Reflections on the 50-year history of the Federation of Asian and Oceanian Biochemists and Molecular Biologists (FAOBMB).

The Federation of Asian and Oceanian Biochemists and Molecular Biologists, Inc. (FAOBMB) celebrates its Golden Jubilee in 2022. Established in August 1972 as a regional grouping of three national societies of biochemists in Australia, India and Japan, it took the name Federation of Asian and Oceanian Biochemists (FAOB). The Federation rapidly grew to encompass another 12 national societies (or groups) of biochemists within 6 years, eventually increasing the number of Constituent Members to 21 by 2014. FAOB soon established regular scientific meetings, including triennial Congresses and annual Symposia; from 1980 FAOB Travel Fellowships enabled regional young scientists to participate in them. In 1992, FAOB was constituted as an Incorporated Association in Victoria, Australia, changing its name 1 year later (yielding the acronym FAOBMB). A printed Newsletter/Bulletin was distributed through each Constituent Society or Group from 1972 to 1999. With the advent of the internet and email in the late 1990s, communication rapidly improved, such that the first webpage of FAOBMB was set up in 1995. From the inception of the Federation, an international journal sponsored by FAOB was foreshadowed but only commenced in 1997, sadly lasting only 6 years. Education in biochemistry and molecular biology became prominent in FAOBMB from the 1990s. In the 21st century, awards to high-achieving scientists and educationists were introduced, the first being the Young Scientist Awards in 2006. The Fellowships program was extended to young educationists in 2018. FAOB(MB) has been supported by the International Union of Biochemistry (and Molecular Biology) almost its entire history, mostly for support of Congresses, Conferences and Symposia, but also for Young Scientist Programs. The most recent challenge to FAOBMB came with the COVID-19 pandemic. Executive Committee and the Constituent Members rapidly adapted to virtual communications for their administrative meetings and Education Symposia, and a memorable Congress was held totally on-line in 2021.

Inhibition of bioenergetics provides novel insights into recruitment of PINK1-dependent neuronal mitophagy.

Contributions of damaged mitochondria to neuropathologies have stimulated interest in mitophagy. We investigated triggers of neuronal mitophagy by disruption of mitochondrial energy metabolism in primary neurons. Mitophagy was examined in cultured murine cerebellar granule cells after inhibition of mitochondrial respiratory chain by drugs rotenone, 3-nitropropionic acid, antimycin A, and potassium cyanide, targeting complexes I, II, III, and IV, respectively. Inhibitor concentrations producing slow cellular demise were determined from analyses of cellular viability, morphology of neuritic damage, plasma membrane permeability, and oxidative phosphorylation. Live cell imaging of dissipation of mitochondrial membrane potential (ΔΨm ) by drugs targeting mitochondrial complexes was referenced to complete depolarization by carbonyl cyanide m-chlorophenyl hydrazone. While inhibition of complexes I, III and IV effected rapid dissipation of ΔΨm , inhibition of complex II using 3-nitropropionic acid led to minimal depolarization of mitochondria. Nonetheless, all respiratory chain inhibitors triggered mitophagy as indicated by increased aggregation of mitochondrially localized PINK1. Mitophagy was further analyzed using a dual fluorescent protein biosensor reporting mitochondrial relocation to acidic lysosomal environment. Significant acidification of mitochondria was observed in neurons treated with rotenone or 3-nitropropionic acid, revealing mitophagy at distal processes. Neurons treated with antimycin A or cyanide failed to show mitochondrial acidification. Minor dissipation of ΔΨm by 3-nitropropionic acid coupled with vigorous triggering of mitophagy suggested depolarization of mitochondria is not a necessary condition to trigger mitophagy. Moreover, weak elicitation of mitophagy by antimycin A, subsequent to loss of ΔΨm , suggested that mitochondrial depolarization is not a sufficient condition for triggering robust neuronal mitophagy. Our findings provide new insight into complexities of mitophagic clearance of neuronal mitochondria.

