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We describe a Rh(I) catalyzed asymmetric ring opening of racemic vinyl cyclopropanes using aryl boronic acids as C-nucleophiles. When ferrocene-based chiral bisphosphines are used as ligands, the products are obtained with regioselectivities typically 99:1 r.r. and ee's generally between 88 and 96%. A wide range of aryl boronic acids can be used, and the products can be converted into a variety of targets. Preliminary mechanistic studies indicate that Zn(OTf) 2 plays a significant role in the reaction by promoting rhodium-ligand complex formation and accelerating the reaction. We expect this method and these mechanistic insights to be useful in the development of new asymmetric methods.

Asymmetric Suzuki-Miyaura cross-couplings with aryl boronic acids and allylic electrophiles are a powerful method to convert racemic mixtures into enantioenriched products. Currently, enantioconvergent allylic arylations are limited to substrates that are symmetrical about the allylic unit, and the absence of strategies to control regio-, E/Z- and enantioselectivity in acyclic allylic systems is a major restriction. Here, using a system capable of either conjugate addition or allylic arylation, we have discovered the structural features and experimental conditions that allow an acyclic system to undergo chemo- and regioselective, enantioconvergent allylic Suzuki-Miyaura-type arylation. A wide variety of boronic acid coupling partners can be used, and both alkyl and aromatic substituents are tolerated on the allylic unit so that a wide variety of structures can be obtained. Preliminary mechanistic studies reveal that the chelating ability of the ester group is crucial to obtaining high regio- and enantioselectivity. Using this method, we were able to synthesize the natural products (S)-curcumene and (S)-4,7-dimethyl-1-tetralone and the clinically used antidepressant sertraline (Zoloft)., (© 2024. The Author(s).)

Huntington's disease (HD) is caused by an expanded CAG trinucleotide repeat in exon 1 of the huntingtin ( HTT ) gene. We report the design of a series of HTT pre-mRNA splicing modulators that lower huntingtin (HTT) protein, including the toxic mutant huntingtin (mHTT), by promoting insertion of a pseudoexon containing a premature termination codon at the exon 49-50 junction. The resulting transcript undergoes nonsense-mediated decay, leading to a reduction of HTT mRNA transcripts and protein levels. The starting benzamide core was modified to pyrazine amide and further optimized to give a potent, CNS-penetrant, and orally bioavailable HTT -splicing modulator 27 . This compound reduced canonical splicing of the HTT RNA exon 49-50 and demonstrated significant HTT-lowering in both human HD stem cells and mouse BACHD models. Compound 27 is a structurally diverse HTT -splicing modulator that may help understand the mechanism of adverse effects such as peripheral neuropathy associated with branaplam.

Germline defects affecting the DNA-binding domain of the transcription factor FLI1 are associated with a bleeding disorder that is characterized by the presence of large, fused α-granules in platelets. We investigated whether the genes showing abnormal expression in FLI1-deficient platelets could be involved in platelet α-granule biogenesis by undertaking transcriptome analysis of control platelets and platelets harboring a DNA-binding variant of FLI1. Our analysis identified 2,276 transcripts that were differentially expressed in FLI1-deficient platelets. Functional annotation clustering of the coding transcripts revealed significant enrichment for gene annotations relating to protein transport, and identified Sorting nexin 24 (SNX24) as a candidate for further investigation. Using an induced pluripotent stem cell-derived megakaryocyte model, SNX24 expression was found to be increased during the early stages of megakaryocyte differentiation and downregulated during proplatelet formation, indicating tight regulatory control during megakaryopoiesis. CRISPR-Cas9 mediated knockout (KO) of SNX24 led to decreased expression of immature megakaryocyte markers, CD41 and CD61, and increased expression of the mature megakaryocyte marker CD42b (P=0.0001), without affecting megakaryocyte polyploidisation, or proplatelet formation. Electron microscopic analysis revealed an increase in empty membrane-bound organelles in SNX24 KO megakaryocytes, a reduction in α-granules and an absence of immature and mature multivesicular bodies, consistent with a defect in the intermediate stage of α-granule maturation. Co-localization studies showed that SNX24 associates with each compartment of α-granule maturation. Reduced expression of CD62P and VWF was observed in SNX24 KO megakaryocytes. We conclude that SNX24 is required for α-granule biogenesis and intracellular trafficking of α-granule cargo within megakaryocytes.

Zoonotic pathogens, such as COVID-19, reside in animal hosts before jumping species to infect humans. The Carnivora, like mink, carry many zoonoses, yet how diversity in host immune genes across species affect pathogen carriage is poorly understood. Here, we describe a progressive evolutionary downregulation of pathogen-sensing inflammasome pathways in Carnivora. This includes the loss of nucleotide-oligomerization domain leucine-rich repeat receptors (NLRs), acquisition of a unique caspase-1/-4 effector fusion protein that processes gasdermin D pore formation without inducing rapid lytic cell death, and the formation of a caspase-8 containing inflammasome that inefficiently processes interleukin-1β. Inflammasomes regulate gut immunity, but the carnivorous diet has antimicrobial properties that could compensate for the loss of these immune pathways. We speculate that the consequences of systemic inflammasome downregulation, however, can impair host sensing of specific pathogens such that they can reside undetected in the Carnivora., Competing Interests: Declaration of interests N.K. and S.W. are employees of Genentech, Z.D. is employed by Nodthera, L.J.H. is employed by Wren Pharmaceuticals, and S.J.W is employed by Cell Press. C.E.B. serves on the Scientific Advisory Board of Lightcast, NodThera, and Related Sciences and consults for Janssen., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Infection of human macrophages with Salmonella enterica serovar Typhimurium ( S. Typhimurium) leads to inflammasome activation. Inflammasomes are multiprotein complexes facilitating caspase-1 activation and subsequent gasdermin D-mediated cell death and IL-1β and IL-18 cytokine release. The NAIP/NLRC4 inflammasome is activated by multiple bacterial protein ligands, including flagellin from the flagellum and the needle protein PrgI from the S. Typhimurium type III secretion system. In this study, we show that transfected ultrapure flagellin from S Typhimurium induced cell death and cytokine secretion in THP-1 cells and primary human monocyte-derived macrophages. In THP-1 cells, NAIP/NLRC4 and NLRP3 played redundant roles in inflammasome activation during infection with S. Typhimurium. Knockout of NAIP or NLRC4 in THP-1 cells revealed that flagellin, but not PrgI, now activated the NLRP3 inflammasome through a reactive oxygen species- and/or cathepsin-dependent mechanism that was independent of caspase-4/5 activity. In conclusion, our data suggest that NLRP3 can be activated by flagellin to act as a "safety net" to maintain inflammasome activation under conditions of suboptimal NAIP/NLRC4 activation, as observed in THP-1 cells, possibly explaining the redundant role of NLRP3 and NAIP/NLRC4 during S. Typhimurium infection., (Copyright © 2021 The Authors.)

