scholarly journals Effects of Trace Metal Profiles Characteristic for Autism on Synapses in Cultured Neurons

2015 ◽  
Vol 2015 ◽  
pp. 1-16 ◽  
Author(s):  
Simone Hagmeyer ◽  
Katharina Mangus ◽  
Tobias M. Boeckers ◽  
Andreas M. Grabrucker
Keyword(s):  
Hippocampal Neurons ◽  
Metal Ion ◽  
Synapse Formation ◽  
Causative Factor ◽  
Autism Spectrum ◽  
Zn Deficiency ◽  
Protein Levels ◽  
Synapse Density ◽  
Profile Characteristic

Various recent studies revealed that biometal dyshomeostasis plays a crucial role in the pathogenesis of neurological disorders such as autism spectrum disorders (ASD). Substantial evidence indicates that disrupted neuronal homeostasis of different metal ions such as Fe, Cu, Pb, Hg, Se, and Zn may mediate synaptic dysfunction and impair synapse formation and maturation. Here, we performedin vitrostudies investigating the consequences of an imbalance of transition metals on glutamatergic synapses of hippocampal neurons. We analyzed whether an imbalance of any one metal ion alters cell health and synapse numbers. Moreover, we evaluated whether a biometal profile characteristic for ASD patients influences synapse formation, maturation, and composition regarding NMDA receptor subunits and Shank proteins. Our results show that an ASD like biometal profile leads to a reduction of NMDAR (NR/Grin/GluN) subunit 1 and 2a, as well as Shank gene expression along with a reduction of synapse density. Additionally, synaptic protein levels of GluN2a and Shanks are reduced. Although Zn supplementation is able to rescue the aforementioned alterations, Zn deficiency is not solely responsible as causative factor. Thus, we conclude that balancing Zn levels in ASD might be a prime target to normalize synaptic alterations caused by biometal dyshomeostasis.

scholarly journals BDNF mobilizes synaptic vesicles and enhances synapse formation by disrupting cadherin–β-catenin interactions

2006 ◽  
Vol 174 (2) ◽  
pp. 289-299 ◽  
Author(s):  
Shernaz X. Bamji ◽  
Beatriz Rico ◽  
Nikole Kimes ◽  
Louis F. Reichardt
Keyword(s):  
Synaptic Vesicle ◽  
Synaptic Vesicles ◽  
Hippocampal Neurons ◽  
Molecular Mechanisms ◽  
Synapse Formation ◽  
Time Lapse ◽  
Synapse Density ◽  
Vesicle Mobility ◽  
Small Clusters ◽  
Vertebrate Central Nervous System

Neurons of the vertebrate central nervous system have the capacity to modify synapse number, morphology, and efficacy in response to activity. Some of these functions can be attributed to activity-induced synthesis and secretion of the neurotrophin brain-derived neurotrophic factor (BDNF); however, the molecular mechanisms by which BDNF mediates these events are still not well understood. Using time-lapse confocal analysis, we show that BDNF mobilizes synaptic vesicles at existing synapses, resulting in small clusters of synaptic vesicles “splitting” away from synaptic sites. We demonstrate that BDNF's ability to mobilize synaptic vesicle clusters depends on the dissociation of cadherin–β-catenin adhesion complexes that occurs after tyrosine phosphorylation of β-catenin. Artificially maintaining cadherin–β-catenin complexes in the presence of BDNF abolishes the BDNF-mediated enhancement of synaptic vesicle mobility, as well as the longer-term BDNF-mediated increase in synapse number. Together, this data demonstrates that the disruption of cadherin–β-catenin complexes is an important molecular event through which BDNF increases synapse density in cultured hippocampal neurons.

