Heterogeneous pools of adult neural stem cells (NSCs) contribute to brain maintenance and regeneration after injury. The balance of NSC activation and quiescence, as well as the induction of lineage-specific transcription factors, may contribute to diversity of neuronal and glial fates. To identify molecular hallmarks governing these characteristics, we performed single-cell sequencing of an unbiased pool of adult subventricular zone NSCs. This analysis identified a discrete, dormant NSC subpopulation that already expresses distinct combinations of lineage-specific transcription factors during homeostasis. Dormant NSCs enter a primed-quiescent state before activation, which is accompanied by downregulation of glycolytic metabolism, Notch, and BMP signaling and a concomitant upregulation of lineage-specific transcription factors and protein synthesis. In response to brain ischemia, interferon gamma signaling induces dormant NSC subpopulations to enter the primed-quiescent state. This study unveils general principles underlying NSC activation and lineage priming and opens potential avenues for regenerative medicine in the brain.

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Adult neural stem cells are generated at embryonic stages by entering a quiescent state that allows their retention into adulthood and thereby maintenance of life-long brain homeostasis. Thus, a tight balance between the quiescence and activation state is instrumental to meet the brain demands for a specific cell type at the correct numbers, at a given time and position. Protein synthesis is the most energy-consuming process within the cell and, not surprisingly, it occurs at low rates in quiescent stem cells. This way quiescent cells adjust to energy constraints and avoid their premature depletion. Stem cell activation is characterized by upregulation of protein synthesis followed by cell division and differentiation. The role of such upregulation as causative or rather a consequence of the activation remains elusive. Here we summarize recent findings connecting stem cell activation to the regulation of protein synthesis, particularly focusing on embryonic and adult neural stem cells of the ventricular zone.

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Multiple processes are involved in gene expression including transcription, translation and stability of mRNAs and proteins. Each of these steps are tightly regulated, affecting the final dynamics of protein abundance. Various regulatory mechanisms exist at the translation step, rendering mRNA levels alone an unreliable indicator of gene expression. In addition, local regulation of mRNA translation has been particularly implicated in neuronal functions, shifting 'translatomics' to the focus of attention in neurobiology. The presented method can be used to bridge transcriptomics and proteomics.Here we describe essential modifications to the technique of polyribosome fractionation, which interrogates the translatome based on the association of actively translated mRNAs to multiple ribosomes and their differential sedimentation in sucrose gradients. Traditionally, working with in vivo samples, particularly of the central nervous system (CNS), has proven challenging due to the restricted amounts of material and the presence of fatty tissue components. In order to address this, the described protocol is specifically optimized for use with minimal amount of CNS material, as demonstrated by the use of single mouse spinal cord and brain. Briefly, CNS tissues are extracted and translating ribosomes are immobilized on mRNAs with cycloheximide. Myelin flotation is then performed to remove lipid rich components. Fractionation is performed on a sucrose gradient where mRNAs are separated according to their ribosomal loading. Isolated fractions are suitable for a range of downstream assays, including new genome wide assay technologies.

13 14 Dedicated to the memory of Bernd Fischer 15 16 SUMMARY 17 The contribution of posttranscriptional regulation of gene expression to neural stem cell 18 differentiation during tissue homeostasis remains elusive. Here we show highly dynamic 19 changes in protein synthesis along differentiation of stem cells to neurons in vivo. 20 Examination of individual transcripts using RiboTag mouse models reveals that neural stem 21 cells efficiently translate abundant transcripts, whereas translation becomes increasingly 22 controlled with the onset of differentiation. Stem cell generation of early neuroblasts involves 23 translational repression of a subset of mRNAs including the stem cell-identity factors Sox2 24 and Pax6 as well as translation machinery components. In silico motif analysis identifies a 25 pyrimidine-rich motif (PRM) in this repressed subset. A drop in mTORC1 activity at the 26 onset of differentiation selectively blocks translation of PRM-containing transcripts. Our data 27 uncovers how a drop in mTORC1 allows robust simultaneous posttranscriptional repression 28homologue O-Propargyl-Puromycin (OPP) into nascent proteins 12 . We confirmed that global 56 protein synthesis is very low in qNSCs and increases dramatically in aNSCs 2 . In 57 addition, we identified a further increase in neurogenic progenitors and a dramatic drop in 58 ENBs back to levels found in qNSC ( Fig. 1b-c). To directly assess global protein synthesis in 59 ENB that exclusively derive from NSCs we used Tlx-CreER-eYFP reporter mice. Tlx-60 recombined population overlapped with the previously characterized GLAST+PROM+ 61 population of NSC, confirming that we are looking at the same subset of cells with Tlx-62 labeling and FACS-isolation approach (Extended data Fig. 1a,b). Reporter-positive ENBs 63 exhibited a dramatic drop in OPP incorporation as compared to NSCs, similar to the drop 64 found in FACS-isolated populations (Extended data Fig. 1c,d). Thereafter, active protein 65 synthesis increases again in LNBs, which in contrast to ENBs are postmitotic and, thus, we 66 hypothesize that this increase potentially relates to the complex process of integration into the 67 neuronal network. Altogether these data support the idea that the need to differentiate and not 68 proliferation dictates the level of protein synthesis as previously reported for HFSCs 8 . From 69LNBs to early and mature neurons active protein synthesis gradually decreases ( Fig. 1 e,f), 70 supposedly because neurons rather need local proteome changes through localized 71 translation 13 . Indeed, a recent study addressing the axonal translatome by ribotagging 72 demonstrates that a subset of mRNAs with key axon-specific functions is highly enriched in 73 axons where they are locally translated 14 . 74 75 RiboTag mouse models target distinct stages of neuronal differentiation 76 To address how transcript-specific translation relates to transcript abundance, we developed a 77 system for parallel assessment of the transcriptome and translatome of NSCs and their 78 progeny ...

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