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Amyotrophic lateral sclerosis (ALS) is an incurable motor neuron disease whose etiology remains unresolved; nonetheless, mutations of superoxide dismutase 1 (SOD1) have been associated with several variants of ALS. Currently available pharmacologic interventions are only symptomatic and palliative in effect; therefore, there is a pressing demand for more effective drugs. This study examined potential therapeutic effects of growth hormone secretagogues (GHSs), a large family of synthetic compounds, as possible candidates for the treatment of ALS. Human neuroblastoma cells expressing the SOD1-G93A mutated protein (SH-SY5Y SOD1G93A cells) were incubated for 24 h with H2O2 (150 µM) in the absence, or presence, of GHS (1 µM), in order to study the protective effect of GHS against increased oxidative stress. The two GHSs examined in this study, hexarelin and JMV2894, protected cells from H2O2-induced cytotoxicity by activating molecules that regulate apoptosis and promote cell survival processes. These findings suggest the possibility of developing new GHS-based anti-oxidant and neuroprotective drugs with improved therapeutic potential. Further investigations are required for the following: (i) to clarify GHS molecular mechanisms of action, and (ii) to envisage the development of new GHSs that may be useful in ALS therapy.

I glicani sono strutture composte da diversi monosaccaridi che formano la porzione di carboidrati tipica delle glicoproteine, dei glicolipidi o dei proteoglicani e sono coinvolti in diverse funzioni fisiologiche. I glicani che sono esposti sulla superficie dei patogeni (pathogen-associated molecular patterns, PAMP) rappresentano un segnale riconosciuto dalle lectine del sistema del complemento, una componente importante del sistema immunitario, tra cui ci sono le ficoline e mannose-binding lectin (MBL). Studi condotti in modelli sperimentali di ictus ischemico e studi clinici nei pazienti hanno dimostrato che MBL contribuisce al danno cerebrale endoteliale attivando cascate di tipo trombo-infiammatorio. Il riconoscimento di segnali di danno (damage-associated molecular patterns, DAMP) esposti sulle cellule endoteliali ischemiche è l’evento che scatena queste cascate tossiche. L’ictus è una delle maggiori cause di mortalità e disabilità permanente nel mondo e dipende dalla rottura (ictus emorragico) o dall’occlusione (ictus ischemico) di un vaso cerebrale. L’ictus ischemico rappresenta il tipo più frequente e si verifica in circa 88% dei casi. Nonostante alcuni progressi nella cura, come l’introduzione della trombectomia meccanica, e nella prevenzione, la patologia rimane un problema medico irrisolto. Sarebbe quindi auspicabile l’identificazione di nuovi target terapeutici. In questo contesto MBL rappresenta un potenziale bersaglio farmacologico. L’obiettivo principale di questa tesi è stato di identificare potenziali inibitori di MBL sviluppando ed utilizzando una robusta sequenza di test in vitro e in vivo. Inizialmente abbiamo utilizzato un approccio in vitro mettendo a punto un saggio con Surface Plasmon Resonance (SPR) che ci ha permesso di identificare i ligandi zuccherini con miglior affinità di legame a MBL. Abbiamo dimostrato che l’aumento della multivalenza dei leganti disponibili, ottenuta avvalendoci di nanoparticelle d’oro funzionalizzate con resi, Glycans are chain-like structures of many monosaccharides which create the carbohydrate portion of glycoconjugates such as a glycoproteins, glycolipids, or proteoglycans, and are involved in many physiological functions. Glycans exposed on the surface of pathogens (pathogen-associated molecular patterns, PAMPs) are recognized by the lectin pathway of the complement system, one of the key components of innate immune system, through carbohydrate-binding lectins, such as mannose binding lectin (MBL) or ficolins. Experimental and clinical evidences indicate that MBL also drives secondary pathogenic thrombo-inflammation on the ischemic vasculature. MBL-mediated pathogenesis is initiated by its binding to the sugar moieties exposed on endothelial cell membranes after brain ischemia (damage-associated molecular patterns, DAMPs), e.g. induced by stroke. Stroke is a major cause of disabilities and death worldwide and occurs by blockage or rupture of a brain artery (ischemic or hemorrhagic stroke), with lack of oxygen supply to brain tissues and sudden death of brain cells. Ischemic stroke is the most common type, i.e. 88% of all the cases. Pathological events on the ischemic vessels include the activation of coagulation, contact/kinin, and complement cascades. However, despite recent substantial progress in prevention and management, stroke still remains a large unmet medical need. Therefore, novel therapeutic strategies are needed and MBL inhibitors have been proposed as potential neuroprotective agents. The main goal of this thesis was to identify potential MBL inhibitors, by developing and exploiting a robust pipeline of preclinical studies. At first, an in vitro Surface Plasmon Resonance (SPR)-based assay was developed to identify MBL ligands/inhibitors and determine their affinities for MBL. The novel assay was extensively characterized and proved to be reliable and convenient. The results obtained with different ligands (e.g. monomeric mannose, a new glycan carrying ni

