• Single-Channel Electrophysiology Reveals a Distinct and Uniform Pore Complex Formed by α-Synuclein Oligomers in Lipid Membranes

      Schmidt, Felix; Levin, Johannes; Kamp, Frits; Kretzschmar, Hans; Giese, Armin; Botzel, Kai; Tsien, Joe Z.; Department of Neurology; College of Graduate Studies (2012-08-3)
      Synucleinopathies such as Parkinson's disease, multiple system atrophy and dementia with Lewy bodies are characterized by deposition of aggregated α-synuclein. Recent findings indicate that pathological oligomers rather than fibrillar aggregates may represent the main toxic protein species. It has been shown that α-synuclein oligomers can increase the conductance of lipid bilayers and, in cell-culture, lead to calcium dyshomeostasis and cell death. In this study, employing a setup for single-channel electrophysiology, we found that addition of iron-induced α-synuclein oligomers resulted in quantized and stepwise increases in bilayer conductance indicating insertion of distinct transmembrane pores. These pores switched between open and closed states depending on clamped voltage revealing a single-pore conductance comparable to that of bacterial porins. Pore conductance was dependent on transmembrane potential and the available cation. The pores stably inserted into the bilayer and could not be removed by buffer exchange. Pore formation could be inhibited by co-incubation with the aggregation inhibitor baicalein. Our findings indicate that iron-induced α-synuclein oligomers can form a uniform and distinct pore species with characteristic electrophysiological properties. Pore formation could be a critical event in the pathogenesis of synucleinopathies and provide a novel structural target for disease-modifying therapy.
    • Focusing on Attention: The Effects of Working Memory Capacity and Load on Selective Attention

      Ahmed, Lubna; de Fockert, Jan W.; Tsien, Joe Z.; Department of Neurology; College of Graduate Studies (2012-08-28)
      Background: Working memory (WM) is imperative for effective selective attention. Distractibility is greater under conditions of high (vs. low) concurrent working memory load (WML), and in individuals with low (vs. high) working memory capacity (WMC). In the current experiments, we recorded the flanker task performance of individuals with high and low WMC during low and high WML, to investigate the combined effect of WML and WMC on selective attention.
    • The Functional Upregulation of Piriform Cortex Is Associated with Cross-Modal Plasticity in Loss of Whisker Tactile Inputs

      Ye, Bing; Huang, Li; Gao, Zilong; Chen, Ping; Ni, Hong; Guan, Sudong; Zhu, Yan; Wang, Jin-Hui; Mei, Lin; Department of Neurology (2012-08-21)
      Background: Cross-modal plasticity is characterized as the hypersensitivity of remaining modalities after a sensory function is lost in rodents, which ensures their awareness to environmental changes. Cellular and molecular mechanisms underlying cross-modal sensory plasticity remain unclear. We aim to study the role of different types of neurons in cross-modal plasticity.
    • Diagnostic Potential of the NMDA Receptor Peptide Assay for Acute Ischemic Stroke

      Dambinova, Svetlana A.; Bettermann, Kerstin; Glynn, Theodore; Tews, Matthew; Olson, David; Weissman, Joseph D.; Sowell, Richard L.; Department of Neurology (2012-07-27)
      Background: The acute assessment of patients with suspected ischemic stroke remains challenging. The use of brain biomarker assays may improve the early diagnosis of ischemic stroke. The main goal of the study was to evaluate whether the NR2 peptide, a product of the proteolytic degradation of N-methyl-D-aspartate (NMDA) receptors, can differentiate acute ischemic stroke (IS) from stroke mimics and persons with vascular risk factors/healthy controls. A possible correlation between biomarker values and lesion sizes was investigated as the secondary objective.
    • Homologous Recombination Mediates Functional Recovery of Dysferlin Deficiency following AAV5 Gene Transfer

      Grose, William E.; Clark, K. Reed; Griffin, Danielle; Malik, Vinod; Shontz, Kimberly M.; Montgomery, Chrystal L.; Lewis, Sarah; Brown, Robert H.; Janssen, Paul M. L.; Mendell, Jerry R.; et al. (2012-06-15)
      The dysferlinopathies comprise a group of untreatable muscle disorders including limb girdle muscular dystrophy type 2B, Miyoshi myopathy, distal anterior compartment syndrome, and rigid spine syndrome. As with other forms of muscular dystrophy, adeno-associated virus (AAV) gene transfer is a particularly auspicious treatment strategy, however the size of the DYSF cDNA (6.5 kb) negates packaging into traditional AAV serotypes known to express well in muscle (i.e. rAAV1, 2, 6, 8, 9). Potential advantages of a full cDNA versus a mini-gene include: maintaining structural-functional protein domains, evading protein misfolding, and avoiding novel epitopes that could be immunogenic. AAV5 has demonstrated unique plasticity with regards to packaging capacity and recombination of virions containing homologous regions of cDNA inserts has been implicated in the generation of full-length transcripts. Herein we show for the first time in vivo that homologous recombination following AAV5.DYSF gene transfer leads to the production of full length transcript and protein. Moreover, gene transfer of full-length dysferlin protein in dysferlin deficient mice resulted in expression levels sufficient to correct functional deficits in the diaphragm and importantly in skeletal muscle membrane repair. Intravascular regional gene transfer through the femoral artery produced high levels of transduction and enabled targeting of specific muscle groups affected by the dysferlinopathies setting the stage for potential translation to clinical trials. We provide proof of principle that AAV5 mediated delivery of dysferlin is a highly promising strategy for treatment of dysferlinopathies and has far-reaching implications for the therapeutic delivery of other large genes.
    • Effects of Kisspeptin1 on Electrical Activity of an Extrahypothalamic Population of Gonadotropin-Releasing Hormone Neurons in Medaka (Oryzias latipes)