Pattern recognition receptors (PRRs) expressed in antigen-presenting cells are thought to shape pathogen-specific immunity by inducing secretion of costimulatory cytokines during T-cell activation, yet data to support this notion in vivo are scarce. Here, we show that the cytosolic PRR Nod-like Receptor CARD 4 (NLRC4) suppresses, rather than facilitates, effector and memory CD4 + T-cell responses against Salmonella in mice. NLRC4 negatively regulates immunological memory by preventing delayed activation of the cytosolic PRR NLR pyrin domain 3 (NLRP3) that would otherwise amplify the production of cytokines important for the generation of Th1 immunity such as intereukin-18. Consistent with a role for NLRC4 in memory immunity, primary challenge with Salmonella expressing flagellin modified to largely evade NLRC4 recognition notably increases protection against lethal rechallenge. This finding suggests flagellin modification to reduce NLRC4 activation enhances protective immunity, which could have important implications for vaccine development against flagellated microbial pathogens.

Copy number variation (CNV) is known to cause all von Willebrand disease (VWD) types, although the associated pathogenic mechanisms involved have not been extensively studied. Notably, in-frame CNV provides a unique opportunity to investigate how specific von Willebrand factor (VWF) domains influence the processing and packaging of the protein. Using multiplex ligation-dependent probe amplification, this study determined the extent to which CNV contributed to VWD in the Molecular and Clinical Markers for the Diagnosis and Management of Type 1 von Willebrand Disease cohort, highlighting in-frame deletions of exons 3, 4-5, 32-34, and 33-34. Heterozygous in vitro recombinant VWF expression demonstrated that, although deletion of exons 3, 32-34, and 33-34 all resulted in significant reductions in total VWF (P < .0001, P < .001, and P < .01, respectively), only deletion of exons 3 and 32-34 had a significant impact on VWF secretion (P < .0001). High-resolution microscopy of heterozygous and homozygous deletions confirmed these observations, indicating that deletion of exons 3 and 32-34 severely impaired pseudo-Weibel-Palade body (WPB) formation, whereas deletion of exons 33-34 did not, with this variant still exhibiting pseudo-WPB formation similar to wild-type VWF. In-frame deletions in VWD, therefore, contribute to pathogenesis via moderate or severe defects in VWF biosynthesis and secretion., (© 2020 by The American Society of Hematology.)

In humans, the majority of sustained traumatic brain injuries (TBIs) are classified as 'mild' and most often a result of a closed head injury (CHI). The effects of a non-penetrating CHI are not benign and may lead to chronic pathology and behavioral dysfunction, which could be worsened by repeated head injury. Clinical-neuropathological correlation studies provide evidence that conversion of tau into abnormally phosphorylated proteotoxic intermediates (p-tau) could be part of the pathophysiology triggered by a single TBI and enhanced by repeated TBIs. However, the link between p-tau and CHI in rodents remains controversial. To address this question experimentally, we induced a single CHI or two CHIs to WT or rTg4510 mice. We found that 2× CHI increased tau phosphorylation in WT mice and rTg4510 mice. Behavioral characterization in WT mice found chronic deficits in the radial arm water maze in 2× CHI mice that had partially resolved in the 1× CHI mice. Moreover, using Manganese-Enhanced Magnetic Resonance Imaging with R1 mapping - a novel functional neuroimaging technique - we found greater deficits in the rTg4510 mice following 2× CHI compared to 1× CHI. To integrate our findings with prior work in the field, we conducted a systematic review of rodent mild repetitive CHI studies. Following Prisma guidelines, we identified 25 original peer-reviewed papers. Results from our experiments, as well as our systematic review, provide compelling evidence that tau phosphorylation is modified by experimental mild TBI studies; however, changes in p-tau levels are not universally reported. Together, our results provide evidence that repetitive TBIs can result in worse and more persistent neurological deficits compared to a single TBI, but the direct link between the worsened outcome and elevated p-tau could not be established., Competing Interests: Declaration of Competing Interest No competing financial interests exist., (Copyright © 2020. Published by Elsevier Inc.)

With the advent of large-scale next-generation sequencing initiatives, there is an increasing importance to interpret and understand the potential phenotypic influence of identified genetic variation and its significance in the human genome. Bioinformatics analyses can provide useful information to assist with variant interpretation. This review provides an overview of tools/resources currently available, and how they can help predict the impact of genetic variation at the deoxyribonucleic acid, ribonucleic acid, and protein level., Competing Interests: None., (Thieme Medical Publishers 333 Seventh Avenue, New York, NY 10001, USA.)

