Use of RIPK1 modulator in treatment of alzheimer's disease

Abstract

Provided is use of an RIPK1 inhibitor in the treatment of Alzheimer's disease. Specifically, provided is a potential treatment method for Alzheimer's disease. The method uses the RIPK1 inhibitor to ameliorate and inhibit brain inflammation and the development of Alzheimer's disease caused by changes in the activity or expression level of myeloid INPP5D or RIPK1 activation, thereby ameliorating symptoms of late-onset Alzheimer's disease, especially early AD progression, and reducing brain inflammation associated with Alzheimer's disease.

Description

Use of RIPK1 modulators in the treatment of Alzheimer's disease Technical Field

[0001] This invention relates to the field of biochemistry, and more specifically, it provides the use of RIPK1 inhibitors in the treatment of Alzheimer's disease. Background Technology

[0002] Advanced Alzheimer's disease (LOAD) is the most prevalent dementia affecting people over 65 years of age. The gradual decline in cognitive function in LOAD patients presents significant personal challenges not only to patients but also to caregivers. Alzheimer's disease has become a huge global health burden, urgently requiring a deeper understanding of its pathological mechanisms to find solutions. Genome-wide association studies (GWAS) have identified numerous genetic risk factors that significantly influence the likelihood of carriers developing LOAD. LOAD risk alleles are enriched in genes highly expressed in microglia, including ApoE, Inpp5D, Trem2, Cd33, Tyrobp, Ptk2b, Spi1, Pld3, Ctsd, Ctsb, Ctsh, Idua, Grn, Rin3, Pilra, Adam17, Maf, and Blnk, indicating that microglia play a crucial role in LOAD; however, the functional relationships among various microglia LOAD risk genes remain unclear. Single nucleotide polymorphisms (SNPs) in the non-coding region of the INPP5D locus have been identified as one of the most common risk factors for LOAD. Levels of full-length INPP5D protein are reduced in the AD brain, and decreased INPP5D activity in microglia is associated with activation of the NLRP3 inflammasome.

[0003] Microglia are highly dynamic immune regulators in the central nervous system, rapidly responding to external signals and the environment by altering their transcriptional profiles. Microglial homeostasis is crucial, as they provide regulators of their response thresholds, types, and intensities, which in turn control microglial activity and function. Changes in microglial homeostasis may help explain why relatively harmless, minor, harmful stimuli in youth become harmful in old age. Microglia exhibit diverse morphologies, ranging from branched at rest to deformed in activated states, and atrophied, fragmented, or tortuous states in disease states; research on the latter morphology has primarily focused on autopsy pathological samples from patients with Alzheimer's disease (AD) and neurodegenerative diseases characterized by TDP-43 pathology. The mechanisms promoting the development of atrophic microglia remain unclear. Recent large-scale microglial transcriptomics studies have further elucidated the complexity of microglia. Based on snRNA transcriptome mapping, up to 16 microglial subtypes have been identified in the human brain. Microglial subtypes regulating homeostasis and neuronal surveillance may be involved in mediating normal homeostasis in the brain, while inflammatory and lipid-processing subtypes have been identified as enriched subtypes and are associated with the pathogenesis of Alzheimer's disease (AD) in humans. Understanding the mechanisms by which microglial homeostasis is restored may be key to developing therapies for AD.

[0004] In conclusion, there is an urgent need in this field to develop more treatments for Alzheimer's disease, as well as corresponding therapeutic components. Summary of the Invention

[0005] The purpose of this invention is to provide a new treatment mechanism for Alzheimer's disease, particularly for advanced Alzheimer's disease.

[0006] Another object of the present invention is to provide the use of RIPK1 inhibitors in the treatment of Alzheimer's disease.

[0007] In a first aspect, the present invention provides the use of a RIPK1 inhibitor, characterized in that it is used to prepare a pharmaceutical composition for the prevention or treatment of Alzheimer's disease; wherein the Alzheimer's disease is selected from the group consisting of: early-onset Alzheimer's disease, late-onset Alzheimer's disease, familial Alzheimer's disease, and sporadic Alzheimer's disease.

[0008] Preferably, the inhibitor treats Alzheimer's disease by inhibiting RIPK1 activation caused by changes in Inpp5d activity or expression levels in the subject, or by RIPK1 activation caused by other reasons.

[0009] In another preferred embodiment, the RIPK1 inhibitor prevents or treats Alzheimer's disease by improving RIPK1-mediated peripheral inflammation and myeloid INPP5D deficiency.

[0010] In another preferred embodiment, the RIPK1 inhibitor treats Alzheimer's disease by reducing the surge in Aβ fiber levels caused by myeloid INPP5D deficiency or other reasons leading to RIPK1 activation.

[0011] In another preferred embodiment, the inhibitor is also used for purposes selected from the group consisting of:

[0012] (1) Reversing atrophic microglial lesions;

[0013] (2) Downregulates the expression of RIPK1-mediated pro-inflammatory cytokines;

[0014] (3) It inhibits the increase in the activation level of astrocytes.

[0015] In another preferred embodiment, the RIPK1 inhibitor is also used to block inflammatory signaling pathways.

[0016] In another preferred embodiment, the RIPK1 inhibitor is also used to regulate AD risk genes.

[0017] In another preferred embodiment, the inhibitor is also used to reverse neuroinflammation caused by Alzheimer's disease.

[0018] In another preferred embodiment, the RIPK1-mediated pro-inflammatory cytokines are selected from the group consisting of TNFα, IL1α, IL1β, IL6, Ccl2, Ccl3, Ccl5, Ccl6, Cxcl12, and IFNγ.

[0019] In another preferred embodiment, the inhibitor is also used to inhibit the expression of a TLR aptamer; preferably, the TLR aptamer is MyD88.

[0020] In another preferred embodiment, the inhibitor is also used to regulate the expression of complement factors in microglia; preferably, the complement factors are selected from the group consisting of C1qa, C1qb, C1qc, C3, Cfh, and Cfp.

[0021] In another preferred embodiment, the inhibitor is also used to suppress the increased expression of inflammasome components in microglia, preferably, the inflammasome components are selected from the group consisting of NLRP3, AIM2, GSDMD and caspase-1.

[0022] In another preferred embodiment, the inhibitor is also used to upregulate the expression of intracellular mediators; preferably, the intracellular mediators are selected from the group consisting of: Irgm1, Irgm2, Ifnar2, Irf8, Ifitm2, and Ifitm3.

[0023] In another preferred embodiment, the inhibitor is also used to suppress the increased expression of ZBP1 and IFNγ in the brain.

[0024] In another preferred embodiment, the inhibitor is also used to inhibit the transcription of Alzheimer's disease risk genes; preferably, the risk genes are selected from the group consisting of: ApoE, Trem2, Tyrobp, Ptk2b, Plcg2, Spi1, Pld3, Ctsd, Ctsb, Ctsh, Idua, Grn, Rin3, Pilra, Adam17, Maf, and Blnk.

[0025] In another preferred embodiment, the inhibitor is also used to improve the infiltration of peripheral T cells into the central nervous system.

[0026] In another preferred embodiment, the inhibitor is also used to downregulate the expression of RIPK1-related cytokine genes; the cytokines are selected from the group consisting of: AD risk factors, pro-inflammatory cytokines, ROS mediators, complement, inflammasomes, and IFNs.

[0027] In another preferred embodiment, the inhibitor is also used to regulate the activity of γ-secretase in neurons.

[0028] In another preferred embodiment, the inhibitor is also used for purposes selected from the group consisting of:

[0029] (1) Improve age-dependent movement disorders;

[0030] (2) Reverse cytoplasmic TDP-43 translocation and neuronal apoptosis.

[0031] In another preferred embodiment, the movement disorder is an Alzheimer's disease-induced movement disorder.

[0032] In another preferred embodiment, the inhibitor is also used to selectively prevent the activation of JNK and c-Jun in INPP5D-deficient BV2 cells.

[0033] In another preferred embodiment, the inhibitor is used to promote the binding of INPP5D to RIPK1.

[0034] In another preferred embodiment, the inhibitor is used to promote the interaction between the SH2 domain of INPP5D and RIPK1.

[0035] In another preferred embodiment, the inhibitor is also used to improve the infiltration of peripheral T cells into the central nervous system.

[0036] In another preferred embodiment, the inhibitor is also used for purposes selected from the group consisting of:

[0037] (1) Inhibits the increase in free Aβ42 fiber levels;

[0038] (2) Improves amyloid deposition in the brain;

[0039] (3) Improves age-dependent peripheral inflammation caused by Inpp5d deficiency;

[0040] (4) Regulate the activity of γ-secretase in neurons;

[0041] (5) Improve sleep disorders induced by Alzheimer's disease;

[0042] (6) Improve learning and memory deficits induced by Alzheimer's disease;

[0043] (7) Block the rapid increase in Aβ fibers promoted by myeloid Inpp5d deletion.

[0044] In another preferred embodiment, the RIPK1 inhibitor is selected from the group consisting of RIPK1-i1, RIPK1-i2, spermidine, acetylated spermidine, or small molecule RIPK1 inhibitors.

[0045] In another preferred embodiment, the small molecule RIPK1 inhibitor is selected from the group consisting of: Nec-1s, DNL788, GDC-8264, ABBV-668, AC-003, Ocadusertib, GSK2982772, GFH-312, Eclitasertib, Oditrasertib, Flizasertib, or small molecule RIPK1 inhibitors selected from the group consisting of:

[0046] Or the small molecule RIPK1 inhibitor may be a compound as shown in Formula I, Formula II or Formula III:

[0047] In the formula:

[0048] for

[0049] M is selected from the following group: chemical bond, O, S, NR 3a CHR 3a or C(R) 3a )2;

[0050] X1 is selected from the following group: CR 2a NR a O, S, CR a N;

[0051] X2 is selected from the following group: CR a N;

[0052] R a Selected from the following group: H, D, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 deuterated alkyl;

[0053] R 1a R 2a Each is independently selected from the following group: H, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 deuterated alkyl; or R1, R2 together with the carbon atom attached to them to form substituted or unsubstituted C3-C6 cycloalkyl, or substituted or unsubstituted 4-6 membered heterocyclic groups;

[0054] Ring A is a group selected from the following group: substituted or unsubstituted C6-C10 aryl, substituted or unsubstituted 5-12 heteroaryl;

[0055] Ring B is a group selected from the following group: substituted or unsubstituted C6-C10 aryl, substituted or unsubstituted 5-12 heteroaryl;

[0056] Wherein, the substitution refers to the hydrogen atom on the substituent group being replaced by one or more (e.g., 2, 3, 4, etc.) substituents selected from the group consisting of: halogen, deuterated, C1-C6 alkoxy, halogenated C1-C6 alkoxy, methyl sulfone, -S(=O)2NH2, oxo(=O), -CN, hydroxyl, -NH2, carboxyl, C2-C6 amide (-C(=O)-N(Rc)2 or -NH-C(=O)(Rc, where Rc is H or a C1-C5 alkyl), C1-C6 alkyl-(C2-C6 amide), or substituted or unsubstituted groups selected from the group consisting of: C1-C6 alkyl, C3-C8 cycloalkyl, C1- C6 amino, C6-C10 aryl, 5-10 heteroaryl having 1-3 heteroatoms selected from N, S and O, 5-12 heterocyclic group having 1-3 heteroatoms selected from N, S and O, -(CH2)-C6-C10 aryl, -(CH2)-(5-10 heteroaryl having 1-3 heteroatoms selected from N, S and O), and the substituent is selected from the group consisting of: halogen, C1-C6 alkyl, C1-C6 alkoxy, oxo, -CN, -NH2, -OH, C6-C10 aryl, C1-C6 amino, C2-C6 amide, 5-10 heteroaryl having 1-3 heteroatoms selected from N, S and O;

[0057] in,

[0058] Ring A is a substituted or unsubstituted 9-10 member nitrogen-containing heteroaryl group, wherein the 9-10 member nitrogen-containing heteroaryl group contains 1, 2, 3 or 4 nitrogen heteroatoms as ring atoms;

[0059] n = 0, 1, or 2;

[0060] R 4b Each is independently selected from the following group: H, CN, halogen, substituted or unsubstituted C. 1-6 Alkyl, -OR b -SRb -N(R) b )2、-C(O)-NR 6b -R b -C(O)-NR 6b -C 1-4 Alkylene-N(R) b )2、-NR 6b -C(O)-R b ;

[0061] Each R b Each is independently selected from the following groups: H, substituted or unsubstituted C. 1-6 Alkyl; or two R b Together with the nitrogen atoms attached to them, they form substituted or unsubstituted 5, 6, or 7-membered heterocyclic alkyl groups, wherein, except for those with R b In addition to the connected N, the heterocyclic alkyl group also contains 0, 1 or 2 other heteroatoms as ring atoms;

[0062] R 6b Selected from the following group: H, OH, substituted or unsubstituted C 1-4 Alkyl, substituted or unsubstituted C 1-4 Alkoxy;

[0063] Cycle B is selected from the following group: substituted or unsubstituted C 6-10 Aryl, substituted or unsubstituted 5-10 heteroaryl groups;

[0064] L 1 and L 2 Each is independently a divalent group selected from the following group:

[0065] none,

[0066] And L 1 and L 2 Not simultaneously equal to none;

[0067] R 1b and R 2b Each is independently selected from the following groups: H, substituted or unsubstituted C. 1-4 alkyl;

[0068] R 3b Selected from the following group: H, OH, substituted or unsubstituted C 1-4 Alkyl, substituted or unsubstituted C 1-4 Alkoxy;

[0069] Ring C is none or Among them, W is independently selected from the following groups: O, S, C, N, C(R) c ), and N(R) d ); Rc Each is independently selected from the following group: H, CN, halogen, substituted or unsubstituted C. 1-6 Alkyl, R d Each is independently selected from the following groups: H, CN, substituted or unsubstituted C. 1-6 alkyl;

[0070] Or, when ring C is L 1 for And L 2 When there is no time, R 3 With L 1 The ring atoms W and L adjacent to the ring C are 1 The -C(O)- groups in the rings together form substituted or unsubstituted 5, 6, or 7-membered saturated heterocycles; wherein, the saturated heterocycles, in addition to being substituted with R, form substituted or unsubstituted 5, 6, or 7-membered saturated heterocycles. 3 In addition to the connected N, there are 0, 1 or 2 other heteroatoms that act as ring atoms;

[0071] R 5 Selected from the following group: H, OH, substituted or unsubstituted C 1-4 Alkyl, substituted or unsubstituted C 1-4 Alkoxy;

[0072] Or when ring C is nonexistent and L 2 for At that time, R 3 and R 5 And the atoms bonded to them together form substituted or unsubstituted 5, 6, or 7-membered saturated heterocycles; wherein, the saturated heterocycles, in addition to being associated with R, form substituted or unsubstituted 5, 6, or 7-membered saturated heterocycles. 3 In addition to the connected N, there are 0, 1 or 2 other heteroatoms that act as ring atoms;

[0073] Ring D is selected from the following group: substituted or unsubstituted C 6-10 Aromatic rings, and substituted or unsubstituted 5-10 membered heteroaryl groups;

[0074] Unless otherwise specified, the term "substitution" refers to the substitution of a hydrogen atom on a group by one or more (e.g., 1, 2, 3, or 4) substituents selected from the group consisting of: oxo (=O), -CN, halogen, nitro, C. 1-6 Alkyl, Halogenated C 1-6 Alkyl groups, -OR, -SR, -S(O)2R, -S(=O)2NR2, -NR2, -COOR, and C groups optionally substituted with R. 6-10 aryl, 5-10 heteroaryl groups with 1-3 heteroatoms selected from N, S and O, optionally substituted by R, and C groups optionally substituted by R 3- 8-cyclic alkyl groups, 5-12-membered heterocyclic alkyl groups having 1-3 heteroatoms selected from N, S, and O, optionally substituted with R, and -C optionally substituted with R.1-4 Alkylene-C 6-10 aryl, -C optionally substituted by R 1-4 Alkylene – a 5-10 membered heteroaryl group having 1-3 heteroatoms selected from N, S, and O, with -C optionally substituted by R. 1-4 Alkylene-C 3-8 Cycloalkyl, -C optionally substituted with R 1-4 Alkylene – 5-12 membered heterocyclic alkyl groups having 1-3 heteroatoms selected from N, S and O;

[0075] R is independently selected from the following groups: H, C 1-6 Alkyl, Halogenated C 1-6 Alkyl, C 1-6 Hydroxyalkyl;

[0076] In the formula:

[0077] X8 is selected from the following group: CH, N, or chemical bonds;

[0078] X7, X3, X4, and X5 are each independently selected from the following groups: CH, N;

[0079] The condition is that the ring formed by X8, X7, X3, X4, and X5 is an aromatic ring;

[0080] M is selected from the following group: O, S, NR 3c CHR 3c or C(R) 3c )2;

[0081] W and U are each independently selected from the following groups: O, S, NR 4c CHR4 or C(R) 4c )2;

[0082] Ring A and ring B are each independently selected from the following group: substituted or unsubstituted C6-C10 aryl groups, substituted or unsubstituted 5-12 heteroaryl groups;

[0083] R 1c and R 2c Each is independently selected from the following group: none, H, substituted or unsubstituted C1-C6 alkyl, halogen;

[0084] And when M is NR 3c CHR 3c or C(R) 3c When R2 and R3 are together with the C or N atom attached to them, as well as -CC(O)-, they can form substituted or unsubstituted 5-7 membered rings.

[0085] R 3c and R 4cSelected from the following group: H, substituted or unsubstituted C1-C6 alkyl groups, halogens;

[0086] R6 is selected from the following group: H, halogen, CN, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C2-C6 olefin, substituted or unsubstituted C2-C6 alkyne, or -CH≡CR5; wherein R5 is selected from the following group: substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C6-C10 aryl, substituted or unsubstituted 5-12-membered heteroaryl, substituted or unsubstituted C3-C8 cycloalkyl, substituted or unsubstituted 4-12-membered heterocyclic group;

[0087] R7 is selected from the following group: H, halogen, CN, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C2-C6 olefin, substituted or unsubstituted C2-C6 alkyne, or -CH≡CR8; wherein R8 is selected from the following group: substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C6-C10 aryl, substituted or unsubstituted 5-12-membered heteroaryl, substituted or unsubstituted C3-C8 cycloalkyl, substituted or unsubstituted 4-12-membered heterocyclic group;

[0088] Wherein, the substitution refers to the hydrogen atom on the substituent group being replaced by one or more (e.g., 2, 3, 4, etc.) substituents selected from the group consisting of: halogen, C1-C6 alkyl, halogenated C1-C6 alkyl, C3-C8 cycloalkyl, halogenated C3-C8 cycloalkyl, C1-C6 alkoxy, halogenated C1-C6 alkoxy, C1-C6 hydroxyalkyl, methyl sulfone, -S(=O)2NH2, oxo(=O), -CN, hydroxyl, -NH2, carboxyl, C2-C6 amide (-C(=O)-N(Rc)2 or -NH-C(=O)(Rc), where Rc is H or C1-C5). Alkyl), C1-C6 alkyl-(C2-C6 amide), C1-C6 amino, deuterated C1-C6 amino, -NHRd (Rd is C3-C8 cycloalkyl, 4-7 membered heterocyclic or heterocyclic substituted with C1-C6 alkyl), C6-C10 aryl, 5-7 membered heteroaryl having 1-3 heteroatoms selected from N, S and O, 4-8 membered heterocyclic having 1-3 heteroatoms selected from N, S and O, 4-7 membered heterocyclic substituted with 1 or 2 Re (Re is halogen, C1-C6 alkyl, C1-C6 amino, -CN, C1-C6 alkoxy or 4-7 membered heterocyclic).

[0089] In another preferred embodiment, the small molecule RIPK1 inhibitor is the inhibitor mentioned below.

[0090] In another preferred embodiment, the inhibitor is administered before, during, or after the patient develops Alzheimer's disease.

[0091] In another preferred embodiment, the inhibitor is administered to the patient before the onset of Alzheimer's disease, preferably to a patient in whom a sudden increase in Aβ fiber levels has been detected.

[0092] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0093] Figure 1. RIPK1-dependent pro-inflammatory cytokines produced by INPP5D-deficient microglia during culture and in vivo aging.

[0094] (A) IBA1ab immunostaining was performed on frozen brain sections of 8-month-old mice with specified genotypes. Representative IBA1+ microglia were visualized and reconstructed in three dimensions using IMARIS (v.10.0, Bitplane).

[0095] (B) Immunofluorescence staining of IBA1 and p-RIPK1(S166) in frozen cortical sections from 8-month-old genotype mice. p-RIPK1(S166) and IBA in the entire cortical section in (A) were quantified using ImageJ. *P<0.05; **P<0.01; ***P<0.001; ns, not significant. Data are expressed as mean ± SEM. Three to four male mice were used for each genotype (four cortical sections per mouse); the images are representative.

[0096] (C) The division process, branch length, and critical radius of microglia for each genotype were quantified. N = 3 mice per genotype (60 microglia were quantified for each genotype). ***P < 0.001.

[0097] (D) Go analysis of RIPK1-dependent upregulated genes in INPP5D gene-deficient microglia.

[0098] (E) Heatmap analysis revealed the expression levels of inflammation-related genes, pro-inflammatory cytokines, and chemokines in neonatal microglia cultures isolated from mice of specified genotypes. Fold change > 1.5; adjusted p < 0.05.

[0099] (F) Quantitative PCR analysis of the expression of certain pro-inflammatory cytokine genes in neonatal microglia cultured from mice of specified genotypes. N = 4. *p < 0.05; **p < 0.01.

[0100] Figure 2. With age, INPP5D deficiency promotes the transcription of multiple pro-inflammatory factors in microglia in a RIPK1-dependent manner.

[0101] (A) A heatmap analysis revealed the expression of 22 ROS-regulating genes in a culture of neonatal microglia with genotype INPP5D. Blue indicates low expression, and red indicates high expression.

[0102] (B) Intracellular ROS expression was detected using CellRox green staining. H2O2 treatment and NAC inhibition were used as positive controls. Microglia were treated with Nec-1s (10M) or a vector for 24 hours. Images were obtained using a high-selectivity content (HSC) microscope, with parameters uniform across all groups. (Right panel) Quantitative analysis shows the percentage of CellRox-positive cells in cultured newborn primary microglia, with or without Nec-1s treatment. Mean ± SEM.

[0103] (C) Using Aif1 (IBA1) as a microglial marker, in situ RNAscope treatment was performed on frozen cortical sections from 10-month-old mice of specified genotypes using the Cybb (NOX2) probe. Cybb expression levels in all cortical sections of the specified genotype mice were determined by H-score. Images were recorded using confocal microscopy. The figures shown are representative images. Four to five male mice of each genotype were used (one to two cortical sections per mouse). Quantification was performed using the HALOFISH module. *P<0.05; **P<0.01; ***P<0.001; ns, not statistically significant. Data are expressed as mean ± SEM.

[0104] (D) Heatmap analysis of the expression of the six complementary genes of the genotypes shown in cultured neonatal microglia. Low expression is shown in blue, and high expression is shown in red.

[0105] (E) Quantitative PCR analysis of C3, C1qa, C1qb and C1qc expression in cultured newborn microglia (N=4).

[0106] (F) Western blot analysis of C3, C1qa, C11qb and C1qc in cultured newborn microglia.

[0107] (GH) Samples used in (C) were treated in situ with RNAscope using C1qa and TNF(G) probes, and C3(H) was treated in situ with RNAscope using the Aif1(IBA1) probe as a microglial marker (G) or with IBA1ab immunostaining as a microglial marker (H). As shown in the right figure, the expression levels of C1qa(G) and C3(H) in whole cortical sections of mice of the specified genotype are expressed as H fractions. Images were recorded by confocal microscopy. Images are representative. 4–5 male mice per genotype (1–2 cortical sections per mouse). Quantification was performed using the HALOFISH module. *P<0.05; **P<0.01; ***P<0.001; ns, no clinical significance. Data are expressed as mean ± SEM.

[0108] (I) Heatmap analysis of 16 TLR regulatory genes expressed in cultured neonatal microglia at specified genotypes. Low expression is shown in blue, and high expression is shown in red.

[0109] (J) IBA1ab immunofluorescence was used as a microglial cell marker, and frozen cortical sections from 10-month-old mice of a specified genotype were treated with the MyD88 probe using in situ RNAscope. The expression level of MyD88 in all cortical sections of the specified genotype mice was determined by H-score (see figure below). Images were recorded by confocal microscopy. The figures shown are representative images. Four to five male mice of each genotype were used (one to two cortical sections per mouse). Quantification was performed using the HALOFISH module. *P<0.05; **P<0.01; ***P<0.001; ns, no clinical significance. Data are presented as Average SEM.

[0110] (K) The heatmap analysis revealed the expression of nine inflammasome-related genes in neonatal microglia cultures of the genotypes shown (genotype color coding is consistent with Figure 1E). Low expression is shown in blue, and high expression is shown in red.

[0111] (L) Quantitative PCR analysis of the expression of Nlrp3, Aim2, Caspase-1, and GSDMD in neonatal microglia cultures of the indicated genotypes. N = 4.

[0112] (M) Western blot analysis of Nlrp3, Aim2, Caspase-1 and GSDMD in cultured newborn microglia with specified genotypes.

