Computational methods and theory for ion channel research.

Ion channels are fundamental biological devices that act as gates in order to ensure selective ion transport across cellular membranes; their operation constitutes the molecular mechanism through which basic biological functions, such as nerve signal transmission and muscle contraction, are carried out. Here, we review recent results in the field of computational research on ion channels, covering theoretical advances, state-of-the-art simulation approaches, and frontline modeling techniques. We also report on few selected applications of continuum and atomistic methods to characterize the mechanisms of permeation, selectivity, and gating in biological and model channels.

Anomalous mobility of a driven active particle in a steady laminar flow.

We study, via extensive numerical simulations, the force-velocity curve of an active particle advected by a steady laminar flow, in the nonlinear response regime. Our model for an active particle relies on a colored noise term that mimics its persistent motion over a time scale [Formula: see text]. We find that the active particle dynamics shows non-trivial effects, such as negative differential and absolute mobility (NDM and ANM, respectively). We explore the space of the model parameters and compare the observed behaviors with those obtained for a passive particle ([Formula: see text]) advected by the same laminar flow. Our results show that the phenomena of NDM and ANM are quite robust with respect to the details of the considered noise: in particular for finite [Formula: see text] a more complex force-velocity relation can be observed.

Sexual dimorphism of AMBRA1-related autistic features in human and mouse.

Ambra1 is linked to autophagy and neurodevelopment. Heterozygous Ambra1 deficiency induces autism-like behavior in a sexually dimorphic manner. Extraordinarily, autistic features are seen in female mice only, combined with stronger Ambra1 protein reduction in brain compared to males. However, significance of AMBRA1 for autistic phenotypes in humans and, apart from behavior, for other autism-typical features, namely early brain enlargement or increased seizure propensity, has remained unexplored. Here we show in two independent human samples that a single normal AMBRA1 genotype, the intronic SNP rs3802890-AA, is associated with autistic features in women, who also display lower AMBRA1 mRNA expression in peripheral blood mononuclear cells relative to female GG carriers. Located within a non-coding RNA, likely relevant for mRNA and protein interaction, rs3802890 (A versus G allele) may affect its stability through modification of folding, as predicted by in silico analysis. Searching for further autism-relevant characteristics in Ambra1+/- mice, we observe reduced interest of female but not male mutants regarding pheromone signals of the respective other gender in the social intellicage set-up. Moreover, altered pentylentetrazol-induced seizure propensity, an in vivo readout of neuronal excitation-inhibition dysbalance, becomes obvious exclusively in female mutants. Magnetic resonance imaging reveals mild prepubertal brain enlargement in both genders, uncoupling enhanced brain dimensions from the primarily female expression of all other autistic phenotypes investigated here. These data support a role of AMBRA1/Ambra1 partial loss-of-function genotypes for female autistic traits. Moreover, they suggest Ambra1 heterozygous mice as a novel multifaceted and construct-valid genetic mouse model for female autism.

Anomalous force-velocity relation of driven inertial tracers in steady laminar flows.

We study the nonlinear response to an external force of an inertial tracer advected by a two-dimensional incompressible laminar flow and subject to thermal noise. In addition to the driving external field F, the main parameters in the system are the noise amplitude [Formula: see text] and the characteristic Stokes time [Formula: see text] of the tracer. The relation velocity vs. force shows interesting effects, such as negative differential mobility (NDM), namely a non-monotonic behavior of the tracer velocity as a function of the applied force, and absolute negative mobility (ANM), i.e. a net motion against the bias. By extensive numerical simulations, we investigate the phase chart in the parameter space of the model, [Formula: see text], identifying the regions where NDM, ANM and more common monotonic behaviors of the force-velocity curve are observed.

Nonlinear Response of Inertial Tracers in Steady Laminar Flows: Differential and Absolute Negative Mobility.

We study the mobility and the diffusion coefficient of an inertial tracer advected by a two-dimensional incompressible laminar flow, in the presence of thermal noise and under the action of an external force. We show, with extensive numerical simulations, that the force-velocity relation for the tracer, in the nonlinear regime, displays complex and rich behaviors, including negative differential and absolute mobility. These effects rely upon a subtle coupling between inertia and applied force that induces the tracer to persist in particular regions of phase space with a velocity opposite to the force. The relevance of this coupling is revisited in the framework of nonequilibrium response theory, applying a generalized Einstein relation to our system. The possibility of experimental observation of these results is also discussed.

