Monoacylglycerol lipase inhibitor JZL184 reduces neuroinflammatory response in APdE9 mice and in adult mouse glial cells.

BACKGROUND: Recently, the role of monoacylglycerol lipase (MAGL) as the principal regulator of simultaneous prostaglandin synthesis and endocannabinoid receptor activation in the CNS was demonstrated. To expand upon previously published research in the field, we observed the effect of the MAGL inhibitor JZL184 during the early-stage proinflammatory response and formation of beta-amyloid (Aß) in the Alzheimer's disease mouse model APdE9. We also investigated its effects in proinflammatory agent - induced astrocytes and microglia isolated from adult mice. FINDINGS: Transgenic APdE9 mice (5 months old) were treated with JZL184 (40 mg/kg) or vehicle every day for 1 month. In vivo binding of the neuroinflammation-related, microglia-specific translocator protein (TSPO) targeting radioligand [(18) F]GE-180 decreased slightly but statistically non-significantly in multiple brain areas compared to vehicle-treated mice. JZL184 treatment induced a significant decrease in expression levels of inflammation-induced, Iba1-immunoreactive microglia in the hippocampus (P < 0.01) and temporal and parietal (P < 0.05) cortices. JZL184 also induced a marked decrease in total Aß burden in the temporal (P < 0.001) and parietal (P < 0.01) cortices and, to some extent, in the hippocampus. Adult microglial and astrocyte cultures pre-treated with JZL184 and then exposed to the neuroinflammation-inducing agents lipopolysaccharide (LPS), interferon-gamma (IFN-γ), and Aß42 had significantly reduced proinflammatory responses compared to cells without JZL184 treatment. CONCLUSIONS: JZL184 decreased the proinflammatory reactions of microglia and reduced the total Aß burden and its precursors in the APdE9 mouse model. It also reduced the proinflammatory responses of microglia and astrocytes isolated from adult mice.

Multiple cellular and molecular mechanisms are involved in human Aß clearance by transplanted adult astrocytes.

Astrocytes and microglia are able to degrade potentially neurotoxic ß-amyloid (Aß) deposits typical for Alzheimer's disease (AD) pathology. Contrary to microglia, astrocytes degrade human Aß from tissue sections in vitro without any additional stimulation, but it has remained unclear whether transplanted astrocytes are able to clear deposited human Aß in vivo. We transplanted adult mouse astrocytes into the hippocampi of transgenic mice mimicking AD and observed their fate, effects on microglial responses, and Aß clearance. After 2-months follow-up time, we discovered a significant reduction in Aß burden compared with AD mice infused with PBS only. The remaining Aß deposits were fragmented and most of the Aß immunoreactivity was seen within the transplanted astrocytes. Concomitant to Aß reduction, both CD68 and CD45 immunoreactivities were significantly upregulated but phagocytic microglia were often surrounding and engulfing Aß burdened, TUNEL-positive astrocytes rather than co-localizing with Aß alone. Astrocytes are known to degrade Aß also by secreting proteases involved in Aß catabolism. To study the contribution of neprilysin (NEP), angiotensin-converting enzyme-1 (ACE-1), and endothelin-converting enzyme-2 (ECE-2) in human Aß clearance, we utilized an ex vivo assay to demonstrate that adult astrocytes respond to human Aß by upregulating NEP expression. Further, incubation of adult astrocytes with known inhibitors of NEP, ACE-1, or ECE-2 significantly inhibited the removal of human Aß from the tissue suggesting an important role for these proteases in Aß clearance by adult astrocytes ex vivo.

Human intravenous immunoglobulin provides protection against Aß toxicity by multiple mechanisms in a mouse model of Alzheimer's disease.

