MFN2 coordinates mitochondria motility with α-tubulin acetylation and this regulation is disrupted in CMT2A.

Mitofusin-2 (MFN2), a large GTPase residing in the mitochondrial outer membrane and mutated in Charcot-Marie-Tooth type 2 disease (CMT2A), is a regulator of mitochondrial fusion and tethering with the ER. The role of MFN2 in mitochondrial transport has however remained elusive. Like MFN2, acetylated microtubules play key roles in mitochondria dynamics. Nevertheless, it is unknown if the α-tubulin acetylation cycle functionally interacts with MFN2. Here, we show that mitochondrial contacts with microtubules are sites of α-tubulin acetylation, which occurs through MFN2-mediated recruitment of α-tubulin acetyltransferase 1 (ATAT1). This activity is critical for MFN2-dependent regulation of mitochondria transport, and axonal degeneration caused by CMT2A MFN2 associated R94W and T105M mutations may depend on the inability to release ATAT1 at sites of mitochondrial contacts with microtubules. Our findings reveal a function for mitochondria in α-tubulin acetylation and suggest that disruption of this activity plays a role in the onset of MFN2-dependent CMT2A.

Mechanisms of gas sensing by internal sensory neurons in Drosophila larvae.

Internal sensory neurons monitor the chemical and physical state of the body, providing critical information to the central nervous system for maintaining homeostasis and survival. A population of larval Drosophila sensory neurons, tracheal dendrite (td) neurons, elaborate dendrites along respiratory organs and may serve as a model for elucidating the cellular and molecular basis of chemosensation by internal neurons. We find that td neurons respond to decreases in O2 levels and increases in CO2 levels. We assessed the roles of atypical soluble guanylyl cyclases (Gycs) and a gustatory receptor (Gr) in mediating these responses. We found that Gyc88E/Gyc89Db were necessary for responses to hypoxia, and that Gr28b was necessary for responses to CO2. Targeted expression of Gr28b isoform c in td neurons rescued responses to CO2 in mutant larvae and also induced ectopic sensitivity to CO2 in the td network. Gas-sensitive td neurons were activated when larvae burrowed for a prolonged duration, demonstrating a natural-like feeding condition in which td neurons are activated. Together, our work identifies two gaseous stimuli that are detected by partially overlapping subsets of internal sensory neurons, and establishes roles for Gyc88E/Gyc89Db in the detection of hypoxia, and Gr28b in the detection of CO2.

Neuromuscular basis of Drosophila larval rolling escape behavior.

When threatened by dangerous or harmful stimuli, animals engage in diverse forms of rapid escape behaviors. In Drosophila larvae, one type of escape response involves C-shaped bending and lateral rolling followed by rapid forward crawling. The sensory circuitry that promotes larval escape has been extensively characterized; however, the motor programs underlying rolling are unknown. Here, we characterize the neuromuscular basis of rolling escape behavior. We used high-speed, volumetric, Swept Confocally Aligned Planar Excitation (SCAPE) microscopy to image muscle activity during larval rolling. Unlike sequential peristaltic muscle contractions that progress from segment to segment during forward and backward crawling, muscle activity progresses circumferentially during bending and rolling escape behavior. We propose that progression of muscular contraction around the larva's circumference results in a transient misalignment between weight and the ground support forces, which generates a torque that induces stabilizing body rotation. Therefore, successive cycles of slight misalignment followed by reactive aligning rotation lead to continuous rolling motion. Supporting our biomechanical model, we found that disrupting the activity of muscle groups undergoing circumferential contraction progression leads to rolling defects. We use EM connectome data to identify premotor to motor connectivity patterns that could drive rolling behavior and perform neural silencing approaches to demonstrate the crucial role of a group of glutamatergic premotor neurons in rolling. Our data reveal body-wide muscle activity patterns and putative premotor circuit organization for execution of the rolling escape response.

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.

MFN2-dependent recruitment of ATAT1 coordinates mitochondria motility with alpha-tubulin acetylation and is disrupted in CMT2A.