[0113] (NO) Samples from (J) were treated in situ using RNAscope probes for Nlrp3 and Aim2, GSDMD, and the RNAscope probe Aif1 (IBA1) was used as a microglial marker. Expression levels of Nlrp3 and Aim2 (N) or GSDMD (O) in whole cortical sections of mice of the specified genotypes were expressed as H-fractions. Images were recorded by confocal microscopy. Images are representative. Four or five male mice were used for each genotype (1-2 cortical sections per mouse). Quantification was performed using the HALOFISH module. *P<0.05; **P<0.01; ***P<0.001; ns, not statistically significant. Data are expressed as mean ± SEM.

[0114] (P) The heatmap analyzed the expression of 19 interferon signaling-related genes in cultured neonatal microglia. Low expression is shown in blue, and high expression is shown in red.

[0115] (Q) Samples identical to those in (J) were treated with in situ RNAscope, using the Igrm1 probe and IBA1ab immunofluorescence as microglial markers. Igrm1 expression levels in whole cortical sections of mice of the specified genotypes are expressed as H fractions. Images were recorded by confocal microscopy. The figures shown are representative images. Four or five male mice were used for each genotype (1–2 cortical sections per mouse). Quantification was performed using the HALOFISH module. *P<0.05; **P<0.01; ***P<0.001; ns, not statistically significant. Data are presented as Average SEM.

[0116] Samples identical to those in (R) and (J) were treated with in situ RNAscope, using the Aif1 (IBA1) RNAscope probe as a microglial marker, and the ZBP1 probe as shown in the figure. ZBP1 expression levels in the entire cortical region of the mice with the indicated genotypes were determined using H-scores. Images were recorded using confocal microscopy. Images are representative. Four to five male mice were used for each genotype (1 to two cortical sections per mouse). Genotypes were color-coded consistently across all figures. Quantification was performed using the HALOFISH module. *P<0.05; **P<0.01; ***P<0.001; ns, not significant. Data are expressed as mean ± SEM.

[0117] Figure 3. INPP5D deficiency promotes RIPK1 kinase-dependent transcription of multiple LOAD risk genes in microglia in response to stress and aging.

[0118] (A) A heatmap analysis was performed on the expression levels of 17 LOAD risk genes with different genotypes in cultured newborn microglia (genotype color coding is shown in Figure 1B). Low expression is shown in blue, and high expression is shown in red.

[0119] (B) Quantitative PCR analysis was performed to determine the expression of the selected LOAD risk genes in neonatal microglia cultures with the indicated genotypes. N = 4.

[0120] (C) Western blot analysis of protein products of selected LOAD risk genes (TREM2, APOE, ADAM17, CTSD, and CTSH) in cultured neonatal microglia with specified genotypes. The right figure shows a semi-quantitative analysis of Western blots using ImageJ, with Actin as an internal control for normalization.

[0121] (DI) The RNAscope probe Aif1 (encoding the gene for IBA1) was used as a microglial marker, and in situ RNAscope treatment was performed using probes for ApoE, Trem2, Plcg2, Ctsb, and Clu. IBA1ab immunostaining was used as a microglial marker, and in situ RNAscope probe treatment was performed on GRN. Quantification was performed on the entire cortical region from 1-2 cortical sections from each mouse (4-5 males per genotype). The expression levels of ApoE (D), Trem2 (E), Ctsb (F), Plcg2 (G), GRN (H), and Clu (I) in the entire cortical region of the specified genotype mouse were quantified using H-scores. Images were recorded by confocal microscopy, and representative images are shown. Quantification was performed using the HALOFISH module. *P<0.05; **P<0.01; ***P<0.001; ns, not clinically significant. Data are expressed as mean ± SEM.

[0122] Figure 4. Gene expression characteristics of microglia regulated by RIPK1 kinase in human AD microglia.

[0123] (A) The bubble chart shows the enrichment analysis of microglia clusters associated with differentially expressed genes regulated by RIPK1 in Sun et al.’s article 202314.

[0124] (B) A heatmap of significantly different clusters enriched in A, namely MG10 inflammation III and MG4 lipid treatment.

[0125] (C) The bubble chart shows the microglia cluster enrichment analysis associated with differentially expressed genes regulated by RIPK1 in the article by Green et al. 202415.

[0126] (D) The heatmap shows the clusters with significant differences in (C), namely Mic.15 inflammation, Mic.12 and Mic.13 lipid treatment.

[0127] Figure 5. Pharmacological inhibition of RIPK1 can block the development of the critical time window caused by Inpp5d deficiency in myeloid mice of 5×FAD.

[0128] A. Administer 5×FAD via drinking water; Inpp5d fl / fl Lyz2 cre / + Male mice (4 to 6 months old) were injected with Nec-1s.

[0129] BF.ELISA analysis of total Aβ42 (B), DEA-soluble Aβ42 (C), DEA-insoluble Aβ42 (D), sarkosyl-soluble Aβ42 (E), and sarkosyl-insoluble Aβ42 (F) in mouse brains treated with the vector or Nec-1s, as shown in (A).

[0130] Representative images (G) and quantification (HI) of Aβ plaque area and size in mice treated with GI, vector, or Nec-1s (A). Reference bar = 200 μm.

[0131] JK. 5×FAD treated with vector or Nec-1s; Inpp5d fl / fl Lyz2 cre / + Representative images (J) and quantification (K) of CD3-positive cells in DG hippocampal sections from male mice are shown in Figure 4A. Reference bar = 5 μm.

[0132] LM. 5×FAD treated with carrier or Nec-1s; Inpp5d fl / fl Lyz2 cre / + Representative images (L) and quantification (M) of dendritic spines in male mouse DG hippocampal neurons (8-10 neurons / mouse), as shown in Figure 6H-I. Reference bar = 5 μm.

[0133] NR. 5×FAD for vector or Nec-1s treatment; Inpp5d fl / fl Lyz2cre / + male mice were used in the Morris water maze test, with escape latency (N), total distance (O), average speed (P), percentage of distance within the target quadrant (Q), and duration within the target quadrant (R) as shown in (A). Each group had n ≥ 14 male mice. Data are expressed as mean ± SEM. Unpaired t-tests were performed; *, P < 0.05; **, P < 0.01.

[0134] Figure 6. INPP5D deficiency in myeloid and microglia promotes RIPK1-dependent myelination disorder and loss of motor neurons in aged mice. After 45 days of treatment with the vector or Nec-1s, the following behavioral tests were performed on 8-month-old mice of the specified genotype:

[0135] (A) Open field test. The total distance traveled, average speed, number of vertical backward steps, and duration of movement in the central area were recorded over one hour for statistical analysis. Each data point represents one mouse. Male mice, Inpp5d. fl / fl WT mice, N=19; Inpp5d fl / fl Lyz2 cre / + Mice, N=18; Inpp5d fl / fl Lyz2 cre / + Mice, N=14. Mean ± SD. *p<0.05,**p<0.01,***p<0.001,ns, no statistical significance.

[0136] (B) Rotation test. Each group consisted of 15 male mice (N=15). Mice were administered the drug or Nec-1s at the indicated dose.

[0137] (C) Grip strength test. Male mice: Inpp5d fl / fl WT mice, N=18; Inpp5d fl / fl Lyz2 cre / + Mice, N=11; Inpp5d fl / fl Lyz2 cre / + Mice, N=13.

[0138] (DE) Immunostaining was performed on paraffin sections of the lumbar spinal cord of 8-month-old mice using ChAT(D) and NeuN(E) immunostaining. Motor neuron counts were quantified (n = 4-5 mice per genotype, male). Mean ± SEM. **P<0.01, ***P<0.001.

[0139] (F) The same samples as in (DE) were prepared, and EM analysis was performed on the myelination of motor axons in the lateral lumbar spinal cord. The percentage of myelinated axons per square millimeter, the average number of axons, and the average axon diameter in the white matter of the ventrolateral lumbar spinal cord of mice of specified genotypes were quantified. Two mice were used for each genotype. Mean ± SEM.

[0140] Figure 7. INPP5D binds directly to RIPK1, inhibiting RIPK1 activation in an SH2-dependent manner.

[0141] (A) WT and sgINPP5DKOBV2 cells were lysed with RIPA buffer after treatment with Nec-1s (10 μM) for 12 hours. The soluble fraction was co-immunoprecipitated with antibodies against p-RIPK1 (S166), p-RIPK1 (S415), and p-RIPK1 (S14 / 15), and then subjected to Western blotting as shown.

[0142] (B) Immunoprecipitate the NP-40 lysates of WTBV2 and sgINPP5DBV2 cells with RIPK1 antibody, and then perform Western blot analysis as shown.

[0143] (C) HEK293T cells were co-transfected with different amounts of HA-INPP5D and Myc-RIPK1 for 12 h, and then lysed after treatment with the vector or Nec-1s (10 μM). The cell lysates were analyzed by Western blot according to the instructions.

[0144] (D) HEK293T cells were co-transfected with Myc-RIPK1 and HA-INPP5D or HA-INPP5D-D676G plasmids for 12 hours, followed by lysis after treatment with the vector or Nec-1s (10 μM) for 12 hours. The lysate was analyzed by Western blot according to the instructions.

[0145] (E) HEK293T cells were co-transfected with HA-RIPK1 and Flag-INPP5D or Flag-△SH2-INPP5D plasmids for 24 hours before cell lysis. Cell lysates were immunoprecipitated with agarose conjugated with anti-HA antibody, and the immune complexes were analyzed by Western blotting.

[0146] (F) HEK293T cells were co-transfected with HA-RIPK1 and Flag-INPP5D or Flag-△SH2-INPP5D plasmids for 24 h, and then the cells were lysed and p-S166RIPK1 was analyzed by Western blot.

[0147] (G) RIPK1KOHEK293T cells were co-transfected with Flag-INPP5D and HA-RIPK1 or HA-RIPK1-Y383F plasmids for 24 hours before cell lysis. Cell lysates were immunoprecipitated with agarose conjugated with anti-HA antibody and analyzed by Western blot as shown.

[0148] (H)RIPK1KOHEK293T cells were transfected with the expression plasmid shown 24 hours prior to harvest. Co-precipitation was performed using anti-HA, followed by Western blotting using anti-Myc antibody to assess the interaction between HA-RIPK1 and Myc-RIPK1.

[0149] (I) Overall structure of the mINPP5D-SH2 / mRIPK1(378-393)-pY383 peptide complex determined by solution NMR. The interaction mode adopts a typical SH2 binding mode. The positively charged pocket formed mainly by R15, R34, and R57 recognizes RIPK1 p-Y383. The hydrophobic pocket formed by Y56, V68, A70, Y89, and V97 wraps residue F386 at the +3 position (p-Y383 is defined as the 0 position). The SH2 domain is shown as an electrostatic surface, the mRIPK1 peptide is shown as a cyan rod, and the phosphotyrosine residues p-Y383 and F386 at the 0 and +3 positions are marked in red.

[0150] (J) A close-up of the interaction between the p-Y383 peptide and the three arginine residues (R15, R34 and R57) of the SH2 domain, showing that R15 and R34 can form hydrogen bonds with the phosphate group of p-Y383 (red dashed line), while the side chain of R57 binds to the aromatic ring of p-Y383 through a cation-π interaction (yellow ring).

[0151] (K)INPP5DSH2 contains a deep hydrophobic pocket formed by side chains Y56, V68, A70, Y89 and V97, which encloses the aromatic ring of F386 at the +3 position of RIPK1.

[0152] (L) Sequence alignment of RIPK1 in different mammalian species shows a conserved aromatic residue at position +3.

[0153] (M) Immunoprecipitation analysis of INPP5D-RIPK1 interaction in RIPK1KOHEK293T cells co-transfected with Flag-INPP5D and HA-RIPK1 or HA-RIPK1-Y386S plasmids 24 hours before cell lysis.

[0154] Figure 8. Inpp5d deficiency in bone marrow of 5×FAD mice promotes age-dependent Aβ spikelet increase.

[0155] A. Total Aβ42 peptide levels extracted from the brains of male mice (3, 5.5, and 8.5 months old) labeled with H using 5 M GuHCl.

[0156] B. Extract the total amount of Aβ40 peptide from the brains of male mice (3 months, 5.5 months, and 8.5 months old) with 5 M Gu HCl using H.

[0157] Total Aβ42 peptide levels in the brains of male mice of genotype C (3, 4, 5, 5.5, 6, 8, 8.5, and 10 months of age).

[0158] D. Compared with age-matched 5×FAD mice (3, 4, 5, 5.5, 6, 8, 8.5, 10 months old), the total content of Aβ42 peptide in the brains of the male mice of the shown genotype was the same as in C, with n = 5 mice in each group.

[0159] DEA insoluble Aβ42 (E) and soluble Aβ42 (F) in the brains of male mice (4, 6, 8 and 10 months old) with the genotype shown in EF.

[0160] G. Frozen mouse brain homogenized with 2% sarkosyl. Representative image of Aβ42 fibers.

[0161] Sarkosyl soluble Aβ42 fibers in the brains of male mice (3, 5.5, and 8.5 months old) with the genotype shown in H.

[0162] I. Sarkosyl insoluble Aβ42 fibers in the brains of male mice (3, 5.5, and 8.5 months old) of the shown genotype, with n = 5 mice in each group.

[0163] Representative images of Aβ plaques (J) in the brains of male mice (4, 6, and 9.5 months of age) shown in JK., and quantification of the percentage of Aβ plaque-positive area (K) stained with 6E10 antibody. Scale bar = 200 μm.

[0164] Quantitative analysis of Aβ plaque size and number in LN.4-month-old (L), 6-month-old (M), and 9.5-month-old (N) mice is shown in Figure J. Data are expressed as mean ± SEM. Unpaired t-tests were performed: *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

[0165] Figure 9. Inpp5d deficiency in bone marrow of 5×FAD mice promotes RIPK1-mediated microglia activation.

[0166] A. Representative images of non-plaque-associated microglia in the hippocampus of 6-month-old male mice of a specified genotype, immunized with IBA1 and CD68 antibodies. Reference bar = 40 μm.

[0167] BC. As in (A), the quantitative analysis of non-plaque-associated microglia, the volume ratio of IBA1+ microglia (B) to CD68 (C) was determined by using Inpp5d fl / fl Volume of indicator microglia in the hippocampus of male WT mice divided by Inpp5d fl / fl The volume of indicator microglia in the hippocampus of male WT mice was derived.

[0168] D. Shore analysis of non-plaque-associated microglial morphology, as in (A). Two-way ANOVA. ***, p<0.001; ****, p<0.0001.

[0169] E. Representative images of microglia associated with hippocampal plaques in male mice, shown in Figure (A), immunostained with antibodies against 6E10, IBA1, and CD68. Reference bar = 20 μm.

[0170] FH. Quantitative analysis of the volume ratios of plaque-associated microglia (IBA1) to (F), CD68 volume (G), and Aβ plaques (H) in the (E) sample. The volume ratios were obtained by dividing the volume of a mouse of a specified color genotype by the volume of a 5 × FAD mouse.

[0171] Representative images (I) and quantification of activated microglia in hippocampal plaque-associated microglia of male IL-1 mice (9.5 months old) showing IBA1 and CD68 immunostaining (J, K) and Aβ plaque 6E10 immunostaining (L). Color representation of IBA1+ microglia and CD68 versus Inpp5d in the hippocampus of male mice. fl / fl The volume ratio (JK) of IBA1+ microglia to CD68 cells in the hippocampus of WT mice. The volume ratio of Aβ plaques was obtained by dividing the hippocampal 6E10+ volume of color indicator genotype mice by the hippocampal 6E10+ volume of 5×FAD mice (L), with n≥5 mice per group. Reference bar = 40 μm.

[0172] Immunostaining with ASC and 6E10 antibodies was used to image (M) and quantify (N) ASC+Aβ plaques in the hippocampus of male mice (9.5 months old) of the indicated genotype. Reference bar = 200 μm. (N) Quantitative analysis of the ASC / Aβ plaque volume ratio, as shown in (M), was performed by dividing the volume of ASC+ / 6E10+ double-positive Aβ plaques by the volume of ASC+ / 6E10+ double-positive Aβ plaques in 5×FAD mice. Data are expressed as mean ± SEM. Unpaired t-tests were performed: *, p < 0.05; **, p < 0.01; ****, p < 0.0001.

[0173] Figure 10. Inpp5d deficiency in bone marrow of 5×FAD mice promotes RIPK1-mediated peripheral inflammation.

[0174] A. with 5×FAD; Inpp5d fl / fl Lyz2 cre / + Compared to male mice, age-matched 5×FAD; Inpp5d fl / fl;Lyz2cre;GO biological process analysis of RIPK1-D138N-rescued DEGs (545 genes) in PBMCs of male Ripk1D138N / D138N mice, and in age-matched 5×FAD;Inpp5d fl / fl Expression of ;Lyz2cre;Ripk1D138N / D138N mice in PBMCs was restored. n = 4 mice (6 months old) / group.

[0175] B. Heatmap analysis of inflammatory signaling pathways and DEGs(A) in AD risk genes.

[0176] CE. Protein levels of Psen1 and ApoE in PBMCs were analyzed by Western blot (C) and ImageJ quantification (DE).

[0177] FL. The levels of CCL12(F), CCL2(G), CSF3(H), IL-16(I), CXCL9(J), IL-6(K), and IL-1β(L) in the plasma of male mice (3.5 or 6 months of age) with specified genotypes were analyzed using Olink proteomics technology, with n = 4 mice per group. Data are expressed as mean ± SEM. Unpaired t-tests were performed: *, p < 0.05; **, p < 0.01; ***, p < 0.001.

[0178] Figure 11. Bone marrow Inpp5d deficiency increases RIPK1-mediated T cell infiltration in the central nervous system.

[0179] AC. Representative images (A) and number of CD3-positive cells in hippocampal dentate gyrus (DG) sections (2 sections / mouse) of specified genotype male mice at 4 months of age (B) and 6 months of age (C). Reference bar = 40 μm.

[0180] D. Representative flow cytometry images analyzing the presence of T cells and B cells in the brain at 6 months of age.

[0181] EF. At 6 months of age, it represents the image (E) and the percentage of CD45high to CD45total (H).

[0182] GJ. Percentage of CD45high in CD45total CD3+ T cells (G), CD45high in CD45total CD4+ T cells (H), CD45high in CD45total CD8+ T cells (I), and CD45high in CD45total CD19+ B cells (J) in 6-month-old male mice. DJ, n≥6 male mice per group. Data are expressed as mean ± SEM. Unpaired t-tests were performed: *, p<0.05; **, p<0.01; ***, p<0.001.

[0183] Figure 12. Myeloid Inpp5d deficiency promotes γ-secretase activity via RIPK1.

[0184] AB. BV2 control cells and Inpp5dKOBV2 cells were incubated with FL-APP-GFP-expressing HEK293T cells for 16 hours using zVAD, Nec-1s, or the γ-secretase inhibitor DAPT. The products were analyzed by Western blotting with anti-GFP (A) and quantified using ImageJ (B).

[0185] CE. Representative images (C) of directly reprogrammed human senescent forebrain cholinergic neurons (normal neurons) and directly reprogrammed human AD forebrain cholinergic neurons (AD neurons) incubated with BV2-control cells and BV2-Inpp5dKO-3# cells for 48 hours and immunostained with Aβ1-42 and MAP2 antibodies. Reference bar = 40 μm.

[0186] (D) Quantitative analysis of Aβ42 point in human neurons directly reprogrammed after 48 hours of treatment with BV2-Inpp5dKO-3# cell culture medium.

[0187] (E) Quantitative analysis of Aβ42 levels in BV2-Inpp5dKO-3# cell culture medium after treatment with directly reprogrammed human neurons for 48 hours. Data are expressed as mean ± SEM. Unpaired t-tests were performed: *, p < 0.05; **, p < 0.01; ****, p < 0.0001.

[0188] Figure 13. Inhibition of RIPK1 kinase can rescue sleep disorders and memory impairment in myeloid Inpp5d-deficient 5×FAD mice.

[0189] A. Heatmap analysis of clock gene expression levels in neurons of specified genotype mice at 5.5 months of age.

[0190] BC. Western blot analysis and ImageJ quantitative analysis of Bmal1 protein levels in the cortex of male mice, as shown in (A).

[0191] Distribution of REM sleep time over 24 hours in D.5.5-month-old mice. Two-way ANOVA.

[0192] Quantitative analysis of the proportion of REM sleep time in E.5.5-month-old mice under light and dark conditions.

[0193] F. Quantitative analysis of the number of REM sleep episodes under light at 5.5 months of age. DF, control group n=3 mice, other groups n≥7 mice per group.

[0194] GI. EGFP-AAV injection strategy (G). Representative images (H) and quantification results (I) of DG hippocampal dendritic spines in genotyped mice (8-10 neurons / mouse). n≥4 male mice per group.

[0195] JM. Training strategy in quadrant 1 (J). Percentage of male mice (5.5 months old) of the indicated genotype in distance to target quadrant (K), duration of target quadrant (L), and frequency of plateau region (M).

[0196] NQ. Training strategy in the third anti-quadrant (N). Same percentage of mouse tests as (JM) in terms of distance to the target quadrant (O), duration in the target quadrant (P), and frequency of plateaus (Q).

[0197] JQ, each group n≥13 male mice. Data are expressed as mean ± SEM. Unpaired t-test, *, p<0.05. **, p<0.01.***, p<0.001.

[0198] Figure 14. RIPK1-dependent pro-inflammatory cytokines produced by Inpp5d-deficient microglia in senescent and stress cultures.

[0199] (A) List of INPP5Dz-score values ​​obtained from siRNA screening of L929 cells.

[0200] (B) L929 cells were transfected with siRNA (control siRNA or INPP5D siRNA) or complementary plasmid (lenti-Vector or Lenti-INPP5D) using LipoJet reagent for 48 hours. Subsequently, cells were treated for 4 hours with TNFα (50 ng / ml), TNFα (20 ng / ml) + SM-164 (50 nM), TNFα (10 ng / ml) + SM-164 (25 nM) + z-VAD.fmk (50 μM), or z-VAD.fmk (100 μM). Cell viability was assessed using the CellTiter-GloATP assay. The knockout efficiency of INPP5D siRNA was confirmed by RT-PCR. ****P < 0.0001, data are expressed as mean ± SEM.

[0201] (C) INPP5D gene knockout BV2 cells were constructed using CRISPR / Cas9 technology. The efficiency of INPP5D gene knockout was verified by Western blotting.

[0202] (D) WT and INPP5D gene knockout BV2 cells were supplemented with wild-type (WT) and enzyme-free D676G-INPP5D via lentivirus. These cells were treated with Nec-1s (10 μM) for 12 h, followed by cell lysis to extract RNA, and the mRNA levels of the cytokines were measured using Q-PCR. ****p<0.0001. Data are presented as mean ± SEM.

[0203] (E) BV2 cells with WT and INPP5D gene knockout were supplemented with wild-type (WT) and enzyme-free D676G-INPP5D via lentivirus. These cells were treated with LPS (20 ng / ml) for 30 min and pretreated with Nec-1s for 12 h. The supernatant was collected for ELISA to detect protein levels of inflammatory factors. ****p<0.0001. Data are presented as mean ± SEM.

[0204] (F) Flowchart of a primary microglia screening protocol for generating potential AD risk genes in vivo via CRISPR / Cas9.

[0205] (G) Primary microglia with the INPP5D gene knocked out were generated. The isolated microglia were then treated with LPS (20 ng / ml) for 12 hours, and the supernatant was collected for ELISA to detect the protein levels of inflammatory factors. **p<0.01, ***p<0.001;

[0206] (H) Schematic diagram of constructing the Inpp5d conditional allele using CRISPR / Cas9 technology.

[0207] (I) Separation of Inpp5d fl / fl ;WT and Inpp5d fl / fl Lyz2 cre / + Primary microglia were then treated with Nec-1s (10 μM) for 24 hours, and the total lysate was collected and Western blotted using the specified N- and C-terminal INPP5D antibodies.

[0208] (J) The distribution of cytokines in the brain and spinal cord of 8-month-old mice was determined by qPCR (n=3, mean ± SEM). *P<0.05, **P<0.01, ***P<0.001.

[0209] (K) Primary microglia of the specified genotype were treated with the vector or Nec-1s (10 μM) for 24 hours, and the supernatant of the primary microglia was collected. The protein levels of inflammatory factors were detected by ELISA. **p<0.01,***p<0.001.

[0210] (L) The distribution of cytokines in the brains of 8-month-old mice (N=3 mice per genotype or treatment) 45 days after Nec-1s administration was detected by qPCR.

[0211] (MN) Frozen cerebral cortex sections specifying genotype and age (M for 2-week-old mice, N for 8-month-old mice). The right side shows the quantification of microglia (IBA1+) and p-RIPK1 (S166).

[0212] Immunostaining of (M) p-RIPK1(S166) and IBA1 in frozen cortical sections from mice on day 14 (P14). Immunofluorescence (IF) signals of p-RIPK1(S166) and IBA1 in two intact cortical sections from each mouse (3–4 males) on day 14 were quantified using the HALO Area Quantification FL module. *P<0.05; **P<0.01; ***P<0.001; ns, no clinical significance. Data are expressed as mean ± SEM. Representative images for each genotype are shown.

[0213] (N) Immunofluorescence staining of IBA1 and p-RIPK1(S166) in frozen cortical sections of 8-month-old genotype mice. ImageJ was used to quantify the signals of IBA1 and p-RIPK1(S166). *P<0.05; **P<0.01; ***P<0.001; ns, no clinical significance. Data are expressed as mean ± SEM. N = 3-4 male mice per group (4 cortical sections per mouse). Representative images for each genotype.