Autophagy regulates satellite cell ability to regenerate normal and dystrophic muscles.

Autophagy is emerging as a key regulatory process during skeletal muscle development, regeneration and homeostasis, and deregulated autophagy has been implicated in muscular disorders and age-related muscle decline. We have monitored autophagy in muscles of mdx mice and human Duchenne muscular dystrophy (DMD) patients at different stages of disease. Our data show that autophagy is activated during the early, compensatory regenerative stages of DMD. A progressive reduction was observed during mdx disease progression, in coincidence with the functional exhaustion of satellite cell-mediated regeneration and accumulation of fibrosis. Moreover, pharmacological manipulation of autophagy can influence disease progression in mdx mice. Of note, studies performed in regenerating muscles of wild-type mice revealed an essential role of autophagy in the activation of satellite cells upon muscle injury. These results support the notion that regeneration-associated autophagy contributes to the early compensatory stage of DMD progression, and interventions that extend activation of autophagy might be beneficial in the treatment of DMD. Thus, autophagy could be a 'disease modifier' targeted by interventions aimed to promote regeneration and delay disease progression in DMD.

Essential versus accessory aspects of cell death: recommendations of the NCCD 2015.

Cells exposed to extreme physicochemical or mechanical stimuli die in an uncontrollable manner, as a result of their immediate structural breakdown. Such an unavoidable variant of cellular demise is generally referred to as 'accidental cell death' (ACD). In most settings, however, cell death is initiated by a genetically encoded apparatus, correlating with the fact that its course can be altered by pharmacologic or genetic interventions. 'Regulated cell death' (RCD) can occur as part of physiologic programs or can be activated once adaptive responses to perturbations of the extracellular or intracellular microenvironment fail. The biochemical phenomena that accompany RCD may be harnessed to classify it into a few subtypes, which often (but not always) exhibit stereotyped morphologic features. Nonetheless, efficiently inhibiting the processes that are commonly thought to cause RCD, such as the activation of executioner caspases in the course of apoptosis, does not exert true cytoprotective effects in the mammalian system, but simply alters the kinetics of cellular demise as it shifts its morphologic and biochemical correlates. Conversely, bona fide cytoprotection can be achieved by inhibiting the transduction of lethal signals in the early phases of the process, when adaptive responses are still operational. Thus, the mechanisms that truly execute RCD may be less understood, less inhibitable and perhaps more homogeneous than previously thought. Here, the Nomenclature Committee on Cell Death formulates a set of recommendations to help scientists and researchers to discriminate between essential and accessory aspects of cell death.

AMBRA1 is able to induce mitophagy via LC3 binding, regardless of PARKIN and p62/SQSTM1.

Damaged mitochondria are eliminated by mitophagy, a selective form of autophagy whose dysfunction associates with neurodegenerative diseases. PINK1, PARKIN and p62/SQTMS1 have been shown to regulate mitophagy, leaving hitherto ill-defined the contribution by key players in 'general' autophagy. In basal conditions, a pool of AMBRA1 - an upstream autophagy regulator and a PARKIN interactor - is present at the mitochondria, where its pro-autophagic activity is inhibited by Bcl-2. Here we show that, upon mitophagy induction, AMBRA1 binds the autophagosome adapter LC3 through a LIR (LC3 interacting region) motif, this interaction being crucial for regulating both canonical PARKIN-dependent and -independent mitochondrial clearance. Moreover, forcing AMBRA1 localization to the outer mitochondrial membrane unleashes a massive PARKIN- and p62-independent but LC3-dependent mitophagy. These results highlight a novel role for AMBRA1 as a powerful mitophagy regulator, through both canonical or noncanonical pathways.

Oxidative stress and autophagy: the clash between damage and metabolic needs.

