Ubiquitylome profiling of Parkin-null brain reveals dysregulation of calcium homeostasis factors ATP1A2, Hippocalcin and GNA11, reflected by altered firing of noradrenergic neurons.

Parkinson's disease (PD) is the second most frequent neurodegenerative disorder in the old population. Among its monogenic variants, a frequent cause is a mutation in the Parkin gene (Prkn). Deficient function of Parkin triggers ubiquitous mitochondrial dysfunction and inflammation in the brain, but it remains unclear how selective neural circuits become vulnerable and finally undergo atrophy. We attempted to go beyond previous work, mostly done in peripheral tumor cells, which identified protein targets of Parkin activity, an ubiquitin E3 ligase. Thus, we now used aged Parkin-knockout (KO) mouse brain for a global quantification of ubiquitylated peptides by mass spectrometry (MS). This approach confirmed the most abundant substrate to be VDAC3, a mitochondrial outer membrane porin that modulates calcium flux, while uncovering also >3-fold dysregulations for neuron-specific factors. Ubiquitylation decreases were prominent for Hippocalcin (HPCA), Calmodulin (CALM1/CALML3), Pyruvate Kinase (PKM2), sodium/potassium-transporting ATPases (ATP1A1/2/3/4), the Rab27A-GTPase activating protein alpha (TBC1D10A) and an ubiquitin ligase adapter (DDB1), while strong increases occurred for calcium transporter ATP2C1 and G-protein subunits G(i)/G(o)/G(Tr). Quantitative immunoblots validated elevated abundance for the electrogenic pump ATP1A2, for HPCA as neuron-specific calcium sensor, which stimulates guanylate cyclases and modifies axonal slow afterhyperpolarization (sAHP), and for the calcium-sensing G-protein GNA11. We assessed if compensatory molecular regulations become insufficient over time, leading to functional deficits. Patch clamp experiments in acute Parkin-KO brain slices indeed revealed alterations of the electrophysiological properties in aged noradrenergic locus coeruleus (LC) neurons. LC neurons of aged Parkin-KO brain showed an acceleration of the spontaneous pacemaker frequency, a reduction in sAHP and shortening of action potential duration, without modulation of KCNQ potassium currents. These findings indicate altered calcium-dependent excitability in a PARK2 model of PD, mediated by diminished turnover of potential Parkin targets such as ATP1A2 and HPCA. The data also identified further novel Parkin substrate candidates like SIRT2, OTUD7B and CUL5. Our elucidation of neuron-specific mechanisms of PD pathogenesis helps to explain the known exceptional susceptibility of noradrenergic and dopaminergic projections to alterations of calcium homeostasis and its mitochondrial buffering.

[Provision of assistive devices in amyotrophic lateral sclerosis. Analysis of 3 years case management in an internet-based supply network]. / Hilfsmittelversorgung bei der amyotrophen Lateralsklerose. Analyse aus 3 Jahren Fallmanagement in einem internetunterstützten Versorgungsnetzwerk.

BACKGROUND: The provision of assistive devices (PAD) is a key element of care in amyotrophic lateral sclerosis (ALS). Since 2011, assistive devices (AD) have been coordinated in an internet-supported care network at university-based ALS centers in Berlin, Bochum, Hannover and Jena. The digitization of PAD processes has facilitated the evaluation of real-life ALS care. OBJECTIVES: Orthotics (OT), augmentative and alternative communication (AAC), supported treadmill (ST) and powered wheelchair (PW) were the PAD groups analyzed for delivery rates (proportion of delivered AD vs. medically indicated AD), rejection by patients and payers and latency of provision of care. RESULTS: Between June 2011 and October 2014 a total of 1479 patients and 12,478 AD were coordinated, among which 3313 PAD were related to OT, AAC, ST or EM. The median delivery rate was 64.3 %. The mean rejection rate by patients was 9.8 % (OT 5.4 %, AAC 9.8 %, ST 10.2 % and PW 15.6 %). Marked differences were noted in the rejection rate by payers and in care provision latency: OT (16.2 %, 68 days, n = 734), AAC (30.4 %, 96 days, n = 392), ST (34.8 %, 113 days, n = 164) and PW (35.6 %, 129 days, n = 259). Analysis of rejection rates showed significant differences among insurers. CONCLUSION: Only two thirds of the medically indicated AD reached the patients. Rejection rates by patients and payers and latency of provision of care were high. The PAD can substantially vary among health insurance companies. The establishment of consented criteria for PAD and their integration into treatment regimens and guidelines are crucial tasks for the future.