[0214] Figure 15. Myeloid INPP5D deficiency promotes the transcription of multiple pro-inflammatory factors mediated by RIPK1 in microglia, and this increase with age.

[0215] (A) Newborn primary microglia of the specified genotype were treated with a vector or Nec-1s (10 μM), and immunofluorescence staining was performed using p-RIPK1(S166) and DAPI to determine the percentage of p-RIPK1(S166)+ microglia on each section (N=5). *p<0.05.**p<0.01.

[0216] (B) Volcano map analysis shows that from Inpp5d fl / fl Lyz2 cre / + The number of differentially expressed genes statistically significant in neonatal microglia isolated from mouse and wild-type mouse cultures (614 genes upregulated, 72 genes downregulated). Fold change > 1.5; adjusted P < 0.05.

[0217] (C) Heatmap analysis showed that RIPK1 inactivation via the Ripk1-D138N mutation restored the expression of 482 out of 614 upregulated genes and 21 out of 72 downregulated genes in (B) to normal levels in WT microglia (DEGs; >1.5-fold change; adjusted P-value: <0.05). Genotypes are indicated by the color on the right and are used consistently throughout this manuscript.

[0218] (DE) In situ RNAscope treatment was performed on frozen cortical sections of young mice (14 days old) with specified genotypes using Cybb or C1qa probes and Aif1RNAscope probes as microglia biomarkers (DE).

[0219] RNAscope was detected in situ using probes for C3 (F), MyD88 (G), Nlrp3 and Aim2 (H), GSDMD (I), Irgm1 (J), IFN, and ZBP1 (K) in (F, G, J). In (F, G, J), IBA1ab immunostaining was used as a biomarker for microglia. In (D, E, H, I, K), the RNAscope probe for Aif1 (IBA1) was used as a biomarker for microglia. Expression levels of the specified probes were determined in the form of H-scores throughout the cortical sections. Images were recorded by confocal microscopy. Images are representative. Four to five male mice were used for each genotype. Quantification was performed using the HALOFISH module. *P<0.05; **P<0.01; ***P<0.001; ns, no clinical significance. Data are expressed as mean ± SEM.

[0220] Figure 16. Myeloid INPP5D deficiency promotes RIPK1 kinase-dependent transcription of multiple LOAD risk genes in microglia in response to stress and aging.

[0221] (AE) Microglia were biomarked using the Aif1RNAscope probe (AC, E) and microglia were biomarked using the GRNRNAscope probe and IBA1ab immunostaining (D). Expression levels of designated genes throughout the cortical sections were quantified as H fractions. Images were recorded using confocal microscopy. The images shown are representative. Four to five male mice of each genotype were used (1 to two cortical sections per mouse). Quantification was performed using the HALOFISH module. *P<0.05; **P<0.01; ***P<0.001; ns, not statistically significant. Data are expressed as mean ± SEM.

[0222] Figure 17. RIPK1 kinase-regulated microglial gene expression characteristics were found in human AD microglia.

[0223] (A) Heatmap of clusters with significant differences in Figure 4A, namely MG5 phagocyte clusters and MG11 antiviral clusters.

[0224] (B) Figure 4C shows a heatmap of gene clusters with significant differences, namely Mic.6: reaction and Mic.14: IFN reaction.

[0225] Figure 18. INPP5D deficiency in human iPSC-derived microglia promotes RIPK1 kinase-dependent transcription of multiple LOAD risk genes and pro-inflammatory factors.

[0226] (A) Immunofluorescence staining of IBA1 and tmem119 in iPSC-derived microglia. The image shown is a representative one.

[0227] (B) iPSC-derived microglia were transfected with control siRNA or INPP5D siRNA for 48 hours, followed by lysis with RIPA buffer and Western blotting. As shown in the figure, phosphorylation of p-S166RIPK1 was analyzed by Western blotting. The knockout efficiency of INPP5D siRNA was confirmed by Western blotting of INPP5D.

[0228] (C) Control group siRNA or INPP5D siRNA was transfected into healthy iPSC-derived microglia for 48 hours, followed by cell lysis with Trizol reagent and large-scale RNA-seq. The volcano plot on the left shows the number of statistically significant differentially expressed genes between siINPP5D and NC (1018 genes upregulated, 1242 genes downregulated). The volcano plot on the right shows the number of statistically significant differentially expressed genes between siINPP5D and siNC treated with Nec-1s (669 genes upregulated, 492 genes downregulated, fold change > 1.5; adjusted P < 0.05). Heatmap analysis shows all differentially expressed genes (DEGs) between siINPP5D and siNC (change > 1.5-fold; adjusted P < 0.05).

[0229] (D) Heatmap analysis showed that inactivation of RIPK1 by Nec-1s treatment restored the expression levels of the 19 LOAD risk genes, cytokine production, ROS metabolism, inflammasome activation, complement, and IFN responses shown. These genes were differentially expressed in the IPSC-derived microglia after the treatment shown. Low expression is shown in blue, and high expression is shown in red.

[0230] Figure 19. Mice lacking INPP5D in myeloid and microglia exhibit age-dependent motor dysfunction mediated by RIPK1.

[0231] (A) Open-field movement and anxiety-related behavioral tests were performed on 6-month-old mice of a specified genotype. Total distance traveled, average speed, number of vertical climbs, and duration of central area movement were recorded during the 1-hour experiment for statistical analysis. Each data point represents one male mouse. Male mice: Inpp5d fl / fl WT mice, N=8; Inpp5d fl / + Lyz2 cre / + Mice, N=14; Inpp5d fl / fl Lyz2cre / + mice, N=7. Mean ± SD. *p<0.05,**p<0.01,***p<0.001, ns, no statistical significance.

[0232] (B) The Y-maze test was performed on mice from the same group as in (A). The total distance traveled, spontaneous alternation performance (SAP), alternating arm return (AAR), and same arm return (SAR) were recorded for each mouse during the 10-minute test period. Each data point represents one male mouse. Male mice, Inpp5d. fl / fl WT mice, N=6; Inpp5d fl / + Lyz2 cre / +Mice, N=16; Inpp5d fl / fl Lyz2cre / + mice, N=6. Mean ± SD. *p<0.05,**p<0.01,***p<0.001, ns, no statistical significance.

[0233] (C) Mice from the same group as in (A) underwent an elevated maze test, showing the percentage of time spent on the open and closed arms within 5 minutes. Male mice: Inpp5d fl / fl WT mice, N=9; Inpp5d fl / + Lyz2 cre / + Mice, N=16; Inpp5d fl / fl Lyz2 cre / + Mice, N=10. Mean ± SD, *p<0.05, **p<0.01, ***p<0.001, ns, no statistical significance.

[0234] (D) The same group of mice as in (A) underwent a rotation test. The average time it took for the mice to fall from the rotating bar was recorded across three experiments. Male mice: Inpp5d fl / fl WT mice, N=14; Inpp5d fl / + Lyz2 cre / + Mice, N=21; Inpp5d fl / fl Lyz2 cre / + Mice, N=8. Mean ± SD, *p<0.05, **p<0.01, ***p<0.001, ns, no significant difference.

[0235] (E) Body weight of the genotype mice shown in this study.

[0236] (F) Perform a tail-lifting and hindlimb clamping test on 8-month-old mice of the specified genotype and take photographs.

[0237] (G)Y maze test. Male mice: Inpp5d fl / fl WT mice, N=19; Inpp5d fl / fl Lyz2 cre / + Mice, N=13; Inpp5d fl / fl Lyz2 cre / + Mice, N=15. Total distance, spontaneous alternation performance (SAP), alternating arm rotation (AAR), and same arm rotation (SAR) were recorded during a 10-minute test for statistical analysis.

[0238] Figure 20. Microglia INPP5D deficiency mice exhibit RIPK1-mediated age-dependent motor dysfunction.

[0239] (A) Adult Inpp5d with or without INPP5D deficiency induced by tamoxifen fl / fl Microglia were isolated from Cx3cr1creERT2 / + mice (8 weeks old) and analyzed by Western blotting.

[0240] (B) Average weight of the mice used in Figures 8E-F.

[0241] (CD) The same brain and spinal cord samples used in Figure 6D were subjected to CD68 (C) and p-RIPK1 (S166) immunostaining.

[0242] (EF) Four-week-old mice with the specified genotype were treated with tamoxifen to induce INPP5D deletion. The mice were then treated with Nec-1s for 45 days and subjected to open field testing at 12 months of age. (E) Total distance traveled during the 1-hour experiment was recorded for statistical analysis. Each data point represents one mouse. Male mice with INPP5D fl / fl WT mice, N=10; Inpp5d fl / fl Cx3cr1 creERT2 / + Mice, N=8; Inpp5d fl / fl Cx3cr1 creERT2 / + Mice, N=6. Mean ± SD. *p<0.05,**p<0.01,***p<0.001,ns, no statistical significance.

[0243] (F) Mice from the same group as (E) underwent a rotation test. Male mice: Inpp5d fl / fl WT mice, N=10; Inpp5d fl / fl Cx3cr1 creERT2 / + Mice, N=8; Inpp5d fl / fl Cx3cr1 creERT2 / + Mice, N=6. Mean ± SD. *p<0.05,**p<0.01,***p<0.001,ns, no statistical significance.

[0244] Figure 21. Mice lacking INPP5D in myeloid and microglia exhibit age-dependent loss of motor neurons mediated by RIPK1.

[0245] (A) Nuclear immunostaining of NeuN in cortical sections using DAPI. The number of NeuN-positive cells in the cortex of mice of specified genotypes was analyzed and quantified (4 or 5 mice of each genotype, 2 sections per mouse, male).

[0246] (B) At 4 weeks of age, mice were induced to lose INPP5D in microglia with tamoxifen and treated with the drug or Nec-1s for 45 days, followed by testing at 12 months of age. Frozen sections of the lumbar spinal cord were immunostained with ChAT and DAPI to stain nucleoli and quantify motor neurons (n ​​= 4 or 5 mice / group, 2 sections per mouse, male). Mean ± SEM. **P < 0.01, ***P < 0.001.

[0247] (C) and (B) mouse lumbar spinal cord sections of the genotypes shown were used for immunostaining of lysed asparagine-3 and DAPI-stained cell nuclei. The number of lysed asparagine-3 positive cells in the sections was counted and quantified (3 mice per genotype, 3 sections per mouse, male). Mean ± SEM. **P<0.01, ***P<0.001.

[0248] (D) Nuclear immunostaining of TDP-43 and DAPI in lumbar spine sections as in (C). The percentage of cells with cytoplasmic TDP-43 inclusion bodies in the lumbar spinal cord of mice of specified genotypes was analyzed and quantified relative to the total DAPI signal (3 mice per genotype, 3 sections per mouse, male).

[0249] (E) Combined immunostaining of TDP-43 and NeuN with DAPI in the nuclei of lumbar spinal cord cells, as shown in (D).

[0250] (F) Immunostaining was performed on frozen sections of the lumbar spinal cord and brain of mice at 8 months of age with pTDP-43 and NeuN. Immunofluorescence (IF) signals of pTDP-43 and NeuN were quantified using the HALO Area Quantification FL module (4–5 male mice). *P<0.05; **P<0.01; ***P<0.001; ns, no clinical significance. Data are expressed as mean ± SEM. Representative images for each genotype are shown in the figure.

[0251] Figure 22. INPP5D directly inhibits the activation of RIPK1 kinase.

[0252] (A) Experimental design for identifying RIPK1 phosphorylation sites by mass spectrometry (MS). Endogenous RIPK1 was isolated from control sgGFPWT BV2 cells, sgINPP5D KOBV2 cells, and sgINPP5D KO cells treated with Nec-1s (10 μM) or the vector for 12 hours by anti-RIPK1 affinity purification and analyzed by mass spectrometry.

[0253] (B) Table shows the changes in RIPK1 phosphorylation sites in control sgGFP and INPP5D gene knockout BV2 cells under Nec-1s treatment or no treatment, as detected by mass spectrometry.

[0254] (C) Control sgGFPWTBV2 cells and sgINPP5DKOBV2 cells were lysed with RIPA buffer after treatment with Nec-1s (10 μM) for 12 hours. Phosphorylation of p-S166 and p-S321RIPK1 was analyzed by Western blotting. The interaction between RIPK1 and INPP5D was analyzed by Western blotting after immunoprecipitation with RIPK1 antibody.

[0255] (D) HEK293T cells were co-transfected with Flag-INPP5D and HA-RIPK1 or HA-RIPK1-Y383F expression plasmids for 24 hours. Cells were then lysed with NP-40 buffer, and the lysate was incubated with purified recombinant protein GST-INPP5D-SH2 (aa1-107). Western blotting was performed using anti-HA antibody to analyze the traction effect of GST.

[0256] (E) HA-RIPK1-p-Y383 or HA-RIPK1-Y383 peptides were loaded onto HA beads as bait proteins to attract recombinant His-SH2-INPP5D (aa1-107). After washing with PBS, the binding proteins were eluted with elution buffer, separated by SDS-PAGE, and stained with Kumasi Brilliant Blue R250.

[0257] (F) Control sgGFPWT BV2 cells and sgINPP5D KOBV2 cells were lysed with NP-40 buffer. Cell lysates were incubated with streptavidin beads loaded with either biotin-RIPK1-p-Y383 or the control biotin-RIPK1-Y383 peptide, and Western blot analysis was performed using an anti-INPP5D antibody. Only biotin-RIPK1-p-Y383 could attract INPP5D.

[0258] (G)RIPK1-p-Y383 peptide binding increased the thermal stability of INPP5D-SH2 (aa1-107) protein. Melting curves of INPP5D-SH2 mixed with phosphorylated (HA-p-Y383) or unphosphorylated (HA-Y383) RIPK1 peptides were measured using a real-time PCR system. In the control experiment, INPP5D-SH2 was mixed with the peptide solution, and the volume of H2O was the same as that of the peptide solution. The bar graph on the left shows that the HA-p-Y383 peptide, rather than the unphosphorylated peptide, increased the melting temperature (Tm) of INPP5D-SH2 by approximately 4.8 degrees Celsius. The right figure shows the Boltzmann melting curves (top) and derivative melting curves (bottom) of the control group (H2O, 5 replicates), the HA-Y383 group (4 replicates), and the HA-p-Y383 group (5 replicates) using the protein thermal transfer software (Applied Biosystems).

[0259] Figure 23. Direct interaction between the SH2 domain at the INPP5DN end and RIPK1.

[0260] (A) Two-dimensional 15N-HSQC spectra recorded using a 600MHz spectrometer were used to monitor the phosphorylation titration at Y383 of the 15N-labeled mINPP5DSH2 domain (residues 6-107) and the synthesized mRIPK1 fragment (residues 378-393). Specific peak shifts indicate a direct physical binding between the SH2 domain and the mRIPK1 fragment.

[0261] The residue-specific chemical shift changes in (B) and (A) reveal specific SH2 regions affected by chemical environmental perturbations and interactions. A threshold of 0.05 ppm (represented by horizontal lines) was used to select residues with significant perturbations for mapping onto the three-dimensional structure in (D).

[0262] (C) An ensemble of 20 low-energy composite structures calculated using NOE constraints shows good convergence of the interacting cores and a backbone rmsd of [value missing].

[0263] (D) Mapping of residues with chemical shift changes greater than 0.05 ppm (red) to representative complex structures in (C). Two key interacting residues, p-Y383 and F386, are shown in the figure.

[0264] (E) Sequence alignment of all reported mouse or human INPP5DSH2 domain binders. RIPK1 differs from other binders in that it has a bulky Tyr or Phe residue at the +3 position.

[0265] Figure 24. Deficiency of myeloid Inpp5d in 5×FAD mice leads to age-dependent Aβ lesions.

[0266] A. As shown in Figures 1A-D, after homogenizing the mouse olfactory bulb and cerebellum in 5M GuHCl, total Aβ40 and Aβ42 were extracted from the mouse brain for ELISA analysis.

[0267] B. Extraction strategy for Aβ42 from mouse brains after homogenization in 0.2% DEA and removal of the olfactory bulb and cerebellum. As shown in Figures 1E-F, DEA-soluble and DEA-insoluble Aβ42 were detected by ELISA.

[0268] Figures C and D show representative images (C) and quantitative data (D) of thioflavin S staining in the hippocampus of genotype-specific male mice (6 months old). Reference bar = 200 μm. Data are expressed as mean ± SEM. Unpaired t-tests were performed; *, p < 0.05; ***, p < 0.001.

[0269] Figure 25. Inpp5d deficiency in bone marrow of 5×FAD mice promotes neuroinflammation.

[0270] AB. Representative images (A) and quantification (B) of non-plaque-associated microglia in the hippocampus of male mice (4 months old) of a specified genotype using IBA1 antibody immunostaining. The volume ratio of IBA1+ microglia was obtained by dividing the volume of IBA1+ microglia in the hippocampus of male mice with Inpp5df / fWT cells by the volume of IBA1+ microglia in the hippocampus of male mice with Inpp5df / fWT cells. Reference bar = 40 μm.

[0271] CG. Representative images (C) and quantification (DG) of hippocampal astrocytes from male mice of specified genotypes immunostained with GFAP antibody. Reference bar = 100 μm. Quantification of GFAP+ area percentage and age-dependent changes (G) in hippocampal astrocytes of 4-month-old (D), 6-month-old (E), and 9.5-month-old (F) mice.

[0272] Data are expressed as mean ± SEM. Unpaired t-tests were performed. **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

[0273] Figure 26. Expression of key genes in PBMCs of RIPK1-D138N-rescued myeloid Inpp5d-deficient 5×FAD mice

[0274] A. The efficiency of knocking out Inpp5d in peritoneal cells of male mice (6 months old) with specified genotypes using Inpp5d c-terminal and n-terminal antibodies, with n ≥ 2 mice per group.

[0275] B. Males with the specified genotype 5×FAD; Inpp5d fl / fl Lyz2cre / + Heatmap analysis of all DGEs (1338) in age-matched male 5×FAD mice PBMCs compared to mice (6 months old), including 793 RIPK1-D138N non-rescue DEGs and 545 RIPK1-D138N rescue DEGs.

[0276] Analysis of GO biological processes by which C.RIPK1-D138N regulates genes related to cell activation.

[0277] Figure 27. The lack of Inpp5d in BV2 cells promotes inflammation similar to the lack of Inpp5d in 5×FAD mice.

[0278] A. Western blot confirmed that the INPP5D gene was knocked out in BV2 cells with Inpp5d gene knockout.

[0279] B. Inpp5dkoBV2 cells were incubated with GFP-beads for 1 hour, and phagocytic activity was quantified by the amount of GFP-beads taken up.

[0280] C. With or without Nec-1s (10 μM), BV2 control cells and BV2-Inpp5d KO-3# cells were treated with z-VAD (50 μM) for the time periods shown in the figure. Western blot analysis was performed on the cell lysates using the specified antibody.

[0281] D. DEGs from BV2-Inpp5dKO-3# cells and BV2 control group cells were mixed with 5×FAD; Inpp5d fl / fl Lyz2 cre / + DEGs in PBMCs of mice and 5×FAD mice were compared.

[0282] E. Perform GO biological process analysis on the overlapping DGEs in (D).

[0283] Heatmap analysis of DEGs in inflammation-related signaling pathways between F.BV2 control cells and BV2-Inpp5d KO-3# cells.

[0284] The G.hAPP-GFP fusion construct. The AICD-GFP fragment is released from APP via sequential cleavage mediated by α, β, and γ secretases.

[0285] H. HEK293T cells expressing hAPP-GFP were incubated with BV2 control cells or Inpp5d KOBV2 cells in the presence of the γ-secretase inhibitor DAPT (20 μM) for 16 hours. The level of AICD-GFP lysis products was detected by Western blotting using anti-GFP.

[0286] Data are expressed as mean ± SEM. Unpaired t-test, *, p<0.05.

[0287] Figure 28. Myeloid Inpp5d-deficient 5×FAD mice showed no defects in circadian rhythms of motor activity or learning at 5-6 months of age.

[0288] A. Heatmap analysis of the expression of 428 genes in neurons of male mice (5.5 months old) with the genotype shown in Figure A. These genes were restored to Inpp5d via the RIPK1-D138N allele. fl / fl WT level.

[0289] B.RIPK1-D138N rescued 428 DEGs from KEGG pathway analysis in A.

[0290] CD. Representative images (C) and quantitative data (D) used to measure the diurnal rhythm of locomotor activity in mice of a specified genotype (5.5 months of age).

[0291] E. At 5.5 months of age, mice of the specified genotype were subjected to electroencephalogram (EEG) measurements of the percentage of awake time over a 24-hour period (12:12 light:12 dark).

[0292] F. Quantitative analysis of the percentage of awake time (E) in mice of a specified genotype.

[0293] Distribution of NREM sleep time percentage in 24-hour (12:12 light-dark ratio) mice of G.5.5 months of age.

[0294] H. Quantitative analysis of the percentage of NREM sleep time (G) in mice of a specified genotype. EH, Inpp5d fl / fl WT, n=3 mice, other groups n≥7 mice.

[0295] I. Spatial learning and memory abilities of male mice (5.5–6 months old) of a specified genotype were assessed using the Morris water maze. Escape latency was quantified over five days of training.

[0296] Total distance (J) and average speed (K) of mice with the genotypes shown in (I) during the test period.

[0297] After L. reversal platform, mice were retrained for five days, and escape latency was recorded.

[0298] The total distance (M) and average speed (N) of the MN genotype mice in the reverse platform test.

[0299] IN. Each group consisted of ≥13 male mice. Data are expressed as mean ± SEM. Two-way ANOVA, ****, P<0.0001. Detailed Implementation

[0300] Through long-term and in-depth research, the inventors discovered that INPP5D deficiency leads to increased peripheral inflammation, promotes abeta production, and increases the production of free abeta fibers, thus exacerbating the pathology of Alzheimer's disease (AD). The RIPK1 inhibitor nec1 or the RIPK1 inactivation mutation RIPK1D138N can effectively improve INPP5D deficiency or inactivation, thereby demonstrating excellent therapeutic effects during the AD pathogenesis window (reducing peripheral and neuroinflammation, reducing abeta production, reducing the amount of free abeta fibers, and improving memory and sleep function in mice). Based on these findings, the inventors completed this invention.

[0301] the term

[0302] In this document, unless otherwise specified, the term "substitution" refers to the substitution of one or more hydrogen atoms on a group by a substituent selected from the group consisting of: halogen, amino, hydroxyl, nitro, cyano, trifluoromethyl, C1-C 12 Alkyl or cycloalkyl, C1-C 12 Alkoxy group, oxygen atom (i.e., =O), unsubstituted or C-substituted 1-4 Alkylamine-substituted C1-C 12 Alkylamine, C2-C6 ester, C2-C6 acyl, C2-C6 amide, thioC1-C 12 Alkyl, carboxyl, C5-C 12 Aryl or heteroaryl, C5-C 12 Heterocyclic group (containing 1-5, preferably 1-3, heteroatoms selected from N, O or S).

[0303] The term "C1-C" 12 "Alkyl" refers to a straight-chain or branched alkyl group having 1 to 12 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, or similar groups. The term "C1-C6 alkyl" has a similar meaning.

[0304] The term "C1-C" 12 "Cycloalkyl" refers to a compound having 1-12 alkyl groups, preferably 3-12 (i.e., C12-12 alkyl groups). 3-12 ) A cycloalkyl group with a carbon atom, such as cyclopropyl, cyclobutyl, cyclopentyl, cycloheptyl, or similar groups.

[0305] The term "C1-C" 12 "Alkoxy" refers to a straight-chain or branched alkoxy group having 1 to 12 carbon atoms, such as methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, sec-butoxy, tert-butoxy, or similar groups.

[0306] The term "halogen" refers to F, Cl, Br, and I.

[0307] The term "C1-C" 12 "Alkylamine group (or alkylamine group)" refers to a C1-C group that has been substituted with an amino group. 12 Alkyl groups, for example, those having "C1-C 12 Alkyl-NH- or (alkyl)2-N- (total number of carbon atoms is 1-12) or -C1-C 12 Groups with the structures "alkylene-NH2", "alkyl-N-alkylene-(total number of carbon atoms 1-12)", or "(alkyl)2-N-alkylene-(total number of carbon atoms 1-12)", such as CH3NH-, C2H5NH-, C3H7NH-, (CH3)2N-, -CH2NH2, -C2H5NH2, -C3H7NH2, -C2H4N(CH3)2, or similar groups. Where C... 1-12 As previously defined, the term "C1-C6 alkylamine" has a similar meaning.

[0308] The term "C2-C6 ester group" refers to a substituent with a structure of "straight-chain or branched alkyl / cycloalkyl / aryl / heteroaryl-carbonyl-oxy-" having 1-5 carbon atoms, such as ethyl ester, propyl ester, butyl ester, or similar groups.

[0309] The term "C1-C6 amide group" refers to a substituent with a structure of "a straight-chain or branched alkyl / cycloalkyl / aryl / heteroaryl-carbonyl-amine-" having 0-5 carbon atoms, such as acetamido, propionamido, butyramido, or similar groups.

[0310] The term "C6-C" 10 "Aryl" refers to a group having 1-12 (preferably 6-10, i.e., C) groups. 6-10 The aryl group of the carbon atom, such as phenyl, naphthyl, etc., may be substituted or unsubstituted.

[0311] The term "5-12-membered heteroaryl" refers to a heteroaryl group having 5-12 ring atoms, including one or more (preferably 1-3) heteroatoms selected from O, S and / or N, preferably a 5-8-membered heteroaryl group. The heteroaryl group may be substituted or unsubstituted.

