Alzheimer’s genetic risk factor CASS4 controls microglial focal adhesion biology and amyloidosis

5xFAD Cass4-/- inhibitory neurons exhibited downregulation of genes and pathways involved in neuronal circuitry.

“Moreover, we observed a downregulation of genes and pathways involved in neuronal circuitry such as Sv2b , Vav3 , Arhgap24 , Asap2 , Elmo1 , and Nrn1 in 5xFAD Cass4 -/-mice relative to 5xFAD littermate controls (Agrawal et al., 2026; Chan et al., 2020; Cowan et al., 2005; Falace et al., 2024; Hale et al., 2011; Kim et al., 2011; Li et al., 2018; Miyamoto et al., 2020; Pugh et al., 2026; Sauzeau et”

• cites Agrawal et al., 2026 sourceSupported
• cites Chan et al., 2020 sourceNot checked — no full text of the source
• cites Cowan et al., 2005 sourceNot checked — no full text of the source

Cass4 is predominantly expressed by microglia in the brain.

“As previously reported, we found that Cass4 is predominantly expressed by microglia in the brain (Fig. S1 G; Lee et al., 2023; Podlesny-Drabiniok et al., 2020).”

• cites Lee et al., 2023 sourceCould not verify

  The Results passages describe APOE expression and microglial gene-expression patterns, but do not address Cass4. A search of our copy for “Cass4” and for predominant expression in microglia found no passage establishing which brain cell type predominantly expresses Cass4. (LLM, conf=0.74)

  Experimental — accuracy not yet measured
• cites Podlesny-Drabiniok et al., 2020 sourceCould not verify

  The Cytoskeleton section identifies CASS4 as an Alzheimer’s-associated gene involved in macrophage migration, but does not say where it is predominantly expressed in the brain. Searches for CASS4 expression and microglial enrichment found no passage making that comparison; the section explicitly discusses microglial expression for ABI3 instead. No page number was available in our copy. (LLM, conf=0.72)

  Experimental — accuracy not yet measured

17% of the 93 differentially phosphorylated proteins are known to be central molecular players in focal adhesion signaling.

“17% are known to be central molecular players in focal adhesion sig­ naling (Fig. 6 D and Fig. S5, A and B; Farhadi and Park, 2025; Fujita et al., 2016; Geiger, 2006; Henderson et al., 2019; Huveneers and Danen, 2009; Islam et al., 2025; Ma”

• cites Farhadi and Park, 2025 sourceSupported
• cites Fujita et al., 2016 sourceCould not verify

  The Abstract reports 17 proteins with abnormal phosphorylation in Alzheimer’s disease models, but does not identify a set of 93 proteins or classify any percentage as central to focal adhesion signaling. The Results section on MARCKS phosphorylation and the targeted searches for focal adhesion, 93 proteins, and 17% likewise provide no passage addressing that classification. Page numbers are unavailable in our copy. (LLM, conf=0.74)

  Experimental — accuracy not yet measured
• cites Geiger, 2006 sourceSupported

Cass4 appears to be expressed only by microglia among brain-resident cell types.

“Of the brain-resident cell types, Cass4 appears to be ex­ pressed only by microglia (Lee et al., 2023; Podlesny-Drabiniok et al., 2020; Zhang et al., 2014).”

• cites Lee et al., 2023 sourceCould not verify

  The Methods section lists cell-type markers, and the Results discuss microglial gene-expression patterns, but neither identifies Cass4 as microglia-specific. Searches for “Cass4” and for its expression across brain-resident cell types found no passage addressing the claim in our copy. (LLM, conf=0.72)

  Experimental — accuracy not yet measured
• cites Podlesny-Drabiniok et al., 2020 sourceCould not verify

  The Cytoskeleton section (page unavailable) discusses CASS4 as an Alzheimer’s-associated locus and its role in macrophage migration, but does not state which brain-resident cells express it. Searches for CASS4 expression and comparisons among brain cell types found no passage establishing that it is expressed only by microglia. (LLM, conf=0.72)

  Experimental — accuracy not yet measured
• cites Zhang et al., 2014 sourceCould not verify

  The Purity of neuronal, glial, and vascular samples and qRT-PCR validation sections discuss cell-type-specific genes, including microglial markers, but do not identify Cass4. Searches for Cass4 and its full gene name found no passage establishing that its expression is restricted to microglia among brain-resident cell types. (LLM, conf=0.72)

  Experimental — accuracy not yet measured

The CASS4 gene locus has consistently been identified in GWAS studies to be associated with an increased risk of developing AD.

