A failure of mitochondrial bioenergetics has been shown to be closely associated with the onset of apoptotic and necrotic neuronal injury. Here, we developed an automated computational model that ...interprets the single-cell fluorescence for tetramethylrhodamine methyl ester (TMRM) as a consequence of changes in either delta psi(m) or delta psi(p), thus allowing for the characterization of responses for populations of single cells and subsequent statistical analysis. Necrotic injury triggered by prolonged glutamate excitation resulted in a rapid monophasic or biphasic loss of delta psi(m) that was closely associated with a loss of delta psi(p) and a rapid decrease in neuronal NADPH and ATP levels. Delayed apoptotic injury, induced by transient glutamate excitation, resulted in a small, reversible decrease in TMRM fluorescence, followed by a sustained hyperpolarization of delta psi(m) as confirmed using the delta psi(p)-sensitive anionic probe DiBAC2(3). This hyperpolarization of delta psi(m) was closely associated with a significant increase in neuronal glucose uptake, NADPH availability, and ATP levels. Statistical analysis of the changes in delta psi(m) or delta psi(p) at a single-cell level revealed two major correlations; those neurons displaying a more pronounced depolarization of delta psi(p) during the initial phase of glutamate excitation entered apoptosis more rapidly, and neurons that displayed a more pronounced hyperpolarization of delta psi(m) after glutamate excitation survived longer. Indeed, those neurons that were tolerant to transient glutamate excitation (18%) showed the most significant increases in delta psi(m). Our results indicate that a hyperpolarization of delta psi(m) is associated with increased glucose uptake, NADPH availability, and survival responses during excitotoxic injury.
Control of Motoneuron Survival by Angiogenin Kieran, Dairin; Sebastia, Jordi; Greenway, Matthew J ...
The Journal of neuroscience,
12/2008, Letnik:
28, Številka:
52
Journal Article
Recenzirano
Odprti dostop
Mutations in the hypoxia-inducible factor angiogenin (ANG) have been identified in Amyotrophic Lateral Sclerosis (ALS) patients, but the potential role of ANG in ALS pathogenesis was undetermined. ...Here we show that angiogenin promotes motoneuron survival both in vitro and in vivo. Angiogenin protected cultured motoneurons against excitotoxic injury in a PI-3-kinase/Akt kinase-dependent manner, whereas knock-down of angiogenin potentiated excitotoxic motoneuron death. Expression of wild-type ANG protected against endoplasmic reticulum (ER) stress-induced and trophic-factor-withdrawal-induced cell death in vitro, whereas the ALS-associated ANG mutant K40I exerted no protective activity and failed to activate Akt-1. In SOD1(G93A) mice angiogenin delivery increased lifespan and motoneuron survival, restored the disease-associated decrease in Akt-1 survival signaling, and reversed a pathophysiological increase in ICAM-1 expression. Our data demonstrate that angiogenin is a key factor in the control of motoneuron survival.
Amyotrophic lateral sclerosis (ALS) presents a poorly understood pathogenesis. Evidence from patients and mutant SOD1 mouse models suggests vascular damage may precede or aggravate motor dysfunction ...in ALS. We have previously shown angiogenin (ANG) treatment enhances motor neuron survival, delays motor dysfunction and prevents vascular regression in the SOD1
ALS model. However, the existence of vascular defects at different stages of disease progression remains to be established in other ALS models. Here, we assessed vascular integrity
throughout different disease stages, and investigated whether ANG treatment reverses vascular regression and prolongs motor neuron survival in the FUS (1-359) mouse model of ALS. Lumbar spinal cord tissue was collected from FUS (1-359) and non-transgenic control mice at postnatal day (P)50, P90 and P120. We found a significant decrease in vascular network density in lumbar spinal cords from FUS (1-359) mice by day 90, at which point motor neuron numbers were unaffected. ANG treatment did not affect survival or counter vascular regression. Endogenous
and
expression were unchanged at P50 and P90; however, we found a significant decrease in miRNA 126 at P50, indicating vascular integrity in FUS mice may be compromised via an alternative pathway. Our study demonstrates that vascular regression occurs before motor neuron degeneration in FUS (1-359) mice, and highlights that heterogeneity in responses to novel ALS therapeutics can already be detected in preclinical mouse models of ALS.This article has an associated First Person interview with the joint first authors of the paper.
