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Subcellular transcriptomes and proteomes of developing axon projections in the cerebral cortex.

ABSTRACT: The development of neural circuits relies on axon projections establishing diverse, yet well-defined, connections between areas of the nervous system. Each projection is formed by growth cones-subcellular specializations at the tips of growing axons, encompassing sets of molecules that control projection-specific growth, guidance, and target selection1. To investigate the set of molecules within native growth cones that form specific connections, here we developed growth cone sorting and subcellular RNA-proteome mapping, an approach that identifies and quantifies local transcriptomes and proteomes from labelled growth cones of single projections in vivo. Using this approach on the developing callosal projection of the mouse cerebral cortex, we mapped molecular enrichments in trans-hemispheric growth cones relative to their parent cell bodies, producing paired subcellular proteomes and transcriptomes from single neuron subtypes directly from the brain. These data provide generalizable proof-of-principle for this approach, and reveal molecular specializations of the growth cone, including accumulations of the growth-regulating kinase mTOR2, together with mRNAs that contain mTOR-dependent motifs3,4. These findings illuminate the relationships between subcellular distributions of RNA and protein in developing projection neurons, and provide a systems-level approach for the discovery of subtype- and stage-specific molecular substrates of circuit wiring, miswiring, and the potential for regeneration.

SUBMITTER: Poulopoulos A

PROVIDER: S-EPMC6484835 | biostudies-literature | 2019 Jan

REPOSITORIES: biostudies-literature

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Subcellular transcriptomes and proteomes of developing axon projections in the cerebral cortex.

Poulopoulos Alexandros A Murphy Alexander J AJ Ozkan Abdulkadir A Davis Patrick P Hatch John J Kirchner Rory R Macklis Jeffrey D JD

Nature 20190109 7739

The development of neural circuits relies on axon projections establishing diverse, yet well-defined, connections between areas of the nervous system. Each projection is formed by growth cones-subcellular specializations at the tips of growing axons, encompassing sets of molecules that control projection-specific growth, guidance, and target selection<sup>1</sup>. To investigate the set of molecules within native growth cones that form specific connections, here we developed growth cone sorting ...[more]

PMID: 30626971

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Project description:BackgroundDocosahexaenoic acid (DHA, 22:6n-3) and arachidonic acid (ARA, 20:4n-6) are the major long chain polyunsaturated fatty acids (LCPUFA) of the central nervous system (CNS). These nutrients are present in most infant formulas at modest levels, intended to support visual and neural development. There are no investigations in primates of the biological consequences of dietary DHA at levels above those present in formulas but within normal breastmilk levels.Methods and findingsTwelve baboons were divided into three formula groups: Control, with no DHA-ARA; "L", LCPUFA, with 0.33%DHA-0.67%ARA; "L3", LCPUFA, with 1.00%DHA-0.67%ARA. All the samples are from the precentral gyrus of cerebral cortex brain regions. At 12 weeks of age, changes in gene expression were detected in 1,108 of 54,000 probe sets (2.05%), with most showing <2-fold change. Gene ontology analysis assigns them to diverse biological functions, notably lipid metabolism and transport, G-protein and signal transduction, development, visual perception, cytoskeleton, peptidases, stress response, transcription regulation, and 400 transcripts having no defined function. PLA2G6, a phospholipase recently associated with infantile neuroaxonal dystrophy, was downregulated in both LCPUFA groups. ELOVL5, a PUFA elongase, was the only LCPUFA biosynthetic enzyme that was differentially expressed. Mitochondrial fatty acid carrier, CPT2, was among several genes associated with mitochondrial fatty acid oxidation to be downregulated by high DHA, while the mitochondrial proton carrier, UCP2, was upregulated. TIMM8A, also known as deafness/dystonia peptide 1, was among several differentially expressed neural development genes. LUM and TIMP3, associated with corneal structure and age-related macular degeneration, respectively, were among visual perception genes influenced by LCPUFA. TIA1, a silencer of COX2 gene translation, is upregulated by high DHA. Ingenuity pathway analysis identified a highly significant nervous system network, with epidermal growth factor receptor (EGFR) as the outstanding interaction partner.ConclusionsThese data indicate that LCPUFA concentrations within the normal range of human breastmilk induce global changes in gene expression across a wide array of processes, in addition to changes in visual and neural function normally associated with formula LCPUFA.

Project description:Cortical networks are shaped by sensory experience and are most susceptible to modifications during critical periods characterized by enhanced plasticity at the structural and functional level. A system particularly well-studied in this context is the mammalian visual system. Plasticity has been documented for the somatodendritic compartment of neurons in detail. A neuronal microdomain not yet studied in this context is the axon initial segment (AIS) located at the proximal axon segment. It is a specific electrogenic axonal domain and the site of action potential (AP) generation. Recent studies showed that structure and function of the AIS can be dynamically regulated. Here we hypothesize that the AIS shows a dynamic regulation during maturation of the visual cortex. We therefore analyzed AIS length development from embryonic day (E) 12.5 to adulthood in mice. A tri-phasic time course of AIS length remodeling during development was observed. AIS first appeared at E14.5 and increased in length throughout the postnatal period to a peak between postnatal day (P) 10 to P15 (eyes open P13-14). Then, AIS length was reduced significantly around the beginning of the critical period for ocular dominance plasticity (CP, P21). Shortest AIS were observed at the peak of the CP (P28), followed by a moderate elongation toward the end of the CP (P35). To test if the dynamic maturation of the AIS is influenced by eye opening (onset of activity), animals were deprived of visual input before and during the CP. Deprivation for 1 week prior to eye opening did not affect AIS length development. However, deprivation from P0 to 28 and P14 to 28 resulted in AIS length distribution similar to the peak at P15. In other words, deprivation from birth prevents the transient shortening of the AIS and maintains an immature AIS length. These results are the first to suggest a dynamic maturation of the AIS in cortical neurons and point to novel mechanisms in the development of neuronal excitability.

Dataset Information

Subcellular transcriptomes and proteomes of developing axon projections in the cerebral cortex.

Publications

Subcellular transcriptomes and proteomes of developing axon projections in the cerebral cortex.

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