“The CASS4 gene locus has consistently been identified in GWAS studies to be associated with an increased risk of developing AD (Lambert et al., 2013; Lin et al., 2017; Ramos de Matos et al., 2018; Schwartzentruber et al., 2021; Wang et al., 2015b).”

• cites Lambert et al., 2013 sourceSupported
• cites Lin et al., 2017 sourceSupported
• cites Ramos de Matos et al., 2018 sourcePartly supported

  The Discussion (page unavailable) says a CASS4 variant had previously been associated with AD in a large meta-analysis, supporting the locus’s connection to AD. But it describes that variant as protective, and this study links it to lower tau biomarker levels; it does not establish that the locus was consistently associated with increased AD risk across GWAS studies. (LLM, conf=0.87)

  Experimental — accuracy not yet measured

Microglia can use their highly dynamic and mobile processes to compact Aβ fibrils into tightly packed aggregates, reducing Aβ aggregate surface area and limiting their potential neurotoxic effects on nearby neurons.

“They can also use their highly dynamic and mobile processes to compact Aβ fibrils into tightly packed aggregates, reducing Aβ aggregate surface area and thereby limiting their potential neurotoxic effects on nearby neurons (Casali et al., 2020; Condello et al., 2015; Zhao et al., 2017).”

• cites Casali et al., 2020 sourcePartly supported

  The Discussion (page unavailable) says microglia envelop plaques with their processes, trim amyloid fibrils to promote compaction, and help protect nearby neurites. It does not describe the processes as highly dynamic and mobile or say that compaction reduces aggregate surface area; it describes a physical barrier and reduced plaque expansion instead. (LLM, conf=0.86)

  Experimental — accuracy not yet measured
• cites Condello et al., 2015 sourcePartly supported

  The Results section (‘Aβ42 hotspots are anti-colocalized with microglia processes’; page unavailable) links microglial coverage to denser amyloid and less soluble Aβ42 binding, while the Discussion proposes that processes may promote fibril compaction and limit nearby neuritic damage. But the paper does not establish reduced aggregate surface area as the mechanism, and its Results say plaque-contacting processes were highly stable, unlike the motile processes away from plaques. The claim therefore overstates both the proposed mechanism and the role of process mobility. (LLM, conf=0.88)

  Experimental — accuracy not yet measured
• cites Zhao et al., 2017 sourcePartly supported

  The Background (page unavailable) describes microglial processes enveloping plaques as a possible barrier to expansion, and the Conclusion (page unavailable) says microglia limit plaque growth and associated neuronal damage. But the paper does not establish the claimed mechanism of dynamic processes compacting Aβ fibrils into tightly packed aggregates, reducing their surface area, and thereby reducing toxicity. (LLM, conf=0.87)

  Experimental — accuracy not yet measured

Numerous SNPs in the CASS4 locus have been linked to AD.

“Although numerous singlenucleotide polymorphisms (SNPs) in the CASS4 locus have been linked to AD (Beck et al., 2014; Bellenguez et al., 2022; EADB et al., 2026; Lambert et al., 2013; Lin et al., 2017; Schwartzentruber et al., 2021)”

• cites Beck et al., 2014 sourcePartly supported

  The Introduction’s genetic-evidence section reports that one CASS4 SNP, rs7274581, was associated with late-onset AD at genome-wide significance. Table 1 likewise lists only that CASS4 SNP. The paper therefore supports a CASS4 SNP association, but not the claim that numerous SNPs in the locus have been linked to AD. (LLM, conf=0.88)

  Experimental — accuracy not yet measured
• cites Bellenguez et al., 2022 sourcePartly supported

