Loss of apoptosis-inducing factor critically affects MIA40 function.

Mitochondrial apoptosis-inducing factor (AIF) influences the oxidative phosphorylation (OXPHOS) system and can be recruited as a mediator of cell death. Pathogenic mutations in the AIFM1 gene cause severe human diseases. Clinical manifestations include inherited peripheral neuropathies, prenatal cerebral abnormalities and progressive mitochondrial encephalomyopathies. In humans, rodents and invertebrates, AIF deficiency results in loss of respiratory complexes and, therefore, impaired OXPHOS. The molecular mechanisms underlying AIF-induced mitochondrial dysfunction remain elusive. Here we show that AIF physically interacts with the oxidoreductase CHCHD4/MIA40. In patient-derived fibroblasts as well as in tissues and glia cells from Harlequin (Hq) mutant mice, AIF deficiency correlates with decreased MIA40 protein levels, without affecting mRNA transcription. Importantly, MIA40 overexpression counteracts loss of respiratory subunits in Hq cells. Together, our findings suggest that MIA40 reduction contributes to the effects of AIF deficiency on OXPHOS, as it may impact on the correct assembly and maintenance of the respiratory subunits. This may be relevant for the development of new therapeutic approaches for AIF-related mitochondrial disorders.

Impaired mitochondrial respiration promotes dendritic branching via the AMPK signaling pathway.

Functional neuronal circuits require a constant remodeling of their network composed of highly interconnected neurons. The plasticity of synapses and the shaping of elaborated dendritic branches are energy demanding and therefore depend on an efficient mitochondrial oxidative phosphorylation (OXPHOS). The spatial and functional regulations of dendritic patterning occur also after cell fate specification; however, the molecular mechanisms underlying this complex process remain elusive. Here, we exploit the changes in dendritic architecture in highly branched neurons as a result of aberrant mitochondrial activity. In sensory neurons of Caenorhabditis elegans, genetic manipulations of mitochondrial complex I subunits cause an unexpected outgrowth of dendritic arbors and ectopic structures. The increased number of dendritic branches is coordinated through a specific signaling cascade rather than as a simple consequence of oxidative stress. On the basis of genetic and pharmacological evidence, we show that OXPHOS deficiency promotes branching through the activation of the AMP-activated protein kinase AMPK and the downstream target phosphoinositide 3-kinase PI3K. Taken together, our findings describe a well-defined signaling pathway that regulates dendritic outgrowth in conditions of compromised OXPHOS and the resulting AMPK activation.

Neurodegenerative processes in Huntington's disease.

Huntington's disease (HD) is a complex and severe disorder characterized by the gradual and the progressive loss of neurons, predominantly in the striatum, which leads to the typical motor and cognitive impairments associated with this pathology. HD is caused by a highly polymorphic CAG trinucleotide repeat expansion in the exon-1 of the gene encoding for huntingtin protein. Since the first discovery of the huntingtin gene, investigations with a consistent number of in-vitro and in-vivo models have provided insights into the toxic events related to the expression of the mutant protein. In this review, we will summarize the progress made in characterizing the signaling pathways that contribute to neuronal degeneration in HD. We will highlight the age-dependent loss of proteostasis that is primarily responsible for the formation of aggregates observed in HD patients. The most promising molecular targets for the development of pharmacological interventions will also be discussed.

Alteration of the nuclear pore complex in Ca(2+)-mediated cell death.

Cell death requires coordinated intracellular signalling before disassembly of cell architecture by degradative enzymes. Although the death signalling cascades that involve the mitochondria, the ER and the plasma membrane have been extensively characterized, only a handful of studies have examined the functional and structural alterations of the nuclear pore complex (NPC) during neuronal death. Here, we show that during excitotoxic neuronal degeneration calpains redistributed across the nuclear envelope and mediated the degradation of NPC components causing altered permeability of the nuclear membrane. In primary dissociated neurons, simultaneous recording of cytosolic [Ca(2+)] and localization of fluorescent proteins showed that the onset of Ca(2+) overload signalled a progressive increase in the diffusion of small reporter molecules across the nuclear envelope. Later, calpain-mediated changes in nuclear pore permeability allowed accumulation of large proteins in the nucleus. Further, in a model of excitotoxic neuronal degeneration in Caenorhabditis elegans, we found similar nuclear changes and redistribution of fluorescent probes across the nuclear membrane in dying neurons. Our findings strongly suggest that increased leakiness of the nuclear barrier affects nucleocytoplasmic transport, alters the localization of proteins across the nuclear envelope and it is likely to be involved in Ca(2+)-dependent cell death, including ischemic neuronal demise.

The plasma membrane Na+/Ca2+ exchanger is cleaved by distinct protease families in neuronal cell death.

Neurodegenerative conditions commonly involve loss of neuronal connectivity, synaptic dysfunction with excessive pruning, and ionic imbalances. These often serve as a prelude to cell death either through the activation of apoptotic or necrotic death routines or excess autophagy. In many instances, a local or generalized Ca2+ deregulation is involved in signaling or executing cell death. We have recently shown that in brain ischemia, and during excitotoxicity triggered by excess glutamate, the irreversible Ca2+ deregulation leading to necrosis is due to calpain-mediated modulation of the plasma membrane Na+/Ca2+ exchanger (NCX). Here we show that the NCX can also be cleaved by caspases in neurons undergoing apoptosis, which suggests that cleavage of the main Ca2+ extrusion pathway is a lethal event in multiple forms of cell death.

