Yeast NDI1 reconfigures neuronal metabolism and prevents the unfolded protein response in mitochondrial complex I deficiency.

Mutations in subunits of the mitochondrial NADH dehydrogenase cause mitochondrial complex I deficiency, a group of severe neurological diseases that can result in death in infancy. The pathogenesis of complex I deficiency remain poorly understood, and as a result there are currently no available treatments. To better understand the underlying mechanisms, we modelled complex I deficiency in Drosophila using knockdown of the mitochondrial complex I subunit ND-75 (NDUFS1) specifically in neurons. Neuronal complex I deficiency causes locomotor defects, seizures and reduced lifespan. At the cellular level, complex I deficiency does not affect ATP levels but leads to mitochondrial morphology defects, reduced endoplasmic reticulum-mitochondria contacts and activation of the endoplasmic reticulum unfolded protein response (UPR) in neurons. Multi-omic analysis shows that complex I deficiency dramatically perturbs mitochondrial metabolism in the brain. We find that expression of the yeast non-proton translocating NADH dehydrogenase NDI1, which reinstates mitochondrial NADH oxidation but not ATP production, restores levels of several key metabolites in the brain in complex I deficiency. Remarkably, NDI1 expression also reinstates endoplasmic reticulum-mitochondria contacts, prevents UPR activation and rescues the behavioural and lifespan phenotypes caused by complex I deficiency. Together, these data show that metabolic disruption due to loss of neuronal NADH dehydrogenase activity cause UPR activation and drive pathogenesis in complex I deficiency.

Phosphorylation of the novel mTOR substrate Unkempt regulates cellular morphogenesis.

Mechanistic target of rapamycin (mTOR) is a protein kinase that integrates multiple inputs to regulate anabolic cellular processes. For example, mTOR complex 1 (mTORC1) has key functions in growth control, autophagy, and metabolism. However, much less is known about the signaling components that act downstream of mTORC1 to regulate cellular morphogenesis. Here, we show that the RNA-binding protein Unkempt, a key regulator of cellular morphogenesis, is a novel substrate of mTORC1. We show that Unkempt phosphorylation is regulated by nutrient levels and growth factors via mTORC1. To analyze Unkempt phosphorylation, we immunoprecipitated Unkempt from cells in the presence or the absence of the mTORC1 inhibitor rapamycin and used mass spectrometry to identify mTORC1-dependent phosphorylated residues. This analysis showed that mTORC1-dependent phosphorylation is concentrated in a serine-rich intrinsically disordered region in the C-terminal half of Unkempt. We also found that Unkempt physically interacts with and is directly phosphorylated by mTORC1 through binding to the regulatory-associated protein of mTOR, Raptor. Furthermore, analysis in the developing brain of mice lacking TSC1 expression showed that phosphorylation of Unkempt is mTORC1 dependent in vivo. Finally, mutation analysis of key serine/threonine residues in the serine-rich region indicates that phosphorylation inhibits the ability of Unkempt to induce a bipolar morphology. Phosphorylation within this serine-rich region thus profoundly affects the ability of Unkempt to regulate cellular morphogenesis. Taken together, our findings reveal a novel molecular link between mTORC1 signaling and cellular morphogenesis.

Stereocontrolled Total Synthesis of Bastimolide B Using Iterative Homologation of Boronic Esters.

Bastimolide B is a polyhydroxy macrolide isolated from marine cyanobacteria displaying antimalarial activity. It features a dense array of hydroxylated stereogenic centers with 1,5-relationships along a hydrocarbon chain. These 1,5-polyols represent a particularly challenging motif for synthesis, as the remote position of the stereocenters hampers stereocontrol. Herein, we present a strategy for 1,5-polyol stereocontrolled synthesis based on iterative boronic ester homologation with enantiopure magnesium carbenoids. By merging boronic ester homologation and transition-metal-catalyzed alkene hydroboration and diboration, the acyclic backbone of bastimolide B was rapidly assembled from readily available building blocks with full control over the remote stereocenters, enabling the total synthesis to be completed in 16 steps (LLS).

The mTOR pathway component Unkempt regulates neural stem cell and neural progenitor cell cycle in the Drosophila central nervous system.