  The GWAS meta-analysis (Table 1; page unavailable) identifies CASS4 as a known Alzheimer’s disease-associated locus and reports a significant association for SNP rs6014724. It does not establish the claim’s stronger point that numerous SNPs in that locus have been linked to AD. (LLM, conf=0.85)

  Experimental — accuracy not yet measured
• cites EADB et al., 2026 sourceCould not verify

  The Main section reports 91 loci associated with Alzheimer’s disease and related dementias, but the passages retrieved for “CASS4” and “CASS4 SNPs” do not identify that locus or describe SNP associations within it. Our copy therefore does not establish the CASS4-specific claim. (LLM, conf=0.70)

  Experimental — accuracy not yet measured

• AssumptionRests on an assumption: Little is currently known about how individual disease-associated mutations impact CASS4 function.

Amyloid pathology is more accelerated and consistent in female 5xFAD mice compared with male 5xFAD mice.

“amyloid pathology is more accelerated and consistent in female 5xFAD mice compared with male 5xFAD mice (Forner et al., 2021; Sasmita et al., 2025).”

• cites Forner et al., 2021 sourcePartly supported

  The Usage Notes state that female 5xFAD mice develop pathology before males, and Technical Validation reports higher cortical plaque numbers in females at several ages. The Usage Notes call plaque pathology reproducible overall, but do not say it is more consistent in females than in males. No page number is available in our copy. (LLM, conf=0.88)

  Experimental — accuracy not yet measured
• cites Sasmita et al., 2025 sourcePartly supported

  The LSM results (p. 3) report approximately twice the plaque burden in female 5xFAD mice compared with age-matched males at six months. That supports greater pathology at that age, but the passage does not establish faster progression or greater consistency in females; the temporal analysis on the same page concerns males. (LLM, conf=0.86)

  Experimental — accuracy not yet measured

Loss of CASS4 impairs microglial mobilization to Aβ plaques.

“Loss of CASS4 impaired microglial mobilization to Aβ plaques and reduced plaque compaction.”

snRNA-seq identified 10 transcriptionally distinct populations from 33,985 nuclei in hippocampi of 5-mo-old 5xFAD and 5xFAD Cass4-/- mice.

“unsupervised clustering of 33,985 nuclei identified 10 transcriptionally distinct populations (Fig. S1 D).”

Full-body Cass4 deletion in 5xFAD mice increases amyloid-β (Aβ) deposition.

“Here, we show that full-body Cass4 deletion in 5xFAD mice increases amyloid-β (Aβ) deposition, neuronal dystrophy, and cognitive deficits.”

5xFAD Cass4-/- mice had increased numbers of ThioS+ Aβ plaques in the hippocampus relative to 5xFAD littermate controls.

“we found that 5xFAD Cass4 -/mice exhibited a greater total volume of ThioS + staining and increased numbers of ThioS + Aβ plaques in the hippocampus relative to 5xFAD littermate controls (Fig. 1, A-C).”

5xFAD Cass4-/- mice had elevated ATG9 staining in the hippocampus and cortex relative to 5xFAD littermate controls.

“In line with our APP puncta data, we found that there was elevated ATG9 staining in the hippocampus and cortex of 5xFAD Cass4 -/mice relative to 5xFAD littermate controls (Fig. 2, C-E).”

Full-body Cass4 deletion in 5xFAD mice increases neuronal dystrophy.

“Here, we show that full-body Cass4 deletion in 5xFAD mice increases amyloid-β (Aβ) deposition, neuronal dystrophy, and cognitive deficits.”

CASS4 deficiency abrogates in vivo microglial recruitment to injury.

“CASS4 deficiency abrogated in vivo microglial recruitment to injury and in vitro wound healing responses, two processes that are centrally orchestrated by focal adhesion biology.”

In inhibitory neurons, there were 213 DEGs between 5xFAD Cass4-/- and 5xFAD mice.

“Here, we identified 213 DEGs between inhibitory neurons isolated from 5xFAD Cass4 -/- and 5xFAD mice (Fig. 2, I and J).”

CASS4 deficiency abrogates in vitro wound healing responses.

“CASS4 deficiency abrogated in vivo microglial recruitment to injury and in vitro wound healing responses, two processes that are centrally orchestrated by focal adhesion biology.”