Cleavage of plasma membrane calcium pumps by caspases: a link between apoptosis and necrosis.

Neuronal death, which follows ischemic injury or is triggered by excitotoxins, can occur by both apoptosis and necrosis. Caspases, which are not directly required for necrotic cell death, are central mediators of the apoptotic program. Here we demonstrate that caspases cleave and inactivate the plasma membrane Ca(2+) pump (PMCA) in neurons and non-neuronal cells undergoing apoptosis. PMCA cleavage impairs intracellular Ca(2+) handling, which results in Ca(2+) overload. Expression of non-cleavable PMCA mutants prevents the disturbance in Ca(2+) handling, slows down the kinetics of apoptosis, and markedly delays secondary cell lysis (necrosis). These findings suggest that caspase-mediated cleavage and inactivation of PMCAs can lead to necrosis, an event that is reduced by caspase inhibitors in brain ischemia.

Effects of PMCA and SERCA pump overexpression on the kinetics of cell Ca(2+) signalling.

The dynamic interactions of the main pathways for active Ca(2+) transport have been analysed in living cells by altering the expression of their components. The plasma membrane (PMCA) and the endoplasmic reticulum (ER) (SERCA) Ca(2+) pumps were transiently overexpressed in CHO cells, and the Ca(2+) homeostasis in the subcellular compartments was investigated using specifically targeted chimaeras of the Ca(2+)- sensitive photoprotein aequorin. In resting cells, overexpression of the PMCA and SERCA pumps caused a reduction and an increase in ER [Ca(2+)] levels, respectively, while no significant differences were detected in cytosolic and mitochondrial [Ca(2+)]. Upon stimulation with an inositol 1,4, 5-trisphosphate (IP(3))-generating agonist, the amplitude of the mitochondrial and cytosolic Ca(2+) rises correlated with the ER [Ca(2+)] only up to a threshold value, above which the feedback inhibition of the IP(3) channel by Ca(2+) appeared to be limiting.

Expression, partial purification and functional properties of themuscle-specific calpain isoform p94.

The muscle-specific calpain isoform p94 has high propensity to autocatalytic degradation, thus no significant amounts of the intact active protein have been available so far. As a result, aspects like its regulation (via Ca2+ and other factors) and its intracellular localization are unknown or obscure. In this work, large amounts of human p94 have been produced in insect cells using a recombinant baculovirus expression system. Although most of the protease was recovered in an insoluble and catalytically inactive form, the soluble fraction contained amounts of intact active p94 adequate for its characterization. His-tagged recombinant p94, obtained by the same expression system, was partially purified as an active product. Both the unmodified and the partially purified His-tagged p94 bound calcium with high affinity, and their autolytic activity required Ca2+. The sensitivity of the catalytic activity of the recombinant protease to Ca2+ was very high. In fact, p94 in soluble cell extracts autolysed to a significant extent even in the presence of submicromolar Ca2+ levels. Thus, in analogy to what demonstrated for the ubiquitous m- and micro-calpain isoforms, intracellular Ca2+ might be one of the factors controlling the activity of this muscle-specific calpain isoform.

Significance of the isolation of Candida species from respiratory samples in critically ill, non-neutropenic patients. An immediate postmortem histologic study.

The diagnosis of pulmonary candidiasis is still controversial. We undertook a prospective study on 25 non-neutropenic, mechanically ventilated (> 72 h) patients who died in our ICU with the aim of assessing the incidence and significance of the isolation of Candida species from quantitative cultures of immediate postmortem lung biopsies and different respiratory sampling techniques. Immediate postmortem respiratory samples (endotracheal aspirate, protected specimen brush [PSB], bronchoalveolar lavage [BAL], blind biopsies [average 14/patient], and bilateral bronchoscopically guided biopsies [two per patient]) were taken from all patients. Lung tissue specimens were histologically examined. Respiratory samples were classified as having Candida or otherwise. Ten (40%) patients had at least one pulmonary biopsy yielding Candida spp. Among these 10 patients with Candida isolates, only two had definite pulmonary candidiasis. A total of 470 microorganisms were isolated from 280 of 375 (77%) lung biopsy samples in all 25 patients. Candida species represented 9% (n = 40) of the isolates, corresponding to 10 patients (40%). In the 10 patients in whom Candida species was isolated from pulmonary biopsies, this was always associated with the isolation of the same microorganism from one of the sampling methods. Quantitative cultures of Candida species from different sampling methods correlated well among each other but could not discriminate the presence from absence of Candida pneumonia. A logistic regression model adjusted for the presence of antibiotics, days of antibiotic treatment, mechanical ventilation period, age, ARDS, parenteral nutrition, and gender did not show any independent risk factor for developing positive pulmonary samples for Candida species. The incidence of Candida isolation from pulmonary biopsies in critically ill mechanically ventilated, non-neutropenic patients who die is high (40%). However, the incidence of definite Candida pneumonia was 8%. We also found that Candida colonization is uniform throughout the different lung regions, and that the presence of Candida in respiratory samples, independently of quantitative cultures, is not a good marker of Candida pneumonia in critically ill, non-neutropenic, non-AIDS patients.