PLCG2 downregulation impairs synaptic function and increases Alzheimer’s disease hallmarks in neuronal cultures

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

Synaptic plasticity in the dentate gyrus is strongly affected in mouse models of AD.

“Synaptic plasticity in the DG is strongly affected in mouse models of AD25,26.”

• cites [25] Jiang, N. et al. Impaired plasticity of intrinsic excitability in the dentate gyrus alters spike transfer in a mouse model of Alzheimer’s disease. Neurobiol. Dis. 154, 105345 (2021). sourceDisputed

  The Results (§3.4; page unavailable) found no significant long-term change in synaptic strength after stimulation in either genotype. The Discussion (§4.3; page unavailable) attributes the altered dentate-gyrus spike transfer in APP/PS1 mice chiefly to plasticity of intrinsic excitability, a nonsynaptic form of plasticity, rather than strongly affected synaptic plasticity. (LLM, conf=0.87)

  Checked by a reasoning model — may be wrong.

  Experimental — accuracy not yet measured
• cites [26] Da Silva, S. V. et al. Hippocampal mossy fibers synapses in CA3 pyramidal cells are altered at an early stage in a mouse model of Alzheimer’s disease. J. Neurosci. 39, 4193–4205 (2019). sourcePartly supported

  The Results section reports strongly diminished short-term plasticity at dentate gyrus mossy-fiber synapses onto CA3 cells in APP/PS1 mice. The Discussion also reports impaired plasticity of DG-evoked inhibitory transmission. These findings concern DG-to-CA3 connections in one mouse model, rather than synaptic plasticity within the dentate gyrus across mouse models of AD. Page numbers were not provided in our copy. (LLM, conf=0.85)

  Experimental — accuracy not yet measured

May combine two claims.

Synaptic fragility/sensitivity was proposed as a pathological trigger depending on genetic risk factors, some of which have already been linked to synaptic mechanisms such as BIN1, FERMT2, PTK2B or CD2AP.

“we proposed synaptic fragility/sensitivity as a pathological trigger10, depending on genetic risk factors, some of which have already been linked to synaptic mechanisms, such as BIN1, FERMT2, PTK2B or CD2AP9–12.”

• cites [10] Eysert, F. et al. Alzheimer’s genetic risk factor FERMT2 (kindlin-2) controls axonal growth and synaptic plasticity in an APP-dependent manner. Mol. Psychiatry 26, 5592–5607 (2021). sourceSupported
• cites [9] Schürmann, B. et al. A novel role for the late-onset Alzheimer’s disease (LOAD)-associated protein Bin1 in regulating postsynaptic trafficking and glutamatergic signaling. Mol. Psychiatry 25, 2000–2016 (2020). sourceSupported
• cites [11] Giralt, A. et al. PTK2B/Pyk2 overexpression improves a mouse model of Alzheimer’s disease. Exp. Neurol. 307, 62–73 (2018). sourceNot checked — no full text of the source
• cites [12] Ojelade, S. A. et al. cindr, the Drosophila homolog of the CD2AP Alzheimer’s disease risk gene, is required for synaptic transmission and proteostasis. Cell Rep. 28, 1799–1813 (2019). sourceSupported

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

Genome-wide association studies and high-throughput sequencing have led to the characterization of 76 loci associated with AD risk.

“leading to the characterization of 76 loci associated with the AD risk6–8”

• cites [6] Bellenguez, C. et al. New insights into the genetic etiology of Alzheimer’s disease and related dementias. Nat. Genet. 54, 412–436 (2022). sourcePartly supported

  The Discussion reports that the GWAS meta-analysis identified 75 independent loci associated with Alzheimer’s disease and related dementias, not 76. The Main section links high-throughput genomic techniques to the discovery of AD-associated loci, but this paper does not report the claimed count of 76. (LLM, conf=0.86)

  Experimental — accuracy not yet measured
• cites [7] Holstege, H. et al. Exome sequencing identifies rare damaging variants in ATP8B4 and ABCA1 as risk factors for Alzheimer’s disease. Nat. Genet. 54, 1786–1794 (2022). sourcePartly supported

  The Main section says GWAS identified 75 AD-risk loci in populations with European ancestry and that DNA sequencing identified additional rare risk variants. It does not support the claimed count of 76 loci. (LLM, conf=0.88)

  Experimental — accuracy not yet measured
• cites [8] Lambert, J. C., Ramirez, A., Grenier-Boley, B. & Bellenguez, C. Step by step: towards a better understanding of the genetic architecture of Alzheimer’s disease. Mol. Psychiatry 28, 2716–2727 (2023). sourceSupported

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

PLCG2 was believed to be almost exclusively expressed in microglia and has been extensively investigated in this cell type.

“this AD risk gene was believed to be almost exclusively expressed in microglia and, as a consequence, it has been extensively investigated in this cell type18–21.”

