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Published in final edited form as: Neuroscience. 2011 Sep 19;199:394–400. doi: 10.1016/j.neuroscience.2011.09.015

Organelles do not Colocalize with mRNA Granules in Post-Ischemic Neurons

Jill T Jamison 1, Jeffrey J Szymanski 1, Donald J DeGracia 1,2
PMCID: PMC3237913  NIHMSID: NIHMS327775  PMID: 21978884

Abstract

Following global brain ischemia and reperfusion, it is well-established that neurons undergo a translation arrest that is reversible in surviving neurons, but irreversible in vulnerable neurons. We previously showed a correlation between translation arrest in reperfused neurons and the presence of granular mRNA-containing structures we termed “mRNA granules.” Here we further characterized the mRNA granules in reperfused neurons by performing colocalization studies using fluorescent in situ hybridization for poly(A) mRNAs and immunofluorescence histochemistry for markers of organelles and mRNA binding proteins. There was no colocalization between the mRNA granules and markers of endoplasmic reticulum, cis- or trans-Golgi apparatus, mitochondria, microtubules, intermediate filaments, 60S ribosomal subunits, or the HuR ligands APRIL and pp32. The mRNA granules colocalized with the neuronal marker NeuN regardless of the relative vulnerability of the neuron type. RNA immunoprecipitation of HuR from the cytoplasmic fraction of 8 hr reperfused forebrains selectively isolated hsp70 mRNA suggesting the mRNA granules are soluble structures. Together, these results rule out several organelle systems and a known HuR pathway as being directly involved in mRNA granule function.

Keywords: brain ischemia and reperfusion, hippocampus, HuR, mRNA granules, translation arrest

INTRODUCTION

It is well-known that all post-ischemic neurons display a translation arrest (TA) which is transient in neurons that will survive the insult. However, the persistance of TA in post-ischemic neurons, such as the hippocampal CA1 region, is one of the strongest predictors of inevitable cell death following brain ischemia and reperfusion (I/R) (Hossmann, 1993). We previously showed that polyadenylated mRNAs [poly(A)] formed granular structures in post-ischemic neurons, which we named “mRNA granules” (Jamison et al. 2008). The presence of mRNA granules correlated precisely with decreased protein synthesis rates in vivo, and hence with the selective vulnerability of CA1 neurons following brain I/R. The mRNA granules colocalized with the mRNA binding proteins poly-A binding protein (PABP) and eukaryotic initiaiton factor 4G, but did not colocalize with the 40S small ribosomal subunit protein S6. Additionally, the mRNA binding protein HuR colocalized with mRNA granules in resistant CA3 neurons, but not vulnerable CA1 neurons, at early (< 16 hr) reperfusion durations, and this correlated with translation of hsp70 mRNA.

To further ascertain the molecular composition of the mRNA granules in vivo, we here describe additional colocalization studies between poly(A) mRNA and markers of intracellular organelles and mRNA regulatory systems. Of all the markers tested, only the neuronal marker NeuN showed colocalization in the form of extra-nuclear granules in post-ischemic neurons. Additionally we show that RNA immunoprecipitation of HuR but not PABP from homogenates of 8 hr reperfused forebrain selectively isolated hsp70 mRNA. Together, these results shed additional light on the identity of the mRNA granule by ruling out a direct involvement with the organelle and mRNA processing systems we tested here.

MATERIALS AND METHODS

Materials

Antisera for α-tubulin (T6199) and neurofilament (NF) H/M (N2912) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Antisera for acidic protein rich in leucine, a HuR accessory protein, (APRIL; ab4224), and cytochrome C oxidase subunit 4, a mitochondrial marker, (COX IV; ab16056) were purchased from Abcam (San Francisco, CA, USA). Antisera for protein disulfide isomerase, a marker of the endoplasmic reticulum (PDI; MA3-019) and and the trans-Golgi marker TGN38 (MA3-063) were purchased from Thermo Scientific (Rockford, IL, USA). A marker of the cis-Golgi, GM130 (610822), was purchased from BD Biosciences (Sparks, MD, USA). Anti- HuR (sc-5261) was purchased from Santa Cruz Biotechnology, Inc (Santa Cruz, CA, USA). Anti-NeuN, used here as a marker for neuronal nuclei, (MAB377) was purchased from Millipore (Billerica, MA, USA). Antisera for pp32, another HuR accessory protein (ADI-905-234-100) was purchased from Enzo Life Sciences (Farmingdale, NY, USA). Ribosomal P antigen(RPA; HPO-0100), a marker of the 60S ribosomal subunit (Bonfa et al., 1988), was purchased from ImmunoVision (Springdale, Arkansas, USA). All other chemicals were reagent grade.

