Abstract
Basal forebrain cholinergic neurons (BFCNs) depend on nerve growth factor (NGF) for their survival/differentiation and innervate cortical and hippocampal regions involved in memory/learning processes. Cholinergic hypofunction and/or degeneration early occurs at prodromal stages of Alzheimer’s disease (AD) neuropathology in correlation with synaptic damages, cognitive decline and behavioral disability. Alteration(s) in ubiquitin-proteasome system (UPS) is also a pivotal AD hallmark but whether it plays a causative, or only a secondary role, in early synaptic failure associated with disease onset remains unclear. We previously reported that impairment of NGF/TrkA signaling pathway in cholinergic-enriched septo-hippocampal primary neurons triggers “dying-back” degenerative processes which occur prior to cell death in concomitance with loss of specific vesicle trafficking proteins, including synapsin I, SNAP-25 and α-synuclein, and with deficit in presynaptic excitatory neurotransmission. Here, we show that in this in vitro neuronal model: (i) UPS stimulation early occurs following neurotrophin starvation (-1 h up to -6 h); (ii) NGF controls the steady-state levels of these three presynaptic proteins by acting on coordinate mechanism(s) of dynamic ubiquitin-C-terminal hydrolase 1 (UCHL-1)-dependent (mono)ubiquitin turnover and UPS-mediated protein degradation. Importantly, changes in miniature excitatory post-synaptic currents (mEPSCs) frequency detected in -6 h NGF-deprived primary neurons are strongly reverted by acute inhibition of UPS and UCHL-1, indicating that NGF tightly controls in vitro the presynaptic efficacy via ubiquitination-mediated pathway(s). Finally, changes in synaptic ubiquitin and selective reduction of presynaptic markers are also found in vivo in cholinergic nerve terminals from hippocampi of transgenic Tg2576 AD mice, even from presymptomatic stages of neuropathology (1-month-old). By demonstrating a crucial role of UPS in the dysregulation of NGF/TrkA signaling on properties of cholinergic synapses, these findings from two well-established cellular and animal AD models provide novel therapeutic targets to contrast early cognitive and synaptic dysfunction associated to selective degeneration of BFCNs occurring in incipient early/middle-stage of disease.
Highlights
We have previously shown that nerve growth factor (NGF) withdrawal induces in vitro an early, selective and reversible structural and functional deterioration of cholinergic presynaptic terminals, just resembling the “dyingback”-like mechanism(s) of cell degeneration occurring in vivo into basal forebrain circuit at the onset of Alzheimer’s disease (AD) neuropathology
Following short-term removal of neurotrophin, we detected a progressive decrease in immunoreactivity of polyubiquitin chains-conjugates (Figures 1A,B) whose degradation -especially of those formed with ubiquitin lysine 48 (K48) linkage- generally relies on the 26S proteasome (Thrower et al, 2000; Grice and Nathan, 2016) and is canonically used as an indirect cellular indicator of activation in proteasomal function(s) (Myeku et al, 2011)
We found out that significant loss of ubiquitin conjugated was inversely correlated with a parallel accumulation of free ubiquitin up to −6 h of neurotrophin withdrawal which is reminiscent of increased proteasomal degradative activity (Emmerich and Cohen, 2015) (Figures 1A,B and Supplementary Figure S1A)
Summary
Basal forebrain cholinergic neurons -whose axons project to cortical mantle and hippocampus where they form the largest part of cholinergic synapses (McKinney et al, 1983; Sofroniew et al, 1990) involved in the regulation of synaptic activity and modulation of memory and attention (Conner et al, 2003, 2005; Hasselmo and Giocomo, 2006)- critically depend on retrograde transport of target-derived NGF for their survival, neurite outgrowth, phenotypic expression and maintenance (Hefti and Weiner, 1986; Hartikka and Hefti, 1988b; Debeir et al, 1999; Cuello et al, 2007). NGF replacement therapy turned out to be an effective disease-modifying treatment to improve the cholinergic deficits in humans affected from mild AD and, to prevent and/or delay the cognitive deterioration of symptomology (Tuszynski et al, 2005; Mufson et al, 2008) In this context, constant adaptations in the delivery mode to CNS and in the drug-design pharmacological approach with the development of TrkA mimetics have recently allowed to achieve significant results in increasing the NGF bioavailability to target neurons and/or in reducing its potential indirect and unwanted side-effects (Mufson et al, 2008) with consequent long-term improvement of the cholinotrophic basal forebrain function affected in AD patients (Mufson et al, 2008). Advancements in NGF basic research are highly relevant for potential therapeutic implications in the field of AD neurodegeneration mainly for the most
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