Publications by authors named "Theo D Palmer"

61 Publications

16p11.2 microdeletion imparts transcriptional alterations in human iPSC-derived models of early neural development.

Elife 2020 11 10;9. Epub 2020 Nov 10.

Department of Neurosurgery and The Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, United States.

Microdeletions and microduplications of the 16p11.2 chromosomal locus are associated with syndromic neurodevelopmental disorders and reciprocal physiological conditions such as macro/microcephaly and high/low body mass index. To facilitate cellular and molecular investigations into these phenotypes, 65 clones of human induced pluripotent stem cells (hiPSCs) were generated from 13 individuals with 16p11.2 copy number variations (CNVs). To ensure these cell lines were suitable for downstream mechanistic investigations, a customizable bioinformatic strategy for the detection of random integration and expression of reprogramming vectors was developed and leveraged towards identifying a subset of 'footprint'-free hiPSC clones. Transcriptomic profiling of cortical neural progenitor cells derived from these hiPSCs identified alterations in gene expression patterns which precede morphological abnormalities reported at later neurodevelopmental stages. Interpreting clinical information-available with the cell lines by request from the Simons Foundation Autism Research Initiative-with this transcriptional data revealed disruptions in gene programs related to both nervous system function and cellular metabolism. As demonstrated by these analyses, this publicly available resource has the potential to serve as a powerful medium for probing the etiology of developmental disorders associated with 16p11.2 CNVs.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.7554/eLife.58178DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7695459PMC
November 2020

Examining Sex Differences in the Human Placental Transcriptome During the First Fetal Androgen Peak.

Reprod Sci 2021 03 4;28(3):801-818. Epub 2020 Nov 4.

Department of Obstetrics and Gynecology, Stanford University School of Medicine, 300 Pasteur Dr. H333, Stanford, CA, 94305-5317, USA.

Sex differences in human placenta exist from early pregnancy to term, however, it is unclear whether these differences are driven solely by sex chromosome complement or are subject to differential sex hormonal regulation. Here, we survey the human chorionic villus (CV) transcriptome for sex-linked signatures from 11 to 16 gestational weeks, corresponding to the first window of increasing testis-derived androgen production in male fetuses. Illumina HiSeq RNA sequencing was performed on Lexogen Quantseq 3' libraries derived from CV biopsies (n = 11 females, n = 12 males). Differential expression (DE) was performed to identify sex-linked transcriptional signatures, followed by chromosome mapping, pathway analysis, predicted protein interaction, and post-hoc linear regressions to identify transcripts that trend over time. We observe 322 transcripts DE between male and female CV from 11 to 16 weeks, with 22 transcripts logFC > 1. Contrary to our predictions, the difference between male and female expression of DE autosomal genes was more pronounced at the earlier gestational ages. In females, we found selective upregulation of extracellular matrix components, along with a number of X-linked genes. In males, DE transcripts centered on chromosome 19, with mitochondrial, immune, and pregnancy maintenance-related transcripts upregulated. Among the highest differentially expressed autosomal genes were CCRL2, LGALS13, and LGALS14, which are known to regulate immune cell interactions. Our results provide insight into sex-linked gene expression in late first and early second trimester developing human placenta and lay the groundwork to understand the mechanistic origins of sex differences in prenatal development.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s43032-020-00355-8DOI Listing
March 2021

An evolutionarily acquired microRNA shapes development of mammalian cortical projections.

Proc Natl Acad Sci U S A 2020 11 2;117(46):29113-29122. Epub 2020 Nov 2.

Department of Neurosurgery, Stanford University, Stanford, CA 94305;

The corticospinal tract is unique to mammals and the corpus callosum is unique to placental mammals (eutherians). The emergence of these structures is thought to underpin the evolutionary acquisition of complex motor and cognitive skills. Corticospinal motor neurons (CSMN) and callosal projection neurons (CPN) are the archetypal projection neurons of the corticospinal tract and corpus callosum, respectively. Although a number of conserved transcriptional regulators of CSMN and CPN development have been identified in vertebrates, none are unique to mammals and most are coexpressed across multiple projection neuron subtypes. Here, we discover 17 CSMN-enriched microRNAs (miRNAs), 15 of which map to a single genomic cluster that is exclusive to eutherians. One of these, miR-409-3p, promotes CSMN subtype identity in part via repression of LMO4, a key transcriptional regulator of CPN development. In vivo, miR-409-3p is sufficient to convert deep-layer CPN into CSMN. This is a demonstration of an evolutionarily acquired miRNA in eutherians that refines cortical projection neuron subtype development. Our findings implicate miRNAs in the eutherians' increase in neuronal subtype and projection diversity, the anatomic underpinnings of their complex behavior.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.2006700117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7682328PMC
November 2020

Aberrant calcium channel splicing drives defects in cortical differentiation in Timothy syndrome.

Elife 2019 12 23;8. Epub 2019 Dec 23.

Department of Neurobiology, Stanford University School of Medicine, Stanford, United States.

The syndromic autism spectrum disorder (ASD) Timothy syndrome (TS) is caused by a point mutation in the alternatively spliced exon 8A of the calcium channel Ca1.2. Using mouse brain and human induced pluripotent stem cells (iPSCs), we provide evidence that the TS mutation prevents a normal developmental switch in Ca1.2 exon utilization, resulting in persistent expression of gain-of-function mutant channels during neuronal differentiation. In iPSC models, the TS mutation reduces the abundance of SATB2-expressing cortical projection neurons, leading to excess CTIP2+ neurons. We show that expression of TS-Ca1.2 channels in the embryonic mouse cortex recapitulates these differentiation defects in a calcium-dependent manner and that Ca1.2 gain-and-loss of function reciprocally regulates the abundance of these neuronal populations. Our findings support the idea that disruption of developmentally regulated calcium channel splicing patterns instructively alters differentiation in the developing cortex, providing important insights into the pathophysiology of a syndromic ASD.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.7554/eLife.51037DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6964969PMC
December 2019

"Females Are Not Just 'Protected' Males": Sex-Specific Vulnerabilities in Placenta and Brain after Prenatal Immune Disruption.

eNeuro 2019 Nov/Dec;6(6). Epub 2019 Nov 7.

