Microglial INPP5D limits plaque formation and glial reactivity in the PSAPP mouse model of Alzheimer's disease

Constitutive deletion or pharmacologic inhibition of SHIP1 increases phagocytosis of various substances, including zymosan in SHIP1 knockout macrophages and Aβ in SHIP1 inhibitor-treated microglia.

“Constitutive deletion or pharmacologic inhibition of SHIP1 increases phagocytosis of various substances, including zymosan in SHIP1 knockout (KO) macrophages and Aβ in SHIP1 inhibitor-treated microglia.12,13”

• cites [12] Pedicone C, Fernandes S, Dungan OM, Pan-SHIP1/2 inhibitors promote microglia effector functions essential for CNS homeostasis. J Cell Sci. 2020: 133. PMC10682645 31780579 sourcePartly supported

  The paper reports increased Aβ1–42 phagocytosis in microglia treated with pan-SHIP1/2 inhibitors (indexed copy, section labeled ABSTRACT; page unavailable). It also says SHIP1 limits macrophage phagocytosis, but does not establish the claimed zymosan result in SHIP1-knockout macrophages. The effective microglial treatments inhibited both SHIP1 and SHIP2; the paper says SHIP1-selective inhibition did not significantly improve microglial phagocytic function. (LLM, conf=0.88)

  Experimental — accuracy not yet measured
• cites [13] Horan KA, Watanabe K-i, Kong AM, Regulation of FcγR-stimulated phagocytosis by the 72-kDa inositol polyphosphate 5-phosphatase: sHIP1, but not the 72-kDa 5-phosphatase, regulates complement receptor 3–mediated phagocytosis by differential recruitment of these 5-phosphatases to the phagocytic cup. Blood. 2007; 110: 4480– 4491. 17682126 sourceNot checked — no full text of the source

SHIP1 inhibits phagocytic signal transduction by TREM2 and TYROBP/DAP12.

“In particular, SHIP1 inhibits phagocytic signal transduction by triggering receptor expressed on myeloid cells 2 (TREM2) and tyrosine kinase binding protein (TYROBP, also known as DAP12).11”

• cites [11] Peng Q, Malhotra S, Torchia JA, Kerr WG, Coggeshall KM, Humphrey MB. TREM2- and DAP12-dependent activation of PI3K requires DAP10 and is inhibited by SHIP1. Sci Signal. 2010; 3: ra38. 20484116 PMC2900152 sourcePartly supported

  The Results section reports that SHIP1 inhibits signaling downstream of TREM2 and DAP12 in preosteoclasts. The Discussion only speculates that SHIP1 may similarly regulate TREM2- and DAP12-mediated phagocytosis; it does not establish inhibition of phagocytic signaling. Page numbers were not provided. (LLM, conf=0.88)

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Conditional Inpp5d knockdown in microglia avoids the developmental effects that can occur in constitutive knockouts.

“This strategy also avoids the developmental effects that can occur in constitutive knockouts.16,17”

• cites [16] Liu Q, Sasaki T, Kozieradzki I, SHIP is a negative regulator of growth factor receptor-mediated PKB/Akt activation and myeloid cell survival. Genes Dev. 1999; 13: 786– 791. 10197978 PMC316591 sourcePartly supported

  The 'Generation of SHIP-deficient mice' section reports that mice lacking SHIP from birth had enlarged spleens apparent by four weeks and progressive abnormalities in myeloid cells. This supports concern about effects of a constitutive knockout, but the paper does not examine conditional Inpp5d knockdown in microglia or show that it avoids developmental effects. Page numbers were not available in our copy. (LLM, conf=0.78)

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• cites [17] Helgason CD, Damen JE, Rosten P, Targeted disruption of SHIP leads to hemopoietic perturbations, lung pathology, and a shortened life span. Genes Dev. 1998; 12: 1610– 1620. 9620849 PMC316868 sourcePartly supported

  The SHIP gene-targeting section describes a germline knockout, and the section on SHIP−/− mice failing to thrive reports reduced body weight and splenomegaly by 4–5 weeks of age. These findings support the concern that constitutive loss has early-life effects, but the paper does not examine microglia or show that conditional knockdown avoids developmental effects. (LLM, conf=0.79)

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INPP5D expression in the brain is restricted to microglia.

“Multiple approaches have highlighted inositol polyphosphate-5-phosphatase D (INPP5D) as an AD risk gene, and its expression in the brain is restricted to microglia.4–6”

• cites [4] Zhang Y, Chen K, Sloan SA, Bennett ML, Scholze AR, O’Keeffe S, An RNA-sequencing transcriptome and splicing database of glia, neurons, and vascular cells of the cerebral cortex. J Neurosci. 2014; 34: 11929– 11947. 25186741 PMC4152602 sourceCould not verify

  The RNA-Seq analysis section describes cell type-specific expression profiles, and the qRT-PCR and in situ hybridization section identifies Olfml3—not INPP5D—as a microglial marker. Searches for INPP5D, Inpp5d, and SHIP1 found no passage in our copy establishing that INPP5D expression is restricted to microglia. (LLM, conf=0.72)

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• cites [5] Zhang Y, Sloan SA, Clarke LE, Caneda C, Plaza CA, Blumenthal PD, Purification and characterization of progenitor and mature human astrocytes reveals transcriptional and functional differences with mouse. Neuron. 2016; 89: 37– 53. 26687838 PMC4707064 sourceCould not verify

  The Results section, “Transcriptomes of acutely purified human neurons, astrocytes, oligodendrocytes, microglia, and endothelial cells” (page unavailable), describes a cell-type expression database. Searches for INPP5D, SHIP1, and microglial expression found no passage stating that INPP5D expression is restricted to microglia. (LLM, conf=0.73)

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• cites [6] Marsh SE, Walker AJ, Kamath T, Dissection of artifactual and confounding glial signatures by single-cell sequencing of mouse and human brain. Nat Neurosci. 2022; 25: 306– 316. 35260865 PMC11645269 sourceCould not verify

  The Results sections on mouse CNS cell types and human postmortem nuclei discuss microglial gene signatures, but do not identify INPP5D or establish where it is expressed. Searches for INPP5D, its full name, SHIP1, and microglia-restricted expression found no passage addressing the claim. (LLM, conf=0.73)

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Cluster 26 overlapped gene sets with previously reported reactive glial substates, including DAM1, DAM2, DAA, PIGs, and IRRAs.