• cites [18] Takalo, M. et al. The Alzheimer’s disease-associated protective Plcγ2-P522R variant promotes immune functions. Mol. Neurodegener. 15, 52 (2020). sourceSupported
• cites [19] Claes, C. et al. 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. 18, 1765–1778 (2022). sourcePartly supported

  The Research in Context section (page unavailable) describes the P522R variant as primarily expressed by microglia in the brain and cites studies in cultured microglia and mice. That supports a microglial research focus, but not the stronger, unqualified claim that PLCG2 is almost exclusively expressed in microglia. The Narrative section also says in vivo studies of the variant had remained limited. (LLM, conf=0.84)

  Experimental — accuracy not yet measured
• cites [20] Tsai, A. P. et al. PLCG2 is associated with the inflammatory response and is induced by amyloid plaques in Alzheimer’s disease. Genome Med. 14, 17 (2022). sourcePartly supported

  The Results section ('Plcg2 expression is increased exclusively in microglia in the brains of 5xFAD mice'; page unavailable) reports that the disease-associated increase in expression was exclusive to microglia in that mouse model. The paper also investigates PLCG2 in human microglia, but it does not establish that PLCG2 was believed to be almost exclusively expressed in microglia generally, or that this belief was why the cell type had been extensively investigated. (LLM, conf=0.83)

  Experimental — accuracy not yet measured
• cites [21] Tsai, A. P. et al. Genetic variants of phospholipase C-γ2 alter the phenotype and function of microglia and confer differential risk for Alzheimer’s disease. Immunity 56, 2121–2136 (2023). sourceSupported

May combine two claims.

These findings support the involvement of nonsense-mediated mRNA decay, as both mutations introduce premature termination codons that meet established criteria for triggering this pathway.

“These findings collectively support the involvement of nonsense-mediated mRNA decay (NMD), as both mutations introduce premature termination codons (PTCs) that meet the established criteria for triggering this pathway28.”

• cites [28] Lejeune, F. Nonsense-mediated mRNA decay, a finely regulated mechanism. Biomedicines 10, 141 (2022). sourcePartly supported

  Section 2.1, “The NMD Reaction” (page unavailable), explains that a premature termination codon can trigger NMD and describes a criterion involving a downstream splicing event at least 50–55 nucleotides away. The review does not identify the two mutations in the citing sentence or establish that either meets that criterion, so it supports the mechanism but not its application to both mutations. (LLM, conf=0.88)

  Experimental — accuracy not yet measured

Classified as probably not a substantial claim.

NRXN3 is the isoform most consistently implicated in AD pathogenesis.

“given that this isoform has been most consistently implicated in AD pathogenesis35–39.”

• cites [35] Martinez-Mir, A. et al. Genetic study of neurexin and neuroligin genes in Alzheimer’s disease. J. Alzheimers Dis. 35, 403–412 (2013). sourceNot checked — no full text of the source
• cites [36] Rahman, M. M., Westermark, G. T., Zetterberg, H., Härd, T. & Sandgren, M. Protofibrillar and fibrillar amyloid-β binding proteins in cerebrospinal fluid. J. Alzheimer Dis. 66, 1053–1064 (2018). sourcePartly supported

  The Novel proteins section identifies neurexin-3 among several neurexins that bound Aβ42 fibrils from AD samples only. The Discussion also mentions neurexin-3β processing, but neither section says NRXN3 is the isoform most consistently implicated in AD pathogenesis. Page numbers were not available in our copy. (LLM, conf=0.86)

  Experimental — accuracy not yet measured
• cites [37] Lee, A. K., Khaled, H., Chofflet, N. & Takahashi, H. Synaptic organizers in Alzheimer’s disease: a classification based on amyloid-β sensitivity. Front. Cell. Neurosci. 14, 281 (2020). sourcePartly supported

  The section “Neurexin: A Novel Binding Partner of Aβ Oligomers” (page unavailable) reports significantly decreased NRXN3 expression in AD patients, supporting an association with AD. But it also says that which neurexin isoforms are mainly affected in AD is not fully understood; it does not establish NRXN3 as the most consistently implicated isoform. (LLM, conf=0.88)

  Experimental — accuracy not yet measured
• cites [38] Hishimoto, A. et al. Neurexin 3 transmembrane and soluble isoform expression and splicing haplotype are associated with neuron inflammasome and Alzheimer’s disease. Alzheimers Res. Ther. 11, 28 (2019). sourcePartly supported

  The Discussion (page not specified) explicitly implicates NRXN3 in AD, and the Results report reduced expression of several NRXN3 isoforms in AD brain samples. The paper does not establish that NRXN3 is the isoform most consistently implicated in AD pathogenesis relative to other isoforms. (LLM, conf=0.86)

  Experimental — accuracy not yet measured
• cites [39] Bot, N., Schweizer, C., Halima Ben, S. & Fraering, P. C. Processing of the synaptic cell adhesion molecule neurexin-3β by Alzheimer disease α- and γ-secretases. J. Biol. Chem. 286, 2762–2773 (2011). sourcePartly supported

  The Results report that Alzheimer’s disease–causing PS1 mutations impair NRXN3β processing. The discussion says its possible contribution to neuronal defects still needs investigation. The paper does not establish NRXN3 as implicated in AD pathogenesis or compare how consistently different neurexin isoforms are implicated. (LLM, conf=0.86)

  Experimental — accuracy not yet measured

May combine two claims.