Mutant SOD1 inhibits ER-Golgi transport in amyotrophic lateral sclerosis.

Cu/Zn-superoxide dismutase is misfolded in familial and sporadic amyotrophic lateral sclerosis, but it is not clear how this triggers endoplasmic reticulum (ER) stress or other pathogenic processes. Here, we demonstrate that mutant SOD1 (mSOD1) is predominantly found in the cytoplasm in neuronal cells. Furthermore, we show that mSOD1 inhibits secretory protein transport from the ER to Golgi apparatus. ER-Golgi transport is linked to ER stress, Golgi fragmentation and axonal transport and we also show that inhibition of ER-Golgi trafficking preceded ER stress, Golgi fragmentation, protein aggregation and apoptosis in cells expressing mSOD1. Restoration of ER-Golgi transport by over-expression of coatomer coat protein II subunit Sar1 protected against inclusion formation and apoptosis, thus linking dysfunction in ER-Golgi transport to cellular pathology. These findings thus link several cellular events in amyotrophic lateral sclerosis into a single mechanism occurring early in mSOD1 expressing cells.

Mitochondrially localised MUL1 is a novel modulator of antiviral signaling.

The innate immune response to virus must be balanced to eliminate infection yet limit damaging inflammation. A critical arm of the antiviral response is launched by the retinoic acid-inducible-gene I (RIG-I) protein. RIG-I is activated by viral RNA then associates with the mitochondrial antiviral signaling (MAVS) protein to subsequently induce potent inflammatory cytokines. Here, we demonstrate the mitochondrial E3 ubiquitin protein ligase 1 (MUL1) is a crucial moderator of RIG-I signaling. MUL1 is localized to the mitochondria where it interacts with MAVS and catalyzes RIG-I post-translational modifications that inhibit RIG-I-dependent cell signaling. Accordingly, depletion of MUL1 potentiated RIG-I mediated nuclear factor-kappa B (NF-κB) and interferon (IFN) ß reporter activity. Moreover, depletion of MUL1 boosted the antiviral response and increased proinflammatory cytokines following challenge with the RNA mimetic poly I:C and Sendai virus. We therefore submit that MUL1 is a novel regulator of the RIG-I-like receptor-dependent antiviral response, that otherwise functions to limit inflammation.

Transitory phases of autophagic death and programmed necrosis during superoxide-induced neuronal cell death.

Neurons can undergo a diverse range of death responses under oxidative stress, encompassing apoptosis (caspase-dependent, programmed cell death) to various forms of caspase-independent death, including necrosis. We recently showed that primary murine cortical neurons exposed acutely to hydrogen peroxide undergo caspase-independent death, both autophagic cell death and programmed necrosis. To determine how oxidative stress induced by superoxide affects the route to cellular demise, we exposed primary cortical neurons to extended superoxide insult (provided by exogenous xanthine and xanthine oxidase in the presence of catalase). Under these conditions, over 24h, the nitroblue tetrazolium-reducing activity (indicative of superoxide) rose significantly during the first 4 to 8h and then declined to background levels. As with hydrogen peroxide, this superoxide insult failed to activate downstream caspases (-3, -7, and -9). Substantial depolarization of mitochondria occurred after 1h, and nuclear morphology changes characteristic of oxidative stress became maximal after 2h. However, death indicated by plasma membrane permeabilization (cellular uptake of propidium iodide) approached maximal levels only after 4h, at which time substantial redistribution to the cytosol of death-associated mitochondrial intermembrane space proteins, notably endonuclease G, had occurred. Applying established criteria for autophagic death (knockdown of Atg7) or programmed necrosis (knockdown of endonuclease G), cells treated with the relevant siRNA showed significant blockade of each type of cell death, 4h after onset of the superoxide flux. Yet at later times, siRNA-mediated knockdown failed to prevent death, monitored by cellular uptake of propidium iodide. We conclude that superoxide initially invokes a diverse programmed caspase-independent death response, involving transient manifestation in parallel of autophagic death and programmed necrosis. Ultimately most neurons become overwhelmed by the consequences of severe oxidative stress and die. This study reveals the multiple phases of neuronal cell death modalities under extended oxidative stress.