“we found overlapping gene sets compared to previously reported, biologically defined reactive glial substates. This included integration with differentially expressed genes (DEGs) from disease associated microglia, both early (DAM1) and late (DAM2),25 disease associated astrocytes (DAA),26 plaque-induced genes (PIGs),27 and our previously identified interferon-responsive reactive astrocytes (IRRAs”

• cites [25] Keren-Shaul H, Spinrad A, Weiner A, A unique microglia type associated with restricting development of Alzheimer’s disease. Cell. 2017; 169: 1276– 1290. 28602351 sourceSupported
• cites [26] Habib N, McCabe C, Medina S, Disease-associated astrocytes in Alzheimer’s disease and aging. Nat Neurosci. 2020; 23: 701– 706. 32341542 PMC9262034 sourceSupported
• cites [27] Chen WT, Lu A, Craessaerts K, Pavie B, Corthout N, Spatial transcriptomics and in situ sequencing to study Alzheimer’s disease. Cell. 2020; 182: 976– 991. 32702314 sourceSupported

Multiple approaches have highlighted INPP5D as an Alzheimer's disease risk gene.

“Multiple approaches have highlighted inositol polyphosphate-5-phosphatase D (INPP5D) as an AD risk gene, and its expression in the brain is restricted to microglia.4–6”

• cites [4] Zhang Y, Chen K, Sloan SA, Bennett ML, Scholze AR, O’Keeffe S, An RNA-sequencing transcriptome and splicing database of glia, neurons, and vascular cells of the cerebral cortex. J Neurosci. 2014; 34: 11929– 11947. 25186741 PMC4152602 sourceCould not verify

  The retrieved passages from the paper’s cell-type expression and splicing sections do not identify INPP5D as an Alzheimer’s disease risk gene. Searches for “INPP5D,” its full name, and Alzheimer’s risk-gene terminology found no passage in our copy addressing that claim; the Alzheimer’s-related passage provided is a reference to a different topic. (LLM, conf=0.75)

  Experimental — accuracy not yet measured
• cites [5] Zhang Y, Sloan SA, Clarke LE, Caneda C, Plaza CA, Blumenthal PD, Purification and characterization of progenitor and mature human astrocytes reveals transcriptional and functional differences with mouse. Neuron. 2016; 89: 37– 53. 26687838 PMC4707064 sourceCould not verify

  The Results sections describe astrocyte and other brain-cell transcriptomes, but the retrieved passages do not identify INPP5D as an Alzheimer’s disease risk gene. Searches for INPP5D and Alzheimer’s genetic susceptibility found no passage in our copy addressing that attribution. (LLM, conf=0.77)

  Experimental — accuracy not yet measured
• cites [6] Marsh SE, Walker AJ, Kamath T, Dissection of artifactual and confounding glial signatures by single-cell sequencing of mouse and human brain. Nat Neurosci. 2022; 25: 306– 316. 35260865 PMC11645269 sourceCould not verify

  The Results section on human postmortem snRNA-seq includes Alzheimer’s disease donors, but does not identify INPP5D as a risk gene. Searches for INPP5D, its full name, SHIP1, and Alzheimer’s risk genes found no passage in our copy addressing that claim. (LLM, conf=0.74)

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INPP5D plays a role in AD pathogenesis.

“INPP5D plays a role in AD pathogenesis, and remains a potential target for microglia-focused AD therapies,15”

• cites [15] Tsai AP, Lin PB-C, Dong C, INPP5D expression is associated with risk for Alzheimer’s disease and induced by plaque-associated microglia. Neurobiology of Disease. 2021; 153: 105303. 33631273 PMC8082515 sourceSupported

The timing of gene and/or total microglial manipulation is critical.

“Notably, the timing of gene and/or total microglial manipulation is also critical,47”

• cites [47] Schoch KM, Ezerskiy LA, Morhaus MM, Acute Trem2 reduction triggers increased microglial phagocytosis, slowing amyloid deposition in mice. Proc Natl Acad Sci U S A. 2021; 118. PMC8271763 34187891 sourcePartly supported

  The Abstract reports that Trem2 reduction reduced amyloid deposition when administered during late-stage pathology but not before or at an early stage, supporting the importance of timing for this gene intervention. The paper does not establish that the timing of total microglial manipulation is critical. (LLM, conf=0.84)

  Experimental — accuracy not yet measured

Constitutive deletion of miR155, which should increase SHIP1, led to an increase in amyloid plaques.

