Heterozygous expression of the Alzheimer’s disease-protective PLCγ2 P522R variant enhances Aβ clearance while preserving synapses

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Evidence suggests CX3CR1 plays an important role in the regulation of synaptic function, with CX3CR1 deficiency resulting in cognitive and LTP impairments indicative of altered synaptic plasticity.

“evidence suggests it plays an important role in the regulation of synaptic function, with CX3CR1 deficiency resulting in cognitive and LTP impairments indicative of altered synaptic plasticity [42, 43].”

• cites [42] Rogers JT et al (2011) CX3CR1 deficiency leads to impairment of hippocampal cognitive function and synaptic plasticity. J Neurosci 31(45):16241–16250 sourceSupported
• cites [43] Maggi L et al (2011) CX(3)CR1 deficiency alters hippocampal-dependent plasticity phenomena blunting the effects of enriched environment. Front Cell Neurosci 5:22 sourceDisputed

  The Discussion reports that under standard housing, CX3CR1-deficient mice had improved hippocampal LTP and spatial memory compared with wild-type mice, contrary to the claimed impairments. It also reports that deficiency blunted the additional benefits of an enriched environment, but that is not the same as impaired LTP and cognition at baseline. (LLM, conf=0.90)

  Checked by a reasoning model — may be wrong.

  Experimental — accuracy not yet measured

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

The P522R variant enhances macrophage phagocytic activity and cytokine release in response to inflammatory stimulation, and may promote microglial activation.

“It has been shown that this altered activity appears to enhance macrophage phagocytic activity and enhance cytokine release in response to inflammatory stimulation, and may promote microglial activation [17]”

• cites [17] Takalo M et al (2020) The Alzheimer’s disease-associated protective Plcgamma2-P522R variant promotes immune functions. Mol Neurodegener 15(1):52 sourceSupported

Classified as probably not a substantial claim. May combine two claims.

The P522R variant is a mild hypermorph that potentiates the enzymatic activity of PLCγ2.

“Recent studies have suggested that the P522R variant is a mild hypermorph [15–17], serving to potentiate the enzymatic activity of the protein”

• cites [15] Maguire E et al (2021) PIP2 depletion and altered endocytosis caused by expression of Alzheimer’s disease-protective variant PLCgamma2 R522. EMBO J 40(17):e105603 sourceSupported
• cites [16] Magno L et al (2019) Alzheimer’s disease phospholipase C-gamma-2 (PLCG2) protective variant is a functional hypermorph. Alzheimer's Res Ther 11:16 sourceSupported
• cites [17] Takalo M et al (2020) The Alzheimer’s disease-associated protective Plcgamma2-P522R variant promotes immune functions. Mol Neurodegener 15(1):52 sourceSupported

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

PLCγ2 acts downstream of TREM2 signalling, modulating microglial functions such as phagocytosis, lipid metabolism, survival and cytokine release.

“Recent studies have identified PLCγ2 downstream of TREM2 signalling, modulating different microglial functions such as phagocytosis, lipid metabolism, survival and cytokine release [14, 15].”

• cites [14] Andreone BJ et al (2020) Alzheimer’s-associated PLCγ2 is a signaling node required for both TREM2 function and the inflammatory response in human microglia. Nat Neurosci 23(8):927–938 sourceNot checked — no full text of the source
• cites [15] Maguire E et al (2021) PIP2 depletion and altered endocytosis caused by expression of Alzheimer’s disease-protective variant PLCgamma2 R522. EMBO J 40(17):e105603 sourceSupported

Classified as probably not a substantial claim. May combine two claims.

Aβ accumulation, whether as insoluble plaques or soluble oligomers, appears to be a primary driver of Alzheimer's disease, particularly linked to synapse dysfunction.

“Despite ongoing debate, Aβ accumulation, whether in the form of insoluble plaques, or smaller soluble oligomers does appear to be a primary driver of the disease process, particularly linked to synapse dysfunction [3].”

• cites [3] Lacor PN et al (2004) Synaptic targeting by Alzheimer’s-related amyloid beta oligomers. J Neurosci 24(45):10191–10200 sourcePartly supported

  The Introduction describes evidence implicating Aβ in Alzheimer’s disease and a hypothesis that soluble oligomers cause early synapse failure. It contrasts that mechanism with the original plaque-related hypothesis, which attributed memory loss to neuron death caused by fibrillar Aβ. The paper therefore supports the soluble-oligomer link to synaptic dysfunction, but not the claim that insoluble plaques are likewise linked to it. (LLM, conf=0.88)

  Experimental — accuracy not yet measured

Probably not a substantial claim: may not pass S3 (load-bearing).

GWAS studies have identified several novel risk-associated coding variants for late-onset Alzheimer's disease, many associated with microglial and immune function.

“GWAS studies have identified several novel risk associated coding variants for late-onset AD (LOAD), many of which are associated with microglial and immune function and further link these cells with AD pathogenesis.”

• Also stated in: § Background · plain

  “Among these identified polymorphic genes are rare coding variants in microglial-associated genes such as triggering receptor expressed on myeloid cell-2 (TREM2), ABI family member 3 (ABI3), and phospholipase C gamma 2 (PLCG2) [10].”

