VGF modifications related to nigrostriatal dopaminergic neurodegeneration induced by the pesticide fipronil in adult male rats.

BACKGROUND: Dopamine is reduced in the brain of rats treated with fipronil, a broad-spectrum insecticide. VGF (no acronym) is a neurotrophin-inducible protein expressed as the 75 kDa form (precursor or pro-VGF) or its truncated peptides. VGF immunostaining has been revealed using an antibody against the C-terminal nonapeptide of the rat pro-VGF in the nerve terminals of the rat substantia nigra, where it was reduced after 6-hydroxydopamine treatment. It is unknown whether pro-VGF and/or its shortened peptides are present in these neurons. Therefore, the aim of this study was first to determine which types of VGF are expressed in the normal substantia nigra (and striatum) and then to determine VGF modulations and whether they occur in parallel with locomotor changes after fipronil injection. METHODS: Rats were divided into two groups that received a unilateral intranigral infusion of either fipronil (25 µg) diluted in dimethyl sulfoxide (DMSO) or DMSO alone, and then were tested for locomotor activity. An untreated group of rats (n=4) was used for identification of the VGF fragments using high performance liquid chromatography-mass spectrometry and western blot, while changes in treated groups (fipronil vs DMSO, each n=6) were investigated by immunohistochemistry using an antibody against the rat pro-VGF C-terminal nonapeptide in parallel with the anti-tyrosine hydroxylase antibody. RESULTS: In untreated rats, the VGF C-terminal antibody identified mostly a 75 kDa band in the substantia nigra and striatum, supporting the finding of high-resolution mass spectrometry, which revealed fragments covering the majority of the pro-VGF sequence. Furthermore, several shortened VGF C-terminal forms (varying from 10 to 55 kDa) were also found by western blot, while high-resolution mass spectrometry revealed a C-terminal peptide overlapping the immunogen used to create the VGF antibody in both substantia nigra and striatum. In the substantia nigra of fipronil-treated rats, immunostaining for tyrosine hydroxylase and VGF was reduced compared to DMSO-treated rat group, and this was related with significant changes in locomotor activity. CONCLUSION: Fipronil has the ability to modulate the production of pro-VGF and/or its C-terminal truncated peptides in the nigrostriatal system indicating its intimate interaction with the dopaminergic neurotransmission and implying a potential function in modulating locomotor activity.

A Pilot Interaction Analysis of Gut Microbiota and Peripheral Markers of Aging in Severe Psychiatric Disorders: A Role for Lachnoclostridium?

Excessive predominance of pathological species in the gut microbiota could increase the production of inflammatory mediators at the gut level and, via modification of the gut-blood barrier, at the systemic level. This pro-inflammatory state could, in turn, increase biological aging that is generally proxied by telomere shortening. In this study, we present findings from a secondary interaction analysis of gut microbiota, aging, and inflammatory marker data from a cohort of patients with different diagnoses of severe mental disorders. We analyzed 15 controls, 35 patients with schizophrenia (SCZ), and 31 patients with major depressive disorder (MDD) recruited among those attending a community mental health center (50 males and 31 females, mean and median age 46.8 and 46.3 years, respectively). We performed 16S rRNA sequencing as well as measurement of telomere length via quantitative fluorescence in situ hybridization and high-sensitivity C-reactive protein. We applied statistical modeling with logistic regression to test for interaction between gut microbiota and these markers. Our results showed statistically significant interactions between telomere length and gut microbiota pointing to the genus Lachnostridium, which remained significantly associated with a reduced likelihood of MDD even after adjustment for a series of covariates. Although exploratory, these findings show that specific gut microbiota signatures overexpressing Lachnoclostridium and interacting with biological aging could modulate the liability for MDD.

Brain-Biomarker Changes in Body Fluids of Patients with Parkinson's Disease.

