Loss of lysosomal ion channel transient receptor potential channel mucolipin-1 (TRPML1) leads to cathepsin B-dependent apoptosis

Mucolipidosis type IV (MLIV) is a lysosomal storage disease caused by mutations in the gene MCOLN1, which codes for the transient receptor potential family ion channel TRPML1. MLIV has an early onset and is characterized by developmental delays, motor and cognitive deficiencies, gastric abnormalities, retinal degeneration, and corneal cloudiness. The degenerative aspects of MLIV have been attributed to cell death, whose mechanisms remain to be delineated in MLIV and in most other storage diseases. Here we report that an acute siRNA-mediated loss of TRPML1 specifically causes a leak of lysosomal protease cathepsin B (CatB) into the cytoplasm. CatB leak is associated with apoptosis, which can be prevented by CatB inhibition. Inhibition of the proapoptotic protein Bax prevents TRPML1 KD-mediated apoptosis but does not prevent cytosolic release of CatB. This is the first evidence of a mechanistic link between acute TRPML1 loss and cell death.     

Cathepsins L and Z are critical in degrading polyglutamine-containing proteins within lysosomes

In neurodegenerative diseases caused by extended polyglutamine (polyQ) sequences in proteins, aggregation-prone polyQ proteins accumulate in intraneuronal inclusions. PolyQ proteins can be degraded by lysosomes or proteasomes. Proteasomes are unable to hydrolyze polyQ repeat sequences, and during breakdown of polyQ proteins, they release polyQ repeat fragments for degradation by other cellular enzymes. This study was undertaken to identify the responsible proteases. Lysosomal extracts (unlike cytosolic enzymes) were found to rapidly hydrolyze polyQ sequences in peptides, proteins, or insoluble aggregates. Using specific inhibitors against lysosomal proteases, enzyme-deficient extracts, and pure cathepsins, we identified cathepsins L and Z as the lysosomal cysteine proteases that digest polyQ proteins and peptides. RNAi for cathepsins L and Z in different cell lines and adult mouse muscles confirmed that they are critical in degrading polyQ proteins (expanded huntingtin exon 1) but not other types of aggregation-prone proteins (e.g. mutant SOD1). Therefore, the activities of these two lysosomal cysteine proteases are important in host defense against toxic accumulation of polyQ proteins.     

Regulator of calcineurin 1 (RCAN1) facilitates neuronal apoptosis through caspase-3 activation

Individuals with Down syndrome (DS) will inevitably develop Alzheimer disease (AD) neuropathology sometime after middle age, which may be attributable to genes triplicated in individuals with DS. The characteristics of AD neuropathology include neuritic plaques, neurofibrillary tangles, and neuronal loss in various brain regions. The mechanism underlying neurodegeneration in AD and DS remains elusive. Regulator of calcineurin 1 (RCAN1) has been implicated in the pathogenesis of DS. Our data show that RCAN1 expression is elevated in the cortex of DS and AD patients. RCAN1 expression can be activated by the stress hormone dexamethasone. A functional glucocorticoid response element was identified in the RCAN1 isoform 1 (RCAN1-1) promoter region, which is able to mediate the up-regulation of RCAN1 expression. Here we show that overexpression of RCAN1-1 in primary neurons activates caspase-9 and caspase-3 and subsequently induces neuronal apoptosis. Furthermore, we found that the neurotoxicity of RCAN1-1 is inhibited by knock-out of caspase-3 in caspase-3(-/-) neurons. Our study provides a novel mechanism by which RCAN1 functions as a mediator of stress- and Aβ-induced neuronal death, and overexpression of RCAN1 due to an extra copy of the RCAN1 gene on chromosome 21 contributes to AD pathogenesis in DS.     

