Molecular Mechanisms of Environmental Enrichment: Impairments in Akt/GSK3β, Neurotrophin-3 and CREB Signaling

<div><p>Experience of mice in a complex environment enhances neurogenesis and synaptic plasticity in the hippocampus of wild type and transgenic mice harboring familial Alzheimer's disease (FAD)-linked APPswe/PS1ΔE9. In FAD mice, this experience also reduces levels of tau hyperphosphorylation and oligomeric β-amyloid. Although environmental enrichment has significant effects on brain plasticity and neuropathology, the molecular mechanisms underlying these effects are unknown. Here we show that environmental enrichment upregulates the Akt pathway, leading to the downregulation of glycogen synthase kinase 3β (GSK3β), in wild type but not FAD mice. Several neurotrophic signaling pathways are activated in the hippocampus of both wild type and FAD mice, including brain derived neurotrophic factor (BDNF) and nerve growth factor (NGF), and this increase is accompanied by the upregulation of the BDNF receptor, tyrosine kinase B (TrkB). Interestingly, neurotrophin-3 (NT-3) is upregulated in the brains of wild type mice but not FAD mice, while insulin growth factor-1 (IGF-1) is upregulated exclusively in the brains of FAD mice. Upregulation of neurotrophins is accompanied by the increase of N-Methyl-D-aspartic acid (NMDA) receptors in the hippocampus following environmental enrichment. Most importantly, we observed a significant increase in levels of cAMP response element- binding (CREB) transcripts in the hippocampus of wild type and FAD mice following environmental enrichment. However, CREB phosphorylation, a critical step for the initiation of learning and memory-required gene transcription, takes place in the hippocampus of wild type but not of FAD mice. These results suggest that experience of wild type mice in a complex environmental upregulates critical signaling that play a major role in learning and memory in the hippocampus. However, in FAD mice, some of these pathways are impaired and cannot be rescued by environmental enrichment.</p></div

PrP induces CK2-mediated phosphorylation of kinesin light chain subunits and detachment of conventional kinesin from membrane cargoes.

<p>(<b>A-B</b>) Quantitative immunoblot analysis of kinesin-1 from two pair of sister axoplasms incubated either with control PrP-Scram or PrP_{<b>106-126</b>} peptides and (<b>C-D</b>) primary embryonic mouse cortical neurons cultured for 3 days in vitro treated for one hour. Antibody 63–90 preferentially recognizes kinesin-1 light chains (KLCs) when they are not phosphorylated by CK2 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188340#pone.0188340.ref027" target="_blank">27</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188340#pone.0188340.ref035" target="_blank">35</a>]. (<b>B and D</b>) Quantitation graph bars show that PrP_{<b>106-126</b>} decreased the immunoreactivity for 63–90 versus control PrP-Scram treated axoplasms and neurons respectively. Note a significant reduction (<b>B</b>) of immunoreactivity when neurons were treated with PrP_{<b>106-126</b>} compared to PrP-Scram; (n = 5, number of independent experiments. p = 0.0313, significance was assessed at P < 0.05). (<b>D</b>) Significant reduction of 63–90 immunoreactivity in axoplasms incubated with PrP_{<b>106-126</b>} compared to control PrP-Scram, (n = 3; number of independent experiments. p = 0.0355, significance was assessed at P < 0.05). (<b>E</b>) Vesicles purified from sister axoplasms by vesicle flotation assays perfused with control PrP-Scram and PrP_{<b>106-126</b>} synthetic peptide were assayed by Western blot for KHC and TrkB. TrkB was used as membrane protein marker and for loading control. (<b>F</b>) Quantitation graph bars shows a significant reduction of kinesin-1 association to purified vesicles in PrP_{<b>106-126</b>} incubated extruded axoplasms compared to control PrP-Scram treated axoplasms, (n = 3, number of independent experiments; significance was assessed at P < 0.05). Taken together, these experiments suggest that PrP_{<b>106-126</b>} increases the intracellular activity of CK2, which in turn results in KLCs phosphorylation and kinesin-1 release from its cargo vesicles.</p

Supplementary Figure 2. PrP Full length (PrP-FL) inhibits fast axonal transport.

Supplementary Figure 2. PrP Full length (PrP-FL) inhibits fast axonal transport

Supplementary Figure. 3 The CK2 pharmacological inhibitor DMAT does not interfere with FAT.