• cites [10] Sims R et al (2017) Rare coding variants in PLCG2, ABI3 and TREM2 implicate microglial-mediated innate immunity in Alzheimer’s disease. Nat Genet 49(9):1373–84 sourceSupported

Classified as probably not a substantial claim.

Microglia are major contributors to molecular and phenotypic changes, including synaptic loss and Aβ clearance, in the Alzheimer's disease brain.

“Converging lines of evidence from human genetics, neuroimaging and post-mortem brain tissue studies suggest that microglia are major contributors to molecular and phenotypic changes, including synaptic loss and Aβ clearance, in the AD brain [4].”

• cites [4] Heneka MT et al (2015) Neuroinflammation in Alzheimer's disease. Lancet Neurol 14(4):388–405 sourcePartly supported

  The Cellular Players > Microglia section links microglial function to Aβ clearance and discusses AD-associated TREM2 and CD33 genetic findings, including a PET observation. It also describes microglial protection and remodeling of synapses, but does not establish that microglia cause synaptic loss in the AD brain; that specific part of the claim is unsupported by the passages found. Page numbers were not available in our copy. (LLM, conf=0.82)

  Experimental — accuracy not yet measured

May combine two claims.

Because P522R appears to be a mild hypermorph, mild changes induced by the variant are likely masked by the larger impact of PLCγ2 over-expression.

“Since the P522R variant appears to be a mild hypermorph [16], it is likely that mild changes induced by the presence of this variant are masked by the larger impact of PLCγ2 over-expression.”

• cites [16] Magno L et al (2019) Alzheimer’s disease phospholipase C-gamma-2 (PLCG2) protective variant is a functional hypermorph. Alzheimer's Res Ther 11:16 sourceSupported

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Increased proton leak is directly related to the prevention of oxidative stress, the inhibition of fatty acid induced mitochondrial damage and the control of oxidative phosphorylation, potentially protecting the cell from excessive damage.

“It has been suggested that increased proton leak is directly related to the prevention of oxidative stress, the inhibition of fatty acid induced mitochondrial damage and the control of oxidative phosphorylation [25], potentially protecting the cell from excessive damage.”

• cites [25] Cheng J et al (2017) Mitochondrial proton leak plays a critical role in pathogenesis of cardiovascular diseases. Adv Exp Med Biol 982:359–370 sourcePartly supported

  The ‘ROS Generation and Proton Leak’ section says enhanced proton leak can reduce ROS production and limit further mitochondrial damage. The section on coupling oxygen consumption to ATP generation explains how proton leak affects oxidative phosphorylation. Our copy does not say that increased leak inhibits fatty-acid-induced mitochondrial damage; the section on ischemia–reperfusion injury also cautions that larger leaks can be harmful. (LLM, conf=0.86)

  Experimental — accuracy not yet measured

Probably not a substantial claim: may not pass S3 (load-bearing).

The P522R variant may prime microglia to adopt an MHC class II state that is reduced in Alzheimer's disease brains.

“priming them to adopt an MHC class II state, that is notably reduced in AD brains [18]”

• cites [18] Claes C et al (2022) The P522R protective variant of PLCG2 promotes the expression of antigen presentation genes by human microglia in an Alzheimer's disease mouse model. Alzheimers Dement https://doi.org/10.1002/alz.12577 sourceSupported

Probably not a substantial claim: may not pass S1 (asserted), S3 (load-bearing).

Aβ may facilitate synaptic pruning by microglia by acting as a tag for targeting synapses.

“It has been suggested that Aβ may facilitate synaptic pruning by microglia by acting as a tag for targeting synapses [24].”

• cites [24] Rajendran L, Paolicelli RC (2018) Microglia-mediated synapse loss in Alzheimer's disease. J Neurosci 38(12):2911–2919 sourceSupported

Probably not a substantial claim: may not pass S3 (load-bearing).

PLCG2 P522R has previously been reported to influence selective cargo uptake, suggesting a shift towards endocytic pathways.

“PLCγ2P522R has previously been reported to influence selective cargo uptake, suggesting a shift towards endocytic pathways [15].”

• cites [15] Maguire E et al (2021) PIP2 depletion and altered endocytosis caused by expression of Alzheimer’s disease-protective variant PLCgamma2 R522. EMBO J 40(17):e105603 sourceSupported

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Microglial ramifications are typically indicative of resting state microglia, which play an active role in the surveillance and monitoring of the brain parenchyma.

“Microglial ramifications are typically indicative of resting state microglia, which play an active role in the surveillance and monitoring of the brain parenchyma, with studies showing that insults such as injury result in reduced ramification [23].”

• cites [23] Morrison H et al (2017) Quantitative microglia analyses reveal diverse morphologic responses in the rat cortex after diffuse brain injury. Sci Rep 7(1):13211 sourceSupported

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

A previous study has suggested that the P522R variant may promote microglial activation, indicating a role for PLCγ2 in microglial activation state.

“A previous study has suggested that the P522R variant may promote microglial activation [17], hence indicating a role for this protein in microglial activation state.”

