ABSTRACT
The emergence of SARS-CoV-2 led to pandemic spread of coronavirus disease 2019 (COVID-19), manifesting with respiratory symptoms and multi-organ dysfunction. Detailed characterization of virus-neutralizing antibodies and target epitopes is needed to understand COVID-19 pathophysiology and guide immunization strategies. Among 598 human monoclonal antibodies (mAbs) from 10 COVID-19 patients, we identified 40 strongly neutralizing mAbs. The most potent mAb, CV07-209, neutralized authentic SARS-CoV-2 with an IC50 value of 3.1 ng/mL. Crystal structures of two mAbs in complex with the SARS-CoV-2 receptor-binding domain at 2.55 and 2.70 Å revealed a direct block of ACE2 attachment. Interestingly, some of the near-germline SARS-CoV-2-neutralizing mAbs reacted with mammalian self-antigens. Prophylactic and therapeutic application of CV07-209 protected hamsters from SARS-CoV-2 infection, weight loss, and lung pathology. Our results show that non-self-reactive virus-neutralizing mAbs elicited during SARS-CoV-2 infection are a promising therapeutic strategy.
Subject(s)
Antibodies, Monoclonal/immunology , Antibodies, Viral/immunology , Betacoronavirus/metabolism , Coronavirus Infections/pathology , Pneumonia, Viral/pathology , Angiotensin-Converting Enzyme 2 , Animals , Antibodies, Monoclonal/therapeutic use , Antibodies, Neutralizing/immunology , Antibodies, Viral/therapeutic use , Antigen-Antibody Reactions , Betacoronavirus/immunology , Betacoronavirus/pathogenicity , Binding Sites , COVID-19 , Coronavirus Infections/drug therapy , Coronavirus Infections/virology , Cricetinae , Crystallography, X-Ray , Disease Models, Animal , Humans , Kinetics , Lung/immunology , Lung/metabolism , Lung/pathology , Mice , Mice, Inbred C57BL , Molecular Dynamics Simulation , Pandemics , Peptidyl-Dipeptidase A/chemistry , Peptidyl-Dipeptidase A/metabolism , Pneumonia, Viral/drug therapy , Pneumonia, Viral/virology , Protein Binding , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/immunology , Spike Glycoprotein, Coronavirus/metabolismABSTRACT
The development of neuroprotective and regenerative therapies in the central nervous system (CNS) poses a major challenge in clinical and basic research. In contrast to the peripheral nervous system, which has a comparatively high intrinsic regenerative capacity, this characteristic is poorly developed in the adult CNS. In this review, some basic growth mechanisms of CNS neurons will be highlighted, as well as factors that prevent successful regeneration after injury. Primarily in the context of glaucoma, preclinical and clinical studies are presented which can improve the understanding of neurodegenerative processes in the optical system and thus provide the basis for current and future therapeutic strategies.
Subject(s)
Central Nervous System , Neuroprotection , Glaucoma , Humans , Nerve Regeneration , NeuronsABSTRACT
Clostridium botulinum C3 transferase (C3bot) ADP-ribosylates rho proteins to change cellular functions in a variety of cell types including astrocytes and neurons. The intermediate filament protein vimentin as well as transmembrane integrins are involved in internalization of C3bot into cells. The exact contribution, however, of these proteins to binding of C3bot to the cell surface and subsequent cellular uptake remains to be unraveled. By comparing primary astrocyte cultures derived from wild-type with Vim-/- mice, we demonstrate that astrocytes lacking vimentin exhibited a delayed ADP-ribosylation of rhoA concurrent with a blunted morphological response. This functional impairment was rescued by the extracellular excess of recombinant vimentin. Binding assays using C3bot harboring a mutated integrin-binding RGD motif (C3bot-G89I) revealed the involvement of integrins in astrocyte binding of C3bot. Axonotrophic effects of C3bot are vimentin dependent and postulate an underlying mechanism entertaining a molecular cross-talk between astrocytes and neurons. We present functional evidence for astrocytic release of vimentin by exosomes using an in vitro scratch wound model. Exosomal vimentin+ particles released from wild-type astrocytes promote the interaction of C3bot with neuronal membranes. This effect vanished when culturing Vim-/- astrocytes. Specificity of these findings was confirmed by recombinant vimentin propagating enhanced binding of C3bot to synaptosomes from rat spinal cord and mouse brain. We hypothesize that vimentin+ exosomes released by reactive astrocytes provide a novel molecular mechanism constituting axonotrophic (neuroprotective) and plasticity augmenting effects of C3bot after spinal cord injury.