Interleukin-1 alpha (IL-1α) is a powerful cytokine that modulates immunity, and requires canonical cleavage by calpain for full activity. Mature IL-1α is produced after inflammasome activation and during cell senescence, but the protease cleaving IL-1α in these contexts is unknown. We show IL-1α is activated by caspase-5 or caspase-11 cleavage at a conserved site. Caspase-5 drives cleaved IL-1α release after human macrophage inflammasome activation, while IL-1α secretion from murine macrophages only requires caspase-11, with IL-1β release needing caspase-11 and caspase-1. Importantly, senescent human cells require caspase-5 for the IL-1α-dependent senescence-associated secretory phenotype (SASP) in vitro, while senescent mouse hepatocytes need caspase-11 for the SASP-driven immune surveillance of senescent cells in vivo. Together, we identify IL-1α as a novel substrate of noncanonical inflammatory caspases and finally provide a mechanism for how IL-1α is activated during senescence. Thus, targeting caspase-5 may reduce inflammation and limit the deleterious effects of accumulated senescent cells during disease and Aging., (© 2019 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.)

Thymic stromal lymphopoietin (TSLP) is a functionally pleotropic cytokine important in immune regulation, and TSLP dysregulation is associated with numerous diseases. TSLP is produced by many cell types, but has predominantly been characterized as a secreted factor from epithelial cells which activates dendritic cells (DC) that subsequently prime T helper (T H ) 2 immunity. However, DC themselves make significant amounts of TSLP in response to microbial products, but the functional role of DC-derived TSLP remains unclear. We show that TSLPR signaling negatively regulates IL-1β production during dectin-1 stimulation of human DC. This regulatory mechanism functions by dampening Syk phosphorylation and is mediated via NADPH oxidase-derived ROS, HIF-1α and pro-IL-1β expression. Considering the profound effect TSLPR signaling has on the metabolic status and the secretome of dectin-1 stimulated DC, these data suggest that autocrine TSLPR signaling could have a fundamental role in modulating immunological effector responses at sites removed from epithelial cell production of TSLP.

Since the concept of the inflammasome was introduced by Martinon, Burns and Tschopp in 2002, there has been an exponential increase in our understanding of how inflammasomes (caspase activating molecular platforms) regulate innate inflammatory responses to infectious microorganisms. Advances in understanding inflammasome biology have been developed using a range of bacterial pathogens. Recent studies investigating inflammasome responses during Chlamydia infection have provided interesting mechanistic insights in to inflammasome activation during intracellular bacterial infection. This review highlights new concepts regulating inflammasome activation to bacterial infections including: interferon-regulated loss of compartmentalisation, mechanisms of canonical and non-canonical inflammasome activation and their relevance to Chlamydia infections are discussed., (Copyright © 2017. Published by Elsevier Masson SAS.)

The innate immune system is a critical component of host defence against microbial pathogens, but effective responses require an ability to distinguish between infectious and non-infectious insult to prevent inappropriate inflammation. Using the important obligate intracellular human pathogen Chlamydia trachomatis; an organism that causes significant immunopathology, we sought to determine critical host and pathogen factors that contribute to the induction of inflammasome activation. We assayed inflammasome activation by immunoblotting and ELISA to detect IL-1β processing and LDH release to determine pyroptosis. Using primary murine bone marrow derived macrophages or human monocyte derived dendritic cells, infected with live or attenuated Chlamydia trachomatis we report that the live organism activates both canonical and non-canonical inflammasomes, but only canonical inflammasomes controlled IL-1β processing which preceded pyroptosis. NADPH oxidase deficient macrophages were permissive to Chlamydia trachomatis replication and displayed elevated type-1 interferon and inflammasome activation. Conversely, attenuated, non-replicating Chlamydia trachomatis, primed but did not activate inflammasomes and stimulated reduced type-1 interferon responses. This suggested bacterial replication or metabolism as important factors that determine interferon responses and inflammasome activation. We identified STING but not cGAS as a central mediator of interferon regulated inflammasome activation. Interestingly, exogenous delivery of a Chlamydia trachomatis metabolite and STING ligand-cyclic di-AMP, recovered inflammasome activation to attenuated bacteria in a STING dependent manner thus indicating that a bacterial metabolite is a key factor initiating inflammasome activation through STING, independent of cGAS. These data suggest a potential mechanism of how the innate immune system can distinguish between infectious and non-infectious insult and instigate appropriate immune responses that could be therapeutically targeted.

