FBS/BSA media concentration determines CCCP's ability to depolarize mitochondria and activate PINK1-PRKN mitophagy.

Mitochondrial quality control is essential for maintaining a healthy population of mitochondria. Two proteins associated with Parkinson disease, the kinase PINK1 and the E3 ubiquitin ligase PRKN, play a central role in the selective degradation of heavily damaged mitochondria (mitophagy), thus avoiding their toxic accumulation. Most of the knowledge on PINK1-PRKN mitophagy comes from in vitro experiments involving the treatment of mammalian cells with high concentrations of mitochondrial uncouplers, such as CCCP. These chemicals have been shown to mediate off target effects, other than mitochondrial depolarization. A matter of controversy between mitochondrial physiologists and cell biologists is the discrepancy between concentrations of CCCP needed to activate mitophagy (usually >10 µM), when compared to the much lower concentrations used to depolarize mitochondria (<1 µM). Thus, there is an urgent need for optimizing the current methods to assess PINK1-PRKN mitophagy in vitro. In this study, we address the utilization of high CCCP concentrations commonly used to activate mitophagy. Combining live fluorescence microscopy and biochemistry, we show that the FBS/BSA in the cell culture medium reduces the ability of CCCP to induce PINK1 accumulation at depolarized mitochondria, subsequent PRKN recruitment and ubiquitin phosphorylation, and ultimately mitochondrial clearance. As a result, high concentrations of CCCP are required to induce mitophagy in FBS/BSA containing media. These data unite mitochondrial physiology and mitophagy studies and are a first step toward a consensus on optimal experimental conditions for PINK1-PRKN mitophagy and mitochondrial physiology investigations to be carried out in parallel. Abbreviations: BSA: bovine serum albumin; CCCP: carbonyl cyanide m-chlorophenylhydrazone; DMEM: dulbecco's Modified Eagle's Medium; DNP: 2,4-dinitrophenol; FBS: fetal bovine serum; FCCP: carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone; GSH: glutathione; HBSS: Hanks' balanced salt solution; mtKeima: mitochondria-targeted monomeric keima-red; PBS: phosphate buffered saline; PD: Parkinson disease; PINK1: PTEN induced kinase 1; POE SHSY5Ys: FLAG-PRKN over-expressing SHSY5Y cells; SDS-PAGE: sodium dodecyl sulfate polyacrylamide gel electrophoresis; TMRM: tetramethylrhodamine methyl ester; WB: western blot; WT: wild-type; ΔΨm: mitochondrial membrane potential.

LRRK2 deficiency induced mitochondrial Ca2+ efflux inhibition can be rescued by Na+/Ca2+/Li+ exchanger upregulation.

Variants of leucine-rich repeat kinase 2 (lrrk2) are associated with an increased risk in developing Parkinson's disease (PD). Mitochondrial dysfunction and specifically mitochondrial Ca2+ handling has been linked to the pathogenesis of PD. Here we describe for the second time a mitochondrial Ca2+ efflux deficiency in a model displaying alterations in a PD-associated risk protein. LRRK2 deletion, inhibition and mutations led to an impaired mitochondrial Ca2+ extrusion via Na+/Ca2+/Li+ exchanger (NCLX) which in turn lowered mitochondrial permeability transition pore (PTP) opening threshold and increased cell death. The mitochondrial membrane potential was found not to be the underlying cause for the Ca2+ extrusion deficiency. NCLX activity was rescued by a direct (phosphomimetic NCLX mutant) and indirect (protein kinase A) activation which in turn elevated the PTP opening threshold. Therefore, at least two PD-associated risk protein pathways appear to converge on NCLX controlling mitochondrial Ca2+ extrusion and therefore mitochondrial health. Since mitochondrial Ca2+ overload has been described in many neurological disorders this study warrants further studies into NCLX as a potential therapeutic target.

α-synuclein oligomers interact with ATP synthase and open the permeability transition pore in Parkinson's disease.

