Microglial-associated responses to comorbid amyloid pathology and hyperhomocysteinemia in an aged knock-in mouse model of Alzheimer's disease.

BACKGROUND: Elevated blood homocysteine levels, termed hyperhomocysteinemia (HHcy), is a prevalent risk factor for Alzheimer's disease (AD) in elderly populations. While dietary supplementation of B-vitamins is a generally effective method to lower homocysteine levels, there is little if any benefit to cognition. In the context of amyloid pathology, dietary-induced HHcy is known to enhance amyloid deposition and certain inflammatory responses. Little is known, however, about whether there is a more specific effect on microglia resulting from combined amyloid and HHcy pathologies. METHODS: The present study used a knock-in mouse model of amyloidosis, aged to 12 months, given 8 weeks of B-vitamin deficiency-induced HHcy to better understand how microglia are affected in this comorbidity context. RESULTS: We found that HHcy-inducing diet increased amyloid plaque burden, altered the neuroinflammatory milieu, and upregulated the expression of multiple damage-associated and "homeostatic" microglial genes. CONCLUSIONS: Taken together, these data indicate complex effects of comorbid pathologies on microglial function that are not driven solely by increased amyloid burden. Given the highly dynamic nature of microglia, their central role in AD pathology, and the frequent occurrence of various comorbidities in AD patients, it is increasingly important to understand how microglia respond to mixed pathological processes.

Development of Novel In Vivo Chemical Probes to Address CNS Protein Kinase Involvement in Synaptic Dysfunction.

Serine-threonine protein kinases are critical to CNS function, yet there is a dearth of highly selective, CNS-active kinase inhibitors for in vivo investigations. Further, prevailing assumptions raise concerns about whether single kinase inhibitors can show in vivo efficacy for CNS pathologies, and debates over viable approaches to the development of safe and efficacious kinase inhibitors are unsettled. It is critical, therefore, that these scientific challenges be addressed in order to test hypotheses about protein kinases in neuropathology progression and the potential for in vivo modulation of their catalytic activity. Identification of molecular targets whose in vivo modulation can attenuate synaptic dysfunction would provide a foundation for future disease-modifying therapeutic development as well as insight into cellular mechanisms. Clinical and preclinical studies suggest a critical link between synaptic dysfunction in neurodegenerative disorders and the activation of p38αMAPK mediated signaling cascades. Activation in both neurons and glia also offers the unusual potential to generate enhanced responses through targeting a single kinase in two distinct cell types involved in pathology progression. However, target validation has been limited by lack of highly selective inhibitors amenable to in vivo use in the CNS. Therefore, we employed high-resolution co-crystallography and pharmacoinformatics to design and develop a novel synthetic, active site targeted, CNS-active, p38αMAPK inhibitor (MW108). Selectivity was demonstrated by large-scale kinome screens, functional GPCR agonist and antagonist analyses of off-target potential, and evaluation of cellular target engagement. In vitro and in vivo assays demonstrated that MW108 ameliorates beta-amyloid induced synaptic and cognitive dysfunction. A serendipitous discovery during co-crystallographic analyses revised prevailing models about active site targeting of inhibitors, providing insights that will facilitate future kinase inhibitor design. Overall, our studies deliver highly selective in vivo probes appropriate for CNS investigations and demonstrate that modulation of p38αMAPK activity can attenuate synaptic dysfunction.

Microglial p38α MAPK is a key regulator of proinflammatory cytokine up-regulation induced by toll-like receptor (TLR) ligands or beta-amyloid (Aß).