Acetylated microtubules play key roles in the regulation of mitochondria dynamics. It has however remained unknown if the machinery controlling mitochondria dynamics functionally interacts with the alpha-tubulin acetylation cycle. Mitofusin-2 (MFN2), a large GTPase residing in the mitochondrial outer membrane and mutated in Charcot-Marie-Tooth type 2 disease (CMT2A), is a regulator of mitochondrial fusion, transport and tethering with the endoplasmic reticulum. The role of MFN2 in regulating mitochondrial transport has however remained elusive. Here we show that mitochondrial contacts with microtubules are sites of alpha-tubulin acetylation, which occurs through the MFN2-mediated recruitment of alpha-tubulin acetyltransferase 1 (ATAT1). We discover that this activity is critical for MFN2-dependent regulation of mitochondria transport, and that axonal degeneration caused by CMT2A MFN2 associated mutations, R94W and T105M, may depend on the inability to release ATAT1 at sites of mitochondrial contacts with microtubules. Our findings reveal a function for mitochondria in regulating acetylated alpha-tubulin and suggest that disruption of the tubulin acetylation cycle play a pathogenic role in the onset of MFN2-dependent CMT2A.

Axon-axon interactions determine modality-specific wiring and subcellular synaptic specificity in a somatosensory circuit.

Synaptic connections between neurons are often formed in precise subcellular regions of dendritic arbors with implications for information processing within neurons. Cell-cell interactions are widely important for circuit wiring; however, their role in subcellular specificity is not well understood. We studied the role of axon-axon interactions in precise targeting and subcellular wiring of Drosophila somatosensory circuitry. Axons of nociceptive and gentle touch neurons terminate in adjacent, non-overlapping layers in the central nervous system (CNS). Nociceptor and touch receptor axons synapse onto distinct dendritic regions of a second-order interneuron, the dendrites of which span these layers, forming touch-specific and nociceptive-specific connectivity. We found that nociceptor ablation elicited extension of touch receptor axons and presynapses into the nociceptor recipient region, supporting a role for axon-axon interactions in somatosensory wiring. Conversely, touch receptor ablation did not lead to expansion of nociceptor axons, consistent with unidirectional axon-axon interactions. Live imaging provided evidence for sequential arborization of nociceptive and touch neuron axons in the CNS. We propose that axon-axon interactions and modality-specific timing of axon targeting play key roles in subcellular connection specificity of somatosensory circuitry.

Neuromuscular Basis of Drosophila larval rolling escape behavior.

When threatened by dangerous or harmful stimuli, animals engage in diverse forms of rapid escape behaviors. In Drosophila larvae, one type of escape response involves C-shaped bending and lateral rolling followed by rapid forward crawling. The sensory circuitry that promotes larval escape has been extensively characterized; however, the motor programs underlying rolling are unknown. Here, we characterize the neuromuscular basis of rolling escape behavior. We used high-speed, volumetric, Swept Confocally-Aligned Planar Excitation (SCAPE) microscopy to image muscle activity during larval rolling. Unlike sequential peristaltic muscle contractions that progress from segment to segment during forward and backward crawling, the muscle activity progresses circumferentially during bending and rolling escape behavior. We propose that progression of muscular contraction around the larval circumference results in a transient misalignment between weight and the ground support forces, which generates a torque that induces stabilizing body rotation. Therefore, successive cycles of slight misalignment followed by reactive aligning rotation lead to continuous rolling motion. Supporting our biomechanical model, we found that disrupting the activity of muscle groups undergoing circumferential contraction progression lead to rolling defects. We use EM connectome data to identify premotor to motor connectivity patterns that could drive rolling behavior, and perform neural silencing approaches to demonstrate the crucial role of a group of glutamatergic premotor neurons in rolling. Our data reveal body-wide muscle activity patterns and putative premotor circuit organization for execution of the rolling escape response.

Dendrite remodeling according to GARP.

Disruptions in membrane trafficking are associated with neurodevelopmental disorders, but underlying pathological mechanisms remain largely unknown. In this issue, O'Brien et al. (2023. J. Cell Biol.https://doi.org/10.1083/jcb.202112108) show how GARP regulates sterol transfer critical for remodeling of dendrites in flies.

The Immunoglobulin Superfamily Member Basigin Is Required for Complex Dendrite Formation in Drosophila.

Coordination of dendrite growth with changes in the surrounding substrate occurs widely in the nervous system and is vital for establishing and maintaining neural circuits. However, the molecular basis of this important developmental process remains poorly understood. To identify potential mediators of neuron-substrate interactions important for dendrite morphogenesis, we undertook an expression pattern-based screen in Drosophila larvae, which revealed many proteins with expression in dendritic arborization (da) sensory neurons and in neurons and their epidermal substrate. We found that reporters for Basigin, a cell surface molecule of the immunoglobulin (Ig) superfamily previously implicated in cell-cell and cell-substrate interactions, are expressed in da sensory neurons and epidermis. Loss of Basigin in da neurons led to defects in morphogenesis of the complex dendrites of class IV da neurons. Classes of sensory neurons with simpler branching patterns were unaffected by loss of Basigin. Structure-function analyses showed that a juxtamembrane KRR motif is critical for this function. Furthermore, knock down of Basigin in the epidermis led to defects in dendrite elaboration of class IV neurons, suggesting a non-autonomous role. Together, our findings support a role for Basigin in complex dendrite morphogenesis and interactions between dendrites and the adjacent epidermis.

