Drugs targeting SHIP2 demonstrate potent antiproliferative effects irrespective of SHIP2 inhibition.

The SH2-containing inositol 5'-phosphatase SHIP2 plays a crucial role in negative regulation of the PI3K/AKT signaling pathway. Putative small molecule inhibitors of SHIP2, AS1949490 and K149 have been reported to elicit a range of beneficial effects in treating or preventing obesity as well as killing cancer cells. However, whether these effects are direct results of SHIP2 inhibition has not been carefully assessed, e.g., in the absence of expression of the protein. Here, we show that these inhibitors alter the PI3K/AKT signaling pathway irrespective of SHIP2 protein expression. Moreover, we found that AS1949490 and K149 alter cell growth in normal and cancer cells lacking both SHIP1 and SHIP2. Overall, our data provide evidence that the antiproliferative effects of AS1949490 and K149 cannot be attributed to SHIP1/2 inhibition.

Discovery and evaluation of novel SHIP-1 inhibitors.

Src Homology 2-containing Inositol 5'-Phosphatase-1 (SHIP-1), encoded by INPP5D, has been identified as an Alzheimer's disease (AD) risk-associated gene through recent genetic and epigenetic studies. SHIP-1 confers AD risk by inhibiting the TREM2 cascade and reducing beneficial microglial cellular processes, including phagocytosis. While several small molecules have been reported to modulate SHIP-1 activity, their limited selectivity and efficacy in advanced models restricted their potential as therapeutic agents or probes for biological studies. Herein, we validated and implemented a high-throughput screening platform to explore new chemotypes that can modulate the phosphatase activity of SHIP-1. We screened 49,260 central nervous system (CNS)-penetrate compounds sourced from commercial vendors using the malachite green-based assay for anti-SHIP-1 activity. Through analysis, prioritization, and validation of the screening hits, we identified three novel types of scaffolds that inhibit the SHIP-1 phosphatase activity with IC50s as low as 46.6 µM. To improve the inhibitory activity of these promising hits, we carried out structure-activity relationship (SAR) studies, resulting in a lead molecule SP3-12 that inhibits SHIP-1 with an IC50 value of 6.1 µM. Kinetic analyses of SP3-12 revealed that its inhibition mechanism is competitive, with a Ki value of 3.2 µM for SHIP-1 and a 7-fold selectivity over SHIP-2. Furthermore, results from testing in a microglial phagocytosis/cell health high content assay indicated that SP3-12 could effectively activate phagocytosis in human microglial clone 3 (HMC3) cells, with an EC50 of 2.0 µM, without cytotoxicity in the dose range. Given its potency, selectivity, and cellular activity, SP3-12 emerges as a promising small molecule inhibitor with potential for investigating the biological functions of SHIP-1.

Exploring a Potential Optimization Route for Peptide Ligands of the Sam Domain from the Lipid Phosphatase Ship2.

The Sam (Sterile alpha motif) domain of the lipid phosphatase Ship2 (Ship2-Sam) is engaged by the Sam domain of the receptor tyrosine kinase EphA2 (EphA2-Sam) and, this interaction is principally linked to procancer effects. Peptides able to hinder the formation of the EphA2-Sam/Ship2-Sam complex could possess therapeutic potential. Herein, by employing the FoldX software suite, we set up an in silico approach to improve the peptide targeting of the so-called Mid Loop interface of Ship2-Sam, representing the EphA2-Sam binding site. Starting from a formerly identified peptide antagonist of the EphA2-Sam/Ship2-Sam association, first, the most stabilizing mutations that could be inserted in each peptide position were predicted. Then, they were combined, producing a list of potentially enhanced Ship2-Sam ligands. A few of the in silico generated peptides were experimentally evaluated. Interaction assays with Ship2-Sam were performed using NMR and BLI (BioLayer Interferometry). In vitro assays were conducted as well to check for cytotoxic effects against both cancerous and healthy cells, and also to assess the capacity to regulate EphA2 degradation. This study undoubtedly enlarges our knowledge on how to properly target EphA2-Sam/Ship2-Sam associations with peptide-based tools and provides a promising strategy that can be used to target any protein-protein interaction.

