Mechanisms underlying the health benefits of intermittent hypoxia conditioning.

Intermittent hypoxia (IH) is commonly associated with pathological conditions, particularly obstructive sleep apnoea. However, IH is also increasingly used to enhance health and performance and is emerging as a potent non-pharmacological intervention against numerous diseases. Whether IH is detrimental or beneficial for health is largely determined by the intensity, duration, number and frequency of the hypoxic exposures and by the specific responses they engender. Adaptive responses to hypoxia protect from future hypoxic or ischaemic insults, improve cellular resilience and functions, and boost mental and physical performance. The cellular and systemic mechanisms producing these benefits are highly complex, and the failure of different components can shift long-term adaptation to maladaptation and the development of pathologies. Rather than discussing in detail the well-characterized individual responses and adaptations to IH, we here aim to summarize and integrate hypoxia-activated mechanisms into a holistic picture of the body's adaptive responses to hypoxia and specifically IH, and demonstrate how these mechanisms might be mobilized for their health benefits while minimizing the risks of hypoxia exposure.

The Kappa Opioid Receptor System in Temporal Lobe Epilepsy.

Temporal lobe epilepsy is considered to be one of the most common and severe forms of focal epilepsies. Patients frequently develop cognitive deficits and emotional blunting along progression of the disease. The high incidence of refractoriness to antiepileptic drugs and a frequent lack of admissibility to surgery pose an unmet medical challenge. In the urgent quest for novel treatment strategies, neuropeptides and their receptors are interesting candidates. However, their therapeutic potential has not yet been fully exploited. This chapter focuses on the functional role of the dynorphins (Dyns) and the kappa opioid receptor (KOR) system in temporal lobe epilepsy and the hippocampus.Genetic polymorphisms in the prepro-dynorphin (pDyn) gene causing lower levels of Dyns in humans and pDyn gene knockout in mice increase the risk to develop epilepsy. This suggests a role of Dyns and KOR as modulators of neuronal excitability. Indeed, KOR agonists induce inhibition of presynaptic neurotransmitter release, as well as postsynaptic hyperpolarization in glutamatergic neurons, both producing anticonvulsant effects.The development of new approaches to modulate the complex KOR signalling cascade (e.g. biased agonism and gene therapy) opens up new exciting therapeutic opportunities with regard to seizure control and epilepsy. Potential adverse side effects of KOR agonists may be minimized through functional selectivity or locally restricted treatment. Preclinical data suggest a high potential of such approaches to control seizures.

Considerations on Using Antibodies for Studying the Dynorphins/Kappa Opioid Receptor System.

Antibodies are important tools for protein and peptide research, including for the kappa opioid receptor (KOR) and dynorphins (Dyns). Well-characterized antibodies are essential for rigorous and reproducible research. However, lack of validation of antibody specificity has been thought to contribute significantly to the reproducibility crisis in biomedical research. Since 2003, many scientific journals have required documentation of validation of antibody specificity and use of knockout mouse tissues as a negative control is strongly recommended. Lack of specificity of antibodies against many G protein-coupled receptors (GPCRs) after extensive testing has been well-documented, but antibodies generated against partial sequences of the KOR have not been similarly investigated. For the dynorphins, differential processing has been described in distinct brain areas, resulting in controversial findings in immunohistochemistry (IHC) when different antibodies were used. In this chapter, we summarized accepted approaches for validation of antibody specificity. We discussed two KOR antibodies most commonly used in IHC and described generation and characterization of KOR antibodies and phospho-KOR specific antibodies in western blotting or immunoblotting (IB). In addition, applying antibodies targeting prodynorphin or mature dynorphin A illustrates the diversity of results obtained regarding the distribution of dynorphins in distinct brain areas.

A Rationale for Hypoxic and Chemical Conditioning in Huntington's Disease.