Parkinson's disease (PD) is an incurable neurodegenerative disease that is rarely diagnosed at an early stage. Although the understanding of PD-related mechanisms has greatly improved over the last decade, the diagnosis of PD is still based on neurological examination through the identification of motor symptoms, including bradykinesia, rigidity, postural instability, and resting tremor. The early phase of PD is characterized by subtle symptoms with a misdiagnosis rate of approximately 16-20%. The difficulty in recognizing early PD has implications for the potential use of novel therapeutic approaches. For this reason, it is important to discover PD brain biomarkers that can indicate early dopaminergic dysfunction through their changes in body fluids, such as saliva, urine, blood, or cerebrospinal fluid (CSF). For the CFS-based test, the invasiveness of sampling is a major limitation, whereas the other body fluids are easier to obtain and could also allow population screening. Following the identification of the crucial role of alpha-synuclein (α-syn) in the pathology of PD, a very large number of studies have summarized its changes in body fluids. However, methodological problems have led to the poor diagnostic/prognostic value of this protein and alternative biomarkers are currently being investigated. The aim of this paper is therefore to summarize studies on protein biomarkers that are alternatives to α-syn, particularly those that change in nigrostriatal areas and in biofluids, with a focus on blood, and, eventually, saliva and urine.

Brain-reactive autoantibodies in neuropsychiatric systemic lupus erythematosus.

Introduction: The pathogenesis of neuropsychiatric systemic lupus erythematosus (NPSLE) is widely unknown, and the role of autoantibodies is still undetermined. Methods: To identify brain-reactive autoantibodies possibly related to NPSLE, immunofluorescence (IF) and transmission electron microscopy (TEM) on rat and human brains were performed. ELISA was used to reveal the presence of known circulating autoantibodies, while western blot (WB) was applied to characterize potential unknown autoantigen(s). Results: We enrolled 209 subjects, including patients affected by SLE (n=69), NPSLE (n=36), Multiple Sclerosis (MS, n=22), and 82 age- and gender-matched healthy donors (HD). Autoantibody reactivity by IF was observed in almost the entire rat brain (cortex, hippocampus, and cerebellum) using sera from NPSLE and SLE patients and was virtually negative in MS and HD. NPSLE showed higher prevalence (OR 2.4; p = 0.047), intensity, and titer of brain-reactive autoantibodies than SLE patients. Most of the patient sera with brain-reactive autoantibodies (75%) also stained human brains. Double staining experiments on rat brains mixing patients' sera with antibodies directed against neuronal (NeuN) or glial markers showed autoantibody reactivity restricted to NeuN-containing neurons. Using TEM, the targets of brain-reactive autoantibodies were located in the nuclei and, to a lesser extent, in the cytoplasm and mitochondria. Given the high degree of colocalization between NeuN and brain-reactive autoantibodies, we assumed NeuN was a possible autoantigen. However, WB analysis with HEK293T cell lysates expressing or not expressing the gene encoding for NeuN protein (RIBFOX3) showed that patients' sera carrying brain-reactive autoantibodies did not recognize the NeuN corresponding band size. Among the panel of NPSLE-associated autoantibodies (e.g., anti-NR2, anti-P-ribosomal protein, antiphospholipid) investigated by ELISA assay, only the anti-ß2-glycoprotein-I (aß2GPI) IgG was exclusively found in those sera containing brain-reactive autoantibodies. Conclusion: In conclusion, SLE and NPSLE patients possess brain-reactive autoantibodies but with higher frequency and titers found in NPSLE patients. Although many target antigens of brain-reactive autoantibodies are still undetermined, they likely include ß2GPI.

Correction: Boiten et al. Pathologically Decreased CSF Levels of Synaptic Marker NPTX2 in DLB Are Correlated with Levels of Alpha-Synuclein and VGF. Cells 2021, 10, 38.

The authors want to notify that they have made the following changes to the author list of the paper [...].

Oxytocin-conjugated saporin injected into the substantia nigra of male rats alters the activity of the nigrostriatal dopaminergic system: A behavioral and neurochemical study.

