Almost all degenerative diseases of the CNS are associated with chronic inflammation. A central step in this process is the activation of brain mononuclear phagocyte cells, called microglia. While it is recognized that healthy neurons and astrocytes regulate the magnitude of microglia-mediated innate immune responses and limit excessive CNS inflammation, the endogenous signals governing this process are not fully understood. In the peripheral nervous system, recent studies suggest that an endogenous 'cholinergic anti-inflammatory pathway' regulates systemic inflammatory responses via a7 nicotinic acetylcholinergic receptors (nAChR) found on blood-borne macrophages. These data led us to investigate whether a similar cholinergic pathway exists in the brain that could regulate microglial activation. Here we report for the first time that cultured microglial cells express a7 nAChR subunit as determined by RT-PCR, western blot, immunofluorescent, and immunochistochemistry analyses. Acetylcholine and nicotine pre-treatment inhibit lipopolysaccharide (LPS)-induced TNF-a release in murine-derived microglial cells, an effect attenuated by a7 selective nicotinic antagonist, a-bungarotoxin. Furthermore, this inhibition appears to be mediated by a reduction in phosphorylation of p44/42 and p38 mitogen-activated protein kinase (MAPK). Though preliminary, our findings suggest the existence of a brain cholinergic pathway that regulates microglial activation through a7 nicotinic receptors. Negative regulation of microglia activation may also represent additional mechanism underlying nicotine's reported neuroprotective properties.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder pathologically characterized by deposition of ␤-amyloid (A␤) peptides as senile plaques in the brain. Recent studies suggest that green tea flavonoids may be used for the prevention and treatment of a variety of neurodegenerative diseases. Here, we report that (-)-epigallocatechin-3-gallate (EGCG), the main polyphenolic constituent of green tea, reduces A␤ generation in both murine neuron-like cells (N2a) transfected with the human "Swedish" mutant amyloid precursor protein (APP) and in primary neurons derived from Swedish mutant APP-overexpressing mice (Tg APP sw line 2576). In concert with these observations, we find that EGCG markedly promotes cleavage of the ␣-C-terminal fragment of APP and elevates the N-terminal APP cleavage product, soluble APP-␣. These cleavage events are associated with elevated ␣-secretase activity and enhanced hydrolysis of tumor necrosis factor ␣-converting enzyme, a primary candidate ␣-secretase. As a validation of these findings in vivo, we treated Tg APP sw transgenic mice overproducing A␤ with EGCG and found decreased A␤ levels and plaques associated with promotion of the nonamyloidogenic ␣-secretase proteolytic pathway. These data raise the possibility that EGCG dietary supplementation may provide effective prophylaxis for AD.

Background: Activated microglial cells have been implicated in a number of neurodegenerative disorders, including Alzheimer's disease (AD), multiple sclerosis (MS), and HIV dementia. It is well known that inflammatory mediators such as nitric oxide (NO), cytokines, and chemokines play an important role in microglial cell-associated neuron cell damage. Our previous studies have shown that CD40 signaling is involved in pathological activation of microglial cells. Many data reveal that cannabinoids mediate suppression of inflammation in vitro and in vivo through stimulation of cannabinoid receptor 2 (CB 2 ).