Neurodegenerative diseases are characterized by adverse cellular environments and pathological alterations causing neurodegeneration in distinct brain regions. This development is triggered or facilitated by conditions such as hypoxia, ischemia or inflammation and is associated with disruptions of fundamental cellular functions, including metabolic and ion homeostasis. Targeting intracellular downstream consequences to specifically reverse these pathological changes proved difficult to translate to clinical settings. Here, we discuss the potential of more holistic approaches with the purpose to re-establish a healthy cellular environment and to promote cellular resilience. We review the involvement of important molecular pathways (e.g., the sphingosine, δ-opioid receptor or N-Methyl-D-aspartate (NMDA) receptor pathways) in neuroprotective hypoxic conditioning effects and how these pathways can be targeted for chemical conditioning. Despite the present scarcity of knowledge on the efficacy of such approaches in neurodegeneration, the specific characteristics of Huntington's disease may make it particularly amenable for such conditioning techniques. Not only do classical features of neurodegenerative diseases like mitochondrial dysfunction, oxidative stress and inflammation support this assumption, but also specific Huntington's disease characteristics: a relatively young age of neurodegeneration, molecular overlap of related pathologies with hypoxic adaptations and sensitivity to brain hypoxia. The aim of this review is to discuss several molecular pathways in relation to hypoxic adaptations that have potential as drug targets in neurodegenerative diseases. We will extract the relevance for Huntington's disease from this knowledge base.

Mitochondrial Respiration Changes in R6/2 Huntington's Disease Model Mice during Aging in a Brain Region Specific Manner.

Mitochondrial dysfunction is crucially involved in aging and neurodegenerative diseases, such as Huntington's Disease (HD). How mitochondria become compromised in HD is poorly understood but instrumental for the development of treatments to prevent or reverse resulting deficits. In this paper, we investigate whether oxidative phosphorylation (OXPHOS) differs across brain regions in juvenile as compared to adult mice and whether such developmental changes might be compromised in the R6/2 mouse model of HD. We study OXPHOS in the striatum, hippocampus, and motor cortex by high resolution respirometry in female wild-type and R6/2 mice of ages corresponding to pre-symptomatic and symptomatic R6/2 mice. We observe a developmental shift in OXPHOS-control parameters that was similar in R6/2 mice, except for cortical succinate-driven respiration. While the LEAK state relative to maximal respiratory capacity was reduced in adult mice in all analyzed brain regions, succinate-driven respiration was reduced only in the striatum and cortex, and NADH-driven respiration was higher as compared to juvenile mice only in the striatum. We demonstrate age-related changes in respirational capacities of different brain regions with subtle deviations in R6/2 mice. Uncovering in situ oxygen conditions and potential substrate limitations during aging and HD disease progression are interesting avenues for future research to understand brain-regional vulnerability in HD.

Restricting calcium currents is required for correct fiber type specification in skeletal muscle.

Skeletal muscle excitation-contraction (EC) coupling is independent of calcium influx. In fact, alternative splicing of the voltage-gated calcium channel CaV1.1 actively suppresses calcium currents in mature muscle. Whether this is necessary for normal development and function of muscle is not known. However, splicing defects that cause aberrant expression of the calcium-conducting developmental CaV1.1e splice variant correlate with muscle weakness in myotonic dystrophy. Here, we deleted CaV1.1 (Cacna1s) exon 29 in mice. These mice displayed normal overall motor performance, although grip force and voluntary running were reduced. Continued expression of the developmental CaV1.1e splice variant in adult mice caused increased calcium influx during EC coupling, altered calcium homeostasis, and spontaneous calcium sparklets in isolated muscle fibers. Contractile force was reduced and endurance enhanced. Key regulators of fiber type specification were dysregulated and the fiber type composition was shifted toward slower fibers. However, oxidative enzyme activity and mitochondrial content declined. These findings indicate that limiting calcium influx during skeletal muscle EC coupling is important for the secondary function of the calcium signal in the activity-dependent regulation of fiber type composition and to prevent muscle disease.

Sprouty2 and -4 hypomorphism promotes neuronal survival and astrocytosis in a mouse model of kainic acid induced neuronal damage.