Phosphoproteomic analysis of purified microglia revealed marked disruption of focal adhesion signaling following Cass4 deletion.

“Phosphoproteomic analysis of purified microglia revealed marked disruption of focal adhesion signaling following Cass4 deletion.”

In the excitatory neuron cluster, there were 2,982 differentially expressed genes between 5xFAD and 5xFAD Cass4-/- excitatory neurons.

“In the excitatory neuron cluster (Fig. 2 F), we identified 2,982 differentially expressed genes (DEGs) between 5xFAD and 5xFAD Cass4 -/- excitatory neurons (Fig. 2 G).”

Excitatory neuron DEGs were spread across pyramidal, granule, and subiculum subpopulations.

“Moreover, we found that the excitatory neuron DEGs were spread across each of the subpopulations (Fig. S1, L-N).”

Loss of CASS4 reduces plaque compaction.

“Loss of CASS4 impaired microglial mobilization to Aβ plaques and reduced plaque compaction.”

Full-body Cass4 deletion in 5xFAD mice increases cognitive deficits.

“Here, we show that full-body Cass4 deletion in 5xFAD mice increases amyloid-β (Aβ) deposition, neuronal dystrophy, and cognitive deficits.”

5xFAD Cass4-/- mice exhibited greater total volume of ThioS+ staining in the hippocampus relative to 5xFAD littermate controls.

“we found that 5xFAD Cass4 -/mice exhibited a greater total volume of ThioS + staining and increased numbers of ThioS + Aβ plaques in the hippocampus relative to 5xFAD littermate controls (Fig. 1, A-C).”

5xFAD Cass4-/- mice had an increase in APP+ dystrophic neurites per plaque in the hippocampus compared with 5xFAD littermate controls.

“We observed an increase in APP + dystrophic neurites per plaque in 5xFAD Cass4 -/mice when compared with 5xFAD littermate controls in the hippocampus (Fig. 2, A and B).”

Cortactin had decreased phosphorylation in 5xFAD Cass4-/- microglia relative to 5xFAD littermate controls.

“This included de­ creased phosphorylation of CrkII, cortactin, and Abl1 in 5xFAD Cass4 -/microglia relative to microglia isolated from 5xFAD littermate controls (Fig. 6 D).”

CASS4-deficient 5xFAD mice show decreased recruitment of IBA1+ processes to Aβ pathology.

“This was most pronounced in our studies, showing poor plaque compaction as well as decreased recruitment of IBA1 + processes to Aβ pathology in CASS4-deficient 5xFAD mice.”

5xFAD Cass4-/- mice had an increased ratio of 6E10:ThioS in the hippocampus and cortex relative to 5xFAD littermate controls.

“we found that 5xFAD Cass4 -/mice had an increased ratio of 6E10:ThioS in the hippocampus and cortex relative to 5xFAD littermate controls (Fig. 4, J and K; and Fig. S3 C).”

5xFAD Cass4-/- mice had lower intensity and sphericity of Aβ plaques in the hippocampus compared with 5xFAD littermate controls.

“we also observed lower intensity and sphericity of Aβ plaques in the hippocampus of 5xFAD Cass4 -/mice when compared with 5xFAD littermate controls (Fig. 4, L-N).”

There were no significant differences in swim speed or total distance traveled between 5xFAD Cass4-/- and 5xFAD mice over the MWM testing paradigm.

“Importantly, we did not detect any significant differences in swim speed or total distance traveled between 5xFAD Cass4 -/and 5xFAD mice over the course of the MWM testing paradigm (Fig. S2, A and B)”

Microglia lacking CASS4 displayed blunted mobilization to the laser injury.

“In stark contrast, microglia lacking CASS4 displayed blunted mobilization to the laser injury (Fig. 6, L and M; and Video 2).”

5-mo-old 5xFAD Cass4 ΔMG mice had a greater total volume of ThioS+ staining in the hippocampus than 5xFAD Cass4 WT littermate controls.