Integrins protect sensory neurons in models of paclitaxel-induced peripheral sensory neuropathy.

Chemotherapy-induced peripheral neuropathy (CIPN) is a major side effect from cancer treatment with no known method for prevention or cure in clinics. CIPN often affects unmyelinated nociceptive sensory terminals. Despite the high prevalence, molecular and cellular mechanisms that lead to CIPN are still poorly understood. Here, we used a genetically tractable Drosophila model and primary sensory neurons isolated from adult mouse to examine the mechanisms underlying CIPN and identify protective pathways. We found that chronic treatment of Drosophila larvae with paclitaxel caused degeneration and altered the branching pattern of nociceptive neurons, and reduced thermal nociceptive responses. We further found that nociceptive neuron-specific overexpression of integrins, which are known to support neuronal maintenance in several systems, conferred protection from paclitaxel-induced cellular and behavioral phenotypes. Live imaging and superresolution approaches provide evidence that paclitaxel treatment causes cellular changes that are consistent with alterations in endosome-mediated trafficking of integrins. Paclitaxel-induced changes in recycling endosomes precede morphological degeneration of nociceptive neuron arbors, which could be prevented by integrin overexpression. We used primary dorsal root ganglia (DRG) neuron cultures to test conservation of integrin-mediated protection. We show that transduction of a human integrin ß-subunit 1 also prevented degeneration following paclitaxel treatment. Furthermore, endogenous levels of surface integrins were decreased in paclitaxel-treated mouse DRG neurons, suggesting that paclitaxel disrupts recycling in vertebrate sensory neurons. Altogether, our study supports conserved mechanisms of paclitaxel-induced perturbation of integrin trafficking and a therapeutic potential of restoring neuronal interactions with the extracellular environment to antagonize paclitaxel-induced toxicity in sensory neurons.

Scarless engineering of the Drosophila genome near any site-specific integration site.

We describe a simple and efficient technique that allows scarless engineering of Drosophila genomic sequences near any landing site containing an inverted attP cassette, such as a MiMIC insertion. This two-step method combines phiC31 integrase-mediated site-specific integration and homing nuclease-mediated resolution of local duplications, efficiently converting the original landing site allele to modified alleles that only have the desired change(s). Dominant markers incorporated into this method allow correct individual flies to be efficiently identified at each step. In principle, single attP sites and FRT sites are also valid landing sites. Given the large and increasing number of landing site lines available in the fly community, this method provides an easy and fast way to efficiently edit the majority of the Drosophila genome in a scarless manner. This technique should also be applicable to other species.

Pathogenic role of delta 2 tubulin in bortezomib-induced peripheral neuropathy.

The pathogenesis of chemotherapy-induced peripheral neuropathy (CIPN) is poorly understood. Here, we report that the CIPN-causing drug bortezomib (Bort) promotes delta 2 tubulin (D2) accumulation while affecting microtubule stability and dynamics in sensory neurons in vitro and in vivo and that the accumulation of D2 is predominant in unmyelinated fibers and a hallmark of bortezomib-induced peripheral neuropathy (BIPN) in humans. Furthermore, while D2 overexpression was sufficient to cause axonopathy and inhibit mitochondria motility, reduction of D2 levels alleviated both axonal degeneration and the loss of mitochondria motility induced by Bort. Together, our data demonstrate that Bort, a compound structurally unrelated to tubulin poisons, affects the tubulin cytoskeleton in sensory neurons in vitro, in vivo, and in human tissue, indicating that the pathogenic mechanisms of seemingly unrelated CIPN drugs may converge on tubulin damage. The results reveal a previously unrecognized pathogenic role for D2 in BIPN that may occur through altered regulation of mitochondria motility.

Characterization of Proprioceptive System Dynamics in Behaving Drosophila Larvae Using High-Speed Volumetric Microscopy.

