FAD Mutations in Amyloid Precursor Protein Do Not Directly Perturb Intracellular Calcium Homeostasis

Disturbances in intracellular calcium homeostasis are likely prominent and causative factors leading to neuronal cell death in Alzheimer's disease (AD). Familial AD (FAD) is early-onset and exhibits autosomal dominant inheritance. FAD-linked mutations have been found in the genes encoding the presenilins and amyloid precursor protein (APP). Several studies have shown that mutated presenilin proteins can directly affect calcium release from intracellular stores independently of Aβ production. Although less well established, there is also evidence that APP may directly modulate intracellular calcium homeostasis. Here, we directly examined whether overexpression of FAD-linked APP mutants alters intracellular calcium dynamics. In contrast to previous studies, we found that overexpression of mutant APP has no effects on basal cytosolic calcium, ER calcium store size or agonist-induced calcium release and subsequent entry. Thus, we conclude that mutated APP associated with FAD has no direct effect on intracellular calcium homeostasis independently of Aβ production

Low Concentrations of Methamphetamine Can Protect Dopaminergic Cells against a Larger Oxidative Stress Injury: Mechanistic Study

Mild stress can protect against a larger insult, a phenomenon termed preconditioning or tolerance. To determine if a low intensity stressor could also protect cells against intense oxidative stress in a model of dopamine deficiency associated with Parkinson disease, we used methamphetamine to provide a mild, preconditioning stress, 6-hydroxydopamine (6-OHDA) as a source of potentially toxic oxidative stress, and MN9D cells as a model of dopamine neurons. We observed that prior exposure to subtoxic concentrations of methamphetamine protected these cells against 6-OHDA toxicity, whereas higher concentrations of methamphetamine exacerbated it. The protection by methamphetamine was accompanied by decreased uptake of both [3H] dopamine and 6-OHDA into the cells, which may have accounted for some of the apparent protection. However, a number of other effects of methamphetamine exposure suggest that the drug also affected basic cellular survival mechanisms. First, although methamphetamine preconditioning decreased basal pERK1/2 and pAkt levels, it enhanced the 6-OHDA-induced increase in these phosphokinases. Second, the apparent increase in pERK1/2 activity was accompanied by increased pMEK1/2 levels and decreased activity of protein phosphatase 2. Third, methamphetamine upregulated the pro-survival protein Bcl-2. Our results suggest that exposure to low concentrations of methamphetamine cause a number of changes in dopamine cells, some of which result in a decrease in their vulnerability to subsequent oxidative stress. These observations may provide insights into the development of new therapies for prevention or treatment of PD

Current Approaches Targeting the Wound Healing Phases to Attenuate Fibrosis and Scarring

Cutaneous fibrosis results from suboptimal wound healing following significant tissue injury such as severe burns, trauma, and major surgeries. Pathologic skin fibrosis results in scars that are disfiguring, limit normal movement, and prevent patient recovery and reintegration into society. While various therapeutic strategies have been used to accelerate wound healing and decrease the incidence of scarring, recent studies have targeted the molecular regulators of each phase of wound healing, including the inflammatory, proliferative, and remodeling phases. Here, we reviewed the most recent literature elucidating molecular pathways that can be targeted to reduce fibrosis with a particular focus on post-burn scarring. Current research targeting inflammatory mediators, the epithelial to mesenchymal transition, and regulators of myofibroblast differentiation shows promising results. However, a multimodal approach addressing all three phases of wound healing may provide the best therapeutic outcome

METH and 6-OHDA induced toxicity in MN9D cells.

<p>(A) MN9D cells were treated with 6-OHDA for 20 min and viability assays performed 24 hr after 6-OHDA removal. 6-OHDA killed MN9D cells in a concentration-dependent fashion with an EC₅₀ of approximately 100 µM. (B) MN9D Cells were treated with the indicated concentrations of METH for 24 hr and viability assays performed 24 hr later. METH affected ATP levels, mitochondrial dehydrogenase activity, and chromatin condensation as assessed by Hoechst staining. The average EC₅₀ for METH for the three different viability assays was between 2 and 3 mM. Data represent means ± SEM of 3–5 independent experiments. *P<0.05, ***p<0.001 compared to controls.</p

METH preconditioning affected MEK1/2 and PP2A.

<p>(A) A representative blot showing that sublethal concentrations of METH decreased the levels of pMEK1/2 and enhanced MEK1/2 activation induced by 6-OHDA. MN9D cells were treated with METH for 24 hr, the medium was changed, and cells treated with 6-OHDA for 20 min. Lysates were collected at 0, 15 min, 60 min, and 24 hr post 6-OHDA treatment. (B) Quantification of panel A in 4 independent experiments. (C) PP2A activity assay was performed as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024722#s2" target="_blank">methods</a> section from cells treated with various METH concentrations for 24 hr. METH pretreatment decreased PP2A activity in a concentration dependent manner, although total cellular PP2A levels were not affected. *p<0.05, **p<0.01 compared to control.</p

Effect of METH preconditioning on MnSOD, CuZnSOD, and Bcl-2 levels.