[0312] The term "5-7 membered heterocycle" refers to a cyclic saturated, partially unsaturated or aromatic group having 5-7 members, wherein the heterocycle has at least one ring atom selected from the group consisting of O, S and / or N.

[0313] The term "5-7 membered heteroaryl" refers to a cyclic aromatic group having 5-7 members, wherein the heterocycle has at least one ring atom selected from the group consisting of O, S and / or N.

[0314] Specifically, expressions in the form "C1-Cn" indicate that the group has 1 to n carbon atoms. For example, expressions in the form "C1-C12" indicate that the group has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 carbon atoms; "C6~C10" indicates that the group has 6, 7, 8, 9 or 10 carbon atoms.

[0315] In this invention, the term "pharmaceuticalally acceptable" refers to a substance that is suitable for use in humans and / or animals without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), i.e., a substance with a reasonable benefit / risk ratio.

[0316] In this invention, the term "effective amount" refers to the amount of a therapeutic agent that treats, alleviates, or prevents a target disease or condition, or the amount that exhibits a detectable therapeutic or preventative effect. The precise effective amount for a given subject depends on that subject's body size and health status, the nature and severity of the condition, and the choice of the therapeutic agent and / or combination of therapeutic agents administered. Therefore, it is useless to pre-specify an accurate effective amount. However, for a given condition, the effective amount can be determined using routine experiments, and a clinician can judge it accordingly.

[0317] Unless otherwise specified, all compounds mentioned in this invention are intended to include all possible optical isomers, such as compounds with a single chirality, or mixtures of various chiral compounds (i.e., racemates). In all compounds of this invention, each chiral carbon atom may optionally be in the R configuration or the S configuration, or a mixture of the R and S configurations.

[0318] As used herein, the term "compound of the invention" refers to a compound of Formula I. The term also includes various crystalline forms, pharmaceutically acceptable salts, hydrates, or solvates of compounds of Formula I.

[0319] As used herein, the term "pharmaceutically acceptable salt" refers to a salt formed by the compounds of the present invention with an acid or base that is suitable for use as a medicine. Pharmaceutically acceptable salts include both inorganic and organic salts. A preferred class of salts are those formed by the compounds of the present invention with an acid. Suitable acids for forming salts include, but are not limited to: inorganic acids such as hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, and phosphoric acid; organic acids such as formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, picric acid, methanesulfonic acid, benzenesulfonic acid, and benzenesulfonic acid; and acidic amino acids such as aspartic acid and glutamic acid.

[0320] RIPK1 inhibitors

[0321] The inventors have discovered that compounds with RIPK1 inhibitory effects exhibit excellent therapeutic effects on neurological dysfunctions, such as the rare disease Rett syndrome. Therefore, patients suffering from neurological dysfunctions can achieve disease or symptom improvement by administering RIPK1 inhibitors. This treatment can be achieved by administering any therapeutic ingredient or method known or unknown in the art that targets RIPK1 expression levels, such as small molecule RIPK1 inhibitors, antibody drugs targeting RIPK1 activity or expression levels, conjugates, or genetic engineering methods. Typical RIPK1 inhibitors can be commercially available clinical small molecule drugs, such as representative RIPK1 inhibitors selected from the following group:

[0322] Other small molecule RIPK1 inhibitors in this field can also be used as RIPK1 inhibitors here.

[0323] In another preferred embodiment, the RIPK1 inhibitor may be the compound described in US2022 / 0213077A1, having the general formula structure shown in Formula I:

[0324] In the formula:

[0325] for

[0326] M is selected from the following group: chemical bond, O, S, NR3, CHR3 or C(R3)2;

[0327] X1 is selected from the following group: CR2, NR, O, S, CR, N;

[0328] X2 is selected from the following group: CR, N;

[0329] R is selected from the following group: H, D, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 deuterated alkyl;

[0330] R1 and R2 are each independently selected from the following group: H, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 deuterated alkyl; or R1 and R2 together with the carbon atom attached to them to form substituted or unsubstituted C3-C6 cycloalkyl, or substituted or unsubstituted 4-6 membered heterocyclic groups.

[0331] Ring A is a group selected from the following group: substituted or unsubstituted C6-C10 aryl, substituted or unsubstituted 5-12 heteroaryl;

[0332] Ring B is a group selected from the following group: substituted or unsubstituted C6-C10 aryl, substituted or unsubstituted 5-12 heteroaryl;

[0333] Wherein, the substitution refers to the hydrogen atom on the substituent group being replaced by one or more (e.g., 2, 3, 4, etc.) substituents selected from the group consisting of: halogen, deuterated, C1-C6 alkoxy, halogenated C1-C6 alkoxy, methyl sulfone, -S(=O)2NH2, oxo(=O), -CN, hydroxyl, -NH2, carboxyl, C2-C6 amide (-C(=O)-N(Rc)2 or -NH-C(=O)(Rc, where Rc is H or a C1-C5 alkyl), C1-C6 alkyl-(C2-C6 amide), or substituted or unsubstituted groups selected from the group consisting of: C1-C6 alkyl, C3-C8 cycloalkyl, C1- C6 amino, C6-C10 aryl, 5-10 heteroaryl having 1-3 heteroatoms selected from N, S and O, 5-12 heterocyclic group having 1-3 heteroatoms selected from N, S and O, -(CH2)-C6-C10 aryl, -(CH2)-(5-10 heteroaryl having 1-3 heteroatoms selected from N, S and O), and the substituent is selected from the group consisting of: halogen, C1-C6 alkyl, C1-C6 alkoxy, oxo, -CN, -NH2, -OH, C6-C10 aryl, C1-C6 amino, C2-C6 amide, 5-10 heteroaryl having 1-3 heteroatoms selected from N, S and O.

[0334] Preferred molecules are shown in US2022 / 0213077A1, the entire contents of which are incorporated herein by reference.

[0335] In another preferred embodiment, the RIPK1 inhibitor may be the compound described in WO2022 / 057787A1, which has the general formula structure shown in Formula II:

[0336] in,

[0337] Ring A is a substituted or unsubstituted 9-10 member nitrogen-containing heteroaryl group, wherein the 9-10 member nitrogen-containing heteroaryl group contains 1, 2, 3 or 4 nitrogen heteroatoms as ring atoms;

[0338] n = 0, 1, or 2;

[0339] R 4 Each is independently selected from the following group: H, CN, halogen, substituted or unsubstituted C. 1-6 Alkyl, -OR b -SR b -N(R) b )2、-C(O)-NR 6 -R b -C(O)-NR 6 -C 1-4 Alkylene-N(R) b )2、-NR6 -C(O)-R b ;

[0340] Each R b Each is independently selected from the following groups: H, substituted or unsubstituted C. 1-6 Alkyl; or two R b Together with the nitrogen atoms attached to them, they form substituted or unsubstituted 5, 6, or 7-membered heterocyclic alkyl groups, wherein, except for those with R b In addition to the connected N, the heterocyclic alkyl group also contains 0, 1 or 2 other heteroatoms as ring atoms;

[0341] R 6 Selected from the following group: H, OH, substituted or unsubstituted C 1-4 Alkyl, substituted or unsubstituted C 1-4 Alkoxy;

[0342] Cycle B is selected from the following group: substituted or unsubstituted C 6-10 Aryl, substituted or unsubstituted 5-10 heteroaryl groups;

[0343] L 1 and L 2 Each is independently a divalent group selected from the following group:

[0344] none,

[0345] And L 1 and L 2 Not simultaneously equal to none;

[0346] R 1 and R 2 Each is independently selected from the following groups: H, substituted or unsubstituted C. 1-4 alkyl;

[0347] R 3 Selected from the following group: H, OH, substituted or unsubstituted C 1-4 Alkyl, substituted or unsubstituted C 1-4 Alkoxy;

[0348] Ring C is none or Among them, W is independently selected from the following groups: O, S, C, N, C(R) c ), and N(R) d ); R c Each is independently selected from the following group: H, CN, halogen, substituted or unsubstituted C. 1-6 Alkyl, R d Each is independently selected from the following groups: H, CN, substituted or unsubstituted C. 1-6 alkyl;

[0349] Or, when ring C is L 1 for And L 2 When there is no time, R 3 With L 1 The ring atoms W and L adjacent to the ring C are 1 The -C(O)- groups in the rings together form substituted or unsubstituted 5, 6, or 7-membered saturated heterocycles; wherein, the saturated heterocycles, in addition to being substituted with R, form substituted or unsubstituted 5, 6, or 7-membered saturated heterocycles. 3 In addition to the connected N, there are 0, 1 or 2 other heteroatoms that act as ring atoms;

[0350] R 5 Selected from the following group: H, OH, substituted or unsubstituted C 1-4 Alkyl, substituted or unsubstituted C 1-4 Alkoxy;

[0351] Or when ring C is nonexistent and L 2 for At that time, R 3 and R 5 And the atoms bonded to them together form substituted or unsubstituted 5, 6, or 7-membered saturated heterocycles; wherein, the saturated heterocycles, in addition to being associated with R, form substituted or unsubstituted 5, 6, or 7-membered saturated heterocycles. 3 In addition to the connected N, there are 0, 1 or 2 other heteroatoms that act as ring atoms;

[0352] Ring D is selected from the following group: substituted or unsubstituted C 6-10 Aromatic rings, and substituted or unsubstituted 5-10 membered heteroaryl groups;

[0353] Unless otherwise specified, the term "substitution" refers to the substitution of a hydrogen atom on a group by one or more (e.g., 1, 2, 3, or 4) substituents selected from the group consisting of: oxo (=O), -CN, halogen, nitro, C. 1-6 Alkyl, Halogenated C 1-6 Alkyl groups, -OR, -SR, -S(O)2R, -S(=O)2NR2, -NR2, -COOR, and C groups optionally substituted with R. 6-10 aryl, 5-10 heteroaryl groups with 1-3 heteroatoms selected from N, S and O, optionally substituted by R, and C groups optionally substituted by R 3- 8-cyclic alkyl groups, 5-12-membered heterocyclic alkyl groups having 1-3 heteroatoms selected from N, S, and O, optionally substituted with R, and -C optionally substituted with R. 1-4 Alkylene-C 6-10 aryl, -C optionally substituted by R 1-4 Alkylene – a 5-10 membered heteroaryl group having 1-3 heteroatoms selected from N, S, and O, with -C optionally substituted by R. 1-4 Alkylene-C 3-8 Cycloalkyl, -C optionally substituted with R1-4 Alkylene – 5-12 membered heterocyclic alkyl groups having 1-3 heteroatoms selected from N, S and O;

[0354] R is independently selected from the following groups: H, C 1-6 Alkyl, Halogenated C 1-6 Alkyl, C 1-6 Hydroxyalkyl.

[0355] Preferred molecules are shown in WO2022 / 057787A1, the entire contents of which are incorporated herein by reference. In addition, another class of preferred compounds is shown in the table below:

[0356] In another preferred embodiment, the RIPK1 inhibitor may be the compound described in WO2025 / 007938A1, having the general formula structure shown in Formula III:

[0357] In the formula:

[0358] X8 is selected from the following group: CH, N, or chemical bonds;

[0359] X7, X3, X4, and X5 are each independently selected from the following groups: CH, N;

[0360] The condition is that the ring formed by X8, X7, X3, X4, and X5 is an aromatic ring;

[0361] M is selected from the following group: O, S, NR 3c CHR 3c or C(R) 3c )2;

[0362] W and U are each independently selected from the following groups: O, S, NR 4c CHR4 or C(R) 4c )2;

[0363] Ring A and ring B are each independently selected from the following group: substituted or unsubstituted C6-C10 aryl groups, substituted or unsubstituted 5-12 heteroaryl groups;

[0364] R 1c and R 2c Each is independently selected from the following group: none, H, substituted or unsubstituted C1-C6 alkyl, halogen;

[0365] And when M is NR 3c CHR 3c or C(R) 3cWhen R2 and R3 are together with the C or N atom attached to them, as well as -CC(O)-, they can form substituted or unsubstituted 5-7 membered rings.

[0366] R 3c and R 4c Selected from the following group: H, substituted or unsubstituted C1-C6 alkyl groups, halogens;

[0367] R6 is selected from the following group: H, halogen, CN, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C2-C6 olefin, substituted or unsubstituted C2-C6 alkyne, or -CH≡CR5; wherein R5 is selected from the following group: substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C6-C10 aryl, substituted or unsubstituted 5-12-membered heteroaryl, substituted or unsubstituted C3-C8 cycloalkyl, substituted or unsubstituted 4-12-membered heterocyclic group;

[0368] R7 is selected from the following group: H, halogen, CN, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C2-C6 olefin, substituted or unsubstituted C2-C6 alkyne, or -CH≡CR8; wherein R8 is selected from the following group: substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C6-C10 aryl, substituted or unsubstituted 5-12-membered heteroaryl, substituted or unsubstituted C3-C8 cycloalkyl, substituted or unsubstituted 4-12-membered heterocyclic group;

[0369] Wherein, the substitution refers to the hydrogen atom on the substituent group being replaced by one or more (e.g., 2, 3, 4, etc.) substituents selected from the group consisting of: halogen, C1-C6 alkyl, halogenated C1-C6 alkyl, C3-C8 cycloalkyl, halogenated C3-C8 cycloalkyl, C1-C6 alkoxy, halogenated C1-C6 alkoxy, C1-C6 hydroxyalkyl, methyl sulfone, -S(=O)2NH2, oxo(=O), -CN, hydroxyl, -NH2, carboxyl, C2-C6 amide (-C(=O)-N(Rc)2 or -NH-C(=O)(Rc), where Rc is H or C1-C5). Alkyl), C1-C6 alkyl-(C2-C6 amide), C1-C6 amino, deuterated C1-C6 amino, -NHRd (Rd is C3-C8 cycloalkyl, 4-7 membered heterocyclic or heterocyclic substituted with C1-C6 alkyl), C6-C10 aryl, 5-7 membered heteroaryl having 1-3 heteroatoms selected from N, S and O, 4-8 membered heterocyclic having 1-3 heteroatoms selected from N, S and O, 4-7 membered heterocyclic substituted with 1 or 2 Re (Re is halogen, C1-C6 alkyl, C1-C6 amino, -CN, C1-C6 alkoxy or 4-7 membered heterocyclic).

[0370] Preferred molecules are shown in WO2025 / 007938A1, the entire contents of which are incorporated herein by reference.

[0371] In addition, other known or unknown RIPK1 small molecule inhibitors in the art can also be used for the purposes of this invention.

[0372] Function of INPP5D in microglia

[0373] The inventors studied the function of INPP5D in microglia and unexpectedly discovered that the absence of INPP5D in cultured new microglia and in vivo senescence promotes the activation of RIPK1, which in turn stimulates the transcription of various microglia LOAD risk genes and pro-inflammatory cytokines, intracellular TLRs, inflammasomes and interferon pro-inflammatory signaling mediators, as well as the production of ROS and complement.

[0374] The inventors also unexpectedly discovered that the RIPK1-regulated gene transcription patterns in Inpp5d-deficient mouse microglia showed a striking similarity to the results of inflammation- and lipid-treated microglia subtypes, both of which have recently been described as key characteristics of human AD microglia. The inventors demonstrated that in INPP5D-deficient aging mice, RIPK1 activation in microglia promotes the development of atrophic microglia, motor neuron death, and motor dysfunction.

[0375] Furthermore, the inventors revealed the structural basis for the direct interaction between the N-terminal SH2 domain of INPP5D and RIPK1 to inhibit its activation, verified the mechanistic link between various LOAD risk factors and different pro-inflammatory signaling mechanisms in microglia, and the role of INPP5D in inhibiting RIPK1-mediated transcriptional responses. INPP5D is a key microglia regulator controlling neuroinflammation and neuronal cell death and is directly related to AD in humans.

[0376] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer. Percentages and parts are by weight unless otherwise stated.

[0377] Example 1: Study on myeloid INPP5D deletion and RIPK1-mediated pro-inflammatory cytokine transcription

[0378] We performed a genome-wide siRNA screening of genes regulating necrosis and identified several key genes that inhibit RIPK1 activation. These genes had negative z-scores, which were used to reveal the role of RIPK1 in mediating neurodegenerative diseases in the context of TNFR1. In this screening, we found that knocking out Inpp5d sensitized necrosis induced by the caspase inhibitor z-VAD.fmk (zscore = -1.58) (Figure 14A). We confirmed this result in L929 cells and found that the RIPK1 inhibitor Nec-1s (a highly specific RIPK1 kinase inhibitor) effectively inhibited cell death in Inpp5d knockout L929 cells treated with the caspase inhibitor zVAD.fmk (Figure 14B). The increased sensitivity of INpp5d knockout L929 cells to zVAD.fmk was rescued through complementation of INPP5D expression. However, in Inpp5d knockout L929 cells, knocking out Inpp5d or supplementing Inpp5d expression had no effect on necrosis induced by TNFα alone, TNFα / SM-164, or TNFα / SM-164 / z-VAD; while Nec-1s still effectively inhibited cell death (Fig. 14B). Since zVAD-induced L929 cell necrosis is mediated by the autocrine production of TNFα21, these results suggest that Inpp5d knockout is sensitive to RIPK1-mediated TNFα production, but may not be directly involved in TNFα signaling downstream of TNFR1.

[0379] We also generated Inpp5d knockout BV2 cells using CRISPR-Cas9 (Fig. 14C). Similarly, we found that Inpp5d knockout BV2 cells also expressed higher levels of inflammatory cytokines such as TNFα, IL6, IL1β, and Cxcl2, which were blocked by Nec-1s treatment (Fig. 14D). Supplementation with wild-type INPP5D or its inactive inositol phosphatase mutant inhibited the production of proliferative cytokines, indicating that its phosphatase activity is not involved in regulating cytokine production. Inpp5d-deficient BV2 cells also showed enhanced IL6 and TNFα production upon LPS stimulation, while Nec-1s treatment or complementation with wild-type INPP5D or its inactive inositol phosphatase mutant reduced IL6 and TNFα production (Fig. 14E). In summary, these results indicate that INPP5D regulates RIPK1-mediated pro-inflammatory cytokine expression in a phosphatase-independent manner.

[0380] Because Inpp5d is highly expressed in microglia, we developed a CRISPR / Cas9-based in vivo rapid gene knockout microglia screening strategy to test the role of INPP5D in the response of primary microglia to inflammatory stimuli (Fig. 14F). This protocol allows us to efficiently generate primary gene knockout microglia within approximately 4–6 weeks. In this screening, we found that Inpp5d knockout microglia exhibited an elevated inflammatory response to LPS stimulation and produced more TNFα, IL6, and IL-1β than WT microglia (Fig. 14G). Since RIPK1 activation is known to promote the expression of TNFα, IL6, and IL-1β in microglia, these results encourage us to further explore the role of RIPK1 in driving microglial inflammatory responses under Inpp5d-deficient conditions in vivo.

[0381] Example 2 verifies the promoting effect of myeloid INPP5D deficiency on RIPK1-mediated age-dependent microglial disease characterized by atrophic microglia.

[0382] The full-length protein encoded by INPP5D is called SHIP1, which contains an SH2 domain at its N-terminus, a lipid phosphatase domain in the center, and three highly phosphorylated Tyr residues at its C-terminus. The Inpp5d gene is known to express multiple isoforms. Transcription of the INPP5D isoform can begin from a downstream intron, unlike the transcription start site in exon 1 targeted by the commonly studied Inpp5d suspended allele (Jax strain number 028255). To create a null allele of Inpp5d, we inserted LoxP around exons 16, 17, and 18, constructing the Inpp5d suspended allele (Inpp5dfl), thereby conditionally removing exons 16, 17, and 18 and stopping translation of downstream exons (Figure 14H). The original P0-Inpp5d... fl / fl Eight generations of backcrossing were performed between mice and the parental C57B6 strain for research. Due to the high expression of Inpp5d in myeloid cells, we produced Inpp5d. fl / fl Lyz2 cre / + Mice were used to remove INPP5D from the myeloid lineage. (Inpp5d) fl / fl Compared to WT microglia, Inpp5d fl / fl Lyz2 cre / + Microglia showed a loss of all INPP5D protein products when analyzed using N-terminal and C-terminal INPP5Dabs, indicating that we generated a null allele (Fig. 14I).

[0383] 8-month-old adult Inpp5d fl / fl Lyz2 cre / +Elevated levels of multiple pro-inflammatory cytokines, including TNFα, IL1β, Cst, and Clec7a, were observed in the brains and spinal cords of mice without stimulation (Fig. 14J). Interestingly, pharmacological inhibition of RIPK1 by Nec-1s suppressed the growth of neonatal primary INPP5D-deficient microglia and adult Inpp5D-deficient microglia. fl / fl Lyz2 cre / + Increased expression of pro-inflammatory cytokines (such as TNFα, IL1β, IL6, and IFNγ) in the mouse brain (Fig. 14K–Fig. 14L). Therefore, myeloid INPP5D deficiency promotes RIPK1-mediated neuroinflammation in vivo.

[0384] We tested Inpp5d fl / fl Lyz2 cre / + Effects of Inpp5d deletion in myeloid mice. Using IBA1 as a biomarker, Inpp5d deletion in 2-week-old newborn mice... fl / fl Lyz2 cre / + Age-matched WT mice or Inpp5d fl / fl Lyz2 cre / + Compared to Ripk1D138N / D138N mice, there was no difference in the number and morphology of microglia in their brains (Fig. 14M). In contrast, Inpp5d... fl / fl Lyz2 cre / + The number of microglia in the mouse cortex increased significantly at 8 months of age, while in Inpp5d fl / fl Lyz2 cre / + In Ripk1D138N / D138N mice, the levels returned to WT (Figure 14N).

[0385] Interestingly, we discovered Inpp5d fl / fl Lyz2 cre / + Microglia in the cortex of 8-month-old mice showed RIPK1 (p-S166) activation, with atrophic morphology exhibiting detachment, fragmentation, tortuosity, and globular protrusions, similar to the atrophic microglia morphology described in human AD pathological samples (Fig. 1A-1B; Fig. 14N). In 8-month-old Inpp5d fl / fl Lyz2 cre / + Partially activated RIPK1 was found in the nuclei of microglia in the brains of mice (Figure 1B). 8-month-old Inpp5d fl / fl Lyz2 cre / + The morphological characteristics of atrophic microglia in the mouse cerebral cortex can be quantified as reduced spatial extension of microglial processes, increased fragmentation and beading of microglial processes (Figure 1C). 8-month-old Inpp5d fl / fl Lyz2 cre / +Microglia in the cortex of Ripk1D138N / D138N mice reverted to the morphology of age-matched WT mice (Fig. 1A-Fig. 1C). Therefore, myeloid Inpp5d deficiency leads to senescence-dependent microglia dysplasia (characterized by RIPK1-mediated atrophic microglia in vivo).

[0386] In summary, the above results indicate that myeloid INPP5D deficiency promotes RIPK1 activation and inflammatory response in cultured microglia, and leads to the development of atrophic microglia during in vivo aging.

[0387] Example 3: Myeloid INPP5D deficiency promotes RIPK1-dependent microglial inflammation.

[0388] In cultured newborn mice Inpp5d fl / fl Lyz2 cre / + RIPK1 activation was observed in microglia, and this activation was inhibited by treatment with the RIPK1 inhibitor Nec-1s (Figure 15A). Therefore, we first used cultured INPP5D-deficient microglia isolated from neonatal mice as a model to investigate the consequences of RIPK1 activation. To characterize the effects of INPP5D deficiency on microglia, we analyzed cultured INPP5D-deficient microglia isolated from neonatal mice. fl / fl WT mice, Inpp5d fl / fl Lyz2 cre / + Mice, Ripk1D138N / D138N mice with RIPK1 kinase death (carrying an endogenous RIPK1 gene knock-in mutation that replaces Asp with Gln in the RIPK1 kinase domain, resulting in kinase inactivation 35), and Inpp5d mice. fl / fl Lyz2 cre / + Ripk1D138N / D138N mice. RNA-Seq data showed that Inpp5d deletion in microglia resulted in significantly differential expression of 686 genes (DEGs; >1.5-fold change, adjusted p-value: <0.05), of which 614 genes were upregulated and 72 genes were downregulated (Fig. 15B). Interestingly, genetic inhibition of RIPK1 kinase using the Ripk1-D138N gene knockout mutation restored the expression of 482 of the 614 upregulated genes and 21 of the 72 downregulated genes to normal levels in WT microglia (DEGs; >1.5-fold change, adjusted p-value: <0.05) (Fig. 15C). GO analysis of genes upregulated in a RIPK1-dependent manner in INPP5D-deficient microglia showed enrichment of regulators of innate immune responses (Fig. 1D). In cultured neonatal Inpp5d mice... fl / fl Lyz2 cre / +In microglia, the transcription of multiple pro-inflammatory cytokines, including TNFα, IL1β, Ccl2, Ccl3, Ccl5, Ccl6, Ccl9, Cxcl10, and Cxcl16, is increased, while Inpp5d, which is silenced by RIPK1 kinase, is also increased. fl / fl Lyz2 cre / + In Ripk1D138N / D138N microglia, inhibition was observed (Figure 1E). Q-PCR confirmed Inpp5d fl / fl Lyz2 cre / + Ripk1D138N / D138N microglia induce the action of pro-inflammatory cytokines TNFα, IL1α, IL1β, IL6, Ccl2, Ccl3, Ccl5, Ccl6, Cxcl12 and IFNγ, and in Inpp5d fl / fl Lyz2 cre / + The Ripk1D138N / D138N microglia exhibit an inhibitory effect (Fig. 1F). Therefore, in vitro culture stress can induce the transcription of multiple pro-inflammatory factors mediated by RIPK1 in newly formed INPP5D-deficient microglia.

