Optimising motor development in the hospitalised infant with CHD: factors contributing to early motor challenges and recommendations for assessment and intervention.

BACKGROUND: Neurodevelopmental challenges are the most prevalent comorbidity associated with a diagnosis of critical CHD, and there is a high incidence of gross and fine motor delays noted in early infancy. The frequency of motor delays in hospitalised infants with critical CHD requires close monitoring from developmental therapies (physical therapists, occupational therapists, and speech-language pathologists) to optimise motor development. Currently, minimal literature defines developmental therapists' role in caring for infants with critical CHD in intensive or acute care hospital units. PURPOSE: This article describes typical infant motor skill development, how the hospital environment and events surrounding early cardiac surgical interventions impact those skills, and how developmental therapists support motor skill acquisition in infants with critical CHD. Recommendations for healthcare professionals and those who provide medical or developmental support in promotion of optimal motor skill development in hospitalised infants with critical CHD are discussed. CONCLUSIONS: Infants with critical CHD requiring neonatal surgical intervention experience interrupted motor skill interactions and developmental trajectories. As part of the interdisciplinary team working in intensive and acute care settings, developmental therapists assess, guide motor intervention, promote optimal motor skill acquisition, and support the infant's overall development.

CCR7 on CD4+ T Cells Plays a Crucial Role in the Induction of Experimental Autoimmune Encephalomyelitis.

Multiple sclerosis (MS) is the most common chronic inflammatory demyelinating disease of the CNS. Myelin-specific CD4+ Th lymphocytes are known to play a major role in both MS and its animal model experimental autoimmune encephalomyelitis (EAE). CCR7 is a critical element for immune cell trafficking and recirculation, that is, lymph node homing, under homeostatic conditions; blocking CCR7+ central memory cells from egress of lymph nodes is a therapeutic approach in MS. To define the effect of CD4+ T cell-specific constitutive deletion of CCR7 in the priming and effector phase in EAE, we used an active EAE approach in T cell reconstituted Rag1-/- mice, as well as adoptive transfer EAE, in which mice received in vitro-primed CCR7-/- or CCR7+/+ myelin Ag TCR-transgenic 2d2 Th17 cells. Two-photon laser scanning microscopy was applied in living anesthetized mice to monitor the trafficking of CCR7-deficient and wild-type CD4+ T cells in inflammatory lesions within the CNS. We demonstrate that CD4+ T cell-specific constitutive deletion of CCR7 led to impaired induction of active EAE. In adoptive transfer EAE, mice receiving in vitro-primed CCR7-/- 2d2 Th17 cells showed similar disease onset as mice adoptively transferred with CCR7+/+ 2d2 Th17 cells. Using two-photon laser scanning microscopy CCR7-/- and CCR7+/+ CD4+ T cells did not reveal differences in motility in either animal model of MS. These findings indicate a crucial role of CCR7 in neuroinflammation during the priming of autoimmune CD4+ T cells but not in the CNS.

Role of IL-17-producing lymphocytes in severity of multiple sclerosis upon natalizumab treatment.

OBJECTIVE: Natalizumab is known to prevent T-helper cells entering the central nervous system (CNS). We hypothesize that more pathogenic T-helper cells are present outside the CNS and a possible relationship to disease severity. METHODS: Characterization and enrichment of human CD4+IL-17+ cells were performed ex vivo using peripheral blood mononuclear cells from natalizumab-treated relapsing-remitting multiple sclerosis (RRMS) patients ( n = 33), untreated RRMS patients ( n = 13), and healthy controls ( n = 33). Magnetic resonance imaging (MRI) scans were performed routinely for patients. RESULTS: Lymphocytes were elevated in peripheral blood of natalizumab-treated patients compared to untreated patients and healthy controls. Whereas group comparison for CD4+IL-17+ numbers also differed, CD4+IFN-γ+ and CD4+IL-22+ counts were not increased. CD4+IL-17+ cells not only expressed but also secreted IL-17. In natalizumab-treated patients, IL-17+ cell frequency was found to correlate with T1-hypointense lesions, but was not an indicator for rebound activity after treatment discontinuation, except in one patient who experienced a fulminant rebound, and interestingly, in whom the highest IL-17+ cell levels were observed. CONCLUSION: Increased lymphocytes and CD4+IL-17+ cells in the blood of RRMS patients receiving natalizumab corroborate the drug's mechanism of action, that is, blocking transmigration to CNS. Correlation between IL-17-expressing lymphocytes and T1-hypointense lesions underlines the important role of these cells in the disease pathology.