Clinical outcomes and predictors of success with Impella weaning in cardiogenic shock: a single-center experience.

Introduction: Cardiogenic shock (CS) is a severe syndrome with poor prognosis. Short-term mechanical circulatory support with Impella devices has emerged as an increasingly therapeutic option, unloading the failing left ventricle (LV) and improving hemodynamic status of affected patients. Impella devices should be used for the shortest time necessary to allow LV recovery because of time-dependent device-related adverse events. The weaning from Impella, however, is mostly performed in the absence of established guidelines, mainly based on the experience of the individual centres. Methods: The aim of this single center study was to retrospectively evaluate whether a multiparametrical assessment before and during Impella weaning could predict successful weaning. The primary study outcome was death occurring during Impella weaning and secondary endpoints included assessment of in-hospital outcomes. Results: Of a total of 45 patients (median age, 60 [51-66] years, 73% male) treated with an Impella device, 37 patients underwent impella weaning/removal and 9 patients (20%) died after the weaning. Non-survivors patients after impella weaning more commonly had a previous history of known heart failure (p = 0.054) and an implanted ICD-CRT (p = 0.01), and were more frequently treated with continuous renal replacement therapy (p = 0.02). In univariable logistic regression analysis, lactates variation (%) during the first 12-24 h of weaning, lactate value after 24 h of weaning, left ventricular ejection fraction (LVEF) at the beginning of weaning, and inotropic score after 24 h from weaning beginning were associated with death. Stepwise multivariable logistic regression identified LVEF at the beginning of weaning and lactates variation (%) in the first 12-24 h from weaning beginning as the most accurate predictors of death after weaning. The ROC analysis indicated 80% accuracy (95% confidence interval = 64%-96%) using the two variables in combination to predict death after weaning from Impella. Conclusions: This single-center experience on Impella weaning in CS showed that two easily accessible parameters as LVEF at the beginning of weaning and lactates variation (%) in the first 12-24 h from weaning begin were the most accurate predictors of death after weaning.

47,X,idic(Y),inv dup(Y): a non-mosaic case of a phenotypically normal boy with two different Y isochromosomes and neocentromere formation.

A de novo aberrant karyotype with 47 chromosomes including 2 different-sized markers was identified during prenatal diagnosis. Fluorescence in situ hybridization (FISH) with a Y painting probe tagged both marker chromosomes which were supposed to be isochromosomes of the short and the long arm, respectively. A normal boy was born in time who shows normal physical and mental development. To characterize both Y markers in detail, we postnatally FISH-mapped a panel of Y chromosomal probes including SHOX (PAR1), TSPY, DYZ3 (Y centromere), UTY, XKRY, CDY, RBMY, DAZ, DYZ1 (Yq12 heterochromatin), SYBL1 (PAR2), and the human telomeric sequence (TTAGGG)(n). The smaller Y marker turned out to be an isochromosome containing an inverted duplication of the entire short arm, the original Y centromere, and parts of the proximal long arm, including AZFa. The bigger Y marker was an isochromosome of the rest of the Y long arm. Despite a clearly visible primary constriction within one of the DAPI- and DYZ1-positive heterochromatic regions, hybridization of DYZ3 detected no Y-specific alphoid sequences in that constriction. Because of its stable mitotic distribution, a de novo formation of a neocentromere has to be assumed.

Neuromyelitis optica spectrum disorders in patients with myasthenia gravis: ten new aquaporin-4 antibody positive cases and a review of the literature.