In the brains of Plcγ2-P522R knock-in mice, Akt1/2 phosphorylation was reduced, casting doubt on the AKT hypothesis.

“in the brains of Plcγ2-P522R knock-in mice, Akt1/2 phosphorylation was reduced, casting doubt on this hypothesis18.”

• cites [18] Takalo, M. et al. The Alzheimer’s disease-associated protective Plcγ2-P522R variant promotes immune functions. Mol. Neurodegener. 15, 52 (2020). sourceSupported

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

Both ADGRL and NRXN pathways are involved in synaptic function and psychiatric disorders.

“Both pathways are involved in synaptic function and psychiatric disorders45–47.”

• cites [45] Sita, L. V., Diniz, G. B., Horta-Junior, J. A. C., Casatti, C. A. & Bittencourt, J. C. Nomenclature and comparative morphology of the teneurin/TCAP/ADGRL protein families. Front. Neurosci. 13, 425 (2019). sourcePartly supported

  The Introduction and Nomenclature section says that latrophilins (ADGRLs) and neurexins interact in a complex involved in synapse formation and maintenance. The paper does not establish that both pathways are involved in psychiatric disorders; its References list an ADHD-related latrophilin study, but provide no corresponding claim about neurexins. (LLM, conf=0.83)

  Experimental — accuracy not yet measured
• cites [46] Bourgeron, T. A synaptic trek to autism. Curr. Opin. Neurobiol. 19, 231–234 (2009). sourceNot checked — no full text of the source
• cites [47] Vitobello, A. et al. ADGRL1 haploinsufficiency causes a variable spectrum of neurodevelopmental disorders in humans and alters synaptic activity and behavior in a mouse model. Am. J. Hum. Genet. 109, 1436–1457 (2022). sourcePartly supported

  The Introduction (page unavailable) identifies NRXN1 as a synaptic protein and neurexins as ADGRL1 binding partners. The Discussion and Results (pages unavailable) link ADGRL1 to altered synaptic activity and to neurodevelopmental and behavioral conditions including ASD and ADHD. Our copy does not link NRXN pathways to psychiatric disorders, so it supports only part of the claim about both pathways. (LLM, conf=0.84)

  Experimental — accuracy not yet measured

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

In AD, PLCG2 has been mostly studied in microglia and shown to be implicated in multiple microglia-related signaling pathways.

“In AD, PLCG2 has been mostly studied in microglia and shown to be implicated in multiple microglia-related signaling pathways18–21.”

• cites [18] Takalo, M. et al. The Alzheimer’s disease-associated protective Plcγ2-P522R variant promotes immune functions. Mol. Neurodegener. 15, 52 (2020). sourceSupported
• cites [19] Claes, C. et al. 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. 18, 1765–1778 (2022). sourceSupported
• cites [20] Tsai, A. P. et al. PLCG2 is associated with the inflammatory response and is induced by amyloid plaques in Alzheimer’s disease. Genome Med. 14, 17 (2022). sourceSupported
• cites [21] Tsai, A. P. et al. Genetic variants of phospholipase C-γ2 alter the phenotype and function of microglia and confer differential risk for Alzheimer’s disease. Immunity 56, 2121–2136 (2023). sourceSupported

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

GSK3β is known to be involved in the regulation of APP metabolism, Tau phosphorylation and synaptic functions, making it a potential link between PLCγ2 and the phenotypes associated with its downregulation.

“GSK3β is already known to be involved in the regulation of APP metabolism, Tau phosphorylation and synaptic functions41–43, making it a potential link between PLCγ2 and the different phenotypes associated with its downregulation.”

• cites [41] Reddy, P. H. Amyloid β-induced glycogen synthase kinase 3β phosphorylated VDAC1 in Alzheimer’s disease: implications for synaptic dysfunction and neuronal damage. Biochim. Biophys. Acta 1832, 1913–1921 (2013). sourceSupported
• cites [42] Hernandez, F., Lucas, J. J. & Avila, J. GSK3 and Tau: two convergence points in Alzheimer’s disease. J. Alzheimers Dis. 33, S141–S144 (2013). sourcePartly supported

  The paper’s Abstract states that GSK3 phosphorylates tau and contributes to amyloid-β production, supporting the tau claim and a related aspect of APP metabolism. It does not address regulation of synaptic functions. The proposed link to PLCγ2 is the citing authors’ inference, not a finding attributed to this source. (LLM, conf=0.86)

  Experimental — accuracy not yet measured
• cites [43] Ly, P. T. T. et al. Inhibition of GSK3β-mediated BACE1 expression reduces Alzheimer-associated phenotypes. J. Clin. Invest. 123, 224–235 (2013). sourceSupported

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

Neurons derived from iPSCs obtained from sporadic AD cases show elevated Tau hyperphosphorylation, increased Aβ levels and GSK3β activation.