DNA-binding proteins from marine bacteria expand the known sequence diversity of TALE-like repeats.

Transcription Activator-Like Effectors (TALEs) of Xanthomonas bacteria are programmable DNA binding proteins with unprecedented target specificity. Comparative studies into TALE repeat structure and function are hindered by the limited sequence variation among TALE repeats. More sequence-diverse TALE-like proteins are known from Ralstonia solanacearum (RipTALs) and Burkholderia rhizoxinica (Bats), but RipTAL and Bat repeats are conserved with those of TALEs around the DNA-binding residue. We study two novel marine-organism TALE-like proteins (MOrTL1 and MOrTL2), the first to date of non-terrestrial origin. We have assessed their DNA-binding properties and modelled repeat structures. We found that repeats from these proteins mediate sequence specific DNA binding conforming to the TALE code, despite low sequence similarity to TALE repeats, and with novel residues around the BSR. However, MOrTL1 repeats show greater sequence discriminating power than MOrTL2 repeats. Sequence alignments show that there are only three residues conserved between repeats of all TALE-like proteins including the two new additions. This conserved motif could prove useful as an identifier for future TALE-likes. Additionally, comparing MOrTL repeats with those of other TALE-likes suggests a common evolutionary origin for the TALEs, RipTALs and Bats.

Generation of HLA-Universal iPSC-Derived Megakaryocytes and Platelets for Survival Under Refractoriness Conditions.

Platelet (PLT) transfusion is indispensable to maintain homeostasis in thrombocytopenic patients. However, PLT transfusion refractoriness is a common life-threatening condition observed in multitransfused patients. The most frequent immune cause for PLT transfusion refractoriness is the presence of alloantibodies specific for human leukocyte antigen (HLA) class I epitopes. Here, we have silenced the expression of HLA class I to generate a stable HLA-universal induced pluripotent stem cell (iPSC) line that can be used as a renewable cell source for the generation of low immunogenic cell products. The expression of HLA class I was silenced by up to 82% and remained stable during iPSC cultivation. In this study, we have focused on the generation of megakaryocytes (MK) and PLTs from a HLA-universal iPSC source under feeder- and xeno-free conditions. On d 19, differentiation rates of MKs and PLTs with means of 58% and 76% were observed, respectively. HLA-universal iPSC-derived MKs showed polyploidy with DNA contents higher than 4n and formed proPLTs. Importantly, differentiated MKs remained silenced for HLA class I expression. HLA-universal MKs produced functional PLTs. Notably, iPSC-derived HLA-universal MKs were capable to escape antibody-mediated complement- and cellular-dependent cytotoxicity. Furthermore, HLA-universal MKs were able to produce PLTs after in vivo transfusion in a mouse model indicating that they might be used as an alternative to PLT transfusion. Thus, in vitro produced low immunogenic MKs and PLTs may become an alternative to PLT donation in PLT-based therapies and an important component in the management of severe alloimmunized patients.

Programmable DNA-binding proteins from Burkholderia provide a fresh perspective on the TALE-like repeat domain.

The tandem repeats of transcription activator like effectors (TALEs) mediate sequence-specific DNA binding using a simple code. Naturally, TALEs are injected by Xanthomonas bacteria into plant cells to manipulate the host transcriptome. In the laboratory TALE DNA binding domains are reprogrammed and used to target a fused functional domain to a genomic locus of choice. Research into the natural diversity of TALE-like proteins may provide resources for the further improvement of current TALE technology. Here we describe TALE-like proteins from the endosymbiotic bacterium Burkholderia rhizoxinica, termed Bat proteins. Bat repeat domains mediate sequence-specific DNA binding with the same code as TALEs, despite less than 40% sequence identity. We show that Bat proteins can be adapted for use as transcription factors and nucleases and that sequence preferences can be reprogrammed. Unlike TALEs, the core repeats of each Bat protein are highly polymorphic. This feature allowed us to explore alternative strategies for the design of custom Bat repeat arrays, providing novel insights into the functional relevance of non-RVD residues. The Bat proteins offer fertile grounds for research into the creation of improved programmable DNA-binding proteins and comparative insights into TALE-like evolution.