Sprouty (Spry) proteins play a key role as negative feedback inhibitors of the Ras/Raf/MAPK/ERK pathway downstream of various receptor tyrosine kinases. Among the four Sprouty isoforms, Spry2 and Spry4 are expressed in the hippocampus. In this study, possible effects of Spry2 and Spry4 hypomorphism on neurodegeneration and seizure thresholds in a mouse model of epileptogenesis was analyzed. The Spry2/4 hypomorphs exhibited stronger ERK activation which was limited to the CA3 pyramidal cell layer and to the hilar region. The seizure threshold of Spry2/4(+/-) mice was significantly reduced at naive state but no difference to wildtype mice was observed 1 month following KA treatment. Histomorphological analysis revealed that dentate granule cell dispersion (GCD) was diminished in Spry2/4(+/-) mice in the subchronic phase after KA injection. Neuronal degeneration was reduced in CA1 and CA3 principal neuron layers as well as in scattered neurons of the contralateral CA1 and hilar regions. Moreover, Spry2/4 reduction resulted in enhanced survival of somatostatin and neuropeptide Y expressing interneurons. GFAP staining intensity and number of reactive astrocytes markedly increased in lesioned areas of Spry2/4(+/-) mice as compared with wildtype mice. Taken together, although the seizure threshold is reduced in naive Spry2/4(+/-) mice, neurodegeneration and GCD is mitigated following KA induced hippocampal lesions, identifying Spry proteins as possible pharmacological targets in brain injuries resulting in neurodegeneration. The present data are consistent with the established functions of the ERK pathway in astrocyte proliferation as well as protection from neuronal cell death and suggest a novel role of Spry proteins in the migration of differentiated neurons.

Metallothioneins and renal ageing.

BACKGROUND: Human lifespan is increasing continuously and about one-third of the population >70 years of age suffers from chronic kidney disease. The pathophysiology of the loss of renal function with ageing is unclear. METHODS: We determined age-associated gene expression changes in zero-hour biopsies of deceased donor kidneys without laboratory signs of impaired renal function, defined as a last serum creatinine >0.96 mg/dL in females and >1.18 mg/dL in males, using microarray technology and the Significance Analysis of Microarrays routine. Expression changes of selected genes were confirmed by quantitative polymerase chain reaction and in situ hybridization and immunohistochemistry for localization of respective mRNA and protein. Functional aspects were examined in vitro. RESULTS: Donors were classified into three age groups (<40, 40-59 and >59 years; Groups 1, 2 and 3, respectively). In Group 3 especially, genes encoding for metallothionein (MT) isoforms were more significantly expressed when compared with Group 1; localization studies revealed predominant staining in renal proximal tubular cells. RPTEC/TERT1 cells overexpressing MT2A were less susceptible towards cadmium chloride-induced cytotoxicity and hypoxia-induced apoptosis, both models for increased generation of reactive oxygen species. CONCLUSIONS: Increased expression of MTs in the kidney with ageing might be a protective mechanism against increased oxidative stress, which is closely related to the ageing process. Our findings indicate that MTs are functionally involved in the pathophysiology of ageing-related processes.

STAM2, a member of the endosome-associated complex ESCRT-0 is highly expressed in neurons.

STAM2 (signal transducing adaptor molecule 2), a subunit of the ESCRT-0 complex, is an endosomal protein acting as a regulator of receptor signaling and trafficking. To analyze STAM2 in the nervous system, its gene expression and protein localization in the mouse brain were identified using three methods: mRNA in situ hybridization, immunohistochemistry, and via lacZ reporter in frame with Stam2 gene using the gene trap mouse line Stam2(Gt1Gaj). STAM2 intracellular localization was analyzed by subcellular fractionation and co-immunofluorescence using confocal microscopy. Stam2 was strongly expressed in the cerebral and cerebellar cortex, hippocampal formation, olfactory bulb, and medial habenula. The majority of STAM2-positive cells co-stained with the neuronal markers. In neurons STAM2 was found in the early endosomes and also in the nucleus. The other members of the ESCRT-0 complex co-localized with STAM2 in the cytoplasm, but they were not present in the nucleus. The newly identified neuron-specific nuclear localization of STAM2, together with its high expression in the brain indicated that STAM2 might have a specific function in the mouse nervous system.