Protein misfolding detected early in pathogenesis of transgenic mouse model of Huntington disease using amyloid seeding assay

Huntington disease (HD) is one of several fatal neurodegenerative disorders associated with misfolded proteins. Here, we report a novel method for the sensitive detection of misfolded huntingtin (HTT) isolated from the brains of transgenic (Tg) mouse models of HD and humans with HD using an amyloid seeding assay (ASA), which is based on the propensity of misfolded proteins to act as a seed and shorten the nucleation-associated lag phase in the kinetics of amyloid formation in vitro. Using synthetic polyglutamine peptides as the substrate for amyloid formation, we found that partially purified misfolded HTT obtained from end-stage brain tissue of two Tg HD mouse models and brain tissue of post-mortem human HD patients was capable of specifically accelerating polyglutamine amyloid formation compared with unseeded reactions and controls. Alzheimer and prion disease brain tissues did not do so, demonstrating the specificity of the ASA. It is unclear whether early intermediates or later conformational species in the protein misfolding process act as seeds in the ASA for HD. However, we were able to detect misfolded protein in the brains of YAC128 mice early in disease pathogenesis (11 weeks of age), whereas large inclusion bodies have not been observed in the brains of these mice by histology until 78 weeks of age, much later in the pathogenic process. The sensitive detection of misfolded HTT protein early in the disease pathogenesis in the YAC128 Tg mouse model strengthens the argument for a causative role of protein misfolding in HD.     

Tau isoform composition influences rate and extent of filament formation

The risk of developing tauopathic neurodegenerative disease depends in part on the levels and composition of six naturally occurring Tau isoforms in human brain. These proteins, which form filamentous aggregates in disease, vary only by the presence or absence of three inserts encoded by alternatively spliced exons 2, 3, and 10 of the Tau gene (MAPT). To determine the contribution of alternatively spliced segments to Tau aggregation propensity, the aggregation kinetics of six unmodified, recombinant human Tau isoforms were examined in vitro using electron microscopy assay methods. Aggregation propensity was then compared at the level of elementary rate constants for nucleation and extension phases. We found that all three alternatively spliced segments modulated Tau aggregation but through differing kinetic mechanisms that could synergize or compete depending on sequence context. Overall, segments encoded by exons 2 and 10 promoted aggregation, whereas the segment encoded by exon 3 depressed it with its efficacy dependent on the presence or absence of a fourth microtubule binding repeat. In general, aggregation propensity correlated with genetic risk reported for multiple tauopathies, implicating aggregation as one candidate mechanism rationalizing the correlation between Tau expression patterns and disease.     

Cysteine proteases bleomycin hydrolase and cathepsin Z mediate N-terminal proteolysis and toxicity of mutant huntingtin

N-terminal proteolysis of huntingtin is thought to be an important mediator of HD pathogenesis. The formation of short N-terminal fragments of huntingtin (cp-1/cp-2, cp-A/cp-B) has been demonstrated in cells and in vivo. We previously mapped the cp-2 cleavage site by mass spectrometry to position Arg167 of huntingtin. The proteolytic enzymes generating short N-terminal fragments of huntingtin remain unknown. To search for such proteases, we conducted a genome-wide screen using an RNA-silencing approach and an assay for huntingtin proteolysis based on the detection of cp-1 and cp-2 fragments by Western blotting. The primary screen was carried out in HEK293 cells, and the secondary screen was carried out in neuronal HT22 cells, transfected in both cases with a construct encoding the N-terminal 511 amino acids of mutant huntingtin. For additional validation of the hits, we employed a complementary assay for proteolysis of huntingtin involving overexpression of individual proteases with huntingtin in two cell lines. The screen identified 11 enzymes, with two major candidates to carry out the cp-2 cleavage, bleomycin hydrolase (BLMH) and cathepsin Z, which are both cysteine proteases of a papain-like structure. Knockdown of either protease reduced cp-2 cleavage, and ameliorated mutant huntingtin induced toxicity, whereas their overexpression increased the cp-2 cleavage. Both proteases partially co-localized with Htt in the cytoplasm and within or in association with early and late endosomes, with some nuclear co-localization observed for cathepsin Z. BLMH and cathepsin Z are expressed in the brain and have been associated previously with neurodegeneration. Our findings further validate the cysteine protease family, and BLMH and cathepsin Z in particular, as potential novel targets for HD therapeutics.     

