• Restricted Morphological and Behavioral Abnormalities following Ablation of β-Actin in the Brain

      Cheever, Thomas R.; Li, Bin; Ervasti, James M.; Mei, Lin; Department of Neurology; College of Graduate Studies (2012-03-5)
      The local translation of β-actin is one mechanism proposed to regulate spatially-restricted actin polymerization crucial for nearly all aspects of neuronal development and function. However, the physiological significance of localized β-actin translation in neurons has not yet been demonstrated in vivo. To investigate the role of β-actin in the mammalian central nervous system (CNS), we characterized brain structure and function in a CNS-specific β-actin knock-out mouse (CNS-ActbKO). β-actin was rapidly ablated in the embryonic mouse brain, but total actin levels were maintained through upregulation of other actin isoforms during development. CNS-ActbKO mice exhibited partial perinatal lethality while survivors presented with surprisingly restricted histological abnormalities localized to the hippocampus and cerebellum. These tissue morphology defects correlated with profound hyperactivity as well as cognitive and maternal behavior impairments. Finally, we also identified localized defects in axonal crossing of the corpus callosum in CNS-ActbKO mice. These restricted defects occurred despite the fact that primary neurons lacking β-actin in culture were morphologically normal. Altogether, we identified novel roles for β-actin in promoting complex CNS tissue architecture while also demonstrating that distinct functions for the ubiquitously expressed β-actin are surprisingly restricted in vivo.
    • Loss of Col3a1, the Gene for Ehlers-Danlos Syndrome Type IV, Results in Neocortical Dyslamination

      Jeong, Sung-Jin; Li, Shihong; Luo, Rong; Strokes, Natalie; Piao, Xianhua; Mei, Lin; Department of Neurology; College of Graduate Studies (2012-01-3)
      It has recently been discovered that Collagen III, the encoded protein of the type IV Ehlers-Danlos Syndrome (EDS) gene, is one of the major constituents of the pial basement membrane (BM) and serves as the ligand for GPR56. Mutations in GPR56 cause a severe human brain malformation called bilateral frontoparietal polymicrogyria, in which neurons transmigrate through the BM causing severe mental retardation and frequent seizures. To further characterize the brain phenotype of Col3a1 knockout mice, we performed a detailed histological analysis. We observed a cobblestone-like cortical malformation, with BM breakdown and marginal zone heterotopias in Col3a1-/- mouse brains. Surprisingly, the pial BM appeared intact at early stages of development but starting as early as embryonic day (E) 11.5, prominent BM defects were observed and accompanied by neuronal overmigration. Although collagen III is expressed in meningeal fibroblasts (MFs), Col3a1-/- MFs present no obvious defects. Furthermore, the expression and posttranslational modification of a-dystroglycan was undisturbed in Col3a1-/- mice. Based on the previous finding that mutations in COL3A1 cause type IV EDS, our study indicates a possible common pathological pathway linking connective tissue diseases and brain malformations.
    • Disease-Associated Mutations Prevent GPR56-Collagen III Interaction

      Luo, Rong; Jin, Zhaohui; Deng, Yiyu; Strokes, Natalie; Piao, Xianhua; Mei, Lin; Department of Neurology (2012-01-4)
      GPR56 is a member of the adhesion G protein-coupled receptor (GPCR) family. Mutations in GPR56 cause a devastating human brain malformation called bilateral frontoparietal polymicrogyria (BFPP). Using the N-terminal fragment of GPR56 (GPR56N) as a probe, we have recently demonstrated that collagen III is the ligand of GPR56 in the developing brain. In this report, we discover a new functional domain in GPR56N, the ligand binding domain. This domain contains four disease-associated mutations and two N-glycosylation sites. Our study reveals that although glycosylation is not required for ligand binding, each of the four disease-associated mutations completely abolish the ligand binding ability of GPR56. Our data indicates that these four single missense mutations cause BFPP mostly by abolishing the ability of GPR56 to bind to its ligand, collagen III, in addition to affecting GPR56 protein surface expression as previously shown.
    • Niclosamide Suppresses Cancer Cell Growth By Inducing Wnt Co-Receptor LRP6 Degradation and Inhibiting the Wnt/β-Catenin Pathway