“Finally, Inpp5d was identified as a target of miR155, and constitutive deletion of miR155, which should increase SHIP1, led to an increase in amyloid plaques.40,41”

• cites [40] Hickman SE, Allison EK. Microglial dysfunction and defective beta-amyloid clearance pathways in aging Alzheimer’s disease mice. J Neurosci. 2008; 28: 8354– 8360. 18701698 PMC2597474 sourceCould not verify

  The Methods section lists the genes measured, and the Results and Discussion describe age-related defects in amyloid clearance in PS1-APP mice. Searches for miR155, Inpp5d, SHIP1, and constitutive deletion found no passage addressing whether miR155 deletion increases amyloid plaques. The paper’s discussion of amyloid accumulation does not establish that specific claim. (LLM, conf=0.77)

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• cites [41] Readhead B, Haure-Mirande JV, Mastroeni D, miR155 regulation of behavior, neuropathology, and cortical transcriptomics in Alzheimer’s disease. Acta Neuropathol. 2020; 140: 295– 315. 32666270 PMC8414561 sourcePartly supported

  The Results section (“Absence of miR155 increases Aß deposition in 8-month-old APP/PSEN1 mice”) reports increased amyloid plaque deposition after constitutive miR155 deletion in APP/PSEN1 mice. The passages from our copy do not establish that the deletion increases SHIP1 or identify Inpp5d as a miR155 target; searches for SHIP1 and Inpp5d did not locate support for those parts of the citing sentence. (LLM, conf=0.86)

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Numerous studies targeting members of the TREM2/TYROBP pathway (Trem2, Tyrobp, and miR155) support beneficial outcomes associated with maintenance and/or restoration of homeostatic functions of microglia.

“Support for beneficial outcomes associated with maintenance and/or restoration of homeostatic functions of microglia comes from the numerous studies targeting the members of this pathway, including Trem2,48,50–52 Tyrobp,53,54 and miR155.41,55”

• cites [48] Krasemann S, Madore C, Cialic R, The TREM2-APOE pathway drives the transcriptional phenotype of dysfunctional microglia in neurodegenerative diseases. Immunity. 2017; 47: 566– 581. 28930663 PMC5719893 sourceSupported
• cites [50] Ulrich JD, Finn MB, Wang Y, Altered microglial response to Aβ plaques in APPPS1-21 mice heterozygous for TREM2. Mol Neurodegener. 2014; 9: 20. 24893973 PMC4049806 sourcePartly supported

  For the Trem2 portion of the citing sentence, the Discussion reports fewer plaque-associated microglia in TREM2-heterozygous mice and suggests that TREM2 supports a beneficial microglial response. But it found no significant difference in Aβ plaque burden and did not test restoration of microglial homeostatic function or demonstrate a beneficial outcome from it. The Tyrobp and miR155 portions have separate citations and are not attributed to this paper. (LLM, conf=0.85)

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• cites [51] Parhizkar S, Arzberger T, Brendel M, Loss of TREM2 function increases amyloid seeding but reduces plaque-associated ApoE. Nat Neurosci. 2019; 22: 191– 204. 30617257 PMC6417433 sourcePartly supported

  The Discussion (page unavailable) says functional TREM2 may protect against early amyloid seeding by enabling microglia to cluster around and clear newly seeded plaques. But it also says TREM2 loss leaves microglia locked in a homeostatic state while reducing plaque clearance. The paper therefore supports a potential benefit of TREM2 function, not a benefit specifically from maintaining or restoring microglial homeostasis. (LLM, conf=0.86)

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• cites [52] Jay TR, Miller CM, Cheng PJ, TREM2 deficiency eliminates TREM2+ inflammatory macrophages and ameliorates pathology in Alzheimer’s disease mouse models. J Exp Med. 2015; 212: 287– 295. 25732305 PMC4354365 sourcePartly supported

  The Results and Discussion report that Trem2 deficiency reduced amyloid pathology, astrocytosis, and MAPT phosphorylation in Alzheimer’s disease mouse models, supporting a beneficial outcome from targeting Trem2. But preservation of a homeostatic microglial phenotype is offered only as a possible explanation; the paper says further studies are needed to determine the mechanism. This citation is attached to Trem2, not to the sentence’s separate Tyrobp and miR155 claims. (LLM, conf=0.84)

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• cites [53] Haure-Mirande JV, Audrain M, Fanutza T, Deficiency of TYROBP, an adapter protein for TREM2 and CR3 receptors, is neuroprotective in a mouse model of early Alzheimer’s pathology. Acta Neuropathol. 2017; 134: 769– 788. 28612290 PMC5645450 sourcePartly supported

  The Discussion (page unavailable) reports that TYROBP deficiency in an early Alzheimer’s mouse model improved learning behavior and normalized some synaptic dysfunction, supporting a beneficial outcome from targeting Tyrobp. It does not establish that the benefit resulted from maintaining or restoring homeostatic microglial functions. (LLM, conf=0.83)

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• cites [54] Haure-Mirande JV, Wang M, Audrain M, Integrative approach to sporadic Alzheimer’s disease: deficiency of TYROBP in cerebral Abeta amyloidosis mouse normalizes clinical phenotype and complement subnetwork molecular pathology without reducing Abeta burden. Mol Psychiatry. 2019; 24: 431– 446. 30283032 PMC6494440 sourceSupported
• cites [41] Readhead B, Haure-Mirande JV, Mastroeni D, miR155 regulation of behavior, neuropathology, and cortical transcriptomics in Alzheimer’s disease. Acta Neuropathol. 2020; 140: 295– 315. 32666270 PMC8414561 sourcePartly supported

  The Discussion (page unavailable) reports that miR155 deletion largely benefited learning and partially rescued synaptic plasticity in APP/PSEN1 mice. It does not establish that these benefits came from maintaining or restoring homeostatic microglial function; the Results (page unavailable) found no change in the measured microglial activation markers. (LLM, conf=0.86)

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• cites [55] Butovsky O, Jedrychowski MP, Cialic R, Targeting miR-155 restores abnormal microglia and attenuates disease in SOD1 mice. Ann Neurol. 2015; 77: 75– 99. 25381879 PMC4432483 sourceSupported

Manipulation of molecules involved in immune/microglial function in either direction may result in detrimental or exacerbated pathological outcomes.

