Perspective role of Substance P in Amyotrophic Lateral Sclerosis: From neuronal vulnerability to neuroprotection.

The neuropeptide Substance P (SP) and its preferred Neurokinin1 Receptor (NK1R) are known to participate in the physiopathology of neurodegenerative diseases and mainly exert a neuroprotective role. In the present work, we have described the involvement of SP and NK1R in Amyotrophic Lateral Sclerosis (ALS). This was demonstrated by the detection of altered levels of SP in the brain, spinal cord and cerebrospinal fluid (CSF) of patients and preclinical models of ALS, and by its ability to inhibit excitotoxicity-induced neurodegeneration in ALS animal models. These data are supported by results indicating an excitatory effect of SP at the motor neuron (MN) level, which promotes locomotor activity. ALS patients are characterized by a differential susceptibility to MNs degeneration, since sphincters and extraocular muscles are classically spared. It is hypothesized that SP may play a role in the maintenance of the ocular system and the innervation of the pelvic floor by contributing directly or indirectly to the selective resistance of this subset of MNs.

ProNGF processing in adult rat tissues and bioactivity of NGF prodomain peptides.

The neurotrophin nerve growth factor (NGF) and its precursor proNGF are both bioactive and exert similar or opposite actions depending on the cell target and its milieu. The balance between NGF and proNGF is crucial for cell and tissue homeostasis and it is considered an indicator of pathological conditions. Proteolytical cleavage of proNGF to the mature form results in different fragments, whose function and/or bioactivity is still unclear. The present study was conducted to investigate the distribution of proNGF fragments derived from endogenous cleavage in brain and peripheral tissues of adult rats in the healthy condition and following inflammatory lipopolysaccharide (LPS) challenge. Different anti-proNGF antibodies were tested and the presence of short peptides corresponding to the prodomain sequence (pdNGFpep) was identified. Processing of proNGF was found to be tissue-specific and accumulation of pdNGFpeps was found in inflamed tissues, mainly in testis, intestine and heart, suggesting a possible correlation between organ functions and a response to insults and/or injury. The bioactivity of pdNGFpep was also demonstrated in vitro by using primary hippocampal neurons. Our study supports a biological function for the NGF precursor prodomain and indicates that short peptides from residues 1-60, differing from the 70-110 sequence, induce apoptosis, thereby opening the way for identification of new molecular targets to study pathological conditions.

Nerve Growth Factor and the Role of Inflammation in Tumor Development.

Nerve growth factor (NGF) plays a dual role both in inflammatory states and cancer, acting both as a pro-inflammatory and oncogenic factor and as an anti-inflammatory and pro-apoptotic mediator in a context-dependent way based on the signaling networks and its interaction with diverse cellular components within the microenvironment. This report aims to provide a summary and subsequent review of the literature on the role of NGF in regulating the inflammatory microenvironment and tumor cell growth, survival, and death. The role of NGF in inflammation and tumorigenesis as a component of the inflammatory system, its interaction with the various components of the respective microenvironments, its ability to cause epigenetic changes, and its role in the treatment of cancer have been highlighted in this paper.

Nerve Growth Factor and Autoimmune Diseases.

NGF plays a crucial immunomodulatory role and increased levels are found in numerous tissues during autoimmune states. NGF directly modulates innate and adaptive immune responses of B and T cells and causes the release of neuropeptides and neurotransmitters controlling the immune system activation in inflamed tissues. Evidence suggests that NGF is involved in the pathogenesis of numerous immune diseases including autoimmune thyroiditis, chronic arthritis, multiple sclerosis, systemic lupus erythematosus, mastocytosis, and chronic granulomatous disease. Furthermore, as NGF levels have been linked to disease severity, it could be considered an optimal early biomarker to identify therapeutic approach efficacy. In conclusion, by gaining insights into how these molecules function and which cells they interact with, future studies can devise targeted therapies to address various neurological, immunological, and other disorders more effectively. This knowledge may pave the way for innovative treatments based on NGF manipulation aimed at improving the quality of life for individuals affected by diseases involving neurotrophins.

