Cells were permeabilized with 0.1% (v/v) Triton X-100/PBS pH 7.4 for 4 min at room temperature. in selected presynaptic and vesicles trafficking proteins including synapsin I, SNAP-25 and -synuclein. This rapid presynaptic dysfunction: (i) precedes the commitment to cell death and is reversible in a time-dependent manner, being suppressed by external 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 ABX-464 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 neuroprotective intervention in affected patients. cholinergic septal neurons favoring its subcellular localization in Golgi compartmentvia downregulation in phosphorylation at the threonine 668 (T668)which, in turn, reduces susceptibility to BACE cleavage and promotes the anti-amyloidogenic processing (Triaca et al., 2016). NGF supply via nasal route has been proved to modulate the secretase levels and, in turn, reduce the amyloid burden in APP/PS1 transgenic mice (Yang et al., 2014) and transgenic mice lacking the APP-TrkA interaction display forebrain damage and cognitive deficits (Matrone et al., 2012). Cholinergic neurons located in the nucleus basalis of Meynert of affected subjects exhibit a great sensibility to undergo neurofibrillary degeneration at early stages of AD neuropathology (Sassin et al., 2000; Mesulam et al., 2004), suggesting that NGF is also able to influence the tau metabolism, in addition to its effects on basal forebrain cholinergic function(s) and on APP processing (Schliebs and Arendt, 2006). Finally, phenotypic knockout of NGF via its antibody-mediated neutralization in adult transgenic AD11 mice causes age-dependent neurodegenerative changes which are reminiscent of human AD pathology characterized by severe deficits in basal forebrain cholinergic neurons, classic histopathological hallmarks including amyloid plaques and tau neurofibrillary tangles in cortical and hippocampal neurons, behavioral deficits (Capsoni et al., 2000). Therefore, in view of the physiopathological relevance of NGF/TrkA ABX-464 signaling dysfunction in triggering the initial AD-type lesions of vulnerable cholinergic basal forebrain population which critically contribute to memory/learning impairment of hippocampal and cortical areas (Mesulam, 2004), the understanding of the earliest molecular events following neurotrophin starvation in cholinergic septo-hippocampal system will support the development of novel disease modifying drugs aimed to slow down the conversion from asymptomatic Mild Cognitive Impairment (MCI) MCI to clinical full-blown dementia (Mufson et al., 2012). Although a crucial involvement of early alterations in the NGF/TrkA system in driving neurodegeneration of basalforebrain at the onset of AD progression has been largely accepted, studies carried out on primary septohippocampal cultures have turned out to be technically challenging mainly due to the scarse yield of the cholinergic and TrkA-positive neuronal population transplanted with consequent difficulties in assessing the specificity, the precise timing and, if possible, the reversibility of any biochemical events triggered by NGF withdrawal. By biochemical, morphological and electrophysiological approaches, here we show that the selective reduction of B27(0.2%), the most widely-used serum-free supplement in culture media, combined with Kcnh6 chronic somministration of NGF (100 ng/ml), added immediately after plating and for 10C12 days (D.I.V.), significantly increases the number of cholinergic neurons in septal primary cultures (+36.36%) at the expense of non-cholinergic, mainly glutamatergic (?56.25%) and GABAergic (?38.45%), populations. The frequency of spontaneous excitatory miniture post-synaptic currents (mEPSCs) is significantly stimulated upon exposure to ectopic NGF in septohippocampal cultures under conditions of low B27(0.2%) media, confirming that a large amount of cholinergic and NGF-responsive neurons are actually enriched following this experimental procedure. Importantly, by taking advantage of this newly-developed neuronal paradigm, we uncover that the withdrawal of NGF induces a progressive ABX-464 deficit in the presynaptic excitatory neurotransmission which occurs in concomitance with a pronounced and time-dependent reduction in several distinct pre-synaptic markers, such as synapsin I, SNAP-25 and -synuclein, and in absence of any sign of neuronal death. This rapid presynaptic dysfunction: (i) is reversible in a time-dependent manner, being suppressed by external administration of NGF within 6 h from its initial withdrawal; (ii) is specific.