Cystatin C Properties Crucial for Uptake and Inhibition of Intracellular Target Enzymes

To elucidate the molecular requirements for cancer cell internalization of the extracellular cysteine protease inhibitor cystatin C, 12 variants of the protein were produced and used for uptake experiments in MCF-7 cells. Variants with alterations in the cysteine cathepsin binding region ((Δ1–10)-, K5A-, R8G-, (R8G,L9G,V10G)-, (R8G,L9G,V10G,W106G)-, and W106G-cystatin C) were internalized to a very low extent compared with the wild-type inhibitor. Substitutions of N39 in the legumain binding region (N39K- and N39A-cystatin C) decreased the internalization and (R24A,R25A)-cystatin C, with substitutions of charged residues not involved in enzyme inhibition, was not taken up at all. Two variants, W106F- and K75A-cystatin C, showed that the internalization can be positively affected by engineering of the cystatin molecule. Microscopy revealed vesicular co-localization of internalized cystatin C with the lysosomal marker proteins cathepsin D and legumain. Activities of both cysteine cathepsins and legumain, possible target enzymes associated with cancer cell invasion and metastasis, were down-regulated in cell homogenates following cystatin C uptake. A positive effect on regulation of intracellular enzyme activity by a cystatin variant selected from uptake properties was illustrated by incubating cells with W106F-cystatin C. This resulted in more efficient down-regulation of intracellular legumain activity than when cells were incubated with wild-type cystatin C. Uptake experiments in prostate cancer cells corroborated that the cystatin C internalization is generally relevant and confirmed an increased uptake of W106F-cystatin C, in PC3 cells. Thus, intracellular cysteine proteases involved in cancer-promoting processes might be controled by cystatin uptake.     

Aggregation of detergent-insoluble tau is involved in neuronal loss but not in synaptic loss

Neurofibrillary tangles (NFTs), which consist of highly phosphorylated tau, are hallmarks of neurodegenerative diseases including Alzheimer disease (AD). In neurodegenerative diseases, neuronal dysfunction due to neuronal loss and synaptic loss accompanies NFT formation, suggesting that a process associated with NFT formation may be involved in neuronal dysfunction. To clarify the relationship between the tau aggregation process and synapse and neuronal loss, we compared two lines of mice expressing human tau with or without an aggregation-prone P301L mutation. P301L tau transgenic (Tg) mice exhibited neuronal loss and produced sarcosyl-insoluble tau in old age but did not exhibit synaptic loss and memory impairment. By contrast, wild-type tau Tg mice neither exhibited neuronal loss nor produced sarcosyl-insoluble tau but did exhibit synaptic loss and memory impairment. Moreover, P301L tau was less phosphorylated than wild-type tau, suggesting that the tau phosphorylation state is involved in synaptic loss, whereas the tau aggregation state is involved in neuronal loss. Finally, increasing concentrations of insoluble tau aggregates leads to the formation of fibrillar tau, which causes NFTs to form.     

Amyloid beta-mediated cell death of cultured hippocampal neurons reveals extensive Tau fragmentation without increased full-length tau phosphorylation