Protein aggregation causes α-synuclein to switch from its physiological role to a pathological toxic gain of function. Under physiological conditions, monomeric α-synuclein improves ATP synthase efficiency. Here, we report that aggregation of monomers generates beta sheet-rich oligomers that localise to the mitochondria in close proximity to several mitochondrial proteins including ATP synthase. Oligomeric α-synuclein impairs complex I-dependent respiration. Oligomers induce selective oxidation of the ATP synthase beta subunit and mitochondrial lipid peroxidation. These oxidation events increase the probability of permeability transition pore (PTP) opening, triggering mitochondrial swelling, and ultimately cell death. Notably, inhibition of oligomer-induced oxidation prevents the pathological induction of PTP. Inducible pluripotent stem cells (iPSC)-derived neurons bearing SNCA triplication, generate α-synuclein aggregates that interact with the ATP synthase and induce PTP opening, leading to neuronal death. This study shows how the transition of α-synuclein from its monomeric to oligomeric structure alters its functional consequences in Parkinson's disease.

Mitochondrial calcium imbalance in Parkinson's disease.

Multiple factors are involved in the mechanism(s) of neuronal loss in neurodegenerative disorders whilst mitochondria are thought to play a central role in neurodegeneration of Parkinson's disease. Mitochondria are vital to cellular functions by supplying energy in form of ATP and affect cell physiology via calcium, ROS and signalling proteins. Changes in mitochondrial calcium homeostasis and ROS overproduction can induce cell death by triggering mitochondrial permeability transition pore opening. One of the major triggers for PTP is mitochondrial calcium overload. Mitochondrial Ca2+ homeostasis is regulated by electrogenic calcium uptake (via Ca2+ uniporter MCU) and efflux (in excitable cells via Na+/Ca2+ exchanger NCLX). NCLX inhibition has been described in a familial form of Parkinson's disease where PINK-1 deficiency leads to a delayed calcium efflux and mitochondrial Ca2+ overload in response to physiological Ca2+ stimulation. Overexpression of NCLX in PINK-1 deficient neurons not only protects against mitochondrial calcium overload and calcium induced cell death but also restores mitochondrial bioenergetics in these neurons. Mitochondrial NCLX might therefore play an important role in the mechanism(s) of neurodegeneration in a variety of neurodegenerative disorders and activation of this exchanger may offer a novel therapeutic target.

Clinical, pathological and functional characterization of riboflavin-responsive neuropathy.

Brown-Vialetto-Van Laere syndrome represents a phenotypic spectrum of motor, sensory, and cranial nerve neuropathy, often with ataxia, optic atrophy and respiratory problems leading to ventilator-dependence. Loss-of-function mutations in two riboflavin transporter genes, SLC52A2 and SLC52A3, have recently been linked to Brown-Vialetto-Van Laere syndrome. However, the genetic frequency, neuropathology and downstream consequences of riboflavin transporter mutations are unclear. By screening a large cohort of 132 patients with early-onset severe sensory, motor and cranial nerve neuropathy we confirmed the strong genetic link between riboflavin transporter mutations and Brown-Vialetto-Van Laere syndrome, identifying 22 pathogenic mutations in SLC52A2 and SLC52A3, 14 of which were novel. Brain and spinal cord neuropathological examination of two cases with SLC52A3 mutations showed classical symmetrical brainstem lesions resembling pathology seen in mitochondrial disease, including severe neuronal loss in the lower cranial nerve nuclei, anterior horns and corresponding nerves, atrophy of the spinothalamic and spinocerebellar tracts and posterior column-medial lemniscus pathways. Mitochondrial dysfunction has previously been implicated in an array of neurodegenerative disorders. Since riboflavin metabolites are critical components of the mitochondrial electron transport chain, we hypothesized that reduced riboflavin transport would result in impaired mitochondrial activity, and confirmed this using in vitro and in vivo models. Electron transport chain complex I and complex II activity were decreased in SLC52A2 patient fibroblasts, while global knockdown of the single Drosophila melanogaster riboflavin transporter homologue revealed reduced levels of riboflavin, downstream metabolites, and electron transport chain complex I activity. This in turn led to abnormal mitochondrial membrane potential, respiratory chain activity and morphology. Riboflavin transporter knockdown in Drosophila also resulted in severely impaired locomotor activity and reduced lifespan, mirroring patient pathology, and these phenotypes could be partially rescued using a novel esterified derivative of riboflavin. Our findings expand the genetic, clinical and neuropathological features of Brown-Vialetto-Van Laere syndrome, implicate mitochondrial dysfunction as a downstream consequence of riboflavin transporter gene defects, and validate riboflavin esters as a potential therapeutic strategy.

Mutations in valosin-containing protein (VCP) decrease ADP/ATP translocation across the mitochondrial membrane and impair energy metabolism in human neurons.

