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Carbon nanotubes (CNTs) are currently under active investigation for their use in several biomedical applications, especially in neurological diseases and nervous system injury due to their electrochemical properties. Nowadays, no CNT-based therapeutic products for internal use appear to be close to the market, due to the still limited knowledge on their fate after delivery to living organisms and, in particular, on their toxicological profile. The purpose of the present work was to address the distribution in the brain parenchyma of two intranasally delivered MWCNTs (MWCNTs 1 and a-MWCNTs 2), different from each other, the first being non electroconductive while the second results in being electroconductive. After intranasal delivery, the presence of CNTs was investigated in several brain areas, discriminating the specific cell types involved in the CNT uptake. We also aimed to verify the neuroprotective potential of the two types of CNTs, delivering them in rats affected by early diabetic encephalopathy and analysing the modulation of nerve growth factor metabolism and the effects of CNTs on the neuronal and glial phenotypes. Our findings showed that both CNT types, when intranasally delivered, reached numerous brain areas and, in particular, the limbic area that plays a crucial role in the development and progression of major neurodegenerative diseases. Furthermore, we demonstrated that electroconductive MWCNTs were able to exert neuroprotective effects through the modulation of a key neurotrophic factor and probably the improvement of neurodegeneration-related gliosis.

The high incidence and increased mortality of COVID-19 make Italy among the most impacted countries by SARS-CoV-2 outbreak. In the beginning of the pandemic, Northern regions accounted for 40% of cases and 45% of deaths from COVID-19 in Italy. Several factors have been suggested to explain the higher incidence and excess mortality from COVID-19 in these regions. It is noticed that Northern Italian regions, and particularly the cities in Po Valley, are the areas with the highest air pollution due to commercial vehicle traffic, industry and a stagnant meteorological condition, with one of the highest levels in Italy and Europe of fine particulate matter 2.5 micron or smaller in size (PM2.5). PM2.5, the major environmental pollutant deriving mainly by factory and automobile exhaust emissions and coal combustion, increases the expression of angiotensin-converting enzyme 2, the epithelial cell entry receptor for SARS-CoV-2, and thus increase the susceptibility to this virus. The epithelial barrier hypothesis proposes that many diverse diseases may rise from the disruption of epithelial barrier of skin, respiratory tract and gastrointestinal system, including allergic diseases, metabolic and autoimmune diseases, and chronic neuropsychiatric conditions. There is evidence of a close correlation between air pollution and airway epithelial barrier dysfunction. Air pollution, causing lung epithelial barrier dysfunction, may contribute to local chronic inflammation, microbiome dysbiosis and impaired antiviral immune response against SARS-CoV-2, all of which contribute to the high incidence and excess mortality from COVID-19. In addition, air pollution and epithelial barrier dysfunction contribute also to the higher prevalence of several comorbidities of COVID-19, such as diabetes, chronic obstructive pulmonary disease and obesity, which have been identified as risk factors for mortality of COVID-19. In this article, on the basis of epidemiological and environmental monitoring data in Northern Italy, it is suggested that epithelial barrier hypothesis may help to understand the excess burden and mortality from COVID-19.

Regenerative capability of the peripheral nervous system after injury is enhanced by Schwann cells (SCs) producing several growth factors. The clinical use of SCs in nerve regeneration strategies is hindered by the necessity of removing a healthy nerve to obtain the therapeutic cells. Adipose-derived stem cells (ASCs) can be chemically differentiated towards a SC-like phenotype (dASCs), and represent a promising alternative to SCs. Their physiology can be further modulated pharmacologically by targeting receptors for neurotransmitters such as acetylcholine (ACh). In this study, we compare the ability of rat dASCs and native SCs to produce NGF in vitro. We also evaluate the ability of muscarinic receptors, in particular the M2 subtype, to modulate NGF production and maturation from the precursor (proNGF) to the mature (mNGF) form. For the first time, we demonstrate that dASCs produce higher basal levels of proNGF and mature NGF compared to SCs. Moreover, muscarinic receptor activation, and in particular M2 subtype stimulation, modulates NGF production and maturation in both SCs and dASCs. Indeed, both cell types express both proNGF A and B isoforms, as well as mNGF. After M2 receptor stimulation, proNGF-B (25 kDa), which is involved in apoptotic processes, is strongly reduced at transcript and protein level. Thus, we demonstrate that dASCs possess a stronger neurotrophic potential compared to SCs. ACh, via M2 muscarinic receptors, contributes to the modulation and maturation of NGF, improving the regenerative properties of dASCs.

The high incidence and increased mortality of COVID-19 make Italy among the most impacted countries by SARS-CoV-2 outbreak. In the beginning of the pandemic, Northern regions accounted for 40% of cases and 45% of deaths from COVID-19 in Italy. Several factors have been suggested to explain the higher incidence and excess mortality from COVID-19 in these regions. It is noticed that Northern Italian regions, and particularly the cities in Po Valley, are the areas with the highest air pollution due to commercial vehicle traffic, industry and a stagnant meteorological condition, with one of the highest levels in Italy and Europe of fine particulate matter 2.5 micron or smaller in size (PM2.5). PM2.5, the major environmental pollutant deriving mainly by factory and automobile exhaust emissions and coal combustion, increases the expression of angiotensin-converting enzyme 2, the epithelial cell entry receptor for SARS-CoV-2, and thus increase the susceptibility to this virus. The epithelial barrier hypothesis proposes that many diverse diseases may rise from the disruption of epithelial barrier of skin, respiratory tract and gastrointestinal system, including allergic diseases, metabolic and autoimmune diseases, and chronic neuropsychiatric conditions. There is evidence of a close correlation between air pollution and airway epithelial barrier dysfunction. Air pollution, causing lung epithelial barrier dysfunction, may contribute to local chronic inflammation, microbiome dysbiosis and impaired antiviral immune response against SARS-CoV-2, all of which contribute to the high incidence and excess mortality from COVID-19. In addition, air pollution and epithelial barrier dysfunction contribute also to the higher prevalence of several comorbidities of COVID-19, such as diabetes, chronic obstructive pulmonary disease and obesity, which have been identified as risk factors for mortality of COVID-19. In this article, on the basis of epidemiological and environmental monitoring data in Northern Italy, it is suggested that epithelial barrier hypothesis may help to understand the excess burden and mortality from COVID-19.

