Caspase-3 mRNA expression in rats with apoptosis of retinal photoreceptor cells

BACKGROUND: Apoptosis of retinal photoreceptor cells is a commonly pathological characteristic of various eye diseases, while caspase-3 is an important regulating gene and plays a key role in apoptosis. OBJECTIVE: To measure the expression of caspase-3 mRNA in rats with apoptosis of retinal photoreceptor cells by using real-time fluorescent quantitative polymerase chain reaction and compare with those of the normal rats. DESIGN: Randomized controlled animal study. SETTING: Zhongshan Ophthalmological Center of Sun Yat-sen University. MATERIALS: A total of 36 female SD rats of 50 days old and clean grade and weighing (150±10) g were provided by Experimental Animal Center of Northern Area of Sun Yat-sen University. All rats were randomly divided into normal control group (n =6) and N-methyl-N-nitrosourea (MNU) group (n =30), and they were observed at 12 hours, 1, 2, 3 and 5 days after model establishment, with 6 rats at each time point. METHODS: The experiment was carried out at Zhongshan Ophthalmological Center, Key Laboratory of Ophthalmology by State Ministry of Education from March to December 2004. Rats in the normal control group were intraperitoneally injected with saline and rats in the MNU group were intraperitoneally injected with 40 mg/kg MNU. And then, retinal photoreceptor injured models were established. At 12 hours, 1, 2, 3 and 5 days after model establishment, the rats were sacrificed for enucleating right eyeballs, isolating retina immediately and extracting total RNA. The expression of caspase-3 mRNA in retina was measured with real-time fluorescent quantitative polymerase chain reaction. MAIN OUTCOME MEASURES: Expression of caspase-3 mRNA in retina of rats in the two groups. RESULTS: A total of 36 SD rats were involved in the final analysis. The expressions of caspase-3 mRNA in the rat retina of both groups at the five time points (12 hours, 1, 2, 3 and 5 days) after model establishment were 1.52×105, 18.35×105, 25.14×105, 29.25×105, 13.72×105 and 12.24×105, respectively. The expression of caspase-3 mRNA in the MNU group increased after 12 hours of intraperitoneal injection, and rose to the top on the 2nd day, which was 19 times as many as that of the normal control group. Then, it decreased gradually and was still 8 times as many as that of the normal control group on the 5th day. CONCLUSION: The expression of caspase-3 mRNA is related to apoptosis of retinal photoreceptor cells, while caspase-3 plays an important role in occurrence and development of apoptosis of retinal photoreceptor cells.     

Endogenous overproduction of β-amyloid induces tau hyperphosphorylation and decreases the solubility of tau in N2a cells

Summary. Although neurofibrillary tangles and senile plaques have been identified as the hallmark pathological changes in the brain of Alzheimer’s disease (AD), the relationship between them is still not fully understood. In the present study, we have studied the effect of endogenously overproduced amyloid β (Aβ) on tau by using wild type amyloid precursor protein (APP) transfected (N2a/APP695), or Swedish mutant APP plus Δ9 deleted presenilin-1 co-transfected (N2a/APPswe.Δ9) and APP vector transfected (N2a/vector) cell lines. We measured the secreted and intracellular Aβ, including Aβ_1–40 and Aβ_1–42, by Sandwich ELISA assay. It was shown that the levels of Aβ were increased time-dependently in N2a/APP695 and N2a/APPswe.Δ9 but not in N2a/vector upon butyric acid (BA) treatment. Compared with N2a/vector cells, tau in N2a/APP695 and N2a/APPswe.Δ9 cells was not extracted by RIPA buffer, and the SDS-extracted tau protein was hyperphosphorylated at Tau-1 and PHF-1 epitopes upon BA treatment. Obvious accumulation of the hyperphosphorylated tau in N2a/APP695 and N2a/APPswe.Δ9 cells was observed at 48 h after BA treatment. The total level of the extracted tau was reduced in N2a/APP695 and N2a/APPswe.Δ9 lines compared with N2a/vector cells by Western blot, and this reduction of total tau was also detected by immunofluorescence staining. No obvious alteration of tau mRNA was observed in both N2a/APP695 and N2a/APPswe.Δ9 cells compared with N2a/vector. This study provides direct evidence demonstrating that endogenously overproduced Aβ not only induces tau hyperphosphorylation but also decreases the level and solubility of tau in N2a cell lines. The first and second authors contributed equally to the paper     

Identification of the miRNA–mRNA regulatory network in multiple sclerosis

Objectives: Multiple sclerosis (MS) is the common neurological disorders in young adults, which affects the central nervous system myelin or oligodendrocytes and results in disability. This study aimed to identify the key miRNAs in blood of patients in MS for better understanding the underlying mechanisms of MS.

