Comparing Automated Morphology Quantification Software on Dendrites of Uninjured and Injured Drosophila Neurons

Dendrites shape inputs and integration of depolarization that controls neuronal activity in the nervous system. Neuron pathologies can damage dendrite architecture and cause abnormalities in morphologies after injury. Dendrite regeneration can be quantified by various parameters, including total dendrite length and number of dendrite branches using manual or automated image analysis approaches. However, manual quantification is tedious and time consuming and automated approaches are often trained using wildtype neurons, making them poorly suited for analysis of genetically manipulated or injured dendrite arbors. In this study, we tested how well automated image analysis software performed on class IV Drosophila neurons, which have several hundred individual dendrite branches. We applied each software to automatically quantify features of uninjured neurons and neurons that regenerated new dendrites after injury. Regenerated arbors exhibit defects across multiple features of dendrite morphology, which makes them challenging for automated pipelines to analyze. We compared the performances of three automated pipelines against manual quantification using Simple Neurite Tracer in ImageJ: one that is commercially available (Imaris) and two developed by independent research groups (DeTerm and Tireless Tracing Genie). Out of the three software tested, we determined that Imaris is the most efficient at reconstructing dendrite architecture, but does not accurately measure total dendrite length even after intensive manual editing. Imaris outperforms both DeTerm and Tireless Tracing Genie for counting dendrite branches, and is better able to recreate previous conclusions from this same dataset. This thorough comparison of strengths and weaknesses of each software demonstrates their utility for analyzing regenerated neuron phenotypes in future studies.

Netrin/UNC-6 triggers actin assembly and non-muscle myosin activity to drive dendrite retraction in the self-avoidance response

SUMMARYDendrite growth is constrained by the self-avoidance response but the downstream pathways that balance these opposing mechanisms are unknown. We have proposed that the diffusible cue UNC-6(Netrin) is captured by UNC-40 (DCC) for a short-range interaction with UNC-5 to trigger self-avoidance in the C. elegans PVD neuron. Here we report that the actin-polymerizing proteins UNC-34(Ena/VASP), WSP-1(WASP), UNC-73(Trio), MIG-10(Lamellipodin) and the Arp2/3 complex effect dendrite retraction in the self-avoidance response mediated by UNC-6(Netrin). The paradoxical idea that actin polymerization results in shorter rather than longer dendrites is explained by our finding that NMY-1 (non-muscle myosin II) is necessary for retraction and could therefore mediate this effect in a contractile mechanism. Our results also show that dendrite length is determined by the antagonistic effects on the actin cytoskeleton of separate sets of effectors for retraction mediated by UNC-6(Netrin) versus outgrowth promoted by the DMA-1 receptor. Thus, our findings suggest that the dendrite length depends on an intrinsic mechanism that balances distinct modes of actin assembly for growth versus retraction.

P.135 Autism-associated mutations in SHANK2 increase synaptic connectivity and dendrite complexity in human neurons

Background: Heterozygous loss-of-function mutations in the synaptic scaffolding gene SHANK2 are strongly associated with autism spectrum disorder (ASD). However, their impact on the function of human neurons is unknown. Derivation of induced pluripotent stem cells (iPSC) from affected individuals permits generation of live neurons to answer this question. Methods: We generated iPSCs by reprogramming dermal fibroblasts of neurotypic and ASD-affected donors. To isolate the effect of SHANK2, we used CRISPR/Cas9 to knock out SHANK2 in control iPSCs and correct a heterozygous nonsense mutation in ASD-affected donor iPSCs. We then derived cortical neurons from SOX1+ neural precursor cells differentiated from these iPSCs. Using a novel assay that overcomes line-to-line variability, we compared neuronal morphology, total synapse number, and electrophysiological properties between SHANK2 mutants and controls. Results: Relative to controls, SHANK2 mutant neurons have increased dendrite complexity, dendrite length, total synapse number (1.5-2-fold), and spontaneous excitatory postsynaptic current (sEPSC) frequency (3-7.6-fold). Conclusions: ASD-associated heterozygous loss-of-function mutations in SHANK2 increase synaptic connectivity among human neurons by increasing synapse number and sEPSC frequency. This is partially supported by increased dendrite length and complexity, providing evidence that SHANK2 functions as a suppressor of dendrite branching during neurodevelopment.

Effect of High-Fat Diet on Peripheral Neuropathy in C57BL/6 Mice

Objective. Dyslipidemia may contribute to the development of peripheral neuropathy, even in prediabetics; however, few studies have evaluated vascular dysfunction and oxidative stress in patients with peripheral neuropathy.Methods. Using high-fat diet- (HFD-) induced prediabetic C57BL/6 mice, we assessed motor and sensory nerve conduction velocity (NCV) using a BIOPAC System and thermal algesia with a Plantar Test (Hargreaves’ method) Analgesia Meter. Intraepidermal nerve fiber density and mean dendrite length were tested following standard protocols. Vascular endothelial growth factor-A (VEGF-A) and 12/15-lipoxygenase (12/15-LOX) were evaluated by immunohistochemistry and Western blot, respectively.Results. HFD-fed mice showed deficits in motor and sensory NCV, thermal hyperalgesia, reduced mean dendrite length, and VEGF-A expression in the plantar skin and increased 12/15-LOX in the sciatic nerve (P<0.05compared with controls).Conclusion. HFD may cause large myelinated nerve and small sensory nerve fiber damage, thus leading to neuropathy. The mean dendrite length may be a more sensitive marker for early detection of peripheral neuropathy. Reduced blood supply to the nerves and increased oxidative stress may contribute to the development and severity of peripheral neuropathy.