Christopher A. McPherson

Alzheimer's Disease (AD)-Like Pathology in Aged Monkeys after Infantile Exposure to Environmental Metal Lead (Pb): Evidence for a Developmental Origin and Environmental Link for AD

The sporadic nature of Alzheimers disease (AD) argues for an environmental link that may drive AD pathogenesis; however, the triggering factors and the period of their action are unknown. Recent studies in rodents have shown that exposure to lead (Pb) during brain development predetermined the expression and regulation of the amyloid precursor protein (APP) and its amyloidogenic β-amyloid (Aβ) product in old age. Here, we report that the expression of AD-related genes [APP, BACE1 (β-site APP cleaving enzyme 1)] as well as their transcriptional regulator (Sp1) were elevated in aged (23-year-old) monkeys exposed to Pb as infants. Furthermore, developmental exposure to Pb altered the levels, characteristics, and intracellular distribution of Aβ staining and amyloid plaques in the frontal association cortex. These latent effects were accompanied by a decrease in DNA methyltransferase activity and higher levels of oxidative damage to DNA, indicating that epigenetic imprinting in early life influenced the expression of AD-related genes and promoted DNA damage and pathogenesis. These data suggest that AD pathogenesis is influenced by early life exposures and argue for both an environmental trigger and a developmental origin of AD.

Microglial M1/M2 polarization and metabolic states

Microglia are critical nervous system‐specific immune cells serving as tissue‐resident macrophages influencing brain development, maintenance of the neural environment, response to injury and repair. As influenced by their environment, microglia assume a diversity of phenotypes and retain the capability to shift functions to maintain tissue homeostasis. In comparison with peripheral macrophages, microglia demonstrate similar and unique features with regards to phenotype polarization, allowing for innate immunological functions. Microglia can be stimulated by LPS or IFN‐γ to an M1 phenotype for expression of pro‐inflammatory cytokines or by IL‐4/IL‐13 to an M2 phenotype for resolution of inflammation and tissue repair. Increasing evidence suggests a role of metabolic reprogramming in the regulation of the innate inflammatory response. Studies using peripheral immune cells demonstrate that polarization to an M1 phenotype is often accompanied by a shift in cells from oxidative phosphorylation to aerobic glycolysis for energy production. More recently, the link between polarization and mitochondrial energy metabolism has been considered in microglia. Under these conditions, energy demands would be associated with functional activities and cell survival and thus, may serve to influence the contribution of microglia activation to various neurodegenerative conditions. This review examines the polarization states of microglia and their relationship to mitochondrial metabolism. Additional supporting experimental data are provided to demonstrate mitochondrial metabolic shifts in primary microglia and the BV‐2 microglia cell line induced under LPS (M1) and IL‐4/IL‐13 (M2) polarization.

Voluntary exercise protects hippocampal neurons from trimethyltin injury: Possible role of interleukin-6 to modulate tumor necrosis factor receptor-mediated neurotoxicity

In the periphery, exercise induces interleukin (IL)-6 to downregulate tumor necrosis factor (TNF), elevate interleukin-1 receptor antagonist (IL-1RA), decreasing inflammation. Exercise also offers neuroprotection and facilitates brain repair. IL-6 production in the hippocampus following exercise suggests the potential of a similar protective role as in the periphery to down-regulate TNFα and inflammation. Using a chemical-induced model of hippocampal dentate granule cell death (trimethyltin, TMT 2.4 mg/kg, ip) dependent upon TNF receptor signaling, we demonstrate neuroprotection in mice with 2 weeks access to running wheel. Exercise attenuated neuronal death and diminished elevations in TNFα, TNF receptor 1, myeloid differentiation primary response gene (MyD) 88, transforming growth factor β, chemokine (C-C motif) ligand 2 (CCL2), and CCL3. Elevated mRNA levels for IL-1α, IL-1RA, occurred with injury and protection. mRNA and protein levels of IL-6 and neuronal expression of IL-6 receptor α, were elevated with injury and protection. Microarray pathway analysis supported an up-regulation of TNFα cell death signaling pathways with TMT and inhibition by exercise. IL-6 pathway recruitment occurred in both conditions. IL-6 downstream signal events differed in the level of STAT3 activation. Exercise did not increase mRNA levels of brain derived neurotrophic factor, nerve growth factor, or glial derived neurotrophic factor. In IL-6 deficient mice, exercise did not attenuate TMT-induced tremor and a diminished level of neuroprotection was observed. These data suggest a contributory role for IL-6 induced by exercise for neuroprotection in the CNS similar to that seen in the periphery.

