The long non-coding RNA nuclear-enriched abundant transcript 1_2 induces paraspeckle formation in the motor neuron during the early phase of amyotrophic lateral sclerosis

Characteristics of NEAT1_2 lncRNA and paraspeckle formation have been elucidated mainly using cultured cells including HeLa cells [16‐20, 28‐34]. To investigate the cellular basis for the pathogenesis of ALS, we initially examined the subcellular localization of WT, mutant and/or truncated forms of TDP-43 and FUS/TLS by exogenously expressing tagged proteins in HeLa cells. Both WT and the 35-kDa C-terminal fragment of TDP-43 protein were widely distributed throughout the nucleus; however, their characteristic aggregates in the nucleus coincided with 93.8 ± 10.6% and 89.7 ± 13.9% of the NEAT1_2 foci, respectively (Figure 1A, B). These findings mean that almost all NEAT1_2 foci overlap with parts of TDP-43-forming nuclear aggregates. Similar enrichment to NEAT1_2 foci was also observed with tagged WT FUS/TLS (96.9 ± 10.2%; Figure 1A) as well as endogenous TDP-43 and FUS/TLS (Additional file 1: Figure S1A). By contrast, the 26-kDa C-terminal fragment of TDP-43 formed few aggregates in the nucleus, and was distributed throughout both the nucleus and the cytosol, as shown in a previous article [8], demonstrating a marked lack of affinity for NEAT1_2 foci (15.8 ± 17.9%; Figure 1A). Additionally, ALS-associated TDP-43 mutants and FUS/TLS mutants were colocalized with NEAT1_2 lncRNA as frequently as WT TDP-43 and WT FUS/TLS (TDP-43^A315T: 93.3 ± 12.7%, TDP-43^A382T: 98.0 ± 8.2%, FUS/TLS^R514S: 95.8 ± 9.3%, and FUS/TLS^P525L: 98.4 ± 5.8%; Additional file 1: Figure S1B, C).

The short form of NEAT1 ncRNA, NEAT1_1, is produced from the 5′-end of NEAT1. Although an in situ hybridization probe targeting NEAT 1_1 ncRNA, that is shown as NEAT1_1/1_2 probe in Additional file 1: Figure S1D (upper), could not precisely distinguish NEAT1_1 foci from NEAT1_2 foci, most NEAT1_1 foci were also colocalized frequently with nuclear aggregates formed by WT TDP-43 and WT FUS/TLS (Additional file 1: Figure S1D, lower).

Another nuclear body, the Cajal body, is related to RNA metabolism; however, NEAT1_2 foci demonstrated a complete different distribution from Cajal bodies labeled with the marker coilin (Figure 2A, upper). Consistent with a previous report that 40% of TDP-43 nuclear bodies overlapped with Cajal bodies [35], endogenous TDP-43 that did not overlap with NEAT1_2 foci overlapped with the Cajal bodies separately (Figure 2A, lower). In light of TDP-43 and FUS/TLS protein colocalization to NEAT1_2 lncRNA, we tested whether endogenous TDP-43 and FUS/TLS bound directly to NEAT1_2 lncRNA. The RNA-protein complex was immunoprecipitated from UV cross-linked HeLa cells using polyclonal anti-TDP-43 and anti-FUS/TLS antibodies with stringent washes in high-salt buffer to spoil protein-protein interactions. The immunoblotting assay verified specific precipitations by using monoclonal anti-TDP-43 and anti-FUS/TLS antibodies (Figure 2B). After bound RNA was isolated, NEAT1_2 lncRNA levels were quantified by reverse transcription (RT) and polymerase chain reaction (PCR). NEAT1_2 lncRNA was enriched in anti-TDP-43 and anti-FUS/TLS immunoprecipitants compared with control IgG immunoprecipitants (Figure 2C). Paraspeckle formation requires NEAT1_2 lncRNA and core paraspeckle proteins, which subsequently recruit other paraspeckle-associated factors and NEAT1_1 ncRNA [19, 29]. Therefore, to determine whether TDP-43 and FUS/TLS formed paraspeckles in cultured cells, immunocytochemistry was performed to examine the intra-nuclear localization of the paraspeckle proteins, PSF and PSP1. Nuclear aggregates of TDP-43 and FUS/TLS colocalized with PSF and PSP1 (Figure 2D). Taking these findings together, NEAT1_2 foci were considered to form paraspeckles with TDP-43 and FUS/TLS.

