scholarly journals Distinctive binding properties of human monoclonal LGI1 autoantibodies determine pathogenic mechanisms

Brain ◽  
2020 ◽  
Vol 143 (6) ◽  
pp. 1731-1745 ◽  
Author(s):  
Melanie Ramberger ◽  
Antonio Berretta ◽  
Jeanne M M Tan ◽  
Bo Sun ◽  
Sophia Michael ◽  
...  
Keyword(s):  
Hippocampal Neurons ◽  
Molecular Mechanisms ◽  
Long Term Potentiation ◽  
Serum Samples ◽  
Pathogenic Potential ◽  
Binding Characteristics ◽  
Domain Specific ◽  
Rodent Brain ◽  
Specific Memory

Abstract Autoantibodies against leucine-rich glioma inactivated 1 (LGI1) are found in patients with limbic encephalitis and focal seizures. Here, we generate patient-derived monoclonal antibodies (mAbs) against LGI1. We explore their sequences and binding characteristics, plus their pathogenic potential using transfected HEK293T cells, rodent neuronal preparations, and behavioural and electrophysiological assessments in vivo after mAb injections into the rodent hippocampus. In live cell-based assays, LGI1 epitope recognition was examined with patient sera (n = 31), CSFs (n = 11), longitudinal serum samples (n = 15), and using mAbs (n = 14) generated from peripheral B cells of two patients. All sera and 9/11 CSFs bound both the leucine-rich repeat (LRR) and the epitempin repeat (EPTP) domains of LGI1, with stable ratios of LRR:EPTP antibody levels over time. By contrast, the mAbs derived from both patients recognized either the LRR or EPTP domain. mAbs against both domain specificities showed varied binding strengths, and marked genetic heterogeneity, with high mutation frequencies. LRR-specific mAbs recognized LGI1 docked to its interaction partners, ADAM22 and ADAM23, bound to rodent brain sections, and induced internalization of the LGI1-ADAM22/23 complex in both HEK293T cells and live hippocampal neurons. By contrast, few EPTP-specific mAbs bound to rodent brain sections or ADAM22/23-docked LGI1, but all inhibited the docking of LGI1 to ADAM22/23. After intrahippocampal injection, and by contrast to the LRR-directed mAbs, the EPTP-directed mAbs showed far less avid binding to brain tissue and were consistently detected in the serum. Post-injection, both domain-specific mAbs abrogated long-term potentiation induction, and LRR-directed antibodies with higher binding strengths induced memory impairment. Taken together, two largely dichotomous populations of LGI1 mAbs with distinct domain binding characteristics exist in the affinity matured peripheral autoantigen-specific memory pools of individuals, both of which have pathogenic potential. In human autoantibody-mediated diseases, the detailed characterization of patient mAbs provides a valuable method to dissect the molecular mechanisms within polyclonal populations.

scholarly journals Neuronal hibernation following hippocampal demyelination

2021 ◽  
Vol 9 (1) ◽  
Author(s):  
Selva Baltan ◽  
Safdar S. Jawaid ◽  
Anthony M. Chomyk ◽  
Grahame J. Kidd ◽  
Jacqueline Chen ◽  
...  
Keyword(s):  
Dendritic Spines ◽  
Hippocampal Neurons ◽  
Molecular Mechanisms ◽  
Three Dimensional ◽  
Long Term Potentiation ◽  
Synaptic Function ◽  
Mammalian Brain ◽  
Gene Transcript ◽  
Ca1 Neurons

