The pathogenesis of Alzheimer's disease (AD) is a critical unsolved question; and although recent studies have demonstrated a strong association between altered brain immune responses and disease progression, the mechanistic cause of neuronal dysfunction and death is unknown. We have previously described the unique CVN-AD mouse model of AD, in which immune-mediated nitric oxide is lowered to mimic human levels, resulting in a mouse model that demonstrates the cardinal features of AD, including amyloid deposition, hyperphosphorylated and aggregated tau, behavioral changes, and age-dependent hippocampal neuronal loss. Using this mouse model, we studied longitudinal changes in brain immunity in relation to neuronal loss and, contrary to the predominant view that AD pathology is driven by proinflammatory factors, we find that the pathology in CVN-AD mice is driven by local immune suppression. Areas of hippocampal neuronal death are associated with the presence of immunosuppressive CD11c ϩ microglia and extracellular arginase, resulting in arginine catabolism and reduced levels of total brain arginine. Pharmacologic disruption of the arginine utilization pathway by an inhibitor of arginase and ornithine decarboxylase protected the mice from AD-like pathology and significantly decreased CD11c expression. Our findings strongly implicate local immune-mediated amino acid catabolism as a novel and potentially critical mechanism mediating the age-dependent and regional loss of neurons in humans with AD.
We have investigated the structural basis for the phenotype of a native rat Slo (rSlo) potassium channel (BK Ca ; KCNMA1) in a rat pituitary cell line, GH 4 C 1 . Opposing regulation of these calcium-and voltage-activated potassium channels by cAMP-and cGMP-dependent protein kinases requires an alternatively spliced exon (strex) of 59 amino acids in the cytoplasmic C terminus of the pore-forming ␣ subunit encoded by rslo. However, inclusion of this cysteine-rich exon produces a 10-fold increase in the sensitivity of the channels to inhibition by oxidation. Inclusion of the strex exon also increases channel sensitivity to stimulation by calcium, but responses in the physiological ranges of calcium and voltage require coassembly with  1 subunits. With strex present, however,  1 subunits only stimulated channels assembled from rSlo ␣ subunits with a truncated N terminus beginning MDALI-. Thus N-terminal variation and strex exon splicing in rSlo interact to produce BK Ca channels with a physiologically relevant phenotype.Only one family of potassium channels (BK Ca ; KCNMA) responds directly to both depolarization and intracellular calcium, which allows them to integrate cellular excitability and provide critical feedback inhibition (1-3). In mammalian tissues, a single gene, slo (KCNMA1), encodes these big conductance, Ca 2ϩ -activated potassium channels (4). Nevertheless, the phenotype of BK Ca channels varies widely between tissues, between cells of the same tissue (5, 6), and in the same cell types under different hormonal environments (7,8). Several factors that contribute individually to this diversity have been identified, including alternative splicing (9, 10), regulatory  subunits (11, 12), and protein phosphorylation (13,14), but the influence of these factors on one another has not been studied in detail. For example, most recombinant forms of BK Ca channels are stimulated by the cAMP-dependent protein kinase (PKA) 1 (15), but the response of BK Ca channels to PKA in a mouse pituitary cell line, AtT20 (16,17), is reversed by insertion of a 59-amino acid exon (strex) at splice site 2 in the C terminus (18). However, most studies of recombinant BK Ca channel regulation by protein phosphorylation have employed a cDNA that begins at the third potential initiator methionine (MDALI-) encoded by the slo gene (19), omitting up to 65 amino acids in the N terminus. Because this extracellular N-terminal sequence of the pore-forming ␣ subunit interacts with the  subunit (20), which also increases calcium sensitivity, we postulated that these structural changes might interact with one another. In addition, we wondered whether some of the variability in the regulation of BK Ca channels by exogenous protein kinases might result from nonspecific effects associated with stimulation of the channel by the sulfhydryl reducing agents (21, 22), which are routinely included in purified preparations of the enzymes. We have previously characterized the regulation of native BK Ca channels by protein phosphorylation in a rat pi...
While the presence of an inflammatory response in AD (Alzheimer's disease) is well known, the data on inflammation are conflicting, suggesting that inflammation either attenuates pathology, exacerbates it or has no effect. Our goal was to more fully characterize the inflammatory response in APP (amyloid precursor protein) transgenic mice with and without disease progression. In addition, we have examined how anti-Aβ (amyloid β-peptide) immunotherapy alters this inflammatory response. We have used quantitative RT–PCR (reverse transcription–PCR) and protein analysis to measure inflammatory responses ranging from pro-inflammatory to anti-inflammatory and repair factors in transgenic mice that develop amyloid deposits only (APPSw) and amyloid deposits with progression to tau pathology and neuron loss [APPSw/NOS2−/− (nitric oxide synthase 2−/−)]. We also examined tissues from previously published immunotherapy studies. These studies were a passive immunization study in APPSw mice and an active vaccination study in APPSw/NOS2−/− mice. Both studies have already been shown to lower amyloid load and improve cognition. We have found that amyloid deposition is associated with high expression of alternative activation and acquired deactivation genes and low expression of pro-inflammatory genes, whereas disease progression is associated with a mixed phenotype including increased levels of some classical activation factors. Immunotherapy targeting amyloid deposition in both mouse models resulted in decreased alternative inflammatory markers and, in the case of passive immunization, a transient increase in pro-inflammatory markers. Our results suggest that an alternative immune response favours retention of amyloid deposits in the brain, and switching away from this state by immunotherapy permits removal of amyloid.