Next, we examined NEAT1_2 distribution in the nervous system of WT control mice in vivo. Some reports have demonstrated that NEAT1_2 expression is abundant in restricted cells including the epithelial cells of the esophagus, forestomach, and surface epithelium of zymogenic region of the stomach in adult mice but not in embryonic stem cells [36]. No previous reports have described NEAT1_2 expression in the spinal cord, aged tissues, or any human tissues. To test the distribution of NEAT1_1 ncRNA and NEAT1_2 lncRNA in each tissue including the nervous system in mice, quantitative RT-PCR was performed using 8-week-old mouse tissue extracts. NEAT1 ncRNA was highly expressed in lung, heart, and kidney (Figure 3A) and markedly dominated by NEAT1_1 expression, consistent with a previous report [36]. Notably, expression levels of NEAT1_1 ncRNA were modest, and NEAT1_2 lncRNA was hardly detectable in the central nervous system. Therefore, we further investigated NEAT1_2 distribution specifically in single spinal motor neurons. In situ hybridization followed by fluorescent immunohistochemistry (RNA-FISH) revealed no NEAT1_2 expression in the nuclei of spinal motor neurons from 8-week-old and 2-y-old mice (Figure 3B). NEAT1_1 ncRNA was expressed in the spinal glial cells of both 8-week-old and 2-y-old mice, and was also expressed at low levels in the spinal motor neurons of both young and aged mice (Additional file 2: Figure S2).

While NEAT1_2 lncRNA interacted with ALS-associated proteins in human cell lines (Figure 1A and B, Figure 2C and Additional file 1: Figure S1A), no NEAT1_2 lncRNA was observed in spinal motor neurons in mice in vivo (Figure 3B). According to these findings, we hypothesized that NEAT1_2 lncRNA has a particular role in motor neurons in sporadic ALS patients. To test this hypothesis, the fresh frozen spinal cords of six sporadic ALS cases (age: 73.2 ± 8.9 y) and six control cases (age: 83.2 ± 4.4 y) were prepared (Table 1). Contrary to the results in mice (Figure 3B), RNA-FISH demonstrated that more than 80% of human spinal motor neurons in ALS cases displayed NEAT1_2 foci in the nuclei (Figure 4A, 5C). Using sense probe as a negative control, the possibility that antisense probe against NEAT1_2 lncRNA recognized other non-specific intranuclear RNAs was excluded in the human spinal cord (Additional file 3 Figure S3). Parts of endogenous TDP-43 aggregates in the nucleus coincided with nearly all NEAT1_2 foci in motor neurons in ALS cases (Figure 4A), consistent with findings in cultured cells (Figure 1A and B, Figure 2C and Additional file 1 Figure S1A).

Next, to test whether NEAT1_2 lncRNA in human motor neurons formed paraspeckle structure, the nuclear distribution pattern of PSP1 was examined with immunohistochemistry and visualized with DAB (Figure 4C). PSP1 was often observed as an aggregated form in the nuclei of motor neurons as well as surrounding glial cells. In addition, RNA-FISH demonstrated that PSF, PSP1, and p54^nrb were colocalized with NEAT1_2 foci in the nuclei of ALS motor neurons (Figure 4D and E, and Additional file 4: Figure S4). In control cases, more than 60% of motor neurons demonstrated no NEAT1_2 foci (Figure 4B, 5C); however, the remaining motor neurons contained NEAT1_2 foci with paraspeckle proteins in the nuclei (Additional file 4: Figure S4). These results suggest that paraspeckle proteins have affinity for NEAT1_2 foci in motor neurons in both ALS and control cases.

