Reduction of mutant huntingtin accumulation and toxicity by lysosomal cathepsins D and B in neurons
© Liang et al; licensee BioMed Central Ltd. 2011
Received: 9 January 2011
Accepted: 1 June 2011
Published: 1 June 2011
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© Liang et al; licensee BioMed Central Ltd. 2011
Received: 9 January 2011
Accepted: 1 June 2011
Published: 1 June 2011
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.
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.
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.
A common feature of neurodegenerative diseases, including Alzheimer's, Parkinson's and Huntington's diseases, is the accumulation of aggregation-prone proteins, such as β-amyloid in Alzheimer's disease, α-synuclein in Parkinson's disease and mutant huntingtin (mHtt) in Huntington's disease . It is generally thought that the response of the neuronal cell to these aggregated proteins determines whether cell death or dysfunction occurs . In this respect the autophagy-lysosomal pathway is particularly important. Lysosomal-mediated macroautophagy is largely responsible for degradation of intracellular damaged or aggregated proteins. The macroautophagy process involves formation of autophagosomes, transportation of damaged or aggregated proteins to the lysosomes, and degradation of these proteins by lysosomal proteases. Because of this capability for high capacity protein degradation inherent in macroautophagy the pathway has been identified as a potential target for the removal of mHtt protein. Previous studies have explored the potential of up-regulating autophagosomal formation by rapamycin, trehalose and lithium, and this resulted in the decreased mHtt aggregation and toxicity in vitro [2, 3]. Recent studies in the context of Alzheimer's disease models have indicated that macroautophagy is a highly efficient process in neurons, and the activities of lysosomal proteins are rate limiting in degrading aggregated proteins . If lysosomal activities are rate limiting, enhancing their activities may alleviate the burden to the proteasomes that are also involved in degradation of huntingtin [5, 6]. Supporting this notion, dysfunction in the lysosomal pathway has long been implicated in aging and neurodegenerative diseases [7–17]. Thus, investigating the impact of enhancing lysosomal proteins on mutant huntingtin accumulation and toxicity is of particular importance.
Lysosomal proteases that are highly expressed in the brain include the aspartate protease Cathepsin D (CathD) and the cysteine protease (CathB) [7–17]. Loss of cathepsins in processing damaged or aggregated proteins has been demonstrated in neurological disorders as well as mouse neurological disease models [7, 18–20]. For example, deficiency of CathB has been shown previously to exacerbate Aβ accumulation in a mouse model for Alzheimer's disease and overexpression of CathB has been shown to reduce Aβ load . In addition, we and others have previously shown that mice with deficient lysosomal CathD exhibited significant α-synuclein accumulation in their brains, indicating a critical role for CathD in mediating α-synuclein metabolism [19, 20]. This is important because α-synuclein mutation and gene amplification is responsible for a small subset of familial Parkinson's disease cases, and α-synuclein is a major component of Lewy bodies in a majority of sporadic Parkinson's disease patients . In vitro, we have shown that overexpression of CathD decreases the level of α-synuclein aggregation and protects against α-synuclein-mediated toxicity [19, 20]. Similarly, in Parkinson's disease research, proteolytic reduction of aggregation-prone and neurotoxic mutant huntingtin is important in Huntington's disease research. Because the huntingtin gene is essential for development , the simple reduction of the huntingtin gene may not be ideal therapeutic strategy. Allelic reduction of mutant huntingtin gene can prevent further production of the product, but lacks the potential to clear accumulated huntingtin toxic protein products. Determining which proteases are neuroprotective and which are detrimental to neurons is critically needed, considering the existence of proteasomes and distinct families of proteases in the cell that are capable of digesting mutant huntingtin to a varying extent [5, 6].
In the present study, we investigated whether enhancing individual lysosomal proteases may be effective in reducing mHtt using a range of molecular approaches. Because full-length mHtt is produced in Huntington's disease patients and may be more relevant to neuron cell death mechanisms , we investigated the effects of lysosomal enzymes on the toxicity of the full-length mHtt in neurons. Our finding indicated that lysosomal CathD and CathB reduced mHtt level, and protected against mHtt toxicity in primary neurons. Furthermore, CathD and CathB neuroprotective effects are dependent on autophagy.
3-methyladenine (3-MA), pepstatin A (PepA) and E64d were purchased from Sigma-Aldrich (St. Louis, MO, USA). Nucleofector Kit was from Lonza (Walkersville, MD, USA). Lipofectamine 2000 was from Invitrogen.
