title: Impaired NGF/TrkA Signaling Causes Early AD-Linked Presynaptic Dysfunction in Cholinergic Primary Neurons.
abstract: Alterations in NGF/TrkA signaling have been suggested to underlie the selective degeneration of the cholinergic basal forebrain neurons occurringin vivoin AD (Counts and Mufson, 2005; Mufson et al., 2008; Niewiadomska et al., 2011) and significant reduction of cognitive decline along with an improvement of cholinergic hypofunction have been found in phase I clinical trial in humans affected from mild AD following therapeutic NGF gene therapy (Tuszynski et al., 2005, 2015). Here, we show that the chronic (10-12 D.I.V.)in vitrotreatment with NGF (100 ng/ml) under conditions of low supplementation (0.2%) with the culturing serum-substitute B27 selectively enriches the basal forebrain cholinergic neurons (+36.36%) at the expense of other non-cholinergic, mainly GABAergic (-38.45%) and glutamatergic (-56.25%), populations. By taking advantage of this newly-developed septo-hippocampal neuronal cultures, our biochemical and electrophysiological investigations demonstrate that the early failure in excitatory neurotransmission following NGF withdrawal is paralleled by concomitant and progressive loss in selected presynaptic and vesicles trafficking proteins including synapsin I, SNAP-25 and α-synuclein. This rapid presynaptic dysfunction: (i) precedes the commitment to cell death and is reversible in a time-dependent manner, being suppressed byde novoexternal administration of NGF within 6 hr from its initial withdrawal; (ii) is specific because it is not accompanied by contextual changes in expression levels of non-synaptic proteins from other subcellular compartments; (ii) is not secondary to axonal degeneration because it is insensible to pharmacological treatment with known microtubule-stabilizing drug such paclitaxel; (iv) involves TrkA-dependent mechanisms because the effects of NGF reapplication are blocked by acute exposure to specific and cell-permeable inhibitor of its high-affinity receptor. Taken together, this study may have important clinical implications in the field of AD neurodegeneration because it: (i) provides new insights on the earliest molecular mechanisms underlying the loss of synaptic/trafficking proteins and, then, of synapes integrity which occurs in vulnerable basal forebrain population at preclinical stages of neuropathology; (ii) offers prime presynaptic-based molecular target to extend the therapeutic time-window of NGF action in the strategy of improving its neuroprotectivein vivointervention in affected patients.
authors.affiliation: Institute of Translational Pharmacology, National Research Council (CNR) Rome, Italy.
authors.forename: Valentina
authors.initials: V
authors.lastname: Latina
authors.affiliation: IRCCS Santa Lucia Foundation Rome, Italy.
authors.forename: Silvia
authors.initials: S
authors.lastname: Caioli
authors.affiliation: IRCCS Santa Lucia FoundationRome, Italy; Department of Systems Medicine, University of Rome Tor VergataRome, Italy.
authors.forename: Cristina
authors.initials: C
authors.lastname: Zona
authors.affiliation: NGF and Molecular Mechanisms of Neurodegenerative Diseases, European Brain Research Institute (EBRI) Rome, Italy.
authors.forename: Maria T
authors.initials: MT
authors.lastname: Ciotti
authors.affiliation: Institute of Translational Pharmacology, National Research Council (CNR)Rome, Italy; NGF and Molecular Mechanisms of Neurodegenerative Diseases, European Brain Research Institute (EBRI)Rome, Italy.
authors.forename: Giuseppina
authors.initials: G
authors.lastname: Amadoro
authors.affiliation: NGF and Molecular Mechanisms of Neurodegenerative Diseases, European Brain Research Institute (EBRI) Rome, Italy.
authors.forename: Pietro
authors.initials: P
authors.lastname: Calissano
keywords: Alzheimer's Disease (AD); basal forebrain; cholinergic neurons; electrophysiological recordings; morphological and biochemical analyses; nerve growth factor (NGF); septo-hippocampal primary cultures; synaptic transmission