A variety of genetic and biochemical evidence suggests that amyloid β (Aβ) oligomers promote downstream errors in Tau action, in turn inducing neuronal dysfunction and cell death in Alzheimer and related dementias. To better understand molecular mechanisms involved in Aβ-mediated neuronal cell death, we have treated primary rat hippocampal cultures with Aβ oligomers and examined the resulting cellular changes occurring before and during the induction of cell death with a focus on altered Tau biochemistry. The most rapid neuronal responses upon Aβ administration are activation of caspase 3/7 and calpain proteases. Aβ also appears to reduce Akt and Erk1/2 kinase activities while increasing GSK3β and Cdk5 activities. Shortly thereafter, substantial Tau degradation begins, generating relatively stable Tau fragments. Only a very small fraction of full-length Tau remains intact after 4 h of Aβ treatment. In conflict with expectations based on suggested increases of GSK3β and Cdk5 activities, Aβ does not cause any major increases in phosphorylation of full-length Tau as assayed by immunoblotting one-dimensional gels with 11 independent site- and phospho-specific anti-Tau antibodies as well as by immunoblotting two-dimensional gels probed with a pan-Tau antibody. There are, however, subtle and transient increases in Tau phosphorylation at 3-4 specific sites before its degradation. Taken together, these data are consistent with the notion that Aβ-mediated neuronal cell death involves the loss of full-length Tau and/or the generation of toxic fragments but does not involve or require hyperphosphorylation of full-length Tau.     

Copper promotes the trafficking of the amyloid precursor protein

Accumulation of the amyloid β peptide in the cortical and hippocampal regions of the brain is a major pathological feature of Alzheimer disease. Amyloid β peptide is generated from the sequential protease cleavage of the amyloid precursor protein (APP). We reported previously that copper increases the level of APP at the cell surface. Here we report that copper, but not iron or zinc, promotes APP trafficking in cultured polarized epithelial cells and neuronal cells. In SH-SY5Y neuronal cells and primary cortical neurons, copper promoted a redistribution of APP from a perinuclear localization to a wider distribution, including neurites. Importantly, a change in APP localization was not attributed to an up-regulation of APP protein synthesis. Using live cell imaging and endocytosis assays, we found that copper promotes an increase in cell surface APP by increasing its exocytosis and reducing its endocytosis, respectively. This study identifies a novel mechanism by which copper regulates the localization and presumably the function of APP, which is of major significance for understanding the role of APP in copper homeostasis and the role of copper in Alzheimer disease.     

Discovery of ABT-957: 1-Benzyl-5-oxopyrrolidine-2-carboxamides as selective calpain inhibitors with enhanced metabolic stability

Aberrant activation of calpain has been observed in various pathophysiological disorders including neurodegenerative diseases such as Alzheimer’s Disease. Here we describe our efforts on ketoamide-based 1-benzyl-5-oxopyrrolidine-2-carboxamides as a novel series of highly selective calpain inhibitors mitigating the metabolic liability of carbonyl reduction. The most advanced compound from this new series, namely A-1212805 (ABT-957, Alicapistat) proceeded to clinical phase I studies.     

Identification of Functional Regions Defining Different Activity in Caspase-3 and Caspase-7 within Cells

Caspases are central to apoptosis, and the principal executioner caspases, caspase-3 and -7, were reported to be similar in activity, primary structure, and three-dimensional structure. Here, we identified different activity in caspase-3 and -7 within cells and examined the relationship between their structure and function using human cells expressing almost equal amounts of exogenous caspase-3, caspase-7, and/or chimeric constructs after down-regulation of endogenous caspase-3 and -7 expression. Caspase-3 (produced in human cells) showed much stronger cleaving activity than caspase-7 against a low molecular weight substrate in vitro dependent on four specific amino acid regions. Within cells, however, an additional three regions were required for caspase-3 to exert much stronger protease activity than caspase-7 against cellular substrates. Three of the former four regions and the latter three regions were shown to form two different three-dimensional structures that were located at the interface of the homodimer of procaspase-7 on opposite sides. In addition, procaspase-3 and -7 revealed specific homodimer-forming activity within cells dependent on five amino acid regions, which were included in the regions critical to the cleaving activity within cells. Thus, human caspase-3 and -7 exhibit differences in protease activity, specific homodimer-forming activity, and three-dimensional structural features, all of which are closely interrelated.     