Involvement of Substance P (SP) and Its Related NK1 Receptor in Primary Sjögren's Syndrome (pSS) Pathogenesis.

Primary Sjögren's Syndrome (pSS) is a systemic autoimmune disease that primarily attacks the lacrimal and salivary glands, resulting in impaired secretory function characterized by xerostomia and xerophthalmia. Patients with pSS have been shown to have impaired salivary gland innervation and altered circulating levels of neuropeptides thought to be a cause of decreased salivation, including substance P (SP). Using Western blot analysis and immunofluorescence studies, we examined the expression levels of SP and its preferred G protein-coupled TK Receptor 1 (NK1R) and apoptosis markers in biopsies of the minor salivary gland (MSG) from pSS patients compared with patients with idiopathic sicca syndrome. We confirmed a quantitative decrease in the amount of SP in the MSG of pSS patients and demonstrated a significant increase in NK1R levels compared with sicca subjects, indicating the involvement of SP fibers and NK1R in the impaired salivary secretion observed in pSS patients. Moreover, the increase in apoptosis (PARP-1 cleavage) in pSS patients was shown to be related to JNK phosphorylation. Since there is no satisfactory therapy for the treatment of secretory hypofunction in pSS patients, the SP pathway may be a new potential diagnostic tool or therapeutic target.

Psycho-Cognitive Profile and NGF and BDNF Levels in Tears and Serum: A Pilot Study in Patients with Graves' Disease.

Nerve Growth Factor (NGF) and Brain derived Neurotrophic Factor (BDNF) mature/precursor imbalance in tears and serum is suggested as a risk factor and symptomatology aggravation in ophthalmology and neuropsychiatric disturbances. Cognitive and mood alterations are reported by patients with Graves' Orbitopathy (GO), indicating neurotrophin alterations might be involved. To address this question, the expression levels of NGF and BDNF and their precursors in serum and tears of GO patients were analyzed and correlated with the ophthalmological and psycho-cognitive symptoms. Hamilton Rating Scale for Anxiety (HAM-A) and Depression (HAM-D), Temperament and Character Inventory (TCI), and Cambridge Neuropsychological Test Automated Battery (CANTAB) test were used as a score. NGF and BDNF levels were measured using ELISA and Western Blot and statistically analyzed for psychiatric/ocular variable trend association. GO patients show memorization time and level of distraction increase, together with high irritability and impulsiveness. HAM-A and CANTAB variables association, and some TCI dimensions are also found. NGF and BDNF expression correlates with ophthalmological symptoms only in tears, while mature/precursor NGF and BDNF correlate with the specific psycho-cognitive variables both in tears and serum. Our study is the first to show that changes in NGF and BDNF processing in tears and serum might profile ocular and cognitive alterations in patients.

Evaluation of the Effectiveness of BNT162b2 Primary Vaccination and Booster Dose to SARS-CoV-2 in Eliciting Stable Mucosal Immunity.

The waning effectiveness of the primary vaccination for SARS-CoV-2 led to administration of an additional booster dose (BD). The efficacy of the BD in stimulating humoral systemic immune response is well established, but its effectiveness on inducing mucosal immune reaction has not yet been reported. To address this issue, we evaluated SARS-CoV-2-specific antibody responses in the serum, saliva, and tears after BNT162b2 (Pfizer/BioNTech, New York, NY, USA) vaccination and BD, as well as after SARS-CoV-2 infection. After two doses of BNT162b2 vaccine, we observed specific serum IgG in 100% and IgA in 97.2% of subjects, associated with mucosal response in both salivary samples (sIgA in 97.2% and IgG(S) in 58.8%) and in tears (sIgA in 77.8% and IgG(S) in 67.7%). BD induced a recovery of the systemic humoral response and of tear sIgA when compared to 6 months of follow-up titers (p < 0.001; p = 0.012). However, sIgA levels in both tears and saliva were significantly lower following BD when compared to patients with prior SARS-CoV-2 infection (p = 0.001 and p = 0.005, respectively). Our results demonstrated that administration of BD restored high serum levels of both IgG and IgA but had a poor effect in stimulating mucosal immunity when compared to prior SARS-CoV-2 infection.

