Functional Impact of Corticotropin-Releasing Factor Exposure on Tau Phosphorylation and Axon Transport.

Stress exposure or increased levels of corticotropin-releasing factor (CRF) induce hippocampal tau phosphorylation (tau-P) in rodent models, a process that is dependent on the type-1 CRF receptor (CRFR1). Although these preclinical studies on stress-induced tau-P provide mechanistic insight for epidemiological work that identifies stress as a risk factor for Alzheimer's disease (AD), the actual impact of stress-induced tau-P on neuronal function remains unclear. To determine the functional consequences of stress-induced tau-P, we developed a novel mouse neuronal cell culture system to explore the impact of acute (0.5hr) and chronic (2hr) CRF treatment on tau-P and integral cell processes such as axon transport. Consistent with in vivo reports, we found that chronic CRF treatment increased tau-P levels and caused globular accumulations of phosphorylated tau in dendritic and axonal processes. Furthermore, while both acute and chronic CRF treatment led to significant reduction in CREB activation and axon transport of brain-derived neurotrophic factor (BDNF), this was not the case with mitochondrial transport. Acute CRF treatment caused increased mitochondrial velocity and distance traveled in neurons, while chronic CRF treatment modestly decreased mitochondrial velocity and greatly increased distance traveled. These results suggest that transport of cellular energetics may take priority over growth factors during stress. Tau-P was required for these changes, as co-treatment of CRF with a GSK kinase inhibitor prevented CRF-induced tau-P and all axon transport changes. Collectively, our results provide mechanistic insight into the consequences of stress peptide-induced tau-P and provide an explanation for how chronic stress via CRF may lead to neuronal vulnerability in AD

Functional Impact of Corticotropin-Releasing Factor Exposure on Tau Phosphorylation and Axon Transport

<div><p>Stress exposure or increased levels of corticotropin-releasing factor (CRF) induce hippocampal tau phosphorylation (tau-P) in rodent models, a process that is dependent on the type-1 CRF receptor (CRFR1). Although these preclinical studies on stress-induced tau-P provide mechanistic insight for epidemiological work that identifies stress as a risk factor for Alzheimer’s disease (AD), the actual impact of stress-induced tau-P on neuronal function remains unclear. To determine the functional consequences of stress-induced tau-P, we developed a novel mouse neuronal cell culture system to explore the impact of acute (0.5hr) and chronic (2hr) CRF treatment on tau-P and integral cell processes such as axon transport. Consistent with in vivo reports, we found that chronic CRF treatment increased tau-P levels and caused globular accumulations of phosphorylated tau in dendritic and axonal processes. Furthermore, while both acute and chronic CRF treatment led to significant reduction in CREB activation and axon transport of brain-derived neurotrophic factor (BDNF), this was not the case with mitochondrial transport. Acute CRF treatment caused increased mitochondrial velocity and distance traveled in neurons, while chronic CRF treatment modestly decreased mitochondrial velocity and greatly increased distance traveled. These results suggest that transport of cellular energetics may take priority over growth factors during stress. Tau-P was required for these changes, as co-treatment of CRF with a GSK kinase inhibitor prevented CRF-induced tau-P and all axon transport changes. Collectively, our results provide mechanistic insight into the consequences of stress peptide-induced tau-P and provide an explanation for how chronic stress via CRF may lead to neuronal vulnerability in AD.</p></div

Impact of stress-induced tau-P on BDNF transport.

<p>Overall velocity of QD-BDNF was reduced in 0.5hr CRF cultures (overall velocity **p = 0.03, retrograde ***p = 0.0002) and 2hr CRF treatment (****p = 0.008); 2hr CRF treated cultures also exhibited reduced anterograde and retrograde velocity (*p = 0.03, **0.0002, respectively); Distance travelled was reduced in 0.5hr CRF treated cultures (overall *p = 0.04, retrograde ***p = 0.01) and 2hr CRF treatment (****p = 0.0001). All compared to vehicle treated controls; <b>(D)</b> Analysis of percent mitochondrial movement at 0.5hr and 2hr revealed no changes with 0.5hr CRF treatment (all p>0.05), though 2hr CRF treatment induced dramatic changes in anterograde (p = 0.001), retrograde (p = 0.0001) and stationary (p = 0.01) movement; <b>(E)</b> As seen in D, CRF treatment reduced retrograde velocity (***p = 0.02); 25mM LiCl treatment increased basal levels of retrograde velocity in vehicle control cultures (*p = 0.03) and prevented CRF-induced reductions in retrograde velocity (p>0.05, vehicle control compared to CRF+LiCl; CRF vs LiCl, *p = 0.03).</p

Stress-induced tau-P and kinase activation.

<p><b>(A)</b> Western blot of PHF-1 and GSK-3 pY²¹⁶ in cultured mouse hippocampal cells exposed to low or high concentrations of stress hormone CRF (1 μM or 10 μM) over a period of 0, 0.5, 2, 4, 8, or 24 hours with β-actin as a loading control. <b>(B)</b> Quantitative analysis of western blots (n = 3). Treatments differ significantly from controls; PHF-1 † (p = 0.01), ††† (p<0.001), *** (p<0.001); Active GSK 3β pY²¹⁶ †† (p = 0.006), ††† (p<0.001), *** (p<0.001). <b>(C)</b> Immunostaining with PHF-1 (green) of neuronal treated with 10 μM CRF and <b>(D)</b> vehicle control cell nuclei with DAPI staining (blue).</p

Activation of CREB Pathways.

<p>Neurons that were cultured in microfluidic chambers were treated with BDNF (50 ng/ml) for 0.5hr. Neurons were fixed and stained for pCREB and the nuclei were stained with Hoechst dye. <b>(A)</b> Representative images are shown (scale bar = 50 μM). <b>(B)</b> Quantitative analysis of the percentage of nuclei that were pCREB-positive (p = 0.005, n = 10 images).</p

Impact of stress-induced tau-P on mitochondria transport.

<p>Kymographs showing axons of cultured mouse hippocampal cells after 2hr treatment; <b>(A)</b> Vehicle control. <b>(B)</b> CRF. <b>(C)</b> Table of quantitative analysis of fluorescent live images acquired from axons of cultured mouse hippocampal cells treated with 10 μM CRF compared to controls; Average velocity of mitochondria 0.5hr (***p = 0.0002), 2hr (*p = 0.02); Distance travelled 0.5hr (*p = 0.04), 2h (*p = 0.01); Density of mitochondria after 0.5hr and 2hr. <b>(D)</b> Percent of mitochondrial movement at 0.5hr and 2hr.</p