Image_2_NGF-Dependent Changes in Ubiquitin Homeostasis Trigger Early Cholinergic Degeneration in Cellular and Animal AD-Model.TIF

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.</p

Image_3_NGF-Dependent Changes in Ubiquitin Homeostasis Trigger Early Cholinergic Degeneration in Cellular and Animal AD-Model.TIF

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.</p

Western blot analysis of Kv 2.1 and Kv4.2 subunit expression in cerebral cortex and hippocampus.

<p>Representative immunoblot of cerebral cortex and hippocampus enriched membrane proteins (50 µg/lane) from (Ctr), Aβ_25–35, Aβ_25–35+SP and SP treated rats. Protein markers are shown at right (in kDa). The immunoreactive signals for <b>a</b>) Kv2.1 and <b>b</b>) Kv4.2 were quantified and normalized against β-actin and expressed as a percentage of control (CTR). Data represent mean (±SEM) from 3 independent experiments. Statistically significant differences were calculated by one-way analysis of variance (ANOVA) for repeated measures followed by Tukey's test for multiple comparisons (**p<0.01 versus Ctr value).</p

Immunofluorescence analysis of Kv1.4 subunit expression in hippocampus and cerebral cortex.

<p>Upper panel. Representative immunofluorescence photomicrographs showing Kv1.4 expression in <b>a</b>) hippocampus and <b>b</b>) frontal cortex after memory tests in the four experimental treatments: Control (Saline), Aβ_25–35-i.c.v. treated rats (Abeta), Aβ_25–35-i.c.v. and SP-i.p. treated rats (Abeta+SP), SP-i.p. treated rats (SP). Brain sections were labeled with the neuronal marker NeuN (green) and with the anti Kv1.4 antibody (red). As shown by the merge channel all neurons are Kv1.4 positive. Note the diffuse increase in Kv1.4 fluorescence intensity in the Abeta group and the decrease in the Abeta+SP group compared to the Control. Scale bar: a) 20 µm; b) 60 µm. Lower panel. Histograms showing image analysis performed on neuronal cytoplasm (first row) and the surrounding neuropil (second row). The indexes used were: total fluorescence intensity, vesicles diameters, and vesicles fluorescence intensity. Data represent means (±S.E.M.) obtained from three independent experiments. Statistically significant differences were calculated by one-way analysis of variance (ANOVA) for repeated measures followed by Tukey's test for multiple comparisons (**p<0.01 versus Saline; #p<0.05, ##p<0.01 versus Aβ_25–35treatment).</p

SP reduced Aβ25–35-induced overexpression of Kv1.4 subunit in rat hippocampal neurons.

<p><b>a</b>) Example of Western blot obtained from hippocampal cultures exposed to 20 µM Aβ_25–35 (Aβ alone or in the presence of SP (100 nM) and analyzed 48 h later using a polyclonal antibody against Kv1.4 subunit. The same blots were stripped and reprobed with an antibody against β-actin as internal control (lower panels). Quantitative analysis is depicted below the blots and was determined by band densitometry analysis considering the values found in CTR cells as 100. Data represent means (±S.E.M.) obtained from 4 independent experiments run in duplicate. (**p<0.001 versus CTR, #p<0.05 versus Aβ_25–35 treatment). <b>b</b>) Representative immunofluorescence photomicrographs showing Kv1.4 expression in primary hippocampal cultures. Note the increase in immunofluorescence in the Aβ_25–35 neurons, as compared to control neurons, reversed by SP treatment. Images were obtained from three independent experiments. Scale bar: 20 µm.</p

Western blot analysis of Kv1.4 subunit expression in hippocampus and cerebral cortex.

<p>Representative immunoblot of (<b>a</b>) hippocampus and (<b>b</b>) cerebral cortex enriched membrane proteins (50 µg/lane) from (Ctr), Aβ_25–35, Aβ_25–35+SP and SP treated rats. Protein markers are shown at right (in kDa). The immunoreactive signals at 97 and 110 kDa were quantified and normalized against β-actin and expressed as a percentage of the control (Ctr). Data represent mean (±SEM) from 5 independent experiments. Statistically significant differences were calculated by one-way analysis of variance (ANOVA) for repeated measures followed by Tukey's test for multiple comparisons (**p<0.01 versus Ctr value; #p<0.05 versus Aβ_25–35 treatment).</p

Neuroprotective effects of SP on memory impairments induced by intracerebroventricular injection of Aβ25–35.

<p>(a) Timeline and experimental design. All animals received an infusion (i.c.v.) of Aβ_25–35 (2 µg/µl; 10 µL injection volume) or its vehicle (PBS 10 µL injection volume) and daily treated (7 days) with SP (50 µg/ml/Kg, i.p.) or its vehicle (saline solution 0.9%, i.p.). On the 31^st day after surgery rats were given a daily training session of 4 trials for 3 consecutive days (days 31^st–33^rd). On the 34^th day after surgery the retention of the spatial training was assessed during a 1 min probe trial. On the 35^th day after surgery rats were given a daily training session of 5 trials for 4 consecutive days (days 35^th–38^th). (b) Mean (±S.E.M.) distance traveled to the escape platform on 4 trials of 3 consecutive days of acquisition learning sessions. (c) Time spent (mean ±S.E.M.) during the 1-minute probe trial in the target quadrant and (d) illustrative paths of all animals for the probe test session. (e) Mean (±S.E.M.) distance traveled to the escape platform on 4 trials of 4 consecutive days of the reversal learning sessions (the hidden platform were relocated in a new position each day). * p<0.05 Aβ_25–35/Sal <i>vs</i> PBS/Sal; # p<0.05 Aβ_25–35/Sal <i>vs</i> PBS/SP; $ p<0.05 Aβ_25–35/Sal <i>vs</i> Aβ_25–35/SP. PBS/Sal, n = 10; PBS/SP, n = 10; Aβ_25–35/Sal n = 12; Aβ_25–35/SP, n = 10.</p