BACKGROUND: Overproduction of proinflammatory cytokines from activated microglia has been implicated as an important contributor to pathophysiology progression in both acute and chronic neurodegenerative diseases. Therefore, it is critical to elucidate intracellular signaling pathways that are significant contributors to cytokine overproduction in microglia exposed to specific stressors, especially pathways amenable to drug interventions. The serine/threonine protein kinase p38α MAPK is a key enzyme in the parallel and convergent intracellular signaling pathways involved in stressor-induced production of IL-1ß and TNFα in peripheral tissues, and is a drug development target for peripheral inflammatory diseases. However, much less is known about the quantitative importance of microglial p38α MAPK in stressor-induced cytokine overproduction, or the potential of microglial p38α MAPK to be a druggable target for CNS disorders. Therefore, we examined the contribution of microglial p38αMAPK to cytokine up-regulation, with a focus on the potential to suppress the cytokine increase by inhibition of the kinase with pharmacological or genetic approaches. METHODS: The microglial cytokine response to TLR ligands 2/3/4/7/8/9 or to Aß1-42 was tested in the presence of a CNS-penetrant p38α MAPK inhibitor, MW01-2-069A-SRM. Primary microglia from mice genetically deficient in p38α MAPK were used to further establish a linkage between microglia p38α MAPK and cytokine overproduction. The in vivo significance was determined by p38α MAPK inhibitor treatment in a LPS-induced model of acute neuroinflammation. RESULTS: Increased IL-1ß and TNFα production by the BV-2 microglial cell line and by primary microglia cultures was inhibited in a concentration-dependent manner by the p38α MAPK-targeted inhibitor. Cellular target engagement was demonstrated by the accompanying decrease in the phosphorylation state of two p38α MAPK protein substrates, MK2 and MSK1. Consistent with the pharmacological findings, microglia from p38α-deficient mice showed a diminished cytokine response to LPS. Further, oral administration of the inhibitor blocked the increase of IL-1ß in the cerebral cortex of mice stressed by intraperitoneal injection of LPS. CONCLUSION: The p38α MAPK pathway is an important contributor to the increased microglial production of proinflammatory cytokines induced by diverse stressors. The results also indicate the feasibility of targeting p38α MAPK to modulate CNS proinflammatory cytokine overproduction.

Effects of aging and dietary restriction on ubiquitination, sumoylation, and the proteasome in the spleen.

In the present study, we demonstrate for the first time that aging increases the levels of ubiquitinated protein in the spleen, and that dietary restriction (DR) significantly reduces these age-related increases in ubiquitinated protein. Sumoylated protein, proteasome subunits, and a protein essential for proteasome biogenesis (POMP1) were also increased with age in the spleen but were not significantly affected by DR. Chymotrypsin-like proteasome activity was elevated in the aged spleen, and was not significantly altered by DR. Together, these data demonstrate for the first time the multiple effects of aging and DR on ubiquitination, sumoylation, and the proteasome in the spleen.

Oxidative stress alters neuronal RNA- and protein-synthesis: Implications for neural viability.

Recent studies have demonstrated that impaired protein synthesis occurs in several neurodegenerative conditions associated with oxidative stress. Studies have also demonstrated that administration of oxidative stressors is sufficient to impair different and discrete regulatory aspects of protein synthesis in neural cells, with the majority of these studies focused on the effects of oxidative stressors towards initiation factors. Currently, little is known with regards to oxidative stress effects on the rates of RNA- and protein-synthesis, or the relationship between oxidant-induced impairments in RNA-/protein-synthesis to subsequent neuron death. In the present study, we demonstrate that administration of an oxidative stressor (hydrogen peroxide) induces a significant and time-dependent decrease in both RNA- and protein-synthesis in primary neurons and neural SH-SY5Y cells. Increases in RNA oxidation and disruption of ribosome complexes were selectively observed following the longer durations of oxidant exposure. Significant correlations between the loss of RNA- and protein-synthesis and the amount of oxidant-induced neuron death were also observed. Interestingly, the addition of a protein synthesis inhibitor (cycloheximide) did not significantly alter the amount of neuron death induced by the oxidative stressor. These data demonstrate that oxidant exposure promotes a time-dependent decrease in both RNA- and protein-synthesis in neurons, and demonstrate a role for elevations in RNA oxidation and ribosome dysfunction as potential mediators of impaired protein synthesis. These data also suggest that there is a complex relationship between the ability of oxidative stressors to modulate RNA- and protein-synthesis, and the ability of oxidative stressors to ultimately induce neuron death.