Saporin conjugated to oxytocin (OXY-SAP) destroys neurons expressing oxytocinergic receptors. When injected unilaterally in the substantia nigra of male rats, OXY-SAP causes a dose-dependent decrease up to 55 % in nigral Tyrosine Hydroxylase (TH)-immunoreactivity compared to control mock peptide BLANK-SAP- and PBS-treated rats or the contralateral substantia nigra. TH decrease was parallel to a dopamine content decrease in the ipsilateral striatum compared to BLANK-SAP- or PBS-treated rats or the contralateral striatum. OXY-SAP-treated rats showed a small but significant increase of locomotor activity 28 days after intranigral injection in the Open field test compared to BLANK-SAP- or PBS-treated rats, in line with an inhibitory role of nigral oxytocin on locomotor activity. OXY-SAP-, but not BLANK-SAP- or PBS-treated rats, also showed marked dose-dependent rotational turning ipsilateral to the injected substantia nigra when challenged with d-amphetamine, but not with apomorphine. Under isoflurane anesthesia OXY-SAP-treated rats showed levels of extracellular dopamine in the dialysate from the ipsilateral striatum only half those of BLANK-SAP- or PBS-treated rats or the contralateral striatum. When treated with d-amphetamine, OXY-SAP_60/120 rats showed increased extracellular dopamine levels in the dialysate from the ipsilateral striatum two third/one third only of those found in BLANK-SAP- or PBS-treated rats or the contralateral striatum, respectively. These results show that OXY-SAP destroys nigrostriatal dopaminergic neurons expressing oxytocin receptors leading to a reduced striatal dopamine function.

TLQP-21 changes in response to a glucose load.

BACKGROUND: The TLQP-21 peptide potentiates glucose-stimulated insulin secretion, hence we investigated its endogenous response to glucose. METHODS: Fasted mice received intraperitoneal glucose (3 g/kg), or saline (controls), and were sacrificed 30 and 120 min later (4 groups, n = 6/group). We investigated TLQP-21 in pancreas and plasma using immunohistochemistry, enzyme-linked immunosorbent assay (ELISA) and high performance liquid chromatography (HPLC), as well as TLQP-21 receptors (gC1q-R and C3a-R1) expression in pancreas by immunohistochemistry. RESULTS: In pancreas, TLQP-immunoreactivity (TLQP-ir.) was shown in insulin-, glucagon- and somatostatin-containing cells. Upon glucose, TLQP-ir. decreased at 30 min (∼40 % vs. controls), while returning to basal values at 120 min. In all groups, C3a-R1 was localized in ∼50 % of TLQP labelled islet cells (mostly central), while gC1q-R was detected in ∼25 % of TLQP cells (mainly peripheral). HPLC fractions of control pancreas extracts, assessed by ELISA, confirmed the presence of a TLQP-21 compatible-form (∼2.5 kDa MW). In plasma, TLQP-ir. increased at 30 min (∼30 %), with highest concentrations at 120 min (both: p<0.05 vs. controls), while HPLC fractions showed an increase in the TLQP-21 compatible form. CONCLUSIONS: Upon hyperglycaemia, TLQP-21 would be released from islets, to enhance insulin secretion but we cannot exclude an autocrine activity which may regulate insulin storage/secretion.

Telomere attrition and inflammatory load in severe psychiatric disorders and in response to psychotropic medications.