BACKGROUND: Purified intravenous immunoglobulin (IVIG) obtained from the plasma of healthy humans is indicated for the treatment of primary immunodeficiency disorders associated with defects in humoral immunity. IVIG contains naturally occurring auto-antibodies, including antibodies (Abs) against ß-amyloid (Aß) peptides accumulating in the brains of Alzheimer's disease (AD) patients. IVIG has been shown to alleviate AD pathology when studied with mildly affected AD patients. Although its mechanisms-of-action have been broadly studied, it remains unresolved how IVIG affects the removal of natively formed brain Aß deposits by primary astrocytes and microglia, two major cell types involved in the neuroinflammatory responses. METHODS: We first determined the effect of IVIG on Aß toxicity in primary neuronal cell culture. The mechanisms-of-action of IVIG in reduction of Aß burden was analyzed with ex vivo assay. We studied whether IVIG solubilizes natively formed Aß deposits from brain sections of APP/PS1 mice or promotes Aß removal by primary glial cells. We determined the role of lysosomal degradation pathway and Aß Abs in the IVIG-promoted reduction of Aß. Finally, we studied the penetration of IVIG into the brain parenchyma and interaction with brain deposits of human Aß in a mouse model of AD in vivo. RESULTS: IVIG was protective against Aß toxicity in a primary mouse hippocampal neuron culture. IVIG modestly inhibited the fibrillization of synthetic Aß1-42 but did not solubilize natively formed brain Aß deposits ex vivo. IVIG enhanced microglia-mediated Aß clearance ex vivo, with a mechanism linked to Aß Abs and lysosomal degradation. The IVIG-enhanced Aß clearance appears specific for microglia since IVIG did not affect Aß clearance by astrocytes. The cellular mechanisms of Aß clearance we observed have potential relevance in vivo since after peripheral administration IVIG penetrated to mouse brain tissue reaching highest concentrations in the hippocampus and bound selectively to Aß deposits in co-localization with microglia. CONCLUSIONS: Our results demonstrate that IVIG promotes recognition and removal of natively formed brain Aß deposits by primary microglia involving natural Aß Abs in IVIG. These findings may have therapeutic relevance in vivo as IVIG penetrates through the blood-brain barrier and specifically binds to Aß deposits in brain parenchyma.

[18F]FMPEP-d2 PET imaging shows age- and genotype-dependent impairments in the availability of cannabinoid receptor 1 in a mouse model of Alzheimer's disease.

Contradictory findings on the role of the type 1 cannabinoid receptor (CB1R) during the pathogenesis of Alzheimer's disease (AD) have been reported. Here, we evaluated the CB1R brain profile in an AD mouse model using longitudinal positron emission tomography with an inverse agonist for CB1R, [18F]FMPEP-d2. APP/PS1-21 and wild-type (n = 8 in each group) mice were repeatedly imaged between 6 to 15 months of age, accompanied by brain autoradiography, western blot, and CB1R immunohistochemistry with additional mice. [18F]FMPEP-d2 positron emission tomography demonstrated lower (p < 0.05) binding ratios in the parietotemporal cortex and hippocampus of APP/PS1-21 mice compared with age-matched wild-type mice. Western blot demonstrated no differences between APP/PS1-21 and wild-type mice in the CB1R abundance, whereas significantly lower (p < 0.05) receptor expression was observed in male than female mice. The results provide the first demonstration that [18F]FMPEP-d2 is a promising imaging tool for AD research in terms of CB1R availability, but not expression. This finding may further facilitate the development of novel therapeutic approaches based on endocannabinoid regulation.

The Anti-Inflammatory Effects of Lipoxygenase and Cyclo-Oxygenase Inhibitors in Inflammation-Induced Human Fetal Glia Cells and the Aß Degradation Capacity of Human Fetal Astrocytes in an Ex vivo Assay.

Chronic inflammation is a common phenomenon present in the background of multiple neurodegenerative diseases, including Alzheimer's disease (AD). The arachidonic acid pathway overproduces proinflammatory eicosanoids during these states and glial cells in the brain gradually lose their vital functions of protecting and supporting neurons. In this study, the role of different key enzymes of the eicosanoid pathway mediating inflammatory responses was examined in vitro and ex vivo using human fetal glial cells. Astrocytes and microglia were exposed to proinflammatory agents i.e., cytokines interleukin 1-ß (IL-1ß) and tumor necrosis factor (TNF-α). ELISA assays were used to examine the effects of inhibitors of key enzymes in the eicosanoid pathway. Inhibitors for 5-lipoxygenase (5-LOX) and cyclo-oxygenase 2 (COX-2) in both cell types and 5-, 12-, and 15-LOX-inhibitor in astrocytes reduced significantly IL-6 secretion, compared to exposed glial cells without inhibitors. The cytokine antibody array showed that especially treatments with 5, -12, and -15 LOX inhibitor in astrocytes, 5-LOX inhibitor in microglia and COX-2 inhibitor in both glial cell types significantly reduced the expression of multiple proinflammatory cytokines. Furthermore, human fetal astrocytes and microglia were cultured on top of AD-affected and control human brain sections for 30 h. According to the immunochemical evaluation of the level of total Aß, astrocytes were very efficient at degrading Aß from AD-affected brain sections ex vivo; simultaneously added enzyme inhibitors did not increase their Aß degradation capabilities. Microglia were not able to reduce the level of total Aß during the 30 h incubation time.

