SEMA4D compromises blood–brain barrier, activates microglia, and inhibits remyelination in neurodegenerative disease

AbstractMultiple sclerosis (MS) is a chronic neuroinflammatory disease characterized by immune cell infiltration of CNS, blood–brain barrier (BBB) breakdown, localized myelin destruction, and progressive neuronal degeneration. There exists a significant need to identify novel therapeutic targets and strategies that effectively and safely disrupt and even reverse disease pathophysiology. Signaling cascades initiated by semaphorin 4D (SEMA4D) induce glial activation, neuronal process collapse, inhibit migration and differentiation of oligodendrocyte precursor cells (OPCs), and disrupt endothelial tight junctions forming the BBB. To target SEMA4D, we generated a monoclonal antibody that recognizes mouse, rat, monkey and human SEMA4D with high affinity and blocks interaction between SEMA4D and its cognate receptors. In vitro, anti-SEMA4D reverses the inhibitory effects of recombinant SEMA4D on OPC survival and differentiation. In vivo, anti-SEMA4D significantly attenuates experimental autoimmune encephalomyelitis in multiple rodent models by preserving BBB integrity and axonal myelination and can be shown to promote migration of OPC to the site of lesions and improve myelin status following chemically-induced demyelination. Our study underscores SEMA4D as a key factor in CNS disease and supports the further development of antibody-based inhibition of SEMA4D as a novel therapeutic strategy for MS and other neurologic diseases with evidence of demyelination and/or compromise to the neurovascular unit

Data Mining A PeopleSoft Database To Assist In Developing Student Retention Interventions

Per a Bellwether Education Partners study (Aldeman, 2015, p. 8), "As of 2013, there were 29.1 million college dropouts versus 24.5 million Americans who dropped out with less than a high school diploma. In pure, raw numbers, college dropouts are a bigger problem than high school dropouts." Conceptually this study is framed within theories of student persistence/attainment and the Knowledge Discovery Process (KDP). This research study developed first time in college (FTIC) and transfer (TRAN) student graduation prediction models by using decision trees and support vector machine (SVM) classification algorithms and identified attributes of students who graduate and do not graduate. Data was collected from the University of Houston’s data warehouse to provide detailed student academic records as the basis for quantitative analysis. The data set included male and female undergraduate students enrolled in the College of Education’s Teaching & Learning Program from 2000-2012 at the University of Houston. These findings may contribute to improving student success and subsequent graduation rates in the College of Education and other colleges across the campus.Curriculum and Instruction, Department o

DJ-1 In Vitro Chaperone Activity and In Vivo Oxidative Stress Protection Activity Require Cysteine 53 but Not Cysteine 106

<div><p>(A) DJ-1 cysteine-to-alanine mutants C106A, C53A, and a triple mutant that harbors mutations at all three cysteines in DJ-1 (C106A/C53A/C46A), as well as L166P, were tested for in vitro chaperone activity by CS aggregation suppression assay.</p> <p>(B) Self-association of DJ-1 cysteine mutants. Murine neuroblastoma CAD cells were transiently cotransfected with Flag-tagged human DJ-1 vectors (either WT or mutant) along with WT YFP-tagged human DJ-1. Lysates were immunoprecipitated with anti-Flag antibodies and probed by Western blotting with an antibody specific for human DJ-1. WT Flag-DJ-1, C106A DJ-1, C53A DJ-1, and C106A/C53A/C46A DJ-1 effectively coprecipitated WT GFP-DJ-1, whereas the L166P mutant Flag-DJ-1 failed to do so. Lysates represent 20% of the input for the immunoprecipitate; Flag-DJ-1 migrates at 22 kDa, and YFP-DJ-1 migrates at 50 kDa.</p> <p>(C) DJ-1-deficient ES cells were transiently transfected with vector alone, WT DJ-1, or DJ-1 cysteine mutants, and exposed to 10 μM H₂O₂ for 15 h followed by MTT assay. The viability of the cells in the absence of drug treatment was not altered by the expression of WT or mutant DJ-1). Data are shown as the mean ± SEM and were analyzed by ANOVA with Fisher's post-hoc test. * <i>p</i> < 0.05</p> <p>(D) Expression levels of WT and mutant forms of DJ-1 were comparable as determined by Western blotting for human DJ-1 and β-actin.</p></div

Sensitivity to oxidative stress in DJ-1-deficient dopamine neurons: an ES- derived cell model of primary Parkinsonism.