Institute for Stem Cell Biology and Regenerative Medicine and Department of Neurosurgery, Stanford University, Stanford, California 94305-5454.

Current perceptions of genetic and environmental vulnerabilities in the developing fetus are biased toward male outcomes. An argument is made that males are more vulnerable to gestational complications and neurodevelopmental disorders, the implication being that an understanding of disrupted development in males is sufficient to understand causal mechanisms that are assumed to be similar but attenuated in females. Here we examine this assumption in the context of immune-driven alterations in fetal brain development and related outcomes in female and male mice. Pregnant C57BL/6 mice were treated with low-dose lipopolysaccharide at embryonic day 12.5. Placental pathology, acute fetal brain inflammation and hypoxia, long-term changes in adult cortex cytoarchitecture, altered densities and ratio of excitatory (Satb2) to inhibitory (parvalbumin) neuronal subtypes, postnatal growth, and behavior outcomes were compared between male and female offspring. We find that while males experience more pronounced placental pathology, fetal brain hypoxia, depleted PV and Satb2 densities, and social and learning-related behavioral abnormalities, females exhibit unique acute inflammatory signaling in fetal brain, postnatal growth delay, opposite alterations in cortical PV densities, changes in juvenile behavior, delayed postnatal body growth, and elevated anxiety-related behavior as adults. While males are more severely impacted by prenatal immune disruption by several measures, females exposed to the same insult exhibit a unique set of vulnerabilities and developmental consequences that is not present in males. Our results clearly outline disparate sex-specific features of prenatal vulnerability to inflammatory insults and warn against the casual extrapolation of male disease mechanisms to females.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1523/ENEURO.0358-19.2019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6838689PMC
June 2020

Human 3D cellular model of hypoxic brain injury of prematurity.

Nat Med 2019 05 6;25(5):784-791. Epub 2019 May 6.

Department of Psychiatry and Behavioral Sciences & Stanford Human Brain Organogenesis Program, Stanford University, Stanford, CA, USA.

Owing to recent medical and technological advances in neonatal care, infants born extremely premature have increased survival rates. After birth, these infants are at high risk of hypoxic episodes because of lung immaturity, hypotension and lack of cerebral-flow regulation, and can develop a severe condition called encephalopathy of prematurity. Over 80% of infants born before post-conception week 25 have moderate-to-severe long-term neurodevelopmental impairments. The susceptible cell types in the cerebral cortex and the molecular mechanisms underlying associated gray-matter defects in premature infants remain unknown. Here we used human three-dimensional brain-region-specific organoids to study the effect of oxygen deprivation on corticogenesis. We identified specific defects in intermediate progenitors, a cortical cell type associated with the expansion of the human cerebral cortex, and showed that these are related to the unfolded protein response and changes. Moreover, we verified these findings in human primary cortical tissue and demonstrated that a small-molecule modulator of the unfolded protein response pathway can prevent the reduction in intermediate progenitors following hypoxia. We anticipate that this human cellular platform will be valuable for studying the environmental and genetic factors underlying injury in the developing human brain.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41591-019-0436-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7020938PMC
May 2019

Characterization of Brain Dysfunction Induced by Bacterial Lipopeptides That Alter Neuronal Activity and Network in Rodent Brains.

J Neurosci 2018 12 31;38(50):10672-10691. Epub 2018 Oct 31.

Biomaterials and Advanced Drug Delivery Laboratory, Stanford University School of Medicine, Palo Alto, California 94304,

The immunopathological states of the brain induced by bacterial lipoproteins have been well characterized by using biochemical and histological assays. However, these studies have limitations in determining functional states of damaged brains involving aberrant synaptic activity and network, which makes it difficult to diagnose brain disorders during bacterial infection. To address this, we investigated the effect of PamCSK (PAM), a synthetic bacterial lipopeptide, on synaptic dysfunction of female mice brains and cultured neurons in parallel. Our functional brain imaging using PET with [F]fluorodeoxyglucose and [F] flumazenil revealed that the brain dysfunction induced by PAM is closely aligned to disruption of neurotransmitter-related neuronal activity and functional correlation in the region of the limbic system rather than to decrease of metabolic activity of neurons in the injection area. This finding was verified by tissue experiments that analyzed synaptic and dendritic alterations in the regions where PET imaging showed abnormal neuronal activity and network. Recording of synaptic activity also revealed that PAM reorganized synaptic distribution and decreased synaptic plasticity in hippocampus. Further study using neuron cultures demonstrated that PAM decreased the number of presynapses and the frequency of miniature EPSCs, which suggests PAM disrupts neuronal function by damaging presynapses exclusively. We also showed that PAM caused aggregation of synapses around dendrites, which may have caused no significant change in expression level of synaptic proteins, whereas synaptic number and function were impaired by PAM. Our findings could provide a useful guide for diagnosis and treatment of brain disorders specific to bacterial infection. It is challenging to diagnose brain disorders caused by bacterial infection because neural damage induced by bacterial products involves nonspecific neurological symptoms, which is rarely detected by laboratory tests with low spatiotemporal resolution. To better understand brain pathology, it is essential to detect functional abnormalities of brain over time. To this end, we investigated characteristic patterns of altered neuronal integrity and functional correlation between various regions in mice brains injected with bacterial lipopeptides using PET with a goal to apply new findings to diagnosis of brain disorder specific to bacterial infection. In addition, we analyzed altered synaptic density and function using both and experimental models to understand how bacterial lipopeptides impair brain function and network.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1523/JNEUROSCI.0825-17.2018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6580656PMC
December 2018

RNA-protein interaction detection in living cells.

