Identification of immune microenvironment subtypes and signature genes for Alzheimer's disease diagnosis and risk prediction based on explainable machine learning.

Background: Using interpretable machine learning, we sought to define the immune microenvironment subtypes and distinctive genes in AD. Methods: ssGSEA, LASSO regression, and WGCNA algorithms were used to evaluate immune state in AD patients. To predict the fate of AD and identify distinctive genes, six machine learning algorithms were developed. The output of machine learning models was interpreted using the SHAP and LIME algorithms. For external validation, four separate GEO databases were used. We estimated the subgroups of the immunological microenvironment using unsupervised clustering. Further research was done on the variations in immunological microenvironment, enhanced functions and pathways, and therapeutic medicines between these subtypes. Finally, the expression of characteristic genes was verified using the AlzData and pan-cancer databases and RT-PCR analysis. Results: It was determined that AD is connected to changes in the immunological microenvironment. WGCNA revealed 31 potential immune genes, of which the greenyellow and blue modules were shown to be most associated with infiltrated immune cells. In the testing set, the XGBoost algorithm had the best performance with an AUC of 0.86 and a P-R value of 0.83. Following the screening of the testing set by machine learning algorithms and the verification of independent datasets, five genes (CXCR4, PPP3R1, HSP90AB1, CXCL10, and S100A12) that were closely associated with AD pathological biomarkers and allowed for the accurate prediction of AD progression were found to be immune microenvironment-related genes. The feature gene-based nomogram may provide clinical advantages to patients. Two immune microenvironment subgroups for AD patients were identified, subtype2 was linked to a metabolic phenotype, subtype1 belonged to the immune-active kind. MK-866 and arachidonyltrifluoromethane were identified as the top treatment agents for subtypes 1 and 2, respectively. These five distinguishing genes were found to be intimately linked to the development of the disease, according to the Alzdata database, pan-cancer research, and RT-PCR analysis. Conclusion: The hub genes associated with the immune microenvironment that are most strongly associated with the progression of pathology in AD are CXCR4, PPP3R1, HSP90AB1, CXCL10, and S100A12. The hypothesized molecular subgroups might offer novel perceptions for individualized AD treatment.

Bone marrow-derived mesenchymal stem cells improve post-ischemia neurological function in rats via the PI3K/AKT/GSK-3ß/CRMP-2 pathway.

BACKGROUND: Transplantation of bone marrow-derived mesenchymal stem cells (BMSCs) is a potential therapy for cerebral ischemia. However, the underlying protective mechanism remains undetermined. Here, we tested the hypothesis that transplantation of BMSCs via intravenous injection can alleviate neurological functional deficits through activating PI3K/AKT signaling pathway after cerebral ischemia in rats. METHODS: A cerebral ischemic rat model was established by the 2 h middle cerebral artery occlusion (MCAO). Twenty-four hours later, BMSCs (1 × 106 in 1 ml PBS) from SD rats were injected into the tail vein. Neurological function was evaluated by modified neurological severity score (mNSS) and modified adhesive removal test before and on d1, d3, d7, d10 and d14 after MCAO. Protein expressions of AKT, GSK-3ß, CRMP-2 and GAP-43 were detected by Western-bolt. NF-200 was detected by immunofluorescence. RESULTS: BMSCs transplantation did not only significantly improve the mNSS score and the adhesive-removal somatosensory test after MCAO, but also increase the density of NF-200 and the expression of p-AKT, pGSK-3ß and GAP-43, while decrease the expression of pCRMP-2. Meanwhile, these effects can be suppressed by LY294002, a specific inhibitor of PI3K/AKT. CONCLUSION: These data suggest that transplantation of BMSCs could promote axon growth and neurological deficit recovery after MCAO, which was associated with activation of PI3K/AKT /GSK-3ß/CRMP-2 signaling pathway.

Restoration of L-OPA1 alleviates acute ischemic stroke injury in rats via inhibiting neuronal apoptosis and preserving mitochondrial function.