Oxidative damage, protein synthesis, and protein degradation in Alzheimer's disease.

A large number of studies has firmly established that increases in oxidative damage occurs in Alzheimer's disease (AD). Such studies have demonstrated that increased in oxidative damage selectively occurs within the brain regions involved in regulating cognitive performance. Studies from our laboratory and others have provided experimental evidence that increased levels of oxidative damage occur in subjects with Mild Cognitive Impairment (MCI), which is believed to be one of the earliest stages of AD, and is a condition which is devoid of dementia or the extensive neurofibrillary pathology and neuritic plaque deposition observed in AD. Together, these data support a role for the accrual of oxidative damage potentially serving as an early event that then initiates the development of cognitive disturbances and pathological features observed in AD. Recent studies from our laboratory have demonstrated that a decline in protein synthesis capabilities occurs in the same brain regions which exhibit increased levels of oxidative damage in MCI and AD subjects. The focus of this review is to describe the large number of studies which suggest protein synthesis may be one of the earliest cellular processes disrupted by oxidative damage in AD. Taken together, these findings have important implications for understanding the molecular and cellular basis of AD, understanding the basis for oxidative stress in AD, and may have important implications for studies involving proteomics and proteolysis in AD.

SOD1 overexpression alters ROS production and reduces neurotoxic inflammatory signaling in microglial cells.

Activation of the oxidative burst is one of the earliest biochemical events in microglial activation, but it is not understood yet how free radicals participate in inflammatory signaling. To determine the role that specific reactive oxygen species play in microglial activation, the levels of SOD1 were manipulated in N9 murine microglia. Stable overexpression of SOD1 caused significant decreases in superoxide and nitric oxide production, with concurrent increases in hydrogen peroxide following LPS. However, LPS-induced activation of NFkappaB, and release of TNFalpha and IL-6 were significantly attenuated in SOD1 overexpressing cells, as was the ability of microglia to induce toxicity in cultured neurons. Conversely, acute inhibition of SOD1 with disulfiram was associated with increased nitric oxide and cytokine release, and increased neurotoxicity. Together, these data suggest that superoxide radicals in microglia play important roles in directing redox-sensitive inflammatory signaling and initiating neurotoxic inflammation.

Proteasome inhibition induces reversible impairments in protein synthesis.

Proteasome inhibition occurs during normal aging and in a variety of age-related diseases, with inhibition of proteasome function sufficient to induce physiological and pathological alterations observed in each of these conditions. It is presumed that proteasome inhibition induces cellular alterations by promoting rapid protein accumulation, as the direct result of impairments in protein removal, which assumes protein synthesis remains relatively unchanged during proteasome inhibition. We conducted experimentation using established proteasome inhibitors and primary rat neuron cultures in order to elucidate whether proteasome inhibition had any effect on neuronal protein synthesis. Proteasome inhibition impaired neuronal protein synthesis, with concentrations of inhibitor necessary to significantly inhibit protein synthesis similar to the concentrations necessary to induce subsequent neuron death. The inhibition of protein synthesis was reversible during the first 6 h of treatment, with the neurotoxicity of proteasome inhibition reversible during the first 12 h of treatment. These studies are the first to demonstrate a potentially important interplay between the proteasome and protein synthesis in neurons, and the first to identify that some effects of proteasome inhibition are reversible in neurons. Together these findings have important implications for understanding proteasome inhibition as a potential contributor to aging and age-related disease.

LMP2 knock-out mice have reduced proteasome activities and increased levels of oxidatively damaged proteins.

The proteasome is a large intracellular protease, composed of multiple subunits, that is present in all eukaryotic cells. Proteasome inhibition is known to occur during normal aging, and is believed to contribute towards an age-related increase in oxidative stress, although at present the mechanisms responsible for mediating age-related changes in proteasome activity have not been elucidated. At present the relationship between proteasome subunit expression, proteasome activity, and protein oxidation during normal aging has not been elucidated. In the present study we observed that the absence of LMP2, a specific proteasome subunit, decreases proteasome activities in both the brain and liver, with increased levels of protein oxidation occurring in both tissues. Results from this study demonstrate for the first time that individual proteasome subunits are important for the regulation of age-related changes in both proteasome activity and protein oxidation.