      Lu, Wenyan; Lin, Cuihong; Roberts, Michael J.; Waud, William R.; Piazza, Gary A.; Li, Yonghe; Mei, Lin; Department of Neurology; College of Graduate Studies (2011-12-16)
      The Wnt/b-catenin signaling pathway is important for tumor initiation and progression. The low density lipoprotein receptor-related protein-6 (LRP6) is an essential Wnt co-receptor for Wnt/b-catenin signaling and represents a promising anticancer target. Recently, the antihelminthic drug, niclosamide was found to inhibit Wnt/b-catenin signaling, although the mechanism was not well defined. We found that niclosamide was able to suppress LRP6 expression and phosphorylation, block Wnt3A-induced b-catenin accumulation, and inhibit Wnt/b-catenin signaling in HEK293 cells. Furthermore, the inhibitory effects of niclosamide on LRP6 expression/phosphorylation and Wnt/b-catenin signaling were conformed in human prostate PC-3 and DU145 and breast MDA-MB-231 and T-47D cancer cells. Moreover, we showed that the mechanism by which niclosamide suppressed LRP6 resulted from increased degradation as evident by a shorter half-life. Finally, we demonstrated that niclosamide was able to induce cancer cell apoptosis, and displayed excellent anticancer activity with IC50 values less than 1 mM for prostate PC-3 and DU145 and breast MDA-MB-231 and T-47D cancer cells. The IC50 values are comparable to those shown to suppress the activities of Wnt/b-catenin signaling in prostate and breast cancer cells. Our data indicate that niclosamide is a unique small molecule Wnt/b-catenin signaling inhibitor targeting the Wnt co-receptor LRP6 on the cell surface, and that niclosamide has a potential to be developed a novel chemopreventive or therapeutic agent for human prostate and breast cancer.
    • Profiling Insulin Like Factor 3 (INSL3) Signaling in Human Osteoblasts

      Ferlin, Alberto; Perilli, Lisa; Gianesello, Lisa; Taglialavoro, Giuseppe; Foresta, Carlo; Mei, Lin; Department of Neurology; College of Graduate Studies (2011-12-28)
      Background: Young men with mutations in the gene for the INSL3 receptor (Relaxin family peptide 2, RXFP2) are at risk of reduced bone mass and osteoporosis. Consistent with the human phenotype, bone analyses of Rxfp2â /â mice showed decreased bone volume, alterations of the trabecular bone, reduced mineralizing surface, bone formation, and osteoclast surface. The aim of this study was to elucidate the INSL3/RXFP2 signaling pathways and targets in human osteoblasts.
    • Genetic Ablation of PLA2G6 in Mice Leads to Cerebellar Atrophy Characterized by Purkinje Cell Loss and Glial Cell Activation

      Zhao, Zhengshan; Wang, Jing; Zhao, Chunying; Bi, Weina; Yue, Zhenyu; Ma, Zhongmin Alex; Mei, Lin; Department of Neurology (2011-10-28)
      Infantile neuroaxonal dystrophy (INAD) is a progressive, autosomal recessive neurodegenerative disease characterized by axonal dystrophy, abnormal iron deposition and cerebellar atrophy. This disease was recently mapped to PLA2G6, which encodes group VI Ca2+-independent phospholipase A2 (iPLA2 or iPLA2b). Here we show that genetic ablation of PLA2G6 in mice (iPLA2b-/-) leads to the development of cerebellar atrophy by the age of 13 months. Atrophied cerebella exhibited significant loss of Purkinje cells, as well as reactive astrogliosis, the activation of microglial cells, and the pronounced upregulation of the pro-inflammatory cytokines tumor necrosis factor-a (TNF-a) and interleukin-1b (IL-1b). Moreover, glial cell activation and the elevation in TNF-a and IL-1b expression occurred before apparent cerebellar atrophy. Our findings indicate that the absence of PLA2G6 causes neuroinflammation and Purkinje cell loss and ultimately leads to cerebellar atrophy. Our study suggests that iPLA2b-/- mice are a valuable model for cerebellar atrophy in INAD and that early antiinflammatory therapy may help slow the progression of cerebellar atrophy in this deadly neurodegenerative disease.
    • Type III Nrg1 Back Signaling Enhances Functional TRPV1 along Sensory Axons Contributing to Basal and Inflammatory Thermal Pain Sensation