Supplementary Figure. 3 The CK2 pharmacological inhibitor DMAT does not interfere with FAT

Figure 1. Full length PrP (PrP-FL) inhibits vesicular fast axonal transport in isolated squid axoplasm.

Figure 1. Full length PrP (PrP-FL) inhibits vesicular fast axonal transport in isolated squid axoplasm

Figure 2. Prion inhibits fast axonal transport of mitochondria in primary hippocampal cultured neurons.

Figure 2. Prion inhibits fast axonal transport of mitochondria in primary hippocampal cultured neurons

Casein kinase 2 mediates PrP-induced fast axonal transport inhibition.

<p>Plots in A-B depict results from vesicle motility assays as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188340#pone.0188340.g001" target="_blank">Fig 1</a>. Co-perfusion experiments of PrP-FL with DMAT, a highly specific CK2 inhibitor, prevent bidirectional FAT inhibition (Compare with <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188340#pone.0188340.g001" target="_blank">Fig 1A</a>). (<b>B</b>) Similarly, co-perfusion of PrP_{<b>106-126</b>} with DMAT prevents the inhibitory effect of PrP_{<b>106-126</b>} on FAT (Compare with <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188340#pone.0188340.g001" target="_blank">Fig 1D</a>). Graphs showing quantification of average rates of anterograde (<b>C</b>) and retrograde (<b>D</b>) FAT obtained 30–50 minutes after co-perfusion of PrP-FL and PrP_{<b>106-126</b>} with DMAT indicated that CK2 plays a key role in PrP-induced FAT inhibition. (<b>E</b>) The stimulatory effect of PrP-FL and PrP_{<b>106-126</b>} peptide on CK2 activity was evaluated <i>in vitro</i> by CK2 kinase assay as described in the experimental section. Bar chart of the phosphorylation kinase activity of CK2 expressed as the incorporation of radioactive inorganic phosphate (P_i) into the synthetic R₃A₂DSD₅ peptide by the recombinant CK2ed in the experimental section. Bar chart of the phosphorylation kinase activity of CK2 expressed as the incorp_{<b>106-126</b>} (Red bar). These results suggest that PrP can activate CK2 directly. C.P.M. stands for counts per minute in arbitrary units. Scintillation counting-based quantitation from 3 independent experiments. *: p<0.0002; **: p<0.0001. Two-tailed P values. (<b>F</b>) Upper panel shows fluorescently labeled mitochondria from axons of 3 DIV neurons treated with either PrP_{<b>106-126</b>} or control PrP_{<b>106-126</b>}-Scram peptides. In the lower panel, kymographs reveal the trajectory of mitochondria motility from neurons incubated with 3μm of either PrP_{<b>106-126</b>} or (<b>G</b>) PrP_{<b>106-126</b>} plus 5μM DMAT for 1 hour. (<b>H</b>) Quantification of average distance traveled by mitochondria as analyzed in (F) and (G) in the anterograde (white) and retrograde (black) direction. Note the lack of inhibitory effect when neurons are co-incubated with 5μM DMAT. (<b>I</b>) Quantification of the percentage of mobile mitochondria analyzed in (F) or (G). Note a reduction of mobile mitochondria in PrP_{<b>106-126</b>} and the dramatic recovery of mitochondria mobility in PrP_{<b>106-126</b>} plus 5μM DMAT co-treated neurons. These pharmacological results suggest that the activation of endogenous axonal CK2 may be responsible for the inhibition of both directions of FAT induced by cellular PrP. (<b>H-I</b>) Mean ±SEM, * p<0.05, total of 22 neurons were analyzed, 11 (PrP_{<b>106-126</b>} treated) and 11 (PrP_{<b>106-126</b>} + DMAT treated). Results were obtained from 3 independent experiments. One-way ANOVA with post-hoc Tukey.</p

Full length PrP (PrP-FL) inhibits fast axonal transport of membrane-bounded organelles in isolated squid axoplasm.