Subject(s)
ADP Ribose Transferases/pharmacology , Astrocytes/metabolism , Botulinum Toxins/pharmacology , Extracellular Vesicles/physiology , Neurons/metabolism , Vimentin/metabolism , ADP Ribose Transferases/metabolism , Animals , Astrocytes/ultrastructure , Botulinum Toxins/metabolism , Cells, Cultured , Disease Models, Animal , Extracellular Vesicles/ultrastructure , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Microscopy, Immunoelectron , Neurons/drug effects , Neurons/ultrastructure , Protein Binding/drug effects , Protein Binding/genetics , Rats , Rats, Inbred Lew , Spinal Cord/cytology , Spinal Cord Injuries/metabolism , Spinal Cord Injuries/pathology , Subcellular Fractions/drug effects , Subcellular Fractions/metabolism , Subcellular Fractions/ultrastructure , Time Factors , Vimentin/geneticsABSTRACT
The Rho ADP-ribosylating C3 exoenzyme (C3bot) is a bacterial protein toxin devoid of a cell-binding or -translocation domain. Nevertheless, C3 can efficiently enter intact cells, including neurons, but the mechanism of C3 binding and uptake is not yet understood. Previously, we identified the intermediate filament vimentin as an extracellular membranous interaction partner of C3. However, uptake of C3 into cells still occurs (although reduced) in the absence of vimentin, indicating involvement of an additional host cell receptor. C3 harbors an Arg-Gly-Asp (RGD) motif, which is the major integrin-binding site, present in a variety of integrin ligands. To check whether the RGD motif of C3 is involved in binding to cells, we performed a competition assay with C3 and RGD peptide or with a monoclonal antibody binding to ß1-integrin subunit and binding assays in different cell lines, primary neurons, and synaptosomes with C3-RGD mutants. Here, we report that preincubation of cells with the GRGDNP peptide strongly reduced C3 binding to cells. Moreover, mutation of the RGD motif reduced C3 binding to intact cells and also to recombinant vimentin. Anti-integrin antibodies also lowered the C3 binding to cells. Our results indicate that the RGD motif of C3 is at least one essential C3 motif for binding to host cells and that integrin is an additional receptor for C3 besides vimentin.
Subject(s)
ADP Ribose Transferases , Botulinum Toxins , Integrin beta1 , Neurons/metabolism , Oligopeptides , Synaptosomes/metabolism , ADP Ribose Transferases/chemistry , ADP Ribose Transferases/pharmacokinetics , ADP Ribose Transferases/pharmacology , Amino Acid Motifs , Animals , Botulinum Toxins/chemistry , Botulinum Toxins/pharmacokinetics , Botulinum Toxins/pharmacology , Cell Line , Integrin beta1/chemistry , Integrin beta1/genetics , Integrin beta1/metabolism , Mice , Vimentin/chemistry , Vimentin/genetics , Vimentin/metabolismABSTRACT
OBJECTIVE: To study the prevalence of autoantibodies to synapsin in patients with psychiatric and neurological disorders and to describe clinical findings in synapsin antibody positive patients. METHODS: Sera of 375 patients with different psychiatric and neurological disorders and sera of 97 healthy controls were screened (dilution 1:320) for anti-synapsin IgG using HEK293 cells transfected with rat synapsin Ia. Positive sera were further analyzed by immunoblots with brain tissue from wild type and synapsin knock out mice and with HEK293 cells transfected with human synapsin Ia and Ib. Binding of synapsin IgG positive sera to primary neurons was studied using murine hippocampal neurons. RESULTS: IgG in serum from 23 (6.1%) of 375 patients, but from none of the 97 healthy controls (p=0.007), bound to rat synapsin Ia transfected cells with a median (range) titer of 1:1000 (1:320-1:100,000). Twelve of the 23 positive sera reacted with a protein of the molecular size of synapsin I in immunoblots of wild type but not of synapsin knock out mouse brain tissue. Out of 19/23 positive sera available for testing, 13 bound to human synapsin Ia and 16 to human synapsin Ib transfected cells. Synapsin IgG positive sera stained fixed and permeabilized murine hippocampal neurons. Synapsin IgG positive patients had various psychiatric and neurological disorders. Tumors were documented in 2 patients (melanoma, small cell lung carcinoma); concomitant anti-neuronal or other autoantibodies were present in 8 patients. CONCLUSIONS: Autoantibodies to human synapsin Ia and Ib are detectable in a proportion of sera from patients with different psychiatric and neurological disorders, warranting further investigation into the potential pathophysiological relevance of these antibodies.