FKBP51 Modulates Hippocampal Size and Function in Post-translational Regulation of Parkin

2021 ◽  
Author(s):  
Bin Qiu ◽  
Zhaohui Zhong ◽  
Shawn Righter ◽  
Yuxue Xu ◽  
Jun Wang ◽  
...  
Keyword(s):  
Hippocampal Neurons ◽  
Translational Regulation ◽  
Disease Onset ◽  
Fold Increase ◽  
Knock Out ◽  
Fk506 Binding Protein ◽  
Protein Levels ◽  
And Function

Abstract FK506-binding protein 51 (encoded by Fkpb51) has been associated with stress-related mental illness. To identify its function, we studied the morphological consequences of Fkbp51 deletion. Artificial Intelligence-assist morphological analysis identified that Fkbp51 knock-out (KO) mice possess more elongated CA and DG but shorter in height in coronal section when compared to WT. Primary cultured Fkbp51 KO hippocampal neurons were shown to exhibit larger dendritic outgrowth than wild-type (WT) controls, pharmacological manipulation experiments suggest that this may occur through regulation of microtubule-associated protein. Both in vitro primary culture and in vivo labeling support that FKBP51 regulates microtubule-associated protein expression. Furthermore, in the absence of differences in mRNA expression, Fkbp51 KO hippocampus exhibited decreases in βIII-tubulin, MAP2, and Tau protein levels, but a greater than 2.5-fold increase in Parkin protein. Overexpression and knock-down FKBP51 demonstrated that FKBP51 negatively regulates Parkin in a dose-dependent and ubiquitin-mediated manner. These results indicate a potential novel post-translational regulatory of Parkin by FKBP51 and significance of their interaction on disease onset.

scholarly journals Deletion of the Nucleotide Exchange Factor Vav3 Enhances Axonal Complexity and Synapse Formation but Tampers Activity of Hippocampal Neuronal Networks In Vitro

2020 ◽  
Vol 21 (3) ◽  
pp. 856
Author(s):  
David Wegrzyn ◽  
Christine Wegrzyn ◽  
Kerry Tedford ◽  
Klaus-Dieter Fischer ◽  
Andreas Faissner
Keyword(s):  
Hippocampal Neurons ◽  
Synapse Formation ◽  
Synergistic Effects ◽  
Neuronal Morphology ◽  
Network Activity ◽  
Nucleotide Exchange ◽  
Double Knockout ◽  
Nucleotide Exchange Factor ◽  
Key Events

Vav proteins activate GTPases of the RhoA subfamily that regulate the cytoskeleton and are involved in adhesion, migration, differentiation, polarity and the cell cycle. While the importance of RhoA GTPases for neuronal morphology is undisputed, their regulation is less well understood. In this perspective, we studied the consequences of the deletion of Vav2, Vav3 and Vav2 and 3 (Vav2−/−, Vav3−/−, Vav2−/−/3−/−) for the development of embryonic hippocampal neurons in vitro. Using an indirect co-culture system of hippocampal neurons with primary wild-type (WT) cortical astrocytes, we analysed axonal and dendritic parameters, structural synapse numbers and the spontaneous network activity via immunocytochemistry and multielectrode array analysis (MEA). Here, we observed a higher process complexity in Vav3−/−, but not in Vav2−/− neurons after three and five days in vitro (DIV). Furthermore, an enhanced synapse formation was observed in Vav3−/− after 14 days in culture. Remarkably, Vav2−/−/3−/− double knockout neurons did not display synergistic effects. Interestingly, these differences were transient and compensated after a cultivation period of 21 days. Network analysis revealed a diminished number of spontaneously occurring action potentials in Vav3−/− neurons after 21 DIV. Based on these results, it appears that Vav3 participates in key events of neuronal differentiation.

scholarly journals RTP801/REDD1 contributes to neuroinflammation severity and memory impairments in Alzheimer’s disease

2021 ◽  
Vol 12 (6) ◽  
Author(s):  
Leticia Pérez-Sisqués ◽  
Anna Sancho-Balsells ◽  
Júlia Solana-Balaguer ◽  
Genís Campoy-Campos ◽  
Marcel Vives-Isern ◽  
...  
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Hippocampal Neurons ◽  
Mrna Levels ◽  
Spine Density ◽  
Memory Impairments ◽  
Protein Levels ◽  
Synaptic Markers ◽  
5Xfad Mice