“we likewise found that 5-mo-old 5xFAD Cass4 ΔMG mice had a greater total volume of ThioS + staining and increased numbers of ThioS + Aβ plaques in the hippocampus relative to 5xFAD Cass4 WT littermate controls (Fig. 5, A-C).”

snRNA-seq analysis uncovered that neuropathology and cognitive dysfunction in these mice were associated with a downregulation of Sv2b, Vav3, Arhgap24, Asap2, Elmo1, and Nrn1.

“and downregulation of Sv2b , Vav3 , Arhgap24 , Asap2 , Elmo1 , and Nrn1”

Abl1 had decreased phosphorylation in 5xFAD Cass4-/- microglia relative to 5xFAD littermate controls.

“This included de­ creased phosphorylation of CrkII, cortactin, and Abl1 in 5xFAD Cass4 -/microglia relative to microglia isolated from 5xFAD littermate controls (Fig. 6 D).”

5-mo-old 5xFAD Cass4 ΔMG mice had increased numbers of ThioS+ Aβ plaques in the hippocampus relative to 5xFAD Cass4 WT littermate controls.

“we likewise found that 5-mo-old 5xFAD Cass4 ΔMG mice had a greater total volume of ThioS + staining and increased numbers of ThioS + Aβ plaques in the hippocampus relative to 5xFAD Cass4 WT littermate controls (Fig. 5, A-C).”

Cass4-deficient BMDMs exhibited reduced migration in response to M-CSF.

“In line with our previous ob­ servations, Cass4deficient BMDMs exhibited reduced migration in response to M-CSF (Fig. 6, I and J).”

5xFAD Cass4 ΔMG mice had elevated numbers of large plaques (>1,000 μm3) in the hippocampus.

“5xFAD Cass4 ΔMG mice also had elevated numbers of large plaques in the hippocampus, which were defined as plaques >1,000 μm 3 in volume (Fig. 5 D).”

snRNA-seq analysis uncovered that neuropathology and cognitive dysfunction in these mice were associated with an upregulation of numerous genes linked to worsened neuronal health and neurodegenerative disease, such as Mid1, Gpr88, Crhr1, Sema5b.

“snRNA-seq analysis in these mice further uncovered that this neuropathology and cognitive dys­ function were associated with an upregulation of numerous genes that have recently been linked to worsened neuronal health and neurodegenerative disease, such as Mid1 , Gpr88 , Crhr1 , Sema5b”

In the full microglia cluster, 98 DEGs were identified between 5xFAD and 5xFAD Cass4-/- microglia.

“For the full microglia cluster, we identified 98 DEGs between 5xFAD and 5xFAD Cass4 -/-microglia (Fig. 6 B).”

Cass4-deficient BMDMs displayed diminished migration in scratch assays when treated with 400 ng/ml osteopontin.

“Additionally, Cass4 -deficient BMDMs also displayed diminished migration in scratch assays when treated with 400 ng/ml osteopontin (Fig. S5, C and D)”

Cass4 deletion in 5-mo-old 5xFAD mice did not appreciably impact the proportion of proliferative Ki67+ CD45int CD11b+ microglia in flow cytometry studies.

“we also found that Cass4 deletion in 5-mo-old 5xFAD mice did not appreciably impact the proportion of proliferative Ki67 + CD45 int CD11b + microglia in flow cytometry studies (Fig. S3, D and E).”

93 proteins were identified to exhibit differential phosphorylation status between 5xFAD and 5xFAD Cass4-/- microglia.

“we observed that of the 93 proteins identified to exhibit differential phosphorylation status between 5xFAD and 5xFAD Cass4 -/microglia, 17% are known to be central molecular players in focal adhesion sig­ naling (Fig. 6 D and Fig. S5, A and B; Farhadi and Park, 2025; Fujita et al., 2016; Geiger, 2006; Henderson et al., 2019; Huveneers and Danen, 2009; Islam et al., 2025; Ma”

• AssumptionRests on an assumption: Focal adhesion biology is regulated predominantly at the posttranslational level.

5xFAD Cass4-/- microglia displayed increased phosphorylation of multiple apical focal adhesion-related proteins such as integrin β3, Src, filamin A, and LYN.