Subject(s)
Autoantibodies/blood , Mental Disorders/immunology , Nervous System Diseases/immunology , Synapsins/blood , Synapsins/immunology , Adolescent , Adult , Aged , Aged, 80 and over , Animals , Child , Female , HEK293 Cells , Hippocampus/metabolism , Humans , Immunoglobulin G/blood , Male , Mental Disorders/blood , Mental Disorders/epidemiology , Mice , Mice, Inbred C57BL , Mice, Knockout , Middle Aged , Nervous System Diseases/blood , Nervous System Diseases/epidemiology , Neurons/metabolism , Prevalence , Rats , Young AdultABSTRACT
The type III intermediate filament protein vimentin was recently identified to mediate binding and uptake of Clostridium botulinum C3 exoenzyme (C3bot) in two cell lines. Here, we used primary neuronal cultures from vimentin knockout (Vim-/- ) mice to study the impact of vimentin on axonal growth and internalization of C3bot. In contrast to wild type, vimentin knockout neurons were insensitive to C3bot. Application of extracellular vimentin to Vim-/- neurons completely restored the growth-promoting effects of C3bot. In line with this uptake of C3bot into Vim-/- neurons was strongly decreased resulting in reduced ADP-ribosylation of RhoA and B as detected by an antibody recognizing selectively ADP-ribosylated RhoA/B. Again, uptake of C3bot into Vim-/- neurons was rescued by addition of extracellular vimentin. In addition, in purified embryonic stem cell-derived motor neurons that are devoid of glial cells C3bot elicited axonotrophic effects confining neuronal vimentin as a binding partner. Primary neuronal cultures from vimentin knockout (KO) mice were used to study the impact of vimentin on axonal growth and internalization of C3bot. In contrast to wild type, vimentin knockout neurons were insensitive to the axonotrophic effects of C3bot. Application of extracellular vimentin (recombinant vimentin) to vimentin KO neurons completely restored the growth-promoting effects of C3bot. In line with this uptake of C3bot into vimentin KO neurons was strongly decreased resulting in reduced ADP-ribosylation of RhoA and B as detected by an antibody recognizing selectively ADP-ribosylated RhoA/B.
Subject(s)
ADP Ribose Transferases/pharmacology , Axons/drug effects , Botulinum Toxins/pharmacology , Vimentin/metabolism , Adenosine Diphosphate Ribose/metabolism , Animals , Cell Line , Genotype , Mice , Mice, 129 Strain , Mice, Knockout , Motor Neurons/drug effects , Motor Neurons/metabolism , Neural Stem Cells/metabolism , Primary Cell Culture , Vimentin/genetics , rho GTP-Binding Proteins/metabolism , rhoA GTP-Binding Protein , rhoB GTP-Binding Protein/metabolismABSTRACT
Synaptic vesicles (SV) are key organelles of neuronal communication. SV are responsible for the storage of neurotransmitters, which are released by Ca(2+)-dependent exocytosis. After release and interaction with postsynaptic receptors, transmitters rapidly diffuse out of the synaptic cleft and are sequestered by plasma membrane transporters (in some cases following enzymatic conversion). SVs undergo endocytosis and are refilled by specific vesicular transmitter transporters different in the various neuronal subtypes. Besides these differences, SVs in general are equipped with a remarkable common set of proteins. Botulinum neurotoxins (BoNTs) inhibit neurotransmitter release from almost all types of neurons by cleaving proteins required for membrane fusion localized either to SVs (synaptobrevin) or to the plasma membrane (SNAP-25 and syntaxin) depending on the BoNT serotype. To enter the neuronal cytoplasm, BoNTs specifically interact with the luminal domain of SV proteins (synaptotagmin or SV2, depending on serotype) transiently exposed during exocytotic membrane fusion and occurring in almost every neuron. Thus, the highly specific interaction with luminal domains of SV proteins commonly expressed on all SV types is one reason why BoNTs exhibit such a high neuronal specificity but attack almost every neuron type.
