Filomena D’Amato

Distribution of VGF peptides in the human cortex and their selective changes in Parkinson's and Alzheimer's diseases

VGF mRNA and its precursor‐derived products are selectively expressed in certain neurons and promptly respond to neurotrophins and to neural/electrical activity. Proteomic studies have previously revealed a reduction in some VGF peptides in the cerebrospinal fluid of patients affected by Alzheimer’s disease and other conditions, suggesting their potential diagnostic and clinical significance. As the presence of VGF peptides within the human cortex has been somewhat elucidated, they were studied postmortem in the frontal, parietal, and temporal cortex areas of control subjects and patients affected by Parkinson’s disease, and in parietal cortex samples from patients with Alzheimer’s disease. We raised antibodies to the C‐/N‐terminal portions of the proVGF precursor protein, to the TPGH and TLQP sequences and to the neuroendocrine regulatory peptide (NERP)‐1, all used for enzyme‐linked immunosorbent assay coupled with gel chromatography and for immunohistochemistry. In the control brain samples, the levels of TPGH and C‐terminus peptides were about 130–200 and 700–2000 pmol g−1, respectively, the N‐terminus and NERP‐1 peptides were less represented (about 10–30 and 4–20 pmol g−1, respectively), and the TLQP peptides were below detection limits. Upon gel chromatography, the VGF antisera mainly revealed small molecular weight forms (i.e. about 0.8–1.3 kDa), whereas VGF immunolocalisation was found within different types of neuron in rat and bovine brain cortices. In the Parkinson’s disease samples, a clear‐cut decrease was revealed in the parietal cortex only, exclusively for TPGH and NERP‐1 peptides, whereas in the Alzheimer’s disease samples, a reduction in all of the VGF peptides was shown. The results suggest the involvement of VGF in the physiological or pathophysiological mechanisms occurring in the parietal cortex of patients with Parkinson’s and Alzheimer’s diseases.

VGF peptides upon osmotic stimuli: Changes in neuroendocrine regulatory peptides 1 and 2 in the hypothalamic–pituitary-axis and plasma

VGF or VGF nerve growth factor inducible is a protein that has been found to play a role in regulating energy homeostasis and metabolism. From VGF precursor derive two neuroendocrine regulatory peptides NERP-1 and NERP-2 that, intracerebroventricular (icv) injected, modulate the antidiuretic hormone (ADH) release. Thus, we investigated possible modulations of the NERPs and other VGF peptides (namely VGF C-terminus, TLQP and PGH) in the hypothalamic-pituitary-axis, adrenal gland and plasma upon osmotic stimuli. The latter tissues were studied using water deprived (WD), salt loaded (SL), rehydrated after salt cargo and control rats by immunohistochemistry and immunoenzymatic assays. The high-performance liquid chromatography ensured the endogenous presence of the two NERPs in both plasma and hypothalamus. Upon dehydration, NERP-1 levels increased in the median eminence (M.E.) only, while using SL rats, the values of both NERPs increased in the M.E. and even in the hypothalamus. Conversely, in the blood of WD and SL rats, the levels of NERP-1 and NERP-2 decreased while, using pituitary from both rat groups, levels of NERP-2 increased and those of NERP-1 decreased. Reduction in the VGF C-terminus peptide levels was observed exclusively in the M.E. (using WD rats) and pituitary (using WD and SL rats), while PGH and TLQP peptide levels never changed in all tissues tested. By immunohistochemistry, the VGF peptides studied (apart from the TLQP peptides) were present in the hypothalamic and pituitary ADH containing neurons of the control rats, while using WD and SL rats, an immunostaining increase was selectively revealed for VGF C-terminus peptides in the hypothalamic neurons that produce ADH. All VGF changes found using SL rats disappeared after only 1h of rehydration. In conclusion, we hypothesize that NERPs may be involved in both autocrine and endocrine mechanisms important for the fluid balance.

VGF changes during the estrous cycle: a novel endocrine role for TLQP peptides?

