In vitro construction of a recombinant human embryonic brain-derived neurotrophin-4 gene and pEGFP-N1 vector

BACKGROUND: Neurotrophin-4 (NT-4) can promote neuronal growth, development, differentiation, maturation, and survival. NT-4 can also improve recovery and regeneration of injured neurons, but cannot pass through the blood-brain barrier, which limits its ac

Creatine and neurotrophin-4/5 promote survival of nitric oxide synthase-expressing interneurons in striatal cultures

Nitric oxide (NO) mediates a variety of physiological functions in the central nervous system and acts as an important developmental regulator. Striatal interneurons expressing neuronal nitric oxide synthase (nNOS) have been described to be relatively spared from the progressive cell loss in Huntington's disease (HD). We have recently shown that creatine, which supports the phosphagen energy system, induces the differentiation of GABAergic cells in cultured striatal tissue. Moreover, neurotrophin-4/5 (NT-4/5) has been found to promote the survival and differentiation of cultured striatal neurons. In the present study, we assessed the effects of creatine and NT-4/5 on nNOS-immunoreactive (-ir) neurons of E14 rat ganglionic eminences grown for 1 week in culture. Chronic administration of creatine [5mM], NT-4/5 [10ng/ml], or a combination of both factors significantly increased numbers of nNOS-ir neurons. NT-4/5 exposure also robustly increased levels of nNOS protein. Interestingly, only NT-4/5 and combined treatment significantly increased general viability but no effects were seen for creatine supplementation alone. In addition, NT-4/5 and combined treatment resulted in a significant larger soma size and number of primary neurites of nNOS-ir neurons while creatine administration alone exerted no effects. Double-immunolabeling studies revealed that all nNOS-ir cells co-localized with GABA. In summary, our findings suggest that creatine and NT-4/5 affect differentiation and/or survival of striatal nNOS-ir GABAergic interneurons. These findings provide novel insights into the biology of developing striatal neurons and highlight the potential of both creatine and NT-4/5 as therapeutics for HD.

BDNF but not NT-4 is required for normal flexion reflex plasticity and function

Neurotrophins can directly modulate the function of diverse types of central nervous system synapses. Brain-derived neurotrophic factor (BDNF) might be released by nociceptors onto spinal neurons and mediate central sensitization associated with chronic pain. We have studied the role of BDNF and neurotrophin-4 (NT-4), both ligands of the trkB tyrosine kinase receptor, in synaptic transmission and reflex plasticity in the mouse spinal cord. We used an in vitro spinal cord preparation to measure monosynaptic and polysynaptic reflexes evoked by primary afferents in BDNF- and NT-4-deficient mice. In situ hybridization studies show that both these neurotrophins are synthesized by sensory neurons, and NT-4, but not BDNF, also is expressed by spinal neurons. BDNF null mutants display selective deficits in the ventral root potential (VRP) evoked by stimulating nociceptive primary afferents whereas the non-nociceptive portion of the VRP remained unaltered. In addition, activity-dependent plasticity of the VRP evoked by repetitive (1 Hz) stimulation of nociceptive primary afferents (termed wind-up) was substantially reduced in BDNF-deficient mice. This plasticity also was reduced in a reversible manner by the protein kinase inhibitor K252a. Although the trkB ligand NT-4 is normally present, reflex properties in NT-4 null mutant mice were normal. Pharmacological studies also indicated that spinal N-methyl-d-aspartate receptor function was unaltered in BDNF-deficient mice. Using immunocytochemistry for markers of nociceptive neurons we found no evidence that their number or connectivity was substantially altered in BDNF-deficient mice. Our data therefore are consistent with a direct role for presynaptic BDNF release from sensory neurons in the modulation of pain-related neurotransmission.

GABAa Receptor Mediated Signalling in the Brain: Inhibition, Shunting and Excitation