Knockdown of prodynorphin gene prevents cognitive decline, reduces anxiety, and rescues loss of group 1 metabotropic glutamate receptor function in aging.

Expression of dynorphin, an endogenous opioid peptide, increases with age and has been associated with memory impairments in rats. In human, prodynorphin (Pdyn) gene polymorphisms might be linked to cognitive function in the elderly. Moreover, elevated dynorphin levels have been reported in postmortem samples from Alzheimer's disease patients. However, the cellular and molecular processes affected by higher dynorphin levels during aging remain unknown. Using Pdyn(-/-) mice, we observed significant changes in the function and expression of Group 1 metabotropic glutamate receptor (mGluR). Compared with age-matched wild-type (WT) littermates, we found increased expression of mGluR1α and mGluR5 in the hippocampus and cortex of old, but not young, Pdyn(-/-) mice. Increased Group 1 mGluR expression in aged Pdyn(-/-) mice was associated with enhanced mGluR-mediated long-term depression, a form of synaptic plasticity. Notably, whereas aged WT mice developed spatial and recognition memory deficits, aged Pdyn(-/-) mice performed similarly as young mice. Pharmacological treatments with 3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide, a positive modulator of mGlu5 receptors, or norbinaltorphimine, an antagonist for dynorphin-targeted κ-opioid receptor, rescued memory in old WT mice. Conversely, mGlu5 receptor antagonist 2-methyl-6-(phenylethynyl)pyridine hydrochloride impaired spatial memory of old Pdyn(-/-) mice. Intact cognition in aged Pdyn(-/-) mice paralleled with increased expression of Group 1 mGluR-related genes Homer 1a and Arc. Finally, aged Pdyn(-/-) mice displayed less anxiety-related behaviors than age-matched WT mice. Together, our results suggest that elevated Pdyn expression during normal aging reduces mGluR expression and signaling, which in turn impairs cognitive functions and increases anxiety.

Direct association of the reticulon protein RTN1A with the ryanodine receptor 2 in neurons.

RTN1A is a reticulon protein with predominant localization in the endoplasmic reticulum (ER). It was previously shown that RTN1A is expressed in neurons of the mammalian central nervous system but functional information remains sparse. To elucidate the neuronal function of RTN1A, we chose to focus our investigation on identifying possible novel binding partners specifically interacting with the unique N-terminus of RTN1A. Using a nonbiased approach involving GST pull-downs and MS analysis, we identified the intracellular calcium release channel ryanodine receptor 2 (RyR2) as a direct binding partner of RTN1A. The RyR2 binding site was localized to a highly conserved 150-amino acid residue region. RTN1A displays high preference for RyR2 binding in vitro and in vivo and both proteins colocalize in hippocampal neurons and Purkinje cells. Moreover, we demonstrate the precise subcellular localization of RTN1A in Purkinje cells and show that RTN1A inhibits RyR channels in [(3)H]ryanodine binding studies on brain synaptosomes. In a functional assay, RTN1A significantly reduced RyR2-mediated Ca(2+) oscillations. Thus, RTN1A and RyR2 might act as functional partners in the regulation of cytosolic Ca(2+) dynamics the in neurons.

A cycloartane glycoside derived from Actaea racemosa L. modulates GABAA receptors and induces pronounced sedation in mice.

