The nucleotide addition cycle of RNA polymerase is controlled by two molecular hinges in the Bridge Helix domain

Abstract Background Cellular RNA polymerases (RNAPs) are complex molecular machines that combine catalysis with concerted conformational changes in the active center. Previous work showed that kinking of a hinge region near the C-terminus of the Bridge Helix (BH-HC) plays a critical role in controlling the catalytic rate. Results Here, new evidence for the existence of an additional hinge region in the amino-terminal portion of the Bridge Helix domain (BH-HN) is presented. The nanomechanical properties of BH-HN emerge as a direct consequence of the highly conserved primary amino acid sequence. Mutations that are predicted to influence its flexibility cause corresponding changes in the rate of the nucleotide addition cycle (NAC). BH-HN displays functional properties that are distinct from BH-HC, suggesting that conformational changes in the Bridge Helix control the NAC via two independent mechanisms. Conclusions The properties of two distinct molecular hinges in the Bridge Helix of RNAP determine the functional contribution of this domain to key stages of the NAC by coordinating conformational changes in surrounding domains.</p

Macrophages in Alzheimer’s disease: the blood-borne identity

Alzheimer’s disease (AD) is a progressive and incurable neurodegenerative disorder clinically characterized by cognitive decline involving loss of memory, reasoning and linguistic ability. The amyloid cascade hypothesis holds that mismetabolism and aggregation of neurotoxic amyloid-β (Aβ) peptides, which are deposited as amyloid plaques, are the central etiological events in AD. Recent evidence from AD mouse models suggests that blood-borne mononuclear phagocytes are capable of infiltrating the brain and restricting β-amyloid plaques, thereby limiting disease progression. These observations raise at least three key questions: (1) what is the cell of origin for macrophages in the AD brain, (2) do blood-borne macrophages impact the pathophysiology of AD and (3) could these enigmatic cells be therapeutically targeted to curb cerebral amyloidosis and thereby slow disease progression? This review begins with a historical perspective of peripheral mononuclear phagocytes in AD, and moves on to critically consider the controversy surrounding their identity as distinct from brain-resident microglia and their potential impact on AD pathology

Neuroprotective function for ramified microglia in hippocampal excitotoxicity

<p>Abstract</p> <p>Background</p> <p>Most of the known functions of microglia, including neurotoxic and neuroprotective properties, are attributed to morphologically-activated microglia. Resting, ramified microglia are suggested to primarily monitor their environment including synapses. Here, we show an active protective role of ramified microglia in excitotoxicity-induced neurodegeneration.</p> <p>Methods</p> <p>Mouse organotypic hippocampal slice cultures were treated with <it>N</it>-methyl-D-aspartic acid (NMDA) to induce excitotoxic neuronal cell death. This procedure was performed in slices containing resting microglia or slices that were chemically or genetically depleted of their endogenous microglia.</p> <p>Results</p> <p>Treatment of mouse organotypic hippocampal slice cultures with 10-50 μM <it>N</it>-methyl-D-aspartic acid (NMDA) induced region-specific excitotoxic neuronal cell death with CA1 neurons being most vulnerable, whereas CA3 and DG neurons were affected less. Ablation of ramified microglia severely enhanced NMDA-induced neuronal cell death in the CA3 and DG region rendering them almost as sensitive as CA1 neurons. Replenishment of microglia-free slices with microglia restored the original resistance of CA3 and DG neurons towards NMDA.</p> <p>Conclusions</p> <p>Our data strongly suggest that ramified microglia not only screen their microenvironment but additionally protect hippocampal neurons under pathological conditions. Morphological activation of ramified microglia is thus not required to influence neuronal survival.</p

Absence of CD14 Delays Progression of Prion Diseases Accompanied by Increased Microglial Activation

Prion diseases are fatal neurodegenerative disorders characterized by accumulation of PrPSc, vacuolation of neurons and neuropil, astrocytosis, and microglial activation. Upregulation of gene expressions of innate immunity-related factors, including complement factors and CD14, is observed in the brains of mice infected with prions even in the early stage of infections. When CD14 knockout (CD14(-/-)) mice were infected intracerebrally with the Chandler and Obihiro prion strains, the mice survived longer than wild-type (WT) mice, suggesting that CD14 influences the progression of the prion disease. Immunofluorescence staining that can distinguish normal prion protein from the disease-specific form of prion protein (PrPSc) revealed that deposition of PrPSc was delayed in CD14(-/-) mice compared with WT mice by the middle stage of the infection. Immunohistochemical staining with Iba1, a marker for activated microglia, showed an increased microglial activation in prion-infected CD14(-/-) mice compared to WT mice. Interestingly, accompanied by the increased microglial activation, anti-inflammatory cytokines interleukin-10 (IL-10) and transforming growth factor beta (TGF-beta) appeared to be expressed earlier in prion-infected CD14(-/-) mice. In contrast, IL-1 beta expression appeared to be reduced in the CD14(-/-) mice in the early stage of infection. Double immunofluorescence staining demonstrated that CD11b- and Iba1-positive microglia mainly produced the anti-inflammatory cytokines, suggesting anti-inflammatory status of microglia in the CD14(-/-) mice in the early stage of infection. These results imply that CD14 plays a role in the disease progression by suppressing anti-inflammatory responses in the brain in the early stage of infection