Since the 1980s, the development of a pharmacology based on nerve growth factor (NGF) has been postulated for the therapy of Alzheimer’s disease (AD). This hypothesis was based on the rescuing effect of the neurotrophin on the cholinergic phenotype of the basal forebrain neurons, primarily compromised during the development of AD. Subsequently, the use of NGF was put forward to treat a broader spectrum of neurological conditions affecting the central nervous system, such as Parkinson’s disease, degenerative retinopathies, severe brain traumas and neurodevelopmental dysfunctions. While supported by solid rational assumptions, the progress of a pharmacology founded on these hypotheses has been hampered by the difficulty of conveying NGF towards the brain parenchyma without resorting to invasive and risky delivery methods. At the end of the last century, it was shown that NGF administered intranasally to the olfactory epithelium was able to spread into the brain parenchyma. Notably, after such delivery, pharmacologically relevant concentration of exogenous NGF was found in brain areas located at considerable distances from the injection site along the rostral-caudal axis. These observations paved the way for preclinical characterization and clinical trials on the efficacy of intranasal NGF for the treatment of neurodegenerative diseases and of the consequences of brain trauma. In this review, a summary of the preclinical and clinical studies published to date will be attempted, as well as a discussion about the mechanisms underlying the efficacy and the possible development of the pharmacology based on intranasal conveyance of NGF to the brain.

Parkinson’s disease (PD) patients lead a sedentary lifestyle, being unable or unwilling to exercise conventionally, due to physical and mental limitations. The aim of this study was to assess the acute effects of a single session of whole-body electromyostimulation (WB-EMS) on the physical performances and serum levels of the neurotrophic factors in PD patients. Ten subjects (aged 72.60 ± 6.82) underwent 20 min of physical activity with superimposed WB-EMS and, after four weeks, the same protocol with no WB-EMS. WB-EMS was conducted with intermittent stimulation, with 4 s WB-EMS/4 s rest, at 85 Hz, 350 μs. A physical fitness assessment and blood samples collection, to evaluate neurotrophic factors’ levels (BDNF, FGF21, proNGF, mNGF), were collected before and after the intervention. The RM-ANOVA showed significant improvements in sit-to-stand (p &lt, 0.01), arm curl (p &lt, 0.01), handgrip (p &lt, 0.01) and soda pop test (p &lt, 0.01) after the WB-EMS intervention. Higher proNFG serum levels were observed in the WB-EMS condition compared to the no WB-EMS after 60 min post-intervention (p = 0.0163). The effect of WB-EMS confirmed the electrostimulation ability to modulate the proNGF quantity. The positive impact of the WB-EMS protocol on physical functioning, and eye–hand coordination, makes this intervention a promising strategy to improve motor and non-motor symptoms in PD patients.

Introduction: Ischemic stroke is an important cause of death and long-term morbidity in children. Viral respiratory infections are emerging as important risk factors for ischemic stroke in this age group of patients. Direct action of virus against cerebral vessels, autoimmune reactivity, and an increased production of cytokines have all been advocated as main factors in determining ischemic stroke. Case report: We report the case of an EBV-induced ischemic stroke in a caucasian 2 year-old female. The cerebrospinal fluid samples showed positivity of polymerase chain reaction for EBV infection, also confirmed by pharyngeal swab culture. Increased levels of interleukin 6 and interleukin 1b were also detected in the cerebrospinal fluid. Discussion: EBV infection has been identified as an important cause of neurological involvement in children. Findings of increased levels of interleukin 6 and interleukin 1b in the cerebrospinal fluid of the child with EBV induced-ischemic stroke seems to validate the role of pro-inflammatory cytokines as crucial mediators of cerebral thrombus formation. Conclusions: We believe that this report can be useful to clarify some pathophysiological mechanisms related to ischemic stroke related to Epstein-Barr Virus (EBV) infection in children.

The precursor of Nerve Growth Factor (proNGF) is the predominant form of NGF in the brain, where its tissue levels are increased in neurodegenerative diseases. proNGF exists in two main splicing variants, the long proNGF-A and the short proNGF-B. We demonstrated that proNGF-B is selectively increased in the hippocampus of rats affected by early diabetic encephalopathy and that native, purified proNGFs elicit different responses when used to stimulate PC12 cells. Therefore, the evaluation of the proNGF-B/proNGF-A ratio may be of important diagnostic and prognostic value in pathologies characterized by dysfunctions of NGF system. To date there is not clear pharmacological characterization of the different proNGFs variants, due to the lack of a proper recombinant proNGF-A. Using a bioinformatics approach, we predicted aminoacid sites involved in proNGF-A intracellular cleavage/conversion into proNGF-B, we cloned and expressed non-cleavable proNGF-A in HeLa cells and pursued a first characterization of their secretion modalities. Finally, we studied the biological effects of different proNGF-A mutants, stimulating PC12 cells with conditioned media from transfected HeLa cells. Based on our results, we propose the A73Y mutation as essential to obtaining an intact proNGF-A, limiting its conversion to proNGF-B. proNGF-A A73Y is probably released in an activity dependent manner and, when supplied to PC12 cells, shows a moderate differentiative capacity opposed to high neuroprotective potential. This preliminary study lays the foundation for future research aimed at uncovering the selective biological activities of proNGF-A and proNGF-B, and at developing pharmacological treatments that target the unbalance of proNGF system, induced by neurodegeneration.

The pandemic caused by SARS-CoV-2 represents an open and unresolved challenge for the global health system. The need to identify drugs that demonstrate efficacy in countering both the mechanisms of interaction of SARS-CoV-2 with host cells and to control the devastating inflammatory phenomena that characterize the late stages of viral infection, requires increasingly urgent answers. The biomedical research approach based on the repurposing of already approved drugs seems to be one of the most viable strategies in this struggle. In this work, by using a computational pharmacology approach and on the basis of what has been recently reported on the potential of nicotinic receptors in countering both phases of COVID-19, we propose a hypothesis aimed at widening the spectrum of pharmacological tools currently available to medical doctors. Our proposal specifically concerns the possibility of using tropisetron, a 5-HT3 receptor antagonist at the same time positive allosteric interactor of the nicotinic alpha-7 receptor, in order to inhibit unexplored virus-host interaction and restore the physiological control mechanisms of the excessive inflammation caused by SARS-CoV-2.