The formation of a complex nervous system requires the coordinated action of progenitor cell proliferation, differentiation and maturation. The Drosophila postembryonic central nervous system provides a powerful model for dissecting the cellular and molecular mechanisms underpinning neurogenesis. We previously identified the conserved zinc finger/RING protein Unkempt (Unk) as a key temporal regulator of neuronal differentiation in the Drosophila developing eye and showed that Unk acts downstream of the mechanistic target of rapamycin (mTOR) pathway together with its binding partner Headcase (Hdc). Here we investigate the role of Unk in Drosophila postembryonic thoracic neurogenesis. The Drosophila central nervous system contains neural stem cells, called neuroblasts, and neural progenitors, known as ganglion mother cells (GMCs). Unk is highly expressed in the central brain and ventral nerve cord but is not required to maintain neuroblast numbers or for the regulation of temporal series factor expression in neuroblasts. However, loss of Unk increases the number of neuroblasts and GMCs in S-phase of the cell cycle, resulting in the overproduction of neurons. We also show that Unk interacts with Hdc through its zinc finger domain. The zinc finger domain is required for the synergistic activity of Unk with Hdc during eye development but is not necessary for the activity of Unk in thoracic neurogenesis. Overall, this study shows that Unk and Hdc are novel negative regulators of neurogenesis in Drosophila and indicates a conserved role of mTOR signalling in nervous system development.

Ras-ERK-ETS inhibition alleviates neuronal mitochondrial dysfunction by reprogramming mitochondrial retrograde signaling.

Mitochondrial dysfunction activates the mitochondrial retrograde signaling pathway, resulting in large scale changes in gene expression. Mitochondrial retrograde signaling in neurons is poorly understood and whether retrograde signaling contributes to cellular dysfunction or is protective is unknown. We show that inhibition of Ras-ERK-ETS signaling partially reverses the retrograde transcriptional response to alleviate neuronal mitochondrial dysfunction. We have developed a novel genetic screen to identify genes that modify mitochondrial dysfunction in Drosophila. Knock-down of one of the genes identified in this screen, the Ras-ERK-ETS pathway transcription factor Aop, alleviates the damaging effects of mitochondrial dysfunction in the nervous system. Inhibition of Ras-ERK-ETS signaling also restores function in Drosophila models of human diseases associated with mitochondrial dysfunction. Importantly, Ras-ERK-ETS pathway inhibition partially reverses the mitochondrial retrograde transcriptional response. Therefore, mitochondrial retrograde signaling likely contributes to neuronal dysfunction through mis-regulation of gene expression.

The role of mTOR signalling in neurogenesis, insights from tuberous sclerosis complex.

Understanding the development and function of the nervous system is one of the foremost aims of current biomedical research. The nervous system is generated during a relatively short period of intense neurogenesis that is orchestrated by a number of key molecular signalling pathways. Even subtle defects in the activity of these molecules can have serious repercussions resulting in neurological, neurodevelopmental and neurocognitive problems including epilepsy, intellectual disability and autism. Tuberous sclerosis complex (TSC) is a monogenic disease characterised by these problems and by the formation of benign tumours in multiple organs, including the brain. TSC is caused by mutations in the TSC1 or TSC2 gene leading to activation of the mechanistic target of rapamycin (mTOR) signalling pathway. A desire to understand the neurological manifestations of TSC has stimulated research into the role of the mTOR pathway in neurogenesis. In this review we describe TSC neurobiology and how the use of animal model systems has provided insights into the roles of mTOR signalling in neuronal differentiation and migration. Recent progress in this field has identified novel mTOR pathway components regulating neuronal differentiation. The roles of mTOR signalling and aberrant neurogenesis in epilepsy are also discussed. Continuing efforts to understand mTOR neurobiology will help to identify new therapeutic targets for TSC and other neurological diseases.

Mitochondrial retrograde signaling regulates neuronal function.