AbstractCognitive dysfunction occurs in greater than 50% of individuals with multiple sclerosis (MS). Hippocampal demyelination is a prominent feature of postmortem MS brains and hippocampal atrophy correlates with cognitive decline in MS patients. Cellular and molecular mechanisms responsible for neuronal dysfunction in demyelinated hippocampi are not fully understood. Here we investigate a mouse model of hippocampal demyelination where twelve weeks of treatment with the oligodendrocyte toxin, cuprizone, demyelinates over 90% of the hippocampus and causes decreased memory/learning. Long-term potentiation (LTP) of hippocampal CA1 pyramidal neurons is considered to be a major cellular readout of learning and memory in the mammalian brain. In acute slices, we establish that hippocampal demyelination abolishes LTP and excitatory post-synaptic potentials of CA1 neurons, while pre-synaptic function of Schaeffer collateral fibers is preserved. Demyelination also reduced Ca2+-mediated firing of hippocampal neurons in vivo. Using three-dimensional electron microscopy, we investigated the number, shape (mushroom, stubby, thin), and post-synaptic densities (PSDs) of dendritic spines that facilitate LTP. Hippocampal demyelination did not alter the number of dendritic spines. Surprisingly, dendritic spines appeared to be more mature in demyelinated hippocampi, with a significant increase in mushroom-shaped spines, more perforated PSDs, and more astrocyte participation in the tripartite synapse. RNA sequencing experiments identified 400 altered transcripts in demyelinated hippocampi. Gene transcripts that regulate myelination, synaptic signaling, astrocyte function, and innate immunity were altered in demyelinated hippocampi. Hippocampal remyelination rescued synaptic transmission, LTP, and the majority of gene transcript changes. We establish that CA1 neurons projecting demyelinated axons silence their dendritic spines and hibernate in a state that may protect the demyelinated axon and facilitates functional recovery following remyelination.

scholarly journals Autophagy is Activated In Vivo during Trimethyltin-Induced Apoptotic Neurodegeneration: A Study in the Rat Hippocampus

2019 ◽  
Vol 21 (1) ◽  
pp. 175 ◽  
Author(s):  
Sabrina Ceccariglia ◽  
Alessandra Alvino ◽  
Aurora Del Fà ◽  
Ornella Parolini ◽  
Fabrizio Michetti ◽  
...  
Keyword(s):  
Western Blotting ◽  
Hippocampal Neurons ◽  
Molecular Mechanisms ◽  
Nuclear Translocation ◽  
Neuronal Degeneration ◽  
Neuronal Loss ◽  
Organotin Compound ◽  
Nuclear Antigen ◽  
Apoptotic Pathway

Trimethyltin (TMT) is an organotin compound known to produce significant and selective neuronal degeneration and reactive astrogliosis in the rodent central nervous system. Autophagy is the main cellular mechanism for degrading and recycling protein aggregates and damaged organelles, which in different stress conditions, such as starvation, generally improves cell survival. Autophagy is documented in several pathologic conditions, including neurodegenerative diseases. This study aimed to investigate the autophagy and apoptosis signaling pathways in hippocampal neurons of TMT-treated (Wistar) rats to explore molecular mechanisms involved in toxicant-induced neuronal injury. The microtubule-associated protein light chain (LC3, autophagosome marker) and sequestosome1 (SQSTM1/p62) (substrate of autophagy-mediated degradation) expressions were examined by Western blotting at different time points after intoxication. The results demonstrate that the LC3 II/I ratio significantly increased at 3 and 5 days, and that p62 levels significantly decreased at 7 and 14 days. Immunofluorescence images of LC3/neuronal nuclear antigen (NeuN) showed numerous strongly positive LC3 neurons throughout the hippocampus at 3 and 5 days. The terminal deoxynucleotidyltransferase dUTP nick end labeling (TUNEL) assay indicated an increase in apoptotic cells starting from 5 days after treatment. In order to clarify apoptotic pathway, immunofluorescence images of apoptosis-inducing factor (AIF)/NeuN did not show nuclear translocation of AIF in neurons. Increased expression of cleaved Caspase-3 was revealed at 5–14 days in all hippocampal regions by Western blotting and immunohistochemistry analyses. These data clearly demonstrate that TMT intoxication induces a marked increase in both autophagy and caspase-dependent apoptosis, and that autophagy occurring just before apoptosis could have a potential role in neuronal loss in this experimental model of neurodegeneration.