Proteasome regulation of oxidative stress in aging and age-related diseases of the CNS.

Proteasome-mediated protein degradation is responsible for a large percentage of bulk protein turnover, particularly the degradation of short-lived and oxidized proteins. Increasing evidence suggests that proteasome inhibition occurs during the aging of the central nervous system (CNS), and in a variety of age-related disorders of the CNS. The focus of this review is to discuss the role of the proteasome as a regulator of oxidative stress, with preservation of proteasome function playing an important role in preventing oxidative stress, and proteasome inhibition playing an important role as a mediator of oxidative stress. In particular, this review will describe experimental evidence that proteasome inhibition is sufficient to induce mitochondrial dysfunction, increase reactive oxygen species generation, elevate RNA and DNA oxidation, and promote protein oxidation. Taken together, these data indicate that the proteasome is an important regulator of oxidative damage in the CNS, and suggest that proteasome inhibition may serve as an important switch for the induction of oxidative stress in the CNS. Additionally we discuss the likelihood that the 20S proteasome and 26S proteasome may play different roles in regulating oxidative stress and neurotoxicity in the aging CNS, and in age-related disorders of the CNS.

Enhanced toxicity to the catecholamine tyramine in polyglutamine transfected SH-SY5Y cells.

Huntington's disease (HD) is a progressive neurodegenerative disorder, of which the pathogenesis is not completely understood. In patients with Huntington's disease, there is a mutation in the gene encoding the protein huntingtin, which results in an expanded polyglutamine sequence leading to degeneration of the basal ganglia. There is mounting evidence that metabolism of the transmitter dopamine by the enzyme monoamine oxidase may contribute to striatal damage in mitochondrial toxin-induced models of HD. In this study, we have examined the role of the catecholamine tyramine in neural SH-SY5Y cells transfected with normal and expanded polyglutamine repeat numbers. Our findings demonstrate that cells containing a pathological number of polyglutamines are more sensitive to tyramine than cells with a non-pathological number. Tyramine-induced cell death was attenuated by MAO inhibitors as well as with catalase and the iron chelator deferoxamine, suggesting that H202 might mediate the observed toxicity. These observations support the notion that the metabolism of dopamine plays a role in neuron death in Huntington's disease.

Estrogen and brain inflammation: effects on microglial expression of MHC, costimulatory molecules and cytokines.

To model the effects of estrogen on adaptive immunity in the brain, we examined the effects of 17beta-estradiol on microglial parameters related to antigen presentation and T cell activation. Specifically, the effects of 17beta-estradiol on basal and LPS-induced surface staining of Class I and II MHC, as well as CD40, CD80, CD86, CD152, CD28, CD8, CD11b, Fas, FasL, and also ERalpha and ERbeta, were examined in N9 microglial cells. Additionally, the effects of 17beta-estradiol on basal and LPS-induced release of cytokines (TNF-alpha, IFN-gamma, IL-2, IL-4, and IL-10) were determined. Data indicate that estrogen increases IL-10 while decreasing TNFalpha and IFNgamma release from resting and LPS-stimulated N9 cells. Additionally, LPS-induced surface staining of MHC Class I, CD40, and CD86 was significantly attenuated by estrogen pretreatment. The basal percentage of cells positive for MHC Class I and II, CD40, and CD152, Fas, and FasL was significantly decreased by estrogen exposure. However, CD8, CD86, CD11b, and CD28 were unaffected by estrogen, and CD80 cell surface staining significantly increased following estrogen exposure. Taken together, these data indicate that estrogen can significantly decrease components of adaptive immunity in microglial cells, and highlight the multi-faceted regulatory effects of estrogen on microglial parameters related to antigen presentation and T cell interaction.

Proteasome inhibition increases DNA and RNA oxidation in astrocyte and neuron cultures.