“However, as we have previously seen in our studies modulating Tyrobp expression, manipulation of molecules involved in immune/microglial function in either direction may result in detrimental or exacerbated pathological outcomes.49,53,54,56”

• cites [49] Audrain M, Haure-Mirande JV, Mleczko J, Reactive or transgenic increase in microglial TYROBP reveals a TREM2-independent TYROBP-APOE link in wild-type and Alzheimer’s-related mice. Alzheimers Dement. 2021; 17: 149– 163. 33314529 PMC7938663 sourceSupported
• cites [53] Haure-Mirande JV, Audrain M, Fanutza T, Deficiency of TYROBP, an adapter protein for TREM2 and CR3 receptors, is neuroprotective in a mouse model of early Alzheimer’s pathology. Acta Neuropathol. 2017; 134: 769– 788. 28612290 PMC5645450 sourcePartly supported

  The Discussion (page unavailable) says reduced TREM2 may be beneficial early but detrimental later, supporting the possibility that an immune-related manipulation can have harmful effects under some conditions. However, this paper found TYROBP deficiency protective in its early Alzheimer’s mouse model and says TYROBP overexpression was beyond its scope. It therefore does not establish detrimental outcomes from manipulating TYROBP in either direction. (LLM, conf=0.82)

  Experimental — accuracy not yet measured
• cites [54] Haure-Mirande JV, Wang M, Audrain M, Integrative approach to sporadic Alzheimer’s disease: deficiency of TYROBP in cerebral Abeta amyloidosis mouse normalizes clinical phenotype and complement subnetwork molecular pathology without reducing Abeta burden. Mol Psychiatry. 2019; 24: 431– 446. 30283032 PMC6494440 sourceSupported
• cites [56] Audrain M, Haure-Mirande JV, Wang M, Integrative approach to sporadic Alzheimer’s disease: deficiency of TYROBP in a tauopathy mouse model reduces C1q and normalizes clinical phenotype while increasing spread and state of phosphorylation of tau. Mol Psychiatry. 2019; 24: 1383– 1397. 30283031 PMC6447470 sourcePartly supported

  The Discussion reports that Tyrobp deletion increased tau phosphorylation and spread in mouse models, supporting the possibility of adverse pathological effects from reducing a microglial molecule. It also reports improved clinical and functional features. This paper does not establish that manipulation in the opposite direction produces detrimental outcomes. (LLM, conf=0.85)

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Phagocytosis was increased in microglia that lacked Inpp5d.

“We found that phagocytosis was increased in microglia that lacked Inpp5d (Figure 3H), in line with previous reports in the literature.12”

• cites [12] Pedicone C, Fernandes S, Dungan OM, Pan-SHIP1/2 inhibitors promote microglia effector functions essential for CNS homeostasis. J Cell Sci. 2020: 133. PMC10682645 31780579 sourcePartly supported

  The passage on dead-neuron uptake (indexed as ABSTRACT; page unavailable) reports increased microglial phagocytosis with a pan-SHIP1/2 inhibitor. It does not report phagocytosis in microglia lacking Inpp5d; only the dual inhibitor, not the SHIP1-selective inhibitor, produced a significant increase in that experiment. (LLM, conf=0.86)

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Microglial function may be adversely affected by Aβ, particularly as a result of chronic exposure to pro-inflammatory cytokines, resulting in increased Aβ accumulation.

“Notably, previous studies have shown that microglial function may be adversely affected by Aβ, particularly as a result of chronic exposure to pro-inflammatory cytokines, resulting in increased Aβ accumulation and potentially underpinning the contradiction between our in vitro and in vivo results.39,40”

• cites [39] Krabbe G, Halle A, Matyash V, Functional impairment of microglia coincides with Beta-amyloid deposition in mice with Alzheimer-like pathology. PLoS One. 2013; 8: e60921. 23577177 PMC3620049 sourcePartly supported

  The Discussion (page unavailable) proposes that chronic exposure to a pro-inflammatory environment may impair microglial function and reports that TNFα reduces Aβ phagocytosis in vitro. It also links impaired phagocytosis with plaque burden, but does not establish that cytokine-driven impairment results in increased Aβ accumulation. (LLM, conf=0.82)

  Experimental — accuracy not yet measured
• cites [40] Hickman SE, Allison EK. Microglial dysfunction and defective beta-amyloid clearance pathways in aging Alzheimer’s disease mice. J Neurosci. 2008; 28: 8354– 8360. 18701698 PMC2597474 sourceSupported

The results are consistent with a role for SHIP1 as a negative regulator of immune signaling.

“were consistent with previous results,12 and with a role for SHIP1 as a negative regulator of immune signaling.”

• cites [12] Pedicone C, Fernandes S, Dungan OM, Pan-SHIP1/2 inhibitors promote microglia effector functions essential for CNS homeostasis. J Cell Sci. 2020: 133. PMC10682645 31780579 sourceSupported

No elevation was detected for OC/6E10 or NU4/6E10 ratios.

“No such elevation was detected for OC/6E10 or NU4/6E10 (Figure S5).”

GO term analysis of the genes induced in Cluster 26 upon Inpp5d knockdown showed enrichment for 'Chemokine signaling pathway'.

