The protective PLCγ2-P522R variant mitigates Alzheimer’s disease-associated pathologies by enhancing beneficial microglial functions

Activated microglia limit AD pathology by clearing and/or compacting β-amyloid and protecting vulnerable neurons from its toxic effects.

“In the context of Alzheimer’s disease (AD), activated microglia limit disease pathology by clearing and/or compacting β-amyloid and protecting vulnerable neurons from its toxic effects [5].”

• cites [5] Keren-Shaul H, Spinrad A, Weiner A, Matcovitch-Natan O, Dvir-Szternfeld R, Ulland TK, et al. A unique microglia type associated with restricting development of alzheimer’s disease. Cell. 2017;169(7):1276-1290.e17. sourcePartly supported

  The Results (page unavailable) show amyloid-containing microglia near plaques, and the Discussion (page unavailable) describes disease-associated microglia as potentially beneficial through phagocytosis and plaque clearance. The paper does not establish that these cells compact amyloid or protect vulnerable neurons from its toxicity; it also frames their disease-limiting role as a suggestion rather than a demonstrated effect. (LLM, conf=0.86)

  Experimental — accuracy not yet measured

Genetic depletion of either TREM2 or PLCγ2 impairs lipid processing and mitochondrial function in human iPSC-derived microglia.

“Genetic depletion of either TREM2 or PLCγ2 similarly impairs lipid processing and mitochondrial function in human iPSC-derived microglia (iMGL) [6, 8].”

• cites [6] Andreone BJ, Przybyla L, Llapashtica C, Rana A, Davis SS, Van Lengerich B, et al. Alzheimer’s-associated PLCγ2 is a signaling node required for both TREM2 function and the inflammatory response in human microglia. Nat Neurosci. 2020;23(8):927–38. sourceNot checked — no full text of the source
• cites [8] Van Lengerich B, Zhan L, Xia D, Chan D, Joy D, Park JI, et al. A TREM2-activating antibody with a blood–brain barrier transport vehicle enhances microglial metabolism in Alzheimer’s disease models. Nat Neurosci. 2023;26(3):416. sourceSupported

PLCγ2-P522R leads to more compacted plaque morphology in 5XFAD model mice.

“In contrast, PLCγ2-P522R was shown to promote microglia-mediated phagocytosis of β-amyloid, leading to more compacted plaque morphology, reduced neuronal damage, and improved working memory in 5XFAD model mice with endogenous microglia carrying the PLCγ2-P522R variant [17].”

• cites [17] Tsai AP, Dong C, Lin PBC, Oblak AL, Viana Di Prisco G, Wang N, et al. Genetic variants of phospholipase C-γ2 alter the phenotype and function of microglia and confer differential risk for Alzheimer’s disease. Immunity. 2023;9:2121–36. sourceSupported

The PLCγ2-P522R variant reduces the risk of AD.

“A rare gain-of-function variant (rs72824905, C > G, p.P522R) was shown to reduce the risk of AD [2].”

• cites [2] Sims R, Van Der Lee SJ, Naj AC, Bellenguez C, et al. Rare coding variants in PLCG2, ABI3, and TREM2 implicate microglial-mediated innate immunity in Alzheimer’s disease. Nat Genet. 2017;49(9):1373–84. sourceSupported

The PLCγ2-P522R variant is a functional hypermorph that mildly increases PLCγ2 enzyme activity.

“PLCγ2-P522R is a functional hypermorph that mildly increases PLCγ2 enzyme activity [3, 6, 7].”

• cites [3] Magno L, Lessard CB, Martins M, Lang V, Cruz P, Asi Y, et al. Alzheimer’s disease phospholipase C-gamma-2 (PLCG2) protective variant is a functional hypermorph. Alz Res Therapy. 2019;11(1):16. sourceSupported
• cites [6] Andreone BJ, Przybyla L, Llapashtica C, Rana A, Davis SS, Van Lengerich B, et al. Alzheimer’s-associated PLCγ2 is a signaling node required for both TREM2 function and the inflammatory response in human microglia. Nat Neurosci. 2020;23(8):927–38. sourceNot checked — no full text of the source
• cites [7] Takalo M, Wittrahm R, Wefers B, Parhizkar S, Jokivarsi K, Kuulasmaa T, et al. The Alzheimer’s disease-associated protective Plcγ2-P522R variant promotes immune functions. Mol Neurodegeneration. 2020;15(1):52. sourceSupported

The PLCγ2-P522R variant increases the survival of mouse bone marrow-derived macrophages upon LPS treatment.

“We previously showed that the PLCγ2-P522R variant increases microglial activation in adult C57BL/6J mice and enhances the acute inflammatory response and increases the survival of mouse bone marrow-derived macrophages upon treatment with lipopolysaccharide (LPS) [7].”

• cites [7] Takalo M, Wittrahm R, Wefers B, Parhizkar S, Jokivarsi K, Kuulasmaa T, et al. The Alzheimer’s disease-associated protective Plcγ2-P522R variant promotes immune functions. Mol Neurodegeneration. 2020;15(1):52. sourcePartly supported

  The Findings report enhanced survival of P522R knock-in bone marrow-derived macrophages after m-CSF withdrawal, not after LPS treatment. In a separate Findings passage, LPS plus IFNγ treatment produced a stronger inflammatory response; it was not reported as a survival test. Page numbers are not available in our copy. (LLM, conf=0.89)

  Experimental — accuracy not yet measured

PLCγ2-P522R improves working memory in 5XFAD model mice.

“In contrast, PLCγ2-P522R was shown to promote microglia-mediated phagocytosis of β-amyloid, leading to more compacted plaque morphology, reduced neuronal damage, and improved working memory in 5XFAD model mice with endogenous microglia carrying the PLCγ2-P522R variant [17].”

• cites [17] Tsai AP, Dong C, Lin PBC, Oblak AL, Viana Di Prisco G, Wang N, et al. Genetic variants of phospholipase C-γ2 alter the phenotype and function of microglia and confer differential risk for Alzheimer’s disease. Immunity. 2023;9:2121–36. sourceSupported

PLCγ2-P522R promotes microglia-mediated phagocytosis of β-amyloid in 5XFAD model mice with endogenous microglia carrying the variant.

“In contrast, PLCγ2-P522R was shown to promote microglia-mediated phagocytosis of β-amyloid, leading to more compacted plaque morphology, reduced neuronal damage, and improved working memory in 5XFAD model mice with endogenous microglia carrying the PLCγ2-P522R variant [17].”

• cites [17] Tsai AP, Dong C, Lin PBC, Oblak AL, Viana Di Prisco G, Wang N, et al. Genetic variants of phospholipase C-γ2 alter the phenotype and function of microglia and confer differential risk for Alzheimer’s disease. Immunity. 2023;9:2121–36. sourcePartly supported

  In “PLCG2 variants affect plaque pathology and microglial uptake of Aβ aggregates” (page unavailable), P522R microglia showed increased uptake of Aβ aggregates, and 5xFAD P522R mice had a shift toward compact plaques. The paper also reports preserved working memory, but the Discussion (page unavailable) presents enhanced phagocytosis as a possible mechanism rather than establishing the sentence’s causal chain; the cited passages do not show reduced neuronal damage. (LLM, conf=0.83)

  Experimental — accuracy not yet measured

PLCG2 is highly expressed in microglia.

“PLCG2 is highly expressed in microglia [3].”

• cites [3] Magno L, Lessard CB, Martins M, Lang V, Cruz P, Asi Y, et al. Alzheimer’s disease phospholipase C-gamma-2 (PLCG2) protective variant is a functional hypermorph. Alz Res Therapy. 2019;11(1):16. sourceSupported

The protective PLCγ2-P522R variant increases microglia activation in WT mice with no β-amyloid pathology.

“We have previously shown that the protective PLCγ2-P522R (rs72824905, p.P522R, Fig. 1A) variant increases microglia activation in WT mice with no β-amyloid pathology [7].”