Animal model

All animal experiments were approved by the Wayne State University Animal Investigation Committee and were conducted following the Guide for the Care and Use of Laboratory Animals (National Research Council, revised 2011). All efforts were made to reduce animal suffering and minimize the total number of animals used. Normothermic global forebrain ischemia of 10 min duration was induced in male Long Evans rats using the bilateral carotid artery (two-vessel) occlusion and hypovolemic hypotension (2VO/HT) model of Smith et al., (1984), as we have previously described (DeGracia et al., 2007; Roberts et al., 2007; Jamison et al., 2008). Exclusion criteria and survival rates were as previously reported (Jamison et al., 2008). Experimental groups (n = 5/group) were: sham-operated, nonischemic controls (NIC), 10 min ischemia and reperfusion durations of: 1 hr (1hR), 8 hr (8hR), 16 hr (16hR), 36 hr (36hR) and 48 hr (48hR).

Tissue Slice Preparation and Double Immunofluorescence/Fluorescent in situ hybridization

At appropriate times, animals were transcardially perfused, brains dissected, and 50 micron slices through the dorsal hippocampus were obtained via vibratome and stored at −20°C in cryostat solution until used, as previously described (Kayali et al., 2005). Double immunofluorescence (IF)/fluorescent in situ hybridization (FISH) was performed exactly as previously described (Jamison et al., 2008), using 50 ng/ml of a 5'-biotinylated 50-mer oligo-dT probe (Integrated DNA Technologies, Inc., Coralville, IA). Antisera dilutions were: α-tubulin, (1:100); APRIL, (1:100); COX IV, (1:50); GM130, (1:100); NeuN, (1:500); NF H/M, (1:300); PDI, (1:200); pp32, (1:250); RPA, (1:5000); TGN38, (1:200).

Validation of antisera stainings included (not shown): (1) loss of signal with omission of primary antisera, (2) graded loss of signal with antisera dilution, and (3) agreement with published descriptions of antisera neuronal staining patterns where available [e.g. endoplasmic reticulum and Golgi (Takeda et al., 2001); NeuN and alpha tubulin (Gu et al., 2009); neurofilament M (Kim et al., 2002); poly(A) mRNAs (Martone et al., 1996)]. We previously validated the specificity of RPA staining (Kayali et al., 2005).

Slides were examined on an Axioplan 2 Imaging System (Carl Zeiss, Oberkochen, Germany) equipped with an ApoTome. Excitation at 488 nm and 568 nm, and emission at 518 nm and 600 nm were used for Alexa 488 (green) and Alexa 555 (red), respectively. Optical sectioning was performed using the X63 oil immersion objective to generate z-stacks as previously described (Jamison et al, 2008). Fluorescent micrographs shown in the figures are orthographic projections of 3.5 micron z-stacks (10 × 0.35 micron optical sections), unless otherwise stated.

Western blot validation of antisera

To further validate antibody specificity, Western blots were performed for each antigen on clarified homogenates of non-ischemic brain. SDS-PAGE gels contained 50 μg protein per lane, determined by Lowry assay. Conditions for each Western blot are listed in Table 1, which includes primary antisera: dilution, incubation buffer, incubation time, incubation temperature, and type of membrane for electroblot transfer, (NC, nitrocellulose; PDVF, polydivinylflouride). The base buffer for all primary antisera incubations was TTBS. Other than the primary antisera incubation conditions, Western blots were performed as previously described (Jamison, 2008).