“GO term analysis of these genes included an enrichment for “Defense response to virus” (Cxcl10, Ifit3, Oas1a, Ccl5, Oasl1), “Type I and II Interferon signaling” (Stat1, Ifit2, Irf5/6, Isg15), and “Chemokine signaling pathway” (Plcb2, Rac2, Ccl9/12,Txnip, H2-T23; Tables S6, S7 in supporting information).”

In Cluster 26, receptor-ligand pairs primarily involved in phagocytosis and lysosome activity were observed, including C1qb-Lrp1, C3-Lrp1, Timp1-Cd63, Pros1-Axl, and Ly86-Cd180.

“In Cluster 26, we observed receptor–ligand pairs primarily involved in phagocytosis and lysosome activity, including (ligand – receptor): C1qb – Lrp1, C3 – Lrp1, Timp1 – Cd63, Pros1 – Axl, and Ly86 – Cd180 (Figure S11, Table S8 in supporting information).”

Cluster 26 had a unique gene expression profile.

“Indeed, Cluster 26 had a unique gene expression profile, but we found overlapping gene sets compared to previously reported, biologically defined reactive glial substates.”

In WT-CO and WT-TAM mice, the number of IBA1+ cells and IBA1 immunoreactive areas in the hippocampus was unchanged after Inpp5d depletion.

“In the WT-CO and WT-TAM mice, the number of IBA1+ cells or IBA1 immunoreactive areas in the hippocampus was unchanged after Inpp5d depletion (Figure 1H–I, Figure S3E).”

muscat uncovered DEGs outside Cluster 26 when comparing WT-TAM to PSAPP-TAM and PSAPP-CO to PSAPP-TAM.

“While we discovered most DEGs in Cluster 26 due to its plaque association (Figure 2F, Figures S7–S9, Tables S1–S5), muscat also uncovered DEGs outside Cluster 26 when comparing WT-TAM to PSAPP-TAM as well as PSAPP-CO to PSAPP-TAM.”

Methoxy-XO4 staining confirmed an increase in amyloid burden associated with Inpp5d/SHIP1 knockdown.

“Staining with the Congo red derivative Methoxy-XO4 (XO4), which binds specifically to β-pleated sheet-containing structures, confirmed the increase in amyloid burden associated with the Inpp5d/SHIP1 knockdown (Figure S3A–B in supporting information).”

Spatial transcriptomics identified a plaque-specific expression profile.

“Spatial transcriptomics identified a plaque-specific expression profile that was extensively altered by Inpp5d knockdown.”

The difference in IBA1+ cells and IBA1 immunoreactive areas between PSAPP-CO and WT-CO mice was not significant.

“There were more IBA1+ cells and IBA1 immunoreactive areas in PSAPP-TAM mice compared to WT-CO mice (Figure 1H–I), whereas the difference between PSAPP-CO and WT-CO was not significant.”

Inpp5d downregulation increases plaque burden in PSAPP mice.

“Herein, we demonstrate that Inpp5d downregulation increases Aβ fibrillization, plaque burden, and number of microglia associated with Aβ plaques in the PSAPP amyloid-depositing mouse model of AD.”

• Also stated in: § Inpp5d knockdown worsened amyloid plaque deposition while increasing recruitment of microglia to the plaque · plain

  “At 6 months of age, the percent area and number of 6E10 immunoreactive deposits were increased by >66% and 73%, respectively (Figure 1D–F; Figure S2) in the hippocampi of PSAPP-TAM compared to PSAPP-CO littermates.”

• Also stated in: § Discussion: · boosted

  “These results demonstrate that conditional Inpp5d downregulation in the PSAPP mouse increases plaque burden and recruitment of microglia to plaques.”

• AssumptionRests on an assumption: The PSAPP mouse model at 6 months recapitulates a preclinical stage of Alzheimer's disease relevant to human disease.
• AssumptionRests on an assumption: Conditional Inpp5d knockdown using Cx3cr1CreER/+ and tamoxifen is specific to microglia and does not confound results through effects on other cell types.

The plaque-associated gene signature overlaps with human Alzheimer's disease gene networks.

“Our plaque-associated gene signature overlaps with human Alzheimer’s disease gene networks.”

Cluster 26 showed a highly cluster-specific gene expression profile.

“Cluster 26 showed a highly cluster-specific gene expression profile as calculated using a Wilcoxon rank sum test comparing the gene expression in Cluster 26 to all other clusters (Figure 2E) and showed that it co-localized with 6E10-positive plaques of adjacent sections (Figure 2F).”

GO term analysis of the genes induced in Cluster 26 upon Inpp5d knockdown showed enrichment for 'Defense response to virus'.

“GO term analysis of these genes included an enrichment for “Defense response to virus” (Cxcl10, Ifit3, Oas1a, Ccl5, Oasl1), “Type I and II Interferon signaling” (Stat1, Ifit2, Irf5/6, Isg15), and “Chemokine signaling pathway” (Plcb2, Rac2, Ccl9/12,Txnip, H2-T23; Tables S6, S7 in supporting information).”

Of the 231 genes induced in Cluster 26 upon Inpp5d knockdown, 126 were specific to PSAPP-TAM plaques.

“Of the 231 genes induced in Cluster 26 upon knockdown of Inpp5d (padj < 0.05, l2f > 1), 126 were specific to PSAPP-TAM plaques.”

DAMs and IRRAs co-localized with Cluster 26.

“While DAMs and IRRAs co-localized with Cluster 26, DAAs had a broad expression throughout the brain, which was not surprising given the many non-specific homeostatic astrocytic genes present within the module.”

The paper recovered 50 of the 57 previously identified PIGs.