      Zhao, Yali; Wayne, Nancy L.; Brann, Darrell W; Department of Neurology; College of Graduate Studies (2012-05-23)
      Kisspeptin (product of the kiss1 gene) is the most potent known activator of the hypothalamo-pituitary-gonadal axis. Both kiss1 and the kisspeptin receptor are highly expressed in the hypothalamus of vertebrates, and low doses of kisspeptin have a robust and long-lasting stimulatory effect on the rate of action potential firing of hypophysiotropic gonadotropin releasing hormone-1 (GnRH1) neurons in mice. Fish have multiple populations of GnRH neurons distinguished by their location in the brain and the GnRH gene that they express. GnRH3 neurons located in the terminal nerve (TN) associated with the olfactory bulb are neuromodulatory and do not play a direct role in regulating pituitary-gonadal function. In medaka fish, the electrical activity of TN-GnRH3 neurons is modulated by visual cues from conspecifics, and is thought to act as a transmitter of information from the external environment to the central nervous system. TN-GnRH3 neurons also play a role in sexual motivation and arousal states, making them an important population of neurons to study for understanding coordination of complex behaviors. We investigated the role of kisspeptin in regulating electrical activity of TN-GnRH3 neurons in adult medaka. Using electrophysiology in an intact brain preparation, we show that a relatively brief treatment with 100 nM of kisspeptin had a long-lasting stimulatory effect on the electrical activity of an extrahypothalamic population of GnRH neurons. Dose-response analysis suggests a relatively narrow activational range of this neuropeptide. Further, blocking action potential firing with tetrodotoxin and blocking synaptic transmission with a low Ca2+/high Mg2+ solution inhibited the stimulatory action of kisspeptin on electrical activity, indicating that kisspeptin is acting indirectly through synaptic regulation to excite TN-GnRH3 neurons. Our findings provide a new perspective on kisspeptin's broader functions within the central nervous system, through its regulation of an extrahypothalamic population of GnRH neurons involved in multiple neuromodulatory functions.
    • Dihydrotestosterone Ameliorates Degeneration in Muscle, Axons and Motoneurons and Improves Motor Function in Amyotrophic Lateral Sclerosis Model Mice

      Yoo, Young-Eun; Ko, Chien-Ping; Mei, Lin; Department of Neurology (2012-05-14)
      Amyotrophic lateral sclerosis (ALS) is a lethal disease characterized by a progressive loss of motoneurons. The clinical symptoms include skeletal muscle weakness and atrophy, which impairs motor performance and eventually leads to respiratory failure. We tested whether dihydrotestosterone (DHT), which has both anabolic effects on muscle and neuroprotective effects on axons and motoneurons, can ameliorate clinical symptoms in ALS. A silastic tube containing DHT crystals was implanted subcutaneously in SOD1-G93A mice at early symptomatic age when decreases in body weight and grip-strength were observed as compared to wild-type mice. DHT-treated SOD1-G93A mice demonstrated ameliorated muscle atrophy and increased body weight, which was associated with stronger grip-strength. DHT treatment increased the expression of insulin-like growth factor-1 in muscle, which can exert myotrophic as well as neurotrophic effects through retrograde transport. DHT treatment attenuated neuromuscular junction denervation, and axonal and motoneuron loss. DHT-treated SOD1-G93A mice demonstrated improvement in motor behavior as assessed by rota-rod and gait analyses, and an increased lifespan. Application of DHT is a relatively simple and non-invasive procedure, which may be translated into therapy to improve the quality of life for ALS patients.
    • Critical Role of NADPH Oxidase in Neuronal Oxidative Damage and Microglia Activation following Traumatic Brain Injury

      Zhang, Quan-Guang; Laird, Melissa D; Han, Dong; Nguyen, Khoi; Scott, Erin L.; Dong, Yan; Dhandapani, Krishnan M.; Brann, Darrell W; Department of Neurology; Institute of Molecular Medicine and Genetics; et al. (2012-04-2)
      Background: Oxidative stress is known to play an important role in the pathology of traumatic brain injury. Mitochondria are thought to be the major source of the damaging reactive oxygen species (ROS) following TBI. However, recent work has revealed that the membrane, via the enzyme NADPH oxidase can also generate the superoxide radical (O2^-), and thereby potentially contribute to the oxidative stress following TBI. The current study thus addressed the potential role of NADPH oxidase in TBI.
    • 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.
    • Wnt proteins regulate acetylcholine receptor clustering in muscle cells

