Stress-induced traps in multilayered structures

The trap parameters of defects in Si/CaF2 multilayered structures were determined from the analysis of optical charging spectroscopy measurements. Two kinds of maxima were observed. Some of them were rather broad, corresponding to "normal" traps, while the others, very sharp, were attributed to stress-induced traps. A procedure of optimal linear smoothing the noisy experimental data has been developed and applied. This procedure is based on finding the minimal value of the relative error with respect to the value of the smoothing window. In order to obtain a better accuracy for the description of the trapping-detrapping process, a Gaussian temperature dependence of the capture crosssections characterizing the stress-induced traps was introduced. Both the normal and the stress-induced traps have been characterized, including some previously considered as only noise features.Comment: 37 pages, 9 figure

Disruption of mitochondrial complex I induces progressive parkinsonism

Loss of functional mitochondrial complex I (MCI) in the dopaminergic neurons of the substantia nigra is a hallmark of Parkinson’s disease1. Yet, whether this change contributes to Parkinson’s disease pathogenesis is unclear2. Here we used intersectional genetics to disrupt the function of MCI in mouse dopaminergic neurons. Disruption of MCI induced a Warburg-like shift in metabolism that enabled neuronal survival, but triggered a progressive loss of the dopaminergic phenotype that was first evident in nigrostriatal axons. This axonal deficit was accompanied by motor learning and fine motor deficits, but not by clear levodopa-responsive parkinsonism—which emerged only after the later loss of dopamine release in the substantia nigra. Thus, MCI dysfunction alone is sufficient to cause progressive, human-like parkinsonism in which the loss of nigral dopamine release makes a critical contribution to motor dysfunction, contrary to the current Parkinson’s disease paradigm.Electron microscopy tissue processing and imaging was performed at the Northwestern University Center for Advanced Microscopy, supported by NCI CCSG P30 CA060553 awarded to the Robert H. Lurie Comprehensive Cancer Center. This study was supported by grants from the Michael J. Fox Foundation (to D.J.S.), the JPB Foundation (to D.J.S.), the IDP Foundation (to D.J.S.), the Flanagan Fellowship (to P.G.-R.) and the European Research Council ERC Advanced Grant PRJ201502629 (to J.L.-B.)

Author Correction: Disruption of mitochondrial complex I induces progressive parkinsonism

In the version of this article initially published, the two bottom-left panels in Extended Data Fig. 8b duplicated the top-left and bottom-right panels of Fig. 4d presenting open field traces in mice. The panels have now been replaced with new images. The errors have been corrected in the online version of the article.Loss of functional mitochondrial complex I (MCI) in the dopaminergic neurons of the substantia nigra is a hallmark of Parkinson’s disease1. Yet, whether this change contributes to Parkinson’s disease pathogenesis is unclear2. Here we used intersectional genetics to disrupt the function of MCI in mouse dopaminergic neurons. Disruption of MCI induced a Warburg-like shift in metabolism that enabled neuronal survival, but triggered a progressive loss of the dopaminergic phenotype that was first evident in nigrostriatal axons. This axonal deficit was accompanied by motor learning and fine motor deficits, but not by clear levodopa-responsive parkinsonism—which emerged only after the later loss of dopamine release in the substantia nigra. Thus, MCI dysfunction alone is sufficient to cause progressive, human-like parkinsonism in which the loss of nigral dopamine release makes a critical contribution to motor dysfunction, contrary to the current Parkinson’s disease paradigm.Electron microscopy tissue processing and imaging was performed at the Northwestern University Center for Advanced Microscopy, supported by NCI CCSG P30 CA060553 awarded to the Robert H. Lurie Comprehensive Cancer Center. This study was supported by grants from the Michael J. Fox Foundation (to D.J.S.), the JPB Foundation (to D.J.S.), the IDP Foundation (to D.J.S.), the Flanagan Fellowship (to P.G.-R.) and the European Research Council ERC Advanced Grant PRJ201502629 (to J.L.-B.).Peer reviewe

PINK1 Defect Causes Mitochondrial Dysfunction, Proteasomal Deficit and α-Synuclein Aggregation in Cell Culture Models of Parkinson's Disease

Mutations in PTEN induced kinase 1 (PINK1), a mitochondrial Ser/Thr kinase, cause an autosomal recessive form of Parkinson's disease (PD), PARK6. Here, we report that PINK1 exists as a dimer in mitochondrial protein complexes that co-migrate with respiratory chain complexes in sucrose gradients. PARK6 related mutations do not affect this dimerization and its associated complexes. Using in vitro cell culture systems, we found that mutant PINK1 or PINK1 knock-down caused deficits in mitochondrial respiration and ATP synthesis. Furthermore, proteasome function is impaired with a loss of PINK1. Importantly, these deficits are accompanied by increased α-synclein aggregation. Our results indicate that it will be important to delineate the relationship between mitochondrial functional deficits, proteasome dysfunction and α-synclein aggregation