Protein kinase RNA activated (PKR) is a crucial mediator of anti-viral responses but is reported to be activated by multiple non-viral stimuli. However, mechanisms underlying PKR activation, particularly in response to bacterial infection, remain poorly understood. We have investigated mechanisms of PKR activation in human primary monocyte-derived dendritic cells in response to infection by Chlamydia trachomatis. Infection resulted in potent activation of PKR that was dependent on TLR4 and MyD88 signalling. NADPH oxidase was dispensable for activation of PKR as cells from chronic granulomatous disease (CGD) patients, or mice that lack NADPH oxidase activity, had equivalent or elevated PKR activation. Significantly, stimulation of cells with endoplasmic reticulum (ER) stress-inducing agents resulted in potent activation of PKR that was blocked by an inhibitor of IRE1α RNAse activity. Crucially, infection resulted in robust IRE1α RNAse activity that was dependent on TLR4 signalling and inhibition of IRE1α RNAse activity prevented PKR activation. Finally, we demonstrate that TLR4/IRE1α mediated PKR activation is required for the enhancement of interferon-β production following C. trachomatis infection. Thus, we provide evidence of a novel mechanism of PKR activation requiring ER stress signalling that occurs as a consequence of TLR4 stimulation during bacterial infection and contributes to inflammatory responses., (Copyright © 2016 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Thymic stromal lymphopoietin (TSLP) produced by epithelial cells acts on dendritic cells (DCs) to drive differentiation of TH 2-cells, and is therefore important in allergic disease pathogenesis. However, DCs themselves make significant amounts of TSLP in response to microbial products, but little is known about the key downstream signals that induce and modulate this TSLP secretion from human DCs. We show that human monocyte derived DC (mDC) secretion of TSLP in response to Candida albicans and β-glucans requires dectin-1, Syk, NF-κB, and p38 MAPK signaling. In addition, TSLP production by mDCs is greatly enhanced by IL-1β, but not TNF-α, in contrast to epithelial cells. Furthermore, TSLP secretion is significantly increased by signals emanating from the endoplasmic reticulum (ER) stress response, specifically the unfolded protein response sensors, inositol-requiring transmembrane kinase/endonuclease 1 and protein kinase R-like ER kinase, which are activated by dectin-1 stimulation. Thus, TSLP production by mDCs requires the integration of signals from dectin-1, the IL-1 receptor, and ER stress signaling pathways. Autocrine TSLP production is likely to play a role in mDC-controlled immune responses at sites removed from epithelial cell production of the cytokine, such as lymphoid tissue., (© 2015 The Authors. European Journal of Immunology published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Introduction: Neuropathological, genetic, and biochemical studies have provided support for the hypothesis that microglia participate in Alzheimer's disease (AD) pathogenesis. Despite the extensive characterization of AD microglia, there are still many unanswered questions, and little is known about microglial morphology in other common forms of age-related dementia: particularly, dementia with Lewy bodies (DLB) and hippocampal sclerosis of aging (HS-Aging). In addition, no prior studies have attempted to compare and contrast the microglia morphology in the hippocampus of various neurodegenerative conditions., Results: Here we studied cases with pathologically-confirmed AD (n = 7), HS-Aging (n = 7), AD + HS-aging (n = 4), DLB (n = 12), and normal (cognitively intact) controls (NC) (n = 9) from the University of Kentucky Alzheimer's Disease Center autopsy cohort. We defined five microglia morphological phenotypes in the autopsy samples: ramified, hypertrophic, dystrophic, rod-shaped, and amoeboid. The Aperio ScanScope digital neuropathological tool was used along with two well-known microglial markers: IBA1 (a marker for both resting and activated microglia) and CD68 (a lysosomal marker in macrophages/microglia associated with phagocytic cells). Hippocampal staining analyses included studies of subregions within the hippocampal formation and nearby white matter. Using these tools and methods, we describe variation in microglial characteristics that show some degree of disease specificity, including, (1) increased microglia density and number in HS-aging and AD + HS-aging; (2) low microglia density in DLB; (3) increased number of dystrophic microglia in HS-aging; and (4) increased proportion of dystrophic to all microglia in DLB., Conclusions: We conclude that variations in morphologies among microglial cells, and cells of macrophage lineage, can help guide future work connecting neuroinflammatory mechanisms with specific neurodegenerative disease subtypes.

Epidemiological studies have associated increased risk of Alzheimer's disease (AD)-related clinical symptoms with a medical history of head injury. Currently, little is known about pathophysiology mechanisms linked to this association. Persistent neuroinflammation is one outcome observed in patients after a single head injury. Neuroinflammation is also present early in relevant brain regions during AD pathology progression. In addition, previous mechanistic studies in animal models link neuroinflammation as a contributor to neuropathology and cognitive impairment in traumatic brain injury (TBI) or AD-related models. Therefore, we explored the potential interplay of neuroinflammatory responses in TBI and AD by analysis of the temporal neuroinflammatory changes after TBI in an AD model, the APP/PS1 knock-in (KI) mouse. Discrete temporal aspects of astrocyte, cytokine, and chemokine responses in the injured KI mice were delayed compared with the injured wild-type mice, with a peak neuroinflammatory response in the injured KI mice occurring at 7 d after injury. The neuroinflammatory responses were more persistent in the injured KI mice, leading to a chronic neuroinflammation. At late time points after injury, KI mice exhibited a significant impairment in radial arm water maze performance compared with sham KI mice or injured wild-type mice. Intervention with a small-molecule experimental therapeutic (MW151) that selectively attenuates proinflammatory cytokine production yielded improved cognitive behavior outcomes, consistent with a link between neuroinflammatory responses and altered risk for AD-associated pathology changes with head injury., (Copyright © 2015 the authors 0270-6474/15/356554-16$15.00/0.)

The first kinase inhibitor drug approval in 2001 initiated a remarkable decade of tyrosine kinase inhibitor drugs for oncology indications, but a void exists for serine/threonine protein kinase inhibitor drugs and central nervous system indications. Stress kinases are of special interest in neurological and neuropsychiatric disorders due to their involvement in synaptic dysfunction and complex disease susceptibility. Clinical and preclinical evidence implicates the stress related kinase p38αMAPK as a potential neurotherapeutic target, but isoform selective p38αMAPK inhibitor candidates are lacking and the mixed kinase inhibitor drugs that are promising in peripheral tissue disease indications have limitations for neurologic indications. Therefore, pursuit of the neurotherapeutic hypothesis requires kinase isoform selective inhibitors with appropriate neuropharmacology features. Synaptic dysfunction disorders offer a potential for enhanced pharmacological efficacy due to stress-induced activation of p38αMAPK in both neurons and glia, the interacting cellular components of the synaptic pathophysiological axis, to be modulated. We report a novel isoform selective p38αMAPK inhibitor, MW01-18-150SRM (=MW150), that is efficacious in suppression of hippocampal-dependent associative and spatial memory deficits in two distinct synaptic dysfunction mouse models. A synthetic scheme for biocompatible product and positive outcomes from pharmacological screens are presented. The high-resolution crystallographic structure of the p38αMAPK/MW150 complex documents active site binding, reveals a potential low energy conformation of the bound inhibitor, and suggests a structural explanation for MW150's exquisite target selectivity. As far as we are aware, MW150 is without precedent as an isoform selective p38MAPK inhibitor or as a kinase inhibitor capable of modulating in vivo stress related behavior.

