Neuronal induction of the immunoproteasome in Huntington's disease

Abstract

Huntington's disease (HD) inclusions are stained with anti-ubiquitin and anti-proteasome antibodies. This, together with proteasome activity studies on transfected cells, suggest that an impairment of the ubiquitin-proteasome system (UPS) may be key in HD pathogenesis. To test whether proteasome activity is impaired in vivo, we performed enzymatic assays for the three peptidase activities of the proteasome in brain extracts from the HD94 conditional mouse model of HD. We found no inhibition of any of the activities, suggesting that if UPS impairment happens in vivo, it is not at the level of the proteasome catalytic core. Intriguingly, the chymotrypsin- and trypsin-like activities increased selectively in the affected and aggregate-containing regions: cortex and striatum. Western blot analysis revealed no difference in total proteasome content whereas an increase in the interferon-inducible subunits of the immunoproteasome, LMP2 and LMP7, was observed. These subunits confer to the proteasome catalytic properties that are optimal for MHC-I peptide presentation. Immunohistochemistry in control mouse brain revealed LMP2 and LMP7 mainly in neurons. Accordingly, their increase in HD94 mice predominantly took place in neurons, and 5% of the ubiquitin-positive cortical aggregates were also LMP2-positive. Ultrastructural analysis of neurons with high level of immunoproteasome subunits revealed signs of neurodegeneration like nuclear indentation or fragmentation and dark cell appearance. The neuronal induction of LMP2 and LMP7 and the associated signs of neurodegeneration were also found in HD postmortem brains. Our results indicate that LMP2 and LMP7 participate in normal neuronal physiology and suggest a role in HD neurodegeneration.

Figure 1.

Increased chymotrypsin-like and trypsin-like proteasome activities in aggregate containing brain regions of the HD94 mice. A, Immunohistochemistry with anti-N-terminal htt (CAG53b) or anti-ubiquitin antibodies revealed the presence of aggregates in the cortex and the striatum, but not in the cerebellum, of HD94 mice. Both nuclear (empty arrows) and neuropil aggregates (black arrows) can be detected. Cx, Cortex; St, striatum; Cb, cerebellum; ML, molecular layer; PCL, Purkinje cell layer; GCL, granule cell layer. Scale bar, 20 μm. B, The chymotrypsin-like, trypsin-like, and post-glutamyl peptidase activities of the proteasome were assayed in brain extracts of control and HD94 mice by incubating with fluorogenic substrates as described in Materials and Methods. The chymotrypsin-like and trypsin-like activities (shown in arbitrary units) were significantly (^*p < 0.01) increased in the cortex and striatum of HD94 mice with respect to control littermates.

Figure 2.

Increased levels of the interferon-inducible subunits of the immunoproteasomes LMP2 and LMP7 in cortex and striatum of HD94 mice. A, Western blot analysis of brain cortical extracts of HD94 mice (HD94) and control littermates (C) with the MCP antibody (raised against native 20 S proteasome), the α1-α7 antibody (that recognizes six of the seven α-subunits of the 20 S proteasome), and the C2 (α6) antibody (raised against the C2 COOH-terminal region and that is able to discriminate the active and latent forms of the proteasome). Membranes are probed with an anti-tubulin antibody to correct for any possible deviation on protein loading. B, Western blot analysis of brain cortical (Cx) and striatal (St) extracts from 14-month-old HD94 mice and control littermates with antibodies against the LMP2 and LMP7 inducible subunits of the immunoproteasome. Membranes are probed with an anti-tubulin antibody to correct for any possible deviation on protein loading. C, Histograms showing the densitometric quantification of LMP2 and LMP7 levels in HD94 versus control samples in 3-, 7-, and 14-month-old mice (^*p < 0.01).

Figure 3.

LMP2 is present in neurons and increases in neurons of HD94 mice in which it can be found in ubiquitinated inclusions. Cortical sections from 14-month-old control (A), or HD94 mice (B, C, E-I). Cortical pyramidal neurons are revealed by immunohistochemistry with the (LMP2-13) LMP2 monoclonal antibody in control mice (A), and this neuronal staining is markedly increased in HD94 mice (B, D). C, The LMP2 polyclonal antibody confirmed the neuronal staining and also revealed LMP2-positive aggregates in the cortex of HD94 mice similar in shape (both ovoid, empty arrows, and spheroid, black arrows) to those detected with anti-ubiquitin antibodies. D, Histogram showing the number of LMP2- or LMP7-immunoreactive (IR) cells in the cortex of control and HD94 mice. E-I, Double immunofluorescence with anti-polyubiquitin (FK-2 antibody) and LMP2 polyclonal antibodies of HD94 cortical sections. E shows the abundance of ubiquitin-positive aggregates in a low-magnification image of the cortex of HD94 mice. White arrowheads in F-I show that a subset of ubiquitin-positive aggregates (F, H) are also LMP2-positive (G, I). Scale bars: (in B) A, B, 50 μm; C, 25 μm; (in E), E, 200 μm; F-I, 50 μm.

Figure 4.

HD94 cortical neurons with heightened levels of LMP2 show signs of neurodegeneration. Immunoelectronmicroscopy images of control (A) or HD94 (B, C) sections stained with LMP2. A, Neuron with cytoplasmic LMP2 staining in the cortex of a control mouse. B, Nuclear indentations (empty arrows) in an HD94 cortical LMP2-positive neuron. C, LMP2-positive dark neuron in the cortex of an HD94 mouse. Black arrows, Patches of reaction product. Scale bar, 1 μm.

Figure 5.

Increased levels of LMP2 and LMP7 in cortex and striatum of HD patients. A, Protein extracts were prepared from cortical and striatal postmortem samples of control and HD cases, resolved by SDS-PAGE, and immunoblotted with anti-LMP2, anti-LMP7, and anti-α-tubulin antibodies. B, Histograms showing the densitometric quantification of the increase in LMP2 and LMP7 in HD versus control samples (^*p < 0.01; ^**p < 0.005).

Figure 6.

The increase in LMP2 and LMP7 in HD brains takes place in neurons and correlates with TUNEL staining in the striatum. A-D, Cortical section from control (A, C) or HD brains (B, D) stained with anti-LMP2 (A, B) or anti-LMP7 (C, D) antibodies. E, F, LMP7 immunohistochemistry in the striatum of a control (E) or HD case (F). Arrows indicate medium-size spiny neurons with faint (black arrows) or strong (empty arrows) LMP7 staining. G, LMP2-immunostained medium-sized spiny neuron in the striatum of an HD case. H, LMP2 and TUNEL double-labeled medium-size spiny neuron in the striatum of an HD case. I, Histogram showing the number of LMP2- or LMP7-immunoreactive (IR) cells in the cortex of control and HD brains. J, Histogram showing the number of LMP2- or LMP7-immunoreactive cells in the striatum of control and HD brains. Scale bars: (in D) A-D, 40 μm; (in F) E, F, 50 μm; (in H) G, H, 10 μm.

Figure 7.

Ultrastructural analysis of LMP7-positive neurons from the cortex of HD patients. Immunoelectronmicroscopy images of cortical section from control (A) or HD human samples (B) stained with LMP7. A, Neuron with cytoplasmic LMP7 staining in the cortex of control subject. B, Nuclear indentations (empty arrows) in LMP7-positive cortical neurons from HD cases. Black arrows, Patches of reaction product. Arrowheads, Lipofuscin accumulations. Scale bar, 1 μm.