In the present study, the expression is knocked down of two downstream signaling components of the PERK pathway, the cytoprotective calcineurin and the pro-apoptotic CHOP, by using specific shRNAs. In opposite ways, these modulate the susceptibility of primary cortical neurons to neurite atrophy after induction of endoplasmic reticulum stress.
The accumulation of unfolded proteins within the endoplasmic reticulum (ER), caused by any stress condition, triggers the unfolded protein response (UPR) through the activation of specialized sensors. UPR attempts first to restore homeostasis; but if damage persists the signaling induces apoptosis.
There is increasing evidence that sustained and unresolved ER stress contributes to many pathological conditions including neurodegenerative diseases. Because the UPR controls cell fate by switching between cytoprotective and apoptotic processes, it is essential to understand the events defining this transition, as well as the elements involved in its modulation.
Recently, we demonstrated that abnormal GM2 ganglioside accumulation causes depletion of ER Ca2+ content, which in turn activates PERK (PKR-like-ER kinase), one of the UPR sensors. Furthermore, PERK signaling participates in the neurite atrophy and apoptosis induced by GM2 accumulation. In this respect, we have established an experimental system that allows us to molecularly modulate the expression of downstream PERK components and thus change vulnerability of neurons to undergo neuritic atrophy.
We performed knockdown of calcineurin (cytoprotective) and CHOP (pro-apoptotic) expression in rat cortical neuronal cultures. Cells were infected with lentivirus-delivered specific shRNA and then treated with GM2 at different times, fixed and immunostained with anti-MAP2 (microtube-associated protein 2) antibody. Later, cell images were recorded using a fluorescence microscope and total neurite outgrowth was evaluated by using the public domain image processing software ImageJ. The inhibition of expression of those PERK signaling components clearly made it possible to either accelerate or delay the neuritic atrophy induced by ER stress.
This approach might be used in cell system models of ER stress to evaluate the vulnerability of neurons to neurite atrophy.
Endoplasmic reticulum (ER) stress is defined as any perturbation that compromises protein-folding capacity in the organelle. The accumulation of unfolded proteins within the ER lumen activates a transduction cascade signal called the unfolded protein response (UPR). This complex signaling pathway is orchestrated by three stress sensors: PERK (protein kinase RNA [PKR]-like ER kinase), IRE1 (inositol-requiring enzyme 1) and ATF6 (activated transcription factor 6). All together attempt to restore homeostasis. But if stress persists, UPR eventually induces cell death by apoptosis1.
PERK, an ER transmembrane protein, upon ER stress, leads the phosphorylation of eukaryotic initiation factor-2 alpha (eIF2α), reducing global protein synthesis and thus protein load in the ER2. We demonstrated that calcineurin A/B (CNA/B), a heterodimer Ca2+ phosphatase, directly binds the cytosolic domain of PERK, increasing its auto-phosphorylation and significantly enhancing inhibition of protein translation and cell viability3,4. Interestingly, CNA/B is abundant in the mammalian brain, distinguishing two isoforms of the subunit A of CN: α and β.
Under sustained ER stress, the PERK signaling pathway is the only UPR branch that remains activated, thus mediating both the pro-survival and the apoptotic response. In the chronic phase, one major downstream event is the induction of the transcription factor, CHOP (CCAAT/enhancer binding protein homologous protein)5. Chronic ER stress is also increasingly recognized as a common contributor to an extensive range of pathological disorders, including neurodegenerative diseases6. It is important to understand how UPR can facilitate cytoprotective signaling instead of cell death 7. However, at present little is known about the exact mechanism controlling the transition between these two UPR phases.
Recently, we found that, in cultured neurons, ganglioside GM2 accumulates in ER membranes and induces luminal calcium depletion. This in turn activates PERK signaling, which mediates neurite atrophy and apoptosis 8. In this study, the GM2 build-up in cultured neurons is used as a cell system model of ER stress-induced neurite atrophy. Specifically, two PERK factor expressions are manipulated, CN-Aα and CHOP, which switches the transition between early/protective events and a chronic/apoptotic phase. To accomplish this, the respective genes are silenced; thus, primary cortical neuron cultures are infected with lentivirus-delivered specific shRNA. Western blot analysis reveals a significant reduction of CN-Aα and CHOP expression levels in comparison with the control cells, which are infected with lentivirus that carry a scrambled shRNA. After this treatment, neurons are subjected to different incubation times of exogenous GM2, fixed, and immunostained with anti-microtubule associated protein 2 (MAP2) antibody9. Images are obtained with an epifluorescence microscope. The total neurite outgrowth is evaluated relative to total cell number.
The animal procedures are performed following approved protocols of the National Institute of Health Guide for the Care and Use of Laboratory Animals. Approval to conduct the study is granted by the Animal Care and Ethics Committee (CICUAL) of INIMEC-CONICET-UNC (Resolution numbers 014/2017 B and 006/2017 A).
1. Primary rat cortical neuron cultures
2. Lentivirus production
NOTE: The oligonucleotides containing the sequences targeting the 3'UTRs of either the CN-Aα isoform or CHOP and with the non-targeting sequences (scrambles) are listed in Table 1.
