Over the past decade, human genome-wide association and expression studies have strongly implicated dysregulation of the innate immune system in the pathogenesis of Alzheimer’s disease (AD). Single cell mRNA sequencing studies have identified innate immune cell subtypes that are minimally present in normal healthy brain, but whose numbers greatly increase in association with AD pathology. These AD pathology-associated immune cells are putatively the locus for the immune-related AD risk. While the prevailing view is that these immune cells arise from transformation of resident brain microglia, studies across several decades and using multiple techniques and strategies suggest instead that the pathology-associated immune cells are bone-marrow derived hematopoietic cells that are recruited into brain. We critically review this translational literature, emphasizing the strengths and limitations of techniques used to address recruitment and the experimental designs employed. We conclude that the aggregate evidence points toward recruitment into brain of innate immune cells of the myeloid dendritic cell lineage. Recruitment of dendritic cells and their role in AD pathogenesis has broad implications for our understanding of the etiology and pathobiology of AD that impact the strategies to develop new, immune system-targeted therapeutics for this devastating disease.

Cerebrovascular alterations are a key feature of Alzheimer's disease (AD) pathogenesis. However, whether vascular damage contributes to synaptic dysfunction and how it synergizes with amyloid pathology to cause neuroinflammation and cognitive decline remain poorly understood. Here, we show that the blood protein fibrinogen induces spine elimination and promotes cognitive deficits mediated by CD11b-CD18 microglia activation. 3D molecular labeling in cleared mouse and human AD brains combined with repetitive in vivo two-photon imaging showed focal fibrinogen deposits associated with loss of dendritic spines independent of amyloid plaques. Fibrinogen-induced spine elimination was prevented by inhibiting reactive oxygen species (ROS) generation or genetic ablation of CD11b. Genetic elimination of the fibrinogen binding motif to CD11b reduced neuroinflammation, synaptic deficits, and cognitive decline in the 5XFAD mouse model of AD. Thus, fibrinogen-induced spine elimination and cognitive decline via CD11b link cerebrovascular damage with immune-mediated neurodegeneration and may have important implications in AD and related conditions.

Activation of innate immunity and deposition of blood-derived fibrin in the central nervous system (CNS) occur in autoimmune and neurodegenerative diseases, including multiple sclerosis (MS) and Alzheimer’s disease (AD). However, the mechanisms that link disruption of the blood–brain barrier (BBB) to neurodegeneration are poorly understood, and exploration of fibrin as a therapeutic target has been limited by its beneficial clotting functions. Here we report the generation of monoclonal antibody 5B8, targeted against the cryptic fibrin epitope γ377–395, to selectively inhibit fibrin-induced inflammation and oxidative stress without interfering with clotting. 5B8 suppressed fibrin-induced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation and the expression of proinflammatory genes. In animal models of MS and AD, 5B8 entered the CNS and bound to parenchymal fibrin, and its therapeutic administration reduced the activation of innate immunity and neurodegeneration. Thus, fibrin-targeting immunotherapy inhibited autoimmunity- and amyloid-driven neurotoxicity and might have clinical benefit without globally suppressing innate immunity or interfering with coagulation in diverse neurological diseases. Fibrin deposition occurs after the blood–brain barrier is breached. Akassoglou and colleagues generate a therapeutic monoclonal antibody that targets a cryptic fibrin epitope to suppress activation of innate immune responses in the CNS and diminish neuroinflammation.

A novel series of tetralin containing amino imidazoles, derived from modification of the corresponding phenyl acetic acid derivatives is described. Replacement of the amide led to identification of a potent series of tetralin-amino imidazoles with robust central efficacy. The reduction of brain Aβ in guinea pigs in the absence of changes in B-cells suggested a potential therapeutic index with respect to APP processing compared with biomarkers of notch related toxicity. Optimization of the FTOC to plasma concentrations at the brain Aβ EC(50) lead to the identification of compound 14f (PF-3084014) which was selected for clinical development.

The synthesis and structure-activity relationship (SAR) of a novel series of di-substituted imidazoles, derived from modification of DAPT, are described. Subsequent optimization led to identification of a highly potent series of inhibitors that contain a β-amine in the imidazole side-chain resulting in a robust in vivo reduction of plasma and brain Aβ in guinea pigs. The therapeutic index between Aβ reductions and changes in B-cell populations were studied for compound 10 h.