Mitochondria are key regulators of cellular homeostasis, and mitochondrial dysfunction is strongly linked to neurodegenerative diseases, including Alzheimer's and Parkinson's. Mitochondria communicate their bioenergetic status to the cell via mitochondrial retrograde signaling. To investigate the role of mitochondrial retrograde signaling in neurons, we induced mitochondrial dysfunction in the Drosophila nervous system. Neuronal mitochondrial dysfunction causes reduced viability, defects in neuronal function, decreased redox potential, and reduced numbers of presynaptic mitochondria and active zones. We find that neuronal mitochondrial dysfunction stimulates a retrograde signaling response that controls the expression of several hundred nuclear genes. We show that the Drosophila hypoxia inducible factor alpha (HIFα) ortholog Similar (Sima) regulates the expression of several of these retrograde genes, suggesting that Sima mediates mitochondrial retrograde signaling. Remarkably, knockdown of Sima restores neuronal function without affecting the primary mitochondrial defect, demonstrating that mitochondrial retrograde signaling is partly responsible for neuronal dysfunction. Sima knockdown also restores function in a Drosophila model of the mitochondrial disease Leigh syndrome and in a Drosophila model of familial Parkinson's disease. Thus, mitochondrial retrograde signaling regulates neuronal activity and can be manipulated to enhance neuronal function, despite mitochondrial impairment.

Unkempt is negatively regulated by mTOR and uncouples neuronal differentiation from growth control.

Neuronal differentiation is exquisitely controlled both spatially and temporally during nervous system development. Defects in the spatiotemporal control of neurogenesis cause incorrect formation of neural networks and lead to neurological disorders such as epilepsy and autism. The mTOR kinase integrates signals from mitogens, nutrients and energy levels to regulate growth, autophagy and metabolism. We previously identified the insulin receptor (InR)/mTOR pathway as a critical regulator of the timing of neuronal differentiation in the Drosophila melanogaster eye. Subsequently, this pathway has been shown to play a conserved role in regulating neurogenesis in vertebrates. However, the factors that mediate the neurogenic role of this pathway are completely unknown. To identify downstream effectors of the InR/mTOR pathway we screened transcriptional targets of mTOR for neuronal differentiation phenotypes in photoreceptor neurons. We identified the conserved gene unkempt (unk), which encodes a zinc finger/RING domain containing protein, as a negative regulator of the timing of photoreceptor differentiation. Loss of unk phenocopies InR/mTOR pathway activation and unk acts downstream of this pathway to regulate neurogenesis. In contrast to InR/mTOR signalling, unk does not regulate growth. unk therefore uncouples the role of the InR/mTOR pathway in neurogenesis from its role in growth control. We also identified the gene headcase (hdc) as a second downstream regulator of the InR/mTOR pathway controlling the timing of neurogenesis. Unk forms a complex with Hdc, and Hdc expression is regulated by unk and InR/mTOR signalling. Co-overexpression of unk and hdc completely suppresses the precocious neuronal differentiation phenotype caused by loss of Tsc1. Thus, Unk and Hdc are the first neurogenic components of the InR/mTOR pathway to be identified. Finally, we show that Unkempt-like is expressed in the developing mouse retina and in neural stem/progenitor cells, suggesting that the role of Unk in neurogenesis may be conserved in mammals.

Dementia in Parkinson's disease is associated with enhanced mitochondrial complex I deficiency.

BACKGROUND: Dementia is a common feature of Parkinson's disease (PD), but the neuropathological changes associated with the development of Parkinson's disease dementia (PDD) are only partially understood. Mitochondrial dysfunction is a hallmark of PD but has not been studied in PDD. METHODS: Molecular and biochemical approaches were used to study mitochondrial activity and quantity in postmortem prefrontal cortex tissue. Tissues from pathologically confirmed PD and PDD patients and from age-matched controls were used to analyze the activity of mitochondrial enzyme complex nicotinamide adenine dinucleotide:ubiquinone oxidoreductase, or complex I (the first enzyme in the mitochondrial respiratory chain), mitochondrial DNA levels, and the expression of mitochondrial proteins. RESULTS: Complex I activity was significantly decreased (27% reduction; analysis of variance with Tukey's post hoc test; P < 0.05) in PDD patients, and mitochondrial DNA levels were also significantly decreased (18% reduction; Kruskal-Wallis analysis of variance with Dunn's multiple comparison test; P < 0.05) in PDD patients compared with controls, but neither was significantly reduced in PD patients. Overall, mitochondrial biogenesis was unaffected in PD or PDD, because the expression of mitochondrial proteins in patients was similar to that in controls. CONCLUSIONS: Patients with PDD have a deficiency in mitochondrial complex I activity and reduced mitochondrial DNA levels in the prefrontal cortex without a change in mitochondrial protein quantity. Therefore, mitochondrial complex I deficiency and reduced mitochondrial DNA in the prefrontal cortex may be a hallmark of dementia in patients with PD.