Application of flow cytometry to determine differential redistribution of cytochrome c and Smac/DIABLO from mitochondria during cell death signaling.

Mitochondrially mediated apoptosis is characterized by redistribution of proteins from mitochondria to cytoplasm following permeabilization of the outer mitochondrial membrane. We applied flow cytometry to quantify simultaneously the redistribution of two apoptogenic proteins, cytochrome c (cyt c) and Smac/DIABLO (Smac). Mammalian cells were treated with digitonin that selectively permeabilizes the plasma membrane. Following fixation, treated cells were infused successively with primary and secondary antibodies (the latter fluorescently tagged) enabling independent detection of cyt c and Smac. Digitonin-treated cells that retain cyt c or Smac in mitochondria generate strong fluorescence signals in flow cytometry. Cells in which cyt c or Smac have transited the outer mitochondrial membrane show greatly reduced fluorescence because the proteins are lost from the digitonin-permeabilized cells. Quantitative flow cytometry revealed that in 143B TK(-) cells treated with staurosporine, cyt c and Smac exit mitochondria asymmetrically, with cyt c redistribution preceding that of Smac. However, in HeLa cells likewise treated, cyt c and Smac exit mitochondria concurrently. Under other conditions of apoptotic induction, for example, 143B TK(-) cells treated with MT-21 (an apoptotic inducer that binds to the mitochondrial adenine nucleotide transporter), redistribution of Smac precedes that of cyt c. The various patterns of redistribution of these proteins were confirmed by immunocytochemical analysis and confocal microscopy. We conclude that flow cytometry can be employed effectively to quantify simultaneously the redistribution of cyt c and Smac from mitochondria to the cytosol. Moreover, differential redistribution of cyt c and Smac occurs under various conditions, thereby reflecting constraints on availability of these proteins to exit mitochondria after permeabilization of the outer membrane.

Bim links ER stress and apoptosis in cells expressing mutant SOD1 associated with amyotrophic lateral sclerosis.

Endoplasmic reticulum (ER) stress is an important pathway to cell death in amyotrophic lateral sclerosis (ALS). We previously demonstrated that ER stress is linked to neurotoxicity associated with formation of inclusions of mutant Cu,Zn-superoxide dismutase 1 (SOD1). Cells bearing mutant inclusions undergo mitochondrial apoptotic signalling. Here, we demonstrate that the BH3-only protein, Bim, is a direct link between ER stress and mitochondrial apoptosis. In the murine neuroblastoma cell line, Neuro2a, bearing mutant SOD1 inclusions, indicators of both ER stress and apoptosis are expressed. Bim knockdown by siRNA significantly reduced nuclear apoptotic features in these inclusion-bearing cells (but did not affect the proportion of cells overall that bear inclusions). Further, both Bax recruitment to mitochondria and cytochrome c redistribution were also decreased under Bim-depletion conditions. However, upregulation of CHOP, a marker of ER stress, was not reduced by Bim knockdown. Significantly, knockdown of CHOP by siRNA reduced the extent of apoptosis in cells bearing mutant SOD1 inclusions. These sequential links between ER stress, CHOP upregulation, and Bim activation of mitochondrial apoptotic signalling indicate a clear pathway to cell death mediated by mutant SOD1.

Identification of a novel gene product that promotes survival of Mycobacterium smegmatis in macrophages.

