Neurotrophin nerve growth factor (NGF) has been demonstrated to upregulate the gene expression of bradykinin receptor 2 (B2R) on sensory neurons, thus facilitating nociceptive signals. The aim of the present study is to investigate the involvement of B2R in the NGF mechanism of action in nonsensory neurons in vitro by using rat mixed cortical primary cultures (CNs) and mouse hippocampal slices, and in vivo in Alzheimer's disease (AD) transgenic mice (5xFAD) chronically treated with NGF. A significant NGF-mediated upregulation of B2R was demonstrated by microarray, Western blot, and immunofluorescence analysis in CNs, indicating microglial cells as the target of this modulation. The B2R involvement in the NGF mechanism of action was also demonstrated by using a selective B2R antagonist which was able to reverse the neuroprotective effect of NGF in CNs, as revealed by viability assay, and the NGF-induced long-term potentiation (LTP) in hippocampal slices. To confirm in vitro observations, B2R upregulation was observed in 5xFAD mouse brain following chronic intranasal NGF treatment. This study demonstrates for the first time that B2R is a key element in the neuroprotective activity and synaptic plasticity mediated by NGF in brain cells.

Reduced levels of Substance P (SP), an endogenous neuropeptide endowed with neuroprotective and anti-apoptotic properties, have been found in brain and spinal fluid of Alzheimer's disease (AD) patients. Potassium (K(+)) channel dysfunction is implicated in AD development and the amyloid-β (Aβ)-induced up-regulation of voltage-gated potassium channel subunits could be considered a significant step in Aβ brain toxicity. The aim of this study was to evaluate whether SP could reduce, in vivo, Aβ-induced overexpression of Kv subunits. Rats were intracerebroventricularly infused with amyloid-β 25-35 (Aβ25-35, 20 µg) peptide. SP (50 µg/Kg, i.p.) was daily administered, for 7 days starting from the day of the surgery. Here we demonstrate that the Aβ infused rats showed impairment in cognitive performances in the Morris water maze task 4 weeks after Aβ25-35 infusion and that this impairing effect was prevented by SP administration. Kv1.4, Kv2.1 and Kv4.2 subunit levels were quantified in hippocampus and in cerebral cortex by Western blot analysis and immunofluorescence. Interestingly, SP reduced Kv1.4 levels overexpressed by Aβ, both in hippocampus and cerebral cortex. Our findings provide in vivo evidence for a neuroprotective activity of systemic administration of SP in a rat model of AD and suggest a possible mechanism underlying this effect.

The peptides encoded by the VGF gene are gaining biomedical interest and are increasingly being scrutinized as biomarkers for human disease. An endocrine/neuromodulatory role for VGF peptides has been suggested but never demonstrated. Furthermore, no study has demonstrated so far the existence of a receptor-mediated mechanism for any VGF peptide. In the present study, we provide a comprehensive in vitro, ex vivo and in vivo identification of a novel pro-lipolytic pathway mediated by the TLQP-21 peptide. We show for the first time that VGF-immunoreactivity is present within sympathetic fibres in the WAT (white adipose tissue) but not in the adipocytes. Furthermore, we identified a saturable receptor-binding activity for the TLQP-21 peptide. The maximum binding capacity for TLQP-21 was higher in the WAT as compared with other tissues, and selectively up-regulated in the adipose tissue of obese mice. TLQP-21 increases lipolysis in murine adipocytes via a mechanism encompassing the activation of noradrenaline/β-adrenergic receptors pathways and dose-dependently decreases adipocytes diameters in two models of obesity. In conclusion, we demonstrated a novel and previously uncharacterized peripheral lipolytic pathway encompassing the VGF peptide TLQP-21. Targeting the sympathetic nerve-adipocytes interaction might prove to be a novel approach for the treatment of obesity-associated metabolic complications.

TLQP-21 (TLQPPASSRRRHFHHALPPAR) is a multifunctional peptide that is involved in the control of physiological functions, including feeding, reproduction, stress responsiveness, and general homeostasis. Despite the huge interest in TLQP-21 biological activity, very little is known about its intracellular mechanisms of action. In microglial cells, TLQP-21 stimulates increases of intracellular Ca2+ that may activate functions, including proliferation, migration, phagocytosis and production of inflammatory molecules. Our aim was to investigate whether JMV5656 (RRRHFHHALPPAR), a novel short analogue of TLQP-21, stimulates intracellular Ca2+ in the N9 microglia cells, and whether this Ca2+ elevation is coupled with the activation Ca2+-sensitive K+ channels. TLQP-21 and JMV5656 induced a sharp, dose-dependent increment in intracellular calcium. In 77% of cells, JMV5656 also caused an increase in the total outward currents, which was blunted by TEA (tetraethyl ammonium chloride), a non-selective blocker of voltage-dependent and Ca2+-activated potassium (K+) channels. Moreover, the effects of ion channel blockers charybdotoxin and iberiotoxin, suggested that multiple calcium-activated K+ channel types drove the outward current stimulated by JMV5656. Additionally, inhibition of JMV5656-stimulated outward currents by NS6180 (4-[[3-(trifluoromethyl)phenyl]methyl]-2H-1,4 benzothiazin-3(4H)-one) and TRAM-34 (triarylmethane-34), indicated that KCa3.1 channels are involved in this JMV5656 mechanisms of action. In summary, we demonstrate that, in N9 microglia cells, the interaction of JMV5656 with the TLQP-21 receptors induced an increase in intracellular Ca2+, and, following extracellular Ca2+ entry, the opening of KCa3.1 channels.

