Longitudinal tracking of neuronal mitochondria delineates PINK1/Parkin-dependent mechanisms of mitochondrial recycling and degradation.

Altered mitochondrial quality control and dynamics may contribute to neurodegenerative diseases, including Parkinson's disease, but we understand little about these processes in neurons. We combined time-lapse microscopy and correlative light and electron microscopy to track individual mitochondria in neurons lacking the fission-promoting protein dynamin-related protein 1 (Drp1) and delineate the kinetics of PINK1-dependent pathways of mitochondrial quality control. Depolarized mitochondria recruit Parkin to the outer mitochondrial membrane, triggering autophagosome formation, rapid lysosomal fusion, and Parkin redistribution. Unexpectedly, these mitolysosomes are dynamic and persist for hours. Some are engulfed by healthy mitochondria, and others are deacidified before bursting. In other cases, Parkin is directly recruited to the matrix of polarized mitochondria. Loss of PINK1 blocks Parkin recruitment, causes LC3 accumulation within mitochondria, and exacerbates Drp1KO toxicity to dopamine neurons. These results define a distinct neuronal mitochondrial life cycle, revealing potential mechanisms of mitochondrial recycling and signaling relevant to neurodegeneration.

Apelinergic system in the kidney: implications for diabetic kidney disease.

The bioactive peptides of the apelinergic system and its receptor APJ have been shown to play a protective role in experimental cardiovascular and diabetic kidney disease (DKD). Mechanisms of this renoprotective effect remain to be elucidated. In this study, we examined the localization of APJ within the normal kidney and its kidney expression in the db/db model of DKD. The effect of hyperglycemia and angiotensin II on APJ was examined in cultured podocytes. In the glomerulus, APJ colocalized with podocyte but not endothelial cell markers. In podocytes stimulated with Pyr1 Apelin-13, a change in the phosphorylation status of the signaling proteins, AKT, ERK, and p70S6K, was observed with an increase 15 min after stimulation. Apelin-13 decreased activity of Caspase-3 in podocytes after high glucose treatment reflecting an antiapoptotic effect of APJ stimulation. In podocytes, APJ mRNA was downregulated in high glucose, when compared to normal glucose conditions and exposure to angiotensin II led to a further significant decrease in APJ mRNA. APJ and preproapelin mRNA levels in kidneys from db/db mice were markedly decreased along with decreased tubular APJ protein by western blotting and immunostaining when compared to db/m controls. In conclusion, the apelinergic system is decreased in kidneys from db/db mice. Within the glomerulus, APJ is mainly localized in podocytes and in this cell type its activation by Apelin-13 abolishes the proapoptotic effect of high glucose, suggesting a potential therapeutic role of apelin and emerging agonists with extended half-life for therapy of DKD.

Cysteine and Aspartyl Proteases Contribute to Protein Digestion in the Gut of Freshwater Planaria.

Proteases perform numerous vital functions in flatworms, many of which are likely to be conserved throughout the phylum Platyhelminthes. Within this phylum are several parasitic worms that are often poorly characterized due to their complex life-cycles and lack of responsiveness to genetic manipulation. The flatworm Schmidtea mediterranea, or planaria, is an ideal model organism to study the complex role of protein digestion due to its simple life cycle and amenability to techniques like RNA interference (RNAi). In this study, we were interested in deconvoluting the digestive protease system that exists in the planarian gut. To do this, we developed an alcohol-induced regurgitation technique to enrich for the gut enzymes in S. mediterranea. Using a panel of fluorescent substrates, we show that this treatment produces a sharp increase in proteolytic activity. These enzymes have broad yet diverse substrate specificity profiles. Proteomic analysis of the gut contents revealed the presence of cysteine and metallo-proteases. However, treatment with class-specific inhibitors showed that aspartyl and cysteine proteases are responsible for the majority of protein digestion. Specific RNAi knockdown of the cathepsin B-like cysteine protease (SmedCB) reduced protein degradation in vivo. Immunohistochemistry and whole-mount in situ hybridization (WISH) confirmed that the full-length and active forms of SmedCB are found in secretory cells surrounding the planaria intestinal lumen. Finally, we show that the knockdown of SmedCB reduces the speed of tissue regeneration. Defining the roles of proteases in planaria can provide insight to functions of conserved proteases in parasitic flatworms, potentially uncovering drug targets in parasites.

Loss of mitochondrial fission depletes axonal mitochondria in midbrain dopamine neurons.