BACKGROUND: Bacteria of the suborder Corynebacterineae include significant human pathogens such as Mycobacterium tuberculosis and M. leprae. Drug resistance in mycobacteria is increasingly common making identification of new antimicrobials a priority. Mycobacteria replicate intracellularly, most commonly within the phagosomes of macrophages, and bacterial proteins essential for intracellular survival and persistence are particularly attractive targets for intervention with new generations of anti-mycobacterial drugs. METHODOLOGY/PRINCIPAL FINDINGS: We have identified a novel gene that, when inactivated, leads to accelerated death of M. smegmatis within a macrophage cell line in the first eight hours following infection. Complementation of the mutant with an intact copy of the gene restored survival to near wild type levels. Gene disruption did not affect growth compared to wild type M. smegmatis in axenic culture or in the presence of low pH or reactive oxygen intermediates, suggesting the growth defect is not related to increased susceptibility to these stresses. The disrupted gene, MSMEG_5817, is conserved in all mycobacteria for which genome sequence information is available, and designated Rv0807 in M. tuberculosis. Although homology searches suggest that MSMEG_5817 is similar to the serine:pyruvate aminotransferase of Brevibacterium linens suggesting a possible role in glyoxylate metabolism, enzymatic assays comparing activity in wild type and mutant strains demonstrated no differences in the capacity to metabolize glyoxylate. CONCLUSIONS/SIGNIFICANCE: MSMEG_5817 is a previously uncharacterized gene that facilitates intracellular survival of mycobacteria. Interference with the function of MSMEG_5817 may provide a novel therapeutic approach for control of mycobacterial pathogens by assisting the host immune system in clearance of persistent intracellular bacteria.

Modulation of cellular Hsp72 levels in undifferentiated and neuron-like SH-SY5Y cells determines resistance to staurosporine-induced apoptosis.

Increased expression of Hsp72 accompanies differentiation of human neuroblastoma SH-SY5Y cells to neuron-like cells. By modulating cellular levels of Hsp72, we demonstrate here its anti-apoptotic activity both in undifferentiated and neuron-like cells. Thermal preconditioning (43°C for 30 min) induced Hsp72, leading to cellular protection against apoptosis induced by a subsequent treatment with staurosporine. Preconditioned staurosporine-treated cells displayed decreased Bax recruitment to mitochondria and subsequent activation, as well as reduced cytochrome c redistribution from mitochondria. The data are consistent with Hsp72 blocking apoptosis upstream of Bax recruitment to mitochondria. Neuron-like cells (with elevated Hsp72) were more resistant to staurosporine by all measured indices of apoptotic signaling. Use of stable transfectants ectopically expressing moderately elevated levels of Hsp72 revealed that such cells in the undifferentiated state showed enhanced resistance to staurosporine-induced apoptosis, which was even more robust after differentiation to neuron-like cells. Overall, the protective effects of differentiation, thermal preconditioning and ectopic Hsp72 expression were additive. The strong inverse correlation between cellular Hsp72 levels and susceptibility to apoptosis support the notion that Hsp72 acts as a significant neuroprotective factor, enabling post-mitotic neurons to withstand potentially lethal stress that induces apoptosis.

Human neuroblastoma SH-SY5Y cells show increased resistance to hyperthermic stress after differentiation, associated with elevated levels of Hsp72.

PURPOSE: Terminally differentiated neurones in the central nervous system need to be protected from stress. We ask here whether differentiation of progenitor cells to neurones is accompanied by up-regulation of Hsp72, with acquisition of enhanced thermotolerance. MATERIALS AND METHODS: Human neuroblastoma SH-SY5Y cells were propagated in an undifferentiated form and subsequently differentiated into neurone-like cells. Thermotolerance tests were carried out by exposure of cells to various temperatures, monitoring nuclear morphology as index of cell death. Abundance of Hsp72 was measured in cell lysates by western immunoblotting. RESULTS: The differentiation of SH-SY5Y cells was accompanied by increased expression of Hsp72. Further, in both cell states, exposure to mild hyperthermic stress (43°C for 30 min) increased Hsp72 expression. After differentiation, SH-SY5Y cells were more resistant to hyperthermic stress compared to their undifferentiated state, correlating with levels of Hsp72. Stable exogenous expression of Hsp72 in SH-SY5Y cells (transfected line 5YHSP72.1, containing mildly elevated levels of Hsp72), led to enhanced resistance to hyperthermic stress. Hsp72 was found to be inducible in undifferentiated 5YHSP72.1 cells; such heat-treated cells displayed enhanced thermotolerance. Treatment of cells with KNK437, a suppressor of Hsp72 induction, resulted in acute thermosensitisation of all cell types tested here. CONCLUSIONS: Hsp72 has a major role in the enhanced hyperthermic resistance acquired during neuronal differentiation of SH-SY5Y cells. These findings model the requirement in intact organisms for highly differentiated neurones to be specially protected against thermal stress.