Nat Methods 2018 03 5;15(3):207-212. Epub 2018 Feb 5.

Program in Epithelial Biology, Stanford University School of Medicine, Stanford, California, USA.

RNA-protein interactions play numerous roles in cellular function and disease. Here we describe RNA-protein interaction detection (RaPID), which uses proximity-dependent protein labeling, based on the BirA* biotin ligase, to rapidly identify the proteins that bind RNA sequences of interest in living cells. RaPID displays utility in multiple applications, including in evaluating protein binding to mutant RNA motifs in human genetic disorders, in uncovering potential post-transcriptional networks in breast cancer, and in discovering essential host proteins that interact with Zika virus RNA. To improve the BirA*-labeling component of RaPID, moreover, a new mutant BirA* was engineered from Bacillus subtilis, termed BASU, that enables >1,000-fold faster kinetics and >30-fold increased signal-to-noise ratio over the prior standard Escherichia coli BirA*, thereby enabling direct study of RNA-protein interactions in living cells on a timescale as short as 1 min.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nmeth.4601DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5886736PMC
March 2018

Adult-generated neurons born during chronic social stress are uniquely adapted to respond to subsequent chronic social stress.

Mol Psychiatry 2019 08 8;24(8):1178-1188. Epub 2018 Jan 8.

Department of Biological Sciences, Stanford University, Stanford, CA, 94305, USA.

Chronic stress is a recognized risk factor for psychiatric and psychological disorders and a potent modulator of adult neurogenesis. Numerous studies have shown that during stress, neurogenesis decreases; however, during the recovery from the stress, neurogenesis increases. Despite the increased number of neurons born after stress, it is unknown if the function and morphology of those neurons are altered. Here we asked whether neurons in adult mice, born during the final 5 days of chronic social stress and matured during recovery from chronic social stress, are similar to neurons born with no stress conditions from a quantitative, functional and morphological perspective, and whether those neurons are uniquely adapted to respond to a subsequent stressful challenge. We observed an increased number of newborn neurons incorporated in the dentate gyrus of the hippocampus during the 10-week post-stress recovery phase. Interestingly, those new neurons were more responsive to subsequent chronic stress, as they showed more of a stress-induced decrease in spine density and branching nodes than in neurons born during a non-stress period. Our results replicate findings that the neuronal survival and incorporation of neurons in the adult dentate gyrus increases after chronic stress and suggest that such neurons are uniquely adapted in the response to future social stressors. This finding provides a potential mechanism for some of the long-term hippocampal effects of stress.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41380-017-0013-1DOI Listing
August 2019

Phosphorylation of αB-crystallin supports reactive astrogliosis in demyelination.

Proc Natl Acad Sci U S A 2017 02 14;114(9):E1745-E1754. Epub 2017 Feb 14.

Department of Neurology and Neurological Sciences, Stanford University School of Medicine, Stanford, CA 94305;

The small heat shock protein αB-crystallin (CRYAB) has been implicated in multiple sclerosis (MS) pathogenesis. Earlier studies have indicated that CRYAB inhibits inflammation and attenuates clinical disease when administered in the experimental autoimmune encephalomyelitis model of MS. In this study, we evaluated the role of CRYAB in primary demyelinating events. Using the cuprizone model of demyelination, a noninflammatory model that allows the analysis of glial responses in MS, we show that endogenous CRYAB expression is associated with increased severity of demyelination. Moreover, we demonstrate a strong correlation between the expression of CRYAB and the extent of reactive astrogliosis in demyelinating areas and in in vitro assays. In addition, we reveal that CRYAB is differentially phosphorylated in astrocytes in active demyelinating MS lesions, as well as in cuprizone-induced lesions, and that this phosphorylation is required for the reactive astrocyte response associated with demyelination. Furthermore, taking a proteomics approach to identify proteins that are bound by the phosphorylated forms of CRYAB in primary cultured astrocytes, we show that there is clear differential binding of protein targets due to the specific phosphorylation of CRYAB. Subsequent Ingenuity Pathway Analysis of these targets reveals implications for intracellular pathways and biological processes that could be affected by these modifications. Together, these findings demonstrate that astrocytes play a pivotal role in demyelination, making them a potential target for therapeutic intervention, and that phosphorylation of CRYAB is a key factor supporting the pathogenic response of astrocytes to oligodendrocyte injury.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.1621314114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5338510PMC
February 2017

Functional Impairment in Miro Degradation and Mitophagy Is a Shared Feature in Familial and Sporadic Parkinson's Disease.

Cell Stem Cell 2016 12 8;19(6):709-724. Epub 2016 Sep 8.

Department of Neurosurgery, Stanford University School of Medicine, Stanford, CA 94305, USA. Electronic address:

Mitochondrial movements are tightly controlled to maintain energy homeostasis and prevent oxidative stress. Miro is an outer mitochondrial membrane protein that anchors mitochondria to microtubule motors and is removed to stop mitochondrial motility as an early step in the clearance of dysfunctional mitochondria. Here, using human induced pluripotent stem cell (iPSC)-derived neurons and other complementary models, we build on a previous connection of Parkinson's disease (PD)-linked PINK1 and Parkin to Miro by showing that a third PD-related protein, LRRK2, promotes Miro removal by forming a complex with Miro. Pathogenic LRRK2G2019S disrupts this function, delaying the arrest of damaged mitochondria and consequently slowing the initiation of mitophagy. Remarkably, partial reduction of Miro levels in LRRK2G2019S human neuron and Drosophila PD models rescues neurodegeneration. Miro degradation and mitochondrial motility are also impaired in sporadic PD patients. We reveal that prolonged retention of Miro, and the downstream consequences that ensue, may constitute a central component of PD pathogenesis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.stem.2016.08.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5135570PMC
December 2016

The Role of the Microenvironmental Niche in Declining Stem-Cell Functions Associated with Biological Aging.