Although the VGF derived peptide TLQP-21 stimulates gonadotropin-releasing hormone (GnRH) and gonadotropin secretion, available data on VGF peptides and reproduction are limited. We used antibodies specific for the two ends of the VGF precursor, and for two VGF derived peptides namely TLQP and PGH, to be used in immunohistochemistry and enzyme-linked immunosorbent assay complemented with gel chromatography. In cycling female rats, VGF C-/N-terminus and PGH peptide antibodies selectively labelled neurones containing either GnRH, or kisspeptin (VGF N-terminus only), pituitary gonadotrophs and lactotrophs, or oocytes (PGH peptides only). Conversely, TLQP peptides were restricted to somatostatin neurones, gonadotrophs, and ovarian granulosa, interstitial and theca cells. TLQP levels were highest, especially in plasma and ovary, with several molecular forms shown in chromatography including one compatible with TLQP-21. Among the cycle phases, TLQP levels were higher during metestrus-diestrus in median eminence and pituitary, while increased in the ovary and decreased in plasma during proestrus. VGF N- and C-terminus peptides also showed modulations over the estrous cycle, in median eminence, pituitary and plasma, while PGH peptides did not. In ovariectomised rats, plasmatic TLQP peptide levels showed distinct reduction suggestive of a major origin from the ovary, while the estrogen-progesterone treatment modulated VGF C-terminus and TLQP peptides in the hypothalamus-pituitary complex. In in vitro hypothalamus, TLQP-21 stimulated release of growth hormone releasing hormone but not of somatostatin. In conclusion, various VGF peptides may regulate the hypothalamus-pituitary complex via specific neuroendocrine mechanisms while TLQP peptides may act at further, multiple levels via endocrine mechanisms involving the ovary.

Photoperiod Regulates vgf-Derived Peptide Processing in Siberian Hamsters

VGF mRNA is induced in specific hypothalamic areas of the Siberian hamster upon exposure to short photoperiods, which is associated with a seasonal decrease in appetite and weight loss. Processing of VGF generates multiple bioactive peptides, so the objective of this study was to determine the profile of the VGF-derived peptides in the brain, pituitary and plasma from Siberian hamsters, and to establish whether differential processing might occur in the short day lean state versus long day fat. Antisera against short sequences at the C- or N- termini of proVGF, as well as against NERP-1, TPGH and TLQP peptides, were used for analyses of tissues, and both immunohistochemistry and enzyme linked immunosorbent assay (ELISA) coupled with high-performance liquid (HPLC) or gel chromatography were carried out. VGF peptide immunoreactivity was found within cortex cholinergic perikarya, in multiple hypothalamic nuclei, including those containing vasopressin, and in pituitary gonadotrophs. ELISA revealed that exposure to short day photoperiod led to a down-regulation of VGF immunoreactivity in the cortex, and a less pronounced decrease in the hypothalamus and pituitary, while the plasma VGF levels were not affected by the photoperiod. HPLC and gel chromatography both confirmed the presence of multiple VGF-derived peptides in these tissues, while gel chromatography showed the presence of the VGF precursor in all tissues tested except for the cortex. These observations are consistent with the view that VGF-derived peptides have pleiotropic actions related to changing photoperiod, possibly by regulating cholinergic systems in the cortex, vasopressin hypothalamic pathways, and the reproductive axis.

VGF Peptide Profiles in Type 2 Diabetic Patients' Plasma and in Obese Mice.