Within central nervous system, the simple division of chemical synaptic transmission to depolarizing excitation mediated by glutamate and hyperpolarizing inhibition mediated by γ-amino butyric acid (GABA), is evidently an oversimplification. The GABAa receptor (GABAaR) mediated responses can be of opposite sign within a single resting cell, due to the compartmentalized distribution of cation chloride cotransporters (CCCs). The K+/Cl- cotransporter 2 (KCC2), member of the CCC family, promotes K+ fuelled Cl- extrusion and sets the reversal potential of GABA evoked anion currents typically slightly below the resting membrane potential. The interesting ionic plasticity property of GABAergic signalling emerges from the short-term and long-term alterations in the intraneuronal concentrations of GABAaR permeable anions (Cl- and HCO3-). The short-term effects arise rapidly (in the time scale of hundreds of milliseconds) and are due to the GABAaR activation dependent shifts in anion gradients, whereas the changes in expression, distribution and kinetic regulation of CCCs are underlying the long-term effects, which may take minutes or even hours to develop. In this Thesis, the differences in the reversal potential of GABAaR mediated responses between dopaminergic and GABAergic cell types, located in the substantia nigra, were shown to be attributable to the differences in the chloride extrusion mechanisms. The stronger inhibitory effect of GABA on GABAergic neurons was due to the cell type specific expression of KCC2 whereas the KCC2 was absent from dopaminergic neurons, leading to a less prominent inhibition brought by GABAaR activation. The levels of KCC2 protein exhibited activity dependent alterations in hippocampal pyramidal neurons. Intense neuronal activity, leading to a massive release of brain derived neurotrophic factor (BDNF) in vivo, or applications of tyrosine receptor kinase B (TrkB) agonists BDNF or neurotrophin-4 in vitro, were shown to down-regulate KCC2 protein levels which led to a reduction in the efficacy of Cl- extrusion. The GABAergic transmission is interestingly involved in an increase of extracellular K+ concentration. A substantial increase in interstitial K+ tends to depolarize the cell membrane. The effects that varying ion gradients had on the generation of biphasic GABAaR mediated responses were addressed, with particular emphasis on the novel idea that the K+/Cl- extrusion via KCC2 is accelerated in response to a rapid accumulation of intracellular Cl-. The KCC2 inhibitor furosemide produced a large reduction in the GABAaR dependent extracellular K+ transients. Thus, paradoxically, both the inefficient KCC2 activity (via increased intracellular Cl-) and efficient KCC2 activity (via increased extracellular K+) may promote excitation.

Neurotrophins and hyperalgesia

Nerve growth factor (NGF), a member of the neurotrophin family, is crucial for survival of nociceptive neurons during development. Recently, it has been shown to play an important role in nociceptive function in adults. NGF is up-regulated after inflammatory injury of the skin. Administration of exogenous NGF either systemically or in the skin causes thermal hyperalgesia within minutes. Mast cells are considered important components in the action of NGF, because prior degranulation abolishes the early NGF-induced component of hyperalgesia. Substances degranulated by mast cells include serotonin, histamine, and NGF. Blockade of histamine receptors does not prevent NGF-induced hyperalgesia. The effects of blocking serotonin receptors are complex and cannot be interpretable uniquely as NGF losing its ability to induce hyperalgesia. To determine whether NGF has a direct effect on dorsal root ganglion neurons, we have begun to investigate the acute effects of NGF on capsaicin responses of small-diameter dorsal root ganglion cells in culture. NGF acutely conditions the response to capsaicin, suggesting that NGF may be important in sensitizing the response of sensory neurons to heat (a process that is thought to operate via the capsaicin receptor VR1). We also have found that ligands for the trkB receptor (brain-derived neurotrophic factor and neurotrophin-4/5) acutely sensitize nociceptive afferents and elicit hyperalgesia. Because brain-derived neurotrophic factor is up-regulated in trkA positive cells after inflammatory injury and is transported anterogradely, we consider it to be a potentially important peripheral component involved in neurotrophin-induced hyperalgesia.

Relation between variants in the neurotrophin receptor gene, NTRK3, and white matter integrity in healthy young adults

The NTRK3 gene (also known as TRKC) encodes a high affinity receptor for the neurotrophin 3'-nucleotidase (NT3), which is implicated in oligodendrocyte and myelin development. We previously found that white matter integrity in young adults is related to common variants in genes encoding neurotrophins and their receptors. This underscores the importance of neurotrophins for white matter development. NTRK3 variants are putative risk factors for schizophrenia, bipolar disorder, and obsessive-compulsive disorder hoarding, suggesting that some NTRK3 variants may affect the brain.To test this, we scanned 392 healthy adult twins and their siblings (mean age, 23.6. ±. 2.2. years; range: 20-29. years) with 105-gradient 4-Tesla diffusion tensor imaging (DTI). We identified 18 single nucleotide polymorphisms (SNPs) in the NTRK3 gene that have been associated with neuropsychiatric disorders. We used a multi-SNP model, adjusting for family relatedness, age, and sex, to relate these variants to voxelwise fractional anisotropy (FA) - a DTI measure of white matter integrity.FA was optimally predicted (based on the highest false discovery rate critical p), by five SNPs (rs1017412, rs2114252, rs16941261, rs3784406, and rs7176429; overall FDR critical p=. 0.028). Gene effects were widespread and included the corpus callosum genu and inferior longitudinal fasciculus - regions implicated in several neuropsychiatric disorders and previously associated with other neurotrophin-related genetic variants in an overlapping sample of subjects. NTRK3 genetic variants, and neurotrophins more generally, may influence white matter integrity in brain regions implicated in neuropsychiatric disorders.