Methods: The publicly available Gene Expression Omnibus data-sets of MS were performed to integrated analysis. miRNA expression and mRNA expression were analyzed in whole blood samples from patients with MS and healthy controls by microarray analysis, Gene Ontology enrichment analyses, Kyoto Encyclopedia of Genes and Genomes pathway analyses, construction of miRNA–mRNA interaction network, and quantitative real-time polymerase reaction.

Results: In patients with MS, microarray analysis identified 45 significantly dysregulated miRNAs and 621 significantly dysregulated mRNAs. 1165 negative correlation pairs of miRNA–mRNA were predicted and used to construct the interaction network. hsa-miR-30a, hsa-miR-93, hsa-miR-20b, and hsa-miR-20a occurred as central hubs regulating 87, 38, 34, and 34 genes. Dysregulated mRNAs were significantly enriched in ribosome, tuberculosis, and pathways in cancer. The verification of qRT-PCR displayed that hsa-miR-328-3p was significantly up-regulated in MS and its target genes RAC2 had the down-regulated tendency in MS. hsa-miR-20a-5p had the up-regulated tendency and the corresponding target gene EIF4EBP2 had the down-regulated tendency in MS compared to healthy controls.

Discussion: hsa-miR-30a, hsa-miR-93, hsa-miR-20b, and hsa-miR-20a might be the key participant in the pathophysiology of MS involved in signaling pathways including ribosome, tuberculosis, and pathways in cancer.     

The role of receptor protein tyrosine phosphatase α in neuronal differentiation of embryonic stem cells

In the present study, we have investigated the function of the receptor protein tyrosine phosphatase α (RPTPα) in the neuronal differentiation of E14-embryonic stem (E14-ES) cells. RNAase protection and western blot analysis revealed that E14-ES cells up regulate RPTPα expression upon neuronal differentiation with retinoic acid. Overexpression of RPTPα, by stable DNA transfection, and subsequent differentiation with retinoic acid, resulted in a temporally enhanced expression of the neuronal markers GAP-43 and NF-164. Electrophysiological experiments demonstrated that RPTPα overexpression also enhanced the development of neurotransmitter responses during differentiation. These results indicate that RPTPα plays an important role in the cascade of molecular events that lead to the formation of neurons.     

Muscarinic agonists reduce tau phosphorylation in non-neuronal cells via GSK-3β inhibition and in neurons

Summary. Muscarinic agonists alter the metabolism of amyloid precursor protein, leading to an increase in α-secretase cleavage and a decreased production of amyloidogenic peptides; suggesting that these compounds might modify the Alzheimer's disease process. A second therapeutic target in AD is the accumulation of stably phosphorylated tau into neurofibrillary tangles; an early event correlating with cognitive impairment. Glycogen synthase kinase-3 (GSK-3β) phosphorylates tau and is inhibited via protein kinase C (PKC). As certain muscarinic receptors are linked to PKC, we examined the effect of a range of agonists on GSK-3β phosphorylation of tau. In neurons a non-specific muscarinic agonist, carbachol, reduced tau phosphorylation. In non-neuronal cells expressing the m1 receptor a range of m1 agonists reduced transiently-expressed tau phosphorylation and altered its microtubule-binding properties. These findings link the two pathological process of AD – APP metabolism and tau phosphorylation – and suggest that muscarinic and other cholinergic compounds might have disease-modifying properties. Received December 10, 1999; accepted March 14, 2000     

Assessment of microRNA-related SNP effects in the 3′ untranslated region of the IL22RA2 risk locus in multiple sclerosis

