Differential activities of the ubiquitin-proteasome system in neurons versus glia may account for the preferential accumulation of misfolded proteins in neurons - PubMed (original) (raw)

Differential activities of the ubiquitin-proteasome system in neurons versus glia may account for the preferential accumulation of misfolded proteins in neurons

Suzanne Tydlacka et al. J Neurosci. 2008.

Abstract

A variety of neurological disorders and polyglutamine (polyQ) diseases are caused by misfolded proteins. The common feature of these diseases is late-onset cellular degeneration that selectively affects neurons in distinct brain regions. polyQ diseases, including Huntington's disease (HD), present a clear case of selective neurodegeneration caused by polyQ expansion-induced protein misfolding, which also leads to predominant inclusions in neuronal nuclei. It remains unclear how these ubiquitously expressed disease proteins selectively kill neurons. In HD, mutant huntingtin accumulates in both neurons and glia, but more neuronal cells display huntingtin aggregates. These aggregates colocalize with components of the ubiquitin-proteasome system (UPS), which plays a critical role in clearing misfolded proteins. Using fluorescent reporters that reflect cellular UPS activity, we found that UPS activity in cultured neurons and glia decreases in a time-dependent manner. Importantly, UPS activity is lower in neurons than in glia and also lower in the nucleus than the cytoplasm. By expressing the UPS reporters in glia and neurons in the mouse brain, we also observed an age-dependent decrease in UPS activity, which is more pronounced in neurons than glial cells. Although brain UPS activities were similar between wild-type and HD 150Q knock-in mice, inhibiting the UPS markedly increases the accumulation of mutant htt in cultured glial cells. These findings suggest that the lower neuronal UPS activity may account for the preferential accumulation of misfolded proteins in neurons, as well as their selective vulnerability.

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Figures

Figure 1.

Different accumulations of mutant htt in neuronal versus glial cells in HD mouse brains. A, EM48 immunohistochemical staining of the striatum (Str) and white matter (WM) of the corpus callosum of wild-type (WT) and HD150Q knock-in (KI) mice at 15 months of age. Note that EM48 labels more neuronal cells in the striatum than glial cells in the corpus callosum. B, EM48 immunohistochemical staining of an R6/2 mouse brain that expresses exon1 mutant htt. Glial cells in the white matter (WM) of the corpus callosum and in neuronal cells of the striatum (Str) show different extents of mutant htt accumulation. The mouse was examined at the age of 12 weeks. Scale bars, 10 μm.

Figure 2.

Expression of proteasomal reporters in neuronal cells. A, Schematic maps of DNA constructs for expressing fluorescent UPS reporters. GFPu is a green fluorescent protein tagged with a CL-1 degron sequence specific for ubiquitination and degradation by the proteasome. RFP serves as a control. Both GFPu and RFP were coexpressed by the cytomegalovirus (CMV) promoter in adenoviral vectors. B, Expression of GFPu/RFP in PC12 cells via adenoviral infection. Inhibiting the proteasome by MG132 (10 μ

for 12 h) increased GFPu signals. C, Western blotting of the infected PC12 cells showing that MG132 treatment increased protein ubiquitination (top blot) and GFPu compared with RFP and tubulin. D, Inhibiting the proteasome by MG132 (10 μ

) also increased GFPu signal in cultured primary neurons that were isolated from rat cerebral cortex and had been infected by adenoviral GFPu/RFP for 48 h. E, The ratio of GFPu to RFP in cultured cortical neurons after MG132 treatment. C, Uninfected control cells. **p < 0.01. F, Western blotting of cultured cortical neurons also showed an increased level of GFPu after proteasome inhibition by MG132. Scale bars, 10 μm.

Figure 3.

Time-dependent increase of GFPu levels in cultured primary neurons. A, Fluorescent images showing a greater increase in GFPu signal (top) than RFP (bottom) in cultured cerebral cortical neurons in a time-dependent manner. The cultured neurons at different days (8, 11, and 17 DIV) were infected by adenoviral GFPu/RFP for 2 d before examination. B, High-magnification graphs showing the expression of GFPu and RFP in the same neuron at 17 DIV. Scale bars, 10 μm. C, Quantification of the ratio (mean + SEM, n = 8–17) of GFPu to RFP in cultured primary neurons. **p < 0.01, ***p < 0.001. D, Western blot analysis showing the increased level of GFPu in old cultured neurons. The same blot was also probed with antibodies to RFP and tubulin.