Individuals with severe psychiatric disorders have a reduced life expectancy compared to the general population. At the biological level, patients with these disorders present features that suggest the involvement of accelerated aging, such as increased circulating inflammatory markers and shorter telomere length (TL). To date, the role of the interplay between inflammation and telomere dynamics in the pathophysiology of severe psychiatric disorders has been scarcely investigated. In this study we measured T-lymphocytes TL with quantitative fluorescent in situ hybridization (Q-FISH) and plasma levels of inflammatory markers in a cohort comprised of 40 patients with bipolar disorder (BD), 41 with schizophrenia (SZ), 37 with major depressive disorder (MDD), and 36 non-psychiatric controls (NPC). TL was shorter in SZ and in MDD compared to NPC, while it was longer in BD (model F6, 137 = 20.128, p = 8.73 × 10-17, effect of diagnosis, F3 = 31.870; p = 1.08 × 10-15). There was no effect of the different classes of psychotropic medications, while duration of treatment with mood stabilizers was associated with longer TL (Partial correlation controlled for age and BMI: correlation coefficient = 0.451; p = 0.001). Levels of high-sensitivity C-Reactive Protein (hsCRP) were higher in SZ compared to NPC (adjusted p = 0.027), and inversely correlated with TL in the whole sample (r = -0.180; p = 0.042). Compared to NPC, patients with treatment resistant (TR) SZ had shorter TL (p = 0.001), while patients with TR MDD had higher levels of tumor necrosis factor-α (TNFα) compared to NPC (p = 0.028) and to non-TR (p = 0.039). Comorbidity with cardio-metabolic disorders did not influence the observed differences in TL, hsCRP, and TNFα among the diagnostic groups. Our study suggests that patients with severe psychiatric disorders present reduced TL and increased inflammation.

Dynamic of TLQP-peptides upon fasting.

BACKGROUND: The VGF-derived TLQP peptides (TLQPp), a new potential drug target for obesity, are expressed in stomach, pancreas, adrenal gland as well as in adipose tissues, and, when exogenously injected, regulate energy expenditure and food intake. However, it is not clear if these peptides physiologically change in these organs in response to fasting. METHODS: Rats were subdivided into four groups: (A) fed ad libitum, (B) fed with restrictions (once a day) (C) fast for 48 h and (D) fast for 48 h and then fed 1 h before sacrifice. Immunosorbent assay was used to possibly reveal TLQPp changes upon fasting in plasma as well as in pancreas, adrenal gland, stomach and adipose tissues. In the latter organs, we also measured the levels of the VGF precursor protein while immunohistochemistry was used to investigate the presence of the TLQP-21 receptors. RESULTS: During fasting, TLQPp were down-regulated in the stomach (45 %), pancreas (47 %), adrenal gland (51 %) and WAT (45.2 %) in parallel with a significant increase in the blood (36.6 %), all versus ad libitum group. In the same organs where the TLQPp were decreased upon fasting, the VGF precursor levels were not changed. In ad libitum rats, TLQP-21 receptors were well represented within the same cells that expressed TLQPp, suggesting an autocrine activity to be better investigated. CONCLUSIONS: During fasting, TLQPp are probably produced and immediately secreted into the blood circulation, until the hypoglycaemia is counteracted.

Identification of novel cerebrospinal fluid biomarker candidates for dementia with Lewy bodies: a proteomic approach.

BACKGROUND: Diagnosis of dementia with Lewy bodies (DLB) is challenging, largely due to a lack of diagnostic tools. Cerebrospinal fluid (CSF) biomarkers have been proven useful in Alzheimer's disease (AD) diagnosis. Here, we aimed to identify novel CSF biomarkers for DLB using a high-throughput proteomic approach. METHODS: We applied liquid chromatography/tandem mass spectrometry with label-free quantification to identify biomarker candidates to individual CSF samples from a well-characterized cohort comprising patients with DLB (n = 20) and controls (n = 20). Validation was performed using (1) the identical proteomic workflow in an independent cohort (n = 30), (2) proteomic data from patients with related neurodegenerative diseases (n = 149) and (3) orthogonal techniques in an extended cohort consisting of DLB patients and controls (n = 76). Additionally, we utilized random forest analysis to identify the subset of candidate markers that best distinguished DLB from all other groups. RESULTS: In total, we identified 1995 proteins. In the discovery cohort, 69 proteins were differentially expressed in DLB compared to controls (p < 0.05). Independent cohort replication confirmed VGF, SCG2, NPTX2, NPTXR, PDYN and PCSK1N as candidate biomarkers for DLB. The downregulation of the candidate biomarkers was somewhat more pronounced in DLB in comparison with related neurodegenerative diseases. Using random forest analysis, we identified a panel of VGF, SCG2 and PDYN to best differentiate between DLB and other clinical groups (accuracy: 0.82 (95%CI: 0.75-0.89)). Moreover, we confirmed the decrease of VGF and NPTX2 in DLB by ELISA and SRM methods. Low CSF levels of all biomarker candidates, except PCSK1N, were associated with more pronounced cognitive decline (0.37 < r < 0.56, all p < 0.01). CONCLUSION: We identified and validated six novel CSF biomarkers for DLB. These biomarkers, particularly when used as a panel, show promise to improve diagnostic accuracy and strengthen the importance of synaptic dysfunction in the pathophysiology of DLB.