Recent large-scale association studies have identified over 100 MS risk loci. One of these MS risk variants is single-nucleotide polymorphism (SNP) rs17066096, located ~14 kb downstream of IL22RA2. IL22RA2 represents a compelling MS candidate gene due to the role of IL-22 in autoimmunity; however, rs17066096 does not map into any known functional element. We assessed whether rs17066096 or a nearby proxy SNP may exert pathogenic effects by affecting microRNA-to-mRNA binding and thus IL22RA2 expression using comprehensive in silico predictions, in vitro reporter assays, and genotyping experiments in 6,722 individuals. In silico screening identified two predicted microRNA binding sites in the 3′UTR of IL22RA2 (for hsa-miR-2278 and hsa-miR-411-5p) encompassing a SNP (rs28366) in moderate linkage disequilibrium with rs17066096 (r ²?=?0.4). The binding of both microRNAs to the IL22RA2 3′UTR was confirmed in vitro, but their binding affinities were not significantly affected by rs28366. Association analyses revealed significant association of rs17066096 and MS risk in our independent German dataset (odds ratio ?=?1.15, P?=?3.48?×?10^?4), but did not indicate rs28366 to be the cause of this signal. While our study provides independent validation of the association between rs17066096 and MS risk, this signal does not appear to be caused by sequence variants affecting microRNA function.     

Co-localization of α-synuclein and phosphorylated tau in neuronal and glial cytoplasmic inclusions in a patient with multiple system atrophy of long duration

Neuronal and glial cytoplasmic inclusions (NCIs and GCIs), which contain alpha-synuclein as a major component, are characteristic cytopathological features of multiple system atrophy (MSA). We report MSA of 19 years' duration in a 73-year-old woman. Her initial symptom was parkinsonism, with dementia appearing about 8 years later. Postmortem examination showed marked atrophy of the frontal and temporal white matter and limbic system, in addition to the pathology typical of MSA. In the limbic system, severe neuronal loss and astrocytosis were observed, and the remaining neurons often had lightly eosinophilic, spherical cytoplasmic inclusions. Interestingly, a double-labeling immunofluorescence study revealed that the NCIs in the dentate gyrus and amygdaloid nucleus, and the GCIs in the frontal and temporal white matter often expressed both alpha-synuclein NACP-5 and phosphorylated tau AT8 epitopes. Double-immunolabeling electron microscopy of the NCIs in the dentate gyrus and the GCIs in the temporal white matter clearly revealed labeling of their constituent granule-associated filaments with NACP-5, and some of them were also labeled with AT8. These findings strongly suggested that some alpha-synuclein filaments were decorated with phosphorylated tau without formation of fibrils such as paired helical filaments. Immunoblotting of sarkosyl-insoluble tau indicated that the accumulated tau consisted mainly of four-repeat tau isoforms of 383 amino acids and 412 amino acids. We consider that the limbic system can be a major site of neurodegeneration in MSA of long duration. The mechanisms of such abnormal tau accumulation in the NCIs and GCIs are unknown.     

Cytokine immunoreactivity in cortical and subcortical neurons in periventricular leukomalacia: are cytokines implicated in neuronal dysfunction in cerebral palsy?

The major neuropathological substrate associated with cerebral palsy (CP) is a form of white matter (WM) injury known as periventricular leukomalacia (PVL). Proinflammatory cytokines were recently shown to be implicated in PVL pathogenesis. Many PVL patients develop cortical and deep gray neuronal dysfunctions such as epilepsy, cognitive deficits and extrapyramidal disorders. The precise nature of the relationship between the WM lesion and the subsequent neuronal disorders is unclear. Cytokines were shown to exert neurotoxicity in experimental models. This raises the need to investigate a possible noxious effect by cytokines on neuronal cortical development. In situ immunohistochemical methods were applied on 22 brains from infants both with PVL (study group) and without PVL (control group) to detect any immunoreactivity for cytokines (TNF-alpha, IL-1beta, IL-6) in cortical and gray matter neurons. While cortical and other neuronal structures in PVL brains did not display noticeable pathological anomalies, strong cytokine immunoreactivity was detected in many neurons in the neocortex, hippocampus, basal ganglia and thalamus. There were, however, regional differences in cytokine labeling. In addition, there was more TNF-alpha staining than IL-1beta; IL-6 was negative. In contrast, neuronal cytokine labeling in the "control" brains was negligible. In conclusion, we report and characterize, for the first time, the in situ immunoreactivity for proinflammatory cytokines in cortical and deep gray neurons in PVL. These findings might provide insights into the neuro-anatomical correlate for the intellectual deficits and the other cortical and deep gray neuronal dysfunctions associated with PVL.     