Neuromodulatory effect of interleukin 1β in the dorsal raphe nucleus on individual differences in aggression

Heightened aggressive behavior is considered as one of the central symptoms of many neuropsychiatric disorders including autism, schizophrenia, and dementia. The consequences of aggression pose a heavy burden on patients and their families and clinicians. Unfortunately, we have limited treatment options for aggression and lack mechanistic insight into the causes of aggression needed to inform new efforts in drug discovery and development. Levels of proinflammatory cytokines in the periphery or cerebrospinal fluid were previously reported to correlate with aggressive traits in humans. However, it is still unknown whether cytokines affect brain circuits to modulate aggression. Here, we examined the functional role of interleukin 1β (IL-1β) in mediating individual differences in aggression using a resident-intruder mouse model. We found that nonaggressive mice exhibit higher levels of IL-1β in the dorsal raphe nucleus (DRN), the major source of forebrain serotonin (5-HT), compared to aggressive mice. We then examined the effect of pharmacological antagonism and viral-mediated gene knockdown of the receptors for IL-1 within the DRN and found that both treatments consistently increased aggressive behavior of male mice. Aggressive mice also exhibited higher c-Fos expression in 5-HT neurons in the DRN compared to nonaggressive mice. In line with these findings, deletion of IL-1 receptor in the DRN enhanced c-Fos expression in 5-HT neurons during aggressive encounters, suggesting that modulation of 5-HT neuronal activity by IL-1β signaling in the DRN controls expression of aggressive behavior

Proteasome function is impaired by reduction of ATP.

<p>Proteasome function is ATP dependent. Fluorescent CFP was fused to degron, a signaling peptide that directs its protein to proteasome for degradation. An increase of fluorescence (open circle) indicates a reduction of proteasome function. ATP production was inhibited by 2-deoxyglucose (2DG), and ATP content was measured with the ATP Assay Kit (Calbiochem) for luminescence (filled circle). A) Increasing dosages of 2DG caused a decrease in ATP production (filled circle) and enhanced proteasome inhibition (open circle). B) Compared to non-treated cells in DMEM, there is a significant proteasome inhibition by 6 mM 2DG (p = 0.0004, ANOVA) and the proteasome inhibitor MG132 (p = 0.0001, ANOVA).</p

Proteasome function is impaired by mutant or loss of PINK1.

<p>Proteasome activity was measured from SH-SY5Y cells expressing mutant PINK1 (A), and PC12 cells expressing siRNA against PINK1 (B, C, D). A) Fluorescence of fluorogenic proteasome substrate Suc-LLVY-AMC (Calbiochem) is positively correlated with proteasome function. No statistically significant changes were detected in proteasome activity between control SH-SY5Y cells and the cells expressing wild type PINK1 (n = 8, p = 0.484, paired student t test). There was a statistically significant decrease of proteasome activity in the SH-SY5Y cells expressing L347P-PINK1 (23% reduction, n = 7, p = 0.018, paired student t test) or in SH-SY5Y cells expressing E417G-PINK1 (19.4% reduction, n = 8, p = 0.012, paired student t test) compared to cells expressing wild type PINK1. MG132, a proteasome inhibitor, was used as a negative control. The bottom panel is a Western analysis of the above samples with the 20S α subunit Ab for normalization. B) Proteasome activity was measured in 20 µg of cell lysate isolated from wild type control PC12 cells (open diamond) or SiPINK1-4 PC12 cell line (filled circle) for 60 min after 30 min incubation. Wild type PC12 cells lysate treated with MG132 (filled triangle) was used as a negative control. The result revealed that the kinetic of proteasome activity monitored over 60 min was markedly decreased in the cells with reduced PINK1. The bottom panel is a Western analysis of the above samples with the 20S α subunit antibody for normalization. C) Histographic presentation for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004597#pone-0004597-g007" target="_blank">Figure 7B</a>. The reduction of PINK1 by siRNA impairs the proteasome activity (31.8% reduction, n = 8, p = 0.01, ANOVA). Experiments were repeated with SiPINK1-2 PC12 cell line, and consistent results were obtained (data not shown). D) PINK1 mediated proteasome activity deficit confirmed by another independent method in the HeLa cells. Compared to control (CFP-de transfection), siRNA against PINK1 (siPINK1) knocked down PINK1 and led to a sigfinicant inhibition of CFP degradation (p = 0.0001, ANOVA) to an extent similar to direct proteasome inhibition by MG132 (p = 0.0014, ANOVA). A scrambled siRNA (siSCR) had no effect (p = 0.876, ANOVA). The RNAi sequences are: GAGAGGUCCAAGCAACUA TT and CCUGGUCGACUACCCUGAU TT.</p

PINK1 constructs and their expressions.