The hallmark of Parkinson's disease (PD) is the selective loss of dopamine neurons in the ventral midbrain. Although the cause of neurodegeneration in PD is unknown, a Mendelian inheritance pattern is observed in rare cases, indicating a genetic factor. Furthermore, pathological analyses of PD substantia nigra have correlated cellular oxidative stress and altered proteasomal function with PD. Homozygous mutations in DJ-1 were recently described in two families with autosomal recessive Parkinsonism, one of which is a large deletion that is likely to lead to loss of function. Here we show that embryonic stem cells deficient in DJ-1 display increased sensitivity to oxidative stress and proteasomal inhibition. The accumulation of reactive oxygen species in toxin-treated DJ-1-deficient cells initially appears normal, but these cells are unable to cope with the consequent damage that ultimately leads to apoptotic death. Furthermore, we find that dopamine neurons derived from in vitro-differentiated DJ-1-deficient embryonic stem cells display decreased survival and increased sensitivity to oxidative stress. These data are consistent with a protective role for DJ-1, and demonstrate the utility of genetically modified embryonic stem cell-derived neurons as cellular models of neuronal disorders

DJ-1 Inhibits Formation of αSyn Protofibrils and Fibrils In Vitro

<div><p>(A) Purified αSyn (200 μM) was incubated for 2 h at 55 °C in the presence of WT DJ-1, L166P mutant DJ-1, GST, or Hsp27 (all at 100 μM). WT DJ-1 inhibits accumulation of αSyn protofibrils in vitro, while L166P mutant DJ-1, GST, and Hsp27 do not.</p> <p>(B) Suppression of αSyn protofibril formation by WT DJ-1 (in triplicate) was quantified as compared to GST (as a negative control) and mutant L166P DJ-1. Data are shown as the mean ± SEM and were analyzed by ANOVA with Fisher's post-hoc test. * <i>p</i> < 0.05.</p> <p>(C) Purified αSyn (200 μM) was incubated for 1 wk at 37 °C in the presence of WT DJ-1, L166P mutant DJ-1, or GST (all at 100 μM). WT DJ-1 inhibits formation of mature Congo red–positive αSyn fibrils. Data are shown as the mean ± SEM and were analyzed by ANOVA with Fisher's post-hoc test. * <i>p</i> < 0.05.</p></div

Overexpression of WT DJ-1 Inhibits Aggregation of αSyn In Vivo

<div><p>(A) CAD murine neuroblastoma cells were transfected with Flag-αSyn along with WT DJ-1, L166P clinical mutant, or vector alone, and were differentiated in vitro via serum withdrawal. Cells were subsequently treated with 2 mM FeCl₂ (Fe), 5 μM lactacystin (LC), or media alone (0). Triton X-100-soluble (Tx-100 sol) and Triton X-100-insoluble (Tx-100 insol) fractions were analyzed by Western blotting. Upon FeCl₂ treatment, αSyn accumulates in the Triton X-100-insoluble fraction, and accumulation of insoluble αSyn is inhibited by overexpression of WT DJ-1 (left) but not the L166P clinical mutant (right).</p> <p>(B) Triton X-100-insoluble αSyn as quantified by NIH Image J of a Western blot (from [A]).</p> <p>(C) Heterozygous (+/–) and DJ-1 deficient (–/–) ES cells were differentiated using the embryoid body protocol. Cells were transfected with Flag-αSyn (F-αSyn), and, after 48 h, treated with 2 mM FeCl₂ or with media alone for 18 h. Cell lysates were analyzed by Western blotting for αSyn or β-actin. In the Triton X-100-soluble fraction (Tx-100 sol), DJ-1 accumulated to a similar extent in the knockout and control cells. In contrast, αSyn accumulation in the insoluble pool (Tx-100 insol) was detectable only in the knockout cells, and this was further promoted by FeCl₂ treatment.</p> <p>(D) CAD cells transfected with Flag-αSyn (F-αSyn) along with WT DJ-1 (or vector alone) were treated with 2 mM FeCl₂ or media alone for 18 h. Triton X-100-soluble cell lysates were immunoprecipitated with a mouse monoclonal antibody for the Flag epitope and Western blotted for DJ-1. FeCl₂ treatment induces association of Flag-αSyn with WT DJ-1. Lysates represent 20% input of the immunoprecipitation (IP α-Flag). The Triton X-100 soluble pool of DJ-1 is reduced by αSyn overexpression (but not vector control), particularly in the context of FeCl₂ treatment (bottom).</p> <p>(E) DJ-1 colocalizes with αSyn in the Triton X-100-insoluble fraction upon FeCl₂ treatment. The Western blot from (A) was stripped and reprobed for DJ-1.</p></div