• cites [7] Takalo M, Wittrahm R, Wefers B, Parhizkar S, Jokivarsi K, Kuulasmaa T, et al. The Alzheimer’s disease-associated protective Plcγ2-P522R variant promotes immune functions. Mol Neurodegeneration. 2020;15(1):52. sourcePartly supported

  The Findings section reports that RNA pathway analysis suggested higher microglial activation in Plcγ2-P522R knock-in mice than in WT littermates. It also says studies with AD-associated pathology are still needed, consistent with the absence of that pathology here. The claim is imprecise in saying the increase occurred in WT mice: the variant-bearing mice were knock-ins, and WT mice were the comparator. (LLM, conf=0.86)

  Experimental — accuracy not yet measured

• AssumptionRests on an assumption: The PLCγ2-P522R variant is protective against Alzheimer's disease in humans.

The PLCγ2-P522R variant enhances the acute inflammatory response in mouse bone marrow-derived macrophages upon LPS treatment.

“We previously showed that the PLCγ2-P522R variant increases microglial activation in adult C57BL/6J mice and enhances the acute inflammatory response and increases the survival of mouse bone marrow-derived macrophages upon treatment with lipopolysaccharide (LPS) [7].”

• cites [7] Takalo M, Wittrahm R, Wefers B, Parhizkar S, Jokivarsi K, Kuulasmaa T, et al. The Alzheimer’s disease-associated protective Plcγ2-P522R variant promotes immune functions. Mol Neurodegeneration. 2020;15(1):52. sourcePartly supported

  The Findings report that bone marrow-derived macrophages from P522R knock-in mice released significantly more inflammatory cytokines than wild-type cells after stimulation. The treatment was LPS together with interferon-gamma (IFNγ), not LPS alone, so the stated treatment condition is incomplete. (LLM, conf=0.88)

  Experimental — accuracy not yet measured

PLCγ2-P522R reduces neuronal damage in 5XFAD model mice.

“In contrast, PLCγ2-P522R was shown to promote microglia-mediated phagocytosis of β-amyloid, leading to more compacted plaque morphology, reduced neuronal damage, and improved working memory in 5XFAD model mice with endogenous microglia carrying the PLCγ2-P522R variant [17].”

• cites [17] Tsai AP, Dong C, Lin PBC, Oblak AL, Viana Di Prisco G, Wang N, et al. Genetic variants of phospholipase C-γ2 alter the phenotype and function of microglia and confer differential risk for Alzheimer’s disease. Immunity. 2023;9:2121–36. sourcePartly supported

  The Discussion reports that PLCG2 P522R ameliorated impaired synaptic function in 5xFAD mice, supporting a protective effect. The paper does not establish reduced neuronal damage as a distinct outcome; its Limitations section says the models cannot determine whether the variants directly affect neuronal function. (LLM, conf=0.82)

  Experimental — accuracy not yet measured

PLCγ2 is expressed primarily in microglia in the brain.

“In the brain, PLCγ2 is expressed primarily in microglia, the resident immune cells of the brain [2, 3].”

• cites [2] Sims R, Van Der Lee SJ, Naj AC, Bellenguez C, et al. Rare coding variants in PLCG2, ABI3, and TREM2 implicate microglial-mediated innate immunity in Alzheimer’s disease. Nat Genet. 2017;49(9):1373–84. sourceSupported
• cites [3] Magno L, Lessard CB, Martins M, Lang V, Cruz P, Asi Y, et al. Alzheimer’s disease phospholipase C-gamma-2 (PLCG2) protective variant is a functional hypermorph. Alz Res Therapy. 2019;11(1):16. sourceSupported

In microglia, PLCγ2 functions as a key downstream effector for TREM2 and TLRs.

“In microglia, PLCγ2 functions as a key downstream effector for triggering receptor expressed on myeloid cells 2 (TREM2) and Toll-like receptors (TLRs) [6, 7].”

• cites [6] Andreone BJ, Przybyla L, Llapashtica C, Rana A, Davis SS, Van Lengerich B, et al. Alzheimer’s-associated PLCγ2 is a signaling node required for both TREM2 function and the inflammatory response in human microglia. Nat Neurosci. 2020;23(8):927–38. sourceNot checked — no full text of the source
• cites [7] Takalo M, Wittrahm R, Wefers B, Parhizkar S, Jokivarsi K, Kuulasmaa T, et al. The Alzheimer’s disease-associated protective Plcγ2-P522R variant promotes immune functions. Mol Neurodegeneration. 2020;15(1):52. sourceSupported

The PLCγ2-P522R variant increases microglial activation in adult C57BL/6J mice.

“We previously showed that the PLCγ2-P522R variant increases microglial activation in adult C57BL/6J mice and enhances the acute inflammatory response and increases the survival of mouse bone marrow-derived macrophages upon treatment with lipopolysaccharide (LPS) [7].”

• cites [7] Takalo M, Wittrahm R, Wefers B, Parhizkar S, Jokivarsi K, Kuulasmaa T, et al. The Alzheimer’s disease-associated protective Plcγ2-P522R variant promotes immune functions. Mol Neurodegeneration. 2020;15(1):52. sourceSupported

The PLCγ2-P522R variant was previously shown to reduce β-amyloid pathology in 5XFAD mice.

“The PLCγ2-P522R variant was previously shown to reduce β-amyloid pathology in 5XFAD mice [2, 17].”

• cites [2] Sims R, Van Der Lee SJ, Naj AC, Bellenguez C, et al. Rare coding variants in PLCG2, ABI3, and TREM2 implicate microglial-mediated innate immunity in Alzheimer’s disease. Nat Genet. 2017;49(9):1373–84. sourceCould not verify

  The paper reports that PLCG2 p.P522R is associated with decreased late-onset Alzheimer’s disease risk in humans (main text, page unavailable). Its discussion of mouse models does not report that this variant reduces β-amyloid pathology in 5XFAD mice; searches for 5XFAD and P522R-related mouse pathology found no such passage. (LLM, conf=0.76)

  Experimental — accuracy not yet measured
• cites [17] Tsai AP, Dong C, Lin PBC, Oblak AL, Viana Di Prisco G, Wang N, et al. Genetic variants of phospholipase C-γ2 alter the phenotype and function of microglia and confer differential risk for Alzheimer’s disease. Immunity. 2023;9:2121–36. sourceSupported

• AssumptionRests on an assumption: The PLCγ2-P522R variant is protective against Alzheimer's disease in humans.

The reduced number of dystrophic neurites indicates decreased β-amyloid-driven axonal damage.

“indicative of decreased β-amyloid-driven axonal damage [44–47]”

• cites [44] Sadleir KR, Kandalepas PC, Buggia-Prévot V, Nicholson DA, Thinakaran G, Vassar R. Presynaptic dystrophic neurites surrounding amyloid plaques are sites of microtubule disruption, BACE1 elevation, and increased Aβ generation in Alzheimer’s disease. Acta Neuropathol. 2016;132(2):235–56. sourceSupported
• cites [45] Wang Y, Ulland TK, Ulrich JD, Song W, Tzaferis JA, Hole JT, et al. TREM2-mediated early microglial response limits diffusion and toxicity of amyloid plaques. J Exp Med. 2016;213(5):667–75. sourceSupported
• cites [46] Li JM, Xue ZQ, Deng SH, Luo XG, Patrylo PR, Rose GW, et al. Amyloid plaque pathogenesis in 5XFAD mouse spinal cord: Retrograde transneuronal modulation after peripheral nerve injury. Neurotox Res. 2013;24(1):1–14. sourcePartly supported

  The Discussion (‘Spinal Aβ deposition largely correlates with glutamatergic axonopathy’; page not provided) identifies dystrophic neurites as axon terminals and reports that their abundance increases alongside Aβ deposits. This supports treating fewer dystrophic neurites as a sign of less axonal pathology, but the paper describes an association, not proof that β-amyloid drives the damage. (LLM, conf=0.84)

  Experimental — accuracy not yet measured
• cites [47] Mabrouk R, Miettinen PO, Tanila H. Most dystrophic neurites in the common 5xFAD Alzheimer mouse model originate from axon terminals. Neurobiol Dis. 2023;15(182): 106150. sourceNot checked — no full text of the source

Previous studies have demonstrated that the PLCγ2-P522R variant increases the phagocytosis of Aβ 1-42 in both mouse microglia and human iPSC models.

“previous studies have demonstrated that the PLCγ2-P522R variant also increases the phagocytosis of Aβ 1–42 in both mouse microglia and human iPSC models [17, 53, 54].”