Increased copy number alterations (CNAs) indicative of chromosomal instability (CIN) have been associated with poor cancer outcome. Here, we study CNAs as potential biomarkers of bevacizumab (BVZ) ...response in metastatic colorectal cancer (mCRC). We cluster 409 mCRCs in three subclusters characterized by different degrees of CIN. Tumors belonging to intermediate-to-high instability clusters have improved outcome following chemotherapy plus BVZ versus chemotherapy alone. In contrast, low instability tumors, which amongst others consist of POLE-mutated and microsatellite-instable tumors, derive no further benefit from BVZ. This is confirmed in 81 mCRC tumors from the phase 2 MoMa study involving BVZ. CNA clusters overlap with CRC consensus molecular subtypes (CMS); CMS2/4 xenografts correspond to intermediate-to-high instability clusters and respond to FOLFOX chemotherapy plus mouse avastin (B20), while CMS1/3 xenografts match with low instability clusters and fail to respond. Overall, we identify copy number load as a novel potential predictive biomarker of BVZ combination therapy.
The miRBase database is the central and official repository for miRNAs and the current release is miRBase version 21.0. Name changes in different miRBase releases cause inconsistencies in miRNA names ...from version to version. When working with only a small number of miRNAs the translation can be done manually. However, with large sets of miRNAs, the necessary correction of such inconsistencies becomes burdensome and error-prone. We developed miRNAmeConverter , available as a Bioconductor R package and web interface that addresses the challenges associated with mature miRNA name inconsistencies. The main algorithm implemented enables high-throughput automatic translation of species-independent mature miRNA names to user selected miRBase versions. The web interface enables users less familiar with R to translate miRNA names given in form of a list or embedded in text and download of the results.
The miRNAmeConverter R package is open source under the Artistic-2.0 license. It is freely available from Bioconductor ( http://bioconductor.org/packages/miRNAmeConverter ). The web interface is based on R Shiny and can be accessed under the URL http://www.systemsmedicineireland.ie/tools/mirna-name-converter/ . The database that miRNAmeConverter depends on is provided by the annotation package miRBaseVersions.db and can be downloaded from Bioconductor ( http://bioconductor.org/packages/miRBaseVersions.db ). Minimum R version 3.3.0 is required.
stefanhaunsberger@rcsi.ie.
Supplementary data are available at Bioinformatics online.
Activation of effector caspases is a final step during apoptosis. Single‐cell imaging studies have demonstrated that this process may occur as a rapid, all‐or‐none response, triggering a complete ...substrate cleavage within 15 min. Based on biochemical data from HeLa cells, we have developed a computational model of apoptosome‐dependent caspase activation that was sufficient to remodel the rapid kinetics of effector caspase activation observed in vivo. Sensitivity analyses predicted a critical role for caspase‐3‐dependent feedback signalling and the X‐linked‐inhibitor‐of‐apoptosis‐protein (XIAP), but a less prominent role for the XIAP antagonist Smac. Single‐cell experiments employing a caspase fluorescence resonance energy transfer substrate verified these model predictions qualitatively and quantitatively. XIAP was predicted to control this all‐or‐none response, with concentrations as high as 0.15 μM enabling, but concentrations >0.30 μM significantly blocking substrate cleavage. Overexpression of XIAP within these threshold concentrations produced cells showing slow effector caspase activation and submaximal substrate cleavage. Our study supports the hypothesis that high levels of XIAP control caspase activation and substrate cleavage, and may promote apoptosis resistance and sublethal caspase activation in vivo.