“we also noted that 5xFAD Cass4 -/microglia displayed increased phosphoryl­ ation of multiple apical focal adhesion-related proteins such as integrin β3, Src, filamin A, and LYN (Fig. 6 D; Bellenguez et al., 2022; Feng and Walsh, 2004; Gao et al., 2021; Kim et al., 2010; Ortiz et al., 2021; Pfaff and Jurdic, 2001; Portugal et al., 2022)”

Microglial processes vigorously converged to the site of laser injury in Ai6+/Cass4 WT mice.

“We observed that microglial processes vigorously converged to the site of laser injury in Ai6 +/Cass4 WT mice (Fig. 6, L and M; and Video 1).”

Cass4-deficient primary microglia exhibited reduced surface area of the focal adhesion protein paxillin in response to laminin-coated coverslips.

“we observed that Cass4deficient primary microglia exhibited reduced surface area of the focal adhesion protein paxillin in response to laminin-coated coverslips (Fig. 6, E and F).”

5xFAD Cass4 ΔMG mice showed no differences in overall IBA1 volume compared with 5xFAD Cass4 WT controls.

“While 5xFAD Cass4 ΔMG mice also showed no differences in overall IBA1 volume (Fig. 5 E)”

Microglia are the sole brain-resident cell type that expresses Cass4.

“as well as the fact that microglia are the sole brain-resident cell type that expresses Cass4 (Fig. S1 G).”

Cass4-deficient primary microglia exhibited reduced paxillin surface area upon treatment with 50 ng/ml M-CSF.

“This reduced surface area was also detected upon treatment with 50 ng/ml M-CSF (Fig. 6, G and H)”

5xFAD Cass4 ΔMG mice showed reduced plaque intensity compared with 5xFAD Cass4 WT controls.

“we observed a similar reduction in plaque sphericity and intensity to that observed in germline Cass4 -deficient mice (Fig. 4, L-M) in 5xFAD Cass4 ΔMG mice when compared with 5xFAD Cass4 WT controls (Fig. 5, F-H).”

Proportions of homeostatic microglia and DAM were equivalent between 5xFAD Cass4-/- and 5xFAD mice in snRNA-seq analysis.

“the proportions of homeostatic microglia and DAM were also found to be equivalent between 5xFAD Cass4 -/- and 5xFAD mice in our snRNA-seq analysis (Fig. S3, I-L).”

CASS4-deficient 5xFAD mice show poor plaque compaction.

“This was most pronounced in our studies, showing poor plaque compaction as well as decreased recruitment of IBA1 + processes to Aβ pathology in CASS4-deficient 5xFAD mice.”

Loss of CASS4 in 5xFAD mice was associated with downregulation of genes linked to contractile actin filament assembly, stress fiber assembly, and regulation of actin filament-based processes.

“analysis in the microglia cluster revealed that loss of CASS4 in 5xFAD mice was associated with a downregulation of genes linked to contractile actin fila­ ment assembly, stress fiber assembly, and regulation of actin filament-based processes (Fig. 6 C; Ahn and Park, 2025; Biermeier et al., 2026; Deveci et al., 2026; Li et al., 2016; Malko et al., 2019; Nobes and Hall, 199”

The M-CSF receptor (P-Y561) had reduced phosphorylation in 5xFAD Cass4-/- microglia.

“Interestingly, one of the proteins that had reduced phosphoryl­ ation in 5xFAD Cass4 -/microglia was the M-CSF receptor (P-Y561) (Fig. 6 D).”

5xFAD Cass4-/- mice had markedly less IBA1 volume in close proximity to Aβ plaques relative to 5xFAD littermate controls.

“Here, we observed markedly less IBA1 volume in close proximity to Aβ plaques in 5xFAD Cass4 -/mice relative to 5xFAD littermate controls (Fig. 4, H and I).”

Multiple processes associated with focal adhesion biology were downregulated in 5xFAD Cass4-/- microglia relative to 5xFAD microglia.

“Pathway analysis of these DEGs identified that multiple pro­ cesses associated with focal adhesion biology were down­ regulated in 5xFAD Cass4 -/-microglia relative to 5xFAD microglia (Fig. 6 C).”

5xFAD Cass4 ΔMG mice showed reduced plaque sphericity compared with 5xFAD Cass4 WT controls.