• cites [17] Tsai AP, Dong C, Lin PBC, Oblak AL, Viana Di Prisco G, Wang N, et al. Genetic variants of phospholipase C-γ2 alter the phenotype and function of microglia and confer differential risk for Alzheimer’s disease. Immunity. 2023;9:2121–36. sourcePartly supported

  In “PLCG2 variants affect plaque pathology and microglial uptake of Aβ aggregates” (page not provided), the paper reports 1.22-fold greater uptake of Aβ1–42 aggregates by primary mouse microglia carrying P522R. The passages about human iPSC-derived microglia do not report a P522R-related increase in Aβ1–42 phagocytosis, so that part of the claim is unsupported by this source. (LLM, conf=0.87)

  Experimental — accuracy not yet measured
• cites [53] Maguire E, Menzies GE, Phillips T, Sasner M, Williams HM, Czubala MA, et al. PIP2 depletion and altered endocytosis caused by expression of Alzheimer’s disease-protective variant PLCγ2 R522. EMBO J. 2021;40(17): e105603. sourcePartly supported

  The Results (Figure 5; page unavailable in our copy) report increased uptake of soluble Aβ1–42 oligomers in R522 mouse microglia and human iPSC-derived microglia, but classify it as endocytosis, not phagocytosis. The same Results section reports reduced phagocytosis of E. coli and zymosan particles in those models. Thus the increased Aβ uptake is supported, but the claimed mechanism is not. (LLM, conf=0.88)

  Experimental — accuracy not yet measured
• cites [54] Solomon S, Sampathkumar NK, Carre I, Mondal M, Chennell G, Vernon AC, et al. Heterozygous expression of the Alzheimer’s disease-protective PLCγ2 P522R variant enhances Aβ clearance while preserving synapses. Cell Mol Life Sci. 2022;79(8):453. sourcePartly supported

  The Results section reports increased Aβ1–42 uptake in human iPSC-derived microglia expressing PLCγ2-P522R. In mouse BV2 microglial cells, however, the increase associated specifically with P522R was mild and not statistically significant (p = 0.3385); increased PLCγ2 expression itself drove the robust uptake effect. The claim therefore overstates the mouse-cell finding. (LLM, conf=0.88)

  Experimental — accuracy not yet measured

Deficiency of PLCγ2 in human iPSC-derived macrophages leads to reduced migration and adhesion, associated with decreased expression of fibronectin and a specific set of integrin genes.

“Deficiency of PLCγ2 in human iPSC-derived macrophages leads to reduced migration and adhesion, which is associated with decreased expression of fibronectin and a specific set of integrin genes [50].”

• cites [50] Obst J, Hall-Roberts HL, Smith TB, Kreuzer M, Magno L, Di Daniel E, et al. PLCγ2 regulates TREM2 signalling and integrin-mediated adhesion and migration of human iPSC-derived macrophages. Sci Rep. 2021;11(1):19842. sourceSupported

The protective PLCγ2-P522R variant may exert similar beneficial effects on microglial functions as those previously reported with a TREM2-activating antibody in different AD disease models.

“Thus, the protective PLCγ2-P522R variant may exert similar beneficial effects on the functions of microglia as those previously reported with a TREM2-activating antibody in different AD disease models [8].”

• cites [8] Van Lengerich B, Zhan L, Xia D, Chan D, Joy D, Park JI, et al. A TREM2-activating antibody with a blood–brain barrier transport vehicle enhances microglial metabolism in Alzheimer’s disease models. Nat Neurosci. 2023;26(3):416. sourceSupported

PLCγ2-P522R KI mouse macrophages respond more efficiently to LPS- and IFNγ-induced inflammation than wild-type cells do.

“We previously showed that PLCγ2-P522R KI mouse macrophages respond more efficiently to LPS- and IFNγ-induced inflammation than wild-type cells do [7].”

• cites [7] Takalo M, Wittrahm R, Wefers B, Parhizkar S, Jokivarsi K, Kuulasmaa T, et al. The Alzheimer’s disease-associated protective Plcγ2-P522R variant promotes immune functions. Mol Neurodegeneration. 2020;15(1):52. sourceSupported

Increasing evidence has shown that microglial activation may be a key driver of the anxiety phenotype in mice.

“increasing evidence has shown that microglial activation may be a key driver of the anxiety phenotype in mice [57].”

• cites [57] Lehmann ML, Weigel TK, Poffenberger CN, Herkenham M. The behavioral sequelae of social defeat require microglia and are driven by oxidative stress in mice. J Neurosci. 2019;39(28):5594–605. sourcePartly supported

  The Results report that microglial depletion prevented anxiety-like behavior after chronic social defeat and infer that microglial activation during that stressor contributes to susceptibility. The Discussion likewise describes microglia as necessary for the stress-induced behavioral effect. The evidence supports the claim for chronic social defeat, but not for anxiety phenotypes in mice generally. (LLM, conf=0.82)

  Experimental — accuracy not yet measured

Depletion of either TREM2 or PLCγ2 has previously been shown to increase lipid accumulation and impair mitochondrial respiration via suppression of lipid regulatory genes, including FABP5.

“depletion of either TREM2 or PLCγ2 has previously been shown to increase lipid accumulation and impair mitochondrial respiration via the suppression of lipid regulatory genes, including FABP5 [6, 8, 61].”

• cites [6] Andreone BJ, Przybyla L, Llapashtica C, Rana A, Davis SS, Van Lengerich B, et al. Alzheimer’s-associated PLCγ2 is a signaling node required for both TREM2 function and the inflammatory response in human microglia. Nat Neurosci. 2020;23(8):927–38. sourceNot checked — no full text of the source
• cites [8] Van Lengerich B, Zhan L, Xia D, Chan D, Joy D, Park JI, et al. A TREM2-activating antibody with a blood–brain barrier transport vehicle enhances microglial metabolism in Alzheimer’s disease models. Nat Neurosci. 2023;26(3):416. sourcePartly supported

  The Results section reports impaired mitochondrial respiration in both TREM2-knockout and PLCG2-knockout microglia. It also describes lipid accumulation with TREM2 loss of function and a failure of antibody-induced triglyceride reduction in PLCG2-knockout cells. The passages do not establish that depletion of either gene increases lipid accumulation through suppression of lipid-regulatory genes, and they do not identify FABP5 as the mechanism. (LLM, conf=0.86)

  Experimental — accuracy not yet measured
• cites [61] Wei W, Zhang L, Xin W, Pan Y, Tatenhorst L, Hao Z, et al. TREM2 regulates microglial lipid droplet formation and represses post-ischemic brain injury. Biomed Pharmacother. 2024;170: 115962. sourcePartly supported

  Section 3.3 reports that TREM2 knockdown increased cholesterol accumulation and downregulated several genes involved in cholesterol clearance and lipid hydrolysis. The passages do not establish the same effects for PLCγ2, impaired mitochondrial respiration, or suppression of FABP5; they also report upregulation of some lipid-synthesis genes. Page numbers were not provided in our copy. (LLM, conf=0.88)

  Experimental — accuracy not yet measured

There was no difference in [18F]FEPPA ligand uptake between the genotypes in 7-month-old mice.

“There was no difference in [18F]FEPPA ligand uptake between the genotypes in 7-month-old mice (Fig. 1B).”

[18F]FEPPA ligand uptake was significantly increased in the pons and hippocampus of 13-month-old A+/Pki/ki mice compared with A+/Pwt/wt mice.

“When the brain regions were examined separately, [18F]FEPPA ligand uptake was significantly increased in the pons (p = 0.013) and hippocampus (p = 0.012) (Fig. 1C).”

The PLCγ2-P522R variant regulated key targets and pathways involved in lipid metabolism, mitochondrial fatty acid oxidation, and inflammatory/interferon signaling in acutely isolated adult mouse microglia and human monocyte-derived microglia-like cells.

“transcriptomic and proteomic analyses revealed that the PLCγ2-P522R variant regulated key targets and pathways involved in lipid metabolism, mitochondrial fatty acid oxidation, and inflammatory/interferon signaling in acutely isolated adult mouse microglia and human monocyte-derived microglia-like cells.”

In 13-month-old animals, [18F]FEPPA ligand uptake was increased in the whole brain of APP/PS1 x PLCγ2-P522R KI (A+/Pki/ki) mice compared with A+/Pwt/wt mice.