[0389] Example 4: Myeloid INPP5D deletion syndrome promotes the transcription of multiple pro-inflammatory factors in microglia in a RIPK1-dependent manner with age.

[0390] We noted that in the cultivation of newborn Inpp5d fl / fl Lyz2 cre / + In microglia, the number of upregulated genes involved in ROS production was significantly increased, while in cultured newborn Inpp5d cells... fl / fl Lyz2 cre / + In Ripk1D138N / D138N microglia, these upregulated genes were suppressed (Fig. 2A). Consistent with the increased expression of ROS-producing genes, cultured newborn Inpp5d cells... fl / fl Lyz2 cre / + CellROX green staining of microglia showed increased ROS production, which was inhibited by the RIPK1 kinase inhibitor Nec-1s (Fig. 2B). In Inpp5d fl / fl In the brains of + and Lyz2Cre / + mice, RNAscope detected increased expression of Cybb encoding NOX2 in situ. NOX2 is a key component of membrane-bound oxidases that can produce superoxide; while in Inpp5d fl / fl Lyz2 cre / + In microglia of the brains of Ripk1D138N / D138N mice, the level of Cybb has returned to WT (Fig. 2C; Fig. 15D).

[0391] After RIPK1 inhibition, newly cultured Inpp5d fl / fl Lyz2 cre / + The expression of multiple complement factors, including C1qa, C1qb, C1qc, C3, Cfh, and Cfp, in Ripk1D138N / D138N microglia was restored to WT levels (Fig. 2D). Cultured newborn Inpp5d... fl / fl Lyz2 cre / + In Ripk1D138N / D138N microglia, the expression of C3, C1qa, C1qb, and C1qc is increased, while Inpp5d... fl / fl Lyz2 cre / + The expression of C3, C1qa, C1qb, and C1qc was suppressed in Ripk1D138N / D138N microglia, a finding further confirmed by Q-PCR and Western blotting (Figure 2E-F). Using RNAscope, we found that Inpp5d... fl / fl Lyz2 cre / + The expression of C1qa and C3 in the brains of mice was elevated at 10 months of age, but not at 2 weeks of age; Inpp5d fl / fl Lyz2 cre / + The elevation of C1qa and C3 in the brains of 10-month-old Ripk1D138N / D138N mice was suppressed (Fig. 2G-Fig. 2H; Fig. 15E-Fig. 15F). Increased complement expression in microglia was associated with increased TNFα expression levels (Fig. 2G).

[0392] Toll-like receptors, nucleotide-binding oligomerization domain-like receptor 3 (NLRP3) inflammasomes, and interferons are key sensors and signal transducers of extracellular and intracellular microbial products and danger signals in the innate immune system. Inflammasomes are considered important intracellular mediators of pro-inflammatory responses in neurodegenerative diseases. Interestingly, we found that in cultured neonatal Inpp5d... fl / fl ; In the training of new students Inpp5d fl / fl Lyz2 cre / + In Ripk1D138N / D138N microglia, cell mediators such as MyD88 are upregulated, while in cultured newborn Inpp5d cells... fl / fl Lyz2 cre / + In Ripk1D138N / D138N microglia, these mediators were restored to WT levels (Fig. 2I). Since MyD88 is a key aptamer for multiple TLRs (including cell surface Toll-like receptors TLR4 and TLR6, and intracellular Toll-like receptors TLR6, TLR7, TLR8, and TLR13), we used RNAscope to detect MyD88 levels in vivo. We found that Inpp5dfl / fl Lyz2 cre / + The expression of MyD88 in the brains of mice increased at 8 months of age, but not at 2 weeks of age; Inpp5d fl / fl Lyz2 cre / + The increase of MyD88 in the brain of Ripk1D138N / D138N mice was suppressed at 10 months of age (Fig. 2J; Fig. 15G).

[0393] In addition to the increase in TLR signaling, the cultured newborn Inpp5d fl / fl Lyz2 cre / + Microglia also showed elevated expression of several key inflammasome components, including NLRP3, AIM2, GSDMD, and caspase-1, and in Inpp5d fl / fl ;Lyz2-cre / +;Ripk1 D138N / D138N Recovery to WT levels in microglia (Fig. 2K). Cultured neonatal Inpp5d fl / fl Lyz2 cre / + Upregulated NLRP3, AIM2, GSDMD, and caspase-1 in Ripk1D138N / D138N microglia were confirmed by Q-PCR and Western blot (Figure 2L-M). We also detected in situ levels of Nlrp3, Aim2, and GSDMD in vivo using RNAscope. In 8-month-old Inpp5d... fl / fl Lyz2 cre / + Elevated levels of Nlrp3, Aim2, and GSDMD were found in mice, while in Inpp5d fl / fl Lyz2 cre / + In Ripk1D138N / D138N mice, RIPK1 inhibitors suppressed the increase in Nlrp3, Aim2, and GSDMD levels (Figure 2N-O). Compared with 2-week-old WT or Ripk1D138N / D138N mice, Inpp5d fl / fl Lyz2 cre / + The levels of Nlrp3, Aim2, and GSDMD in the mouse cortex remained unchanged (Figure 15H-J).

[0394] New students trained Inpp5d fl / fl Lyz2 cre / + Microglia also showed elevated expression of many key intracellular mediators of interferon, such as Irgm1, Irgm2, Ifnar2, Irf8, Ifitm2, and Ifitm3, which were expressed in cultured newborn Inpp5d cells. fl / fl Lyz2 cre / +In Ripk1D138N / D138N microglia, levels returned to WT (Figure 2P). Using RNAscope, we found that Inpp5d... fl / fl Lyz2 cre / + Irgm1 expression was elevated in the brains of 10-month-old mice, but not at 2-week-old mice; Inpp5d fl / fl Lyz2 cre / + In 10-month-old Ripk1D138N / D138N mice, Irgm1 expression was suppressed in the brain (Fig. 2Q; Fig. 15J). Furthermore, we also examined Inpp5d... fl / fl Lyz2 cre / + ZBP1 expression in the mouse brain; ZBP1 is an important sensor and activator of the innate immune response. We investigated this expression in 10-month-old Inpp5d mice. fl / fl Lyz2 cre / + Increased expression of ZBP1 and IFNγ was detected in the brains of mice, but not at 2 weeks of age; it was not detected in 10-month-old Inpp5d mice. fl / fl Lyz2 cre / + In the brains of Ripk1D138N / D138N mice, the increase of ZBP1 and IFNγ was inhibited (Fig. 2R; Fig. 15K).

[0395] In summary, these results indicate that myeloid INPP5D loss in the brains of aging mice promotes RIPK1-mediated transcription of multiple intracellular PAMP and DAMP signaling mediators, including TLRs, inflammasomes, and interferons.

[0396] Example 5: Myeloid INPP5D deficiency promotes RIPK1 kinase-dependent transcription of multiple LOAD risk genes in microglia during stress and aging.

[0397] Unexpectedly, we discovered 17 LOAD risk genes, including ApoE, Trem2, Tyrobp, Ptk2b, Plcg2, Spi1, Pld3, Ctsd, Ctsb, Ctsh, Idua, Grn, Rin3, Pilra, Adam17, Maf, and Blnk, in cultured newborn Inpp5d. fl / fl Lyz2 cre / + Upregulated genes in microglia, these genes are in Inpp5d fl / fl Lyz2 cre / +In Ripk1D138N / D138N microglia, levels returned to normal WT (Fig. 3A). We confirmed the upregulation of ApoE, Trem2, Adam17, Ctsb, Ctsd, Ctsh, and Plcg2 by Q-PCR, and confirmed the levels of ApoE, Trem2, Adam17, Ctsh, and Ctsd in cultured newborn Inpp5d cells by Western blotting. fl / fl Lyz2 cre / + Upregulation in microglia, and Inpp5d fl / fl Lyz2 cre / + Inhibition of Ripk1D138N / D138N in microglia (Fig. 3B-Fig. 3C). Therefore, the absence of INPP5D in cultured primary microglia can stimulate the transcription of multiple LOAD risk genes in a RIPK1-dependent manner.

[0398] Next, we used RNAscope to identify the effect of INPP5D deficiency in myeloid cells on the expression of LOAD risk genes in vivo. We found that at 10 months of age, Inpp5d... fl / fl Lyz2 cre / + The expression of apolipoprotein E and Trem2 was elevated in the mouse cortex, but not at 2 weeks of age (Fig. 3D-3E; Fig. 16A). In 10-month-old Inpp5d mice... fl / fl Lyz2 cre / + In Ripk1D138N / D138N mice, RIPK1 kinase inhibitors suppressed the upregulation of ApoE and Trem2.

[0399] We also used RNAscope to identify the expression of three other LOAD risk genes, Plcg2, Ctsb, and GRN, in vivo. We found that Inpp5d fl / fl Lyz2 cre / + In mice at 10 months of age, the expression of Plcg2, Ctsb, and GRN in the cortex was elevated, but not at 2 weeks of age (Figs. 3F-3H; 16B-16D). In 8-month-old Inpp5d mice... fl / fl Lyz2 cre / + In Ripk1D138N / D138N mice, RIPK1 kinase inhibitors suppressed the upregulation of Plcg2, Ctsb, and GRN.

[0400] We also investigated the effect of INPP5D loss in myeloid cells on Clu expression, another load risk gene that encodes a chaperone protein whose expression is not limited to microglia. RNAscope analysis showed that at 10 months of age, Inpp5d... fl / fl Lyz2 cre / +Clu expression was elevated in the mouse cerebral cortex, but not at 2 weeks of age (Fig. 3I; Fig. 16E). In Inpp5d fl / fl Lyz2 cre / + At 10 months of age, RIPK1 inhibitors also inhibited Clu at Inpp5d fl / fl Lyz2 cre / + Expression of RIPK1 in the brains of Ripk1D138N / D138N mice. Therefore, with age, myeloid INPP5D deficiency may stimulate the expression of multiple RIPK1-dependent load risk factors in the brain.

[0401] Example 6: RIPK1-regulated microglia gene expression characteristics were discovered in human AD microglia.

[0402] Recent mononuclear transcriptomic studies have revealed a wealth of new insights into the dynamics of microglia during the progression of AD pathology in humans. Therefore, we compared the transcriptomic analysis of RIPK1-regulated genes in INPP5D-deficient microglia with the microglia subtypes identified in these two studies. The MIT study of human AD microglia transcriptomics conducted by Li-Huei Tsai and Manolis Kellis's group analyzed 443 human subjects, covering different brain regions and pathological AD states, and identified 12 microglia transcriptional states (MG1-12). Compared to the 12 microglia subtypes found in the human AD brain, the RIPK1-regulated gene expression patterns of mouse myeloid Inpp5d-deficient microglia identified in this study showed statistically high similarity to the inflammatory cluster III (MG10), phagocytic cluster (MG5), and antiviral cluster (MG11) of microglia in human AD. Most interestingly, the lipid-associated microglia cluster (MG4) was highly enriched in human AD and showed the most significant positive correlation with tangles, amyloid, Braak scores, and cognitive decline (Figs. 4A-4B; Fig. 17A). We also compared the human AD microglia transcriptomics study by Green et al., who performed a comprehensive snRNA-seq analysis of the prefrontal cortex of 437 elderly individuals, identifying 16 microglia subpopulations. Green et al. identified two lipid-processing-associated microglia subtypes (Mic.12 and Mic.13), which were closely associated with amyloid-β syndrome and significant cognitive decline in human AD subjects. Interestingly, in mouse Inpp5d-deficient microglia, RIPK1-regulated gene expression was highly enriched in two microglia subtypes associated with lipid processing in human AD (Mic.12 and Mic.13), as well as the Mic.15 (inflammation), Mic.14 (IFN), and Mic.6 (reaction) microglia subtypes (Fig. 4C–4D; Fig. 17B). These data suggest that RIPK1-regulated microglia gene transcription may be involved in the pathological progression of human AD.

[0403] We also investigated the effects of INPP5D deficiency on human iPSC-derived microglia. Similar to mouse INPP5D-deficient microglia, INPP5D deficiency in human iPSC-derived microglia also promoted RIPK1 (p-S166) activation, while Nec-1s treatment inhibited RIPK1 activation (Fig. 18A-18B). INPP5D deficiency in human iPSC-derived microglia led to increased expression of 1018 genes; of these, the expression of 492 genes was inhibited by Nec-1s treatment (Fig. 18C). In INPP5D-deficient human iPSC-derived microglia, RIPK1 upregulated genes included AD risk factors, pro-inflammatory cytokines, ROS mediators, complement, inflammasomes, and IFNs, similar to the situation in INPP5D-deficient mouse microglia (Fig. 18D). The loss of INPP5D in human iPSC-derived microglia also led to a reduction in the expression of 1242 genes; the expression of 669 of these genes was rescued by Nec-1s treatment (Fig. 18C). Therefore, the function of microglia RIPK1 in mediating the AD inflammatory pathway can also be confirmed in human iPSC-derived microglia.

[0404] Example 7: Myeloid INPP5D deficiency promotes RIPK1-mediated age-related motor dysfunction.

[0405] The above data indicate that INPP5D deficiency in microglia can promote the production of ROS and various pro-inflammatory factors in a RIPK1-dependent manner. We then assessed the potential contribution of RIPK1-regulated inflammatory processes in microglia to neuronal dysfunction in a non-cell-autonomous manner. We first analyzed data from 6-month-old Inpp5d cells... fl / fl Lyz2 cre / + Mice were observed to exhibit behaviors in open areas, the Y-maze, and elevated mazes, but no abnormalities were found (Figs. 19A-D). Regardless of whether they were treated with Nec-1s, Inpp5d fl / fl WT mice and Inpp5d fl / fl Lyz2 cre / + There was no difference in body weight among the mice (Fig. 19E). Therefore, myeloid INPP5D loss does not impair juvenile Inpp5d. fl / fl Lyz2 cre / + Motor function in mice.

[0406] Starting from 8 months of age, Inpp5d fl / fl Lyz2 cre / + Mice showed defects in the hindlimb clamping test (Fig. 19F). Therefore, we studied older Inpp5d mice. fl / fl Lyz2 cre / +Further motor function tests were conducted on the mice, and the effects of treatment with the RIPK1 inhibitor Nec-1s were investigated. We found that 8-month-old Inpp5d mice... fl / fl Lyz2 cre / + Mice exhibited motor dysfunction, including decreased total distance traveled, average speed, and vertical crawling ability in the open field test, increased tendency to fall on the merry-go-round, and decreased grip strength in the extreme point test, but no difference in duration of movement in the central zone during the open field test (Figs. 19A-19C). Oral administration of Nec-1s suppressed the motor dysfunctions observed in these tests. These results suggest that myeloid INPP5D deficiency can lead to age-related INpp5d deficiency. fl / fl Lyz2 cre / + Mice exhibited RIPK1-mediated motor dysfunction.

[0407] For 8-month-old Inpp5d fl / fl Lyz2 cre / + Mice and Inpp5d fl / fl Further spatial learning and memory assessments were conducted on the offspring of WT mice using the elevated Y-maze test. Eight-month-old Inpp5d mice... fl / fl Lyz2 cre / + Mice exhibited insufficient walking distance in the elevated Y-maze test, a condition salvaged by Nec-1s treatment. However, no significant differences were found in learning and memory parameters such as SAP, AAR, and SAR in the Y-maze test (Figure 19G). Compared with Inpp5d fl / fl Compared to WT mice, Inpp5d fl / fl Lyz2 cre / + There was no difference in the time or number of times mice entered the open or closed arm. Therefore, at 8 months of age, Inpp5d fl / fl Lyz2 cre / + Myeloid INPP5D deficiency in mice is sufficient to induce motor dysfunction, but may not be sufficient to cause spatial learning and memory impairment.

[0408] Cx3cr1 creERT2 Alleles have been used to remove genes of interest in the central nervous system in a microglia-specific manner. Therefore, we generated Inpp5d. fl / fl Cx3cr1 creERT2 / + Mice were used to test the effects of induced deletion of Inpp5d in microglia on adult mice (Figs. 20A-20B). Induced deletion of INPP5D also induced Inpp5d in 8-month-old adult mice. fl / fl Cx3cr1 creERT2 / +RIPK1 and microglia activation were observed in mice, while Nec-1s treatment inhibited both activations (Figs. 20C-20D). Tamoxifen-induced INPP5D loss also reduced the activity of 12-month-old Inpp5d cells. fl / fl Cx3cr1 creERT2 / + The locomotor function of mice was also tested using rotation and open-field tests (Figs. 20E-20F). Tamoxifen-induced Inpp5d fl / fl Cx3cr1 creERT2 / + In mice, INPP5D deficiency or Nec-1s treatment did not affect their body weight (Figure 20B). Importantly, after 45 days of treatment with Nec-1s, the Inpp5D levels in 12-month-old mice decreased. fl / fl Cx3cr1 creERT2 / + The mice's motor function was also restored (Fig. 20E-Fig. 20F).

[0409] These data indicate that the loss of INPP5D in microglia alone promotes age-dependent microglia activation and dyskinesia, while blocking RIPK1 kinase inhibits age-dependent microglia activation and dyskinesia.

[0410] Example 8: Myeloid INPP5D loss promotes RIPK1-dependent motor neuron loss.

[0411] Inpp5d fl / fl The age-dependent impairment of motor function in Lyz2cre mice prompted us to investigate the histological pathology of the spinal cord. This study focused on 8-month-old Inpp5d mice. fl / fl Lyz2 cre / + Quantitative analysis of ChAT-positive motor neurons in the L1-L4 segments of the mouse spinal cord showed that, in age-matched littermate Inpp5d mice... fl / fl Compared to WT mice, the number of motor neurons was reduced (Fig. 6D). Age-matched littermate Inpp5d mice... fl / fl Compared to WT mice, 8-month-old Inpp5d fl / fl Lyz2 cre / + The number of NeuN-positive neurons in the spinal cord and cortex of mice was also reduced (Fig. 6E; Fig. 21A). After oral administration of the RIPK1 kinase inhibitor Nec-1s and 45 days of RIPK1 activity inhibition using the D138N gene, the number of 8-month-old Inpp5d neurons was significantly reduced. fl / fl Lyz2 cre / + The loss of ChAT-positive motor neurons and NeuN-positive neurons in the mouse spinal cord was inhibited (Fig. 6D-6E; Fig. 21A).

[0412] Furthermore, we investigated the effect of INPP5D loss on myelination. We observed that in Inpp5d...fl / fl .Lyz2 cre / + In the white matter of the ventrolateral spinal cord of mice, the number of myelinated axons was significantly reduced. Myelin sheath desquamation led to abnormal myelination, manifested as a decreased g-ratio (axon diameter / axon plus myelin diameter), a decrease in the number of smaller diameter axons, and an increase in the number of larger diameter axons; Lyz2 cre / + Mice and age-matched littermates of Inpp5d fl / fl Compared to WT mice (Figure 6F), inhibition of RIPK1 kinase by Nec-1s also significantly reduced the number of 8-month-old Inpp5d mice. fl / fl Lyz2 cre / + Disorders of myelination in the mouse spinal cord.

[0413] We found that tamoxifen-induced 12-month-old Inpp5d fl / fl Cx3cr1 creERT2 / + Loss of INPP5D expression in mouse microglia also leads to Caspase-3 activation and motor neuron loss in the spinal cord, which is related to the aging of Inpp5D cells. fl / fl Lyz2 cre / + The situation was the same in mice (Figs. 21B-21C). Importantly, in Inpp5d... fl / fl Cx3cr1 creERT2 / + In vivo injection of Nec-1s in mice for 45 days was able to salvage motor neuron loss and apoptosis.

[0414] The presence of cytoplasmic TDP-43 inclusion bodies in the central nervous system is a major neuropathological feature of human neurodegenerative diseases. We observed this in 12-month-old Inpp5d... fl / fl Cx3cr1 creERT2 / + Activation of RIPK1 (p-S166) and cytoplasmic inclusions of TDP-43 and p-TDP-43 were found in the mouse spinal cord and brain. This was because tamoxifen induced the loss of INPP5D in microglia, and drug inhibition of RIPK1 kinase activity suppressed this loss (Fig. 22D-Fig. 22F). Interestingly, TDP-43 cytoplasmic inclusions were primarily found in neurons. These data suggest that the loss of INPP5D in microglia leads to a RIPK1-dependent neuroinflammatory response, which in turn promotes cytoplasmic TDP-43 translocation and neuronal apoptosis in a non-cell-autonomous manner.

[0415] Example 9: INPP5D inhibits RIPK1 activity through direct interaction.

[0416] Since the above results indicate that RIPK1 kinase is activated in INPP5D-deficient microglia, we then investigated the mechanism by which INPP5D inhibits RIPK1 kinase activation. The full-length protein encoded by INPP5D is called SHIP1, which contains an SH2 domain at its N-terminus, a lipid phosphatase domain in the center, and three highly phosphorylated Tyr residues at its C-terminus. Because RIPK1 activation is regulated by phosphorylation, we used mass spectrometry to perform a comprehensive analysis of the phosphorylation sites and levels of RIPK1 in WT and INPP5DKOBV2 cells (Figure 22A). Our data showed a significant increase in phosphorylation at many sites on RIPK1 in INPP5DKOBV2 cells. The sites with the most increased phosphorylation were S595 and Y383. In addition, minor changes in phosphorylation were detected at S430, S336, S332, S452, S321, Y309, S25, S360, S6, S14, and S415 (Figure 22B). We confirmed using a RIPK1 site-specific phosphorylation antibody that INPP5D gene knockout led to increased phosphorylation of S166, S321, S14 / S15, and S415 residues (Fig. 6A). RIPK1 kinase inhibitors Nec-1s effectively suppressed the signaling of p-S14 / S15 and p-S166, biomarkers of RIPK1 activation, but had no effect on p-S321 and p-S415, which are mediated by other kinases such as TAK1, IKKs, and MK2 (Fig. 6A). Furthermore, we noted increased phosphorylation of JNK and c-Jun (S63) after INPP5D knockout in BV2 cells, which Nec-1s reduced. However, Nec-1s had no effect on the activation of Akt in BV2 cells (as shown by p-S473Akt) (Fig. 6A). These results indicate that INPP5D deficiency leads to the loss of the inhibitory mechanism for RIPK1 activation. Inhibiting RIPK1 kinase in INPP5D-deficient BV2 cells prevents the activation of JNK and c-Jun, but does not affect AKT activation, which is known to be regulated by the PtdIns(3,4,5)P3 phosphatase activity of the INPP5D protein product SHIP1.

[0417] We further investigated the interaction between INPP5D and RIPK1. The interaction between endogenous INPP5D and RIPK1 was detected by co-immunoprecipitation (Fig. 6B). Notably, co-expression of INPP5D and RIPK1 inhibited RIPK1 activation in 293T cells in a dose-dependent manner (Fig. 6C). The inhibition of RIPK1 activation by INPP5D was independent of its lipid phosphatase activity, as the INPP5D mutant with the inactivating mutation D676G at the active site of the lipid phosphatase still reduced RIPK1 activation (Fig. 6D). Therefore, the inhibition of RIPK1 kinase activity by INPP5D may not require its inhibition of SHIP1 lipid phosphatase activity. In INPP5DKOBV2 cells, RIPK1 was activated, as shown by p-S166, and Nec-1s inhibited it (Fig. 22C).

[0418] INPP5D encodes SHIP1, which contains an N-terminal SH2 (Src homology 2) domain, an inositol phosphatase domain, and two C-terminal protein-protein interaction domains. To investigate the interaction between INPP5D and RIPK1, we tested different truncated mutants of INPP5D and found that simply removing the SH2 domain canceled its binding to RIPK1 (Figure 6E). Therefore, the INPP5D mutant with the ΔSH2 domain could not inhibit RIPK1 activation (Figure 6F). Thus, the SH2 domain of INPP5D is crucial for mediating its interaction with RIPK1 and inhibiting RIPK1 activation.

[0419] The SH2 domain is an important protein-protein interaction module that mediates cellular signaling cascades by binding to specific phosphotyrosine residues in target proteins. Y383RIPK1 is known to be phosphorylated by JAK1 in the IFNγ pathway. Interestingly, in our mass spectrometry analysis, p-Y383 was the most phosphorylated Tyr under INPP5D deficiency conditions (Figure 22B). We then investigated the role of p-Y383RIPK1 in INPP5D-RIPK1 recognition. Compared to WTRIPK1, the interaction between INPP5D and the Y383FRIPK1 mutant was significantly reduced, indicating that phosphorylation of Y383 in RIPK1 is crucial for binding to INPP5D. Furthermore, INPP5D expression decreased the level of p-S166 in WTRIPK1 but did not decrease the level of Y383FRIPK1, suggesting that the binding of INPP5D to RIPK1 is essential for inhibiting RIPK1 kinase activation.