Inhibition of the EphA2-Sam/Ship2-Sam Association through Peptide Ligands: Studying the Combined Effect of Charge and Aromatic Character.

The Sam (sterile alpha motif) domain from the lipid phosphatase Ship2 binds the Sam domain from the EphA2 receptor to negatively regulate receptor endocytosis and degradation. This interaction is primarily linked to pro-oncogenic effects. We report on the design and evaluation of EphA2-Sam/Ship2-Sam peptide inhibitors provided with positive charges and different aromatic characters. Starting from the sequence of previously identified Ship2-Sam targeting peptides, an in silico approach was set up to predict higher affinity peptide ligands. A few peptides were experimentally tested through an interdisciplinary approach. Interaction studies were performed by nuclear magnetic resonance spectroscopy and biolayer interferometry. 3D models of Ship2-Sam/peptide complexes were predicted by AlphaFold2. Cell-based assays were carried out to investigate whether such peptide sequences might have an influence on EphA2 signaling. The approach led to the identification of novel Ship2-Sam ligands and shed further light on original approaches to design inhibitors of the Ship2-Sam/EphA2-Sam interaction.

Helicobacter pylori East Asian type CagA hijacks more SHIP2 by its EPIYA-D motif to potentiate the oncogenicity.

CagA is a significant oncogenic factor injected into host cells by Helicobacter pylori, which is divided into two subtypes: East Asian type (CagAE), characterized by the EPIYA-D motif, and western type (CagAW), harboring the EPIYA-C motif. CagAE has been reported to have higher carcinogenicity than CagAW, although the underlying reason is not fully understood. SHIP2 is an intracellular phosphatase that can be recruited by CagA to perturb the homeostasis of intracellular signaling pathways. In this study, we found that SHIP2 contributes to the higher oncogenicity of CagAE. Co-Immunoprecipitation and Pull-down assays showed that CagAE bind more SHIP2 than CagAW. Immunofluorescence staining showed that a higher amount of SHIP2 recruited by CagAE to the plasma membrane catalyzes the conversion of PI(3,4,5)P3 into PI(3,4)P2. This alteration causes higher activation of Akt signaling, which results in enhanced IL-8 secretion, migration, and invasion of the infected cells. SPR analysis showed that this stronger interaction between CagAE and SHIP2 stems from the higher affinity between the EPIYA-D motif of CagAE and the SH2 domain of SHIP2. Structural analysis revealed the crucial role of the Phe residue at the Y + 5 position in EPIYA-D. After mutating Phe of CagAE into Asp (the corresponding residue in the EPIYA-C motif) or Ala, the activation of downstream Akt signaling was reduced and the malignant transformation of infected cells was alleviated. These findings revealed that CagAE hijacks SHIP2 through its EPIYA-D motif to enhance its carcinogenicity, which provides a better understanding of the higher oncogenic risk of H. pylori CagAE.

Ikaros sets the threshold for negative B-cell selection by regulation of the signaling strength of the AKT pathway.

Inhibitory phosphatases, such as the inositol-5-phosphatase SHIP1 could potentially contribute to B-cell acute lymphoblastic leukemia (B-ALL) by raising the threshold for activation of the autoimmunity checkpoint, allowing malignant cells with strong oncogenic B-cell receptor signaling to escape negative selection. Here, we show that SHIP1 is differentially expressed across B-ALL subtypes and that high versus low SHIP1 expression is associated with specific B-ALL subgroups. In particular, we found high SHIP1 expression in both, Philadelphia chromosome (Ph)-positive and ETV6-RUNX1-rearranged B-ALL cells. As demonstrated by targeted knockdown of SHIP1 by RNA interference, proliferation of B-ALL cells in vitro and their tumorigenic spread in vivo depended in part on SHIP1 expression. We investigated the regulation of SHIP1, as an important antagonist of the AKT signaling pathway, by the B-cell-specific transcription factor Ikaros. Targeted restoration of Ikaros and pharmacological inhibition of the antagonistic casein kinase 2, led to a strong reduction in SHIP1 expression and at the same time to a significant inhibition of AKT activation and cell growth. Importantly, the tumor suppressive function of Ikaros was enhanced by a SHIP1-dependent additive effect. Furthermore, our study shows that all three AKT isoforms contribute to the pro-mitogenic and anti-apoptotic signaling in B-ALL cells. Conversely, hyperactivation of a single AKT isoform is sufficient to induce negative selection by increased oxidative stress. In summary, our study demonstrates the regulatory function of Ikaros on SHIP1 expression in B-ALL and highlights the relevance of sustained SHIP1 expression to prevent cells with hyperactivated PI3K/AKT/mTOR signaling from undergoing negative selection.