BACKGROUND: Neuromyelitis optica (NMO, Devic syndrome) and myasthenia gravis (MG) are rare antibody-mediated autoimmune disorders. Concurrent incidence has been reported in only few patients, mostly non-Caucasians. OBJECTIVE: To report on ten Caucasian patients with NMO spectrum disorders (NMOSD) and MG and to provide a comprehensive review of the literature. METHOD: Retrospective study. RESULTS: In total, 26 patients (m:f = 1:12; Caucasian in 12) with MG (generalized in 17) and NMOSD (NMO in 21, longitudinally extensive transverse myelitis in five) were identified from the authors' own files (n = 10) and the previous literature (n = 16). MG preceded NMOSD in 24/25 cases (96%). AQP4-Ab were tested in 20 patients and were positive in 17 (85%). Twenty out of 25 patients (80%) had been treated with thymectomy or thymic irradiation, which preceded NMOSD in all cases (median latency, 12 years; range, 0.3-32). At last follow-up, complete remission of MG was reported in 15/22 (68%), and MG was well controlled with pyridostigmine in three. Co-existing autoimmune disorders or autoimmune antibodies were reported in 17 patients. CONCLUSION: Our study demonstrates that i) AQP4-Ab-positive NMOSD are more commonly associated with MG in Caucasians than previously thought; ii) MG precedes NMOSD in most cases, often by more than a decade; iii) NMOSD almost exclusively occur in females with juvenile or early-onset MG; and iv) MG frequently takes an unusually mild course in patients with NMOSD. A history of thymectomy could be a possible risk factor for the later development of NMOSD. We recommend testing for AQP4-Ab in MG patients presenting with atypical motor or optic symptoms.

Differential RNA cleavage and polyadenylation of the glutamate transporter EAAT2 in the human brain.

We cloned four novel transcripts of the excitatory amino acid transporter 2, named EAAT2/3UT1-4, resulting from differential cleavage and polyadenylation. Tandem poly (A) sites were found to be functional at 72, 654, 973 nucleotides and more than 2 kb downstream of the stop codon. A tissue-specific expression was identified for 3'-variants of the EAAT2 RNA, most prominently for EAAT2/3UT4 (hippocampus>cortex>>cerebellum>thalamus) as demonstrated by Northern blot analysis and quantitative PCR. We conclude, that alternative poly (A) selection may contribute to the reported differential EAAT2 protein expression under normal and diseased conditions.

5'-heterogeneity of the human excitatory amino acid transporter cDNA EAAT2 (GLT-1).

TWO novel transcripts of the human excitatory amino acid transporter EAAT2 (GLT-1) were cloned using PCR cloning techniques. Comparative sequence analysis of the 5'-untranslated region (UTR) of five EAAT2 transcripts led to the definition of five putative 5'-untranslated exons that are combined in a variable manner. Alternative splicing is a likely mechanism for the 5' complexity of the EAAT2 cDNA. The potential functional meaning of this finding such as differential expression of the EAAT2 transcripts in the central nervous system remains to be demonstrated.

Alternative splicing of the glutamate transporter EAAT2 (GLT-1).

The human glutamate transporter EAAT2 (GLT-1) is of major importance for synaptic glutamate reuptake, and reportedly, a candidate gene for neurodegenerative diseases such as amyotrophic lateral sclerosis, Alzheimer's disease and epilepsy. Here we report the polymerase chain reaction (PCR) cloning of two novel EAAT2 transcripts, named EAAT2-C1 and EAAT2-C2, which originate from alternative splicing of the human EAAT2 gene. EAAT2-C1 results from skipping of the protein coding exon eight. In contrast, EAAT2-C2 is characterized by usage of internal splice sites in the exons five and six. The splicing events lead to a deletion of 45 and 107 amino acids, respectively, located in the C-terminal and central part of the putative protein.

The RNA of the glutamate transporter EAAT2 is variably spliced in amyotrophic lateral sclerosis and normal individuals.

Impaired re-uptake of synaptic glutamate, and a reduced expression of the glutamate transporter EAAT2 have been found in the motor cortex of patients with amyotrophic lateral sclerosis (ALS). Two splice forms of the EAAT2 RNA resulting from retention of intronic sequences (EAAT2/Int) and deletion of one protein coding exon (EAAT2/C1) have been reported to account for the EAAT2 protein loss in ALS. In this study we investigated the presence of two known (EAAT2/C1; EAAT2/Int) and three novel (EAAT2/C2-4) EAAT2 RNA in motor cortex of 17 ALS cases and 11 controls. Reverse transcription and PCR were carried out to amplify the complementary DNA of the complete and variably spliced EAAT2 transcripts. Nested PCR was followed to generate amplicons specific for EAAT2/C1-4 and EAAT2/Int. EAAT2/Int was detected in 59% of ALS specimens as compared to 36% of controls showing a trend but no statistical significance of a more frequent expression in ALS (Type I error 24.6%). EAAT2/C1-4 were found to be equally expressed in ALS patients and controls. Our results indicate that the involvement of EAAT2 transcripts in ALS is unlikely to be primary, and more complex than previously recognized. Alterations of quantitative expression of distinct EAAT2 splice forms in ALS cannot be excluded from this study and remain to be investigated.