Differences in telomere length between patients with bipolar disorder and controls are influenced by lithium treatment.

Aim: To assess the role of lithium treatment in the relationship between bipolar disorder (BD) and leukocyte telomere length (LTL). Materials & methods: We compared LTL between 131 patients with BD, with or without a history of lithium treatment, and 336 controls. We tested the association between genetically determined LTL and BD in two large genome-wide association datasets. Results: Patients with BD with a history lithium treatment showed longer LTL compared with never-treated patients (p = 0.015), and similar LTL compared with controls. Patients never treated with lithium showed shorter LTL compared with controls (p = 0.029). Mendelian randomization analysis showed no association between BD and genetically determined LTL. Conclusion: Our data support previous findings showing that long-term lithium treatment might protect against telomere shortening.

The pesticide fipronil injected into the substantia nigra of male rats decreases striatal dopamine content: A neurochemical, immunohistochemical and behavioral study.

Experimental evidence shows that the phenylpyrazole pesticide fipronil exerts neurotoxic effects at central level in rodents, and in particular on nigrostriatal dopaminergic neurons, whose degeneration is well known to cause motor and non-motor deficits in animals and in humans. In order to characterize better the central neurotoxic effect of fipronil, we injected fipronil (15 and 25 µg) dissolved in dimethyl sulfoxide (DMSO) unilaterally into the substantia nigra of male rats. Male rats injected with DMSO unilaterally into the substantia nigra were used as controls. Control and fipronil-treated rats were then tested in different motor (i.e., open field arena, rotarod, tail flick) and non motor tests (novel object recognition, social interaction) 15 days after injection. A systemic challenge dose of the dopamine-agonist apomorphine was also used to study the presence of a rotational behavior. Sixteen days after fipronil or DMSO injection into the substantia nigra, rats were sacrificed, and either striatal dopamine content or substantia nigra tyrosine hydroxylase (TH) immunoreactivity were measured. The results confirm that the unilateral injection of fipronil into the substantia nigra caused the degeneration of nigrostriatal dopaminergic neurons, which leads to a decrease around 50 % in striatal dopamine content and substantia nigra TH imunoreactivity. This occurred together with changes in motor activity and coordination, and in nociception but not in recognition memory and in social interaction, as revealed by the results of the behavioral experiments performed in fipronil-treated rats compared to vehicle-treated rats 15 days after treatment, as found with other compounds that destroy nigrostriatal dopaminergic neurons.

A multidisciplinary approach to mental illness: do inflammation, telomere length and microbiota form a loop? A protocol for a cross-sectional study on the complex relationship between inflammation, telomere length, gut microbiota and psychiatric disorders.

INTRODUCTION: Severe psychiatric disorders are typically associated with a significant reduction in life expectancy compared with the general population. Among the different hypotheses formulated to explain this observation, accelerated ageing has been increasingly recognised as the main culprit. At the same time, telomere shortening is becoming widely accepted as a proxy molecular marker of ageing. The present study aims to fill a gap in the literature by better defining the complex interaction/s between inflammation, age-related comorbidities, telomere shortening and gut microbiota in psychiatric disorders. METHODS AND ANALYSIS: A cross-sectional study is proposed, recruiting 40 patients for each of three different diagnostic categories (bipolar disorder, schizophrenia and major depressive disorder) treated at the Section of Psychiatry and at the Unit of Clinical Pharmacology of the University Hospital Agency of Cagliari (Italy), compared with 40 age-matched and sex-matched non-psychiatric controls. Each group includes individuals suffering, or not, from age-related comorbidities, to account for the impact of these medical conditions on the biological make-up of recruited patients. The inflammatory state, microbiota composition and telomere length (TL) are assessed. ETHICS AND DISSEMINATION: The study protocol was approved by the Ethics Committee of the University Hospital Agency of Cagliari (PG/2018/11693, 5 September 2018). The study is conducted in accordance with the principles of good clinical practice and the Declaration of Helsinki, and in compliance with the relevant Italian national legislation. Written, informed consent is obtained from all participants. Participation in the study is on a voluntary basis only. Patients will be part of the dissemination phase of the study results, during which a local conference will be organised and families of patients will also be involved. Moreover, findings will be published in one or more research papers and presented at national and international conferences, in posters or oral communications.