To address the possible involvement of VGF peptides in obesity and diabetes, we studied type 2 diabetes (T2D) and obese patients, and high-fat diet induced obese mice. Two VGF peptides (NAPP-19 and QQET-30) were identified in human plasma by HPLC-ESI-MS. The VGF C-terminus, the above two cleaved peptides, and the TLQP-21 related peptide/s were studied using ELISA and immunohistochemistry. In euglycemic patients, plasma NAPPE and TLQP like peptides were significantly reduced with obesity (74±10 vs. 167±28, and 92±10 vs. 191±19 pmol/ml, mean+SEM, n = 10 and 6, obese vs. normal BMI, respectively, p<0.03). Upon a standard glucose load, a distinct response was shown for VGF C-terminus, TLQP and QQET-like (ERVW immunoreactive) peptides in euglycemic normal BMI patients, but was virtually abolished in euglycemic obese, and in T2D patients independently of BMI. High-fat diet induced obese mice showed reduced plasma VGF C-terminus, NAPPE and QQET-like (ERVW) peptide/s (3±0.2 vs. 4.6±0.3, 22±3.5 vs. 34±1.3, and 48±7 vs. 100±7 pmol/ml, mean+SEM, n = 8/group, obese vs. slim, respectively, p<0.03), with a loss of the response to glucose for all VGF peptides studied. In immunohistochemistry, TLQP and/or VGF C-terminus antibodies labelled VGF containing perikarya in mouse celiac ganglia, pancreatic islet cells and thin beaded nerve fibres in brown adipose tissues, with fewer in white adipose tissue. Upon the glucose load, tyrosine hydroxylase and VGF C-terminus immunoreactive axons became apparent in pancreatic islets of slim animals, but not in obese animals. Alltogether, a significant loss of VGF peptide immunoreactivity and/or their response to glucose was demonstrated in obese patients, with or without T2D, in parallel with a similar loss in high-fat diet induced obese mice. An involvement of VGF in metabolic regulations, including those of brown and/or white adipose tissues is underlined, and may point out specific VGF peptides as potential targets for diagnosis and/or treatment.

Pituitary gonadotropins and autoimmunity

Autoimmune disease occurs when the body produces an inappropriate immune response against its own tissues producing antibodies, called autoantibodies, reacting to specific antigens. Studies regarding the presence of an autoimmune process specifically involving gonadotropins date from over than 20 years ago, when antibodies to gonadotropic-secreting cells were found by immunofluorescence in sera from a group of patients affected by cryptorchidism. Later on, antibodies detected by the same technique, and directed to the same cells were also found at high titer in sera from patients affected by hypogonadotropic hypogonadism, Kallmanns syndrome, lymphocytic hypophysitis with isolated gonadotropin deficiency, as well as autoimmune polyendocrine syndrome. Concerning the autoimmune target/s within the gonadotropic cells, rarely autoantibodies were found labeling gonadotropins while in a large number of cases, auto-antigens remained to be identified. Since pituitary gonadotropins are fundamental for the sexual maturity and reproductive mechanisms, patients with infertility were largely investigated by enzyme-linked immunosorbent assay for the presence of circulating antibodies likely interfering with gonadotropin activity. In infertile women, autoantibodies to gonadotropins were found related to ovarian autoimmunity, ovarian disorders that cause infertility and also associated with in vitro fertilization treatments. In infertile men, autoantibodies to gonadotropins may alter the testicular spermatogenesis and cause apoptosis of the spermatogenic cells. In conclusion, circulating antibodies were found labeling gonadotropic cells and/or gonadotropins, and in both cases they could create dysfunctions in gonadotropin related mechanism. The intriguing question of what can cause the production of such autoantibodies is not clear yet.

Profiles of VGF Peptides in the Rat Brain and Their Modulations after Phencyclidine Treatment

From the VGF precursor protein originate several low molecular weight peptides, whose distribution in the brain and blood circulation is not entirely known. Among the VGF peptides, those containing the N-terminus portion were altered in the cerebro-spinal fluid (CSF) and hypothalamus of schizophrenia patients. “Hence, we aimed to better investigate the involvement of the VGF peptides in schizophrenia by studying their localization in the brain regions relevant for the disease, and revealing their possible modulations in response to certain neuronal alterations occurring in schizophrenia”. We produced antibodies against different VGF peptides encompassing the N-terminus, but also C-terminus-, TLQP-, GGGE- peptide sequences, and the so named NERP-3 and -4. These antibodies were used to carry out specific ELISA and immunolocalization studies while mass spectrometry (MS) analysis was also performed to recognize the intact brain VGF fragments. We used a schizophrenia rat model, in which alterations in the prepulse inhibition (PPI) of the acoustic startle response occurred after PCP treatment. In normal rats, all the VGF peptides studied were distributed in the brain areas examined including hypothalamus, prefrontal cortex, hippocampus, accumbens and amygdaloid nuclei and also in the plasma. By liquid chromatography-high resolution mass, we identified different intact VGF peptide fragments, including those encompassing the N-terminus and the NERPs. PCP treatment caused behavioral changes that closely mimic schizophrenia, estimated by us as a disruption of PPI of the acoustic startle response. The PCP treatment also induced selective changes in the VGF peptide levels within certain brain areas. Indeed, an increase in VGF C-terminus and TLQP peptides was revealed in the prefrontal cortex (p < 0.01) where they were localized within parvoalbumin and tyrosine hydroxylase (TH) containing neurons, respectively. Conversely, in the nucleus accumbens, PCP treatment produced a down-regulation in the levels of VGF C-terminus-, N-terminus- and GGGE- peptides (p < 0.01), expressed in GABAergic- (C-terminus/GGGE) and somatostatin- (N-terminus) neurons. These results confirm that VGF peptides are widely distributed in the brain and modulated in specific areas involved in schizophrenia.