Background: Evidence from clinical studies and preclinical animal models suggests that proinflammatory cytokine overproduction is a potential driving force for pathology progression in traumatic brain injury (TBI). This raises the possibility that selective targeting of the overactive cytokine response, a component of the neuroinflammation that contributes to neuronal dysfunction, may be a useful therapeutic approach. MW151 is a CNS-penetrant, small molecule experimental therapeutic that selectively restores injury- or disease-induced overproduction of proinflammatory cytokines towards homeostasis. We previously reported that MW151 administered post-injury (p.i.) is efficacious in a closed head injury (CHI) model of diffuse TBI in mice. Here we test dose dependence of MW151 to suppress the target mechanism (proinflammatory cytokine up-regulation), and explore the therapeutic window for MW151 efficacy., Methods: We examined suppression of the acute cytokine surge when MW151 was administered at different times post-injury and the dose-dependence of cytokine suppression. We also tested a more prolonged treatment with MW151 over the first 7 days post-injury and measured the effects on cognitive impairment and glial activation., Results: MW151 administered up to 6 h post-injury suppressed the acute cytokine surge, in a dose-dependent manner. Administration of MW151 over the first 7 days post-injury rescues the CHI-induced cognitive impairment and reduces glial activation in the focus area of the CHI., Conclusions: Our results identify a clinically relevant time window post-CHI during which MW151 effectively restores cytokine production back towards normal, with a resultant attenuation of downstream cognitive impairment.

The calcium/calmodulin-dependent protein kinase II (CaMKII) is abundant in the brain, where it makes important contributions to synaptic organization and homeostasis, including playing an essential role in synaptic plasticity and memory. Four genes encode isoforms of CaMKII (α, β, δ, γ), with CaMKIIα and CaMKIIβ highly expressed in the brain. Decades of molecular and cellular research, as well as the use of a large number of CaMKIIα mutant mouse lines, have provided insight into the pivotal roles of CaMKIIα in brain plasticity and cognition. However, less is known about the CaMKIIβ isoform. We report the development and extensive behavioral and phenotypic characterization of a CaMKIIβ knockout (KO) mouse. The CaMKIIβ KO mouse was found to be smaller at weaning, with an altered body mass composition. The CaMKIIβ KO mouse showed ataxia, impaired forelimb grip strength, and deficits in the rotorod, balance beam and running wheel tasks. Interestingly, the CaMKIIβ KO mouse exhibited reduced anxiety in the elevated plus maze and open field tests. The CaMKIIβ KO mouse also showed cognitive impairment in the novel object recognition task. Our results provide a comprehensive behavioral characterization of mice deficient in the β isoform of CaMKII. The neurologic phenotypes and the construction of the genotype suggest the utility of this KO mouse strain for future studies of CaMKIIβ in brain structure, function and development.

The goal of this review is to discuss how behavioral tests in mice relate to the pathological and neuropsychological features seen in human Alzheimer's disease (AD), and present a comprehensive analysis of the temporal progression of behavioral impairments in commonly used AD mouse models that contain mutations in amyloid precursor protein (APP). We begin with a brief overview of the neuropathological changes seen in the AD brain and an outline of some of the clinical neuropsychological assessments used to measure cognitive deficits associated with the disease. This is followed by a critical assessment of behavioral tasks that are used in AD mice to model the cognitive changes seen in the human disease. Behavioral tests discussed include spatial memory tests [Morris water maze (MWM), radial arm water maze (RAWM), Barnes maze], associative learning tasks (passive avoidance, fear conditioning), alternation tasks (Y-Maze/T-Maze), recognition memory tasks (Novel Object Recognition), attentional tasks (3 and 5 choice serial reaction time), set-shifting tasks, and reversal learning tasks. We discuss the strengths and weaknesses of each of these behavioral tasks, and how they may correlate with clinical assessments in humans. Finally, the temporal progression of both cognitive and non-cognitive deficits in 10 AD mouse models (PDAPP, TG2576, APP23, TgCRND8, J20, APP/PS1, TG2576 + PS1 (M146L), APP/PS1 KI, 5×FAD, and 3×Tg-AD) are discussed in detail. Mouse models of AD and the behavioral tasks used in conjunction with those models are immensely important in contributing to our knowledge of disease progression and are a useful tool to study AD pathophysiology and the resulting cognitive deficits. However, investigators need to be aware of the potential weaknesses of the available preclinical models in terms of their ability to model cognitive changes observed in human AD. It is our hope that this review will assist investigators in selecting an appropriate mouse model, and accompanying behavioral paradigms to investigate different aspects of AD pathology and disease progression.

Introduction: Mutations in proteolipid protein (PLP), the most abundant myelin protein in the CNS, cause the X-linked dysmyelinating leukodystrophies, Pelizaeus-Merzbacher disease (PMD) and spastic paraplegia type 2 (SPG2). Point mutations, deletion, and duplication of the PLP1 gene cause PMD/SPG2 with varying clinical presentation. Deletion of an intronic splicing enhancer (ISEdel) within intron 3 of the PLP1 gene is associated with a mild form of PMD. Clinical and preclinical studies have indicated that mutations in myelin proteins, including PLP, can induce neuroinflammation, but the temporal and spatial onset of the reactive glia response in a clinically relevant mild form of PMD has not been defined., Methods: A PLP-ISEdel knockin mouse was used to examine the behavioral and neuroinflammatory consequences of a deletion within intron 3 of the PLP gene, at two time points (two and four months old) early in the pathological progression. Mice were characterized functionally using the open field task, elevated plus maze, and nesting behavior. Quantitative neuropathological analysis was for markers of astrocytes (GFAP), microglia (IBA1, CD68, MHCII) and axons (APP). The Aperio ScanScope was used to generate a digital, high magnification photomicrograph of entire brain sections. These digital slides were used to quantify the immunohistochemical staining in ten different brain regions to assess the regional heterogeneity in the reactive astrocyte and microglial response., Results: The PLP-ISEdel mice exhibited behavioral deficits in the open field and nesting behavior at two months, which did not worsen by four months of age. A marker of axonal injury (APP) increased from two months to four months of age. Striking was the robust reactive astrocyte and microglia response which was also progressive. In the two-month-old mice, the astrocyte and microglia reactivity was most apparent in white matter rich regions of the brain. By four months of age the gliosis had become widespread and included both white as well as gray matter regions of the brain., Conclusions: Our results indicate, along with other preclinical models of PMD, that an early reactive glia response occurs following mutations in the PLP gene, which may represent a potentially clinically relevant, oligodendrocyte-independent therapeutic target for PMD.