This review discusses current knowledge of the complex interactions between amyloid-beta (A beta) peptide, the receptor for advanced glycation endproducts (RAGE), and inflammatory mediators, focusing on the roles of such interactions in the pathogenesis of Alzheimer's disease. As a ubiquitous cell-surface receptor, RAGE demonstrates enhanced expression in an A beta-rich environment; the effects of RAGE on microglia, the blood-brain barrier and neurons are mediated through various signaling pathways. Relevant preclinical models illustrate that the A beta-RAGE interaction amplifies neuronal stress and the accumulation of A beta, impairs memory and learning, and exaggerates neuroinflammation. These findings suggest that RAGE may mediate a common proinflammatory pathway in neurodegenerative disorders.

The neuritic plaque is a hallmark pathology of Alzheimer's disease (AD), which contains a core composed of Aβ peptide in a stacked β-pleated sheet conformation (“Aβ-amyloid”) bound by activated microglia. Amyloid cores are associated with dystrophic neurites, while non-amyloid brain Aβ deposits have little adverse effect on surrounding neuropil. As prevalence of dystrophic neurites in AD brain correlates with severity of dementia, preventing the appearance of Aβ in its amyloid conformation is a potential therapeutic strategy. Most efforts to model amyloid fibril formation in vitro initiate spontaneous fibril formation by stirring Aβ peptide under highly controlled conditions. However, the ultrastructural relationship between microglia and amyloid fibrils in AD brain suggests that microglia play an active role in the “refolding” of Aβ into amyloid. We investigated this hypothesis by adding freshly solubilized Aβ to plate-bound rat microglia, primary human monocytes and macrophages, differentiated THP-1 cells, and the IC21 mouse peritoneal macrophage line. All cell types were able to refold Aβ peptide into an amyloid conformation as demonstrated by thioflavin-S staining. This effect is specific to cells of monocytoid lineage, including macrophage-differentiated embryonic stem cells, and did not occur in neurons, undifferentiated embryonic stem cells, or H4 cells. In the absence of cells, extensive Aβ precipitates form on the tissue culture plastic (measured using the Aβ-specific antibody 4G8) but these precipitates are nearly devoid of thioflavin-S fluorescence. The amyloidogenic factor does not appear to be soluble as Aβ incubation in microglia-conditioned media does not recapitulate the effect. Transmission electron microscopy of cultures showed clusters of electron-dense fibrils in cytoplasmic channels that often terminated in clathrin-coated heads, a similar pattern to that observed in AD brain and transgenic models. These data support the hypothesis that neuritic plaque formation is an active microglia-mediated process.

The receptor for advanced glycation end products (RAGE) is a member of the immunoglobulin supergene family that can bind to a range of functionally and structurally diverse ligands. The amyloid beta (Aβ) peptides 1–40 and 1–42 are key ligands implicated in the pathology of neurodegenerative Alzheimer's disease and bind to RAGE with nanomolar affinity. Furthermore, RAGE expression is upregulated in Alzheimer's brains compared with age-matched controls. Using Translational Technology™, we have discovered small molecules that inhibit the binding of Aβ peptides to RAGE. Here we report preclinical data on the mechanisms of these small-molecule inhibitors to further characterize the role of RAGE in the pathophysiology of Alzheimer's disease. Other effects of the small-molecule inhibitors include preventing interaction of RAGE with additional RAGE ligands including S100B, HMGB1, and CML, indicating a common binding site for these ligands on RAGE. We have also demonstrated inhibition of ligand-mediated signaling in different RAGE-expressing human cell types, for example, the RAGE ligand-mediated release of cytokines from the human monocytic cell line THP-1 was attenuated by small-molecule ligands at potencies similar to the in-vitro assays. Neurotoxicity caused by treating SH-SY5Y neuroblastoma cells with micromolar concentrations of Aβ peptide was also blocked by these small molecules. PF-04494700 (formerly TTP488) is the most advanced small-molecule RAGE antagonist in development and has demonstrated efficacy in two peripheral models of inflammation, delayed type hypersensitivity, and splenic amyloid formation. Moreover, PF-04494700 achieved high brain exposure and, when dosed chronically, strongly attenuated Aβ deposition and cognitive deficits in a transgenic Aβ-depositing mouse model. These data confirm and extend previous reports implicating RAGE in the pathogenesis of Alzheimer's disease and indicate that small-molecule RAGE antagonists are a viable therapeutic avenue for Alzheimer's disease.