Increased levels of nucleic acid oxidation have been described as part of normal brain aging and have been demonstrated to occur in multiple neurological disorders. The basis for increased nucleic acid oxidation in each of these conditions is presently unknown. Proteasome inhibition occurs in a host of neurodegenerative conditions and likely contributes to increased levels of oxidative damage and neurotoxicity. In the present study we demonstrate for the first time the ability of proteasome inhibition to increase the level of nucleic acid oxidation in primary neuron and astrocyte cultures. Administration of proteasome inhibitors (MG262, MG115) at concentrations that do not induce neuron death in the first 24 h of treatment, dramatically increase the levels of 8-hydroxy-2'-deoxyguanosine (8-OHdG) and 8-hydroxyguanosine (8OHG) immunoreactivity in both cell types. Neurons underwent larger increases in nucleic acid oxidation compared to astrocyte cultures. While both DNA and RNA oxidation were observed following proteasome inhibition, RNA appeared to undergo a greater degree of oxidation than DNA. Both 18S and 28S ribosomal RNA were dramatically decreased following proteasome inhibition. Interestingly, an accumulation of unprocessed and/or cross-linked RNA species was observed following proteasome inhibition. Taken together, these data indicate the ability of proteasome inhibition to increase the levels of nucleic acid oxidation in both neurons and astrocytes, and suggest that proteasome inhibition may have deleterious effects on transcription and translation in both neurons and glia.

Autophagy, proteasomes, lipofuscin, and oxidative stress in the aging brain.

In order to successfully respond to stress all cells rely on the ability of the proteasomal and lysosomal proteolytic pathways to continually maintain protein turnover. Increasing evidence suggests that as part of normal aging there are age-related impairments in protein turnover by the proteasomal proteolytic pathway, and perturbations of the lysosomal proteolytic pathway. Furthermore, with numerous studies suggest an elevated level of a specialized form of lysosomal proteolysis (autophagy or macroautophagy) occurs during the aging of multiple cell types. Age-related alterations in proteolysis are believed to contribute to a wide variety of neuropathological manifestations including elevations in protein oxidation, protein aggregation, and cytotoxicity. Within the brain altered protein turnover is believed to contribute to elevations in multiple forms of protein aggregation ranging from tangle and Lewy body formation, to lipofuscin-ceroid accumulation. In this review we discuss and summarize evidence for proteolytic alterations occurring in the aging brain, the contribution of oxidative stress to disruption of protein turnover during normal aging, the evidence for cross-talk between the proteasome and lysosomal proteolytic pathways in the brain, and explore the contribution of altered proteolysis as a mediator of oxidative stress, neuropathology, and neurotoxicity in the aging brain.

Proteasome inhibition alters neural mitochondrial homeostasis and mitochondria turnover.

Inhibition of proteasome activity occurs in normal aging and in a wide variety of neurodegenerative conditions including Alzheimer's disease and Parkinson's disease. Although each of these conditions is also associated with mitochondrial dysfunction potentially mediated by proteasome inhibition, the relationship between proteasome inhibition and the loss of mitochondrial homeostasis in each of these conditions has not been fully elucidated. In this study, we conducted experimentation in order to begin to develop a more complete understanding of the effects proteasome inhibition has on neural mitochondrial homeostasis. Mitochondria within neural SH-SY5Y cells exposed to low level proteasome inhibition possessed similar morphological features and similar rates of electron transport chain activity under basal conditions as compared with untreated neural cultures of equal passage number. Despite such similarities, maximal complex I and complex II activities were dramatically reduced in neural cells subject to proteasome inhibition. Proteasome inhibition also increased mitochondrial reactive oxygen species production, reduced intramitochondrial protein translation, and increased cellular dependence on glycolysis. Finally, whereas proteasome inhibition generated cells that consistently possessed mitochondria located in close proximity to lysosomes with mitochondria present in the cellular debris located within autophagosomes, increased levels of lipofuscin suggest that impairments in mitochondrial turnover may occur following proteasome inhibition. Taken together, these data demonstrate that proteasome inhibition dramatically alters specific aspects of neural mitochondrial homeostasis and alters lysosomal-mediated degradation of mitochondria with both of these alterations potentially contributing to aging and age-related disease in the nervous system.