RNA-seq pinpoints a Xanthomonas TAL-effector activated resistance gene in a large-crop genome.

Transcription activator-like effector (TALE) proteins of the plant pathogenic bacterial genus Xanthomonas bind to and transcriptionally activate host susceptibility genes, promoting disease. Plant immune systems have taken advantage of this mechanism by evolving TALE binding sites upstream of resistance (R) genes. For example, the pepper Bs3 and rice Xa27 genes are hypersensitive reaction plant R genes that are transcriptionally activated by corresponding TALEs. Both R genes have a hallmark expression pattern in which their transcripts are detectable only in the presence and not the absence of the corresponding TALE. By transcriptome profiling using next-generation sequencing (RNA-seq), we tested whether we could avoid laborious positional cloning for the isolation of TALE-induced R genes. In a proof-of-principle experiment, RNA-seq was used to identify a candidate for Bs4C, an R gene from pepper that mediates recognition of the Xanthomonas TALE protein AvrBs4. We identified one major Bs4C candidate transcript by RNA-seq that was expressed exclusively in the presence of AvrBs4. Complementation studies confirmed that the candidate corresponds to the Bs4C gene and that an AvrBs4 binding site in the Bs4C promoter directs its transcriptional activation. Comparison of Bs4C with a nonfunctional allele that is unable to recognize AvrBs4 revealed a 2-bp polymorphism within the TALE binding site of the Bs4C promoter. Bs4C encodes a structurally unique R protein and Bs4C-like genes that are present in many solanaceous genomes seem to be as tightly regulated as pepper Bs4C. These findings demonstrate that TALE-specific R genes can be cloned from large-genome crops with a highly efficient RNA-seq approach.

Comparison of propofol pharmacokinetic and pharmacodynamic models for awake craniotomy: A prospective observational study.

BACKGROUND: Anaesthesia for awake craniotomy aims for an unconscious patient at the beginning and end of surgery but a rapidly awakening and responsive patient during the awake period. Therefore, an accurate pharmacokinetic/pharmacodynamic (PK/PD) model for propofol is required to tailor depth of anaesthesia. OBJECTIVE: To compare the predictive performances of the Marsh and the Schnider PK/PD models during awake craniotomy. DESIGN: A prospective observational study. SETTING: Single university hospital from February 2009 to May 2010. PATIENTS: Twelve patients undergoing elective awake craniotomy for resection of brain tumour or epileptogenic areas. INTERVENTION: Arterial blood samples were drawn at intervals and the propofol plasma concentration was determined. MAIN OUTCOME MEASURES: The prediction error, bias [median prediction error (MDPE)] and inaccuracy [median absolute prediction error (MDAPE)] of the Marsh and the Schnider models were calculated. The secondary endpoint was the prediction probability PK, by which changes in the propofol effect-site concentration (as derived from simultaneous PK/PD modelling) predicted changes in anaesthetic depth (measured by the bispectral index). RESULTS: The Marsh model was associated with a significantly (P = 0.05) higher inaccuracy (MDAPE 28.9 ± 12.0%) than the Schnider model (MDAPE 21.5 ± 7.7%) and tended to reach a higher bias (MDPE Marsh -11.7 ± 14.3%, MDPE Schnider -5.4 ± 20.7%, P = 0.09). MDAPE was outside of accepted limits in six (Marsh model) and two (Schnider model) of 12 patients. The prediction probability was comparable between the Marsh (PK 0.798 ± 0.056) and the Schnider model (PK 0.787 ± 0.055), but after adjusting the models to each individual patient, the Schnider model achieved significantly higher prediction probabilities (PK 0.807 ± 0.056, P = 0.05). CONCLUSION: When using the 'asleep-awake-asleep' anaesthetic technique during awake craniotomy, we advocate using the PK/PD model proposed by Schnider. Due to considerable interindividual variation, additional monitoring of anaesthetic depth is recommended. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT 01128465.

NECAB2 is an endosomal protein important for striatal function.