Amyotrophic lateral sclerosis (ALS) arises from neuronal death due to complex interactions of genetic, molecular, and environmental factors. Currently, only two drugs, riluzole and edaravone, have been approved to slow the progression of this disease. However, ghrelin and other ligands of the GHS-R1a receptor have demonstrated interesting neuroprotective activities that could be exploited in this pathology. Ghrelin, a 28-amino acid hormone, primarily synthesized and secreted by oxyntic cells in the stomach wall, binds to the pituitary GHS-R1a and stimulates GH secretion; in addition, ghrelin is endowed with multiple extra endocrine bioactivities. Native ghrelin requires esterification with octanoic acid for binding to the GHS-R1a receptor; however, this esterified form is very labile and represents less than 10% of circulating ghrelin. A large number of synthetic compounds, the growth hormone secretagogues (GHS) encompassing short peptides, peptoids, and non-peptidic moieties, are capable of mimicking several biological activities of ghrelin, including stimulation of GH release, appetite, and elevation of blood IGF-I levels. GHS have demonstrated neuroprotective and anticonvulsant effects in experimental models of pathologies both in vitro and in vivo. To illustrate, some GHS, currently under evaluation by regulatory agencies for the treatment of human cachexia, have a good safety profile and are safe for human use. Collectively, evidence suggests that ghrelin and cognate GHS may constitute potential therapies for ALS.

This study investigated modifications of microRNA expression profiles in knee synovial fluid of patients with osteoarthritis (OA) and rupture of the anterior cruciate ligament (ACL). Twelve microRNAs (26a-5p, 27a-3p, let7a-5p, 140-5p, 146-5p, 155-5p, 16-5p,186-5p, 199a-3p, 210-3p, 205-5p, and 30b-5p) were measured by real-time quantitative polymerase chain reaction (RT-qPCR) in synovial fluids obtained from 30 patients with ACL tear and 18 patients with knee OA. These 12 miRNAs were chosen on the basis of their involvement in pathological processes of bone and cartilage. Our results show that miR-26a-5p, miR-186-5p, and miR-30b-5p were expressed in the majority of OA and ACL tear samples, whereas miR-199a-3p, miR-210-3p, and miR-205-5p were detectable only in a few samples. Interestingly, miR-140-5p was expressed in only one sample of thirty in the ACL tear group. miR-140-5p has been proposed to modulate two genes (BGN and COL5A1100) that are involved in ligamentous homeostasis; their altered expression could be linked with ACL rupture susceptibility. The expression of miR-30b-5p was higher in OA and chronic ACL groups compared to acute ACL samples. We provide evidence that specific miRNAs could be detected not only in synovial fluid of patients with OA, but also in post-traumatic ACL tears.