<p>A) A schematic depiction of PINK1 constructs. Full length wild type, L347P-, or E417G- PINK1 tagged with Flag, V5, or GFP are indicated. Several truncated PINK1 tagged with Flag or V5 are also depicted. M stands for mitochondrial targeting sequence. B) Confirmation of the expression of the above constructs in HEK 293 cells. HEK293 cells were transfected by various PINK1 constructs, and their lysates were analyzed by Western blots with Flag antibody (Left panel), V5 antibody (the middle panel) or GFP antibody (the right Panel). Lane 1–10 are lysates from cells transfected by plasmids with the same numbering as shown in A). The lysates from the cells transfected with the empty cloning vector without PINK1 insert were used as controls (labeled as C). The results demonstrated that the expression of all constructs yielded recombinant PINK1 proteins with expected molecular weights.</p

Recombinant and endogenous PINK1 are associated with protein complexes in mitochondria.

<p>In all experiments, adenoviruses of PINK1-Flag, L347P-PINK1-Flag, E417G-PINK1-Flag or Del 245 PINK1-Flag were infected into SH-SY5Y cells. A) Western blot analysis of PINK1 sub-cellular distributions with anti-Flag Ab. The two forms of PINK1 (a 64 kD full length protein and a truncated form of 50 kD, presumably a proteolytic product) are present in mitochondria and cytosol. The L347P, E417G or Del 245 mutant PINK1 did not affect this distribution. S: cytosolic fraction; M: mitochondrial fraction. B) PINK1 is associated with protein complexes. Mitochondrial proteins were sub-fractionated by 15% to 35% discontinuous sucrose gradient, from which fractions 1–10 were collected from top (lighter proteins) to bottom (heavier proteins or complexes). They were subjected to SDS-PAGE, and Western analyses with anti-Flag Ab for PINK1 (the top three panels); anti-39 kD protein Ab for complex I (4^th panel); anti-70 kD protein Ab for complex II (5^th panel); anti-core 2 Ab for complex III (6^th panel); and anti-cox I Ab for complex IV (7^th panel). No PINK1 was observed in lane 1 and 2, the fractions that contained proteins of the sizes for monomeric PINK1. Instead, PINK1 was associated with protein complexes ranging from 130–900 kD, which co-migrated with ETC complexes. The L347P and E417G mutations did not affect the PINK1 association and distribution of these complexes. More importantly, anti-human PINK1antibodies (Novus) detected endogenous PINK1 in SH-SY5Y cells with similar distribution along the sucrose gradient (bottom panel).</p

Loss of PINK1 impairs OXPHOS function in PC12 cells with reduced PINK1 expression.

<p>Normal respiration is impaired in the PC12 cell lines with PINK1 knocked-down by RNAi. A) PINK1 mRNA is significantly reduced in two stable cell lines expressing PINK1 siRNA. Compared to the wild type control, there is an 81.7% and 91% reduction in PINK1 mRNA in SiPINK1-2 and SiPINK1-4 cell line. PINK1 mRNA is significantly reduced in two stable cell lines expressing PINK1 siRNA. The level of PINK1 mRNA is normalized to GAPDH. B) Oxygen consumption is significantly reduced in both SiPINK1-2 (22.4% reduction, n = 10, p<0.05, ANOVA) and SiPINK1-4 (33.1% reduction, n = 11, p<0.01, ANOVA) cell lines compared to that of control cells. SiPINK1-2: 46.38±3.5SE; SiPINK1-4: 39.96±1.93SE; control cell: 59.70±2.1SE. C) The respiratory deficit in SiPINK1-4 cells can be partially rescued by wild type (n = 7, p = 0.008, student T test) but not E417G-PINK1 (n = 4, p = 0.76, student T test) or Del 245 PINK1 (n = 3, p = 0.1, student T test). Control: 60 ±3.38SE; SiPINK1-4: 37±2.32SE; SiPINK1-4/wt-PINK1: 47.9±2.39SE; SiPINK1/E417G: 38.53±3.35SE; SiPINK1/Del 245: 28.63±4.75SE. D) With glutamate/malate as substrates, ATP synthesis rate was significantly reduced in both SiPINK1-2 (41.3% reduction, n = 7, p<0.01, ANOVA) and SiPINK1-4 (29.8% reduction, n = 8, p<0.01, ANOVA) cell lines compared to that of control cell. SiPINK1-2: 8.73±0.99SE; SiPINK1-4:10.59±0.69SE; control cells: 14.883±0.78SE. E) Western analysis of the samples used for the rescued experiment shown in (C) with Tim 23 Ab. The result demonstrates that equal amount of mitochondria is present in all the samples subject to respiration experiment. F) Equal amount of mitochondria were used for the experiments shown in B and D as demonstrated by identical Tim 23 in all the samples.</p