Targeting Nrf2 to Suppress Ferroptosis and Mitochondrial Dysfunction in Neurodegeneration.

Ferroptosis is a newly described form of regulated cell death, distinct from apoptosis, necroptosis and other forms of cell death. Ferroptosis is induced by disruption of glutathione synthesis or inhibition of glutathione peroxidase 4, exacerbated by iron, and prevented by radical scavengers such as ferrostatin-1, liproxstatin-1, and endogenous vitamin E. Ferroptosis terminates with mitochondrial dysfunction and toxic lipid peroxidation. Although conclusive identification of ferroptosis in vivo is challenging, several salient and very well established features of neurodegenerative diseases are consistent with ferroptosis, including lipid peroxidation, mitochondrial disruption and iron dysregulation. Accordingly, interest in the role of ferroptosis in neurodegeneration is escalating and specific evidence is rapidly emerging. One aspect that has thus far received little attention is the antioxidant transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2). This transcription factor regulates hundreds of genes, of which many are either directly or indirectly involved in modulating ferroptosis, including metabolism of glutathione, iron and lipids, and mitochondrial function. This potentially positions Nrf2 as a key deterministic component modulating the onset and outcomes of ferroptotic stress. The minimal direct evidence currently available is consistent with this and indicates that Nrf2 may be critical for protection against ferroptosis. In contrast, abundant evidence demonstrates that enhancing Nrf2 signaling is potently neuroprotective in models of neurodegeneration, although the exact mechanism by which this is achieved is unclear. Further studies are required to determine to extent to which the neuroprotective effects of Nrf2 activation involve the prevention of ferroptosis.

The evolving biology of microglia in Alzheimer's disease.

Alzheimer's disease (AD) is typified by a robust microglial-mediated inflammatory response within the brain. Indeed, microglial accumulation around plaques in AD is one of the classical hallmarks of the disease pathology. Although microglia have the capacity to remove ß-amyloid deposits and alleviate disease pathology, they fail to do so. Instead, they become chronically activated and promote inflammation-mediated impairment of cognition and cytotoxicity. However, if microglial function could be altered to engage their phagocytic response, promote their tissue maintenance functions, and prevent release of factors that promote tissue damage, this could provide therapeutic benefit. This review is focused on the current knowledge of microglial homeostatic mechanisms in AD, and mechanisms involved in the regulation of microglial phenotype in this context.

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 Alzheimer's 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.

Minocycline reduces engraftment and activation of bone marrow-derived cells but sustains their phagocytic activity in a mouse model of Alzheimer's disease.

Bone marrow (BM)-derived monocytes contribute to the development of microglial reaction around beta-amyloid (Abeta) plaques in Alzheimer's disease (AD) and possibly clear Abeta. Therefore, it is of great importance to separate the proinflammatory actions of monocytic cells from Abeta phagocytic effects. We used minocycline (mino) to systemically downregulate microglial activation and studied proliferation, expression of markers for activated microglia, and Abeta removal in vitro and in vivo. Mino did not affect proliferation or phagocytic activity of BM-derived cells toward Abeta in vitro. Intrahippocampal LPS injection used to induce inflammation and increase recruitment of BM cells from periphery, reduced Abeta burden in BM-transplanted AD transgenic mice. All engrafted cells expressed CD45, approximately 50% expressed Iba-1, and <0.5% of these cells expressed CD3e. About 40% of the engrafted cells were mitotically active. LPS increased immunoreactivity for Iba-1, MHC II, a marker of antigen presenting cells, and CD68, a marker of lysosomal activity. The endogenous microglia largely contributed to these LPS-induced immunoreactivities. Mino reduced the engraftment of BM-derived cells and blocked the LPS-induced MHC II and Iba-1 immunoreactivities, but did not prevent the increased CD68-immunoreactivity or the reduced Abeta burden. Importantly, mino did not block the association of eGFP-positive cells with Abeta deposits and the percentage of mitotically active BM-derived cells. In conclusion, mino reduces overall inflammatory potential of BM-derived monocytic cells without preventing their phagocytic activity. The separation of harmful activation of microglia/monocytic cells from their Abeta clearing mechanism may hold important therapeutic potential.

Pyrrolidine dithiocarbamate activates Akt and improves spatial learning in APP/PS1 mice without affecting beta-amyloid burden.

Pyrrolidine dithiocarbamate (PDTC) is a clinically tolerated inhibitor of nuclear factor-kappaB (NF-kappaB), 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 3beta (GSK-3beta). Because chronic inflammation, oxidative stress, and increased GSK-3beta activity are features of Alzheimer's 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 beta-amyloid burden or gliosis. Moreover, marked oxidative stress and activation of NF-kappaB were not part of the brain pathology. Instead, the phosphorylated form of GSK-3beta was decreased in the AD mouse brain, and PDTC treatment increased the phosphorylation of Akt and GSK-3beta. Also, PDTC treatment increased the copper concentration in the brain. In addition, PDTC rescued cultured hippocampal neurons from the toxicity of oligomeric Abeta 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.

BACKGROUND: 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). METHODS: We investigated whether there is any significant difference between the efficiencies of U6 and H1 in LV-mediated RNAi using green fluorescent protein (GFP) as a target gene by flow cytometry and real-time reverse-transcription polymerase chain reaction (RT-PCR) in endothelial cells. Also, we compared the efficiencies of U6 and H1 in the GFP transgenic mouse brain after stereotactic LV injection. RESULTS: We show that the U6 promoter is more efficient than H1 in GFP silencing in vitro, leading to 80% GFP knockdown at an average of one integrated vector genome per target cell genome. The silencing is persistent for several months. In addition, the U6 promoter is superior to H1 in vivo and leads to stable GFP knockdown in mouse brain for at least 9 months. CONCLUSIONS: These results show that LV-mediated RNAi is a powerful gene-silencing method for the long-term inhibition of gene expression in vitro and in vivo.

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 Alzheimer's 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.

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 minocycline's 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.