In Alzheimer's disease (AD) brain, inflammatory activation regulates protein levels of amyloid-beta-peptide (A beta) and phosphorylated tau (p-tau), as well as neurodegeneration; however, the regulatory mechanisms remain unclear. We constructed APP- and tau-transgenic AD mice with deletion of IKK beta specifically in neurons, and observed that IKK beta deficiency reduced cerebral A beta and p-tau, and modified inflammatory activation in both AD mice. However, neuronal deficiency of IKK beta decreased apoptosis and maintained synaptic proteins (e.g., PSD-95 and Munc18-1) in the brain and improved cognitive function only in APP-transgenic mice, but not in tau-transgenic mice. Additionally, IKK beta deficiency decreased BACE1 protein and activity in APP-transgenic mouse brain and cultured SH-SY5Y cells. IKK beta deficiency increased expression of PP2A catalytic subunit isoform A, an enzyme dephosphorylating cerebral p-tau, in the brain of tau-transgenic mice. Interestingly, deficiency of IKK beta in neurons enhanced autophagy as indicated by the increased ratio of LC3B-II/I in brains of both APP- and tau-transgenic mice. Thus, IKK beta deficiency in neurons ameliorates AD-associated pathology in APP- and tau-transgenic mice, perhaps by decreasing A beta production, increasing p-tau dephosphorylation, and promoting autophagy-mediated degradation of BACE1 and p-tau aggregates in the brain. However, IKK beta deficiency differently protects neurons in APP- and tau-transgenic mice. Further studies are needed, particularly in the context of interaction between A beta and p-tau, before IKK beta/NF-kappa B can be targeted for AD therapies.

Abstract Background: Alzheimer’s disease (AD) is pathologically characterized by extracellular deposition of amyloid β peptide (Aβ), intracellular neurofibrillary tangles (composed mainly of phosphorylated tau [p-tau]) and neuroinflammation. The pathogenic role of inflammatory activation in AD has been extensively studied; however, the underlying mechanisms remain unclear. In this project, we investigated how neuronal IKKβ/NF-kB regulates AD-associated pathologies in APP- and tau-transgenic AD mice. Methods: APP- and tau-transgenic mice were cross-bred with ikbkb-floxed and nex-cre knock-in mice to generate AD models with deletion of IKKβ specifically in neurons. After assessing cognitive function with the Morris water maze test, mice were analyzed for Aβ and p-tau levels, microglial numbers, transcription of inflammatory genes, apoptosis and synaptic protein levels in the brain by histological, biochemical and molecular biology methods. To explore pathogenic mechanisms, we analyzed the activity and/or protein levels of: 1) β- and γ-secretases, 2) tau-phosphorylating (e.g., GSK3β and p38α-MAPK) and dephosphorylating enzymes (e.g., PP2A and PP2B), and 3) autophagy-related proteins (e.g., LC3B, beclin1 and SQSTM1/p62). In addition, IKKβ-knockdown and wild-type SH-SY5Y cell lines were created to verify the in vivo results.Results: In APP-transgenic mice, neuronal deficiency of IKKβ decreased Aβ load, inflammatory activation, and apoptosis in the brain, and improved cognitive function and maintenance of synaptic proteins (e.g., PSD-95). IKKβ deficiency decreased BACE1 activity and protein in the brain and cultured neuronal cells. In tau-transgenic mice, neuronal deficiency of IKKβ decreased p-tau, shifted pro- to anti-inflammatory activation and inhibited autophagy in the brain. IKKβ deficiency increased expression of PP2A catalytic subunit isoform A, an enzyme dephosphorylating cerebral p-tau. However, deficiency of IKKβ in neurons did not alter the cognitive function and even increased apoptosis in the brain.Conclusions: Deficiency of IKKβ in neurons attenuates Aβ and p-tau loads in the brains of AD mice. As possible molecular mechanisms, IKKβ deficiency decreases BACE1 activity, thereby reducing Aβ production, and increases PP2A expression, promoting p-tau dephosphorylation. However, IKKβ deficiency protects neurons only in APP-transgenic mice, but not in tau-transgenic AD mice. Further studies are needed before IKKβ/NF-kB can be targeted for AD therapies.