Pro-nerve growth factor (proNGF) is the predominant form of NGF in the brain and its levels increase in neurodegenerative diseases. The balance between NGF receptors may explain the contradictory biological activities of proNGF. However, the specific role of the two main proNGF variants is mostly unexplored. proNGF-A is prevalently expressed in healthy brain, while proNGF-B content increases in the neuro-degenerating brain. Recently we have investigated in vitro the biological action of native mouse proNGF variants. To gain further insights into the specific functions of the two proNGFs, here we intranasally delivered mouse-derived proNGF-A and proNGF-B to the brain parenchyma of healthy and diabetic rats, the latter characterized by dysfunction in spatial learning and memory, in the septo-hippocampal circuitry and by relative increase in proNGF-B hippocampal levels. Exogenous proNGF-B induces depression of hippocampal DG-LTP and impairment of hippocampal neurogenesis in healthy animals, with concomitant decrease in basal forebrain cholinergic neurons and cholinergic fibers projecting to the hippocampus. proNGF-A, while ineffective in healthy animals, rescues the diabetes-induced impairment in DG-LTP and hippocampal neurogenesis, promoting the concomitant recovery of the basal forebrain cholinergic phenotype. Our experimental evidences suggest that the balance between different proNGFs may influence the development and progression of neurodegenerative diseases.

The ongoing COVID-19 pandemic still requires fast and effective efforts from all fronts, including epidemiology, clinical practice, molecular medicine, and pharmacology. A comprehensive molecular framework of the disease is needed to better understand its pathological mechanisms, and to design successful treatments able to slow down and stop the impressive pace of the outbreak and harsh clinical symptomatology, possibly via the use of readily available, off-the-shelf drugs. This work engages in providing a wider picture of the human molecular landscape of the SARS-CoV-2 infection via a network medicine approach as the ground for a drug repurposing strategy. Grounding on prior knowledge such as experimentally validated host proteins known to be viral interactors, tissue-specific gene expression data, and using network analysis techniques such as network propagation and connectivity significance, the host molecular reaction network to the viral invasion is explored and exploited to infer and prioritize candidate target genes, and finally to propose drugs to be repurposed for the treatment of COVID-19. Ranks of potential target genes have been obtained for coherent groups of tissues/organs, potential and distinct sites of interaction between the virus and the organism. The normalization and the aggregation of the different scores allowed to define a preliminary, restricted list of genes candidates as pharmacological targets for drug repurposing, with the aim of contrasting different phases of the virus infection and viral replication cycle.

A single bout of low-frequency electroacupuncture (EA) causing muscle contractions increases whole-body glucose uptake in insulin-resistant rats. We explored the underlying mechanism of this finding and whether it can be translated into clinical settings. Changes in glucose infusion rate (GIR) were measured by euglycemic-hyperinsulinemic clamp during and after 45 min of low-frequency EA in 21 overweight/obese women with polycystic ovary syndrome (PCOS) and 21 controls matched for age, weight, and body mass index (experiment 1) and in rats receiving autonomic receptor blockers (experiment 2). GIR was higher after EA in controls and women with PCOS. Plasma serotonin levels and homovanillic acid, markers of vagal activity, decreased in both controls and patients with PCOS. Adipose tissue expression of pro-nerve growth factor (NGF) decreased, and the mature NGF/proNGF ratio increased after EA in PCOS, but not in controls, suggesting increased sympathetic-driven adipose tissue metabolism. Administration of alpha-/beta-adrenergic receptor blockers in rats blocked the increase in GIR in response to EA. Muscarinic and dopamine receptor antagonist also blocked the response but with slower onset. In conclusion, a single bout of EA increases whole-body glucose uptake by activation of the sympathetic and partly the parasympathetic nervous systems, which could have important clinical implications for the treatment of insulin resistance.-Benrick, A., Kokosar, M., Hu, M., Larsson, M., Maliqueo, M., Marcondes, R. R., Soligo, M., Protto, V., Jerlhag, E., Sazonova, A., Behre, C. J., Hojlund, K., Thoren, P., Stener-Victorin, E. Autonomic nervous system activation mediates the increase in whole-body glucose uptake in response to electroacupuncture.

Traumatic spinal cord injury has dramatic consequences and a huge social impact. We propose a new mouse model of spinal trauma that induces a complete paralysis of hindlimbs, still observable 30 days after injury. The contusion, performed without laminectomy and deriving from the pressure exerted directly on the bone, mimics more closely many features of spinal injury in humans. Spinal cord was injured at thoracic level 10 (T10) in adult anesthetized female CD1 mice, mounted on stereotaxic apparatus and connected to a precision impactor device. Following severe injury, we evaluated motor and sensory functions, and histological/morphological features of spinal tissue at different time points. Moreover, we studied the effects of early and subchronic administration of Docosahexaenoic acid, investigating functional responses, structural changes proximal and distal to the lesion in primary and secondary injury phases, proteome modulation in injured spinal cord. Docosahexaenoic acid was able i) to restore behavioural responses and ii) to induce pro-regenerative effects and neuroprotective action against demyelination, apoptosis and neuroinflammation. Considering the urgent health challenge represented by spinal injury, this new and reliable mouse model together with the positive effects of docosahexaenoic acid provide important translational implications for promising therapeutic approaches for spinal cord injuries.