Disruptions in mitochondrial dynamics may contribute to the selective degeneration of dopamine (DA) neurons in Parkinson's disease (PD). However, little is known about the normal functions of mitochondrial dynamics in these neurons, especially in axons where degeneration begins, and this makes it difficult to understand the disease process. To study one aspect of mitochondrial dynamics-mitochondrial fission-in mouse DA neurons, we deleted the central fission protein dynamin-related protein 1 (Drp1). Drp1 loss rapidly eliminates the DA terminals in the caudate-putamen and causes cell bodies in the midbrain to degenerate and lose α-synuclein. Without Drp1, mitochondrial mass dramatically decreases, especially in axons, where the mitochondrial movement becomes uncoordinated. However, in the ventral tegmental area (VTA), a subset of midbrain DA neurons characterized by small hyperpolarization-activated cation currents (Ih) is spared, despite near complete loss of their axonal mitochondria. Drp1 is thus critical for targeting mitochondria to the nerve terminal, and a disruption in mitochondrial fission can contribute to the preferential death of nigrostriatal DA neurons.

Foxc1 is required by pericytes during fetal brain angiogenesis.

Brain pericytes play a critical role in blood vessel stability and blood-brain barrier maturation. Despite this, how brain pericytes function in these different capacities is only beginning to be understood. Here we show that the forkhead transcription factor Foxc1 is expressed by brain pericytes during development and is critical for pericyte regulation of vascular development in the fetal brain. Conditional deletion of Foxc1 from pericytes and vascular smooth muscle cells leads to late-gestation cerebral micro-hemorrhages as well as pericyte and endothelial cell hyperplasia due to increased proliferation of both cell types. Conditional Foxc1 mutants do not have widespread defects in BBB maturation, though focal breakdown of BBB integrity is observed in large, dysplastic vessels. qPCR profiling of brain microvessels isolated from conditional mutants showed alterations in pericyte-expressed proteoglycans while other genes previously implicated in pericyte-endothelial cell interactions were unchanged. Collectively these data point towards an important role for Foxc1 in certain brain pericyte functions (e.g. vessel morphogenesis) but not others (e.g. barriergenesis).

Investigation of the proteolytic functions of an expanded cercarial elastase gene family in Schistosoma mansoni.

BACKGROUND: Cercarial elastase is the major invasive larval protease in Schistosoma mansoni, a parasitic blood fluke, and is essential for host skin invasion. Genome sequence analysis reveals a greatly expanded family of cercarial elastase gene isoforms in Schistosoma mansoni. This expansion appears to be unique to S. mansoni, and it is unknown whether gene duplication has led to divergent protease function. METHODS: Profiling of transcript and protein expression patterns reveals that cercarial elastase isoforms are similarly expressed throughout the S. mansoni life cycle. Computational modeling predicts key differences in the substrate-binding pockets of various cercarial elastase isoforms, suggesting a diversification of substrate preferences compared with the ancestral gene of the family. In addition, active site labeling of SmCE reveals that it is activated prior to exit of the parasite from its intermediate snail host. CONCLUSIONS: The expansion of the cercarial gene family in S. mansoni is likely to be an example of gene dosage. In addition to its critical role in human skin penetration, data presented here suggests a novel role for the protease in egress from the intermediate snail host. This study demonstrates how enzyme activity-based analysis complements genomic and proteomic studies, and is key in elucidating proteolytic function.

The Trypanosoma cruzi protease cruzain mediates immune evasion.

Trypanosoma cruzi is the causative agent of Chagas' disease. Novel chemotherapy with the drug K11777 targets the major cysteine protease cruzain and disrupts amastigote intracellular development. Nevertheless, the biological role of the protease in infection and pathogenesis remains unclear as cruzain gene knockout failed due to genetic redundancy. A role for the T. cruzi cysteine protease cruzain in immune evasion was elucidated in a comparative study of parental wild type- and cruzain-deficient parasites. Wild type T. cruzi did not activate host macrophages during early infection (<60 min) and no increase in ∼P iκB was detected. The signaling factor NF-κB P65 colocalized with cruzain on the cell surface of intracellular wild type parasites, and was proteolytically cleaved. No significant IL-12 expression occurred in macrophages infected with wild type T. cruzi and treated with LPS and BFA, confirming impairment of macrophage activation pathways. In contrast, cruzain-deficient parasites induced macrophage activation, detectable iκB phosphorylation, and nuclear NF-κB P65 localization. These parasites were unable to develop intracellularly and survive within macrophages. IL 12 expression levels in macrophages infected with cruzain-deficient T. cruzi were comparable to LPS activated controls. Thus cruzain hinders macrophage activation during the early (<60 min) stages of infection, by interruption of the NF-κB P65 mediated signaling pathway. These early events allow T. cruzi survival and replication, and may lead to the spread of infection in acute Chagas' disease.

Homeodomain interacting protein kinase 2 regulates postnatal development of enteric dopaminergic neurons and glia via BMP signaling.

