Targeting ferroptosis: A novel therapeutic strategy for the treatment of mitochondrial disease-related epilepsy.

BACKGROUND: Mitochondrial disease is a family of genetic disorders characterized by defects in the generation and regulation of energy. Epilepsy is a common symptom of mitochondrial disease, and in the vast majority of cases, refractory to commonly used antiepileptic drugs. Ferroptosis is a recently-described form of iron- and lipid-dependent regulated cell death associated with glutathione depletion and production of lipid peroxides by lipoxygenase enzymes. Activation of the ferroptosis pathway has been implicated in a growing number of disorders, including epilepsy. Given that ferroptosis is regulated by balancing the activities of glutathione peroxidase-4 (GPX4) and 15-lipoxygenase (15-LO), targeting these enzymes may provide a rational therapeutic strategy to modulate seizure. The clinical-stage therapeutic vatiquinone (EPI-743, α-tocotrienol quinone) was reported to reduce seizure frequency and associated morbidity in children with the mitochondrial disorder pontocerebellar hypoplasia type 6. We sought to elucidate the molecular mechanism of EPI-743 and explore the potential of targeting 15-LO to treat additional mitochondrial disease-associated epilepsies. METHODS: Primary fibroblasts and B-lymphocytes derived from patients with mitochondrial disease-associated epilepsy were cultured under standardized conditions. Ferroptosis was induced by treatment with the irreversible GPX4 inhibitor RSL3 or a combination of pharmacological glutathione depletion and excess iron. EPI-743 was co-administered and endpoints, including cell viability and 15-LO-dependent lipid oxidation, were measured. RESULTS: EPI-743 potently prevented ferroptosis in patient cells representing five distinct pediatric disease syndromes with associated epilepsy. Cytoprotection was preceded by a dose-dependent decrease in general lipid oxidation and the specific 15-LO product 15-hydroxyeicosatetraenoic acid (15-HETE). CONCLUSIONS: These findings support the continued clinical evaluation of EPI-743 as a therapeutic agent for PCH6 and other mitochondrial diseases with associated epilepsy.

Rapid Assembly of Presynaptic Materials behind the Growth Cone in Dopaminergic Neurons Is Mediated by Precise Regulation of Axonal Transport.

The proper assembly of neural circuits depends on the process of synaptogenesis, or the formation of synapses between partner neurons. Using the dopaminergic PDE neurons in C. elegans, we developed an in vivo system to study the earliest steps of the formation of en passant presynaptic specializations behind an extending growth cone. We find that presynaptic materials coalesce into puncta in as little as a few minutes and that both synaptic vesicle (SV) and active zone (AZ) proteins arrive nearly simultaneously at the nascent sites of synapse formation. We show that precise regulation of UNC-104/Kinesin-3 determines the distribution of SV proteins along the axon. The localization of AZ proteins to en passant puncta, however, is largely independent of the major axonal kinesins: UNC-104/Kinesin-3 and UNC-116/Kinesin-1. Moreover, AZ proteins play a crucial role in recruiting and tethering SV precursors (SVPs).

The THO Complex Coordinates Transcripts for Synapse Development and Dopamine Neuron Survival.

Synaptic vesicle and active zone proteins are required for synaptogenesis. The molecular mechanisms for coordinated synthesis of these proteins are not understood. Using forward genetic screens, we identified the conserved THO nuclear export complex (THOC) as an important regulator of presynapse development in C. elegans dopaminergic neurons. In THOC mutants, synaptic messenger RNAs are retained in the nucleus, resulting in dramatic decrease of synaptic protein expression, near complete loss of synapses, and compromised dopamine function. CRE binding protein (CREB) interacts with THOC to mark synaptic transcripts for efficient nuclear export. Deletion of Thoc5, a THOC subunit, in mouse dopaminergic neurons causes severe defects in synapse maintenance and subsequent neuronal death in the substantia nigra compacta. These cellular defects lead to abrogated dopamine release, ataxia, and animal death. Together, our results argue that nuclear export mechanisms can select specific mRNAs and be a rate-limiting step for neuronal differentiation and survival.

Local inhibition of microtubule dynamics by dynein is required for neuronal cargo distribution.

Abnormal axonal transport is associated with neuronal disease. We identified a role for DHC-1, the C. elegans dynein heavy chain, in maintaining neuronal cargo distribution. Surprisingly, this does not involve dynein's role as a retrograde motor in cargo transport, hinging instead on its ability to inhibit microtubule (MT) dynamics. Neuronal MTs are highly static, yet the mechanisms and functional significance of this property are not well understood. In disease-mimicking dhc-1 alleles, excessive MT growth and collapse occur at the dendrite tip, resulting in the formation of aberrant MT loops. These unstable MTs act as cargo traps, leading to ectopic accumulations of cargo and reduced availability of cargo at normal locations. Our data suggest that an anchored dynein pool interacts with plus-end-out MTs to stabilize MTs and allow efficient retrograde transport. These results identify functional significance for neuronal MT stability and suggest a mechanism for cellular dysfunction in dynein-linked disease.

