Reduction of mutant huntingtin accumulation and toxicity by lysosomal cathepsins D and B in neurons

Abstract

Background: Huntington's disease is caused by aggregation of mutant huntingtin (mHtt) protein containing more than a 36 polyQ repeat. Upregulation of macroautophagy was suggested as a neuroprotective strategy to degrade mutant huntingtin. However, macroautophagy initiation has been shown to be highly efficient in neurons whereas lysosomal activities are rate limiting. The role of the lysosomal and other proteases in Huntington is not clear. Some studies suggest that certain protease activities may contribute to toxicity whereas others are consistent with protection. These discrepancies may be due to a number of mechanisms including distinct effects of the specific intermediate digestion products of mutant huntingtin generated by different proteases. These observations suggested a critical need to investigate the consequence of upregulation of individual lysosomal enzyme in mutant huntingtin accumulation and toxicity.

Results: In this study, we used molecular approaches to enhance lysosomal protease activities and examined their effects on mutant huntingtin level and toxicity. We found that enhanced expression of lysosomal cathepsins D and B resulted in their increased enzymatic activities and reduced both full-length and fragmented huntingtin in transfected HEK cells. Furthermore, enhanced expression of cathepsin D or B protected against mutant huntingtin toxicity in primary neurons, and their neuroprotection is dependent on macroautophagy.

Conclusions: These observations demonstrate a neuroprotective effect of enhancing lysosomal cathepsins in reducing mutant huntingtin level and toxicity in transfected cells. They highlight the potential importance of neuroprotection mediated by cathepsin D or B through macroautophagy.

Figure 1

Cathepsin D (CathD) and B (CathB) reduce Htt loads in HEK cells. HEK cells transfected by lipofectamine with different Htt and cathepsin cDNA constructs were harvested 48 hr later for real-time quantitative RT-PCR, western blot and enzymatic activity analyses. A. Transfection of cathepsin D (CathD) and B (CathB) increases their expression in HEK cells. HEK cells were transfected with 23QHtt, 23QHtt plus CathD, 23QHtt plus CathB, 145QmHtt, 145QmHtt plus CathD, and 145QmHtt plus CathB constructs for 48 hr. Western blot analyses were performed with anti-CathD and anti-CathB antibodies. β-actin western blots were used as loading controls. Pre-pro forms of CathD are 49 and 50 kD. Mature CathD is 32 kD. Mature CathB is 27 kD. Relative expression levels were quantified by band intensity. Student t-test was performed. *p < 0.05 compared to without cathepsin transfection. B. Increase of CathD and CathB enzymatic activities after transfection. HEK cells were transfected with CathD and CathB, and as described in A. CathD and CathB activities were assayed by CathD or CathB activity assay kit. *p < 0.05 compared to without cathepsin transfection. (C-E) Transfection of CathD and CathB reduce transfected Htt levels. HEK cells were transfected with 23QHtt, 23QHtt plus CathD, 23QHtt plus CathB, 145QmHtt, 145QmHtt plus CathD, and 145QmHtt plus CathB constructs. Western blot analyses were performed first with (C) 1C2 antibody that is specific for the polyQ of 145QmHtt, and then with (D) EM48 that preferentially recognizes the aggregates or (E) Ab2166 antibody that recognizes both 23Q and 145QHtt. β-actin western blots were used as loading controls. *p < 0.05 compared to without cathepsin transfection. †p < 0.05 compared to 23Q transfection.

Figure 2

Cathepsin D (CathD) and B (CathB) inhibitors exacerbate 145QmHtt-induced cell death in cortical neurons. A. 145QmHtt transfected neurons exhibited increased cell death. Primary cortical neurons were transfected with 23QHtt and 145QmHtt constructs. At 9 DIV, neurons were immunostained by anti-Htt Ab2166 and nuclei were stained by Hoechst. Arrows point to the pyknotic nuclei. Scale bar = 10 micron. B. 3-MA, pepstatin A and E64d exacerbated 145QmHtt-induced neuron death. The combined use of pepA and E64d further exacerbated 145Q-mhtt-induced neuron death. Primary cortical neurons (9 DIV) were treated with 3-MA (10mM), CathD and B inhibitors, pepstatin A (15 μM) and E64d (30 μM), respectively, for 24 hr. Con = empty vector control. Neuronal cell death was scored by counting pyknotic nuclei after Hoescht staining. There is no statistic significant difference in 23QHtt transfected neurons in the absence or presence of any of the chemicals. *p < 0.05 Student t-test compared to no inhibitor. †p < 0.05 compared to 23Q transfection. **p < 0.05 Student t-test compared to either pepA or E64d alone.

