proNGF/NGF mixtures induce gene expression changes in PC12 cells that neither singly produces.

BACKGROUND: Growing evidence shows that, in vivo, the precursor of Nerve Growth Factor (NGF), proNGF, displays biological activities different from those of its mature NGF counterpart, mediated by distinct, and somewhat complementary, receptor binding properties. NGF and proNGF induce distinct transcriptional signatures in target cells, highlighting their different bioactivities. In vivo, proNGF and mature NGF coexist. It was proposed that the relative proNGF/NGF ratio is important for their biological outcomes, especially in pathological conditions, since proNGF, the principal form of NGF in Central Nervous System (CNS), is increased in Alzheimer's disease brains. These observations raise a relevant question: does proNGF, in the presence of NGF, influence the NGF transcriptional response and viceversa? In order to understand the specific proNGF effect on NGF activity, depending on the relative proNGF/NGF concentration, we investigated whether proNGF affects the pattern of well-known NGF-regulated mRNAs. RESULTS: To test any influence of proNGF on pure NGF expression fingerprinting, the expression level of a set of candidate genes was analysed by qReal-Time PCR in rat adrenal pheochromocytoma cell line PC12, treated with a mixture of NGF and proNGF recombinant proteins, in different stoichiometric ratios. These candidates were selected amongst a set of genes well-known as being rapidly induced by NGF treatment. We found that, when PC12 cells are treated with proNGF/NGF mixtures, a unique pattern of gene expression, which does not overlap with that deriving from treatment with either proNGF or NGF alone, is induced. The specific effect is also dependent on the stoichiometric composition of the mixture. The proNGF/NGF equimolar mixture seems to partially neutralize the specific effects of the proNGF or NGF individual treatments, showing a weaker overall response, compared to the individual contributions of NGF and proNGF alone. CONCLUSIONS: Using gene expression as a functional read-out, our data demonstrate that the relative availability of NGF and proNGF in vivo might modulate the biological outcome of these ligands.

Direct intracellular selection and biochemical characterization of a recombinant anti-proNGF single chain antibody fragment.

proNGF, the precursor of the neurotrophin NGF, is widely expressed in central and peripheral nervous system. Its physiological functions are still largely unknown, although it emerged from studies in the last decade that proNGF has additional and distinct functions with respect to NGF, besides acting chaperone-like for NGF folding during its biogenesis. The regulation of proNGF/NGF ratio represents a crucial process for homeostasis of brain and other tissues, and understanding the molecular aspects of these differences is important. We report the selection and characterization of a recombinant monoclonal anti-proNGF antibody in single chain Fv fragment (scFv) format. The selection exploited the Intracellular Antibody Capture Technology (IACT), starting from a naïve mouse SPLINT (Single Pot Library of INTracellular antibodies) library. This antibody (scFv FPro10) was expressed recombinantly in Escherichia coli, was proven to be highly soluble and stable, and thoroughly characterized from the biochemical-biophysical point of view. scFv FPro10 displays high affinity and specificity for proNGF, showing no cross-reactivity with other pro-neurotrophins. A structural model was obtained by SAXS. scFv FPro10 represents a new tool to be exploited for the selective immunoanalysis of proNGF, both in vitro and in vivo, and might help in understanding the molecular function of proNGF in neurodegeneration.

A study of classroom seat (dis)comfort: Relationships between body movements, center of pressure on the seat, and lower limbs' sensations.

The aim of this work is to define a new method that helps researchers to analyze perceptions of (dis)comfort in dynamic conditions. Recent studies pay considerable attention to body movements, mobility, and stability to measure comfort or discomfort when seated. Most of these discuss the relations between subjective comfort/discomfort and objective measurements (e.g. body pressure distribution, body movement and EMG) for short- and medium-term sitting. The present analysis took place in a classroom of the Industrial Engineering Department at the University of Salerno. The participants included 25 students (12 females and 13 males), who were observed during classroom hours. The students were invited to sit at a combo-desk and were free to perform different combinations of movements while writing and listening. These activities required that they adapt their body movements, as the combo-desk was fixed to the floor. A pressure pad was used to detect pressure at interface and center of pressure's changes, allowing for the bodies' motion data to be recorded. The aim was to identify the correct threshold to be used for movement detection and to investigate correlations between the number of movements and the perceived (dis)comfort. The study also identifies those body parts that have the greatest effect on (dis)comfort perception.