Background: Our previous studies1 showed high expression levels of p75NTR, the nerve growth factor (NGF) receptor, in mononuclear cells (MNCs) obtained from blood and synovial fluids of patients with juvenile idiopathic arthritis (JIA) and rheumatoid arthritis (RA). p75NTR binds with high affinity proNGF, the immature form of NGF whose concentration, as we recently demonstrated2, is extremely high in the synovial fluids of arthritis patients. In ex vivo experiments we demonstrated that recombinant proNGF increases inflammatory cytokine production in patient MNCs, an effect that was abolished using p75NTR inhibitors2. We also found that synovial fibroblasts (SFs) represent the main source of proNGF in the inflamed synoviae. At present it is not known whether proNGF can influence the activity of SFs that are key effector cells in synovia inflammation producing inflammatory mediators that regulate chondrocytes and osteoblasts activation. Objectives: To investigate the mechanisms regulating p75NTR expression in synovial fibroblasts of arthritis patients and to evaluate the effects of its inhibition on the inflammatory response. Methods: SFs from arthritis patients were used to study the activity of proNGF/p75NTR axis. SFs from osteoarthritis patiens (OA) and skin fibroblasts from healthy donors (HD) were used as controls. p75NTR, NGF, and cytokine transcripts were evaluated by quantitative PCR (qPCR). Protein expression of p75NTR, NGF, and proNGF were analyzed by Western Blot. ELISAs were used to evaluate NGF, proNGF and cytokine concentration in supernatants and synovial fluids. p75NTR was inhibited using LM11A-31, a synthetic inhibitor that blocks the binding between p75NTR and its specific ligand proNGF. Results: mRNA and protein expression of p75NTR were up-regulated in arthritis SFs compared to OA SFs and skin fibroblasts. In vitro stimulation with recombinant cytokines (IL-1β, TNF, IL-6), TLR-ligands (such as LPS), and JIA synovial fluids, containing a mixture of inflammatory mediators, strongly increased p75NTR mRNA expression in arthritis SFs. Interestingly, after stimulation we observed that also OA SFs and HD fibroblasts up-regulated p75NTR transcript, suggesting that p75NTR levels are modulated by the inflammatory mediators. SFs of arthritis patients spontaneously produced proNGF and its release was further enhanced by all the above-mentioned inflammatory stimuli, albeit with different ability. Thus patient SFs can both produce proNGF and bind it through p75NTR. The inhibition of proNGF binding to p75NTR using LM11A-31 in arthritis SFs, activated with inflammatory cytokines or with synovial fluids, significantly reduced the expression and release of pro-inflammatory cytokine. Conclusion: In addition to inducing p75NTR up-regulation, inflammatory stimuli increase the release of proNGF in arthritis SFs. Autocrine proNGF binds to p75NTR and further enhances pro-inflammatory cytokine production, creating a vicious circle that amplifies the inflammatory response. Blocking the binding of endogenous proNGF to its receptor p75NTR strongly reduces the production of inflammatory mediators and prospects the use of p75NTR inhibitors as a new therapeutic approach to chronic arthritis. References [1] Prencipe, et al., Nerve Growth Factor Down regulates Inflammatory Response in Human Monocytes through TrkA, J Immunol. 2014 [2] Minnone, et al., ProNGF-p75NTR axis plays a proinflammatory role in inflamed joints: a novel pathogenic mechanism in chronic arthritis, RMD Open. 2017 Disclosure of Interests: Luciapia Farina: None declared, Gaetana Minnone : None declared, Marzia Soligo : None declared, Luigi Manni : None declared, Gian Marco Moneta: None declared, Ivan Caiello: None declared, Luigi Manzo : None declared, Fabrizio De Benedetti Grant/research support from: Abbvie, SOBI, Novimmune, Roche, Novartis, Sanofi, Pfizer, Luisa Bracci-Laudiero: None declared

Diabetes induces early sufferance in the cholinergic septo-hippocampal system, characterized by deficits in learning and memory, reduced hippocampal plasticity and abnormal pro-nerve growth factor (proNGF) release from hippocampal cells, all linked to dysfunctions in the muscarinic cholinergic modulation of hippocampal physiology. These alterations are associated with dysregulation of several cholinergic markers, such as the NGF receptor system and the acetylcholine biosynthetic enzyme choline-acetyl transferase (ChAT), in the medial septum and its target, the hippocampus. Controlled and repeated sensory stimulation by electroacupuncture has been proven effective in counteracting the consequences of diabetes on cholinergic system physiology in the brain. Here, we used a well-established Type 1 diabetes model, obtained by injecting young adult male rats with streptozotocin, to induce sufferance in the septo-hippocampal system. We then evaluated the effects of a 3-week treatment with low-frequency electroacupuncture on: (a) the expression and protein distribution of proNGF in the hippocampus, (b) the tissue distribution and content of NGF receptors in the medial septum, (c) the neuronal cholinergic and glial phenotype in the septo-hippocampal circuitry. Twice-a-week treatment with low-frequency electroacupuncture normalized, in both hippocampus and medial septum, the ratio between the neurotrophic NGF and its neurotoxic counterpart, the precursor proNGF. Electroacupuncture regulated the balance between the two major proNGF variants (proNGF-A and proNGF-B) at both gene expression and protein synthesis levels. In addition, electroacupuncture recovered to basal level the pro-neurotrophic NGF receptor tropomyosin receptor kinase-A content, down-regulated in medial septum cholinergic neurons by diabetes. Electroacupuncture also regulated ChAT content in medial septum neurons and its anterograde transport toward the hippocampus. Our data indicate that repeated sensory stimulation can positively affect brain circuits involved in learning and memory, reverting early impairment induced by diabetes development. Electroacupuncture could exert its effects on the septo-hippocampal cholinergic neurotransmission in diabetic rats, not only by rescuing the hippocampal muscarinic responsivity, as previously described, but also normalizing acetylcholine biosynthesis and NGF metabolism in the hippocampus.

BACKGROUND AND LIMITATIONS: Impaired wound healing (WH) and chronic inflammation are hallmarks of non-communicable diseases (NCDs). However, despite WH being a recognized player in NCDs, mainstream therapies focus on (un)targeted damping of the inflammatory response, leaving WH largely unaddressed, owing to three main factors. The first is the complexity of the pathway that links inflammation and wound healing; the second is the dual nature, local and systemic, of WH; and the third is the limited acknowledgement of genetic and contingent causes that disrupt physiologic progression of WH. PROPOSED APPROACH: Here, in the frame of Predictive, Preventive, and Personalized Medicine (PPPM), we integrate and revisit current literature to offer a novel systemic view on the cues that can impact on the fate (acute or chronic inflammation) of WH, beyond the compartmentalization of medical disciplines and with the support of advanced computational biology. CONCLUSIONS: This shall open to a broader understanding of the causes for WH going awry, offering new operational criteria for patients’ stratification (prediction and personalization). While this may also offer improved options for targeted prevention, we will envisage new therapeutic strategies to reboot and/or boost WH, to enable its progression across its physiological phases, the first of which is a transient acute inflammatory response versus the chronic low-grade inflammation characteristic of NCDs.