Table 1.

antisera dilution 1° buffer time temperature membrane
APRIL 1:500 5% milk 1 hr 25° C PVDF
a-tubulin 1:1000 10% milk 1 hr 25° C PVDF
COX IV 1:150 5% milk overnight 4° C PVDF
GM130 1:500 5% milk 1 hr 25° C PVDF
NeuN 1:1000 5% milk overnight 4° C NC
NFH/M 1:1000 5% milk overnight 4° C NC
PDI 1:1000 2% milk overnight 4° C NC
pp32 1:333 - overnight 4° C NC
ribo P 1:300 5% milk overnight 4° C NC
TGN38 1:250 5% milk overnight 4° C PVDF
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RNA Immunoprecipitation (RIP)

RIP was based on the method of Keene et al. (2006) and Baroni et al. (2008). 8hR or NIC animals were sacrificed and brains rapidly removed. Whole forebrain was dissected at 4°C and homoginzed on ice in 5:1 (w/v) of 50 mM HEPES pH 7.5, 150 mM NaCl, 1% (v/v) Triton X-100, 10% (v/v) glycerol, 1 mM MgCl2, 1 mM EGTA, 80 U/mL RNase inhibitor (Ambion, Austin, TX, USA), 0.2% ribonucleoside vanadyl complexes (Sigma, St. Loius, MO, USA), and 1:85 protease inhibitor cocktail (Sigma). Centrifugation of the 2500 g post-nuclear supernatant at 25,000 g generated a cytoplasmic supernatant, and 600 μg of supernatant protein was precleared with 1 μL of an unrelated antibody (Lamin AlC, Santa Cruz Biotech, Santa Cruz, CA, USA) plus 20 μL Protein A-Sepharose beads (Invitrogen, Carlsbad, CA, USA). Precleared supernatants were rotated 16 hr, 4°C in 15 μL Protein A-Sepharose prebound with 16 μg HuR antiserum (Santa Cruz). Beads were washed ×3 in sterile phosphate buffered saline. RNA was extracted from precipitated protein using TRIzol reagent (Invitrogen) and reverse transcriptase PCR was performed for gapdh (5'-ACAAGATGGTGAAGGTCGGTGTGA-3', 5'-TTGTCATTGAGAGCAATGCCAGCC-3'; 1.0 kb product) and hsp70 mRNA (5'-TCTTGGTTGCCAACACCCAAATCC -3', 5'-AAAGGTCACTGCTAGCTCCGTGTT -3'; 0.5 kb product) using 2 μg total RNA according to vendor instructions (Roche, Boulder, CO, USA). Amplification products were run on Tris-acetic acid- EDTA-1% agarose gels and visualized by SYBR gold (Invitrogen). For some IP reactions, RNA was not extracted and beads were boiled in Laemmli buffer, run on SDS-PAGE gels, and western blotted for HuR or PABP using methods previously described (Jamison, et al., 2008).

RESULTS

Double IF/FISH

We previously showed that 10 min of 2VO/HT forebrain ischemia in rat caused selective CA1 cell death at 3 days reperfusion and that mRNA granules were present in CA1 neurons to 48 hr reperfusion and in CA3 neurons to 16 hr of reperfusion (Jamison et al., 2008). Identical results were obtained in the present study across the reperfusion time course (data not shown). Figures 1 and 2 illustrate representative photomicrographs of double IF/FISH staining in NIC and 1hR CA1 and CA3. We show only 1hR samples because the qualitative pattern of colocalization (or lack thereof) for a given antigen with the mRNA granules did not change over the reperfusion time course. Additionally, similar colocalization patterns were seen in cerebral cortical, thalamic and hilar neurons that evidenced mRNA granules following reperfusion (data not shown).

Figure 1.

Figure 1

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Representative photomicrographs of poly(A) mRNAs and organelle markers as indicated in the figure for nonischemic control (NIC) and following one hour of reperfusion (1hR) in hippocampal layers CA1 and CA3. All images were aquired under ×63 oil immersion and are orthographic projections of ten sequential 0.35 micron optical slices. Scale bar in lower right most panel is 10 microns and applies to each image. Each image is a 1/3rd crop from the original photomicrograph.

Figure 2.

Figure 2

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Representative photomicrographs of poly(A) mRNAs and mRNA binding systems as indicated in the figure for nonischemic control (NIC) and following one hour of reperfusion (1hR) in hippocampal layers CA1 and CA3. All images were aquired under ×63 oil immersion and are orthographic projections of ten sequential 0.35 micron optical slices. Scale bar in lower right most panel is 10 microns and applies to each image. Each image is a 1/3rd crop from the original photomicrograph.