“However, in 13-month-old animals, increased [18F]FEPPA ligand uptake was observed in the whole brain (p = 0.032) of APP/PS1 x PLCγ2-P522R KI (hereafter referred to as A+/Pki/ki) mice compared with A+/Pwt/wt mice.”

The area of microglia around β-amyloid plaques was increased in mice carrying the PLCγ2-P522R variant.

“The area of the microglia around β-amyloid plaques was also increased in mice carrying the PLCγ2-P522R variant”

The PLCγ2-P522R variant promoted anxiety in female APP/PS1 mice.

“Moreover, the PLCγ2-P522R variant promoted anxiety in these mice.”

β-amyloid plaque-associated neuronal dystrophy was reduced in mice carrying the PLCγ2-P522R variant.

“while β-amyloid plaque-associated neuronal dystrophy and the levels of certain cytokines, including IL-6 and IL-1β, were reduced.”

The PLCγ2-P522R variant increased mitochondrial respiration in human iPSC-derived microglia.

“Finally, the PLCγ2-P522R variant also increased mitochondrial respiration in human iPSC-derived microglia.”

The PLCγ2-P522R variant decreased β-amyloid plaque count in female APP/PS1 mice.

“The PLCγ2-P522R variant decreased β-amyloid plaque count and coverage in female APP/PS1 mice.”

In cultured mouse primary microglia, the PLCγ2-P522R variant reduced the size of lipid droplets.

“In cultured mouse primary microglia, the PLCγ2-P522R variant reduced the size of lipid droplets.”

Levels of certain cytokines, including IL-6 and IL-1β, were reduced in mice carrying the PLCγ2-P522R variant.

“while β-amyloid plaque-associated neuronal dystrophy and the levels of certain cytokines, including IL-6 and IL-1β, were reduced.”

The PLCγ2-P522R variant decreased β-amyloid plaque coverage in female APP/PS1 mice.

“The PLCγ2-P522R variant decreased β-amyloid plaque count and coverage in female APP/PS1 mice.”

No differences in the colocalization of 6E10 within the IBA1+ area were detected between the mice with different genotypes.

“Furthermore, no differences in the colocalization of 6E10 within the IBA1+ area were detected between the mice with different genotypes (Fig. 4B and Supplementary Figures, S4B)”

• AssumptionRests on an assumption: The APP/PS1 mouse model recapitulates Alzheimer's disease-associated β-amyloid pathology relevant to human AD.

No DEGs or enriched biological pathways were detected in the temporo-occipital cortex or isolated CD11b+ microglia when A+/Pki/ki were compared with A+/Pwt/wt mice.

“In fact, no DEGs or enriched biological pathways were detected in the temporo-occipital cortex (Supplementary Figures, Figure S6D) or isolated CD11b+ microglia (Supplementary Figures, Figure S6E) when A+/Pki/ki were compared with A+/Pwt/wt mice.”

No difference was found in the IBA1+ signal overlapping the β-amyloid plaque area itself, either in the entorhinal cortex or hippocampus.

“No difference was found in the IBA1+ signal overlapping the β-amyloid plaque area itself, either in the entorhinal cortex or hippocampus.”

• AssumptionRests on an assumption: The APP/PS1 mouse model recapitulates Alzheimer's disease-associated β-amyloid pathology relevant to human AD.

The Pki/ki mice had a stronger behavioral response (crab-like walking backward) to foot shock than did the Pwt/wt mice.

“In fact, the Pki/ki mice had a stronger behavioral response (crab-like walking backward) to foot shock than did the Pwt/wt mice (Fig. 7H).”

Several genes encoding PI3K subunits, Pik3cd, Pik3cg, Pik3r5, Pik3ap1, Pik3r1, and Pik3r4, were downregulated.

“Interestingly, several genes encoding PI3K subunits, Pik3cd, Pik3cg, Pik3r5, Pik3ap1, Pik3r1, and Pik3r4, were downregulated (Supplementary Figures, Figure S8C).”

The PLCγ2-P522R variant does not promote DAM signature in female APP/PS1 mice.

“PLCγ2-P522R variant does not increase β-amyloid plaque-associated APOE or promote DAM signature in the female APP/PS1 mice”

IL-1β levels were lower in the hippocampus of A+/Pki/ki mice than in A+/Pwt/wt mice (p = 0.021).

“while those of IL-1β (p = 0.021) and IL-8 (p = 0.026) were lower in the hippocampus.”

APP/PS1 wild-type (-) A-/Pki/ki mice had a lower weight than the A-/Pwt/wt littermates did (p = 0.024).

“such that the APP/PS1 wild-type (-) A-/Pki/ki mice had a lower weight than the A-/Pwt/wt littermates did (p = 0.024)”

Pki/ki and Pwt/wt mice did not differ in their motor abilities on the rotating rod.

“Given that Pki/ki and Pwt/wt mice did not differ in their motor abilities on the rotating rod (Fig. 7F) and that they had similar pain thresholds (Fig. 7G)”

Upregulated genes within the FA metabolism pathway included genes linked to LD lipolysis and FA synthesis (Mgll, Fasn, Fads2, Fabp7, Fabp5, Fabp3).

“The upregulated genes within the FA metabolism pathway included, e.g., genes linked to LD lipolysis and FA synthesis (Mgll, Fasn, Fads2, Fabp7, Fabp5, Fabp3, Fig. 8B; Supplementary Figures, Figure S8C).”

Calcium signaling, fatty acid metabolism, cholesterol homeostasis, and oxidative phosphorylation were prominently enriched in the upregulated DEGs in PLCγ2-P522R KI microglia.

“Among these pathways, calcium signaling, fatty acid (FA) metabolism, cholesterol homeostasis, and oxidative phosphorylation were prominently enriched in the upregulated DEGs in PLCγ2-P522R KI microglia”

A similar reduction in β-amyloid plaque number or coverage was not detected in the hippocampus of the same mice.

“However, a similar reduction was not detected in the hippocampus of the same mice (Supplementary Figures, Figure S2A).”

• AssumptionRests on an assumption: The APP/PS1 mouse model recapitulates Alzheimer's disease-associated β-amyloid pathology relevant to human AD.

The PLCγ2-P522R variant does not increase β-amyloid plaque-associated APOE in female APP/PS1 mice.

“PLCγ2-P522R variant does not increase β-amyloid plaque-associated APOE or promote DAM signature in the female APP/PS1 mice”

Total area of diffuse β-amyloid and composition of β-amyloid plaques, as indicated by percentage of compact (X-34) and diffuse (6E10) β-amyloid plaques, remain unaltered in entorhinal cortex of APP/PS1xPLCγ2-P522R (A+/Pki/ki) mice compared with APP/PS1 (A+/Pwt/wt) mice.

“Total area (µm2) of diffuse β-amyloid (6E10) and composition β-amyloid plaques, as indicated by the percentage of compact (X-34) and diffuse (6E10) β-amyloid plaques, remaining unaltered in the entorhinal cortex of the APP/PS1xPLCγ2-P522R (A+/Pki/ki) mice compared with the APP/PS1 (A+/Pwt/wt) mice.”

A similar decrease in the p-Tau/total Tau ratio in the tissue lysates was not detected.

“Taken together, these findings suggest that the PLCγ2-P522R variant decreases the formation of dystrophic neurites around β-amyloid plaques, as shown by N-terminal APP staining, whereas a similar decrease in the p-Tau/total Tau ratio in the tissue lysates was not detected.”

There were no differences in the levels of full-length APP, APP C-terminal fragments (C83 and C99), or soluble APPα and -β in the temporo-occipital cortex or hippocampus of A+/Pki/ki and A+/Pwt/wt mice according to Western blot analyses.

“Importantly, there were no differences in the levels of full-length APP (APPtot), APP C-terminal fragments (C83 and C99, Fig. 2C and Supplementary Figures, S2C, left panel), or soluble APPα and -β (sAPPα and -β, Fig. 2C and Supplementary Figures, S2C, right panel) in the temporo-occipital cortex or hippocampus according to Western blot analyses of these mice”

• AssumptionRests on an assumption: The APP/PS1 mouse model recapitulates Alzheimer's disease-associated β-amyloid pathology relevant to human AD.