Neurotrophin-3 gene polymorphisms and schizophrenia:no evidence for linkage or association

It has been suggested on the basis of neuropathological and epidemiological evidence that schizophrenia is, at least in part, a neurodevelopmental illness. Some patients show abnormalities in cell position in the medial temporal lobes of their brains. Neurotrophin-3 is one of many proteins essential for the proper growth and development of the nervous system. Therefore the finding of a polymorphism near the promoter region of the gene, alleles of which were associated with the disease, prompted us to attempt replication. In a linkage and association analysis of the same polymorphism using familial schizophrenics and population controls we found no evidence to support the finding. We conclude that mutations or polymorphisms at this gene are unlikely to be involved in the genetic aetiology of schizophrenia.

Altered Neurotrophin, Neuropeptide, Cytokines and Nitric Oxide Levels in Autism

Introduction: Modifications in neurotrophins, neuropeptides, cytokines and nitric oxide (NO) levels in autism may represent different biological aspects of the disease. In the present study we investigate simultaneously all these variables as an attempt to clarify their interrelationships in autism. Methods: Plasma levels of vasoactive intestinal peptide (VIP), neurotrophin-3 (NT-3), cytokines and nitric oxide (NO) were determined in children with DSM-IV autistic disorder (n = 24) and in age- and gender-matched healthy controls (n = 24). VIP, NT-3, IFN-gamma and IL-1 beta levels were measured by ELISA, TNF-alpha, IL-10, IL-6, IL-4, IL-2 were evaluated by flow cytometry, and NO by Griess reaction. Results: Plasma levels of VIP, IFN-gamma and NO were significantly higher and NT-3 plasma levels were significantly lower in children with autism, compared to the healthy subjects. In children with autism there was a positive correlation between plasma levels of NO and IFN-gamma. Discussion: Our results indicate the presence of altered levels of neurotrophin and neuropeptide in infantile autism and provide additional evidence that higher levels of IFN-gamma may be associated with increased oxidative stress in autism.

Neurotrophin 3 rescues neuronal precursors from apoptosis and promotes neuronal differentiation in the embryonic metanephric kidney.

We analyzed the developmental regulation and role of the neurotrophins during metanephric kidney morphogenesis. RNase protection assay revealed the presence of nerve growth factor, neurotrophin 3 (NT-3), and brain-derived neurotrophic factor mRNAs and the regulation of their expression during embryonic development of rat metanephros. NT-3 induced differentiation (neurite outgrowth) and survival (inhibition of apoptosis) of the neuronal precursors in cultured nephrogenic mesenchymes and neuronal differentiation in cultured whole kidneys, whereas NT-4/5, brain-derived neurotrophic factor, and nerve growth factor were without effect. The neurotrophins did not trigger tubular differentiation of isolated nephrogenic cells, which underwent apoptosis when cultured with or without the neurotrophins. NT-3 is thus an inducer of differentiation and a survival factor for renal neuronal cells, but none of the neurotrophins is a morphogen in kidney tubule induction.

Survival, differentiation, and neuroprotective mechanisms of human stem cells complexed with neurotrophin-3-releasing pharmacologically active microcarriers in an ex vivo model of Parkinson's disease

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A new low temperature synthesis route of fraipontite (Zn,Al)3(Si,Al)2O5(OH)4

A low temperature synthesis method based on the decomposition of urea at 90°C in water has been developed to synthesise fraipontite. This material is characterised by a basal reflection 001 at 7.44 Å. The trioctahedral nature of the fraipontite is shown by the presence of a 06l band around 1.54 Å, while a minor band around 1.51 Å indicates some cation ordering between Zn and Al resulting in Al-rich areas with a more dioctahedral nature. TEM and IR indicate that no separate kaolinite phase is present. An increase in the Al content however, did result in the formation of some SiO2 in the form of quartz. Minor impurities of carbonate salts were observed during the synthesis caused by to the formation of CO32- during the decomposition of urea.