“neurons derived from iPSCs obtained from sporadic AD cases also show elevated Tau hyperphosphorylation, increased Aβ levels and GSK3β activation44.”

• cites [44] Ochalek, A. et al. Neurons derived from sporadic Alzheimer’s disease iPSCs reveal elevated TAU hyperphosphorylation, increased amyloid levels, and GSK3B activation. Alzheimer Res. Ther. 9, 90 (2017). sourceSupported

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

These results are consistent with previous reports suggesting that PLCγ2 modulates Tau pathology and disease progression in patients with mild cognitive impairment.

“These results are consistent with previous reports suggesting that PLCγ2 modulates Tau pathology and disease progression in patients with mild cognitive impairment49.”

• cites [49] Kleineidam, L. et al. PLCG2 protective variant p.P522R modulates Tau pathology and disease progression in patients with mild cognitive impairment. Acta Neuropathol. 139, 1025–1044 (2020). sourceSupported

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

Neurexins have been shown to regulate APP synaptic functions and/or modulate Aβ-induced synaptotoxicity.

“neurexins have been shown to regulate APP synaptic functions and/or modulate Aβ-induced synaptotoxicity36,37,48.”

• cites [36] Rahman, M. M., Westermark, G. T., Zetterberg, H., Härd, T. & Sandgren, M. Protofibrillar and fibrillar amyloid-β binding proteins in cerebrospinal fluid. J. Alzheimer Dis. 66, 1053–1064 (2018). sourcePartly supported

  The Discussion reports that neurexins bind APP, and the Novel proteins section identifies neurexins binding Aβ42 fibrils in AD cerebrospinal-fluid samples (page numbers unavailable). Neither passage shows that neurexins regulate APP synaptic functions or modulate Aβ-induced synaptotoxicity; binding alone does not establish those functional effects. (LLM, conf=0.83)

  Experimental — accuracy not yet measured
• cites [37] Lee, A. K., Khaled, H., Chofflet, N. & Takahashi, H. Synaptic organizers in Alzheimer’s disease: a classification based on amyloid-β sensitivity. Front. Cell. Neurosci. 14, 281 (2020). sourcePartly supported

  The section “Neurexin: A Novel Binding Partner of Aβ Oligomers” (page unavailable) reports that Aβ oligomers reduce axonal β-neurexin and impair neurexin-mediated presynaptic assembly, supporting a role in Aβ-induced synaptic dysfunction. The passages do not establish that neurexins regulate APP synaptic functions; the paper instead identifies an APP interaction for LRRTM3. (LLM, conf=0.84)

  Experimental — accuracy not yet measured
• cites [48] Cvetkovska, V. et al. Neurexin-β mediates the synaptogenic activity of amyloid precursor protein. J. Neurosci. 42, 8936–8947 (2022). sourceNot checked — no full text of the source

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

In ref. 23, cells were already annotated with cell types.

“In ref. 23, cells were already annotated with cell types.”

• cites [23] Leng, K. et al. Molecular characterization of selectively vulnerable neurons in Alzheimer’s disease. Nat. Neurosci. 24, 276–287 (2021). sourceSupported

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

The first snRNA-seq dataset was performed on temporal cortex tissue, and the second was performed on entorhinal cortex and superior frontal gyrus.

“The former was performed on temporal cortex tissue, and the latter was performed on entorhinal cortex and superior frontal gyrus.”

• cites [22] Li, Z. et al. APOE genotype determines cell-type-specific pathological landscape of Alzheimer’s disease. Neuron 113, 1380–1397 (2025). sourcePartly supported

  The STAR Methods (page unavailable) describe isolating nuclei from frozen temporal cortex for this paper’s snRNA-seq work, supporting the former dataset’s tissue location. The passages in our copy do not establish that a second dataset was performed on entorhinal cortex and superior frontal gyrus; a search for those regions found no supporting passage. (LLM, conf=0.84)

  Experimental — accuracy not yet measured
• cites [23] Leng, K. et al. Molecular characterization of selectively vulnerable neurons in Alzheimer’s disease. Nat. Neurosci. 24, 276–287 (2021). sourceSupported

May combine two claims.

Plcg2 downregulation in mouse dentate gyrus neurons consistently disrupted dendritic morphology and synaptic function.

“we found that Plcg2 downregulation in mouse dentate gyrus neurons consistently disrupted dendritic morphology and synaptic function.”