Blastocystis Legumain Is Localized on the Cell Surface, and Specific Inhibition of Its Activity Implicates a Pro-survival Role for the Enzyme

Programmed cell death (PCD) is crucial for cellular growth and development in multicellular organisms. Although distinct PCD features have been described for unicellular eukaryotes, homology searches have failed to reveal clear PCD-related orthologues among these organisms. Our previous studies revealed that a surface-reactive monoclonal antibody (mAb) 1D5 could induce multiple PCD pathways in the protozoan Blastocystis. In this study, we identified, by two-dimensional gel electrophoresis and mass spectrometry, the target of mAb 1D5 as a surface-localized legumain, an asparagine endopeptidase that is usually found in lysosomal/acidic compartments of other organisms. Recombinant Blastocystis legumain displayed biphasic pH optima in substrate assays, with peaks at pH 4 and 7.5. Activity of Blastocystis legumain was greatly inhibited by the legumain-specific inhibitor carbobenzyloxy-Ala-Ala-AAsn-epoxycarboxylate ethyl ester (APE-RR) (where AAsn is aza-asparagine) and moderately inhibited by mAb 1D5, cystatin, and caspase-1 inhibitor. Interestingly, inhibition of legumain activity induced PCD in Blastocystis, observed by increased externalization of phosphatidylserine residues and in situ DNA fragmentation. In contrast to plants, in which legumains have been shown to play a pro-death role, legumain appears to display a pro-survival role in Blastocystis.     

Proteolytic Activation of the Essential Parasitophorous Vacuole Cysteine Protease SERA6 Accompanies Malaria Parasite Egress from Its Host Erythrocyte

The malaria parasite replicates within an intraerythrocytic parasitophorous vacuole (PV). The PV and host cell membranes eventually rupture, releasing merozoites in a process called egress. Certain inhibitors of serine and cysteine proteases block egress, indicating a crucial role for proteases. The Plasmodium falciparum genome encodes nine serine-repeat antigens (SERAs), each of which contains a central domain homologous to the papain-like (clan CA, family C1) protease family. SERA5 and SERA6 are indispensable in blood-stage parasites, but the function of neither is known. Here we show that SERA6 localizes to the PV where it is precisely cleaved just prior to egress by an essential serine protease called PfSUB1. Mutations that replace the predicted catalytic Cys of SERA6, or that block SERA6 processing by PfSUB1, could not be stably introduced into the parasite genomic sera6 locus, indicating that SERA6 is an essential enzyme and that processing is important for its function. We demonstrate that cleavage of SERA6 by PfSUB1 converts it to an active cysteine protease. Our observations reveal a proteolytic activation step in the malarial PV that may be required for release of the parasite from its host erythrocyte.     

Caspase-1 promiscuity is counterbalanced by rapid inactivation of processed enzyme

Members of the caspase family of cysteine proteases coordinate the highly disparate processes of apoptosis and inflammation. However, although hundreds of substrates for the apoptosis effector caspases (caspase-3 and caspase-7) have been identified, only two confirmed substrates for the key inflammatory protease (caspase-1) are known. Whether this reflects intrinsic differences in the substrate specificity of inflammatory versus apoptotic caspases or their relative abundance in vivo is unknown. To address this issue, we have compared the specificity of caspases-1, -3, and -7 toward peptide and protein substrates. Contrary to expectation, caspase-1 displayed concentration-dependent promiscuity toward a variety of substrates, suggesting that caspase-1 specificity is maintained by restricting its abundance. Although endogenous concentrations of caspase-1 were found to be similar to caspase-3, processed caspase-1 was found to be much more labile, with a half-life of ~9 min. This contrasted sharply with the active forms of caspase-3 and caspase-7, which exhibited half-lives of 8 and 11 h, respectively. We propose that the high degree of substrate specificity displayed by caspase-1 is maintained through rapid spontaneous inactivation of this protease.