3. Lentivirus infection
NOTE: Perform the lentivirus generation procedure in a Class II laminar flow hood.
4. Primary neuron cultures stressed with ganglioside GM2 accumulation and immunocytochemistry using anti-MAP2 antibody
5. Neurite atrophy analysis
Here, we address the question of whether silencing two PERK downstream components affects the transition phase of UPR in an ER stress cell model.To achieve this, we silence the CN-Aα gene as well as the CHOP gene by two specific shRNA sequences for each (Table 1) in primary neuron cell culture for 1 day10. The expression is analyzed by Western blotting (Figure 1 and Figure 2). A clear inhibition is observed of ER stress-mediated CN-Aα and CHOP increase in knockdown cells, but not in control cells (shRNA scrambles). It should be noted that the basal CN expression level is not affected with this treatment condition.
We also examine the possible link of GM2 accumulation-induced ER stress with neuronal degeneration by performing MAP2 immunostaining of primary neuronal cultures (Figure 3 and Figure 4).
Neurite atrophy is analyzed as total neurite outgrowth relative to the total cell number. This increases significantly after inducing ER stress by incubation of GM2 at 16-48 h. Interestingly, silencing of CN-Aα expression significantly enhances neurite atrophy, particularly at 16 h of GM2 accumulation, relative to GM2-untreated groups (Figure 3). Thus, CN-Aα knockdown accelerated the degeneration processes in neurons, corroborating the pro-survival effect of CN during the early phase of UPR. Conversely, CHOP knockdown resulted in significantly diminished neurite atrophy relative to controls, specifically at 16-24 h (Figure 4).
Figure 1. Data analysis for CN-Aα silencing efficiency. Knockdown is evaluated by Western blot analysis using antibody against CN-Aα and GADPH (loading control). The primary antibody is visualized by near-infrared fluorescence imaging system. Numbers below represent relative intensity ratio between CN-Aα and GADPH bands. This figure has been republished from Virgolini, M.J. et al.4. Please click here to view a larger version of this figure.
Figure 2. Data analysis for CHOP silencing efficiency. Knockdown is evaluated by Western blot analysis using an antibody against CHOP and β actin (loading control). The primary antibody is visualized and then the data are analyzed as indicated in Figure 1. This figure has been republished from Virgolini, M.J. et al.4. Please click here to view a larger version of this figure.
Figure 3. Data analysis for neuritic atrophy imaging in CN-Aα knockdown cells. (Top) Cultured primary neurons are loaded with GM2. Representative images of MAP2 immunostained cells are shown under control and experimental conditions. Images are recorded with an epifluorescence-inverted microscope equipped with a CCD-camera. Scale bars: 150 µm (regular images), 75 µm (magnified images). (Bottom) Histogram (mean ± SEM) represents neurite outgrowth with respect to total cells, analyzed by ImagJ plug-ins. *** and ###: p 0.0001 for comparison with the control, from one-way ANOVA. This figure has been republished from Virgolini, M.J. et al.4.
Figure 4. (Top) Data analysis for neuritic atrophy imaging in CHOP silencing cells. Primary neuron cultures are treated as indicated in Figure 3(top). (Bottom) Histograms and statistical notations as in Figure 3(Bottom) except ##: p≤ 0.001. This figure has been republished from Virgolini, M.J. et al.4. Please click here to view a larger version of this figure.
Constructs | Sequence 5’ to 3’ shRNA |
CN-Aα 1 | AATTGCCAGGAATTGGATTCAGTTTCTCGA GAAACTGAATCCAATTCCTGGCTTTTTTTAT |
CN-Aα 2 | AATTCGCCAACCTTAACTCCATCAACTCGAG TTGATGGAGTTAAGGTTGGCGTTTTTTTAT |
CN-Aα Scrb | AATTGAGTGAATTGTCGCTCTAAGTCTCGAG ACTTAGAGCGACAATTCACTCTTTTTTTAT |
CHOP 1 | AATTGGTCCTGTCCTCAGATGAAATCTCGAG ATTTCATCTGAGGACAGGACCTTTTTTTAT |
CHOP 2 | AATTTGAAGAGAACGAGCGGCTCAACTCGA GTTGAGCCGCTCGTTCTCTTCATTTTTTTAT |
CHOP Scrb | AATTGAAGAGAGAAAGCGAACAATACTCGAG TATTGTTCGCTTTCTCTCTTCTTTTTTTAT |
Table 1. Forward construct sequences inserted to pKLG0.3 viral vector. Scrb: scramble.
We describe an experimental system that enables molecular modulation of the transition from survival to apoptotic UPR phases in a neuronal cell model.
For a proper analysis of neurite atrophy, it is essential to obtain primary neuron cultures with numerous, long, highly branched processes9,11. This facilitates the examination of neuron process extension, allowing the clear differences between treatments to be detected. It is important to note that, if primary cortical neuron cultures at 7-8 days in vitro already show high neurite outgrowth, these may be used to induce stress and perform the subsequent analysis, instead of 15 days in vitro cultures.