Regio- and Stereoselective Homologation of 1,2-Bis(Boronic Esters): Stereocontrolled Synthesis of 1,3-Diols and Sch 725674.

1,2-Bis(boronic esters), derived from the enantioselective diboration of terminal alkenes, can be selectively homologated at the primary boronic ester by using enantioenriched primary/secondary lithiated carbamates or benzoates to give 1,3-bis(boronic esters), which can be subsequently oxidized to the corresponding secondary-secondary and secondary-tertiary 1,3-diols with full stereocontrol. The transformation was applied to a concise total synthesis of the 14-membered macrolactone, Sch 725674. The nine-step synthetic route also features a novel desymmetrizing enantioselective diboration of a divinyl carbinol derivative and high-yielding late-stage cross-metathesis and Yamaguchi macrolactonization reactions.

Concerted control of gliogenesis by InR/TOR and FGF signalling in the Drosophila post-embryonic brain.

Glial cells are essential for the development and function of the nervous system. In the mammalian brain, vast numbers of glia of several different functional types are generated during late embryonic and early foetal development. However, the molecular cues that instruct gliogenesis and determine glial cell type are poorly understood. During post-embryonic development, the number of glia in the Drosophila larval brain increases dramatically, potentially providing a powerful model for understanding gliogenesis. Using glial-specific clonal analysis we find that perineural glia and cortex glia proliferate extensively through symmetric cell division in the post-embryonic brain. Using pan-glial inhibition and loss-of-function clonal analysis we find that Insulin-like receptor (InR)/Target of rapamycin (TOR) signalling is required for the proliferation of perineural glia. Fibroblast growth factor (FGF) signalling is also required for perineural glia proliferation and acts synergistically with the InR/TOR pathway. Cortex glia require InR in part, but not downstream components of the TOR pathway, for proliferation. Moreover, cortex glia absolutely require FGF signalling, such that inhibition of the FGF pathway almost completely blocks the generation of cortex glia. Neuronal expression of the FGF receptor ligand Pyramus is also required for the generation of cortex glia, suggesting a mechanism whereby neuronal FGF expression coordinates neurogenesis and cortex gliogenesis. In summary, we have identified two major pathways that control perineural and cortex gliogenesis in the post-embryonic brain and have shown that the molecular circuitry required is lineage specific.

A Drosophila model of mitochondrial disease phenotypic heterogeneity.

Mutations in genes that affect mitochondrial function cause primary mitochondrial diseases. Mitochondrial diseases are highly heterogeneous and even patients with the same mitochondrial disease can exhibit broad phenotypic heterogeneity, which is poorly understood. Mutations in subunits of mitochondrial respiratory complex I cause complex I deficiency, which can result in severe neurological symptoms and death in infancy. However, some complex I deficiency patients present with much milder symptoms. The most common nuclear gene mutated in complex I deficiency is the highly conserved core subunit NDUFS1. To model the phenotypic heterogeneity in complex I deficiency, we used RNAi lines targeting the Drosophila NDUFS1 homolog ND-75 with different efficiencies. Strong knockdown of ND-75 in Drosophila neurons resulted in severe behavioural phenotypes, reduced lifespan, altered mitochondrial morphology, reduced endoplasmic reticulum (ER)-mitochondria contacts and activation of the unfolded protein response (UPR). By contrast, weak ND-75 knockdown caused much milder behavioural phenotypes and changes in mitochondrial morphology. Moreover, weak ND-75 did not alter ER-mitochondria contacts or activate the UPR. Weak and strong ND-75 knockdown resulted in overlapping but distinct transcriptional responses in the brain, with weak knockdown specifically affecting proteosome activity and immune response genes. Metabolism was also differentially affected by weak and strong ND-75 knockdown including gamma-aminobutyric acid (GABA) levels, which may contribute to neuronal dysfunction in ND-75 knockdown flies. Several metabolic processes were only affected by strong ND-75 knockdown including the pentose phosphate pathway and the metabolite 2-hydroxyglutarate (2-HG), suggesting 2-HG as a candidate biomarker of severe neurological mitochondrial disease. Thus, our Drosophila model provides the means to dissect the mechanisms underlying phenotypic heterogeneity in mitochondrial disease.