<p>(<b>A</b>) Schematic representation of different PrP constructs and peptides used in this work. The PrP central domain (CD) is indicated in the top graph in red. Note that the truncated PrP (PrP-ΔCD) lacks most of the PrP CD. Two PrP peptides of 21 amino acids were used, one that corresponds to the amino acids 106 to 126 (PrP_{<b>106-126)</b>}, and the other is the corresponding scrambled control peptide (PrP-Scram). Plots in <b>B, C, D</b> and <b>E</b> represent results from vesicle motility assays in isolated extruded squid axoplasms perfused with different PrP constructs. Blue arrowheads and blue line represent fast axonal transport (FAT) rates of kinesin-1 driven vesicles moving in the anterograde direction and the red arrows and red lines represent retrograde dynein-mediated FAT rates. Lines represent the best fit exponential of rates for vesicles moving in the anterograde blue arrows and retrograde red arrows directions over time in axoplasms. (<b>B</b>) Perfusion with 2μM of PrP-FL showed a marked reduction of anterograde and retrograde FAT soon after perfusion, compared to perfusing X/2 buffer alone [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188340#pone.0188340.ref048" target="_blank">48</a>] (data not shown in this manuscript). (<b>C</b>) Perfusion of a PrP full length construct lacking amino acids 111 to 134 (PrP-ΔCD) showed not effect on FAT <b>(D)</b> Perfusion with PrP_{<b>106-126</b>}, a 21 amino acid peptide corresponding to the PrP CD inhibited bidirectional FAT with a profiles of inhibition almost identical to the one induced by PrP-FL. (<b>E</b>) Perfusion of the PrP_{<b>106-126</b>}-Scram control peptide encompassing the same amino acids but arranged in a scrambled order did not alter FAT. Graphs showing quantitation of average rates of anterograde (<b>F</b>) and retrograde (<b>G</b>) FAT obtained 30–50 minutes after PrP perfusion indicating that when PrP-FL and its 21 amino acid peptide corresponding to the central domain of PrP-FL are perfused they induce bidirectional FAT inhibition. Letter “n” represents the number of independent axoplasms perfused per construct. Light blue and green dots in graphs F and G represent outlier values.</p

Prion inhibits fast axonal transport of mitochondria in mammalian cultured neurons.

<p>The effects of Prion on mitochondria mobility was analyzed in 3 days <i>in vitro</i> rat embryonic primary hippocampal neurons by time-lapse microscopy. (<b>A</b>) Upper panel shows fluorescently labeled mitochondria from axons of neurons treated with PrP_{<b>106-126</b>} or control PrP_{<b>106-126</b>}-Scram. In the lower panel, kymographs reveal the trajectory of mitochondria motility from neurons incubated for 1 hour with 3μm PrP_{<b>106-126</b>} versus PrP_106-126-Scram control peptide (<b>B</b>). Kymographs were obtained from images in the upper panel. Scale bar in the X-axis equals 30μm and in the Y-axis equals 60 seconds. (<b>C</b>) Quantification of the distance traveled by mitochondria analyzed in (<b>B)</b> in the anterograde (white) and retrograde (black) direction. (<b>D</b>) Quantification of the percentage of moving mitochondria in neurons treated with 3μm PrP_{<b>106-126</b>} compared to control PrP_{<b>106-126</b>}-Scram, or non-treated control neurons (Ctrl). (<b>C-D</b>) Mean ±SEM, * p<0.05, total of 26 neurons were analyzed, 7 (PrP-Scram treated), 8 (Control un-treated), 11 (PrP_{<b>106-126</b>} treated). A total of 143 mitochondria were analyzed in figures C and D; 57 mitochondria were analyzed in scramble treated neurons 26 (Not mobile), 12 (anterograde direction), 19 (retrograde direction); 86 mitochondria were analyzed in PrP_106-126 treated neurons, 58 (not mobile), 7 (anterograde direction), 21 (retrograde direction). Results were obtained from 3 independent experiments. One-way ANOVA with post-hoc Tukey.</p

Proposed molecular mechanism for PrP-induced FAT inhibition.

<p>Pharmacological data showed here indicates that PrP-induced FAT inhibition is mediated by activation of endogenous tetrameric CK2αβ and subsequent phosphorylation of KLCs. Phosphorylation of KLCs (red letter P) promotes the detachment of conventional kinesin from its transported vesicular cargoes. Our experimental data suggests that both PrP-FL and its central domain (CD) peptide of 21 amino acids PrP_{<b>106-126</b>} inhibit FAT with an identical profile of inhibition.</p