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.

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 LPSinduced expression of interleukin (IL)-6 and inducible nitric oxide synthase mRNAs, as well as the secretion of cytokines IL-6, tumour necrosis factor-a 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-jB) inhibitors caffeic acid phenethyl ester and helenalin, demonstrating that the NF-jB signalling pathway is involved. The DNA-binding activity of the NF-jB 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-jB 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. Keywords: acetylation, Alzheimer, epigenetics, histone deacetylase, neurodegeneration, nuclear factor kappa B. Abbreviations used: CAPE, caffeic acid phenethyl ester; FBS, fetal bovine serum; HDAC, histone deacetylase; IL, interleukin; iNOS, inducible nitric oxide synthase; LDH, lactate dehydrogenase; LPS, lipopolysaccharide; M344, 4-dimethylamino-N-(6-hydroxycarbamoylhexyl)-benzamide; MIP, macrophage inflammatory protein; NF-jB, nuclear factor kappa B; NO, nitric oxide; SAHA, suberoylanilide hydroxamic acid; SDS, sodium dodecyl sulfate; TNF, tumour necrosis factor; TSA, trichostatin A.

BackgroundPurified 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 β-amyloid (Aβ) 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 Aβ deposits by primary astrocytes and microglia, two major cell types involved in the neuroinflammatory responses.MethodsWe first determined the effect of IVIG on Aβ toxicity in primary neuronal cell culture. The mechanisms-of-action of IVIG in reduction of Aβ burden was analyzed with ex vivo assay. We studied whether IVIG solubilizes natively formed Aβ deposits from brain sections of APP/PS1 mice or promotes Aβ removal by primary glial cells. We determined the role of lysosomal degradation pathway and Aβ Abs in the IVIG-promoted reduction of Aβ. Finally, we studied the penetration of IVIG into the brain parenchyma and interaction with brain deposits of human Aβ in a mouse model of AD in vivo.ResultsIVIG was protective against Aβ toxicity in a primary mouse hippocampal neuron culture. IVIG modestly inhibited the fibrillization of synthetic Aβ1-42 but did not solubilize natively formed brain Aβ deposits ex vivo. IVIG enhanced microglia-mediated Aβ clearance ex vivo, with a mechanism linked to Aβ Abs and lysosomal degradation. The IVIG-enhanced Aβ clearance appears specific for microglia since IVIG did not affect Aβ clearance by astrocytes. The cellular mechanisms of Aβ 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 Aβ deposits in co-localization with microglia.ConclusionsOur results demonstrate that IVIG promotes recognition and removal of natively formed brain Aβ deposits by primary microglia involving natural Aβ Abs in IVIG. These findings may have therapeutic relevance in vivo as IVIG penetrates through the blood-brain barrier and specifically binds to Aβ deposits in brain parenchyma.

Pyrrolidine dithiocarbamate (PDTC) is an antioxidant and inhibitor of transcription factor nuclear factor kappa-B (NFjB). Because the role of NF-jB in brain injury is controversial and another NF-jB 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-jB activation in the ischaemic brain as determined by reduced DNA binding and nuclear translocation of NF-jB in neurons. PDTC had anti-inflammatory effect by preventing induction of NF-jB-regulated proinflammatory genes. In ischaemic rats, NF-jB 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-jB. The clinically approved PDTC and its analogues may act as antiinflammatories and may be safe therapies in stroke with a wide time window. Keywords: cytokines, inflammation, neuroimmunology, rodent. Stroke is the third leading cause of death in industrialized countries and a major cause of severe disability in the elderly (Centers for Disease Control 1992; Wolf et al. 1997). There is no other acute stroke therapy than intravenous thrombolysis and it is safe and effective only for a fraction of the patients (Bednar and Gross 1999;Lindsberg and Kaste 2003). Even though animal research conducted on acute ischaemic injury has revealed several pathophysiological cascades contributing to brain infarction, no breakthroughs in developing clinically relevant stroke therapy have been achieved. One factor is that laboratory studies on a single animal species or stroke model are not sufficient for modelling more variable human strokes (STAIR 1999). Another valid reason is that the compounds, which are protective in animal models, have a limited therapeutic time window or toxic secondary effects in humans (STAIR 1999).Reactive oxygen species (ROS) and inflammation are involved in human stroke and play a crucial role in animal models of stroke during the first days after the onset of ischaemia ( Abbreviations used: COX-2, cyclo-oxygenase-2; DDTC, diethyldithiocarbamate; div, days in vitro; FBS-HI, fetal bovine serum heat inactivated; IjB, inhibitor of NF-jB; IL-1b, interleukin-1b; iNOS, inducible nitric oxide synthase; LDH, lactate dehydrogenase; MAPKs, mitogen-activated protein kinases; MCA, middle cerebral artery; MCAO, middle cerebral artery occlusion; MEM, minimal essential medium; NF-jB, nuclear factor kappa-B; NMDA, N-methyl-D-asparatate; PDTC, pyrrolidine dithiocarbamate; ROS, reactive oxygen species; SHR, spontaneously ...

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