Traumatic spinal cord injury has dramatic consequences and a huge social impact. We propose a new mouse model of spinal trauma that induces a complete paralysis of hindlimbs, still observable 30 days after injury. The contusion, performed without laminectomy and deriving from the pressure exerted directly on the bone, mimics more closely many features of spinal injury in humans. Spinal cord was injured at thoracic level 10 (T10) in adult anesthetized female CD1 mice, mounted on stereotaxic apparatus and connected to a precision impactor device. Following severe injury, we evaluated motor and sensory functions, and histological/morphological features of spinal tissue at different time points. Moreover, we studied the effects of early and subchronic administration of Docosahexaenoic acid, investigating functional responses, structural changes proximal and distal to the lesion in primary and secondary injury phases, proteome modulation in injured spinal cord. Docosahexaenoic acid was able i) to restore behavioural responses and ii) to induce pro-regenerative effects and neuroprotective action against demyelination, apoptosis and neuroinflammation. Considering the urgent health challenge represented by spinal injury, this new and reliable mouse model together with the positive effects of docosahexaenoic acid provide important translational implications for promising therapeutic approaches for spinal cord injuries.

Carbon nanotubes (CNTs) are gaining increasing importance in the biomedical field. Among several biomedical applications, they represent a versatile tool to deliver or increase the yield in delivering therapeutics to selected targets. CNTs have proven to be attractive as potential therapeutics or vehicle to target blood-brain-barrier-protected brain cells. The possibility to deliver drugs to the brain parenchyma by the olfactory route has been recently reported. To date, the delivery of CNTs to the brain by intranasal administration has never been attempted. The main aim of the present study was a preliminary characterization of pharmacokinetic and pharmacodynamic parameters of two types of carbon nanotubes (CNTs) delivered to the brain parenchyma via the novel and unexplored olfactory route. To achieve this general aim, we studied tissue and cellular distribution of intranasally-delivered multi-walled carbon nanotube-11 (MWNT-11, not electroconductive) and annealed-MWNT-12 (aMWNT-12, electroconductive) in healthy and early diabetic rats, the latter known to be affected by mild cognitive impairment and neurodegeneration. We also attempted a preliminary characterization of the effects of CNTs on the metabolism of the neurotrophin nerve growth factor (NGF), known to play a central role in the development and progression of major neurodegenerative diseases, including diabetic encephalopathy. Our study, mainly performed by confocal microscopy, revealed that intranasally-delivered CNTs consistently diffuse into the brain parenchyma, reaching at least medial regions of the brain. CNTs were internalized by both neurons and glia, both expressing the GABA-ergic marker GAD, in all the regions reached by the nanomaterial. The tissue and cell distribution of MWNT-11 and aMWNT-12 in the brain of healthy and diabetic rats did not present qualitative macroscopic variances. However, significant differences were found when the effects of the treatments were evaluated on mNGF/proNGF protein levels and the glial/neuronal phenotypes in the hippocampus, caused both by the induction of diabetes and by the different CNT used as therapeutic. Overall, our data indicated that aMWNT-12 was able to elicit a modulation of the neurotrophic milieu and a control over the surrounding reactive gliosis similar to what observed when other neuroprotective therapies were applied in the same experimental model. Overall, our preliminary studies strongly support the need for building a clear and complete description and knowledge of the pharmacokinetic and pharmacodynamic features of CNTs after their delivery to the brain via the intranasal route.

The present work aimed to explore the innovative hypothesis that different transcript/protein variants of a pro-neurotrophin may generate different biological outcomes in a cellular system. Nerve growth factor (NGF) is important in the development and progression of neurodegenerative and cancer conditions. Mature NGF (mNGF) originates from a precursor, proNGF, produced in mouse in two major variants, proNGF-A and proNGF-B. Different receptors bind mNGF and proNGF, generating neurotrophic or neurotoxic outcomes. It is known that dysregulation in the proNGF/mNGF ratio and in NGF-receptors expression affects brain homeostasis. To date, however, the specific roles of the two major proNGF variants remain unexplored. Here we attempted a first characterization of the possible differential effects of proNGF-A and proNGF-B on viability, differentiation and endogenous ngf gene expression in the PC12 cell line. We also investigated the differential involvement of NGF receptors in the actions of proNGF. We found that native mouse mNGF, proNGF-A and proNGF-B elicited different effects on PC12 cell survival and differentiation. Only mNGF and proNGF-A promoted neurotrophic responses when all NGF receptors are exposed at the cell surface. Tropomyosine receptor kinase A (TrkA) blockade inhibited cell differentiation, regardless of which NGF was added to culture media. Only proNGF-A exerted a pro-survival effect when TrkA was inhibited. Conversely, proNGF-B exerted differentiative effects when the p75 neurotrophin receptor (p75NTR) was antagonized. Stimulation with NGF variants differentially regulated the autocrine production of distinct proNgf mRNA. Overall, our findings suggest that mNGF and proNGF-A may elicit similar neurotrophic effects, not necessarily linked to activation of the same NGF-receptor, while the action of proNGF-B may be determined by the NGF-receptors balance. Thus, the proposed involvement of proNGF/NGF on the development and progression of neurodegenerative and tumor conditions may depend on the NGF-receptors balance, on specific NGF trancript expression and on the proNGF protein variant ratio.

Paediatric optic pathway gliomas are low-grade brain tumours characterized by slow progression and invalidating visual loss. Presently there is no strategy to prevent visual loss in this kind of tumour. This study evaluated the effects of nerve growth factor administration in protecting visual function in patients with optic pathway glioma-related visual impairment. A prospective randomized double-blind phase II clinical trial was conducted in 18 optic pathway glioma patients, aged from 2 to 23 years, with stable disease and severe visual loss. Ten patients were randomly assigned to receive a single 10-day course of 0.5 mg murine nerve growth factor as eye drops, while eight patients received placebo. All patients were evaluated before and after treatment, testing visual acuity, visual field, visual-evoked potentials, optic coherence tomography, electroretinographic photopic negative response, and magnetic resonance imaging. Post-treatment evaluations were repeated at 15, 30, 90, and 180 days Brain magnetic resonance imaging was performed at baseline and at 180 days. Treatment with nerve growth factor led to statistically significant improvements in objective electrophysiological parameters (electroretinographic photopic negative response amplitude at 180 days and visual-evoked potentials at 30 days), which were not observed in placebo-treated patients. Furthermore, in patients in whom visual fields could still be measured, visual field worsening was only observed in placebo-treated cases, while three of four nerve growth factor-treated subjects showed significant visual field enlargement. This corresponded to improved visually guided behaviour, as reported by the patients and/or the caregivers. There was no evidence of side effects related to nerve growth factor treatment. Nerve growth factor eye drop administration appears a safe, easy and effective strategy for the treatment of visual loss associated with optic pathway gliomas.