Alteration of gene expression and protein solubility of the PI 5-phosphatase SHIP2 are correlated with Alzheimer's disease pathology progression.

A recent large genome-wide association study has identified EGFR (encoding the epidermal growth factor EGFR) as a new genetic risk factor for late-onset AD. SHIP2, encoded by INPPL1, is taking part in the signalling and interactome of several growth factor receptors, such as the EGFR. While INPPL1 has been identified as one of the most significant genes whose RNA expression correlates with cognitive decline, the potential alteration of SHIP2 expression and localization during the progression of AD remains largely unknown. Here we report that gene expression of both EGFR and INPPL1 was upregulated in AD brains. SHIP2 immunoreactivity was predominantly detected in plaque-associated astrocytes and dystrophic neurites and its increase was correlated with amyloid load in the brain of human AD and of 5xFAD transgenic mouse model of AD. While mRNA of INPPL1 was increased in AD, SHIP2 protein undergoes a significant solubility change being depleted from the soluble fraction of AD brain homogenates and co-enriched with EGFR in the insoluble fraction. Using FRET-based flow cytometry biosensor assay for tau-tau interaction, overexpression of SHIP2 significantly increased the FRET signal while siRNA-mediated downexpression of SHIP2 significantly decreased FRET signal. Genetic association analyses suggest that some variants in INPPL1 locus are associated with the level of CSF pTau. Our data support the hypothesis that SHIP2 is an intermediate key player of EGFR and AD pathology linking amyloid and tau pathologies in human AD.

SHIP1 modulation and proteome characterization of microglia.

Understanding microglial states in the aging brain has become crucial, especially with the discovery of numerous Alzheimer's disease (AD) risk and protective variants in genes such as INPP5D and TREM2, which are essential to microglia function in AD. Here we present a thorough examination of microglia-like cells and primary mouse microglia at the proteome and transcriptome levels to illuminate the roles these genes and the proteins they encode play in various cell states. First, we compared the proteome profiles of wildtype and INPP5D (SHIP1) knockout primary microglia. Our findings revealed significant proteome alterations only in the homozygous SHIP1 knockout, revealing its impact on the microglial proteome. Additionally, we compared the proteome and transcriptome profiles of commonly used in vitro microglia BV2 and HMC3 cells with primary mouse microglia. Our results demonstrated a substantial similarity between the proteome of BV2 and mouse primary cells, while notable differences were observed between BV2 and human HMC3. Lastly, we conducted targeted lipidomic analysis to quantify different phosphatidylinositols (PIs) species, which are direct SHIP1 targets, in the HMC3 and BV2 cells. This in-depth omics analysis of both mouse and human microglia enhances our systematic understanding of these microglia models. SIGNIFICANCE: Given the growing urgency of comprehending microglial function in the context of neurodegenerative diseases and the substantial therapeutic implications associated with SHIP1 modulation, we firmly believe that our study, through a rigorous and comprehensive proteomics, transcriptomics and targeted lipidomic analysis of microglia, contributes to the systematic understanding of microglial function in the context of neurodegenerative diseases.

The Functional Roles of the Src Homology 2 Domain-Containing Inositol 5-Phosphatases SHIP1 and SHIP2 in the Pathogenesis of Human Diseases.