Glucocorticoid acts on a putative G protein-coupled receptor to rapidly regulate the activity of NMDA receptors in hippocampal neurons

2012 ◽  
Vol 302 (7) ◽  
pp. E747-E758 ◽  
Author(s):  
Yanmin Zhang ◽  
Hui Sheng ◽  
Jinshun Qi ◽  
Bei Ma ◽  
Jihu Sun ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Nmda Receptors ◽  
G Protein ◽  
Hippocampal Neurons ◽  
Long Term Potentiation ◽  
G Protein Coupled Receptor ◽  
Protein Activity ◽  
Ca1 Region ◽  
G Protein Coupled

Glucocorticoids (GCs) have been demonstrated to act through both genomic and nongenomic mechanisms. The present study demonstrated that corticosterone rapidly suppressed the activity of N-methyl-d-aspartate (NMDA) receptors in cultured hippocampal neurons. The effect was maintained with corticosterone conjugated to bovine serum albumin and blocked by inhibition of G protein activity with intracellular GDP-β-S application. Corticosterone increased GTP-bound Gs protein and cyclic AMP (cAMP) production, activated phospholipase Cβ3 (PLC-β3), and induced inositol-1,4,5-triphosphate (IP3) production. Blocking PLC and the downstream cascades with PLC inhibitor, IP3 receptor antagonist, Ca2+ chelator, and protein kinase C (PKC) inhibitors prevented the actions of corticosterone. Blocking adenylate cyclase (AC) and protein kinase A (PKA) caused a decrease in NMDA-evoked currents. Application of corticosterone partly reversed the inhibition of NMDA currents caused by blockage of AC and PKA. Intracerebroventricular administration of corticosterone significantly suppressed long-term potentiation (LTP) in the CA1 region of the hippocampus within 30 min in vivo, implicating the possibly physiological significance of rapid effects of GC on NMDA receptors. Taken together, our results indicate that GCs act on a putative G protein-coupled receptor to activate multiple signaling pathways in hippocampal neurons, and the rapid suppression of NMDA activity by GCs is dependent on PLC and downstream signaling.

scholarly journals c-Jun NH2-terminal Kinase (JNK)-interacting Protein-3 (JIP3) Regulates Neuronal Axon Elongation in a Kinesin- and JNK-dependent Manner

2013 ◽  
Vol 288 (20) ◽  
pp. 14531-14543 ◽  
Author(s):  
Tao Sun ◽  
Nuo Yu ◽  
Lu-Kai Zhai ◽  
Na Li ◽  
Chao Zhang ◽  
...  
Keyword(s):  
Cortical Neurons ◽  
Hippocampal Neurons ◽  
Molecular Mechanisms ◽  
Neuronal Development ◽  
Dependent Manner ◽  
Loss Of Function ◽  
Anterograde Transport ◽  
Interacting Protein ◽  
Axon Elongation

The development of neuronal polarity is essential for the establishment of the accurate patterning of neuronal circuits in the brain. However, little is known about the underlying molecular mechanisms that control rapid axon elongation during neuronal development. Here, we report that c-Jun NH2-terminal kinase (JNK)-interacting protein-3 (JIP3) is highly expressed at axon tips during the critical period for axon development. Using gain- and loss-of-function approaches, immunofluorescence analysis, and in utero electroporation, we find that JIP3 can enhance axon elongation in primary hippocampal neurons and cortical neurons in vivo. We further demonstrate that JIP3 promotes axon elongation in a kinesin- and JNK-dependent manner using several deletion mutants of JIP3. Next, we demonstrate that the successful transportation of JIP3 to axon tips by kinesin is a prerequisite for enhancing JNK phosphorylation in this area and therefore promotes axon elongation, constituting a novel mechanism for coupling JIP3 anterograde transport with JNK signaling at the distal axons and axon elongation. Finally, our immunofluorescence data suggest that the activation of JNK at axon tips facilitates axon elongation by modulating cofilin activity and actin filament dynamics. These findings may have important implications for our understanding of neuronal axon elongation during development.