23-O-Acetylshengmanol 3-O-ß-D-xylopyranoside (Ac-SM) isolated from Actaea racemosa L.-an herbal remedy for the treatment of mild menopausal disorders-has been recently identified as a novel efficacious modulator of GABAA receptors composed of α1-, ß2-, and γ2S-subunits. In the present study, we analyzed a potential subunit-selective modulation of GABA-induced chloride currents (IGABA) at GABA concentrations eliciting 3-8% of the maximal GABA response (EC3-8) through nine GABAA receptor isoforms expressed in Xenopus laevis oocytes by Ac-SM with two-microelectrode voltage clamp and behavioral effects 30 minutes after intraperitoneal application in a mouse model. Efficacy of IGABA enhancement by Ac-SM displayed a mild α-subunit dependence with α2ß2γ2S (maximal IGABA potentiation [Emax] = 1454 ± 97%) and α5ß2γ2S (Emax = 1408 ± 87%) receptors being most efficaciously modulated, followed by slightly weaker IGABA enhancement through α1ß2γ2S (Emax = 1187 ± 166%), α3ß2γ2S (Emax = 1174 ± 218%), and α6ß2γ2S (Emax = 1171 ± 274%) receptors and less pronounced effects on receptors composed of α4ß2γ2S (Emax = 752 ± 53%) subunits, whereas potency was not affected by the subunit composition (EC50 values ranging from α1ß2γ2S = 35.4 ± 12.3 µM to α5ß2γ2S = 50.9 ± 11.8 µM). Replacing ß2- with ß1- or ß3-subunits as well as omitting the γ2S-subunit affected neither efficacy nor potency of IGABA enhancement by Ac-SM. Ac-SM shifted the GABA concentration-response curve toward higher GABA sensitivity (about 3-fold) and significantly increased the maximal GABA response by 44 ± 13%, indicating a pharmacological profile distinct from a pure allosteric GABAA receptor modulator. In mice, Ac-SM significantly reduced anxiety-related behavior in the elevated plus maze test at a dose of 0.6 mg/kg, total ambulation in the open field test at doses ≥6 mg/kg, stress-induced hyperthermia at doses ≥0.6 mg/kg, and significantly elevated seizure threshold at doses ≥20 mg/kg body weight. High efficacy and long biologic half-life of Ac-SM suggest that potential cumulative sedative side effects upon repetitive intake of A. racemosa L. preparations might not be negligible.

GPER1 (GPR30) knockout mice display reduced anxiety and altered stress response in a sex and paradigm dependent manner.

The putative estrogen receptor GPER1 (the former orphan receptor GPR30) is discussed to be involved in emotional and cognitive functions and stress control. We recently described the induction of anxiety-like effects by the GPER1 agonist G-1 upon systemic injection into mice. To contribute to a better understanding of the role of GPER1 in anxiety and stress, we investigated germ-line GPER1 deficient mice. Our experiments revealed marked differences between the sexes. A mild but consistent phenotype of increased exploratory drive was observed in the home cage, the elevated plus maze and the light-dark choice test in male GPER1 KO mice. In contrast, female GPER1-KO mice displayed a less pronounced phenotype in these tests. Estrous-stage dependent mild anxiolytic-like effects were observed solely in the open field test. Notably, we observed a strong shift in acute stress coping behavior in the tail suspension test and basal corticosterone levels in different phases of the estrous cycle in female GPER1-KO mice. Our data, in line with previous reports, suggest that GPER1 is involved in anxiety and stress control. Surprisingly, its effects appear to be stronger in male than female mice.

Possible role of dynorphins in Alzheimer's disease and age-related cognitive deficits.

BACKGROUND/AIMS: Expression of dynorphin, an endogenous opioid peptide, increases with age and has been associated with cognitive deficits in rodents. Elevated dynorphin levels have been reported in postmortem samples from Alzheimer's disease (AD) patients, and prodynorphin (PDYN) gene polymorphisms might be linked to cognitive function in the elderly. Activation of κ-opioid receptors by dynorphins has been associated with stress-related memory impairments. Interestingly, these peptides can also modulate glutamate neurotransmission and may affect synaptic plasticity underlying memory formation. N-methyl-D-aspartate (NMDA) and α-amino-3-hydroxy-5-methyl-4-isoxazol-propionate (AMPA) ionotropic glutamate receptor levels generally decrease with aging, and their function is impaired in AD. METHODS: Here, we compared the impact of aging on ionotropic glutamate receptor levels in the hippocampal formation of wild-type (WT) and Pdyn knock-out (KO) mice. RESULTS: We observed a significant reduction in GluR1 and GluR2 AMPA receptor subunits in the hippocampal formation of 18- to 25-month-old WT mice in comparison with 6-month-old mice. Conversely, the GluR1 protein level was maintained in old Pdyn KO mice, and the NMDA NR2B subunit level was increased by 42% when compared to old WT animals. CONCLUSIONS: These results suggest that elevated dynorphin expression occurring during aging and AD may mediate cognitive deficits by altering the glutamatergic system integrity.

