Ruth F. Deighton

Neuronal development is promoted by weakened intrinsic antioxidant defences due to epigenetic repression of Nrf2

Forebrain neurons have weak intrinsic antioxidant defences compared with astrocytes, but the molecular basis and purpose of this is poorly understood. We show that early in mouse cortical neuronal development in vitro and in vivo, expression of the master-regulator of antioxidant genes, transcription factor NF-E2-related-factor-2 (Nrf2), is repressed by epigenetic inactivation of its promoter. Consequently, in contrast to astrocytes or young neurons, maturing neurons possess negligible Nrf2-dependent antioxidant defences, and exhibit no transcriptional responses to Nrf2 activators, or to ablation of Nrf2s inhibitor Keap1. Neuronal Nrf2 inactivation seems to be required for proper development: in maturing neurons, ectopic Nrf2 expression inhibits neurite outgrowth and aborization, and electrophysiological maturation, including synaptogenesis. These defects arise because Nrf2 activity buffers neuronal redox status, inhibiting maturation processes dependent on redox-sensitive JNK and Wnt pathways. Thus, developmental epigenetic Nrf2 repression weakens neuronal antioxidant defences but is necessary to create an environment that supports neuronal development.

Glioma Pathophysiology: Insights Emerging from Proteomics

Proteomics is increasingly employed in both neurological and oncological research to provide insight into the molecular basis of disease but rarely has a coherent, novel pathophysiological insight emerged. Gliomas account for >50% of adult primary intracranial tumors, with malignant gliomas (anaplastic astrocytomas and glioblastoma multiforme) being the most common. In glioma, the application of proteomic technology has identified altered protein expression but without consistency of these alterations or their biological significance being established. A systematic review of multiple independent proteomic analyses of glioma has demonstrated alterations of 99 different proteins. Importantly 10 of the 99 proteins found differentially expressed in glioma [PHB, Hsp20, serum albumin, epidermal growth factor receptor (EGFR), EA‐15, RhoGDI, APOA1, GFAP, HSP70, PDIA3] were identified in multiple publications. An assessment of protein–protein interactions between these proteins compiled using novel web‐based technology, revealed a robust and cohesive network for glioblastoma. The protein network discovered (containing TP53 and RB1 at its core) compliments recent findings in genomic studies of malignant glioma. The novel perspective provided by network analysis indicates that the potential of this technology to explore crucial aspects of glioma pathophysiology can now be realized but only if the conceptual and technical limitations highlighted in this review are addressed.

The Role of Mitochondria in Glioma Pathophysiology

It has long been recognised that malignant tumours favour aerobic glycolysis to generate ATP and contain abnormalities of the intrinsic, mitochondria-dependent, apoptotic pathway, suggesting the involvement of dysfunctional mitochondria in tumour pathophysiology. However, the mechanisms underlying such processes in gliomas are poorly understood. Few recent studies have evaluated mitochondrial ultrastructure and proteomics in the pathophysiology of malignant gliomas. However, aberrant energy metabolism has been reported in gliomas and mitochondrial dysfunction links to glioma apoptotic signalling have been observed. Mitochondrial structural abnormalities and dysfunction in malignant gliomas is a neglected area of research. Definition of abnormalities in mitochondrial proteomics, membrane potential regulation, energy metabolism and intrinsic apoptotic pathway signalling in gliomas may open novel therapeutic opportunities.

Proteomic analysis of mitochondria in APOE transgenic mice and in response to an ischemic challenge