PCR Duplication: A One-Step Cloning-Free Method to Generate Duplicated Chromosomal Loci and Interference-Free Expression Reporters in Yeast.

Here, we report on a novel PCR targeting-based strategy called 'PCR duplication' that enables targeted duplications of genomic regions in the yeast genome using a simple PCR-based approach. To demonstrate its application we first duplicated the promoter of the FAR1 gene in yeast and simultaneously inserted a GFP downstream of it. This created a reporter for promoter activity while leaving the FAR1 gene fully intact. In another experiment, we used PCR duplication to increase the dosage of a gene in a discrete manner, from 1× to 2x. Using TUB4, the gene encoding for the yeast γ-tubulin, we validated that this led to corresponding increases in the levels of mRNA and protein. PCR duplication is an easy one-step procedure that can be adapted in different ways to permit rapid, disturbance-free investigation of various genomic regulatory elements without the need for ex vivo cloning.

Axon and dendritic trafficking.

Neuronal trafficking is crucial to the formation and dynamics of presynaptic and postsynaptic structures and the development and maintenance of axonal and dendritic processes. The mechanism for delivering specific organelles and synaptic molecules in axons and dendrites primarily depends on molecular motor proteins that move along the cytoskeleton. Adaptor proteins, regulatory molecules and local signaling pathways provide additional layers of specificity and control over bidirectional movement, polarized transport and cargo delivery. Here we review recent advances and emerging concepts related to the transport machinery of crucial neuronal components, such as mitochondria and presynaptic cargoes, and the mechanisms that modulate their polarized axo-dendritic sorting and synaptic delivery.

In vivo neuron-wide analysis of synaptic vesicle precursor trafficking.

During synapse development, synaptic proteins must be targeted to sites of presynaptic release. Directed transport as well as local sequestration of synaptic vesicle precursors (SVPs), membranous organelles containing many synaptic proteins, might contribute to this process. Using neuron-wide time-lapse microscopy, we studied SVP dynamics in the DA9 motor neuron in Caenorhabditis elegans. SVP transport was highly dynamic and bi-directional throughout the entire neuron, including the dendrite. While SVP trafficking was anterogradely biased in axonal segments prior to the synaptic domain, directionality of SVP movement was stochastic in the dendrite and distal axon. Furthermore, frequency of movement and speed were variable between different compartments. These data provide evidence that SVP transport is differentially regulated in distinct neuronal domains. It also suggests that polarized SVP transport in concert with local vesicle capturing is necessary for accurate presynapse formation and maintenance. SVP trafficking analysis of two hypomorphs for UNC-104/KIF1A in combination with mathematical modeling identified directionality of movement, entry of SVPs into the axon as well as axonal speeds as the important determinants of steady-state SVP distributions. Furthermore, detailed dissection of speed distributions for wild-type and unc-104/kif1a mutant animals revealed an unexpected role for UNC-104/KIF1A in dendritic SVP trafficking.

The balance between capture and dissociation of presynaptic proteins controls the spatial distribution of synapses.

The location, size, and number of synapses critically influence the specificity and strength of neural connections. In axons, synaptic vesicle (SV) and active zone (AZ) proteins are transported by molecular motors and accumulate at discrete presynaptic loci. Little is known about the mechanisms coordinating presynaptic protein transport and deposition to achieve proper distribution of synaptic material. Here we show that SV and AZ proteins exhibit extensive cotransport and undergo frequent pauses. At the axonal and synaptic pause sites, the balance between the capture and dissociation of mobile transport packets determines the extent of presynaptic assembly. The small G protein ARL-8 inhibits assembly by promoting dissociation, while a JNK kinase pathway and AZ assembly proteins inhibit dissociation. Furthermore, ARL-8 directly binds to the UNC-104/KIF1A motor to limit the capture efficiency. Together, molecular regulation of the dichotomy between axonal trafficking and local assembly controls vital aspects of synapse formation and maintenance.

Genetic dissection of synaptic specificity.

Nervous systems are built of a myriad of neurons connected by an even larger number of synapses. While it has been long known that neurons specifically select their synaptic partners among many possible choices during development, we only begin to understand how they make those decisions. Recent findings have started to elucidate the molecular mechanisms underlying synaptic target selection including positive as well as negative cues from synaptic partners, intermediate targets and surrounding tissues. Furthermore, emerging evidence suggests that synaptic connections are not only formed among specific sets of neurons, but also targeted to specific subcellular domains. Finally, spatial and temporal transcriptional regulation of these molecular cues represents an additional, versatile mechanism to provide wiring specificity.