The thesis deals with the effects and mechanisms of electroacupuncture therapy on hippocampal functional and molecular dysregulations induced in rats by early experimental diabetes. In the first part of the thesis, the candidate Marzia Soligo studied the impact of the early diabetic encephalopathy on the hippocampal physiology, in particular targeting the alterations of pro-nerve growth factor (proNGF) secretion, the functionality of muscarinic receptors M1 and M2 subtypes and the effects of electroacupuncture in counteracting the diabetes-induced dysregulations. The data obtained have been published in two referenced papers and indicate that the development and progression of diabetes generate a mild cognitive impairment reducing the functions of the septo-hippocampal cholinergic circuitry, depressing neural plasticity and inducing proNGF accumulation in the brain. Furthermore, the data demonstrate that the physical therapy, electroacupuncture, probably normalizing the altered M1-AChR functionality, counteracts the diabetes-induced deleterious effects on hippocampal physiology by ameliorating hippocampal-related memory functions, recovering the impaired long-term potentiation at the dentate gyrus (DG-LTP) and normalizing the activity-dependent release of proNGF. Therefore, it was hypothesized that a physical therapy based on repetitive sensory stimulation could promote hippocampal neural activity and conceivably improve the diabetes-induced cognitive impairment, in turn regulating the metabolism of key trophic factors such as proNGF/mNGF. In the second part of the thesis, the candidate focused her attention on the role that proNGF played on the hippocampal physiology, shifting the attention to the balance between the two relative parts of the total proNGF (proNGF-A and proNGF-B). The data suggested a differential modulation of the two proNGFs and a selective challenge of the proNGF-B to the pro-apoptotic receptor complex p75NTR/sortilin in diabetic animals, which were normalized by electroacupuncture. Based on these evidences, the candidate analysed the impact that alteration in the physiological balance between the two main proNGF protein variants, obtained by treatments with purified proNGF variants, could have in vitro and in normal and degenerating diabetic brain. The data obtained suggest that proNGF-B is the protein variant responsible for the observed neurodegenerative changes and that electroacupuncture effects could relies on the normalization of the proNGF-A/B balance in diabetic brain. The candidate suggests, based on her overall observation, a novel and intriguing update of the actual model for the proNGF metabolism. Moreover, she obtained evidence for the existence of the predicted proNGF longer splicing variant (proNGF-A) in rat tissues, by a novel RT-PCR assay.

Since its discovery, nerve growth factor (NGF) has long occupied a critical role in developmental and adult neuro-biology for its many important regulatory functions on the survival, growth and differentiation of nerve cells in the peripheral and central nervous system. NGF is the first discovered member of a family of neurotrophic factors, collectively indicated as neurotrophins, (which include brain-derived neurotrophic factor, neurotrophin-3 and neurotrophin 4/5). NGF was discov-ered for its action on the survival and differentiation of selected populations of peripheral neurons. Since then, an enormous number of basic and human studies were undertaken to explore the role of purified NGF to prevent the death of NGF-receptive cells. These studies revealed that NGF possesses important therapeutic properties, after topical administration, on human cutaneous pressure ulcer, corneal ulcers, glaucoma, retinal maculopathy, Retinitis Pigmentosa and in pediatric optic gliomas and brain traumas. The aim of this review is to present our previous, recent and ongoing clinical studies on the ther-apeutic properties of NGF.

The nerve growth factor (NGF) is a neurotrophin produced as a precursor proNGF. Both proNGF and mature NGF (mNGF) are biologically active. mNGF, challenging the tropomyosin receptor A (TrkA) or to the p75 neurotrophin receptor (p75NTR)-TrkA complex, exerts neurotrophic effects. ProNGF can bind p75NRT/TrkA and/or the p75NRT-Sortilin complex, respectively eliciting neurotrophic and neurotoxic effects. We aimed to characterize the role of the two main murine proNGF variants, proNGF-A and proNGF-B, studying their biological effects on PC12 cells. To this aim, we first characterized the receptors phenotype of the PC12 cells, maintained in different culture conditions and then we studied the effect of proNGF variants on cell viability and differentiation. The presence of all of the possible mNGF/proNGF receptors on cell surface correlated with cell cycle phase and was promoted by serum starvation and/or by pretreatment with mNGF (priming). Both mNGF and proNGF-A promoted cell survival and differentiation in primed PC12 cells, while proNGF-B displayed neurotoxic properties. The neurotrophic effects of mNGF and proNGF-A were TrkA-dependent, while the blockade of p75NRT counteracted or even reverted the effects of proNGF-B. Our results suggest that proNGF-B and mNGF/proNGF-A preferentially challenge different receptor complexes, eliciting specific and opposite biological effects in PC12 cells.

Objective To identify the role of mature nerve growth factor (mNGF), its immature form proNGF and their receptors in arthritis inflammation. Methods Real-time PCR, western blot and ELISA were performed to evaluate NGF, proNGF, their receptor and cytokine expression in synovial tissue and cells of patients with juvenile idiopathic arthritis (JIA) and rheumatoid arthritis (RA), and controls. Results proNGF and not mNGF is the prevalent form measured in synovial fluids of patients with JIA and RA with synovial fibroblasts as a major source of proNGF in the inflamed synoviae. p75NTR, the specific receptor for proNGF, is the NGF receptor most expressed in mononuclear cells of patients with JIA, while TrkA is the prevalent receptor in healthy donors. In ex vivo experiments the effects of proNGF differ from those of mNGF, suggesting that the balance of p75NTR and TrkA expression represents a critical factor in regulating mNGF/proNGF functions, determining which intracellular pathways and biological activities are triggered. Contrary to NGF, proNGF administration increased inflammatory cytokines but not interleukin (IL)-10 expression, inducing a stronger activation of p38 and JNK pathways. proNGF effects depend on its binding to p75NTR, as inhibition of p75NTR with neutralising antibodies or LM11A-31 abolished proNGF-induced production of IL-6 in patients’ mononuclear cells, while inhibition of TrkA did not. There is a correlation in patients with arthritis between high p75NTR levels and severity of clinical symptoms. Conclusions Our data suggest that an active proNGF-p75NTR axis promotes proinflammatory mechanisms contributing to chronic tissue inflammation, and that the use of p75NTR inhibitors may represent a new therapeutic approach in chronic arthritis.