Introduction: Acute respiratory distress syndrome (ARDS) is an acute form of diffuse lung injury characterized by (i) an intense inflammatory response, (ii) increased pulmonary vascular permeability, and (iii) the loss of respiratory pulmonary tissue. In this article we explore the therapeutic potential of hexarelin, a synthetic hexapeptide growth hormone secretagogue (GHS), in an experimental model of ARDS. Hexarelin has anti-inflammatory properties and demonstrates cardiovascular-protective activities including the inhibition of cardiomyocyte apoptosis and cardiac fibrosis, both of which may involve the angiotensin-converting enzyme (ACE) system.Methods: In our experimental model, ARDS was induced by the instillation of 100 mM HCI into the right bronchus; these mice were treated with hexarelin (320 pg/kg, ip) before (Pre) or after (Post) HCI challenge, or with vehicle. Respiratory system compliance, blood gas analysis, and differential cell counts in a selective bronchoalveolar lavage (BAL) were determined 6 or 24 hours after HCI instillation. In an extended study, mice were observed for a subsequent 14 days in order to assess lung fibrosis.Results: Hexarelin induced a significant improvement in lung compliance and a reduction of the number of total immune cells in BAL 24 hours after HCI instillation, accompanied with a lower recruitment of neutrophils compared with the vehicle group. At day 14, hexarelin-treated mice presented with less pulmonary collagen deposition compared with vehicle-treated controls.Conclusions: Our data suggest that hexarelin can inhibit the early phase of the inflammatory response in a murine model of HCI-induced ARDS, thereby blunting lung remodeling processes and fibrotic development.

VGF gene encodes for a neuropeptide precursor of 68 kDa composed by 615 (human) and 617 (rat, mice) residues, expressed prevalently in the central nervous system (CNS), but also in the peripheral nervous system (PNS) and in various endocrine cells. This precursor undergoes proteolytic cleavage, generating a family of peptides different in length and biological activity. Among them, TLQP-21, a peptide of 21 amino acids, has been widely investigated for its relevant endocrine and extraendocrine activities. The complement complement C3a receptor-1 (C3aR1) has been suggested as the TLQP-21 receptor and, in different cell lines, its activation by TLQP-21 induces an increase of intracellular Ca2+. This effect relies both on Ca2+ release from the endoplasmic reticulum (ER) and extracellular Ca2+ entry. The latter depends on stromal interaction molecules (STIM)-Orai1 interaction or transient receptor potential channel (TRPC) involvement. After Ca2+ entry, the activation of outward K+-Ca2+-dependent currents, mainly the KCa3.1 currents, provides a membrane polarizing influence which offset the depolarizing action of Ca2+ elevation and indirectly maintains the driving force for optimal Ca2+ increase in the cytosol. In this review, we address the main endocrine and extraendocrine actions displayed by TLQP-21, highlighting recent findings on its mechanism of action and its potential in different pathological conditions.

Growth hormone secretagogues (GHS) are a family of synthetic molecules, first discovered in the late 1970s for their ability to stimulate growth hormone (GH) release. Many effects of GHS are mediated by binding to GHS-R1a, the receptor for the endogenous hormone ghrelin, a 28-amino acid peptide isolated from the stomach. Besides endocrine functions, both ghrelin and GHS are endowed with some relevant extraendocrine properties, including stimulation of food intake, anticonvulsant and anti-inflammatory effects, and protection of muscle tissue in different pathological conditions. In particular, ghrelin and GHS inhibit cardiomyocyte and endothelial cell apoptosis and improve cardiac left ventricular function during ischemia-reperfusion injury. Moreover, in a model of cisplatin-induced cachexia, GHS protect skeletal muscle from mitochondrial damage and improve lean mass recovery. Most of these effects are mediated by GHS ability to preserve intracellular Ca2+ homeostasis. In this review, we address the muscle-specific protective effects of GHS mediated by Ca2+ regulation, but also highlight recent findings of their therapeutic potential in pathological conditions characterized by skeletal or cardiac muscle impairment.