Cold Spring Harb Perspect Med 2015 Dec 1;5(12). Epub 2015 Dec 1.

Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, California 94305 Department of Neurosurgery, Stanford University School of Medicine, Stanford, California 94305.

Aging is strongly correlated with decreases in neurogenesis, the process by which neural stem and progenitor cells proliferate and differentiate into new neurons. In addition to stem-cell-intrinsic factors that change within the aging stem-cell pool, recent evidence emphasizes new roles for systemic and microenvironmental factors in modulating the neurogenic niche. This article focuses on new insights gained through the use of heterochronic parabiosis models, in which an old mouse and a young circulatory system are joined. By studying the brains of both young and old mice, researchers are beginning to uncover circulating proneurogenic "youthful" factors and "aging" factors that decrease stem-cell activity and neurogenesis. Ultimately, the identification of factors that influence stem-cell aging may lead to strategies that slow or even reverse age-related decreases in neural-stem-cell (NSC) function and neurogenesis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1101/cshperspect.a025874DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4665032PMC
December 2015

Aging-like changes in the transcriptome of irradiated microglia.

Glia 2015 May 17;63(5):754-67. Epub 2015 Feb 17.

Department of Neurosurgery, Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, California.

Whole brain irradiation remains important in the management of brain tumors. Although necessary for improving survival outcomes, cranial irradiation also results in cognitive decline in long-term survivors. A chronic inflammatory state characterized by microglial activation has been implicated in radiation-induced brain injury. We here provide the first comprehensive transcriptional profile of irradiated microglia. Fluorescence-activated cell sorting was used to isolate CD11b+ microglia from the hippocampi of C57BL/6 and Balb/c mice 1 month after 10 Gy cranial irradiation. Affymetrix gene expression profiles were evaluated using linear modeling and rank product analyses. One month after irradiation, a conserved irradiation signature across strains was identified, comprising 448 and 85 differentially up- and downregulated genes, respectively. Gene set enrichment analysis demonstrated enrichment for inflammation, including M1 macrophage-associated genes, but also an unexpected enrichment for extracellular matrix and blood coagulation-related gene sets, in contrast previously described microglial states. Weighted gene coexpression network analysis confirmed these findings and further revealed alterations in mitochondrial function. The RNA-seq transcriptome of microglia 24-h postradiation proved similar to the 1-month transcriptome, but additionally featured alterations in apoptotic and lysosomal gene expression. Reanalysis of published aging mouse microglia transcriptome data demonstrated striking similarity to the 1-month irradiated microglia transcriptome, suggesting that shared mechanisms may underlie aging and chronic irradiation-induced cognitive decline. GLIA 2015;63:754-767.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/glia.22782DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4625786PMC
May 2015

Stress and glucocorticoids promote oligodendrogenesis in the adult hippocampus.

Mol Psychiatry 2014 Dec 11;19(12):1275-1283. Epub 2014 Feb 11.

Helen Wills Neuroscience Institute, University of California, Berkeley, Berkeley, CA.

Stress can exert long-lasting changes on the brain that contribute to vulnerability to mental illness, yet mechanisms underlying this long-term vulnerability are not well understood. We hypothesized that stress may alter the production of oligodendrocytes in the adult brain, providing a cellular and structural basis for stress-related disorders. We found that immobilization stress decreased neurogenesis and increased oligodendrogenesis in the dentate gyrus (DG) of the adult rat hippocampus and that injections of the rat glucocorticoid stress hormone corticosterone (cort) were sufficient to replicate this effect. The DG contains a unique population of multipotent neural stem cells (NSCs) that give rise to adult newborn neurons, but oligodendrogenic potential has not been demonstrated in vivo. We used a nestin-CreER/YFP transgenic mouse line for lineage tracing and found that cort induces oligodendrogenesis from nestin-expressing NSCs in vivo. Using hippocampal NSCs cultured in vitro, we further showed that exposure to cort induced a pro-oligodendrogenic transcriptional program and resulted in an increase in oligodendrogenesis and decrease in neurogenesis, which was prevented by genetic blockade of glucocorticoid receptor (GR). Together, these results suggest a novel model in which stress may alter hippocampal function by promoting oligodendrogenesis, thereby altering the cellular composition and white matter structure.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/mp.2013.190DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4128957PMC
December 2014

Stereotypical alterations in cortical patterning are associated with maternal illness-induced placental dysfunction.

J Neurosci 2013 Oct;33(43):16874-88

Stanford University, Institute for Stem Cell Biology and Regenerative Medicine, Stanford, California 94305-5454.

We have previously shown in mice that cytokine-mediated damage to the placenta can temporarily limit the flow of nutrients and oxygen to the fetus. The placental vulnerability is pronounced before embryonic day 11, when even mild immune challenge results in fetal loss. As gestation progresses, the placenta becomes increasingly resilient to maternal inflammation, but there is a narrow window in gestation when the placenta is still vulnerable to immune challenge yet resistant enough to allow for fetal survival. This gestational window correlates with early cortical neurogenesis in the fetal brain. Here, we show that maternal illness during this period selectively alters the abundance and laminar positioning of neuronal subtypes influenced by the Tbr1, Satb2, and Ctip2/Fezf2 patterning axis. The disturbances also lead to a laminar imbalance in the proportions of projection neurons and interneurons in the adult and are sufficient to cause changes in social behavior and cognition. These data illustrate how the timing of an illness-related placental vulnerability causes developmental alterations in neuroanatomical systems and behaviors that are relevant to autism spectrum disorders.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1523/JNEUROSCI.4654-12.2013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3807021PMC
October 2013

Neuronal Rac1 is required for learning-evoked neurogenesis.