International audience; We explored the mechanisms underlying microglia cell-carbon nanotube interactions in order to investigate whether electrical properties of Carbon-Nanotubes (CNTs) could affect microglia brain cells function and phenotype. We analyzed the effects induced by highly electro-conductive Multi-Walled-Carbon-Nanotubes (a-MWCNTs), on microglia cells from rat brain cortex and compared the results with those obtained with as prepared not conductive MWCNTs (MWCNTs) and redox-active Double-Walled-Carbon-Nanotubes (DWCNTs). Cell viability and CNT capacity to stimulate the release of nitric oxide (NO), pro-inflammatory (IL-1b, TNF-a) and anti-inflammatory (IL-10, TGF-b1) cytokines and neurotrophic factors (mNGF) were assessed. Electro-conductive MWCNTs, besides not being cytotoxic, were shown to stimulate, at 24 h cell exposure, classical "M100 microglia activation phenotype, increasing significantly the release of the main pro-inflammatory cytokines. Conversely, after 48 h cell exposure, they induced the transition from classical "M100 to alternative "M200 microglia phenotype, supported by anti-inflammatory cytokines and neuroprotective factor mNGF release. The analysis of cell morphology change, by tubulin and CD-206 þ labelling showed that M2 phenotype was much more expressed at 48 h in cells exposed to a-MWCNTs than in untreated cells. Our data suggest that the intrinsic electrical properties of CNTs could be exploited to modulate microglia phenotype and function stimulating microglia anti-inflammatory potential.

CORSO DI LAUREA MAGISTRALE IN BIOTECNOLOGIE MEDICHE TESI DI LAUREA SPERIMENTALE

The muscarinic receptor response to acetylcholine regulates the hippocampal-related learning, memory, neural plasticity and the production and processing of the pro-nerve growth factor (proNGF) by hippocampal cells. The development and progression of diabetes generate a mild cognitive impairment reducing the functions of the septo-hippocampal cholinergic circuitry, depressing neural plasticity and inducing proNGF accumulation in the brain. Here we demonstrate, in a rat model of early type-1 diabetes, that a physical therapy, the electroacupuncture, counteracts the diabetes-induced deleterious effects on hippocampal physiology by ameliorating hippocampal-related memory functions; recovering the impaired long-term potentiation at the dentate gyrus (DG-LTP) and the lowered expression of the vesicular glutamate transporter 1; normalizing the activity-dependent release of proNGF in diabetic rat hippocampus. Electroacupuncture exerted its therapeutic effects by regulating the expression and activity of M1- and M2-acetylcholine muscarinic receptors subtypes in the dentate gyrus of hippocampus. Our results suggest that a physical therapy based on repetitive sensory stimulation could promote hippocampal neural activity, neuronal metabolism and functions, and conceivably improve the diabetes-induced cognitive impairment. Our data can support the setup of therapeutic protocols based on a better integration between physical therapies and pharmacology for the cure of diabetes-associated neurodegeneration and possibly for Alzheimer’s disease.

BACKGROUND: Nerve growth factor (NGF) promotes neural recovery after experimental traumatic brain injury (TBI) supporting neuronal growth, differentiation and survival of brain cells and up-regulating the neurogenesis-associated protein Doublecortin (DCX). Only a few studies reported NGF administration in paediatric patients with severe TBI. METHODS: A four-year-old boy in a persistent unresponsive wakefulness syndrome (UWS) was treated with intranasal murine NGF administration 6 months after severe TBI. The patient received four cycles of intranasal NGF (0.1 mg/kg, twice a day for 10 consecutive days). RESULTS: NGF administration improved functional [Positron Emission Tomography/Computed Tomography (PET/CT); Single photon emission/Computed Tomography (SPECT/CT) and Magnetic Resonance Imaging (MRI)] assessment, electrophysiological [Electroencephalogram (EEG) and Visual Evoked Potential (VEP)] studies and clinical conditions. He showed improvements in voluntary movements, facial mimicry, phonation, attention and verbal comprehension, ability to cry, cough reflex, oral motility, feeding capacity, and bowel and urinary functions. After NGF administration, raised levels of both NGF and DCX were found in the cerebrospinal fluid of the patient. No side effects were reported. CONCLUSIONS: Although further studies are needed for better understanding the neuroprotective role of this neurotrophin, intranasal NGF administration appears to be a promising and safe rescuing strategy treatment in children with neurological impairment after TBI.

The purpose of this work was to investigate whether, by intranasal administration, the nerve growth factor bypasses the blood-brain barrier and turns over the spinal cord neurons and if such therapeutic approach could be of value in the treatment of spinal cord injury. Adult Sprague-Dawley rats with intact and injured spinal cord received daily intranasal nerve growth factor administration in both nostrils for 1 day or for 3 consecutive weeks. We found an increased content of nerve growth factor and enhanced expression of nerve growth factor receptor in the spinal cord 24 hours after a single intranasal administration of nerve growth factor in healthy rats, while daily treatment for 3 weeks in a model of spinal cord injury improved the deficits in locomotor behaviour and increased spinal content of both nerve growth factor and nerve growth factor receptors. These outcomes suggest that the intranasal nerve growth factor bypasses blood-brain barrier and affects spinal cord neurons in spinal cord injury. They also suggest exploiting the possible therapeutic role of intranasally delivered nerve growth factor for the neuroprotection of damaged spinal nerve cells.

