Oxygen is essential to the human life and life of all aerobic organisms. The complete oxidation of nutrients for the biological energy supply is one of the most important prerequisites for the formation of higher life forms. However, cells that benefit from oxidative respiration also suffer from reactive oxygen species because they adapted to oxygen as an energy source. Healthy cells balance the formation and elimination of reactive oxygen species thereby creating and keeping reactive oxygen species-homeostasis. When the concentration of free radicals exceeds a critical level and homeostasis is disturbed, oxidative stress occurs leading to damage of multiple cellular molecules and compartments. Therefore, oxidative stress plays an important role in the physiology and pathology of various diseases. Often, the antioxidant protection system becomes pathologically unbalanced in the genesis of several diseases, leading to functional losses of the organism, as in the case of amyotrophic lateral sclerosis, or cells develop metabolic mechanisms to use this system as protection against external influences, such as in the case of glioblastoma cells. Either way, understanding the underlying deregulated mechanisms of the oxidative protection system would allow the development of novel treatment strategies for various diseases. Thus, regardless of the direction in which the reactive oxygen species-homeostasis disequilibrate, the focus should be on the oxidative protection system.

Amyotrophic lateral sclerosis is a devastating motor neuron disease and to this day not curable. While 5-10% of patients inherit the disease (familiar ALS), up to 95% of patients are diagnosed with the sporadic form (sALS). ALS is characterized by the degeneration of upper motor neurons in the cerebral cortex and of lower motor neurons in the brainstem and spinal cord. The wobbler mouse resembles almost all phenotypical hallmarks of human sALS patients and is therefore an excellent motor neuron disease model. The motor neuron disease of the wobbler mouse develops over a time course of around 40 days and can be divided into three phases: p0, presymptomatic; p20, early clinical; and p40, stable clinical phase. Recent findings suggest an essential implication of the NAD-producing enzyme Nmnat2 in neurodegeneration as well as maintenance of healthy axons. Here, we were able to show a significant downregulation of both gene and protein expression of Nmnat2 in the spinal cord of the wobbler mice at the stable clinical phase. The product of the enzyme NAD is also significantly reduced, and the values of the reactive oxygen species are significantly increased in the spinal cord of the wobbler mouse at p40. Thus, the deregulated expression of Nmnat2 appears to have a great influence on the cellular stress in the spinal cord of wobbler mice.

Vascular endothelial growth factor (VEGF) is well known as the growth factor with wide-ranging functions even in the central nervous system (CNS). Presently, most attention is given to the investigation of its role in neuronal protection, growth and maturation processes, whereby most effects are mediated through VEGF receptor 2 (VEGFR-2). The purpose of our current study is to provide new insights into the impact of VEGF on immature and mature Purkinje cells (PCs) in accordance with maturity and related receptor expression. Therefore, to expand our knowledge of VEGF effects in PCs development and associated VEGFR-2 expression, we used cultivated organotypic cerebellar slice cultures in immunohistochemical or microinjection studies, followed by confocal laser scanning microscopy (CLSM) and morphometric analysis. Additionally, we incorporated in our study the method of laser microdissection, followed by quantitative polymerase chain reaction (qPCR). For the first time we could show the age-dependent VEGF sensitivity of PCs with the largest promoting effects being on dendritic length and cell soma size in neonatal and juvenile stages. Once mature, PCs were no longer susceptible to VEGF stimulation. Analysis of VEGFR-2 expression revealed its presence in PCs throughout development, which underlined its mediating functions in neuronal cells.

Ionotropic glutamate receptors are the most important excitatory receptors in the central nervous system, and their impairment can lead to multiple neuronal diseases. Here, we show that glutamate-induced currents in oocytes expressing GluA1 are increased by coexpression of the schizophrenia-associated phosphoinositide kinase PIP5K2A. This effect was due to enhanced membrane abundance and was blunted by a point mutation (N251S) in PIP5K2A. An increase in GluA1 currents was also observed upon acute injection of PI(4,5)P2, the main product of PIP5K2A. By expression of wild-type and mutant PIP5K2A in human embryonic kidney cells, we were able to provide evidence of impaired kinase activity of the mutant PIP5K2A. We defined the region K813–K823 of GluA1 as critical for the PI(4,5)P2 effect by performing an alanine scan that suggested PI(4,5)P2 binding to this area. A PIP strip assay revealed PI(4,5)P2 binding to the C-terminal GluA1 peptide. The present observations disclose a novel mechanism in the regulation of GluA1.Electronic supplementary materialThe online version of this article (doi:10.1007/s00424-013-1424-8) contains supplementary material, which is available to authorized users.

