p75NTR ectodomain is a physiological neuroprotective molecule against amyloid-beta toxicity in the brain of Alzheimer's disease.

In Alzheimer's disease (AD), neurodegenerative signals such as amyloid-beta (Aß) and the precursors of neurotrophins, outbalance neurotrophic signals, causing synaptic dysfunction and neurodegeneration. The neurotrophin receptor p75 (p75NTR) is a receptor of Aß and mediates Aß-induced neurodegenerative signals. The shedding of its ectodomain from the cell surface is physiologically regulated; however, the function of the diffusible p75NTR ectodomain (p75ECD) after shedding remains largely not known. Here, we show that p75ECD levels in cerebrospinal fluid and in the brains of Alzheimer's patients and amyloid-beta precursor protein (APP)/PS1 transgenic mice were significantly reduced, due to inhibition of the sheddase-tumor necrosis factor-alpha-converting enzyme by Aß. Restoration of p75ECD to the normal level by brain delivery of the gene encoding human p75ECD before or after Aß deposition in the brain of APP/PS1 mice reversed the behavioral deficits and AD-type pathologies, such as Aß deposit, apoptotic events, neuroinflammation, Tau phosphorylation and loss of dendritic spine, neuronal structures and synaptic proteins. Furthermore, p75ECD can also reduce amyloidogenesis by suppressing ß-secretase expression and activities. Our data demonstrate that p75ECD is a physiologically neuroprotective molecule against Aß toxicity and would be a novel therapeutic target and biomarker for AD.

Preventing activation of receptor for advanced glycation endproducts in Alzheimer's disease.

Receptor for advanced glycation endproducts (RAGE), a member of the immunoglobulin superfamily, is a multi-ligand, cell surface receptor expressed by neurons, microglia, astrocytes, cerebral endothelial cells, pericytes, and smooth muscle cells. At least three major types of the RAGE isoforms (full length, C-truncated, and N-truncated) are present in human brains as a result of alternative splicing. Differential expression of each isoform may play a regulatory role in the physiological and pathophysiological functions of RAGE. Analysis of RAGE expression in non-demented and Alzheimer's disease (AD) brains indicated that increases in RAGE protein and percentage of RAGE-expressing microglia paralleled the severity of disease. Ligands for RAGE in AD include amyloid beta peptide (Abeta), S100/calgranulins, advanced glycation endproduct-modified proteins, and amphoterin. Collective evidence from in vitro and in vivo studies supports that RAGE plays multiple roles in the pathogenesis of AD. The major features of RAGE activation in contributing to AD result from its interaction with Abeta, from the positive feedback mechanisms driven by excess amounts of Abeta, and combined with sustained elevated RAGE expression. The adverse consequences of RAGE interaction with Abeta include perturbation of neuronal properties and functions, amplification of glial inflammatory responses, elevation of oxidative stress and amyloidosis, increased Abeta influx at the blood brain barrier and vascular dysfunction, and induction of autoantibodies. In this article, we will review recent advances of RAGE and RAGE activation based on findings from cell cultures, animal models, and human brains. The potential for targeting RAGE mechanisms as therapeutic strategies for AD will be discussed.

What happens to microglial TREM2 in Alzheimer's disease: Immunoregulatory turned into immunopathogenic?

Microglia play major roles in initiation, coordination and execution of innate immunity in the brain. In the adult brain, these include maintenance of homeostasis, neuron and tissue repair, and eliminating infectious agents, apoptotic cells, and misfolded proteins. Some of these activities are accompanied by inflammatory reactions; and others are performed with no inflammatory effects. Under normal conditions, triggering receptor expressed on myeloid cells 2 (TREM2) belongs to the second category. It pairs with the adaptor protein DNAX-activating protein of 12kDa (DAP12) to induce phagocytosis of apoptotic neurons without inflammatory responses, and to regulate Toll-like receptor-mediated inflammatory responses, and microglial activation. Although ligands for TREM2 are largely unknown, the mitochondrial heat shock protein 60, expressed on cell surface of apoptotic neurons, is a specific ligand that activates TREM2-mediated phagocytosis by microglia. TREM2 also phagocytoses amyloid beta peptide in cultured cells. Several TREM2 mutations have been identified recently that increase the risk of Alzheimer's disease, Frontotemporal dementia, Parkinson's disease, and amyotrophic lateral sclerosis. Some of these mutations cause impaired proteolysis of full-length TREM2 at the plasma membrane to different degrees. The defects in the intramembrane cleavage result in dysfunction of phagocytosis signaling. The association of TREM2 mutations with neurodegenerative disease also calls for the understanding of the biology and pathological role of non-mutated TREM2 on human brains and microglia. This review provides a summary of current literature in TREM2 and DAP12 from several aspects, and proposes a theory that loss of TREM2 functions might contribute to the immunopathogenic role of microglia in Alzheimer's disease.