Aim Insulin sensitivity is ~40% lower in women with polycystic ovary syndrome (PCOS) than in controls. We tested the hypothesis that 5weeks of electroacupuncture treatment improves glucose regulation and androgen levels in overweight/obese women with PCOS. Material and Methods Seventeen women with PCOS, aged 18 to 38years, with a body mass index (BMI) ≥25 kg/m2 and diagnosed with PCOS were included in this experimental and feasibility study and subjected to five weeks of electroacupuncture treatments three times/week. The primary outcome was changes in whole‐body glucose homeostasis measured by euglycemic hyperinsulinemic clamp before and after the intervention. Secondary outcome were changes in HbA1c, circulating catecholamines, adipocyte size and adipose tissue expression of sex steroids and nerve growth factor (NGF). Results No significant change in glucose homeostasis was observed, but HbA1c decreased by 9.5% (p=0.004), circulating testosterone decreased by 22% (p=0.0007) and dihydrotestosterone decreased by 12% (p=0.007). The two vagal activity markers of plasma serotonin levels and the dopamine metabolite homovanillic acid decreased by 21% (p=0.027) and 20% (p=0.011), respectively. Adipose tissue concentrations of testosterone decreased by 18% (p=0.049), and androstenedione decreased by 13% (p=0.035), and mature NGF/proNGF ratio, a marker of sympathetic activity, increased (p=0.04). These changes occurred without changes in anthropometrics. Conclusion Five weeks of electroacupuncture treatment improves HbA1c and circulating and adipose tissue androgens in women with PCOS. This effect is mediated, at least in part, via modulation of vagal activity and adipose tissue sympathetic activity. Based on these findings, we have recently initiated a randomized controlled study (NTC02647827). Electroacupuncture improves HbA1c in PCOS

Nerve growth factor (NGF) is essential for the survival and functional maintenance of forebrain cholinergic neurons projecting mainly to the cortex and hippocampus. NGF is produced in these brain areas but while mature NGF (mNGF) has a survival/differentiative effect its precursor proNGF elicits apoptosis in cholinergic neurons. Impaired neurotransmission, loss of cholinergic phenotype and abnormal NGF content characterize the cholinergic circuitries in animal models of diabetic encephalopathy (DE). It is not known whether defective production or maturation of NGF could play a key role in cholinergic neurodegeneration in DE. Quantification of the mNGF/proNGF ratio is therefore needed to characterize the development and progression of NGF-related neuronal diseases. In our work, we aimed at developing ELISA methods to measure either mNGF or proNGF tissue concentration; and to define the mNGF/proNGF ratio in the rat cortex and hippocampus during the early stage of streptozotocin-induced type 1 diabetes. Using commercially available NGF ELISA kits and antibodies, we set up ELISAs for human and rat mNGF and proNGF. We then analyzed the mNGF/proNGF ratio in the cortex and hippocampus of DE rats and found that it decreased in both tissues starting from the fourth week after diabetes induction. In diabetic brain the increase in proNGF involves accumulation of the isoforms with molecular weights of 50 and 34 kDa. Our study for the first time specifically quantifies the absolute content of mature and proNGF and the mNGF/proNGF ratio in brain tissues, suggesting that early progression of experimental DE is characterized by defective maturation of NGF.

Rationale: Diabetic Encephalopathy (DE) affects neurogenesis of the hippocampus (HP) dentate gyrus subgranular zone (SGZ)1,2 probably dysregulating the neurotrophic proNGF/mature-NGF (mNGF) system3, whose activity is regulated by the cholinergic neurotransmission from medial septum (MS)4. Electroacupuncture (EA) is effective in modulating proNGF/NGF balance and activity in the brain cholinergic system of DE rats3. Objectives: To study the effects of EA on the SGZ neurogenesis and on NGF receptors (TrkA and p75NTR) presence and activity in the MS in a rat model of DE. Materials and Methods: DE was induced in young adult rats by intraperitoneal streptozotocin (STZ). One week after STZ, EA treatment (burst frequency of 2 Hz, intensity 1.0-1.5 mA) was started and repeated twice a week for 3 weeks. At the end of the treatments, brain tissues representative of the cholinergic circuitry (HP and MS) were collected. Brain sections containing SGZ and MS regions were also collected. We analyzed: the mNGF/proNGF in HP by ELISA5; the number of doublecortin (DCX)-positive cells in SGZ by confocal microscopy; the distribution of p75NTR and TrkA in MS, together with markers of cholinergic phenotype and neuronal activity, by confocal microscopy; the activation of p75NTR pro-apoptotic signaling in MS by Western blot for the JNK/pospho-JNK system. Results: proNGF/mNGF ratio in HP was increased by STZ and normalized by EA. STZ also decreased and EA normalized the SGZ neurogenesis measured as number of DCX-positive cells. The number of cells immune-stained for p75NTR and TrkA in MS were increased and decreased respectively after STZ, while EA restored control levels. In MS, the EA reduced JNK phosphorylation, index of p75NTR-mediated pro-apoptotic activation, which increased after STZ. Conclusions: EA, probably normalizing the acetylcholine-mediated neurotransmission, stimulates the SGZ neurogenesis modulating the balance of proNGF/NGF, the cellular content of its receptors system and inhibiting p75NTR pro-apoptotic signal cascade.

Rationale: Diabetic encephalopathy (DE) affects the function of brain cholinergic circuits1-3 and dysregulates the neurotrophic proNGF/NGF system1,3. Electroacupuncture (EA) modulates NGF and NGF receptors expression in an animal model of DE1. Objectives: To study, in experimental DE, EA effects on: long-term potentiation (LTP) in the dentate gyrus (DG); proNGF/NGF release in the hippocampus (HP); p75 neurotropin receptor (p75NTR) challenge by proNGF. Materials and Methods: DE was induced in young adult rats by streptozotocin (STZ). One week after STZ injection, EA (burst frequency of 2 Hz, intensity 1.0-1.5 mA) was started and repeated twice a week for 3 weeks. At the end of the treatments, HP tissues were collected. In HP slices from control, STZ and STZ/EA rats, we analyzed field excitatory post-synaptic potentials and LTP in the DG after electrical stimulation of the medial perforant path and proNGF isoforms release in the HP slices superfused and stimulated by the muscarinic agonist carbachol (CCh). In HP tissues we studied: the proNGF/NGF content by Western blot and ELISA4 and the role of different proNGF isoform in challenging the pro-apoptotic p75NTR by co-immunoprecipitation experiments. Results: STZ depressed and EA normalized the perforant pathway-mediated LTP. proNGF increased in diabetic rats and EA normalized pro-neurotrophin content in whole HP lysates. In superfusates, proNGF increased after CCh in STZ rats and the release was sustained over 60 minutes after stimulation. EA decreased basal proNGF release and counteracted the sustained release. The enhancement of p75NTR interaction with the 25 kDa proNGF in diabetic brain was counteracted by EA. Conclusions: EA in DE rats is effective in restoring synaptic plasticity in brain areas that produce and release proNGF. Diabetes affects and EA normalizes the production and release of proNGF, suggesting a possible mechanism based on the ability of EA to modulate the activity of the cholinergic brain.