Regulation des Kainatrezeptors GluK2 durch NDRG und SGK1

Ionotrope Glutamatrezeptoren spielen eine wichtige Rolle bei der Funktion von Astrozyten. Der Kainatrezeptor GluK2, der in Neuronen und Astrozyten exprimiert wird, steht unter Regulation der SGK-Signalkaskade, wie bereits in Neuronen gezeigt wurde. In dieser Arbeit wurde der Effekt von SGK1 auf GluK2 in Astrozyten untersucht. Dabei konnte gezeigt werden, dass die Proteine NDRG1 und NDRG2, keinen direkten Einfluss auf die GluK2-Stromamplitude bei heterologer Expression haben. Wenn jedoch NDRG2 mit GluK2 und SGK1 koexprimiert wird, wird die stimulierende Wirkung von SGK1 auf GluK2 unterdrückt. So konnte ein neuer Rückkopplungs-Mechanismus aufgedeckt werden, wobei der GluK2-Stimulation durch SGK1 durch eine parallele Phosphorylierung von NDRG2 gegenreguliert wird. Diese Regulation des GluK2 durch SGK1 und NDRG2 in Astrozyten könnte eine wichtige Rolle bei der Gliotransmission, Modulation der Genexpression oder bei Regulation der Exozytose von tPA spielen

Little helpers or mean rogue—role of microglia in animal models of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is one of the most common neurodegenerative diseases, causing degeneration of both upper and lower motor neurons in the central nervous system (CNS). ALS patients suffer from hyperreflexia, spasticity, paralysis and muscle atrophy and typically die due to respiratory failure 1–5 years after disease onset. In addition to the degeneration of motor neurons on the cellular level, ALS has been associated with neuroinflammation, such as microgliosis. Microglial activation in ALS can either be protective or degenerative to the neurons. Among others, mutations in superoxide dismutase 1 (SOD1), chromosome 9 open reading frame 72 (C9Orf72), transactive response DNA binding protein (TDP) 43 and vacuolar protein sorting-associated protein 54 (VPS54) genes have been associated with ALS. Here, we describe the dual role and functionality of microglia in four different in vivo ALS models and search for the lowest common denominator with respect to the role of microglia in the highly heterogeneous disease of ALS

Caffeine and NAD+NAD^{+} improve motor neural integrity of dissociated wobbler cells in vitro

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

miR-129-5p and miR-130a-3p regulate VEGFR-2 expression in sensory and motor neurons during development

The wide-ranging influence of vascular endothelial growth factor (VEGF) within the central (CNS) and peripheral nervous system (PNS), for example through effects on axonal growth or neuronal cell survival, is mainly mediated by VEGF receptor 2 (VEGFR-2). However, the regulation of VEGFR-2 expression during development is not yet well understood. As microRNAs are considered to be key players during neuronal maturation and regenerative processes, we identified the two microRNAs (miRNAs)—miR-129-5p and miR-130a-3p—that may have an impact on VEGFR-2 expression in young and mature sensory and lower motor neurons. The expression level of VEGFR-2 was analyzed by using in situ hybridization, RT-qPCR, Western blot, and immunohistochemistry in developing rats. microRNAs were validated within the spinal cord and dorsal root ganglia. To unveil the molecular impact of our candidate microRNAs, dissociated cell cultures of sensory and lower motor neurons were transfected with mimics and inhibitors. We depicted age-dependent VEGFR-2 expression in sensory and lower motor neurons. In detail, in lower motor neurons, VEGFR-2 expression was significantly reduced during maturation, in conjunction with an increased level of miR-129-5p. In sensory dorsal root ganglia, VEGFR-2 expression increased during maturation and was accompanied by an overexpression of miR-130a-3p. In a second step, the functional significance of these microRNAs with respect to VEGFR-2 expression was proven. Whereas miR-129-5p seems to decrease VEGFR-2 expression in a direct manner in the CNS, miR-130a-3p might indirectly control VEGFR-2 expression in the PNS. A detailed understanding of genetic VEGFR-2 expression control might promote new strategies for the treatment of severe neurological diseases like ischemia or peripheral nerve injury

ROS scavengers decrease γ\gammaH2ax spots in motor neuronal nuclei of ALS model mice in vitro\textit {in vitro}

$\bf Background:$ Amyotrophic lateral sclerosis (ALS) is an incurable neurodegenerative disease characterized by the loss of motor neurons in cerebral cortex, brainstem and spinal cord. Numerous studies have demonstrated signs of oxidative stress in postmortem neuronal tissue, cerebrospinal fluid, plasma and urine of ALS patients, without focusing on the specific processes within motor neurons. Thus, we aimed to investigate the relevance of reactive oxygen species (ROS) detoxification mechanisms and its consequences on the formation of toxic/lethal DNA double strand breaks (DSBs) in the ALS model of the Wobbler mouse. $\bf Methods:$ Live cell imaging in dissociated motor neuronal cultures was used to investigate the production of ROS using Dihydroethidium (DHE). The expression levels of ROS detoxifying molecules were investigated by qPCR as well as Western blots. Furthermore, the expression levels of DNA damage response proteins p53bp1 and H2ax were investigated using qPCR and immunofluorescence staining. Proof-of-principle experiments using ROS scavengers were performed $\textit {in vitro}$ to decipher the influence of ROS on the formation of DNA double strand breaks quantifying the $\gamma$H2ax spots formation. $\bf Results:$ Here, we verified an elevated ROS-level in spinal motor neurons of symptomatic Wobbler mice $\textit {in vitro}$. As a result, an increased number of DNA damage response proteins p53bp1 and $\gamma$H2ax in dissociated motor neurons of the spinal cord of Wobbler mice was observed. Furthermore, we found a significantly altered expression of several antioxidant molecules in the spinal cord of Wobbler mice, suggesting a deficit in ROS detoxification mechanisms. This hypothesis could be verified by using ROS scavenger molecules $\textit {in vitro}$ to reduce the number of $\gamma$H2ax foci in dissociated motor neurons and thus counteract the harmful effects of ROS. $\bf Conclusion:$ Our data indicate that maintenance of redox homeostasis may play a key role in the therapy of the neurodegenerative disease ALS. Our results underline a necessity for multimodal treatment approaches to prolong the average lifespan of motor neurons and thus slow down the progression of the disease, since a focused intervention in one pathomechanism seems to be insufficient in ALS therapy

