate receptor (NMDAR) has previously been identified as a receptor for fibrillar α-syn, and some work has shown that when this interaction occurs on microglia, the NMDA activation of MAPK is blocked ^[51][100]. The binding of aggregated α-syn to Cd11b on microglia is important for the activation of NADPH oxidase (NOX2) ^[52][101]. The purinergic receptor P2X7 has been shown to interact with both WT and A53T α-syn as well on microglia and activate the p47-PHOX pathway via PI3K/AKT activity, which increases intracellular ROS generation ^[53][102]. The FcγRIIB receptor on microglia has been shown to bind to aggregated α-syn, leading to increased SHP-1 activation and the inhibition of phagocytosis ^[54][103]. Lastly, the receptor for advanced glycation end products (RAGE) has an alkaline region that was recently found to bind to the acidic C-terminus of α-syn, with preferential binding of α-syn fibrils over other forms ^[55][104]. This binding to α-syn fibrils induces neuroinflammation that is reduced in RAGE KO models and with RAGE receptor inhibitors, such as FPS-ZM1. In fact, RAGE, similar to Lag3, is in the immunoglobulin superfamily and physiologically acts as a pattern-recognition receptor on microglia. Further studies should investigate whether there are any other effects of these interactions on microglia and whether receptors such as NMDAR, Cd11b, P2X7, FcγRIIB, or RAGE, could serve as therapeutic targets.

Some receptors have been shown to be important for α-syn interactions in other cell types but have yet to be shown as significant α-syn receptors in microglia. For example, heparan sulfate proteoglycans (HSPGs) are important receptors for the internalization of α-syn by oligodendrocytes, but they have been shown to be far less important for microglia ^[56][105]. Neurexins, APLP1, and PrPc have been well studied and proven to interact with α-syn, but they each have very low expression in microglia ^[37][38][86,87]. The a3 subunit of the Na+/K+-ATPase has also not been studied as an α-syn receptor in microglia. The findings involving key microglial receptors for α-syn are summarized in Table 1.

Receptor	Known Interactor?	Aggregation State of α-Syn Known to Interact with Receptor	Downstream Effect
TLR	Yes	Monomeric and aggregated forms	Phagocytosis of α-syn Secretion of ROS and pro-inflammatory cytokines Synucleinphagy
Lag3	Yes, but not yet involving microglia	Aggregated forms	Unknown
TREM2	Unknown	Not yet known	Unknown, but likely survival, phagocytosis and proliferation
mGluR5	Yes	Monomeric and aggregated forms	Neuroprotection Dampens immune response
NMDAR	Yes	Aggregated forms	Decreased homeostatic microglial activity
Cd11b	Yes	Aggregated forms	Increased microglial oxidative stress
P2X7	Yes	Aggregated forms	Increased microglial oxidative stress
FcγRIIB	Yes	Aggregated forms	Inhibition of phagocytosis
RAGE	Yes	Monomeric and preferential binding of aggregated forms	Neuroinflammation evidenced by secretion of TNF-α, IL-1β, and IL-6