A γ-secretase inhibitor, but not a γ-secretase modulator, induced defects in BDNF axonal trafficking and signaling: evidence for a role for APP.

Clues to Alzheimer disease (AD) pathogenesis come from a variety of different sources including studies of clinical and neuropathological features, biomarkers, genomics and animal and cellular models. An important role for amyloid precursor protein (APP) and its processing has emerged and considerable interest has been directed at the hypothesis that Aβ peptides induce changes central to pathogenesis. Accordingly, molecules that reduce the levels of Aβ peptides have been discovered such as γ-secretase inhibitors (GSIs) and modulators (GSMs). GSIs and GSMs reduce Aβ levels through very different mechanisms. However, GSIs, but not GSMs, markedly increase the levels of APP CTFs that are increasingly viewed as disrupting neuronal function. Here, we evaluated the effects of GSIs and GSMs on a number of neuronal phenotypes possibly relevant to their use in treatment of AD. We report that GSI disrupted retrograde axonal trafficking of brain-derived neurotrophic factor (BDNF), suppressed BDNF-induced downstream signaling pathways and induced changes in the distribution within neuronal processes of mitochondria and synaptic vesicles. In contrast, treatment with a novel class of GSMs had no significant effect on these measures. Since knockdown of APP by specific siRNA prevented GSI-induced changes in BDNF axonal trafficking and signaling, we concluded that GSI effects on APP processing were responsible, at least in part, for BDNF trafficking and signaling deficits. Our findings argue that with respect to anti-amyloid treatments, even an APP-specific GSI may have deleterious effects and GSMs may serve as a better alternative

Spatiotemporal regulation of axonal growth and protein synthesis by BDNF in hippocampal neurons

Over the past few decades, brain-derived neurotrophic factor (BDNF)— a member of a small family of secreted proteins called neurotrophins— has emerged as a crucial regulator of almost all stages of neuronal circuit development, including axonal specification and neurite growth. Nonetheless, details regarding the spatiotemporal regulation of BDNF-induced axonal growth and underlying mechanisms remain poorly understood. Here, we show that BDNF signals locally in distal axons of hippocampal neurons to rapidly increase axonal elongation rates, whereas BDNF signaling in somatodendritic compartments does not increase distal axon growth. We found that BDNF signals through TrkB receptors in axons to locally activate the mTOR/S6K signaling pathway and intra-axonal protein synthesis. Our findings point to a mechanism whereby local BDNF signaling in axons induces: (1) an early growth response, noticeable within the first 10 min of axonal exposure to BDNF, which requires intra-axonal protein synthesis and local mTOR activity, and (2) a sustained growth response, which occurs 60 min or longer after initial exposure to BDNF, and requires new transcription and translation in neuronal cell bodies. Given the requirement for new transcription to sustain BDNF-induced axonal growth, in Chapter 3 we explored transcriptional changes following BDNF stimulation of hippocampal neurons in mass cultures or compartmentalized microfluidic cultures. We describe dynamic changes in the expression of select transcripts resulting from global and/or axonal BDNF signaling. Taken together, these results provide mechanistic insights into BDNF-induced axonal growth in hippocampal neurons

Identification and quantitative analyses of microRNAs located in the distal axons of sympathetic neurons

microRNAs (miRNAs) constitute a novel class of small, noncoding RNAs that act as negative post-transcriptional regulators of gene expression. Although the nervous system is a prominent site of miRNA expression, little is known about the spatial expression profiles of miRNAs in neurons. Here, we employed compartmentalized Campenot cell culture chambers to obtain a pure axonal RNA fraction of superior cervical ganglia (SCG) neurons, and determined the miRNA expression levels in these subcellular structural domains by microarray analysis and by real-time reverse-transcription polymerase chain reaction. The data revealed stable expression of a number of mature miRNAs that were enriched in the axons and presynaptic nerve terminals. Among the 130 miRNAs identified in the axon, miR-15b, miR-16, miR-204, and miR-221 were found to be highly abundant in distal axons as compared with the cell bodies of primary sympathetic neurons. Moreover, a number of miRNAs encoded by a common primary transcript (pri-miRNA) were differentially expressed in the distal axons, suggesting that there is a differential subcellular transport of miRNAs derived from the same coding region of the genome. Taken together, the data provide an important resource for future studies on the regulation of axonal protein synthesis and the role played by miRNAs in the maintenance of axonal structure and function as well as neuronal growth and development

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 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

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 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

A γ-secretase inhibitor, but not a γ-secretase modulator, induced defects in BDNF axonal trafficking and signaling: evidence for a role for APP.

Clues to Alzheimer disease (AD) pathogenesis come from a variety of different sources including studies of clinical and neuropathological features, biomarkers, genomics and animal and cellular models. An important role for amyloid precursor protein (APP) and its processing has emerged and considerable interest has been directed at the hypothesis that Aβ peptides induce changes central to pathogenesis. Accordingly, molecules that reduce the levels of Aβ peptides have been discovered such as γ-secretase inhibitors (GSIs) and modulators (GSMs). GSIs and GSMs reduce Aβ levels through very different mechanisms. However, GSIs, but not GSMs, markedly increase the levels of APP CTFs that are increasingly viewed as disrupting neuronal function. Here, we evaluated the effects of GSIs and GSMs on a number of neuronal phenotypes possibly relevant to their use in treatment of AD. We report that GSI disrupted retrograde axonal trafficking of brain-derived neurotrophic factor (BDNF), suppressed BDNF-induced downstream signaling pathways and induced changes in the distribution within neuronal processes of mitochondria and synaptic vesicles. In contrast, treatment with a novel class of GSMs had no significant effect on these measures. Since knockdown of APP by specific siRNA prevented GSI-induced changes in BDNF axonal trafficking and signaling, we concluded that GSI effects on APP processing were responsible, at least in part, for BDNF trafficking and signaling deficits. Our findings argue that with respect to anti-amyloid treatments, even an APP-specific GSI may have deleterious effects and GSMs may serve as a better alternative