To test whether paraspeckles in the motor neuron were formed significantly in sporadic ALS, occurrence rates of NEAT1_2 lncRNA in human motor neurons were quantified. All motor neurons were regarded as the subjects of the number count in the ventral horns of several spinal cord slices at a constant thickness of 14 μm. The motor neurons were definitively-distinguishable from surrounding glia cells according to the morphology, specific structures including lipofuscin, or the size that is more than approximately 35 μm. To evaluate occurrence rates of NEAT1_2 lncRNA at the same stage of TDP-43 distribution, we subdivided the pathological stages of ALS spinal motor neurons into four classes (Table 2, Figure 5A). At Stage 0, TDP-43 is normally distributed within the nucleus. Since TDP-43 is seen in the cytoplasm or the nucleus of the motor neuron degrades, stage I, II and III indicate pathological stages in ALS. The nucleus degrades but is still recognized at stage I. In stage II, the nuclear TDP-43 was not recognized any more, however, the plasma membrane seems still retained. Stage III is the final stage, where the plasma membrane disappeared and TDP-43 skein-like inclusions are left behind. ALS cases demonstrated motor neurons at all pathological stages, in contrast to control cases in which all motor neurons were limited to stage 0 (Figure 5B). In overall counting of the motor neurons in the ventral horn, there was no significant difference in the NEAT1_2 lncRNA appearance rate between in ALS and control cases (Additional file 5: Figure S5). Only at stage 0, however, the occurrence of NEAT1_2 expression in motor neurons was significantly increased in ALS cases compared to that in control cases (85.6 ± 11.0% and 35.1 ± 22.6%, respectively; Figure 5C). The positive rate of NEAT1_2 foci in motor neurons was higher at the early stage of ALS than at the advanced stage (Figure 5D). This finding suggests that NEAT1_2 lncRNA appeared preferentially in spinal motor neurons at the earliest point of ALS.

The nuclear distribution pattern of NEAT1_2 lncRNA in the human nervous system has not been previously reported, and its function remains largely unknown. To observe NEAT1_2 localization in human spinal motor neurons in detail, electron microscopic analysis combined with in situ hybridization (EM-ISH) was performed. In the spinal cords of human ALS cases, NEAT1_2 lncRNA was localized to IGAZ in a halo-like fashion, suggesting that NEAT1_2 lncRNA formed the characteristic structure of paraspeckles in the nuclei of spinal motor neurons in ALS cases (Figure 6B-a and -b, and Additional file 6: Figure S6C), consistent with previous articles [17, 28, 30]. By contrast, NEAT1_2-labeled gold particles around IGAZ were rare in control cases (Figure 6B-c and -d). The IGAZ has been considered to be identical to a paraspeckle [17, 28‐30], indicating that the formation of IGAZ depends on NEAT1_2 lncRNA. IGAZ-like zone without NEAT1_2 granules in Figure 6B-d suggests that a human motor neuron has a moderately electron-dense intranuclear structure besides paraspeckles. Alternatively, independent of NEAT1_2 lncRNA, IGAZ may exist in the nucleus as a scaffold prior to the recruitment of NEAT1_2 lncRNA to form a complete paraspeckle. NEAT1_1/1_2 probe, detecting both NEAT1_1 ncRNA and NEAT1_2 lncRNA (Additional file 1: Figure S1D, upper), was observed in the nuclei of spinal motor neurons in control cases as frequently as those in ALS cases (Additional file 6: Figure S6A-a, -b, -c and -d). Notably, halo-shaped aggregation patterns formed by NEAT1_1/1_2 ncRNAs were found throughout nuclei in ALS spinal motor neurons (Additional file 6: Figure S6A-b) but were observed rarely in control cases (Additional file 6: Figure S6A-c and -d). Even when diluted NEAT1_1/1_2 probe is used, both central (Additional file 6: Figure S6B-a) and halo-like (Additional file 6: Figure S6B-b) patterns of aggregation were observed in a HeLa cell. This suggests that NEAT1_1 ncRNA may show the central accumulation pattern independently of the IGAZ margin; however, it could demonstrate the halo-shaped pattern depending on NEAT1_2 lncRNA. Taken these findings together, NEAT1_2 lncRNA forms paraspeckles specifically around IGAZ in the nuclei of motor neurons in ALS cases, which may affect the distribution of NEAT1_1 ncRNA.