J Neurosci 2013 Jul;33(30):12229-41

Institute for Stem Cell Biology and Regenerative Medicine, Stanford University, Stanford, California 94305, USA.

Hippocampus-dependent learning and memory relies on synaptic plasticity as well as network adaptations provided by the addition of adult-born neurons. We have previously shown that activity-induced intracellular signaling through the Rho family small GTPase Rac1 is necessary in forebrain projection neurons for normal synaptic plasticity in vivo, and here we show that selective loss of neuronal Rac1 also impairs the learning-evoked increase in neurogenesis in the adult mouse hippocampus. Earlier work has indicated that experience elevates the abundance of adult-born neurons in the hippocampus primarily by enhancing the survival of neurons produced just before the learning event. Loss of Rac1 in mature projection neurons did reduce learning-evoked neurogenesis but, contrary to our expectations, these effects were not mediated by altering the survival of young neurons in the hippocampus. Instead, loss of neuronal Rac1 activation selectively impaired a learning-evoked increase in the proliferation and accumulation of neural precursors generated during the learning event itself. This indicates that experience-induced alterations in neurogenesis can be mechanistically resolved into two effects: (1) the well documented but Rac1-independent signaling cascade that enhances the survival of young postmitotic neurons; and (2) a previously unrecognized Rac1-dependent signaling cascade that stimulates the proliferative production and retention of new neurons generated during learning itself.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1523/JNEUROSCI.2939-12.2013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3721836PMC
July 2013

PET imaging of stroke-induced neuroinflammation in mice using [18F]PBR06.

Mol Imaging Biol 2014 Feb 9;16(1):109-17. Epub 2013 Jul 9.

Department of Radiation Oncology, Stanford University School of Medicine, Stanford, CA, USA.

Purpose: The purpose of this study is to evaluate the 18 kDa translocator protein (TSPO) radioligand [(18)F]N-fluoroacetyl-N-(2,5-dimethoxybenzyl)-2-phenoxyaniline ([(18)F]PBR06) as a positron emission tomography (PET) imaging biomarker of stroke-induced neuroinflammation in a rodent model.

Procedures: Stroke was induced by transient middle cerebral artery occlusion in Balb/c mice. Dynamic PET/CT imaging with displacement and preblocking using PK111195 was performed 3 days later. PET data were correlated with immunohistochemistry (IHC) for the activated microglial markers TSPO and CD68 and with autoradiography.

Results: [(18)F]PBR06 accumulation peaked within the first 5 min postinjection, then decreased gradually, remaining significantly higher in infarct compared to noninfarct regions. Displacement or preblocking with PK11195 eliminated the difference in [(18)F]PBR06 uptake between infarct and noninfarct regions. Autoradiography and IHC correlated well spatially with uptake on PET.

Conclusions: [(18)F]PBR06 PET specifically images TSPO in microglial neuroinflammation in a mouse model of stroke and shows promise for imaging and monitoring microglial activation/neuroinflammation in other disease models.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s11307-013-0664-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4141125PMC
February 2014

Natural killer cell-activating receptor NKG2D mediates innate immune targeting of allogeneic neural progenitor cell grafts.

Stem Cells 2013 Sep;31(9):1829-39

Program in Immunology Stanford University School of Medicine University of Colorado, Boulder, Colorado, USA.

Cell replacement therapy holds promise for a number of untreatable neurological or psychiatric diseases but the immunogenicity of cellular grafts remains controversial. Emerging stem cell and reprogramming technologies can be used to generate autologous grafts that minimize immunological concerns but autologous grafts may carry an underlying genetic vulnerability that reduces graft efficacy or survival. Healthy allogeneic grafts are an attractive and commercially scalable alternative if immunological variables can be controlled. Stem cells and immature neural progenitor cells (NPC) do not express major histocompatibility complex (MHC) antigens and can evade adaptive immune surveillance. Nevertheless, in an experimental murine model, allogeneic NPCs do not survive and differentiate as well as syngeneic grafts, even when traditional immunosuppressive treatments are used. In this study, we show that natural killer (NK) cells recognize the lack of self-MHC antigens on NPCs and pose a barrier to NPC transplantation. NK cells readily target both syngeneic and allogeneic NPC, and killing is modulated primarily by NK-inhibiting "self" class I MHC and NK-activating NKG2D-ligand expression. The absence of NKG2D signaling in NK cells significantly improves NPC-derived neuron survival and differentiation. These data illustrate the importance of innate immune mechanisms in graft outcome and the potential value of identifying and targeting NK cell-activating ligands that may be expressed by stem cell derived grafts.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/stem.1422DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4199832PMC
September 2013

Lineage tracing with Axin2 reveals distinct developmental and adult populations of Wnt/β-catenin-responsive neural stem cells.

Proc Natl Acad Sci U S A 2013 Apr 15;110(18):7324-9. Epub 2013 Apr 15.

Department of Developmental Biology, Stanford University, Stanford, CA 94305, USA.