Heterogeneity of microglia and TNF signaling as determinants for neuronal death or survival

Microglia do not constitute a single, uniform cell population, but rather comprise cells with varied phenotypes, some which are beneficial and others that may require active regulatory control. Thus, gaining a better understanding of the heterogeneity of resident microglia responses will contribute to any interpretation regarding the impact of any such response in the brain. Microglia are the primary source of the pro-inflammatory cytokine, tumor necrosis factor (TNF) that can initiate various effects through the activation of membrane receptors. The TNF p55 receptor contains a death domain and activation normally leads to cellular apoptosis; however, under specific conditions, receptor activation can also lead to the activation of NF-kappaB and contribute to cell survival. These divergent outcomes have been linked to receptor localization with receptor internalization leading to cell death and membrane localization supporting cell survival. A second TNF receptor, TNF p75 receptor, is normally linked to cell growth and survival, however, it can cooperate with the p55 receptor and contribute to cell death. Thus, while an elevation in TNFalpha in the brain is often considered an indicator of microglia activation and neuroinflammation, a number of factors come into play to determine the final outcome. Data are reviewed demonstrating that heterogeneity in morphological response of microglia and the expression of TNFalpha and TNF receptors are critical in identifying and characterizing neurotoxic events as they relate to neuroinflammation, neuronal damage and in stimulating neuroprotection.

Tumor necrosis factor p55 and p75 receptors are involved in chemical-induced apoptosis of dentate granule neurons

Localized tumor necrosis factor‐α (TNFα) elevation has diverse effects in brain injury often attributed to signaling via TNFp55 or TNFp75 receptors. Both dentate granule cells and CA pyramidal cells express TNF receptors (TNFR) at low levels in a punctate pattern. Using a model to induce selective death of dentate granule cells (trimethyltin; 2 mg/kg, i.p.), neuronal apoptosis [terminal deoxynucleotidyl transferase‐mediated dUTP‐biotin in situ end labeling, active caspase 3 (AC3)] was accompanied by amoeboid microglia and elevated TNFα mRNA levels. TNFp55R (55 kDa type‐1 TNFR) and TNFp75R (75 kDa type‐2 TNFR) immunoreactivity in AC3+ neurons displayed a pattern suggestive of receptor internalization and a temporal sequence of expression of TNFp55R followed by TNFp75R associated with the progression of apoptosis. A distinct ramified microglia response occurred around CA1 neurons and healthy dentate neurons that displayed an increase in the normal punctate pattern of TNFRs. Neuronal damage was decreased with i.c.v. injection of TNFα antibody and in TNFp55R−/−p75R−/− mice that showed higher constitutive mRNA levels for interleukin (IL‐1α), macrophage inflammatory protein 1‐α (MIP‐1α), TNFα, transforming growth factor β1, Fas, and TNFRSF6‐assoicated via death domain (FADD). TNFp75R−/− mice showed exacerbated injury and elevated mRNA levels for IL‐1α, MIP‐1α, and TNFα. In TNFp55R−/− mice, constitutive mRNA levels for TNFα, IL‐6, caspase 8, FADD, and Fas‐associated phosphatase were higher; IL‐1α, MIP‐1α, and transforming growth factor β1 lower. The mice displayed exacerbated neuronal death, delayed microglia response, increased FADD and TNFp75R mRNA levels, and co‐expression of TNFp75R in AC3+ neurons. The data demonstrate TNFR‐mediated apoptotic death of dentate granule neurons utilizing both TNFRs and suggest a TNFp75R‐mediated apoptosis in the absence of normal TNFp55R activity.