Autophagic activity in cortical neurons under acute oxidative stress directly contributes to cell death.

Primary neurons undergo insult-dependent programmed cell death. We examined autophagy as a process contributing to cell death in cortical neurons after treatment with either hydrogen peroxide (H(2)O(2)) or staurosporine. Although caspase-9 activation and cleavage of procaspase-3 were significant following staurosporine treatment, neither was observed following H(2)O(2) treatment, indicating a non-apoptotic death. Autophagic activity increased rapidly with H(2)O(2), but slowly with staurosporine, as quantified by processing of endogenous LC3. Autophagic induction by both stressors increased the abundance of fluorescent puncta formed by GFP-LC3, which could be blocked by 3-methyladenine. Significantly, such inhibition of autophagy blocked cell death induced by H(2)O(2) but not staurosporine. Suppression of Atg7 inhibited cell death by H(2)O(2), but not staurosporine, whereas suppression of Beclin 1 prevented cell death by both treatments, suggesting it has a complex role regulating both apoptosis and autophagy. We conclude that autophagic mechanisms are activated in an insult-dependent manner and that H(2)O(2) induces autophagic cell death.

Oxidative stress: emerging mitochondrial and cellular themes and variations in neuronal injury.

Oxidative stress plays a central role in neuronal injury and cell death in acute and chronic pathological conditions. The cellular responses to oxidative stress embrace changes in mitochondria and other organelles, notably endoplasmic reticulum, and can lead to a number of cell death paradigms, which cover a spectrum from apoptosis to necrosis and include autophagy. In Alzheimer's disease, and other pathologies including Parkinson's disease, protein aggregation provides further cellular stresses that can initiate or feed into the pathways to cell death engendered by oxidative stress. Specific attention is paid here to mitochondrial dysfunction and programmed cell death, and the diverse modes of cell death mediated by mitochondria under oxidative stress. Novel insights into cellular responses to neuronal oxidative stress from a range of different stressors can be gained by detailed transcriptomics analyses. Such studies at the cellular level provide the key for understanding the molecular and cellular pathways whereby neurons respond to oxidative stress and undergo injury and death. These considerations underpin the development of detailed knowledge in more complex integrated systems, up to the intact human bearing the neuropathology, facilitating therapeutic advances.

Multifaceted deaths orchestrated by mitochondria in neurones.

Neurones undergo diverse forms of cell death depending on the nature and severity of the stress. These death outcomes are now classified into various types of programmed cell death, including apoptosis, autophagy and necrosis. Each of these pathways can run in parallel and all have mitochondria as a central feature. Recruitment of mitochondria into cell death signalling involves either (or both) induction of specific death responses through release of apoptogenic proteins into the cytosol, or perturbation in function leading to loss of mitochondrial energization and ATP synthesis. Cross-talk between these signalling pathways, particularly downstream of mitochondria, determines the resultant pattern of cell death. The differential recruitment of specific death pathways depends on the timing of engagement of mitochondrial signalling. Other influences on programmed cell death pathways occur through stress of the endoplasmic reticulum and the associated ubiquitin-proteasome system normally handling potentially neurotoxic protein aggregates. Based upon contemporary evidence apoptosis is a relatively rare in the mature brain whereas the contribution of programmed necrosis to various neuropathologies has been underestimated. The death outcomes that neurones exhibit during acute or chronic injury or pathological conditions considered here (oxidative stress, hypoxic-ischaemic injury, amyotrophic lateral sclerosis, Parkinson's and Huntington's diseases) fall within a spectrum of the diverse death types across the apoptosis-necrosis continuum. Indeed, dying or dead neurones may simultaneously manifest characteristics of more than one type of death pathway. Understanding neuronal death pathways and their cross-talk not only informs the detailed pathobiology but also suggests novel therapeutic strategies.