Understanding the pathophysiological mechanisms underlying Alzheimer disease (AD) relies on knowledge of disease onset and the sequence of development of brain pathologies. We present a comprehensive analysis of early and progressive changes in a mouse model that demonstrates a full spectrum of characteristic AD-like pathologies. This model demonstrates an altered immune redox state reminiscent of the human disease and capitalizes on data indicating critical differences between human and mouse immune responses, particularly in nitric oxide (NO) levels produced by immune activation of the NOS2 gene. Using the APPSwDI+/+/mNos2−/− (CVN-AD) mouse strain, we show a sequence of pathological events leading to neurodegeneration that include pathologically hyperphosphorylated tau in the perforant pathway at 6 weeks of age progressing to insoluble tau, the early appearance of β-amyloid peptides in perivascular deposits around blood vessels in brain regions known to be vulnerable in AD and progression to damage and overt loss in select vulnerable neuronal populations in these regions. The role of species differences between hNOS2 and mNos2 was supported by generating mice in which the human NOS2 gene replaced mNos2. When crossed to CVN-AD mice, pathological characteristics of this new strain (APPSwDI+/−/huNOS2t +/+g/mNos2−/−) mimicked the pathological phenotypes found in the CVN-AD strain.
Lithium is an anti-psychotic that has been shown to prevent the hyperphosphorylation of tau protein through the inhibition of glycogen-synthase kinase 3-beta (GSK3β). We recently developed a mouse model that progresses from amyloid pathology to tau pathology and neurodegeneration due to the genetic deletion of NOS2 in an APP transgenic mouse; the APPSwDI/NOS2−/− mouse. Because this mouse develops tau pathology, amyloid pathology and neuronal loss we were interested in the effect anti-tau therapy would have on amyloid pathology, learning and memory. We administered lithium in the diets of APPSwDI/NOS2−/− mice for a period of eight months, followed by water maze testing at 12 months of age, immediately prior to sacrifice. We found that lithium significantly lowered hyperphosphorylated tau levels as measured by Western blot and immunocytochemistry. However, we found no apparent neuroprotection, no effect on spatial memory deficits and an increase in histological amyloid deposition. Aβ levels measured biochemically were unaltered. We also found that lithium significantly altered the neuroinflammatory phenotype of the brain, resulting in enhanced alternative inflammatory response while concurrently lowering the classical inflammatory response. Our data suggest that lithium may be beneficial for the treatment of tauopathies but may not be beneficial for the treatment of Alzheimer's disease.
Alzheimer’s disease (AD) is a complex neurodegenerative process that involves altered brain immune, neuronal and metabolic functions. Understanding the underlying mechanisms has relied on mouse models that mimic components of AD pathology. We used gel-free, label-free LC/MS/MS to quantify protein and phosphopeptide levels in brains of APPSwDI/NOS2−/− (CVN-AD) mice. CVN-AD mice show a full spectrum of AD-like pathology, including amyloid deposition, hyperphosphorylated and aggregated tau and neuronal loss that worsens with age. Tryptic digests, with or without phosphopeptide enrichment on an automated titanium dioxide LC system, were separated by on-line two-dimensional LC and analyzed on a Waters Synapt G2 HDMS, yielding relative expression for >950 proteins and >1100 phosphopeptides. Among differentially expressed proteins were known markers found in humans with AD, including GFAP and C1Q. Phosphorylation of connexin 43, not previously described in AD, was increased at 42 weeks, consistent with dysregulation of gap junctions and activation of astrocytes. Additional alterations in phosphoproteins suggests dysregulation of mitochondria, synaptic transmission, vesicle trafficking and innate immune pathways. These data validate the CVN-AD mouse model of AD, identify novel disease and age-related changes in the brain during disease progression and demonstrate the utility of integrating unbiased and phosphoproteomics for understanding disease processes in AD.