AbstractRTP801/REDD1 is a stress-regulated protein whose upregulation is necessary and sufficient to trigger neuronal death. Its downregulation in Parkinson’s and Huntington’s disease models ameliorates the pathological phenotypes. In the context of Alzheimer’s disease (AD), the coding gene for RTP801, DDIT4, is responsive to Aβ and modulates its cytotoxicity in vitro. Also, RTP801 mRNA levels are increased in AD patients’ lymphocytes. However, the involvement of RTP801 in the pathophysiology of AD has not been yet tested. Here, we demonstrate that RTP801 levels are increased in postmortem hippocampal samples from AD patients. Interestingly, RTP801 protein levels correlated with both Braak and Thal stages of the disease and with GFAP expression. RTP801 levels are also upregulated in hippocampal synaptosomal fractions obtained from murine 5xFAD and rTg4510 mice models of the disease. A local RTP801 knockdown in the 5xFAD hippocampal neurons with shRNA-containing AAV particles ameliorates cognitive deficits in 7-month-old animals. Upon RTP801 silencing in the 5xFAD mice, no major changes were detected in hippocampal synaptic markers or spine density. Importantly, we found an unanticipated recovery of several gliosis hallmarks and inflammasome key proteins upon neuronal RTP801 downregulation in the 5xFAD mice. Altogether our results suggest that RTP801 could be a potential future target for theranostic studies since it could be a biomarker of neuroinflammation and neurotoxicity severity of the disease and, at the same time, a promising therapeutic target in the treatment of AD.

scholarly journals Superfluous Role of Mammalian Septins 3 and 5 in Neuronal Development and Synaptic Transmission

2008 ◽  
Vol 28 (23) ◽  
pp. 7012-7029 ◽  
Author(s):  
Christopher W. Tsang ◽  
Michael Fedchyshyn ◽  
John Harrison ◽  
Hong Xie ◽  
Jing Xue ◽  
...  
Keyword(s):  
Synaptic Transmission ◽  
Synaptic Vesicle ◽  
Hippocampal Neurons ◽  
Neuronal Development ◽  
Synapse Formation ◽  
Vesicle Traffic ◽  
Axon Development ◽  
Central Synapses ◽  
Synaptic Vesicle Fusion

ABSTRACT The septin family of GTPases, first identified for their roles in cell division, are also expressed in postmitotic tissues. SEPT3 (G-septin) and SEPT5 (CDCrel-1) are highly expressed in neurons, enriched in presynaptic terminals, and associated with synaptic vesicles. These characteristics suggest that SEPT3 or SEPT5 might be important for synapse formation, maturation, or synaptic vesicle traffic. Since Sept5 −/− mice do not show any overt neurological phenotypes, we generated Sept3 −/− and Sept3 −/− Sept5 −/− mice and found that SEPT3 and SEPT5 are not essential for development, fertility, or viability. Changes in the expression of septins were noted in the absence of SEPT3, SEPT5, and both septins. SEPT5 association with other septins in brain tissue was unaffected by the removal of SEPT3. No abnormalities were observed in the gross morphology and synapses of the hippocampus. Similarly, axon development and synapse formation were unaffected in vitro. In cultured hippocampal neurons, the size of the recycling synaptic vesicle pool was unaltered in the absence of SEPT3. Furthermore, synaptic transmission at two different central synapses was not significantly affected in Sept3 −/− Sept5 −/− mice. These results indicate that SEPT3 and SEPT5 are dispensable for neuronal development as well as for synaptic vesicle fusion and recycling.

scholarly journals Absence of Thyroid Hormone Induced Delayed Dendritic Arborization in Mouse Primary Hippocampal Neurons Through Insufficient Expression of Brain-Derived Neurotrophic Factor

2021 ◽  
Vol 12 ◽  
Author(s):  
Hiroyuki Yajima ◽  
Izuki Amano ◽  
Sumiyasu Ishii ◽  
Tetsushi Sadakata ◽  
Wataru Miyazaki ◽  
...  
Keyword(s):  
Thyroid Hormone ◽  
Neurotrophic Factor ◽  
Hippocampal Neurons ◽  
Brain Derived Neurotrophic Factor ◽  
Mrna Levels ◽  
Protein Levels ◽  
Primary Hippocampal Neurons ◽  
Developmental Impairment