Autophagy is a catabolic process aimed at recycling cellular components and damaged organelles in response to diverse conditions of stress, such as nutrient deprivation, viral infection and genotoxic stress. A growing amount of evidence in recent years argues for oxidative stress acting as the converging point of these stimuli, with reactive oxygen species (ROS) and reactive nitrogen species (RNS) being among the main intracellular signal transducers sustaining autophagy. This review aims at providing novel insight into the regulatory pathways of autophagy in response to glucose and amino acid deprivation, as well as their tight interconnection with metabolic networks and redox homeostasis. The role of oxidative and nitrosative stress in autophagy is also discussed in the light of its being harmful for both cellular biomolecules and signal mediator through reversible posttranslational modifications of thiol-containing proteins. The redox-independent relationship between autophagy and antioxidant response, occurring through the p62/Keap1/Nrf2 pathway, is also addressed in order to provide a wide perspective upon the interconnection between autophagy and oxidative stress. Herein, we also attempt to afford an overview of the complex crosstalk between autophagy and DNA damage response (DDR), focusing on the main pathways activated upon ROS and RNS overproduction. Along these lines, the direct and indirect role of autophagy in DDR is dissected in depth.

The autophagy regulators Ambra1 and Beclin 1 are required for adult neurogenesis in the brain subventricular zone.

Autophagy is a conserved proteolytic mechanism required for maintaining cellular homeostasis. The role of this process in vertebrate neural development is related to metabolic needs and stress responses, even though the importance of its progression has been observed in a number of circumstances, both in embryonic and in postnatal differentiating tissues. Here we show that the proautophagic proteins Ambra1 and Beclin 1, involved in the initial steps of autophagosome formation, are highly expressed in the adult subventricular zone (SVZ), whereas their downregulation in adult neural stem cells in vitro leads to a decrease in cell proliferation, an increase in basal apoptosis and an augmented sensitivity to DNA-damage-induced death. Further, Beclin 1 heterozygosis in vivo results in a significant reduction of proliferating cells and immature neurons in the SVZ, accompanied by a marked increase in apoptotic cell death. In sum, we propose that Ambra1- and Beclin 1-mediated autophagy plays a crucial role in adult neurogenesis, by controlling the survival of neural precursor cells.

Proteolysis of Ambra1 during apoptosis has a role in the inhibition of the autophagic pro-survival response.

Under stress conditions, pro-survival and pro-death processes are concomitantly activated and the final outcome depends on the complex crosstalk between these pathways. In most cases, autophagy functions as an early-induced cytoprotective response, favoring stress adaptation by removing damaged subcellular constituents. Moreover, several lines of evidence suggest that autophagy inactivation by the apoptotic machinery is a crucial event for cell death execution. Here we show that apoptotic stimuli induce a rapid decrease in the level of the autophagic factor Activating Molecule in Beclin1-Regulated Autophagy (Ambra1). Ambra1 degradation is prevented by concomitant inhibition of caspases and calpains. By both in vitro and in vivo approaches, we demonstrate that caspases are responsible for Ambra1 cleavage at the D482 site, whereas calpains are involved in complete Ambra1 degradation. Finally, we show that Ambra1 levels are critical for the rate of apoptosis induction. RNA interference-mediated Ambra1 downregulation further sensitizes cells to apoptotic stimuli, while Ambra1 overexpression and, more efficiently, a caspase non-cleavable mutant counteract cell death by prolonging autophagy induction. We conclude that Ambra1 is an important target of apoptotic proteases resulting in the dismantling of the autophagic machinery and the accomplishment of the cell death program.

Type 2 transglutaminase is involved in the autophagy-dependent clearance of ubiquitinated proteins.

Eukaryotic cells are equipped with an efficient quality control system to selectively eliminate misfolded and damaged proteins, and organelles. Abnormal polypeptides that escape from proteasome-dependent degradation and aggregate in the cytosol can be transported via microtubules to inclusion bodies called 'aggresomes', where misfolded proteins are confined and degraded by autophagy. Here, we show that Type 2 transglutaminase (TG2) knockout mice display impaired autophagy and accumulate ubiquitinated protein aggregates upon starvation. Furthermore, p62-dependent peroxisome degradation is also impaired in the absence of TG2. We also demonstrate that, under cellular stressful conditions, TG2 physically interacts with p62 and they are localized in cytosolic protein aggregates, which are then recruited into autophagosomes, where TG2 is degraded. Interestingly, the enzyme's crosslinking activity is activated during autophagy and its inhibition leads to the accumulation of ubiquitinated proteins. Taken together, these data indicate that the TG2 transamidating activity has an important role in the assembly of protein aggregates, as well as in the clearance of damaged organelles by macroautophagy.