scholarly journals EphrinB2 regulates VEGFR2 during dendritogenesis and hippocampal circuitry development

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Eva Harde ◽  
LaShae Nicholson ◽  
Beatriz Furones Cuadrado ◽  
Diane Bissen ◽  
Sylvia Wigge ◽  
...  
Keyword(s):  
Hippocampal Neurons ◽  
Long Term Potentiation ◽  
Dendritic Arborization ◽  
Angiogenic Factor ◽  
Vascular Endothelial ◽  
Term Potentiation ◽  
Endothelial Growth Factor ◽  
Spine Morphogenesis ◽  
Hippocampal Circuitry

Vascular endothelial growth factor (VEGF) is an angiogenic factor that play important roles in the nervous system, although it is still unclear which receptors transduce those signals in neurons. Here, we show that in the developing hippocampus VEGFR2 (also known as KDR or FLK1) is expressed specifically in the CA3 region and it is required for dendritic arborization and spine morphogenesis in hippocampal neurons. Mice lacking VEGFR2 in neurons (Nes-cre Kdrlox/-) show decreased dendritic arbors and spines as well as a reduction in long-term potentiation (LTP) at the associational-commissural – CA3 synapses. Mechanistically, VEGFR2 internalization is required for VEGF-induced spine maturation. In analogy to endothelial cells, ephrinB2 controls VEGFR2 internalization in neurons. VEGFR2-ephrinB2 compound mice (Nes-cre Kdrlox/+ Efnb2lox/+) show reduced dendritic branching, reduced spine head size and impaired LTP. Our results demonstrate the functional crosstalk of VEGFR2 and ephrinB2 in vivo to control dendritic arborization, spine morphogenesis and hippocampal circuitry development.

Abundant GFP Expression and LTP in Hippocampal Acute Slices by In Vivo Injection of Sindbis Virus

2001 ◽  
Vol 86 (2) ◽  
pp. 1037-1042 ◽  
Author(s):  
Massimo D'Apuzzo ◽  
Georgia Mandolesi ◽  
Gerald Reis ◽  
Erin M. Schuman
Keyword(s):  
Hippocampal Neurons ◽  
Sindbis Virus ◽  
Fluorescent Protein ◽  
Hippocampal Slices ◽  
Pyramidal Cells ◽  
Long Term Potentiation ◽  
Neuronal Structure ◽  
Adult Rats ◽  
Acute Hippocampal Slices

Virus-mediated gene transfer into neurons is a powerful tool for the analysis of neuronal structure and function. Recombinant sindbis virus has been previously used to study protein function in hippocampal neuron cultures as well as in hippocampal organotypic slice cultures. Nevertheless, some concern still exists about the physiological relevance of these cultured preparations. Acute hippocampal slices are a widely used preparation for the study of synaptic transmission, but currently recombinant gene delivery is usually achieved only through time-consuming transgenic techniques. In this study, we show that a subregion of the CA1 area in acute hippocampal slices can be specifically altered to express a gene of interest. A sindbis virus vector carrying an enhanced green fluorescent protein (EGFP) reporter was injected in vivo into the hippocampus of adult rats. After 18 h, rats were killed, and acute hippocampal slices, infected in the CA1 field, were analyzed morphologically and electrophysiologically. Infected slices showed healthy and stable electrophysiological responses as well as long-term potentiation. In addition, infected pyramidal cells were readily recognized in living slices by two-photon imaging. Specifically, the introduction of an EGFP-Actin fusion protein greatly enhanced the detection of fine processes and dendritic spines. We propose this technique as an efficient tool for studying gene function in adult hippocampal neurons.