Trophic factor signaling is important for the migration, differentiation, and survival of enteric neurons during development. The mechanisms that regulate the maturation of enteric neurons in postnatal life, however, are poorly understood. Here, we show that transcriptional cofactor HIPK2 (homeodomain interacting protein kinase 2) is required for the maturation of enteric neurons and for regulating gliogenesis during postnatal development. Mice lacking HIPK2 display a spectrum of gastrointestinal (GI) phenotypes, including distention of colon and slowed GI transit time. Although loss of HIPK2 does not affect the enteric neurons in prenatal development, a progressive loss of enteric neurons occurs during postnatal life in Hipk2(-/-) mutant mice that preferentially affects the dopaminergic population of neurons in the caudal region of the intestine. The mechanism by which HIPK2 regulates postnatal enteric neuron development appears to involve the response of enteric neurons to bone morphogenetic proteins (BMPs). Specifically, compared to wild type mice, a larger proportion of enteric neurons in Hipk2(-/-) mutants have an abnormally high level of phosphorylated Smad1/5/8. Consistent with the ability of BMP signaling to promote gliogenesis, Hipk2(-/-) mutants show a significant increase in glia in the enteric nervous system. In addition, numbers of autophagosomes are increased in enteric neurons in Hipk2(-/-) mutants, and synaptic maturation is arrested. These results reveal a new role for HIPK2 as an important transcriptional cofactor that regulates the BMP signaling pathway in the maintenance of enteric neurons and glia, and further suggest that HIPK2 and its associated signaling mechanisms may be therapeutically altered to promote postnatal neuronal maturation.

A contiguous compartment functions as endoplasmic reticulum and endosome/lysosome in Giardia lamblia.

The dynamic evolution of organelle compartmentalization in eukaryotes and how strictly compartmentalization is maintained are matters of ongoing debate. While the endoplasmic reticulum (ER) is classically envisioned as the site of protein cotranslational translocation, it has recently been proposed to have pluripotent functions. Using transfected reporter constructs, organelle-specific markers, and functional enzyme assays, we now show that in an early-diverging protozoan, Giardia lamblia, endocytosis and subsequent degradation of exogenous proteins occur in the ER or in an adjacent and communicating compartment. The Giardia endomembrane system is simple compared to those of typical eukaryotes. It lacks peroxisomes, a classical Golgi apparatus, and canonical lysosomes. Giardia orthologues of mammalian lysosomal proteases function within an ER-like tubulovesicular compartment, which itself can dynamically communicate with clathrin-containing vacuoles at the periphery of the cell to receive endocytosed proteins. These primitive characteristics support Giardia's proposed early branching and could serve as a model to study the compartmentalization of endocytic and lysosomal functions into organelles distinct from the ER. This system also may have functional similarity to the retrograde transport of toxins and major histocompatibility complex class I function in the ER of mammals.

PLIC proteins or ubiquilins regulate autophagy-dependent cell survival during nutrient starvation.

Ubiquilins (UBQLNs) are adaptor proteins thought to deliver ubiquitinated substrates to proteasomes. Here, we show a role for UBQLN in autophagy: enforced expression of UBQLN protects cells from starvation-induced death, whereas depletion of UBQLN renders cells more susceptible. The UBQLN protective effect requires the autophagy-related genes ATG5 and ATG7, two essential components of autophagy. The ubiquitin-associated domain of UBQLN mediates both its association with autophagosomes and its protective effect against starvation. Depletion of UBQLN delays the delivery of autophagosomes to lysosomes. This study identifies a new role for UBQLN in regulating the maturation of autophagy, expanding the involvement of ubiquitin-related proteins in this process.

Differential use of protease families for invasion by schistosome cercariae.

Schistosomes are parasitic platyhelminths (flatworms) of birds and mammals. As a parasitic disease of humans, schistosomiasis ranks second only to malaria in global importance. Schistosome larvae (cercariae) must invade and penetrate skin as an initial step to successful infection of the vertebrate host. Proteolytic enzymes secreted from the acetabular glands of cercariae contribute significantly to the invasion process. In this comparative study, we analyzed protease activities secreted by cercariae of Schistosoma mansoni, Schistosoma japonicum and Schistosomatium douthitti. Using protease-family specific, irreversible active-site probes, fluorogenic peptidyl substrates, immuno-histochemistry and high-resolution mass spectrometry, considerable species differences were noted in the quantity and character of proteases. Serine proteases, the most abundant enzymes secreted by S. mansoni cercariae, were not identified in S. japonicum. In contrast, the acetabular gland contents of S. japonicum cercariae had a 40-fold greater cathepsin B-like activity than those of S. mansoni. Based on the present data and previous reports, we propose that cysteine proteases represent an archetypal tool for tissue invasion among primitive metazoa and the use of serine proteases arose later in schistosome evolution. Computational analysis of serine protease phylogeny revealed an extraordinarily distant relationship between S. mansoni serine proteases and other members of the Clan PA family S1 proteases.