Aβ exacerbates the neuronal dysfunction caused by human tau expression in a Drosophila model of Alzheimer's disease

Alzheimer's disease (AD) is characterised by neurofibrillary tangles composed of hyper-phosphorylated tau, and neuritic plaques composed of misfolded amyloid peptide (Aβ₄₂). It is generally believed that the hyper-phosphorylated tau and oligomeric Aβ₄₂ are responsible for the neuronal dysfunction and cognitive impairments that underlie the early stages of AD, but the mechanism by which they interact in the pathogenic process is not clear. Mounting evidence suggests that Aβ₄₂ pathology lies upstream of hyper-phosphorylated tau pathology. Similarly much is being learnt about how each protein affects neuronal function. However, the impact that either pathological protein has on neuronal dysfunction caused by the other is not extensively studied. We have investigated this in a Drosophila model of AD in which we express both phosphorylated human tau (tau_wt) and oligomeric Aβ₄₂. We find that expression of tau_wt causes neuronal dysfunction by disrupting axonal transport and synaptic structure, and that this leads to behavioural impairments and reduced lifespan. Co-expression of Aβ₄₂ with tau_wt increases tau phosphorylation and exacerbates all these tau-mediated phenotypes. Treatment of tau_wt/Aβ₄₂ and flies with LiCl ameliorates the exacerbating effect of Aβ₄₂, suggesting that GSK-3β may be involved in the mechanism by which Aβ₄₂ and tau_wt interact to cause neuronal dysfunction. Conversely to the effect of Aβ₄₂, mimicking the wingless signalling pathway by co-expression of dishevelled with tau_wt reduces tau phosphorylation and suppresses the tau-mediated phenotypes. It is therefore possible to speculate that the mechanism by which Aβ₄₂ interacts with tau in the pathogenesis of AD is by down-regulating endogenous wnt signalling.     

The effect of amyloidosis-β and ageing on proliferation of neuronal progenitor cells in APP-transgenic mouse hippocampus and in culture

Stimulation of endogenous neurogenesis and transplantation of neuronal progenitors (NPs) are considered in therapy of neuronal loss associated with ageing and in neurodegenerative diseases with amyloidosis-beta, for example, Alzheimer's disease and Down syndrome. However, the influence of brain environment altered by ageing and deposits of amyloid-beta on proliferation of endogenous and transplanted NPs and their maturation into neurons is not understood. We studied the effect of ageing and development of amyloidosis-beta on proliferation of NPs (1) in the granular layer of dentate gyrus in the hippocampi of APP-transgenic mice (Tg9291) before and after development of amyloidosis-beta, that is, in mice aged 2-4 months and 9-12 months, respectively, and in age-matched controls; and (2) in culture of NPs isolated from brains of control and Tg9291 mice, aged 3 and 9 months. We found that the number of proliferating NPs was reduced in 9-12-months-old mice, in both control and Tg9291, as compared to 2-4-months-old mice. However, the 9-12-months-old Tg9291 mice with amyloid-beta deposits had significantly more proliferating NPs than the age-matched controls. NPs proliferation in culture did not depend on the age, presence of APP-transgene, and amyloidosis-beta in donors. The results indicate that the local brain environment influences proliferation of NPs, and development of amyloidosis-beta in the neurogenic regions attenuates the age-associated reduction of proliferation of NPs. Identification of the responsible mechanisms may be important for development of a successful therapy of neurodegeneration caused by amyloidosis-beta.     