• cites [17] Takalo M et al (2020) The Alzheimer’s disease-associated protective Plcgamma2-P522R variant promotes immune functions. Mol Neurodegener 15(1):52 sourceSupported

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Other studies suggest that P522R impairs phagocytic clearance while enhancing endocytic clearance.

“Conversely however, other studies have suggested that in fact phagocytic clearance is impaired, while endocytic clearance is enhanced [15]”

• cites [15] Maguire E et al (2021) PIP2 depletion and altered endocytosis caused by expression of Alzheimer’s disease-protective variant PLCgamma2 R522. EMBO J 40(17):e105603 sourceSupported

Classified as probably not a substantial claim. May combine two claims.

Microglia-mediated synaptic pruning is a fundamental physiological process during development, but its proposed reactivation during ageing has deleterious consequences and may account for much of the memory loss and cognitive decline observed in AD.

“While microglia-mediated synaptic pruning is a fundamental physiological process during development, its proposed reactivation during ageing has deleterious consequences and may account for much of the memory loss and cognitive decline observed in AD [24].”

• cites [24] Rajendran L, Paolicelli RC (2018) Microglia-mediated synapse loss in Alzheimer's disease. J Neurosci 38(12):2911–2919 sourcePartly supported

  Split into 2 atoms — 1 confirmed, 1 partially supported, 0 disputed, 0 misattributed, 0 unverifiable. Expand the claim to see per-atom verdicts.

  Experimental — accuracy not yet measured

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

A growing body of evidence suggests that lipid metabolism is crucial to fuel microglial functions such as phagocytosis, and can vary depending upon the activation status of the cell.

“A growing body of evidence suggests that lipid metabolism is crucial to fuel microglial functions such as phagocytosis, and can vary depending upon the activation status of the cell (reviewed in [46]).”

• cites [46] Yang S et al (2021) Microglia reprogram metabolic profiles for phenotype and function changes in central nervous system. Neurobiol Dis 152:105290 sourceSupported

Classified as probably not a substantial claim.

In the central nervous system, microglia shape neuronal synaptic connections and strength during development through the removal of excess synapses.

“In the central nervous system, microglia shape neuronal synaptic connections and strength during development, through the removal of excess synapses [37].”

• cites [37] Paolicelli RC et al (2011) Synaptic pruning by microglia is necessary for normal brain development. Science 1456–1458 sourceSupported

Classified as probably not a substantial claim. May combine two claims.

These heterozygous cells may be 'primed' to more readily respond to an inflammatory challenge, and can therefore more efficiently uptake and phagocytose targets such as Aβ when required.

“This may suggest that these heterozygous cells are ‘primed’ to more readily respond to an inflammatory challenge, and can, therefore, more efficiently uptake and phagocytose targets such as Aβ when required, a hypothesis supported by recent work suggesting that PLCγ2P522R may promote microglial activation [17].”

• cites [17] Takalo M et al (2020) The Alzheimer’s disease-associated protective Plcgamma2-P522R variant promotes immune functions. Mol Neurodegener 15(1):52 sourceSupported

Probably not a substantial claim: may not pass S3 (load-bearing).

Increased expression of CX3CR1 attenuates microglial inflammatory responses to LPS.

“Certainly increased expression of CX3CR1 attenuates microglial inflammatory responses to LPS (liposaccharide) [44]”

• cites [44] Inoue K et al (2021) Modulation of inflammatory responses by fractalkine signaling in microglia. PLoS ONE 16(5):e0252118 sourceSupported

Probably not a substantial claim: may not pass S3 (load-bearing).

Several other AD risk polymorphisms, such as the R47H TREM2 mutation, alter microglial phagocytic capacity, leading to reduced clearance of Aβ aggregates and neuronal decline.

“Several other polymorphisms implicated as risk factors in AD, such as the R47H TREM2 mutation, result in alterations to microglial phagocytic capacity that lead to reduced clearance of Aβ aggregates and neuronal decline [27, 28].”

• cites [27] Arcuri C et al (2017) The pathophysiological role of microglia in dynamic surveillance, phagocytosis and structural remodeling of the developing CNS. Front Mol Neurosci 10:191 sourcePartly supported

  The Phagocytosis section associates TREM2 mutations with AD risk, and the Neurodevelopmental Diseases section says that nonfunctional TREM2 or DAP12 in AD patients impairs amyloid engulfment and is associated with dementia. The copy does not establish that the specific R47H variant causes this impaired clearance or neuronal decline; its explicit R47H reference concerns ALS. No page numbers are available in the indexed copy. (LLM, conf=0.86)

  Experimental — accuracy not yet measured
• cites [28] Zhao Y et al (2018) TREM2 is a receptor for β-amyloid that mediates microglial function. Neuron 97(5):1023–1031 sourcePartly supported

  The Introduction identifies R47H as an AD-risk variant, and the Results (‘TREM2 Binds to Aβ Oligomers’; page unavailable) show that it compromises TREM2 binding to Aβ oligomers. But the Results on Aβ degradation report that TREM2 deletion had little effect on microglial phagocytic uptake, while reducing degradation after uptake. The paper does not establish that R47H causes reduced clearance of Aβ aggregates or consequent neuronal decline. (LLM, conf=0.90)

  Experimental — accuracy not yet measured

Classified as probably not a substantial claim. May combine two claims.