The DNA repair complex Ku70/86 modulates Apaf1 expression upon DNA damage.

Apaf1 is a key regulator of the mitochondrial intrinsic pathway of apoptosis, as it activates executioner caspases by forming the apoptotic machinery apoptosome. Its genetic regulation and its post-translational modification are crucial under the various conditions where apoptosis occurs. Here we describe Ku70/86, a mediator of non-homologous end-joining pathway of DNA repair, as a novel regulator of Apaf1 transcription. Through analysing different Apaf1 promoter mutants, we identified an element repressing the Apaf1 promoter. We demonstrated that Ku70/86 is a nuclear factor able to bind this repressing element and downregulating Apaf1 transcription. We also found that Ku70/86 interaction with Apaf1 promoter is dynamically modulated upon DNA damage. The effect of this binding is a downregulation of Apaf1 expression immediately following the damage to DNA; conversely, we observed Apaf1 upregulation and apoptosis activation when Ku70/86 unleashes the Apaf1-repressing element. Therefore, besides regulating DNA repair, our results suggest that Ku70/86 binds to the Apaf1 promoter and represses its activity. This may help to inhibit the apoptosome pathway of cell death and contribute to regulate cell survival.

A brain-specific isoform of mitochondrial apoptosis-inducing factor: AIF2.

Apoptosis-inducing factor (AIF) has important supportive as well as potentially lethal roles in neurons. Under normal physiological conditions, AIF is a vital redox-active mitochondrial enzyme, whereas in pathological situations, it translocates from mitochondria to the nuclei of injured neurons and mediates apoptotic chromatin condensation and cell death. In this study, we reveal the existence of a brain-specific isoform of AIF, AIF2, whose expression increases as neuronal precursor cells differentiate. AIF2 arises from the utilization of the alternative exon 2b, yet uses the same remaining 15 exons as the ubiquitous AIF1 isoform. AIF1 and AIF2 are similarly imported to mitochondria in which they anchor to the inner membrane facing the intermembrane space. However, the mitochondrial inner membrane sorting signal encoded in the exon 2b of AIF2 is more hydrophobic than that of AIF1, indicating a stronger membrane anchorage of AIF2 than AIF1. AIF2 is more difficult to be desorbed from mitochondria than AIF1 on exposure to non-ionic detergents or basic pH. Furthermore, AIF2 dimerizes with AIF1, thereby preventing its release from mitochondria. Conversely, it is conceivable that a neuron-specific AIF isoform, AIF2, may have been 'designed' to be retained in mitochondria and to minimize its potential neurotoxic activity.

Neuronal caspase-3 signaling: not only cell death.

Caspases are a family of cysteinyl aspartate-specific proteases that are highly conserved in multicellular organisms and function as central regulators of apoptosis. A member of this family, caspase-3, has been identified as a key mediator of apoptosis in neuronal cells. Recent studies in snail, fly and rat suggest that caspase-3 also functions as a regulatory molecule in neurogenesis and synaptic activity. In this study, in addition to providing an overview of the mechanism of caspase-3 activation, we review genetic and pharmacological studies of apoptotic and nonapoptotic functions of caspase-3 and discuss the regulatory mechanism of caspase-3 for executing nonapoptotic functions in the central nervous system. Knowledge of biochemical pathway(s) for nonapoptotic activation and modulation of caspase-3 has potential implications for the understanding of synaptic failure in the pathophysiology of neurological disorders. Fine-tuning of caspase-3 lays down a new challenge in identifying pharmacological avenues for treatment of many neurological disorders.

Clinical outcome of sacral neuromodulation in incomplete spinal cord-injured patients suffering from neurogenic bowel dysfunctions.