This tendency was further accentuated by the APP/PS1+ genotype.

“This tendency was further accentuated by the APP/PS1+ genotype.”

Core DEGs within the calcium signaling pathway included Itpr1, Itpka, Camk4, Camk2a and Camk2b.

“Closer examination revealed that the core DEGs within the calcium signaling pathway included Itpr1, Itpka, Camk4, Camk2a and Camk2b”

There were no differences between mice with different genotypes in the levels of sTREM2 in the temporo-occipital and hippocampal lysates.

“There were no differences between mice with different genotypes in the levels of sTREM2 in these brain areas (Supplementary Figures, Figure S6C)”

The IBA1+ area was increased within the 0-40 μm radius from the β-amyloid plaque outline in the entorhinal cortex and hippocampus of A+/Pki/ki mice compared with A+/Pwt/wt mice.

“The IBA1+ area was increased within the 0–40 µm radius from the β-amyloid plaque outline in the entorhinal cortex (p = 0.03, Fig. 3A) and hippocampus (p = 0.037, Supplementary Figures, Figure S3A) of A+/Pki/ki mice compared with A+/Pwt/wt mice.”

• AssumptionRests on an assumption: The APP/PS1 mouse model recapitulates Alzheimer's disease-associated β-amyloid pathology relevant to human AD.

In the light-dark box, ANOVA revealed a main effect of both the PLCγ2-P522R (p = 0.03) and APP/PS1 (p = 0.04) genotypes and their interaction (p = 0.004).

“In this test, ANOVA revealed a main effect of both the PLCγ2-P522R (p = 0.03) and APP/PS1 (p = 0.04) genotypes and their interaction (p = 0.004).”

Western blot analysis did not reveal significant differences in the ratio of p-Tau/total Tau between A+/Pki/ki and A+/Pwt/wt mice in the tissue lysates studied.

“Consequently, Western blot analysis did not reveal significant differences in the ratio of p-Tau/total Tau between A+/Pki/ki and A+/Pwt/wt mice in the tissue lysates studied.”

The average size of individual X-34+ β-amyloid plaques did not differ between the genotypes in either the entorhinal cortex or the hippocampus.

“The average size of individual X-34+ β-amyloid plaques did not differ between the genotypes in either the entorhinal cortex (Fig. 2A) or the hippocampus (Supplementary Figures, Figure S2A).”

• AssumptionRests on an assumption: The APP/PS1 mouse model recapitulates Alzheimer's disease-associated β-amyloid pathology relevant to human AD.

There were no significant changes in the levels of soluble and insoluble Aβ40 and Aβ42 in the hippocampus of A+/Pki/ki mice compared with those in the hippocampus of A+/Pwt/wt mice.

“Similarly, there were no significant changes in the levels of soluble and insoluble Aβ40 and Aβ42 in the hippocampus of A+/Pki/ki mice compared with those in the hippocampus of A+/Pwt/wt mice (Supplementary Figures, Figure S2B).”

• AssumptionRests on an assumption: The APP/PS1 mouse model recapitulates Alzheimer's disease-associated β-amyloid pathology relevant to human AD.

No differences in APOE+ area overlapping or within a 10 μm radius from the plaque outline were found between A+/Pki/ki and A+/Pwt/wt mice in the entorhinal cortex or in the hippocampus.

“We found no differences in the APOE+ area overlapping or within a 10 µm radius from the plaque outline (Supplementary Figures, Figure S1D) between A+/Pki/ki and A+/Pwt/wt mice in the entorhinal cortex (Fig. 6A) or in the hippocampus (Supplementary Figures, Figure S6A).”

The 22C11-positive area was decreased within the β-amyloid plaque area (p = 0.033) in entorhinal cortices of A+/Pki/ki mice compared with A+/Pwt/wt mice when plaque size was normalized.

“The 22C11-positive area was decreased within the β-amyloid plaque area (p = 0.033), and a similar decreasing trend was observed within the 0–14 µm radius from the β-amyloid plaque outline in the entorhinal cortices of the A+/Pki/ki mice compared with the A+/Pwt/wt mice when the plaque size was normalized.”

A similar decreasing trend in 22C11-positive area was observed within the 0-14 μm radius from the β-amyloid plaque outline in entorhinal cortices of A+/Pki/ki mice compared with A+/Pwt/wt mice when plaque size was normalized.

“The 22C11-positive area was decreased within the β-amyloid plaque area (p = 0.033), and a similar decreasing trend was observed within the 0–14 µm radius from the β-amyloid plaque outline in the entorhinal cortices of the A+/Pki/ki mice compared with the A+/Pwt/wt mice when the plaque size was normalized.”

In the hippocampus, the 22C11+ area was significantly decreased within the 0-14 μm radius from the plaque outline (p = 0.010).

“In the hippocampus, the 22C11+ area was significantly decreased within the 0–14 µm radius from the plaque outline (p = 0.010) (Supplementary Figures, Figure S4C).”

Soluble and insoluble APOE levels in the temporo-occipital cortex and hippocampus did not differ between the mice with different genotypes.

“In line with these findings, soluble and insoluble APOE levels in the temporo-occipital cortex and hippocampus did not differ between the mice with different genotypes (Supplementary Figures, Figure S6B).”

The PLCγ2-P522R variant did not influence the ambulatory distance traveled in the spontaneous activity test.

“Given that the PLCγ2-P522R variant did not influence the ambulatory distance traveled in the spontaneous activity test (Fig. 7B), the observed weight reduction was not related to increased activity.”

The calcium signaling pathway, fatty acid metabolism, and oxidative phosphorylation-related pathways are activated in acutely isolated microglia from male mouse carrying the PLCγ2-P522R variant.

“The calcium signaling pathway, fatty acid metabolism, and oxidative phosphorylation-related pathways are activated in acutely isolated microglia from male mouse carrying the PLCγ2-P522R variant”

APP/PS1+ mice exhibited strongly impaired learning ability in terms of escape latency (p < 0.001).

“whereas APP/PS1+ mice exhibited strongly impaired learning ability in terms of escape latency (p < 0.001).”

The time spent in the center of the light-dark box was significantly shorter in the Pki/ki than in the Pwt/wt mice, regardless of the APP/PS1 genotype (p = 0.011).

“However, the time spent in the center of the light–dark box was significantly shorter in the Pki/ki than in the Pwt/wt mice, regardless of the APP/PS1 genotype (p = 0.011, Fig. 7C)”

IL-12-p70 levels were significantly lower in the cortex of A+/Pki/ki mice than in A+/Pwt/wt mice (p = 0.049).

“The levels of IL-12-p70 (p70 refers to an active heterodimer, p = 0.049) and IL-6 (p = 0.003) in A+/Pki/ki mice were significantly lower in the cortex”

The PLCγ2-P522R variant did not influence spatial learning or memory in the Morris water maze in either APP/PS1- or APP/PS1+ mice.

“Finally, the PLCγ2-P522R variant did not influence spatial learning or memory in the Morris water maze in either APP/PS1- or APP/PS1+ mice (Fig. 7I)”

IL-8 levels were lower in the hippocampus of A+/Pki/ki mice than in A+/Pwt/wt mice (p = 0.026).

“while those of IL-1β (p = 0.021) and IL-8 (p = 0.026) were lower in the hippocampus.”

Neuroinflammation is alleviated in the brain tissue of female APP/PS1 mice carrying the PLCγ2-P522R variant.

“Neuroinflammation is alleviated in the brain tissue of female APP/PS1 mice carrying the PLCγ2-P522R variant”

RNA sequencing revealed dramatically different RNA signatures between male PLCγ2-P522R KI and WT microglia, including 3719 DEGs (FDR 0.05).

“RNA sequencing revealed dramatically different RNA signatures between male PLCγ2-P522R KI and WT microglia, including 3719 DEGs (FDR 0.05) (Supplementary Figures, Figure S8A).”

Calcium-sensitive MEF2 transcription factor family members Mef2a, Mef2c, and Mef2d were downregulated in PLCγ2-P522R KI microglia.

“whereas the calcium-sensitive MEF2 transcription factor family members Mef2a, Mef2c, and Mef2d were downregulated in PLCγ2-P522R KI microglia (Fig. 8B; Supplementary Figures, Figure S8C).”