Mutations in the gene encoding valosin-containing protein (VCP) lead to multisystem proteinopathies including frontotemporal dementia. We have previously shown that patient-derived VCP mutant fibroblasts exhibit lower mitochondrial membrane potential, uncoupled respiration, and reduced ATP levels. This study addresses the underlying basis for mitochondrial uncoupling using VCP knockdown neuroblastoma cell lines, induced pluripotent stem cells (iPSCs), and iPSC-derived cortical neurons from patients with pathogenic mutations in VCP Using fluorescent live cell imaging and respiration analysis we demonstrate a VCP mutation/knockdown-induced dysregulation in the adenine nucleotide translocase, which results in a slower rate of ADP or ATP translocation across the mitochondrial membranes. This deregulation can explain the mitochondrial uncoupling and lower ATP levels in VCP mutation-bearing neurons via reduced ADP availability for ATP synthesis. This study provides evidence for a role of adenine nucleotide translocase in the mechanism underlying altered mitochondrial function in VCP-related degeneration, and this new insight may inform efforts to better understand and manage neurodegenerative disease and other proteinopathies.

Monomeric Alpha-Synuclein Exerts a Physiological Role on Brain ATP Synthase.

Misfolded α-synuclein is a key factor in the pathogenesis of Parkinson's disease (PD). However, knowledge about a physiological role for the native, unfolded α-synuclein is limited. Using brains of mice lacking α-, ß-, and γ-synuclein, we report that extracellular monomeric α-synuclein enters neurons and localizes to mitochondria, interacts with ATP synthase subunit α, and modulates ATP synthase function. Using a combination of biochemical, live-cell imaging and mitochondrial respiration analysis, we found that brain mitochondria of α-, ß-, and γ-synuclein knock-out mice are uncoupled, as characterized by increased mitochondrial respiration and reduced mitochondrial membrane potential. Furthermore, synuclein deficiency results in reduced ATP synthase efficiency and lower ATP levels. Exogenous application of low unfolded α-synuclein concentrations is able to increase the ATP synthase activity that rescues the mitochondrial phenotypes observed in synuclein deficiency. Overall, the data suggest that α-synuclein is a previously unrecognized physiological regulator of mitochondrial bioenergetics through its ability to interact with ATP synthase and increase its efficiency. This may be of particular importance in times of stress or PD mutations leading to energy depletion and neuronal cell toxicity. SIGNIFICANCE STATEMENT: Misfolded α-synuclein aggregations in the form of Lewy bodies have been shown to be a pathological hallmark in histological staining of Parkinson's disease (PD) patient brains. It is known that misfolded α-synuclein is a key driver in PD pathogenesis, but the physiological role of unfolded monomeric α-synuclein remains unclear. Using neuronal cocultures and isolated brain mitochondria of α-, ß-, and γ-synuclein knock-out mice and monomeric α-synuclein, this current study shows that α-synuclein in its unfolded monomeric form improves ATP synthase efficiency and mitochondrial function. The ability of monomeric α-synuclein to enhance ATP synthase efficiency under physiological conditions may be of importance when α-synuclein undergoes the misfolding and aggregation reported in PD.

Ca2+ is a key factor in α-synuclein-induced neurotoxicity.

Aggregation of α-synuclein leads to the formation of oligomeric intermediates that can interact with membranes to form pores. However, it is unknown how this leads to cell toxicity in Parkinson's disease. We investigated the species-specific effects of α-synuclein on Ca(2+) signalling in primary neurons and astrocytes using live neuronal imaging and electrophysiology on artificial membranes. We demonstrate that α-synuclein induces an increase in basal intracellular Ca(2+) in its unfolded monomeric state as well as in its oligomeric state. Electrophysiology of artificial membranes demonstrated that α-synuclein monomers induce irregular ionic currents, whereas α-synuclein oligomers induce rare discrete channel formation events. Despite the ability of monomeric α-synuclein to affect Ca(2+) signalling, it is only the oligomeric form of α-synuclein that induces cell death. Oligomer-induced cell death was abolished by the exclusion of extracellular Ca(2+), which prevented the α-synuclein-induced Ca(2+) dysregulation. The findings of this study confirm that α-synuclein interacts with membranes to affect Ca(2+) signalling in a structure-specific manner and the oligomeric ß-sheet-rich α-synuclein species ultimately leads to Ca(2+) dysregulation and Ca(2+)-dependent cell death.