Applicability of [11C]PIB micro-PET imaging for in vivo follow-up of anti-amyloid treatment effects in APP23 mouse model.

In this study, we evaluated the anti-amyloid effect of functionalized nanoliposomes (mApoE-PA-LIP) in a mouse model of Alzheimer's disease with use of positron emission tomography and ß-amyloid (Aß)-targeted tracer [11C]Pittsburgh compound B ([11C]PIB). APP23 mice were injected with mApoE-PA-LIP or saline (3 times per week for 3 weeks) and [11C]PIB imaging was performed at baseline, after the treatment and after 3 months follow-up period, accompanied by Aß immunohistochemistry and ELISA. After the treatment, [11C]PIB binding ratios between mApoE-PA-LIP and saline groups were equivalent in all analyzed brain regions; however, in the saline group, binding ratios increased from the baseline, whereas no increase was detected in the mApoE-PA-LIP group. During the additional follow-up, [11C]PIB binding increased significantly from baseline in both groups, and binding ratios correlated with the immunohistochemically defined Aß load. This study further supports the use of [11C]PIB positron emission tomography imaging as a biomarker of Aß deposition in APP23 mice and highlights the benefits of noninvasive follow-up, that is, using baseline data for animal stratification and normalization of treatment effects to baseline values, for future anti-amyloid treatment studies.

Antioxidant pyrrolidine dithiocarbamate activates Akt-GSK signaling and is neuroprotective in neonatal hypoxia-ischemia.

Pyrrolidine dithiocarbamate (PDTC), an antioxidant and inhibitor of transcription factor nuclear factor kappa-B (NF-kappaB), has been reported to reduce inflammation and apoptosis. Because PDTC was recently found to protect in various models of adult brain ischemia with a wide therapeutic time window, we tested the effect of PDTC in a rodent model of neonatal hypoxia-ischemia (HI) brain injury. T2-weighed magnetic resonance imaging (T2-MRI) 7 days after the insult showed that a single PDTC (50 mg/kg) injection 2.5 h after the HI reduced the mean brain infarct size by 59%. PDTC reduced the HI-induced dephosphorylation of Akt and glycogen synthase kinase-3beta (GSK-3beta), expression of cleaved caspase-3, and nuclear translocation of NF-kappaB in the neonatal brain. PDTC targeted directly neurons, as PDTC reduced hypoxia-reoxygenation-induced cell death in pure hippocampal neuronal cultures. It is suggested that in addition to the previously indicated NF-kappaB inhibition as a protective mechanism of PDTC treatment, PDTC may reduce HI-induced brain injury at least partially by acting as an antioxidant, which reduces the Akt-GSK-3beta pathway of apoptotic cell death. The clinically approved PDTC and its analogues may be beneficial after HI insults with a reasonable time window.

Transplanted astrocytes internalize deposited beta-amyloid peptides in a transgenic mouse model of Alzheimer's disease.