The multigenic program underlying neuronal apoptosis is mostly unknown. To study the program, we used genome-scale screening by oligonucleotide microarrays during serum and potassium deprivation-induced apoptosis of cerebellar granule neurons. From the 8740 genes interrogated by the arrays, 423 genes were found to be regulated at both the transcriptional and the posttranscriptional level and segregated into distinct clusters. Semantic clustering based on gene ontologies showed coordinated expression of genes with common biological functions and metabolic pathways. Among the genes implicated in apoptotic cerebellar granule neurons, 70 were in common with those differentially expressed in cortical neurons exposed to amyloid beta-protein, indicating the existence of common mechanisms responsible for neuronal cell death. Our results offer a genomic view of the changes that accompany neuronal apoptosis and yield new insights into the underlying molecular basis.

Among mediators of inflammation, chemokines play a pivotal role in the neuroinflammatory process related to Alzheimer's disease (AD). The chemokine Bv8/prokineticin 2 (PROK2) is a critical player in inflammatory and neuroinflammatory diseases and has been demonstrated to be involved in Aβ toxicity. The aim of the present study was to extend the research to rats chronically intracerebroventricularly (i.c.v.) injected with Aβ, to an AD transgenic mouse model, and subsequently to AD patients, mainly with the aim of detecting a potential biomarker. Real-time PCR and immunofluorescence analysis were used to evaluate Prokineticin-2 (PROK2) mRNA and the corresponding protein levels in both animal and human AD brain extracts, and the ELISA test was used to measure the amount of PROK2 in the serum of AD patients. We demonstrated a significant upregulation of PROK2 levels in brain tissues of Aβ1-42 i.c.v. injected rats, transgenic AD mice (Tg2576), and in the hippocampus of AD patients. Additionally, through a pilot study, an approximate twofold increase of PROK2 levels has been proved in the serum of AD patients, compared to the control subjects, identifying a potential blood-based biomarker of the disease.

TLQP-21, a VGF-encoded peptide is emerging as a novel target for obesity-associated disorders. TLQP-21 is found in the sympathetic nerve terminals in the adipose tissue and targets the G-protein-coupled receptor complement-3a receptor1 (C3aR1). The mechanisms of TLQP-21-induced receptor activation remain unexplored. Here, we report that TLQP-21 is intrinsically disordered and undergoes a disorder-to-order transition, adopting an α-helical conformation upon targeting cells expressing the C3aR1. We determined that the hot spots for TLQP-21 are located at the C terminus, with mutations in the last four amino acids progressively reducing the bioactivity and, a single site mutation (R21A) or C-terminal amidation abolishing its function completely. Additionally, the human TLQP-21 sequence carrying a S20A substitution activates the human C3aR1 receptor with lower potency compared to the rodent sequence. These studies reveal the mechanism of action of TLQP-21 and provide molecular templates for designing agonists and antagonists to modulate C3aR1 functions.

VGF is a propeptide of 617 amino acids expressed throughout the central and the peripheral nervous system. VGF and peptides derived from its processing have been found in dense core vesicles and are released from neuronal and neuroendocrine cells via the regulated secretory pathway. Among VGF-derived neuropeptides, TLQP-21 (VGF556-576) has raised a huge interest and is one of most studied. TLQP-21 is a multifunctional neuropeptide involved in the control of several physiological functions, potentially including energy homeostasis, pain modulation, stress responsiveness and reproduction. Although little information is available about its receptor and the intracellular mechanisms mediating its biological effects, recent reports suggest that TLQP-21 may bind to the complement receptors C3aR1 and/or gC1qR. The first aim of this study was to ascertain the existence and nature of TLQP-21 binding sites in CHO cells. Secondly, we endeavored to characterize the ligand binding to these sites by using a small panel of VGF-derived peptides. And finally, we investigated the influence of TLQP-21 on selected intracellular signaling pathways. We report that CHO cells express a single class of saturable and specific binding sites for TLQP-21 with an affinity and capacity of Kd = 0.55 ± 0.05 × 10-9 M and Bmax = 81.7 ± 3.9 fmol/mg protein, respectively. Among the many bioactive products derived from the C-terminal region of VGF that we tested, TLQP-21 was the most potent in stimulating intracellular calcium mobilization in CHO cells; this effect is primarily due to its C-terminal fragment (HFHH-10). TLQP-21 induced rapid and transient dephosphorylation of phospholipase Cγ1 and phospholipase A2. Generation of IP3 and diacylglycerol was crucial for TLQP-21 bioactivity. In conclusion, our results suggest that the receptor stimulated by TLQP-21 belongs to the family of the Gq-coupled receptors, and its activation first increases membrane-lipid derived second messengers which thereby induce the mobilization of Ca2+ from the endoplasmic reticulum followed by a slower store-operated Ca2+ entry from outside the cell.

Most cells activate intracellular signalling to recover from heat damage. An increase of temperature, known as HS (heat shock), induces two major signalling events: the transcriptional induction of HSPs (heat-shock proteins) and the activation of the MAPK (mitogen-activated protein kinase) cascade. We performed the present study to examine the effects of HS, induced by different experimental conditions, on various kinases [ERK (extracellular-signal-regulated kinase), JNK (c-Jun N-terminal kinase), p38, Akt, AMPK (AMP-activated protein kinase) and PKC (protein kinase C)]. We investigated by Western blot analysis the phosphorylation of MAPK as a measure of cellular responsiveness to heat shift (37°C) and mild HS (40°C) in different cell lines. The results of the study indicate that every cell line responded to heat shift, and to a greater extent to HS, increasing ERK and JNK phosphorylation, whereas variable effects on activation or inhibition of PKC, AMPK, Akt and p38 were observed. Besides the implications of intracellular signalling activated by heat variations, these data may be of technical relevance, indicating possible sources of error due to different experimental temperature conditions.