scholarly journals CRISPR/Cas9 mediated intersectional knockout of GSK3β in D2 receptor expressing mPFC neurons reveals contributions to emotional regulation

2019 ◽  
Author(s):  
Jivan Khlghatyan ◽  
Jean-Martin Beaulieu
Keyword(s):  
Emotional Regulation ◽  
Large Scale ◽  
Dopamine D2 Receptor ◽  
Glycogen Synthase ◽  
D2 Receptor ◽  
Dependent Manner ◽  
Targeted Deletion ◽  
Conventional Animal ◽  
Cortical Regions ◽  
The Brain

AbstractBackgroundGlycogen synthase kinase 3β (GSK3β) regulates neurodevelopment, synaptic plasticity as well as mood, cognition, social interaction, and depressive-like behaviors. Inhibition of GSK3β is a shared consequence of treatment by lithium, SSRIs, ketamine and antipsychotics. GSK3β activity is regulated by dopamine D2 receptor signaling and can be inhibited by psychoactive drugs in a D2 receptor dependent manner. Functions of GSK3β in striatal D2 neurons has been studied extensively. However, GSK3β is ubiquitously expressed in the brain and D2 receptor expressing cells are distributed as a mosaic in multiple cortical regions. This complicates the interrogation of GSK3β functions in cortical D2 cells in a circuit defined manner using conventional animal models.MethodsWe have used a CRISPR/Cas9 mediated intersectional approach to achieve targeted deletion of GSK3β in D2 expressing neurons of the adult medial prefrontal cortex (mPFC).ResultsIsolation and analysis of ribosome associated RNA specifically from mPFC D2 neurons lacking GSK3β demonstrated large scale translatome alterations. Deletion of GSK3β in mPFC D2 neurons revealed its contribution to anxiety-related, cognitive, and social behaviors.ConclusionsOur results underscore the viability of intersectional knockout approach to study functions of a ubiquitous gene in a network defined fashion while uncovering a contribution of GSK3β expressed in mPFC D2 neurons in the regulation of behavioral dimensions related to mood and emotions. This advances our understanding of GSK3β action at a brain circuit level and can potentially lead to the development of circuit selective therapeutics.

scholarly journals Proteomic analysis identifies the E3 ubiquitin ligase Pdzrn3 as a regulatory target of Wnt5a-Ror signaling

2021 ◽  
Vol 118 (25) ◽  
pp. e2104944118
Author(s):  
Sara E. Konopelski Snavely ◽  
Michael W. Susman ◽  
Ryan C. Kunz ◽  
Jia Tan ◽  
Srisathya Srinivasan ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
Large Scale ◽  
Glycogen Synthase ◽  
E3 Ubiquitin Ligase ◽  
Ubiquitin Proteasome System ◽  
Dependent Manner ◽  
Casein Kinase 1 ◽  
Downstream Signaling ◽  
Proteomic Screen ◽  
Responsive Element

Wnt5a-Ror signaling is a conserved pathway that regulates morphogenetic processes during vertebrate development [R. T. Moon et al., Development 119, 97–111 (1993); I. Oishi et al., Genes Cells 8, 645–654 (2003)], but its downstream signaling events remain poorly understood. Through a large-scale proteomic screen in mouse embryonic fibroblasts, we identified the E3 ubiquitin ligase Pdzrn3 as a regulatory target of the Wnt5a-Ror pathway. Upon pathway activation, Pdzrn3 is degraded in a β-catenin–independent, ubiquitin-proteasome system–dependent manner. We developed a flow cytometry-based reporter to monitor Pdzrn3 abundance and delineated a signaling cascade involving Frizzled, Dishevelled, Casein kinase 1, and Glycogen synthase kinase 3 that regulates Pdzrn3 stability. Epistatically, Pdzrn3 is regulated independently of Kif26b, another Wnt5a-Ror effector. Wnt5a-dependent degradation of Pdzrn3 requires phosphorylation of three conserved amino acids within its C-terminal LNX3H domain [M. Flynn, O. Saha, P. Young, BMC Evol. Biol. 11, 235 (2011)], which acts as a bona fide Wnt5a-responsive element. Importantly, this phospho-dependent degradation is essential for Wnt5a-Ror modulation of cell migration. Collectively, this work establishes a Wnt5a-Ror cell morphogenetic cascade involving Pdzrn3 phosphorylation and degradation.

scholarly journals Scale-Free Coupled Dynamics in Brain Networks Captured by Bivariate Focus-Based Multifractal Analysis

2021 ◽  
Vol 11 ◽  
Author(s):  
Orestis Stylianou ◽  
Frigyes Samuel Racz ◽  
Andras Eke ◽  
Peter Mukli
Keyword(s):  
Resting State ◽  
Functional Coupling ◽  
Dependent Manner ◽  
Coupled Dynamics ◽  
Scale Free ◽  
Neural Processes ◽  
Eeg Recordings ◽  
Cortical Regions ◽  
The Brain