“We also recovered 50 of the 57 previously identified PIGs and found that the PIG module co-localized with plaques and Cluster 26 (Figure 2H).”

Tamoxifen injection alone drove a modest number of DEGs in WT-CO versus WT-TAM mice.

“Given the recent evidence for Cre-mediated activation of microglia,30 we calculated genes that were changed in WT-CO versus WT-TAM mice and found that while tamoxifen injection alone drove a modest numbers of DEGs, they did not fall into categories associated with interferon signaling (Figure S10, Table S2).”

Cluster 26 was exclusively expressed in PSAPP mice.

“We found that one cluster, Cluster 26, was exclusively expressed in PSAPP mice (Figure 2D).”

The A11/6E10 ratio was increased by approximately 50% in the brains of the various groups.

“However, epitomic characterization23 of the Aβ conformers (i.e., A11, OC, NU4) present in the brains of the various groups (Figure S5B) revealed a ≈50% increase in A11/6E10 ratio (i.e., ratio of prefibrillar oligomer content to total Aβ content).”

The Cluster 26 expression signature and cluster-specific DEG list were significantly enriched in the subnetwork within a path length of 6 of INPP5D in the parahippocampal gyrus region Bayesian network (up to 16-fold enrichment, adjusted P = 3.1E-30).

“When projecting the Cluster 26 expression signature and cluster-specific DEG list onto the network neighborhood of INPP5D in the parahippocampal gyrus region BN, we found that both were significantly enriched in the subnetwork within a path length of 6 of INPP5D (up to 16-fold enrichment, adjusted P-value = 3.1E-30; Table S9 in supporting information; Figure 3A).”

There was no difference in the size distribution of individual amyloid plaques between PSAPP-TAM and PSAPP-CO mice.

“There was no difference in the size distribution of individual amyloid plaques in PSAPP-TAM compared to PSAPP-CO mice (Figure 1G).”

Cluster 26 co-localized with 6E10-positive plaques of adjacent sections.

“Cluster 26 showed a highly cluster-specific gene expression profile as calculated using a Wilcoxon rank sum test comparing the gene expression in Cluster 26 to all other clusters (Figure 2E) and showed that it co-localized with 6E10-positive plaques of adjacent sections (Figure 2F).”

Most DEGs were discovered in Cluster 26 due to its plaque association.

“While we discovered most DEGs in Cluster 26 due to its plaque association (Figure 2F, Figures S7–S9, Tables S1–S5), muscat also uncovered DEGs outside Cluster 26 when comparing WT-TAM to PSAPP-TAM as well as PSAPP-CO to PSAPP-TAM.”

There was a spatially constrained increase in microglia associated with Aβ plaques in PSAPP-TAM mice.

“Most striking was a spatially constrained increase in microglia associated with Aβ plaques (Figure 1C,J–L).”

The DEGs driven by tamoxifen injection alone in WT-CO versus WT-TAM mice did not fall into categories associated with interferon signaling.

“Given the recent evidence for Cre-mediated activation of microglia,30 we calculated genes that were changed in WT-CO versus WT-TAM mice and found that while tamoxifen injection alone drove a modest numbers of DEGs, they did not fall into categories associated with interferon signaling (Figure S10, Table S2).”

The plaque-specific expression profile was extensively altered by Inpp5d knockdown.

“Spatial transcriptomics identified a plaque-specific expression profile that was extensively altered by Inpp5d knockdown.”

CST7 (cystatin F) is a novel marker of plaques.

“Spatial transcriptomics highlighted an extended gene expression signature associated with plaques and identified CST7 (cystatin F) as a novel marker of plaques.”

There were more IBA1+ cells and IBA1 immunoreactive areas in PSAPP-TAM mice compared with WT-CO mice.

“There were more IBA1+ cells and IBA1 immunoreactive areas in PSAPP-TAM mice compared to WT-CO mice (Figure 1H–I), whereas the difference between PSAPP-CO and WT-CO was not significant.”

DAM genes such as Gpnmb, Trem2, and Tyrobp were among the DEGs present in the network neighborhood of INPP5D.

“DAM genes like Gpnmb, Trem2, and Tyrobp were among the DEGs present in the network neighborhood of INPP5D.”

Inpp5d downregulation increases the number of microglia associated with Aβ plaques in PSAPP mice.

“Herein, we demonstrate that Inpp5d downregulation increases Aβ fibrillization, plaque burden, and number of microglia associated with Aβ plaques in the PSAPP amyloid-depositing mouse model of AD.”

• Also stated in: § Inpp5d knockdown worsened amyloid plaque deposition while increasing recruitment of microglia to the plaque · plain

  “Most striking was a spatially constrained increase in microglia associated with Aβ plaques (Figure 1C,J–L).”

• Also stated in: § Discussion: · boosted

  “These results demonstrate that conditional Inpp5d downregulation in the PSAPP mouse increases plaque burden and recruitment of microglia to plaques.”

• AssumptionRests on an assumption: The PSAPP mouse model at 6 months recapitulates a preclinical stage of Alzheimer's disease relevant to human disease.
• AssumptionRests on an assumption: Conditional Inpp5d knockdown using Cx3cr1CreER/+ and tamoxifen is specific to microglia and does not confound results through effects on other cell types.

Inpp5d knockdown did not significantly increase GFAP+ cell numbers or immunoreactive areas in the hippocampus.

“We did not observe a significant increase of glial fibrillary acidic protein (GFAP)+ cell numbers or immunoreactive areas in the hippocampus in association with Inpp5d knockdown (Figure S3A, C–D).”

231 genes were induced in Cluster 26 upon knockdown of Inpp5d (padj < 0.05, l2f > 1).