Alpha-Synuclein Oligomers Interact with Metal Ions to Induce Oxidative Stress and Neuronal Death in Parkinson's Disease.

AIMS: Protein aggregation and oxidative stress are both key pathogenic processes in Parkinson's disease, although the mechanism by which misfolded proteins induce oxidative stress and neuronal death remains unknown. In this study, we describe how aggregation of alpha-synuclein (α-S) from its monomeric form to its soluble oligomeric state results in aberrant free radical production and neuronal toxicity. RESULTS: We first demonstrate excessive free radical production in a human induced pluripotent stem-derived α-S triplication model at basal levels and on application of picomolar doses of ß-sheet-rich α-S oligomers. We probed the effects of different structural species of α-S in wild-type rat neuronal cultures and show that both oligomeric and fibrillar forms of α-S are capable of generating free radical production, but that only the oligomeric form results in reduction of endogenous glutathione and subsequent neuronal toxicity. We dissected the mechanism of oligomer-induced free radical production and found that it was interestingly independent of several known cellular enzymatic sources. INNOVATION: The oligomer-induced reactive oxygen species (ROS) production was entirely dependent on the presence of free metal ions as addition of metal chelators was able to block oligomer-induced ROS production and prevent oligomer-induced neuronal death. CONCLUSION: Our findings further support the causative role of soluble amyloid oligomers in triggering neurodegeneration and shed light into the mechanisms by which these species cause neuronal damage, which, we show here, can be amenable to modulation through the use of metal chelation.

PKA Phosphorylation of NCLX Reverses Mitochondrial Calcium Overload and Depolarization, Promoting Survival of PINK1-Deficient Dopaminergic Neurons.

Mitochondrial Ca(2+) overload is a critical, preceding event in neuronal damage encountered during neurodegenerative and ischemic insults. We found that loss of PTEN-induced putative kinase 1 (PINK1) function, implicated in Parkinson disease, inhibits the mitochondrial Na(+)/Ca(2+) exchanger (NCLX), leading to impaired mitochondrial Ca(2+) extrusion. NCLX activity was, however, fully rescued by activation of the protein kinase A (PKA) pathway. We further show that PKA rescues NCLX activity by phosphorylating serine 258, a putative regulatory NCLX site. Remarkably, a constitutively active phosphomimetic mutant of NCLX (NCLX(S258D)) prevents mitochondrial Ca(2+) overload and mitochondrial depolarization in PINK1 knockout neurons, thereby enhancing neuronal survival. Our results identify an mitochondrial Ca(2+) transport regulatory pathway that protects against mitochondrial Ca(2+) overload. Because mitochondrial Ca(2+) dyshomeostasis is a prominent feature of multiple disorders, the link between NCLX and PKA may offer a therapeutic target.

A missense mutation in KCTD17 causes autosomal dominant myoclonus-dystonia.

Myoclonus-dystonia (M-D) is a rare movement disorder characterized by a combination of non-epileptic myoclonic jerks and dystonia. SGCE mutations represent a major cause for familial M-D being responsible for 30%-50% of cases. After excluding SGCE mutations, we identified through a combination of linkage analysis and whole-exome sequencing KCTD17 c.434 G>A p.(Arg145His) as the only segregating variant in a dominant British pedigree with seven subjects affected by M-D. A subsequent screening in a cohort of M-D cases without mutations in SGCE revealed the same KCTD17 variant in a German family. The clinical presentation of the KCTD17-mutated cases was distinct from the phenotype usually observed in M-D due to SGCE mutations. All cases initially presented with mild myoclonus affecting the upper limbs. Dystonia showed a progressive course, with increasing severity of symptoms and spreading from the cranio-cervical region to other sites. KCTD17 is abundantly expressed in all brain regions with the highest expression in the putamen. Weighted gene co-expression network analysis, based on mRNA expression profile of brain samples from neuropathologically healthy individuals, showed that KCTD17 is part of a putamen gene network, which is significantly enriched for dystonia genes. Functional annotation of the network showed an over-representation of genes involved in post-synaptic dopaminergic transmission. Functional studies in mutation bearing fibroblasts demonstrated abnormalities in endoplasmic reticulum-dependent calcium signaling. In conclusion, we demonstrate that the KCTD17 c.434 G>A p.(Arg145His) mutation causes autosomal dominant M-D. Further functional studies are warranted to further characterize the nature of KCTD17 contribution to the molecular pathogenesis of M-D.