Age-related decline in stress responses of human myocardium may not be explained by changes in mtDNA.

Elderly patients undergoing cardiac surgery are more likely to suffer postoperative heart failure than younger patients. This phenomenon is mirrored by an age-related loss of mitochondrial function and by an in vitro loss of myocardial contractile force following a stress. To examine the possibility that loss of mtDNA integrity may be responsible, we quantified representative age-associated mtDNA mutations (mtDNA(4977) and mtDNA(A3243G)) and mtDNA copy number using quantitative polymerase chain reaction in atrial samples obtained during cardiac surgery. The myocardium underwent organ bath contractility testing before and after either an ischaemic or hypoxic stress. We found that with age, recovery of developed force after either stressor significantly declined (p<0.0001). The abundance of mtDNA(4977) correlated weakly with loss of contractility (R(2)=0.09, p=0.047). However, the abundance level was low (average 0.0075% of total mtDNA) and the correlation disappeared when age was included in a multivariate analysis. Neither the abundance of mtDNA(A3243G) nor mtDNA copy number correlated with reduced recovery of developed force after stress. We conclude that, although mtDNA mutations (as exemplified by mtDNA(4977)) accumulate in the ageing heart, they are unlikely to make a major contribution to loss of contractile function.

Oxidative stress triggers neuronal caspase-independent death: endonuclease G involvement in programmed cell death-type III.

To characterize neuronal death, primary cortical neurons (C57/Black 6 J mice) were exposed to hydrogen peroxide (H2O2) and staurosporine. Both caused cell shrinkage, nuclear condensation, DNA fragmentation and loss of plasma membrane integrity. Neither treatment induced caspase-7 activity, but caspase-3 was activated by staurosporine but not H2O2. Each treatment caused redistribution from mitochondria of both endonuclease G (Endo G) and cytochrome c. Neurons knocked down for Endo G expression using siRNA showed reduction in both nuclear condensation and DNA fragmentation after treatment with H2O2, but not staurosporine. Endo G suppression protected cells against H2O2-induced cell death, while staurosporine-induced death was merely delayed. We conclude that staurosporine induces apoptosis in these neurons, but severe oxidative stress leads to Endo G-dependent death, in the absence of caspase activation (programmed cell death-type III). Therefore, oxidative stress triggers in neurons a form of necrosis that is a systematic cellular response subject to molecular regulation.

GABAergic striatal neurons exhibit caspase-independent, mitochondrially mediated programmed cell death.

GABAergic striatal neurons are compromised in basal ganglia pathologies and we analysed how insult nature determined their patterns of injury and recruitment of the intrinsic mitochondrial pathway during programmed cell death (PCD). Stressors affecting targets implicated in striatal neurodegeneration [3-morpholinylsydnoneimine (SIN-1), 3-nitropropionic acid (3-NP), NMDA, 3,5-dihydroxyphenylglycine (DHPG), and staurosporine (STS)] were compared in cultured GABAergic neurons from murine striatum by analyzing the progression of injury and its correlation with mitochondrial involvement, the redistribution of intermembrane space (IMS) proteins, and patterns of protease activation. Stressors produced PCD exhibiting slow-onset kinetics with time-dependent annexin-V labeling and eventual DNA fragmentation. IMS proteins including cytochrome c were differentially distributed, although stressors except STS produced early redistribution of apoptosis-inducing factor and Omi, suggestive of early recruitment of both caspase-dependent and caspase-independent signaling. In general, Bax mobilization to mitochondria appeared to promote IMS protein redistribution. Caspase 3 activation was prominent after STS, whereas NMDA and SIN-1 produced mainly calpain activation, and 3-NP and DHPG elicited a mixed profile of protease activation. PCD and redistribution of IMS proteins in striatal GABAergic neurons were canonical and insult-dependent, reflecting differential interplay between the caspase cascade and alternate cell death pathways.