Deletion of CX3CR1 results in an increased inflammatory response and phagocytic activity.

“while deletion results in an increased inflammatory response and phagocytic activity [45].”

• cites [45] Murai N, Mitalipova M, Jaenisch R (2020) Functional analysis of CX3CR1 in human induced pluripotent stem (iPS) cell-derived microglia-like cells. Eur J Neurosci 52(7):3667–3678 sourceSupported

Probably not a substantial claim: may not pass S3 (load-bearing).

A significant gain of function of PLCγ2 can lead to deleterious impacts on the immune system.

“These clearly indicate that a significant gain of function of PLCγ2 can lead to deleterious impacts on the immune system.”

• cites [29] Novice T et al (2020) A germline mutation in the C2 domain of PLCgamma2 associated with gain-of-function expands the phenotype for PLCG2-related diseases. J Clin Immunol 40(2):267–276 sourceSupported
• cites [30] Kutukculer N et al (2021) Four diseases, PLAID, APLAID, FCAS3 and CVID and one gene (PHOSPHOLIPASE C, GAMMA-2; PLCG2): striking clinical phenotypic overlap and difference. Clin Case Rep 9(4):2023–2031 sourceSupported
• cites [31] Ombrello MJ et al (2012) Cold urticaria, immunodeficiency, and autoimmunity related to PLCG2 deletions. N Engl J Med 366(4):330–338 sourceSupported
• cites [32] Zhou Q et al (2012) A hypermorphic missense mutation in PLCG2, encoding phospholipase Cgamma2, causes a dominantly inherited autoinflammatory disease with immunodeficiency. Am J Hum Genet 91(4):713–720 sourceNot checked — no full text of the source
• cites [33] Neves JF et al (2018) Novel PLCG2 mutation in a patient with APLAID and Cutis Laxa. Front Immunol 9:2863 sourceSupported
• cites [34] Deza G et al (2019) Acquired cold urticaria vs. autoinflammatory diseases, genetic and clinical profile and differential diagnosis: study of a cohort of patients in a Tertiary Reference Centre. Acta Derm Venereol 99(12):1071–1077 sourcePartly supported

  The Discussion (page unavailable) describes immune harms associated with PLAID, including recurrent infections, hypogammaglobulinaemia and autoimmunity. However, the paper does not identify significant gain of function of PLCγ2 as their cause; its Results found no pathogenic or likely pathogenic PLCG2 variants in the studied cohort. (LLM, conf=0.82)

  Experimental — accuracy not yet measured
• cites [35] Bogaert DJ et al (2016) Genes associated with common variable immunodeficiency: one diagnosis to rule them all? J Med Genet 53(9):575–590 sourceSupported
• cites [36] Chae JJ et al (2015) Connecting two pathways through Ca2+ signalling: NLRP3 inflammasome activation induced by a hypermorphic PLCG2 mutation. Arthritis Rheumatol (Hoboken, N.J.) 563–567 sourceSupported

Classified as probably not a substantial claim. May combine two claims.

IL-10 is known to inhibit the synthesis of pro-inflammatory cytokines, and may promote the differentiation of microglia into the anti-inflammatory state (M2 or homeostatic state).

“which is known to inhibit the synthesis of pro-inflammatory cytokines, and may promote the differentiation of microglia into the anti-inflammatory state, classically known as M2 or homeostatic state [40].”

• cites [40] Laffer B et al (2019) Loss of IL-10 promotes differentiation of microglia to a M1 phenotype. Front Cell Neurosci 13:430 sourceSupported

Probably not a substantial claim: may not pass S3 (load-bearing).

Individuals with MCI who carry the PLCγ2P522R variant had better cognitive performance even in the presence of the APOE4 AD risk gene.

“It has recently been reported that individuals with MCI (mild cognitive impairment), who carry the PLCγ2P522R variant had better cognitive performance even in the presence of the APOE4 AD risk gene [38].”

• cites [38] Kleineidam L et al (2020) PLCG2 protective variant p.P522R modulates tau pathology and disease progression in patients with mild cognitive impairment. Acta Neuropathol 39(6):1025–1044 sourcePartly supported

  The Results section reports slower cognitive decline among MCI patients carrying p.P522R and compares its effect in the presence of APOE-ε4. But it says the variant *may* initially counteract APOE-ε4’s detrimental effect, at least in part; the claim presents better cognitive performance in those carrying both variants as a definite finding. No page number was provided for the indexed passage. (LLM, conf=0.82)

  Experimental — accuracy not yet measured

Probably not a substantial claim: may not pass S3 (load-bearing).

Proinflammatory environments and signalling are a prominent observation in AD brain pathology.

“Proinflammatory environments and signalling are a prominent observation in AD brain pathology [39].”

• cites [39] Heneka MT et al (2013) NLRP3 is activated in Alzheimer's disease and contributes to pathology in APP/PS1 mice. Nature 674–678 sourceSupported

Probably not a substantial claim: may not pass S3 (load-bearing).

A broad spectrum of gene expression changes have also been reported in homozygous PLCγ2P522R knock-in mice.

“A broad spectrum of gene expression changes have also been reported in homozygous PLCγ2P522R knock-in mice [17].”

