Lentiviral vector-mediated overexpression of Klotho in the brain improves Alzheimer's disease-like pathology and cognitive deficits in mice.

Alzheimer's disease (AD) is the most common type of senile dementia. The antiaging gene Klotho is reported to decline in the brain of patients and animals with AD. However, the role of Klotho in the progression of AD remains elusive. The present study explored the effects and underlying mechanism of Klotho in a mouse model of AD. The upregulation of cerebral Klotho expression was mediated by an intracerebroventricular injection of a lentiviral vector that encoded Klotho (LV-KL) in 7-month-old amyloid precursor protein/presenilin 1 transgenic mice. Three months later, LV-KL significantly induced Klotho overexpression in the brain and effectively ameliorated cognitive deficit and AD-like pathology in amyloid precursor protein/presenilin 1 mice. LV-KL induced autophagy activation and protein kinase B/mammalian target of rapamycin inhibition both in AD mice and BV2 murine microglia. These results suggest that the upregulation of Klotho expression in the brain may promote the autophagic clearance of amyloid beta and protect against cognitive deficits in AD mice. These findings highlight the preventive and therapeutic potential of Klotho for the treatment of AD.

Lentivirus-mediated klotho up-regulation improves aging-related memory deficits and oxidative stress in senescence-accelerated mouse prone-8 mice.

AIMS: Oxidative stress caused by aging aggravates neuropathological changes and cognitive deficits. Klotho, an anti-aging protein, shows an anti-oxidative effect. The aims of the present study were to determine the potential therapeutic effect of klotho in aging-related neuropathological changes and memory impairments in senescence-accelerated mouse prone-8 (SAMP8) mice, and identify the potential mechanism of these neuroprotective effects. MATERIALS AND METHODS: A lentivirus was used to deliver and sustain the expression of klotho. The lentiviral vectors were injected into the bilateral lateral ventricles of 7-month-old SAMP8 mice or age-matched SAMR1 mice. Three months later, the Y-maze alternation task and passive avoidance task were used to assess the memory deficits of the mice. In situ hybridization, immunohistochemistry, immunofluorescence, Nissl staining, quantitative real-time PCR and Western blot assays were applied in the following research. KEY FINDINGS: Our results showed that 3 months after injection of the lentiviral vectors encoding the full-length klotho gene, the expression of klotho in the brain was significantly increased in 10-month-old SAMP8 mice. This treatment reduced memory deficits, neuronal loss, synaptic damage and 4-HNE levels but increased mitochondrial manganese-superoxide dismutase (Mn-SOD) and catalase (CAT) expression. Moreover, the up-regulation of klotho expression decreased Akt and Forkhead box class O1 (FoxO1) phosphorylation. SIGNIFICANCE: The present study provides a novel approach for klotho gene therapy and demonstrates that direct up-regulation of klotho in the brain might improve aging-related memory impairments and decrease oxidative stress. The underlying mechanism of this effect likely involves the inhibition of the Akt/FoxO1 pathway.

Klotho upregulation contributes to the neuroprotection of ligustilide against cerebral ischemic injury in mice.

Klotho, an aging-suppressor gene, encodes a protein that potentially acts as a neuroprotective factor. Our previous studies showed that ligustilide minimizes the cognitive dysfunction and brain damage induced by cerebral ischemia; however, the underlying mechanisms remain unclear. This study aims to investigate whether klotho is involved in the protective effects of ligustilide against cerebral ischemic injury in mice. Cerebral ischemia was induced by bilateral common carotid arterial occlusion. Neurobehavioral tests as well as Nissl and Fluoro-Jade B staining were used to evaluate the protective effects of ligustilide in cerebral ischemia, and Western blotting and ELISA approaches were used to investigate the underlying mechanisms. Administration of ligustilide prevented the development of neurological deficits and reduced neuronal loss in the hippocampal CA1 region and the caudate putamen after cerebral ischemia. The protective effects were associated with inhibition of the RIG-I/NF-κB p65 and Akt/FoxO1 pathways and with prevention of inflammation and oxidative stress in the brain. Further, downregulation of klotho could attenuate the neuroprotection of ligustilide against cerebral ischemic injury. Ligustilide exerted neuroprotective effects in mice after cerebral ischemia by regulating anti-inflammatory and anti-oxidant signaling pathways. Furthermore, klotho upregulation contributes to the neuroprotection of LIG against cerebral ischemic injury. These results indicated that ligustilide may be a promising therapeutic agent for the treatment of cerebral ischemia.