La malattia di Alzheimer (AD), la forma più comune di demenza senile, è caratterizzata da una progressiva degenerazione del sistema nervoso centrale (SNC) che conduce ad un declino della funzione cognitiva e a perdita di memoria. I tratti caratteristici dell’AD includono placche extracellulari di beta amiloide (Abeta), grovigli neurofibrillari (NFTs), neuriti distrofici, morte neuronale e attivazione gliale. In base a quanto affermato dall’ “Ipotesi della cascata della Abeta” - la teoria più corroborata nell’ambito degli studi sull’AD negli ultimi decenni – la neuroinfiammazione veniva supposta come un processo caratterizzante solo le fasi tardive della malattia, e considerata come una mera risposta secondaria agli eventi patologici indotti dall’ A beta. Recentemente, nuovi studi preclinici, epidemiologici e genetici hanno dimostrato un coinvolgimento del sistema immunitario molto più precoce, comportando una rivalutazione del ruolo della principale cellula dell’immunità innata del cervello: la microglia. Dal momento che non vi è cura per l’AD, questi studi hanno motivato lo sviluppo di strategie terapeutiche con l’obiettivo di rallentare i processi degenerativi attraverso un’azione sulle cellule microgliali, in virtù del loro ruolo nell’orchestrare la neuroinfiammazione nelle malattie neurodegenerative. Le cellule staminali mesenchimali (MSC) sono cellule staminali multipotenti che negli ultimi decenni sono emerse per la loro capacità di migliorare il decorso patologico in diversi modelli di malattie neurodegenerative, grazie alla loro attività paracrina, che in gran parte dipende dal rilascio di vescicole extracellulari (EV). Le EV - strutture lipidiche importanti per la comunicazione intercellulare - si sono dimostrate essere mediatori di molti effetti benefici indotti dalle MSC, come l’immunomodulazione. Nello specifico, il concetto che le intrinseche abilità immunoregolatorie delle MSC sono fortemente potenziate dall’ambiente in cui si trovano, ha por, Alzheimer’s disease (AD), the most common form of age-related dementia, is characterized by a progressive degeneration of the central nervous system (CNS) that leads to a gradual decline of cognitive functions and memory loss. Neuropathological hallmarks of AD include extracellular beta-amyloid plaques, derived from the altered processing of amyloid precursor protein (APP), neurofibrillary tangles (NFTs, intraneuronal aggregates of hyperphosphorylated and misfolded tau), dystrophic neurites, neuronal loss and glial activation. According to the “Amyloid cascade hypothesis” - the most validated theory in the field of AD for the past few decades - neuroinflammation was assumed to occur only in the late stages of the disease, being considered as a mere secondary response to Abeta-induced pathophysiological events. Recently, new preclinical, epidemiological and genetic studies have demonstrated a much earlier involvement of immune system-related actions, leading to a reassessment of the role of the principal innate immune entities of the brain, that are microglia cells. Since there is still no cure for AD, these studies motivated the design of innovative therapeutic strategies aiming at slowing down degenerative processes by targeting microglia cells, in virtue of their main recognized role in orchestrating neuroinflammatory process in neurodegenerative diseases, including AD. Mesenchymal stem cells (MSCs) are adult multipotent stem cells that over the last decades have been demonstrated to show improvement in various model of neurodegenerative pathologies, thanks to their paracrine ability that is largely dependent on the secretion of extracellular vesicles (EVs). EVs - membrane bound entities known to be important players in intercellular communication - have emerged as mediator of multiple MSC beneficial effects, including immunomodulation. Particularly, the concept that intrinsic immunomoregulatory abilities of MSCs are strongly influenced and strengthened by the env