• cites [17] Takalo M et al (2020) The Alzheimer’s disease-associated protective Plcgamma2-P522R variant promotes immune functions. Mol Neurodegener 15(1):52 sourceSupported

Probably not a substantial claim: may not pass S3 (load-bearing).

Ca2+ currents have been implicated in the regulation of microglial motility.

“Ca2+ currents have also been implicated in the regulation of microglial motility (reviewed in [50]).”

• cites [50] Umpierre AD, Wu LJ (2021) How microglia sense and regulate neuronal activity. Glia 69(7):1637–1653 sourceSupported

May combine two claims.

Heterozygous expression of the PLCG2 P522R variant results in increased microglial clearance of Aβ while preserving synapses.

“Heterozygous expression of the P522R variant resulted in increased microglial clearance of Aβ, while preserving synapses.”

• Also stated in: § PLCγ2P522R affects synaptic pruning in hiPSC-derived cortical neuron and microglia co-culture · boosted

  “These results collectively establish that heterozygous PLCγ2P522R expression enhances the clearance and modulation of Aβ peptides while sparing synapses in a co-culture setup, providing a possible mechanism for AD protection.”

Probably not a substantial claim: may not pass S3 (load-bearing).

In BV2 cells, PLCγ2 P522R expression shows a non-significant mild enhancement of Aβ uptake (p = 0.3385).

“and a non-significant mild enhancement (p = 0.3385) was seen in PLCγ2P522R expressing cells”

May combine two claims.

Synaptosome uptake is significantly reduced in PLCγ2 P522R variants with a dose-dependent reduction between PLCγ2HET and PLCγ2HOM variant cells.

“By contrast, synaptosome uptake was significantly reduced in PLCγ2P522R variants with a dose-dependent reduction between PLCγ2HET and PLCγ2HOM variant cells (Fig. 1F, G).”

Probably not a substantial claim: may not pass S3 (load-bearing).

In resting hiPSC-derived microglia, a set of additional genes (CD68, P2RY12, NLRP3, CYP1A1, CYP1B1, TMEM119) show a trend towards increased expression in PLCγ2HET cells compared to PLCγ2WT cells, but these do not reach statistical significance.

“A small number of additional genes also showed a trend towards increased expression in PLCγ2HET cells, although these did not reach significance (CD68; p = 0.1656, P2RY12; p = 0.1739, NLRP3; p = 0.1498, CYP1A1; p = 0.1209, CYP1B1; p = 0.1219, TMEM119; p = 0.1739).”

May combine two claims.

PLCG2 P522R has a potential impact on microglial mitochondrial function and ATP production, which may contribute to maintenance of microglial metabolic fitness.

“We show a potential impact of PLCγ2P522R on microglia mitochondrial function and ATP production, which may contribute to maintenance of microglia metabolic fitness.”

May combine two claims.

In BV2 cells over-expressing WT or P522R PLCγ2, over-expression of the protein irrespective of variant resulted in a marked increase in zymosan uptake and reduced uptake of both dextran substrates.

“Similar studies in BV2 cells over expressing either WT or P522R PLCγ2 showed that over-expression of the protein, irrespective of the variant, resulted in a marked increase in zymosan uptake, and reduced uptake of both the dextran substrates”

May combine two claims.

Aβ accumulation is predominately observed in subcellular compartments such as lysosomes, while synaptosomes are internalised by control but appear predominantly bound to the cellular membrane in P225R mutants.

“Furthermore, Aβ accumulation was predominately observed in subcellular compartments, such as lysosomes, while synaptosomes were internalised by the control, but appeared predominantly bound to the cellular membrane in P225R mutants (S. Figure 2).”

May combine two claims.

3D image analysis showed increased Aβ uptake by PLCγ2 microglia variants compared to controls, with no clear significant difference between the PLCγ2HET and PLCγ2HOM lines.

“3D Image analysis showed increased Aβ uptake by PLCγ2 microglia variants compared to controls, with no clear significant difference between the PLCγ2HET and PLCγ2HOM lines (Fig. 2A, B).”

May combine two claims.

In resting hiPSC-derived microglia, there are no significant differences in gene expression between PLCγ2HET and PLCγ2HOM cells, but there is a trend towards significant differences in RAB5 (p=0.1737), CX3CR1 (p=0.1221) and IL-10 (p=0.0753).

“Although we did not see any significant differences between PLCγ2HET and PLCγ2HOM cells, there was a trend towards significant differences in RAB5 (p = 0.1737), CX3CR1 (p = 0.1221) and Il-10 (p = 0.0753), highlighting the potential importance of these genes in the functional impact of the PLCγ2P522r variant.”

May combine two claims.

In co-culture supernatant, all conditions containing microglia recorded reduced Aβ level compared to neuronal cells in the absence of microglia, but only cultures containing PLCγ2HET microglia reached significance (p = 0.036).

“While all conditions containing microglia recorded reduced Aβ level compared to neuronal cells in the absence of microglia, only cultures containing microglia heterozygous for the PLCγ2P522R variant reached significance (p = 0.036)”

Probably not a substantial claim: may not pass S3 (load-bearing).

PLCγ2HET microglia have slightly higher lysotracker levels than PLCγ2HOM microglia.