Microglia are thought to play an active role in the formation of neuritic plaques in Alzheimer's disease (AD). We have previously demonstrated that microglia and other cells of monocytoid lineage rapidly convert amorphous Aβ deposits into amyloid (assessed by thioflavin-S fluorescence and electron microscopy) using an in-vitro model. This study aimed to further characterize our in-vitro model of microglia-facilitated amyloidogenesis (MFA) to determine the factors regulating its activity. We therefore profiled several agents including cytokines, dexamethasone and HMG-CoA reductase inhibitors (statins). The Th2-promoting cytokines TGFβ and IL-4 both block MFA with low nanomolar potency. This effect is unlikely to be due to direct interaction with Aβ, which is present at 10 μM. The synthetic glucocorticoid receptor agonist dexamethasone also potently blocks MFA but, in contrast to TGFβ, also inhibits Aβ internalization and clearance by microglia (monitored by cell-associated Aβ immunostaining). The effect of dexamethasone on microglial interaction with Aβ is similar to the cytoskeleton-disrupting agent cytochalasin D, which also blocks both MFA and uptake/degradation of Aβ. The profile of these mechanisms in vitro may have relevance in vivo as: 1) overexpression of TGFβ in the brains of hAPP Aβ-depositing transgenic mice greatly decreases formation of parenchymal thioflavin-positive Aβ deposits, consistent with blockade of MFA in vitro; and 2) central administration of dexamethasone is reported to increase levels of brain Aβ by decreasing its clearance, consistent with the blocked clearance of Aβ by dexamethasone observed in cultured microglia. Several statins were also found to inhibit MFA. This inhibition was reversed by mevalonate, indicating involvement of the cholesterol metabolic pathway in regulation of MFA. Recent literature has indicated that chronic statin treatment may suppress the expression of key immune signaling molecules on microglia in AD brain, suggesting central immunomodulatory effects of statins. These data indicate that multiple mechanisms may contribute to MFA. Modulation of these mechanisms may prevent a microglial phenotype that leads to neuritic plaque formation.

PURPOSE:To present the findings of an outbreak of toxic anterior segment syndrome (TASS).SETTING:Six states, 7 ophthalmology surgical centers, United States.METHODS:Cases were identified through electronic communication networks and via reports to a national TASS referral center. Information on the procedure, details of instrument reprocessing, and products used during cataract surgery were also collected. Medications used during the procedures were tested for endotoxin using a kinetic assay.RESULTS:The search identified 112 case patients (median age 74 years) from 7 centers from July 19, 2005, through November 28, 2005. Common presenting clinical features included blurred vision (60%), anterior segment inflammation (49%), and cell deposition (56%). Of the patients, 100 (89%) had been exposed to a single brand of balanced salt solution manufactured by Cytosol Laboratories and distributed by Advanced Medical Optics as AMO Endosol. Two patients continued to have residual symptoms. There were no reports of significant breaches in sterile technique or instrument reprocessing. Of 14 balanced salt solution lots, 5 (35%) had levels exceeding the endotoxin limit (0.5 EU/mL). Based on these findings, the balanced salt solution product was withdrawn, resulting in a termination of the outbreak.CONCLUSIONS:This is the first known report of an outbreak of TASS caused by intrinsic contamination of a product with endotoxin. Ophthalmologists and epidemiologists should be aware of TASS and its common causes. To facilitate investigations of adverse outcomes such as TASS, those performing cataract surgeries should document the type and lot numbers of products used intraoperatively.

Postoperative cognitive dysfunction, confusion, and delirium are common after general anesthesia in the elderly, with symptoms persisting for months or years in some patients. Even middle-aged patients are likely to have postoperative cognitive dysfunction for months after surgery, and Alzheimer's disease (AD) patients appear to be particularly at risk of deterioration after anesthesia. Several investigators have thus examined whether general anesthesia is associated with AD, with some studies suggesting that exposure to anesthetics may increase the risk of AD. However, little is known on the biochemical consequences of anesthesia on pathogenic pathways in vivo. Here, we investigated the effect of anesthesia on tau phosphorylation and amyloid precursor protein (APP) metabolism in mouse brain. We found that, regardless of the anesthetic used, anesthesia induced rapid and massive hyperphosphorylation of tau, rapid and prolonged hypothermia, inhibition of Ser/Thr PP2A (protein phosphatase 2A), but no changes in APP metabolism or Abeta (beta-amyloid peptide) accumulation. Reestablishing normothermia during anesthesia completely rescued tau phosphorylation to normal levels. Our results indicate that changes in tau phosphorylation were not a result of anesthesia per se, but a consequence of anesthesia-induced hypothermia, which led to inhibition of phosphatase activity and subsequent hyperphosphorylation of tau. These findings call for careful monitoring of core temperature during anesthesia in laboratory animals to avoid artifactual elevation of protein phosphorylation. Furthermore, a thorough examination of the effect of anesthesia-induced hypothermia on the risk and progression of AD is warranted.