Thyroid hormone (TH) plays important roles in the developing brain. TH deficiency in early life leads to severe developmental impairment in the hippocampus. However, the mechanisms of TH action in the developing hippocampus are still largely unknown. In this study, we generated 3,5,3’-tri-iodo-l-thyronine (T3)-free neuronal supplement, based on the composition of neuronal supplement 21 (NS21), to examine the effect of TH in the developing hippocampus using primary cultured neurons. Effects of TH on neurons were compared between cultures in this T3-free culture medium (-T3 group) and a medium in which T3 was added (+T3 group). Morphometric analysis and RT-qPCR were performed on 7, 10, and 14 days in vitro (DIV). On 10 DIV, a decreased dendrite arborization in -T3 group was observed. Such difference was not observed on 7 and 14 DIV. Brain-derived neurotrophic factor (Bdnf) mRNA levels also decreased significantly in -T3 group on 10 DIV. We then confirmed protein levels of phosphorylated neurotrophic tyrosine kinase type 2 (NTRK2, TRKB), which is a receptor for BDNF, on 10 DIV by immunocytochemistry and Western blot analysis. Phosphorylated NTRK2 levels significantly decreased in -T3 group compared to +T3 group on 10 DIV. Considering the role of BDNF on neurodevelopment, we examined its involvement by adding BDNF on 8 and 9 DIV. Addition of 10 ng/ml BDNF recovered the suppressed dendrite arborization induced by T3 deficiency on 10 DIV. We show that the lack of TH induces a developmental delay in primary hippocampal neurons, likely caused through a decreased Bdnf expression. Thus, BDNF may play a role in TH-regulated dendritogenesis.

scholarly journals The Interaction of TRAF6 With Neuroplastin Promotes Spinogenesis During Early Neuronal Development

2020 ◽  
Vol 8 ◽  
Author(s):  
Sampath Kumar Vemula ◽  
Ayse Malci ◽  
Lennart Junge ◽  
Anne-Christin Lehmann ◽  
Ramya Rama ◽  
...  
Keyword(s):  
Neuronal Development ◽  
Synapse Formation ◽  
Tumor Necrosis Factor Receptor ◽  
Physical Interaction ◽  
Binding Assays ◽  
C Terminus ◽  
Synapse Density ◽  
Pharmacological Blockade ◽  
Necrosis Factor

Correct brain wiring depends on reliable synapse formation. Nevertheless, signaling codes promoting synaptogenesis are not fully understood. Here, we report a spinogenic mechanism that operates during neuronal development and is based on the interaction of tumor necrosis factor receptor-associated factor 6 (TRAF6) with the synaptic cell adhesion molecule neuroplastin. The interaction between these proteins was predicted in silico and verified by co-immunoprecipitation in extracts from rat brain and co-transfected HEK cells. Binding assays show physical interaction between neuroplastin’s C-terminus and the TRAF-C domain of TRAF6 with a Kd value of 88 μM. As the two proteins co-localize in primordial dendritic protrusions, we used young cultures of rat and mouse as well as neuroplastin-deficient mouse neurons and showed with mutagenesis, knock-down, and pharmacological blockade that TRAF6 is required by neuroplastin to promote early spinogenesis during in vitro days 6-9, but not later. Time-framed TRAF6 blockade during days 6–9 reduced mEPSC amplitude, number of postsynaptic sites, synapse density and neuronal activity as neurons mature. Our data unravel a new molecular liaison that may emerge during a specific window of the neuronal development to determine excitatory synapse density in the rodent brain.

scholarly journals Transient oxytocin signaling primes the development and function of excitatory hippocampal neurons

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Silvia Ripamonti ◽  
Mateusz C Ambrozkiewicz ◽  
Francesca Guzzi ◽  
Marta Gravati ◽  
Gerardo Biella ◽  
...  
Keyword(s):  
Hippocampal Neurons ◽  
Pyramidal Neurons ◽  
Hippocampal Pyramidal Neurons ◽  
Excitatory Transmission ◽  
Oxytocin Receptors ◽  
Synapse Density ◽  
Protein Components ◽  
And Function

Beyond its role in parturition and lactation, oxytocin influences higher brain processes that control social behavior of mammals, and perturbed oxytocin signaling has been linked to the pathogenesis of several psychiatric disorders. However, it is still largely unknown how oxytocin exactly regulates neuronal function. We show that early, transient oxytocin exposure in vitro inhibits the development of hippocampal glutamatergic neurons, leading to reduced dendrite complexity, synapse density, and excitatory transmission, while sparing GABAergic neurons. Conversely, genetic elimination of oxytocin receptors increases the expression of protein components of excitatory synapses and excitatory synaptic transmission in vitro. In vivo, oxytocin-receptor-deficient hippocampal pyramidal neurons develop more complex dendrites, which leads to increased spine number and reduced γ-oscillations. These results indicate that oxytocin controls the development of hippocampal excitatory neurons and contributes to the maintenance of a physiological excitation/inhibition balance, whose disruption can cause neurobehavioral disturbances.