Figure 1 shows double-labeling of poly(A) mRNAs and antisera detecting intracellular organelles. Only the antisera for PDI showed slight colocalization with poly(A) in the NIC samples as indicated by the slight yellowish hue of the NIC cytoplasm (Figure 1A, NIC CA1 and CA3), likely indicative of endoplasmic reticulum-localized translation. However, at 1hR, the mRNA granules were distinctly green against the red PDI staining, thus the mRNA granules did not colocalize with endoplasmic reticulum (Figure 1A, 1hR CA1 and CA3). Markers of cis and trans- Golgi apparatus, GM130 and TGN38, respectively, showed no colocalization in NICs or at 1hR (Figures 1B and 1C). A lack of colocalization also held for the mitochondria marker COX IV (Figure 1D), and two cytoskeleton components, α-tubulin (Figure 1E) and NF-H/M (Figure 1F). Thus, the mRNA granules did not colocalize with any of the organelle markers tested.

Figure 2 shows antigens representing mRNA binding systems. In NICs, the large (60S) ribosomal subunit, as marked by RPA, showed a diffuse cytoplasmic colocalization with poly(A) mRNAs (Figure 2A), as would be expected for cells active in protein synthesis. However, the mRNA granules did not colocalize with RPA in reperfused samples. We previously showed a lack of colocalization of mRNA granules with the 40S marker S6 following brain I/R (Jamison et al., 2008). As it is well-known that polysomes are fully dissociated at the 1hR time point (Hossmann, 1993; Martin de la Vega et al., 2001), our results indicate that the dissociated ribosomal subunits are not associated with the mRNA granules.

We also previously showed that the mRNA binding protein HuR colocalized with mRNA granules in CA3 but not CA1 pyramidal neurons at 1hR (Jamison et al, 2008). Steitz and colleagues (Gallouzi et al., 2001) have shown that HuR interacts with two protein ligands, APRIL and pp32, during CRM1-dependent nucleocytoplasmic transport of HuR-bound mRNAs. We observed no colocalization of APRIL (Figure 2B) or pp32 (Figure 2C) with the mRNA granules in the reperfused samples. Unexpectedly, the well-known neuronal nuclear marker NeuN strongly colocalized with the mRNA granules in both CA1 and CA3 during reperfusion (Figure 2D).

For all of the antisera used above, Western blots on brain homogenate proteins produced single bands at the correct molecular weights (Figure 3E). The only exception was RPA which produced several bands, but this result is expected because RPA cross reacts with many of the large ribosomal subunit proteins on Western blots (Lin et al., 1982).

Figure 3.

Figure 3

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RNA immunoprecipitation of HuR and PABP. (A) and (D) Western blots of immunoprecipitations of HuR and PABP, respectively. “Input” is 75 mg total protein of cytoplasmic fraction; “sham” and “8hR” are IP results from respective experimental groups; “-input” is IP reaction run in absence of cytoplasmic fraction for HuR. Molecular weights are as indicated, and HuR and PABP bands indicated as labeled. (B) and (C) Agarose gels (1%) of PCR amplification products (30 cycles) after extraction of nucleic acids from HuR and PABP IPs, respectively. “g” amd “h” indicate amplification with primers for gapdh and hsp70 mRNAs, respectively. “bp”, base pair weight standards. (E) Western blots validating the specificity of the antisera used for microscope analysis. Positions of molecular weight standards are indicates to the left. Abbreviations for anti-sera are given in Materials and Methods.

RIP Results

RIP of HuR or PABP was performed on cytoplasmic fractions from forebrain homogenates of NIC and 8hR samples (Figure 3A–D). When mRNA was extracted and the cDNA amplified via probes for either gapdh or hsp70, HuR RIPs selectively brought down hsp70 mRNA but not gapdh. PABP RIPs brought down both hsp70 and gapdh mRNA. Thus HuR demonstrated selective mRNA binding compared to PABP.

DISCUSSION

Because transient mRNA structures tend to be labile upon cell disruption (Mili & Steitz, 2004; Kedersha and Anderson, 2007), here we primarily used histochemical methods to further assess the colocalization properties of the mRNA granules formed in reperfused neurons. The mRNA granules present in the reperfused CA1 and CA3 neurons did not colocalize with markers of major intracellular organelles or cytoskeleton. Nor did the mRNA granules colocalize with a marker of the 60S subunit. In combination with our previous result showing mRNA granules did not colocalize with 40S subunits (Jamison et al., 2008), we conclude that poly(A) mRNAs are sequestered away from both ribosomal subunits following polysome dissociation during the initial hours of brain reperfusion. This stands in contrast to stress granules, which contain a modified form of the 40S subunit (Anderson and Kedersha, 2006). In spite of mRNA granules in CA3 neurons colocalizing with HuR at 1hR (Jamison et al. 2008), there was no colocalization with the HuR accessory proteins APRIL and pp32, suggesting that the function of HuR in the mRNA granules may be distinct from its role in nuclear to cytoplasmic transport of mRNAs mediated in conjunction with APRIL and pp32 (Gallouzi et al., 2001).

