Transneuronal Dpr12/DIP-δ interactions facilitate compartmentalized dopaminergic innervation of Drosophila mushroom body axons.

The mechanisms controlling wiring of neuronal networks are not completely understood. The stereotypic architecture of the Drosophila mushroom body (MB) offers a unique system to study circuit assembly. The adult medial MB γ-lobe is comprised of a long bundle of axons that wire with specific modulatory and output neurons in a tiled manner, defining five distinct zones. We found that the immunoglobulin superfamily protein Dpr12 is cell-autonomously required in γ-neurons for their developmental regrowth into the distal γ4/5 zones, where both Dpr12 and its interacting protein, DIP-δ, are enriched. DIP-δ functions in a subset of dopaminergic neurons that wire with γ-neurons within the γ4/5 zone. During metamorphosis, these dopaminergic projections arrive to the γ4/5 zone prior to γ-axons, suggesting that γ-axons extend through a prepatterned region. Thus, Dpr12/DIP-δ transneuronal interaction is required for γ4/5 zone formation. Our study sheds light onto molecular and cellular mechanisms underlying circuit formation within subcellular resolution.

Developmental axon regrowth and primary neuron sprouting utilize distinct actin elongation factors.

Intrinsic neurite growth potential is a key determinant of neuronal regeneration efficiency following injury. The stereotypical remodeling of Drosophila γ-neurons includes developmental regrowth of pruned axons to form adult specific connections, thereby offering a unique system to uncover growth potential regulators. Motivated by the dynamic expression in remodeling γ-neurons, we focus here on the role of actin elongation factors as potential regulators of developmental axon regrowth. We found that regrowth in vivo requires the actin elongation factors Ena and profilin, but not the formins that are expressed in γ-neurons. In contrast, primary γ-neuron sprouting in vitro requires profilin and the formin DAAM, but not Ena. Furthermore, we demonstrate that DAAM can compensate for the loss of Ena in vivo. Similarly, DAAM mutants express invariably high levels of Ena in vitro. Thus, we show that different linear actin elongation factors function in distinct contexts even within the same cell type and that they can partially compensate for each other.

Tissue-specific (ts)CRISPR as an efficient strategy for in vivo screening in Drosophila.

Gene editing by CRISPR/Cas9 is commonly used to generate germline mutations or perform in vitro screens, but applicability for in vivo screening has so far been limited. Recently, it was shown that in Drosophila, Cas9 expression could be limited to a desired group of cells, allowing tissue-specific mutagenesis. Here, we thoroughly characterize tissue-specific (ts)CRISPR within the complex neuronal system of the Drosophila mushroom body. We report the generation of a library of gRNA-expressing plasmids and fly lines using optimized tools, which provides a valuable resource to the fly community. We demonstrate the application of our library in a large-scale in vivo screen, which reveals insights into developmental neuronal remodeling.

Combining Developmental and Perturbation-Seq Uncovers Transcriptional Modules Orchestrating Neuronal Remodeling.

Developmental neuronal remodeling is an evolutionarily conserved mechanism required for precise wiring of nervous systems. Despite its fundamental role in neurodevelopment and proposed contribution to various neuropsychiatric disorders, the underlying mechanisms are largely unknown. Here, we uncover the fine temporal transcriptional landscape of Drosophila mushroom body γ neurons undergoing stereotypical remodeling. Our data reveal rapid and dramatic changes in the transcriptional landscape during development. Focusing on DNA binding proteins, we identify eleven that are required for remodeling. Furthermore, we sequence developing γ neurons perturbed for three key transcription factors required for pruning. We describe a hierarchical network featuring positive and negative feedback loops. Superimposing the perturbation-seq on the developmental expression atlas highlights a framework of transcriptional modules that together drive remodeling. Overall, this study provides a broad and detailed molecular insight into the complex regulatory dynamics of developmental remodeling and thus offers a pipeline to dissect developmental processes via RNA profiling.