“with PLCγ2HET having slightly higher levels than PLCγ2HOM (Fig. 1C, E)”

Probably not a substantial claim: may not pass S3 (load-bearing).

We identified a number of additional genes that trended towards a difference between PLCγ2HET and PLCγ2WT cells.

“we identified a number of additional genes that trended towards a difference between PLCγ2HET and PLCγ2WT cells”

May combine two claims.

The presence of the P522R variant increased microglial uptake of the Aβ peptide, accompanied by a reduction in synapse pruning.

“We were able to show that the presence of the P522R variant increased microglial uptake of the Aβ peptide, accompanied by a reduction in synapse pruning, two key pathological processes in AD.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

PLCγ2HOM microglia have slightly reduced Ca2+ levels compared to PLCγ2WT cells, but this does not reach significance (p=0.1528).

“Indeed, PLCγ2HOM microglia had slightly reduced Ca2+ levels compared to PLCγ2WT cells, although this did not reach significance (p = 0.1528).”

May combine two claims.

While no significant changes were observed between PLCG2HET and PLCG2HOM cells, Il-10, CX3CR1 and RAB5 showed trends towards enhanced expression in PLCG2HET cells.

“while no significant changes were observed between PLCG2HET and PLCG2HOM cells, Il-10, CX3CR1 and RAB5 showed trends towards enhanced expression in PLCG2HET cells.”

May combine two claims.

In the current study, heterozygous PLCγ2P522R induced increases in the expression of genes associated with phagocyte maturation (RAB5, RAB7) and various aspects of lipid metabolism (LIPA, APOE, PLIN2) in resting state microglia, an effect not seen in homozygous cells.

“In the current study, heterozygous PLCγ2P522R induced increases in the expression of genes associated with phagocyte maturation (RAB5, RAB7) and various aspects of lipid metabolism (LIPA, APOE, PLIN2) in resting state microglia, an effect which is not seen in homozygous cells.”

May combine two claims.

A number of the processes assessed appeared to be dose-dependently modified by heterozygous vs. homozygous expression of the PLCγ2P522R variant, while other processes were preferentially impacted only in heterozygous cells.

“while a number of the processes we assessed did appear to be ‘dose dependently’ modified by heterozygous vs. homozygous expression of the PLCγ2P522R variant, other processes were preferentially impacted only in heterozygous cells.”

May combine two claims.

PLCγ2P522R microglia selectively spare synaptosomes while maintaining efficient clearance of Aβ when treated concurrently with both biological cargoes.

“these PLCγ2P522R microglia selectively spare synaptosomes while maintaining efficient clearance of Aβ when treated concurrently with both biological cargoes.”

May combine two claims.

We did not see significant changes in the basal expression of TREM2 or TLR4, or functionally associated proteins such as NLRP3 in our cells.

“we did not see significant changes in the basal expression of TREM2 or TLR4, or functionally associated proteins such as NLRP3 in our cells.”

Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

A number of resting state transcriptional changes are associated with this altered function.

“A number of resting state transcriptional changes are associated with this altered function”

May combine two claims.

PLCγ2HET cells demonstrated enhanced mobility compared to PLCγ2WT cells, including both whole cell movement and surveillance.

“Consistent with the increase, we see in intracellular Ca2+ in PLCγ2HET cells, they also demonstrated enhanced mobility compared to PLCγ2WT cells.”

• Also stated in: § Discussion · plain

  “Both whole cell movement, detected via nuclear tracking, as well as surveillance, detected via assessing cytoplasmic movement alone, were enhanced.”

May combine two claims.

Microglia expressing PLCγ2P522R in general show enhanced mitochondrial respiration as well as significantly higher ATP production, which was not associated with increases in mitochondrial number but rather changes in mitochondrial function.

“Our findings indicate that microglia expressing PLCγ2P522R in general show enhanced mitochondrial respiration as well as significantly higher ATP production, which was not associated with increases in mitochondrial number but rather changes in mitochondrial function.”

May combine two claims.

PLCγ2HOM cells showed increased surveillance, but their nuclear movement did not differ from PLCγ2WT cells.

“PLCγ2HOM cells also showed increased surveillance, but their nuclear movement did not differ from PLCγ2WT cells”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

PLCγ2 is a PIP2 metabolising enzyme recruited to the cell membrane upon activation by tyrosine kinase and modulates immune signals impacting cell fate and function.

“The protein product of PLCG2 (PLCγ2) is a PIP2 metabolising enzyme that is recruited to the cell membrane upon activation by tyrosine kinase and modulates immune signals impacting cell fate and function, both in the brain and periphery [12, 13].”

• cites [12] Rhee SG (2001) Regulation of phosphoinositide-specific phospholipase C. Annu Rev Biochem 70:281–312 sourceNot checked — no full text of the source
• cites [13] Patterson RL et al (2002) Phospholipase C-γ is required for agonist-induced Ca2+ entry. Cell 111(4):529–541 sourceNot checked — no full text of the source

Probably not a substantial claim: may not pass S3 (load-bearing).

PLCγ2 plays a role in TLR4 signalling independent of its role in the TREM2 pathway.