Several targets related to cell motility and phagocytosis were slightly upregulated in A+/Pki/ki microglia compared with A+/Pwt/wt microglia, but these results were not statistically significant.

“Although several targets related to cell motility and phagocytosis were slightly upregulated in A+/Pki/ki microglia compared with A+/Pwt/wt microglia, these results were not statistically significant (Supplementary Figures, Figure S7A and S7B).”

The same body weight difference was not detected among the APP/PS1 hemizygous (+) mice.

“but the same was not detected among the APP/PS1 hemizygous (+) mice (Fig. 7A).”

IL-6 levels were significantly lower in the cortex of A+/Pki/ki mice than in A+/Pwt/wt mice (p = 0.003).

“The levels of IL-12-p70 (p70 refers to an active heterodimer, p = 0.049) and IL-6 (p = 0.003) in A+/Pki/ki mice were significantly lower in the cortex”

The area of 22C11-labeled dystrophic neurites around β-amyloid plaques strongly correlates with plaque size (r = 0.8113, p < 0.0001).

“The area (µm2) of 22C11-labeled dystrophic neurites around β-amyloid plaques strongly correlates with plaque size (r = 0.8113, ****p < 0.0001).”

Gene enrichment analysis revealed several enriched biological pathways in the upregulated and downregulated DEGs between PLCγ2-P522R KI microglia and WT microglia.

“Gene enrichment analysis revealed several enriched biological pathways in the upregulated and downregulated DEGs between PLCγ2-P522R KI microglia and WT microglia (Fig. 8A).”

Pki/ki and Pwt/wt mice had similar pain thresholds.

“Given that Pki/ki and Pwt/wt mice did not differ in their motor abilities on the rotating rod (Fig. 7F) and that they had similar pain thresholds (Fig. 7G)”

A+/Pki/ki mice presented lower levels of several inflammatory markers in both the temporo-occipital cortex and hippocampus than did A+/Pwt/wt mice.

“In general, A+/Pki/ki mice presented lower levels of several inflammatory markers in both the temporo-occipital cortex and hippocampus than did A+/Pwt/wt mice (Fig. 5).”

Pathways related to the inflammatory response and the interferon-α (IFN-α) response were enriched in the downregulated DEGs.

“whereas pathways related to the inflammatory response and the interferon-α (IFN-α) response were enriched in the downregulated DEGs.”

The soluble and insoluble levels of Aβ40 and Aβ42 in the ELISA did not significantly differ in the temporo-occipital cortex between A+/Pki/ki and A+/Pwt/wt mice.

“The soluble and insoluble levels of Aβ40 and Aβ42 in the ELISA did not significantly differ in the temporo-occipital cortex between A+/Pki/ki and A+/Pwt/wt mice (Fig. 2B).”

• AssumptionRests on an assumption: The APP/PS1 mouse model recapitulates Alzheimer's disease-associated β-amyloid pathology relevant to human AD.

The expression of both Apoe and Trem2 in cortical tissue and CD11b+ microglia did not differ between the A+/Pki/ki and A+/Pwt/wt mice.

“The expression of both Apoe and Trem2 in cortical tissue and CD11b+ microglia did not differ between the A+/Pki/ki and A+/Pwt/wt mice, suggesting that the DAM signature is not upregulated in APP/PS1 mice carrying the PLCγ2-P522R variant (Fig. 6B and C).”

Body weight showed an APP/PS1 x PLCγ2-P522R genotype interaction (p = 0.04).

“First, the body weight showed an APP/PS1 x PLCγ2-P522R genotype interaction (p = 0.04)”

PLCγ2-P522R variant decreases β-amyloid plaque-associated dystrophic neurites in female APP/PS1 mice.

“PLCγ2-P522R variant decreases β-amyloid plaque-associated dystrophic neurites in female APP/PS1 mice.”

In acutely isolated microglia from 13-month-old female A+/Pki/ki and A+/Pwt/wt mice, no significant changes were observed in pathway enrichment analysis for cell motility genes.

“Although we did not observe significant changes in the pathway enrichment analysis”

The size of the LDs was clearly smaller in PLCγ2-P522R KI microglia than in WT microglia (p < 0.0001) upon myelin treatment.

“However, the size of the LDs was clearly smaller in PLCγ2-P522R KI microglia than in WT microglia (p < 0.0001) upon myelin treatment.”

Maximal respiration was greater in PLCγ2-P522R GG/APOE3/3 iMGLs than in all the other groups (CC/APOE3/3 p = 0.001, CC/APOE4/4 p < 0.0001, CG/APOE4 p < 0.0001).

“Specifically, maximal respiration was greater in PLCγ2-P522R GG/APOE3/3 iMGLs than in all the other groups (CC/APOE3/3 p = 0.001, CC/APOE4/4 p < 0.0001, CG/APOE4 p < 0.0001) (Fig. 10F).”

This decrease was associated with decreased formation of β-amyloid plaque-associated neuronal dystrophy, as shown by 22C11 IHC staining.

“This decrease was associated with increased microglial activation and microglial area around β-amyloid plaques as well as decreased formation of β-amyloid plaque-associated neuronal dystrophy, as shown by 22C11 IHC staining.”

In the studied mice, the number of dystrophic neurites was reduced.

“which was accompanied by a reduced number of dystrophic neurites”

Increased microglial PET signals were observed in the brain tissue of 13-month-old female mice.

“Together with increased microglial PET signals in the brain tissue of 13-month-old female mice”

Proteomics analysis of microglia acutely isolated from 13-month-old male PLCγ2-P522R and wild-type mice without the APP/PS1 background revealed upregulation of genes involved in microglial phagocytosis of pathogens in PLCγ2-P522R microglia.

“proteomics analysis of microglia acutely isolated from 13-month-old male PLCγ2-P522R and wild-type mice without the APP/PS1 background revealed the upregulation of genes involved in microglial phagocytosis of pathogens in PLCγ2-P522R microglia.”

LPS had the most robust effect on gene expression, resulting in 236 DEGs in CG cells compared with CC cells.

“LPS had the most robust effect on gene expression, resulting in 236 DEGs in CG cells compared with CC cells (Fig. 10C).”

A similar but nonsignificant trend in LD size was detected in LPS-treated microglia.

“A similar but nonsignificant trend was also detected in LPS-treated microglia.”

In the current study, the number of X-34-positive compact plaques was reduced in the brain tissues of 13-month-old female APP/PS1 mice carrying the PLCγ2-P522R variant.

“In the current study, the number of X-34-positive compact plaques was reduced in the brain tissues of 13-month-old female APP/PS1 mice carrying the PLCγ2-P522R variant.”

Several mitochondrial complex I, complex II, complex III, and complex IV genes as well as genes encoding other mitochondrial enzymes involved in FA oxidation were upregulated.

“Furthermore, several mitochondrial complex I (Ndufa1, Ndufa2, Ndufc2, Ndufb8, Ndufs6, Ndufa4, Ndufa8, Ndufa5, Ndufa3, and Ndufb6), complex II (Sdha), complex III (Uqcrh, Uqcrq, and Uqcr10), and complex IV (Cox5a, Cox7a2, Cox7b, Cox6a1, Cox4i1, Cox6c, Cox5b, Cox6b1, and Cox7c) genes as well as genes encoding other mitochondrial enzymes involved in FA oxidation (FAO, e.g., Eci1) were upregulated.”

The PLCγ2-P522R variant decreases the β-amyloid plaque count and coverage in the brain tissue of female APP/PS1 mice.

“In line with these findings, we now demonstrate that PLCγ2-P522R variant decreases the β-amyloid plaque count and coverage in the brain tissue of female APP/PS1 mice, which is a less aggressive mouse model of AD as compared to 5XFAD mouse model.”

The core enriched genes in the oxidative phosphorylation pathway included ACADVL, NDUFB7, ATP5MC1, and FXN.

“The core enriched genes in the oxidative phosphorylation pathway included ACADVL, an enzyme regulating FA oxidation; the mitochondrial complex I gene; NDUFB7; mitochondrial ATP synthase; ATP5MC1; and FXN, a gene associated with mitochondrial iron transport and respiration (Fig. 10E).”