“we observed a similar reduction in plaque sphericity and intensity to that observed in germline Cass4 -deficient mice (Fig. 4, L-M) in 5xFAD Cass4 ΔMG mice when compared with 5xFAD Cass4 WT controls (Fig. 5, F-H).”

There were no appreciable differences in uptake of methoxy-X04+ Aβ species by CD45int CD11b+ microglia from 5-mo-old 5xFAD and 5xFAD Cass4-/- mice.

“we did not observe appreciable differences in the uptake of methoxy-X04 + Aβ species by CD45 int CD11b + microglia from 5-mo-old 5xFAD and 5xFAD Cass4 -/mice (Fig. S3 H).”

CrkII had decreased phosphorylation in 5xFAD Cass4-/- microglia relative to 5xFAD littermate controls.

“This included de­ creased phosphorylation of CrkII, cortactin, and Abl1 in 5xFAD Cass4 -/microglia relative to microglia isolated from 5xFAD littermate controls (Fig. 6 D).”

Microglial-targeted deletion of Cass4 leads to increased levels of hippocampal ATG9 staining in 5xFAD Cass4 ΔMG mice.

“we also found that microglial-targeted deletion of Cass4 leads to increased levels of hippocampal ATG9 staining in 5xFAD Cass4 ΔMG mice (Fig. 5, I and J)”

Genetic ablation of Cass4 in 5xFAD mice leads to impaired macrophage recovery following scratch injury.

“Genetic ablation of Cass4 in 5xFAD mice leads to altered focal adhesion signaling in microglia and impaired macrophage recovery fol­ lowing scratch injury.”

The absence of CASS4 did not alter microglial ability to take on a DAM phenotype.

“While the absence of CASS4 did not alter microglial numbers or their ability to take on a DAM phenotype”

CASS4-deficient microglia display defective mobilization to Aβ plaques.

“we found that CASS4-deficient microglia display defective mobilization to Aβ plaques”

Genetic ablation of Cass4 in 5xFAD mice leads to altered focal adhesion signaling in microglia.

“Genetic ablation of Cass4 in 5xFAD mice leads to altered focal adhesion signaling in microglia and impaired macrophage recovery fol­ lowing scratch injury.”

• AssumptionRests on an assumption: Proteins marked with green asterisks are known to interact with the focal adhesion protein p130cas, and proteins marked with pink asterisks function in focal adhesion and actin pathways.

The absence of CASS4 did not alter microglial numbers.

“While the absence of CASS4 did not alter microglial numbers or their ability to take on a DAM phenotype”

Loss of CASS4 in 5xFAD mice leads to a greater level of neuronal dystrophy.

“These collective findings point toward a greater level of neuronal dystrophy upon loss of CASS4 in 5xFAD mice.”

Deletion of Cass4 leads to increased Aβ deposition in the brain.

“These data suggest that deletion of Cass4 leads to increased Aβ deposition in the brain.”

CASS4 may represent a therapeutic target for AD.

“Together, these findings identify CASS4 as a critical regulator of microglial function and focal adhesion biology and suggest that CASS4 may represent a therapeutic target for AD.”

• AssumptionRests on an assumption: CASS4's role in microglial function and focal adhesion biology is conserved between mice and humans.

Focal adhesion biology is an important player in AD.

“These col­ lective studies, coupled with the CASS4 findings presented in this manuscript, help to identify focal adhesion biology as an important player in AD.”

Microglia-targeted deletion of Cass4 recapitulates the amyloidosis and neuritic dystrophy phenotypes seen in full-body CASS4-deficient 5xFAD mice.

“These data suggest that microglia-targeted deletion of Cass4 recapitulates the amyloidosis and neuritic dystrophy phenotypes seen in full-body CASS4-deficient 5xFAD mice (Figs. 1, 2, and 4)”

Loss of CASS4 reduces microglial responses to M-CSF.

“suggesting that loss of CASS4 reduces microglial responses to M-CSF.”

The increased Aβ load and defective plaque compaction seen in 5xFAD Cass4-/- mice are more likely to be contributing to a brain environment that promotes neuronal pathology and dysfunction.