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

Downregulation of Syp significantly decreased burst frequency in primary neuronal cultures.

“As a positive control, the downregulation of Syp significantly decreased burst frequency, a readout for neuronal activity”

May combine two claims.

Plcg2 downregulation did not change the complexity index or total branch number in mouse dentate gyrus granule cells.

“but not in the complexity index (P = 0.24) or a total branch number (P = 0.52”

The quote only partly states this claim.

Differential expression analysis revealed a higher number of DEGs in shPLCG2-hNCs than in the other model.

“Differential expression analysis revealed a higher number of differentially expressed genes (DEGs) in shPLCG2-hNCs (Fig. 6c).”

May combine two claims.

In snRNA-seq of shPLCG2-hNCs versus shNT-hNCs, the proportion of inhibitory neurons was higher in shPLCG2-hNCs, and this change was mirrored to a lower extent in PLCG2R953*-hNCs.

“we observed a higher proportion of inhibitory neurons in shPLCG2-hNCs (Fig. 6b), a change mirrored in PLCG2R953*-hNCs to a lower extent.”

• AssumptionRests on an assumption: The snRNA-seq cell-type proportions reflect the actual proportions of cell types in the hNC cultures.

May combine two claims.

No significant decrease in AKT phosphorylation at S473 was observed in PLCG2R953*-hNCs, although a similar trend was noted.

“No significant decrease in AKT phosphorylation at S473 was observed, although a similar trend was noted (−45%, P = 0.057).”

• AssumptionRests on an assumption: Human neuronal cultures derived from human induced pluripotent stem cells and differentiated for 4 weeks are a valid model of human neuronal synaptic function and AD hallmarks.

May combine two claims.

The R953* variant leads to increased Tau phosphorylation and Aβ levels.

“we also observed that the R953* variant leads to increased Tau phosphorylation and Aβ levels”

May combine two claims.

Differential analysis revealed 18 and 10 altered L/R signaling pathways in shPLCG2-hNCs versus shNT-hNCs and PLCG2R953*-hNCs versus PLCG2WT-hNCs, respectively, with three main pathways depending on NRXN, ADGRL and PTPR, upregulated or downregulated in both models in neurons.

“Differential analysis revealed 18 and 10 altered L/R signaling pathways in shPLCG2-hNCs versus shNT-hNCs and PLCG2R953*-hNCs versus PLCG2WT-hNCs, respectively, with three main pathways depending on NRXN, ADGRL and PTPR, upregulated or downregulated in both models in neurons (Fig. 6f,g and Supplementary Fig. 7).”

• Also stated in: § Discussion · plain

  “This revealed that ADGRL signaling and NRXN signaling were preferentially affected within neurons across both models.”

• AssumptionRests on an assumption: CellChat-inferred ligand–receptor interactions reflect actual intercellular communication in the hNC cultures.

May combine two claims.

PLCG2/Plcγ2 downregulation impacts synaptic density and neuronal electrophysiological properties in four distinct experimental models and with different shRNAs.

“we demonstrated that PLCγ2/Plcγ2 downregulation impacts synaptic density and neuronal electrophysiological properties using four distinct experimental models (and different shRNAs).”

May combine two claims.

On average, 727 ligand-receptor interactions were inferred per sample, grouped into 89 signaling pathways, with excitatory-inhibitory neuron interaction being strongest.

“On average, 727 ligand–receptor (L/R) interactions were inferred per sample, grouped into 89 signaling pathways, with excitatory–inhibitory neuron interaction being strongest (Supplementary Fig. 7b).”

• AssumptionRests on an assumption: CellChat-inferred ligand–receptor interactions reflect actual intercellular communication in the hNC cultures.

May combine two claims.

Among genes with the strongest impact on synaptic density in the HCS, PLCG2 was one of the best hits, despite the absence of microglia in the HCS.

“Among the genes with the strongest impact on synaptic density, PLCG2 was one of the best hits (despite the absence of microglia in our HCS).”

• AssumptionRests on an assumption: The hNC models recapitulate relevant aspects of human AD neuronal pathophysiology.

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

Laminin signaling was downregulated during communication between VLMCs and astrocytes, although less consistently.

“Notably, we also observed that laminin signaling was downregulated during communication between vascular leptomeningeal cells (VLMCs) and astrocytes, although less consistently (Supplementary Fig. 7c).”

• AssumptionRests on an assumption: CellChat-inferred ligand–receptor interactions reflect actual intercellular communication in the hNC cultures.

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

From the 23 clusters, the analysis identified astrocytes (four subpopulations), excitatory neurons, inhibitory neurons, NPCs, VLMCs and fibroblasts.

“From these 23 clusters, we identified—astrocytes, subdivided into four subpopulations (steady state—clusters 1, 6, 7 and 18; proliferative—clusters 4 and 8; immature—cluster 10; with VLMC potential—clusters 13 and 22); excitatory neurons (clusters 12, 19 and 20); inhibitory neurons (clusters 5, 14 and 17); NPCs (cluster 9); VLMCs (cluster 11) and fibroblasts (cluster 21).”