Subject(s)
Botulinum Toxins/metabolism , Neurotoxins/metabolism , Synaptic Vesicles/metabolism , Animals , Botulinum Toxins/toxicity , Botulism/microbiology , Botulism/physiopathology , Cell Membrane/metabolism , Clostridium botulinum/pathogenicity , Exocytosis , Membrane Fusion , Membrane Glycoproteins/metabolism , Mice , Nerve Tissue Proteins/metabolism , Neurons/metabolism , Neurons/microbiology , Neurotoxins/toxicity , Protein Transport , Proteolysis , Synaptic Transmission , Synaptosomal-Associated Protein 25/metabolism , Synaptotagmins/metabolismABSTRACT
Autoantibodies against proteins in the brain are increasingly considered as a potential cause of cognitive decline, not only in subacute autoimmune encephalopathies but also in slowly progressing impairment of memory in patients with classical neurodegenerative dementias. In this retrospective cohort study of 161 well-characterized patients with different forms of dementia and 34 controls, we determined the prevalence of immunoglobulin (Ig) G and IgA autoantibodies to brain proteins using unbiased immunofluorescence staining of unfixed murine brain sections. Autoantibodies were detected in 21.1% of dementia patients and in 2.9% of gender-matched controls, with higher frequencies in vascular dementia (42%), Alzheimer's disease (30%), dementia of unknown cause (25%), and subjective cognitive impairment (16.7%). Underlying antigens involved glial fibrillary acidic protein (GFAP), glycine receptor, and Rho GTPase activating protein 26 (ARHGAP26), but also a range of yet undetermined epitopes on neurons, myelinated fiber tracts, choroid plexus, glial cells, and blood vessels. Antibody-positive patients were younger than antibody-negative patients but did not differ in the extent of cognitive impairment, epidemiological and clinical factors, or comorbidities. Further research is needed to understand the potential contribution to disease progression and symptomatology, and to determine the antigenic targets of dementia-associated autoantibodies.
ABSTRACT
OBJECTIVE: To determine the presence and kinetics of antibodies against synaptic proteins in patients with herpes simplex virus encephalitis (HSE). METHODS: Retrospective analysis of 44 patients with polymerase chain reaction-proven HSE for the presence of a large panel of onconeuronal and synaptic receptor antibodies. The effect of patients' serum was studied in cultures of primary mouse hippocampal neurons. RESULTS: N-Methyl-D-aspartate receptor (NMDAR) antibodies of the immunoglobulin (Ig) subtypes IgA, IgG, or IgM were detected in 13 of 44 patients (30%) in the course of HSE, suggesting secondary autoimmune mechanisms. NMDAR antibodies were often present at hospital admission, but in some patients developed after the first week of HSE. Antibody-positive sera resulted in downregulation of synaptic marker proteins in hippocampal neurons. INTERPRETATION: Some patients with HSE develop IgA, IgG, or IgM autoantibodies against NMDAR. Sera from these patients alter the density of neuronal synaptic markers, suggesting a potential pathogenic disease-modifying effect. These findings have implications for the understanding of autoimmunity in infectious diseases, and prospective studies should reveal whether the subgroup of patients with HSE and NMDAR antibodies may benefit from immunotherapy. .