[0420] Next, we identified the direct interaction between the SH2 domain of INPP5D and RIPK1 in vitro. We incubated 293T cell lysates transfected with WT-RIPK1 or Y383F-RIPK1 expression vectors using recombinant GST-INPP5D-SH2. We found that GST-INPP5D-SH2 reduced WT-RIPK1, but its binding rate to Y383F-RIPK1 was significantly decreased (Fig. 22D). Recombinant GST-INPP5D-SH2 also attracted endogenous RIPK1 (Fig. 22E). Furthermore, biotin-Y383RIPK1 peptide incubation with WTBV2 cell lysates reduced INPP5D, but non-phosphorylated biotin-Y383 peptide did not (Fig. 22F). Since RIPK1 activation is mediated by its dimerization, we then tested the effect of INPP5D expression on RIPK1 dimerization. We found that INPP5D expression reduced RIPK1 dimerization and dimerization-induced RIPK1 activation (as shown by p-RIPK1(S166)). Therefore, the binding of INPP5D to RIPK1 may compete with RIPK1 homodimerization, which is crucial for RIPK1 kinase activation. We also used differential scanning calorimetry to detect the binding of INPP5D to RIPK1 and found that treatment with the pY383-RIPK1 peptide increased the melting temperature of the INPP5D-SH2 protein, while the Y383-RIPK1 peptide had no such effect (Figure 22G). This result further highlights the importance of pY383RIPK1 in mediating its direct binding to the INPP5DSH2 domain.

[0421] Next, we used solution NMR spectroscopy to investigate the interaction between the mouse INPP5DSH2 domain and the RIPK1pY383 peptide. NMR titration showed that after the addition of the RIPK1pY383 peptide, 15 Many N-labeled INPP5D-SH2 proteins 15 The N-HSQC peak shifts in a dose-dependent manner (Figure 23A). A series of triple resonance experiments were used to investigate this shift. 15 After assigning residue-specific values ​​to the N-HSQC peak, we found that two regions of the SH2 domain (residues 55-58 and 68-77) were strongly perturbed, indicating that the RIPK1pY383 peptide can specifically interact with two regions of the INPP5DSH2 domain.

[0422] To elucidate the structural basis of the specific binding of mouse INPP5D to RIPK1, we used NMR-derived intermolecular distance constraint to determine the solution structure of the mINPP5DSH2 domain complex with the mRIPK1 peptide fragment (residues 378-393) and the p-Y383 complex (Fig. 23B). The p-Y383-mRIPK1 peptide adopts an extended conformation, binding to two major pockets in the SH2 domain (Fig. 23C-Fig. 23D). Residue p-Y383 (position 0) corresponds to a highly positively charged pocket composed of R15, R34, and R57; its phosphate group forms hydrogen bonds with the guanidino groups of R15 and R34, while its aromatic ring forms a cation-π interaction with the side chain of R57. In the other pocket, residue F386 at the +3 position inserts into a deep hydrophobic cavity composed of Y56, V68, A70, Y89, and V97. In fact, sequence alignment revealed that the aromatic residue at position +3 in RIPK1 is highly conserved across mammalian species. The bulky aromatic residues Phe or Tyr at position +3 in RIPK1, compared to the Tyr / Val-rich pocket in the mINPP5DSH2 domain, are more consistent with other reported SH2 conjugates, most of which contain Ala / Val / Leu at position +3 (Figure 23E). Furthermore, we found that mutating F386 to serine affects the specific binding of RIPK1 to INPP5D. Therefore, both structural analysis and mutagenesis studies support a model that the interaction between INPP5D and RIPK1 depends on the simultaneous recognition of Y383 phosphorylation and the bulky aromatic residue at position +3 by its SH2 domain.

[0423] Example 10: In 5×FAD mice, myeloid Inpp5d deficiency promotes a surge in RIPK1-regulated age-specific Aβ production.

[0424] Inpp5d is highly selectively expressed in myeloid cells such as microglia and macrophages. We investigated the effect of myeloid INPP5D on Aβ deposition using C57BL / 6J hemi-heterozygous 5×FAD mice (MMRRCstock34848). These mice exhibit low levels of Aβ plaques and glial lesions in early childhood (3–6 months) and late-stage amyloid deposition and neuroinflammation in older mice (9 months and beyond). We also investigated the effect of Inpp5d-deficient female myeloid mice. fl / fl Lyz2 cre / + Mice were crossed with male 5×FAD mice to produce 5×FAD; Inpp5d fl / fl Lyz2 cre / + Mice. All experiments were conducted in male mice, and the mutant APP / PS1 transgene was inherited from male mice to avoid sex differences in phenotypic development. Inpp5d mice of different ages were extracted with 5M GuHCl (a chaotic strong denaturant). fl / flWT mice, 5×FAD mice, 5×FAD; Inpp5d fl / fl Lyz2 cre / + Mice and 5×FAD; Inpp5d fl / fl Lyz2 cre / + The total levels of Aβ40 and Aβ42 in the brain lysate of Ripk1D138N / D138N mice were determined, and the total levels of Aβ42 and Aβ40 were measured using Aβ40 / 42 enzyme-linked immunosorbent assay (Figs. 8A-8B and 24A). Interestingly, the levels of Aβ40 and Aβ42 in the brain lysate of age-matched 5× FAD mice were also measured. fl / fl Lyz2 cre Compared to mice, we detected 5×FAD at approximately 5.5 months of age; Inpp5d fl / fl Lyz2 cre The level of total Aβ42 in the mouse brain showed a striking and dramatic increase, reaching approximately 23 μg / brain, an increase of more than 6-fold, reaching approximately 23 μg / brain (the entire brain after removing the olfactory bulb and cerebellum) (Fig. 8A). This was observed in age-matched 5×FAD; Inpp5d mice. fl / fl Lyz2 cre / + In Ripk1D138N / D138N mice (carrying a RIPK1 kinase inactivation mutant allele at the endogenous RIPK1 locus), this rapid increase in Aβ42 levels was suppressed to the level of 5×FAD mice. Compared with age-matched male 5×FAD mice, 5×FAD mice around 5.5 months of age... fl / fl Lyz2 cre / + The total level of Aβ40 in the mouse brain also increased dramatically in a RIPK1-dependent manner, but its level (approximately 750 ng / brain) was much lower than that of Aβ42 (Figure 1B).

[0425] To determine whether myeloid INPP5D deficiency in 5×FAD mice promotes peak Aβ production around 5.5 months of age, or simply accelerates age-dependent Aβ production, we analyzed another group of 5×FAD mice and 5×FAD;Inpp5d mice at a higher temporal resolution. fl / fl Lyz2 cre / + Aβ levels in mice at 3, 4, 5, 5.5, 6, 8, 8.5, and 10 months of age. We found that 5×FAD;Inpp5d fl / fl Lyz2 cre / +Mice did indeed exhibit a surge in Aβ levels between 5 and 5.5 months of age, which then subsided around 6 months of age (Fig. 8C, D). Therefore, there is a critical time window between 5 and 5.5 months of age where myeloid INPP5D deficiency in 5×FAD mice promotes a rapid increase in total Aβ42 / Aβ40 levels. This surge in Aβ depends on myeloid INPP5D deficiency and RIPK1 activity, as in 5×FAD mice or 5×FAD;Inpp5d fl / fl Lyz2 cre / + This increase was not detected in either Ripk1D138N / D138N mice.

[0426] To determine whether this surge in Aβ42 was primarily due to monomers or fibers, we then analyzed Aβ42 levels using two additional extraction methods employing 0.2% DEA (diethanolamine) as a mild detergent. The DEA-soluble fraction consisted mainly of Aβ42 monomers and oligomers, while the DEA-insoluble fraction was primarily Aβ42 fibers. These fibers were further dissolved in 5 M GuHCl before being used for Aβ ELISA (Figure 24B). Age-matched 5× FAD; Inpp5d fl / fl Lyz2 cre / + Compared to mice, 5×FAD;Inpp5d mice aged 4 to 5 months fl / fl Lyz2 cre / + The level of DEA-insoluble Aβ42 fibers in the mouse brain also showed a dramatic increase of more than 13-fold, reaching greater than 9 μg / brain. In age-matched 5× FAD; Inpp5d fl / fl Lyz2 cre / + In Ripk1D138N / D138N mice, inhibition of RIPK1 reduced the level of DEA-insoluble Aβ42 fibers to approximately 2.7 μg / brain, comparable to the level in 5×FAD mice, but did not reduce the level of DEA-soluble Aβ42 (Fig. 8E-F). These results suggest that in 5×FAD mice, myeloid INPP5D deficiency primarily promotes RIPK1-regulated Aβ42 fibers.

[0427] To determine the biochemical properties of the Aβ42 fibers present in the spike, we also analyzed the Aβ42 fibers treated with 2% sarkosyl extract. After centrifugation at 166,000 x g, abundant Aβ42 fibers were found in the sarkosyl-soluble fraction (Fig. 8G, white triangles). 5×FAD; Inpp5d fl / fl Lyz2 cre / + Mice aged 5-6 months showed a dramatic increase in the amount of soluble Sarkosyl Aβ42 fibers compared to age-matched male 5×FAD mice; while in age-matched 5×FAD mice... fl / fl Lyz2cre / + In Ripk1D138N / D138N mice, inhibition of RIPK1 also blocked the surge in sarkosyl-soluble Aβ42 fibers (Fig. 8H). Conversely, 5×FAD;Inpp5d fl / fl Lyz2 cre / + The level of sarkosyl-insoluble Aβ42 fibers in mice was not significantly increased (Fig. 8I). At 5×FAD; Inpp5d fl / fl Lyz2 cre / + In Ripk1D138N / D138N mice, inhibition of RIPK1 slightly decreased sarkosyl-insoluble Aβ42 fiber levels at 6 months of age (Figure 8I). In summary, these data suggest that 5×FAD;Inpp5d fl / fl Lyz2 cre / + The loss of myeloid Inpp5d in mice may be a key time window in the pathological evolution of AD, during which the production of free Aβ fibers is significantly stimulated in a RIPK1-dependent manner (Figure 1C-D) and is lost.

[0428] Therefore, even with strong transgenic expression of mutant APP and PS1 driven by the neuronal Thy-1 promoter in 5×FAD mice, myeloid INPP5D-deficient RIPK1 activation could drive a further surge in Aβ fibers at 5-6 months of age. These results indicate that Aβ fiber generation involves interactions between neurons and myeloid non-neuronal cells during AD pathogenesis. Specifically, RIPK1 activation in myeloid cells may lead to a defined time window during AD pathological evolution when Aβ fiber levels are highly stimulated in a non-cellular autonomous manner (critical time window). fl / fl Lyz2 cre / + Inpp5d fl / fl Lyz2 cre / + Inpp5d fl / fl Lyz2 cre / +

[0429] Example 11: Age-specific dynamic changes in amyloid plaques in 5×FAD mice with Inpp5d deficiency in myeloid lineage

[0430] Accumulation of Aβ42 and related Aβ peptides leads to the formation of extracellular amyloid plaques, a major pathological feature of Alzheimer's disease (AD). We then used the 6E10 antibody to treat 5×FAD mice and 5×FAD;Inpp5d mice. fl / fl Lyz2 cre / + Mice and 5×FAD; Inpp5d fl / fl Lyz2 cre / +Ripk1D138N / D138N mice at 4, 6, and 9.5 months of age (Figs. 8J and 8K). At 4 months of age, 5×FAD mice, 5×FAD; Inpp5d fl / fl Lyz2 cre / + Mice and 5×FAD; Inpp5d fl / fl Lyz2 cre / + The data from Ripk1D138N / D138N mice are comparable to those from published studies of age-matched, semi-heterozygous 5×FAD mice with a C57BL / 6J background. (Inpp5d) fl / fl Lyz2 cre / +

[0431] At 6 months, 5×FAD; Inpp5d fl / fl Lyz2 cre / + The number of amyloid plaques in the brains of mice was significantly reduced compared to age-matched 5×FAD mice (Figs. 8J, 8K, 8M and 24C, 8D), which coincides with the critical time window of the surge in Aβ42 fibers (Figs. 8C-8D). Compared to 5×FAD; Inpp5d fl / fl Lyz2 cre / + Compared to mice, at 5×FAD;Inpp5d fl / fl Lyz2 cre / + Inhibition of RIPK1 in 6-month-old Ripk1D138N / D138N mice prevented a transient decrease in the number of amyloid plaques. (Inpp5d) fl / fl Lyz2 cre / + And the level of amyloid plaques was made comparable to that of age-matched 5×FAD mice (Fig. 8J, Fig. 8K, and Fig. 8M). Inpp5d fl / fl Lyz2 cre / + Inpp5d fl / fl Lyz2 cre / + Inpp5d fl / fl Lyz2 cre / + These results indicate that RIPK1 kinase activation occurs at 5×FAD;Inpp5d fl / fl Lyz2 cre / + It plays a key role in the rapid increase of free Aβ42 fibers in mice and the transient reduction of amyloid plaques at 5-6 months of age.

[0432] 5×FAD; Inpp5d fl / fl Lyz2 cre / + The reduction in amyloid plaques observed in mice at 6 months of age was transient, as the 5×FAD;Inpp5d mice at 9.5 months of age showed a decrease. fl / fl Lyz2 cre / +The total amount of amyloid plaques in the brains of mice was even higher than that in age-matched 5×FAD mice (Fig. 8J, Fig. 8K, and Fig. 8N). Inhibition of 9.5-month-old 5×FAD mice; Inpp5d fl / fl Lyz2 cre / + In Ripk1D138N / D138N mice, RIPK1 kinase can reduce amyloid plaque levels to levels consistent with those in age-matched 5×FAD mice (Fig. 8G, Fig. 8H, Fig. 8K). Therefore, myeloid Inpp5d deficiency leads to RIPK1 kinase activation, resulting in a surge in most free Aβ42 fibers within a critical time window of approximately 5-6 months of age, ultimately leading to increased levels in older 5×FAD mice. fl / fl Lyz2 cre / + Increased amyloid deposition levels were observed in mice. These data suggest that RIPK1 and myeloid INPP5D deficiency in 5×FAD mice exhibit considerable age specificity in the non-cellular autonomous regulation of amyloid deposition. (5×FAD; Inpp5d) fl / fl Lyz2 cre / + The transient decrease in amyloid plaque number in mice around 6 months of age may be due to activation of microglia phagocytic activity, as microglia INPP5D deficiency has been shown to enhance microglia activation and recruitment of Aβ plaques, as well as Aβ phagocytosis, in 5×FAD mice. However, the phagocytic activity of activated microglia in clearing Aβ plaques is limited, similar to the higher amyloid load in older (9.5 months) mice due to myeloid INPP5D deficiency in 5×FAD mice, which is similar to the increased Aβ plaque load in PSAPP mice with conditional INPP5D knockout of microglia. Therefore, in 5×FAD; Inpp5d fl / fl Lyz2 cre / + In Ripk1D138N / D138N mice, RIPK1 inhibition can promote Inpp5d fl / fl Lyz2 cre / + Microglial homeostasis.

[0433] Example 12 Regulation of neuroinflammation in myeloid Inpp5d-deficient 5×FAD mice by RIPK1

[0434] Next, we used ionized calcium-binding aptamer protein-1 (IBA1) and CD68 immunohistochemical staining to assess hippocampal microglia activation in 4, 6, and 9.5-month-old 5×FAD mice with and without myeloid Inpp5d deletion and RIPK1 activity. At 4 months of age, 5×FAD; Inpp5d fl / fl Lyz2 cre / +The volume of microglia associated with non-amyloid plaques in the hippocampus of mice was not different from that in age-matched 5×FAD mice, indicating that myeloid Inpp5d deficiency does not promote microglial activation in the central nervous system at 4 months of age (Fig. 25A, Fig. 25B). By 6 months of age, we found that the volume of microglia associated with non-amyloid plaques in the hippocampus of mice was not different from that in age-matched 5×FAD Inpp5d mice. fl / fl Lyz2 cre / + Compared to mice, 5×FAD; Inpp5d fl / fl Lyz2 cre / + Activation of non-plaque-associated microglia was more pronounced in the mouse hippocampus, manifested by increased IBA1+ microglia volume, CD68 cell volume, and branching complexity (Figs. 9A-9D). 5×FAD; Inpp5d fl / fl Lyz2 cre / + In Ripk1D138N / D138N mice at 6 months of age, non-plaque-associated microglial activation was reduced to the level of 5×FAD mice (Fig. 9A-Fig. 9D). Therefore, in 6-month-old 5×FAD mice... fl / fl Lyz2 cre / + In mice, myeloid INPP5D deficiency promotes RIPK1-dependent microglial activation, but not in 4-month-old mice.

[0435] Inpp5d fl / fl Lyz2 cre / + Inpp5d fl / fl Lyz2 cre / + Inpp5d fl / fl Lyz2 cre / + Inpp5d fl / fl Lyz2 cre / + Inpp5d fl / fl Lyz2 cre / + Inpp5d fl / fl Lyz2 cre / + We also examined the activation of plaque-associated microglia. We found 5×FAD;Inpp5d fl / fl Lyz2 cre / + At 6 months of age, the level of plaque-associated microglia in mice was elevated, while the level of 5×FAD in Inpp5d mice was higher than that in age-matched mice. fl / fl Lyz2 cre / + Ripk1D138N / D138N mice showed a decrease (Figs. 9E-9G). Compared with age-matched 5×FAD mice, 9.5-month-old 5×FAD;Inpp5d fl / fl Lyz2 cre / + The level of plaque-associated microglia in mice was also increased, and in age-matched 5×FAD;Inpp5d fl / fl Lyz2cre / + In Ripk1D138N / D138N mice, inhibition of RIPK1 also reduced plaque-associated microglia levels (Fig. 9I-Fig. 9K). Therefore, RIPK1 activation in myeloid INPP5D-deficient 5×FAD mice plays a crucial role in promoting microglia recruitment from amyloid plaques, as inhibition of RIPK1 reduced microglia in 6-month and 9.5-month-old 5×FAD mice. fl / fl Lyz2 cre / + Activation of plaque-associated microglia in mice.

[0436] We then describe the changes in the morphology and number of amyloid plaques with age. (In 5×FAD; Inpp5d) fl / fl Lyz2 cre / + In the hippocampus of mice, the content of 6E10+Aβ plaques was reduced at 6 months of age compared with age-matched 5×FAD mice, while at 9.5 months of age, the Aβ plaques were loosely shaped and larger in size compared with age-matched 5×FAD mice (Figs. 9E, 9H, 9I, and 9L). Conversely, in 5×FAD mice, the content of 6E10+Aβ plaques was reduced compared with age-matched 5×FAD mice (Figs. 9E, 9H, 9I, and 9L). fl / fl Lyz2 cre / + The size of 6E10+Aβ plaques in Ripk1D138N / D138N mice increased at 6 months of age, but remained at 9.5 months of age consistent with age-matched 5×FAD; Inpp5d fl / fl Lyz2 cre Compared to mice, they are smaller and more compact, resembling age-matched 5×FAD mice (Figs. 9E, 9H, 9I, and 9L). These results demonstrate the effectiveness of RIPK1 in 5×FAD; Inpp5d mice. fl / fl Lyz2 cre A crucial role in the dynamic evolution of Aβ plaques in the mouse hippocampus: Activation of myeloid RIPK1 may promote the phagocytic activity of early-activated microglia around 6 months of age, leading to the clearance of Aβ plaques. However, the ability of activated microglia to clear Aβ plaques is limited. Our results suggest that aged activated microglia may primarily mediate harmful neuroinflammation, thereby promoting Aβ plaque deposition.

[0437] Activation of NLRP3 and ASC expression in microglia is associated with promoting amyloid deposition in early human Alzheimer's disease (AD). Since we found that myeloid Inpp5d deficiency promotes NLRP3 inflammasome expression in microglia, we next evaluated ASC spots associated with Aβ plaques. We found that in 9.5-month-old 5×FAD patients, Inpp5d... fl / fl Lyz2 cre / +The volume ratio of ASC+ / 6E10+ amyloid plaques in the hippocampus of mice was increased compared to 5×FAD mice, while inhibition of RIPK1 reduced the volume of ASC+ / 6E10+ amyloid plaques in 5×FAD mice; Inpp5d fl / fl Lyz2 cre / + The ratio of this RIPK1 in Ripk1D138N / D138N mice decreased to the level in 5×FAD mice (Fig. 9M, Fig. 9N). This result reveals that RIPK1 activation mediated by INPP5D loss in myeloid cells promotes Aβ plaque deposition in aged 5×FAD mice. fl / fl Lyz2 cre / +

[0438] We also assessed astrocyte activation in mice aged 4 to 9.5 months (Fig. 25C–Fig. 25G). At 4 months of age, 5×FAD; Inpp5d fl / fl Lyz2 cre / + The activation level of astrocytes in the hippocampus of mice was similar to that in 5×FAD mice (Fig. 25C, Fig. 25D) Inpp5d fl / fl Lyz2 cre / + In 5×FAD mice, astrocyte activation in the hippocampus increased at 6 months and 9.5 months of age, and in 5×FAD;Inpp5d fl / fl Lyz2 cre / + Further enhancement was observed in mice, with 5×FAD;Inpp5d fl / fl Lyz2 cre / + The Ripk1D138N / D138N mice decreased to the level of 5×FAD mice at 9.5 months of age, but did not decrease at 6 months of age (Fig. 25E-Fig. 25G). These results indicate that in 5×FAD; Inpp5d fl / fl Lyz2 cre / + In Ripk1D138N / D138N mice, myeloid Inpp5d deficiency promotes a sustained age-dependent increase in astrocyte activation, while inhibition of RIPK1 mitigates this increase.

[0439] In summary, these results indicate that in 5×FAD;Inpp5d fl / fl Lyz2 cre / + In mice, the increase in peak Aβ around 5.5 months of age precedes microglial activation. (5×FAD; Inpp5d) fl / fl Lyz2 cre / + Microglia in the mouse brain are activated around 6 months of age, suggesting that the temporary decrease in Aβ plaque levels during this period may be due to enhanced phagocytic activity of activated microglia. However, the ability of activated microglia to clear amyloid plaques is limited, especially in older mice (5×FAD;Inpp5d). fl / flLyz2 cre / + In mice, the number of plaques was even higher at 9.5 months of age than in 5×FAD mice. Inhibition of 5×FAD; Inpp5d fl / fl Lyz2 cre / + RIPK1 in Ripk1D138N / D138N mice maintained amyloid plaque levels in 6-month-old and 9.5-month-old mice comparable to those in 5×FAD mice. These data suggest that in 5×FAD mice... fl / fl Lyz2 cre / + In mice, there is a non-cell-autonomous interaction between RIPK1-regulated neuroinflammation and Aβ generation and deposition mediated by neuronal γ-secretase activity.

[0440] Example 13 Myeloid Inpp5d deletion promotes RIPK1-mediated peripheral inflammation and T cell infiltration

[0441] Because INPP5D is highly expressed in myeloid cells such as microglia and macrophages, we next investigated the contribution of peripheral inflammation, as it is considered a contributing factor to AD. Therefore, we analyzed INPP5D levels from 6-month-old cells using RNA-seq. fl / fl WT mice, 5×FAD mice, 5×FAD; Inpp5d fl / fl Lyz2 cre / + Mice and 5×FAD; Inpp5d fl / fl Lyz2 cre / + Peripheral blood mononuclear cells (PBMCs) isolated from Ripk1D138N / D138N mice.

[0442] Inpp5d fl / fl Inpp5d fl / fl Lyz2 cre / + Inpp5d fl / fl Inpp5d fl / fl Inpp5d fl / fl Lyz2 cre / + Inpp5d fl / fl Lyz2 cre / + Inpp5d fl / fl Lyz2 cre / + Inpp5d fl / fl Lyz2 cre / + Inpp5d fl / fl Lyz2 cre / + Inpp5d fl / fl Inpp5d fl / fl Lyz2 cre / + Inpp5d fl / fl Inpp5dfl / fl Inpp5d fl / fl Lyz2 cre / + Inpp5d fl / fl Lyz2 cre / + Inpp5d fl / fl Inpp5d fl / fl Lyz2 cre / + Inpp5d f l / fl Lyz2 cre / + With Inpp5d fl / fl Compared to WT mice, 5×FAD; Inpp5d fl / fl Lyz2 cre / + and 5×FAD; Inpp5d fl / fl Lyz2 cre Inpp5d expression was also decreased in peritoneal cells of Ripk1D138N / D138N mice (Fig. 26A). Cluster analysis heatmap showed that 5×FAD and 5×FAD;Inpp5d fl / fl Lyz2 cre There were 1338 DEGs in the peripheral PBMCs of mice; interestingly, inhibiting RIPK1 resulted in 5× FAD; Inpp5d fl / fl Lyz2 cre / + Gene expression patterns of 545 DEGs in peripheral PBMCs of Ripk1D138N / D138N mice were restored to levels close to those of 5×FAD and WT mice (Fig. 26B). GOBP (Biological Process Clustering) analysis showed that the restored DEGs in RIPK1-D138N were mainly enriched in immune system processes, as the number of upregulated genes in immune system processes increased sharply in 5×FAD mice; age-matched Inpp5d fl / fl Compared with 5×FAD mice, WT mice showed a significant increase in the number of upregulated genes in immune system processes, while 5×FAD mice showed a significantly higher number of upregulated genes. fl / fl Lyz2 cre / + The effect was reduced in Ripk1D138N / D138N mice (Figure 10A). Inpp5d fl / fl Inpp5d fl / fl Lyz2 cre / + Inpp5d fl / fl Lyz2 cre / + In particular, we found that in 5×FAD;Inpp5d fl / fl Lyz2 cre / +In the PBMCs of Ripk1D138N / D138N mice, gene expression in the TNF signaling pathway, NF-κB signaling pathway, NOD-like receptor signaling pathway, complement and coagulation cascade pathways was increased, and four AD risk factor genes were upregulated; Inhibition of Inpp5d fl / fl Lyz2 cre / + After RIPK1, 5×FAD; Inpp5d fl / fl Lyz2 cre / + The expression of four AD risk factor genes was reduced in PBMCs of Ripk1D138N / D138N mice (Fig. 10B). Notably, in 5×FAD;Inpp5d fl / fl Lyz2 cre / + In the PBMCs of Ripk1D138N / D138N mice, the expression of the LOAD risk factor ApoE and the EOAD gene PSEN1 was also increased, while the expression of 5×FAD was reduced. fl / fl Lyz2 cre / + After RIPK1 was introduced into Ripk1D138N / D138N mice, its expression was restored to Inpp5d. fl / fl Levels in WT mice (Fig. 10C-Fig. 10E). Therefore, myeloid Inpp5d deficiency also induces a RIPK1-dependent peripheral inflammatory response. Furthermore, 5×FAD;Inpp5d fl / fl Lyz2 cre / + Apolipoprotein E and PSEN1 in mouse peripheral PBMCs are upregulated in a RIPK1-dependent manner, which may provide a link between LOAD and EOAD, two different forms of AD.