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

The estimated ambient RNA level was 14% on average (min = 5.1%, max = 34%) across samples.

“The estimated ambient level was 14% on average (min = 5.1%, max = 34%) across samples.”

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

A total of 23 clusters were identified with a mix of samples within each cluster.

“A total of 23 clusters were identified (Supplementary Fig. 5c) with mix of samples within each cluster (Supplementary Fig. 5d,g).”

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

Cluster 0 contained a mix of excitatory and inhibitory neurons and was subdivided into two clusters based on expression of SLC17A6 and GAD1/GAD2.

“Cluster 0 contained a mix of excitatory and inhibitory neurons and was therefore subdivided into two clusters based on the expression of glutamate transporter SLC17A6 and the GABAergic enzymes GAD1/GAD2.”

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

We identified three participants in the gnomAD control cohort who carried any predicted PLCG2 LoF SNV or indel variants.

“We identified three participants therein who carried any predicted PLCG2 LoF SNV or indel variants.”

May combine two claims.

PLCγ2 downregulation could increase AD risk by impairing synaptic functions and by increasing Aβ levels and Tau phosphorylation in neurons.

“In conclusion, PLCγ2 downregulation could increase AD risk by impairing synaptic functions and by increasing Aβ levels and Tau phosphorylation in neurons.”

• AssumptionRests on an assumption: Rodent primary neuronal cultures and mouse dentate gyrus neurons are adequate models for human AD-related synaptic pathology.

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

The proportion of risk attributable to genetic susceptibility factors for AD has been estimated to be between 60% and 80% in twin studies.

“Because the proportion of risk attributable to genetic susceptibility factors for AD has been estimated to be between 60% and 80% in twin studies5”

• cites [5] Gatz, M. et al. Role of genes and environments for explaining Alzheimer disease. Arch. Gen. Psychiatry 63, 168–174 (2006). sourceNot checked — no full text of the source

May combine two claims.

Both LoF variants, PLCG2 p.Q816* and p.R953*, likely trigger NMD, leading to reduced mRNA and protein levels of PLCG2/PLCγ2.

“These results suggest that both LoF variants, PLCG2 p.Q816* and PLCG2 p.R953*, likely trigger NMD, leading to reduced mRNA and protein levels of PLCG2/PLCγ2.”

May combine two claims.

Downregulation of Plcg2 selectively decreases the amount of both postsynaptic AMPARs and NMDARs, as well as the NMDAR/AMPAR ratio at synapses in dentate gyrus cells.

“Our data suggest that the downregulation of Plcg2 selectively decreases the amount of both postsynaptic AMPARs and NMDARs, as well as the NMDAR/AMPAR ratio at synapses in DG cells.”

May combine two claims.

PLCG2 downregulation may be deleterious and favor the development of AD through synaptic impairment, increased Aβ levels and Tau hyperphosphorylation.

“our abovementioned data suggest that PLCG2 downregulation may be deleterious and favor the development of AD through synaptic impairment, increased Aβ levels and Tau hyperphosphorylation.”

• AssumptionRests on an assumption: Human neuronal cultures derived from human induced pluripotent stem cells and differentiated for 4 weeks are a valid model of human neuronal synaptic function and AD hallmarks.
• AssumptionRests on an assumption: Rodent primary neuronal cultures and mouse dentate gyrus neurons are adequate models for human AD-related synaptic pathology.

May combine two claims.

PLCG2 downregulation impacts synaptic density and function in vitro in hNCs.

“In conclusion and in accordance with our results observed in the rodent brain, PLCG2 downregulation impacts synaptic density and function in vitro in hNCs.”

• AssumptionRests on an assumption: Human neuronal cultures derived from human induced pluripotent stem cells and differentiated for 4 weeks are a valid model of human neuronal synaptic function and AD hallmarks.
• AssumptionRests on an assumption: Rodent primary neuronal cultures and mouse dentate gyrus neurons are adequate models for human AD-related synaptic pathology.

May combine two claims.

Plcg2 downregulation markedly impairs dendritic morphology and spine density in dentate gyrus granule cells in situ.

“Overall, in congruence with our findings in vitro, Plcg2 downregulation markedly impairs dendritic morphology and spine density in DG GCs in situ.”

May combine two claims.

Plcg2 downregulation leads to a substantial decrease in input resistance and neuronal excitability.

“Overall, Plcg2 downregulation leads to a substantial decrease in input resistance and neuronal excitability.”

May combine two claims.

The higher number of DEGs in shPLCG2-hNCs may be attributed to the more pronounced reduction in PLCG2 expression compared with the PLCG2R953*-hNCs model, although model-specific factors likely also have a role.