Trimethyltin-induced neurogenesis in the murine hippocampus

Neurogenesis continues to occur in the mature rodent brain with one of the most prominent sources for new neurons being the subgranular layer (SGL) of the dentate gyrus (DG) in the hippocampus. A number of factors can stimulate this process including synaptic activity and injury. To determine if this process would occur upon a direct injury to the dentate region, we exposed young, 21 day old male CD-1 mice to the hippocampal toxicant, trimethyltin (TMT). An acute i.p. injection of TMT (2 mg/kg) produced extensive damage and loss of dentate granule neurons within 72 h. This active period of degeneration was accompanied by an increase in the generation of progenitor cells within the SGL as identified by BrdU uptake and Ki-67 immunostaining. As additional markers for neurogenesis, both nestin and doublecortin showed increased staining patterns within the blades of the dentate. In these young weanling mice, the level of proliferation was sufficient to significantly repopulate the dentate region by 4 weeks post-TMT, suggesting a high level of regenerative potential. Our data indicate a significant level of neurogenesis occurring during the active process of degeneration and in an environment of microglia activation. The TMT-induced injury offers a model system for further examination of the process of neurogenesis, neural adaptation, and the influence of inflammatory factors and glia interactions.

Lifespan Profiles of Alzheimer's Disease-Associated Genes and Products in Monkeys and Mice

Alzheimers disease (AD) is characterized by plaques of amyloid-beta (Abeta) peptide, cleaved from amyloid-beta protein precursor (AbetaPP). Our hypothesis is that lifespan profiles of AD-associated mRNA and protein levels in monkeys would differ from mice and that differential lifespan expression profiles would be useful to understand human AD pathogenesis. We compared profiles of AbetaPP mRNA, AbetaPP protein, and Abeta levels in rodents and primates. We also tracked a transcriptional regulator of the AbetaPP gene, specificity protein 1 (SP1), and the beta amyloid precursor cleaving enzyme (BACE1). In mice, AbetaPP and SP1 mRNA and their protein products were elevated late in life; Abeta levels declined in old age. In monkeys, SP1, AbetaPP, and BACE1 mRNA declined in old age, while protein products and Abeta levels rose. Proteolytic processing in both species did not match production of Abeta. In primates, AbetaPP and SP1 mRNA levels coordinate, but an inverse relationship exists with corresponding protein products as well as Abeta levels. Comparison of human DNA and mRNA sequences to monkey and mouse counterparts revealed structural features that may explain differences in transcriptional and translational processing. These findings are important for selecting appropriate models for AD and other age-related diseases.

The type 1 interleukin 1 receptor is not required for the death of murine hippocampal dentate granule cells and microglia activation

Alterations in inflammatory process, neuronal death, and glia response have been observed under manipulation of interleukin-1 (IL-1) and subsequent signaling through the type 1 IL-1 receptor (IL-1R1). To investigate the influence of IL-1R1 activation in the pathophysiology of a chemical-induced injury to the murine hippocampus, we examined the level and pattern of neuronal death and neuroinflammation in male weanling mice exposed to trimethyltin hydroxide (2.0 mg TMT/kg, i.p.). Dentate granule cell death occurred at 6 h post-TMT as detected by active caspase 3 immunostaining and presence of lectin positive microglia. The severity of neuronal death and microglia response increased by 12-24 h with elevations in mRNA levels for TNFalpha and IL-1alpha. In IL-1R1 null (IL-1R1-/-) mice, the pattern and severity of neuronal death at 24 or 72 h post-TMT was similar as compared to wildtype (WT) mice. In both groups, mRNA levels for TNFalpha and MIP-1alpha were elevated, no significant change was seen in either IL-1alpha or IL-1beta, and the early activation of microglia, including their ability to progress to a phagocytic phenotype, was maintained. Compared to WT mice, IL-1R1-/- mice displayed a limited glial fibrillary acidic protein (GFAP) astrocytic response, as well as a preferential induction in mRNA levels of Fas signaling components. Cumulatively, these results indicate that IL-1R1 activation is not necessary for TMT-induced death of dentate granule neurons or local activation of microglia; however, IL-1R1 signaling is involved in mediating the structural response of astrocytes to injury and may regulate apoptotic mechanisms via Fas signaling components.

