Date of Award

8-2010

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Biochemistry and Cellular and Molecular Biology

Major Professor

Cynthia B. Peterson

Committee Members

Rebecca Prosser, Jim Hall, Jae Park, Matthew Cooper

Abstract

Mammalian circadian rhythms are controlled by a clock located in the suprachiasmatic nucleus (SCN). The mechanisms through which light phase-shifts the SCN circadian clock are similar to those underlying memory formation and long-term potentiation (LTP). Several secreted proteins, including tissue-type plasminogen activator (tPA), plasminogen, and brain-derived neurotrophic factor (BDNF), have been implicated in this process. These same proteins are important for photic phase-shifts of the SCN circadian clock. Early night glutamate application to SCN containing brain slices resets the circadian clock. Our experiments find that the endogenous tPA inhibitor, plasminogen activator inhibitor 1(PAI-1), blocked these shifts in slices from wildtype mice but not mice lacking its stabilizing protein, vitronectin (VN). Plasmin, but not plasminogen, prevented inhibition by PAI-1. Both plasmin and active BDNF reversed alpha2-antiplasmin inhibition of glutamate-induced shifts. alpha2-Antiplasmin decreased the conversion of inactive to active BDNF in the SCN. Both tPA and BDNF allowed daytime glutamate-induced phase-resetting. Together, these data are the first to demonstrate expression of these proteases in the SCN, their involvement in modulating photic phase-shifts, and their activation of BDNF in the SCN, a potential ‘gating’ mechanism for photic phase-resetting.

Using western-blot analyses of SCN tissue maintained in vitro, we find higher tPA, plasmin and mBDNF levels in the SCN at night vs. the day. Also, in vitro glutamate treatment of SCN tissue during early night increases tPA levels to ~2.5 times control levels, while similar treatments during late night and mid-day do not alter tPA expression. Glutamate treatment in the early night does not alter PAI-1, plasmin and BDNF levels. Co-treatment with glutamate and PAI-1 decreases plasmin levels (vs. glutamate treatment alone), while co-treatment with glutamate and alpha2-antiplasmin decreases the amount of pro- and mBDNF in the SCN relative to glutamate treatment alone. We also show that mBDNF levels are significantly lower in tPA knockout mice during both day and night. Together, these results support circadian clock modulation of BDNF and fibrinolytic protein levels in the SCN. They also suggest that glutamate modulates tPA expression in the SCN, while tPA and plasmin modulate BDNF expression.

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