Apolipoprotein E (APOE)-ɛ4 is associated with a deleterious outcome after ischemic brain injury, which may involve abnormal regulation of mitochondrial function. We have assessed the mitochondrial proteomic response of APOE-ɛ3 and APOE-ɛ4 transgenic mice to transient global ischemic injury in the hippocampus. A genotype-dependent increase in ApoE levels in mitochondria was observed after ischemia, with APOE-ɛ4 mice showing significantly greater increases than APOE-ɛ3 mice. Quantitative analysis of the mitochondria-enriched fractions was performed using liquid-chromatography mass spectrometry coupled to label-free analysis. Of the 1,067 identified proteins, 274 were mitochondria associated. Mitochondrial protein expression was significantly different between genotypes under basal conditions as well as in response to global ischemia. A total of 12 mitochondrial proteins (including respiratory chain proteins NDUFA11, NDUFS3, NDUF5B, ATP5J, as well as ETFA, CYB5B, ATP6V1A, HSPA1B, OXR1, GLUL, IARS2, and PHYHIPL) were significantly altered with respect to genotype, global ischemia, or their interaction (P<0.01). A compelling interactome, created using proteins found to be significantly modulated by global ischemia (P<0.05), involved proteins that regulate energy production and oxidative stress. Thus, APOE genotype has a differential effect on the mitochondrial protein expression in the absence and presence of an injury, which may underlie the differing genotype susceptibility.

Proteomic analysis of gliomas

Primary malignant brain tumours (anaplastic glioma and glioblastoma) display heterogenous histopathology and diverse genetic abnormalities. These tumours remain incurable with no significant improvement in median survival times in the last 20 years, despite significant technological advances in surgery and radiotherapy, and mechanistic insights into their aetiology. Recent clinical trials suggest molecular characterization of tumours is essential in guiding both therapy and predicting prognosis. Genetic insight into tumour biology and increasingly proteomic technology has opened new avenues for novel applied clinical research. Protein expression in human malignant glioma and matched normal brain tissues can now be reliably analysed using quantitative proteomic techniques, the most accessible of which is two-dimensional gel electrophoresis (2DGE) and matrix-assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometry from which differentially expressed proteins can be identified and characterized. The potential of using differential proteomic profiling in gliomas to identify prognostic markers and to gain insight into tumour biology is currently being investigated. The current status of proteomic technology, its application to gliomas and the utility of such translational studies is reviewed.

Network generation enhances interpretation of proteomic data from induced apoptosis

Thirteen proteins (identified with 2‐D gels and MALDI‐TOF MS) are significantly altered during staurosporine‐induced apoptosis in SH‐SY5Y cells. To gain further insight into the integrated cellular response to apoptosis, we have investigated whether a network can be generated of direct and indirect interactions between these 13 proteins. A network that contains 12 out of the 13 proteins was generated using Ingenuity Pathway Analysis (IPA) and this network is dominated (89%) by direct protein−protein interactions. This network scored 34 with IPA. Bootstrapping 1000 random lists of 13 proteins suggested that the frequency of this score occurring by chance was 1 in 500. We examined whether subsets of proteins such as HSPs, which were elevated after staurosporine, had a disproportionate impact on the network generated. There was no evidence that any subset of 8 from the 13 proteins contributed disproportionately to the network. Network generation, using IPA, identified common features (such as endoplasmic reticular stress protein interactions) in apoptotic studies from different laboratories. The generation of protein interaction networks clearly enhances the interpretation of proteomic data, but only when interpreted cautiously, particularly in respect of statistical analyses.

Adaptive changes in the neuronal proteome: mitochondrial energy production, endoplasmic reticulum stress, and ribosomal dysfunction in the cellular response to metabolic stress

Impaired energy metabolism in neurons is integral to a range of neurodegenerative diseases, from Alzheimers disease to stroke. To investigate the complex molecular changes underpinning cellular adaptation to metabolic stress, we have defined the proteomic response of the SH-SY5Y human neuroblastoma cell line after exposure to a metabolic challenge of oxygen glucose deprivation (OGD) in vitro. A total of 958 proteins across multiple subcellular compartments were detected and quantified by label-free liquid chromatography mass spectrometry. The levels of 130 proteins were significantly increased (P < 0.01) after OGD and the levels of 63 proteins were significantly decreased (P < 0.01) while expression of the majority of proteins (765) was not altered. Network analysis identified novel protein–protein interactomes involved with mitochondrial energy production, protein folding, and protein degradation, indicative of coherent and integrated proteomic responses to the metabolic challenge. Approximately one third (61) of the differentially expressed proteins was associated with the endoplasmic reticulum and mitochondria. Electron microscopic analysis of these subcellular structures showed morphologic changes consistent with the identified proteomic alterations. Our investigation of the global cellular response to a metabolic challenge clearly shows the considerable adaptive capacity of the proteome to a slowly evolving metabolic challenge.