Since the discovery of neural stem cells in the mammalian brain, there has been significant interest in understanding their contribution to tissue homeostasis at both the cellular and molecular level. Wnt/β-catenin signaling is crucial for development of the central nervous system and has been implicated in stem cell maintenance in multiple tissues. Based on this, we hypothesized that the Wnt pathway likely controls neural stem cell maintenance and differentiation along the entire developmental continuum. To test this, we performed lineage tracing experiments using the recently developed tamoxifen-inducible Cre at Axin2 mouse strain to follow the developmental fate of Wnt/β-catenin-responsive cells in both the embryonic and postnatal mouse brain. From as early as embryonic day 8.5 onwards, Axin2(+) cells can give rise to spatially and functionally restricted populations of adult neural stem cells in the subventricular zone. Similarly, progeny from Axin2(+) cells labeled from E12.5 contribute to both the subventricular zone and the dentate gyrus of the hippocampus. Labeling in the postnatal brain, in turn, demonstrates the persistence of long-lived, Wnt/β-catenin-responsive stem cells in both of these sites. These results demonstrate the continued importance of Wnt/β-catenin signaling for neural stem and progenitor cell formation and function throughout developmental time.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.1305411110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3645553PMC
April 2013

Differential roles of TNFR1 and TNFR2 signaling in adult hippocampal neurogenesis.

Brain Behav Immun 2013 May 9;30:45-53. Epub 2013 Feb 9.

Stanford University, Institute for Stem Cell Biology and Regenerative Medicine, Lorry I. Lokey Stem Cell Building, G1141, 265 Campus Drive, Stanford, CA 94305, United States.

Tumor necrosis factor alpha (TNFα) is a potent inhibitor of neurogenesis in vitro but here we show that TNFα signaling has both positive and negative effects on neurogenesis in vivo and is required to moderate the negative impact of cranial irradiation on hippocampal neurogenesis. In vitro, basal levels of TNFα signaling through TNFR2 are required for normal neural progenitor cell proliferation while basal signaling through TNFR1 impairs neural progenitor proliferation. TNFR1 also mediates further reductions in proliferation and elevated cell death following exposure to recombinant TNFα. In vivo, TNFR1(-/-) and TNFα(-/-) animals have elevated baseline neurogenesis in the hippocampus, whereas absence of TNFR2 decreases baseline neurogenesis. TNFα is also implicated in defects in neurogenesis that follow radiation injury but we find that loss of TNFR1 has no protective effects on neurogenesis and loss of TNFα or TNFR2 worsened the effects of radiation injury on neurogenesis. We conclude that the immunomodulatory signaling of TNFα mediated by TNFR2 is more significant to radiation injury outcome than the proinflammatory signaling mediated through TNFR1.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bbi.2013.01.083DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3641155PMC
May 2013

PPARγ activation prevents impairments in spatial memory and neurogenesis following transient illness.

Brain Behav Immun 2013 Mar 26;29:28-38. Epub 2012 Oct 26.

Institute for Stem Cell Biology and Regenerative Medicine, Stanford University, Stanford, CA 94305, USA. Electronic address:

The detrimental effects of illness on cognition are familiar to virtually everyone. Some effects resolve quickly while others may linger after the illness resolves. We found that a transient immune response stimulated by lipopolysaccharide (LPS) compromised hippocampal neurogenesis and impaired hippocampus-dependent spatial memory. The immune event caused an ∼50% reduction in the number of neurons generated during the illness and the onset of the memory impairment was delayed and coincided with the time when neurons generated during the illness would have become functional within the hippocampus. Broad spectrum non-steroidal anti-inflammatory drugs attenuated these effects but selective Cox-2 inhibition was ineffective while PPARγ activation was surprisingly effective at protecting both neurogenesis and memory from the effects of LPS-produced transient illness. These data may highlight novel mechanisms behind chronic inflammatory and neuroinflammatory episodes that are known to compromise hippocampus-dependent forms of learning and memory.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bbi.2012.10.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3570721PMC
March 2013

Absence of CCL2 is sufficient to restore hippocampal neurogenesis following cranial irradiation.

Brain Behav Immun 2013 May 3;30:33-44. Epub 2012 Oct 3.

Stanford University, Institute for Stem Cell Biology and Regenerative Medicine, Stanford, CA 94305-5454, USA.

Cranial irradiation for the treatment of brain tumors causes a delayed and progressive cognitive decline that is pronounced in young patients. Dysregulation of neural stem and progenitor cells is thought to contribute to these effects by altering early childhood brain development. Earlier work has shown that irradiation creates a chronic neuroinflammatory state that severely and selectively impairs postnatal and adult neurogenesis. Here we show that irradiation induces a transient non-classical cytokine response with selective upregulation of CCL2/monocyte chemoattractant protein-1 (MCP-1). Absence of CCL2 signaling in the hours after irradiation is alone sufficient to attenuate chronic microglia activation and allow the recovery of neurogenesis in the weeks following irradiation. This identifies CCL2 signaling as a potential clinical target for moderating the long-term defects in neural stem cell function following cranial radiation in children.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bbi.2012.09.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3556199PMC
May 2013

Adult neural progenitor cells reactivate superbursting in mature neural networks.

Exp Neurol 2012 Mar 14;234(1):20-30. Epub 2011 Dec 14.