The src homology 2 domain-containing inositol 5-phosphatases SHIP1 and SHIP2 are two proteins involved in intracellular signaling pathways and have been linked to the pathogenesis of several diseases. Both protein paralogs are well known for their involvement in the formation of various kinds of cancer. SHIP1, which is expressed predominantly in hematopoietic cells, has been implicated as a tumor suppressor in leukemogenesis especially in myeloid leukemia, whereas SHIP2, which is expressed ubiquitously, has been implicated as an oncogene in a wider variety of cancer types and is suggested to be involved in the process of metastasis of carcinoma cells. However, there are numerous other diseases, such as inflammatory diseases as well as allergic responses, Alzheimer's disease, and stroke, in which SHIP1 can play a role. Moreover, SHIP2 overexpression was shown to correlate with opsismodysplasia and Alzheimer's disease, as well as metabolic diseases. The SHIP1-inhibitor 3-α-aminocholestane (3AC), and SHIP1-activators, such as AQX-435 and AQX-1125, and SHIP2-inhibitors, such as K161 and AS1949490, have been developed and partly tested in clinical trials, which indicates the importance of the SHIP-paralogs as possible targets in the therapy of those diseases. The aim of this article is to provide an overview of the current knowledge about the involvement of SHIP proteins in the pathogenesis of cancer and other human diseases and to create awareness that SHIP1 and SHIP2 are more than just tumor suppressors and oncogenes.

ORF3a of SARS-CoV-2 modulates PI3K/AKT signaling in human lung epithelial cells via hsa-miR-155-5p.

SARS-CoV-2 infection results in cytokine burst, leading to proinflammatory responses in lungs of COVID-19 patients. SARS-CoV-2 ORF3a triggers the generation of proinflammatory cytokines. However, the underlying mechanism of dysregulation of proinflammatory responses is not well understood. We studied the role of microRNA in the generation of proinflammatory responses as a bystander effect of SARS-CoV-2 ORF3a in human lung epithelial cells. We observed upregulation of hsa-miR-155-5p in SARS-CoV-2 ORF3a transfected human lung epithelial cells, which led to the reduced expression of SHIP1. This resulted in phosphorylation of AKT and NF-κB, which further led to the increased expression of the proinflammatory cytokines IL-6 and TNF-α. Additionally, overexpression and knockdown studies of hsa-miR-155-5p were performed to confirm the role of hsa-miR-155-5p in the regulation of the SHIP1. We demonstrated that hsa-miR-155-5p modulates the proinflammatory response by activating the PI3K/AKT pathway through the inhibition of SHIP1 in SARS-CoV-2 ORF3a transfected human lung epithelial cells.

SHIP inhibition mediates select TREM2-induced microglial functions.

Microglia play a pivotal role in the pathology of Alzheimer's Disease (AD), with the Triggering Receptor Expressed on Myeloid cells 2 (TREM2) central to their neuroprotective functions. The R47H variant of TREM2 has emerged as a significant genetic risk factor for AD, leading to a loss-of-function phenotype in mouse AD models. This study elucidates the roles of TREM2 in human microglia-like HMC3 cells and the regulation of these functions by SH2-containing inositol-5'-phosphatase 1 (SHIP1). Using stable cell lines expressing wild-type TREM2, the R47H variant, and TREM2-deficient lines, we found that functional TREM2 is essential for the phagocytosis of Aß, lysosomal capacity, and mitochondrial activity. Notably, the R47H variant displayed increased phagocytic activity towards apoptotic neurons. Introducing SHIP1, known to modulate TREM2 signaling in other cells, revealed its role as a negative regulator of these TREM2-mediated functions. Moreover, pharmacological inhibition of both SHIP1 and its isoform SHIP2 amplified Aß phagocytosis and lysosomal capacity, independently of TREM2 or SHIP1 expression, suggesting a potential regulatory role for SHIP2 in these functions. The absence of TREM2, combined with the presence of both SHIP isoforms, suppressed mitochondrial activity. However, pan-SHIP1/2 inhibition enhanced mitochondrial function in these cells. In summary, our findings offer a deeper understanding of the relationship between TREM2 variants and SHIP1 in microglial functions, and emphasize the therapeutic potential of targeting the TREM2 and SHIP1 pathways in microglia for neurodegenerative diseases.

[Microglia differential genes and their functions in paraquat-induced Parkinson's disease-like in mice's brains based on single-cell RNA sequencing].

