FBXL4 suppresses mitophagy by restricting the accumulation of NIX and BNIP3 mitophagy receptors.

To maintain both mitochondrial quality and quantity, cells selectively remove damaged or excessive mitochondria through mitophagy, which is a specialised form of autophagy. Mitophagy is induced in response to diverse conditions, including hypoxia, cellular differentiation and mitochondrial damage. However, the mechanisms that govern the removal of specific dysfunctional mitochondria under steady-state conditions to fine-tune mitochondrial content are not well understood. Here, we report that SCFFBXL4 , an SKP1/CUL1/F-box protein ubiquitin ligase complex, localises to the mitochondrial outer membrane in unstressed cells and mediates the constitutive ubiquitylation and degradation of the mitophagy receptors NIX and BNIP3 to suppress basal levels of mitophagy. We demonstrate that the pathogenic variants of FBXL4 that cause encephalopathic mtDNA depletion syndrome (MTDPS13) do not efficiently interact with the core SCF ubiquitin ligase machinery or mediate the degradation of NIX and BNIP3. Thus, we reveal a molecular mechanism whereby FBXL4 actively suppresses mitophagy by preventing NIX and BNIP3 accumulation. We propose that the dysregulation of NIX and BNIP3 turnover causes excessive basal mitophagy in FBXL4-associated mtDNA depletion syndrome.

Mitochondrial fusion and altered beta-oxidation drive muscle wasting in a Drosophila cachexia model.

Cancer cachexia is a tumour-induced wasting syndrome, characterised by extreme loss of skeletal muscle. Defective mitochondria can contribute to muscle wasting; however, the underlying mechanisms remain unclear. Using a Drosophila larval model of cancer cachexia, we observed enlarged and dysfunctional muscle mitochondria. Morphological changes were accompanied by upregulation of beta-oxidation proteins and depletion of muscle glycogen and lipid stores. Muscle lipid stores were also decreased in Colon-26 adenocarcinoma mouse muscle samples, and expression of the beta-oxidation gene CPT1A was negatively associated with muscle quality in cachectic patients. Mechanistically, mitochondrial defects result from reduced muscle insulin signalling, downstream of tumour-secreted insulin growth factor binding protein (IGFBP) homologue ImpL2. Strikingly, muscle-specific inhibition of Forkhead box O (FOXO), mitochondrial fusion, or beta-oxidation in tumour-bearing animals preserved muscle integrity. Finally, dietary supplementation with nicotinamide or lipids, improved muscle health in tumour-bearing animals. Overall, our work demonstrates that muscle FOXO, mitochondria dynamics/beta-oxidation and lipid utilisation are key regulators of muscle wasting in cancer cachexia.

PPTC7 antagonizes mitophagy by promoting BNIP3 and NIX degradation via SCFFBXL4.

Mitophagy must be carefully regulated to ensure that cells maintain appropriate numbers of functional mitochondria. The SCFFBXL4 ubiquitin ligase complex suppresses mitophagy by controlling the degradation of BNIP3 and NIX mitophagy receptors, and FBXL4 mutations result in mitochondrial disease as a consequence of elevated mitophagy. Here, we reveal that the mitochondrial phosphatase PPTC7 is an essential cofactor for SCFFBXL4-mediated destruction of BNIP3 and NIX, suppressing both steady-state and induced mitophagy. Disruption of the phosphatase activity of PPTC7 does not influence BNIP3 and NIX turnover. Rather, a pool of PPTC7 on the mitochondrial outer membrane acts as an adaptor linking BNIP3 and NIX to FBXL4, facilitating the turnover of these mitophagy receptors. PPTC7 accumulates on the outer mitochondrial membrane in response to mitophagy induction or the absence of FBXL4, suggesting a homoeostatic feedback mechanism that attenuates high levels of mitophagy. We mapped critical residues required for PPTC7-BNIP3/NIX and PPTC7-FBXL4 interactions and their disruption interferes with both BNIP3/NIX degradation and mitophagy suppression. Collectively, these findings delineate a complex regulatory mechanism that restricts BNIP3/NIX-induced mitophagy.

The association of microcephaly protein WDR62 with CPAP/IFT88 is required for cilia formation and neocortical development.