Synaptic signaling depends on ATP generated by mitochondria. Dysfunctional mitochondria shift the redox balance towards a more oxidative environment. Due to extensive connectivity, the striatum is especially vulnerable to mitochondrial dysfunction. We found that neuronal calcium-binding protein 2 (NECAB2) plays a role in striatal function and mitochondrial homeostasis. NECAB2 is a predominantly endosomal striatal protein which partially colocalizes with mitochondria. This colocalization is enhanced by mild oxidative stress. Global knockout of Necab2 in the mouse results in increased superoxide levels, increased DNA oxidation and reduced levels of the antioxidant glutathione which correlates with an altered mitochondrial shape and function. Striatal mitochondria from Necab2 knockout mice are more abundant and smaller and characterized by a reduced spare capacity suggestive of intrinsic uncoupling respectively mitochondrial dysfunction. In line with this, we also found an altered stress-induced interaction of endosomes with mitochondria in Necab2 knockout striatal cultures. The predominance of dysfunctional mitochondria and the pro-oxidative redox milieu correlates with a loss of striatal synapses and behavioral changes characteristic of striatal dysfunction like reduced motivation and altered sensory gating. Together this suggests an involvement of NECAB2 in an endosomal pathway of mitochondrial stress response important for striatal function.

Association of cognitive dietary restraint and disinhibition with prediabetes--cross-sectional and longitudinal data of a feasibility study in German employees.

OBJECTIVE: To investigate the impact of eating behaviour traits on central obesity, prediabetes and associated major dietary food patterns. DESIGN: Assessment of eating behaviour was based on the revised German version of the Three-Eating Factor Questionnaire using cross-sectional and longitudinal data of a feasibility study in employees. Data on lifestyle and nutrition were obtained by validated self-administered questionnaires. Baseline characteristics were analysed by the univariate χ2 test or the Mann-Whitney test. To quantify correlations linear regression analysis was used. SETTING: The Delay of Impaired Glucose Tolerance by a Healthy Lifestyle Trial (DELIGHT), which investigated measures to prevent type 2 diabetes mellitus in 2004-2008. SUBJECTS: Employees (21-64 years, 127 men, 157 women) with elevated waist circumference (men ≥ 94 cm, women ≥ 80 cm) of five medium-sized companies in northern Germany. RESULTS: At baseline (T0), BMI but particularly waist circumference showed a strong inverse correlation with flexible control (P < 0.0001) and a positive correlation with disinhibition (P < 0.0001) and rigid control (P = 0.063). Flexible control was also significantly inversely related to fasting plasma glucose (P = 0.040), energy intake (P < 0.0001), intake of meat and meat products (P = 0.0001), and positively associated with intake of fruit and vegetables (P < 0.0001) at baseline (T0). Changes in flexible control within the first year of intervention (T1 v. T0) predicted changes in central obesity (P < 0.0001) and fasting plasma glucose (P = 0.025). CONCLUSIONS: DELIGHT shows that flexible control characterizes individuals with a higher dietary quality, a lower waist circumference and a lower glucose level. Enhancing flexible control more than rigid control, and decreasing disinhibition, seems beneficial in terms of central adiposity and glucose levels.

Chronic social stress disrupts the intracellular redistribution of brain hexokinase 3 induced by shifts in peripheral glucose levels.

Chronic stress has the potential to impair health and may increase the vulnerability for psychiatric disorders. Emerging evidence suggests that specific neurometabolic dysfunctions play a role herein. In mice, chronic social defeat (CSD) stress reduces cerebral glucose uptake despite hyperglycemia. We hypothesized that this metabolic decoupling would be reflected by changes in contact sites between mitochondria and the endoplasmic reticulum, important intracellular nutrient sensors, and signaling hubs. We thus analyzed the proteome of their biochemical counterparts, mitochondria-associated membranes (MAMs) from whole brain tissue obtained from CSD and control mice. This revealed a lack of the glucose-metabolizing enzyme hexokinase 3 (HK3) in MAMs from CSD mice. In controls, HK3 protein abundance in MAMs and also in striatal synaptosomes correlated positively with peripheral blood glucose levels, but this connection was lost in CSD. We conclude that the ability of HK3 to traffic to sites of need, such as MAMs or synapses, is abolished upon CSD and surmise that this contributes to a cellular dysfunction instigated by chronic stress. KEY MESSAGES : Chronic social defeat (CSD) alters brain glucose metabolism CSD depletes hexokinase 3 (HK3) from mitochondria-associated membranes (MAMs) CSD results in loss of positive correlation between blood glucose and HK3 in MAMs and synaptosomes.