Proteomics analysis of microglia acutely isolated from 13-month-old male PLCγ2-P522R KI and wild-type mice without the APP/PS1 background revealed significant upregulation of focal adhesion-associated proteins in PLCγ2-P522R microglia.

“proteomics analysis of microglia acutely isolated from 13-month-old male PLCγ2-P522R KI and wild-type mice without the APP/PS1 background revealed significant upregulation of focal adhesion-associated proteins in PLCγ2-P522R microglia.”

In female A+/Pki/ki mice, the area of microglia around β-amyloid plaques was significantly increased.

“we detected a significant increase in the area of microglia around β-amyloid plaques”

• Also stated in: § Discussion · plain

  “the observed increase in the area of microglia around β-amyloid plaques in female A+/Pki/ki mice”

Downregulated DEGs in PLCγ2-P522R KI microglia related to the inflammatory response, TLR signaling and the IFN-α response included Tlr9, Tlr3, P2rx4, P2rx7, and Casp8.

“Finally, the downregulated DEGs in PLCγ2-P522R KI microglia related to the inflammatory response, TLR signaling and the IFN-α response included Tlr9, Tlr3, P2rx4, P2rx7, and Casp8 (Fig. 8B).”

The mitochondrial OCR significantly differed between the genotypes (p < 0.0001).

“In general, the mitochondrial OCR significantly differed between the genotypes (p < 0.0001).”

No significant changes were found in the levels of IL-6 and TNF-α in the conditioned medium of LPS-treated MDMi cells.

“However, no significant changes were found in the levels of either of the cytokines (Supplementary Figures, Figure S9A).”

Similar RNA-seq analysis of CD11b+ microglia from female mice did not yield as prominent results as microglia from male mice did.

“Conversely, similar RNA-seq analysis of CD11b+ microglia from female mice did not yield as prominent results as microglia from male mice did (Supplementary Figures, Figure S8E).”

The PLCγ2-P522R variant did not promote transcriptional changes in the bulk cortex or in isolated CD11b+ microglia of APP/PS1 mice.

“the PLCγ2-P522R variant did not promote transcriptional changes in the bulk cortex or in isolated CD11b+ microglia of APP/PS1 mice.”

Signaling pathways related to the inflammatory response were suppressed in these models.

“Conversely, signaling pathways related to the inflammatory response were suppressed in these models.”

Proteomic analysis revealed 34 differentially expressed proteins (DEPs, FDR 0.05), 14 of which were downregulated and 20 of which were upregulated in male PLCγ2-P522R KI microglia compared with WT microglia.

“Proteomic analysis revealed 34 differentially expressed proteins (DEPs, FDR 0.05), 14 of which were downregulated and 20 of which were upregulated in male PLCγ2-P522R KI microglia compared with WT microglia (Fig. 8C; Supplementary Figures, Figure S8B).”

Several genes linked to cell motility showed a subtle increase in their expression in PLCγ2-P522R microglia.

“several genes linked to cell motility showed a subtle increase in their expression in PLCγ2-P522R microglia.”

Proteins related to FA oxidation were mildly upregulated in PLCγ2-P522R KI microglia, similar to what was observed at the gene expression level.

“In contrast, proteins related to FA oxidation were mildly upregulated in PLCγ2-P522R KI microglia, which was similar to what was observed at the gene expression level (Supplementary Figures, Figure S8D).”

In acutely isolated microglia from 13-month-old female A+/Pki/ki and A+/Pwt/wt mice, there was a subtle increase in the expression of phagocytosis genes in PLCγ2-P522R microglia, although this change was not statistically significant in the pathway enrichment analysis.

“This analysis also revealed a subtle increase in the expression of phagocytosis genes in PLCγ2-P522R microglia, although this change was not statistically significant in the pathway enrichment analysis.”

In contrast to gene expression, core enriched proteins related to oxidative phosphorylation were downregulated in the proteomics dataset.

“In contrast to gene expression, core enriched proteins related to oxidative phosphorylation were downregulated in the proteomics dataset (Fig. 8C).”

No significant changes were detected in the p-Tau/total Tau ratio in the tissue lysates studied.

“we did not detect significant changes in other well-established markers of neuronal dystrophy, such as the p-Tau/total Tau ratio in the tissue lysates we studied.”

Genotype had no effect on the percentage of LD-accumulating microglia.

“whereas genotype had no effect on the percentage of LD-accumulating microglia (Fig. 9).”

PLCγ2-P522R did not increase mitochondrial respiration in the APOE4/4 background.

“However, PLCγ2-P522R did not increase mitochondrial respiration in the APOE4/4 background.”

RNA sequencing revealed that several genes encoding PI3K subunits were downregulated in adult PLCγ2-P522R KI mouse microglia compared with WT microglia.

“RNA sequencing revealed that several genes encoding PI3K subunits were downregulated in adult PLCγ2-P522R KI mouse microglia compared with WT microglia.”

During activity monitoring, PLCγ2-P522R mice avoided the center of the arena more than the control mice did.

“during activity monitoring, the PLCγ2-P522R mice avoided the center of the arena more than the control mice did.”

There was no evident improvement in learning and memory functions in the APP/PS1 mice carrying PLCγ2-P522R.

“there was no evident improvement in learning and memory functions in the APP/PS1 mice.”

Omics-based analyses of male PLCγ2-P522R microglia without the APP/PS1 background revealed more pronounced effects on PLCγ2-P522R microglia than female PLCγ2-P522R microglia.

“omics-based analyses of male PLCγ2-P522R microglia without the APP/PS1 background revealed more pronounced effects on PLCγ2-P522R microglia than female PLCγ2-P522R microglia.”

Reduced accumulation of LDs was observed in PLCγ2-P522R KI mouse microglia upon myelin and, to a lesser extent, LPS treatment.

“Reduced accumulation of LDs was observed in the present study in PLCγ2-P522R KI mouse microglia upon myelin and, to a lesser extent, LPS treatment”

Several DEGs (e.g., Fabp3, Fabp5, Fabp7, Mgll, and Eci1) and biological pathways (β-oxidation and FAO) identified from RNA sequencing and proteomics data directly indicate enhanced lipid metabolism and FAO in acutely isolated adult PLCγ2-P522R mouse microglia.

“several DEGs (e.g., Fabp3, Fabp5, Fabp7, Mgll, and Eci1) and biological pathways (β-oxidation and FAO) identified from RNA sequencing and proteomics data directly indicate enhanced lipid metabolism and FAO in acutely isolated adult PLCγ2-P522R mouse microglia.”

The total distance traversed in a new test box did not differ between the genotypes.

“The total distance traversed in a new test box also did not differ.”

Before passive avoidance memory testing, PLCγ2-P522R mice spent significantly less time in the illuminated chamber than the other groups did.

“before passive avoidance memory testing, we observed that the PLCγ2-P522R mice spent significantly less time in the illuminated chamber than the other groups did.”

After receiving foot shock, PLCγ2-P522R mice walked backward in a crab-like manner.

“after receiving foot shock, PLCγ2-P522R mice walked backward in a crab-like manner”

PLCγ2-P522R reduced β-amyloid deposition and associated neuronal pathologies in APP/PS1 mice.

“Although PLCγ2-P522R reduced β-amyloid deposition and associated neuronal pathologies”

The threshold to react to an electric foot shock did not differ between the genotypes.

“The threshold to react to an electric foot shock did not differ between the genotypes.”

Several genes encoding fatty acid-binding proteins (Fabp3, Fabp5, and Fabp7) were found to be upregulated in PLCγ2-P522R mouse microglia.

“several genes encoding fatty acid-binding proteins (Fabp3, Fabp5, and Fabp7), which play a role in lipid droplet lipolysis and the transport of free fatty acids to mitochondria for energy production [62], were found to be upregulated in PLCγ2-P522R mouse microglia.”

The most robust manifestation of the behavioral phenotype associated with PLCγ2-P522R was observed in the passive avoidance task.

“The most robust manifestation of the behavioral phenotype associated with PLCγ2-P522R was observed in the passive avoidance task.”

The PLCγ2-P522R variant exerts protective effects against β-amyloid pathologies in APP/PS1 mice.

“These findings suggest that the PLCγ2-P522R variant exerts protective effects against β-amyloid and neuronal pathologies by increasing microglial responsiveness to β-amyloid plaques in APP/PS1 mice.”