Indirubin-3′-monoxime suppresses amyloid-beta-induced apoptosis by inhibiting tau hyperphosphorylation

Indirubin-3′-monoxime is an effective inhibitor of cyclin-dependent protein kinases, and may play an obligate role in neuronal apoptosis in Alzheimer's disease. Here, we found that indirubin-3′-monoxime improved the morphology and increased the survival rate of SHSY5 Y cells exposed to amyloid-beta 25–35(Aβ25–35), and also suppressed apoptosis by reducing tau phosphorylation at Ser199 and Thr205. Furthermore, indirubin-3′-monoxime inhibited phosphorylation of glycogen synthase kinase-3β(GSK-3β). Our results suggest that indirubin-3′-monoxime reduced Aβ25–35-induced apoptosis by suppressing tau hyperphosphorylation via a GSK-3β-mediated mechanism. Indirubin-3′-monoxime is a promising drug candidate for Alzheimer's disease.     

Variant in the 3′ region of SNCA associated with Parkinson’s disease and serum α-synuclein levels

Parkinson's disease (PD) is the second most common neurodegenerative disorder. The presence of Lewy bodies is a major pathological change of PD. α-synuclein is the main component of Lewy bodies and is encoded by the SNCA gene. Mutations in the SNCA gene mainly result in rare familial forms of PD, while genetic variability in the SNCA gene modulates susceptibility to sporadic PD. Recent studies have suggested that levels of α-synuclein in extracellular biological fluid are associated with PD and implicated α-synuclein as a potential biomarker for PD diagnosis and severity. We studied serum α-synuclein concentration and two polymorphic variants of SNCA (Rep1 and rs11931074) in 110 sporadic PD patients and 136 controls. We further explored the influence of the two polymorphisms on the expression levels of serum α-synuclein. Soluble α-synuclein was detected in serum in all subjects, with no statistically significant difference between PD patients and controls (p?=?0.611). Different Rep1 alleles and genotypes did not influence the expression of serum α-synuclein. The frequency of allele T of rs11931074 was significantly elevated in PD patients (p?=?0.041), and was correlated with decreased serum α-synuclein in both dominant (p?=?0.011) and additive (p?=?0.008) models of association.     

SNCA 3′UTR genetic variants in patients with Parkinson’s disease and REM sleep behavior disorder

REM sleep behavior disorder (RBD) is an early marker of Parkinson’s disease (PD); however, it is still unclear which patients with RBD will eventually develop PD. Single nucleotide polymorphisms (SNPs) in the 3′untranslated region (3′UTR) of alpha-synuclein (SNCA) have been associated with PD, but at present, no data is available about RBD. The 3′UTR hosts regulatory regions involved in gene expression control, such as microRNA binding sites. The aim of this study was to determine RBD specific genetic features associated to an increased risk of progression to PD, by sequencing of the SNCA-3′UTR in patients with “idiopathic” RBD (iRBD) and in patients with PD. We recruited 113 consecutive patients with a diagnosis of iRBD (56 patients) or PD (with or without RBD, 57 patients). Sequencing of SNCA-3′UTR was performed on genomic DNA extracted from peripheral blood samples. Bioinformatic analyses were carried out to predict the potential effect of the identified genetic variants on microRNA binding. We found three SNCA-3′UTR SNPs (rs356165, rs3857053, rs1045722) to be more frequent in PD patients than in iRBD patients (p = 0.014, 0.008, and 0.008, respectively). Four new or previously reported but not annotated specific genetic variants (KP876057, KP876056, NM_000345.3:c*860T>A, NM_000345.3:c*2320A>T) have been observed in the RBD population. The in silico approach highlighted that these variants could affect microRNA-mediated gene expression control. Our data show specific SNPs in the SNCA-3′UTR that may bear a risk for RBD to be associated with PD. Moreover, new genetic variants were identified in patients with iRBD.     

Encapsulated VEGF-secreting cells enhance proliferation of neuronal progenitors in the hippocampus of AβPP/Ps1 mice