Synaptic and Extrasynaptic NMDA Receptor NR2 Subunits in Cultured Hippocampal Neurons

2006 ◽  
Vol 95 (3) ◽  
pp. 1727-1734 ◽  
Author(s):  
Christopher G. Thomas ◽  
Ashleigh J. Miller ◽  
Gary L. Westbrook
Keyword(s):  
Nmda Receptors ◽  
Hippocampal Neurons ◽  
Synapse Formation ◽  
Whole Cell ◽  
Extrasynaptic Receptors ◽  
C Terminus ◽  
Specific Antagonist ◽  
Nr2 Subunits ◽  
Cultured Hippocampal Neurons

Early in development, neurons only express NR1/NR2B-containing N-methyl-d-aspartate (NMDA) receptors. Later, NR2A subunits are upregulated during a period of rapid synapse formation. This pattern is often interpreted to indicate that NR2A-containing receptors are synaptic and that NR2B-containing receptors are extrasynaptic. We re-examined this issue using whole cell recordings in cultured hippocampal neurons. As expected, the inhibition of whole cell currents by the NR2B-specific antagonist, ifenprodil, progressively decreased from 69.5 ± 2.4% [6 days in vitro (DIV)] to 54.9 ± 2.6% (8 DIV), before reaching a plateau in the second week (42.5 ± 2%, 12–19 DIV). In NR2A−/− neurons, which express only NR1/NR2B-containing NMDA receptors, autaptic excitatory postsynaptic currents (EPSCs; ≥12 DIV) were more sensitive to ifenprodil and decayed more slowly than EPSCs in wild-type neurons. Thus NR2B-containing receptors were not excluded from synapses. We blocked synaptic NMDA receptors with MK-801 during evoked transmitter release, thus allowing us to isolate extrasynaptic receptors. Ifenprodil inhibition of this extrasynaptic population was highly variable in different neurons. Furthermore, extrasynaptic receptors in autaptic cultures were only partially blocked by ifenprodil, indicating that NR2A-containing receptors are not exclusively confined to the synapse. Extrasynaptic NR2A-containing receptors were also detected in NR2A−/− neurons transfected with full-length NR2A. Truncation of the NR2A C terminus did not eliminate synaptic expression of NR2A-containing receptors. Our results indicate that NR2A- and NR2B-containing receptors can be located in either synaptic or extrasynaptic compartments.

scholarly journals Systematic quantification of synapses in primary neuronal culture

2020 ◽  
Author(s):  
Peter Verstraelen ◽  
Gerardo Garcia ◽  
Marlies Verschuuren ◽  
Bob Asselbergh ◽  
Rony Nuydens ◽  
...  
Keyword(s):  
Synapse Formation ◽  
Dynamic Range ◽  
Cortical Cell ◽  
Neuronal Culture ◽  
Maturity Stage ◽  
Primary Neuronal Culture ◽  
Synaptic Markers ◽  
Synapse Density ◽  
Cortical Cell Cultures

AbstractA vast set of neurological disorders is associated with impaired synaptic connectivity. Therefore, modulation of synapse formation could have therapeutic relevance. However, the high density and small size of synapses make their quantification a challenging task. To improve the reliability of synapse-oriented drug screens, we evaluated a panel of synapse-targeting antibodies for their labeling specificity on hippocampal and cortical cell cultures using quantitative immunofluorescence microscopy. For those antibodies that passed multiparametric validation, we assessed pairwise colocalization, an often-used readout for established synapses. We found that even when two pan-synaptic markers were used, the overlap was incomplete, and the presence of spurious signals limited the dynamic range. To circumvent this problem, we implemented a proximity ligation-based approach, that only leads to a signal when two pre- and postsynaptic markers are sufficiently close. We demonstrate that this approach can be applied to different synaptic marker combinations and can be successfully used for quantification of synapse density in cultures of different maturity stage in healthy or pathological conditions. Thus, the unbiased analysis of synapse labeling and exploitation of resident protein proximity, allows increasing the sensitivity of synapse quantifications in neuronal culture and therefore represents a valuable extension of the analytical toolset for in vitro synapse screens.

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