Neuroprotective effect of a novel gastrodin derivative against ischemic brain injury: involvement of peroxiredoxin and TLR4 signaling inhibition.

The inhibition of extracellular inflammatory peroxiredoxin (Prx) signaling appears to be a potential therapeutic strategy for neuroinflammatory injury after acute ischemic stroke. Gastrodin (Gas) is a phenolic glycoside that is used for the treatment of cerebral ischemia, accompanied by regulation of the autoimmune inflammatory response. The present study investigated the neuroprotective effects of Gas and its derivative, Gas-D, with a focus on the potential mechanism associated with inflammatory Prx-Toll-like receptor 4 (TLR4) signaling. Gas-D significantly inhibited Prx1-, Prx2-, and Prx4-induced inflammatory responses in RAW264.7 macrophages and H2O2-mediated oxidative injury in SH-SY5Y nerve cells. In rats, intraperitoneal Gas-D administration 10 h after reperfusion following 2-h middle cerebral artery occlusion (MCAO) ameliorated neurological deficits, brain infarction, and neuropathological alterations, including neuron loss, astrocyte and microglia/macrophage activation, T-lymphocyte invasion, and lipid peroxidation. Delayed Gas-D treatment significantly inhibited postischemic Prx1/2/4 expression and spillage, TLR4 signaling activation, and inflammatory mediator production. In contrast, Gas had no significant effects in either cell model or in MCAO rats under the same conditions. These results indicate that Gas-D may be a drug candidate with an extended therapeutic time window that blocks inflammatory responses and attenuates the expression and secretome of inflammatory Prxs in acute ischemic stroke.

Neuroprotective Effect of Ligustilide through Induction of α-Secretase Processing of Both APP and Klotho in a Mouse Model of Alzheimer's Disease.

Emerging evidence suggests that alpha-processing single transmembrane proteins, amyloid precursor protein (APP) and anti-aging protein Klotho, are likely to be involved in the progression of Alzheimer's disease (AD). The natural phthalide Ligustilide (LIG) has been demonstrated to protect against aging- and amyloid-ß (Aß)-induced brain dysfunction in animal models. The present study is to investigate the effects of LIG on cognitive deficits and metabolism of both APP and Klotho and its underlying mechanism in AD double-transgenic (APP/PS1) mice and cultured human cells. Our results show that treatment with LIG significantly ameliorated memory impairment and Aß levels and plaques burden. Specifically, LIG might act as a potent enhancer of α-secretase, disintegrin, and metalloprotease 10 (ADAM10), leading to upregulation of alpha-processing of both APP and Klotho and subsequent increases in the levels of both soluble APP fragment (sAPPα) and soluble Klotho (sKL) with inhibition of IGF-1/Akt/mTOR signaling in AD mice and cultured cells. Moreover, the specific ADAM10 inhibitor (G1254023X) effectively reversed LIG-induced alpha-processing of both APP and Klotho in vitro, while Klotho gene knockdown by small interfering RNA significantly blunted LIG-mediated inhibition of IGF-1/Akt/mTOR signaling in vitro. Taken together with the reported neuroprotective effects of both sAPPα and sKL as well as autophagy induction by Akt/mTOR pathway inhibition, our findings suggest that neuroprotection of LIG against AD is associated with induction alpha-processing of APP and Klotho and potential Aß clearance. Whether LIG might induce Aß autophagic clearance and the underlying mechanisms need to be further studied.

Protective Effect of Klotho against Ischemic Brain Injury Is Associated with Inhibition of RIG-I/NF-κB Signaling.

Aging is the greatest independent risk factor for the occurrence of stroke and poor outcomes, at least partially through progressive increases in oxidative stress and inflammation with advanced age. Klotho is an antiaging gene, the expression of which declines with age. Klotho may protect against neuronal oxidative damage that is induced by glutamate. The present study investigated the effects of Klotho overexpression and knockdown by an intracerebroventricular injection of a lentiviral vector that encoded murine Klotho (LV-KL) or rat Klotho short-hairpin RNA (LV-KL shRNA) on cerebral ischemia injury and the underlying anti-neuroinflammatory mechanism. The overexpression of Klotho induced by LV-KL significantly improved neurobehavioral deficits and increased the number of live neurons in the hippocampal CA1 and caudate putamen subregions 72 h after cerebral hypoperfusion that was induced by transient bilateral common carotid artery occlusion (2VO) in mice. The overexpression of Klotho significantly decreased the immunoreactivity of glial fibrillary acidic protein and ionized calcium binding adaptor molecule-1, the expression of retinoic-acid-inducible gene-I, the nuclear translocation of nuclear factor-κB, and the production of proinflammatory cytokines (tumor necrosis factor α and interleukin-6) in 2VO mice. The knockdown of Klotho mediated by LV-KL shRNA in the brain exacerbated neurological dysfunction and cerebral infarct after 22 h of reperfusion following 2 h middle cerebral artery occlusion in rats. These findings suggest that Klotho itself or enhancers of Klotho may compensate for its aging-related decline, thus providing a promising therapeutic approach for acute ischemic stroke during advanced age.