HeLa human carcinoma cells were maintained in Dulbecco’s modified Eagle’s medium (Gibco, Grand Island, NY) supplemented with 10% fetal bovine serum as described previously [8, 47]. Transfection was performed using GeneJuice Transfection Reagent (Novagen, Madison, WI) according to the manufacturer’s instructions. To transfect HeLa cells, cells were grown to 60–80% confluence on 0.001% poly-L-lysine (PLL)-coated coverslips or 8-chamber slides (Chamber slide II, Iwaki Glass, Tokyo, Japan). Forty-eight hours after transfection, cells were fixed in 4% paraformaldehyde (PFA) overnight at 4°C for in situ hybridization or for 10 min at room temperature for immunofluorescence staining. Cells were washed with phosphate-buffered saline (PBS), and total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA) for quantitative RT-PCR.

Among primary antibodies, mouse monoclonal anti-V5 (1:500, R960-25) was purchased from Invitrogen. Rabbit polyclonal anti-V5 (1:500, A190-120A) and rabbit polyclonal anti-FUS/TLS (1:250, A300-302A) were from Bethyl Labs (Montgomery, TX). Rabbit polyclonal anti-TDP-43 (1:250, 10782-2-AP) was from Proteintech (Chicago, IL). These polyclonal antibodies directed against FUS/TLS and TDP-43 were used for immunochemistry and immunoprecipitation. Mouse monoclonal antibodies, anti-TDP-43 (1:1,000, H00023435-M01) from Abnova (Walnut, CA) and anti-FUS/TLS (1:1,000, 4H11, sc-47711) from Santa Cruz Biotechnology (Santa Cruz, CA), were used for immunoblotting. Rabbit polyclonal anti-fluorescein isothiocyanate (FITC) (1:500, ab19491), mouse monoclonal anti-digoxigenin (DIG) IgG1 (1:500, H8, ab420), and mouse monoclonal anti-coilin IgG2b (1:500, IH10, ab87913) were from Abcam (Cambridge, MA). Mouse monoclonal anti-β-actin (1:10,000, AC-15, A1978) and anti-PSF (1:200, B92, P2860) antibodies were from Sigma (St. Louis, MO). Mouse monoclonal anti-p54^nrb (1:200, 3/p54nrb, 611279) was from BD Transduction Laboratories (San Diego, CA). Rabbit polyclonal anti-PSP1 (1:500) was generated by T. H. The secondary antibodies used for immunofluorescence, Alexa Fluor 488 goat anti-rabbit IgG (A-11034), Alexa Fluor 488 goat anti-mouse IgG1 (A-21121), Alexa Fluor 488 goat anti-mouse IgG2b (A-21141), Alexa Fluor 555 goat anti-rabbit IgG (A-21429), Alexa Fluor 555 goat anti-mouse IgG (A-21424), Alexa Fluor 555 goat anti-mouse IgG1 (A-21127), and Alexa Fluor 647 goat anti-mouse IgG2b (A-21242), were purchased from Life Technologies (Carlsbad, CA).

Plasmids containing the V5-tagged series of TDP-43 and FUS/TLS were kindly provided by Dr. Daisuke Ito (Keio University) [8, 9].