Anti-huntingtin monoclonal antibody 2166 (Ab2166), anti-polyglutamine expansion monoclonal antibody MAB1574 (clone 1C2) and anti-huntingtin monoclonal antibody (clone mEM48) were purchased from Millipore (Temecula, CA, USA). Anti-cathepsin D and Anti-cathepsin B antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-LAMP1 was from Novus Biologicals (Littleton CO, USA). Anti-LC3 antibody was from Sigma-Aldrich (St. Louis, MO, USA). Anti-β-actin monoclonal antibody was from Sigma-Aldrich. Fluorescent Alexa 488 (anti-mouse), fluorescent Alexa 568 (anti-goat) and fluorescent Alexa 488 (anti-rabbit) secondary antibodies were from Invitrogen (Carlsbad, CA, USA). Horseradish peroxidase-labeled secondary antibodies for enhanced chemiluminescence system detection were from Pierce (Rockford, IL USA).
Full-length Htt with 23 or 145 polyQ repeats using pcDNA vector were purchased from Invitrogen. Plasmids encoding human cathepsin D and B in pCMV-5a expression vectors were purchased from Origene.
HEK293 cells were grown in Dulbecco's modified Eagle's medium (DMEM) (Invitrogen) supplemented with 10% fetal bovine serum (FBS) (Atlanta Biologicals), and 100 U/ml penicillin/streptomycin (Invitrogen) at 37°C, and 5% CO2. Transfections were performed using lipofectamine 2000 according to the manufacturer's instructions. Cell death was measured by trypan blue exclusion, MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) colorimetric and Calcein AM viability assays.
The animal studies have been approved by the University of Alabama at Birmingham IACUC. Primary cortical neurons were obtained from embryonic day 18 (E18) embryos. Timed-pregnant Sprague-Dawley rats (Charles River Laboratories, Wilmington, MA, USA) were sacrificed by CO2 inhalation and embryos were collected in a Petri dish and placed on ice. Dissections were performed in ice cold Hanks' balanced sodium salts (without Ca2+ and Mg2+). Cerebral cortices were isolated and collected in a 15 ml Falcon tube. The tissues were incubated for 15 min at 37°C with papain (Worthington). The tissues were mechanically dissociated with a fire-polished Pasteur pipette. Cells were finally concentrated by centrifugation at 25°C for 5 min at 1000 × g and resuspended in Neurobasal medium containing 2% B27 supplement (Invitrogen), 1% Pen-Strep (10,000 U/ml, 10,000 μg/ml) and 0.5 mM L-glutamine. Electroporation was performed using Rat Nucleofector Kit with Nucleofector II machine. Cells were then plated in multiwell 24-well or 6-well plates coated with 0.1 mg/ml Poly-L-Lysine (Sigma). The cultures were kept in a humid incubator (5% CO2, 37°C) and half of the medium was changed once a week.
Cells on coverslips were washed by room temperature PBS and fixed in 4% paraformaldehyde for 45 min. After washing with PBS, the fixed cells were then permeabilized with PBS-T (0.1% Triton X-100 in PBS) for 3 min. The cells on coverslips were blocked in PBS supplemented with 3% BSA for 1 hour at room temperature. The cells were then incubated with a primary antibody in blocking solution overnight at 4°C and then washed 3 times with PBS. The secondary antibody conjugated to fluorophores was incubated for 1 hr at room temperature. Before mounting in fluoromount G on microscope slides, coverslips were incubated in 2 μg/ml Hoechst 33342 to label nuclear DNA. Stained cells were visualized by a fluorescent microscope (Leica Microsystems, Wetzlar, Germany). Staining was visualized on a Zeiss Axiocam CCD camera on a 100 W Axioscope bright field and fluorescence microscope.
For the cell death assay, primary cortical neurons were electroporated the day of plating with the rat neuron nucleofector kit (Lonza) and then plated in 24-well plates with coverslips. At 9 DIV (day in vitro), cells were fixed and immunostained with mAb2166 antibody as above, and nuclei counter-stained by Hoechst to identify degenerated neurons by counting mAb2166-positive neurons with nuclei shrinkage or fragmentation. We selected 15-20 random fields for each coverslip to take images for quantifying cell death. Each graph represents three independent experiments. Data are expressed as the percentage of neuronal cell death.