Effect of in-seat exercising on comfort perception of airplane passengers.

Sitting still for extended periods of time can lead to physical discomfort and even serious health risks. Due to safety regulations, reducing passenger' sitting time in aircrafts is not feasible. This paper presents the results of a laboratory study, in where an interactive airplane seat was compared with a current economy class seat. Participants used both seats for 3.5 h, and performed significantly more in-seat movements when using the interactive seating system. Furthermore, this interactive seat predominantly lead to significantly better comfort experiences and reduced discomfort experiences, however no significant differences have been found in self-reported localized musculoskeletal discomfort. Passengers indicated that they would prefer this interactive seat over a standard aircraft seat.

ProNGF Drives Localized and Cell Selective Parvalbumin Interneuron and Perineuronal Net Depletion in the Dentate Gyrus of Transgenic Mice.

ProNGF, the precursor of mature Nerve Growth Factor (NGF), is the most abundant NGF form in the brain and increases markedly in the cortex in Alzheimer's Disease (AD), relative to mature NGF. A large body of evidence shows that the actions of ProNGF and mature NGF are often conflicting, depending on the receptors expressed in target cells. TgproNGF#3 mice, expressing furin-cleavage resistant proNGF in CNS neurons, directly reveal consequences of increased proNGF levels on brain homeostasis. Their phenotype clearly indicates that proNGF can be a driver of neurodegeneration, including severe learning and memory behavioral deficits, cholinergic deficits, and diffuse immunoreactivity for A-beta and A-beta-oligomers. In aged TgproNGF#3 mice spontaneous epileptic-like events are detected in entorhinal cortex-hippocampal slices, suggesting occurrence of excitatory/inhibitory (E/I) imbalance. In this paper, we investigate the molecular events linking increased proNGF levels to the epileptiform activity detected in hippocampal slices. The occurrence of spontaneous epileptiform discharges in the hippocampal network in TgproNGF#3 mice suggests an impaired inhibitory interneuron homeostasis. In the present study, we detect the onset of hippocampal epileptiform events at 1-month of age. Later, we observe a regional- and cellular-selective Parvalbumin interneuron and perineuronal net (PNN) depletion in the dentate gyrus (DG), but not in other hippocampal regions of TgproNGF#3 mice. These results demonstrate that, in the hippocampus, the DG is selectively vulnerable to altered proNGF/NGF signaling. Parvalbumin interneuron depletion is also observed in the amygdala, a region strongly connected to the hippocampus and likewise receiving cholinergic afferences. Transcriptome analysis of TgproNGF#3 hippocampus reveals a proNGF signature with broad down-regulation of transcription. The most affected mRNAs modulated at early times belong to synaptic transmission and plasticity and extracellular matrix (ECM) gene families. Moreover, alterations in the expression of selected BDNF splice variants were observed. Our results provide further mechanistic insights into the vicious negative cycle linking proNGF and neurodegeneration, confirming the regulation of E/I homeostasis as a crucial mediating mechanism.

Apoptotic effect of caspase-3 cleaved tau in hippocampal neurons and its potentiation by tau FTDP-mutation N279K.