Fixed cells or tissues on PLL-coated glass slides were treated with 0.2 N HCl for 20 min, followed by 3 μg/ml proteinase K (PCR grade; Roche, Indianapolis, IN) at 37°C for 3 min (in the case of cell samples) or 7 min (in the case of tissue samples). After acetylation in a solution consisting of 1.5% triethanolamine, 0.25% acetic anhydride, and 0.25% HCl, the hybridization reaction was carried out with DIG- or FITC-labeled probes (1 μg/ml) for 16 h at 55°C in hybridization buffer (50% formamide, 2× SSC, 1× Denhardt’s solution, 5% dextran sulfate, 10 mM ethylenediaminetetraacetic acid (EDTA), and 0.01% Tween 20). Samples were washed twice at 55°C for 30 min with a solution consisting of 50% formamide, 2× SSC, and 0.01% Tween 20, and then 10 μg/ml RNase A was added at 37°C for 1 h in buffer (0.5 M NaCl, 10 mM Tris (pH 8.0), 1 mM EDTA, and 0.01% Tween 20). They were washed in 2× SSC wash buffer with 0.01% Tween 20 at 55°C for 30 min, followed by washing in 0.2× SSC wash buffer with 0.01% Tween 20 at 55°C for 30 min. After additional washing in Tris-buffered saline (TBS, pH 7.6) and blocking in a buffer consisting of blocking reagent (Roche 11096176001), 0.1 M maleate, 0.15 M NaCl, and 0.01% Tween 20 in TBS, DIG- or FITC-labeled probes were detected by standard immunohisto-/immunocytochemical procedures using antibodies against DIG, FITC, and other proteins.

In preparation for DIG- or FITC-labeled RNA probes, cDNA fragments were amplified using M13 forward (FW) and reverse (RV) primers with the AV089414 EST clone as a template for the mouse NEAT1_1/1_2 probe. cDNA fragments were subcloned into pCRII (Invitrogen) for the mouse NEAT1_2 probe after amplification using mNEAT1_2 FW/RV primers with the BAC clone RP23-209P9 as a template [36]. For the human NEAT1_1/1_2 probe and NEAT1_2 probe, cDNA fragments were obtained using hNEAT1 FW/RV primers and hNEAT1_2 FW/RV primers, respectively, using a cDNA library derived from HeLa cells as a template and were subcloned into pCRII-TOPO (Invitrogen) and pGEM-T Easy vectors (Promega, Madison, WI). Each primer is shown in Additional file 8: Table S1. Both antisense and negative control sense RNA probes were prepared using a DIG/FITC RNA labeling mix (1277073/1685619, Roche), RNasin Plus RNase Inhibitor (N2611, Promega), and T3, T7 or SP6 RNA polymerase (P2083/ P2075/P1085, respectively, Promega) according to the manufacturer’s instructions. The usability of each probe was finally characterized by Northern blot.

CLIP assay was performed as described previously [48, 49]. Briefly, HeLa cells were UV cross-linked at 254 nm (UV-B) with 600 J/cm² in a UV Stratalinker 1800 crosslinker (Stratagene, La Jolla, CA); lysed in wash buffer containing 1× PBS, 0.1% sodium dodecyl sulfate (SDS), 0.5% deoxycholate, and 0.5% NP-40; supplemented with 0.015 U/μl RNasein Plus (N261, Promega) and RQ RNase-Free DNase (M610A, Promega); and immunoprecipitated for 2 h at 4°C with 5 μg polyclonal anti-TDP-43, anti-FUS/TLS, and rabbit IgG control (AB-105-C, R&D Systems, Minneapolis, MN) antibodies bound in advance to Dynabeads Protein G (100.04 D, Invitrogen). Immunoprecipitated materials were washed as follows: twice with wash buffer described above for 5 min; twice with 5× PBS, 0.1% SDS, 0.5% deoxycholate, and 0.5% NP-40 for 5 min (using high-salt wash buffer to completely remove indirect protein-RNA interactions); and twice with 50 mM Tris-HCl (pH 7.4), 10 mM MgCl₂, and 0.5% NP-40 for 5 min. The immunoprecipitated protein-bead complexes were removed from RNA by proteinase K (13731196, Roche) digestion. RNA was then isolated by phenol/chloroform extraction and treated again with DNase I before the RT reaction was performed as described below. Bound RNAs were evaluated by PCR assay using the qhNEAT1_2 FW/RV primers listed in Additional file 8: Table S1. The PCR reaction was performed in a 50 μl reaction mixture containing 0.2 μM of each primer, 0.25 mM dNTP mix (Takara, Otsu, Japan), 5 μl 10× PCR buffer, and 1 μl Advantage 2 Polymerase mix (639201, Clontech, Mountain View, CA). PCR amplification began with a denaturation step at 95°C for 1 min, followed by 25, 33, or 40 cycles of denaturation at 95°C for 10 s, annealing at 64°C for 30 s, and extension at 68°C for 30 s.