Total cellular extracts were collected in lysis buffer containing 50 mM Tris-HCl pH7.4, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100 and supplemented with protease inhibitor mixture (Roche). Homogenates were centrifuged at 10,000 × g for 15 min at 4°C. Protein concentrations were determined by detergent-compatible protein assay (Bio-Rad). Protein extracts were mixed with 5 × sample loading buffer and boiled for 5 min. Thirty to fifty micrograms of protein was resolved on 12% SDS-PAGE gel and transferred to nitrocellulose membranes. Membranes were blocked in 5% non-fat dry milk or 5% horse serum (for cathepsin D antibody detection) in TBST (50 mM Tris-HCl, 150 mM NaCl, pH7.4, 0.1% Tween 20) for 30 min at room temperature. The membranes were then incubated overnight at 4°C with primary antibodies: anti-mAb2166 1:1000; anti-1C2 1:5000; anti-EM48 1:1000; anti-cathepsin D 1:1000; anti-cathepsin B 1:1000; anti-LC3 1:1000 or anti-actin 1:5000. The membranes were then washed 4 times with TBST and incubated with horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. After washing for 40 min with TBST, the membranes were developed using enhanced chemiluminescence (ECL) substrate kit. We used U-Scan-IT software to quantify the western blot band intensity.
CathD activity was measured using an assay kit from Sigma. Cells were lysed in 20 mM MES pH6.8 containing 80 mM NaCl, 1 mM MgCl2, 2 mM EGTA, 10 mM NaH2PO4, proteinase inhibitor cocktail and phosphotase inhibitor cocktail. Ten μg of cell lysate were assayed in a 96-well plate according to protocol described by the manufacturer. CathB activity was measured using a kit from Biovision. Cells were lysed with the cell lysis buffer supplied with the kit. CathB activity was measured in a 96-well plate according to manufacturer's instruction.
Total RNA was extracted using TRIZOL reagent from Invitrogen. cDNA was synthesized using an iScript cDNA synthesis kit(BioRad) following the manufacturer's instructions.
Real-time PCR was performed to determine the mRNA levels of Htt and CathD and CathB. Primer sequences were: GAPDH, 5'- GCCAAAAGGGTCATCATCTC-3' and 5'GGCCATCCACAGTCTTCT-3'; CathD, 5'-TTCCCGAGGTGCTCAAGAACTACA -3'and 5'-TGTCGAAGACGACTGTGAAGCACT -3'; CathB, 5'-AAGCTTCGATGCACGGGAACAA-3' and 5'- ATGCAGATCCGGTCAGAGAT-3'.
Htt, 5'- CTCATTTCTCCGTCAGCACA-3' and 5'- CAAAGCTTCACAGCATCCAA-3'.
The mRNA level of GAPDH served as an internal control.
Data are reported as means ± SEM. Comparisons between two groups were performed with unpaired Student's t-tests. Comparisons among multiple groups or between two groups at multiple time-points were performed by either one-way or two-way analysis of variance, as appropriate. A p value of less than 0.05 was considered statistically significant.
Understanding the mechanisms of clearance of toxic mutant huntingtin is essential in order to explore therapeutic strategies against Huntington's disease. Here we examined the role of enhancing lysosomal CathB or D regarding the consequence of whether they are detrimental or protective against mutant huntingtin toxicity. We found that overexpression of full-length Htt with 23 polyQ (23QHtt) and mHtt with 145 polyQ (145QmHtt) had no effects on cell viability in HEK cells. This allowed an assessment of the ability of CathB and D to process huntingtin protein in the absence of the confounding effects of cell death. Prior studies indicated that CathD is more effective in reducing α-synuclein levels than CathB [19, 20]. Interestingly, in our studies, both proteases were capable of processing mHtt to an equivalent extent. This may be due to the much bigger size of the mHtt protein that allows for more proteolytic cleavage sites compared to α-synuclein. It is interesting to note that endogenous htt levels are not significantly decreased by CathD or CathB transfection. Prior studies demonstrated that endogenous normal htt plays an important role in development , although its exact role in neurons has not been well-established. Our observation that increased CathD and CathB only reduce excessive htt, without affecting basal level endogenous htt, is most likely because only in the case of overproduction of htt is the lysosomal system engaged for clearance of this protein. This would be consistent with a potential benefit of increasing these lysosomal activities for reduction of toxic proteins without reducing endogenous functional proteins. mHtt can be digested by proteasomes, caspases, cathepsins and gamma-secretase in a variety of cell models [5, 6, 27–29]. For example, inhibiting proteasomes by inhibitors such as ALLN and lactacystin was shown to promote the accumulation of NH2-terminal Htt fragments in both HEK293 cells and in brain tissues. Moreover, the endogenous NH2-terminal mutant Htt fragments accumulated in the brains by proteasome inhibition [5, 6, 27–29]. The Autophagy-lysosome pathway is a key clearance pathway for mHtt fragments. Induction of autophagy protects against polyglutamine toxicity and inhibition of autophagy has the converse effects [5, 6, 27–29]. Our observations indicate that in vitro, up-regulation of CathD or CathB does not lead to increased smaller mHtt fragments, but reduced overall full length and smaller fragments, consistent with a function of the lysosomal enzymes in complete rather than partial degradation of mHtt.