J. Crayton Pruitt Family Department of Biomedical Engineering, University of Florida, Gainesville, FL 32611, USA.

Behavioral recovery in animal models of human CNS syndromes suggests that transplanted stem cell derivatives can augment damaged neural networks but the mechanisms behind potentiated recovery remain elusive. Here we use microelectrode array (MEA) technology to document neural activity and network integration as rat primary neurons and rat hippocampal neural progenitor cells (NPCs) differentiate and mature. The natural transition from neuroblast to functional excitatory neuron consists of intermediate phases of differentiation characterized by coupled activity. High-frequency network-wide bursting or "superbursting" is a hallmark of early plasticity that is ultimately refined into mature stable neural network activity. Microelectrode array (MEA)-plated neurons transition through this stage of coupled superbursting before establishing mature neuronal phenotypes in vitro. When plated alone, adult rat hippocampal NPC-derived neurons fail to establish the synchronized bursting activity that neurons in primary and embryonic stem cell-derived cultures readily form. However, adult rat hippocampal NPCs evoke re-emergent superbursting in electrophysiologically mature rat primary neural cultures. Developmental superbursting is thought to accompany transient states of heightened plasticity both in culture preparations and across brain regions. Future work exploring whether NPCs can re-stimulate developmental states in injury models would be an interesting test of their regenerative potential.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.expneurol.2011.12.009DOI Listing
March 2012

Using iPSC-derived neurons to uncover cellular phenotypes associated with Timothy syndrome.

Nat Med 2011 Nov 27;17(12):1657-62. Epub 2011 Nov 27.

Department of Neurobiology, Stanford University School of Medicine, Stanford, California, USA.

Monogenic neurodevelopmental disorders provide key insights into the pathogenesis of disease and help us understand how specific genes control the development of the human brain. Timothy syndrome is caused by a missense mutation in the L-type calcium channel Ca(v)1.2 that is associated with developmental delay and autism. We generated cortical neuronal precursor cells and neurons from induced pluripotent stem cells derived from individuals with Timothy syndrome. Cells from these individuals have defects in calcium (Ca(2+)) signaling and activity-dependent gene expression. They also show abnormalities in differentiation, including decreased expression of genes that are expressed in lower cortical layers and in callosal projection neurons. In addition, neurons derived from individuals with Timothy syndrome show abnormal expression of tyrosine hydroxylase and increased production of norepinephrine and dopamine. This phenotype can be reversed by treatment with roscovitine, a cyclin-dependent kinase inhibitor and atypical L-type-channel blocker. These findings provide strong evidence that Ca(v)1.2 regulates the differentiation of cortical neurons in humans and offer new insights into the causes of autism in individuals with Timothy syndrome.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nm.2576DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3517299PMC
November 2011

SNCA triplication Parkinson's patient's iPSC-derived DA neurons accumulate α-synuclein and are susceptible to oxidative stress.

PLoS One 2011 16;6(11):e26159. Epub 2011 Nov 16.

Department of Bioengineering, Stanford University, Stanford, California, United States of America.

Parkinson's disease (PD) is an incurable age-related neurodegenerative disorder affecting both the central and peripheral nervous systems. Although common, the etiology of PD remains poorly understood. Genetic studies infer that the disease results from a complex interaction between genetics and environment and there is growing evidence that PD may represent a constellation of diseases with overlapping yet distinct underlying mechanisms. Novel clinical approaches will require a better understanding of the mechanisms at work within an individual as well as methods to identify the specific array of mechanisms that have contributed to the disease. Induced pluripotent stem cell (iPSC) strategies provide an opportunity to directly study the affected neuronal subtypes in a given patient. Here we report the generation of iPSC-derived midbrain dopaminergic neurons from a patient with a triplication in the α-synuclein gene (SNCA). We observed that the iPSCs readily differentiated into functional neurons. Importantly, the PD-affected line exhibited disease-related phenotypes in culture: accumulation of α-synuclein, inherent overexpression of markers of oxidative stress, and sensitivity to peroxide induced oxidative stress. These findings show that the dominantly-acting PD mutation is intrinsically capable of perturbing normal cell function in culture and confirm that these features reflect, at least in part, a cell autonomous disease process that is independent of exposure to the entire complexity of the diseased brain.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0026159PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3217921PMC
March 2012

The CCR2/CCL2 interaction mediates the transendothelial recruitment of intravascularly delivered neural stem cells to the ischemic brain.

Stroke 2011 Oct 11;42(10):2923-31. Epub 2011 Aug 11.

Department of Neurosurgery, Stanford Stroke Center, Stanford University, School of Medicine, 300 Pasteur Drive R211, Stanford, CA 94305-5327, USA.

Background And Purpose: The inflammatory response is a critical component of ischemic stroke. In addition to its physiological role, the mechanisms behind transendothelial recruitment of immune cells also offer a unique therapeutic opportunity for translational stem cell therapies. Recent reports have demonstrated homing of neural stem cells (NSC) into the injured brain areas after intravascular delivery. However, the mechanisms underlying the process of transendothelial recruitment remain largely unknown. Here we describe the critical role of the chemokine CCL2 and its receptor CCR2 in targeted homing of NSC after ischemia.

Methods: Twenty-four hours after induction of stroke using the hypoxia-ischemia model in mice CCR2+/+ and CCR2-/- reporter NSC were intra-arterially delivered. Histology and bioluminescence imaging were used to investigate NSC homing to the ischemic brain. Functional outcome was assessed with the horizontal ladder test.

Results: Using NSC isolated from CCR2+/+ and CCR2-/- mice, we show that receptor deficiency significantly impaired transendothelial diapedesis specifically in response to CCL2. Accordingly, wild-type NSC injected into CCL2-/- mice exhibited significantly decreased homing. Bioluminescence imaging showed robust recruitment of CCR2+/+ cells within 6 hours after transplantation in contrast to CCR2-/- cells. Mice receiving CCR2+/+ grafts after ischemic injury showed a significantly improved recovery of neurological deficits as compared to animals with transplantation of CCR2-/- NSC.

Conclusions: The CCL2/CCR2 interaction is critical for transendothelial recruitment of intravascularly delivered NSC in response to ischemic injury. This finding could have significant implications in advancing minimally invasive intravascular therapeutics for regenerative medicine or cell-based drug delivery systems for central nervous system diseases.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1161/STROKEAHA.110.606368DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3371396PMC
October 2011

Transplanted stem cell-secreted vascular endothelial growth factor effects poststroke recovery, inflammation, and vascular repair.