Cells and UV cross-linked immunoprecipitates after stringent washes with the high-salt wash buffer were lysed in cold lysis buffer (50mM Tris–HCl, pH 7.4, 150 mM NaCl, 0.5% Nonidet P-40, 0.5% sodium deoxycholate, 0.25% SDS, 5mM EDTA and Complete, EDTA-free Protease Inhibitor Cocktail Tablets (1873580, Roche)). The input cell lysate was briefly sonicated and lysed samples were separated via reducing SDS-PAGE on a 4–20% Tris-glycine gradient gel (Invitrogen). Proteins were then transferred onto a polyvinylidene difluoride membrane (Millipore, Billerica, MA). The membrane was incubated with primary antibodies, followed by horseradish peroxidase-conjugated secondary antibodies. Proteins were visualized using electrochemiluminescence (ECL) detection reagent (GE Healthcare, Milwaukee, WI) and an ImageQuant LAS 4000 digital imaging system (GE Healthcare).

Fixed cells or tissues were permeabilized in 0.2% Triton X-100 for 10 min. After blocking for nonspecific binding in TNB blocking buffer (NEL702, Perkin Elmer, Norwalk, CT), samples were incubated with primary antibodies, washed three times in PBS with 0.1% Tween-20, and incubated with secondary antibodies described above. Immunofluorescent images were obtained using a confocal microscope LSM700 (Carl Zeiss, Oberkochen, Germany).

Paraffin-embedded tissue sections were deparaffinized, pretreated with 0.3% H₂O₂ for 30 min, boiled in citrate solution (pH 6.0), and reacted with TNB blocking buffer, anti-PSP1 primary antibody, and biotin-conjugated donkey anti-rabbit IgG secondary antibody (711-066-152, Jackson Immunoresearch, West Grove, PA). Signals were enhanced with the Vectastain ABC kit (Vector Laboratories, Burlingame, CA). Visualization was performed with DAB (Wako, Osaka, Japan) and 1:20,000 dilution of saturated H₂O₂ to observe PSP1 immunopositivity in the nucleus of the motor neuron. The figures were examined using AxioVision imaging software (Carl Zeiss).

Total RNA was extracted from TRIzol reagent-treated samples using an RNAspin Mini RNA Isolation kit (74106, Qiagen, Germantown, MD) after chloroform/phenol extraction [14]. After treatment with DNase I, RT was carried out using Ready-To-Go You-Prime First-Strand Beads (27-9264-01, GE Healthcare) according to the manufacturer’s instructions. The resultant cDNA was measured by quantitative PCR with SYBR Premix Ex Taq (Perfect Real Time) (RR041A, Takara) in the Mx3000p system (Stratagene) using Rox as a reference and the following primers: qmNEAT1 FW/RV for mouse NEAT1_1/1_2 cDNA, qmNEAT1_2 FW/RV for mouse NEAT1_2 cDNA, and qmActin FW/RV for mouse β-actin cDNA. All primer sequences are listed in Additional file 8: Table S1. Primers for PCR to detect NEAT1_1 cDNA alone could not be designed; thus, the amount of NEAT1_1 expression was calculated by subtracting the copy number of NEAT 1_2 cDNA from that of total NEAT1 cDNA. The reaction was performed at 95°C for 10 min, followed by 50 cycles at 95°C for 30 s, 60°C for 1 min, and 72°C for 30 s after serial dilutions ranging from 10¹⁰, 10⁸, 10⁶, 10⁴, 10², to 10¹ copies per 1 μl of DNA solution were prepared as standard samples by subcloning each PCR product into the Zero Blunt TOPO PCR Cloning Kit (K2800, Invitrogen).

Statistical significance was determined using unpaired Student’s t-test. P < 0.05 was considered statistically significant. Error bars represent the standard deviation of the mean.