NGF Prevents Loss of TrkA/VEGFR2 Cells, and VEGF Isoform Dysregulation in the Retina of Adult Diabetic Rats.

Among the factors involved in diabetic retinopathy (DR), nerve growth factor (NGF) and vascular endothelial growth factor A (VEGFA) have been shown to affect both neuronal survival and vascular function, suggesting that their crosstalk might influence DR outcomes. To address this question, the administration of eye drops containing NGF (ed-NGF) to adult Sprague Dawley rats receiving streptozotocin (STZ) intraperitoneal injection was used as an experimental paradigm to investigate NGF modulation of VEGFA and its receptor VEGFR2 expression. We show that ed-NGF treatment prevents the histological and vascular alterations in STZ retina, VEGFR2 expression decreased in GCL and INL, and preserved the co-expression of VEGFR2 and NGF-tropomyosin-related kinase A (TrkA) receptor in retinal ganglion cells (RGCs). The WB analysis confirmed the NGF effect on VEGFR2 expression and activation, and showed a recovery of VEGF isoform dysregulation by suppressing STZ-induced VEGFA121 expression. Reduction in inflammatory and pro-apoptotic intracellular signals were also found in STZ+NGF retina. These findings suggest that ed-NGF administration might favor neuroretina protection, and in turn counteract the vascular impairment by regulating VEGFR2 and/or VEGFA isoform expression during the early stages of the disease. The possibility that an increase in the NGF availability might contribute to the switch from the proangiogenic/apoptotic to the neuroprotective action of VEGF is discussed.

Topical nerve growth factor prevents neurodegenerative and vascular stages of diabetic retinopathy.

Specific and effective preventive treatment for diabetic retinopathy (DR) is presently unavailable, mostly because the early stages of the complication have been, until recently, poorly understood. The recent demonstration that the vascular phase of DR is preceded and possibly caused by the neurodegeneration of retinal ganglion cells suggests that DR could, at least theoretically, be prevented through an early neuroprotective approach. The aims of our study were to clarify the natural history of diabetes-driven retinal neurodegeneration and to verify the possibility to prevent DR using topical nerve growth factor (NGF). The results of the study show that retinal neurodegeneration, characterized by the loss of retinal ganglion cells represents a relatively early phenomenon of diabetes (between 5 and 16 weeks of age), which tends to be self-limiting in the long run. Neurodegeneration is followed by the development of DR-related vascular dysfunctions, as confirmed by the development of acellular capillaries and the loss of retinal pericytes. Both retinal neurodegeneration and subsequent vascular dysfunction can be successfully prevented by topical NGF administration. These findings suggest that: 1) The first stage of DR consists in a self-limiting retinal neurodegeneration 2) The demonstrated effectiveness of topical NGF in the prevention of DR could be rapidly translated into clinical practice.

Pilot Investigation on p75ICD Expression in Laryngeal Squamous Cell Carcinoma.

We investigated the p75 Neurotrophin Receptor (p75NTR) expression and cleavage product p75NTR Intracellular Domain (p75ICD) as potential oncogenic and metastatic markers in human Laryngeal Squamous Cell Carcinoma (LSCC). p75NTR is highly expressed in Cancer Stem Cells (CSCs) of the laryngeal epithelia and it has been proposed as a marker for stemness, cell migration, and chemo-resistance in different squamous carcinomas. To investigate the clinical significance of p75NTR cleavage products in solid tumors, full-length and cleaved p75NTR expression was analyzed in laryngeal primary tumors from different-stage LSCC patients, diagnosed at the Policlinico Umberto I Hospital. Molecular and histological techniques were used to detect the expressions of p75NTR and p75ICD, and ATP Binding Cassette Subfamily G Member 2 (ABCG2), a CSC marker. We found regulated p75NTR cleavage during squamous epithelial tumor progression and tissue invasion. Our preliminary investigation suggests p75ICD expression and localization as possible features of tumorigenesis and metastaticity. Its co-localization with ABCG2 in squamous cells in the parenchyma invaded by the tumor formation allows us to hypothesize p75NTR and p75ICD roles in tumor invasion and CSC spreading in LSCC patients. These data might represent a starting point for a comprehensive analysis of p75NTR cleavage and of its clinical relevance as a potential molecular LSCC signature, possibly helping diagnosis, and improving prognosis and personalized therapy.