“It also appears to play a role in TLR4 signalling independent of its role in the TREM2 pathway [14]”

• cites [14] Andreone BJ et al (2020) Alzheimer’s-associated PLCγ2 is a signaling node required for both TREM2 function and the inflammatory response in human microglia. Nat Neurosci 23(8):927–938 sourceNot checked — no full text of the source

May combine two claims.

Heterozygous PLCγ2 P522R expression enhances clearance and modulation of Aβ peptides while sparing synapses in a co-culture setup, providing a possible mechanism for Alzheimer's disease protection.

“These results collectively establish that heterozygous PLCγ2P522R expression enhances the clearance and modulation of Aβ peptides while sparing synapses in a co-culture setup, providing a possible mechanism for AD protection.”

Classified as probably not a substantial claim. May combine two claims.

Conversion of 522P to 522R was confirmed by DNA sequencing and positive colonies were identified for downstream assays.

“To confirm conversion of 522P to 522R, DNA was extracted using DNeasy Blood & Tissue (69506, QIAGEN) and sent for sequencing (Eurofins Genomics) (sequencing primers are available in supplementary methods Table 1) and positive colonies were identified to be used in downstream assays.”

Classified as probably not a substantial claim. May combine two claims.

The PLCγ2 WT locus was authenticated through sequencing and the PLCγ2HET and PLCγ2HOM hiPSC lines were generated on the parent BIONi10-C WT background using CRISPR editing.

“The PLCγ2 WT locus was authenticated through sequencing (Supplementary Table 1) and the PLCγ2HET and PLCγ2HOM hiPSC lines were generated on the parent BIONi10-C WT background in house using CRISPR editing.”

May combine two claims.

PLCG2WT, PLCG2HET and PLCG2HOM hiPSC-derived microglia did not display obvious phenotypic or karyotypic defects and robustly expressed microglia/macrophage-associated markers including TMEM119 and IBA1.

“Upon differentiation, PLCγ2WT, PLCγ2HET and PLCγ2HOM did not display any obvious phenotypic or karyotypic defects and robustly expressed microglia/macrophage-associated markers including TMEM119 and IBA1 (S. Figure 1).”

Classified as probably not a substantial claim.

PLCγ2 activity can induce calcium influx from the ER through the binding of IP3 to its receptor IP3R.

“PLCγ2 activity can induce calcium influx from the ER through the binding of IP3 to its receptor in the ER, IP3R [26].”

• cites [26] Kadamur G, Ross EM (2013) Mammalian phospholipase C. Annual Rev Physiol 75:127–54 sourceNot checked — no full text of the source

May combine two claims.

PLCγ2 has a novel role in mitochondrial function, indicating better microglial metabolic fitness.

“In conclusion, we identified a novel role of PLCγ2 on mitochondrial function, which indicates better microglial metabolic fitness.”

• AssumptionRests on an assumption: The bioenergetic profile of microglia is directly linked to their functional state.

Probably not a substantial claim: may not pass S3 (load-bearing).

This massive cytoplasmic Ca2+ influx is critical for various downstream signalling pathways that modulate numerous microglial functions.

“This massive cytoplasmic Ca2+ influx is critical for various downstream signalling pathways that modulate numerous microglial functions.”

• cites [26] Kadamur G, Ross EM (2013) Mammalian phospholipase C. Annual Rev Physiol 75:127–54 sourceNot checked — no full text of the source

Classified as probably not a substantial claim. May combine two claims.

LOAD develops over many years, rather than arising as a result of a single acute challenge, thus these low level ongoing changes likely reflect the chronic impact of the variant on general microglial function.

“LOAD develops over many years, rather than arising as a result of a single acute challenge, thus these low level ongoing changes likely reflect the chronic impact of the variant on general microglial function.”

• AssumptionRests on an assumption: LOAD develops over many years, rather than arising as a result of a single acute challenge.

May combine two claims.

Significant changes in gene expression in our cells were primarily observed when comparing PLCG2HET and PLCG2WT cells, and under our current experimental conditions, we cannot rule out an impact of CRISPR editing per se on this expression profile.

“It is important to note that significant changes in gene expression in our cells were primarily observed when comparing PLCG2HET and PLCG2WT cells, and under our current experimental conditions, we cannot rule out an impact of CRISPR editing per se on this expression profile.”

Probably not a substantial claim: may not pass S3 (load-bearing).

It is possible that the increase in CX3CR1 observed in resting state PLCγ2HET microglia is linked to the reduction in synapse phagocytosis seen in these cells.

“It is, therefore, possible that the increase in CX3CR1 we observe in resting state PLCγ2HET microglia is linked to the reduction in synapse phagocytosis seen in these cells”

• AssumptionRests on an assumption: Within the CNS, CX3CR1 is expressed exclusively by microglia, and binds to the neuronally expressed chemokine CX3CL1 (fractalkine).

Probably not a substantial claim: may not pass S3 (load-bearing).

These additional genes may indicate a subset of genes 'primed' to respond to immune challenge.

“that may indicate a subset of genes ‘primed’ to respond to immune challenge.”

Probably not a substantial claim: may not pass S3 (load-bearing).

The enhanced cytoplasmic motility is potentially indicative of increased surveillance activity.