Regulation by the RNA-binding protein Unkempt at its effector interface.

How RNA-binding proteins (RBPs) convey regulatory instructions to the core effectors of RNA processing is unclear. Here, we document the existence and functions of a multivalent RBP-effector interface. We show that the effector interface of a conserved RBP with an essential role in metazoan development, Unkempt, is mediated by a novel type of 'dual-purpose' peptide motifs that can contact two different surfaces of interacting proteins. Unexpectedly, we find that the multivalent contacts do not merely serve effector recruitment but are required for the accuracy of RNA recognition by Unkempt. Systems analyses reveal that multivalent RBP-effector contacts can repurpose the principal activity of an effector for a different function, as we demonstrate for the reuse of the central eukaryotic mRNA decay factor CCR4-NOT in translational control. Our study establishes the molecular assembly and functional principles of an RBP-effector interface.

Regioselective Synthesis of Fully Substituted Fused Pyrroles through an Oxidant-Free Multicomponent Reaction.

The synthesis of fully substituted fused pyrroles through a multicomponent reaction between a thioamide, an aldehyde, and ammonium acetate is described. This process improves on a route commonly employed in the patent literature by avoiding the use of potentially hazardous oxidants, which cause the formation of side products and require a stringent process of derisking to be utilized on scale. The reaction proceeds under mild conditions, displays excellent functional group tolerance, and facilitates diversification through multiple vectors.

Iterative synthesis of 1,3-polyboronic esters with high stereocontrol and application to the synthesis of bahamaolide A.

Polyketide natural products often contain common repeat motifs, for example, propionate, acetate and deoxypropionate, and so can be synthesized by iterative processes. We report here a highly efficient iterative strategy for the synthesis of polyacetates based on boronic ester homologation that does not require functional group manipulation between iterations. This process involves sequential asymmetric diboration of a terminal alkene, forming a 1,2-bis(boronic ester), followed by regio- and stereoselective homologation of the primary boronic ester with a butenyl metallated carbenoid to generate a 1,3-bis(boronic ester). Each transformation independently controls the stereochemical configuration, making the process highly versatile, and the sequence can be iterated prior to stereospecific oxidation of the 1,3-polyboronic ester to yield the 1,3-polyol. This methodology has been applied to a 14-step synthesis of the oxopolyene macrolide bahamaolide A, and the versatility of the 1,3-polyboronic esters has been demonstrated in various stereospecific transformations, leading to polyalkenes, -alkynes, -ketones and -aromatics with full stereocontrol.

A paradigm for regulation at the effector interface with RNA-binding proteins.

RNA-binding proteins (RBPs) are key regulators of gene expression, but how RBPs convey regulatory instructions to the core effectors of RNA processing is unclear. Here we document the existence and functions of a multivalent RBP-effector interface. We show that the effector interface of a deeply conserved RBP with an essential role in metazoan development, Unkempt, is mediated by a novel type of 'dual-purpose' peptide motifs that can contact two different surfaces of interacting proteins. Unexpectedly, we find that the multivalent contacts do not merely serve effector recruitment but are required for the accuracy of RNA recognition by the recruiting RBP. Systems analyses reveal that multivalent RBP-effector contacts can repurpose the principal activity of an effector for a different function, as we demonstrate for reuse of the central eukaryotic mRNA decay factor CCR4-NOT in translational control. Our study establishes the molecular assembly and functional principles of an RBP-effector interface, with implications for the evolution and function of RBP-operated regulatory networks.

Mitochondrial DNA Transport in Drosophila Neurons.

Mitochondria are essential organelles that generate energy and play vital roles in cellular metabolism. The small circular mitochondrial genome encodes key components of the mitochondrial respiratory apparatus. Depletion of, or mutations in mitochondrial DNA (mtDNA) cause mitochondrial dysfunction and disease. mtDNA is packaged into nucleoids, which are transported throughout the cell within mitochondria. Efficient transport of nucleoids is essential in neurons, where mitochondrial function is required locally at synapses. Here I describe methods for visualization of nucleoids in Drosophila neurons using a GFP fusion of the mitochondrial transcription factor TFAM. TFAM-GFP, together with mCherry-labeled mitochondria, was used to visualize nucleoids in fixed larval segmental nerves. I also describe how these tools can be used for live imaging of nucleoid dynamics. Using Drosophila as a model system, these methods will enable further characterization and analysis of nucleoid dynamics in neurons.