Objective: To analyze the differential genes and related signaling pathways of microglia subpopulations in Parkinson's disease (PD) -like mouse brains induced by paraquat (PQ) based on single-cell RNA sequencing, and provide clues to elucidate the mechanism of PQ-induced PD-like changes in the brain of animals. Methods: In September 2021, six male 6-week-old C57BL/6 mice were randomly divided into control group and experimental group (three mice in each group) . The mice were injected with saline, 10.0 mg/kg PQ intraperitoneally, once every three days, and 10 consecutive injections were used for modeling. After infection, the brains of mice were taken and 10×Genomics single-cell RNA sequencing was performed. Microglia subpopulations were screened based on gene expression characteristics, and Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed. The differential genes of microglia subpopulations between the experimental group and control group were further screened, and functional enrichment analysis was performed using bioinformatics tools. Mouse microglia (BV2 cells) were treated with 0, 60, 90 µmol/L PQ solution, respectively. And real-time fluorescence quantitative PCR experiments were conducted to validate the expressions of differential genes hexokinase 2 (Hk2) , ATPase H+ Transporting V0 Subunit B (Atp6v0b) and Neuregulin 1 (Nrg1) . Results: Cluster 7 and Cluster 20 were identified as microglia subpopulations based on the signature genes inositol polyphosphate-5-phosphatase d, Inpp5d (Inpp5d) and transforming growth factor beta receptor 1 (Tgfbr1) , and they reflected the microglia-activated M2 phenotype. The bioinformatics analysis showed that the characteristic genes of identified microglia subpopulations were enriched in endocytosis. In terms of molecular function, it mainly enriched in transmembrane receptor protein kinase activity and cytokine binding. The up-regulated genes of Cluster 7 were mainly enriched in lysosomal pathway, endocytosis pathway, and down-regulated genes were mainly enriched in neurodegenerative disease and other signaling pathways. The up-regulated genes of Cluster 20 were mainly enriched in signaling pathways related to PD, and down-regulated genes were mainly enriched in cyclic adenosine 3', 5'-monophosphate (cAMP) signaling pathways, neurological development, synaptic function and other signaling pathways. The results of real-time fluorescence quantitative PCR showed that the expressions of Hk2 mRNA and Atp6v0b mRNA increased and the expression of Nrg1 mRNA decreased in the 90 µmol/L PQ-treated BV2 cells compared with the 0 µmol/L, and the differences were statistically significant (P<0.05) . Conclusion: Microglia are activated in the PQ-induced PD-like mouse model and polarized toward the M2 phenotype. And their functions are associated with lysosomal (endocytosis) , synaptic functions and the regulation of PD-related pathways.

Proinflammatory phenotype of B10 and B10pro cells elicited by TNF-α in rheumatoid arthritis.

OBJECTIVES: B10 and B10pro cells suppress immune responses via secreting interleukin (IL)-10. However, their regulators and underlying mechanisms, especially in human autoimmune diseases, are elusive. This study aimed to address these questions in rheumatoid arthritis (RA), one of the most common highly disabling autoimmune diseases. METHODS: The frequencies and functions of B10 and B10pro cells in healthy individuals and patients with RA were first analysed. The effects of proinflammatory cytokines, particularly tumour necrosis factor (TNF)-α on the quantity, stability and pathogenic phenotype of these cells, were then assessed in patients with RA before and after anti-TNF therapy. The underlying mechanisms were further investigated by scRNA-seq database reanalysis, transcriptome sequencing, TNF-α-/- and B cell-specific SHIP-1-/- mouse disease model studies. RESULTS: TNF-α was a key determinant for B10 cells. TNF-α elicited the proinflammatory feature of B10 and B10pro cells by downregulating IL-10, and upregulating interferon-γ and IL-17A. In patients with RA, B10 and B10pro cells were impaired with exacerbated proinflammatory phenotype, while anti-TNF therapy potently restored their frequencies and immunosuppressive functions, consistent with the increased B10 cells in TNF-α-/- mice. Mechanistically, TNF-α diminished B10 and B10pro cells by inhibiting their glycolysis and proliferation. TNF-α also regulated the phosphatidylinositol phosphate signalling of B10 and B10pro cells and dampened the expression of SHIP-1, a dominant phosphatidylinositol phosphatase regulator of these cells. CONCLUSIONS: TNF-α provoked the proinflammatory phenotype of B10 and B10pro cells by disturbing SHIP-1 in RA, contributing to the disease development. Reinstating the immunosuppressive property of B10 and B10pro cells might represent novel therapeutic approaches for RA.