Subject(s)
Antibodies/blood , Antibodies/cerebrospinal fluid , Encephalitis, Herpes Simplex/blood , Encephalitis, Herpes Simplex/cerebrospinal fluid , Receptors, N-Methyl-D-Aspartate/immunology , Adolescent , Adult , Aged , Animals , Antibodies/classification , Cells, Cultured , Child , Embryo, Mammalian , Female , Hippocampus/cytology , Humans , Magnetic Resonance Imaging , Male , Mice , Middle Aged , Neurons/metabolism , Receptors, N-Methyl-D-Aspartate/genetics , Receptors, N-Methyl-D-Aspartate/metabolism , Synapsins/metabolism , Transfection , Young AdultABSTRACT
Objective: To study the prevalence of autoantibodies to glial and neuronal antigens with a focus on glial acidic fibrillary protein (GFAP) in patients with dementia. Methods: Sera of 127 patients with different forms of dementia and sera of 82 age-matched patients with various neurological diseases except for dementia, as well as sera from 15 age-matched healthy controls were analyzed for anti-glial or anti-neuronal IgG using 1) primary murine embryonic hippocampus cell cultures, 2) murine brain sections, 3) immunoblotting on mouse brain homogenates and 4) astrocyte cultures. Sera reacting with astrocytes in hippocampus cell cultures were further analyzed using HEK293 cells transfected with human GFAP. Results: IgG in serum from 45 of 127 (35.5%) patients with dementia but only 8 of 97 (8.2%, p ≤ 0.001) controls bound to either glial or neuronal structures in cultured murine hippocampus cells. In these cultures antibodies to astrocytes were detected in 35 of 127 (27.5%) of the dementia patients, whereas in controls antibodies to astrocytes were detected in 4 sera only (4.1%, p ≤ 0.001). Among the sera exhibiting reactivity to astrocytes, 14 of 35 (40%) showed immunoreaction to HEK293 cells transfected with GFAP in dementia patients, representing 11% of all sera. Within the 4 immunoreactive control sera reacting with astrocytes one reacted with GFAP (1.0% of total immunoreactivity, p = 0.003). Conclusions: Autoantibodies to glial epitopes in general and to GFAP in particular are more frequent in patients with dementia than in age-matched controls without dementia, thus indicating the need for further investigations regarding the potential pathophysiological relevance of these antibodies.
ABSTRACT
Introduction: The antibody repertoire from CSF-derived antibody-secreting cells and memory B-cells in patients with encephalitis contains a considerable number of antibodies that do not target the disease-defining autoantigen such as the GABA or NMDA receptors. This study focuses on the functional relevance of autoantibodies to brain blood vessels in patients with GABAA and NMDA receptor encephalitis. Methods: We tested 149 human monoclonal IgG antibodies from the cerebrospinal fluid of six patients with different forms of autoimmune encephalitis on murine brain sections for reactivity to blood vessels using immunohistochemistry. Positive candidates were tested for reactivity with purified brain blood vessels, effects on transendothelial electrical resistance (TEER), and expression of tight junction proteins as well as gene regulation using human brain microvascular endothelial hCMEC/D3 cells as in vitro blood-brain barrier model. One blood-vessel reactive antibody was infused intrathecally by pump injection in mice to study in vivo binding and effects on tight junction proteins such as Occludin. Target protein identification was addressed using transfected HEK293 cells. Results: Six antibodies reacted with brain blood vessels, three were from the same patient with GABAAR encephalitis, and the other three were from different patients with NMDAR encephalitis. One antibody from an NMDAR encephalitis patient, mAb 011-138, also reacted with cerebellar Purkinje cells. In this case, treatment of hCMEC/D3 cells resulted in decreased TEER, reduced Occludin expression, and mRNA levels. Functional relevance in vivo was confirmed as Occludin downregulation was observed in mAb 011-138-infused animals. Unconventional Myosin-X was identified as a novel autoimmune target for this antibody. Discussion: We conclude that autoantibodies to blood vessels occur in autoimmune encephalitis patients and might contribute to a disruption of the blood-brain barrier thereby suggesting a potential pathophysiological relevance of these antibodies.
ABSTRACT
Maternal autoantibodies can be transmitted diaplacentally, with potentially deleterious effects on neurodevelopment. Synapsin 1 (SYN1) is a neuronal protein that is important for synaptic communication and neuronal plasticity. While monoallelic loss of function (LoF) variants in the SYN1 gene result in X-linked intellectual disability (ID), learning disabilities, epilepsy, behavioral problems, and macrocephaly, the effect of SYN1 autoantibodies on neurodevelopment remains unclear. We recruited a clinical cohort of 208 mothers and their children with neurologic abnormalities and analyzed the role of maternal SYN1 autoantibodies. We identified seropositivity in 9.6% of mothers, and seropositivity was associated with an increased risk for ID and behavioral problems. Furthermore, children more frequently had epilepsy, macrocephaly, and developmental delay, in line with the SYN1 LoF phenotype. Whether SYN1 autoantibodies have a direct pathogenic effect on neurodevelopment or serve as biomarkers requires functional experiments.