Introduction: Despite the extensive mechanistic and pathological characterization of the amyloid precursor protein (APP)/presenilin-1 (PS-1) knock-in mouse model of Alzheimer's disease (AD), very little is known about the AD-relevant behavioral deficits in this model. Characterization of the baseline behavioral performance in a variety of functional tasks and identification of the temporal onset of behavioral impairments are important to provide a foundation for future preclinical testing of AD therapeutics. Here we perform a comprehensive behavioral characterization of this model, discuss how the observed behavior correlates with the mechanistic and pathological observations of others, and compare this model with other commonly used AD mouse models., Methods: FOUR DIFFERENT GROUPS OF MICE RANGING ACROSS THE LIFESPAN OF THIS MODEL (TEST GROUPS: 7, 11, 15, and 24 months old) were run in a behavioral test battery consisting of tasks to assess motor function (grip strength, rotor rod, beam walk, open field ambulatory movement), anxiety-related behavior (open field time spent in peripheral zone vs. center zone, elevated plus maze), and cognitive function (novel object recognition, radial arm water maze)., Results: There were no differences in motor function or anxiety-related behavior between APP/PS-1 knock-in mice and wild-type counterpart mice for any age group. Cognitive deficits in both recognition memory (novel object recognition) and spatial reference memory (radial arm water maze) became apparent for the knock-in animals as the disease progressed., Conclusion: This is the first reported comprehensive behavioral analysis of the APP/PS1 knock-in mouse model of AD. The lack of motor/coordination deficits or abnormal anxiety levels, coupled with the age/disease-related cognitive decline and high physiological relevance of this model, make it well suited for utilization in preclinical testing of AD-relevant therapeutics.

The onset of parturition is associated with a number of proinflammatory mediators that are themselves regulated by the nuclear factor κB (NF-κB) family of transcription factors. In this context, we previously reported that the RelA NF-κB subunit represses transcription and mRNA expression of the proquiescent Gαs gene in human myometrial cells following stimulation with the proinflammatory cytokine TNF. In the present study, we initially defined the functional consequence of this on myometrial contractility. Here we show that, contrary to our initial expectations, TNF did not induce myometrial contractility but did inhibit the relaxation produced by the histone deacetylase inhibitor trichostatin A, an effect that in turn was abolished by the NF-κB inhibitor N(4)-[2-(4-phenoxyphenyl)ethyl]-4,6-quinazolinediamine. This result suggested a role for TNF in regulating Gαs expression via activating NF-κB and modifying histone acetylation associated with the promoter region of the gene. In this context, we show that the -837 to -618 region of the endogenous Gαs promoter is occupied by cAMP-response element-binding protein (CREB), Egr-1, and Sp1 transcription factors and that CREB-binding protein (CBP) transcriptional complexes form within this region where they induce histone acetylation, resulting in increased Gαs expression. TNF, acting via NF-κB, did not change the levels of CREB, Sp1, or Egr-1 binding to the Gαs promoter, but it induced a significant reduction in the level of CBP. This was associated with increased levels of histone deacetylase-1 and surprisingly an increase in H4K8 acetylation. The latter is discussed herein.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder that gradually destroys a person's memory. Substantial evidence suggests that amyloid beta (Aβ) and the receptor for advanced glycation endproducts (RAGE) play an important and often deleterious role in the pathogenesis of AD. RAGE facilitates the translocation of Aβ from the periphery into the brain, mediates the Aβ-induced neurotoxicity, and enhances the release of pro-inflammatory cytokines increasing the inflammatory response. In addition, soluble forms of RAGE (sRAGE) and Aβ bind together in the periphery forming high molecular weight complexes that are more highly immunogenic and less neurotoxic than Aβ1-42 alone. We show here that there are elevated anti-RAGE and anti-Aβ titers (in a near 1:1 relationship) in samples analyzed from human AD patients, aged non-human primates, and AD transgenic mice (APPSWE-PS1). We show that an in vitro prepared RAGE/Aβ complex induces a greater immunogenic response (increased anti-Aβ1-42 and anti-RAGE antibody titers) in both human peripheral blood mononuclear cells (PBMCs) and immunized Balb-C mice than does either Aβ1-42 or RAGE alone. Further, pretreatment with endogenous anti-RAGE antibodies isolated from our transgenic APPSWE-PS1 mice can prevent Aβ1-42-induced neurotoxicity in cultured primary rat cortical neurons. Finally, we examine the effectiveness of an orally administered vaccine of either RAGE/Aβ complex or Aβ1-42 alone in improving cognitive function in our AD transgenic mice. Our results to date support the hypothesis that a protein complex vaccine that targets both RAGE and Aβ1-42 will provide a more effective treatment for AD than vaccination with Aβ1-42 alone.