“Cytoplasmic motility was also significantly enhanced in PLCγ2HET cells, potentially indicative of increased surveillance activity (Fig. 6D).”

May combine two claims.

PLCγ2HET microglia were more active in both surveillance and migration compared to PLCγ2WT and PLCγ2HOM microglia.

“Our findings indicate that PLCγ2HET microglia were more active in both surveillance and migration compared to PLCγ2WT and PLCγ2HOM microglia, and this could be tied to their housekeeping Ca2+ level.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Loss of PLCG2 results in a spectrum of gene expression changes in microglia both at rest, and in response to TLR stimulation.

“Loss of PLCG2 results in a spectrum of gene expression changes in microglia both at rest, and in response to TLR stimulation [14]”

• cites [14] Andreone BJ et al (2020) Alzheimer’s-associated PLCγ2 is a signaling node required for both TREM2 function and the inflammatory response in human microglia. Nat Neurosci 23(8):927–938 sourceNot checked — no full text of the source

May combine two claims.

The impact of PLCγ2P522R is dependent on gene dose, with heterozygous cells overall showing a more beneficial profile than homozygous cells.

“Crucially, we have shown that the impact of PLCγ2P522R is dependent on gene dose, with heterozygous cells overall showing a more beneficial profile than homozygous cells.”

Classified as probably not a substantial claim. May combine two claims.

Previous studies have shown that PLCG2 acts downstream of TREM2, and knock out of either protein results in lipid accumulation deficits and a failure to activate key lipid processing genes such as LIPA and APOC1, as well as a generalised alteration in the overall lipidome of the cells.

“Previous studies have shown that PLCG2 acts downstream of TREM2, and knock out of either protein results in lipid accumulation deficits and a failure to activate key lipid processing genes such as LIPA and APOC1 [14], as well as a generalised alteration in the overall lipidome of the cells.”

• cites [14] Andreone BJ et al (2020) Alzheimer’s-associated PLCγ2 is a signaling node required for both TREM2 function and the inflammatory response in human microglia. Nat Neurosci 23(8):927–938 sourceNot checked — no full text of the source

May combine two claims.

The protective nature of the PLCγ2P522R variant in AD may reflect a selective increase in microglial clearance of Aβ, coupled with a preservation of synapses.

“Our study is the first to suggest that the protective nature of the PLCγ2P522R variant in AD may reflect a selective increase in microglial clearance of Aβ, coupled with a preservation of synapses.”

• AssumptionRests on an assumption: The PLCγ2P522R variant is protective in Alzheimer's disease.

Probably not a substantial claim: may not pass S2 (checkable), S3 (load-bearing).

Other as yet unidentified factors may play a key role in modulating microglial cell energy homeostasis.

“This separation between Ca2+ levels and mitochondrial performance suggests that other as yet unidentified factors may play a key role in modulating microglial cell energy homeostasis.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

While in a resting state, microglia use mitochondrial respiration, specifically OXPHOS, as their main source of energy, although resting state is a misconception, since microglia are never quiescent, rather they are constantly engaged in parenchymal surveillance.

“While in a resting state, microglia use mitochondrial respiration, specifically OXPHOS (oxidative phosphorylation) as their main source of energy, although resting state is a misconception, since microglia are never quiescent, rather they are constantly engaged in parenchymal surveillance [48].”

• cites [48] Nimmerjahn A, Kirchhoff F, Helmchen F (2005) Neuroscience: resting microglial cells are highly dynamic surveillants of brain parenchyma in vivo. Sci 308:1314–1318 sourceNot checked — no full text of the source

May combine two claims.

The TLR induced profile appears distinctly different from that seen with TREM2 knockout, suggesting that PLCG2 may interact independently with both TREM2 and TLR linked pathways.

“this TLR induced profile appears distinctly different from that seen with TREM2 knockout, suggesting that PLCG2 may interact independently with both TREM2 and TLR linked pathways”

Probably not a substantial claim: may not pass S3 (load-bearing).

To maintain brain homeostasis and surveillance, microglia require energy-demanding cytoskeleton remodelling, necessary for extending and retracting their ramified processes, to constantly scan their surroundings.

“To maintain brain homeostasis and surveillance, microglia require energy-demanding cytoskeleton remodelling, necessary for extending and retracting their ramified processes, to constantly scan their surroundings [47, 48].”

• cites [47] Davalos D et al (2005) ATP mediates rapid microglial response to local brain injury in vivo. Nat Neurosci 8(6):752–758 sourceNot checked — no full text of the source
• cites [48] Nimmerjahn A, Kirchhoff F, Helmchen F (2005) Neuroscience: resting microglial cells are highly dynamic surveillants of brain parenchyma in vivo. Sci 308:1314–1318 sourceNot checked — no full text of the source

May combine two claims.

PLCγ2P522R may enhance basal microglial functioning, leading to a relative protection against the accumulation of pathogenic proteins seen in both healthy ageing and AD.

“suggesting that PLCγ2P522R may enhance basal microglial functioning, leading to a relative protection against the accumulation of pathogenic proteins seen in both healthy ageing and AD.”

• AssumptionRests on an assumption: The PLCγ2P522R variant is protective in Alzheimer's disease.