Pathologically Decreased CSF Levels of Synaptic Marker NPTX2 in DLB Are Correlated with Levels of Alpha-Synuclein and VGF.

Background: Dementia with Lewy bodies (DLB) is a neurodegenerative disease where synaptic loss and reduced synaptic integrity are important neuropathological substrates. Neuronal Pentraxin 2(NPTX2) is a synaptic protein that drives the GABAergic inhibitory circuit. Our aim was to examine if NPTX2 cerebral spinal fluid (CSF) levels in DLB patients were altered and how these levels related to other synaptic protein levels and to cognitive function and decline. Methods: NPTX2, VGF, and α-synuclein levels were determined in CSF of cognitive healthy (n = 27), DLB (n = 48), and AD (n = 20) subjects. Multiple cognitive domains were tested, and data were compared using linear models. Results: Decreased NPTX2 levels were observed in DLB (median = 474) and AD (median = 453) compared to cognitive healthy subjects (median = 773). Strong correlations between NPTX2, VGF, and α-synuclein were observed dependent on diagnosis. Combined, these markers had a high differentiating power between DLB and cognitive healthy subjects (AUC = 0.944). Clinically, NPTX2 levels related to global cognitive function and cognitive decline in the visual spatial domain. Conclusion: NPTX2 CSF levels were reduced in DLB and closely correlated to decreased VGF and α-synuclein CSF levels. CSF NPTX2 levels in DLB related to decreased functioning in the visual spatial domain.

VGF peptides as novel biomarkers in Parkinson's disease.

Parkinson's disease (PD) is characterized by a progressive degeneration of dopaminergic neurons in the substantia nigra (SN). At disease onset, a diagnosis is often difficult. VGF peptides are abundant in the SN and peripheral circulation; hence, we investigate whether their plasma profile may reflect the brain dopamine reduction. Using antibodies against the VGF C-terminal portion, we analyzed the rat brain and human plasma, with immunohistochemistry and ELISA. Rats were unilaterally lesioned with 6-hyroxydopamine and sacrificed either 3 or 6 weeks later with or without levodopa treatment. Plasma samples were obtained from PD patients, either at the time of diagnosis (group 1, drug naïve, n = 23) or upon dopamine replacement (group 2, 1-6 years, n = 24; group 3, > 6 years, n = 16), compared with age-matched control subjects (group 4, n = 21). Assessment of the olfactory function was carried out in group 2 using the "Sniffin' Sticks" test. VGF immunoreactivity was present in GABAergic neurons and, on the lesioned side, it was reduced at 3 weeks and abolished at 6 weeks after lesion. Conversely, upon levopoda, VGF labeling was restored. In PD patients, VGF levels were reduced at the time of diagnosis (1504 ± 587 vs. 643 ± 348 pmol/mL, means ± S.E.M: control vs. naïve; p < 0.05) but were comparable with the controls after long-term drug treatment (> 6 years). A linear correlation was demonstrated between VGF immunoreactivity and disease duration, levodopa equivalent dose and olfactory dysfunction. Plasma VGF levels may represent a useful biomarker, especially in the early stages of PD.

VGF Peptides in Cerebrospinal Fluid of Patients with Dementia with Lewy Bodies.