Pathological changes in the microtubule associated protein tau are a major hallmark of many human dementias collectively defined as tauopathies. In familiar frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17), several mutations in the tau gene have been identified showing that primary malfunction of tau can lead to neurodegeneration. In addition to mutation at genetic level, a number of post-translational modifications of tau occur in tauopathies, including abnormal phosphorylation and aberrant proteolysis described in Alzheimer's Disease (AD). The presence of cleaved tau in AD neurons is associated with expression of markers for neuronal death. According to our previous work, tau is a substrate for the apoptotic protease caspase-3 that turns tau itself into an effector of apoptosis (tau cleaved at D-421), generating a positive-feedback loop that is self-propagating. Cleavage of tau by caspase-3 was recently confirmed to occur in AD brain as an early event. Here we show the apoptotic properties of tau fragment tau151-421 in primary cultures of rat hippocampal neurons; such cellular model is of special interest considering the selective vulnerability of hippocampal neurones in AD. The apoptotic capacity of tau151-421 is markedly enhanced by both treatment with amyloid peptide Abeta25-35, and the FTDP-17 tau mutation N279K.

Bystander effect on brain tissue of mesoangioblasts producing neurotrophins.

Neurotrophic factors (NTFs) are involved in the regulation of neuronal survival and function and, thus, may be used to treat neurological diseases associated with neuronal death. A major hurdle for their clinical application is the delivery mode. We describe here a new strategy based on the use of progenitor cells called mesoangioblasts (MABs). MABs can be isolated from postnatal mesoderm tissues and, because of a high adhesin-dependent migratory capacity, can reach perivascular targets especially in damaged areas. We generated genetically modified MABs producing nerve growth factor (MABs-NGF) or brain-derived neurotrophic factor (MABs-BDNF) and assessed their bystander effects in vitro using PC12 cells, primary cultures, and organotypic cultures of adult hippocampal slices. MABs-NGF-conditioned medium induced differentiation of PC12 cells, while MABs-BDNF-conditioned medium increased viability of cultured neurons and slices. Slices cultured with MABs-BDNF medium also better retained their morphology and functional connections, and all these effects were abolished by the TrkB kinase blocker K252a or the BDNF scavenger TrkB-IgG. Interestingly, the amount of BDNF released by MABs-BDNF produced greater effects than an identical amount of recombinant BDNF, suggesting that other NTFs produced by MABs synergize with BDNF. Thus, MABs can be an effective vehicle for NTF delivery, promoting differentiation, survival, and functionality of neurons. In summary, MABs hold distinct advantages over other currently evaluated approaches for NTF delivery in the CNS, including synergy of MAB-produced NTF with the neurotrophins. Since MABs may be capable of homing into damaged brain areas, they represent a conceptually novel, promising therapeutic approach to treat neurodegenerative diseases.

NGF and proNGF regulate functionally distinct mRNAs in PC12 cells: an early gene expression profiling.

The biological activities of NGF and of its precursor proNGF are quite distinct, due to different receptor binding profiles, but little is known about how proNGF regulates gene expression. Whether proNGF is a purely pro-apoptotic molecule and/or simply a "less potent NGF" is still a matter of debate. We performed experiments to address this question, by verifying whether a proNGF specific transcriptional signature, distinct from that of NGF, could be identified. To this aim, we studied gene expression regulation by proNGF and NGF in PC12 cells incubated for 1 and 4 hours with recombinant NGF and proNGF, in its wild-type or in a furin-cleavage resistant form. mRNA expression profiles were analyzed by whole genome microarrays at early time points, in order to identify specific profiles of NGF and proNGF. Clear differences between the mRNA profiles modulated by the three neurotrophin forms were identified. NGF and proNGF modulate remarkably distinct mRNA expression patterns, with the gene expression profile regulated by NGF being significantly more complex than that by proNGF, both in terms of the total number of differentially expressed mRNAs and of the gene families involved. Moreover, while the total number of genes modulated by NGF increases dramatically with time, that by proNGFs is unchanged or reduced. We identified a subset of regulated genes that could be ascribed to a "pure proNGF" signalling, distinct from the "pure NGF" one. We also conclude that the composition of mixed NGF and proNGF samples, when the two proteins coexist, influences the profile of gene expression. Based on this comparison of the gene expression profiles regulated by NGF and its proNGF precursor, we conclude that the two proteins activate largely distinct transcriptional programs and that the ratio of NGF to proNGF in vivo can profoundly influence the pattern of regulated mRNAs.