Alzheimer's disease (AD), the most common cause of dementia in the elderly, is pathologically characterized by extracellular deposition of amyloid-β peptides (Aβ) and microglia-dominated inflammatory activation in the brain. p38α-MAPK is activated in both neurons and microglia. How p38α-MAPK in microglia contributes to AD pathogenesis remains unclear. In this study, we conditionally knocked out p38α-MAPK in all myeloid cells or specifically in microglia of APP-transgenic mice, and examined animals for AD-associated pathologies (i.e., cognitive deficits, Aβ pathology, and neuroinflammation) and individual microglia for their inflammatory activation and Aβ internalization at different disease stages (e.g., at 4 and 9 months of age). Our experiments showed that p38α-MAPK-deficient myeloid cells were more effective than p38α-MAPK-deficient microglia in reducing cerebral Aβ and neuronal impairment in APP-transgenic mice. Deficiency of p38α-MAPK in myeloid cells inhibited inflammatory activation of individual microglia at 4 months but enhanced it at 9 months. Inflammatory activation promoted microglial internalization of Aβ. Interestingly, p38α-MAPK-deficient myeloid cells reduced IL-17a-expressing CD4-positive lymphocytes in 9 but not 4-month-old APP-transgenic mice. By cross-breeding APP-transgenic mice with Il-17a-knockout mice, we observed that IL-17a deficiency potentially activated microglia and reduced Aβ deposition in the brain as shown in 9-month-old myeloid p38α-MAPK-deficient AD mice. Thus, p38α-MAPK deficiency in all myeloid cells, but not only in microglia, prevents AD progression. IL-17a-expressing lymphocytes may partially mediate the pathogenic role of p38α-MAPK in peripheral myeloid cells. Our study supports p38α-MAPK as a therapeutic target for AD patients.

Amyloid beta peptide (A beta) is the major pathogenic molecule in Alzheimer's disease (AD). BACE1 enzyme is essential for the generation of A beta. Deficiency of p38 alpha-MAPK in neurons increases lysosomal degradation of BACE1 and decreases A beta deposition in the brain of APP-transgenic mice. However, the mechanisms mediating effects of p38 alpha-MAPK are largely unknown. In this study, we used APP-transgenic mice and cultured neurons and observed that deletion of p38 alpha-MAPK specifically in neurons decreased phosphorylation of Snapin at serine, increased retrograde transportation of BACE1 in axons and reduced BACE1 at synaptic terminals, which suggests that p38 alpha-MAPK deficiency promotes axonal transportation of BACE1 from its predominant locations, axonal terminals, to lysosomes in the cell body. In vitro kinase assay revealed that p38 alpha-MAPK directly phosphorylates Snapin. By further performing mass spectrometry analysis and site-directed mutagenic experiments in SH-SY5Y cell lines, we identified serine residue 112 as a p38 alpha-MAPK-phosphorylating site on Snapin. Replacement of serine 112 with alanine did abolish p38 alpha-MAPK knockdown-induced reduction of BACE1 activity and protein level, and transportation to lysosomes in SH-SY5Y cells. Taken together, our study suggests that activation of p38 alpha-MAPK phosphorylates Snapin and inhibits the retrograde transportation of BACE1 in axons, which might exaggerate amyloid pathology in AD brain.

