Inflammatory genes are upregulated in expanded ataxin-3-expressing cell lines and spinocerebellar ataxia type 3 brains

Abstract

Spinocerebellar ataxia type 3 (SCA3) is a polyglutamine disorder caused by a CAG repeat expansion in the coding region of a gene encoding ataxin-3. To study putative alterations of gene expression induced by expanded ataxin-3, we performed PCR-based cDNA subtractive hybridization in a cell culture model of SCA3. In rat mesencephalic CSM14.1 cells stably expressing expanded ataxin-3, we found a significant upregulation of mRNAs encoding the endopeptidase matrix metalloproteinase 2 (MMP-2), the transmembrane protein amyloid precursor protein, the interleukin-1 receptor-related Fos-inducible transcript, and the cytokine stromal cell-derived factor 1alpha (SDF1alpha). Immunohistochemical studies of the corresponding or associated proteins in human SCA3 brain tissue confirmed these findings, showing increased expression of MMP-2 and amyloid beta-protein (Abeta) in pontine neurons containing nuclear inclusions. In addition, extracellular Abeta-immunoreactive deposits were detected in human SCA3 pons. Furthermore, pontine neurons of SCA3 brains strongly expressed the antiinflammatory interleukin-1 receptor antagonist, the proinflammatory cytokine interleukin-1beta, and the proinflammatory chemokine SDF1. Finally, increased numbers of reactive astrocytes and activated microglial cells were found in SCA3 pons. These results suggest that inflammatory processes are involved in the pathogenesis of SCA3.

Fig. 1.

Upregulated genes in CSM14.1 cell lines expressing expanded ataxin-3. For each identified gene, the altered expression was analyzed by Northern blot analyses of total RNA isolated from two individual clonal cell lines (clones 1 and 2) of each stable transfection (SCA3-Q23 and SCA3-Q70) and the mock-transfected control cell line (control) after 7 d of induced expression. Furthermore, differential expression of the identified genes was analyzed in both proliferating and neuronally differentiated cells at 33 and 39°C, respectively. A, Substantially altered mRNA levels were found for transcripts encoding MMP-2,APP, Fit-1S, and SDF1α in both Q70 clonal lines. Northern blot analysis was performed using 10 μg of RNA and the indicated cDNAs as probes. Equal loading of RNA in each lane is shown by methylene blue staining of 28S and 18S rRNA species (two representative images are shown). B, Northern (top panels) and Western blot analysis (bottom panels) showing the corresponding transgene levels of the respective SCA3 mRNAs and ataxin-3 protein isoforms in the different clonal SCA3 cell lines, respectively. The nonexpanded and expanded human full-length SCA3 mRNAs were detected as ∼1.4 and 1.5 kb transcripts, respectively, and were not present in the mock-transfected control cell line. The corresponding recombinant ataxin-3 isoforms were detected as 55 and 47 kDa immunoreactive bands for expanded and nonexpanded human full-length ataxin-3, respectively. The endogenous rat ataxin-3 appeared as 45 and 43 kDa immunoreactive bands.

Fig. 2.

Time course analysis of altered MMP-2gene expression in nonexpanded and expanded ataxin-3-expressing SCA3 cell lines. SCA-Q23 and SCA3-Q70 cells were cultured at 33°C (A) and 39°C (B) in either tetracycline-free medium for 1, 7, 14, and 21 d to induce or in tetracycline-containing medium for 1 d (1*) to suppress expression of the recombinant ataxin-3 isoforms. At the indicated times, total RNA and concentrated medium were prepared simultaneously from cultured cells and analyzed by Northern blot and gelatin zymography, respectively. Northern blot analysis (top panels) was performed using 10 μg of RNA of each sample and the MMP-2-specific cDNA probe detecting MMP-2mRNA as a single 3.0 kb band. Equal loading of RNA in each lane was verified by methylene blue staining of 28S and 18S RNA species (data not shown). Gelatin zymography (bottom panels) was performed using 50 μg of protein of concentrated medium from each condition. Cleared proteolytic zones indicated the presence of gelatinases at their respective molecular weights and were assigned to the latent form of pro MMP-2 with 68 kDa, the intermediate and fully activated form of MMP-2 with 64 and 62 kDa, respectively.

Fig. 3.

Increased cytoplasmic expression of MMP-2 and Aβ in pontine neurons containing ubiquitinated intranuclear inclusions in SCA3 pons. MMP-2 immunohistochemical staining of control pons (A) compared with diseased pons (B) showed several pontine neurons with strong cytoplasmic immunoreactivity (B andinset). Coimmunofluorescence staining of SCA3 pons sections using anti-ubiquitin (C, red) and anti-MMP-2 (D, green) demonstrated that a ubiqitinated NI (red) is present within the nucleus of the same MMP-2-positive neuron as confirmed by merging the images (E, yellow). Aβ immunohistochemical staining of control pons (F) compared with diseased pons (G, H) showed a large densely immunoreactive deposit (G andinset) and several pontine neurons with a distinct cytoplasmic staining (H and inset). Coimmunofluorescence staining of SCA3 pons sections using anti-ubiquitin (I, green) and anti-Aβ (J, red) demonstrated that ubiqitinated NIs (green) are present within the nucleus of the same Aβ-positive neuron as confirmed by merging the images (K, yellow). Tissue sections presented inA, B, F, G, and H were counterstained with hematoxylin. White arrowheads indicate the position of ubiquitinated NIs. Scale bars: A, B, F–H, 100 μm; C–E, I–K, insets, 10 μm.

Fig. 4.

Altered expression of the inflammatory mediators IL-1ra, IL-1β, and SDF1 and increased numbers of CD68- and IL-1β-immunoreactive glial cells in pons sections of diseased SCA3 brain. Comparison of IL-1ra-immunostained control (A) and disease pons sections (B, C) revealed a significantly increased cytoplasmic staining of pontine neurons (B and inset) and several immunoreactive plaque-like structures (C) in SCA3 patients. Comparison of IL-1β-immunostained control (D) and disease pons sections (E) showed an enhanced staining of pontine neurons (E andinset) in SCA3 patients. Immunohistochemical analysis using anti-SDF1 antibodies showed an intense staining of pontine neurons in SCA3 cases (G) that was also present in controls but to a much lesser extent (F). Immunostaining for CD68 revealed several positive perineuronal cells displaying a typical morphology of activated microglia in SCA3 pons (I), whereas CD68-positive cells were less frequently in controls (H). Immunohistochemical analysis against IL-1β showed a significant increase of reactive astrocytes in SCA3 pons (K) compared with control pons section (J). Tissue sections were counterstained with hematoxylin. Scale bars: A–K, 100 μm; insets, 10 μm.