Proprioceptors provide feedback about body position that is essential for coordinated movement. Proprioceptive sensing of the position of rigid joints has been described in detail in several systems; however, it is not known how animals with a flexible skeleton encode their body positions. Understanding how diverse larval body positions are dynamically encoded requires knowledge of proprioceptor activity patterns in vivo during natural movement. Here we used high-speed volumetric swept confocally aligned planar excitation (SCAPE) microscopy in crawling Drosophila larvae to simultaneously track the position, deformation, and intracellular calcium activity of their multidendritic proprioceptors. Most proprioceptive neurons were found to activate during segment contraction, although one subtype was activated by extension. During cycles of segment contraction and extension, different proprioceptor types exhibited sequential activity, providing a continuum of position encoding during all phases of crawling. This sequential activity was related to the dynamics of each neuron's terminal processes, and could endow each proprioceptor with a specific role in monitoring different aspects of body-wall deformation. We demonstrate this deformation encoding both during progression of contraction waves during locomotion as well as during less stereotyped, asymmetric exploration behavior. Our results provide powerful new insights into the body-wide neuronal dynamics of the proprioceptive system in crawling Drosophila, and demonstrate the utility of our SCAPE microscopy approach for characterization of neural encoding throughout the nervous system of a freely behaving animal.

Nociceptive interneurons control modular motor pathways to promote escape behavior in Drosophila.

Rapid and efficient escape behaviors in response to noxious sensory stimuli are essential for protection and survival. Yet, how noxious stimuli are transformed to coordinated escape behaviors remains poorly understood. In Drosophila larvae, noxious stimuli trigger sequential body bending and corkscrew-like rolling behavior. We identified a population of interneurons in the nerve cord of Drosophila, termed Down-and-Back (DnB) neurons, that are activated by noxious heat, promote nociceptive behavior, and are required for robust escape responses to noxious stimuli. Electron microscopic circuit reconstruction shows that DnBs are targets of nociceptive and mechanosensory neurons, are directly presynaptic to pre-motor circuits, and link indirectly to Goro rolling command-like neurons. DnB activation promotes activity in Goro neurons, and coincident inactivation of Goro neurons prevents the rolling sequence but leaves intact body bending motor responses. Thus, activity from nociceptors to DnB interneurons coordinates modular elements of nociceptive escape behavior.

Diversity of Internal Sensory Neuron Axon Projection Patterns Is Controlled by the POU-Domain Protein Pdm3 in Drosophila Larvae.

Internal sensory neurons innervate body organs and provide information about internal state to the CNS to maintain physiological homeostasis. Despite their conservation across species, the anatomy, circuitry, and development of internal sensory systems are still relatively poorly understood. A largely unstudied population of larval Drosophila sensory neurons, termed tracheal dendrite (td) neurons, innervate internal respiratory organs and may serve as a model for understanding the sensing of internal states. Here, we characterize the peripheral anatomy, central axon projection, and diversity of td sensory neurons. We provide evidence for prominent expression of specific gustatory receptor genes in distinct populations of td neurons, suggesting novel chemosensory functions. We identify two anatomically distinct classes of td neurons. The axons of one class project to the subesophageal zone (SEZ) in the brain, whereas the other terminates in the ventral nerve cord (VNC). We identify expression and a developmental role of the POU-homeodomain transcription factor Pdm3 in regulating the axon extension and terminal targeting of SEZ-projecting td neurons. Remarkably, ectopic Pdm3 expression is alone sufficient to switch VNC-targeting axons to SEZ targets, and to induce the formation of putative synapses in these ectopic target zones. Our data thus define distinct classes of td neurons, and identify a molecular factor that contributes to diversification of axon targeting. These results introduce a tractable model to elucidate molecular and circuit mechanisms underlying sensory processing of internal body status and physiological homeostasis.SIGNIFICANCE STATEMENT How interoceptive sensory circuits develop, including how sensory neurons diversify and target distinct central regions, is still poorly understood, despite the importance of these sensory systems for maintaining physiological homeostasis. Here, we characterize classes of Drosophila internal sensory neurons (td neurons) and uncover diverse axonal projections and expression of chemosensory receptor genes. We categorize td neurons into two classes based on dichotomous axon target regions, and identify the expression and role of the transcription factor Pdm3 in mediating td axon targeting to one of these target regions. Our results provide an entry point into studying internal sensory circuit development and function, and establish Pdm3 as a regulator of interoceptive axon targeting.

A Drosophila model of Fragile X syndrome exhibits defects in phagocytosis by innate immune cells.