Subject(s)
Autoantibodies , Epilepsy , Intellectual Disability , Humans , Neurons/metabolism , Phenotype , Synapsins/genetics , Synapsins/metabolismABSTRACT
Anti-neuronal autoantibodies can be transplacentally transferred during pregnancy and may cause detrimental effects on fetal development. It is unclear whether autoantibodies against synapsin-I, one of the most abundant synaptic proteins, are associated with developmental abnormalities in humans. We recruited a cohort of 263 pregnant women and detected serum synapsin-I IgG autoantibodies in 13.3% using cell-based assays. Seropositivity was strongly associated with abnormalities of fetal development including structural defects, intrauterine growth retardation, amniotic fluid disorders and neuropsychiatric developmental diseases in previous children (odds ratios of 3-6.5). Autoantibodies reached the fetal circulation and were mainly of IgG1/IgG3 subclasses. They bound to conformational and linear synapsin-I epitopes, five distinct epitopes were identified using peptide microarrays. The findings indicate that synapsin-I autoantibodies may be clinically useful biomarkers or even directly participate in the disease process of neurodevelopmental disorders, thus being potentially amenable to antibody-targeting interventional strategies in the future.
ABSTRACT
C3 ADP-ribosyltransferase is a valuable tool to study Rho-dependent cellular processes. In the current study we investigated the impact of enzyme-deficient peptides derived from Clostridium botulinum C3 transferase in the context of neuronal process elongation and branching, synaptic connectivity, and putative beneficial effects on functional outcome following traumatic injury to the CNS. By screening a range of peptidic fragments, we identified three short peptides from C3bot that promoted axon and dendrite outgrowth in cultivated hippocampal neurons. Furthermore, one of these fragments, a 26-amino acid peptide covering the residues 156-181 enhanced synaptic connectivity in primary hippocampal culture. This peptide was also effective to foster axon outgrowth and re-innervation in organotypical brain slice culture. To evaluate the potential of the 26mer to foster repair mechanisms after CNS injury we applied this peptide to mice subjected to spinal cord injury by either compression impact or hemisection. A single local administration at the site of the lesion improved locomotor recovery. In addition, histological analysis revealed an increased serotonergic input to lumbar motoneurons in treated compared with control mice. Pull-down assays showed that lesion-induced up-regulation of RhoA activity within the spinal cord was largely blocked by C3bot peptides despite the lack of enzymatic activity.
Subject(s)
ADP Ribose Transferases/chemistry , ADP Ribose Transferases/pharmacology , Botulinum Toxins/chemistry , Botulinum Toxins/pharmacology , Neurons/cytology , Spinal Cord Injuries/drug therapy , Spinal Cord Regeneration/drug effects , Synapses/drug effects , Animals , Animals, Newborn , Cells, Cultured , Disease Models, Animal , Embryo, Mammalian , GABAergic Neurons/drug effects , GABAergic Neurons/physiology , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Hippocampus/cytology , Locomotion/drug effects , Mice , Mice, Inbred BALB C , Organ Culture Techniques , Peptides/pharmacology , Peptides/therapeutic use , Serotonergic Neurons/drug effects , Serotonergic Neurons/physiology , Serotonin/metabolism , Spinal Cord Injuries/etiology , Spinal Cord Injuries/pathology , Time Factors , Transfection , Versicans/metabolism , Vesicular Inhibitory Amino Acid Transport Proteins/metabolism , rho GTP-Binding Proteins/metabolismABSTRACT
Functional recovery and regeneration of corticospinal tract (CST) fibers following spinal cord injury by compression or dorsal hemisection in mice was monitored after application of the enzyme-deficient Clostridium botulinum C3-protein-derived 29-amino-acid fragment C3bot(154-182). This peptide significantly improved locomotor restoration in both injury models as assessed by the open-field Basso Mouse Scale for locomotion test and Rotarod treadmill experiments. These data were supported by tracing studies showing an enhanced regenerative growth of CST fibers in treated animals as visualized by anterograde tracing. Additionally, C3bot(154-182) stimulated regenerative growth of raphespinal fibers and improved serotonergic input to lumbar alpha-motoneurons. These in vivo data were confirmed by in vitro data, showing an enhanced axon outgrowth of alpha-motoneurons and hippocampal neurons cultivated on normal or growth-inhibitory substrates after application of C3bot(154-182). The observed effects were probably caused by a non-enzymatic downregulation of active RhoA by the C3 peptide as indicated by pull-down experiments. By contrast, C3bot(154-182) did not induce neurite outgrowth in primary cultures of dorsal root ganglion cells. In conclusion, C3bot(154-182) represents a novel, promising tool to foster axonal protection and/or repair, as well as functional recovery after traumatic CNS injury.
