Cofilin Regulates Filopodial Structure and Flexibility in Neuronal Growth Cones

Filopodia are actin-rich cytoskeletal protrusions at the leading edge of motile cells. In neuronal growth cones they function as antennae, guiding axonal growth toward the appropriate cellular targets. Proper brain development relies on robust axonal guidance mechanisms, so it is imperative to understand how the actin cytoskeleton functions in remodeling to meet the demands of growth cone exploration. Here we show by cryo-electron tomography and fluorescence imaging that filopodia in neuronal growth cones switch between fascin-linked and cofilin-decorated states, and that this transition regulates the exclusion of fascin from the cofilactin bundle at the filopodial base by hyper-twisting individual filaments and rearranging their packing. Additionally, we show that cofilactin bundles contribute to the flexibility of filopodial actin networks, thus, likely regulating the efficiency of targeted neurite outgrowth.

Mitochondrial enzyme GPT2 regulates metabolic mechanisms required for neuron growth and motor function in vivo

The metabolic needs for postnatal growth of the human nervous system are vast. Recessive loss-of-function mutations in the mitochondrial enzyme glutamate pyruvate transaminase 2 (GPT2) in humans cause postnatal undergrowth of brain, and cognitive and motor disability. We demonstrate that GPT2 governs critical metabolic mechanisms in neurons required for neuronal growth and survival. These metabolic processes include neuronal alanine synthesis and anaplerosis, the replenishment of tricarboxylic acid (TCA) cycle intermediates. We performed metabolomics across postnatal development in Gpt2-null mouse brain to identify the trajectory of dysregulated metabolic pathways: alterations in alanine occur earliest; followed by reduced TCA cycle intermediates and reduced pyruvate; followed by elevations in glycolytic intermediates and amino acids. Neuron-specific deletion of GPT2 in mice is sufficient to cause motor abnormalities and death pre-weaning, a phenotype identical to the germline Gpt2-null mouse. Alanine biosynthesis is profoundly impeded in Gpt2-null neurons. Exogenous alanine is necessary for Gpt2-null neuronal survival in vitro, but is not needed for Gpt2-null astrocytes. Dietary alanine supplementation in Gpt2-null mice enhances animal survival, and improves the metabolic profile of Gpt2-null brain, but does not alone appear to correct motor function. In surviving Gpt2-null animals, we observe smaller upper and lower motor neurons in vivo. We also observe selective death of lower motor neurons in vivo with worsening motor behavior with age. In conclusion, these studies of the pathophysiology of GPT2 Deficiency have identified metabolic mechanisms required for neuronal growth and that potentially underlie selective neuronal vulnerabilities in motor neurons.

Label-free monitoring of 3D cortical neuronal growth in vitro using optical diffraction tomography

The highly complex central nervous systems of mammals are often studied using three-dimensional (3D) in vitro primary neuronal cultures. A coupled confocal microscopy and immunofluorescence labeling are widely utilized for visualizing the 3D structures of neurons. However, this requires fixation of the neurons and is not suitable for monitoring an identical sample at multiple time points. Thus, we propose a label-free monitoring method for 3D neuronal growth based on refractive index tomograms obtained by optical diffraction tomography. The 3D morphology of the neurons was clearly visualized, and the developmental processes of neurite outgrowth in 3D spaces were analyzed for individual neurons.