Talks in this session:
- Rafalski: Sirt1 in adult neural stem cells
- Charville: Non-random chromosome segregation in skeletal muscle precursor cells
- Xie: Connecting molecular markers and morphological changes to the lifespan of individual yeast cells
Victoria Rafalski (Stanford; Brunet lab) — Sirt1 in adult neural stem cells
Cognitive decline occurs with age: speed of processing, working memory, and long-term memory all decline. Presumably cell loss is partially to blame – not only loss of neurons, but also other types of cells (e.g., oligodendrocytes). Neural stem cells (NSC) can regenerate lost cells to some extent, but their ability to do so diminishes with age.
The Brunet lab is looking at the idea that pathways that control lifespan in “lower” organisms (worms; yeast) may be involved in regenerative capacity in “higher” organisms (us; mice). Rafalski’s work is focusing on the now-famous SIRT1. SIRT1 is downregulated over the course of differentiation, so there’s a smoking gun – but is there a causative relationship between SIRT1 downregulation and loss of regenerative capacity in NSCs?
Rafalski has constructed a mouse with a brain-specific deletion of SIRT1. Her metabolic labeling experiments show that loss of SIRT1 results in increased NSC proliferation in part of the brain called dentate gyrus – leading to the hypothesis that SIRT1 prevents the premature proliferative exhaustion of the NSC pool – in other words, SIRT1 prevents early cell division in order to preserve replicative capacity for late life. She also asked whether SIRT1 plays a role in differentiation. Loss of SIRT1 increases the number of oligodendrocytes, probably because in the absence of SIRT1 there are more oligodendrocyte precursors in the brain.
Overall, the findings point toward a role for SIRT1 in maintaining regenerative capacity in the brain. Hopefully, future experiments will explore the functional role of this pathway in maintenance of cognitive function throughout the aging – e.g., do mice that lack neuronal SIRT1 undergo more rapid cognitive decline than wildtype? (From previously published work on whole-organism knockdowns, it appears that the mice do indeed have memory deficits.)
- Moving from neural stem cells to muscle stem cells…
Greg Charville (Stanford; Rando lab) — Non-random chromosome segregation in skeletal muscle precursor cells
Satellite cells are committed adult muscle stem cells. Under normal conditions they are senescent, but upon injury they rapidly proliferate into myoblasts, which in turn beget muscle.
Proliferating muscle precursor cells divide asymmetrically, to regenerate the satellite cell and produce a new myoblast. During this division, chromosomes are also segregated asymmetrically. Charville used a clever and subtle metabolic labeling approach to demonstrate that newly synthesized chromosomes are preferentially segregated to one of the two sister nuclei generated in this asymmetric division.
Why does this happen? Charville explored the hypothesis that the nonrandom segregation was a function of persistent DNA damage. Activated muscle precursor cells exhibit replication-associated DNA damage, and the markers of DNA damage localize asymmetrically in the sister nuclei. The Numb protein, a pro-differentiation factor that also is an inhibitor of the Notch pathway, cosegregates with markers of DNA damage. Numb stabilizes p53, so this protein could be orchestrating the more robust DNA damage response required in the more damaged sister nucleus. It is not yet known how these asymmetries influence the cells’ ultimate fate (in the sense of differentiation).
Overall, Charville hypothesizes that this phenomenon serves to maintain the genomic integrity of the stem cell population.
- And now on to a (somewhat) simpler system…
Zhengwei Xie (UCSF; Li lab) — Connecting molecular markers and morphological changes to the lifespan of individual yeast cells
Yeast have proven an important model system in the study of aging; budding yeast undergo asymmetric divisions in which the mother (old) and daughter (new) can be distinguished, allowing a study of replicative aging in a genetically tractable system.
Xie has developed a microfluidic system for studying yeast aging. Mother cells are immobilized with streptavidin, while daughter cells are washed away; this allows the direct observation of an aging population of mother cells – how many daughters does each mother produce? What is the division timing? The system also allows Xie to measure lifespan in an automated manner, and simultaneously follow fluorescently labeled proteins, cell morphology, and staining for a variety of other phenotypes (ROS, mitochondria).
Using this system, Xie has shown that lifespan is negatively correlated with the activity of the HSP104 promoter, in particular with the levels of a specific transcriptional factor that acts on that promoter. He has also observed progressive mitochondrial abnormalities arising in old mother cells: Old mothers contain “blobs” that contain mitochondrial protein markers but not mitochondrial DNA.
The microfluidics system is very powerful, allowing temporal sequencing of molecular events in single cells. Exploiting this power, Xie demonstrated that the HSP104 promoter is induced after the appearance of the mitochondrial blobs, suggesting that the high HSP104 activity may be a marker of a stressed or moribund cell. Indeed, cells with damaged mitochondria appear to have elevated levels of reactive oxygen species (ROS).