Figure 4.

Expression of GFPu/RFP in cultured astrocytes at different days of culture. A, Fluorescent images of astrocytes that had been cultured for different days (4, 8, 12, 16, and 20) and then infected by adenoviral GFPu/RFP for 2 d. Note that GFPu signals increased only slightly or remained at similar levels in older astrocytes. Scale bar, 10 μm. B, Fluorescent images of mixed cultured cells containing neurons (arrows) and astrocytes that had been cultured for 16 or 20 d and then infected by adenoviral GFPu/RFP for 2 d. Note that GFPu signal (green) is higher in neuronal cells than in glial cells.

Figure 5.

Quantitative analysis of proteasomal activities in cultured neuronal and glial cells. A, The ratios (mean + SEM, n = 8–18) of GFPu to RFP in cultured neurons or astrocytes at different culturing days. B, Biochemical assays of chymotrypsin-like and trypsin-like activities (mean + SEM) of cultured neurons and astrocytes that had been cultured for various days (4–20 d). **p < 0.01, ***p < 0.001.

Figure 6.

Targeting GFPu/RFP to the nucleus and cytoplasm of cultured cells. A, NLS–GFPu/RFP (top) or NES–GFPu/RFP (bottom) were transfected into cultured rat brain cortical neurons at 8 DIV. Note that nuclear NLS–GFPu is more intense than cytoplasmic NES–GFPu. B, Cultured astrocytes at 20 DIV were infected with adenoviral NLS–GFPu/RFP (top) or NES–GFPu/RFP (bottom). Note that nuclear NLS–GFPu signal is also more intense than cytoplasmic NES–GFPu. In A and B, merged images show the Hoechst-stained nuclei (blue). Scale bars, 10 μm. C, Quantification of the ratios (mean + SEM, n = 10–16) for NLS–GFPu/RFP in the nucleus and NES–GFPu/RFP in the cytoplasm in glia and neurons. **p < 0.01.

Figure 7.

Expression of GFPu/RFP reporters in mouse brains. Stereotaxic injection of adenoviral GFPu/RFP into the white matter of the corpus callosum (glia) and the striatum (neuron) to examine their expression in glial and neuronal cells, respectively, in mice at different ages (4, 12, and 24 months). A, Low-magnification micrographs showing RFP and GFPu signals in the injected regions. Note that the neuronal level of GFPu is higher than the glial GFPu level, suggesting lower UPS activity in neuronal cells. B, High-magnification micrographs (630×) showing the distribution of GFPu and RFP in the infected neurons (top) and glial cells (bottom). Arrows indicate neuronal or glial nuclei that were labeled by Hoechst dye. Scale bars, 10 μm.

Figure 8.

Differential UPS activities in neurons and glia. A, Quantification of the ratios of adenoviral GFPu to RFP in the infected neurons and glial cells in the brains of mice at 4, 12, and 24 months of age. The ratios of GFPu to RFP in glial cells are lower than neuronal GFPu/RFP in the striatum and brain cortex at different ages. *p < 0.05, **p < 0.01, ***p < 0.001. B, The ratios of GFPu to RFP in the glia and striatal neurons in the adenoviral GFPu/RFP-injected brains of wild-type (WT) and HD150Q knock-in (KI) mice at the age of 2, 12, and 24 months. No significant difference in the ratios of GFPu to RFP was seen between wild-type and HD150Q KI cells, although the ratio was lower in glia than neurons and increased in the old mouse brains. C, Cultured astrocytes (8 DIV) from wild-type and HD150Q KI mice were treated with MG132 (10 μ

) for 12 h. The accumulation of mutant htt and its fragments, which were recognized by an antibody (1C2) specific to expanded polyQ tracts, was increased by using MG132 to inhibit the proteasome. The blots were also probed with anti-tubulin.

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Differential activities of the ubiquitin-proteasome system in neurons versus glia may account for the preferential accumulation of misfolded proteins in neurons - PubMed (original) (raw)