Report of the ad-hoc consultation on aging and immunization for a future WHO research agenda on life-course immunization.

WHO convened a meeting of around 30 experts to address the topic of aging and immunization in March 2011 in Geneva. The purpose of the meeting was to develop a global research agenda to eventually inform WHO policy recommendations regarding immunization beyond childhood and into old age. This issue is becoming more critical, since the population aged 60 and above will reach two billion people - three-quarters of whom will be in developing countries - in the next 40 years. The meeting reviewed current knowledge and gaps in information about: (1) the epidemiology of infectious diseases in the elderly in developed and developing countries and their contribution to disability in old age; (2) the deterioration of the immune system with age ("immune senescence") and possible ways to measure and counteract it; and (3) immunization approaches to maintain or improve health in older persons. These approaches include the concept of a "life-course vaccination" schedule to help sustain immunity to vaccine-preventable diseases beyond childhood and into old age; strategies to strengthen older persons' responses to vaccines (e.g., by adding adjuvants to vaccines, increasing vaccine dosage, and intradermal vaccine administration); and the possible development of new vaccines targeted specifically for older adults. Participants proposed priority research topics as well as strategies to facilitate and coordinate the research, including the establishment of networks of collaborators, with WHO playing a key coordinating role.

Neurturin evokes MAPK-dependent upregulation of Egr4 and KCC2 in developing neurons.

The K-Cl cotransporter KCC2 plays a crucial role in the functional development of GABA(A)-mediated responses rendering GABA hyperpolarizing in adult neurons. We have previously shown that BDNF upregulates KCC2 in immature neurons through the transcription factor Egr4. The effect of BDNF on Egr4 and KCC2 was shown to be dependent on the activation of ERK1/2. Here we demonstrate that the trophic factor neurturin can also trigger Egr4 expression and upregulate KCC2 in an ERK1/2-dependent manner. These results show that Egr4 is an important component in the mechanism for trophic factor-mediated upregulation of KCC2 in immature neurons involving the activation of specific intracellular pathways common to BDNF and Neurturin.

Early growth response 4 mediates BDNF induction of potassium chloride cotransporter 2 transcription.

A major event in the maturation of CNS GABAergic transmission is the qualitative change in GABA(A)-mediated responses from depolarizing to hyperpolarizing. In cortical regions, this is attributed to the increased expression of potassium chloride cotransporter 2b (KCC2b), the main isoform of the neuron-specific K-Cl cotransporter KCC2. We have previously shown that transcription factor early growth response 4 (Egr4) can activate the KCC2b promoter. Here we demonstrate that in immature hippocampal neurons BDNF robustly induces ERK1/2 (extracellular signal-regulated kinase 1/2)-dependent Egr4 expression and rapid Egr4-dependent activation of the KCC2b promoter. The subsequent increase in KCC2b mRNA contributes to the expression of total KCC2 protein levels. These results indicate that Egr4 is an important component in the mechanism of BDNF-dependent KCC2 gene regulation via the ERK1/2 pathway in immature neurons.

RNA looping by PTB: Evidence using FRET and NMR spectroscopy for a role in splicing repression.

Alternative splicing plays an important role in generating proteome diversity. The polypyrimidine tract-binding protein (PTB) is a key alternative splicing factor involved in exon repression. It has been proposed that PTB acts by looping out exons flanked by pyrimidine tracts. We present fluorescence, NMR, and in vivo splicing data in support of a role of PTB in inducing RNA loops. We show that the RNA recognition motifs (RRMs) 3 and 4 of PTB can bind two distant pyrimidine tracts and bring their 5' and 3' ends in close proximity, thus looping the RNA. Efficient looping requires an intervening sequence of 15 nucleotides or longer between the pyrimidine tracts. RRM3 and RRM4 bind the 5' and the 3' pyrimidine tracts, respectively, in a specific directionality and work synergistically for efficient splicing repression in vivo.

Posttraumatic GABA(A)-mediated [Ca2+]i increase is essential for the induction of brain-derived neurotrophic factor-dependent survival of mature central neurons.

