Translatome Analyses Using Conditional Ribosomal Tagging in GABAergic Interneurons and Other Sparse Cell Types

Abstract

GABAergic interneurons comprise a small but diverse subset of neurons in the mammalian brain that tightly regulate neuronal circuit maturation and information flow and, ultimately, behavior. Because of their centrality in the etiology of numerous neurological disorders, examining the molecular architecture of these neurons under different physiological scenarios has piqued the interest of the broader neuroscience community. The last few years have seen an explosion in next-generation sequencing (NGS) approaches aimed at identifying genetic and state-dependent subtypes in neuronal diversity. Although several approaches are employed to address neuronal molecular diversity, ribosomal tagging has emerged at the forefront of identifying the translatomes of neuronal subtypes. This approach primarily relies on Cre recombinase-driven expression of hemagglutinin A (HA)-tagged RiboTag mice exclusively in the neuronal subtype of interest. This allows the immunoprecipitation of cell-type-specific, ribosome-engaged mRNA, expressed both in the soma and the neuronal processes, for targeted quantitative real-time PCR (qRT-PCR) or high-throughput RNA sequencing analyses. Here we detail the typical technical caveats associated with successful application of the RiboTag technique for analyzing GABAergic interneurons, and in theory other sparse cell types, in the central nervous system. Published 2020. U.S. Government. Basic Protocol 1: Breeding mice to obtain RiboTag homozygosity Support Protocol 1: Detection of ectopic Cre recombinase expression Basic Protocol 2: The RiboTag assay Support Protocol 2: Real-time quantitative PCR (qRT-PCR) assay of RiboTag-derived cell-type-specific RNA Support Protocol 3: Construction of cell-type-specific RNA-seq library Support Protocol 4: Secondary analyses of RiboTag-derived RNA-seq dataset.

Keywords: GABAergic; RiboTag; Translation affinity purification TRAP RNA-seq; immunoprecipitation; interneuron.

Figure 1.. In silico analysis of Rpl22 expression in adult hippocampal different celltypes (obtained from Saunders et al., 2018).

(A) Indicates the expression of Rpl22 in global cell classes, namely the neurons, and non-neurons. (B) Indicates Rpl22 expression in GABAergic interneuron subtypes derived from the CGE. (C) Indicates the expression in GABAergic interneuron subtypes derived from the MGE. The heatmaps indicate the log normalized expression levels of Rpl22 ranging from minimum of no expression (indicated in blue), to maximum expression (indicated in red). Abbreviations: CA1/2/3, Cornu Ammonis subfields in the hippocampus; DG, Dentate Gyrus; Pyr, pyramidal neurons; CGE, caudal ganglionic eminence; MGE, medial ganglionic eminence; Interneuron subtype markers: CCK, cholecystokinin; CHAT, choline acetyltransferase; NGFC, neurogliaform; VIP, vasoactive intestinal polypeptide; SST, somatostatin; PVALB, parvalbumin; PTHLH, parathyroid hormonelike hormone; CHODL, chondrolectin.

Figure 2.. Schematic overview of the breeding strategy and Ribotag assay in Nkx2.1 cre driver line.

(A) Cre-negative Floxed Ribotag homozygous male mouse are bred with Nkx2.1 Cre positive, mouse containing Ai14 reporter female mouse to obtain a F1 progeny that are heterozygous for Rpl22-HA allele. All litters with ectopic Ai14 expression indicating a faulty Nkx2.1 expression, are to be discarded for further breeding or experiments (B) F1 progeny that are heterozygous for the alleles are bred to generate the F2 progeny. Only the Cre-genotyping positive, Ai14 positive, Ribotag homozygotes or heterozygotes are used for subsequent experiments and for future breeding (right). Cre negative and animals containing WT Rpl22 are used as negative control, for the Ribotag assays (left). (C) An example agarose gel electrophoresis indicating the PCR amplification of Rpl22^WT/WT, Rpl22^HA/WT, Rpl22^HA/HA, alleles. (D) An outline of the Ribotag assay. Tissues obtained from the animals indicates in (B, C) are homogenized and the MGE-expressed, Rpl22-HA-associated mRNA are immunoprecipitated and sequenced in parallel with the bulk mRNA.