In a previous proteomic study, we identified the neurosecretory protein VGF (VGF) as a potential biomarker for dementia with Lewy bodies (DLB). Here, we extended the study of VGF by comparing levels in cerebrospinal fluid (CSF) from 44 DLB patients, 20 Alzheimer's disease (AD) patients, and 22 cognitively normal controls selected from the Amsterdam Dementia Cohort. CSF was analyzed using two orthogonal analytical methods: (1) In-house-developed quantitative ELISA and (2) selected reaction monitoring (SRM). We further addressed associations of VGF with other CSF biomarkers and cognition. VGF levels were lower in CSF from patients with DLB compared to either AD patients or controls. VGF was positively correlated with CSF tau and α-synuclein (0.55 < r < 0.75), but not with Aß1-42. In DLB patients, low VGF levels were related to a more advanced cognitive decline at time of first presentation, whereas high levels of VGF were associated with steeper subsequent longitudinal cognitive decline. Hence, CSF VGF levels were lower in DLB compared to both AD and controls across different analytical methods. The strong associations with cognitive decline further points out VGF as a possible disease stage or prognostic marker for DLB.

Photoperiodic changes in adiposity increase sensitivity of female Siberian hamsters to systemic VGF derived peptide TLQP-21.

TLQP-21, a peptide encoded by the highly conserved vgf gene, is expressed in neuroendocrine cells and has been the most prominent VGF-derived peptide studied in relation to control of energy balance. The recent discovery that TLQP-21 is the natural agonist for the complement 3a receptor 1 (C3aR1) has revived interest in this peptide as a potential drug target for obesity. We have investigated its function in Siberian hamsters (Phodopus sungorus), a rodent that displays natural seasonal changes in body weight and adiposity as an adaptation to survive winter. We have previously shown that intracerebroventricular administration of TLQP-21 reduced food intake and body weight in hamsters in their long-day fat state. The aim of our current study was to determine the systemic actions of TLQP-21 on food intake, energy expenditure and body weight, and to establish whether adiposity affected these responses. Peripheral infusion of TLQP-21 (1mg/kg/day for 7 days) in lean hamsters exposed to short photoperiods (SP) reduced cumulative food intake in the home cage (p<0.05), and intake when measured in metabolic cages (P<0.01). Energy expenditure was significantly increased (p<0.001) by TLQP-21 infusion, this was associated with a significant increase in uncoupling protein 1 mRNA in brown adipose tissue (BAT) (p<0.05), and body weight was significantly reduced (p<0.05). These effects of systemic TLQP-21 treatment were not observed in hamsters exposed to long photoperiod (LP) with a fat phenotype. C3aR1 mRNA and protein were abundantly expressed in the hypothalamus, brown and white adipose tissue in hamsters, but changes in expression cannot explain the differential response to TLQP-21 in lean and fat hamsters.

Oxytocin induces penile erection and yawning when injected into the bed nucleus of the stria terminalis: A microdialysis and immunohistochemical study.

Oxytocin (5, 20 and 100 ng) injected unilaterally into the bed nucleus of the stria terminalis (BNST) of male rats stereotaxically implanted with a microinjection cannula coupled to a microdialysis probe, induces penile erection and yawning that occur concomitantly with a dose-dependent increase in the extracellular concentration of glutamic acid, dopamine and its main metabolite 3,4-dihydroxyphenilacetic acid (DOPAC), and nitrites (NO2-) in the dialysate obtained from the BNST by intracerebral microdialysis. The responses induced by oxytocin (100 ng) were all abolished by the oxytocin receptor antagonist d(CH2)5Tyr(Me)2-Orn8-vasotocin (1 µg), and reduced by CNQX (1 µg), a competitive antagonist of the AMPA receptors, both given into the BNST 25 min before oxytocin. In contrast, (+) MK-801 (1 µg), a non-competitive antagonist of NMDA receptors, and SCH 23390 (1 µg), a selective dopamine D1 receptor antagonist, reduced penile erection and yawning, but not glutamic acid and dopamine increases in the BNST dialysate induced by oxytocin. Immunohistochemistry revealed oxytocin-labelled neuronal structures in close proximity to tyrosine hydroxylase-labelled neurons or nitric oxide synthase-labelled cell bodies surrounded by intense vesicular glutamate transporter1-stained synapses in BNST sections where oxytocin injections induce the above responses. Together, these findings show that oxytocin injected into the BNST induces penile erection and yawning by activating not only the glutamatergic (and nitrergic) but also the dopaminergic neurotransmission, leading in turn to the activation of neural pathways mediating penile erection and yawning.