Hypoxia signaling controls postnatal changes in cardiac mitochondrial morphology and function.

Fetal cardiomyocyte adaptation to low levels of oxygen in utero is incompletely understood, and is of interest as hypoxia tolerance is lost after birth, leading to vulnerability of adult cardiomyocytes. It is known that cardiac mitochondrial morphology, number and function change significantly following birth, although the underlying molecular mechanisms and physiological stimuli are undefined. Here we show that the decrease in cardiomyocyte HIF-signaling in cardiomyocytes immediately after birth acts as a physiological switch driving mitochondrial fusion and increased postnatal mitochondrial biogenesis. We also investigated mechanisms of ATP generation in embryonic cardiac mitochondria. We found that embryonic cardiac cardiomyocytes rely on both glycolysis and the tricarboxylic acid cycle to generate ATP, and that the balance between these two metabolic pathways in the heart is controlled around birth by the reduction in HIF signaling. We therefore propose that the increase in ambient oxygen encountered by the neonate at birth acts as a key physiological stimulus to cardiac mitochondrial adaptation.

An ancestral non-proteolytic role for presenilin proteins in multicellular development of the social amoeba Dictyostelium discoideum.

Mutations in either of two presenilin genes can cause familial Alzheimer's disease. Presenilins have both proteolysis-dependent functions, as components of the γ-secretase complex, and proteolysis-independent functions in signalling. In this study, we investigate a conserved function of human presenilins in the development of the simple model organism Dictyostelium discoideum. We show that the block in Dictyostelium development caused by the ablation of both Dictyostelium presenilins is rescued by the expression of human presenilin 1, restoring the terminal differentiation of multiple cell types. This developmental role is independent of proteolytic activity, because the mutation of both catalytic aspartates does not affect presenilin ability to rescue development, and the ablation of nicastrin, a γ-secretase component that is crucial for proteolytic activity, does not block development. The role of presenilins during Dictyostelium development is therefore independent of their proteolytic activity. However, presenilin loss in Dictyostelium results in elevated cyclic AMP (cAMP) levels and enhanced stimulation-induced calcium release, suggesting that presenilins regulate these intracellular signalling pathways. Our data suggest that presenilin proteins perform an ancient non-proteolytic role in regulating intracellular signalling and development, and that Dictyostelium is a useful model for analysing human presenilin function.

Nrf2 affects the efficiency of mitochondrial fatty acid oxidation.

Transcription factor Nrf2 (NF-E2 p45-related factor 2) regulates the cellular redox homoeostasis and cytoprotective responses, allowing adaptation and survival under conditions of stress. The significance of Nrf2 in intermediary metabolism is also beginning to be recognized. Thus this transcription factor negatively affects fatty acid synthesis. However, the effect of Nrf2 on fatty acid oxidation is currently unknown. In the present paper, we report that the mitochondrial oxidation of long-chain (palmitic) and short-chain (hexanoic) fatty acids is depressed in the absence of Nrf2 and accelerated when Nrf2 is constitutively active. Addition of fatty acids stimulates respiration in heart and liver mitochondria isolated from wild-type mice. This effect is significantly weaker when Nrf2 is deleted, whereas it is stronger when Nrf2 activity is constitutively high. In the absence of glucose, addition of fatty acids differentially affects the production of ATP in mouse embryonic fibroblasts from wild-type, Nrf2-knockout and Keap1 (Kelch-like ECH-associated protein 1)-knockout mice. In acute tissue slices, the rate of regeneration of FADH2 is reduced when Nrf2 is absent. This metabolic role of Nrf2 on fatty acid oxidation has implications for chronic disease conditions including cancer, metabolic syndrome and neurodegeneration.

Loss of PINK1 increases the heart's vulnerability to ischemia-reperfusion injury.

