Tarja Malm

Bone-marrow-derived cells contribute to the recruitment of microglial cells in response to beta-amyloid deposition in APP/PS1 double transgenic Alzheimer mice

The role of microglia recruited from bone marrow (BM) into the CNS during the progression of Alzheimers disease (AD) is poorly understood. To investigate whether beta-amyloid (Abeta) associated microglia are derived from blood monocytes, we transplanted BM cells from enhanced green fluorescent protein expressing mice into young or old transgenic AD mice and determined the engraftment of BM-derived cells into the brain and their relative distribution near Abeta deposits. When young transgenic mice were transplanted before the onset of AD-like pathology and the brains analyzed 6.5 months later, the number of engrafted cells was significantly higher than in age-matched wild type mice. Moreover, the number of BM-derived cells associated with Abeta was significantly higher than in old transgenic mice transplanted after the establishment of AD-like pathology. Local inflammation caused by intrahippocampal lipopolysaccharide injection significantly increased the engraftment of BM-derived cells in old AD mice and decreased the hippocampal Abeta burden. These results suggest that infiltration of BM-derived monocytic cells into the brain contributes to the development of microglial reaction in AD.

Intrahippocampal injection of a lentiviral vector expressing Nrf2 improves spatial learning in a mouse model of Alzheimer's disease

The amyloid hypothesis of Alzheimers disease (AD) postulates that amyloid-β (Aβ) deposition and neurotoxicity play a causative role in AD; oxidative injury is thought to be central in the pathogenesis. An endogenous defense system against oxidative stress is induced by binding of the transcription factor nuclear factor E2-related factor 2 (Nrf2) to the antioxidant response element (ARE) enhancer sequence. The Nrf2-ARE pathway is activated in response to reactive oxygen species to trigger the simultaneous expression of numerous protective enzymes and scavengers. To exploit the Nrf2-ARE pathway therapeutically, we delivered Nrf2 bilaterally into the hippocampus of 9-month-old transgenic AD mice (APP/PS1 mice) using a lentiviral vector encoding human Nrf2. The data indicate that significant reductions in spatial learning deficits of aged APP/PS1 mice in a Morris Water Maze can be achieved by modulating levels of Nrf2 in the brain. Memory improvement in APP/PS1 mice after Nrf2 transduction shifts the balance between soluble and insoluble Aβ toward an insoluble Aβ pool without concomitant change in total brain Aβ burden. Nrf2 gene transfer is associated with a robust reduction in astrocytic but not microglial activation and induction of Nrf2 target gene heme oxygenase 1, indicating overall activation of the Nrf2-ARE pathway in hippocampal neurons 6 months after injection. Results warrant further exploration of the Nrf2-ARE pathway for treatment of AD and suggest that the Nrf2-ARE pathway may represent a potential therapeutic strategy to pursue in AD in humans, particularly in view of the multiple mechanisms by which Nrf2 can exert its protective effects.

Hypoxia induces microRNA miR-210 in vitro and in vivo ephrin-A3 and neuronal pentraxin 1 are potentially regulated by miR-210.

Shortage of oxygen is one of the prime stress conditions in tissues. In this study, we looked for microRNAs expressed during hypoxia and showed that miR‐210 expression was upregulated in response to hypoxia in vitro and in vivo. An active form of the HIF‐1α induced the expression of miR‐210, showing the involvement of the HIF‐1 signaling pathway in miR‐210 gene transcription. Furthermore, miR‐210 was shown to bind to the predicted target sites of ephrin‐A3 or neuronal pentraxin 1, causing repression in luciferase reporter activity. Contrary to the microRNA‐mediated repression hypothesis, ephrin‐A3 was expressed at very high levels in post‐ischemic mouse hippocampus in vivo. Thus, the regulatory effects of miR‐210 on its targets in vivo need to be further characterized.

Nuclear factor erythroid 2-related factor 2 protects against beta amyloid

Nuclear factor erythroid 2-related factor 2 (Nrf2) coordinates the up-regulation of cytoprotective genes via the antioxidant response element (ARE). In the pathogenesis of Alzheimers disease (AD) current evidence supports the role of oxidative stress. Considering the protective role of Nrf2 against oxidative injury, we studied Nrf2 and Nrf2-ARE target genes in transgenic AD mice and tested whether Nrf2 could confer neuroprotection against amyloid-beta peptides (Abeta). Nrf2-ARE pathway was attenuated in APP/PS1 transgenic mouse brain at the time of Abeta deposition. Boosting the activity of the Nrf2-ARE pathway by tert-butylhydroquinone treatment or adenoviral Nrf2 gene transfer protected against Abeta toxicity. This neuroprotection was associated with increased expression of Nrf2 target genes and reduced phosphorylation of p66Shc, a marker of increased susceptibility for oxidative stress. The findings suggest that the Nrf2-ARE pathway may be impaired in AD and that induction of the Nrf2-ARE defence mechanism may prevent or delay AD-like pathology.

