To shift or not to shift: Low density lipoprotein receptor-related protein 1 and the plasminogen activators gate phase shifting in the mammalian circadian clock

Here, we present data supporting low density lipoprotein receptor-related protein-1 (LRP- 1) and urokinase plasminogen activator (uPA) involvment in the suprachiasmatic nucleus (SCN), the primary mammalian circadian pacemaker. Previous work using extracellular recordings of SCN neurons in ex vivo hypothalamic slices demonstrated that tissue-type plasminogen activator (tPA) gates glutamate-induced phase shifts via plasmin-dependent maturation of brain derived neurotrophic factor (BDNF) and subsequent tropomysin receptor kinase B (TrkB) receptor activation. Here, we find first, that tPA knockout mice (tPA^−/−; B6.129S2-Plattm1Mlg/J) exhibit minimal phase shifting deficits in vivo and in vitro, and that uPA compensates for the lack of tPA to enable phase shifts in these mice. Intriguingly, the data support tPA, but not uPA, acting via BDNF maturation, suggesting functional compensation achieved through differential mechanisms. Second, we find that LRP-1 also regulates SCN phase shifting. Inhibiting LRP-1 with receptor associated protein (RAP) or anti-LRP-1 antibody prevents glutamate-induced phase delays and advances in neuronal activity rhythms in vitro at ZT16 and ZT23, respectively. We then turned our attention to potential interactions between tPA and LRP-1, and through three lines of evidence demonstrate that tPA proteolytic activity is not necessary for LRP-1’s permissive effect on phase shifting: 1) RAP inhibits phase shifts in tPA^-/- SCN, 2) inhibiting LRP-1 does not impact BDNF maturation, or 3) Trk receptor phosphorylation on Y680/681. Suprisingly, inhibiting LRP-1 with RAP changes N-Methyl_D-aspartic acid receptor (NMDAR) phosphorylation patterns in the SCN in vitro, by decreasing phosphorylation on S1480 of NR2B subunits. Finally, we evaluated uPA and tPA expression and proteolytic activity across the circadian day, and LRP-1 expression and phosphorylation patterns. We find evidence of circadian rhythms in tPA expression but not proteolytic activity, no rhythms in uPA expression or proteolytic activity, and potential diurnal variations in αLRP-1 but not βLRP-1 subunits. Additionally, uPA activity and βLRP-1 expression exhibit changes that correlate with the time slices are maintained in vitro, suggesting that a response to slicing injury may occlude an accurate view of expression patterns in the SCN in vitro. Collectively, the data presented here implicate uPA and LRP-1 in the processes gating glutamate-induced phase shifts in the SCN.