scholarly journals Shisa7 phosphorylation regulates GABAergic transmission and neurodevelopmental behaviors

2021 ◽  
Author(s):  
Kunwei Wu ◽  
Ryan David Shepard ◽  
David Castellano ◽  
Qingjun Tian ◽  
Lijin Dong ◽  
...  
Keyword(s):  
Neurodevelopmental Disorders ◽  
Hippocampal Neurons ◽  
Molecular Mechanisms ◽  
Critical Role ◽  
Phosphorylation Site ◽  
Long Term Potentiation ◽  
Surface Expression ◽  
Tonic Inhibition ◽  
Gaba A

GABA-A receptors (GABAARs) are crucial for development and regulation of the central nervous system. Altered GABAergic signaling is hypothesized to be involved in the pathophysiology of neurodevelopmental disorders. Nevertheless, how aberrant cellular and molecular mechanisms affect GABAARs in these diseases remain elusive. Recently, we identified Shisa7 as a GABAAR auxiliary subunit that modulates GABAAR trafficking, kinetics, and pharmacology, and discovered a phosphorylation site in Shisa7 (S405) critical for extrasynaptic a5-GABAAR trafficking and tonic inhibition. However, the role of S405 phosphorylation in the regulation of synaptic inhibition, plasticity, and behavior remains unknown. Here, we found that expression of a phospho-null mutant (Shisa7 S405A) in heterologous cells and neurons diminishes a2-GABAAR trafficking. Subsequently, we generate a Shisa7 S405A knock-in (KI) mouse line that displays reduced surface expression of GABAARs in hippocampal neurons. Importantly, both synaptic and tonic inhibition are decreased in KI mice. Moreover, chemically induced inhibitory long-term potentiation is impaired, highlighting a critical role of Shisa7 S405 in GABAergic plasticity. Lastly, KI mice exhibit enhanced locomotor activity and grooming associated with neurodevelopmental disorders. Collectively, our study reveals a phosphorylation site critical for Shisa7-dependent trafficking of synaptic and extrasynaptic GABAARs which contributes to behavioral endophenotypes displayed in neurodevelopmental disorders.

The cellular and molecular basis of in vivo synaptic plasticity in rodents

2020 ◽  
Vol 318 (6) ◽  
pp. C1264-C1283
Author(s):  
Juliette E. Cheyne ◽  
Johanna M. Montgomery
Keyword(s):  
Synaptic Plasticity ◽  
Molecular Mechanisms ◽  
Long Term Potentiation ◽  
Synaptic Strength ◽  
Network Activity ◽  
In Vivo Studies ◽  
Sensory Stimuli ◽  
External Stimulation ◽  
The Brain

Plasticity within the neuronal networks of the brain underlies the ability to learn and retain new information. The initial discovery of synaptic plasticity occurred by measuring synaptic strength in vivo, applying external stimulation and observing an increase in synaptic strength termed long-term potentiation (LTP). Many of the molecular pathways involved in LTP and other forms of synaptic plasticity were subsequently uncovered in vitro. Over the last few decades, technological advances in recording and imaging in live animals have seen many of these molecular mechanisms confirmed in vivo, including structural changes both pre- and postsynaptically, changes in synaptic strength, and changes in neuronal excitability. A well-studied aspect of neuronal plasticity is the capacity of the brain to adapt to its environment, gained by comparing the brains of deprived and experienced animals in vivo, and in direct response to sensory stimuli. Multiple in vivo studies have also strongly linked plastic changes to memory by interfering with the expression of plasticity and by manipulating memory engrams. Plasticity in vivo also occurs in the absence of any form of external stimulation, i.e., during spontaneous network activity occurring with brain development. However, there is still much to learn about how plasticity is induced during natural learning and how this is altered in neurological disorders.

scholarly journals 2-Pentadecyl-2-oxazoline ameliorates memory impairment and depression-like behaviour in neuropathic mice: possible role of adrenergic alpha2- and H3 histamine autoreceptors

