Control of T-shaped Bifurcation by Multiple Guidance Mechanisms during Dorsal Funiculus Development in the Spinal Cord.

The dorsal funiculus in the spinal cord relays somatosensory information to the brain. It is made of T-shaped bifurcation of dorsal root ganglion (DRG) sensory axons. Our previous study has shown that Slit signaling is required for proper guidance during bifurcation, but loss of Slit does not affect all DRG axons. Here, we examined the role of the extracellular molecule Netrin-1 (Ntn1). Using wholemount staining with tissue clearing, we showed that mice lacking Ntn1 have axons escaping from the dorsal funiculus at the time of bifurcation. Genetic labeling confirmed that these misprojecting axons come from DRG neurons. Single axon analysis showed that the defect does not affect bifurcation but rather alters turning angles. To distinguish their guidance functions, we examined mice with triple deletion of Ntn1, Slit2, and Slit2 and found a completely disorganized dorsal funiculus. Comparing mice with different genotypes using immunolabeling and single axon tracing revealed additive guidance defects, demonstrating the independent roles of Ntn1 and Slit. Moreover, the same defects were observed in embryos lacking their cognate receptors. These in vivo studies thus demonstrate the presence of multi-factorial guidance mechanisms that ensure proper formation of a common branched axonal structure during spinal cord development.

Gbx2 controls amacrine cell dendrite stratification through Robo1/2 receptors.

Within the neuronal classes of the retina, amacrine cells (ACs) exhibit the greatest neuronal diversity in morphology and function. We show that the selective expression of the transcription factor Gbx2 is required for cell fate specification and dendritic stratification of an individual AC subtype in the mouse retina. We identify Robo1 and Robo2 as downstream effectors that when deleted, phenocopy the dendritic misprojections seen in Gbx2 mutants. Slit1 and Slit2, the ligands of Robo receptors, are localized to the OFF layers of the inner plexiform layer where we observe the dendritic misprojections in both Gbx2 and Robo1/2 mutants. We show that Robo receptors also are required for the proper dendritic stratification of additional AC subtypes, such as Vglut3+ ACs. These results show both that Gbx2 functions as a terminal selector in a single AC subtype and identify Slit-Robo signaling as a developmental mechanism for ON-OFF pathway segregation in the retina.

Selective axonal transport through branch junctions is directed by growth cone signaling and mediated by KIF1/kinesin-3 motors.

Development and function of nerve cells rely on the orchestration of microtubule-based transport from the cell body into distal axonal terminals. Neurons often have highly elaborate branches innervating multiple targets, but how protein or membrane cargos navigate through branch junctions to specific branch targets is unknown. Here, we demonstrate that anterograde transport of membrane vesicles through axonal branch junctions is highly selective, which is influenced by branch length and more strongly by growth cone motility. Using an optogenetic tool, we demonstrate that signaling from the growth cone can rapidly direct transport through branch junctions. We further demonstrate that such transport selectivity is differentially regulated for different vesicles and mediated by the KIF1/kinesin-3 family motors. We propose that this transport regulation through branch junctions could broadly impact neuronal development, function, and regeneration.

Synaptic dysfunction induced by glycine-alanine dipeptides in C9orf72-ALS/FTD is rescued by SV2 replenishment.

The most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) is an intronic hexanucleotide repeat expansion in the C9orf72 gene. In disease, RNA transcripts containing this expanded region undergo repeat-associated non-AUG translation to produce dipeptide repeat proteins (DPRs), which are detected in brain and spinal cord of patients and are neurotoxic both in vitro and in vivo paradigms. We reveal here a novel pathogenic mechanism for the most abundantly detected DPR in ALS/FTD autopsy tissues, poly-glycine-alanine (GA). Previously, we showed motor dysfunction in a GA mouse model without loss of motor neurons. Here, we demonstrate that mobile GA aggregates are present within neurites, evoke a reduction in synaptic vesicle-associated protein 2 (SV2), and alter Ca2+ influx and synaptic vesicle release. These phenotypes could be corrected by restoring SV2 levels. In GA mice, loss of SV2 was observed without reduction of motor neuron number. Notably, reduction in SV2 was seen in cortical and motor neurons derived from patient induced pluripotent stem cell lines, suggesting synaptic alterations also occur in patients.