GDAP1 loss of function inhibits the mitochondrial pyruvate dehydrogenase complex by altering the actin cytoskeleton.

Charcot-Marie-Tooth (CMT) disease 4A is an autosomal-recessive polyneuropathy caused by mutations of ganglioside-induced differentiation-associated protein 1 (GDAP1), a putative glutathione transferase, which affects mitochondrial shape and alters cellular Ca2+ homeostasis. Here, we identify the underlying mechanism. We found that patient-derived motoneurons and GDAP1 knockdown SH-SY5Y cells display two phenotypes: more tubular mitochondria and a metabolism characterized by glutamine dependence and fewer cytosolic lipid droplets. GDAP1 interacts with the actin-depolymerizing protein Cofilin-1 and beta-tubulin in a redox-dependent manner, suggesting a role for actin signaling. Consistently, GDAP1 loss causes less F-actin close to mitochondria, which restricts mitochondrial localization of the fission factor dynamin-related protein 1, instigating tubularity. GDAP1 silencing also disrupts mitochondria-ER contact sites. These changes result in lower mitochondrial Ca2+ levels and inhibition of the pyruvate dehydrogenase complex, explaining the metabolic changes upon GDAP1 loss of function. Together, our findings reconcile GDAP1-associated phenotypes and implicate disrupted actin signaling in CMT4A pathophysiology.

Preferential Partitioning of Rumen-Protected n-3 and n-6 Fatty Acids into Functionally Different Adipose Tissues.

Lipids are stored at various sites inside the body as adipose tissue (AT). These include subcutaneous, abdominal, and intermuscular locations. The AT substantially differ in their metabolic function. It is, however, unclear whether AT have specific requirements for individual essential n-3 and n-6 fatty acids (FA). If so, control mechanisms would partition FA from the blood. To investigate the hypothesis of a selective FA incorporation, 18 beef heifers were fed diets supplemented with 60 g/kg diet with lipids from either fish oil (FO) or sunflower oil (SO). The lipids had partially been rumen-protected to ruminal biohydrogenation of n-3 and n-6 FA. The AT analyzed for n-3 and n-6 FA by gas chromatography were obtained from pericardial, longissimus thoracis (LT) intermuscular, perirenal, and subcutaneous sites. The greatest proportions of n-3 and n-6 FA were found in the pericardial AT. Despite generally low abundance, n-3 FA proportions increased with FO compared to SO supplementation in all AT, but to a different extent. No such partitioning was found for the n-6 FA when supplementing SO. Concomitantly, the n-6/n-3 FA ratio was reduced with FO in all AT, except in the pericardial AT. The latter has specific metabolic functions and thus appears to be quite resistant to diet-induced changes in FA profile in order to maintain its function. The present findings showed the special role of specific n-3 and n-6 FA in bovine AT.

Redox Modifications of Proteins of the Mitochondrial Fusion and Fission Machinery.

Mitochondrial fusion and fission tailors the mitochondrial shape to changes in cellular homeostasis. Players of this process are the mitofusins, which regulate fusion of the outer mitochondrial membrane, and the fission protein DRP1. Upon specific stimuli, DRP1 translocates to the mitochondria, where it interacts with its receptors FIS1, MFF, and MID49/51. Another fission factor of clinical relevance is GDAP1. Here, we identify and discuss cysteine residues of these proteins that are conserved in phylogenetically distant organisms and which represent potential sites of posttranslational redox modifications. We reveal that worms and flies possess only a single mitofusin, which in vertebrates diverged into MFN1 and MFN2. All mitofusins contain four conserved cysteines in addition to cysteine 684 in MFN2, a site involved in mitochondrial hyperfusion. DRP1 and FIS1 are also evolutionarily conserved but only DRP1 contains four conserved cysteine residues besides cysteine 644, a specific site of nitrosylation. MFF and MID49/51 are only present in the vertebrate lineage. GDAP1 is missing in the nematode genome and contains no conserved cysteine residues. Our analysis suggests that the function of the evolutionarily oldest proteins of the mitochondrial fusion and fission machinery, the mitofusins and DRP1 but not FIS1, might be altered by redox modifications.

Transmembrane BAX Inhibitor-1 Motif Containing Protein 5 (TMBIM5) Sustains Mitochondrial Structure, Shape, and Function by Impacting the Mitochondrial Protein Synthesis Machinery.

