classification-neural-stem-cell-activation-state-vitro-using

Classification of Neural Stem Cell Activation State <em>In Vitro</em> Using Autofluorescence

Neural stem cells (NSCs) divide and produce newborn neurons in the adult brain through a process called adult neurogenesis. Adult NSCs are primarily ...

University of Wisconsin-Madison

The molecular basis for the neural stem cell quiescence-to-activation transition has become an important focus in the study of adult neurogenesis. Recently in Cell, Kalamakis et al. (2019) show that aged neural stem cells face greater barriers to exiting quiescence, imposed by the niche through inflammation and altered Wnt signaling.

Stem Cell Aging? Blame It on the Niche.

Maintaining a healthy proteome throughout life is critical for proper somatic stem cell function, but the complexities of the stem cell response to increases in damaged or aggregated proteins remain unclear. Here we demonstrate that adult neural stem cells (NSCs) utilize aggresomes to recover from disrupted proteostasis and describe a novel function for the intermediate filament vimentin in proteostasis as a spatial coordinator of proteasomes to the aggresome. In the absence of vimentin, NSCs have a reduced capacity to exit quiescence, a time when NSCs are required to clear a wave of aggregated proteins, and demonstrate an early age-dependent decline in proliferation and neurogenesis. Taken together, these data reveal a significant role of vimentin and aggresomes in the regulation of proteostasis during quiescent NSC activation.

Vimentin Coordinates Protein Turnover at the Aggresome during Neural Stem Cell Quiescence Exit.

Intermediate filaments (IFs) perform a diverse set of well-known functions including providing structural support for the cell and resistance to mechanical stress, yet recent evidence has revealed unexpected roles for IFs as stress response proteins. Previously, it was shown that the type III IF protein vimentin forms cage-like structures around centrosome-associated proteins destined for degradation, structures referred to as aggresomes, suggesting a role for vimentin in protein turnover. However, vimentin's function at the aggresome has remained largely understudied. In a recent report, vimentin was shown to be dispensable for aggresome formation, but played a critical role in protein turnover at the aggresome through localizing proteostasis-related machineries, such as proteasomes, to the aggresome. Here, we review evidence for vimentin's function in proteostasis and highlight the organismal implications of these findings.

Vimentin's side gig: Regulating cellular proteostasis in mammalian systems.

Although exogenous overexpression of a protein fused to a fluorescent tag can provide insight for the protein's function, it also can produce artifacts attributed to its upregulation and may not fully report the endogenous regulation of the protein of interest. To circumvent these issues, we adapted a protocol to label endogenous proteins with fluorescent tags in primary adult mouse neural stem cells . Here, we describe reagent construction, reagent delivery, and a screening strategy to isolate edited cells. For complete details on the use and execution of this protocol, please refer to Morrow et al. (2020).

Fluorescent tagging of endogenous proteins with CRISPR/Cas9 in primary mouse neural stem cells.

Metabolism in the Midwest: research from the Midwest Aging Consortium at the 49 Annual Meeting of the American Aging Association.

The aggresome is a protein turnover system in which proteins are trafficked along microtubules to the centrosome for degradation. Despite extensive focus on aggresomes in immortalized cell lines, it remains unclear if the aggresome is conserved in all primary cells and all cell-states. Here we examined the aggresome in primary adult mouse dermal fibroblasts shifted into four distinct cell-states. We found that in response to proteasome inhibition, quiescent and immortalized fibroblasts formed aggresomes, whereas proliferating and senescent fibroblasts did not. Using this model, we generated a resource to provide a characterization of the proteostasis networks in which the aggresome is used and transcriptomic features associated with the presence or absence of aggresome formation. Using this resource, we validate a previously reported role for p38 MAPK signaling in aggresome formation and identify TAK1 as a novel driver of aggresome formation upstream of p38 MAPKs. Together, our data demonstrate that the aggresome is a non-universal protein degradation system which can be used cell-state specifically and provide a resource for studying aggresome formation and function.

Adult fibroblasts use aggresomes only in distinct cell-states.

Neural stem cells (NSCs) must exit quiescence to produce neurons; however, our understanding of this process remains constrained by the technical limitations of current technologies. Fluorescence lifetime imaging (FLIM) of autofluorescent metabolic cofactors has been used in other cell types to study shifts in cell states driven by metabolic remodeling that change the optical properties of these endogenous fluorophores. Using this non-destructive, live-cell, and label-free strategy, we found that quiescent NSCs (qNSCs) and activated NSCs (aNSCs) have unique autofluorescence profiles. Specifically, qNSCs display an enrichment of autofluorescence localizing to a subset of lysosomes, which can be used as a graded marker of NSC quiescence to predict cell behavior at single-cell resolution. Coupling autofluorescence imaging with single-cell RNA sequencing, we provide resources revealing transcriptional features linked to deep quiescence and rapid NSC activation. Together, we describe an approach for tracking mouse NSC activation state and expand our understanding of adult neurogenesis.

Article	Year
Stem Cell Aging? Blame It on the Niche. Cell stem cell\| PubMed ID: 30849364	2019
Vimentin Coordinates Protein Turnover at the Aggresome during Neural Stem Cell Quiescence Exit. Cell stem cell\| PubMed ID: 32109376	2020
Vimentin's side gig: Regulating cellular proteostasis in mammalian systems. Cytoskeleton (Hoboken, N.J.)\| PubMed ID: 33190414	2020
Fluorescent tagging of endogenous proteins with CRISPR/Cas9 in primary mouse neural stem cells. STAR protocols\| PubMed ID: 34430917	2021
Metabolism in the Midwest: research from the Midwest Aging Consortium at the 49 Annual Meeting of the American Aging Association. GeroScience\| PubMed ID: 34714522	2022
Adult fibroblasts use aggresomes only in distinct cell-states. Scientific reports\| PubMed ID: 36056070	2022
Autofluorescence is a biomarker of neural stem cell activation state. Cell stem cell\| PubMed ID: 38521057	2024

Christopher S. Morrow

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