L'acido Gamma Amino butirrico, noto come GABA, rappresenta il principale neurotrasmettitore inibitorio del Sistema Nervoso nell'individuo adulto. Tuttavia, durante le precoci fasi di sviluppo, il GABA ha attività depolarizzante ed eccitatoria, fondamentale per il successivo e corretto sviluppo della rete. Il "GABA switch" rappresenta quindi un evento critico dello sviluppo neuronale, caratterizzato da uno shift funzionale della trasmissione GABAergica, da eccitatoria a inibitoria, le cui alterazioni sono comuni caratteristiche dei disordini del neurosviluppo, solitamente associati a disabilità cognitive e deficit comportamentali. Anche se i meccanismi molecolari del switch del GABA sono stati ampiamente descritti, nuovi regolatori di questo evento sono continuamente individuati e caratterizzati. È noto che le Cellule Staminali Mesenchimali (MSC) rappresentano buoni candidati a fini terapeutici, data la loro influenza positiva nella neuroprotezione verso le malattie neurologiche e immuno-mediate. Tuttavia, sempre più crescenti evidenze stanno dimostrando che le vescicole extracellulari (EVs) derivate da MSCs potrebbero rappresentare delle candidate migliori rispetto all'utilizzo di cellule per possibili applicazioni cliniche, mostrando un miglior profilo di sicurezza e utilizzo, e minori effetti collaterali. Tra le molecole immunomodulatorie, notevoli studi considerano l'Interleuchina 6 (IL-6) come un nuovo fattore neurotrofico, nonostante il suo ruolo ben descritto nelle malattie dello sviluppo neurologico, come l'autismo. Combinando tecniche funzionali (imaging di calcio e cloro) e approcci molecolari (RT-PCR), abbiamo osservato che le MSC-EVs, ma non le MSCs promuovono il GABA switch e aumentano l'espressione dei marcatori sinaptici inibitori del GABA. Allo stesso modo, l'esposizione precoce di IL-6 nei neuroni ippocampali, accelera il GABA switch modulando la trasmissione GABAergica e sovraregolando l'espressione di KCC2, in modo STAT-3- dipendente. Studi in, The neuronal “GABAergic switch” represents a critical event that occurs early in life before birth, during brain development, characterized by the excitatory-to-inhibitory transition of the GABAergic transmission. Impairments in the accomplishment of this event have been associated to a remarkable excitation/inhibition network imbalance, usually linked to cognitive disabilities and behavioural deficits, typical hallmarks of neurodevelopmental disorders. Even though molecular mechanisms of the GABA switch have been widely described, novel regulators of this event are being continuously characterized. It is well known that mesenchymal stem cells (MSCs) represent good candidates for therapeutic interventions, given their positive roles in neuroprotection against immune-mediated and neurological diseases. However, raising evidences are considering MSC-derived extracellular vesicles (EVs) better candidates than the whole cells for clinical applications, bearing more safety and less side effects. Among the immunomodulatory molecules, increasing studies consider the cytokine Interleukin 6 (IL-6) as a novel trophic factor, despite its well described role in neurodevelopmental diseases, such as autism. By taking advantage of a combination of functional (calcium and chloride imaging) and molecular approaches (RT-PCRs), we found that MSC-EVs but not MSCs accelerated the timing of the GABA switch and boosted the expression of the GABA inhibitory synaptic markers. Likewise, IL-6 early exposure in neurons accelerated the timing of the GABA switch by enhancing the GABAergic transmission and upregulating the expression of KCC2, in a STAT3- dependent manner. Given several evidences suggesting the presence of IL-6 within the MSC-MV cargo it is possible to speculate about their synergistic action when combined. All these data open the possibility to harness such system as a new therapeutical approach, for delivering safe and nontoxic organelles to those pathological conditions charact

Objectives. To understand brain ischemia injury contribution to Alzheimer’s disease (AD) onset, looking for post-translational modifications, turnover alterations and AD hallmarks secretion such as tau and amyloid precursor protein (APP), associated to autophagy activation. Methods. Rat cultured hippocampal neurons are subjected to oxygen glucose deprivation (OGD) followed by normal culture condition restoration (R), mimicking an ischemia/reperfusion (I/R) event. Afterwards AD hallmarks and autophagy markers expression and secretion are analyzed in the cells lysate and in microvescicles (MVs) isolated from culture medium. Confocal microscopy is employed to evaluate the potential re- and co-localization of tau and autophagic markers after treatment. Results. In our cellular model subjected to OGD/R, we observed tau and APP post-translational and protein level modifications in parallel to the autophagic markers (LC3II and beclin-1) expression alterations. Moreover, OGD/R treatment elicits a time-dependent increase in tau and APP C-terminal fragments (CTF) secretion by means of a heterogeneous MV population, including a group of LC3II positive vescicles. Preliminary data obtained by confocal microscopy suggest that LC3 might undergo re-localization after OGD/R treatment. Conclusions. The obtained results suggest that OGD treatment leads to multiple unconventional secretion mechanisms for tau and APP CTFs, in which autophagy activation seems to partially outcome in the exophagy secretory pathway. Although exophagy might be a defensive mechanism to prevent intracellular accumulation of misfolded proteins under conventional autophagy alteration, the secretion of misprocessed proteins has been recently associated to dendritic degeneration and neuronal death becoming a possible link to Alzheimer’s disease.