Urinary Ethyl Glucuronide for the Assessment of Alcohol Consumption During Pregnancy: Comparison between Biochemical Data and Screening Questionnaires.

BACKGROUND: Ethyl glucuronide (EtG) is a metabolite of ethanol used as a marker of alcohol drinking and is identified in urine. Gestational alcohol drinking harms the fetus, so disclosing any form of use and abuse of this substance during pregnancy is crucial. Many discovery methods have been planned to overcome this question, including using screening questionnaires as the AUDIT-C, T-ACE/TACER-3, and TWEAK. AIM: The aim and novelties of this study were to compare biochemical data from urinary EtG assays (cut-off 100 ng/mL for risking drinking behavior) with the outcome of questionnaires and of a food diary routinely used in our hospital; moreover, for the first time, we analyzed in pregnant women the EtG values normalized by the amount of creatinine excreted according to methods previously established. METHODS: Random urine samples were collected from 309 pregnant women immediately after being interviewed. EtG was quantified using an enzyme immunoassay, and urinary creatinine was assessed using an enzymatic colorimetric method. Women who had not exhaustively answered one of the questionnaires or refused to provide urine samples were excluded. Finally, 309 women were considered for this study. Urine creatinine measurements were performed to determine if urine dilution might have resulted in false negatives in the challenge study. In order to accomplish this objective, as urinary creatinine concentrations are, on average, approximately 1 mg/mL, we used a normalized value of 100 ng EtG/mg Creatinine. RESULTS: Our data show that 20.4% of the pregnant women in the study were over the established normalized cut-off value. Poor to null concordance (unweighted k < 0.2) was found between EtG data and the screening interviews showed, on average, lower levels of alcohol consumption. CONCLUSION: This study provides evidence that the assessment of maternal alcohol consumption during pregnancy, only indirectly estimated with questionnaires and food diary, can produce misleading results.

Gender differences in ultradian serum levels of NGF and BDNF correlate with psychophysical traits in healthy humans.

We aimed at investigating the gender and/or ultradian pattern of serum levels of the Nerve Growth Factor (NGF) and Brain-Derived Neurotrophic Factor (BDNF). Blood samples were collected at the 8.00, 13.00 and 20.00 hours of the day in healthy men and women, and the neurotrophins concentration was measured in the serum by ELISA. A further aim of the study was to evaluate whether or not the NGF/BDNF variations might be related to specific physiological or psychological traits as mood, feeling good and feeling rested, sexual desire and energy. Heart rate and blood pressure were also monitored at the same hours in each enrolled subject. The anxiety (STAI-T and STAI-S score) and sleeping quality were once evaluated in the morning too. We found that serum BDNF increases in men and decreases in women from morning to evening, while NGF shows a similar ultradian profile between men and women, but with higher concentrations in women. Both neurotrophins also show gender-related associations with psychophysiological variables. High NGF levels correlated with a high score for all the psychological variables in men, but with a low score in women. An inverse correlation was found between BDNF and energy and sexual desire in women, while no correlations were found in men. These data disclose that the condition of well-being (or activity/arousal status) is featured by an increasing NGF profile in men and a negative BDNF/NGF trend in women. The clinical relevance of the present data is discussed.

What substance P might tell us about the prognosis and mechanism of Parkinson's disease?