Microglial activation is a hall marker of Alzheimer’s disease (AD); its pathogenic role and regulating mechanisms are unclear. p38α-MAPK, a stress-responding kinase, is activated in AD brain in early disease stages. In APP-transgenic mice, we deleted p38α-MAPK in whole myeloid cells from birth or specifically in microglia from 9 months, and analysed AD pathology at the age of 4, 9 and 12 months. In both experimental settings, p38α-MAPK deficiency decreased cerebral Aβ and improved cognitive function of AD mice; however, p38α-MAPK-deficient myeloid cells were more effective than p38α-MAPK-deficient microglia in preventing AD pathogenesis. Deficiency of p38α-MAPK in myeloid cells inhibited the inflammatory activation of individual microglia by 4 months, but enhanced it by 9 months. Inflammatory activation was essential for p38α-MAPK deficiency to promote microglial internalization of Aβ. Interestingly, p38α-MAPK deficiency in peripheral myeloid cells reduced il-17a transcription in CD4-positive spleen cells. By cross-breeding APP-transgenic mice and IL-17a knockout mice, we further observed that IL-17a deficiency activated microglia and decreased Aβ deposits in AD mouse brain. Thus, p38α-MAPK deficiency in myeloid cells prevents AD pathogenesis, perhaps through reducing IL-17a-expressing T lymphocytes, and promoting Aβ clearance in the brain. Our study supports p38α-MAPK as a novel target for AD therapy.

Alzheimer's disease (AD) is the leading cause of dementia with very limited therapeutic options. Amyloid β (Aβ) and phosphorylated Tau (p‐Tau) are key pathogenic molecules in AD. P38α‐MAPK is specifically activated in AD lesion sites. However, its effects on AD pathogenesis, especially on p‐Tau‐associated brain pathology, and the underlying molecular mechanisms remain unclear. We mated human APP‐transgenic mice and human P301S Tau‐transgenic mice with mapk14‐floxed and neuron‐specific Cre‐knock‐in mice. We observed that deletion of p38α‐MAPK specifically in neurons improves the cognitive function of both 9‐month‐old APP and Tau‐transgenic AD mice, which is associated with decreased Aβ and p‐Tau load in the brain. We further used next‐generation sequencing to analyze the gene transcription in brains of p38α‐MAPK deficient and wild‐type APP‐transgenic mice, which indicated that deletion of p38α‐MAPK regulates the transcription of calcium homeostasis‐related genes, especially downregulates the expression of grin2a, a gene encoding NMDAR subunit NR2A. Cell culture experiments further verified that deletion of p38α‐MAPK inhibits NMDA‐triggered calcium influx and neuronal apoptosis. Our systemic studies of AD pathogenic mechanisms using both APP‐ and Tau‐transgenic mice suggested that deletion of neuronal p38α‐MAPK attenuates AD‐associated brain pathology and protects neurons in AD pathogenesis. This study supports p38α‐MAPK as a novel target for AD therapy.

BACKGROUND/AIMS:Multiple sclerosis (MS) is one of the most common autoimmune disorders of the central nervous system (CNS) and the leading cause of neurological disability among young adults in the Western world. We have previously shown that the acid sphingomyelinase plays an important role in the pathogenesis of experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis.METHODS:We induced adoptively transferred EAE in wildtype and acid sphingomyelinase-deficient mice. In addition, we immunized mice with MOGaa35-55 to induce active EAE and treated the mice with amitriptyline, a functional inhibitor of the acid sphingomyelinase. We investigated symptoms of EAE, blood-brain barrier integrity and neuroinflammation.RESULTS:In the model of adoptively transferred EAE we demonstrate that expression of acid sphingomyelinase in the recipients rather than on transferred encephalitogenic T cells contributes to the clinical development of EAE symptoms. To test if pharmacological targeting of acid sphingomyelinase can be explored for the development of novel therapies for MS, we inhibited acid sphingomyelinase with amitriptyline in mice in which EAE was induced by active immunization. We demonstrate that pharmacological inhibition of acid sphingomyelinase using amitriptyline protects against the development of EAE and markedly attenuates the characteristic detrimental neuroinflammatory response.CONCLUSION:The studies identify the acid sphingomyelinase as a novel therapeutic target for treating MS patients.