VGF Involvement on Parkinson’s disease

Parkinson’s disease (PD) is a neurological disorder characterized by a progressive degeneration of dopaminergic neurons of the substantia nigra, that results in dopamine depletion in the striatum with a deregulation of a number of substances/ neurotrasmitters. A previous study demonstrated a clear-cut decrease of certain peptides derived from the VGF gene in the parietal cortex from patients affected by PD. Hence, we used an animal model of PD, and plasma samples from PD patients and control subjects to investigate the role of VGF in PD. Rats (Sprague Dawley) were treated with injections of 6 hydroxydopamine into the medial forebrain bundle, anesthetized and perfused with 4% paraformaldehyde 3 weeks (group 1: n= 8) or 6 weeks (group 2: n=17) after injection. In addition, subcutaneous injections of L-DOPA (6mg/ Kg) were carried out one week before the sacrifice in rats from group 1 and 2 (n=4 and n=6, respectively). We raised antibodies to the C-terminal end of the human VGF sequence, for immunohistochemistry (IHC) and enzyme-linked immunosorbent assay (ELISA, IC50 = 15 pmol/ml). For pilot experiments, human plasma samples were taken from subjects affected by PD (n=13, age range: 51–84 years), and from age matched controls (n=10), to be used for ELISA experiments. We analysed the substantia nigra (SN) of the PD rats using VGF-C-terminus antiserum in double staining with antibodies to glutamate decarboxylase (GAD), tyrosine hydroxylase (TH), serotonin and Substance P. In the control side, VGF-C-terminus peptides were found in neurone terminals/axons within the pars compacta (SNC) and reticulata (SNR) of the substantia nigra, co-localised with GAD and substance P (but not with serotonin or TH) and in closed connection with TH cell bodies of the SNC. In the PD side, while the GAD staining remains unaffected, VGF-C-terminus and substance P immnureactivities disappeared (compared to the control side) in a large population of neurone terminals of both SNC and SNR, and in relation with the TH decrease. When L-DOPA was applied to the animals, VGF-C-terminus immunoreactivity on the PD side returned comparable to the control side. By ELISA, high concentrations of proVGF, in the order of 600-2000 pmol/ml were shown in the plasma from controls with a significant decrease in PD samples (p=0,0029). In conclusion, using the animal models, we showed here for the first time that VGF is involved into nigro-striatal circuits and down-regulated in PD. Besides, of a great interest, is the finding that a deregulation of VGF can occur also in humans.

VGF and Type II Diabetes: Decrease of VGF fragment levels in adipose tissues of diet-induced obese mice