“Of the 231 genes induced in Cluster 26 upon knockdown of Inpp5d (padj < 0.05, l2f > 1), 126 were specific to PSAPP-TAM plaques.”

Western blotting showed no obvious effect of any manipulation or parameter on brain levels of PSD95 or synaptophysin in female WT-CO, WT-TAM, PSAPP-CO, and PSAPP-TAM mice at age 6 months.

“Western blotting of protein extracts from the hippocampi of female WT-CO, WT-TAM, PSAPP-CO, and PSAPP-TAM mice at age 6 months using anti-PSD95 and anti-synaptophysin antibodies showed no obvious effect of any manipulation or parameter on brain levels of either synaptic marker (Figure S5 in supporting information).”

At 6 months of age, the number of 6E10 immunoreactive deposits in the hippocampi was increased by 73% in PSAPP-TAM mice compared with PSAPP-CO littermates.

“At 6 months of age, the percent area and number of 6E10 immunoreactive deposits were increased by >66% and 73%, respectively (Figure 1D–F; Figure S2) in the hippocampi of PSAPP-TAM compared to PSAPP-CO littermates.”

GO term analysis of the genes induced in Cluster 26 upon Inpp5d knockdown showed enrichment for 'Type I and II Interferon signaling'.

“GO term analysis of these genes included an enrichment for “Defense response to virus” (Cxcl10, Ifit3, Oas1a, Ccl5, Oasl1), “Type I and II Interferon signaling” (Stat1, Ifit2, Irf5/6, Isg15), and “Chemokine signaling pathway” (Plcb2, Rac2, Ccl9/12,Txnip, H2-T23; Tables S6, S7 in supporting information).”

Inpp5d knockdown extensively altered the gene expression profile in Cluster 26.

“We found that the gene expression profile in Cluster 26 was extensively altered by the knockdown of Inpp5d (Figure 2I).”

At 6 months of age, the percent area of 6E10 immunoreactive deposits in the hippocampi was increased by more than 66% in PSAPP-TAM mice compared with PSAPP-CO littermates.

“At 6 months of age, the percent area and number of 6E10 immunoreactive deposits were increased by >66% and 73%, respectively (Figure 1D–F; Figure S2) in the hippocampi of PSAPP-TAM compared to PSAPP-CO littermates.”

In the current PSAPP mouse cohorts, Cst7 remained upregulated in both CO- and TAM-treated mice.

“In our current PSAPP mice cohorts, Cst7 remained upregulated in both CO- and TAM-treated mice, suggesting that CST7 might be an excellent marker of plaques.”

Cst7 is specific to Cluster 26.

“We next probed for the gene Cst7 (cystatin F), which is specific to Cluster 26 and which has previously been identified in DAMs and PIGs (Figures 2E,H, 3C–G).[25,27]”

Conditional Inpp5d downregulation in microglia of the adult PSAPP mouse increases recruitment of microglia to plaques.

“Here we report that conditional Inpp5d downregulation in microglia of the adult PSAPP mouse increases plaque burden and recruitment of microglia to plaques.”

We observed increased plaque compaction.

“However, we observed increased plaque deposition and compaction.”

All the same factors altered by Trem2 manipulation are impacted by Inpp5d knockdown.

“All the same factors altered by Trem2 manipulation are impacted by Inpp5d knockdown.”

CST7 protein was exclusively present around plaques in PSAPP brains.

“We confirmed the specificity of Cst7 using immunohistochemistry and show that the CST7 protein was exclusively present around plaques in PSAPP brains (Figure 3G), leading us to propose that it is a novel marker for Aβ plaques.”

Conditional Inpp5d downregulation in microglia of the adult PSAPP mouse increases plaque burden.

“Here we report that conditional Inpp5d downregulation in microglia of the adult PSAPP mouse increases plaque burden and recruitment of microglia to plaques.”

• Also stated in: § DISCUSSION · plain

  “However, we observed increased plaque deposition and compaction.”

Epitomic characterization of hippocampal extracts revealed a ≈50% increase in the A11/6E10 ratio (ratio of prefibrillar oligomer content to total Aβ content) after Inpp5d knockout.

“However, epitomic characterization of these same extracts revealed a ≈50% increase in A11/6E10 ratio (i.e., ratio of prefibrillar oligomer content to total Aβ content).”

The study was unable to associate the altered pathology with any detectable changes in brain function.

“a caveat is our inability to associate this altered pathology with any detectable changes in brain function”

Cross-species gene regulatory network analysis highlighted crossover of the INPP5D/SHIP1 pathway in human AD.

“We also took advantage of cross-species gene regulatory network analysis to highlight crossover of the INPP5D/SHIP1 pathway in human AD, as some aspects of plaque pathology can be species-specific.”

Manipulating microglial SHIP1 has a profound impact on the amyloidosis and neuroinflammation of AD.

“Nonetheless, we show here that manipulating microglial SHIP1 has a profound impact on the amyloidosis and neuroinflammation of AD.”

• AssumptionRests on an assumption: The PSAPP mouse model is a valid model of Alzheimer's disease pathology.

Plaque burden increased after inhibition of SHIP1/Inpp5d knockdown.

“but in fact, plaque burden increased.”

The functional phagocytosis assay showed increased bead phagocytosis by constitutive Inpp5d KO primary microglia.

“our functional phagocytosis assay showing increased bead phagocytosis by constitutive Inpp5d KO primary microglia”

Inpp5d downregulation increases Aβ fibrillization in PSAPP mice.