Strategies involved in mesenchymal stem cell (MSC) differentiation toward neuronal cells for screening purposes are characterized by quality and quantity issues. Differentiated cells are often scarce with respect to starting undifferentiated population, and the differentiation process is usually quite long, with high risk of contamination and low yield efficiency. Here, we describe a novel simple method to induce direct differentiation of MSCs into neuronal cells, without neurosphere formation. Differentiated cells are characterized by clear morphological changes, expression of neuronal specific markers, showing functional response to depolarizing stimuli and electrophysiological properties similar to those of developing neurons. The method described here represents a valuable tool for future strategies aimed at personalized screening of therapeutic agents in vitro.

Bone marrow-derived mesenchymal stem cells (MSCs) account for a small population of cells of the non-hematopoietic component of bone marrow. MSCs are multipotent stem cells endowed with neurotrophic potential combined to immunological properties, making them a promising therapeutic tool for neurodegenerative disorders. Although the mechanisms by which they act are still largely unknown, trans-differentiation, paracrine and autocrine actions have been hypothesized. Here we focus on the study of the effects exerted by rat MSCs on CNS neurons and oligodendrocytes by using a simplified in vitro co-culture system that precludes any direct contact between different cell types. The analysis of hippocampal synaptogenesis, synaptic vesicle recycling and electrical activity show that MSCs by themselves, efficiently support morphological and functional neuronal differentiation. Our observations demonstrate that MSCs selectively and directly increased hippocampal GABAergic presynapses and inhibitory transmission. In fact, this increment correlated to a higher expression of the potassium/chloride KCC2 cotransporter and to an enhancement of both the frequency and the amplitude of mIPSC and sIPSC. The decreased of GABA synapses following the treatment with a widely used Trk-neurotrophin receptor blocker, K252a, and the more specific TrkB receptor bodies prompt for the involvement of the brain derived neurotrophic factor (BDNF) in mediating such effects. The involvement of this neurotrophin is also strengthened by test ELISA on the culture medium collected from MSC-neuron co-cultures in which an higher BDNF concentration was detected, when compared to astrocyte-neuron co-cultures. The results obtained indicate that MSC-secreted factors induce glial-dependent neuronal survival and directly trigger an augmented GABAergic transmission in hippocampal cultures, highlighting a new effect by which MSCs could cooperate in CNS repair. Additionally, MSCs have been described to improve the cl

Bone marrow-derived mesenchymal stem cells (MSCs) are multipotent stem cells endowed with neurotrophic potential combined with immunological properties, making them a promising therapeutic tool for neurodegenerative disorders. However, the mechanisms through which MSCs promote the neurological recovery following injury or inflammation are still largely unknown, although cell replacement and paracrine mechanisms have been hypothesized. In order to find out what are the mechanisms of the trophic action of MSCs, as compared to glial cells, on CNS neurons, we set up a co-culture system where rat MSCs (or cortical astrocytes) were used as a feeding layer for hippocampal neurons without any direct contact between the two cell types. The analysis of hippocampal synaptogenesis, synaptic vesicle recycling and electrical activity show that MSCs were capable to support morphological and functional neuronal differentiation. The proliferation of hippocampal glial cells induced by the release of bioactive substance(s) from MSCs was necessary for neuronal survival. Furthermore, MSCs selectively increased hippocampal GABAergic pre-synapses. This effect was paralleled with a higher expression of the potassium/chloride KCC2 co-transporter and increased frequency and amplitude of mIPSCs and sIPSCs. The enhancement of GABA synapses was impaired by the treatment with K252a, a Trk/neurotrophin receptor blocker, and by TrkB receptor bodies hence suggesting the involvement of BDNF as a mediator of such effects. The results obtained here indicate that MSC-secreted factors induce glial-dependent neuronal survival and trigger an augmented GABAergic transmission in hippocampal cultures, highlighting a new effect by which MSCs could promote CNS repair. Our results suggest that MSCs may be useful in those neurological disorders characterized by an impairment of excitation versus inhibition balance.