“Instead, the increased Aβ load and defective plaque com­ paction seen in 5xFAD Cass4 -/-mice are more likely to be contributing to a brain environment that promotes neuronal pathology and dysfunction.”

The absence of CASS4 in microglia alters focal adhesion signaling.

“Altogether, these results indicate that the absence of CASS4 in microglia alters focal adhesion signaling”

CASS4 is unlikely to be impacting neural health through cell-intrinsic roles in neurons.

“Therefore, CASS4 is unlikely to be impacting neural health through cell-intrinsic roles in neu­ rons.”

Cass4-/- microglia may have dampened migratory responses.

“and consequently have dampened migratory re­ sponses.”

CASS4 is centrally involved in coordinating in vivo microglial mobilization to laser injury.

“our findings suggest that CASS4 is centrally involved in coordinating in vivo microglial mobilization to laser injury”

CASS4 is involved in regulating focal adhesion signaling and related processes such as wound healing and microglial mobilization to injury.

“Our studies also suggest that CASS4 is involved in regulating focal adhesion signaling and related processes such as wound healing and microglial mobilization to injury.”

Cass4-/- microglia may have reduced signaling capacity through the M-CSF pathway.

“Our observation of reduced P-Y561 suggests that Cass4 -/- mi­ croglia may have reduced signaling capacity through the M-CSF pathway”

CASS4 functions in CNS-resident macrophages to impact amyloidosis.

“and further indicate that CASS4 functions in CNS-resident macrophages to impact amyloidosis.”

The deficits in cognitive flexibility observed in 5xFAD Cass4-/- mice are in response to Aβ pathology.

“which suggests that the deficits in cognitive flexibility observed in 5xFAD Cass4 -/-mice are in response to Aβ pathology.”

Loss of CASS4 in 5xFAD mice causes exacerbated neuritic dystrophy.

“Our studies also revealed that loss of CASS4 in 5xFAD mice causes defective cognitive flexibility and exacerbated neuritic dystrophy.”

Increased hippocampal ATG9 staining in 5xFAD Cass4 ΔMG mice is indicative of worsened neuronal health.

“which is indicative of worsened neuronal health.”

CASS4 has a novel role in regulating the ability of microglia to contain and encapsulate Aβ plaques.

“Our studies highlight a novel role for CASS4 in regulating the ability of microglia to contain and encapsulate Aβ plaques.”

The absence of CASS4 in microglia results in impairments to cellular processes known to be regulated by focal adhesions, such as microglia and macrophage mobilization to injury and wound healing.

“and results in impairments to cellular processes that are known to be regulated by focal adhesions, such as microglia and macro­ phage mobilization to injury and wound healing.”

Loss of CASS4 in 5xFAD mice causes defective cognitive flexibility.

“Our studies also revealed that loss of CASS4 in 5xFAD mice causes defective cognitive flexibility and exacerbated neuritic dystrophy.”

Loss of Cass4 in the 5xFAD mouse model of AD leads to exacerbated neuronal dystrophy.

“Here, we show that loss of Cass4 in the 5xFAD mouse model of AD leads to worsened Aβ pathology, exacerbated neuronal dystrophy, and defective cognitive flexibility.”

Loss of Cass4 in the 5xFAD mouse model of AD leads to defective cognitive flexibility.

“Here, we show that loss of Cass4 in the 5xFAD mouse model of AD leads to worsened Aβ pathology, exacerbated neuronal dystrophy, and defective cognitive flexibility.”

Loss of Cass4 in the 5xFAD mouse model of AD leads to worsened Aβ pathology.

“Here, we show that loss of Cass4 in the 5xFAD mouse model of AD leads to worsened Aβ pathology, exacerbated neuronal dystrophy, and defective cognitive flexibility.”

Defective mobilization of CASS4-deficient microglia leads to poor Aβ plaque compaction.

“which leads to poor Aβ plaque compaction and the generation of more toxic con­ formations of Aβ aggregates.”

Defective mobilization of CASS4-deficient microglia leads to generation of more toxic conformations of Aβ aggregates.

“which leads to poor Aβ plaque compaction and the generation of more toxic con­ formations of Aβ aggregates.”