“Herein, we demonstrate that Inpp5d downregulation increases Aβ fibrillization, plaque burden, and number of microglia associated with Aβ plaques in the PSAPP amyloid-depositing mouse model of AD.”

• Also stated in: § Inpp5d knockdown worsened amyloid plaque deposition while increasing recruitment of microglia to the plaque · plain

  “However, epitomic characterization23 of the Aβ conformers (i.e., A11, OC, NU4) present in the brains of the various groups (Figure S5B) revealed a ≈50% increase in A11/6E10 ratio (i.e., ratio of prefibrillar oligomer content to total Aβ content).”

• AssumptionRests on an assumption: The PSAPP mouse model at 6 months recapitulates a preclinical stage of Alzheimer's disease relevant to human disease.
• AssumptionRests on an assumption: Conditional Inpp5d knockdown using Cx3cr1CreER/+ and tamoxifen is specific to microglia and does not confound results through effects on other cell types.

INPP5D encodes a dual-specificity phosphatase that can dephosphorylate both phospholipids and phosphoproteins.

“The inositol polyphosphate-5-phosphatase D (INPP5D) gene encodes a dual-specificity phosphatase that can dephosphorylate both phospholipids and phosphoproteins.”

• Also stated in: § BACKGROUND · plain

  “INPP5D is a dual-specificity phosphatase that can dephosphorylate both phospholipids and phosphoproteins.9,10”

• cites [9] Stenmark H, Aasland R, Driscoll PC. The phosphatidylinositol 3-phosphate-binding FYVE finger. FEBS Lett. 2002; 513: 77– 84. 11911884 sourceNot checked — no full text of the source
• cites [10] Rohrschneider LR, Fuller JF, Wolf I, Liu Y, Lucas DM. Structure, function, and biology of SHIP proteins. Genes Dev. 2000; 14: 505– 520. 10716940 sourceNot checked — no full text of the source

SHIP1 negatively regulates targets such as immune cell surface receptors by catalyzing the conversion of PI(3,4,5)P3 to PI(3,4)P2.

“SHIP1 negatively regulates targets such as immune cell surface receptors by catalyzing the conversion of phosphatidylinositol (3,4,5)-trisphosphate (PI[3,4,5]P3) to phosphatidylinositol (3,4)-bisphosphate (PI[3,4]P2).”

• cites [10] Rohrschneider LR, Fuller JF, Wolf I, Liu Y, Lucas DM. Structure, function, and biology of SHIP proteins. Genes Dev. 2000; 14: 505– 520. 10716940 sourceNot checked — no full text of the source

Increased INPP5D levels associated with inheritance of the common variant rs35349669 confer increased risk for late-onset sporadic Alzheimer's disease.

“Current evidence suggests that increased INPP5D levels associated with inheritance of the common variant rs35349669 confer increased risk for LOAD.7”

• cites [7] Yoshino Y, Yamazaki K, Ozaki Y, INPP5D mRNA expression and cognitive decline in Japanese Alzheimer’s disease subjects. J Alzheimers Dis. 2017; 58: 687– 694. 28482637 sourceNot checked — no full text of the source

Cst7 is a marker for Aβ plaques.

“In our current PSAPP mice cohorts, Cst7 remained upregulated in both CO- and TAM-treated mice, suggesting that CST7 might be an excellent marker of plaques.”

• Also stated in: § Novel plaque marker Cst7 was limited to plaque-associated microglia · boosted

  “We confirmed the specificity of Cst7 using immunohistochemistry and show that the CST7 protein was exclusively present around plaques in PSAPP brains (Figure 3G), leading us to propose that it is a novel marker for Aβ plaques.”

The observed increase in plaque burden was not predicted by the apparent effect of common INPP5D polymorphisms.

“This result was not predicted by the apparent effect of the common INPP5D polymorphisms, given that the rs35349669 single nucleotide polymorphism (SNP) confers AD risk,7 while rs61068452 is apparently protective against AD.8”

Decrease of SHIP1 function after initiation of AD amyloid pathology leads to an increase in plaque-associated microglial gene expression.

“These data imply that decrease of SHIP1 function after initiation of AD amyloid pathology leads to an increase in plaque burden and plaque-associated microglial gene expression.”

Cluster 26 genes are relatively specific as putative plaque markers.

“Further analysis of the spatial transcriptomics data also confirmed the relative specificity of Cluster 26 genes as putative plaque markers, showing that Cst7 was expressed in 100% of plaque spots and only 8% of non-plaque spots (Figure 3F).”

Decrease of SHIP1 function after initiation of AD amyloid pathology leads to an increase in plaque burden.

“These data imply that decrease of SHIP1 function after initiation of AD amyloid pathology leads to an increase in plaque burden and plaque-associated microglial gene expression.”

Inhibition of SHIP1 once disease is initiated might not lead to a clinical benefit.

“This may have therapeutic implications, in that inhibition of SHIP1 once disease is initiated might not lead to a clinical benefit.”

• AssumptionRests on an assumption: Increased plaque burden is detrimental/pathological.

The existence of DEGs in regions without plaques after Inpp5d knockdown indicates that, at least within the time period of this study, these transcriptomic changes do not always result in plaque deposition.

“The existence of DEGs in regions without plaques after the knockdown of Inpp5d indicates that (at least within the time period of this study) these transcriptomic changes do not always result in plaque deposition.”

Microglial function and/or dysfunction is a central contributor to early AD pathology.

“This, combined with the GWAS-associated SNPs in INPP5D, highlights microglial function and/or dysfunction as a central contributor to early AD pathology.”

• AssumptionRests on an assumption: The PSAPP mouse model is a valid model of Alzheimer's disease pathology.