Apoptosis Signal-Regulating Kinase 1 Mediates MPTP Toxicity and Regulates Glial Activation

Apoptosis signal-regulating kinase 1 (ASK1), a member of the mitogen-activated protein kinase 3 family, is activated by oxidative stress. The death-signaling pathway mediated by ASK1 is inhibited by DJ-1, which is linked to recessively inherited Parkinson's disease (PD). Considering that DJ-1 deficiency exacerbates the toxicity of the mitochondrial complex I inhibitor 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), we sought to investigate the direct role and mechanism of ASK1 in MPTP-induced dopamine neuron toxicity. In the present study, we found that MPTP administration to wild-type mice activates ASK1 in the midbrain. In ASK1 null mice, MPTP-induced motor impairment was less profound, and striatal dopamine content and nigral dopamine neuron counts were relatively preserved compared to wild-type littermates. Further, microglia and astrocyte activation seen in wild-type mice challenged with MPTP was markedly attenuated in ASK1−/− mice. These data suggest that ASK1 is a key player in MPTP-induced glial activation linking oxidative stress with neuroinflammation, two well recognized pathogenetic factors in PD. These findings demonstrate that ASK1 is an important effector of MPTP-induced toxicity and suggest that inhibiting this kinase is a plausible therapeutic strategy for protecting dopamine neurons in PD

Dopamine depletion causes fragmented clustering of neurons in the sensorimotor striatum: Evidence of lasting reorganization of corticostriatal input

Firing during sensorimotor exam was used to categorize single neurons in the lateral striatum of awake, unrestrained rats. Five rats received unilateral injection of 6-hydroxydopamine (6-OHDA) into the medial forebrain bundle to deplete striatal dopamine (DA; >98% depletion, postmortem assay). Three months after treatment, rats exhibited exaggerated rotational behavior induced by L-dihydroxyphenylalanine (L-DOPA) and contralateral sensory neglect. Electrode track “depth profiles” on the DA-depleted side showed fragmented clustering of neurons related to sensorimotor activity of single body parts (SBP neurons). Clusters were smaller than normal, and more SBP neurons were observed in isolation, outside of clusters. More body parts were represented per unit volume. No recovery in these measures was observed up to one year post lesion. Overall distributions of neurons related to different body parts were not altered. The fragmentation of SBP clusters after DA depletion indicates that a percentage of striatal SBP neurons switched responsiveness from one body part to one or more different body parts. Because the specific firing that characterizes striatal SBP neurons is mediated by corticostriatal inputs (Liles and Updyke [1985] Brain Res. 339:245–255), the data indicate that DA depletion resulted in a reorganization of corticostriatal connections, perhaps via unmasking or sprouting of connections to adjacent clusters of striatal neurons. After reorganization, sensory activity in a localized body part activates striatal neurons that have switched to that body part. In turn, switched signals sent from basal ganglia to premotor and motor neurons, which likely retain their original connections, would create mismatches in these normally precise topographic connections. Switched signals could partially explain parkinsonian deficits in motor functions involving somatosensory guidance and their intractability to L-DOPA therapy—particularly if the switching involves sprouting. J. Comp. Neurol. 452:24–37, 2002. © 2002 Wiley-Liss, Inc

Dopamine depletion causes fragmented clustering of neurons in the sensorimotor striatum: Evidence of lasting reorganization of corticostriatal input

Firing during sensorimotor exam was used to categorize single neurons in the lateral striatum of awake, unrestrained rats. Five rats received unilateral injection of 6-hydroxydopamine (6-OHDA) into the medial forebrain bundle to deplete striatal dopamine (DA; >98% depletion, postmortem assay). Three months after treatment, rats exhibited exaggerated rotational behavior induced by L-dihydroxyphenylalanine (L-DOPA) and contralateral sensory neglect. Electrode track “depth profiles” on the DA-depleted side showed fragmented clustering of neurons related to sensorimotor activity of single body parts (SBP neurons). Clusters were smaller than normal, and more SBP neurons were observed in isolation, outside of clusters. More body parts were represented per unit volume. No recovery in these measures was observed up to one year post lesion. Overall distributions of neurons related to different body parts were not altered. The fragmentation of SBP clusters after DA depletion indicates that a percentage of striatal SBP neurons switched responsiveness from one body part to one or more different body parts. Because the specific firing that characterizes striatal SBP neurons is mediated by corticostriatal inputs (Liles and Updyke [1985] Brain Res. 339:245–255), the data indicate that DA depletion resulted in a reorganization of corticostriatal connections, perhaps via unmasking or sprouting of connections to adjacent clusters of striatal neurons. After reorganization, sensory activity in a localized body part activates striatal neurons that have switched to that body part. In turn, switched signals sent from basal ganglia to premotor and motor neurons, which likely retain their original connections, would create mismatches in these normally precise topographic connections. Switched signals could partially explain parkinsonian deficits in motor functions involving somatosensory guidance and their intractability to L-DOPA therapy—particularly if the switching involves sprouting. J. Comp. Neurol. 452:24–37, 2002. © 2002 Wiley-Liss, Inc