      Zhang, Bin; Liang, Chuan; Bates, Ryan; Yin, Yiming; Xiong, Wen-Cheng; Mei, Lin; Department of Neurology; Institute of Molecular Medicine and Genetics (2012-02-6)
      Background: The neuromuscular junction (NMJ) is a cholinergic synapse that rapidly conveys signals from motoneurons to muscle cells and exhibits a high degree of subcellular specialization characteristic of chemical synapses. NMJ formation requires agrin and its coreceptors LRP4 and MuSK. Increasing evidence indicates that Wnt signaling regulates NMJ formation in Drosophila, C. elegans and zebrafish.
    • a-Calcium Calmodulin Kinase II Modulates the Temporal Structure of Hippocampal Bursting Patterns

      Cho, Jeiwon; Bhatt, Rushi; Elgersma, Ype; Silva, Alcino J.; Tsien, Joe Z.; Department of Neurology; College of Graduate Studies (2012-02-20)
      The alpha calcium calmodulin kinase II (a-CaMKII) is known to play a key role in CA1/CA3 synaptic plasticity, hippocampal place cell stability and spatial learning. Additionally, there is evidence from hippocampal electrophysiological slice studies that this kinase has a role in regulating ion channels that control neuronal excitability. Here, we report in vivo single unit studies, with a-CaMKII mutant mice, in which threonine 305 was replaced with an aspartate (a-CaMKIIT305D mutants), that indicate that this kinase modulates spike patterns in hippocampal pyramidal neurons. Previous studies showed that a- CaMKIIT305D mutants have abnormalities in both hippocampal LTP and hippocampal-dependent learning. We found that besides decreased place cell stability, which could be caused by their LTP impairments, the hippocampal CA1 spike patterns of a-CaMKIIT305D mutants were profoundly abnormal. Although overall firing rate, and overall burst frequency were not significantly altered in these mutants, inter-burst intervals, mean number of intra-burst spikes, ratio of intra-burst spikes to total spikes, and mean intra-burst intervals were significantly altered. In particular, the intra burst intervals of place cells in a- CaMKIIT305D mutants showed higher variability than controls. These results provide in vivo evidence that besides its wellknown function in synaptic plasticity, a-CaMKII, and in particular its inhibitory phosphorylation at threonine 305, also have a role in shaping the temporal structure of hippocampal burst patterns. These results suggest that some of the molecular processes involved in acquiring information may also shape the patterns used to encode this information.
    • 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.
    • 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.
    • Preoperative Evaluation with fMRI of Patients with Intracranial Gliomas

      Kapsalakis, Ioannis Z.; Kapsalaki, Eftychia Z.; Gotsis, Efstathios D.; Verganelakis, Dimitrios; Toulas, Panagiotis; Hadjigeorgiou, Georgios; Chung, Indug; Fezoulidis, Ioannis; Papadimitriou, Alexandros; Robinson, Joe Sam; et al. (2012--2012)
    • 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.
    • 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.
    • Sex-independent neuroprotection with minocycline after experimental thromboembolic stroke

      Hoda, Md Nasrul; Li, Weiguo; Ahmad, Ajmal; Ogbi, Safia; Zemskova, Marina A; Johnson, Maribeth H.; Ergul, Adviye; Hill, William D; Hess, David C.; Sazonova, Irina Y; et al. (2011-12-16)
      Background: Minocycline provides neurovascular protection reducing acute cerebral injury. However, it is unclear whether minocycline is effective in females. We tested minocycline in both sexes and aged animals using a novel embolic stroke model in mice that closely mimics acute thromboembolic stroke in humans.
    • Lactate Produced by Glycogenolysis in Astrocytes Regulates Memory Processing

      Newman, Lori A.; Korol, Donna L.; Gold, Paul E.; Brann, Darrell W; Department of Neurology; College of Graduate Studies (2011-12-13)
      When administered either systemically or centrally, glucose is a potent enhancer of memory processes. Measures of glucose levels in extracellular fluid in the rat hippocampus during memory tests reveal that these levels are dynamic, decreasing in response to memory tasks and loads; exogenous glucose blocks these decreases and enhances memory. The present experiments test the hypothesis that glucose enhancement of memory is mediated by glycogen storage and then metabolism to lactate in astrocytes, which provide lactate to neurons as an energy substrate. Sensitive bioprobes were used to measure brain glucose and lactate levels in 1-sec samples. Extracellular glucose decreased and lactate increased while rats performed a spatial working memory task. Intrahippocampal infusions of lactate enhanced memory in this task. In addition, pharmacological inhibition of astrocytic glycogenolysis impaired memory and this impairment was reversed by administration of lactate or glucose, both of which can provide lactate to neurons in the absence of glycogenolysis. Pharmacological block of the monocarboxylate transporter responsible for lactate uptake into neurons also impaired memory and this impairment was not reversed by either glucose or lactate. These findings support the view that astrocytes regulate memory formation by controlling the provision of lactate to support neuronal functions.
    • 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)