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

RACK1_Y246F induces accumulation of β-actin mRNA in growth cones of cortical cells and reduces its translation.

<p><b>A</b>, Growth cones of cortical cells co-transfected with Flag-ZBP1_wt and GFP-RACK1_Y246F show increased levels of β-actin mRNA as measured by Q-FISH. The method to quantify the signal is reported in supplemental information and the values are summarized in the histogram in the left. <b>B</b>, The translation of β-actin protein in growth cones of cortical neurons co-transfected with Flag-ZBP1_wt and GFP-RACK1_Y246F was reduced, as indicated by in Q-IF. All data are reported as mean ± s.e.m. Significance was set as p≤0.05. When significance was adjusted, it is referred to as alpha. β-tubulin immunofluorescence is shown as a marker for growth cone morphology. Scale bar 10 µm.</p

RACK1_Y246F impairs the binding of ZBP1 and Src kinase to ribosomes and reduces the release and translation of β-actin mRNA.

<p><b>A</b>, ZBP1 and Src are less abundant in ribosomal profile fractions collected from stable GFP-RACK1_Y246F overexpressing cells, transfected with Flag-ZBP1 cDNA. The histograms in <b>B</b> and in <b>C</b> summarize the ratio of density values between Flag-ZBP1 <b>B</b> or Src <b>C</b> and GFP-RACK1_wt or GFP-RACK1_Y246F immunoblots in 2–4 ribosomal fractions. <b>D</b>, The level of β-actin mRNA, measured by qRT-PCR, on RNA isolated from non translating fractions (fractions 2–3 of ribosomal profile reported in <b>A</b>) and from polysomal fractions (fractions 5–8) was decreased in GFP-RACK1_Y246F/Flag-ZBP1 cells, as compared to that of GFP-RACK1_wt/Flag-ZBP1 expressing cells. <b>E</b>, GFP-RACK1_Y246F induced an increase of β-actin mRNA associated to Flag-ZBP1, as measured by qRT-PCR on RNA isolated from Flag-ZBP1 immunoprecipitation of GFP-RACK1_Y246F/Flag-ZBP1 cells. In <b>D</b> and <b>E</b>, the values were normalized to those of 18S rRNA and the data are graphed as mean ± S.D.* = p<0,05, # and § = p<0,01 second <i>t-test</i> student. In A and D the transfection efficiency was normalized with respect to the amount of Actin protein (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035034#pone.0035034.s003" target="_blank">Figure S3</a>). In E, the transfection efficiency was normalized with respect to immunoprecipitated Flag-ZBP1 proteins as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035034#pone-0035034-g002" target="_blank">Figure 2B</a>.</p

RACK1 localizes on RNA transport granules in cortical neurons.

<p><b>A</b>, RACK1 appears in granular forms in rat cortical neurons immunostained with anti-RACK1(<i>green</i>) and DAPI (<i>blue</i>). Scale bar 20 µm. <i>Enlarged view</i> indicates neurite of cortical cells. <b>B</b>, RACK1 also appears in granular forms <i>in vivo</i>. Hippocampal tissue of adult mouse immunostained with RACK1 (<i>green</i>). <i>Enlarged view</i> shows the soma of neurons. <i>Arrows</i> indicate the granules stained by RACK1 antibody. Scale bar 20 µm. <b>C</b>, RACK1 colocalizes with endogenous ZBP1 transport RNPs. Cortical neurons were immunostained with RACK1 (<i>red</i>) and ZBP1 (<i>green</i>) antibodies. Scale bar 40 µm. <i>Arrows</i> in enlarged images indicate granules where RACK1 co-localizes with endogenous ZBP1 along neurites. <b>D</b>, RACK1 and GFP-ZBP1 co-localize on transport RNPs of GFP-ZBP1 transfected neurons. Cortical cells were transfected with GFP-ZBP1 cDNA and after 24 hours fixed and processed for anti-RACK1. The <i>arrows</i> in e<i>nlarged view (</i><b><i>a</i></b><i>)</i> indicate the granules where GFP-ZBP1 and RACK1 colocalize. Scale bar 20 µm.</p

The dendritic branching out induced by GFP-RACK1wt.

<p><b>A</b>, Immunofluorescence for GFP in cortical neurons transfected with GFP, or GFP-RACK1_wt or GFP-RACK1_Y246F. GFP-RACK1_Y246F reduced the dendritic arbors indeced by GFP-RACK1_wt overxpression Scale bar 20 µm <b>B</b>, Graphic reporting the values of dendritic branching seen in <b>A</b>. The values were measured as means of the number of neurite intersections measured by Sholl analysis. Data are graphed as mean ± S.D.</p

Molecular mechanism of RACK1/ZBP1 complex regulating the release and translation of β-actin mRNA.

<p>A, Schematic figure showing the interaction of RACK1 with the β-actin mRNA/ZBP1 complex through its Src binding site (Y246) on 40S ribosome subunit of RNPs (<i>left</i>). In presence of the mutation Y246F (<i>right</i>), RACK1 on ribosomes fails to recruit the β-actin mRNA/ZBP1 complex and Src. This impairment blocks the release of β-actin mRNA from ZBP1 and its translation. B, Src can be activated by BDNF or by PACAP treatments. In BDNF stimulation, Src, activated through TrkB, may directly bind, and phosphorylate, both free or ribosomal bound RACK1 and free β-actin mRNA/ZBP1 complex. Next, RACK1 and β-Actin mRNA/ZBP1 associate on ribosomes and the β-actin mRNA is released to be translated. Instead, PACAP-activated Src stimulates PKA kinase which in turn may induce the dissociation of the β-actin mRNA/ZBP1-RACK1 complex by phosphorylating ZBP1.</p

Src kinase regulates the RACK1/ZBP1 complex and the Y246 of RACK1 is critical for the binding with ZBP1.

<p><b>A</b>, Endogenous RACK1 interacts with Flag-ZBP1 in an anti-Flag immunoprecipitation assay from total lysate of neuroblastoma Flag-ZBP1-transfected cells. Western blotting for endogenous RACK1 and Flag-ZBP1 on proteins eluted from anti-Flag immunoprecipitation assay. Flag transfected cells were used as negative control. Input represents 5% of total lysate. <b>B</b> and <b>C</b>, Src activity regulates the RACK1-ZBP1 complex formation. <b>B</b>, db-cAMP or PACAP treatments of Flag-ZBP1 transfected cells reduced the binding between RACK1 and Flag-ZBP1, whereas PP2 restored the binding, as in untreated cells. <b>C</b> BDNF treatments increased the binding of Flag-ZBP1 to RACK1 in Flag-ZBP1 transfected cells. Src inhibition by Src inhibitor PP2 reduced the RACK1-ZBP1 complex stimulated by BDNF. The density value of immnoprecipitated RACK1 is normalized to that of immunoprecipitated Flag-ZBP1 and summarized in both graphics. Data are graphed as means plus S.D. <b>D</b>, The Src binding and phosphorylation site (Y246) of RACK1 is critical for complex formation. Flag-ZBP1 protein co-immunoprecipitated with GFP-RACK1_wt and GFP-RACK1_Y246F in immunopreciptation assays using anti-GFP antibody, but in the presence of GFP-RACK1_Y246F the binding was reduced. The figures are representative of three independent experiments. The density value of co-immnoprecipitated Flag-ZBP1 is normalized to that of immnoprecipitated GFP-RACK1_wt or GFP-RACK1_Y246F and summarized in both graphic. Data are graphed as means plus S.D.</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