A shift of GABA(A)-mediated responses from hyperpolarizing to depolarizing after neuronal injury leads to GABA(A)-mediated increase in [Ca2+](i). In addition, central neurons become dependent on BDNF for survival. Whether these two mechanisms are causally interrelated is an open question. Here, we show in lesioned CA3 hippocampal neurons in vitro and in axotomized corticospinal neurons in vivo that posttraumatic downregulation of the neuron-specific K-Cl cotransporter KCC2 leads to intracellular chloride accumulation by the Na-K-2Cl cotransporter NKCC1, resulting in GABA-induced [Ca2+](i) transients. This mechanism is required by a population of neurons to survive in a BDNF-dependent manner after injury, because blocking GABA(A)-depolarization with the NKCC1 inhibitor bumetanide prevents the loss of neurons on BDNF withdrawal. The resurgence of KCC2 expression during recovery coincides with loss of BDNF dependency for survival. This is likely mediated through BDNF itself, because injured neurons reverse their response to this neurotrophin by switching the BDNF-induced downregulation of KCC2 to upregulation.

Mechanism of activity-dependent downregulation of the neuron-specific K-Cl cotransporter KCC2.

GABA-mediated fast-hyperpolarizing inhibition depends on extrusion of chloride by the neuron-specific K-Cl cotransporter, KCC2. Here we show that sustained interictal-like activity in hippocampal slices downregulates KCC2 mRNA and protein expression in CA1 pyramidal neurons, which leads to a reduced capacity for neuronal Cl- extrusion. This effect is mediated by endogenous BDNF acting on tyrosine receptor kinase B (TrkB), with down-stream cascades involving both Shc/FRS-2 (src homology 2 domain containing transforming protein/FGF receptor substrate 2) and PLCgamma (phospholipase Cgamma)-cAMP response element-binding protein signaling. The plasmalemmal KCC2 has a very high rate of turnover, with a time frame that suggests a novel role for changes in KCC2 expression in diverse manifestations of neuronal plasticity. A downregulation of KCC2 may be a general early response involved in various kinds of neuronal trauma.

A novel method for monitoring surface membrane trafficking on hippocampal acute slice preparation.

Protein trafficking has attracted considerable attention as a potential regulator of neuronal plasticity. Therefore, it is of interest to study the mechanism involved in protein trafficking in experimental paradigms commonly used in this context. Here, we present a method for cell surface protein biotinylation in the acute hippocampal slice, the most commonly used preparation for electrophysiological recordings. We validated this procedure with two previously characterized cell surface receptors, glutamate receptor subunit A (GluR A) and the transferrin receptor (TfR). We observed a glutamate-dependent increase in the degradation of surface GluR A, whereas the TfR did not show significant degradation in the time window used. In addition, the presented method offers the opportunity to study processes such as internalisation and recycling, and can also be applied to examine the effect of normal and pathological patterns of activity on membrane protein trafficking in commonly used preparations for electrophysiological recordings.

BDNF-induced TrkB activation down-regulates the K+-Cl- cotransporter KCC2 and impairs neuronal Cl- extrusion.

Pathophysiological activity and various kinds of traumatic insults are known to have deleterious long-term effects on neuronal Cl- regulation, which can lead to a suppression of fast postsynaptic GABAergic responses. Brain-derived neurotrophic factor (BDNF) increases neuronal excitability through a conjunction of mechanisms that include regulation of the efficacy of GABAergic transmission. Here, we show that exposure of rat hippocampal slice cultures and acute slices to exogenous BDNF or neurotrophin-4 produces a TrkB-mediated fall in the neuron-specific K+-Cl- cotransporter KCC2 mRNA and protein, as well as a consequent impairment in neuronal Cl- extrusion capacity. After kindling-induced seizures in vivo, the expression of KCC2 is down-regulated in the mouse hippocampus with a spatiotemporal profile complementary to the up-regulation of TrkB and BDNF. The present data demonstrate a novel mechanism whereby BDNF/TrkB signaling suppresses chloride-dependent fast GABAergic inhibition, which most likely contributes to the well-known role of TrkB-activated signaling cascades in the induction and establishment of epileptic activity.