Expression of suppressor of cytokine signaling genes in human elderly and Alzheimer's disease brains and human microglia.

Multiple cellular systems exist to prevent uncontrolled inflammation in brain tissues; the suppressor of cytokine signaling (SOCS) proteins have key roles in these processes. SOCS proteins are involved in restricting cellular signaling pathways by enhancing the degradation of activated receptors and removing the stimuli for continued activation. There are eight separate SOCS genes that code for proteins with similar structures and properties. All SOCS proteins can reduce signaling of activated transcription factors Janus kinase (JAK) and signal transducer and activator of transcription (STAT), but they also regulate many other signaling pathways. SOCS-1 and SOCS-3 have particular roles in regulating inflammatory processes. Chronic inflammation is a key feature of the pathology present in Alzheimer's disease (AD)-affected brains resulting from responses to amyloid plaques or neurofibrillary tangles, the pathological hallmarks of AD. The goal of this study was to examine SOCS gene expression in human non-demented (ND) and AD brains and in human brain-derived microglia to determine if AD-related pathology resulted in a deficit of these critical molecules. We demonstrated that SOCS-1, SOCS-2, SOCS-3 and cytokine-inducible SH2 containing protein (CIS) mRNA expression was increased in amyloid beta peptide (Aß)- and inflammatory-stimulated microglia, while SOCS-6 mRNA expression was decreased by both types of treatments. Using human brain samples from the temporal cortex from ND and AD cases, SOCS-1 through SOCS-7 and CIS mRNA and SOCS-1 through SOCS-7 protein could be detected constitutively in ND and AD human brain samples. Although, the expression of key SOCS genes did not change to a large extent as a result of AD pathology, there were significantly increased levels of SOCS-2, SOCS-3 and CIS mRNA and increased protein levels of SOCS-4 and SOCS-7 in AD brains. In summary, there was no evidence of a deficit of these key inflammatory regulating proteins in aged or AD brains.

Inflammation, A beta deposition, and neurofibrillary tangle formation as correlates of Alzheimer's disease neurodegeneration.

We evaluated entorhinal cortex and superior frontal gyrus for hallmarks of Alzheimer's disease (AD) pathology, including inflammation, in three patient sets: AD patients, nondemented elderly patients with few or no neurofibrillary tangles (NFTs) and amyloid beta peptide (A beta) deposits, i.e. normal controls (NC), and nondemented elderly patients with profuse entorhinal cortex NFTs and neocortical A beta deposits, i.e. high pathology controls (HPC). Membrane attack complex (C5b-9) immunoreactivity and immune activation of microglia (MHCII expression) were used as general markers for inflammation. Compared to NC patients, AD patients exhibited significant cortical synapse loss, A beta deposition, NFT formation, and inflammation. HPC patients also had significantly elevated A beta deposition and NFT formation, but there was no evidence of synapse loss and little or no evidence of inflammation. Across patients and brain regions the measures of inflammation each accounted for significant percentages of the variance in synaptophysin immunoreactivity and each was more highly correlated with synapse estimates than NFT formation or A beta deposition.

Modeling microglial activation in Alzheimer's disease with human postmortem microglial cultures.