During CNS development neurons undergo directional migration to achieve their adult localizations. To study neuronal migration, we used a model cell line of immortalized murine neurons (gonadotropin-releasing hormone expressing neurons; GN11), enriched with caveolins and caveolae invaginations that show in vitro chemotaxis upon serum exposure. Cholesterol depletion with methyl-beta-cyclodextrin induced the loss of caveolae and the inhibition of chemotaxis, thus suggesting that GN11 migration depends upon the structural integrity of caveolae. Polarization of proteins and organelles is a hallmark of cell migration. Accordingly, GN11 cells transmigrating through filter pores exhibited a polarized distribution of caveolin-1 isoform (cav-1) in the leading processes. In contrast, during two-dimensional migration cav-1 and caveolae polarized at the trailing edge. As caveolae are enriched with signaling molecules, we suggest that asymmetrical localization of caveolae may spatially orient GN11 neurons to incoming migratory signals thereby transducing them into directional migration.

Botulinum neurotoxins (BoNTs), proteases specific for the SNARE proteins, are used to study the molecular machinery supporting exocytosis and are used to treat human diseases characterized by cholinergic hyperactivity. The recent extension of the use of BoNTs to central nervous system (CNS) pathologies prompted the study of their traffic in central neurons. We used fluorescent BoNT/A and BoNT/E to study the penetration, the translocation and the catalytic action of these toxins in excitatory and inhibitory neurons. We show that BoNT/A and BoNT/E, besides preferentially inhibiting synaptic vesicle recycling at glutamatergic relative to Gamma-aminobutyric acid (GABA)-ergic neurons, are more efficient in impairing the release of excitatory than inhibitory neurotransmitter from brain synaptosomes. This differential effect does not result from a defective penetration of the toxin in line with the presence of the BoNT/A receptor, synaptic vesicle protein 2 (SV2), in both types of neurons. Interestingly, exogenous expression of SNAP-25 in GABAergic neurons confers sensitivity to BoNT/A. These results indicate that the expression of the toxin substrate, and not the toxin penetration, most likely accounts for the distinct effects of the two neurotoxins at the two types of terminals and support the use of BoNTs for the therapy of CNS diseases caused by the altered activity of selected neuronal populations.

Synaptosomal associated protein of 25 kDa (SNAP-25) is a component of the soluble N-ethylmaleimide-sensitive fusion protein (NSF) attachment protein receptor (SNARE) complex which plays a central role in synaptic vesicle exocytosis. We have previously demonstrated that adult rat hippocampal GABAergic synapses, both in culture and in brain, are virtually devoid of SNAP-25 immunoreactivity and are less sensitive to the action of botulinum toxin type A, which cleaves this SNARE protein [Neuron 41 (2004) 599]. In the present study, we extend our findings to the adult mouse hippocampus and we also provide demonstration that hippocampal inhibitory synapses lacking SNAP-25 labeling belong to parvalbumin-, calretinin- and cholecystokinin-positive interneurons. A partial colocalization between SNAP-25 and glutamic acid decarboxylase is instead detectable in developing mouse hippocampus at P0 and, at a lesser extent, at P5. In rat embryonic hippocampal cultures at early developmental stages, SNAP-25 immunoreactivity is detectable in a percentage of GABAergic neurons, which progressively reduces with time in culture. Consistent with the presence of the substrate, botulinum toxin type A is partially effective in inhibiting synaptic vesicle recycling in immature GABAergic neurons. Since SNAP-25, beside its role as a SNARE protein, is involved in additional processes, such as neurite outgrowth and regulation of calcium dynamics, the presence of higher levels of the protein at specific stages of neuronal differentiation may have implications for the construction and for the functional properties of brain circuits.