OBJECTIVES: Mutations in PTEN inducible kinase-1 (PINK1) induce mitochondrial dysfunction in dopaminergic neurons resulting in an inherited form of Parkinson's disease. Although PINK1 is present in the heart its exact role there is unclear. We hypothesized that PINK1 protects the heart against acute ischemia reperfusion injury (IRI) by preventing mitochondrial dysfunction. METHODS AND RESULTS: Over-expressing PINK1 in HL-1 cardiac cells reduced cell death following simulated IRI (29.2±5.2% PINK1 versus 49.0±2.4% control; N = 320 cells/group P<0.05), and delayed the onset of mitochondrial permeability transition pore (MPTP) opening (by 1.3 fold; P<0.05). Hearts excised from PINK1+/+, PINK1+/- and PINK1-/- mice were subjected to 35 minutes regional ischemia followed by 30 minutes reperfusion. Interestingly, myocardial infarct size was increased in PINK1-/- hearts compared to PINK1+/+ hearts with an intermediate infarct size in PINK1+/- hearts (25.1±2.0% PINK1+/+, 38.9±3.4% PINK1+/- versus 51.5±4.3% PINK1-/- hearts; N>5 animals/group; P<0.05). Cardiomyocytes isolated from PINK1-/- hearts had a lower resting mitochondrial membrane potential, had inhibited mitochondrial respiration, generated more oxidative stress during simulated IRI, and underwent rigor contracture more rapidly in response to an uncoupler when compared to PINK1+/+ cells suggesting mitochondrial dysfunction in hearts deficient in PINK1. CONCLUSIONS: We show that the loss of PINK1 increases the heart's vulnerability to ischemia-reperfusion injury. This may be due, in part, to increased mitochondrial dysfunction. These findings implicate PINK1 as a novel target for cardioprotection.

Molecular pharmacology in a simple model system: implicating MAP kinase and phosphoinositide signalling in bipolar disorder.

Understanding the mechanisms of drug action has been the primary focus for pharmacological researchers, traditionally using rodent models. However, non-sentient model systems are now increasingly being used as an alternative approach to better understand drug action or targets. One of these model systems, the social amoeba Dictyostelium, enables the rapid ablation or over-expression of genes, and the subsequent use of isogenic cell culture for the analysis of cell signalling pathways in pharmacological research. The model also supports an increasingly important ethical view of research, involving the reduction, replacement and refinement of animals in biomedical research. This review outlines the use of Dictyostelium in understanding the pharmacological action of two commonly used bipolar disorder treatments (valproic acid and lithium). Both of these compounds regulate mitogen activated protein (MAP) kinase and inositol phospholipid-based signalling by unknown means. Analysis of the molecular pathways targeted by these drugs in Dictyostelium and translation of discoveries to animal systems has helped to further understand the molecular mechanisms of these bipolar disorder treatments.

Protein kinase C signalling during miracidium to mother sporocyst development in the helminth parasite, Schistosoma mansoni.

For schistosomes, development of the miracidium to mother sporocyst within a compatible molluscan host requires considerable physiological and morphological changes by the parasite. The molecular mechanisms controlling such development have not been explored extensively. To begin to elucidate the importance of kinase-mediated signal transduction to this process, the phosphorylation (activation) of protein kinase C (PKC) in larval stages of Schistosoma mansoni undergoing in vitro transformation was explored. Mining of the S. mansoni genomic database revealed two S. mansoni PKC proteins with high homology to human PKCbeta and containing the conserved autophosphorylation (activation) site represented by serine 660 of human PKCbeta(II). Western blotting with anti-phosphospecific antibodies directed to this site demonstrated that miracidia freshly-hatched from eggs possessed PKC (78kDa) which was phosphorylated (activated) when miracidia were exposed to phorbol ester, and dephosphorylated (inhibited) following exposure to the PKC inhibitor GF109203X. Miracidia treated with the phospholipase C (PLC) inhibitor U73122 also displayed decreased PKC phosphorylation. S. mansoni PKC was phosphorylated during the initial 24h development of miracidia into mother sporocysts; after 31h and 48h development, phosphorylation was reduced by 72% and 86%, respectively. Confocal microscopy of miracidia revealed phosphorylated PKC associated with the neural mass, excretory vesicle, tegument, ciliated plates, terebratorium and germinal cells; in larvae undergoing transformation for 31h, phosphorylated PKC was only occasionally detected, being present in regions likely corresponding to the ridge cyton. Inhibition of PKC in miracidia by GF109230X resulted in accelerated transformation, particularly to the postmiracidium stage; ciliated plates were also shed from developing larvae more rapidly. These results highlight the dynamic nature of PKC signalling during S. mansoni postembryonic development and support a role for active PKC in restricting transformation of S. mansoni miracidia into mother sporocysts.