Background: Dimebolin (latrepirdine), a compound with multiple potential drug targets, is being evaluated in clinical trials for the treatment of Alzheimer's disease (AD) and preliminary results suggest it can slow the disease process. There is also evidence that dimebolin directly improves aspects of cognition. Here we examined the acute effect of dimebolin on components of working memory in non-human primates, young adult (11-17 years old) and aged (20-31 years old) rhesus macaques., Experimental Approach: The effects of dimebolin (3.9-118 µg kg(-1)) on working memory, as measured by performance on delayed matching-to-sample (DMTS), were examined in the normal young adult monkeys and aged adult monkeys. All the monkeys studied were proficient in the performance of a computer-assisted DMTS task. In a subsequent experiment in the same subjects, dimebolin was administered 15 min before a cognitively-impairing dose (20 µg kg(-1)) of scopolamine., Key Results: In both the young adult and aged monkeys, dimebolin significantly increased the DMTS task accuracies. In young adults, the task improvement was associated with long (retention/retrieval) delay trials, and a protracted enhancement was observed for sessions run 24 h post administration of a single dose. Dimebolin did not significantly attenuate the scopolamine-induced impairment. In the aged monkeys, dimebolin significantly improved the reduced task accuracies associated with long delay intervals., Conclusions and Implications: Here we demonstrated that dimebolin is able to improve components of working memory in monkeys and to induce a protracted response for at least 24 h after administration of a single dose., (© 2011 Georgia Health Sciences University. British Journal of Pharmacology © 2011 The British Pharmacological Society.)

An unfortunate result of the rapid rise in geriatric populations worldwide is the increasing prevalence of age-related cognitive disorders such as Alzheimer's disease (AD). AD is a devastating neurodegenerative illness that is characterized by a profound impairment of cognitive function, marked physical disability, and an enormous economic burden on the afflicted individual, caregivers, and society in general. The rise in elderly populations is also resulting in an increase in individuals with related (potentially treatable) conditions such as "Mild Cognitive Impairment" (MCI) which is characterized by a less severe (but abnormal) level of cognitive impairment and a high-risk for developing dementia. Even in the absence of a diagnosable disorder of cognition (e.g., AD and MCI), the perception of increased forgetfulness and declining mental function is a clear source of apprehension in the elderly. This is a valid concern given that even a modest impairment of cognitive function is likely to be associated with significant disability in a rapidly evolving, technology-based society. Unfortunately, the currently available therapies designed to improve cognition (i.e., for AD and other forms of dementia) are limited by modest efficacy and adverse side effects, and their effects on cognitive function are not sustained over time. Accordingly, it is incumbent on the scientific community to develop safer and more effective therapies that improve and/or sustain cognitive function in the elderly allowing them to remain mentally active and productive for as long as possible. As diagnostic criteria for memory disorders evolve, the demand for pro-cognitive therapeutic agents is likely to surpass AD and dementia to include MCI and potentially even less severe forms of memory decline. The purpose of this review is to provide an overview of the contemporary therapeutic targets and preclinical pharmacologic approaches (with representative drug examples) designed to enhance memory function., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Peripheral blood monocytes represent the rapid response component of mononuclear phagocyte host defense, generating vigorous but finite antibacterial responses. We investigated the fate of highly purified primary human monocytes following phagocytosis of different bacteria. Exposure to high bacterial loads resulted in rapid loss of cell viability and decreased functional competence. Cell death typically involved classical apoptosis. Exposure to high numbers of Escherichia coli and Klebsiella pneumoniae induced nonapoptotic death with loss of cell membrane integrity, marked disruption of phagolysosomes, and caspase-1 activation, while a subset of cells also released caspase-1-regulated extracellular traps. Classical apoptosis increased if extracellular bacterial replication was reduced and decreased if intracellular ATP levels were reduced during these infections. Both classical apoptosis and the alternative forms of cell death allowed monocytes, whose functional competence was exhausted, to downregulate reactive oxygen species and proinflammatory cytokine responses. In contrast, sustained stimulation of glycolytic metabolism and mitochondrial oxidative phosphorylation, with associated hypoxia inducible factor-1alpha upregulation, maintained intracellular ATP levels and prolonged monocyte functional longevity, as assessed by maintenance of phagocytosis, reactive oxygen species production, and proinflammatory cytokine generation. Monocyte innate responses to bacteria are short-lived and are limited by an intrinsic program of apoptosis, a response that is subverted by overwhelming infection with E. coli and K. pneumoniae or bacterial stimulation of cell metabolism. In this regard, the fate of monocytes following bacterial challenge more closely resembles neutrophils than macrophages.

The immunoglobulins (IgGs) for beta amyloid (Abeta) and receptors for the advanced glycation end products (RAGE) have previously been shown to be related to memory and language measures in a mixed neurological sample of older adults. In this study, we examined group differences in Abeta and RAGE IgGs, as well as the relationship between both IgGs and cognitive performance in nondiabetic older adults with normal cognition, mild cognitive impairment (MCI), and probable Alzheimer's disease (AD). We found RAGE and Abeta levels to be elevated in some AD participants, leading to significant AD-control group differences. While there was an overall correlation between both IgG levels and global cognition across all three groups, this relationship was largely attributable to group differences in cognition, highlighted by considerable variability within groups in the relationship between IgG levels and cognition. While findings do not support a consistent relationship between cognition and either IgG, further research with larger samples is needed to better characterize cognitive differences between AD participants with high versus low Abeta and RAGE titers.