Subject(s)
ADP Ribose Transferases/pharmacology , Botulinum Toxins/pharmacology , Clostridium botulinum/metabolism , Motor Neurons/drug effects , Nerve Regeneration , Peptide Fragments/pharmacology , Spinal Cord Injuries/physiopathology , Spinal Cord/drug effects , Animals , Cell Growth Processes/drug effects , Cells, Cultured , Humans , Mice , Mice, Inbred BALB C , Motor Activity/drug effects , Motor Neurons/metabolism , Motor Neurons/pathology , Pyramidal Tracts/drug effects , Pyramidal Tracts/physiology , Recovery of Function , Serotonin/genetics , Serotonin/metabolism , Spinal Cord/metabolism , Spinal Cord/pathology , Spinal Cord/surgery , Spinal Cord Injuries/drug therapy , rho GTP-Binding Proteins/genetics , rho GTP-Binding Proteins/metabolism , rhoA GTP-Binding ProteinABSTRACT
In primary murine hippocampal neurons we investigated the regulation of EAAT3-mediated glutamate transport by the Clostridium botulinum C3 transferase C3bot and a 26mer peptide derived from full length protein. Incubation with either enzyme-competent C3bot or enzyme-deficient C3bot156-181 peptide resulted in the upregulation of glutamate uptake by up to 22% compared to untreated cells. A similar enhancement of glutamate transport was also achieved by the classical phorbol-ester-mediated activation of protein kinase C subtypes. Yet comparable, effects elicited by C3 preparations seemed not to rely on PKCα, γ, ε, or ζ activation. Blocking of tyrosine phosphorylation by tyrosine kinase inhibitors prevented the observed effect mediated by C3bot and C3bot 26mer. By using biochemical and molecular biological assays we could rule out that the observed C3bot and C3bot 26mer-mediated effects solely resulted from enhanced transporter expression or translocation to the neuronal surface but was rather mediated by transporter phosphorylation at tyrosine residues that was found to be significantly enhanced following incubation with either full length protein or the 26mer C3 peptide.
ABSTRACT
Large-conductance Ca(2+) -activated (BK) potassium channels are centrally involved in neurovascular coupling, immunity, and neural transmission. The ability to be synergistically activated by membrane depolarization, different ligands and intracellular Ca(2+) links intracellular signaling and membrane excitability. The diverse physiological functions of BK channels crucially depend on regulatory ß subunits. Although first studies characterized the neuronal distribution of BKß subunits in the rodent brain, it is largely unknown which ß subunit proteins are expressed in astrocytes and thus mediate these regulatory effects. We therefore analyzed the expression of BKß subunits in rat and mouse brain and glial cell cultures. A monospecific polyclonal antibody against the BKß4 channel subunit was raised, affinity-purified and extensively characterized. BKß4 and to a lesser degree BKß1 transcripts and protein were detected in several astrocytic populations and cultured cells. Particularly strong BKß4 immunostaining was detected in astrocytic progenitors derived from the subventricular zone. The overlapping expression of BKα and BKß4 in astrocytes implies a functional relationship and suggests that BKß4 is an important accessory ß subunit for astrocytic BK channels. In addition, BKß4 might exert effects independent of the α subunit as functional heterologous co-expression of Nav1.6 and BKß4 resulted in reduced Nav1.6 sodium currents. Thus, BKß4 expression in astrocytes likely participates in regulating astrocytic voltage gradients and maintaining K(+) homeostasis, hence enabling astrocytes to fulfill their complex regulatory influence on proper brain function.