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Serena Boccella ◽  
Francesca Guida ◽  
Monica Iannotta ◽  
Fabio Arturo Iannotti ◽  
Rosmara Infantino ◽  
...  
Keyword(s):  
Neuropathic Pain ◽  
Nerve Injury ◽  
Molecular Mechanisms ◽  
Day Treatment ◽  
Long Term Potentiation ◽  
Dopamine Level ◽  
Term Potentiation ◽  
Report Data ◽  
Target Molecules

AbstractNeuropathic pain (NP) remains an untreatable disease due to the complex pathophysiology that involves the whole pain neuraxis including the forebrain. Sensory dysfunctions such as allodynia and hyperalgesia are only part of the symptoms associated with neuropathic pain that extend to memory and affectivity deficits. The development of multi-target molecules might be a promising therapeutic strategy against the symptoms associated with NP. 2-pentadecyl-2-oxazoline (PEA-OXA) is a plant-derived agent, which has shown effectiveness against chronic pain and associated neuropsychiatric disorders. The molecular mechanisms by which PEA-OXA exerts its effects are, however, only partially known. In the current study, we show that PEA-OXA, besides being an alpha2 adrenergic receptor antagonist, also acts as a modulator at histamine H3 receptors, and report data on its effects on sensory, affective and cognitive symptoms associated with the spared nerve injury (SNI) model of neuropathic pain in mice. Treatment for 14 days with PEA-OXA after the onset of the symptoms associated with neuropathic pain resulted in the following effects: (i) allodynia was decreased; (ii) affective/cognitive impairment associated with SNI (depression, spatial, and working memories) was counteracted; (iii) long-term potentiation in vivo in the lateral entorhinal cortex-dentate gyrus (perforant pathway, LPP) was ameliorated, (iv) hippocampal glutamate, GABA, histamine, norepinephrine and dopamine level alterations after peripheral nerve injury were reversed, (v) expression level of the TH positive neurons in the Locus Coeruleus were normalized. Thus, a 16-day treatment with PEA-OXA alleviates the sensory, emotional, cognitive, electrophysiological and neurochemical alterations associated with SNI-induced neuropathic pain.

scholarly journals Phosphorylation of CRMP2 by Cdk5 Regulates Dendritic Spine Development of Cortical Neuron in the Mouse Hippocampus

2016 ◽  
Vol 2016 ◽  
pp. 1-7 ◽  
Author(s):  
Xiaohua Jin ◽  
Kodai Sasamoto ◽  
Jun Nagai ◽  
Yuki Yamazaki ◽  
Kenta Saito ◽  
...  
Keyword(s):  
Mouse Brain ◽  
Dendritic Spines ◽  
Dendritic Spine ◽  
Cortical Neurons ◽  
Hippocampal Neurons ◽  
Molecular Mechanisms ◽  
Brain Functions ◽  
Spine Development ◽  
Mouse Hippocampus

Proper density and morphology of dendritic spines are important for higher brain functions such as learning and memory. However, our knowledge about molecular mechanisms that regulate the　development and maintenance of dendritic spines is limited. We recently reported that cyclin-dependent kinase 5 (Cdk5) is required for the development and maintenance of dendritic spines of cortical neurons in the mouse brain. Previousin vitrostudies have suggested the involvement of Cdk5 substrates in the formation of dendritic spines; however, their role in spine development has not been testedin vivo. Here, we demonstrate that Cdk5 phosphorylates collapsin response mediator protein 2 (CRMP2) in the dendritic spines of cultured hippocampal neurons andin vivoin the mouse brain. When we eliminated CRMP2 phosphorylation inCRMP2KI/KImice, the densities of dendritic spines significantly decreased in hippocampal CA1 pyramidal neurons in the mouse brain. These results indicate that phosphorylation of CRMP2 by Cdk5 is important for dendritic spine development in cortical neurons in the mouse hippocampus.

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