Proteomic analysis of cellular models of neurodegeneration and mitochondrial dysfunction

Mitochondrial dysfunction is thought to contribute to neurodegenerative processes. As an example, dysfunction of complex I of the electron transport chain has been observed in Parkinson’s disease patients and 1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine (MPTP), a complex I inhibitor, produces a Parkinsonian state in mammals. The aims of the present study were to determine the effects of MPTP on the mitochondrial proteome in a cellular model using mouse N2a neuroblastoma cells and to identify novel biomarkers of MPTP-induced toxicity. The enrichment of mitochondria and the presence of cross-contamination from other subcellular components were monitored using a range of molecular markers. Mitochondrial proteins were then fractionated using an optimised 2-dimensional gel electrophoresis (2DE) protocol and the reproducibility of the method was investigated. A preliminary study comparing the mitochondrial proteome profile from two different states of mouse N2a neuroblastoma cells, mitotic and differentiated, was undertaken to establish whether differentiation of cells had major effects on the mitochondrial proteome. Since nine proteins showed changes in levels, which included stress-70 protein and aconitase, it was decided that differentiation did affect the mitochondrial proteome: hence, differentiated cells were used for further studies. The effects of different concentrations (0 to 5 mM) and time-points (0 to 48 hours) of MPTP on plasma membrane integrity, cellular metabolic activity, cellular ATP concentration, mitochondrial potential, cytochrome c release and a variety of caspase activities were investigated. From this study, sub-cytotoxic and cytotoxic concentrations were defined and 1 mM MPTP for 24 hours was chosen as an example of a sub-cytotoxic concentration for further analysis. Using the previously optimised protocol for 2DE, mitochondrial preparations from differentiated N2a neuroblastoma cells treated with 1 mM MPTP for 24 hours were fractionated and compared to controls. Up to 32 proteins showed changes in protein levels, of which 10 were identified by peptide mass fingerprinting. Increases in the levels of 60 kDa heat shock protein (Hsp60), heat shock cognate 71 kDa protein (Hsc70), glutamate oxaloacetate 2 (GOT2) and voltage-dependent anion channel 1 (VDAC1) were validated as potential markers of MPTP-induced toxicity using western blot analysis. In parallel, a study of the mitochondrial phosphoproteome was undertaken. Despite the limitations of detection methods, a change in the phosphorylation status of a few mitochondrial proteins was observed following MPTP treatment, notably potential increased phosphorylation of Hsc70 and Hsp60. Further analysis was undertaken in order to gain a better understanding of the increase in VDAC1 levels following sub-cytotoxic treatments with MPTP. Although VDAC1 protein levels were increasing in a dose- and time-dependent manner, no mRNA upregulation was observed. Similarly, the use of other inhibitors of the electron transfer chain led to increased VDAC1 protein levels but no change in mRNA. Finally, modulations in VDAC1 phosphorylation were observed following MPTP-induced toxicity, further implicating the channel in mitochondrial dysfunction. To conclude, studies with this cellular model implicate the involvement of several mitochondrial pathways in the MPTP-induced Parkinsonian syndrome. In particular, alterations to VDAC1 may represent a novel target of neurodegeneration

Alterations in the mitochondrial proteome of neuroblastoma cells 2 in response to complex 1 inhibition

Increasing evidence points to mitochondrial dysfunction in Parkinson's disease (PD) associated with complex I dysfunction, but the exact pathways which lead to cell death have not been resolved. 2D-gel electrophoresis profiles of isolated mitochondria from neuroblastoma cells treated with subcytotoxic concentrations of l-methyl-4-phenyl-l,2,3,6-tetrahydropyridine (MPTP), a well-characterized complex I inhibitor, were assessed to identify associated targets. Up to 27 differentially expressed proteins were observed, of which 16 were identified using peptide mass fingerprinting. Changes in protein levels were validated by immunoprobing ID blots, confirming increases in heat shock cognate 71 kDa (Hsc70), 60 kDa heat shock protein (Hsp60), fumarase, glutamate oxaloacetate transaminase 2, ATP synthase subunit d, and voltage-dependent anion-channel 1 (VDACl). Immunoprobing of 2D blots revealed isoform changes in Hsc70, Hsp60, and VDACl. Subcytoxic concentrations of MPTP modulated a host of mitochondrial proteins including chaperones, metabolic enzymes, oxidative phosphorylation-related proteins, an inner mitochondrial protein (mitofilin), and an outer mitochondrial membrane protein (VDACl). Early changes in chaperones suggest a regulated link between complex 1 inhibition and protein folding. VDACl, a multifunctional protein, may have a key role in signaling between mitochondria and the rest of the cell prior to cell death. Our work provides new important information of relevance to PD

Depleted circulatory complement-lysis inhibitor (CLI) in childhood cerebral malaria returns to normal with convalescence