Figure 3

Cathepsin D (CathD) and B (CathB) expression and localization in primary neurons. A. Western blot analyses of CathD and CathB protein levels after transfection. Primary cortical neurons were transfected with 23QHtt, 23QHtt plus CathD, 23QHtt plus CathB, 145QmHtt, 145QmHtt plus CathD and 145QmHtt plus CathB constructs. Western blot analyses were performed with anti-CathD and anti-CathB antibodies. β-actin western blots were used as loading controls. Relative expression levels were quantified by band intensity. Positions of molecular weight markers were indicated. Quantification of the western blots are shown in the bar graphs. *p < 0.05 compared to without cathepsin transfection. B. Analyses of CathD and CathB enzymatic activities after transfection. Primary cortical neurons were transfected with CathD, CathB and as described in A. CathD and CathB activities were assayed by CathD or CathB activity assay kit. *p < 0.05 compared to without cathepsin transfection. C and D. Colocalization of transfected CathD (C) and CathB (D) with LAMP-1. CathD and CathB transfected neurons were examined by co-immunostaining of LAMP-1 and CathD or CathB. Yellow colored cytoplasmic spots are indicative of co-localization of transfected cathepsins and LAMP-1. Scale bar = 10 micron.

Figure 4

Cathepsin D (CathD) and B (CathB) reduce mHtt-induced toxicity in primary cortical neurons. A. Neurons were transfected with full length 23QHtt or 145QmHtt, either alone or co-transfected with CathD or CathB. Con = empty vector control. Cell death was increased by 145QmHtt and reduced by co-transfection with CathD or CathB. *p < 0.05 compared between 145QmHtt alone versus 145QmHtt co-transfection with CathD or CathB. †p < 0.05 compared between 145QmHtt and 23QHtt transfected cells. B. Blockade of autophagy reduces the effects of CathD and CathB in neuroprotection against 145QmHtt induced cortical neuron death. In 23QHtt transfected neurons, overexpression of CathD or CathB, or 3-MA inhibition alone did not cause neuron death. However, in these 23QHtt transfected neurons when autophagy is blocked by 3-MA, increasing CathD or CathB increased cell death. In 145QmHtt transfected neurons, CathD and CathB reduced 145QmHtt-induced neuron death. When autophagy is blocked by 3-MA, 145QmHtt is more toxic, and CathD or CathB enhancement could no longer reduce 145QmHtt-induced cell death. Con = empty vector control. †p < 0.05 compared between 23QHtt transfected versus 145QmHtt transfected cells. *p < 0.05 compared between 145Q and 145Q+CathD or CathB transfected cells. #p < 0.05 compared between transfected cells with 3-MA treatment and without 3-MA treatment. C. 145QmHtt increases LC3 II/LC3 I ratio. Primary neurons were transfected with 23QHtt, 23QHtt plus CathD, 23QHtt plus CathB, 145QmHtt, 145QmHtt plus CathD, and 145QmHtt plus CathB constructs. Western blot analyses were performed with an anti-LC3 antibody. β-actin western blots were used as loading controls. Immunoreactive bands were quantified and shown in the graphs. *p < 0.05 compared between 145QmHtt and 23QHtt transfection.

Figure 5

A model for neuroprotective effects of lysosomal cathepsins D and B against mHtt toxicity. A. In Huntington's disease conditions, mHtt cannot be completely degraded and accumulates, leading to neurotoxicity. 3-MA inhibits autophagy activity and exacerbates mHtt-induced neurotoxicity. B. Increasing lysosomal cathepsins D and B reduces mHtt accumulation, and confers neuroprotection. Neuroprotection is dependent on autophagy, since 3-MA reduces cells survival even in the presence of CathD or B.