Alzheimer's disease (AD) is one of the most devastating neurodegenerative disorders. The neuropathological hallmarks include extracellular senile plaques consisting of deposited beta-amyloid (Abeta) peptides and intraneuronal neurofibrillary tangles. Neuroinflammation and activation of astrocytes are also well-established features of AD neuropathology; however, the relationships between astrocytes and Abeta deposition remain unclear. Previous studies have shown that adult mouse astrocytes internalize and degrade Abeta deposits in brain sections prepared from human amyloid precursor protein (APP) transgenic mice. In the present study, we demonstrate that cultured adult, but not neonatal mouse astrocytes, respond morphologically and degrade Abeta deposits present in human AD brain. We also transplanted astrocytes isolated from enhanced green fluorescent protein expressing adult and neonatal mice into the hippocampi of human Abeta plaque-bearing transgenic APPSwe+PS1dE9 (APdE9) mice and their wild-type littermates and followed the migration and localization of these astrocytes by confocal microscopy upto 7 days after transplantation. Posttransplantation the astrocytes localized as aggregates or thin strings of many cells within the hippocampi of APdE9 and wild-type mice and showed limited migration from the injection site. Interestingly, most of the transplanted astrocytes were found near Abeta deposits in the hippocampi of APdE9 mice. In contrast to findings in ex vivo degradation assay, confocal microscopy revealed that both adult and neonatal transplanted astrocytes internalized human Abeta immunoreactive material in vivo. These results support the role of astrocytes as active Abeta clearing cells in the CNS that may have important implications for future development of therapeutic strategies for AD.

Pyrrolidine dithiocarbamate inhibits translocation of nuclear factor kappa-B in neurons and protects against brain ischaemia with a wide therapeutic time window.

Pyrrolidine dithiocarbamate (PDTC) is an antioxidant and inhibitor of transcription factor nuclear factor kappa-B (NF-kappa B). Because the role of NF-kappa B in brain injury is controversial and another NF-kappa B inhibiting thiocarbamate, DDTC, was recently shown to increase ischaemic brain damage, we investigated the effect of PDTC on transient brain ischaemia. Ischaemia was induced by occlusion of the middle cerebral artery (MCAO). In Wistar rats, the PDTC treatment started even 6 h after MCAO reduced the infarction volume by 48%. PDTC protected against MCAO also in spontaneously hypertensive rats and against forebrain ischaemia in Mongolian gerbils. PDTC prevented NF-kappa B activation in the ischaemic brain as determined by reduced DNA binding and nuclear translocation of NF-kappa B in neurons. PDTC had anti-inflammatory effect by preventing induction of NF-kappa B-regulated pro-inflammatory genes. In ischaemic rats, NF-kappa B was localized in cyclo-oxygenase-2-immunoreactive neurons. Blood cytokine levels were not altered by ischaemia or PDTC. When cultured neurons were exposed to an excitotoxin, no production of reactive oxygen species was detected, but PDTC provided protection and prevented nuclear translocation of NF-kappa B. The clinically approved PDTC and its analogues may act as anti-inflammatories and may be safe therapies in stroke with a wide time window.

Regulation of microglial inflammatory response by histone deacetylase inhibitors.

The activation of microglial cells is involved in the pathogenesis of a variety of neurodegenerative diseases, stroke and traumatic brain injuries. Recent studies suggest that protein acetylation can affect the extent of inflammatory responses. Our aim was to elucidate whether histone deacetylase inhibitors, inducers of protein hyperacetylation, regulate the inflammatory response in neural models of inflammation in vitro and whether neurone-glia interactions affect this regulation. Interestingly, we observed that histone deacetylase inhibitors, such as trichostatin A (TSA) and suberoylanilide hydroxamic acid, strongly potentiated the lipopolysaccharide (LPS)-induced inflammatory response in murine N9 and rat primary microglial cells as well in neural co-cultures and hippocampal slice cultures. TSA clearly potentiated the LPS-induced expression of interleukin (IL)-6 and inducible nitric oxide synthase mRNAs, as well as the secretion of cytokines IL-6, tumour necrosis factor-alpha and macrophage inflammatory protein (MIP)-2, and nitric oxide (NO). Co-culture and slice culture experiments showed that the presence of astrocytes and neurones did not stimulate or prevent the pro-inflammatory potentiation induced by histone deacetylase inhibitor in microglial cells. The potentiation of cytokine and NO responses was blocked by the nuclear factor kappa B (NF-kappa B) inhibitors caffeic acid phenethyl ester and helenalin, demonstrating that the NF-kappa B signalling pathway is involved. The DNA-binding activity of the NF-kappa B complex was strongly increased by LPS treatment but not enhanced by TSA. This suggests that potentiation of the inflammatory response is not dependent on the level of cytoplasmic NF-kappa B activation or DNA-binding activity but that site of action may be at the level of transcriptional regulation. Our results suggest that environmental stresses, ageing, diet and diseases that regulate protein acetylation status may also affect the inflammatory response.