While most connectivity studies investigate functional connectivity (FC) in a scale-dependent manner, coupled neural processes may also exhibit broadband dynamics, manifesting as power-law scaling of their measures of interdependence. Here we introduce the bivariate focus-based multifractal (BFMF) analysis as a robust tool for capturing such scale-free relations and use resting-state electroencephalography (EEG) recordings of 12 subjects to demonstrate its performance in reconstructing physiological networks. BFMF was employed to characterize broadband FC between 62 cortical regions in a pairwise manner, with all investigated connections being tested for true bivariate multifractality. EEG channels were also grouped to represent the activity of six resting-state networks (RSNs) in the brain, thus allowing for the analysis of within- and between- RSNs connectivity, separately. Most connections featured true bivariate multifractality, which could be attributed to the genuine scale-free coupling of neural dynamics. Bivariate multifractality showed a characteristic topology over the cortex that was highly concordant among subjects. Long-term autocorrelation was higher in within-RSNs, while the degree of multifractality was generally found stronger in between-RSNs connections. These results offer statistical evidence of the bivariate multifractal nature of functional coupling in the brain and validate BFMF as a robust method to capture such scale-independent coupled dynamics.

scholarly journals Presence of a D2 receptor truncated protein in the brain of the D2 receptor functional knockout mouse, revisiting its molecular and neurochemical features

2019 ◽  
Author(s):  
Natalia Sánchez ◽  
Montserrat Olivares-Costa ◽  
Marcela P González ◽  
Angélica P Escobar ◽  
Rodrigo Meza ◽  
...  
Keyword(s):  
Knockout Mice ◽  
Knockout Mouse ◽  
Transmembrane Domain ◽  
Dopamine D2 Receptor ◽  
D2 Receptor ◽  
Wild Type ◽  
Intracellular Loop ◽  
Protein Protein Interaction ◽  
Acute Injection ◽  
The Brain

AbstractNull mice for the dopamine D2 receptor (D2R) have been instrumental in understanding the function of this protein in the central nervous system. Several lines of D2R knockout mice have been generated, which share some characteristics but differ in others. The D2R functional knockout mouse, first described in 1997, is functionally null for D2R-mediated signaling but the Drd2 gene was interrupted at the most extreme distal end leaving open the question about whether transcript and protein are produced. We decided to determine if there are D2R transcripts, the characteristics of these transcripts and whether they are translated in the brain of D2R functional knockout mice. Sequence analysis of 3’ Rapid Amplification of cDNA Ends showed that D2R functional knockout mice express transcripts that lack only the exon eight. Immunofluorescence showed D2R-like protein in the brain of the knockout mice. As previously reported, D2R functional knockout mice are hypoactive and insensitive to the D2R agonist quinpirole (QNP). However, the heterozygous showed locomotor activity and response to QNP similar to the wild-type mice. Intriguingly, microdialysis experiments showed that heterozygous mice, such as knockouts, have half the normal levels of synaptic dopamine in the striatum. However, heterozygous mice responded similarly to wild-type mice to an acute injection of QNP, showing a 50% decrease in synaptic dopamine. In conclusion, D2R functional knockout mice express transcripts that lead to a truncated D2R protein that lacks from the sixth transmembrane domain to the C-terminal end but retains the third intracellular loop. We discuss the implications of this truncated D2R coexisting with the native D2R that may explain the unexpected outcomes observed in the heterozygous. Finally, we suggest that the D2R functional knockout mouse can be a useful model for studying protein-protein interaction and trafficking of D2R.

Dopamine D2 receptor activator quinpirole protects against trypsinogen activation during acute pancreatitis via upregulating HSP70

2020 ◽  
Vol 318 (6) ◽  
pp. G1000-G1012
Author(s):  
Xin Ye ◽  
Xiao Han ◽  
Bin Li ◽  
Juanjuan Dai ◽  
Zengkai Wu ◽  
...  
Keyword(s):  
Acute Pancreatitis ◽  
Dopamine D2 Receptor ◽  
D2 Receptor ◽  
Pancreatic Injury ◽  
Dependent Manner ◽  
Trypsinogen Activation ◽  
Mechanistic Insight ◽  
Receptor Activator

The current study demonstrated that activation of DRD2 by quinpirole protects against trypsinogen activation in the in vitro and in vivo setting of acute pancreatitis by upregulating HSP70 and restoring lysosomal degradation via a PP2A-dependent manner, therefore leading to reduced pancreatic injury. These findings provide a new mechanistic insight on the protective effect of DRD2 activation in acute pancreatitis.