“This may be attributed to the more pronounced reduction in PLCG2 expression”

May combine two claims.

PLCG2 downregulation is associated with increased Aβ levels and Tau phosphorylation, the latter potentially through the AKT/GSK3β axis.

“Altogether, these data indicate that PLCG2 downregulation is associated with increased Aβ levels and Tau phosphorylation, the latter potentially through the AKT/GSK3β axis.”

• AssumptionRests on an assumption: Phosphorylation of GSK3β at Y216 and AKT at S473 indicates the active forms of these kinases.
• AssumptionRests on an assumption: Human neuronal cultures derived from human induced pluripotent stem cells and differentiated for 4 weeks are a valid model of human neuronal synaptic function and AD hallmarks.

May combine two claims.

Diacylglycerol-driven PKC activation can enhance AKT signaling, and loss of PLCγ2 function may thus reduce AKT activity, as observed.

“Diacylglycerol-driven PKC activation can enhance AKT signaling and loss of PLCγ2 function may thus reduce AKT activity, as observed.”

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

Written informed consent was obtained from study participants or, for those with substantial cognitive impairment, from a caregiver, legal guardian or other proxy, and the study protocols for all populations were reviewed and approved by the appropriate local institutional review boards.

“Written informed consent was obtained from study participants or, for those with substantial cognitive impairment, from a caregiver, legal guardian or other proxy, and the study protocols for all populations were reviewed and approved by the appropriate local institutional review boards51.”

• cites [51] McKhann, G. et al. Clinical diagnosis of Alzheimer’s disease: report of the NINCDS-ADRDA Work Group under the auspices of Department of Health and Human Services Task Force on Alzheimer’s Disease. Neurology 34, 939–944 (1984). sourceNot checked — no full text of the source

May combine two claims.

This signaling cascade may promote increased Aβ secretion, Tau hyperphosphorylation and synaptic dysfunction.

“This may promote increased Aβ secretion, Tau hyperphosphorylation and synaptic dysfunction.”

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

The ADGEN study was approved by the Research Ethics Committee of Northern Savo (approval 420/13.02.00/2016).

“Research Ethics Committee of Northern Savo has approved the ADGEN study (approval 420/13.02.00/2016).”

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

It cannot be ruled out that PLCG2 downregulation in astrocytes contributes to the observed phenotypes.

“we cannot rule out the possibility that PLCG2 downregulation in astrocytes contributes to them.”

May combine two claims.

The results suggest that PLCG2 downregulation perturbs neuronal and synaptic functions, at least partly through neuron-neuron communication, with NRXN/ADGRL signaling emerging as a key candidate pathway.

“Altogether, these results suggest that PLCG2 downregulation perturbs neuronal and synaptic functions, at least partly through neuron–neuron communication, with NRXN/ADGRL signaling emerging as a key candidate pathway.”

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

The HCS was performed thrice using three independent neuronal cultures.

“the HCS was performed thrice using three independent neuronal cultures.”

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

Assay quality was assessed using β score, and only plates with a β score >1 were analyzed.

“Assay quality was assessed using β score calculated according to β=(μshNT−μshSyp)/(σshNT²+σshSyp²), where μ and σ are the mean and s.d. of Syp density in control wells treated with shNT and shSyp, and only plates with a β score >1 were analyzed.”

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

All animal experiments were approved by the local ethical committee of the Institut Pasteur de Lille and according to the regulations of the University of Bordeaux/Centre National de la Recherche Scientifique Animal Care and Use Committee.

“All animal experiments were approved by the local ethical committee of the Institut Pasteur de Lille and according to the regulations of the University of Bordeaux/Centre National de la Recherche Scientifique Animal Care and Use Committee.”

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

The difference in maturation may impact the observed modifications in phenotypes following PLCG2 downregulation.

“Thus, we cannot exclude that this difference in maturation impacts the observed modifications in phenotypes following PLCG2 downregulation.”

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

The investigators were not blinded to allocation during the experiments or the assessment of outcomes, except when generating the snRNA-seq data and performing electrophysiological recordings of mouse DG slices.

“The investigators were not blinded to allocation during the experiments or the assessment of outcomes, except when generating the snRNA-seq data and performing electrophysiological recordings of mouse DG slices.”

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

Cluster 16, expressing markers of more than one cell type and with greater RNA content than others, was deemed doublet cells and removed.

“Cluster 16, expressing markers of more than one cell type and with greater RNA content than others, was deemed doublet cells and removed.”

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

A total of 12 donors were excluded for both DLPFC and MTG regions.

“A total of 12 donors were excluded for both DLPFC and MTG regions.”

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

Data analysis for electrophysiological recordings was performed blindly to the experimental conditions, except when recordings were made from noninfected cells.

“Data analysis for electrophysiological recordings was performed blindly to the experimental conditions, except when recordings were made from noninfected cells.”