Alzheimer's disease (AD) is a uniquely human disorder. Despite intense research, the lack of availability of model systems has hindered AD studies though in recent years transgenic mouse models have been produced, which develop AD-like amyloid beta peptide (Abeta) plaques. For the study of inflammatory changes in AD brains, these transgenic mice may have limitations due to differences in the innate immune system of humans and rodents. Many studies of inflammatory processes in AD have focused on the role of activated microglia. Over the last 8 years, our research has focused on the properties of human microglia cultured from brain tissues of AD and non-demented (ND) individuals. As these are the cells observed to be activated in AD tissues, they represent a useful system for modeling the inflammatory components of AD. In this review, we summarize data by our group and others on the use of microglia for AD-related inflammatory research, with emphasis on results using human postmortem brain microglia. A range of products have been shown to be produced by human postmortem microglia, both constitutively and in response to treatment with Abeta, including proinflammatory cytokines such as interleukin (IL)-1beta, IL-6, tumor necrosis factor (TNF) alpha, and macrophage colony stimulating factor (M-CSF), along with complement proteins, especially C1q, superoxide radicals and neurotoxic factors. In our studies, we have demonstrated that there was a significant difference between AD and ND microglia in terms of their secretion of M-CSF and C1q. We also discuss the role of putative Abeta microglial receptors, particular recent data showing a role for the receptor for advanced glycation endproducts (RAGE) in mediating the responses of human microglia to Abeta. Finally, our studies on the use of an Abeta spot paradigm to model microglia interactions with plaques demonstrated that many of the features of AD inflammation can be modeled with postmortem brain derived microglia.

Characterization of glial cultures from rapid autopsies of Alzheimer's and control patients.

We have developed isolated and mixed cultures of microglia, astrocytes, and oligodendrocytes from rapid (mean of 2 h 55 min) autopsies of nondemented elderly patients and patients with Alzheimer's disease, Parkinson's disease, and multiple sclerosis. Cultures were derived from both the corpus callosum (CC) and superior frontal gyrus (SFG). Cultured microglia phagocytosed latex beads, were reactive for Dil-acetylated low density lipoprotein, were immunoreactive for CD68 and major histocompatibility complex II markers, and were not immunoreactive for fibroblast, astrocyte, or oligodendrocyte markers. Cultured astrocytes included fibrous and protoplasmic types, were immunoreactive for GFAP, and were not immunoreactive for fibroblast, microglia, or oligodendrocyte markers. Cultured oligodendrocytes were poorly adherent, were slow to develop, were immunoreactive for galactocerebroside, and were not immunoreactive for fibroblast, microglia, or astrocyte markers. Because they are readily manipulated under controlled experimental conditions, and because they permit immediate access to individual cells and sets of cells from patients who have actually suffered the disease, these cultures may provide an important new tool for unravelling the etiology and pathogenesis of human CNS disorders.

Gene expression profiling of amyloid beta peptide-stimulated human post-mortem brain microglia.

Activation of microglia is a central part of the chronic inflammatory processes in Alzheimer's disease (AD). In the brains of AD patients, activated microglia are associated with amyloid beta (Abeta) peptide plaques. A number of previous studies have shown that aggregated synthetic Abeta peptide activates cultured microglia to produce a range inflammatory products. The full extent of the inflammatory response still remains to be determined. In this study, gene array technology was employed to investigate in a more extensive manner the consequences of microglial activation by Abeta peptide. RNA was prepared from pooled samples of cortical human microglia isolated from post-mortem cases and incubated with a low dose (2.5 microM) of Abeta1-42 (or peptide solvent) for 24 h. This material was used to prepare cDNA probes, which were used to detect the differential pattern of expressed genes on a 1,176 Clontech membrane gene array. Results obtained showed that 104 genes were either upregulated or downregulated by 1.67 fold or greater. The most highly induced genes belonged to the chemokine family with interleukin-8 (IL-8) expression being increased by 11.7 fold. Interestingly, many of the highly induced genes had been identified as being responsive to activation by the transcription factor NF-kappaB. A number of genes were downregulated. Thymosin beta, prothymosin alpha and parathymosin, all belonging to the same gene family, were downregulated. To validate these semi-quantitative results, the expression of intercellular adhesion molecule-1 (ICAM-1) and rhoB were measured by RT-PCR in samples of cDNA derived from Abeta and control stimulated human cortical microglia. These results confirm the usefulness of the gene array approach for studying Abeta-mediated inflammatory processes.