Morphological plasticity of emerging purkinje cells in response to exogenous VEGF

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

Vascular endothelial growth factor, irradiation, and axitinib have diverse effects on motility and proliferation of glioblastoma multiforme cells

Glioblastoma multiforme (GBM) is the most common primary brain tumor. It is highly aggressive with an unfavorable prognosis for the patients despite therapies including surgery, irradiation, and chemotherapy. One important characteristic of highly vascu-larized GBM is the strong expression of vascular endothelial growth factor (VEGF). VEGF has become a new target in the treatment of GBM, and targeted therapies such as the VEGF-receptor blocker axitinib are in clinical trials. Most studies focus on VEGF-induced angiogenesis, but only very few investigations analyze autocrine or paracrine effects of VEGF on the tumor cells. In this study, we examined the impact of VEGF, irradiation, and axitinib on cell proliferation and cell motility in human GBM cell lines U-251 and U-373. VEGF receptor 2 was shown to be expressed within both cell lines by using PCR and immunochemistry. Moreover, we performed 24-h videography to analyze motility, and a viability assay for cell proliferation. We observed increasing effects of VEGF and irradiation on cell motility in both cell lines, as well as strong inhibiting effects on cellular motility by VEGF-receptor blockade using axitinib. Moreover, axitinib diminished irradiation induced accelerating effects. While VEGF stimulation or irradiation did not affect cell proliferation, axitinib significantly decreased cell proliferation in both cell lines. Therefore, the impairment of VEGF signaling might have a crucial role in the treatment of GBM

Vascular endothelial growth factor, irradiation, and axitinib have diverse effects on motility and proliferation of glioblastoma multiforme cells

Glioblastoma multiforme (GBM) is the most common primary brain tumor. It is highly aggressive with an unfavorable prognosis for the patients despite therapies including surgery, irradiation, and chemotherapy. One important characteristic of highly vascularized GBM is the strong expression of vascular endothelial growth factor (VEGF). VEGF has become a new target in the treatment of GBM, and targeted therapies such as the VEGF-receptor blocker axitinib are in clinical trials. Most studies focus on VEGF-induced angiogenesis, but only very few investigations analyze autocrine or paracrine effects of VEGF on the tumor cells. In this study, we examined the impact of VEGF, irradiation, and axitinib on cell proliferation and cell motility in human GBM cell lines U-251 and U-373. VEGF receptor 2 was shown to be expressed within both cell lines by using PCR and immunochemistry. Moreover, we performed 24-h videography to analyze motility, and a viability assay for cell proliferation. We observed increasing effects of VEGF and irradiation on cell motility in both cell lines, as well as strong inhibiting effects on cellular motility by VEGF-receptor blockade using axitinib. Moreover, axitinib diminished irradiation induced accelerating effects. While VEGF stimulation or irradiation did not affect cell proliferation, axitinib significantly decreased cell proliferation in both cell lines. Therefore, the impairment of VEGF signaling might have a crucial role in the treatment of GBM

Deregulated miR-29b-3p\textit {miR-29b-3p} correlates with tissue-specific activation of intrinsic apoptosis in an animal model of amyotrophic lateral sclerosis

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 $\textit {miR-29b-3p}$ 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 $\textit {miR-29b-3p}$ 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 $\textit {miR-29b-3p}$ might regulate antiapoptotic survival mechanisms in CNS areas that are not affected by neurodegeneration in the wobbler mouse ALS model

Neuroprotective effects of VEGF in the enteric nervous system

Although the enteric nervous system (ENS) functions largely autonomously as part of the peripheral nervous system (PNS), it is connected to the central nervous system (CNS) via the gut–brain axis. In many neurodegenerative diseases, pathological changes occur in addition to gastrointestinal symptoms, such as alpha-synuclein aggregates in Parkinson’s disease, which are found early in the ENS. In both the CNS and PNS, vascular endothelial growth factor (VEGF) mediates neuroprotective and neuroregenerative effects. Since the ENS with its close connection to the microbiome and the immune system is discussed as the origin of neurodegenerative diseases, it is necessary to investigate the possibly positive effects of VEGF on enteric neurons. Using laser microdissection and subsequent quantitative RT-PCR as well as immunohistochemistry, for the first time we were able to detect and localize VEGF receptor expression in rat myenteric neurons of different ages. Furthermore, we demonstrate direct neuroprotective effects of VEGF in the ENS in cell cultures. Thus, our results suggest a promising approach regarding neuroprotection, as the use of VEGF (may) prevent neuronal damage in the ENS