Spatio-temporal expression of HOX genes in human hindgut development.

BACKGROUND: Hox genes belong to a highly conserved subgroup of the homeobox gene superfamily. Studies of animal models have emphasized their role in defining the body plan by their coordinated expression along the body axis during ontogeny. Although an important role of HOX genes in human development is assumed, little is known about their expression during human ontogenesis. Therefore, we investigated the expression of the nine most posterior members of the HOXA, HOXB, HOXC, and HOXD clusters in embryonic hindgut between weeks 6 to 12 and in adult rectal tissue. RESULTS: Applying in situ hybridization and immunohistochemistry, we observed expression of HOXA11, HOXA13, HOXD12, and HOXD13 in developmental week 6. However, expression of HOXD12 faded during weeks 7 and 8, and then became increasingly re-expressed during week 9 in humans. With the exception of HOXD13, all expressed HOX genes dropped below detection limits in week 11. Adult rectal tissue displayed distinct HOXA11, HOXA13, HOXD12, and HOXD13 expression patterns within the rectal layers. CONCLUSIONS: Our data suggest a strict spatio-temporal regulation of HOX gene expression during human development, supporting the idea of their role as key regulators. Nonetheless, the expression pattern of distinct HOX genes differs markedly from animal models.

Characterization of the intrahippocampal kainic acid model in female mice with a special focus on seizure suppression by antiseizure medications.

Despite special challenges in the medical treatment of women with epilepsy, in particular preclinical animal studies were focused on males for decades and females have only recently moved into the focus of scientific interest. The intrahippocampal kainic acid (IHKA) mouse model of temporal lobe epilepsy (TLE) is one of the most studied models in males reproducing electroencephalographic (EEG) and histopathological features of human TLE. Hippocampal paroxysmal discharges (HPDs) were described as drug resistant focal seizures in males. Here, we investigated the IHKA model in female mice, in particular drug-resistance of HPDs and the influence of antiseizure medications (ASMs) on the power spectrum. After injecting kainic acid (KA) unilaterally into the hippocampus of female mice, we monitored the development of epileptiform activity by local field potential (LFP) recordings. Subsequently, we evaluated the effect of the commonly prescribed ASMs lamotrigine (LTG), oxcarbazepine (OXC) and levetiracetam (LEV), as well as the benzodiazepine diazepam (DZP) with a focus on HPDs and power spectral analysis and assessed neuropathological alterations of the hippocampus. In the IHKA model, female mice replicated key features of human TLE as previously described in males. Importantly, HPDs in female mice did not respond to commonly prescribed ASMs in line with the drug-resistance in males, thus representing a suitable model of drug-resistant seizures. Intriguingly, we observed an increased occurrence of generalized seizures after LTG. Power spectral analysis revealed a pronounced increase in the delta frequency range after the higher dose of 30 mg/kg LTG. DZP abolished HPDs and caused a marked reduction over a wide frequency range (delta, theta, and alpha) of the power spectrum. By characterizing the IHKA model of TLE in female mice we address an important gap in basic research. Considering the special challenges complicating the therapeutic management of epilepsy in women, inclusion of females in preclinical studies is imperative. A well-characterized female model is a prerequisite for the development of novel therapeutic strategies tailored to sex-specific needs and for studies on the effect of epilepsy and ASMs during pregnancy.

Dimethyl sulfoxide's impact on epileptiform activity in a mouse model of chronic temporal lobe epilepsy.

