A novel PSMB8 isoform associated with multiple sclerosis lesions induces P-body formation

Abstract

Introduction: Multiple sclerosis (MS) is an inflammatory and demyelinating disease of the central nervous system (CNS). Current therapies primarily target the inflammatory component of the disease and are highly effective in early stages of MS while limited therapies have an effect in the more chronic progressive stages of MS where resident glia have a larger role. MS lesions tend to be inflammatory even after the initial peripheral immune cell invasion has subsided and this inflammation is known to cause alternative splicing events.

Methods: We used qPCR of normal-appearing white matter and white matter lesions from postmortem MS tissue, in vitro studies, and immunostaining in MS tissue to investigate the alternative splicing of one gene known to be important during recovery in an animal model of MS, PSMB8.

Results: We found a novel, intron-retained isoform which has not been annotated, upregulated specifically in MS patient white matter lesions. We found that this novel isoform activates the nonsense-mediated decay pathway in primary human astrocytes, the most populous glial cell in the CNS, and is then degraded. Overexpression of this isoform in astrocytes leads to an increased number of processing bodies in vitro, the primary site of mRNA decay. Finally, we demonstrated that MS white matter lesions have a higher burden of processing bodies compared to normal-appearing white matter, predominantly in GFAP-positive astrocytes.

Discussion: The increase in alternative splicing of the PSMB8 gene, the stress that this alternative splicing causes, and the observation that processing bodies are increased in white matter lesions suggests that the lesion microenvironment may lead to increased alternative splicing of many genes. This alternative splicing may blunt the protective or reparative responses of resident glia in and around white matter lesions in MS patients.

Keywords: alternative splicing; astrocytes; multiple sclerosis; neuroinflammation; nonsense-mediated decay.

Figure 1

PSMB8 undergoes alternative splicing in the MS WML. (A) Representative gel image of cDNA amplified with primers corresponding to exon 1B and exon 6 of PSMB8. cDNA was amplified from NAWM (left lane) or WML (right lane) to visualize the canonical PSMB8 isoform (918 bp) and a higher molecular weight isoform (1,076 bp, red box). (B) qPCR for total exon 1B was performed on cDNA from NAWM and WMLs. PSMB8 expression is shown as a fold change calculated from the ΔΔCt of PSMB8 to GAPDH and WML to NAWM. One sample was excluded due to lack of amplification in GAPDH. (C) i2R-PSMB8 was quantified in the same samples. The i2R-PSMB8 expression is shown as a fold change calculated from the ΔΔCt of i2R-PSMB8 to total exon 1B containing PSMB8 and WML to NAWM. Total exon 1B-containing PSMB8 was used for normalization rather than GAPDH as it is a better representation of how the splicing changes within the lesion. Data representative of two replicate experiments from the same cDNA. n = 5–6 patients with matched NAWM and WML samples. One WML was analyzed per patient. Significance was determined using a one-sample t-test. **p < 0.01.

Figure 2

i2R-PSMB8 induces P-body formation and leads to phosphorylation of UPF1. Human primary cortical astrocytes were transfected with vectors encoding either (A) intron 2 retained (i2R) or (B) full-length (FL) PSMB8 and co-transfected with a vector encoding green fluorescent protein (GFP) to identify positively transfected cells. (C) GFP⁺ cells were identified and the automated counting of the number of EDC4⁺ puncta (white) was performed using ImageJ. Scale bars (red) are 10 μm. Data representative of two independent experiments. Each point represents an individual transfected GFP⁺ cell. (D) Representative western blot images of phospho-UPF1 (top) and total UPF1 (bottom) of lysates from primary human cortical astrocytes transfected with vectors encoding either intron 2 retained (i2R) or full-length (FL) PSMB8. (E) The ratio of pUPF1 to total UPF1 was quantified. Data are from three independent experiments. Significance was determined by unpaired t test. *p < 0.05; ***p < 0.001.

Figure 3

i2R-PSMB8 undergoes NMD in astrocytes. qPCR measurements of intron 2 retained (i2R) or full-length (FL) PSMB8 transcript after the addition of cycloheximide. Data are normalized to time 0 and are representative of two independent experiments. Significance was determined by two-way ANOVA. F_3,16 = 18.09, p < 0.0001.

Figure 4

MS WMLs exhibit substantial EDC4 condensation. Representative merged confocal images of either (A) white matter lesion or (B) normal-appearing white matter stained for GFAP (red) and EDC4 (white). (Ai,ii) Single channel images of the representative chronic active lesion from MS patient 88. (Aiii) A representative merged 10X confocal image of the chronic active lesion characterization labeled for MHCII (Blue), MBP (Green), GFAP (Red), and EDC4 (White). (Bi,ii) Single channel images of the representative chronic active lesion from MS patient 88. (Biii) A representative merged 10X confocal image of tissue from patient 88 labeled for MHCII (Blue), MBP (Green), GFAP (Red), and EDC4 (White). Scale bars (yellow) in (A) and (B) are 10 μm. Scale bars (yellow) in (Aiii) and (Biii) are 100 μm. (C) 3D volumetric reconstruction of (A) showing buildup of EDC4 (white) in astrocytes (red; GFAP). Yellow arrowheads indicate EDC4-laden astrocytes. Scale bar is 10 μm. (D) Quantification of percent of astrocyte cell bodies laden with EDC4 per high powered field, data are representative of three patients, matched within patient, and 1–2 lesions were analyzed per patient. Statistics by two-sample t test. *p < 0.05.