Pharmacological treatment of ALS.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease resulting from chronic and selective loss of motor neurons in the brain and spinal cord. In 1993, the etiology of ALS has been clarified for a small sub-group of patients with an autosomal-dominant form of this disease. About 10 percent of familial ALS patients have been associated with more than 50 mutations of the gene of the Cu/Zn superoxide dismutase (SOD1). Mutations in the SOD1 gene account for 1 percent of all ALS patients and have therefore limited epidemological and clinical relevance; however, they are of fundamental importance for the understanding of the ALS pathogenesis, and the development of neuroprotective strategies. In two double-blind and placebo-controlled studies the membrane stabilisator riluzole has been shown to be the first neuroprotective compound with a significant effect on survival of ALS patients. The neuroprotective approach reduced therapeutic nihilism in ALS and is a first step in the treatment of this devastating disease.

Evaluation of the metabolic diversity of microbial communities in four different filter layers of a constructed wetland with vertical flow by Biolog analysis.

The community-level substrate utilization test based on direct incubation of environmental samples in Biolog EcoPlates is a suitable and sensitive tool to characterize microbial communities. The aim of this study was to investigate the influence of plant roots and soil structure on the metabolic diversity of microorganisms in a constructed wetland with vertical flow. Sediment samples were taken from different filter depths representing specific filter layers. The color development representing the substrate utilization was measured with the samples over a period of 10 days. The average well color development (AWCD) for all carbon sources was calculated as an indicator of total activity and in order to compensate the influence of the inoculum's density on the color development in the plates. After transformation by dividing by the AWCD, the optical density data were analysed by principal component analysis (PCA). An analysis of the kinetic profile of the AWCD was carried out to increase the analytical power of the method. The corrected data have been successfully fit to the logistic growth equation. Three kinetic model parameters, the asymptote (K), the exponential rate of color change (p) and the time to the midpoint of the exponential portion of the curve (s), were used for statistical analysis of the physiological profile of the microbial community in the different filter layers of the constructed wetland. We found out that in the upper two horizons, which were rooted most densely, mainly easily degradable materials like specific carbohydrates were utilized, while in the lower layers, where only single roots occur, more biochemically inert compounds, e.g. 2-hydroxy benzoic acid, were utilized. Furthermore it could be shown that microorganisms in the surface layer benefited from the plant litter because they can utilize decay products of these. In the lower filter layers specialists took advantage because they had to cope with the biochemically inert materials and the lower nutrient supply.

[Elective termination of respiratory therapy in amyotrophic lateral sclerosis]. / Elektive Termination der Beatmungstherapie bei der amyotrophen Lateralsklerose.

BACKGROUND: Due to the growing use of artificial respiration in amyotrophic lateral sclerosis (ALS), physicians are increasingly confronted with patients seeking discontinuation of therapy. Yet there are few systematic investigations of the withdrawal of ventilation therapy. PATIENTS AND METHODS: In a retrospective investigation of nine German ALS patients, clinical data were recorded from the discontinuation of noninvasive ventilation (n=4) and mechanical ventilation (n=5). RESULTS: In cases of residual spontaneous breathing, intensified symptom control of dyspnea and anxiety was possible with intravenous morphine sulfate at a low dose rate (10 mg/h) but high cumulative dose (185-380 mg). The terminal phase after removing the mask was protracted (22:10 h to 28:00 h). In cases of minimal or absent spontaneous breathing the disconnection was realized in deep sedation, which required a moderate total dose of morphine sulfate (120 mg) but a high dosage rate (up to 300 mg/h). The terminal phase in deep sedation was short (15-80 min). CONCLUSION: The elective termination of ventilation requires differentiated pharmacologic palliative care. More controlled studies are required in order to establish evidence-based guidelines for the termination of ventilation.