Background/Aims: Acquired as well as inherited channelopathies are disorders that are caused by altered ion channel function. A family of channels whose malfunction is associated with different channelopathies is the Kv7 K⁺ channel family; and restoration of normal Kv7 channel function by small molecule modulators is a promising approach for treatment of these often fatal diseases. Methods: Here, we show the modulation of Kv7 channels by the natural compound Rottlerin heterologously expressed in Xenopus laevis oocytes and on iPSC cardiomyocytes overexpressing Kv7.1 channels. Results: We show that currents carried by Kv7.1 (EC₅₀ = 1.48 μM), Kv7.1/KCNE1 (EC₅₀ = 4.9 μM), and Kv7.4 (EC₅₀ = 0.148 μM) are strongly enhanced by the compound, whereas Kv7.2, Kv7.2/Kv7.3, and Kv7.5 are not sensitive to Rottlerin. Studies on Kv7.1/KCNE1 mutants and in silico modelling indicate that Rottlerin binds to the R-L3-activator site. Rottlerin mediated activation of Kv7.1/KCNE1 channels might be a promising approach in long QT syndrome. As a proof of concept, we show that Rottlerin shortens cardiac repolarisation in iPSC-derived cardiomyocytes expressing Kv7.1.Conclusion: Rottlerin or an optimized derivative holds a potential as QT interval correcting drug.

Amyotrophic lateral sclerosis (ALS) is a common degenerative disease of the central nervous system concerning a progressive loss of upper and lower motor neurons. While 5%–10% of patients are diagnosed with the inherited form of the disease, the vast majority of patients suffer from the less characterized sporadic form of ALS (sALS). As the wobbler mouse and the ALS show striking similarities in view of phenotypical attributes, the mouse is rated as an animal model for the disease. Recent investigations show the importance of nicotinamide adenine dinucleotide (NAD+) and its producing enzyme nicotinic acid mononucleotide transferase 2 (Nmnat2) for neurodegeneration as well as for the preservation of health of the neuronal cells. Furthermore, it is newly determined that these molecules show significant downregulations in the spinal cord of wobbler mice in the stable phase of disease development. Here, we were able to prove a positive benefit on affected motor neurons from an additional NAD+ supply as well as an increase in the Nmnat2 level through caffeine treatment in cells in vitro. In addition, first assumptions about the importance of endogenous and exogenous factors that have an influence on the wellbeing of motor nerve cells in the model of ALS can be considered.

Amyotrophic lateral sclerosis (ALS) is one of the most common incurable motor neuron disorders in adults. The majority of all ALS cases occur sporadically (sALS). Symptoms of ALS are caused by a progressive degeneration of motor neurons located in the motor cortex and spinal cord. The question arises why motor neurons selectively degenerate in ALS, while other cells and systems appear to be spared the disease. Members of the intrinsic apoptotic pathway are frequent targets of altered microRNA expression. Therefore, microRNAs and their effects on cell survival are subject of controversial debates. In this study, we investigated the expression of numerous members of the intrinsic apoptotic cascade by qPCR, western blot, and immunostaining in two different regions of the CNS of wobbler mice. Further we addressed the expression of miR-29b-3p targeting BMF, Bax, and, Bak, members of the apoptotic pathway. We show a tissue-specific differential expression of BMF, Bax, and cleaved-Caspase 3 in wobbler mice. An opposing regulation of miR-29b-3p expression in the cerebellum and cervical spinal cord of wobbler mice suggests different mechanisms regulating the intrinsic apoptotic pathway. Based on our findings, it could be speculated that miR-29b-3p might regulate antiapoptotic survival mechanisms in CNS areas that are not affected by neurodegeneration in the wobbler mouse ALS model.

Amyotrophic lateral sclerosis (ALS) is a common neurodegenerative disease that affects motor neurons in the spinal cord and motor cortex. Various mouse models have been used to investigate the progression of the pathology of sporadic and familial ALS. Degeneration in the spinal cord and motor cortex in the Wobbler mouse model of sporadic ALS have been documented, but alterations of the cerebellum during disease progression have not been well characterized. We analyzed neurodegeneration and inflammatory responses in the cerebellar cortex of preclinical (p20), clinical (p40), and late (p60) stages in these mice. We did not identify evidence of neuron cell death, but we observed an inflammatory response detected by IL1B and TNFA expression by quantitative PCR, increased activated microglia and astrocytosis by immunohistochemistry, and ultrastructural abnormalities in the cerebella of Wobbler mice at late stages. These alterations may be caused by protein aggregations and variations in the distribution of cytoskeletal proteins; they might be reflected in the early manifestation of head tremor, which precedes motor deficits in these mice. Thus, we conclude that, in addition to the motor cortex and spinal cord, the cerebellum is affected by neurodegenerative and inflammatory processes in the Wobbler mouse model of ALS.