Microglial activation in the brain tissue of APP/PS1 mice carrying the PLCγ2-P522R variant moderately increases at 13 months of age.

“These findings suggest that microglial activation in the brain tissue of APP/PS1 mice carrying the PLCγ2-P522R variant moderately increases at 13 months of age.”

• AssumptionRests on an assumption: The APP/PS1 mouse model recapitulates Alzheimer's disease-associated β-amyloid pathology relevant to human AD.

The protective effects of the PLCγ2-P522R variant are mediated by increasing microglial responsiveness to β-amyloid plaques in APP/PS1 mice.

“These findings suggest that the PLCγ2-P522R variant exerts protective effects against β-amyloid and neuronal pathologies by increasing microglial responsiveness to β-amyloid plaques in APP/PS1 mice.”

• AssumptionRests on an assumption: The area of microglia around β-amyloid plaques reflects microglial responsiveness to plaques.

The PLCγ2-P522R variant exerts protective effects against neuronal pathologies in APP/PS1 mice.

“These findings suggest that the PLCγ2-P522R variant exerts protective effects against β-amyloid and neuronal pathologies by increasing microglial responsiveness to β-amyloid plaques in APP/PS1 mice.”

The protective effects of the PLCγ2-P522R variant are potentially associated with increased metabolic capacity of microglia.

“The changes observed in lipid/fatty acid and mitochondrial metabolism revealed by the omics and metabolic assessments of mouse and human microglial models suggest that the protective effects of the PLCγ2-P522R variant are potentially associated with increased metabolic capacity of microglia.”

The PLCγ2-P522R variant decreases the formation of dystrophic neurites around β-amyloid plaques, as shown by N-terminal APP staining.

“Taken together, these findings suggest that the PLCγ2-P522R variant decreases the formation of dystrophic neurites around β-amyloid plaques, as shown by N-terminal APP staining, whereas a similar decrease in the p-Tau/total Tau ratio in the tissue lysates was not detected.”

The PLCγ2-P522R variant promotes anxiety in female APP/PS1 mice carrying the PLCγ2-P522R variant.

“The PLCγ2-P522R variant promotes anxiety in female APP/PS1 mice carrying the PLCγ2-P522R variant”

Despite increased microglial activation, the PLCγ2-P522R variant does not increase chronic β-amyloid-associated neuroinflammation in the APP/PS1 mouse brain upon aging.

“These findings suggest that despite increased microglial activation, the PLCγ2-P522R variant does not increase chronic β-amyloid-associated neuroinflammation in the APP/PS1 mouse brain upon aging.”

The PLCγ2-P522R variant increases the avoidance of open places.

“suggesting that the PLCγ2-P522R variant increases the avoidance of open places.”

PLCγ2-P522R variant-driven microglial activation does not promote astrocyte activation in the brain tissue of 13-month-old APP/PS1 mice.

“suggesting that PLCγ2-P522R variant-driven microglial activation does not promote astrocyte activation in the brain tissue of 13-month-old APP/PS1 mice.”

• AssumptionRests on an assumption: The APP/PS1 mouse model recapitulates Alzheimer's disease-associated β-amyloid pathology relevant to human AD.

Rushing into the dark chamber behavior is not a result of memory dysfunction related to the foot shock in the Pki/ki mice.

“This finding suggests that rushing into the dark chamber behavior is not a result of memory dysfunction related to the foot shock in the Pki/ki mice.”

The DAM signature is not upregulated in APP/PS1 mice carrying the PLCγ2-P522R variant.

“The expression of both Apoe and Trem2 in cortical tissue and CD11b+ microglia did not differ between the A+/Pki/ki and A+/Pwt/wt mice, suggesting that the DAM signature is not upregulated in APP/PS1 mice carrying the PLCγ2-P522R variant (Fig. 6B and C).”

TREM2 shedding is not promoted in the mice carrying the PLCγ2-P522R variant.

“suggesting that TREM2 shedding is not promoted in the mice carrying the PLCγ2-P522R variant.”

The histochemically determined decrease in the β-amyloid pathology of A+/Pki/ki mice was not due to alterations in APP stability or processing.

“suggesting that the histochemically determined decrease in the β-amyloid pathology of A+/Pki/ki mice was not due to alterations in APP stability or processing.”

• AssumptionRests on an assumption: The APP/PS1 mouse model recapitulates Alzheimer's disease-associated β-amyloid pathology relevant to human AD.

The observed weight reduction was not related to increased activity.

“Given that the PLCγ2-P522R variant did not influence the ambulatory distance traveled in the spontaneous activity test (Fig. 7B), the observed weight reduction was not related to increased activity.”

The results of the RNA and proteomic analyses suggest that pathways related to calcium signaling, the inflammatory/IFN response, mitochondrial oxidative metabolism (FAO), and lipid/FA metabolism are modulated in acutely isolated microglia from male mice carrying the PLCγ2-P522R variant.

“Together, the results of the RNA and proteomic analyses suggest that pathways related to calcium signaling, the inflammatory/IFN response, mitochondrial oxidative metabolism (FAO), and lipid/FA metabolism are modulated in acutely isolated microglia from male mice carrying the PLCγ2-P522R variant.”

PLCγ2-P522R KI reduces the buildup of LDs in microglia under conditions of cellular stress.

“These findings suggest that PLCγ2-P522R KI reduces the buildup of LDs in microglia under conditions of cellular stress.”

The observed increase in the area of microglia around β-amyloid plaques in female A+/Pki/ki mice could be due to increased clustering of activated microglia around β-amyloid plaques.

“Collectively, these findings suggest that the observed increase in the area of microglia around β-amyloid plaques in female A+/Pki/ki mice could be due to the increased clustering of activated microglia around β-amyloid plaques.”

The PLCγ2-P522R variant improves mitochondrial function in human microglial models.

“PLCγ2-P522R variant improves mitochondrial function in human microglial models”

At the mechanistic level, the PLCγ2-P522R variant upregulated pathways related to lipid/FA metabolism and mitochondrial function in several different PLCγ2-P522R mouse and human microglial models.

“Importantly, at the mechanistic level, the PLCγ2-P522R variant upregulated pathways related to lipid/FA metabolism and mitochondrial function in several different PLCγ2-P522R mouse and human microglial models.”

Together with findings in mouse CD11b+ microglia, these data confirm a central role for PLCγ2-P522R in the regulation of genes associated with mitochondrial function and FA oxidation as well as inflammatory/IFN responses.

“Together with findings in mouse CD11b+ microglia, these data confirm a central role for PLCγ2-P522R in the regulation of genes associated with mitochondrial function and FA oxidation as well as inflammatory/IFN responses.”

The PLCγ2-P522R variant improves lipid processing in mouse primary microglia.

“PLCγ2-P522R variant improves lipid processing in mouse primary microglia”

Enhanced barrier formation around β-amyloid plaques protects the surrounding neuronal population.

“This then leads to enhanced barrier formation around β-amyloid plaques, which protects the surrounding neuronal population.”

Changes in anxiety level remain the most plausible explanation for the passive avoidance test results.

“When these issues are excluded, changes in anxiety level remain the most plausible explanation for the passive avoidance test results.”

The PLCγ2-P522R variant has not been associated with any autoimmune diseases or cancers in previous studies.

“the PLCγ2-P522R variant has not been associated with any autoimmune diseases or cancers in previous studies”

The current findings are primarily based on female A+/Pki/ki mice.

“Importantly, our current findings are primarily based on female A+/Pki/ki mice.”

The protective PLCγ2-P522R variant cannot fully counteract the negative effects of APOE4 on mitochondrial respiration.

“These findings indicate that the protective PLCγ2-P522R variant cannot fully counteract the negative effects of APOE4 on mitochondrial respiration.”

The protective effects of the PLCγ2-P522R variant upon AD-associated stress could be a consequence of slower metabolic deterioration related to microglial aging.

“our findings suggest that the protective effects of the PLCγ2-P522R variant upon AD-associated stress could be a consequence of slower metabolic deterioration related to microglial aging.”

The PLCγ2-P522R variant enhances lipid metabolism under different stress conditions.

“suggesting that the PLCγ2-P522R variant enhances lipid metabolism under different stress conditions.”