      Canetta, Sarah E.; Luca, Edlira; Pertot, Elyse; Role, Lorna W.; Talmage, David A.; Mei, Lin; Department of Neurology; College of Graduate Studies (2011-09-20)
      Type III Nrg1, a member of the Nrg1 family of signaling proteins, is expressed in sensory neurons, where it can signal in a bi-directional manner via interactions with the ErbB family of receptor tyrosine kinases (ErbB RTKs)
    • Molecular Basis of NDM-1, a New Antibiotic Resistance Determinant

      Liang, Zhongjie; Li, Lianchun; Wang, Yuanyuan; Chen, Limin; Kong, Xiangqian; Hong, Yao; Lan, Lefu; Zheng, Mingyue; Guang-Yang, Cai; Liu, Hong; et al. (2011-08-24)
      The New Delhi Metallo-b-lactamase (NDM-1) was first reported in 2009 in a Swedish patient. A recent study reported that Klebsiella pneumonia NDM-1 positive strain or Escherichia coli NDM-1 positive strain was highly resistant to all antibiotics tested except tigecycline and colistin. These can no longer be relied on to treat infections and therefore, NDM-1 now becomes potentially a major global health threat.
    • BDNF Facilitates L-LTP Maintenance in the Absence of Protein Synthesis through PKMf

      Mei, Fan; Nagappan, Guhan; Ke, Yang; Sacktor, Todd C.; Lu, Bai; Mei, Lin; Department of Neurology (2011-06-29)
      Late-phase long term potentiation (L-LTP) is thought to be the cellular basis for long-term memory (LTM). While LTM as well as L-LTP is known to depend on transcription and translation, it is unclear why brain-derived neurotrophic factor (BDNF) could sustain L-LTP when protein synthesis is inhibited. The persistently active protein kinase f (PKMf) is the only molecule implicated in perpetuating L-LTP maintenance. Here, in mouse acute brain slices, we show that inhibition of PKMf reversed BDNF-dependent form of L-LTP. While BDNF did not alter the steady-state level of PKMf, BDNF together with the L-LTP inducing theta-burst stimulation (TBS) increased PKMf level even without protein synthesis. Finally, in the absence of de novo protein synthesis, BDNF maintained TBS-induced PKMf at a sufficient level. These results suggest that BDNF sustains L-LTP through PKMf in a protein synthesis-independent manner, revealing an unexpected link between BDNF and PKMf.
    • SHP-2 Promotes the Maturation of Oligodendrocyte Precursor Cells Through Akt and ERK1/2 Signaling In Vitro

      Liu, Xiujie; Li, Yuanyuan; Zhang, Yong; Lu, Yan; Guo, Wei; Liu, Peng; Zhou, Jiazhen; Xiang, Zhenghua; He, Cheng; Department of Neurology; et al. (2011-06-20)
      Background: Oligodendrocyte precursor cells (OPCs) differentiate into oligodendrocytes (OLs), which are responsible for myelination. Myelin is essential for saltatory nerve conduction in the vertebrate nervous system. However, the molecular mechanisms of maturation and myelination by oligodendrocytes remain elusive.
    • Factors Underlying the Early Limb Muscle Weakness in Acute Quadriplegic Myopathy Using an Experimental ICU Porcine Model

      Ochala, Julien; Ahlbeck, Karsten; Radell, Peter J.; Eriksson, Lars I.; Larsson, Lars; Mei, Lin; Department of Neurology (2011-06-14)
      The basic mechanisms underlying acquired generalized muscle weakness and paralysis in critically ill patients remain poorly understood and may be related to prolonged mechanical ventilation/immobilization (MV) or to other triggering factors such as sepsis, systemic corticosteroid (CS) treatment and administration of neuromuscular blocking agents (NMBA). The present study aims at exploring the relative importance of these factors by using a unique porcine model. Piglets were all exposed to MV together with different combinations of endotoxin-induced sepsis, CS and NMBA for five days. Peroneal motor nerve conduction velocity and amplitude of the compound muscle action potential (CMAP) as well as biceps femoris muscle biopsy specimens were obtained immediately after anesthesia on the first day and at the end of the 5-day experimental period. Results showed that peroneal nerve motor conduction velocity is unaffected whereas the size of the CMAP decreases independently of the type of intervention, in all groups after 5 days. Otherwise, despite a preserved size, muscle fibre specific force (maximum force normalized to cross-sectional area) decreased dramatically for animals exposed to MV in combination with CS or/and sepsis. These results suggest that the rapid declines in CMAP amplitude and in force generation capacity are triggered by independent mechanisms with significant clinical and therapeutic implications.
    • Binge-Pattern Alcohol Exposure during Puberty Induces Long-Term Changes in HPA Axis Reactivity