Regulation of inositol 5-phosphatase activity by the C2 domain of SHIP1 and SHIP2.

SHIP1, an inositol 5-phosphatase, plays a central role in cellular signaling. As such, it has been implicated in many conditions. Exploiting SHIP1 as a drug target will require structural knowledge and the design of selective small molecules. We have determined apo, and magnesium and phosphate-bound structures of the phosphatase and C2 domains of SHIP1. The C2 domains of SHIP1 and the related SHIP2 modulate the activity of the phosphatase domain. To understand the mechanism, we performed activity assays, hydrogen-deuterium exchange mass spectrometry, and molecular dynamics on SHIP1 and SHIP2. Our findings demonstrate that the influence of the C2 domain is more pronounced for SHIP2 than SHIP1. We determined 91 structures of SHIP1 with fragments bound, with some near the interface between the two domains. We performed a mass spectrometry screen and determined four structures with covalent fragments. These structures could act as starting points for the development of potent, selective probes.

Actin-binding protein profilin1 is an important determinant of cellular phosphoinositide control.

Membrane polyphosphoinositides (PPIs) are lipid-signaling molecules that undergo metabolic turnover and influence a diverse range of cellular functions. PPIs regulate the activity and/or spatial localization of a number of actin-binding proteins (ABPs) through direct interactions; however, it is much less clear whether ABPs could also be an integral part in regulating PPI signaling. In this study, we show that ABP profilin1 (Pfn1) is an important molecular determinant of the cellular content of PI(4,5)P2 (the most abundant PPI in cells). In growth factor (EGF) stimulation setting, Pfn1 depletion does not impact PI(4,5)P2 hydrolysis but enhances plasma membrane (PM) enrichment of PPIs that are produced downstream of activated PI3-kinase, including PI(3,4,5)P3 and PI(3,4)P2, the latter consistent with increased PM recruitment of SH2-containing inositol 5' phosphatase (SHIP2) (a key enzyme for PI(3,4)P2 biosynthesis). Although Pfn1 binds to PPIs in vitro, our data suggest that Pfn1's affinity to PPIs and PM presence in actual cells, if at all, is negligible, suggesting that Pfn1 is unlikely to directly compete with SHIP2 for binding to PM PPIs. Additionally, we provide evidence for Pfn1's interaction with SHIP2 in cells and modulation of this interaction upon EGF stimulation, raising an alternative possibility of Pfn1 binding as a potential restrictive mechanism for PM recruitment of SHIP2. In conclusion, our findings challenge the dogma of Pfn1's binding to PM by PPI interaction, uncover a previously unrecognized role of Pfn1 in PI(4,5)P2 homeostasis and provide a new mechanistic avenue of how an ABP could potentially impact PI3K signaling byproducts in cells through lipid phosphatase control.

INPP5D regulates inflammasome activation in human microglia.

Microglia and neuroinflammation play an important role in the development and progression of Alzheimer's disease (AD). Inositol polyphosphate-5-phosphatase D (INPP5D/SHIP1) is a myeloid-expressed gene genetically-associated with AD. Through unbiased analyses of RNA and protein profiles in INPP5D-disrupted iPSC-derived human microglia, we find that reduction in INPP5D activity is associated with molecular profiles consistent with disrupted autophagy and inflammasome activation. These findings are validated through targeted pharmacological experiments which demonstrate that reduced INPP5D activity induces the formation of the NLRP3 inflammasome, cleavage of CASP1, and secretion of IL-1ß and IL-18. Further, in-depth analyses of human brain tissue across hundreds of individuals using a multi-analytic approach provides evidence that a reduction in function of INPP5D in microglia results in inflammasome activation in AD. These findings provide insights into the molecular mechanisms underlying microglia-mediated processes in AD and highlight the inflammasome as a potential therapeutic target for modulating INPP5D-mediated vulnerability to AD.

Microglial function, INPP5D/SHIP1 signaling, and NLRP3 inflammasome activation: implications for Alzheimer's disease.