Inflammation and Alzheimer's disease pathogenesis.

Appreciation of the role that inflammatory mediators play in Alzheimer's disease (AD) pathogenesis continues to be hampered by two related misconceptions. The first is that to be pathogenically significant a neurodegenerative mechanism must be primary. The second is that inflammation merely occurs to clear the detritis of already existent pathology. The present review addresses these issues by showing that 1) inflammatory molecules and mechanisms are uniquely present or significantly elevated in the AD brain, 2) inflammation may be a necessary component of AD pathogenesis, 3) inflammation may be sufficient to cause AD neurodegeneration, and 4) retrospective and direct clinical trials suggest a therapeutic benefit of conventional antiinflammatory medications in slowing the progress or even delaying the onset of AD.

Molecular and cellular characterization of the membrane attack complex, C5b-9, in Alzheimer's disease.

The membrane attack complex, C5b-9, is of considerable importance in many inflammatory reactions. It is the terminal, cytolytic component of both classical and alternative pathway activation, and its presence presupposes other potentially destructive complement constituents, including anaphylotoxins and opsonins. We have characterized C5b-9 and its C9 constituent in the Alzheimer's disease (AD) and nondemented elderly (ND) brain using immunohistochemistry at the light and electron microscopic levels, Western blot analysis, and the reverse transcriptase polymerase chain reaction. We have also conducted in vitro ELISA assays of amyloid beta-peptide-stimulated SC5b-9 production. C5b-9 is abundantly present in Alzheimer's disease cortex, associated with neurofibrillary tangle containing neurons, dystrophic neurites within neuritic plaques, and neuropil threads, but is weakly detected, if at all, in nondemented elderly cortex under the same conditions. Staining of Alzheimer's disease sections is abolished both by deletion of primary antibody or preabsorption with purified SC5b-9.

Association cortex, cerebellum, and serum concentrations of C1q and factor B in Alzheimer's disease.

Concentrations of C1q, the first subcomponent of the classical complement pathway, were assayed by Western blot analysis of sera and brain homogenates from Alzheimer's disease (AD) and nondemented (ND) control patients. Immunoreactive serum C1q concentrations did not differ in the two groups, whereas AD superior frontal gyrus exhibited nearly 4-fold more immunoreactive C1q than ND superior frontal gyrus. Cerebellar C1q concentrations were significantly lower than those in superior frontal gyrus, and ND cerebellar C1q was lowest of all. Parallel immunohistochemical experiments showed a linkage between the extent of beta-amyloid immunoreactivity or AD pathology in a structure and the extent of C1q immunoreactivity. These data support and extend the hypothesis that complement mediated processes are related to beta-amyloid deposition and may be involved in the pathogenesis of AD.

Microglial chemotaxis, activation, and phagocytosis of amyloid beta-peptide as linked phenomena in Alzheimer's disease.

Microglia are widely held to play important pathophysiologic roles in Alzheimer's disease (AD). On exposure to amyloid beta peptide (A beta) they exhibit chemotactic, phagocytic, phenotypic and secretory responses consistent with scavenger cell activity in a localized inflammatory setting. Because AD microglial chemotaxis, phagocytosis, and secretory activity have common, tightly linked soluble intermediaries (e.g., cytokines, chemokines), cell surface intermediaries (e.g., receptors, opsonins), and stimuli (e.g., highly inert A beta deposits and exposed neurofibrilly tangles), the mechanisms for microglial clearance of A beta are necessarily coupled to localized inflammatory mechanisms that can be cytotoxic to nearby tissue. This presents a critical dilemma for strategies to remove A beta by enhancing micoglial activation--a dilemma that warrants substantial further investigation.

Neural tract tracing using Di-I: a review and a new method to make fast Di-I faster in human brain.