Cl-NQTrp Alleviates Tauopathy Symptoms in a Model Organism through the Inhibition of Tau Aggregation-Engendered Toxicity.

Alzheimer's disease (AD) is the most abundant tauopathy and is characterized by Aß-derived plaques and tau-derived tangles, resulting from the unfolding of the corresponding monomeric subunits into ordered ß-sheet oligomers and fibrils. Intervening in the toxic aggregation process is a promising therapeutic approach, but, to date, a disease-modifying therapy is neither available for AD nor for other tauopathies. Along these lines, we have previously demonstrated that a small naphthoquinone-tryptophan hybrid, termed NQTrp, is an effective modulator of tauopathy in vitro and in vivo. However, NQTrp is difficult to synthesize and is not very stable. Therefore, we tested whether a more stable and easier-to-synthesize modified version of NQTrp, containing a Cl ion, namely Cl-NQTrp, is also an effective inhibitor of tau aggregation in vitro and in vivo. Cl-NQTrp was previously shown to efficiently inhibit the aggregation of various amyloidogenic proteins and peptides. We demonstrate that Cl-NQTrp inhibits the in vitro assembly of PHF6, the aggregation-prone fragment of tau, and alleviates tauopathy symptoms in a transgenic Drosophila model through the inhibition of tau aggregation-engendered toxicity. These results suggest that Cl-NQTrp could be a unique potential therapeutic for AD since it targets aggregation of both Aß and tau.

Naphthoquinone-Tryptophan Hybrid Inhibits Aggregation of the Tau-Derived Peptide PHF6 and Reduces Neurotoxicity.

Tauopathies, such as Alzheimer's disease (AD), are a group of disorders characterized neuropathologically by intracellular toxic accumulations of abnormal protein aggregates formed by misfolding of the microtubule-associated protein tau. Since protein self-assembly appears to be an initial key step in the pathology of this group of diseases, intervening in this process can be both a prophylactic measure and a means for modifying the course of the disease for therapeutic purposes. We and others have shown that aromatic small molecules can be effective inhibitors of aggregation of various protein assemblies, by binding to the aromatic core in aggregation-prone motifs and preventing their self-assembly. Specifically, we have designed a series of small aromatic naphthoquinone-tryptophan hybrid molecules as candidate aggregation inhibitors of ß -sheet based assembly and demonstrated their efficacy toward inhibiting aggregation of the amyloid-ß peptide, another culprit of AD, as well as of various other aggregative proteins involved in other protein misfolding diseases. Here we tested whether a leading naphthoquinone-tryptophan hybrid molecule, namely NQTrp, can be repurposed as an inhibitor of the aggregation of the tau protein in vitro and in vivo. We show that the molecule inhibits the in vitro assembly of PHF6, the aggregation-prone fragment of tau protein, reduces hyperphosphorylated tau deposits and ameliorates tauopathy-related behavioral defect in an established transgenic Drosophila model expressing human tau. We suggest that NQTrp, or optimized versions of it, could act as novel disease modifying drugs for AD and other tauopathies.

Nitric Oxide as a Switching Mechanism between Axon Degeneration and Regrowth during Developmental Remodeling.

During development, neurons switch among growth states, such as initial axon outgrowth, axon pruning, and regrowth. By studying the stereotypic remodeling of the Drosophila mushroom body (MB), we found that the heme-binding nuclear receptor E75 is dispensable for initial axon outgrowth of MB γ neurons but is required for their developmental regrowth. Genetic experiments and pharmacological manipulations on ex-vivo-cultured brains indicate that neuronally generated nitric oxide (NO) promotes pruning but inhibits regrowth. We found that high NO levels inhibit the physical interaction between the E75 and UNF nuclear receptors, likely accounting for its repression of regrowth. Additionally, NO synthase (NOS) activity is downregulated at the onset of regrowth, at least partially, by short inhibitory NOS isoforms encoded within the NOS locus, indicating how NO production could be developmentally regulated. Taken together, these results suggest that NO signaling provides a switching mechanism between the degenerative and regenerative states of neuronal remodeling.