The neuropeptide substance P (SP) plays an important role in neurodegenerative disorders, among which Parkinson's disease (PD). In the present work we have reviewed the involvement of SP and its preferred receptor (NK1-R) in motor and non-motor PD symptoms, in both PD animal models and patients. Despite PD is primarily a motor disorder, non-motor abnormalities, including olfactory deficits and gastrointestinal dysfunctions, can represent diagnostic PD predictors, according to the hypothesis that the olfactory and the enteric nervous system represent starting points of neurodegeneration, ascending to the brain via the sympathetic fibers and the vagus nerve. In PD patients, the α-synuclein aggregates in the olfactory bulb and the gastrointestinal tract, as well as in the dorsal motor nucleus of the vagus nerve often co-localize with SP, indicating SP-positive neurons as highly vulnerable sites of degeneration. Considering the involvement of the SP/NK1-R in both the periphery and specific brain areas, this system might represent a neuronal substrate for the symptom and disease progression, as well as a therapeutic target for PD.

NGF Eye Administration Recovers the TrkB and Glutamate/GABA Marker Deficit in the Adult Visual Cortex Following Optic Nerve Crush.

Eye-drop recombinant human nerve growth factor (ed-rhNGF) has proved to recover the retina and optic nerve damage in animal models, including the unilateral optic nerve crush (ONC), and to improve visual acuity in humans. These data, associated with evidence that ed-rhNGF stimulates the brain derived neurotrophic factor (BDNF) in retina and cortex, suggests that NGF might exert retino-fugal effects by affecting BDNF and its receptor TrkB. To address these questions, their expression and relationship with the GABAergic and glutamatergic transmission markers, GAD65 and GAD67, vesicular inhibitory amino acid transporter (VGAT), and vesicular glutamate transporters 1 and 2 (VGLUT-1 and VGLUT-2) were investigated in adult ONC rats contralateral and ipsilateral visual cortex (VCx). Ed-rhNGF recovers the ONC-induced alteration of GABAergic and glutamatergic markers in contralateral VCx, induces an upregulation of TrkB, which is positively correlated with BDNF precursor (proBDNF) decrease in both VCx sides, and strongly enhances TrkB+ cell soma and neuronal endings surrounded by GAD65 immuno-reactive afferents. These findings contribute to enlarging the knowledge on the mechanism of actions and cellular targets of exogenously administrated NGF, and suggest that ed-rhNGF might act by potentiating the activity-dependent TrkB expression in GAD+ cells in VCx following retina damage and/or ONC.

Enzymatic Spermine Metabolites Induce Apoptosis Associated with Increase of p53, caspase-3 and miR-34a in Both Neuroblastoma Cells, SJNKP and the N-Myc-Amplified Form IMR5.

Neuroblastoma (NB) is a common malignant solid tumor in children and accounts for 15% of childhood cancer mortality. Amplification of the N-Myc oncogene is a well-established poor prognostic marker in NB patients and strongly correlates with higher tumor aggression and resistance to treatment. New therapies for patients with N-Myc-amplified NB need to be developed. After treating NB cells with BSAO/SPM, the detection of apoptosis was determined after annexin V-FITC labeling and DNA staining with propidium iodide. The mitochondrial membrane potential activity was checked, labeling cells with the probe JC-1 dye. We analyzed, by real-time RT-PCR, the transcript of genes involved in the apoptotic process, to determine possible down- or upregulation of mRNAs after the treatment on SJNKP and the N-Myc-amplified IMR5 cell lines with BSAO/SPM. The experiments were carried out considering the proapoptotic genes Tp53 and caspase-3. After treatment with BSAO/SPM, both cell lines displayed increased mRNA levels for all these proapoptotic genes. Western blotting analysis with PARP and caspase-3 antibody support that BSAO/SPM treatment induces high levels of apoptosis in cells. The major conclusion is that BSAO/SPM treatment leads to antiproliferative and cytotoxic activity of both NB cell lines, associated with activation of apoptosis.

Nerve Growth Factor in Alcohol Use Disorders.

The nerve growth factor (NGF) belongs to the family of neurotrophic factors. Initially discovered as a signaling molecule involved in the survival, protection, differentiation, and proliferation of sympathetic and peripheral sensory neurons, it also participates in the regulation of the immune system and endocrine system. NGF biological activity is due to the binding of two classes of receptors: the tropomyosin-related kinase A (TrkA) and the low-affinity NGF pan-neurotrophin receptor p75. Alcohol Use Disorders (AUD) are one of the most frequent mental disorders in developed countries, characterized by heavy drinking, despite the negative effects of alcohol on brain development and cognitive functions that cause individual's work, medical, legal, educational, and social life problems. In addition, alcohol consumption during pregnancy disrupts the development of the fetal brain causing a wide range of neurobehavioral outcomes collectively known as fetal alcohol spectrum disorders (FASD). The rationale of this review is to describe crucial findings on the role of NGF in humans and animals, when exposed to prenatal, chronic alcohol consumption, and on binge drinking.