Loss of ionic homeostasis during excitotoxic stress depletes ATP levels and activates the AMP-activated protein kinase (AMPK), re-establishing energy production by increased expression of glucose ...transporters on the plasma membrane. Here, we develop a computational model to test whether this AMPK-mediated glucose import can rapidly restore ATP levels following a transient excitotoxic insult. We demonstrate that a highly compact model, comprising a minimal set of critical reactions, can closely resemble the rapid dynamics and cell-to-cell heterogeneity of ATP levels and AMPK activity, as confirmed by single-cell fluorescence microscopy in rat primary cerebellar neurons exposed to glutamate excitotoxicity. The model further correctly predicted an excitotoxicity-induced elevation of intracellular glucose, and well resembled the delayed recovery and cell-to-cell heterogeneity of experimentally measured glucose dynamics. The model also predicted necrotic bioenergetic collapse and altered calcium dynamics following more severe excitotoxic insults. In conclusion, our data suggest that a minimal set of critical reactions may determine the acute bioenergetic response to transient excitotoxicity and that an AMPK-mediated increase in intracellular glucose may be sufficient to rapidly recover ATP levels following an excitotoxic insult.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Mitochondrial alterations have been associated with the cytotoxic effect of 6-hydroxydopamine (6-OHDA), a widely used toxin to study Parkinson's disease. In previous work, we have demonstrated that ...6-OHDA increases mitochondrial membrane permeability leading to cytochrome c release, but the precise mechanisms involved in this process remain unknown. Herein we studied the mechanism of increased mitochondrial permeability of SH-SY5Y neuroblastoma cells in response to 6-OHDA. Cytochrome c release induced by 6-OHDA occurred, in both SH-SY5Y cells and primary cultures, in the absence of mitochondrial swelling or a decrease in mitochondrial calcein fluorescence, suggesting little involvement of the mitochondrial permeability transition pore in this process. In contrast, 6-OHDA-induced cell death was associated with a significant translocation of the pro-apoptotic Bax protein from the cytosol to mitochondria and with a significant induction of the BH3-only protein PUMA. Experiments in mouse embryonic fibroblasts deficient in Bax or PUMA demonstrated a role for both proteins in 6-OHDA-induced apoptosis. Although 6-OHDA elevated both total and nuclear p53 protein levels, activation of p53 was not essential for subsequent cell death. In contrast, we found that p38 mitogen-activated protein kinase (MAPK) was activated early during 6-OHDA-induced apoptosis, and that treatment with the p38 MAPK inhibitor SKF86002 potently inhibited PUMA induction, green fluorescent protein-Bax redistribution and apoptosis in response to 6-OHDA. These data demonstrate a critical involvement of p38 MAPK, PUMA, and Bax in 6-OHDA-induced apoptosis.
There is currently an urgent need to identify factors predictive of immunogenicity in colorectal cancer (CRC). Mucinous CRC is a distinct histological subtype of CRC, associated with a poor response ...to chemotherapy. Recent evidence suggests the commensal facultative anaerobe
Fusobacterium
may be especially prevalent in mucinous CRC. The objectives of this study were to assess the association of
Fusobacterium
abundance with immune cell composition and prognosis in mucinous CRC. Our study included two independent colorectal cancer patient cohorts, The Cancer Genome Atlas (TCGA) cohort, and a cohort of rectal cancers from the Beaumont RCSI Cancer Centre (BRCC). Multiplexed immunofluorescence staining of a tumour microarray (TMA) from the BRCC cohort was undertaken using Cell DIVE technology. Our cohorts included 87 cases (13.3%) of mucinous and 565 cases (86.7%) of non-mucinous CRC. Mucinous CRC in the TCGA dataset was associated with an increased proportion of CD8 + lymphocytes (
p
= 0.018), regulatory T-cells (
p
= 0.001) and M2 macrophages (
p
= 0.001). In the BRCC cohort, mucinous RC was associated with enhanced CD8 + lymphocyte (
p
= 0.022), regulatory T-cell (
p
= 0.047), and B-cell (
p
= 0.025) counts. High
Fusobacterium
abundance was associated with an increased proportion of CD4 + lymphocytes (
p
= 0.031) and M1 macrophages (
p
= 0.006), whilst M2 macrophages (
p
= 0.043) were under-represented in this cohort. Patients with increased
Fusobacterium
relative abundance in our mucinous CRC TCGA cohort tended to have better clinical outcomes (DSS: likelihood ratio
p
= 0.04, logrank
p
= 0.052).
Fusobacterium
abundance may be associated with improved outcomes in mucinous CRC, possibly due to a modulatory effect on the host immune response.
Key messages
• Increased
Fusobacterium
relative abundance was not found to be associated with microsatellite instability in mucinous CRC.
• Increased
Fusobacterium
relative abundance was associated with an M2/M1 macrophage switch, which is especially significant in mucinous CRC, where M2 macrophages are overexpressed.
• Increased
Fusobacterium
relative abundance was associated with a significant improvement in disease specific survival in mucinous CRC.
• Our findings were validated at a protein level within our own in house mucinous and non-mucinous rectal cancer cohorts.
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