VGF encodes a precursor protein processed in various neuropeptides, one of these involved in metabolic mechanisms and named TLQP-21, stimulates the autonomic activation of adipose tissue and reduces the early phase of diet-induced obesity in mouse. In immunohistochemistry (ICC) and ELISA, we used antisera to short sequences at C-/N-terminus of the VGF precursor, and of several VGF-derived peptides. Plasma of type II diabetes (DMII) was taken from patients with different body mass index (BMI): (i) BMI =18-24, (ii) BMI=25-29, (iii) BMI=30-39, in parallel with samples from healthy subjects (iiii) matched with BMI of group “i” (n=10 samples/ group). Male CD1 mice were fed with standard chow (group-a: controls), or with standard chow plus 36% lard (group-b: obese) for 16 weeks (n=24 animals/group). For glucose tolerance test, 12 mice from groups a and b, respectively, received i.p. injection of glucose (3g/kg), and were sacrificed after 120 minutes. White and brown adipose tissues (WAT and BAT, respectively) were extracted (n=9 animals/group) while for ICC, mice (~4/group) were perfused with 4% paraformaldehyde. In plasma of DM II patients, we found a clear decrease of VGF C-t, in parallel with the increase of BMI (iiii=1200±70, i=1100±85, ii=760±60, iii=550±40 pmol/ml, mean±SEM). In WAT, through ELISA, we found a significant decrease for VGF C-t in group-b respect to group-a (2±0.5 vs. 5±2*, pmol/g, mean±SEM, *p<0.03). A similar trend was observed in BAT, not only for VGF C-t, but also for N-t, TLQP, and RVW peptides (groups b vs. a, respectively: 10±3 vs. 22±7**, 9±3 vs. 18±4**, 210±75 vs. 420±95**, and 38±11 vs. 70±10*, pmol/g, means±SEM, *p<0.007,**p<0.02). Moreover, exclusively in BAT of group-a but not of group-b, we measured a great increase of VGF C-t, N-t and RVW peptide levels after glucose load (values taken before and after glucose load, respectively: 22±7 vs 35±5**, 18±4 vs. 30±7 **and 70±10 vs. 95±10*, pmol/g, means±SEM, *p<0.02,**p<0.04). In pancreas, BAT and WAT tissues was showed an extended colocalization of VGF C-t peptides with sympathetic fibers, while TLQP peptides were largely restricted to noradrenergic perikaria in celiac ganglia of both control and obese mice. In conclusions, more than one VGF fragment could be important for peripheral activation of autonomic nervous system.

VGF-peptides in the Siberian hamster

vgf is one of the few genes selectively induced in the hypothalamus of Siberian hamsters upon their typical change from an obese phenotype (long day adaptation, during summer) to a lean, catabolic phenotype (short day, or winter adaptation). In fact, the i.c.v. injection of the VGF-derived peptide TLQP-21 caused hypophagia and a decrease in body weight in long day hamster. Hence, we studied VGF multi-peptide profiles in brain cortex and hypothalamus of (male) Siberian hamsters, in the long day (LD) versus short day (SD) adapted state. Specific antisera were produced against short sequences at the C- or N-termini of VGF, and of several known/predicted VGF-derived products: TLQP, NERP-1, and PGH peptides, and used in immunohistochemistry (IHC) and ELISA. Hamsters were perfused with 4% paraformaldehyde (n= 4/group) for IHC or used for tissue sampling and extraction (n= 7/group). In IHC, VGF C- and N- terminus peptides were brightly labelled, as well as most abundant. They were found in both perikarya and axons, in different layers of brain cortex and in multiple hypothalamic areas, including the paraventricular (PVN), suprachiasmatic (SCN), supraoptic (SON) and arcuate nuclei, the lateral and anterior hypothalamic areas, and the median eminence (ME). TLQP peptides were largely restricted to SCN perikarya, and ME axons, while PGH and NERP-1 peptides were revealed in perikarya of the brain cortex, in ME axons, and certain perikarya of PVN and SON (NERP-1 only). Most VGF peptides studied were well represented in tissue extracts of hypothalamus and cortex, VGF C- and N- terminus peptides being again most abundant (hypothalamus: 1.8±0.3 and 10.9±0.6; cortex: 0.7±0.1 and 5±0.3 nmol/g, mean±SEM, C- and N-terminus, respectively, LD animals). A selective decrease in certain VGF peptides was revealed in SD animals, compared to LD ones, so that NERP-1 peptides were decreased in hypothalamus and cortex (61.3±12.7% and 45.8±11.1% of LD animals, respectively, mean±SEM, p<0.04), PGH peptides were reduced in hypothalamus (24.8±12.7% of LD group, mean±SEM, p<0.02), and both TLQP and N-terminus peptides in the brain cortex (31.8±10.9% and 41.5±10.8% of LD animals, respectively, mean±SEM, p<0.02). In conclusion, VGF peptides were well represented in the Siberian hamster brain, with a distinct, apparently selective modulation in the hypothalamus and brain cortex. A regionally specific, differential post-translational processing of the VGF precursor may be implicated. Supported by a RAS grant (Regione Autonoma Sardegna, PO FSE 2007-1013, L.R. 7/2007).