Moreover, the inhibition percentage of ER stress-mediated CHOP overexpression should be about 65-70% or higher. Importantly, silencing of the CN-Aα expression level should affect only the ER stress-induced CN-Aα expression increase without modifying its basal level. This is to minimize the impact on CN functions not associated with PERK signaling12,13.
With respect to the neurite atrophy analysis procedure, this paper presents an alternative methodology that requires less manual work than specific ImageJ plug-ins that help to trace neurites. Commonly, these require that processes be individually traced for each neuron, while the steps followed in this paper trace the processes of multiple neurons at once using 5 sets of common accessible ImageJ commands. Both plug ins and this protocol enable the value of all the processes in the sample image to be quantified.
We previously demonstrated a novel cytoprotective role for the ubiquitous CN in the early phase of UPR, triggered either by pharmacological tools or ischemic processes3,4,8. Others and we described the impact of the inhibition of CHOP expression on cell survival in different systems8,11,14. Here we propose a methodology in which manipulating the expression of any of these genes enables to opposite modulate the beginning of the neuritic atrophy process in a cellular model of ER stress to be modulated in the opposite mode.
We envision that this methodology can be used to evaluate the susceptibility of neurons to undergo neuritic atrophy in different cell system models of ER stress and thus, contribute to understanding the molecular bases of the transition between acute and chronic phases of UPR in diverse neurodegenerative disease models.
The authors have nothing to disclose.
We thank Dr. Gonzalo Quasollo for his invaluable help with imaging and Dr. Andrea Pellegrini for cell culture technical support.
This research was supported by grants from: the National Institute of Health, USA (#RO1AG058778-01A1, Subaward Agreement No 165148/165147 between UTHSCSA-Instituto Investigación Médica M y M Ferreyra) and from the National Agency of Agencia Nacional de Scientific and Technological Promotion, Argentina (ANPCyT, PICT 2017 #0618).
Alexa Fluor 488 anti-Mouse | Thermo Fisher Scientific | #R37120 | |
anti-CHOP | Thermo Fisher | # MA1 – 250 | |
Anti-CN-Aα | Millipore | # 07-067 | |
Anti-GM2 | Matreya | #1961 | |
anti-MAP2 | Sigma Aldrich | # M2320 | |
anti-β-actin | Thermo Fisher | # PA1 – 183 | |
aprotinin | Santa Cruz Biotechnology | #3595 | |
Axiovert 200 epifluorescence microscope | Zeiss | ||
B27 supplement | Life Technologies | #17504944 | |
Dulbecco's Modified Eagle's Medium (DMEM) | Life Technologies | #11966025 | |
EcoRI | Promega | #R6011 | |
Fetal Calf Serum (FCS) | Life Technologies | #16000044 | |
Fine-tippeds forceps style #5 | Dumont | ||
Forcep style #3 | Dumont | ||
HEK 293 | ATCC | #CRL-1573 | |
IRDye 680 CW secondary antibody | LI-COR Biosciences | #92632221 | |
IRDye 680 secondary antibody | LI-COR Biosciences | #92632220 | |
IRDye 800 CW secondary antibody | LI-COR Biosciences | #92632210 | |
IRDye 800 CW secondary antibody | LI-COR Biosciences | #92632211 | |
lentiviral envelope plasmid pMD2.G | Addgene | #12259 | |
lentiviral packing plasmid psPAX2 | Addgene | #12260 | |
lentiviral vector pLKO.3G | Addgene | #14748 | |
Leupeptin hemisulfate | Santa Cruz Biotechnology | #295358 | |
Lipofectamine LTX & Plus Reagent (plasmid transfection reagent) | Life Technologies | #A12621 | |
MISSION shRNA | Sigma Aldrich | ||
Monosialoganglioside GM2 | Matreya | #1502 | |
NanoDrop 2000 | Thermo Scientific | ||
Neurobasal Medium | Life Technologies | #21103049 | |
Nitrocellulose membrane 0.45 µm | BIO-RAD | #1620115 | |
Odyssey infrared imaging system | LI-COR Bioscience | ||
OneShot Top 10 | Life Technology | #C404010 | |
Opti-MEM (Reduced serum media) | Life Technologies | #105802 | |
PacI | BioLabs | #R0547S | |
penicillin-streptomycin | Life Technologies | #15140122 | |
Pepstatin A | Santa Cruz Biotechnology | #45036 | |
phenylmethylsulfonyl fluoride | Santa Cruz Biotechnology | #329-98-6 | |
Poly-L-lysine | sigma aldrich | P#2636 | |
Straight sharp small spring scissors | Fine Science Tools | ||
T4 DNA Ligase | Promega | #M1801 | |
Trypsin-EDTA 0.25 % | Life Technologies | #25200056 | |
Vibra-Cell Ultrasonic Liquid Processor (VCX 130) | Sonics | ||
Wizard plus SV Minipreps DNA purification system | Promega | #A1330 |