Alterations in cyclin A, B, and D1 in mouse dentate gyrus following TMT-induced hippocampal damage

The interactions of glia and neurons during injury and subsequent neurodegeneration are a subject of interest both in disease and chemical-induced brain injury. One such model is the prototypical hippocampal toxicant trimethyltin (TMT). An acute injection of TMT (2.0 mg/kg, i.p.) to postnatal day 21 CD-1 male mice produced neuronal necrosis and loss of dentate granule cells, astrocyte hypertrophy, and microglia activation in the hippocampus within 24 h. Neuronal necrosis and microglia differentiation to a phagocytic phenotype is temporally correlated with peak elevations in TNF-α, cyclin A2, cyclin B1 and cyclin D1 at 72 h post-TMT. TNF-α mRNA levels were significantly elevated in the hippocampus by 12 h and remained elevated for 72 h. mRNA levels for cyclin A2 and cyclin B1 were elevated by approximately 2-fold at 72 h. Immunohistochemistry suggested a cellular localization of cyclin A to microglia in the region of neuronal necrosis in the dentate, cyclin B in glial cells in juxtaposition to neurons in the hilus of the hippocampus and cyclin D1 to non-glial cells in the dentate. mRNA levels for cyclin D1 were elevated approximately 1.5-fold by 72 h as determined by RNase protection assay. No changes were seen in mRNA levels for cyclins E, F, G1, G2, H or I nor cyclin dependent kinases. These elevations are not associated with proliferation of microglia as determined by BrdU incorporation and Ki-67 immunohistochemistry. Upregulation of cell cycle genes was associated with cellular processes other than proliferation and may contribute to the differentiation of microglia to a phagocytic phenotype. These data suggest an integrated role for cell cycle regulation of neural cells in the manifestation of hippocampal pathophysiology.

Interleukin-6 (IL-6) receptor/IL-6 fusion protein (Hyper IL-6) effects on the neonatal mouse brain: possible role for IL-6 trans-signaling in brain development and functional neurobehavioral outcomes.

Adverse neurodevelopmental outcomes are linked to perinatal production of inflammatory mediators, including interleukin 6 (IL-6). While a pivotal role for maternal elevation in IL-6 has been established in determining neurobehavioral outcomes in the offspring and considered the primary target mediating the fetal inflammatory response, questions remain as to the specific actions of IL-6 on the developing brain. CD-1 male mice received a subdural injection of the bioactive fusion protein, hyper IL-6 (HIL-6) on postnatal-day (PND)4 and assessed from preweaning until adulthood. Immunohistochemical evaluation of astrocytes and microglia and mRNA levels for pro-inflammatory cytokines and host response genes indicated no evidence of an acute neuroinflammatory injury response. HIL-6 accelerated motor development and increased reactivity to stimulation and number of entries in a light/dark chamber, decreased ability to learn to withhold a response in passive avoidance, and effected deficits in social novelty behavior. No changes were observed in motor activity, pre-pulse startle inhibition, or learning and memory in the Morris water maze or radial arm maze, as have been reported for models of more severe developmental neuroinflammation. In young animals, mRNA levels for MBP and PLP/DM20 decreased and less complexity of MBP processes in the cortex was evident by immunohistochemistry. The non-hydroxy cerebroside fraction of cerebral lipids was increased. These results provide evidence for selective effects of IL-6 signaling, particularly trans-signaling, in the developing brain in the absence of a general neuroinflammatory response. These data contribute to our further understanding of the multiple aspects of IL-6 signaling in the developing brain.