[The calcaneus as the site of manifestation for osteoporosis-associated fractures: age- and sex-specific changes in calcaneal morphology correlate with the incidence and severity of intra-articular calcaneal fractures]. / Der Kalkaneus als Manifestationsort osteoporoseassoziierter Frakturen: Alters- und geschlechtsabhängige Veränderungen der Kalkaneusmorphologie korrelieren zur Inzidenz und Schwere intraartikulärer Kalkaneusfrakturen.

BACKGROUND: While it is recognized that trauma energy at the time of injury is an important factor in the pathogenesis and severity of calcaneal fractures, the possible role of changes in calcaneal microarchitecture remains largely undefined. The purpose of this study was to determine whether the calcaneal bone structure changes with age and to address if local bone mass is of clinical relevance in respect to the occurrence and complexity of calcaneal fractures. MATERIAL AND METHODS: The radiographic and clinical data of 182 patients with intra-articular calcaneal fractures were analyzed to provide correlative clinical evidence for a relation between local bone mass and fractures of the calcaneus. To measure bone mass, 60 calcanei were harvested from 30 age- and gender-matched patients at autopsy. RESULTS: The average age at the time of fracture was higher in females (46.0+/-18.3 years) than in males (39.9+/-13.9 years). Furthermore, the relative frequency of fractures during aging shifted from males to females and the frequency of compound fractures was higher in females (65%) than in males (48%). The calcaneal bone mass was significantly reduced by 19% in older females (female symbol 20-40 years: 292 mg/cm(3); female symbol 61-80 years: 237 mg/cm(3); p<0.05). CONCLUSION: The calcaneus displayed age- and gender-related changes in its microarchitecture that are known to reduce the biomechanical stability of trabecular bone. These results suggest that bone mass and structure are risk factors in respect to the occurrence and severity of calcaneal fractures.

[Progressive muscle atrophy. A rarely diagnosed variant of amyotrophic lateral sclerosis]. / Progressive Muskelatrophie. Eine unterdiagnostizierte Variante der amyotrophen Lateralsklerose.

Progressive muscle atrophy (PMA) is a degenerative disease of the lower motor neuron. The course of the illness and the fatal prognosis correspond to those of amyotrophic lateral sclerosis (ALS). Neuropathologic and genetic findings support categorizing PMA within the spectrum of ALS, even though no clinical sign of a disorder of the upper motor neuron is demonstrable. The diagnosis of PMA is based on advanced extremity pareses and atrophies with a high progression rate. Respiratory insufficiency is determinative of the prognosis. Absent or late affection of bulbar functions is characteristic of the disease. Intraneuronal bunina bodies and ubiquitine-positive inclusions, which are established morphologic characteristics of ALS, are found post mortem. The treatment options of riluzol medication, respiratory therapy, and nutrition are analogous to those for typical ALS.

The p150 subunit of dynactin (DCTN1) gene in multiple sclerosis.

OBJECTIVES: Mutations in the p150 subunit of the axonal transport protein dynactin (DCTN1) have been reported in patients with amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Given the common features of neurodegeneration in multiple sclerosis (MS), FTD and ALS, sequence variants of the DCTN1 gene may be a contributory factor to neurodegeneration in MS. METHODS: We investigated a total of 200 MS patients and 200 controls. A total of 100 patients had a relapsing-remitting form of MS, 100 cases were primary progressive. Sequence alterations were screened for in the coding region of DCTN1 using heteroduplex and sequence analyses. RESULTS: Two heterozygous missense mutations (T1249I, I196V) were found in two healthy control subjects. No mutations were identified in 200 MS patients. The frequency of a known single nucleotide polymorphism (R495Q) was not significantly different between patients and controls. CONCLUSION: The results indicate that the DCTN1 gene is probably not influencing susceptibility to neurodegeneration in MS.

[Pathophysiology and pathomorphology of osteoporosis]. / Pathophysiologie und Pathomorphologie der Osteoporose.