Introduction: The alpha(2)-adrenergic receptor agonist clonidine was examined for its ability to improve working memory in monkeys., Materials and Methods: Clonidine (0.116-34.8 microg/kg) was administered to six pigtail macaques in their performance of a computer-assisted delayed matching-to-sample (DMTS) task., Results and Discussion: During DMTS sessions initiated 1 hour after dosing, there was a slight improvement in mean task accuracy (long delay trials; 0.116-microg/kg). On the following day, there was continued and added improvement in accuracies associated with the long delay trials. On the day following 1.16-microg/kg, the entire memory retention curve was shifted to the right of vehicle. When the animals were again tested 48 hours after dosing (no pretreatment), these two patterns of task enhancement were continued and enhanced. Mean task accuracy associated with long delay trials was significantly increased by 14.2% trials correct when animals were originally treated with 0.116-microg/kg of clonidine. Mean task accuracy associated with medium delay trials was significantly increased by 11.8% trials correct when animals were treated with 1.16-microg/kg. On the sixth day after clonidine, task accuracies were still significantly improved during medium delay trials after 0.116-microg/kg. Median sample and choice latencies were not significantly influenced by clonidine treatment. These findings are consistent with the ability of clonidine to induce a protracted improvement in aspects of working memory., Conclusion: Early (attentional) and late (retention) components of memory appeared to be differentially sensitive to the dose of clonidine. Central alpha(2)-adrenergic receptors should be considered legitimate drug targets for future compound development for cognition enhancement.

Rationale: The scopolamine-reversal model is enjoying a resurgence of interest in clinical studies as a reversible pharmacological model for Alzheimer's disease (AD). The cognitive impairment associated with scopolamine is similar to that in AD. The scopolamine model is not simply a cholinergic model, as it can be reversed by drugs that are noncholinergic cognition-enhancing agents., Objectives: The objective of the study was to determine relevance of computer-assisted operant-conditioning tasks in the scopolamine-reversal model in rats and monkeys., Materials and Methods: Rats were evaluated for their acquisition of a spatial reference memory task in the Morris water maze. A separate cohort was proficient in performance of an automated delayed stimulus discrimination task (DSDT). Rhesus monkeys were proficient in the performance of an automated delayed matching-to-sample task (DMTS)., Results: The AD drug donepezil was evaluated for its ability to reverse the decrements in accuracy induced by scopolamine administration in all three tasks. In the DSDT and DMTS tasks, the effects of donepezil were delay (retention interval)-dependent, affecting primarily short delay trials. Donepezil produced significant but partial reversals of the scopolamine-induced impairment in task accuracies after 2 mg/kg in the water maze, after 1 mg/kg in the DSDT, and after 50 microg/kg in the DMTS task., Conclusions: The two operant-conditioning tasks (DSDT and DMTS) provided data most in keeping with those reported in clinical studies with these drugs. The model applied to nonhuman primates provides an excellent transitional model for new cognition-enhancing drugs before clinical trials.

JWB1-84-1 is one of 50 tertiary amine analogs of choline synthesized with expectation that they would be high potency compounds for cytoprotection. As one of the more potent analogs in this regard, JWB1-84-1, a piperazine derivative, was selected for testing as a cognition-enhancing agent. The compound was evaluated for efficacy in Alzheimer's disease transgenic mice (B6C3-Tg(APPswe, PSEN1dE9)85Dbo/J). A separate cohort of mice (AD Tg) were first subjected to a behavioral test battery in which the transgenic strain was compared with the wild-type strain. AD Tg mice were shown to exhibit specific deficits in the acquisition of a working memory (5-trial/session radial arm water maze, RAWM) task at a time when the animals exhibited maximal cerebral amyloid burden. JWB1-84-1 produced a dose-dependent decrease in the number of errors made by well trained AD-Tg mice the RAWM task that was maximal after the 20 microg/kg dose. Aged macaques (20-32 y) were trained to proficiency in their performance of a computer-assisted delayed matching-to-sample task. Vehicle (normal saline) or JWB1-84-1 (5-150 microg/kg, i.m.) was administered 10 min before the initiating of testing. On average, JWB1-84-1 treatment significantly improved task accuracy after all but the lowest dose. The maximal degree of improvement was attained after animals received the 100 microg/kg dose. The drug's effects were restricted primarily to Medium and Long delay trials - the most difficult portions of the task, which were improved by up to 18% above control. In young macaques JWB1-84-1 treatment also significantly reversed the decrements in task accuracy associated with the random presentation of a task distractor. Thus JWB1-84-1exhibits the potential for treating the cognitive symptoms associated with neurodegenerative diseases and attention deficit disorders. Its cytoprotective action might also work to slow the progression of Alzheimer's disease.

Interleukin-8/CXCL8 (IL-8) is a chemokine and angiogenic factor. Recently, IL-8 was identified as an autocrine growth factor in several human cancers. Here, we investigated the expression and function of IL-8 in lung cancer cells. The expressions of IL-8 and its receptors, CXCR1 and CXCR2, were examined in a panel of non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC) cell lines. Using reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay, we found that all NSCLC cell lines tested produced modest or high levels of IL-8 (up to 51 ng ml(-1) 10(6) cells(-1)). Expression of CXCR1 and CXCR2 was found by RT-PCR and flow cytometry in two out of three cell lines. In contrast, SCLC cell lines produced very low or undetectable levels of IL-8, but expressed CXCR1 and CXCR2. We next investigated whether IL-8 could act as an autocrine growth factor in two NSCLC cell lines (H460 and MOR/P) expressing both IL-8 and its receptors. We found that cell proliferation was attenuated by anti-IL-8 neutralising antibody to 71 and 76% in H460 and MOR/P, respectively (P<0.05). Exogenous IL-8 significantly stimulated cell proliferation in four SCLC cell lines tested in a dose-dependent fashion. Cell proliferation was increased by between 18% (P<0.05) and 37% (P<0.05). Stimulation of cell proliferation by IL-8 was also demonstrated by analysis of proliferating cell nuclear antigen expression and cell cycle in H69 cells. Furthermore, we investigated which receptor(s) mediated the mitogenic function of IL-8 in lung cancer cells. We found that cell proliferation was significantly reduced by anti-CXCR1 antibody but not by anti-CXCR2 antibody. In conclusion, IL-8 can act as an autocrine and/or paracrine growth factor for lung cancer cells, and the mitogenic function of IL-8 in lung cancer is mediated mainly by CXCR1 receptor.