Tran Tien Phuoc, Budnik Bogdan, Froberg John E, Macklis Jeffrey D
Department of Stem Cell and Regenerative Biology, and Center for Brain Science, Harvard University, Cambridge, MA, USA.
Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA, USA.
bioRxiv. 2024 Dec 28:2024.12.22.629968. doi: 10.1101/2024.12.22.629968.
Diverse subtypes of cortical projection neurons (PN) form long-range axonal projections that are responsible for distinct sensory, motor, cognitive, and behavioral functions. Translational control has been identified at multiple stages of PN development, but how translational regulation contributes to formation of distinct, subtype-specific long-range circuits is poorly understood. Ribosomal complexes (RCs) exhibit variations of their component proteins, with an increasing set of examples that confer specialized translational control. Here, we directly compare the protein compositions of RCs from two closely related cortical neuron subtypes-cortical output "subcerebral PN" (SCPN) and interhemispheric "callosal PN" (CPN)- during establishment of their distinct axonal connectivity. Using retrograde labeling of subtype-specific somata, purification by fluorescence-activated cell sorting, ribosome immunoprecipitation, and ultra-low-input mass spectrometry, we identify distinct protein compositions of RCs from these two subtypes. Strikingly, we identify 16 associated proteins reliably and exclusively detected only in RCs of SCPN. 10 of these proteins have known interaction with components of ribosomes; we further validated ribosome interaction with protein kinase C epsilon (PRKCE), a candidate with roles in synaptogenesis. PRKCE and a subset of SCPN-specific candidate ribosome-associated proteins also exhibit enriched gene expression by SCPN. Together, these results indicate that ribosomal complexes exhibit subtype-specific protein composition in distinct subtypes of cortical projection neurons during development, and identify potential candidates for further investigation of function in translational regulation involved in subtype-specific circuit formation.
皮质投射神经元（PN）的不同亚型形成长距离轴突投射，这些投射负责不同的感觉、运动、认知和行为功能。在PN发育的多个阶段已确定存在翻译控制，但翻译调控如何促成不同的、亚型特异性长距离回路的形成却知之甚少。核糖体复合物（RCs）的组成蛋白存在差异，越来越多的例子表明其具有特殊的翻译控制作用。在这里，我们在两种密切相关的皮质神经元亚型——皮质输出“大脑下PN”（SCPN）和半球间“胼胝体PN”（CPN）——建立其独特的轴突连接期间，直接比较了它们的核糖体复合物的蛋白质组成。通过对亚型特异性胞体进行逆行标记、利用荧光激活细胞分选进行纯化、核糖体免疫沉淀和超微量质谱分析，我们确定了这两种亚型的核糖体复合物的不同蛋白质组成。令人惊讶的是，我们鉴定出16种相关蛋白，它们仅在SCPN的核糖体复合物中可靠且专门被检测到。其中10种蛋白已知与核糖体成分相互作用；我们进一步验证了核糖体与蛋白激酶Cε（PRKCE）的相互作用，PRKCE是一种在突触发生中起作用的候选蛋白。PRKCE和一部分SCPN特异性候选核糖体相关蛋白在SCPN中也表现出基因表达富集。总之，这些结果表明，在发育过程中，核糖体复合物在皮质投射神经元的不同亚型中表现出亚型特异性蛋白质组成，并确定了在参与亚型特异性回路形成的翻译调控功能的进一步研究中的潜在候选蛋白。