Unexpectedly, the mRNA granules strongly colocalized with NeuN outside of the nucleus following brain I/R (Figure 2D). This result is consistent with a recent study that identified NeuN as an mRNA splicing factor, FOX-3 (Kim et al., 2009). The colocalization of NeuN and the mRNA granules suggests NeuN may function similarly to other mRNA binding proteins such as HuR (Gorospe, 2003) or TIA-1 (Anderson and Kedersha, 2006) that reside in the nucleus under normal conditions, but export into the cytoplasm under conditions of cell stress to contribute to the genetic reprogramming to a stress response phenotype (DeGracia et al. 2008).

The RIP experiments show HuR selectively bound hsp70 mRNA compared to PABP. It has been reported that hsp70 mRNA contains an adenine and uridine rich element (ARE) in its 3'-untranslated region (Zhao et al., 2002). The ARE sequence is the binding target for HuR (Chen et al., 2002). HuR binding to ARE-containing mRNAs, such as c-fos, contribute to their stabilization and selective translation (Fan and Steitz, 1998). On the other hand, PABP binds any mRNA with a poly(A) tail of sufficient length (Mangus et al., 2003), and is expected to pull down a variety of mRNAs. For the purpose of the present report, the isolation of hsp70 mRNA following HuR RIP from the soluble fraction is consistent with the lack of colocalization of the mRNA granules with the organelle systems we tested.

In conclusion, the present study confirms our previous observation of mRNA granules (Jamison et al., 2008), and extended these to show that the mRNA granules: (1) do not colocalize with several major intracellular organelle systems, and (2) are sequestered from both ribosomal subunits. Further, we reported the unexpected finding that NeuN strongly colocalized with the mRNA granules. The present observations support the that mRNA regulation plays an important role in the post-ischemic response of all neurons to ischemic stress (DeGracia et al., 2008). Understanding the intrinsic response of the neurons will be crucial to developing effective therapies to combat ischemic brain damage as occurs following cardiac arrest and resusciation and stroke, therapies which to this point have remained frustratingly elusive.

Highlights

  1. mRNA granules formed after brain ischemia do not colocalize with organelles.

  2. mRNA granules formed after brain ischemia do not colocalize with ribosomes.

  3. RNA immunoprecipitation of HuR co-precipitates hsp70 mRNA.

ACKNOWLEDGEMENTS

We thank Jie Wang for her assistance with the 2VO/HT model. This work was sponsored by NIH NINDS Grant No. NS057167 (D.J.D.), a Ruth L. Kirschstein National Research Service Award, NS063651 (J.J.S.), and a Thomas C. Rumble Fellowship, Wayne State University (J.T.J.).

Abbreviations used

2VO/HT

bilateral carotid artery occlusion and hypovolemic hypotension

40S

small ribosomal subunit

APRIL

acidic protein rich in leucine

ARE

adenine and uridine rich element

CA

Cornu Ammonis (Ammon's horn)

COX IV

cytochrome c oxidase subunit IV

FISH

fluorescence in situ hybridization

FOX3

homolouge 3 of feminizing locus on X protein 1

gapdh

mRNA for glyceraldehyde-3-phosphate dehydrogenase

GM130

Golgi matrix protein 130

hsp70

mRNA for 70 kDa inducible heat shock protein

HuR

also called HuA or ELAV

I/R

ischemia and reperfusion

IF

immunofluorescence histochemistry

NeuN

neuronal nuclei

NF H/M

neurofilament heavy and medium chain

NIC

sham-operated, non-ischemic control

PABP

poly(A) binding protein

PDI

protein disulfide isomerase

poly(A)

poly-adenylated mRNAs

pp32

polypeptide 32 kDa

RIP

RNA immunoprecipitation

S6

small ribosomal subunit protein 6

TA

translation arrest

TGN38

trans Golgi network 38

TIA-1

T cell internal antigen

Footnotes

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