A multienzyme network functions in intestinal protein digestion by a platyhelminth parasite.

Proteases frequently function not only as individual enzymes but also in cascades or networks. A notable evolutionary switch occurred in one such protease network that is involved in protein digestion in the intestine. In vertebrates, this is largely the work of trypsin family serine proteases, whereas in invertebrates, cysteine proteases of the papain family and aspartic proteases assume the role. Utilizing a combination of protease class-specific inhibitors and RNA interference, we deconvoluted such a network of major endopeptidases functioning in invertebrate intestinal protein digestion, using the parasitic helminth, Schistosoma mansoni as an experimental model. We show that initial degradation of host blood proteins is ordered, occasionally redundant, and substrate-specific. Although inhibition of parasite cathepsin D had a greater effect on primary cleavage of hemoglobin, inhibition of cathepsin B predominated in albumin degradation. Nevertheless, in both cases, inhibitor combinations were synergistic. An asparaginyl endopeptidase (legumain) also synergized with cathepsin B and L in protein digestion, either by zymogen activation or facilitating substrate cleavage. This protease network operates optimally in acidic pH compartments either in the gut lumen or in vacuoles of the intestinal lining cells. Defining the role of each of these major enzymes now provides a clearer understanding of the function of a complex protease network that is conserved throughout invertebrate evolution. It also provides insights into which of these proteases are logical targets for development of chemotherapy for schistosomiasis, a major global health problem.

Functional expression and characterization of Schistosoma mansoni cathepsin B and its trans-activation by an endogenous asparaginyl endopeptidase.

Peptidases are essential for the establishment and survival of the medically important parasite, Schistosoma mansoni. This helminth expresses a number of gut-associated peptidases that degrade host blood proteins, including hemoglobin, as a means of nutrition. Using irreversible affinity probes, we demonstrate that S. mansoni cathepsin B-like endopeptidase 1 (SmCB1) is the most abundant papain family cysteine peptidase in both the parasite gut and somatic extracts. SmCB1 zymogen (SmCB1pm) was functionally expressed in Pichia pastoris (4-11mgl(-1)). Monospecific and immunoselected antibodies raised against SmCB1pm localized the enzyme exclusively to the gut lumen and surrounding gastrodermis of adult worms. Recombinant SmCB1pm was unable to catalyze its activation, even at low pH. However, recombinant S. mansoni asparaginyl endopeptidase (SmAE), another gut-associated cysteine peptidase, processed and activated SmCB1pm in trans. Consistent with the known specificity of AEs, processing occurred on the carboxyl side of an asparagine residue, two residues upstream of the start of the mature SmCB1 sequence. The remaining pro-region dipeptide was removed by rat cathepsin C (dipeptidyl-peptidase I)-an action conceivably performed by an endogenous cathepsin C in vivo. The activated recombinant SmCB1 is biochemically identical to the native enzyme with respect to dipeptidyl substrate kinetics and pH profiles. Also, the serum proteins, hemoglobin, serum albumin, IgG, and alpha-2 macroglobulin were efficiently degraded. Further, a novel application of an assay to measure the peptidyl carboxypeptidase activity of SmCB1 and other cathepsins B was developed using the synthetic substrate benzoyl-glycinyl-histidinyl-leucine (Bz-Gly-His-Leu). This study characterizes the major digestive cysteine peptidase in schistosomes and defines novel trans-processing events required to activate the SmCB1 zymogen in vitro which may facilitate the digestive process in vivo.

SmCB2, a novel tegumental cathepsin B from adult Schistosoma mansoni.

Papain-like cysteine endopeptidases have been recognized as potential targets for chemotherapy and serodiagnostic reagents in infections with the human parasitic helminth Schistosoma. A novel cathepsin B endopeptidase from adult S. mansoni has been isolated and characterized. The enzyme is termed SmCB2 to distinguish it from the first recorded schistosome cathepsin B, SmCB1, also known as Sm31. A rapid and convenient protocol involving anion exchange and affinity chromatography is described for the isolation of SmCB1 and SmCB2 from the same parasite starting material. SmCB2 has been functionally expressed in and purified from Pichia pastoris. Both native and recombinant SmCB2 migrate similarly (33 kDa) by SDS-PAGE. Both display strict acidic pH activity profiles and similar K(m) and k(cat) for dipeptidyl amidomethylcoumarin substrates. We conclude that the recombinant enzyme is properly folded. The S(2) subsite specificity of recombinant SmCB2 exhibits the preferences Phe>Leu>Val>>Arg. By immunoblotting with anti-SmCB2 IgG, a 33 kDa protein was identified in soluble extracts of male schistosomes. By immunohistochemistry, SmCB2 was localized in the tegumental tubercles and parenchyma of males with less product being visualized in the parenchyma of females. The enzyme may be lysosomal and function at the host parasite-interface.