Stem Cells 2011 Feb;29(2):274-85

Department of Neurosurgery and Stanford Stroke Center, Stanford Institute for Neuro-Innovation and Translational Neurosciences, Stanford University School of Medicine, Stanford, California 94305-5487, USA.

Cell transplantation offers a novel therapeutic strategy for stroke; however, how transplanted cells function in vivo is poorly understood. We show for the first time that after subacute transplantation into the ischemic brain of human central nervous system stem cells grown as neurospheres (hCNS-SCns), the stem cell-secreted factor, human vascular endothelial growth factor (hVEGF), is necessary for cell-induced functional recovery. We correlate this functional recovery to hVEGF-induced effects on the host brain including multiple facets of vascular repair and its unexpected suppression of the inflammatory response. We found that transplanted hCNS-SCns affected multiple parameters in the brain with different kinetics: early improvement in blood-brain barrier integrity and suppression of inflammation was followed by a delayed spatiotemporal regulated increase in neovascularization. These events coincided with a bimodal pattern of functional recovery, with, an early recovery independent of neovascularization, and a delayed hVEGF-dependent recovery coincident with neovascularization. Therefore, cell transplantation therapy offers an exciting multimodal strategy for brain repair in stroke and potentially other disorders with a vascular or inflammatory component.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/stem.584DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3524414PMC
February 2011

Placental TNF-α signaling in illness-induced complications of pregnancy.

Am J Pathol 2011 Jun;178(6):2802-10

Institute for Stem Cell Biology and Regenerative Medicine and Department of Neurosurgery, Stanford University, Stanford, California, USA.

Maternal infections are implicated in a variety of complications during pregnancy, including pregnancy loss, prematurity, and increased risk of neurodevelopmental disorders in the child. Here, we show in mice that even mild innate immune activation by low-dose lipopolysaccharide in early pregnancy causes hemorrhages in the placenta and increases the risk of pregnancy loss. Surviving fetuses exhibit hypoxia in the brain and impaired fetal neurogenesis. Maternal Toll-like receptor 4 signaling is a critical mediator of this process, and its activation is accompanied by elevated proinflammatory cytokines in the placenta. We evaluated the role of tumor necrosis factor-α (TNF-α) signaling and show that TNF receptor 1 (TNFR1) is necessary for the illness-induced placental pathology, accompanying fetal hypoxia, and neuroproliferative defects in the fetal brain. We also show that placental TNFR1 in the absence of maternal TNFR1 is sufficient for placental pathology to develop and that a clinically relevant TNF-α antagonist prevents placental pathology and fetal loss. Our observations suggest that the placenta is highly sensitive to proinflammatory signaling in early pregnancy and that TNF-α is an effective target for preventing illness-related placental defects and related risks to the fetus and fetal brain development.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ajpath.2011.02.042DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3124299PMC
June 2011

MHC mismatch inhibits neurogenesis and neuron maturation in stem cell allografts.

PLoS One 2011 Mar 30;6(3):e14787. Epub 2011 Mar 30.

Department of Neurosurgery, Stanford Institute for Stem Cell Biology and Regenerative Medicine, Stanford, California, United States of America.

Background: The role of histocompatibility and immune recognition in stem cell transplant therapy has been controversial, with many reports arguing that undifferentiated stem cells are protected from immune recognition due to the absence of major histocompatibility complex (MHC) markers. This argument is even more persuasive in transplantation into the central nervous system (CNS) where the graft rejection response is minimal.

Methodology/principal Findings: In this study, we evaluate graft survival and neuron production in perfectly matched vs. strongly mismatched neural stem cells transplanted into the hippocampus in mice. Although allogeneic cells survive, we observe that MHC-mismatch decreases surviving cell numbers and strongly inhibits the differentiation and retention of graft-derived as well as endogenously produced new neurons. Immune suppression with cyclosporine-A did not improve outcome but non-steroidal anti-inflammatory drugs, indomethacin or rosiglitazone, were able to restore allogeneic neuron production, integration and retention to the level of syngeneic grafts.

Conclusions/significance: These results suggest an important but unsuspected role for innate, rather than adaptive, immunity in the survival and function of MHC-mismatched cellular grafts in the CNS.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0014787PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3068158PMC
March 2011

LRRK2 mutant iPSC-derived DA neurons demonstrate increased susceptibility to oxidative stress.

Cell Stem Cell 2011 Mar;8(3):267-80

Institute for Stem Cell Biology and Regenerative Medicine, Stanford University, Stanford, CA 94305, USA.

Studies of Parkinson's disease (PD) have been hindered by lack of access to affected human dopaminergic (DA) neurons. Here, we report generation of induced pluripotent stem cells that carry the p.G2019S mutation (G2019S-iPSCs) in the Leucine-Rich Repeat Kinase-2 (LRRK2) gene, the most common PD-related mutation, and their differentiation into DA neurons. The high penetrance of the LRRK2 mutation and its clinical resemblance to sporadic PD suggest that these cells could provide a valuable platform for disease analysis and drug development. We found that DA neurons derived from G2019S-iPSCs showed increased expression of key oxidative stress-response genes and α-synuclein protein. The mutant neurons were also more sensitive to caspase-3 activation and cell death caused by exposure to stress agents, such as hydrogen peroxide, MG-132, and 6-hydroxydopamine, than control DA neurons. This enhanced stress sensitivity is consistent with existing understanding of early PD phenotypes and represents a potential therapeutic target.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.stem.2011.01.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3578553PMC
March 2011