      Przybycien-Szymanska, Magdalena M.; Mott, Natasha N.; Paul, Caitlin R.; Gillespie, Roberta A.; Pak, Toni R.; Brann, Darrell W; Department of Neurology; College of Graduate Studies (2011-04-13)
      Adolescence is a dynamic and important period of brain development however, little is known about the long-term neurobiological consequences of alcohol consumption during puberty. Our previous studies showed that binge-pattern ethanol (EtOH) treatment during pubertal development negatively dysregulated the responsiveness of the hypothalamo-pituitary-adrenal (HPA) axis, as manifested by alterations in corticotrophin-releasing hormone (CRH), arginine vasopressin (AVP), and corticosterone (CORT) during this time period. Thus, the primary goal of this study was to determine whether these observed changes in important central regulators of the stress response were permanent or transient. In this study, juvenile male Wistar rats were treated with a binge-pattern EtOH treatment paradigm or saline alone for 8 days. The animals were left undisturbed until adulthood when they received a second round of treatments consisting of saline alone, a single dose of EtOH, or a second binge-pattern treatment paradigm. The results showed that pubertal binge-pattern EtOH exposure induced striking long-lasting alterations of many HPA axis parameters. Overall, our data provide strong evidence that binge-pattern EtOH exposure during pubertal maturation has long-term detrimental effects for the healthy development of the HPA axis.
    • Cell-Type Specific Expression of a Dominant Negative PKA Mutation in Mice

      Willis, Brandon S.; Niswender, Colleen M.; Su, Thomas; Amieux, Paul S.; McKnight, G. Stanley; Mei, Lin; Department of Neurology (2011-04-12)
      We employed the Cre recombinase/loxP system to create a mouse line in which PKA activity can be inhibited in any celltype that expresses Cre recombinase. The mouse line carries a mutant Prkar1a allele encoding a glycine to aspartate substitution at position 324 in the carboxy-terminal cAMP-binding domain (site B). This mutation produces a dominant negative RIa regulatory subunit (RIaB) and leads to inhibition of PKA activity. Insertion of a loxP-flanked neomycin cassette in the intron preceding the site B mutation prevents expression of the mutant RIaB allele until Cre-mediated excision of the cassette occurs. Embryonic stem cells expressing RIaB demonstrated a reduction in PKA activity and inhibition of cAMPresponsive gene expression. Mice expressing RIaB in hepatocytes exhibited reduced PKA activity, normal fasting induced gene expression, and enhanced glucose disposal. Activation of the RIaB allele in vivo provides a novel system for the analysis of PKA function in physiology.
    • Two Distinct Integrin-Mediated Mechanisms Contribute to Apical Lumen Formation in Epithelial Cells

      Myllymaki, Satu Marja; Teravainen, Terhi Piritta; Manninen, Aki; Mei, Lin; Department of Neurology; College of Graduate Studies (2011-05-6)
      Background: Formation of apical compartments underlies the morphogenesis of most epithelial organs during development. The extracellular matrix (ECM), particularly the basement membrane (BM), plays an important role in orienting the apico-basal polarity and thereby the positioning of apical lumens. Integrins have been recognized as essential mediators of matrix-derived polarity signals. The importance of b1-integrins in epithelial polarization is well established but the significance of the accompanying a-subunits have not been analyzed in detail.
    • HER2 Targeted Molecular MR Imaging Using a De Novo Designed Protein Contrast Agent