Reduction of Total Brain and Cerebellum Volumes Associated With Neuronal Autoantibodies in Patients With APECED.

Context: In autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), autoantibodies (AutoAbs) labeling brain neurons were reported; conversely, brain MRI alterations associated with these AutoAbs were never reported. Objectives: To describe brain alterations in APECED and to correlate them with AutoAbs against glutamic acid decarboxylase (GAD), tyrosine hydroxylase (TH), and 5-tryptophan hydroxylase (5-HT) neurons. Design and Participants: Fourteen Sardinian patients with APECED and age-matched control subjects were recruited for MRI analysis and blood sampling to detect AutoAbs to GAD, TH, and 5-HT neurons by using rat brain sections. The majority of patients (n = 12) were investigated for AutoAbs a decade earlier, and 7 of 12 were positive for AutoAbs to GAD and TH neurons. Main Outcomes: Patients with APECED had smaller cerebellum and gray matter volumes, with a ventricular enlargement and a total cerebrospinal fluid (CSF) increase, compared with controls (P < 0.01). In 11 of 14 patients, brain abnormalities were associated with AutoAbs to GAD or TH neurons (titer 1:100 to 15,000) that had persisted for 10 years in 7 of 11 patients. AutoAbs to 5-HT neurons were revealed in all patients with AutoAbs to TH neurons. A decrease in whole brain and cerebellum volumes (P = 0.028) was associated with AutoAbs to GAD neurons, and a CSF increase was associated with AutoAbs to GAD and TH/5-HT neurons (P < 0.05). HLA alleles did not appear to be involved in neuronal autoimmunity. Conclusions: Brain alterations and neuronal AutoAbs were observed in 78.6% of Sardinian patients with APECED, suggesting a brain autoimmune reaction. Prolonged clinical follow-up must be conducted for the possible appearance of clinical neurologic consequences.

TLQP Peptides in Amyotrophic Lateral Sclerosis: Possible Blood Biomarkers with a Neuroprotective Role.

While the VGF-derived TLQP peptides have been shown to prevent neuronal apoptosis, and to act on synaptic strengthening, their involvement in Amyotrophic Lateral Sclerosis (ALS) remains unclarified. We studied human ALS patients' plasma (taken at early to late disease stages) and primary fibroblast cultures (patients vs controls), in parallel with SOD1-G93A transgenic mice (taken at pre-, early- and late symptomatic stages) and the mouse motor neuron cell line (NSC-34) treated with Sodium Arsenite (SA) to induce oxidative stress. TLQP peptides were measured by enzyme-linked immunosorbent assay, in parallel with gel chromatography characterization, while their localization was studied by immunohistochemistry. In controls, TLQP peptides, including forms compatible with TLQP-21 and 62, were revealed in plasma and spinal cord motor neurons, as well as in fibroblasts and NSC-34 cells. TLQP peptides were reduced in ALS patients' plasma starting in the early disease stage (14% of controls) and remaining so at the late stage (16% of controls). In mice, a comparable pattern of reduction was shown (vs wild type), in both plasma and spinal cord already in the pre-symptomatic phase (about 26% and 70%, respectively). Similarly, the levels of TLQP peptides were reduced in ALS fibroblasts (31% of controls) and in the NSC-34 treated with Sodium Arsenite (53% of decrease), however, the exogeneous TLQP-21 improved cell viability (SA-treated cells with TLQP-21, vs SA-treated cells only: about 83% vs. 75%). Hence, TLQP peptides, reduced upon oxidative stress, are suggested as blood biomarkers, while TLQP-21 exerts a neuroprotective activity.