Klotho Protects Dopaminergic Neuron Oxidant-Induced Degeneration by Modulating ASK1 and p38 MAPK Signaling Pathways

<div><p>Klotho transgenic mice exhibit resistance to oxidative stress as measured by their urinal levels of 8-hydroxy-2-deoxyguanosine, albeit this anti-oxidant defense mechanism has not been locally investigated in the brain. Here, we tested the hypothesis that the reactive oxygen species (ROS)-sensitive apoptosis signal-regulating kinase 1 (ASK1)/p38 MAPK pathway regulates stress levels in the brain of these mice and showed that: 1) the ratio of free ASK1 to thioredoxin (Trx)-bound ASK1 is relatively lower in the transgenic brain whereas the reverse is true for the Klotho knockout mice; 2) the reduced p38 activation level in the transgene corresponds to higher level of ASK1-bound Trx, while the KO mice showed elevated p38 activation and lower level of–bound Trx; and 3) that 14-3-3ζ is hyper phosphorylated (Ser-58) in the transgene which correlated with increased monomer forms. In addition, we evaluated the <i>in vivo</i> robustness of the protection by challenging the brains of Klotho transgenic mice with a neurotoxin, MPTP and analyzed for residual neuron numbers and integrity in the substantia nigra pars compacta. Our results show that Klotho overexpression significantly protects dopaminergic neurons against oxidative damage, partly by modulating p38 MAPK activation level. Our data highlight the importance of ASK1/p38 MAPK pathway in the brain and identify Klotho as a possible anti-oxidant effector.</p></div

Engrailed-2 (En2) deletion produces multiple neurodevelopmental defects in monoamine systems, forebrain structures and neurogenesis and behavior

Many genes involved in brain development have been associated with human neurodevelopmental disorders, but underlying pathophysiological mechanisms remain undefined. Human genetic and mouse behavioral analyses suggest that ENGRAILED-2 (EN2) contributes to neurodevelopmental disorders, especially autism spectrum disorder. In mouse, En2 exhibits dynamic spatiotemporal expression in embryonic mid-hindbrain regions where monoamine neurons emerge. Considering their importance in neuropsychiatric disorders, we characterized monoamine systems in relation to forebrain neurogenesis in En2-knockout (En2-KO) mice. Transmitter levels of serotonin, dopamine and norepinephrine (NE) were dysregulated from Postnatal day 7 (P7) to P21 in En2-KO, though NE exhibited the greatest abnormalities. While NE levels were reduced ∼35% in forebrain, they were increased 40 -: 75% in hindbrain and cerebellum, and these patterns paralleled changes in locus coeruleus (LC) fiber innervation, respectively. Although En2 promoter was active in Embryonic day 14.5 -: 15.5 LC neurons, expression diminished thereafter and gene deletion did not alter brainstem NE neuron numbers. Significantly, in parallel with reduced NE levels, En2-KO forebrain regions exhibited reduced growth, particularly hippocampus, where P21 dentate gyrus granule neurons were decreased 16%, suggesting abnormal neurogenesis. Indeed, hippocampal neurogenic regions showed increased cell death (+77%) and unexpectedly, increased proliferation. Excess proliferation was restricted to early Sox2/Tbr2 progenitors whereas increased apoptosis occurred in differentiating (Dcx) neuroblasts, accompanied by reduced newborn neuron survival. Abnormal neurogenesis may reflect NE deficits because intra-hippocampal injections of β-adrenergic agonists reversed cell death. These studies suggest that disruption of hindbrain patterning genes can alter monoamine system development and thereby produce forebrain defects that are relevant to human neurodevelopmental disorders

Close-up view of images of 2D-GE extracted from Fig 2.

<p>(A) Plots of phosphorylation levels of ProQ diamond stained 14-3-3 proteins. The Pro-Q diamond stained gel images in Fig 3B (a-d) are close-up views of areas of spots in WT1, <i>kl/kl</i>, WT2 and EFmKL48 full gels respectively within pI 4.3–4.8. Plots are pooled data from two replicate gels and deviations are shown. Representative ProQ diamond gel images and their corresponding positions in SyproRuby-stained protein gels are shown together to normalize for levels of protein load. Digitized values of stained spots used for the plots are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139914#pone.0139914.s003" target="_blank">S3 Table</a>.</p

Summary of seven protein spots of interest identified by mass spectrometry on 2D gels of Klotho mutant and wild type mice.

<p>^<i>a</i>Spots 1–3 were identified based on their differential phosphorylation [KO mice (klkl) vs. wild type control) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139914#pone.0139914.g002" target="_blank">Fig 2</a>; spots 4 and 5 were downregulated in klkl vs. wild type; spots 6 and 7 were upregulated in klkl mice.</p><p>*expt., experimentally obtained p<i>I</i>s and M<i>r</i>s are those analyzed from 2D gels; deter., determined pIs and Mrs values are based on primary sequence data and were obtained from literature. DHLA, dihydrolipoamide</p><p>Summary of seven protein spots of interest identified by mass spectrometry on 2D gels of Klotho mutant and wild type mice.</p