Role of the proteasome in protein oxidation and neural viability following low-level oxidative stress.

Numerous studies suggest that proteasome inhibition may play a causal role in mediating the increased levels of protein oxidation and neuron death observed in conditions associated with oxidative stress. In the present study we demonstrate that administration of non-toxic levels of oxidative stress does not result in impairment of 20S/26S proteasome activity, and actually increases the expression of specific proteasome subunits. Non-toxic levels of oxidative stress were observed to elevate the amount of protein oxidation in the presence of preserved proteasomal function, suggesting that proteasome inhibition may not mediate increases in protein oxidation following low-level oxidative stress. Preserving basal proteasome function appears to be critical to preventing the neurotoxicity of low-level oxidative stress, based on the ability of proteasome inhibitor treatment to exacerbate oxidative stress toxicity. Taken together, these data indicate that maintaining neural proteasome function may be critical to preventing neurotoxicity, but not the increase in protein oxidation, following low-level oxidative stress.

Characterization of chronic low-level proteasome inhibition on neural homeostasis.

Increasing evidence suggests that proteasome inhibition plays a causal role in promoting the neurodegeneration and neuron death observed in multiple disorders, including Alzheimer's disease (AD) and Parkinson's disease (PD). The ability of severe and acute inhibition of proteasome function to induce neuron death and neuropathology similar to that observed in AD and PD is well documented. However, at present the effects of chronic low-level proteasome inhibition on neural homeostasis has not been elucidated. In order to determine the effects of chronic low-level proteasome inhibition on neural homeostasis, we conducted studies in individual colonies of neural SH-SY5Y cells that were isolated following continual exposure to low concentrations (100 nm) of the proteasome inhibitor MG115. Clonal cell lines appeared morphologically similar to control cultures but exhibited significantly different rates of both proliferation and differentiation. Elevated levels of protein oxidation and protein insolubility were observed in clonal cell lines, with all clonal cell lines being more resistant to neural death induced by serum withdrawal and oxidative stress. Interestingly, clonal cell lines demonstrated evidence for increased macroautophagy, suggesting that chronic low-level proteasome inhibition may cause an excessive activation of the lysosomal system. Taken together, these data indicate that chronic low-level proteasome inhibition has multiple effects on neural homeostasis, and suggests that studying the effects of chronic low-level proteasome inhibition may be useful in understanding the relationship between protein oxidation, protein insolubility, proteasome function, macroautophagy and neural viability in AD and PD.

Polyglutamine expansion, protein aggregation, proteasome activity, and neural survival.

Huntington's disease (HD) is one of eight established triplet repeat neurodegenerative disorders, which are collectively caused by the genetic expansion of polyglutamine repeats. While the mechanism(s) by which polyglutamine expansion causes neurodegeneration in each of these disorders is being intensely investigated, the underlying cause of polyglutamine toxicity has not been fully elucidated. A number of studies have focused on the potential role of protein aggregation and disruption of the proteasome proteolytic pathway in polyglutamine-mediated neurodegeneration. However, at present it is not clear whether polyglutamine-mediated protein aggregation is sufficient to induce cell death, nor has it been clearly determined whether proteasome inhibition precedes, coincides, or occurs as the result of the formation of polyglutamine-associated protein aggregation. To address these important components of polyglutamine toxicity, in the present study we utilized neural SH-SY5Y cells stably transfected with polyglutamine-green fluorescent protein constructs to examine the effects of polyglutamine expansion on protein aggregation, proteasome activity, and neural cell survival. Data from the present study demonstrate that polyglutamine expansion does not dramatically impair proteasome activity or elevate protein aggregate formation under basal conditions, but does significantly impair the ability of the proteasome to respond to stress, and increases stress-induced protein aggregation following stress, all in the absence of neural cell death.