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 occurring in vivo in 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 vitro treatment 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 by de novo 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 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 in vivo intervention in affected patients.

The Copper(II)-Assisted Connection between NGF and BDNF by Means of Nerve Growth Factor-Mimicking Short Peptides

“…By Western blotting analysis on whole-cell lysates ( Figure S3), we found out that the loss in protein amounts of synapsin I following 6h NGF starvation was significantly suppressed by the external application of increasing concentration of NGF(1-14) (5-10 µM) in a dose-dependent manner (** p < 0.01, *** p < 0.0001 versus −6h, respectively), mimicking the rescue action of the wild-type NGF (* p < 0.05 versus −6h). Similar results were also found for SNAP-25 (* p < 0.05, **p < 0.01 versus −6h, respectively) and α-synuclein (data not shown), the other two presynaptic proteins whose expression levels we previously reported to be strongly suppressed in −6h NGF-deprived cultures by de novo external re-application of NGF [51]. Taken togethr, these findings demonstrate that NGF (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14) is endowed with the same functional phenotype of its wild-type holoprotein and is able, in vitro, to control the cholinergic presynaptic stability of mature primary neurons, just as reported for full-length NGF [51].…”

“…Septal neurons were prepared from embryonic day 17/18 (E17/18), as previously described [51]. Chronic, 10-12 days in vitro treatment (D.I.V.…”

“…Chronic, 10-12 days in vitro treatment (D.I.V. ), with NGF (100 ng/mL) under conditions of low supplementation (0.2%) with the culturing serum-substitute B27 selectively enriched the basal forebrain NGF-dependent cholinergic neurons (+36.36%) at the expense of other non-cholinergic populations, mainly GABAergic (−38.45%) and glutamatergic (−56.25%) [51]. Briefly, embryos were surgically removed, and septo-hippocampal areas were dissected from the cerebral tissue in ice-cold Hanks' balanced salt solution (HBSS, Gibco, Life Technologies), freed of meninges, digested with 0.25% trypsin for 15 min at 37 • C, dissociated by trituration, and seeded as follows: 2 × 10 6 cells on poly-l-lysine (SIGMA)-coated plates (BD Falcon, Durham, NC, USA; 353001) for biochemistry analyses and 1 × 10 5 cells on glass coverslips in 24-well plates (BD Falcon; 351147) for immunofluorescence analyses.…”

“…In order to ascertain that NGF (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14) peptides were also biologically active in post-mitotic cellular environment, by inducing physiologically significant responses that resemble those evoked by its whole parental counterpart in terminally-differentiated primary neurons, we took advantage of a well-characterized NGF-dependent in vitro neuronal model such as cholinergic septo-hippocampal cultures [51].…”

Neuroprotective Effect of Nerve Growth Factor Loaded in Porous Silicon Nanostructures in an Alzheimer's Disease Model and Potential Delivery to the Brain

“…Aβ induces toxicity via p75 binding, thus, NGF introduction competes with the activation of the p75 receptor and has a highly neuroprotective influence, reducing cell degeneration and inducing regeneration . Moreover, impaired NGF signaling is linked to the loss of central cholinergic functions …”