Mechanistic target of rapamycin signaling in human nervous system development and disease.

Mechanistic target of rapamycin (mTOR) is a highly conserved serine/threonine kinase that regulates fundamental cellular processes including growth control, autophagy and metabolism. mTOR has key functions in nervous system development and mis-regulation of mTOR signaling causes aberrant neurodevelopment and neurological diseases, collectively called mTORopathies. In this mini review we discuss recent studies that have deepened our understanding of the key roles of the mTOR pathway in human nervous system development and disease. Recent advances in single-cell transcriptomics have been exploited to reveal specific roles for mTOR signaling in human cortical development that may have contributed to the evolutionary divergence from our primate ancestors. Cerebral organoid technology has been utilized to show that mTOR signaling is active in and regulates outer radial glial cells (RGCs), a population of neural stem cells that distinguish the human developing cortex. mTOR signaling has a well-established role in hamartoma syndromes such as tuberous sclerosis complex (TSC) and other mTORopathies. New ultra-sensitive techniques for identification of somatic mTOR pathway mutations have shed light on the neurodevelopmental origin and phenotypic heterogeneity seen in mTORopathy patients. These emerging studies suggest that mTOR signaling may facilitate developmental processes specific to human cortical development but also, when mis-regulated, cause cortical malformations and neurological disease.

The conserved translocase Tim17 prevents mitochondrial DNA loss.

Maintenance of an intact mitochondrial genome is essential for oxidative phosphorylation in all eukaryotes. Depletion of mitochondrial genome copy number can have severe pathological consequences due to loss of respiratory capacity. In Saccharomyces cerevisiae, several bifunctional metabolic enzymes have been shown to be required for mitochondrial DNA (mtDNA) maintenance. For example, Ilv5 is required for branched chain amino acid biosynthesis and mtDNA stability. We have identified OXA1 and TIM17 as novel multicopy suppressors of mtDNA instability in ilv5 cells. In addition, overexpression of TIM17, but not OXA1, prevents the complete loss of mtDNA in cells lacking the TFAM homologue Abf2. Introduction of the disease-associated A3243G mutant mtDNA into human NT2 teratocarcinoma cells frequently causes mtDNA loss. Yet when human TIM17A is overexpressed in NT2 cybrids carrying A3243G mtDNA, the proportion of cybrid clones maintaining mtDNA increases significantly. TIM17A overexpression results in long-term mtDNA stabilization, since NT2 cybrids overexpressing TIM17A maintain mtDNA at levels similar to controls for several months. Tim17 is a conserved suppressor of mtDNA instability and is the first factor to be identified that can prevent mtDNA loss in a human cellular model of mitochondrial disease.

The zinc finger/RING domain protein Unkempt regulates cognitive flexibility.

Correct orchestration of nervous system development is a profound challenge that involves coordination of complex molecular and cellular processes. Mechanistic target of rapamycin (mTOR) signaling is a key regulator of nervous system development and synaptic function. The mTOR kinase is a hub for sensing inputs including growth factor signaling, nutrients and energy levels. Activation of mTOR signaling causes diseases with severe neurological manifestations, such as tuberous sclerosis complex and focal cortical dysplasia. However, the molecular mechanisms by which mTOR signaling regulates nervous system development and function are poorly understood. Unkempt is a conserved zinc finger/RING domain protein that regulates neurogenesis downstream of mTOR signaling in Drosophila. Unkempt also directly interacts with the mTOR complex I component Raptor. Here we describe the generation and characterisation of mice with a conditional knockout of Unkempt (UnkcKO) in the nervous system. Loss of Unkempt reduces Raptor protein levels in the embryonic nervous system but does not affect downstream mTORC1 targets. We also show that nervous system development occurs normally in UnkcKO mice. However, we find that Unkempt is expressed in the adult cerebellum and hippocampus and behavioural analyses show that UnkcKO mice have improved memory formation and cognitive flexibility to re-learn. Further understanding of the role of Unkempt in the nervous system will provide novel mechanistic insight into the role of mTOR signaling in learning and memory.