Nerve Growth Factor, Stress and Diseases.

Stress is a constant threat for homeostasis and is represented by different extrinsic and intrinsic stimuli (stressors, Hans Selye's "noxious agents"), such as aggressive behavior, fear, diseases, physical activity, drugs, surgical injury, and environmental and physiological changes. Our organisms respond to stress by activating the adaptive stress system to activate compensatory responses for restoring homeostasis. Nerve Growth Factor (NGF) was discovered as a signaling molecule involved in survival, protection, differentiation, and proliferation of sympathetic and peripheral sensory neurons. NGF mediates stress with an important role in translating environmental stimuli into physiological and pathological feedbacks since NGF levels undergo important variations after exposure to stressful events. Psychological stress, lifestyle stress, and oxidative stress are well known to increase the risk of mental disorders such as schizophrenia, major depressive disorders, bipolar disorder, alcohol use disorders and metabolic disorders such as metabolic syndrome. This review reports recent works describing the activity of NGF in mental and metabolic disorders related to stress.

ProNGF/p75NTR Axis Drives Fiber Type Specification by Inducing the Fast-Glycolytic Phenotype in Mouse Skeletal Muscle Cells.

Despite its undisputable role in the homeostatic regulation of the nervous system, the nerve growth factor (NGF) also governs the relevant cellular processes in other tissues and organs. In this study, we aimed at assessing the expression and the putative involvement of NGF signaling in skeletal muscle physiology. To reach this objective, we employed satellite cell-derived myoblasts as an in vitro culture model. In vivo experiments were performed on Tibialis anterior from wild-type mice and an mdx mouse model of Duchenne muscular dystrophy. Targets of interest were mainly assessed by means of morphological, Western blot and qRT-PCR analysis. The results show that proNGF is involved in myogenic differentiation. Importantly, the proNGF/p75NTR pathway orchestrates a slow-to-fast fiber type transition by counteracting the expression of slow myosin heavy chain and that of oxidative markers. Concurrently, proNGF/p75NTR activation facilitates the induction of fast myosin heavy chain and of fast/glycolytic markers. Furthermore, we also provided evidence that the oxidative metabolism is impaired in mdx mice, and that these alterations are paralleled by a prominent buildup of proNGF and p75NTR. These findings underline that the proNGF/p75NTR pathway may play a crucial role in fiber type determination and suggest its prospective modulation as an innovative therapeutic approach to counteract muscle disorders.

Vagus nerve stimulation and Neurotrophins: a biological psychiatric perspective.

Since 2004, vagus nerve stimulation (VNS) has been used in treatment-resistant or treatment-intolerant depressive episodes. Today, VNS is suggested as possible therapy for a larger spectrum of psychiatric disorders, including schizophrenia, obsessive compulsive disorders, and panic disorders. Despite a large body of literature supports the application of VNS in patients' treatment, the exact mechanism of action of VNS remains not fully understood. In the present study, the major knowledges on the brain areas and neuronal pathways regulating neuroimmune and autonomic response subserving VNS effects are reviewed. Furthermore, the involvement of the neurotrophins (NTs) Nerve Growth Factor (NGF) and Brain Derived Neurotrophic Factor (BDNF) in vagus nerve (VN) physiology and stimulation is revised. The data on brain NGF/BDNF synthesis and in turn on the activity-dependent plasticity, connectivity rearrangement and neurogenesis, are presented and discussed as potential biomarkers for optimizing stimulatory parameters for VNS. A vagus nerve-neurotrophin interaction model in the brain is finally proposed as a working hypothesis for future studies addressed to understand pathophysiology of psychiatric disturbance.