Recently, we have shown that green tea polyphenol (؊)-epigallocatechin-3-gallate (EGCG) exerts a beneficial role on reducing brain A␤ levels, resulting in mitigation of cerebral amyloidosis in a mouse model of Alzheimer disease. EGCG seems to accomplish this by modulating amyloid precursor protein (APP) processing, resulting in enhanced cleavage of the ␣-COOH-terminal fragment (␣-CTF) of APP and corresponding elevation of the NH 2 -terminal APP product, soluble APP-␣ (sAPP-␣). These beneficial effects were associated with increased ␣-secretase cleavage activity, but no significant alteration in ␤-or ␥-secretase activities. To gain insight into the molecular mechanism whereby EGCG modulates APP processing, we evaluated the involvement of three candidate ␣-secretase enzymes, a-disintegrin and metalloprotease ( Proteolytic processing of amyloid precursor protein (APP)4 to form amyloid-␤ (A␤) peptides are implicated in the pathogenesis and progression of Alzheimer disease (AD) (1-4). A␤ peptides from 40 -42 amino acids in length are constituents of senile plaques in AD brain and exhibit direct or indirect neurotoxic effects (5). Processing of APP is accomplished by enzymes known as secretases. Whereas non-amyloidogenic ␣-secretase cleavage produces the amino-terminal product named soluble APP␣ (sAPP-␣) and the carboxyl-terminal fragment (CTF) ␣-CTF (also known as C83), the action of amyloidogenic ␤-secretase on APP results in the amino-terminal product sAPP-␤ and the carboxyl-terminal product ␤-CTF (also known as C99). Subsequent ␥-secretase complex cleavage of ␤-CTF yields ␥-CTF (also known as C57), and releases A␤ (6 -10). Promotion of ␣-secretase processing leads to both a reduction in A␤ and an increase in sAPP-␣, a protein that exhibits neuroprotective properties (11)(12)(13)(14). A number of studies have sought to discern the molecular identity of ␣-secretase, with the hope of targeting such enzyme(s) to modulate A␤ production (15,16).A number of reports have implicated members of the a-disintegrin and metalloprotease (ADAM) family, a family of zinc metalloproteases including ADAM9, -10, and -17, as putative ␣-secretase candidates (15-17). Lammich and colleagues (18) first described the ability of ADAM10 to act as an ␣-secretase, whereas Buxbaum and co-workers (19) reported that ADAM17 contributes to ␣-secretase processing of APP. Others have demonstrated the ability of ADAM9 to promote ␣-secretase cleavage (20). However, Asai and colleagues (17) reported that ADAM9, -10, and -17 all have roles in the processing of APP to sAPP-␣ in vitro. In cerebrospinal fluid from AD patients, ADAM10 and corresponding sAPP/␣-CTFs are decreased (21,22). Moreover, ADAM10 is also decreased in AD and Down syndrome brains (23). A report by Lopez-Perez and colleagues (24) implicates ADAM10 as a contributor to constitutive sAPP-␣ production, whereas ADAM17 (also known as tumor necrosis factor-␣ converting enzyme, TACE) is implicated in a regulated mechanism of sAPP-␣ production (24). Recently, Postina and colleagues (25) showed th...

Although considered an immunologically privileged site, the central nervous system (CNS) can display significant inflammatory responses, which may play a pathogenic role in a number of neurological diseases. Microglia appear to be particularly important for initiating and sustaining CNS inflammation. These cells exist in a quiescent form in the normal CNS, but acquire macrophage-like properties (including active phagocytosis, upregulation of proteins necessary for antigen presentation, and production of proinflammatory cytokines) after stimulation with inflammatory substances such as lipopolysaccharide (LPS). Recent studies have focused on elucidating the role of neurons in the regulation of microglial inflammatory responses. In the present study, we demonstrate, using neuron-microglial cocultures, that neurons are capable of inhibiting LPS-induced tumor necrosis factor-alpha (TNF-alpha) production by microglia. This inhibition appears to be dependent on secretion of substances at axon terminals, as treatment with the presynaptic calcium channel blocker omega-conotoxin abolishes this inhibitory effect. Moreover, we show that conditioned medium from neuronal cultures similarly inhibits microglial TNF-alpha production, which provides additional evidence that neurons secrete inhibitory substances. We previously demonstrated that the transmembrane protein-tyrosine phosphatase CD45 plays an important role in negatively regulating microglial activation. The recent characterization of CD22 as an endogenous ligand of this receptor led us to investigate whether neurons express this protein. Indeed, we were able to demonstrate CD22 mRNA and protein expression in cultured neurons and mouse brain, using reverse transcriptase-polymerase chain reaction and antibody-based techniques. Furthermore, we show that neurons secrete CD22, which functions as an inhibitor of microglial proinflammatory cytokine production.