STUDY DESIGN: Retrospective study. OBJECTIVES: Efficacy and safety of sacral neuromodulation (SNM) in incomplete spinal cord-injured patients (SCIPs) affected by chronic neurogenic bowel symptoms (NBSs). SETTING: Neurourology Department. Primary to tertiary care. METHODS: Retrospective non-blinded study without controls. Thirty-nine SCIPs were submitted to temporary stimulation for NBS. Permanent implantation was carried out if both their NBSs improved and the Wexner questionnaire scores were reduced by at least 50% during the first stage compared with that at baseline. Outcome measures included episodes of fecal incontinence and number of evacuations per week, as well as the Wexner score and the Short Form 36 (SF-36) Health Survey questionnaire. RESULTS: Twenty-three SCIPs were submitted to definitive SNM, maintaining their clinical benefits after permanent implantation with a median follow-up of 38 months. The length of time since neurological diagnosis to SNM therapy represents the only factor related to the success of the implantation, P<0.05. In subjects with constipation (12), the median number of evacuations shifted from 1.65 to 4.98 per week, whereas the Wexner score changed from 19.91 to 6.82 in the final checkup with P<0.05. In subjects with fecal incontinence (11), the median number of episodes per week in the final follow-up was 1.32 compared with 4.55 pre-SNM. The general and mental health of both groups was measured with the SF-36 questionnaire and consistently showed statistical improvement (P<0.05).Anorectal manometry showed no important variation compared with baseline. There were no major complications. CONCLUSIONS: SNM therapy should be considered for the treatment of NBS for select patients with incomplete spinal cord injury when conservative treatments fail.

Sexual life of males over 50 years of age with spinal-cord lesions of at least 20 years.

BACKGROUND: To assess over the past year the sexuality of male patients with spinal-cord injury (SCI) over 50 years of age with spinal lesions of at least 20 years. METHODS: Subjects were stratified in two groups: 44 individuals under 60 years and 55 individuals over 60 years. A detailed sexual anamnesis was taken for all, and the SF-36 Health Survey questionnaire was completed, with questions 13 and 14 concerning overall sexual life answered through the International Index of Erectile Function. RESULTS: The younger group reached a median score of +50 for each domain of the SF-36; however, the differences between the two groups are not statistically relevant. The physical domain of the SF-36 showed a median score of 43.2 for the younger versus 41.1 for the older, whereas the mental domain showed 44.8 and 43.1, respectively. In the first group, 29/44 (65.9) individuals claimed erectile dysfunction versus 43/55 (78.1%) in the second, whereas 34/44 (77.2%) of the first group reported having sexual intercourse versus 13/55 (23.6%) with P<0.01 (chi2 test). In the 2nd, 29/55 (52.7%) individuals reported physical intimacy without sexual intercourse. For each group, the overall sexual satisfaction is statistically correlated to the duration of the relationship (linear progression test P<0.05). CONCLUSIONS: Median quality of life was high. Sexual intercourse is fundamental only for males of the first group. Most aging couples presented a different way of thinking about sex without the need for intercourse. Physicians should acquire knowledge about sexuality and aging in SCI patients. SPONSORSHIP: This study was not sponsored.

Faf1 is expressed during neurodevelopment and is involved in Apaf1-dependent caspase-3 activation in proneural cells.

Fas-associated factor 1 (Faf1) has been described as a Fas-binding pro-apoptotic protein and as a component of the death-inducing signaling complex (DISC) in Fas-mediated apoptosis. Faf1 is able to potentiate Fas-induced apoptosis in several cell lines, although its specific functions are still not clear. Here we show that Faf1 is highly expressed in several areas of the developing telencephalon. Its expression pattern appears to be dynamic at different embryonic stages and to be progressively confined within limited territories. To decipher the specific role of Faf1 in developing brain, we used cDNA over-expression and mRNA down-regulation experiments to modulate Faf1 expression in telencephalic neural precursor cells, and we showed that in neural cell death Faf1 acts as a Fas-independent apoptotic enhancer. Moreover, we found that Faf1 protein level is down-regulated during apoptosis in a caspase- and Apaf1-dependent manner.

The involvement of cell death and survival in neural tube defects: a distinct role for apoptosis and autophagy?

Neural tube defects (NTDs), such as spina bifida (SB) or exencephaly, are common congenital malformations leading to infant mortality or severe disability. The etiology of NTDs is multifactorial with a strong genetic component. More than 70 NTD mouse models have been reported, suggesting the involvement of distinct pathogenetic mechanisms, including faulty cell death regulation. In this review, we focus on the contribution of functional genomics in elucidating the role of apoptosis and autophagy genes in neurodevelopment. On the basis of compared phenotypical analysis, here we discuss the relative importance of a tuned control of both apoptosome-mediated cell death and basal autophagy for regulating the correct morphogenesis and cell number in developing central nervous system (CNS). The pharmacological modulation of genes involved in these processes may thus represent a novel strategy for interfering with the occurrence of NTDs.