BACKGROUND: Cerebral malaria (CM), is a life-threatening childhood malaria syndrome with high mortality. CM is associated with impaired consciousness and neurological damage. It is not fully understood, as yet, why some children develop CM. Presented here is an observation from longitudinal studies on CM in a paediatric cohort of children from a large, densely-populated and malaria holoendemic, sub-Saharan, West African metropolis. METHODS: Plasma samples were collected from a cohort of children with CM, severe malarial anaemia (SMA), uncomplicated malaria (UM), non-malaria positive healthy community controls (CC), and coma and anemic patients without malaria, as disease controls (DC). Proteomic two-dimensional difference gel electrophoresis (2D-DIGE) and mass spectrometry were used in a discovery cohort to identify plasma proteins that might be discriminatory among these clinical groups. The circulatory levels of identified proteins of interest were quantified by ELISA in a prospective validation cohort. RESULTS: The proteome analysis revealed differential abundance of circulatory complement-lysis inhibitor (CLI), also known as Clusterin (CLU). CLI circulatory level was low at hospital admission in all children presenting with CM and recovered to normal level during convalescence (p < 0.0001). At acute onset, circulatory level of CLI in the CM group significantly discriminates CM from the UM, SMA, DC and CC groups. CONCLUSIONS: The CLI circulatory level is low in all patients in the CM group at admission, but recovers through convalescence. The level of CLI at acute onset may be a specific discriminatory marker of CM. This work suggests that CLI may play a role in the pathophysiology of CM and may be useful in the diagnosis and follow-up of children presenting with CM

A mutant wfs1 zebrafish model of Wolfram syndrome manifesting visual dysfunction and developmental delay

Funder: Fight for Sight UK; doi: http://dx.doi.org/10.13039/501100000615Funder: Wellcome Trust; doi: http://dx.doi.org/10.13039/100004440Abstract: Wolfram syndrome (WS) is an ultra-rare progressive neurodegenerative disorder defined by early-onset diabetes mellitus and optic atrophy. The majority of patients harbour recessive mutations in the WFS1 gene, which encodes for Wolframin, a transmembrane endoplasmic reticulum protein. There is limited availability of human ocular and brain tissues, and there are few animal models for WS that replicate the neuropathology and clinical phenotype seen in this disorder. We, therefore, characterised two wfs1 zebrafish knockout models harbouring nonsense wfs1a and wfs1b mutations. Both homozygous mutant wfs1a−/− and wfs1b−/− embryos showed significant morphological abnormalities in early development. The wfs1b−/− zebrafish exhibited a more pronounced neurodegenerative phenotype with delayed neuronal development, progressive loss of retinal ganglion cells and clear evidence of visual dysfunction on functional testing. At 12 months of age, wfs1b−/− zebrafish had a significantly lower RGC density per 100 μm2 (mean ± standard deviation; 19 ± 1.7) compared with wild-type (WT) zebrafish (25 ± 2.3, p < 0.001). The optokinetic response for wfs1b−/− zebrafish was significantly reduced at 8 and 16 rpm testing speeds at both 4 and 12 months of age compared with WT zebrafish. An upregulation of the unfolded protein response was observed in mutant zebrafish indicative of increased endoplasmic reticulum stress. Mutant wfs1b−/− zebrafish exhibit some of the key features seen in patients with WS, providing a versatile and cost-effective in vivo model that can be used to further investigate the underlying pathophysiology of WS and potential therapeutic interventions

Disturbed mitochondrial dynamics and neurodegenerative disorders

Mitochondria form a highly interconnected tubular network throughout the cell via a dynamic process, with mitochondrial segments fusing and breaking apart continuously. Strong evidence has emerged to implicate disturbed mitochondrial fusion and fission as central pathological components underpinning a number of childhood and adult-onset neurodegenerative disorders. Several proteins that regulate the morphology of the mitochondrial network have been identified, the most widely studied of which are optic atrophy 1 and mitofusin 2. Pathogenic mutations that disrupt these two pro-fusion proteins cause autosomal dominant optic atrophy and axonal Charcot-Marie-Tooth disease type 2A, respectively. These disorders predominantly affect specialized neurons that require precise shuttling of mitochondria over long axonal distances. Considerable insight has also been gained by carefully dissecting the deleterious consequences of imbalances in mitochondrial fusion and fission on respiratory chain function, mitochondrial quality control (mitophagy), and programmed cell death. Interestingly, these cellular processes are also implicated in more-common complex neurodegenerative disorders, such as Alzheimer disease and Parkinson disease, indicating a common pathological thread and a close relationship with mitochondrial structure, function and localization. Understanding how these fundamental processes become disrupted will prove crucial to the development of therapies for the growing number of neurodegenerative disorders linked to disturbed mitochondrial dynamic