Fragile X syndrome, the most common known monogenic cause of autism, results from the loss of FMR1, a conserved, ubiquitously expressed RNA-binding protein. Recent evidence suggests that Fragile X syndrome and other types of autism are associated with immune system defects. We found that Drosophila melanogaster Fmr1 mutants exhibit increased sensitivity to bacterial infection and decreased phagocytosis of bacteria by systemic immune cells. Using tissue-specific RNAi-mediated knockdown, we showed that Fmr1 plays a cell-autonomous role in the phagocytosis of bacteria. Fmr1 mutants also exhibit delays in two processes that require phagocytosis by glial cells, the immune cells in the brain: neuronal clearance after injury in adults and the development of the mushroom body, a brain structure required for learning and memory. Delayed neuronal clearance is associated with reduced recruitment of activated glia to the site of injury. These results suggest a previously unrecognized role for Fmr1 in regulating the activation of phagocytic immune cells both in the body and the brain.

Dendritic diversification through transcription factor-mediated suppression of alternative morphologies.

Neurons display a striking degree of functional and morphological diversity, and the developmental mechanisms that underlie diversification are of significant interest for understanding neural circuit assembly and function. We find that the morphology of Drosophila sensory neurons is diversified through a series of suppressive transcriptional interactions involving the POU domain transcription factors Pdm1 (Nubbin) and Pdm2, the homeodomain transcription factor Cut, and the transcriptional regulators Scalloped and Vestigial. Pdm1 and Pdm2 are expressed in a subset of proprioceptive sensory neurons and function to inhibit dendrite growth and branching. A subset of touch receptors show a capacity to express Pdm1/2, but Cut represses this expression and promotes more complex dendritic arbors. Levels of Cut expression are diversified in distinct sensory neurons by selective expression of Scalloped and Vestigial. Different levels of Cut impact dendritic complexity and, consistent with this, we show that Scalloped and Vestigial suppress terminal dendritic branching. This transcriptional hierarchy therefore acts to suppress alternative morphologies to diversify three distinct types of somatosensory neurons.

Swept confocally-aligned planar excitation (SCAPE) microscopy for high speed volumetric imaging of behaving organisms.

We report a new 3D microscopy technique that allows volumetric imaging of living samples at ultra-high speeds: Swept, confocally-aligned planar excitation (SCAPE) microscopy. While confocal and two-photon microscopy have revolutionized biomedical research, current implementations are costly, complex and limited in their ability to image 3D volumes at high speeds. Light-sheet microscopy techniques using two-objective, orthogonal illumination and detection require a highly constrained sample geometry, and either physical sample translation or complex synchronization of illumination and detection planes. In contrast, SCAPE microscopy acquires images using an angled, swept light-sheet in a single-objective, en-face geometry. Unique confocal descanning and image rotation optics map this moving plane onto a stationary high-speed camera, permitting completely translationless 3D imaging of intact samples at rates exceeding 20 volumes per second. We demonstrate SCAPE microscopy by imaging spontaneous neuronal firing in the intact brain of awake behaving mice, as well as freely moving transgenic Drosophila larvae.

Development of the embryonic and larval peripheral nervous system of Drosophila.

The peripheral nervous system (PNS) of embryonic and larval stage Drosophila consists of diverse types of sensory neurons positioned along the body wall. Sensory neurons, and associated end organs, show highly stereotyped locations and morphologies. Many powerful genetic tools for gene manipulation available in Drosophila make the PNS an advantageous system for elucidating basic principles of neural development. Studies of the Drosophila PNS have provided key insights into molecular mechanisms of cell fate specification, asymmetric cell division, and dendritic morphogenesis. A canonical lineage gives rise to sensory neurons and associated organs, and cells within this lineage are diversified through asymmetric cell divisions. Newly specified sensory neurons develop specific dendritic patterns, which are controlled by numerous factors including transcriptional regulators, interactions with neighboring neurons, and intracellular trafficking systems. In addition, sensory axons show modality specific terminations in the central nervous system, which are patterned by secreted ligands and their receptors expressed by sensory axons. Modality-specific axon projections are critical for coordinated larval behaviors. We review the molecular basis for PNS development and address some of the instances in which the mechanisms and molecules identified are conserved in vertebrate development.

SAX-7 and menorin light the path for dendrite morphogenesis.

Environmental and cellular cues pattern dendritic growth and direct dendrites to their targets. However, little is known about the signals regulating interactions with the surrounding substrate. Dong et al. and Salzberg et al. now identify a tripartite ligand-receptor complex that conveys cues from the substrate necessary for the patterning of complex dendrites in C. elegans.