Oxidative and excitotoxic insults exert differential effects on spinal motoneurons and astrocytic glutamate transporters: Implications for the role of astrogliosis in amyotrophic lateral sclerosis.

In amyotrophic lateral sclerosis (ALS) non-neuronal cells play key roles in disease etiology and loss of motoneurons via noncell-autonomous mechanisms. Reactive astrogliosis and dysfunctional transporters for L-glutamate [excitatory amino acid transporters, (EAATs)] are hallmarks of ALS pathology. Here, we describe mechanistic insights into ALS pathology involving EAAT-associated homeostasis in response to a destructive milieu, in which oxidative stress and excitotoxicity induce respectively astrogliosis and motoneuron injury. Using an in vitro neuronal-glial culture of embryonic mouse spinal cord, we demonstrate that EAAT activity was maintained initially, despite a loss of cellular viability induced by exposure to oxidative [3-morpholinosydnonimine chloride (SIN-1)] and excitotoxic [(S)-5-fluorowillardiine (FW)] conditions. This homeostatic response of EAAT function involved no change in the cell surface expression of EAAT1/2 at 0.5-4 h, but rather alterations in kinetic properties. Over this time-frame, EAAT1/2 both became more widespread across astrocytic arbors in concert with increased expression of glial fibrillary acidic protein (GFAP), although at 8-24 h there was gliotoxicity, especially with SIN-1 rather than FW. An opposite picture was found for motoneurons where FW, not SIN-1, produced early and extensive neuritic shrinkage and blebbing (> or =0.5 h) with somata loss from 2 h. We postulate that EAATs play an early homeostatic and protective role in the pathologic milieu. Moreover, the differential profiles of injury produced by oxidative and excitotoxic insults identify two distinct phases of injury which parallel important aspects of the pathology of ALS.

Recruitment of mitochondria into apoptotic signaling correlates with the presence of inclusions formed by amyotrophic lateral sclerosis-associated SOD1 mutations.

Mutations in Cu, Zn-superoxide dismutase 1 (SOD1) are associated with degeneration of motor neurons in the disease, familial amyotrophic lateral sclerosis. Intracellular protein inclusions containing mutant SOD1 (mSOD1) are associated with disease but it is unclear whether they are neuroprotective or cytotoxic. We report here that the formation of mSOD1 inclusions in a motor neuron-like cell line (NSC-34) strongly correlates with apoptosis via the mitochondrial death pathway. Applying confocal microscopic analyses, we observed changes in nuclear morphology and activation of caspase 3 specifically in cells expressing mSOD1 A4V or G85R inclusions. Furthermore, markers of mitochondrial apoptosis (activation and recruitment of Bax, and cytochrome c redistribution) were observed in 30% of cells bearing mSOD1 inclusions but not in cells expressing dispersed SOD1. In the presence of additional apoptotic challenges (staurosporine, etoposide, and hydrogen peroxide), cells bearing mSOD1 inclusions were susceptible to further apoptosis suggesting they were in a pro-apoptotic state, thus confirming that inclusions are linked to toxicity. Surprisingly, cells displaying dispersed SOD1 [both wildtype (WT) and mutant] were protected against apoptosis upstream of mitochondrial apoptotic signaling, induced by all agents tested. This protection against apoptosis was unrelated to SOD1 enzymatic activity because the G85R that lacks enzymatic function protected cells similarly to both WT SOD1 and A4V that possesses WT-like activity. These findings demonstrate new aspects of SOD1 in relation to cellular viability; specifically, mSOD1 can be either neuroprotective or cytotoxic depending on its aggregation state.