WDR62 mutations that result in protein loss, truncation or single amino-acid substitutions are causative for human microcephaly, indicating critical roles in cell expansion required for brain development. WDR62 missense mutations that retain protein expression represent partial loss-of-function mutants that may therefore provide specific insights into radial glial cell processes critical for brain growth. Here we utilized CRISPR/Cas9 approaches to generate three strains of WDR62 mutant mice; WDR62 V66M/V66M and WDR62R439H/R439H mice recapitulate conserved missense mutations found in humans with microcephaly, with the third strain being a null allele (WDR62stop/stop). Each of these mutations resulted in embryonic lethality to varying degrees and gross morphological defects consistent with ciliopathies (dwarfism, anophthalmia and microcephaly). We find that WDR62 mutant proteins (V66M and R439H) localize to the basal body but fail to recruit CPAP. As a consequence, we observe deficient recruitment of IFT88, a protein that is required for cilia formation. This underpins the maintenance of radial glia as WDR62 mutations caused premature differentiation of radial glia resulting in reduced generation of neurons and cortical thinning. These findings highlight the important role of the primary cilium in neocortical expansion and implicate ciliary dysfunction as underlying the pathology of MCPH2 patients.

Vision in Drosophila: seeing the world through a model's eyes.

The fruit fly, Drosophila melanogaster, has been used for decades as a genetic model for unraveling mechanisms of development and behavior. In order to efficiently assign gene functions to cellular and behavioral processes, early measures were often necessarily simple. Much of what is known of developmental pathways was based on disrupting highly regular structures, such as patterns of cells in the eye. Similarly, reliable visual behaviors such as phototaxis and motion responses provided a solid foundation for dissecting vision. Researchers have recently begun to examine how this model organism responds to more complex or naturalistic stimuli by designing novel paradigms that more closely mimic visual behavior in the wild. Alongside these advances, the development of brain-recording strategies allied with novel genetic tools has brought about a new era of Drosophila vision research where neuronal activity can be related to behavior in the natural world.

Drosophila larval motor patterning relies on regulated alternative splicing of Dscam2.

Recent studies capitalizing on the newly complete nanometer-resolution Drosophila larval connectome have made significant advances in identifying the structural basis of motor patterning. However, the molecular mechanisms utilized by neurons to wire these circuits remain poorly understood. In this study we explore how cell-specific expression of two Dscam2 isoforms, which mediate isoform-specific homophilic binding, contributes to motor patterning and output of Drosophila larvae. Ablating Dscam2 isoform diversity resulted in impaired locomotion. Electrophysiological assessment at the neuromuscular junction during fictive locomotion indicated that this behavioral defect was largely caused by weaker bouts of motor neuron activity. Morphological analyses of single motor neurons using MultiColour FlpOut revealed severe errors in dendrite arborization and assessment of cholinergic and GABAergic projections to the motor domain revealed altered morphology of interneuron processes. Loss of Dscam2 did not affect locomotor output, motor neuron activation or dendrite targeting. Our findings thus suggest that locomotor circuit phenotypes arise specifically from inappropriate Dscam2 interactions between premotor interneurons and motor neurons when they express the same isoform. Indeed, we report here that first-order premotor interneurons express Dscam2A. Since motor neurons express Dscam2B, our results provide evidence that Dscam2 isoform expression alternates between synaptic partners in the nerve cord. Our study demonstrates the importance of cell-specific alternative splicing in establishing the circuitry that underlies neuromotor patterning without inducing unwanted intercellular interactions.

Dscam2 mediates axonal tiling in the Drosophila visual system.

Sensory processing centres in both the vertebrate and the invertebrate brain are often organized into reiterated columns, thus facilitating an internal topographic representation of the external world. Cells within each column are arranged in a stereotyped fashion and form precise patterns of synaptic connections within discrete layers. These connections are largely confined to a single column, thereby preserving the spatial information from the periphery. Other neurons integrate this information by connecting to multiple columns. Restricting axons to columns is conceptually similar to tiling. Axons and dendrites of neighbouring neurons of the same class use tiling to form complete, yet non-overlapping, receptive fields. It is thought that, at the molecular level, cell-surface proteins mediate tiling through contact-dependent repulsive interactions, but proteins serving this function have not yet been identified. Here we show that the immunoglobulin superfamily member Dscam2 restricts the connections formed by L1 lamina neurons to columns in the Drosophila visual system. Our data support a model in which Dscam2 homophilic interactions mediate repulsion between neurites of L1 cells in neighbouring columns. We propose that Dscam2 is a tiling receptor for L1 neurons.