Osteoporosis is a disease that leads to fragility fractures due to loss of bone mass and bone microstructure. This review presents an update on the fundamental pathophysiologic and pathomorphologic mechanisms of bone loss situations. Pathomorphologic characteristics such as perforations and microcallus formations are explained. The physiologic relevance of the remodeling process as well as its control by local-paracrine, systemic-endocrine and central-neural signaling pathways is discussed. Furthermore the role of hormones such as estrogen, FSH and leptin, of transcription-factors such as Runx2 and osterix and as well as that of the wnt signaling pathway for bone cell differentiation and function is presented. On the basis of current knowledge osteoporosis can be diagnosed, treated and fractures can be prevented. However, it is likely that new and even more effective diagnostic and therapeutic strategies will emerge as our understanding of the remodeling process that controls osteoblast and osteoclast function increases.

Possible gender-dependent association of vascular endothelial growth factor (VEGF) gene and ALS.

Individuals homozygous for haplotypes -2578-A/-1154-A/-634-G or -2578-A/-1154-G/-634-G in the promoter/5'UTR of the VEGF gene have a 1.8-fold increased risk of ALS in several European populations. We did not observe any significant association with single markers, or haplotype pairs, in a German sample of 580 sporadic ALS patients and 628 controls. However, the promoter SNP-1154 (rs1570360) was associated with affection status in women (p = 0.036), suggesting that the VEGF effect may be dependent on the sex ratio of the sample.

[Treatment of traumatic rotatory atlanto-axial subluxation in childhood. Case report and literature review]. / Behandlung der traumatisch rotatorischen atlantoaxialen Dislokation im Kindesalter. Fallvorstellung und Literaturübersicht.

Cervical spine injuries are rare in children. They tend to occur predominantly in the region of atlanto-axial articulation. Even traumatic rotatory atlanto-axial subluxation (AAD) has been described in the literature, however, there is no consistent therapeutic protocol. We report on a 4 year old girl with an acute traumatic rotatory atlanto-axial subluxation treated with closed reduction and retention in a hard-collar. Control CT scans and physical examination after 6 weeks revealed an excellent outcome without any signs of recurrence. As there is a correlation between outcome and increasing length of the dislocation-therapy interval, optimal management of acute rotatory atlanto-axial subluxation depends on early diagnosis with plain radiography and CT scans. Timely diagnosed cases may be treated successfully with closed reduction and cervical immobilisation in a hard-collar. Failure to obtain reduction and recurrence are an appropriate indication for surgical intervention.

[Prediction of outcome in critically ill patients using haemodynamic and oxyphoretic parameters]. / Predizione dell'esito dei pazienti critici utilizzando i parametri emodinamici ed ossiforetici.

OBJECTIVE: To find a predictive model for short time mortality and survival of haemodinamically instable patients. DESIGN: Prospective study on two consecutive series of critically ill patients admitted to ICU. SUBJECTS: 115 critically ill patients, subdivided in two series of 83 (47 survivors and 36 non survivors after 20 days from admission) and 32 patients (19 survivors and 13 non survivors), respectively. INTERVENTIONTS: All the patients have been monitored with a Swan-Ganz catheter. MEASUREMENTS: Oxyphoretic parameters were measured at 7 different times (T): T0 (start), T1 (after 12 hours from T0, T2 (24 hrs), T3 (48 hrs), T4 (72 hrs), T5 (96 hrs) and T6 (120 hrs). A total number of 401 recorded values of the first patients' series were used to create the predictive model for outcome (0 non survivors, 1 survivors). Stepwise logistic regression was used to create the model. Model calibration and discrimination was assessed. The model was validated using data collected from the second series. RESULTS: The probability of survival after 20 days was Pr (survive) = e(logit)/1+e(logit), with "logit" = -5,106+(-6,58E-02 SVI)+(-2,76E-03 SVR)+(0,1379 LVSWI)+(0,8933 Hb)+(-3,25E-02 VO2I)+(9,09E-02 O2ER)+(-7,89E-02 SHUNT)+PAT. For medical patients PAT = -0.568; for surgical patients PAT = -1.0525 and for politrauma PAT = 1.593. Goodness-of-fit test showed a good calibration: chi 2 = 4.267, p = 0.832. The area under the ROC curve was 0.831. The model used in the validation data set, with 199 recorded values, also showed a good calibration and discrimination (Goodness-of-fit test: chi 2 = 15.65, p = 0.111; area under the curve 0.798). CONCLUSIONS: The mathematical model we found has been validated also in the second series and the discrimination capability increases with time. Using this model we can evaluate the probability of survival at every time. Its application at different times permits a better evaluation of haemodynamically instable patient trend.