GDF15 is a dynamic biomarker of the integrated stress response in the central nervous system.

AIM: Characterize Growth Differentiation Factor 15 (GDF15) as a secreted biomarker of the integrated stress response (ISR) within the central nervous system (CNS). METHODS: We determined GDF15 levels utilizing in vitro and in vivo neuronal systems wherein the ISR was activated. Primarily, we used the murine model of vanishing white matter disease (VWMD), a neurological disease driven by persistent ISR in the CNS, to establish a link between levels of GDF15 in the cerebrospinal fluid (CSF) and ISR gene expression signature in the CNS. GDF15 was also determined in the CSF of VWM patients. RESULTS: GDF15 expression was increased concomitant to ISR activation in stress-induced primary astrocytes as well as in retinal ganglion cells following optic nerve crush, while treatment with 2Bact, a specific eIF2B activator, suppressed both the ISR and GDF15. In the VWMD model, CSF GDF15 levels corresponded with the magnitude of the ISR and were reduced by 2BAct. In VWM patients, mean CSF GDF15 was elevated >20-fold as compared to healthy controls, whereas plasma GDF15 was undifferentiated. CONCLUSIONS: These data suggest that CSF GDF15 is a dynamic marker of ISR activation in the CNS and may serve as a pharmacodynamic biomarker for ISR-modulating therapies.

Mapping interactions between the CRAC activation domain and CC1 regulating the activity of the ER Ca2+ sensor STIM1.

Stromal interaction molecule 1 (STIM1) is a widely expressed protein that functions as the endoplasmic reticulum (ER) Ca2+ sensor and activator of Orai1 channels. In resting cells with replete Ca2+ stores, an inhibitory clamp formed by the coiled-coil 1 (CC1) domain interacting with the CRAC-activation domain (CAD) of STIM1 helps keep STIM1 in a quiescent state. Following depletion of ER Ca2+ stores, the brake is released, allowing CAD to extend away from the ER membrane and enabling it to activate Orai1 channels. However, the molecular determinants of CC1-CAD interactions that enforce the inhibitory clamp are incompletely understood. Here, we performed Ala mutagenesis in conjunction with live-cell FRET analysis to examine residues in CC1 and CAD that regulate the inhibitory clamp. Our results indicate that in addition to previously identified hotspots in CC1⍺1 and CC3, several hydrophobic residues in CC2 and the apex region of CAD are critical for CC1-CAD interactions. Mutations in these residues loosen the CC1-CAD inhibitory clamp to release CAD from CC1 in cells with replete Ca2+ stores. By contrast, altering the hydrophobic residues L265 and L273 strengthens the clamp to prevent STIM1 activation. Inclusion of the inactivation domain of STIM1 helps stabilize CC1-CAD interaction in several mutants to prevent spontaneous STIM1 activation. In addition, R426C, a human disease-linked mutation in CC3, affects the clamp but also impairs Orai1 binding to inhibit CRAC channel activation. These results identify the CC2, apex, and inactivation domain regions of STIM1 as important determinants of STIM1 activation.

Orai1 Channels Are Essential for Amplification of Glutamate-Evoked Ca2+ Signals in Dendritic Spines to Regulate Working and Associative Memory.

Store-operated Orai1 calcium channels function as highly Ca2+-selective ion channels and are broadly expressed in many tissues including the central nervous system, but their contributions to cognitive processing are largely unknown. Here, we report that many measures of synaptic, cellular, and behavioral models of learning are markedly attenuated in mice lacking Orai1 in forebrain excitatory neurons. Results with focal glutamate uncaging in hippocampal neurons support an essential role of Orai1 channels in amplifying NMDA-receptor-induced dendritic Ca2+ transients that drive activity-dependent spine morphogenesis and long-term potentiation at Schaffer collateral-CA1 synapses. Consistent with these signaling roles, mice lacking Orai1 in pyramidal neurons (but not interneurons) exhibit striking deficits in working and associative memory tasks. These findings identify Orai1 channels as essential regulators of dendritic spine Ca2+ signaling, synaptic plasticity, and cognition.

Mechanical stress activates NMDA receptors in the absence of agonists.

While studying the physiological response of primary rat astrocytes to fluid shear stress in a model of traumatic brain injury (TBI), we found that shear stress induced Ca2+ entry. The influx was inhibited by MK-801, a specific pore blocker of N-Methyl-D-aspartic acid receptor (NMDAR) channels, and this occurred in the absence of agonists. Other NMDA open channel blockers ketamine and memantine showed a similar effect. The competitive glutamate antagonists AP5 and GluN2B-selective inhibitor ifenprodil reduced NMDA-activated currents, but had no effect on the mechanically induced Ca2+ influx. Extracellular Mg2+ at 2 mM did not significantly affect the shear induced Ca2+ influx, but at 10 mM it produced significant inhibition. Patch clamp experiments showed mechanical activation of NMDAR and inhibition by MK-801. The mechanical sensitivity of NMDARs may play a role in the normal physiology of fluid flow in the glymphatic system and it has obvious relevance to TBI.

A Threshold Shear Force for Calcium Influx in an Astrocyte Model of Traumatic Brain Injury.

Traumatic brain injury (TBI) refers to brain damage resulting from external mechanical force, such as a blast or crash. Our current understanding of TBI is derived mainly from in vivo studies that show measurable biological effects on neurons sampled after TBI. Little is known about the early responses of brain cells during stimuli and which features of the stimulus are most critical to cell injury. We generated defined shear stress in a microfluidic chamber using a fast pressure servo and examined the intracellular Ca(2+) levels in cultured adult astrocytes. Shear stress increased intracellular Ca(2+) depending on the magnitude, duration, and rise time of the stimulus. Square pulses with a fast rise time (∼2 ms) caused transient increases in intracellular Ca(2+), but when the rise time was extended to 20 ms, the response was much less. The threshold for a response is a matrix of multiple parameters. Cells can integrate the effect of shear force from repeated challenges: A pulse train of 10 narrow pulses (11.5 dyn/cm(2) and 10 ms wide) resulted in a 4-fold increase in Ca(2+) relative to a single pulse of the same amplitude 100 ms wide. The Ca(2+) increase was eliminated in Ca(2+)-free media, but was observed after depleting the intracellular Ca(2+) stores with thapsigargin suggesting the need for a Ca(2+) influx. The Ca(2+) influx was inhibited by extracellular Gd(3+), a nonspecific inhibitor of mechanosensitive ion channels, but it was not affected by the more specific inhibitor, GsMTx4. The voltage-gated channel blockers, nifedipine, diltiazem, and verapamil, were also ineffective. The data show that the mechanically induced Ca(2+) influx commonly associated with neuron models for TBI is also present in astrocytes, and there is a viscoelastic/plastic coupling of shear stress to the Ca(2+) influx. The site of Ca(2+) influx has yet to be determined.