In Syrian hamster embryo (SHE) fibroblasts, epidermal growth factor receptor (EGFR) tyrosine kinase activity regulates the metabolism of endogenous linoleic acid to (13S)-hydroperoxyoctadecadienoic acid (13S)-HPODE). (13S)-HPODE stimulates EGF-dependent mito-genesis in a SHE cell phenotype, which expresses tumor suppressor genes (supB ؉ ), but was not effective in a variant that does not express these suppressor genes (supB ؊ ). In the present study, we have investigated the potential effects of this lipid metabolite on the EGFR signaling pathways in these two SHE cell lines. Treatment of quiescent SHE cells with EGF produced a rapid, transient increase in the tyrosine phosphorylation of EGFR. Dependence on EGF concentration for EGFR tyrosine phosphorylation was similar in both SHE cell lines, but a more prolonged phosphorylation was detected in the supB ؊ variant. Incubation of supB ؉ cells with (13S)-HPODE and EGF increased EGFR autophosphorylation and tyrosine phosphorylation on several signaling proteins with Src homology-2 domains including GTPase-activating protein. The lipid metabolite did not significantly alter EGF-dependent tyrosine phosphorylation in the supB ؊ variant. Tyrosine phosphorylation of mitogen-activated protein (MAP) kinase was also measured. The addition of (13S)-HPODE increased the extent and duration of MAP kinase tyrosine phosphorylation in supB ؉ cells but not in the supB ؊ variant. MAP kinase activity in supB ؉ cells, as measured in immunoprecipitates from cells after the addition of EGF, was increased by the presence of (13S)-HPODE. The addition of (13S)-HPODE did not directly alter EGFR kinase activity or the internalization of the EGFR. However, the addition of (13S)-HPODE to supB ؉ cells extended the tyrosine phosphorylation of the EGFR in response to EGF. The dephosphorylation of the EGFR was measured directly, and a slower rate was observed in the supB ؊ compared with the supB ؉ cells. Incubation of the supB ؉ cells with (13S)-HPODE attenuated the dephosphorylation of the EGFR. Thus, (13S)-HPODE stimulates EGF-dependent mitogenesis and up-regulation of EGF-dependent tyrosine phosphorylation by inhibiting the dephosphorylation of the EGFR. This study shows that a metabolite of an essential dietary fatty acid, linoleic acid, can modulate tyrosine phosphorylation and activity of key signal transduction proteins in a growth factor mitogenic pathway.Several lines of evidence suggest that metabolism of the cis-polyunsaturated fatty acids, arachidonic acid and linoleic acid, by prostaglandin H synthase and lipoxygenases generate metabolites that modulate the EGF 1 mitogenic signal in fibroblasts. In Balb/c 3T3 fibroblasts, mitogenic stimulation by EGF induced the formation of prostaglandin E 2 (1) and the expression of c-myc (2). Inhibition of prostaglandin H synthase partially blocked both mitogenesis and c-myc expression, which was restored and enhanced by the addition of exogenous prostaglandins. In these studies lipoxygenase inhibitors were very effective inhibitors of mitogenesis...
BackgroundMouse models are used in the study of human disease. Despite well-known homologies, the difference in immune response between mice and humans impacts the application of data derived from mice to human disease outcomes. Nitric oxide synthase-2 (NOS2) is a key gene that displays species-specific outcomes via altered regulation of the gene promoter and via post-transcriptional mechanisms in humans that are not found in mice. The resulting levels of NO produced by activation of human NOS2 are different from the levels of NO produced by mouse Nos2. Since both tissue redox environment and immune responsiveness are regulated by the level of NO and its interactions, we investigated the significance of mouse and human differences on brain oxidative stress and on immune activation in HuNOS2tg/mNos2-/- mice that express the entire human NOS2 gene and that lack a functional mNos2 compared to wild type (WT) mice that express normal mNos2.Methods/resultsSimilarly to human, brain tissue from HuNOS2tg/mNos2-/- mice showed the presence of a NOS2 gene 3′UTR binding site. We also identified miRNA-939, the binding partner for this site, in mouse brain lysates and further demonstrated reduced levels of nitric oxide (NO) typical of the human immune response on injection with lipopolysaccharide (LPS). HuNOS2tg/mNos2-/- brain samples were probed for characteristic differences in redox and immune gene profiles compared to WT mice using gene arrays. Selected genes were also compared against mNos2-/- brain lysates. Reconstitution of the human NOS2 gene significantly altered genes that encode multiple anti-oxidant proteins, oxidases, DNA repair, mitochondrial proteins and redox regulated immune proteins. Expression levels of typical pro-inflammatory, anti-inflammatory and chemokine genes were not significantly different with the exception of increased TNFα and Ccr1 mRNA expression in the HuNOS2tg/mNos2-/- mice compared to WT or mNos2-/- mice.ConclusionsNO is a principle factor in establishing the tissue redox environment and changes in NO levels impact oxidative stress and immunity, both of which are primary characteristics of neurodegenerative diseases. The HuNOS2tg/mNos2-/- mice provide a potentially useful mechanism to address critical species- specific immune differences that can impact the study of human diseases.
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