[0443] To further confirm peripheral inflammation, samples were collected from Inpp5d mice at 3.5 months and 6 months of age. fl / fl WT mice, 5×FAD mice, 5×FAD; Inpp5d fl / fl Lyz2 cre Mice and 5×FAD; Inpp5d fl / fl Lyz2 cre / + Plasma was collected from Ripk1D138N / D138N mice, and Olink proteomics analysis was performed on 48 cytokines. At 3.5 months of age, there were no differences in cytokine levels among the groups. However, 5×FAD;Inpp5d fl / fl Lyz2 cre / + The levels of pro-inflammatory cytokines and chemokines in the plasma of mice at 6 months of age, including CCL12, CCL2, CSF3, CXCL9, IL1β, IL6, and IL16, were compared with those at 3.5 months of age and age-matched Inpp5 d. fl / flLevels were increased in both WT and 5×FAD mice (Figs. 10F-10L). In age-matched 5×FAD mice; Inpp5d fl / fl Lyz2 cre / + In Ripk1D138N / D138N mice, all of these increased levels of pro-inflammatory cytokines and chemokines were reduced to levels seen in 5×FAD mice (Fig. 10F-Fig. 10L). Therefore, myeloid Inpp5d deficiency in 5×FAD mice also promotes age-dependent peripheral inflammation.

[0444] Interestingly, we observed that DEGs rescued by RIPK1-D138N in PBMCs (Fig. 26B) were enriched in genes involved in leukocyte and T cell activation processes (Fig. 26C), suggesting that Inpp5d deletion in the myeloid lineage of 5×FAD mice may promote peripheral T cell activation via RIPK1. Increasing evidence suggests that T cell activation can enhance its infiltration into the central nervous system in neurodegenerative diseases. To investigate whether increased T cell activation promotes T cell infiltration into the central nervous system in a RIPK1-regulated manner, we used a T cell-specific CD3 antibody against Inpp5d... fl / fl WT mice, 5×FAD mice, 5×FAD; Inpp5d fl / fl Lyz2 cre / + Mice and 5×FAD; Inpp5d fl / fl Lyz2 cre / + Hippocampal sections stained from Ripk1D138N / D138N mice at 4 and 6 months of age. Results analysis showed that, compared with Inpp5d... fl / fl Compared to WT mice, 5×FAD mice showed an increase in the number of CD3+ T cells in the dentate gyrus (DG). fl / fl Lyz2 cre / + The number of CD3+ T cells in Ripk1D138N / D138N mice was further increased; Inpp5d fl / fl Lyz2 cre / + RIPK1 levels in Ripk1D138N / D138N mice decreased to those in 5×FAD mice (Figures 11A-11C). Notably, in 5×FAD mice, Inpp5d... fl / fl Lyz2 cre / + The mice showed increased T-cell hippocampal infiltration as early as 4 months of age.

[0445] We further analyzed the characteristics of T cell and B cell infiltration in the central nervous system of 6-month-old mice using flow cytometry (Figure 11D). The results showed that, in age-matched Inpp5d mice... fl / flWT mice (4.57%), 5×FAD; Inpp5d fl / fl Lyz2 cre / + The percentage of CD45-overexpressing immune cells in the mouse central nervous system (15.23%) was the same as that of age-matched Inpp5d cells. fl / fl Three times that of WT mice (Figs. 11E and 11F). In 5×FAD; Inpp5d fl / fl Lyz2 cre / + In Ripk1D138N / D138N mice, inhibition of RIPK1 restored the proportion of CD45-overexpressing cells in the central nervous system (4.71%) to age-matched Inpp5d levels. fl / fl Levels in WT mice.

[0446] We further analyzed the subclasses of CD45-highly expressing immune cells, including CD3+ T cells, CD4+ T cells, CD8+ T cells, and CD19+ B cells (Fig. 10G-Fig. 10J). We found that in age-matched 5×FAD mice and Inpp5d... fl / fl Compared to WT mice, 5×FAD; Inpp5d fl / fl Lyz2 cre / + The number of CD3+ T cells and CD4+ T cells in the mouse central nervous system was significantly increased, but the number of CD8+ T cells and CD19+ B cells was not increased (Fig. 10G-Fig. 10J). Inhibition of RIPK1 reduced 5×FAD; Inpp5d fl / fl Lyz2 cre / + The number of CD3+ T cells, CD4+ T cells, and CD8+ T cells infiltrating the central nervous system of Ripk1D138N / D138N mice was observed, but the number of CD19+ B cells was not affected (Fig. 11G-Fig. 11J). Therefore, RIPK1 activation induced by the loss of myeloid Inpp5d in 5xFAD mice not only promotes neuroinflammation but also enhances the infiltration of peripheral T cells into the central nervous system.

[0447] Example 14: Myeloid Inpp5d Deletion Promotes γ-Secretase Activity

[0448] Myeloid Inpp5d deletion syndrome can cause 5×FAD in infants around 5.5 months old; Inpp5d fl / fl Lyz2 cre / +A strikingly dramatic increase in the total human Aβ42 / 40 in mice exceeded levels against the 5×FAD background, suggesting that the non-cellular autonomous interaction between myeloid Inpp5d deficiency and γ-secretase activity may be a key event in AD pathogenesis. We reconstructed the interaction between myeloid Inpp5d deficiency and γ-secretase activity. APP proteolysis involves initial cleavage by β-secretase to generate a 99-residue C-terminal fragment (APP-C99), followed by successive cleavage by γ-secretase, releasing the APP intracellular domain (AICD) and β-amyloid peptide (Aβ). HEK293T cells express endogenous γ-secretase and were therefore used to detect biochemical events associated with APP proteolysis, such as γ-secretase-mediated AICD generation. The AICD level in APP-expressing 293T cells was used as an indicator of γ-secretase activity. To verify the hypothesis that myeloid Inpp5d deletion is associated with non-cellular autonomous regulation of neuronal γ-secretase activity, we constructed two Inpp5dKOBV2 cell lines (Inpp5d KO-3# and Inpp5d KO-10#) using CRISPR-Cas9. Western blotting identified Inpp5d knockout and RIPK1 kinase activation (Figure 27C), and phagocytosis experiments with GFP microbeads confirmed enhanced phagocytic activity (Figure 27B). RNA sequencing and DGE enrichment results showed increased inflammatory levels, including enrichment of DEGs related to immune system biological processes and upregulation of the NF-κB / NOD-like receptor signaling pathway, cytokine-cytokine receptor interaction / coagulation factor signaling pathway, complement, and coagulation cascade pathways (Figures 27D-27F). Notably, there were 186 DEGs and 5×FAD between Inpp5dKO BV2 cells and control BV2 cells; Inpp5d fl / fl Lyz2 cre / + DEGs overlap in PBMCs of mice and 5×FAD mice, and these DEGs are also enriched in immune-related biological processes. Therefore, the inflammatory process activated by INPP5DKO in BV2 cells is associated with 5×FAD;Inpp5d fl / fl Lyz2 cre / + The inflammatory process is similar in mouse PBMCs.

[0449] Next, the effects of hAPP-GFP-expressing 293T cell control and Inpp5d KO BV2 cell culture media on AICD production were examined. Unsurprisingly, the γ-secretase inhibitor DAPT inhibited AICD-GFP production, but the caspase inhibitor zVAD.fmk did not (Fig. 12A; Fig. 27G, Fig. 27H). We found that treatment with culture media modulated with Inpp5d KO BV2 cells increased AICD-GFP protein levels compared to treatment with culture media modulated with control BV2 cells, and Nec-1s prevented the increase in AICD-GFP levels (Fig. 12A, Fig. 12B). These results indicate that myeloid Inpp5d deletion can stimulate γ-secretase activity in a RIPK1-dependent, non-cell-autonomous manner.

[0450] Next, we examined the effects of the culture medium for control groups and Inpp5dKO BV2 cells on directly transdifferentiated forebrain basal cholinergic neurons (normal neurons) and forebrain basal cholinergic neurons (AD neurons) from elderly patients. We found that culturing normal neurons in Inpp5dKO BV2 cell culture medium increased the production of intracellular Aβ spots, which was inhibited by Nec-1s treatment (Fig. 12C-12D). Compared to normal neurons, culture in Inpp5dKO BV2 cell culture medium further enhanced the production of intracellular Aβ spots in AD neurons, while the effect of Nec-1s was weakened (Fig. 12E). We also determined the effect of the Inpp5dKO BV2 cell culture medium on Aβ release from human neurons. We found that the culture medium of Inpp5dKO BV2 cells also increased the release of Aβ from normal neurons, while Nec-1s did not inhibit Aβ release; however, the culture medium of Inpp5dKO BV2 cells further stimulated the release of Aβ from AD neurons, while Nec-1s inhibited the release of Aβ from AD neurons (Figure 12E). These results suggest that RIPK1 may also non-cellularly regulate the activity of γ-secretase in neurons, which may explain why myeloid Inpp5d deficiency leads to a surge in Aβ fiber production in 5xFAD mice.

[0451] Example 15: Inpp5d deletion in the myeloid lineage of 5×FAD mice disrupts REM sleep.

[0452] To investigate the functional effects of myeloid Inpp5d deletion on neurons, we used 5.5-month-old 5×FAD mice and 5×FAD;Inpp5d fl / fl Lyz2 cre / + Mice and 5×FAD; Inpp5d fl / fl Lyz2 cre / +Neurons isolated from Ripk1D138N / D138N mice were analyzed using RNA-seq, revealing 1531 DEGs, including 428 RIPK1-regulated DEGs (Figure 28A). Interestingly, the RIPK1-D138N-rescued DEGs were enriched in circadian rhythm pathways, with Arntl(Bmal1) showing the highest concentration at 5× FAD; Inpp5d fl / fl Lyz2 cre / + Downregulated in mouse neurons, while Dbp, Per2, Per3, Nr1d1, and Nr1d2 were upregulated at 5×FAD; Inpp5d fl / fl Lyz2 cre / + Transcriptional upregulation was observed in neurons of mice; furthermore, in age-matched 5×FAD mice; Inpp5d fl / fl Lyz2 cre / + In neurons of Ripk1D138N / D138N mice, their expression levels were restored to Inpp5d. fl / fl Levels in WT mice (Fig. 13A, Fig. 28B). We confirmed the 5×FAD;Inpp5d fl / fl Lyz2 cre / + Bmal1 protein levels in the mouse hippocampus decreased at 5.5 months of age, while at age-matched 5×FAD; Inpp5d fl / fl Lyz2 cre / + In the cortex of Ripk1D138N / D138N mice, Bmal1 protein levels recovered to WT levels (Fig. 13B, Fig. 13C). However, in 5×FAD; Inpp5d fl / fl Lyz2 cre / + No changes were found in the circadian rhythm of mouse activity in LD (12 hours light / 12 hours dark) and DD (24 hours dark) (Figs. 28C-28D), suggesting that the effect of neuroinflammation on the interference of circadian rhythm gene expression may be independent of the suprachiasmatic nucleus (SCN).

[0453] Bmal1 (Brainandmusclearnt-like, or Arntl) is a bHLH / PAS domain transcription factor, central to the circadian rhythm transcription / translation feedback loop, and is also involved in sleep regulation. Bmal1 knockout in macaques and mice results in sleep disturbances and mental disorders. Sleep deprivation reduces Bmal1 expression and is also known to promote the production of Aβ42 and Aβ40. Therefore, we next characterized sleep using EEG / EMG, and the results showed that in 5-month-old Inpp5d... fl / fl WT mice, 5×FAD mice, 5×FAD; Inpp5d fl / fl Lyz2 cre / +Mice and 5×FAD; Inpp5d fl / fl Lyz2 cre / + The ratio of total sleep to wake time was similar in Ripk1D138N / D138N mice (Figs. 28E-28H). However, we found that compared with age-matched 5×FAD mice, 5.5-month-old 5×FAD;Inpp5d fl / fl Lyz2 cre / + The percentage of time spent in rapid eye movement (REM) sleep (primarily occurring during the photoperiod) and the frequency of REM sleep in mice were reduced (Fig. 13D–Fig. 13F). Interestingly, this was achieved by inhibiting RIPK1, 5×FAD;Inpp5d fl / fl Lyz2 cre / + The amount and pattern of REM sleep were restored in Ripk1D138N / D138N mice (Figs. 13D-13F). All these results indicate that myeloid Inpp5d deficiency in 5×FAD mice disrupts REM sleep in a RIPK1-dependent manner. fl / fl Lyz2 cre / + Inpp5d fl / fl Lyz2 cre / + Inpp5d fl / fl Lyz2 cre / + .

[0454] Example 16: Inhibition of RIPK1 can salvage 5×FAD; Inpp5d fl / fl Lyz2 cre / + Learning and memory deficits in mice

[0455] The loss of dendritic spines in hippocampal neurons has been proven to be closely associated with cognitive decline in AD patients. To investigate the effect of myeloid Inpp5d loss on hippocampal neuronal dendritic spines, we labeled individual hippocampal neurons with AAV9-TRE-fDIO-EGFP-RIES-TTA and AAV9-hSyn-Flp-P2A-mCherry adeno-associated virus and quantitatively analyzed Inpp5d... fl / fl WT, 5×FAD, 5×FAD; Inpp5d fl / fl Lyz2 cre Mice and 5.5-month-old 5×FAD; Inpp5d fl / fl Lyz2 cre / + Number of dendritic spines in neurons of Ripk1D138N / D138N mice. Age-matched Inpp5d fl / fl Compared to WT mice, we detected a reduction in the number of dendritic spines in the brains of 5×FAD mice, and myeloid Inpp5d deletion further accelerated the loss of dendritic spines in 5×FAD mice; compared with 5×FAD; Inpp5d fl / fl Lyz2cre / + Compared with mice, it inhibited 5×FAD; Inpp5d fl / fl Lyz2 cre / + RIPK1 in Ripk1D138N / D138N mice prevents the loss of dendritic spines (Fig. 13E-Fig. 13G). These results suggest that RIPK1-driven activation of microglia and astrocytes can promote the early loss of neuronal dendritic spines.

[0456] Next, we used the Morris water maze to evaluate Inpp5d. fl / fl WT mice, 5×FAD mice, 5×FAD; Inpp5d fl / fl Lyz2 cre / + Mice and 5×FAD; Inpp5d fl / fl Lyz2 cre / + In Ripk1D138N / D138N mice aged 5.5-6 months, the absence of only myeloid Inpp5d at this age did not affect motor function (Xie et al., 2024). All mice underwent five consecutive days of training until each mouse could successfully locate the escape platform within 60 seconds. During the training process, Inpp5d... fl / fl The latency period of WT mice was shorter than that of 5×FAD mice and 5×FAD; Inpp5d fl / fl Lyz2 cre / + Mice and 5×FAD; Inpp5d fl / fl Lyz2 cre The short latency period in mice indicates that, at this age, mice with a 5×FAD background exhibit learning disabilities similar to previously observed (Fig. 28I). In the exploratory experiment, we found no difference in total distance and average speed among all tested mice, suggesting no motor impairment (Fig. 28J, Fig. 28K). Compared to age-matched 5×FAD mice, 5×FAD; Inpp5d fl / fl Lyz2 cre / + Mice exhibited significantly reduced time and distance in the target quadrant, indicating a memory deficit (Figs. 13J-13L). In the spatial reversal test, which involved moving the platform to the opposite quadrant, all mice underwent further training for 5 days, 4 times per day (Fig. 28L). Total distance and average speed again showed no difference among all mice in the reversal detection test (Figs. 28M, 28N). 5×FAD; Inpp5d fl / fl Lyz2 cre / + The time and distance spent by mice in the target quadrant decreased again, further demonstrating memory deficit in the mice (Fig. 13N-Fig. 13P). Furthermore, we recorded the frequency with which mice crossed the platform during the reversal exploration test. We found that 5×FAD; Inpp5d fl / flLyz2 cre / + The mice exhibited a reduced frequency of crossing the plateau region in the reversal exploration test, indicating a memory deficit (Fig. 13M, Fig. 13Q). In all these tests, 6-month-old 5×FAD mice compared with Inpp5d... fl / fl Compared to WT mice, there were no differences except for an increased latency period (Figure 28I), indicating that 5×FAD mice exhibited impaired learning ability at this stage, but had not yet developed memory deficits. Therefore, 5×FAD; Inpp5d fl / fl Lyz2 cre / + Myeloid Inpp5d deficiency in mice promotes memory impairment. Inhibition of 5×FAD; Inpp5d fl / fl Lyz2 cre / + RIPK1 in Ripk1D138N / D138N mice completely alleviated the 6-month memory deficit caused by the loss of Inpp5d in the lower myeloid lineage in 5×FAD mice (Fig. 13J-Fig. 13O). These results indicate that the loss of Inpp5d in the myeloid lineage at 6 months of age in 5×FAD mice leads to spatial memory deficits, and that blocking RIPK1 kinase can inhibit this deficit.

[0457] Example 17: Pharmacological inhibition of RIPK1 can block the sharp increase in Aβ fibers caused by myeloid Inpp5d deficiency.

[0458] To investigate the effect of specifically blocking RIPK1 kinase activity during development in a critical time window of rapid Aβ fibril level increase, we used 5×FAD;Inpp5d fl / fl Lyz2 cre / +Mice were administered the RIPK1 kinase inhibitor Nec-1s via drinking water from 4 to 6 months of age (Fig. 5A). Two months after treatment, mouse brains were harvested and homogenized, and Aβ42 levels were analyzed by ELISA. Administration of Nec-1s reduced the levels of total Aβ42 and DEA-insoluble Aβ42, but did not significantly reduce the level of DEA-soluble Aβ42, which was approximately 1,600 times lower than that of insoluble Aβ42 (Fig. 5B-5D). Nec-1s also reduced the level of sarkosyl-soluble Aβ42, but did not reduce the level of sarkosyl-insoluble Aβ42 (Fig. 5E, Fig. 5F). These results indicate that the RIPK1 inhibitor Nec-1s can reduce the surge in free Aβ42 fiber levels induced by myeloid Inpp5d deficiency, but may not affect pre-deposited amyloid fibers in 5xFAD mice. Administration of Nec-1s resulted in a slight increase in the number of Aβ plaques, similar to the results for the RIPK1-D138N allele (Fig. 5G-Fig. 5I). This result suggests that Nec-1s treatment may lead to a slight increase in Aβ peptide deposition in myeloid Inpp5d-deficient 5×FAD mice during a dramatic approximately 6-fold increase within a critical time window. We also found that in 5×FAD;Inpp5d... fl / fl Lyz2 cre / + At 6 months of age, treatment with Nec-1s reduced microglial activation, as indicated by microglial volume, CD68 volume, and branching complexity, and reduced CD3+ T cell infiltration in the hippocampus (Fig. 5J-Fig. 5K). Therefore, the RIPK1 inhibitor Nec-1s can effectively alleviate inflammation induced by myeloid Inpp5d deficiency.

[0459] We then explored whether pharmacological inhibition of RIPK1 with Nec-1s within a critical time window could improve 5×FAD; Inpp5d fl / fl Lyz2 cre / + Learning and memory abilities in mice. Administer 5×FAD; Inpp5d fl / fl Lyz2 cre / + Feeding mice with Nec-1s inhibited RIPK1 activity and increased the number of dendritic spines in neurons at 6 months of age (Fig. 5L, Fig. 5M). We found that administering 5×FAD; Inpp5d fl / fl Lyz2 cre+ Nec-1s treatment in mice had no effect on motor function (measured by total distance and speed), but it increased the time mice spent in the target quadrant of the Morris water maze (measured by distance and time) (Figs. 5N-5R). In summary, our results indicate that Nec-1s treatment during the critical time window reduces the number of free Aβ42 fibers and enhances 5×FAD;Inpp5d fl / flLyz2 cre / + Spatial learning and memory abilities in mice.

[0460] discuss

[0461] The enrichment of microglia in numerous LOAD risk genes highlights their importance in the pathogenesis of Alzheimer's disease (AD). Since RIPK1 has been identified as a key mediator of inflammatory responses, this project aimed to explore the interaction between RIPK1 and LOAD risk genes. Our initial screening identified the role of INPP5D (a common LOAD risk factor) in regulating RIPK1-mediated inflammatory responses, an role independent of TNFR1. We found that INPP5D binds directly to RIPK1 in a SH2 domain-dependent manner, thereby controlling its activation under stress and aging. Unexpectedly, we found that the loss of INPP5D in microglia promotes the transcription of multiple RIPK1-mediated LOAD risk genes, suggesting that the regulation of RIPK1 activation in microglia may be a core mechanism for various LOAD risk factors. Our study indicates that myeloid INPP5D loss is crucial for promoting RIPK1-dependent expression of these LOAD risk genes in vivo. Therefore, our research reveals previously overlooked potential links between different LOAD risk factors in the context of aging. Interestingly, Prater et al., in another transcriptomic study of human microglia involving 12 AD patients and 10 controls, found an increase in cluster 6 of microglia in the brains of human AD patients. Cluster 6 contained decreased Inpp5d levels and upregulated levels of 26 LOAD risk genes. Therefore, the microglia cluster 6 found by Prater et al. in human AD may be regulated by RIPK1, in a manner similar to the regulation defined in this study of mouse INPP5D-deficient microglia. The RIPK1-dependent mediating of increased transcription of multiple AD risk factors in microglia with aging due to INPP5D deficiency in microglia may mechanistically explain why aging is the greatest risk factor for Alzheimer's disease.

[0462] Many inflammatory signaling pathways, such as inflammasomes, complement, ROS, and pro-inflammatory cytokines, are involved in mediating inflammatory signaling pathways in human Alzheimer's disease (AD). We found that INPP5D deficiency in microglia promotes RIPK1-dependent transcription of various pro-inflammatory cytokines, the complement system, and intracellular ROS promoters, as well as several key intracellular mediators of the pro-inflammatory response, including TLR signaling, inflammasomes, and interferon signaling.

[0463] Microglia are resident immune cells in the brain with homeostatic functions, responding to factors that disrupt this balance by initiating inflammatory responses. In particular, inflammatory microglia subtypes, such as MG2 and MG8 (identified by Sun et al.) and inflammatory Mic.15 and inflammatory Mic.MG10 (identified by Green et al.), are considered more inflammatory than MG2 and MG8. Sun et al. reported that MG10 exhibited greater transcriptional changes in early AD in humans, suggesting that RIPK1-mediated neuroinflammation may control the early pathological progression of AD. Since RIPK1 activation in microglia can drive the expression of multiple pro-inflammatory pathways, sufficiently strong activation of any of these inflammatory responses in a suitable aging context could promote AD pathogenesis. Furthermore, RIPK1-regulated microglia gene signatures are enriched in the antiviral subtype (MG11), the IFN subtype (Mic.14), and the reactive subtype (Mic.6), suggesting that infectious pathogens long suspected of playing a role in AD pathogenesis are also involved.

[0464] Altered lipid metabolism in glial cells, particularly changes in apolipoprotein lipidation, has been identified as being associated with Alzheimer's disease (AD). Lipid-processing microglia are enriched in human AD and are highly significantly positively correlated with pathological progression, including tangles, amyloidosis, Braak scores, and cognitive decline. Microglial lipid metabolism is involved in microglial phagocytic activity, which is involved in the clearance of amyloid plaques. Similarly, RIPK1-regulated microglial gene signatures have been found in the phagocytic microglial subtype (MG5) of human AD. Therefore, dysregulation of RIPK1-controlled microglial lipid processing and phagocytic activity may contribute to the deposition of amyloid plaques.

[0465] While previous research on RIPK1 has largely focused on its role in downstream TNFR1-mediated apoptosis and necrosis, our findings highlight its crucial role as a transcriptional regulator, capable of controlling microglia independently of TNFR1 under stress and during aging. We propose that INPP5D provides a key balancer for RIPK1 activation in microglia, and that RIPK1 activation, in turn, modulates the thresholds for activating multiple intracellular signaling pathways by regulating gene transcription, thereby generating pro-inflammatory responses. Our results indicate that INPP5D deficiency lowers the activation threshold for microglia initiation during aging, as INPP5D deficiency in microglia leads to motor neuron lesions in aged mice, while younger mice show no significant lesions. In this regard, we found RIPK1-dependent upregulation of GAL3 and GRN in Inpp5d-deficient microglia, two well-known ALS genes. ACSL5, an enzyme involved in long-chain fatty acid metabolism, has recently been identified as an ALS gene, with increased transcription in ALS patients. Therefore, RIPK1 activation may not only be a key molecular event in this age-related microglial priming response, which is associated with AD, but may also have implications for the pathogenesis of ALS.