The Transmembrane Bax Inhibitor-1 motif (TMBIM)-containing protein family is evolutionarily conserved and has been implicated in cell death susceptibility. The only member with a mitochondrial localization is TMBIM5 (also known as GHITM or MICS1), which affects cristae organization and associates with the Parkinson's disease-associated protein CHCHD2 in the inner mitochondrial membrane. We here used CRISPR-Cas9-mediated knockout HAP1 cells to shed further light on the function of TMBIM5 in physiology and cell death susceptibility. We found that compared to wild type, TMBIM5-knockout cells were smaller and had a slower proliferation rate. In these cells, mitochondria were more fragmented with a vacuolar cristae structure. In addition, the mitochondrial membrane potential was reduced and respiration was attenuated, leading to a reduced mitochondrial ATP generation. TMBIM5 did not associate with Mic10 and Mic60, which are proteins of the mitochondrial contact site and cristae organizing system (MICOS), nor did TMBIM5 knockout affect their expression levels. TMBIM5-knockout cells were more sensitive to apoptosis elicited by staurosporine and BH3 mimetic inhibitors of Bcl-2 and Bcl-XL. An unbiased proteomic comparison identified a dramatic downregulation of proteins involved in the mitochondrial protein synthesis machinery in TMBIM5-knockout cells. We conclude that TMBIM5 is important to maintain the mitochondrial structure and function possibly through the control of mitochondrial biogenesis.

Effect of Implementing a Bundled-Payment Program for Heart Failure at a Safety-Net Community Hospital.

Safety net community hospitals face unique challenges when entering risk-based contracts. The financial viability of such programs in these settings has not been well studied. This study analyzed a bundled-payment program for congestive heart failure at one such facility. To assess financial performance, the authors calculated the net patient payment by quarter after bundle implementation, and also compared the leading cost drivers before and after bundle implementation, specifically the next site of care and readmission rate. After 21 months of participating in the bundle, the program has saved money, been financially feasible, and generated positive returns for the hospital. Admission to skilled nursing facilities decreased from 21.3% to 16.0% after bundle implementation. The readmission rate was not significantly different, but trended downward. This study shows that safety net community hospitals can successfully participate in a bundled-payment program. For heart failure patients, decreasing admission to skilled nursing facilities and lowering the readmission rate are essential for program success.

Partitioning of Rumen-Protected n-3 and n-6 Fatty Acids is Organ-Specific in Growing Angus Heifers.

Dietary polyunsaturated fatty acids (PUFA), especially n-3 and n-6 fatty acids (FA), play an important role in the regulation of FA metabolism in all mammals. However, FA metabolism differs between different organs, suggesting a distinct partitioning of highly relevant FA. For the present study in cattle, a novel technology was applied to overcome rumen biohydrogenation of dietary unsaturated FA. Angus heifers were fed a straw-based diet supplemented for 8 weeks with 450 g/day of rumen-protected oil, either from fish (FO) or sunflower (SO). The FA composition in blood and five important organs, namely heart, kidney, liver, lung, and spleen, was examined. In blood, proportions of polyunsaturated FA were increased by supplementing FO compared to SO. The largest increase of eicosapentaenoic acid (EPA) proportion was found with FO instead of SO in the kidney, the lowest in the lung. Docosahexaenoic acid (DHA) was increased more in the liver than in kidney, lung, and spleen. The heart incorporated seven times more EPA than DHA, which is more than all other organs and described here for the first time in ruminants. In addition, the heart had the highest proportions of α-linolenic acid (18:3n-3) and linoleic acid (18:2n-6) of all organs. The proportions of polyunsaturated FA in the lung and spleen were exceptionally low compared to heart, liver, and kidney. In conclusion, it was shown that the response to FO in the distribution of dietary n-3 FA was organ-specific while proportions of n-6 FA were quite inert with respect to the type of oil supplemented.

The Charcot-Marie Tooth Disease Mutation R94Q in MFN2 Decreases ATP Production but Increases Mitochondrial Respiration under Conditions of Mild Oxidative Stress.