Regulated alternative splicing of Dscam2 is required for somatosensory circuit wiring.

Axon and dendrite placement and connectivity is guided by a wide range of secreted and surface molecules in the developing nervous system. Nevertheless, the extraordinary complexity of connections in the brain requires that this repertoire be further diversified to precisely and uniquely regulate cell-cell interactions. One important mechanism for molecular diversification is alternative splicing. Drosophila Down syndrome cell adhesion molecule (Dscam2) undergoes cell type-specific alternative splicing to produce two isoform-specific homophilic binding proteins. Regulated alternative splicing of Dscam2 is important for dendrite and axon patterning, but how this translates to circuit wiring and animal behavior is not well understood. Here, we examined the role of cell-type specific expression of Dscam2 isoforms in regulating synaptic partner selection in the larval somatosensory system. We found that synaptic partners in the nociceptive circuit express different Dscam2 isoforms. Forcing synaptic partners to express a common isoform resulted in nociceptive axon patterning defects and attenuated nocifensive behaviors, indicating that a role for Dscam2 alternative splicing is to ensure that synaptic partners do not express matching isoforms. These results point to a model in which regulated alternative splicing of Dscam2 across populations of neurons restricts connectivity to specific partners and prevents inappropriate synaptic connections.

Dscam-mediated repulsion controls tiling and self-avoidance.

Recent studies have uncovered the molecular basis of self-avoidance and tiling, two fundamental principles required for the formation of neural circuits. Both of these wiring strategies are established through homophilic repulsion between Dscam proteins expressed on opposing cell surfaces. In Drosophila, Dscam1 mediates self-avoidance, whereas Dscam2 mediates tiling. By contrast, phenotypes in the retina of the DSCAM mutant mouse indicate that DSCAM functions in both self-avoidance and tiling. These findings suggest that homophilic recognition molecules that have classically been defined as adhesive may also function as repulsive cues and that Dscam proteins specialize in this function.

Elevated levels of Drosophila Wdr62 promote glial cell growth and proliferation through AURKA signalling to AKT and MYC.

WD40-Repeat Protein 62 (WDR62) is required to maintain neural and glial cell populations during embryonic brain growth. Although elevated expression of WDR62 is frequently associated with several tumour types, potential effects of excess WDR62 on proliferative growth remain undefined. Here, we demonstrate that glia specific overexpression of WDR62 in Drosophila larval brains resulted in increased cell size, over-proliferation and increased brain volume, without overt disruption of tissue organization. We further demonstrate WDR62 promoted over-proliferation and brain overgrowth by activating AURKA and pAKT signalling to increase MYC function in glial cells. Together these data suggest WDR62 normally functions in the glial lineage to activate oncogenic signalling networks, promoting proliferation and brain overgrowth.

Dscam2 suppresses synaptic strength through a PI3K-dependent endosomal pathway.

Dscam2 is a cell surface protein required for neuronal development in Drosophila; it can promote neural wiring through homophilic recognition that leads to either adhesion or repulsion between neurites. Here, we report that Dscam2 also plays a post-developmental role in suppressing synaptic strength. This function is dependent on one of two distinct extracellular isoforms of the protein and is autonomous to motor neurons. We link the PI3K enhancer, Centaurin gamma 1A, to the Dscam2-dependent regulation of synaptic strength and show that changes in phosphoinositide levels correlate with changes in endosomal compartments that have previously been associated with synaptic strength. Using transmission electron microscopy, we find an increase in synaptic vesicles at Dscam2 mutant active zones, providing a rationale for the increase in synaptic strength. Our study provides the first evidence that Dscam2 can regulate synaptic physiology and highlights how diverse roles of alternative protein isoforms can contribute to unique aspects of brain development and function.

Deterministic splicing of Dscam2 is regulated by Muscleblind.