Recent genetic studies on Alzheimer's disease (AD) have brought microglia under the spotlight, as loci associated with AD risk are enriched in genes expressed in microglia. Several of these genes have been recognized for their central roles in microglial functions. Increasing evidence suggests that SHIP1, the protein encoded by the AD-associated gene INPP5D, is an important regulator of microglial phagocytosis and immune response. A recent study from our group identified SHIP1 as a negative regulator of the NLRP3 inflammasome in human iPSC-derived microglial cells (iMGs). In addition, we found evidence for a connection between SHIP1 activity and inflammasome activation in the AD brain. The NLRP3 inflammasome is a multiprotein complex that induces the secretion of pro-inflammatory cytokines as part of innate immune responses against pathogens and endogenous damage signals. Previously published studies have suggested that the NLRP3 inflammasome is activated in AD and contributes to AD-related pathology. Here, we provide an overview of the current understanding of the microglial NLRP3 inflammasome in the context of AD-related inflammation. We then review the known intracellular functions of SHIP1, including its role in phosphoinositide signaling, interactions with microglial phagocytic receptors such as TREM2 and evidence for its intersection with NLRP3 inflammasome signaling. Through rigorous examination of the intricate connections between microglial signaling pathways across several experimental systems and postmortem analyses, the field will be better equipped to tailor newly emerging therapeutic strategies targeting microglia in neurodegenerative diseases.

Regulation of Microglial Signaling by Lyn and SHIP-1 in the Steady-State Adult Mouse Brain.

Chronic neuroinflammation and glial activation are associated with the development of many neurodegenerative diseases and neuropsychological disorders. Recent evidence suggests that the protein tyrosine kinase Lyn and the lipid phosphatase SH2 domain-containing inositol 5' phosphatase-1 (SHIP-1) regulate neuroimmunological responses, but their homeostatic roles remain unclear. The current study investigated the roles of Lyn and SHIP-1 in microglial responses in the steady-state adult mouse brain. Young adult Lyn-/- and SHIP-1-/- mice underwent a series of neurobehavior tests and postmortem brain analyses. The microglial phenotype and activation state were examined by immunofluorescence and flow cytometry, and neuroimmune responses were assessed using gene expression analysis. Lyn-/- mice had an unaltered behavioral phenotype, neuroimmune response, and microglial phenotype, while SHIP-1-/- mice demonstrated reduced explorative activity and exhibited microglia with elevated activation markers but reduced granularity. In addition, expression of several neuroinflammatory genes was increased in SHIP-1-/- mice. In response to LPS stimulation ex vivo, the microglia from both Lyn-/- and SHIP-1-/- showed evidence of hyper-activity with augmented TNF-α production. Together, these findings demonstrate that both Lyn and SHIP-1 have the propensity to control microglial responses, but only SHIP-1 regulates neuroinflammation and microglial activation in the steady-state adult brain, while Lyn activity appears dispensable for maintaining brain homeostasis.

INPP5D/SHIP1: Expression, Regulation and Roles in Alzheimer's Disease Pathophysiology.

Alzheimer's disease (AD) is the most common form of dementia, accounting for approximately 38.5 million cases of all-cause dementia. Over 60% of these individuals live in low- and middle-income countries and are the worst affected, especially by its deleterious effects on the productivity of both patients and caregivers. Numerous risk factors for the disease have been identified and our understanding of gene-environment interactions have shed light on several gene variants that contribute to the most common, sporadic form of AD. Microglial cells, the innate immune cells of the central nervous system (CNS), have long been established as guardians of the brain by providing neuroprotection and maintaining cellular homeostasis. A protein with a myriad of effects on various important signaling pathways that is expressed in microglia is the Src Homology 2 (SH2) domain-containing Inositol 5' Phosphatase 1 (SHIP1) protein. Encoded by the INPP5D (Inositol Polyphosphate-5-Phosphatase D) gene, SHIP1 has diminutive effects on most microglia signaling processes. Polymorphisms of the INPP5D gene have been found to be associated with a significantly increased risk of AD. Several studies have elucidated mechanistic processes by which SHIP1 exerts its perturbations on signaling processes in peripheral immune cells. However, current knowledge of the controllers of INPP5D/SHIP1 expression and the idiosyncrasies of its influences on signaling processes in microglia and their relevance to AD pathophysiology is limited. In this review, we summarize these discoveries and discuss the potential of leveraging INPP5D/SHIP1 as a therapeutic target for Alzheimer's disease.