Differences in Proinflammatory Property of Six Subtypes of Peroxiredoxins and Anti-Inflammatory Effect of Ligustilide in Macrophages.

BACKGROUND: Peroxiredoxins (Prxs) are proposed to function as damage-associated molecular patterns (DAMPs) and contribute to post-ischemic neuroinflammation and brain injury by activating Toll-like receptor (TLR) 4 at the acute and subacute phases after ischemic stroke. However, there are few studies concerning the inflammatory profiles of six distinct subtypes of Prxs (Prx1-Prx6). Our previous study demonstrated that the protective effect of ligustilide (LIG) against cerebral ischemia was associated with inhibition of neuroinflammatory response and Prx/TLR4 signaling in rats. Herein, the present study explored the inflammatory members of Prxs and the effect of LIG on their inflammatory responses in macrophages. METHODOLOGY/PRINCIPAL FINDINGS: The murine RAW264.7 macrophages were treated with each of exogenous recombinant Prxs at a range of 1 to 50 nM for 24 h. The WST-1 test showed that Prx3 exhibited a significant cytotoxicity, whereas the rest five Prxs did not affect cellular viability. The quantitative measurements with spectrometry or ELISA indicated that three subtypes, Prx1, Prx2 and Prx4, increased production of proinflammatory mediators, including nitric oxide (NO) metabolites, tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) in a concentration-dependent manner. Immunostaining demonstrated that 20 nM Prx1, Prx2 or Prx4 significantly increased expression of TLR4 and iNOS and nuclear translocation of NF-κB p65. However, Prx5 and Prx6 showed no poinflammatory effect in macrophages. Remarkably, LIG treatment effectively inhibited the inflammatory response induced by Prx1, Prx2 and Prx4. CONCLUSION: Three members of Prxs, Prx1, Prx2 and Prx4, are inflammatory DAMPs that induce TLR4 activation and inflammatory response in macrophages, which is effectively inhibited by LIG. These results suggest that inflammatory Prxs-activated macrophages may provide a novel cellular model for screening the potential inhibitors of DAMPs-associated inflammatory diseases such as stroke. Moreover, selective blocking strategies targeting the inflammatory subtypes of Prxs probably provide promising therapeutic approaches with a prolonged time window for stroke.

[Construction and identification of SPARC shRNA lentiviral vector and its expression in human SKM-1 cells].

AIM: To construct a lentiviral vector expressing small-hairpin RNA(shRNA) targeting SPARC gene and investigate its silenced effect on SPARC in human myelodysplastic syndromes(MDS) cell line SKM-1. METHODS: The targeting sequence of SPARC gene which can be effectively silenced in RNA interference was confirmed in our previous study. The designed and synthesized single-stranded primers were annealed to double-stranded oligo sequences and subcloned into linear pGCSIL-GFP lentiviral plasmid digested by enzyme Age I and EcoR I to produce GC-shSPARC lentiviraL vector. After being identified by PCR and sequencing, plasmids GC-shSPARC with pHelper 1.0 and pHelper 2.0 were cotransfected into 293T cells to package lentiviral particles. The recombinant lentiviral vector was transfected into human SKM-1 cells, transfection efficiency was evaluated with expression of green fluorescent protein(GFP) determined by fluorescent microscope. Expression of SPARC in SKM-1 cells was detected using RT-PCR and Western blotting. RESULTS: A recombinant lentiviral vector, GC-shSPARC, expressing shRNAs targeting SPARC gene was constructed and confirmed by DNA sequencing. The recombinant lentivirus was harvested from 293T cells with a viral titer of 1×10(9); TU/mL. GFP was observed in the 70% of SKM-1 cells after transfection. Expression of SPARC mRNA and protein was significantly reduced in the GC-shSPARC transfected group than that in the control group (P<0.05). CONCLUSION: The lentivirus RNAi vector targeting SPARC has been successfully constructed, and can effectively inhibit the expression of SPARC in SKM-1 cell line, which shed light on the foundation for researching the inhibition of SPARC siRNA target against human MDS cells proliferation, induction apoptosis and gene therapy.