Pyrrolidine Dithiocarbamate Activates Akt and Improves Spatial Learning in APP/PS1 Mice without Affecting β-Amyloid Burden

Pyrrolidine dithiocarbamate (PDTC) is a clinically tolerated inhibitor of nuclear factor-κB (NF-κB), antioxidant and antiinflammatory agent, which provides protection in brain ischemia models. In neonatal hypoxia–ischemia model, PDTC activates Akt and reduces activation of glycogen synthase kinase 3β (GSK-3β). Because chronic inflammation, oxidative stress, and increased GSK-3β activity are features of Alzheimers disease (AD) pathology, we tested whether PDTC reduces brain pathology and improves cognitive function in a transgenic animal model of AD. A 7 month oral treatment with PDTC prevented the decline in cognition in AD mice without altering β-amyloid burden or gliosis. Moreover, marked oxidative stress and activation of NF-κB were not part of the brain pathology. Instead, the phosphorylated form of GSK-3β was decreased in the AD mouse brain, and PDTC treatment increased the phosphorylation of Akt and GSK-3β. Also, PDTC treatment increased the copper concentration in the brain. In addition, PDTC rescued cultured hippocampal neurons from the toxicity of oligomeric Aβ and reduced tau phosphorylation in the hippocampus of AD mice. Finally, astrocytic glutamate transporter GLT-1, known to be regulated by Akt pathway, was decreased in the transgenic AD mice but upregulated back to the wild-type levels by PDTC treatment. Thus, PDTC may improve spatial learning in AD by interfering with Akt–GSK pathway both in neurons and astrocytes. Because PDTC is capable of transferring external Cu2+ into a cell, and, in turn, Cu2+ is able to activate Akt, we hypothesize that PDTC provides the beneficial effect in transgenic AD mice through Cu2+-activated Akt pathway.

Stable RNA interference: comparison of U6 and H1 promoters in endothelial cells and in mouse brain

RNA interference (RNAi) is a post‐transcriptional RNA degradation process, which has become a very useful tool in gene function studies and gene therapy applications. Long‐term cellular expression of small interfering RNA (siRNA) molecules required for many gene therapy applications can be achieved by lentiviral vectors (LVs). The two most commonly used promoters to drive the short hairpin RNA (shRNA) expression are the human U6 small nuclear promoter (U6) and the human H1 promoter (H1).

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

Alzheimers disease (AD) is one of the most devastating neurodegenerative disorders. The neuropathological hallmarks include extracellular senile plaques consisting of deposited β‐amyloid (Aβ) peptides and intraneuronal neurofibrillary tangles. Neuroinflammation and activation of astrocytes are also well‐established features of AD neuropathology; however, the relationships between astrocytes and Aβ deposition remain unclear. Previous studies have shown that adult mouse astrocytes internalize and degrade Aβ 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 Aβ 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 Aβ 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 Aβ 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 Aβ immunoreactive material in vivo. These results support the role of astrocytes as active Aβ clearing cells in the CNS that may have important implications for future development of therapeutic strategies for AD.

Age-related decrease in stimulated glutamate release and vesicular glutamate transporters in APP/PS1 transgenic and wild-type mice

We assessed baseline and KCl‐stimulated glutamate release by using microdialysis in freely moving young adult (7 months) and middle‐aged (17 months) transgenic mice carrying mutated human amyloid precursor protein and presenilin genes (APdE9 mice) and their wild‐type littermates. In addition, we assessed the age‐related development of amyloid pathology and spatial memory impaired in the water maze and changes in glutamate transporters. APdE9 mice showed gradual spatial memory impairment between 6 and 15 months of age. The stimulated glutamate release declined very robustly in 17‐month‐old APdE9 mice as compared to 7‐month‐old APdE9 mice. This age‐dependent decrease in stimulated glutamate release was also evident in wild‐type mice, although it was not as robust as in APdE9 mice. When compared to individual baselines, all aged wild‐type mice showed 25% or greater increase in glutamate release upon KCl stimulation, but none of the aged APdE9 mice. There was an age‐dependent decline in VGLUT1 levels, but not in the levels of VGLUT2, GLT‐1 or synaptophysin. Astrocyte activation as measured by glial acidic fibrillary protein was increased in middle‐aged APdE9 mice. Blunted pre‐synaptic glutamate response may contribute to memory deficit in middle‐aged APdE9 mice.

Minocycline protects against permanent cerebral ischemia in wild type but not in matrix metalloprotease-9-deficient mice

Minocycline is protective in models of transient middle cerebral artery occlusion (MCAO). We studied whether minocycline and doxycycline, another tetracycline derivative, provide protection in permanent MCAO. Because minocycline inhibits matrix metalloprotease-9 (MMP-9), we also compared minocyclines protective effect in wild type (wt) and MMP-9 knock-out (ko) mice. Wt FVB/N, Balb/C, and two lines of MMP-9 ko and their wt C57Bl/6 control mice were subjected to 24- or 72-hour permanent MCAO. Drug administration was started either 12 hours before or 2 hours after the onset of MCAO. Infarct size was determined by triphenyltetrazolium staining or T2-weighted MRI. Zymography was used to study the expression of MMPs. In wt strains, tetracycline treatments started before MCAO reduced the infarct size by 25% to 50%, whereas the treatment started after MCAO was not protective. Minocycline inhibited ischemia-provoked pro-MMP-9 induction in wt mice, but was not protective in MMP-9 ko mice. Pro-MMP-2 was induced by MCAO in wt and MMP-9 ko mice. MCAO-induced pro-MMP-2 was downregulated by minocycline treatment in wt mice but remained in MMP-9 ko mice at the same level as in saline-treated wt mice. Tetracyclines are protective in permanent MCAO when the treatment is started before the insult. Minocycline may provide protection by interfering with MMPs.

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

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 Alzheimers 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.