In the quest for novel treatments for patients with drug-resistant seizures, poor water solubility of potential drug candidates is a frequent obstacle. Literature indicated that the highly efficient solvent dimethyl sulfoxide (DMSO) may have a confounding influence in epilepsy research, reporting both pro- and antiepileptic effects. In this study, we aim to clarify the effects of DMSO on epileptiform activity in one of the most frequently studied models of chronic epilepsy, the intrahippocampal kainic acid (IHKA) mouse model, and in a model of acute seizures. We show that 100 % DMSO (in a volume of 1.5 µl/g corresponding to 1651 mg/kg) causes a significant short-term anti-seizure effect in epileptic IHKA mice of both sexes, but does not affect the threshold of acute seizures induced by pentylenetetrazol (PTZ). These findings highlight that the choice of solvent and appropriate vehicle control is crucial to minimize undesirable misleading effects and that drug candidates exclusively soluble in 100 % DMSO need to be modified for better solubility already at initial testing.

Evaluation of the SH-SY5Y cell line as an in vitro model for potency testing of a neuropeptide-expressing AAV vector.

Viral vectors have become important tools for basic research and clinical gene therapy over the past years. However, in vitro testing of vector-derived transgene function can be challenging when specific post-translational modifications are needed for biological activity. Similarly, neuropeptide precursors need to be processed to yield mature neuropeptides. SH-SY5Y is a human neuroblastoma cell line commonly used due to its ability to differentiate into specific neuronal subtypes. In this study, we evaluate the suitability of SH-SY5Y cells in a potency assay for neuropeptide-expressing adeno-associated virus (AAV) vectors. We looked at the impact of neuronal differentiation and compared single-stranded (ss) AAV and self-complementary (sc) AAV transduction at increasing MOIs, RNA transcription kinetics, as well as protein expression and mature neuropeptide production. SH-SY5Y cells proved highly transducible with AAV1 already at low MOIs in the undifferentiated state and even better after neuronal differentiation. Readouts were GFP or neuropeptide mRNA expression. Production of mature neuropeptides was poor in undifferentiated cells. By contrast, differentiated cells produced and sequestered mature neuropeptides into the medium in a MOI-dependent manner.

Sprouty2 and -4 regulate axon outgrowth by hippocampal neurons.

Sprouty proteins act as negative feedback inhibitors of fibroblast growth factor (FGF) signaling. FGFs belong to the neurotrophic factors and are involved in axonal growth during development and repair. We investigated the expression of Sprouty isoforms in hippocampal neurons as well as the regulation of Sprouty2 and -4 during development and their role in axon growth. Sprouty2 and -4 were located in the nucleus, the cytoplasm, in dendrites, and axons of hippocampal neurons concentrated in growth cones. During development in vivo and differentiation in vitro, expression of Sprouty2 and -4 was gradually downregulated in hippocampal neurons. Between 5 and 24 days in culture expression of both Sprouty isoforms was reduced by 70%. In vivo expression of Sprouty2 was reduced by 79% and of Sprouty4 by 93% on postnatal day 14 compared to embryonic day 16.5. Downregulation of Sprouty2 and -4 by shRNAs strongly promoted elongative axon growth by cultured hippocampal neurons, which was further increased by FGF-2 treatment. In addition, FGF-2 reduced expression of Sprouty2 by 33% and of Sprouty4 by 44%. Together, our results imply that Sprouty2 and -4 are downregulated in the hippocampus during postnatal brain development and that they can act as regulators of developmental axon growth.

Novel mutation in potassium channel related gene KCTD7 and progressive myoclonic epilepsy.

Progressive myoclonic epilepsy (PME) is a heterogeneous group of epilepsies characterized by myoclonus, seizures and progressive neurological symptoms. The index patient was a 6-year old boy showing early-onset therapy resistant PME and severe developmental delay. Genome-wide linkage analysis identified several candidate regions. The potassium channel tetramerization domain containing 7 gene (KCTD7) in the 7q11.21 linkage region emerged as a suitable candidate. Sequence analysis revealed a novel homozygous missense mutation (p.R94W) in a highly conserved segment of exon 2. This is the second family with PME caused by KCTD7 mutations, hence KCTD7 mutations might be a recurrent cause of PME.