The use of Di-I in tract-tracing is briefly reviewed and a novel delayed-fixation approach to neural tract-tracing in the postmortem human adult brain is reported. Using the new approach, fast Di-I, a highly lipophilic fluorescent dye was injected into a particular region or nucleus and labelled tracts were followed for distances of some 20-40 mm. The procedure required approximately 36 h, yielding dye penetration rates of 1.0 mm/h or more. This contrasts with previous Di-I, silver impregnation, and horseradish peroxidase protocols, where the tracer penetration rate is typically 0.003 mm/h or less, and the distance traversed amounts to only a few mm even after months of incubation. The new method hinges on the simple consideration that aldehyde fixation, which is normally employed prior to administration of the marker, crosslinks membrane proteins and impedes dye diffusion. The short postmortem samples used in our protocol permit delaying fixation until after the dye has had time to penetrate, dramatically increasing the length and scope of neural circuits that can be traced. Using these methods, for example, we have confirmed the presence of an ipsilateral olivocerebellar climbing fiber projection in the human.

An immunohistochemical and fine-structural analysis of peptidergic hypothalamic neurosecretory axon regeneration into the leptomeninges of the rat.

Regeneration of severed hypothalamic peptidergic neurosecretory axons into the ventral pia-arachnoid was observed in rats at the light microscopic and fine-structural levels. A temporal increase occurred in the number of neurophysin-positive axons regenerating into the leptomeninges for distances up to 3.3 mm by 40 days post-lesioning. A consistent pattern of parallel, meshed and clustered axons, occurring either singly or in bundles, was present within the connective tissue, while plexus and bundles were observed in association with leptomeningeal blood vessels. Axons were characterized by preterminal and terminal dilatations. Neurosecretory granulated vesicles occurred throughout axons. The presence of microvesicles at contact points with basal lamina suggests the possibility of hormone release. Most axons were arranged as fascicles associated closely with basal lamina-bounded support cells whose thin lamellar processes wrapped single axons or fascicles of axons. We conclude, therefore, that cellular and intercellular leptomeningeal microenvironments support and sustain the growth and regeneration of transected neurosecretory axons.

Consequences of Aberrant Insulin Regulation in the Brain: Can Treating Diabetes be Effective for Alzheimer's Disease.

There is an urgent need for new ways to treat Alzheimer's disease (AD), the most common cause of dementia in the elderly. Current therapies are modestly effective at treating the symptoms, and do not significantly alter the course of the disease. Over the years, a range of epidemiological and experimental studies have demonstrated interactions between diabetes mellitus and AD. As both diseases are leading causes of morbidity and mortality in the elderly and are frequent co-morbid conditions, it has raised the possibility that treating diabetes might be effective in slowing AD. This is currently being attempted with drugs such as the insulin sensitizer rosiglitazone. These two diseases share many clinical and biochemical features, such as elevated oxidative stress, vascular dysfunction, amyloidogenesis and impaired glucose metabolism suggesting common pathogenic mechanisms. The main thrust of this review will be to explore the evidence from a pathological point of view to determine whether diabetes can cause or exacerbate AD. This was supported by a number of animal models of AD that have been shown to have enhanced pathology when diabetic conditions were induced. The one drawback in linking diabetes and insulin to AD has been the postmortem studies of diabetic brains demonstrating that AD pathology was not increased; in fact decreased pathology has often been reported. In addition, diabetes induces its own distinct features of neuropathology different from AD. There are common pathological features to be considered including vascular abnormalities, a major feature arising from diabetes; there is increasing evidence that vascular abnormalities can contribute to AD. The most important common mechanism between insulin-resistant (type II) diabetes and AD could be impaired insulin signaling; a form of toxic amyloid can damage neuronal insulin receptors and affect insulin signaling and cell survival. It has even been suggested that AD could be considered as "type 3 diabetes" since insulin can be produced in brain. Another common feature of diabetes and AD are increased advanced glycation endproduct-modified proteins are found in diabetes and in the AD brain; the receptor for advanced glycation endproducts plays a prominent role in both diseases. In addition, a major role for insulin degrading enzyme in the degradation of Aß peptide has been identified. Although clinical trials of certain types of diabetic medications for treatment of AD have been conducted, further understanding the common pathological processes of diabetes and AD are needed to determine whether these diseases share common therapeutic targets.

Investigations with cultured human microglia on pathogenic mechanisms of Alzheimer's disease and other neurodegenerative diseases.