Subject(s)
Astrocytes/metabolism , Brain/metabolism , Large-Conductance Calcium-Activated Potassium Channels/physiology , Animals , Astrocytes/cytology , Brain/cytology , CHO Cells , Cells, Cultured , Cricetinae , Cricetulus , HEK293 Cells , Homeostasis/genetics , Humans , Large-Conductance Calcium-Activated Potassium Channels/chemistry , Large-Conductance Calcium-Activated Potassium Channels/genetics , Mice , Mice, Inbred C57BL , Oocytes , Rats , Rats, Wistar , Xenopus laevisABSTRACT
Small GTPases of the Rho family play versatile roles in the formation and development of axons and dendrites, effects often studied by the Rho-inactivating C3 transferase (C3bot) from Clostridium botulinum. Recently, we reported that transferase-deficient C3bot also exerted axonotrophic activity. Using overlapping peptides from the C3bot sequence, we identified a small peptide of 29 amino acids (covering residues 154-182) from the C-terminal region of C3bot that promotes both axonal and dendritic growth, as well as branching of hippocampal neurons, at submicromolar concentrations. Several C3bot constructs, including the short peptide, enhanced the number of axonal segments from mid- to higher-order segments. C3bot(154-182) also increased the number of synaptophysin-expressing terminals, up-regulated various synaptic proteins, and functionally increased the glutamate uptake. Staining against the vesicular glutamate and GABA transporters further revealed that the effect was attributable to a higher number of glutamatergic and GABAergic inputs on proximal dendrites of enhanced green fluorescent protein (EGFP)-transfected neurons. Using organotypical slice cultures, we also detected trophic effects of C3bot(154-182) on length and density of outgrowing fibers from the entorhinal cortex that were comparable to the effects elicited by full-length C3bot. In addition, an enhanced reinnervation was observed in a hippocampal-entorhinal lesion model. In summary, the neurotrophic effect of C3bot is executed by a C-terminal peptide fragment covering aa 154-182 of C3; it triggers dendritic and axonal growth and branching as well as increased synaptic connectivity. In contrast to full-length C3, this C3 peptide selectively acts on neurons but not on glial cells.
Subject(s)
ADP Ribose Transferases/metabolism , Amino Acids/pharmacology , Axons/physiology , Botulinum Toxins/metabolism , Dendrites/physiology , Neurons/physiology , ADP Ribose Transferases/chemistry , ADP Ribose Transferases/genetics , Amino Acids/chemistry , Animals , Axons/drug effects , Axons/metabolism , Biomarkers/metabolism , Botulinum Toxins/chemistry , Botulinum Toxins/genetics , Cells, Cultured , Clostridium botulinum/genetics , Clostridium botulinum/metabolism , Coculture Techniques , Dendrites/drug effects , Dendrites/genetics , Dendrites/metabolism , Embryo, Mammalian , Glutathione Transferase/metabolism , Green Fluorescent Proteins/metabolism , Hippocampus/cytology , Hippocampus/embryology , Immunohistochemistry , Mice , Mice, Inbred Strains , Microtubule-Associated Proteins/metabolism , Molecular Weight , Neurofilament Proteins/chemistry , Neurofilament Proteins/metabolism , Neurons/drug effects , Neurons/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Time FactorsABSTRACT
The current study investigates the neurotrophic effects of Clostridium botulinum C3 transferase (C3bot) on highly purified, glia-free, GABAergic, and glutamatergic neurons. Incubation with nanomolar concentrations of C3bot promotes dendrite formation as well as dendritic and axonal outgrowth in rat GABAergic neurons. A comparison of C3bot effects on sorted mouse GABAergic and glutamatergic neurons obtained from newly established NexCre;Ai9xVGAT Venus mice revealed a higher sensitivity of GABAergic cells to axonotrophic and dendritic effects of C3bot in terms of process length and branch formation. Protein biochemical analysis of known C3bot binding partners revealed comparable amounts of ß1 integrin in both cell types but a higher expression of vimentin in GABAergic neurons. Accordingly, binding of C3bot to GABAergic neurons was stronger than binding to glutamatergic neurons. A combinatory treatment of glutamatergic neurons with C3bot and vimentin raised the amount of bound C3bot to levels comparable to the ones in GABAergic neurons, thereby confirming the specificity of effects. Overall, different surface vimentin levels between GABAergic and glutamatergic neurons exist that mediate neurotrophic C3bot effects.
ABSTRACT
An amendment to this paper has been published and can be accessed via a link at the top of the paper.