An Arf-like small G protein, ARL-8, promotes the axonal transport of presynaptic cargoes by suppressing vesicle aggregation.

Presynaptic assembly requires the packaging of requisite proteins into vesicular cargoes in the cell soma, their long-distance microtubule-dependent transport down the axon, and, finally, their reconstitution into functional complexes at prespecified sites. Despite the identification of several molecules that contribute to these events, the regulatory mechanisms defining such discrete states remain elusive. We report the characterization of an Arf-like small G protein, ARL-8, required during this process. arl-8 mutants prematurely accumulate presynaptic cargoes within the proximal axon of several neuronal classes, with a corresponding failure to assemble presynapses distally. This proximal accumulation requires the activity of several molecules known to catalyze presynaptic assembly. Dynamic imaging studies reveal that arl-8 mutant vesicles exhibit an increased tendency to form immotile aggregates during transport. Together, these results suggest that arl-8 promotes a trafficking identity for presynaptic cargoes, facilitating their efficient transport by repressing premature self-association.

Two cyclin-dependent kinase pathways are essential for polarized trafficking of presynaptic components.

Polarized trafficking of synaptic proteins to axons and dendrites is crucial to neuronal function. Through forward genetic analysis in C. elegans, we identified a cyclin (CYY-1) and a cyclin-dependent Pctaire kinase (PCT-1) necessary for targeting presynaptic components to the axon. Another cyclin-dependent kinase, CDK-5, and its activator p35, act in parallel to and partially redundantly with the CYY-1/PCT-1 pathway. Synaptic vesicles and active zone proteins mostly mislocalize to dendrites in animals defective for both PCT-1 and CDK-5 pathways. Unlike the kinesin-3 motor, unc-104/Kif1a mutant, cyy-1 cdk-5 double mutants have no reduction in anterogradely moving synaptic vesicle precursors (SVPs) as observed by dynamic imaging. Instead, the number of retrogradely moving SVPs is dramatically increased. Furthermore, this mislocalization defect is suppressed by disrupting the retrograde motor, the cytoplasmic dynein complex. Thus, PCT-1 and CDK-5 pathways direct polarized trafficking of presynaptic components by inhibiting dynein-mediated retrograde transport and setting the balance between anterograde and retrograde motors.

The SpoMBe pathway drives membrane bending necessary for cytokinesis and spore formation in yeast meiosis.

Precise control over organelle shapes is essential for cellular organization and morphogenesis. During yeast meiosis, prospore membranes (PSMs) constitute bell-shaped organelles that enwrap the postmeiotic nuclei leading to the cellularization of the mother cell's cytoplasm and to spore formation. Here, we analysed how the PSMs acquire their curved bell-shaped structure. We discovered that two antagonizing forces ensure PSM shaping and proper closure during cytokinesis. The Ssp1p-containing coat at the leading edge of the PSM generates a pushing force, which is counteracted by a novel pathway, the spore membrane-bending pathway (SpoMBe). Using genetics, we found that Sma2p and Spo1p, a phospholipase, as well as several GPI-anchored proteins belong to the SpoMBe pathway. They exert a force all along the membrane, responsible for membrane bending during PSM biogenesis and for PSM closure during cytokinesis. We showed that the SpoMBe pathway involves asymmetric distribution of Sma2p and does not involve a GPI-protein-containing matrix. Rather, repulsive forces generated by asymmetrically distributed and dynamically moving GPI-proteins are suggested as the membrane-bending principle.

Spatial regulation of Fus3 MAP kinase activity through a reaction-diffusion mechanism in yeast pheromone signalling.

Signal transduction through mitogen-activated protein kinase (MAPK) cascades is thought to occur through the assembly of macromolecular complexes. We quantified the abundance of complexes in the cytoplasm among the MAPKs Ste11, Ste7, Fus3 and the scaffold protein Ste5 in yeast pheromone signalling using fluorescence cross-correlation spectroscopy (FCCS). Significant complex concentrations were observed that remained unchanged on pheromone stimulation, demonstrating that global changes in complex abundances do not contribute to the transmission of signal through the cytoplasm. On the other hand, investigation of the distribution of active Fus3 (Fus3(PP)) across the cytoplasm using fluorescence lifetime imaging microscopy (FLIM) revealed a gradient of Fus3(PP) activity emanating from the tip of the mating projection. Spatial partitioning of Fus3 activating kinases to this site and deactivating phosphatases in the cytoplasm maintain this Fus3(PP)-activity distribution. Propagation of signalling from the shmoo is, therefore, spatially constrained by a gradient-generating reaction-diffusion mechanism.