Talks in this session:
- Choy: Intracellular trafficking and processing of amyloid precursor protein
- Kown: Age-associated decline in immune function; new role of SIRT1 in regulatory T cells
- Pan: Regulation of p53 and ageing by SnoN
- Grueter: Disruption of the lipid synthesis gene, DGAT1, extends longevity
Regina Choy (Berkeley; Shekman lab) — Intracellular trafficking and processing of amyloid precursor protein
The talk began with a review of the proteolytic processing of amyloid precursor protein (APP) into Aß peptides. Choy emphasized that it is important to have a balance between the amyloidogenic and non-amyloidogenic pathways – a bias toward amyloidogenesis places one at risk for Alzheimer’s disease (AD).
The big question: Where is Aß being produced inside the cells? (What are the possible intracellular sites of Aß peptide production? Where is it actually happening). The approach: study of APP trafficking. The goal: Insights into regulation of Aß production and its relationship to AD.
Building on evidence that the primary site of Aß is the endosome, Choy performed RNAi knockdowns of the endosomal sorting machinery (ESCRT complexes as well as the ATPase VPS4). Knockdown of early components in endosomal sorting result in decreased Aß production, but knocking down the later components or VPS4 results in an increase in Aß production. Together with immunofluorescence results, these findings suggest that Aß production happens after APP leaves the early endosome. Surprisingly, however, APP does not colocalize with early endosome markers in the VPS4 knockdown – in fact, it ends up getting rerouted to the TGN. This raises the possibility that Aß production may happen after APP recycles through the TGN.
More beautiful immunofluorescence data followed, bolstering the recycling hypothesis and leading Choy to conclude in favor of a model in which the primary site of Aß production is in the TGN.
Hye-Sook Kown (Gladstone; Ott lab) — Age-associated decline in immune function; new role of SIRT1 in regulatory T cells
Regulatory T cells (Treg) maintain immune tolerance, i.e., they stop the rest of the immune system from attacking the body. They accomplish this by suppressing differentiation of naive cells and the activation of effector cells. This, in turn, helps to prevent autoimmune disease and graft rejection. However, Treg cells increase their activity during aging, which might make elderly people more susceptible to infection.
Treg activity is regulated by FoxP3, which is in turn modified by acetylation that is regulated by SIRT1. Kown used mass spec to identify the specific acetylation sites on FoxP3; she found three, and raising specific antibodies against the acetylated peptides.
Inhibition of SIRT1, a deacetylase, enhances acetylation of FoxP3 at a specific site in both Jurkat T cells and mouse inducible Treg (iTreg) cells. The acetylated protein is stabilized and active, promoting Treg differentiation and survival in a variety of cell culture and in vivo assays.
Thus, by downregulating the activity of Treg cells, SIRT1 promotes a more active immune system: lower iTreg activity promotes increased differentiation of naive T cells and activation of Th1, Th2 and Th17 effector cells. In older people where SIRT1 levels are lower, higher Treg activity may result in a less responsive immune system and higher susceptibility to infection.
In questions, I asked whether SIRT1 inhibition could therefore be used to prevent autoimmune disease – the short answer is “yes”; this has advantages over expanding Treg populations ex vivo, which sometimes results in loss of FoxP3 expression.
Deng Pan (Berkeley; Luo lab) — Regulation of p53 and ageing by SnoN
Starts off with a review of the cancer-aging hypothesis, i.e., the idea that the anticancer activity of tumor suppressors like p53 have a cost: apoptosis and senescence of damaged cells ultimately reduces the regenerative capacity of tissues, contributing to age-related decline in tissue function.
Pan has focused on SnoN, an inhibitor of TGFß/Smad signaling, using a knock-in mouse in which SnoN can no longer bind the Smad promoter. Using this system, he demonstrated that SnoN can function as a tumor suppressor by activating p53-dependent senescence.
SnoN can interact with the PML-p53 pathway; the SnoN protein is a component of PML-nuclear bodies, which in turn activate p53. There are several ways to activate p53: stabilization (i.e., preventing ubiquitination); antiprepression, and promoter-specific activation. How specifically is SnoN activating p53?
Using pulldown assays, Pan showed that SnoN can directly bind to p53, in a manner that does not depend on PML. This binding stabilizes p53, probably because SnoN competes with Mdm2 (which ubiquitinates p53, targeting it for destruction). The working model is that SnoN is a stress transducer that communicates information about cellular stress to the p53 pathway.
The knock-in mice showed premature aging-related phenotypes, including kyphosis and hair loss, as well as higher levels of senescent and apoptotic cells.
Carrie Grueter (Gladstone; Farese lab) — Disruption of the lipid synthesis gene, DGAT1, extends longevity
Given how much we know about fat and lifespan, it is perhaps surprising that very few longevity studies have focused on mice with modified lipid metabolism. To remedy this omission, Carrie Grueter has been studying the effect of the DGAT1 (diacylglycerol O-acyltransferase) knockout on phenotypes including lifespan. (DGAT is involved in triglyceride synthesis.)
Hypothesis: Leanness, with a concomitant improvement in metabolism, will extend longevity.
DGAT-deficient mice use more oxygen than wildtype siblings, but do not consume proportionally more food. The knockout mice are protected from the age-related increase in fat mass, as well as age-related increases in inflammation. (Not surprising since abdominal fat is associated with chronic inflammation.) The knockouts exhibit decreased serum IGF-I levels.
The payoff: DGAT knockouts live 25% longer than wildtype. There’s a cost: according to Grueter’s data, DGAT-KO have trouble lactating and therefore have decreased fecundity. Furthermore, the knockouts are bad at surviving short-term calorie restriction: half the mice fail to survive a 48-hour fast, probably because their core body temperatures plummet in the absence of stored fat to burn – the lethality can be rescued by group-housing the mice with wildtype animals or by raising the temperature to 30°C.
So in sum, the hypothesis enumerated above seems to hold, at least when calories are abundant – but when times are tough, it’s nice to have a little bit of extra padding.