Alternative splicing increases the proteome diversity crucial for establishing the complex circuitry between trillions of neurons. To provide individual cells with different repertoires of protein isoforms, however, this process must be regulated. Previously, we found that the mutually exclusive alternative splicing of Drosophila Dscam2 produces two isoforms (A and B) with unique binding properties. This splicing event is cell type specific, and the transmembrane proteins that it generates are crucial for the development of axons, dendrites, and synapses. Here, we show that Muscleblind (Mbl) controls Dscam2 alternative splicing. Mbl represses isoform A and promotes the selection of isoform B. Mbl mutants exhibit phenotypes also observed in flies engineered to express a single Dscam2 isoform. Consistent with this, mbl expression is cell type specific and correlates with the splicing of isoform B. Our study demonstrates how the regulated expression of a splicing factor is sufficient to provide neurons with unique protein isoforms crucial for development.

Strategies for assembling columns and layers in the Drosophila visual system.

A striking feature of neural circuit structure is the arrangement of neurons into regularly spaced ensembles (i.e. columns) and neural connections into parallel layers. These patterns of organization are thought to underlie precise synaptic connectivity and provide a basis for the parallel processing of information. In this article we discuss in detail specific findings that contribute to a framework for understanding how columns and layers are assembled in the Drosophila visual system, and discuss their broader implications.

Regulated Alternative Splicing of Drosophila Dscam2 Is Necessary for Attaining the Appropriate Number of Photoreceptor Synapses.

How the brain makes trillions of synaptic connections using a genome of only 20,000 genes is a major question in modern neuroscience. Alternative splicing is one mechanism that can increase the number of proteins produced by each gene, but its role in regulating synapse formation is poorly understood. In Drosophila, photoreceptors form a synapse with multiple postsynaptic elements including lamina neurons L1 and L2. L1 and L2 express distinct isoforms of the homophilic repulsive protein Dscam2, and since these isoforms cannot bind to each other, cell-specific expression has been proposed to be necessary for preventing repulsive interactions that could disrupt the synapse. Here, we show that the number of synapses are reduced in flies that express only one isoform, and L1 and L2 dendritic morphology is perturbed. We propose that these defects result from inappropriate interactions between L1 and L2 dendrites. We conclude that regulated Dscam2 alternative splicing is necessary for the proper assembly of photoreceptor synapses.

Glial-Specific Functions of Microcephaly Protein WDR62 and Interaction with the Mitotic Kinase AURKA Are Essential for Drosophila Brain Growth.

The second most commonly mutated gene in primary microcephaly (MCPH) patients is wd40-repeat protein 62 (wdr62), but the relative contribution of WDR62 function to the growth of major brain lineages is unknown. Here, we use Drosophila models to dissect lineage-specific WDR62 function(s). Interestingly, although neural stem cell (neuroblast)-specific depletion of WDR62 significantly decreased neuroblast number, brain size was unchanged. In contrast, glial lineage-specific WDR62 depletion significantly decreased brain volume. Moreover, loss of function in glia not only decreased the glial population but also non-autonomously caused neuroblast loss. We further demonstrated that WDR62 controls brain growth through lineage-specific interactions with master mitotic signaling kinase, AURKA. Depletion of AURKA in neuroblasts drives brain overgrowth, which was suppressed by WDR62 co-depletion. In contrast, glial-specific depletion of AURKA significantly decreased brain volume, which was further decreased by WDR62 co-depletion. Thus, dissecting relative contributions of MCPH factors to individual neural lineages will be critical for understanding complex diseases such as microcephaly.

Dscam Proteins Direct Dendritic Targeting through Adhesion.

Cell recognition molecules are key regulators of neural circuit assembly. The Dscam family of recognition molecules in Drosophila has been shown to regulate interactions between neurons through homophilic repulsion. This is exemplified by Dscam1 and Dscam2, which together repel dendrites of lamina neurons, L1 and L2, in the visual system. By contrast, here we show that Dscam2 directs dendritic targeting of another lamina neuron, L4, through homophilic adhesion. Through live imaging and genetic mosaics to dissect interactions between specific cells, we show that Dscam2 is required in L4 and its target cells for correct dendritic targeting. In a genetic screen, we identified Dscam4 as another regulator of L4 targeting which acts with Dscam2 in the same pathway to regulate this process. This ensures tiling of the lamina neuropil through heterotypic interactions. Thus, different combinations of Dscam proteins act through distinct mechanisms in closely related neurons to pattern neural circuits.

Dscam2 affects visual perception in Drosophila melanogaster.

