NEAT1 is overexpressed in Parkinson's disease substantia nigra and confers drug-inducible neuroprotection from oxidative stress

Abstract

Recent reports attribute numerous regulatory functions to the nuclear paraspeckle-forming long noncoding RNA, nuclear enriched assembly transcript 1 (NEAT1), but the implications of its involvement in Parkinson's disease (PD) remain controversial. To address this issue, we assessed NEAT1 expression levels and cell type patterns in the substantia nigra (SN) from 53 donors with and without PD, as well as in interference tissue culture tests followed by multiple in-house and web-available models of PD. PCR quantification identified elevated levels of NEAT1 expression in the PD SN compared with control brains, an elevation that was reproducible across a multitude of disease models. In situ RNA hybridization supported neuron-specific formation of NEAT1-based paraspeckles at the SN and demonstrated coincreases of NEAT1 and paraspeckles in cultured cells under paraquat (PQ)-induced oxidative stress. Furthermore, neuroprotective agents, including fenofibrate and simvastatin, induced NEAT1 up-regulation, whereas RNA interference-mediated depletion of NEAT1 exacerbated death of PQ-exposed cells in a leucine-rich repeat kinase 2-mediated manner. Our findings highlight a novel protective role for NEAT1 in PD and suggest a previously unknown mechanism for the neuroprotective traits of widely used preventive therapeutics.-Simchovitz, A., Hanan, M., Niederhoffer, N., Madrer, N., Yayon, N., Bennett, E. R., Greenberg, D. S., Kadener, S., Soreq, H. NEAT1 is overexpressed in Parkinson's disease substantia nigra and confers drug-inducible neuroprotection from oxidative stress.

Keywords: lncRNA; neurodegeneration; paraspeckles.

Figure 1

Nuclear paraspeckles are formed in SN dopaminergic neurons. A) Intranuclear PSPC1 aggregates in TH-expressing cells (see Supplemental Movie S1). Scale bar, 10 μm. B) Human SN brain slices costained for TH and PSPC1 or NEAT1 show nuclear PSPC1 and NEAT1 staining of paraspeckles in dopaminergic neurons (arrows). Scale bar, 10 μm. C) Costaining of these slices as above reveals lack of TH and paraspeckle staining in support cells. Scale bar, 10 μm. D–F) Quantification of paraspeckle staining, showing a higher proportion of paraspeckle-forming nuclei in TH-positive compared with TH-negative cells for both PSPC1 (D), NEAT1 (E), and a higher number of paraspeckles per nucleus in TH-positive compared with TH-negative cells (F). ****P < 0.0001 (Fisher’s exact test or Mann-Whitney U test).

Figure 2

NEAT1 is elevated in the human parkinsonian SN and in a plethora of cellular and murine models of PD, increasing paraspeckle formation. A) NEAT1 origin on chromosome 11 (Chr. 11), splice variants, and the corresponding qPCR primers for variant analysis, not drawn to scale. B) mRNA levels for NEAT1 in a cohort of human SN samples (control n = 24; PD n = 29), determined by qPCR, measuring either both variants or the long variant alone (solid bars: controls, striped bars: PD; percent median of control group). C) qPCR-measured NEAT1 mRNA levels in control (Cont) and PQ-treated HEK-293T cells. Elevation is seen for the long variant only [n.s., not significant (P < 0.001 and P = 0.28, respectively), Welch’s t test]. D) qPCR-measured NEAT1 mRNA levels in control, PQ-exposed, and tBHP-exposed SH-SY5Y cells E–G) Confocal microscopy imaging and quantification of HEK-293T cells with single molecule RNA FISH targeting the long variant of NEAT1 and costained with DAPI for nuclear localization. Quantification (ImageJ) of paraspeckles per nucleus in control and treated cells. Note PQ-induced increase in the median number of paraspeckles per nucleus (E). No difference in the number of nuclei containing paraspeckles between control and PQ-treated cells [n.s., not significant (P = 0.57), χ² test] (F). Confocal microscopy images (G). Scale bar, 5 μm. Note nuclear location of paraspeckles in both control and PQ-treated nuclei and lack of extranuclear staining. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (Welch’s t test or Mann-Whitney U test).

Figure 3

NEAT1 knockdown (KD) abolishes paraspeckles and exposes HEK-293T and SH-SY5Y cells to PQ damage mediated by LRRK2 kinase activity. A) Scheme outlining the experimental procedure of the MTT assays. B) Confocal microscopy imaging of RNA FISH in HEK-293T cells, targeting both variants of NEAT1, and costained with DAPI for nuclear localization. Note nuclear paraspeckles (marked by arrows) in both control and knockdown cells, yet less prominent staining after knockdown. Scale bar, 10 μm. C) Quantification of nuclei that contain paraspeckles, showing a smaller proportion of paraspeckle-forming nuclei after NEAT1 knockdown). D) Quantification (ImageJ) of paraspeckles per nucleus in control and NEAT1 knockdown cells shows a decrease in the number of paraspeckles per nucleus. E) qPCR quantification of NEAT1 knockdown in HEK-293T cells; siRNA pools target and down-regulate both NEAT1 variants. F) qPCR quantification of NEAT1 knockdown in SH-SY5Y cells; 3 different long variant–specific LNA GapmeRs (G₁–G₃) target and down-regulate the long but not the short NEAT1 variant. G) Cell viability under NEAT1 knockdown was determined by MTT assay and shows exacerbated death of HEK-293T cells exposed to 2 mM PQ, which is abolished under treatment with the LRRK2 inhibitor PF-06447475 [n.s., not significant (P = 0.07), for NEAT1-induced change in cell mortality]. H) Cell viability under NEAT1 knockdown, determined by MTT assay, and showing exacerbated death of SH-SY5Y cells treated with 1 mM PQ, which is abolished under treatment with the LRRK2 inhibitor PF-06447475 (n.s., P = 0.49). I) Cell viability as above for SH-SY5Y cells exposed to 20 μM tBHP. *P < 0.05 (for long variant quantification, Welch’s t test), **P < 0.01 (Welch’s t test or Mann-Whitney U test), ***P < 0.001 (χ² test).

Figure 4

Various drugs induce up-regulation of the paraspeckle-forming long NEAT1 variant in multiple cell types. A) Bar plots depicting the expression profile of NEAT1 in cerebrocortical cultures under treatment with various drugs; data presented as z score for the difference between a single exposure to each drug and the average of 8 control samples (GSE110256); note the PPAR-α agonists marked in green and simvastatin in brown. B) Boxplot depicting microarray data of fenofibrate-induced up-regulation of NEAT1 in the murine liver (GSE68480, significance according to GEO microarray analysis algorithm). C) Boxplot depicting RNA-Seq data showing simvastatin-induced up-regulation of NEAT1 in HUVECs (HUVEC; GSE85799). CPM, counts per million. D) NEAT1 levels in SH-SY5Y cells treated with 80 μM fenofibrate or 10 μM simvastatin and compared with DMSO control, evaluated by qPCR using primers specific to the long variant (left) and for both variants (right) *P < 0.05 (Welch’s t test; n = 3/group), ****P < 0.0001 (significance according to edgeR statistical analysis).