Charcot-Marie tooth disease is a hereditary polyneuropathy caused by mutations in Mitofusin-2 (MFN2), a GTPase in the outer mitochondrial membrane involved in the regulation of mitochondrial fusion and bioenergetics. Autosomal-dominant inheritance of a R94Q mutation in MFN2 causes the axonal subtype 2A2A which is characterized by early onset and progressive atrophy of distal muscles caused by motoneuronal degeneration. Here, we studied mitochondrial shape, respiration, cytosolic, and mitochondrial ATP content as well as mitochondrial quality control in MFN2-deficient fibroblasts stably expressing wildtype or R94Q MFN2. Under normal culture conditions, R94Q cells had slightly more fragmented mitochondria but a similar mitochondrial oxygen consumption, membrane potential, and ATP production as wildtype cells. However, when inducing mild oxidative stress 24 h before analysis using 100 µM hydrogen peroxide, R94Q cells exhibited significantly increased respiration but decreased mitochondrial ATP production. This was accompanied by increased glucose uptake and an up-regulation of hexokinase 1 and pyruvate kinase M2, suggesting increased pyruvate shuttling into mitochondria. Interestingly, these changes coincided with decreased levels of PINK1/Parkin-mediated mitophagy in R94Q cells. We conclude that mitochondria harboring the disease-causing R94Q mutation in MFN2 are more susceptible to oxidative stress, which causes uncoupling of respiration and ATP production possibly by a less efficient mitochondrial quality control.

Neurturin, a member of the glial cell line-derived neurotrophic factor family, affects the development of acetylcholinesterase-positive cells in a three-dimensional model system of retinogenesis.

The glial cell line-derived neurotrophic factor (GDNF) family consists of the four ligands GDNF, neurturin (NRTN), artemin and persephin, which bind to the four co-receptors GDNF family receptor alpha1-4 and control through the activation of the receptor tyrosin kinase Ret several developmental processes. The purpose of this study was to analyse the expression and the influence of NRTN in the developing retina. We used retinospheres, a three-dimensional model system of the developing chicken retina. The expression of NRTN and the GDNF family receptor alpha 2 increased during development. Furthermore, expression was comparable in retinae and retinospheres. Analysis of signalling pathways influenced by NRTN in retinospheres showed activation of phosphatidylinositol-3 kinase and mitogen-activated protein kinase (MAPK). Activation of MAPK could be localised in cells of the innermost rows of the inner nuclear layer which were predominantly acetylcholinesterase-positive cells. Exogenous application of NRTN increased the amount of acetylcholinesterase-positive cells within the retinospheres at late culture stages. Additionally, we could show that Müller glia cells did not express the GFRalpha2 receptor and were probably not involved in NRTN signalling. Therefore, we conclude that NRTN directly participates in regulatory processes concerning the differentiation of acetylcholinesterase-positive cells in the chicken retina.

Exogenous application of persephin influences phosphatidylinositol-3 kinase and MAPK/ERK signalling and enhances proliferation during early development in retinospheres.

Persephin (PSPN), a member of the glial cell line-derived neurotrophic factor family, and its implication in the retina is not well understood but might be an interesting therapeutic target for degenerative diseases. Although, PSPN is lost in the chicken during evolution, its target, the GDNF family receptor alpha 4 (GFRalpha4), is still expressed in a temporal and spatial pattern in the developing retina. We used this "knockout-precondition" to study the bioactivity and the effect of exogenous PSPN application and subsequent GFRalpha activation during retinal development in vitro without impairments of endogenous PSPN. Retinospheres, derived from dissociated chicken retina of embryonic day 6, were treated with PSPN and intracellular signalling was monitored. Additionally, PSPN was added during cultivation of the retinospheres and immunhistochemical stainings and Western blotting were performed to evaluate changes in proliferation, apoptosis and differentiation. Exogenous applied PSPN enhanced phosphatidylinositol-3-kinase (PI-3K) signalling and decreased signalling of mitogen-activated protein kinases (MAPK). Most importantly early retinal proliferation was enhanced and glutamine synthetase expression was decreased whereas differentiation of major retinal cell types was not changed. In contrast to GDNF, PSPN is exclusively influencing early progenitors whereas differentiation is not effected and seems to be regulated through PSPN-independent mechanisms. Since the binding site of PSPN and therefore the target of potential therapeuticals, is well conserved among species and is with high probability not able to bind other members of the GDNF-family, these results might be assigned to other species including mammals and humans.