The cuprizone animal model, also known as the toxic demyelination model, is a well-reproducible model of demyelination- and remyelination in mice, and has been useful in studying important aspect of human demyelinating diseases, including multiple sclerosis. In this study, we investigated the role of acid sphingomyelinase in demyelination and myelin repair by inducing acute and chronic demyelination with 5- or 12-week cuprizone treatment, followed by a 2-week cuprizone withdrawal phase to allow myelin repair. Sphingolipids, in particular ceramide and the enzyme acid sphingomyelinase, which generates ceramide from sphingomyelin, seem to be involved in astrocyte activation and neuronal damage in multiple sclerosis. We used immunohistochemistry to study glial reaction and oligodendrocyte distribution in acid sphingomyelinase deficient mice and wild-type C57BL/6J littermates at various time intervals after demyelination and remyelination. Axonal injury was quantified using amyloid precursor protein and synaptophysin, and gene expression and protein levels were measured using gene analysis and Western blotting, respectively. Our results show that mice lacking acid sphingomyelinase had a significant increase in myelin recovery and a significantly higher oligodendrocyte cell count after 2 weeks remyelination compared to wild-type littermates. Detrimental astroglial distribution was also significantly reduced in acid sphingomyelinase deficient animals. We obtained similar results in experiments using amitriptyline to inhibit acid sphingomyelinase. These findings suggest that acid sphingomyelinase plays a significant role in myelin repair, and its inhibition by amitriptyline may constitute a novel therapeutic approach for multiple sclerosis patients.

In experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS), peripherally developed myelin-reactive T lymphocytes stimulate myeloid cells (ie, microglia and infiltrated macrophages) to trigger an inflammatory reaction in the central nervous system, resulting in demyelination and neurodegeneration. IκB kinase β (IKKβ) is a kinase that modulates transcription of inflammatory genes. To investigate the pathogenic role of IKKβ in MS, we developed strains in which IKKβ was conditionally ablated in myeloid cells and established active or passive EAE in these animals. Deficiency of IKKβ in myeloid cells ameliorated EAE symptoms and suppressed neuroinflammation, as shown by decreased infiltration of T lymphocytes and macrophages and reduced inflammatory gene transcription in the spinal cord at the peak or end stage of EAE. Myeloid deficiency of IKKβ also reduced the transcription of Rorc or Il17 genes in T lymphocytes isolated from lymph nodes, spleen, and spinal cord of EAE mice. Moreover, cultured splenocytes isolated from myeloid IKKβ-deficient EAE mice released less IL-17, interferon-γ, and granulocyte-macrophage colony-stimulating factor after treatment with myelin peptide than splenocytes from IKKβ wild-type EAE mice. Thus, deficiency of myeloid IKKβ attenuates the severity of EAE by inhibiting both the neuroinflammatory activity and the activation of encephalitogenic T lymphocytes. These results suggest IKKβ may be a potential target for MS patients, especially when neuroinflammation is the primary problem.

Amyloid β (Aβ) damages neurons and triggers microglial inflammatory activation in the Alzheimer disease (AD) brain. BACE1 is the primary enzyme in Aβ generation. Neuroinflammation potentially up-regulates BACE1 expression and increases Aβ production. In Alzheimer amyloid precursor protein-transgenic mice and SH-SY5Y cell models, we specifically knocked out or knocked down gene expression of mapk14, which encodes p38α MAPK, a kinase sensitive to inflammatory and oxidative stimuli. Using immunological and biochemical methods, we observed that reduction of p38α MAPK expression facilitated the lysosomal degradation of BACE1, decreased BACE1 protein and activity, and subsequently attenuated Aβ generation in the AD mouse brain. Inhibition of p38α MAPK also enhanced autophagy. Blocking autophagy by treating cells with 3-methyladenine or overexpressing dominant-negative ATG5 abolished the deficiency of the p38α MAPK-induced BACE1 protein reduction in cultured cells. Thus, our study demonstrates that p38α MAPK plays a critical role in the regulation of BACE1 degradation and Aβ generation in AD pathogenesis.