Urokinase‐type plasminogen activator modulates mammalian circadian clock phase regulation in tissue‐type plasminogen activator knockout mice

Abstract

Glutamate phase shifts the circadian clock in the mammalian suprachiasmatic nucleus (SCN) by activating NMDA receptors. Tissue-type plasminogen activator (tPA) gates phase shifts by activating plasmin to generate m(ature) BDNF, which binds TrkB receptors allowing clock phase shifts. Here, we investigate phase shifting in tPA knockout (tPA ; B6.129S2-Plat /J) mice, and identify urokinase-type plasminogen activator (uPA) as an additional circadian clock regulator. Behavioral activity rhythms in tPA mice entrain to a light-dark (LD) cycle and phase shift in response to nocturnal light pulses with no apparent loss in sensitivity. When the LD cycle is inverted, tPA mice take significantly longer to entrain than C57BL/6J wild-type (WT) mice. SCN brain slices from tPA mice exhibit entrained neuronal activity rhythms and phase shift in response to nocturnal glutamate with no change in dose-dependency. Pre-treating slices with the tPA/uPA inhibitor, plasminogen activator inhibitor-1 (PAI-1), inhibits glutamate-induced phase delays in tPA slices. Selective inhibition of uPA with UK122 prevents glutamate-induced phase resetting in tPA but not WT SCN slices. tPA expression is higher at night than the day in WT SCN, while uPA expression remains constant in WT and tPA slices. Casein-plasminogen zymography reveals that neither tPA nor uPA total proteolytic activity is under circadian control in WT or tPA SCN. Finally, tPA SCN tissue has lower mBDNF levels than WT tissue, while UK122 does not affect mBDNF levels in either strain. Together, these results suggest that either tPA or uPA can support photic/glutamatergic phase shifts of the SCN circadian clock, possibly acting through distinct mechanisms.

Circadian rhythm and sleep-wake systems share the dynamic extracellular synaptic milieu

“…One of the first indications that extracellular proteases influence the SCN circadian clock was data showing that the plasminogen activating cascade regulates SCN clock phase resetting. Proteins within this enzymatic cascade, including tPA, uPA, PAI-1, vitronectin, plasminogen, and plasmin are all expressed in the SCN (Mou et al, 2009, Cooper et al, 2017). tPA expression in the SCN is rhythmic, with high levels at night and low levels during the day (Cooper et al, 2017).…”

Regulation of Locomotor activity in fed, fasted, and food-restricted mice lacking tissue-type plasminogen activator

“…We previously reported that tPA −/− mice took longer to adjust to a 12-h shift of the LD cycle than did tPA +/+ mice [ 12 ]. However, these shifts represent only the phase delaying effects of light.…”

“…The loss of tPA should have a significant impact on the ability of animals to entrain to light cycles, through reduced BDNF levels [ 6 , 9 ]. However, recent data suggests that urokinase-type plasminogen activity may substitute for tPA in the tPA −/− mice [ 12 ]. Our initial finding was that tPA −/− mice had reduced nocturnal wheel-running under a standard 12:12 LD cycle.…”

“…We interpret this to mean that the residual mBDNF remaining is sufficient to allow for normal entrainment of the circadian clock by light. This residual BDNF is produced by the activation of plasmin by enzymes other than tPA, such as urokinase-type plasminogen activator [ 12 ] or other enzymes that perform this function such as kallikreins [ 23 ].…”

“…These findings suggest that tPA activity is important for regulating glutamate induced phase shifts. Surprisingly, mice that lack tPA have normal free-running periods and normal phase-shifting responses to light pulses, though they do show slower entrainment to a large shift of the light-dark cycle [ 12 ]. This suggests that any deficiency in entrainment in these mice is modest.…”