<p>(A) MN9D cells were treated with 0.5 mM METH for 24 hr and the cells lysates were analyzed for MnSOD, CuZnSOD, and Bcl-2 levels using Western blot analysis. Densitometry analysis showed that METH preconditioning did not affect MnSOD and CuZnSOD but significantly increases Bcl-2 levels. (B) METH increases Bcl-2 in basal conditions and in the presence of the ERK1/2 inhibitor U0126 but has no effects on Bcl-2 levels if the cells are pretreated with the Akt inhibitor LY294002. α-tubulin used as a loading control. Data are means ± ESM (N = 3). *p<0.05 compared to control.</p

METH affects dopamine transporter function.

<p>(A) MN9D cells were treated with different concentration of METH for 24 hr and ³H-DA uptake assayed with METH still in the media as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024722#s2" target="_blank">methods</a> section. (B) MN9D cells were treated with METH for 24 hr, the media was changed, the cells were then treated with vehicle or 6-OHDA (100 µM) for 20 min, and DA uptake assayed immediately after. (C) MN9D cells were treated with METH for 24 hr, the media was changed and cells treated with 100 µM 6-OHDA or vehicle. The cells were collected at different time points to analyze 6-OHDA content using HPLC-EC. Data are presented as means ± SEM (N = 3). *p<0.05, **p<0.01 compared to 0 mM METH, #p<0.05 compared to 0.5 mM METH.</p

Sub-lethal concentrations of METH protected MN9D cells against 6-OHDA toxicity.

<p>Cells were treated with the indicated concentrations of METH for 24 hr. The medium was then changed, the cells treated with 100 µM 6-OHDA for 20 min and viability assays performed 24 hr after 6-OHDA removal. Sub-toxic concentrations of METH protected ATP levels (A) and mitochondrial dehydrogenase activity (B), and prevented chromatin condensation of Hoechst stained cells (C). The photomicrographs in Panel (C-I) show control cells lacking any sign of nuclear condensation or chromatin clumping, In panel (C-II) MN9D cells have lifted off the plate after 6-OHDA exposure, presumably due to cell death. Arrows point to dying cells with high chromatin condensation. In panel (C-III) METH pretreated cells were protected against 6-OHDA toxicity (C-III). Data represent means ± SEM (N = 3). *P<0.05 compared to 0 mM METH/100 µM 6-OHDA, #p<0.01compared to 0 mM METH/0 µM 6-OHDA.</p

METH preconditioning enhanced Akt but not JNK activation in response to 6-OHDA.

<p>MN9D cells were treated with a sub-lethal concentration of METH for 24 hr and then exposed to 100 µM 6-OHDA for 20 min before the cells were lysed at 15 min, 30 min, 1 hr, 6 hr, and 24 hr post 6-OHDA. (A) METH decreased pAkt levels as shown for the 15 and 30 min time points. (B) METH preconditioning potentiates Akt activation in response to 6-OHDA and no effect was seen on total Akt levels. (C) Quantification of pAkt compared to total Akt in 4 independent experiments. (D, E) METH preconditioning has no effect on basal or 6-OHDA induced phosphorylation of JNK. α-tubulin was used to control for protein loading. Data are means ± SEM (N = 3). *p<0.05 compared to control.</p

METH decreased basal pERK1/2 levels and potentiated ERK1/2 phosphorylation induced by 6-OHDA.

<p>(A) Western blot analysis showing pERK levels at different time points after exposure to sub-lethal concentration of METH (0.5 mM) for up to 24 hr. Lysates were collected at 15 min, 30 min, 1 hr, 6 hr, and 24 hr. Shown are the 15 min and 30 min time points. (B) pERK1/2 levels decreased by 15 min post METH exposure and lasted for up to 24 hr. METH decreased activation of ERK 1/2 in a concentration-dependent manner (0, 0.5, and 1 mM at the 24 hr time point) and enhanced the activation of ERK1/2 after 6-OHDA exposure. This activation lasted from 15 to at least 24 hr. (C) Quantification of ERK1/2 activation in response to 6-OHDA. (D) MN9D cells were treated with sub-lethal concentration of METH (0.5 mM) for 24 hr, the medium was removed and the cells treated with the MEK1/2 inhibitor U0126 (10 µM) for 1 hr before and during the 20 min 6-OHDA exposure. Western blot analysis confirmed the activation of ERK1/2 by 6-OHDA and its enhancement by METH preconditioning and confirmed that U0126 blocked the phosphorylation of ERK1/2. (E) Viability assay looking at ATP levels and performed 24 hr post 6-OHDA treatment showed that METH protected MN9D cells against 6-OHDA toxicity and U0126 abolishes this protection. α-tubulin was used as a loading control. Data are presented as means ± SEM (N = 3). *p<0.05, **p<0.01 compared to vehicle and ^#p<0.05 compared to lane 10.</p