Background: It is well known that most neurodegenerative diseases are associated with microglia-mediated inflammation. Our previous research demonstrates that the CD40 signaling is critically involved in microglia-related immune responses in the brain. For example, it is well known that the activation of the signal transducer and activator of transcription (STAT) signaling pathway plays a central role in interferon-gamma (IFN-γ)-induced microglial CD40 expression. We and others have previously reported that microglial CD40 expression is significantly induced by IFN-γ and amyloid-β (Aβ) peptide. Recent studies have shown that certain flavonoids possess antiinflammatory and neuroprotective properties distinct from their well-known anti-oxidant effects. In particular, flavonoids, apigenin and luteolin have been found to be effective CD40 immunomodulators.

BackgroundAmyotrophic lateral sclerosis (ALS) is a neurodegenerative disease affecting upper and lower motor neurons in the CNS and leading to paralysis and death. There are currently no effective treatments for ALS due to the complexity and heterogeneity of factors involved in motor neuron degeneration. A complex of interrelated effectors have been identified in ALS, yet systemic factors indicating and/or reflecting pathological disease developments are uncertain. The purpose of the study was to identify humoral effectors as potential biomarkers during disease progression.MethodsThirteen clinically definite ALS patients and seven non-neurological controls enrolled in the study. Peripheral blood samples were obtained from each ALS patient and control at two visits separated by 6 months. The Revised ALS Functional Rating Scale (ALSFRS-R) was used to evaluate overall ALS-patient functional status at each visit. Eleven humoral factors were analyzed in sera. Cytokine levels (GM-CSF, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, and TNF-α) were determined using the Bio-Rad Bio-Plex® Luminex 200 multiplex assay system. Nitrite, a breakdown product of NO, was quantified using a Griess Reagent System. Glutathione (GSH) concentrations were measured using a Glutathione Fluorometric Assay Kit.ResultsALS patients had ALSFRS-R scores of 30.5 ± 1.9 on their first visit and 27.3 ± 2.7 on the second visit, indicating slight disease progression. Serum multiplex cytokine panels revealed statistically significant changes in IL-2, IL-5, IL-6, and IL-8 levels in ALS patients depending on disease status at each visit. Nitrite serum levels trended upwards in ALS patients while serum GSH concentrations were drastically decreased in sera from ALS patients versus controls at both visits.ConclusionsOur results demonstrated a systemic pro-inflammatory state and impaired antioxidant system in ALS patients during disease progression. Increased levels of pro-inflammatory IL-6, IL-8, and nitrite and significantly decreased endogenous antioxidant GSH levels could identify these humoral constituents as systemic biomarkers for ALS. However, systemic changes in IL-2, IL-5, and IL-6 levels determined between visits in ALS patients might indicate adaptive immune system responses dependent on current disease stage. These novel findings, showing dynamic changes in humoral effectors during disease progression, could be important for development of an effective treatment for ALS.

Modulation of immune/inflammatory responses by diverse strategies including amyloid-beta (Abeta) immunization, nonsteroidal anti-inflammatory drugs, and manipulation of microglial activation states has been shown to reduce Alzheimer's disease (AD)-like pathology and cognitive deficits in AD transgenic mouse models. Human umbilical cord blood cells (HUCBCs) have unique immunomodulatory potential. We wished to test whether these cells might alter AD-like pathology after infusion into the PSAPP mouse model of AD. Here, we report a marked reduction in Abeta levels/beta-amyloid plaques and associated astrocytosis following multiple low-dose infusions of HUCBCs. HUCBC infusions also reduced cerebral vascular Abeta deposits in the Tg2576 AD mouse model. Interestingly, these effects were associated with suppression of the CD40-CD40L interaction, as evidenced by decreased circulating and brain soluble CD40L (sCD40L), elevated systemic immunoglobulin M (IgM) levels, attenuated CD40L-induced inflammatory responses, and reduced surface expression of CD40 on microglia. Importantly, deficiency in CD40 abolishes the effect of HUCBCs on elevated plasma Abeta levels. Moreover, microglia isolated from HUCBC-infused PSAPP mice demonstrated increased phagocytosis of Abeta. Furthermore, sera from HUCBC-infused PSAPP mice significantly increased microglial phagocytosis of the Abeta1-42 peptide while inhibiting interferon-gammainduced microglial CD40 expression. Increased microglial phagocytic activity in this scenario was inhibited by addition of recombinant CD40L protein. These data suggest that HUCBC infusion mitigates AD-like pathology by disrupting CD40L activity.