mHtt was shown to affect protein levels and maturation of CathD in optineurin/Rab8-dependent trafficking pathway from post-Golgi to lysosomes . However, in our studies, CathD and CathB mature enzymes are produced both in the absence and presence of mHtt. And the CathD and CathB enzymatic activities are similar in the absence and presence of mHtt. Furthermore, exogenously expressed CathD and CathB are localized to the lysosomes. There could be a number of reasons for these differences between ours and the prior studies, including: a) different cells and constructs for the expression of mHtt; and b) overexpressed CathD and CathB in our studies reduced the levels of mHtt below the levels needed to block protein trafficking.
145QmHtt over-expression in primary cortical neurons resulted in increased cytotoxicity which was further enhanced by the inhibitors of cathepsin B and D. 3-methyladenine (3-MA), which inhibits autophagosome formation due to its inhibitory effects on the VPS34-Beclin complex, had a similar effect on mHtt toxicity as the cathepsin inhibitors. The fact that the combined effects of cathepsin B and D inhibition were greater than either alone and similar to 3-MA suggest that both proteases make a significant contribution to mHtt degradation.
Prior studies have analyzed mTOR-dependent and independent pathways in stimulating macroautophagy . Enhancing macroautophagy may be beneficial to degradation of mHtt aggregates [2, 3, 31–34]. Rapamycin induces autophagosome formation and reduces mHtt aggregates, albeit this appears to be a somewhat inefficient process . One explanation is that autophagosomal activity is efficient in healthy neurons, such that accumulation of autophagosomes is a rare event . In contrast, lysosomal protease activities seem to be rate-limiting and easily disturbed as evidenced in a variety of lysosomal diseases , and their activities decline with age [10, 24, 35]. Recent studies also demonstrated that up-regulation of lysosomal biogenesis reduced mHtt levels in cell lines , highlighting the importance of the lysosomes. Surprisingly, we did not observe LC3 II/LC3 I conversion increase in CathD and CathB transfected neurons, one explanation may be the autophagy flux is too fast to measure LC3 II accumulation in CathD and CathB transfected neurons. Interestingly, our studies also observed that LC3 II/LC3 I ratio is upregulated in primary neuron cultures transfected with CathD and CathB in the presence of 23QHtt. This observation suggests a feedback regulation by signals from the lysosome to the machinery regulating autophagosomal formation. Recent studies suggest that mTOR may be in a complex on the lysosomal membrane and can serve as a signal for autophagy initiation . Future studies will critically investigate how lysosomal activities influence autophagy initiation through either an mTOR-dependent or an mTOR-independent pathway. In the HdhQ200 knock-in mouse model, mHtt aggregates colocalize with LC3 positive staining, suggesting accumulation of mHtt within uncleared autophagosomes . Consisting with this observation, our studies also showed 145QmHtt transfected neurons had increased LC3 II/LC3 I ratio compared to 23QHtt transfected neurons. CathD or CathB transfected neurons that express 145QmHtt exhibit reduced LC3 II/LC3 I ratio, suggesting CathD and CathB play a role in helping clear the accumulated autophagosomes in these neurons. Future studies will also need to critically investigate whether CathD and CathB can serve not only to attenuate nascent mHtt aggregate formation, but also to enhance clearance of existing pre-formed aggregates .
days in vitro
This work was supported by a grant from the CHDI foundation, NIHR01-NS064090 and a VA merit award to Dr. Jianhua Zhang, and by UAB Neuroscience Core Facilities (NS47466 and NS57098). We are grateful to Drs. Michelle Gray, Mathieu Lesort, Paula Dietrich, Ioannis Dragatsis, Deanna Marchionini and Victor Darley-Usmar for technical help and discussions.
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