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

One replicate of shPLCG2 (shPLCG2_4) had a strong mitochondrial percentage (median = 3% while all other samples had <1%) and low RNA and gene counts; this replicate and the associated control (shNT_4) were excluded from the analysis.

“We observed at this stage that one replicate of shPLCG2 (shPLCG2_4) had a strong mitochondrial percentage (median = 3% while all other samples had <1%) and low RNA and gene counts; we thus excluded this replicate as well as the associated control (shNT_4) from the analysis.”

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

MOhms and the experiment were discarded if it changed by >20%.

“MOhms and the experiment were discarded if it changed by >20%.”

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

No statistical method was used to predetermine sample size.

“No statistical method was used to predetermine sample size.”

Classified as probably not a substantial claim.

DEGs were identified according to adjusted P value <0.05 and |log2(FC)| > 0.5, using pseudobulk DESeq2 analysis, adjusted for average mitochondrial RNA percentage, library batch and ambient RNA level.

“DEGs were identified according to adjusted P value <0.05 and |log2(FC)| > 0.5, using pseudobulk DESeq2 analysis, adjusted for average mitochondrial RNA percentage, library batch and ambient RNA level.”

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

The snRNA-seq analyses used 140,163 high-quality nuclei from 14 hNCs, with n = 3 biological replicates per group for the shPLCG2 versus shNT model and n = 4 biological replicates per group for the R953* versus WT model.

“Results from the 140,163 transcriptomically profiled high-quality nuclei from 14 hNCs; n = 3 biological replicates per group for shPLCG2 versus shNT model (control = shNT), and n = 4 biological replicates per group for R953* versus WT model (control = WT).”

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

At least two devices were used for each experimental condition and at least three independent cultures were performed.

“At least two devices were used for each experimental condition and at least three independent cultures were performed.”

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

The access resistance was <20%.

“The access resistance was <20%.”

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

SoupX::adjustCounts() was used to correct the gene expression matrix, and the ambient RNA level was used as a covariate to ensure no impact on downstream differential analysis.

“We then used SoupX::adjustCounts() to correct the gene expression matrix accordingly, and we further ensured no impact on downstream differential analysis using the ambient RNA level as a covariate.”

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

Outliers in the HCS of synaptic markers in PNCs were identified using the ±3 median absolute deviation method.

“Outliers in the HCS of synaptic markers in PNCs were identified using the ±3 median absolute deviation method.”

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

The ADGEN cohort AD patients were examined at the Department of Neurology, Kuopio University Hospital, and all fulfilled the NINCDS-ADRDA criteria for probable AD.

“These AD patients were examined at the Department of Neurology, Kuopio University Hospital, and they all fulfilled the NINCDS-ADRDA criteria for probable AD51.”

• cites [51] McKhann, G. et al. Clinical diagnosis of Alzheimer’s disease: report of the NINCDS-ADRDA Work Group under the auspices of Department of Health and Human Services Task Force on Alzheimer’s Disease. Neurology 34, 939–944 (1984). sourceNot checked — no full text of the source

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

Images failing to meet QC criteria, specifically those with <300 synaptophysin puncta or a HOMER1/Syp ratio outside 0.5-5.0, were excluded from subsequent analysis.

“Images failing to meet QC criteria were excluded from subsequent analysis, specifically those with <300 synaptophysin puncta or a HOMER1/Syp ratio outside the range of 0.5–5.0.”

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

The ROUT method (Q = 1%) was applied to identify outliers in the electrophysiological recording dataset of mouse DG slices.

“The ROUT method (Q = 1%) was applied to identify outliers in the electrophysiological recording dataset of mouse DG slices.”

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

Clusters 2, 3 and 15 having low RNA content, high mitochondrial RNA percentage, low cell-type identity and/or expressing stress markers were filtered out.

“Clusters 2, 3 and 15 having low RNA content, high mitochondrial RNA percentage, low cell-type identity and/or expressing stress markers (chaperone/heat shock protein gene family, stress granules TIA1) were filtered out as well (Supplementary Fig. 5e).”

Classified as probably not a substantial claim.

Wells with shRNA-induced toxicity, defined by normalized network area <0.4, were excluded from the analysis.

“Wells with shRNA-induced toxicity, as defined by normalized network area of <0.4, were excluded from the analysis.”

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

The experiments were not randomized.

“The experiments were not randomized.”

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

No data were excluded from analyses elsewhere.

“No data were excluded from analyses elsewhere.”

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

For the quantification of synaptic markers in PNCs and hNCs on microfluidic devices and for MEA analyses, any data point falling outside the interval calculated by Q1 − 1.5× IQR and Q3 + 1.5× IQR was considered an outlier and excluded from the analysis.

“For the quantification of synaptic markers in PNCs and hNCs on microfluidic devices and for MEA analyses, any data point falling outside the interval calculated by Q1 − 1.5× IQR and Q3 + 1.5× IQR was considered an outlier and excluded from the analysis.”