Inflammation-mediated mechanisms for human neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease (PD) have evolved from being on the fringe of medical hypotheses to mainstream thinking. Pioneering immunopathology studies with human brain tissues identified microglia associated with neuropathologic hallmarks of these diseases. As activated macrophages were known to produce many potential toxic products, this gave rise to the hypothesis that activated microglia (brain resident macrophages) could be contributing to the degeneration of key target neurons in these diseases, as well as potential vascular dysfunction. Studies with microglia derived from different sources, including human brains, have confirmed that activated microglia can mediate neuronal cell death. Based on these theories, a number of human clinical trials with antiinflammatory agents have been carried out on AD patients. Results to date have indicated a lack of effectiveness at slowing disease progression and have begun to cast doubt on the significance of inflammation in AD. It has been shown recently that activating microglia through immunization of amyloid plaque-developing mice with amyloid beta peptide (Abeta) has promise as a therapeutic strategy and despite some setbacks, has potential as a treatment for AD patients. This article will consider experimental data with microglia to determine whether the additional targets need to be investigated. The use of human microglia cultures, in particular those derived from elderly diseased human brains, offers an experimental system that can closely model the cell type activated in human neurodegenerative diseases. Experimental data produced by our laboratory and others is reviewed to determine the contribution of this unique experimental model to understanding disease mechanisms and possibly discovering new therapeutic targets.

Fine structural characteristics of neurophysin-positive perivascular plexus that develop in the rat hypothalamus following interruption of the hypothalamo-neurohypophysial tract.

Transection of neurosecretory axons of the hypothalamo-neurohypophysial tract within the hypothalamus by stereotactic grafts of various tissues or knife cuts induced the development of neurophysin-positive plexus around arterioles, venules and capillaries in the vicinity of these grafts or cuts. These plexus ranged from single axons to densely woven networks and tended to increase progressively with time after experimental intervention. At the fine structural level, typical neurosecretory axon profiles were either abutting the perivascular connective tissue space or located within it. They were usually accompanied by astrocyte processes or microglial cells. Many of these axons had extensive contact with the surrounding basal lamina at which point clusters of microvesicles reminiscent of axon terminals in the neural lobe were present.

Neurosecretory axon regeneration into intrahypothalamic neural lobe allografts: neurophysin immunohistochemistry and fine structure.

Neural lobe allografts placed stereotactically into the hypothalamo-neurohypophysial tract between the paraventricular and supraoptic nuclei were investigated between 5 and 70 days post-transplantation (dpt). They contained temporally increasing numbers of neurophysin-positive axons. At the fine structural level, endogenous neurosecretory axons had virtually disappeared from successful (vascularized) grafts by 5 dpt. At this time, single host neurosecretory axons and especially numerous growth cones were associated with pituicyte processes and/or scalloped basal lamina scaffolds. Axon terminals containing neurosecretory granulated vesicles and microvesicles were present only occasionally at 5 dpt but became much more numerous subsequently. These terminals were associated with pituicytes and abutted the parenchymal basal lamina of pericapillary connective tissue spaces. In addition, beginning at 10 dpt, neurolemmocyte-like cells were associated with neurosecretory axons. At 70 dpt, the fine structural characteristics of grafted neural lobes were virtually indistinguishable from those of intact controls, except for the presence of occasional areas of more extensive connective tissue, nonfenestrated capillaries and neurolemmocyte-like cells.

Peptidergic neurosecretory axons regenerate into sciatic nerve autografts in the rat hypothalamus.

Pieces of intact or degenerated sciatic nerves autografted into contact with transected neurosecretory axons within the hypothalamus were invaded by neurophysin-positive axons. With increasing time after grafting, increasing numbers of axons were present in both types of grafts, but grafts of degenerated sciatic nerves always contained more axons. At the fine-structural level typical neurosecretory as well as nonneurosecretory axons were usually associated with basal lamina-enclosed neurolemmocyte processes; occasional axons occurred among collagen fibrils or within basal lamina scaffolds. Profiles with the fine structural characteristics of axon terminals were present by 20 days after transplantation.