[0466] The presence of atrophic microglia in autopsy pathological samples from patients with neurodegenerative diseases suggests that neurodegenerative processes play a crucial role in promoting microglia dysfunction, and that dysfunctional microglia contribute to the development of neurodegenerative diseases. Our study indicates that upregulated multiple pro-inflammatory stress signaling pathways may be a mechanism promoting the development of atrophic microglia. Since INPP5D deficiency also promotes the transcriptional upregulation of multiple sensors and signaling mediators (including TLRs, inflammasomes, complement system, and interferons) in pathological and injury-related molecular patterns (PAMPs and DAMPs), our results suggest that microglia INPP5D controls RIPK1 activation, thereby modulating the overall host defense response from a state of equilibrium to atrophy. The sustained activation of multiple pro-inflammatory pathways may ultimately lead to the development of atrophic microglia, thereby impairing the nervous system. Our findings suggest that motor neurons may be most sensitive to the presence of atrophic microglia, as the long axons of motor neurons are known to be a particular challenge in maintaining normal homeostasis. However, our findings do not rule out the possibility that INPP5D deficiency may lead to additional neurological dysfunction in older mice. With the significant increase in human lifespan, we anticipate that a reduction in INPP5D during aging may increase the risk of various neurodegenerative diseases, particularly Alzheimer's disease.

[0467] The pro-inflammatory response of the central nervous system is mediated by the innate immune system, which initiates inflammatory responses through various signaling pathways, such as pattern recognition receptors (PRRs), including Toll-like receptors (TLRs), nucleotide-binding oligomerization domain (NOD)-leucine-rich repeat receptors (NLRs), interferons, and secreted complement proteins. While these important mediators of neuroinflammation have been shown to be activated in aging and age-related neurodegenerative diseases such as Alzheimer's disease, the mechanisms of integration and coordination of these different signaling processes remain unclear. Myeloid INPP5D loss promotes RIPK1-mediated upregulation of pathological and damage-associated molecular pattern sensors (PAMPs and DAMPs), including TLRs and inflammasomes, both of which are involved in mediating neurodegenerative diseases. These receptors recognize and bind to PAMPs and DAMPs (such as misfolded proteins like Aβ and p-Tau, as well as intracellular components released from dying cells), thereby initiating downstream signaling, which may include intracellular and extracellular mediators. The loss of INPP5D in microglia promotes the transcription of two key inflammasomes mediated by RIPK1, namely NLRP3 and AIM2. NLRP3 encodes an intracellular sensor for detecting a range of microbial patterns, endogenous danger signals, and environmental stimuli, while AIM2 encodes a membrane dsDNA sensor for defense against bacterial and viral infections. A common downstream consequence of NLRP3 and AIM2 inflammasome signaling is the activation of pre-cleavage IL1β from caspase-1, leading to the production of mature IL1β, which is released through membrane pores formed by cleaved GSDMD. Therefore, INPP5D loss in microglia may not only promote upstream inflammasome signal transduction by facilitating the transcription of NLRP3 and AIM2, but also downstream mediators of inflammasome signal transduction, including caspase-1, IL1β, and GSDMD. In conclusion, INPP5D loss may enhance the overall ability of microglia to sense various danger signals and activate downstream pro-inflammatory responses in a RIPK1-dependent manner.

[0468] Our research indicates that microglial INPP5D deficiency leads to multiple hallmarks of neurodegeneration, including the production of pro-inflammatory cytokines and interferon, activation of the ROS and complement system, and non-cellular autonomous neuronal damage manifested by neuronal cytoplasmic TDP-43 translocation and neuronal apoptosis. IFNγ, a regulator of microglial proliferation, promotes both protective and pathological effects in the central nervous system, such as TLR signaling, oxidative stress, neurological dysfunction, and cell death. The binding of SH2-INPP5D to pY383RIPK1 may be a key factor in JAK1-controlled RIPK1 kinase activation in the interferon pathway. Although acute complement activation helps maintain central nervous system homeostasis through the clearance of infection and cellular debris, synaptic pruning, and BBB integrity, persistent complement activation has been considered a contributing factor to neurodegenerative diseases such as multiple sclerosis, Parkinson's disease, and Alzheimer's disease. High levels of complement activation have been observed in the brains of Alzheimer's patients.

[0469] In this invention,

[0470] We found that myeloid Inpp5d deletion in 5×FAD mice promotes a dramatic increase in RIPK1-mediated age-specific free Aβ fibers. This surge in free Aβ fibers has important functional significance because it leads to REM sleep apnea, hippocampal dendritic spines, and memory impairment—pathological features commonly found in human Alzheimer's disease (AD). Our findings highlight the pathological role of free Aβ fibers in mediating AD pathogenesis. Inhibition of RIPK1 through gene therapy and pharmacology reduces free Aβ fibers, thereby reducing and delaying the onset of memory and REM sleep apnea dysfunction while maintaining higher levels of Aβ plaques. Since Aβ plaques have been found in post-mortem neuropathology studies of brain samples from elderly individuals without dementia, and even in individuals with autosomal dominant mutations in APP and PS1 / 2 leading to EOAD, low levels of Aβ deposition may not interfere with normal cognition.

[0471] The dramatic increase in Aβ fibers and the decrease in Aβ plaques were both transient, observed only at 5–6 months of age, and both were RIPK1-dependent. These results indicate that in myeloid INPP5D-deficient 5×FAD mice, age-specific stimulation of neuroinflammation by RIPK1 can stimulate neurons to produce Aβ fibers and temporarily reduce Aβ plaques within this critical time window. Since the mutant APP / PS1 transgene in 5×FAD mice is driven by the neuronal Thy-1 promoter, the fact that myeloid INPP5D deletion promotes a transient spike in free Aβ fibers likely demonstrates the non-cellular autonomous stimulation of γ-secretase activity by myeloid RIPK1 activity. The myeloid regulatory mechanism of neuronal γ-secretase will be an important avenue for further research. Our results suggest that the dramatic increase in free Aβ fiber levels in Inpp5d-deficient 5×FAD mice defines a key molecular event that mediates the transition from chronic inflammation resulting from low Aβ levels to the onset of AD cognitive impairment. Therefore, our study may provide a model for older individuals and early-stage EOAD patients with amyloid deposition but no dementia, where non-cellular autonomous stimulation of γ-secretase activity may lead to the pathogenesis of clinical AD. Since pharmacological inhibition of RIPK1 within a critical time window was sufficient to salvage cognitive decline, our results suggest that activation of RIPK1 kinase may play a significant role in promoting the pathogenesis of AD dementia.

[0472] Our findings suggest that while the surge in Aβ fibers driven by RIPK1-regulated inflammation is transient, activation of RIPK1 by INPP5D deficiency in the medulla leads to persistent neuroinflammation, ultimately increasing Aβ plaque formation in aged 5×FAD mice. The transient nature of surge Aβ production and the eventual accumulation of higher Aβ levels are reminiscent of an inflammatory response. Generally, the purpose of an inflammatory response is to eliminate or reduce disruptive sources, re-establish balance, and restore function. However, in this case, the persistent inflammatory state (Aβ fiber and Aβ plaque formation resulting from the APP / PS1 mutant transgene) shifts the system to a different set point, as occurs during chronic inflammation, characterized by an increased Aβ burden.

[0473] Mechanistically, we found that myeloid Inpp5d deficiency in 5×FAD mice promotes RIPK1-mediated peripheral inflammation and T cell infiltration in the central nervous system. T cells interact with neuronal γ-secretase, promoting the production of free Aβ fibers. Our results demonstrate the role of chronic peripheral inflammation in promoting AD pathogenesis. The role of LOAD risk genes in regulating central nervous system inflammation has been extensively studied. Here, we discovered that the LOAD risk factor INPP5D regulates RIPK1 activation in the peripheral immune system to control T cell infiltration in 5×FAD mice; Inpp5d fl / fl Lyz2 cre / + Evidence from the mouse central nervous system. Microglia-mediated T-cell infiltration has recently been confirmed in human AD. Our data suggest that LOAD risk genes play a peripheral role in promoting central nervous system inflammation and AD pathogenesis. Because Aβ plaques have been found in post-mortem neuropathological studies of brain samples from elderly individuals without dementia, and also decades before the onset of neurological and cognitive decline in patients with familial EOAD gene mutations, our results indicate that key peripheral events may simultaneously promote the pathogenesis of both LOAD and EOAD. Inpp5d fl / fl Lyz2 cre / +

[0474] The dramatic increase in Aβ in the brains of myeloid INPP5D-deficient 5xFAD mice may mimic a central nervous system (CNS) infection event, which has been identified as a contributing factor to the pathogenesis of Alzheimer's disease (AD). Specific microorganisms, particularly herpes simplex virus type 1 (HSV1), Chlamydia pneumoniae, fungi, and several spirochetes, are associated with the etiology of AD. Gamma-secretase is involved in viral membrane fusion early in human papillomavirus (HPV) infection, and the increased PSEN1 expression observed in our study also correlates with host immune response activation mimicking pathogen invasion. Aβ has been described as possessing antibacterial and antiviral properties. Therefore, 5×FAD;Inpp5d fl / fl Lyz2 cre / + The age-dependent increase in peripheral inflammation in mice may mimic peripheral infection events, ultimately triggering a defensive response in the central nervous system by stimulating Aβ production. Similar to infection events, 5×FAD;Inpp5d fl / fl Lyz2 cre / +The increase in Aβ in the mouse brain is also transient and eventually controlled. Many transient infections produce long-term consequences that are difficult to understand, such as postviral syndrome, including "long amyloidosis." In this case, a transient surge of Aβ in a human body that already has a sufficient amount of amyloid deposits, possibly due to the presence of LOAD risk factors or other incidental infectious events, can have long-term consequences, leading to cognitive impairment and the onset of AD.

[0475] Decreased sleep quality is associated with the pathology of attention deficit disorder (ADD), and reduced REM sleep is particularly associated with the risk of developing dementia. Insufficient sleep throughout the night reduces the expression of BMAL1, CRY1, and PER2 in human plasma leukocytes. Under sleep deprivation or stress, the expression levels of clock genes can become temporally decoupled from their local circadian oscillations without interfering with the oscillation of the master clock (SCN). Our study shows that myeloid Inpp5d deficiency in 5×FAD leads to sleep disturbances, manifested as defects in the asynchronous quality of REM sleep EEG, one of a series of clinical and neurological features observed in the early stages of AD. Sleep promotes synaptic plasticity and remodeling processes, crucial for translating waking experiences into consolidated memories. Growing evidence suggests that sleep disturbances can lead to cognitive decline and promote Aβ lesions. REM sleep disturbances predict longitudinal decline in cognitive function as well as mood and pattern states, both of which clinically affect AD.

[0476] Similar sleep disorders have been found in carriers of the APOE4 allele, who exhibited significantly shortened REM sleep duration, and the APOE4 allele is the most significant genetic risk factor for LOAD (Loss in Attention Deficit Hyperactivity Disorder). Studies have found that, compared to controls with normal sleep, sleep-deprived participants produced 30% more Aβ42 and Aβ40 than at baseline. Sleep deprivation also reduces Bmal1 expression. Therefore, chronic inflammation and sleep disorders may have a causal relationship, jointly increasing the risk of attention deficit disorder by leading to a net decrease in neuronal firing rate, particularly during REM sleep.

[0477] Our results indicate that microglia activation in myeloid Inpp5d-deficient 5×FAD mice leads to Aβ42 activation and the formation of loosely packaged 6E10+ amyloid plaques, while inhibition of RIPK1 kinase activity blocks AD plaque formation. In 6-month-old 5×FAD;Inpp5d fl / fl Lyz2 cre / + The loosely packed 6E10+ amyloid plaques observed in the hippocampus and cortex of mice may resemble diffuse amyloid plaques commonly seen in the brains of early-stage Alzheimer's disease (AD) patients. However, amyloid deposition in 5×FAD mice begins at 2 months of age, 5×FAD;Inpp5d.fl / fl In Lyz2cre mice around 6 months of age, a sharp increase in Aβ42 and the formation of loosely packed 6E10+ amyloid plaques are sufficient to promote dendritic spine reduction and impair memory. Therefore, in 5xFAD mice, pseudoinfection events induced by myelin INPP5D loss during aging can stimulate Aβ, transforming pre-existing benign amyloid deposits into malignant amyloid deposits, thereby leading to impaired normal neuronal function. Since inhibiting RIPK1 kinase can suppress the transformation of amyloid deposits from benign to malignant, our data suggest the possibility of targeting RIPK1 to delay or inhibit the onset of AD.

[0478] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. The use of a RIPK1 inhibitor, characterized in that, A pharmaceutical composition for the preparation of a drug for the prevention or treatment of Alzheimer's disease; wherein the Alzheimer's disease is selected from the group consisting of: early-onset Alzheimer's disease, late-onset Alzheimer's disease, familial Alzheimer's disease, and sporadic Alzheimer's disease; Preferably, the inhibitor treats Alzheimer's disease by inhibiting RIPK1 activation caused by changes in Inpp5d activity or expression levels in the subject, or by RIPK1 activation caused by other reasons.

2. The use as described in claim 1, characterized in that, The inhibitor is also used for purposes selected from the group consisting of: (1) Reversing atrophic microglial lesions; (2) Downregulates the expression of RIPK1-mediated pro-inflammatory cytokines; (3) It inhibits the increase in the activation level of astrocytes.

3. The use as described in claim 1, characterized in that, The inhibitor is also used to inhibit the transcription of Alzheimer's disease risk genes; preferably, the risk genes are selected from the group consisting of: ApoE, Trem2, Tyrobp, Ptk2b, Plcg2, Spi1, Pld3, Ctsd, Ctsb, Ctsh, Idua, Grn, Rin3, Pilra, Adam17, Maf, and Blnk.

4. The use as described in claim 1, characterized in that, The inhibitor is also used to downregulate the expression of RIPK1-related cytokine genes; the cytokines are selected from the following group: AD risk factors, pro-inflammatory cytokines, ROS mediators, complement, inflammasomes, and IFNs.

5. The use as described in claim 1, characterized in that, The inhibitor is also used for purposes selected from the group consisting of: (1) Improve age-dependent movement disorders; (2) Reverse cytoplasmic TDP-43 translocation and neuronal apoptosis.

6. The use as described in claim 1, characterized in that, The inhibitor is also used for purposes selected from the group consisting of: (1) Inhibits the increase in free Aβ42 fiber levels; (2) Improves amyloid deposition in the brain; (3) Improves age-dependent peripheral inflammation caused by Inpp5d deficiency; (4) Regulate the activity of γ-secretase in neurons; (5) Improve sleep disorders induced by Alzheimer's disease; (6) Improve learning and memory deficits induced by Alzheimer's disease; (7) Block the rapid increase in Aβ fibers promoted by myeloid Inpp5d deletion.

7. The use as described in claim 1, characterized in that, The RIPK1 inhibitor is selected from the following group: RIPK1-i1, RIPK1-i2, spermidine, acetylated spermidine, or small molecule RIPK1 inhibitors.

8. The use as described in claim 7, characterized in that, The small molecule RIPK1 inhibitors mentioned are selected from the following group: Nec-1s, DNL788, GDC-8264, ABBV-668, AC-003, Ocadusertib, GSK2982772, GFH-312, Eclitasertib, Oditrasertib, Flizasertib, or small molecule RIPK1 inhibitors selected from the following group: Or the small molecule RIPK1 inhibitor may be a compound as shown in Formula I, Formula II or Formula III: In the formula: for or M is selected from the following group: chemical bond, O, S, NR 3a CHR 3a or C(R) 3a )2; X1 is selected from the following group: CR 2a NR a O, S, CR a N; X2 is selected from the following group: CR a N; R a Selected from the following group: H, D, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 deuterated alkyl; R 1a R 2a Each is independently selected from the following group: H, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 deuterated alkyl; or R1, R2 together with the carbon atom attached to them to form substituted or unsubstituted C3-C6 cycloalkyl, or substituted or unsubstituted 4-6 membered heterocyclic groups; Ring A is a group selected from the following group: substituted or unsubstituted C6-C10 aryl, substituted or unsubstituted 5-12 heteroaryl; Ring B is a group selected from the following group: substituted or unsubstituted C6-C10 aryl, substituted or unsubstituted 5-12 heteroaryl; Wherein, the substitution refers to the hydrogen atom on the substituent group being replaced by one or more (e.g., 2, 3, 4, etc.) substituents selected from the group consisting of: halogen, deuterated, C1-C6 alkoxy, halogenated C1-C6 alkoxy, methyl sulfone, -S(=O)2NH2, oxo(=O), -CN, hydroxyl, -NH2, carboxyl, C2-C6 amide (-C(=O)-N(Rc)2 or -NH-C(=O)(Rc, where Rc is H or a C1-C5 alkyl), C1-C6 alkyl-(C2-C6 amide), or substituted or unsubstituted groups selected from the group consisting of: C1-C6 alkyl, C3-C8 cycloalkyl, C1- C6 amino, C6-C10 aryl, 5-10 heteroaryl having 1-3 heteroatoms selected from N, S and O, 5-12 heterocyclic group having 1-3 heteroatoms selected from N, S and O, -(CH2)-C6-C10 aryl, -(CH2)-(5-10 heteroaryl having 1-3 heteroatoms selected from N, S and O), and the substituent is selected from the group consisting of: halogen, C1-C6 alkyl, C1-C6 alkoxy, oxo, -CN, -NH2, -OH, C6-C10 aryl, C1-C6 amino, C2-C6 amide, 5-10 heteroaryl having 1-3 heteroatoms selected from N, S and O; in, Ring A is a substituted or unsubstituted 9-10 member nitrogen-containing heteroaryl group, wherein the 9-10 member nitrogen-containing heteroaryl group contains 1, 2, 3 or 4 nitrogen heteroatoms as ring atoms; n = 0, 1, or 2; R 4b Each is independently selected from the following group: H, CN, halogen, substituted or unsubstituted C. 1-6 Alkyl, -OR b -SR b -N(R) b )2、-C(O)-NR 6b -R b -C(O)-NR 6b -C 1-4 Alkylene-N(R) b )2、-NR 6b -C(O)-R b ; Each R b Each is independently selected from the following groups: H, substituted or unsubstituted C. 1-6 Alkyl; or two R b Together with the nitrogen atoms attached to them, they form substituted or unsubstituted 5, 6, or 7-membered heterocyclic alkyl groups, wherein, except for those with R b In addition to the connected N, the heterocyclic alkyl group also contains 0, 1 or 2 other heteroatoms as ring atoms; R 6b Selected from the following group: H, OH, substituted or unsubstituted C 1-4 Alkyl, substituted or unsubstituted C 1-4 Alkoxy; Cycle B is selected from the following group: substituted or unsubstituted C 6-10 Aryl, substituted or unsubstituted 5-10 heteroaryl groups; L 1 and L 2 Each is independently a divalent group selected from the following group: none, And L 1 and L 2 Not simultaneously equal to none; R 1b and R 2b Each is independently selected from the following groups: H, substituted or unsubstituted C. 1-4 alkyl; R 3b Selected from the following group: H, OH, substituted or unsubstituted C 1-4 Alkyl, substituted or unsubstituted C 1-4 Alkoxy; Ring C is none or Among them, W is independently selected from the following groups: O, S, C, N, C(R) c ), and N(R) d ); R c Each is independently selected from the following group: H, CN, halogen, substituted or unsubstituted C. 1-6 Alkyl, R d Each is independently selected from the following groups: H, CN, substituted or unsubstituted C. 1-6 alkyl; Or, when ring C is L 1 for And L 2 When there is no time, R 3 With L 1 The ring atoms W and L adjacent to the ring C are 1 The -C(O)- groups in the rings together form substituted or unsubstituted 5, 6, or 7-membered saturated heterocycles; wherein, the saturated heterocycles, in addition to being substituted with R, form substituted or unsubstituted 5, 6, or 7-membered saturated heterocycles. 3 In addition to the connected N, there are 0, 1 or 2 other heteroatoms that act as ring atoms; R 5 Selected from the following group: H, OH, substituted or unsubstituted C 1-4 Alkyl, substituted or unsubstituted C 1-4 Alkoxy; Or when ring C is nonexistent and L 2 for At that time, R 3 and R 5 And the atoms bonded to them together form substituted or unsubstituted 5, 6, or 7-membered saturated heterocycles; wherein, the saturated heterocycles, in addition to being associated with R, form substituted or unsubstituted 5, 6, or 7-membered saturated heterocycles. 3 In addition to the connected N, there are 0, 1 or 2 other heteroatoms that act as ring atoms; Ring D is selected from the following group: substituted or unsubstituted C 6-10 Aromatic rings, and substituted or unsubstituted 5-10 membered heteroaryl groups; Unless otherwise specified, the term "substitution" refers to the substitution of a hydrogen atom on a group by one or more (e.g., 1, 2, 3, or 4) substituents selected from the group consisting of: oxo (=O), -CN, halogen, nitro, C. 1-6 Alkyl, Halogenated C 1-6 Alkyl groups, -OR, -SR, -S(O)2R, -S(=O)2NR2, -NR2, -COOR, and C groups optionally substituted with R. 6-10 aryl, 5-10 heteroaryl groups with 1-3 heteroatoms selected from N, S and O, optionally substituted by R, and C groups optionally substituted by R 3- 8-cyclic alkyl groups, 5-12-membered heterocyclic alkyl groups having 1-3 heteroatoms selected from N, S, and O, optionally substituted with R, and -C optionally substituted with R. 1-4 Alkylene-C 6-10 aryl, -C optionally substituted by R 1-4 Alkylene – a 5-10 membered heteroaryl group having 1-3 heteroatoms selected from N, S, and O, with -C optionally substituted by R. 1-4 Alkylene-C 3-8 Cycloalkyl, -C optionally substituted with R 1-4 Alkylene – 5-12 membered heterocyclic alkyl groups having 1-3 heteroatoms selected from N, S and O; R is independently selected from the following groups: H, C 1-6 Alkyl, Halogenated C 1-6 Alkyl, C 1-6 Hydroxyalkyl; In the formula: X8 is selected from the following group: CH, N, or chemical bonds; X7, X3, X4, and X5 are each independently selected from the following groups: CH, N; The condition is that the ring formed by X8, X7, X3, X4, and X5 is an aromatic ring; M is selected from the following group: O, S, NR 3c CHR 3c or C(R) 3c )2; W and U are each independently selected from the following groups: O, S, NR 4c CHR4 or C(R) 4c )2; Ring A and ring B are each independently selected from the following group: substituted or unsubstituted C6-C10 aryl groups, substituted or unsubstituted 5-12 heteroaryl groups; R 1c and R 2c Each is independently selected from the following group: none, H, substituted or unsubstituted C1-C6 alkyl, halogen; And when M is NR 3c CHR 3c or C(R) 3c When R2 and R3 are together with the C or N atom attached to them, as well as -CC(O)-, they can form substituted or unsubstituted 5-7 membered rings. R 3c and R 4c Selected from the following group: H, substituted or unsubstituted C1-C6 alkyl groups, halogens; R6 is selected from the following group: H, halogen, CN, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C2-C6 olefin, substituted or unsubstituted C2-C6 alkyne, or -CH≡CR5; wherein R5 is selected from the following group: substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C6-C10 aryl, substituted or unsubstituted 5-12-membered heteroaryl, substituted or unsubstituted C3-C8 cycloalkyl, substituted or unsubstituted 4-12-membered heterocyclic group; R7 is selected from the following group: H, halogen, CN, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C2-C6 olefin, substituted or unsubstituted C2-C6 alkyne, or -CH≡CR8; wherein R8 is selected from the following group: substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C6-C10 aryl, substituted or unsubstituted 5-12-membered heteroaryl, substituted or unsubstituted C3-C8 cycloalkyl, substituted or unsubstituted 4-12-membered heterocyclic group; Wherein, the substitution refers to the hydrogen atom on the substituent group being replaced by one or more (e.g., 2, 3, 4, etc.) substituents selected from the group consisting of: halogen, C1-C6 alkyl, halogenated C1-C6 alkyl, C3-C8 cycloalkyl, halogenated C3-C8 cycloalkyl, C1-C6 alkoxy, halogenated C1-C6 alkoxy, C1-C6 hydroxyalkyl, methyl sulfone, -S(=O)2NH2, oxo(=O), -CN, hydroxyl, -NH2, carboxyl, C2-C6 amide (-C(=O)-N(Rc)2 or -NH-C(=O)(Rc), where Rc is H or C1-C5). Alkyl), C1-C6 alkyl-(C2-C6 amide), C1-C6 amino, deuterated C1-C6 amino, -NHRd (Rd is C3-C8 cycloalkyl, 4-7 membered heterocyclic or heterocyclic substituted with C1-C6 alkyl), C6-C10 aryl, 5-7 membered heteroaryl having 1-3 heteroatoms selected from N, S and O, 4-8 membered heterocyclic having 1-3 heteroatoms selected from N, S and O, 4-7 membered heterocyclic substituted with 1 or 2 Re (Re is halogen, C1-C6 alkyl, C1-C6 amino, -CN, C1-C6 alkoxy or 4-7 membered heterocyclic).

9. The use as described in claim 7, characterized in that, The small molecule RIPK1 inhibitors mentioned are selected from the following group:

10. The use as described in claim 1, characterized in that, The inhibitors are administered before, during, or after a patient develops Alzheimer's disease.

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