      Qiao, Jingjuan; Li, Shunyi; Wei, Lixia; Jiang, Jie; Long, Robert; Mao, Hui; Wei, Ling; Wang, Liya; Yang, Hua; Grossniklaus, Hans E.; et al. (2011-03-24)
      The application of magnetic resonance imaging (MRI) to non-invasively assess disease biomarkers has been hampered by the lack of desired contrast agents with high relaxivity, targeting capability, and optimized pharmacokinetics. We have developed a novel MR imaging probe targeting to HER2, a biomarker for various cancer types and a drug target for anti-cancer therapies. This multimodal HER20targeted MR imaging probe integrates a de novo designed protein contrast agent with a high affinity HER2 affibody and a near IR fluorescent dye. Our probe can differentially monitor tumors with different expression levels of HER2 in both human cell lines and xenograft mice models. In addition to its 100-fold higher dose efficiency compared to clinically approved non-targeting contrast agent DTPA, our developed agent also exhibits advantages in crossing the endothelial boundary, tissue distribution, and tumor tissue retention over reported contrast agents as demonstrated by even distribution of the imaging probe across the entire tumor mass. This contrast agent will provide a powerful tool for quantitative assessment of molecular markers, and improved resolution for diagnosis, prognosis and drug discovery.
    • Novel Mouse Model Reveals Distinct Activity-Dependent and -Independent Contributions to Synapse Development

      Pacifici, Pier Giorgio; Peter, Christoph; Yampolsky, Pessah; Koenen, Michael; McArdle, Joseph J.; Witzemann, Veit; Mei, Lin; Department of Neurology; College of Graduate Studies (2011-01-31)
      The balanced action of both pre- and postsynaptic organizers regulates the formation of neuromuscular junctions (NMJ). The precise mechanisms that control the regional specialization of acetylcholine receptor (AChR) aggregation, guide ingrowing axons and contribute to correct synaptic patterning are unknown. Synaptic activity is of central importance and to understand synaptogenesis, it is necessary to distinguish between activity-dependent and activity-independent processes. By engineering a mutated fetal AChR subunit, we used homologous recombination to develop a mouse line that expresses AChR with massively reduced open probability during embryonic development. Through histological and immunochemical methods as well as electrophysiological techniques, we observed that endplate anatomy and distribution are severely aberrant and innervation patterns are completely disrupted. Nonetheless, in the absence of activity AChRs form postsynaptic specializations attracting motor axons and permitting generation of multiple nerve/muscle contacts on individual fibers. This process is not restricted to a specialized central zone of the diaphragm and proceeds throughout embryonic development. Phenotypes can be attributed to separate activity-dependent and -independent pathways. The correct patterning of synaptic connections, prevention of multiple contacts and control of nerve growth require AChR-mediated activity. In contrast, myotube survival and acetylcholine-mediated dispersal of AChRs are maintained even in the absence of AChR-mediated activity. Because mouse models in which acetylcholine is entirely absent do not display similar effects, we conclude that acetylcholine binding to the AChR initiates activity-dependent and activity-independent pathways whereby the AChR modulates formation of the NMJ.
    • The Actin Binding Domain of bI-Spectrin Regulates the Morphological and Functional Dynamics of Dendritic Spines

      Nestor, Michael W.; Cai, Xiang; Stone, Michele R.; Bloch, Robert J.; Thompson, Scott M.; Mei, Lin; Department of Neurology (2011-01-31)
      Actin microfilaments regulate the size, shape and mobility of dendritic spines and are in turn regulated by actin binding proteins and small GTPases. The bI isoform of spectrin, a protein that links the actin cytoskeleton to membrane proteins, is present in spines. To understand its function, we expressed its actin-binding domain (ABD) in CA1 pyramidal neurons in hippocampal slice cultures. The ABD of bI-spectrin bundled actin in principal dendrites and was concentrated in dendritic spines, where it significantly increased the size of the spine head. These effects were not observed after expression of homologous ABDs of utrophin, dystrophin, and a-actinin. Treatment of slice cultures with latrunculin-B significantly decreased spine head size and decreased actin-GFP fluorescence in cells expressing the ABD of a-actinin, but not the ABD of bI-spectrin, suggesting that its presence inhibits actin depolymerization. We also observed an increase in the area of GFPtagged PSD-95 in the spine head and an increase in the amplitude of mEPSCs at spines expressing the ABD of bI-spectrin. The effects of the bI-spectrin ABD on spine size and mEPSC amplitude were mimicked by expressing wild-type Rac3, a small GTPase that co-immunoprecipitates specifically with bI-spectrin in extracts of cultured cortical neurons. Spine size was normal in cells co-expressing a dominant negative Rac3 construct with the bI-spectrin ABD. We suggest that bI-spectrin is a synaptic protein that can modulate both the morphological and functional dynamics of dendritic spines, perhaps via interaction with actin and Rac3.
    • Forced Notch Signaling Inhibits Commissural Axon Outgrowth in the Developing Chick Central Nerve System

