Peptidomimetics hold a great promise as therapeutic agents for neurodegenerative disorders. We previously described a Nerve Growth Factor (NGF)-like peptide, now named BB14, which was found to act as a strong TrkA agonist and to be effective in the sciatic nerve injury model of neuropathic pain. In this report we present the effects of BB14 in reducing reactive astrocytosis and reverting neuroplastic changes of the glutamate/GABAergic circuitry in the lumbar spinal cord following spared nerve injury (SNI) of the sciatic nerve. Immunohistochemical analysis of spinal cord sections revealed that SNI was associated with increased microglial (Iba1) and astrocytic (GFAP) responses, indicative of reactive gliosis. These changes were paralleled by (i) decreased glial aminoacid transporters (GLT1 and GlyT1) and increased levels of (ii) neuronal glutamate transporter EAAC1, (iii) neuronal vesicular GABA transporter (vGAT) and (iv) the GABAergic neuron marker GAD65/67. A remarkable increase of the Glutamate/GABA ratio and the reduction of glutathione (GSH) levels were also indicative of modifications of glial function in neuroprotection. All these molecular changes were found to be linked to an alteration of endogenous NGF metabolism, as demonstrated by decreased levels of mature NGF, increase of proNGF and increased activity of NGF-degrading methallo-proteinases (MMPs). Biochemical alterations and SNI-related neuropathic behavior, characterized by allodynia and hyperalgesia, were reversed by 7-days i.t. administration of the NGF-like peptide BB14, as well as by increasing endogenous NGF levels by i.t. infusion of GM6001, a MMPs inhibitor. All together, while confirming the correlation between reactive astrogliosis and perturbation of synaptic circuitry in the SNI model of peripheral nerve injury, these data strongly support the beneficial effect of BB14 in reducing reactive astrogliosis and restoring synaptic homeostasis under pathological conditions linked to alteration of NGF availability and signaling, thereby suggesting a potential role of BB14 as a therapeutic agent.
Recently, the drug discovery process has been greatly implemented by new biotechnological approaches, such as in silico library screening and molecular modeling, which allow to predict properties and activities of new chemical entities. These tools proved to be particularly useful for neurotrophin-based interventions in neurodegenerative diseases, which currently lack of effective therapies. Neurotrophins (NGF, BDNF, NT-3 and NT-4/5) regulate several functions in distinct classes of neurons through interaction with two types of receptors: the tyrosine kinase receptors (TrkA, TrkB and TrkC) and the common p75 NTR receptor. While Trk activation is essential for neuronal survival, differentiation and synaptic function, the p75 NTR signaling is more complex: depending on cellular environment, expression of co-receptors (Nykjaer et al., 2004) and ligands availability (mature neurotrophins or their precursors, such as proNGF and proBDNF), the p75 NTR receptor can transduce signals that are either pro-survival, pro-differentiation, or pro-apoptotic (Chao et al., 2006). Functional mimicry of specific pharmacophores has allowed the development of small peptide or non-peptide molecules functioning as ligands of specific neurotrophin receptors and/or mimicking selected components of the neurotrophin signaling that modulate specific ligand/receptors functions. Likewise, several molecules mimicking specific neurotrophin signalings have been reported to be a valuable tool to overcome limitations of native proteins as pharmacological agents (Longo et al., 1997, Maliartchouk et al., 2000, Massa et al., 2010, Peleshok and Saragovi, 2006, Yang et al., 2008, Zaccaro et al., 2005).
Our previous studies, based on crystallographic structures of the NGF/TrkA complex (McDonald and Chao, 1995, Wiesmann et al., 1999), as well as site-specific mutagenesis (Kullander et al., 1997, Rydén and Ibànez, 1997, Shih et al., 1994) and molecular modeling studies, led to development of several new NGF-like molecules. Among them, the NGF-mimetic peptide, now named BB14, displayed TrkA agonist activity and, like the native NGF protein, was found to be effective in reducing biochemical and structural modifications linked to reactive astrogliosis in the chronic constriction injury (CCI) model of peripheral neuropathy (Colangelo et al., 2008).
The astrocytic network actively influences neuronal and synaptic properties and plasticity by providing metabolic support to neurons and participating to reuptake and release of aminoacid transmitters (Allen and Barres, 2009, Giaume et al., 2010, Hamilton and Attwell, 2010, Henneberger et al., 2010, Ni and Parpura, 2009, Rouach et al., 2008). Phenotypic changes of reactive astrocytes (Pekny and Nilsson, 2005) impair neuron-astrocyte cross-talk and synaptic efficacy due to changes of glutamate clearance and extracellular levels of gliotransmitters, which modulate N-methyl-D-aspartate (NMDA) receptor-mediated synaptic transmission (Panatier et al. 2006). These alterations are believed to contribute to progression of neurodegenerative diseases (Cirillo et al., 2010a, Giovannoni et al., 2007, Lobsiger and Cleveland, 2007).
We previously reported that glial changes following peripheral nerve injury were associated with increased sprouting of primary afferent nociceptive fibers (C and A-δ fibers) entering the spinal cord (Colangelo et al., 2008) and down-regulation of glial aminoacid transporters (Cavaliere et al., 2007, Cirillo et al., 2011), suggesting a strict correlation between neuro-glial plasticity changes and peripheral sensitization (Giaume et al., 2010, Scholz and Woolf, 2007, Todd, 2010).
Superficial laminae of the dorsal horns of spinal cord represent a nodal point for modulation and integration of peripheral sensory stimuli through complex networks involving glutamate receptors and local inhibitory GABAergic interneurons (Meisner et al., 2010). In the spared nerve injury (SNI) model of peripheral nerve lesion, we recently found, in agreement with previous reports (Polgár and Todd, 2008), that nerve injury was able to perturb spinal synaptic circuitry by producing profound morpho-functional changes affecting neuro-glial interaction and the glutamatergic/GABAergic system (Cirillo et al., 2011).
Based on our previous findings regarding the ability of intrathecal (i.t.) administration of NGF or the NGF-like peptide BB14 to restrain reactive gliosis (Cirillo et al., 2010b, Cirillo et al., 2011, Colangelo et al., 2008), we evaluated the effect of this molecule in restoring spinal synaptic homeostasis by modulating the expression of glial (GLT1 and GlyT1) and neuronal excitatory aminoacid transporters (EAAC1). To further characterize the role of BB14 in reverting the effects of decreased NGF availability, we also compared the efficacy of BB14 with the effect of increasing endogenous NGF by inhibiting its proteolytic degradation by the MMPs system (Bruno and Cuello, 2006). We show that both i.t. BB14 supply and GM6001-mediated inhibition of MMPs restored neuronal circuitry and metabolic homeostasis, thus providing a strong evidence of the efficacy of this NGF-mimetic molecule in targeting pathological conditions linked to alteration of NGF availability/signaling and mechanisms of reactive astrogliosis.
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Adult (250–300 g; Charles River, Calco, Italy) male Sprague Dawley rats (n=40) were used. Rats were maintained on a 12/12 h light/dark cycle and allowed free access to food and water. Each animal was housed under specific pathogen-free conditions in iron-sheet cages with solid floors covered with 4–6 cm of sawdust. Cages with thin-plate floors were avoided on the assumption that they would exacerbate the discomfort arising from the affected hind paw (Bai et al., 1999). All surgerier and