DJ-1 Inhibits Formation of αSyn Intracytoplasmic Inclusions

<div><p>(A–L) CAD murine neuroblastoma cells were transfected with WT DJ-1 (A–F), L166P DJ-1 (G–I) or vector control (J–L), along with Flag-αSyn (D–L) or vector control (A–C) and differentiated in vitro by serum withdrawal for 72 h. Cells were fixed and stained with a mouse monoclonal antibody for αSyn and ToPro3, a nuclear dye, and images were obtained by confocal microscopy. Transfection of Flag-αSyn induced formation of intracytoplasmic inclusions (arrows). Scale bar, 20 μm.</p> <p>(M) Quantification of cells with inclusions was performed on ten random images from each of three wells per condition. Images were quantified by an observer blinded to the experiment. A significantly lower percentage of cells harbor inclusions in the context of WT DJ-1 overexpression. Aggregation is expressed as the percentage of cells containing αSyn aggregates per frame. Total cell number per frame, as determined by ToPro3 staining, did not differ significantly (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020362#sg003" target="_blank">Figure S3</a>). Data are shown as the mean ± SEM, and were analyzed by ANOVA with Fisher's post-hoc test. * <i>p</i> < 0.05.</p> <p>(N–S) Cells were fixed and stained with a monoclonal antibody for αSyn and a polyclonal antibody that recognizes both transfected human DJ-1 and endogenous murine DJ-1. DJ-1 does not appear to colocalize with the αSyn aggregates. Scale bar, 20 μm.</p></div

DJ-1 Inhibits Formation of NFL Intracytoplasmic Inclusions

<div><p>(A–L) CAD cells were transfected with an aggregation-prone mutant NFL (Q333P) plasmid, as well as WT human DJ-1 plasmid (that also harbors GFP; E–H), L166P mutant DJ-1 (that also harbors GFP; I–L), or control GFP vector (A–D). After 72 h in culture, cells were fixed and stained with a mouse monoclonal antibody for NFL and ToPro3, a nuclear dye. Scale bar, 100 μm.</p> <p>(M–R) CAD cell transfectants, as above, were fixed and stained with a polyclonal antibody for NFL (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020362#pbio-0020362-Perez-Olle1" target="_blank">Perez-Olle et al. 2002</a>) along with a mouse monoclonal antibody specific for the transfected human DJ-1. Scale bar, 20 μm.</p> <p>(S) Quantification of CAD cell NFL aggregates was performed using confocal microscopy. Images from tenrandomly selected fields in each of three wells were quantified for the presence of aggregates for each condition and presented as a percentage of total cells per field. Total cell number was determined by ToPro3 nuclear staining and did not differ significantly (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020362#sg003" target="_blank">Figure S3</a>). Data are shown as the mean ± SEM and were analyzed by ANOVA with Fisher's post-hoc test. * <i>p</i> < 0.05.</p></div

DJ-1 Is a Redox-Dependent Molecular Chaperone That Inhibits α-Synuclein Aggregate Formation

<div><p>Parkinson's disease (PD) pathology is characterized by the degeneration of midbrain dopamine neurons (DNs) ultimately leading to a progressive movement disorder in patients. The etiology of DN loss in sporadic PD is unknown, although it is hypothesized that aberrant protein aggregation and cellular oxidative stress may promote DN degeneration. Homozygous mutations in <em>DJ-1</em> were recently described in two families with autosomal recessive inherited PD (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020362#pbio-0020362-Bonifati1">Bonifati et al. 2003</a>). In a companion article (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020362#pbio-0020362-Martinat1">Martinat et al. 2004</a>), we show that mutations in DJ-1 alter the cellular response to oxidative stress and proteasomal inhibition. Here we show that DJ-1 functions as a redox-sensitive molecular chaperone that is activated in an oxidative cytoplasmic environment. We further demonstrate that DJ-1 chaperone activity in vivo extends to α-synuclein, a protein implicated in PD pathogenesis.</p> </div