      Shi, Ming; Liu, Zhirong; Lv, Yonggang; Zheng, Minhua; Du, Fang; Zhao, Gang; Huang, Ying; Chen, Jiayin; Han, Hua; Ding, Yuqiang; et al. (2011-01-21)
      Background: A collection of in vitro evidence has demonstrated that Notch signaling plays a key role in the growth of neurites in differentiated neurons. However, the effects of Notch signaling on axon outgrowth in an in vivo condition remain largely unknown.
    • Synaptic Neurotransmission Depression in Ventral Tegmental Dopamine Neurons and Cannabinoid-Associated Addictive Learning

      Liu, Zhiqiang; Han, Jing; Jia, Lintao; Maillet, Jean-Christian; Bai, Guang; Xu, Lin; Jia, Zhengping; Zheng, Qiaohua; Zhang, Wandong; Monette, Robert; et al. (2010-12-20)
      Drug addiction is an association of compulsive drug use with long-term associative learning/memory. Multiple forms of learning/memory are primarily subserved by activity- or experience-dependent synaptic long-term potentiation (LTP) and long-term depression (LTD). Recent studies suggest LTP expression in locally activated glutamate synapses onto dopamine neurons (local Glu-DA synapses) of the midbrain ventral tegmental area (VTA) following a single or chronic exposure to many drugs of abuse, whereas a single exposure to cannabinoid did not significantly affect synaptic plasticity at these synapses. It is unknown whether chronic exposure of cannabis (marijuana or cannabinoids), the most commonly used illicit drug worldwide, induce LTP or LTD at these synapses. More importantly, whether such alterations in VTA synaptic plasticity causatively contribute to drug addictive behavior has not previously been addressed. Here we show in rats that chronic cannabinoid exposure activates VTA cannabinoid CB1 receptors to induce transient neurotransmission depression at VTA local Glu-DA synapses through activation of NMDA receptors and subsequent endocytosis of AMPA receptor GluR2 subunits. A GluR2-derived peptide blocks cannabinoid-induced VTA synaptic depression and conditioned place preference, i.e., learning to associate drug exposure with environmental cues. These data not only provide the first evidence, to our knowledge, that NMDA receptor-dependent synaptic depression at VTA dopamine circuitry requires GluR2 endocytosis, but also suggest an essential contribution of such synaptic depression to cannabinoid-associated addictive learning, in addition to pointing to novel pharmacological strategies for the treatment of cannabis addiction.
    • Operant Sensation Seeking Requires Metabotropic Glutamate Receptor 5 (mGluR5)

      Olsen, Christopher M.; Childs, Daniel S.; Stanwood, Gregg D.; Winder, Danny G.; Tsien, Joe Z.; Department of Neurology; College of Graduate Studies (2010-11-30)
      Pharmacological and genetic studies have suggested that the metabotropic glutamate receptor 5 (mGluR5) is critically involved in mediating the reinforcing effects of drugs of abuse, but not food. The purpose of this study was to use mGluR5 knockout (KO), heterozygous (Het), and wildtype (WT) mice to determine if mGluR5 modulates operant sensation seeking (OSS), an operant task that uses varied sensory stimuli as a reinforcer. We found that mGluR5 KO mice had significantly reduced OSS responding relative to WT mice, while Het mice displayed a paradoxical increase in OSS responding. Neither KO nor Het mice exhibited altered operant responding for food as a reinforcer. Further, we assessed mGluR5 KO, Het and WT mice across a battery of cocaine locomotor, place preference and anxiety related tests. Although KO mice showed expected differences in some locomotor and anxiety measures, Het mice either exhibited no phenotype or an intermediate one. In total, these data demonstrate a key role for mGluR5 in OSS, indicating an important role for this receptor in reinforcement-based behavior.