Vascular endothelial growth factor (VEGF) promotes neurogenesis in the adult hippocampus, but the way in which this process occurs in the Alzheimer's disease (AD) brain is still unknown. We examined the proliferation of neuronal precursors with an ex vivo approach, using encapsulated VEGF secreting cells, in AβPP/PS1 mice, a mouse model of AD. Overexpression of VEGF and VEGF receptor flk-1 was observed in the cerebral cortex from VEGF microcapsules-treated AβPP/PS1 mice at 1, 3 and 6 months after VEGF-microcapsule implantation. Stereological counting of 5-bromodeoxyuridine positive cells revealed that encapsulated VEGF secreting cells significantly enhanced cellular proliferation in the hippocampal dentate gyrus (DG). The number of neuronal precursors in VEGF microcapsules-treated AβPP/PS1 mice was also greater, and this effect remains after 6 months. We also confirmed that encapsulated VEGF secreting cells also stimulated angiogenesis in the cerebral cortex and hippocampal dentate gyrus. In addition, we found that VEGF-microcapsule treatment was associated with a depressed expression and activity of acetylcholinesterase in the hippocampus of AβPP/PS1 mice, a similar pattern as first-line medications for the treatment of AD. We conclude that stereologically-implanted VEGF-microcapsules exert an acute and long-standing neurotrophic effects, and could be utilized to improve potential therapies to control the progression of AD.     

Neuroprotective effects of ZL006 in Aβ_(1–42)-treated neuronal cells

Amyloid beta(Aβ)-induced neurotoxicity and oxidative stress plays an important role in the pathogenesis of Alzheimer's disease(AD). ZL006 is shown to reduce over-produced nitric oxide and oxidative stress in ischemic stroke by interrupting the interaction of neuronal nitric oxide synthase and postsynaptic density protein 95. However, few studies are reported on the role of ZL006 in AD. To investigate whether ZL006 exerted neuroprotective effects in AD, we used Aβ_(1–42) to treat primary cortical neurons and N2a neuroblastoma cells as an in vitro model of AD. Cortical neurons were incubated with ZL006 or dimethyl sulfoxide for 2 hours and treated with Aβ_(1–42) or NH3·H_2O for another 24 hours. The results of cell counting Kit-8(CCK-8) assay and calcein-acetoxymethylester/propidium iodide staining showed that ZL006 pretreatment rescued the neuronal death induced by Aβ_(1–42). Fluorescence and western blot assay were used to detect oxidative stress and apoptosis-related proteins in each group of cells. Results showed that ZL006 pretreatment decreased neuronal apoptosis and oxidative stress induced by Aβ_(1–42). The results of CCK8 assay showed that inhibition of Akt or NF-E2-related factor 2(Nrf2) in cortical neurons abolished the protective effects of ZL006. Moreover, similar results were also observed in N2a neuroblastoma cells. ZL006 inhibited N2a cell death and oxidative stress induced by Aβ_(1–42), while inhibition of Akt or Nrf2 abolished the protective effect of ZL006. These results demonstrated that ZL006 reduced Aβ_(1–42)-induced neuronal damage and oxidative stress, and the mechanisms might be associated with the activation of Akt/Nrf2/heme oxygenase-1 signaling pathways.     

Drug discovery targeted to the Alzheimer’s APP mRNA 5′-untranslated region

We screened for drugs that specifically interact with the 5′-untranslated region of the mRNA encoding the Alzheimer’s amyloid precursor protein (APP). Our goal was to use newly discovered APP 5′ UTR directed compounds to limit amyloid-β (Aβ)-peptide output in cell culture systems. The APP 5′ UTR folds into a stable RNA secondary structure (Gibbs free energy: ΔG=−54.9 kcal/mol) and is an important regulator of the amount of APP translated in response to IL-1 (Nilsson et al., 1998; Rogers et al., 1999) and iron (Rogers et al., 2002). Seventeen drug “hits” were identified from a library of 1,200 FDA preapproved drugs (Rogers et al., 2002). Six of the original 17 compounds were validated for their capacity to suppress reporter gene expression in stable neuroblastoma transfectants expressing the dicistronic reporter construct shown in Fig. 2. These six leads suppressed APP 5′ UTR driven luciferase translation while causing no effect on the translation of dicistronic GFP gene translated from a viral IRES (negative control to ensure specificity during drug screens). In this report, we show that paroxetine (serotonin reuptake blocker) and dimercaptopropanol (Hg chelator) exerted significant effects on APP expression (steady-state levels of APP), whereas Azithromycin altered APP processing. None of these three compounds altered APLP-1 expression. In the future, we will identify further novel compounds that influence Aβ levels, either via translation inhibition or by changing the activity of proteins coupled between APP translation and APP processing.