Figure 3.. Validation of Rpl22^HA/HA expression in the hippocampus of Nkx2.1 Cre mice.

(A) tdTomato Ai14 and (B) HA expression in Nkx2.1 Cre positive, homozygous Ribotag (Rpl22^HA/HA) / MGERibo^homo mouse line. (C) High fidelity of HA-immunostain co-expressed with tdTomato signal across all fields of hippocampus (Scale bars 200μM. Higher magnification images in CA3 subfield indicated by the white squares (Scale bars 50μM). White arrow heads indicate minimal tdTomato signal that does not contain HA-immunostaining. Abbreviations: s.o., Stratum oriens; s.p., Stratum pyramidale; s.r., Stratum radiatum; s.l-m., Stratum lacunosum-moleculare; s.m., Stratum moleculare; S.gr., Stratum granulosum; h., Hilus.

Figure 4.. Optimizing the conditions for Ribotag assay based on ribosomal integrity.

Bioanalyzer traces, measuring the RNA Integrity (RIN) Values of RNA obtained from the bulk hippocampal tissue (Input/total RNA fraction), in Green box, left; vs. hippocampal MGE interneuron-specific RNA (Rpl22-HA immunoprecipitation fraction), in Purple box, right. Comparing quality of RNA in Nkx2.1 Cre^Positive Ribotag^Heterozygous animals between (A) tissue processed after flash-freezing in liquid nitrogen Vs, (B) tissue processed from fresh animals. Comparing quality of RNA between tissue processed fresh in Nkx2.1 Cre^Positive (B) Ribotag^Heterozygous animals Vs, (C) Ribotag^Homozygouss animals. (D) Negative control for the Ribotag assay using Nkx2.1 Cre^Negative Ribotag^Homozygouss animals. Abbreviations: RIN, RNA Integrity; IP, immunoprecipitation; Ribo, Ribotag; MGE, Medial ganglionic eminence.

Figure 5.. Validation of the hippocampal MGE translatome between Ribo^het and Ribo^homo alleles using iDEP9.0.

(A) Principal component analysis plot comparing the Bulk RNA vs MGE-specific Ribotag-associated RNA across different genotypes. (B) Differentially up and downregulated genes according to DeSEq2 at a stringent FDR <0.01 and fold difference >2, in the MGE-specific Ribotag-associated translatome. (C) MGE-translatome-enriched, top 25 genes obtained from Rpl22^HA/HA RNAsequencing. Genes highlighted in green are established MGE-enriched positive controls (see Supplemental Table. 1 for the complete list of differentially enriched genes identified in the present Ribo^homo Ribotag assay).

Figure 6.. Expression of positive and negative control genes in the hippocampus of control and Nkx2.1-Ribotag mouse.

Ratio of normalized expression reads between the MGE-enriched Rpl22-HA immunoprecipitated and bulk tissue in logarithmic scale (A) established genes known to be enriched in all MGE-derived interneurons (B) established genes known to be enriched different subtypes of MGE-derived interneurons (PV, SST and NGFC), (C) established genes that are not enriched in MGE-derived interneurons, but in CGE-derived interneurons or pyramidal neurons or microglia.

Figure 7.. Understanding the results.

(A) MGE-translatome: bulk transcriptome Log2. enrichment ratio of hippocampal-expressed control genes, Ai14, Lhx6, Nkx2.1 and Dlx6. (B) Raw normalized read counts of the same control genes, shown in normal scale (left) and log-scale (right), to demonstrate the high MGE-expression of Ai14 reporter and Lhx6, but a low MGE-expression of Nkx2.1 and Dlx6 in the adult mouse hippocampus.