Dscam2, a cell surface protein that mediates cellular repulsion, plays a crucial role in the development of the Drosophila melanogaster visual system. Dscam2 generates boundaries between neighboring modules in the fly optic lobe; in Dscam2 mutants this visual system modularity is compromised. Although developmental wiring defects have been well described in the Dscam2 mutant, behavioral consequences have not been investigated. To address this, we examined the visual behavior of Dscam2 mutant flies. Using a phototaxis assay, we ascertained that these flies are not blind, but have a reduced phototaxic response. Through population-based and single fly optomotor assays, we found that Dscam2 mutant flies can track motion but that their response is opposite to control flies under defined experimental conditions. In a fixation paradigm, which allows tethered flies to control the angular position of a visual stimulus, mutant flies' responses were diametrically opposed to those seen in control flies. These data suggest that modest changes in the modularity of the fly visual system in the Dscam2 mutant can dramatically change the perception of specific visual cues and modify behavior.

Neuronal cell-type-specific alternative splicing: A mechanism for specifying connections in the brain?

Alternative splicing (AS) allows a single gene to generate multiple protein isoforms. It has been hypothesized that AS plays a role in brain wiring by increasing the number of cell recognition molecules necessary for forming connections between neurons. Many studies have characterized isoform expression patterns of various genes in the brain, but very few have addressed whether specific isoforms play a functional role in neuronal wiring. In our recent work, we reported the cell-type-specific AS of the cell recognition molecule Dscam2. Exclusive expression of Dscam2 isoforms allows tightly associated neurons to signal repulsion selectively within the same cell-types, without interfering with one another. We show that preventing cell-specific isoform expression in 2 closely associated neurons disrupts their axon terminal morphology. We propose that the requirement for isoform specificity extends to synapses and discuss experiments that can test this directly. Factors that regulate Dscam2 cell-type-specific AS likely regulate the splicing of many genes involved in neurodevelopment. These regulators of alternative splicing may act broadly to control many genes involved in the development of specific neuron types. Identifying these factors is a key step in understanding how AS contributes to the brain connectome.

Cell-specific alternative splicing of Drosophila Dscam2 is crucial for proper neuronal wiring.

How a finite number of genes specify a seemingly infinite number of neuronal connections is a central question in neurobiology. Alternative splicing has been proposed to increase proteome diversity in the brain. Here we show that cell-specific alternative splicing of a cell-surface protein is crucial for neuronal wiring. Down syndrome cell adhesion molecule 2 (Dscam2) is a conserved homophilic binding protein that can induce repulsion between opposing neurons. In the fly visual system, L1 and L2 neurons both require Dscam2 repulsion, but paradoxically, they also physically contact each other. We found that the cell-specific expression of two biochemically distinct alternative isoforms of Dscam2 prevents these cells from repelling each other. Phenotypes were observed in the axon terminals of L1 and L2 when they expressed the incorrect isoform, demonstrating a requirement for distinct isoforms. We conclude that cell-specific alternative splicing is a mechanism for achieving proper connectivity between neurons.

Combinatorial rules of precursor specification underlying olfactory neuron diversity.

BACKGROUND: Sensory neuron diversity ensures optimal detection of the external world and is a hallmark of sensory systems. An extreme example is the olfactory system, as individual olfactory receptor neurons (ORNs) adopt unique sensory identities by typically expressing a single receptor gene from a large genomic repertoire. In Drosophila, about 50 different ORN classes are generated from a field of precursor cells, giving rise to spatially restricted and distinct clusters of ORNs on the olfactory appendages. Developmental strategies spawning ORN diversity from an initially homogeneous population of precursors are largely unknown. RESULTS: Here we unravel the nested and binary logic of the combinatorial code that patterns the decision landscape of precursor states underlying ORN diversity in the Drosophila olfactory system. The transcription factor Rotund (Rn) is a critical component of this code that is expressed in a subset of ORN precursors. Addition of Rn to preexisting transcription factors that assign zonal identities to precursors on the antenna subdivides each zone and almost exponentially increases ORN diversity by branching off novel precursor fates from default ones within each zone. In rn mutants, rn-positive ORN classes are converted to rn-negative ones in a zone-specific manner. CONCLUSIONS: We provide a model describing how nested and binary changes in combinations of transcription factors could coordinate and pattern a large number of distinct precursor identities within a population to modulate the level of ORN diversity during development and evolution.