The peptide Buforin II (BF2) displays lethal activity toward a wide range of pathogens. Many antimicrobial peptides kill cells by disrupting membranes or forming membrane pores. In contrast, BF2 kills target cells without apparent membrane disruption. The hypothesis is that BF2 diffuses across cell membranes and binds to DNA. To understand BF2’s antimicrobial action, its membrane translocation mechanism must be elucidated. We are investigating the effects of lipid composition on BF2’s translocation using vesicle assays and molecular dynamics (MD) simulations. BF2’s ability to translocate across neutral phosphatidylcholine (PC) or negative phosphatidylglycerol (PG) membranes has been probed with an assay in which fluorescence resonance energy transfer (FRET) is used to visualize the movement of BF2 into vesicles encapsulating trypsin. A similar assay omitting trypsin is used to measure binding of BF2 to PC and PG vesicles. We have run MD simulations of BF2 with PC and PG membranes to obtain molecular-level insight into these interactions. The data show that BF2 crosses pure PG bilayers, but not PC bilayers. However, the binding curves for PG and PC are similar, indicating that BF2 binds comparably to both surfaces. Therefore, although BF2 is similarly attracted to PC and PG membranes, the negative charge of PG membranes is crucial for translocation.

There is extensive evidence that changes in immune system activation accompany the pathological changes of Alzheimer's disease (AD), but a mechanistic understanding of how the immune system actually participates in disease pathogenesis is still largely lacking. Because of the complexity of the immunological response, and the difficulty in identifying the key molecular players that underlie any given immunological response, expanding our understanding of the immunological response in AD beyond its descriptive stages has not been a straightforward exercise. The development of transgenic animals that form deposits of Abeta peptide in their brains has provided an unexpected dividend to those interested in the immunological response characterizing AD. Several of these transgenic models develop structures greatly resembling neuritic plaques, a hallmark feature of AD brain that is also a focal point of the immunological response occurring in AD. Genetic and pharmacological manipulation of these Abeta-depositing transgenic mice is providing some intriguing and unexpected insights into the role of innate and adaptive immune mechanisms in the pathogenesis of AD. This review will discuss immunological perspectives that have arisen from research using Abeta-depositing transgenic mice, and place these perspectives in the context of epidemiological and genetic studies that have previously suggested a role for the immune system in AD. The emerging story affirms the likely role of innate and adaptive immune mechanisms in the pathogenesis of AD, but provides a cautionary note as to the difficulties that are likely to face potential immunomodulatory therapies due to the dualistic beneficial and detrimental roles that immune mechanisms appear to play in AD.

The synthesis and structure-activity relationships of a series of 6-phenyl-2-aminopyridines that potently and selectively inhibit the neuronal isoform of nitric oxide synthase (nNOS) are described. Compound 14bi from this series exhibits potent in vivo activity in harmaline-induced cGMP formation in rat cerebellum, a functional model of nNOS inhibition, and in the PCP-induced hypermotility model in the rat. These results suggest that 14bi may be a useful reagent for evaluating potential therapeutic applications of nNOS inhibitors in the central nervous system.

AbstractFor Abstract see ChemInform Abstract in Full Text.

Hyperphosphorylation of microtubule-associated proteins such as tau and neurofilament may underlie the cytoskeletal abnormalities and neuronal death seen in several neurodegenerative diseases including Alzheimer's disease. One potential mechanism of microtubule-associated protein hyperphosphorylation is augmented activity of protein kinases known to associate with microtubules, such as cdk5 or GSK3beta. Here we show that tau and neurofilament are hyperphosphorylated in transgenic mice that overexpress human p25, an activator of cdk5. The p25 transgenic mice display silver-positive neurons using the Bielschowsky stain. Disturbances in neuronal cytoskeletal organization are apparent at the ultrastructural level. These changes are localized predominantly to the amygdala, thalamus/hypothalamus, and cortex. The p25 transgenic mice display increased spontaneous locomotor activity and differences from control in the elevated plus-maze test. The overexpression of an activator of cdk5 in transgenic mice results in increased cdk5 activity that is sufficient to produce hyperphosphorylation of tau and neurofilament as well as cytoskeletal disruptions reminiscent of Alzheimer's disease and other neurodegenerative diseases.