BACKGROUND: Ischemic stroke can induce changes in mitochondrial morphology and function. As a regulatory gene in mitochondria, optic atrophy 1 (OPA1) plays a pivotal role in the regulation of mitochondrial dynamics and other related functions. However, its roles in cerebral ischemia-related conditions are barely understood. METHODS: Cultured rat primary cortical neurons were respectively transfected with OPA1-v1ΔS1-encoding and OPA1-v1-encoding lentivirus before exposure to 2-h oxygen-glucose deprivation (OGD) and subsequent reoxygenation (OGD/R). Adult male SD rats received an intracranial injection of AAV-OPA1-v1ΔS1 and were subjected to 90 min of transient middle cerebral artery occlusion (tMCAO) followed by reperfusion. OPA1 expression and function were detected by in vitro and in vivo assays. RESULTS: OPA1 was excessively cleaved after cerebral ischemia/reperfusion injury, both in vitro and in vivo. Under OGD/R condition, compared with that of the LV-OPA1-v1-treated group, the expression of OPA1-v1ΔS1 efficiently restored L-OPA1 level and alleviated neuronal death and mitochondrial morphological damage. Meanwhile, the expression of OPA1-v1ΔS1 markedly improved cerebral ischemia/reperfusion-induced motor function damage, attenuated brain infarct volume, neuronal apoptosis, mitochondrial bioenergetics deficits, oxidative stress, and restored the morphology of mitochondrial cristae and mitochondrial length. It also preserved the mitochondrial integrity and reinforced the mtDNA content and expression of mitochondrial biogenesis factors in ischemic rats. INTERPRETATION: Our results demonstrate that the stabilization of L-OPA1 protects ischemic brains by reducing neuronal apoptosis and preserving mitochondrial function, suggesting its significance as a promising therapeutic target for stroke prevention and treatment.

IL-10 produces a dual effect on OGD-induced neuronal apoptosis of cultured cortical neurons via the NF-κB pathway.

As a classic immunoregulatory cytokine, interleukin-10 (IL-10) can provide in vivo and in vitro neuroprotection respectively during cerebral ischemia and after the oxygen-glucose deprivation (OGD)-induced injury. However, its role in cortical neuronal survival at different post-ischemic phases remains unclear. The current study found that IL-10 had distinct effects on the neuronal apoptosis at different OGD stages: at an early stage after OGD, IL-10 promoted the OGD-induced neuronal apoptosis in the cultured primary cortical neurons by activating p65 subunit, which up-regulated Bax expression and down-regulated Bcl-xL expression; at a late OGD stage, however, it attenuated the OGD-induced neuronal apoptosis by activating c-Rel, which up-regulated Bcl-xL expression and down-regulated Bax expression. The early-stage pro-apoptosis and late-stage anti-apoptosis were both partly abolished by PDTC, an NF-κB inhibitor, and promoted by PMA, an NF-κB activator. The optimal anti-apoptotic effect appeared when the cultured neurons were treated with IL-10 at 9-24 h after OGD. Taken together, our findings suggest that IL-10 exerts a dual effect on the survival of the cultured neurons by activating the NF-κB pathway at different stages after OGD injury and that PMA treatment at a late stage can facilitate the IL-10-conferred neuroprotection against OGD-induced neuronal injury.

Netrin-1 Promotes Synaptic Formation and Axonal Regeneration via JNK1/c-Jun Pathway after the Middle Cerebral Artery Occlusion.

As a secreted axon guidance molecule, Netrin-1 has been documented to be a neuroprotective factor, which can reduce infarct volume, promote angiogenesis and anti-apoptosis after stroke in rodents. However, its role in axonal regeneration and synaptic formation after cerebral ischemic injury, and the related underlying mechanisms remain blurred. In this study, we used Adeno-associated vectors carrying Netrin-1 gene (AAV-NT-1) to up-regulate the expression level of Netrin-1 in rats' brain after middle cerebral artery occlusion (MCAO). We found that the up-regulated level of Netrin-1 and its receptor DCC promoted axonal regeneration and synaptic formation; the overexpression of Netrin-1 activated the JNK1 signaling pathway; these effects were partially reduced when JNK1 signaling pathway was inhibited by SP600125 (JNK specific inhibitor). Taken together, these findings suggest that Netrin-1 can facilitate the synaptic formation and axonal regeneration via the JNK1 signaling pathway after cerebral ischemia, thus promoting the recovery of neural functions.

Netrin-1 Prevents Rat Primary Cortical Neurons from Apoptosis via the DCC/ERK Pathway.

In the nervous system, Netrin-1 serves as a neural guide, mediating the neuronal development. However, it remains blurred whether Netrin-1 can protect neurons from apoptosis induced by cerebral stroke. In the current study, the cultured rat primary cortical neurons were transfected with Netrin-1-encoding lentivirus before the oxygen-glucose-deprivation (OGD) treatment. Cell death and apoptosis were evaluated by lactate dehydrogenase (LDH) release and flow cytometry. We found that Netrin-1 attenuated OGD-induced cell death and neuronal apoptosis at 24 h after OGD treatment, and that the overexpression of Netrin-1 activated the ERK signaling pathway. These effects were partly abolished by blocking its receptor deleted in colorectal cancer (DCC) or U0126, an inhibitor of the ERK signaling pathway. Netrin-1 overexpression in neurons elevated the expression of DCC, on mRNA level and protein level. Netrin-1 also reduced DNA damage. Taken together, our findings suggest that Netrin-1 attenuates cell death and neuronal apoptosis via the DCC/ERK signaling pathway in the cultured primary cortical neurons after OGD injury, which may involve the mediation of DNA damage in the neurons.