Nrf2 target genes can be controlled by neuronal activity in the absence of Nrf2 and astrocytes

The transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) is a master regulator of antioxidant genes, and its activation in astrocytes confers noncell-autonomous neuroprotection. A recent interesting and provocative study published in PNAS proposes that neuronal activity stabilizes Nrf2 expression in astrocytes, leading to astrocytic Nrf2-mediated gene expression (1). However, we show here that it is possible for neuronal activity to activate expression of known Nrf2 target genes independently of both Nrf2 and of astrocytes. Habas et al. (1) observe that in mixed neuronal/astrocyte rat hippocampal cultures, electrical activity induced by GABAA receptor inhibition in the presence of the K+ channel antagonist 4-aminopyridine induces the known Nrf2 target genes Gclc (glutamate-cysteine ligase, catalytic subunit) and Nqo1 (NAD(P)H dehydrogenase, quinone 1). The authors attribute this gene induction to activation of Nrf2 signaling in astrocytes because it was stronger in a mixed culture (17% astrocytes) than in a neuronal culture with fewer (4%) astrocytes. However, the presence of a significant number of astrocytes in both preparations makes it difficult to make definitive conclusions. Moreover, in the absence of Nrf2 knockout/knock-down studies, it is not completely clear whether the induction of Gclc and Nqo1 was Nrf2-dependent, particularly as Nrf2 target genes can be controlled by other transcription factors. For example, we and colleagues have studied the transcriptional regulation of sulfiredoxin 1 ( Srxn1 ) and xCT (2, … [↵][1]2To whom correspondence should be addressed. E-mail: Giles.Hardingham{at}ed.ac.uk. [1]: #xref-corresp-1-1

The utility of functional interaction and cluster analysis in CNS proteomics.

Proteomic studies offer enormous potential for gaining insight into cellular dynamics and disease processes. An immediate challenge for enhancing the utility of proteomics in translational research lies in methods of handling and interpreting the large datasets generated. Publications rarely extend beyond lists of proteins, putatively altered derived from basic statistics. Here we describe two additional distinct approaches (with particular strengths and limitations) that will enhance the analysis of proteomic datasets. Arithmetic and functional cluster analyses have been performed on proteins found differentially regulated in human glioma. These two approaches highlight (i) subgroups of proteins that may be co-regulated and play a role in glioma pathophysiology, and (ii) functional protein interactions that may improve comprehension of the biological mechanisms involved. A coherent proteomic strategy which involves both arithmetic and functional clustering, (together with careful consideration of conceptual limitations), is imperative for quantitative proteomics to deliver and advance the biological understanding of disease of the CNS. A strategy which combines arithmetic analysis and bioinformatics of protein-protein interactions is both generally applicable and will facilitate the interpretation of proteomic data.

Proteomic data in meningiomas: post-proteomic analysis can reveal novel pathophysiological pathways

Meningiomas account for approximately 20% of adult primary intracranial tumours. WHO I meningiomas are the most common and are generally benign, but can progress, recur or transform to WHO II or WHO III grades over many years. A systematic review of multiple independent shotgun proteomic analyses of meningioma was performed to provide insight into underlying disease pathways. Shotgun proteomics has been conducted in seven meningioma related studies but there is considerable variation in aims, methodology, statistical power and the use of control tissue between these studies. Fifteen proteins which are different between WHO I and WHO II meningiomas and nine proteins which are different between WHO II and WHO III meningiomas have been described but without a view of their biological significance. Network analysis of proteins different between WHO I and WHO II meningiomas provided a coherent hypothesis for the involvement of these proteins in meningioma. Western blot analyses of meningioma tissue provided a measure of support for a core component in the network (involving VDAC2, APOA1 and HNF4α) but highlighted intrinsic difficulty of proteomic and biochemical analysis of meningiomas (as a consequence of gross alterations in tissue composition). Systematic review of shotgun proteomics and network analysis provides insight into meningioma pathophysiology despite the many barriers and difficulties that are inherent to this type of study.