scholarly journals Knockout or Knock-in? A Truncated D2 Receptor Protein Is Expressed in the Brain of Functional D2 Receptor Knockout Mice

NeuroSci ◽  
2021 ◽  
Vol 2 (2) ◽  
pp. 193-206
Author(s):  
Natalia Sánchez ◽  
Montserrat Olivares-Costa ◽  
Marcela P González ◽  
Roberto Munita ◽  
Angélica P Escobar ◽  
...  
Keyword(s):  
Golgi Apparatus ◽  
Mouse Strain ◽  
Knockout Mice ◽  
Biochemical Characterization ◽  
Transmembrane Domain ◽  
Dopamine D2 Receptor ◽  
Intracellular Localization ◽  
D2 Receptor ◽  
Receptor Protein ◽  
The Brain

Null mice for the dopamine D2 receptor (D2R) have been instrumental in understanding the function of this protein. For our research, we obtained the functional D2R knockout mouse strain described initially in 1997. Surprisingly, our biochemical characterization showed that this mouse strain is not a true knockout. We determined by sequence analysis of the rapid 3′ amplification of cDNA ends that functional D2R knockout mice express transcripts that lack only the eighth exon. Furthermore, immunofluorescence assays showed a D2R-like protein in the brain of functional D2R knockout mice. We verified by immunofluorescence that the recombinant truncated D2R is expressed in HEK293T cells, showing intracellular localization, colocalizing in the Golgi apparatus and the endoplasmic reticulum, but with less presence in the Golgi apparatus compared to the native D2R. As previously reported, functional D2R knockout mice are hypoactive and insensitive to the D2R agonist quinpirole. Concordantly, microdialysis studies confirmed that functional D2R knockout mice have lower extracellular dopamine levels in the striatum than the native mice. In conclusion, functional D2R knockout mice express transcripts that lead to a truncated D2R protein lacking from the sixth transmembrane domain to the C-terminus. We share these findings to avoid future confusion and the community considers this mouse strain in D2R traffic and protein–protein interaction studies.

scholarly journals Obesity Is Associated with Decreased  -Opioid But Unaltered Dopamine D2 Receptor Availability in the Brain

2015 ◽  
Vol 35 (9) ◽  
pp. 3959-3965 ◽  
Author(s):  
H. K. Karlsson ◽  
L. Tuominen ◽  
J. J. Tuulari ◽  
J. Hirvonen ◽  
R. Parkkola ◽  
...  
Keyword(s):  
Dopamine D2 Receptor ◽  
D2 Receptor ◽  
Dopamine D2 ◽  
The Brain

scholarly journals The Brain Cytoplasmic RNA BC1 Regulates Dopamine D2 Receptor-Mediated Transmission in the Striatum

2007 ◽  
Vol 27 (33) ◽  
pp. 8885-8892 ◽  
Author(s):  
D. Centonze ◽  
S. Rossi ◽  
I. Napoli ◽  
V. Mercaldo ◽  
C. Lacoux ◽  
...  
Keyword(s):  
Dopamine D2 Receptor ◽  
D2 Receptor ◽  
Dopamine D2 ◽  
Cytoplasmic Rna ◽  
The Brain

scholarly journals Presynaptic vesicular accumulation is required for antipsychotic efficacy in psychotic-like rats

2020 ◽  
Vol 35 (1) ◽  
pp. 65-77
Author(s):  
Taygun C Uzuneser ◽  
Eva-Maria Weiss ◽  
Jana Dahlmanns ◽  
Liubov S Kalinichenko ◽  
Davide Amato ◽  
...  
Keyword(s):  
Hippocampal Neurons ◽  
Time Course ◽  
Dopamine D2 Receptor ◽  
D2 Receptor ◽  
Feedback Mechanism ◽  
Therapeutic Effects ◽  
Dependent Manner ◽  
Therapeutic Action ◽  
Dopamine D2 ◽  
New Strategy

Background: The therapeutic effects of antipsychotic drugs (APDs) are mainly attributed to their postsynaptic inhibitory functions on the dopamine D2 receptor, which, however, cannot explain the delayed onset of full therapeutic efficacy. It was previously shown that APDs accumulate in presynaptic vesicles during chronic treatment and are released like neurotransmitters in an activity-dependent manner triggering an auto-inhibitory feedback mechanism. Although closely mirroring therapeutic action onset, the functional consequence of the APD accumulation process remained unclear. Aims: Here we tested whether the accumulation of the APD haloperidol (HAL) is required for full therapeutic action in psychotic-like rats. Methods: We designed a HAL analog compound (HAL-F), which lacks the accumulation property of HAL, but retains its postsynaptic inhibitory action on dopamine D2 receptors. Results/outcomes: By perfusing LysoTracker fluorophore-stained cultured hippocampal neurons, we confirmed the accumulation of HAL and the non-accumulation of HAL-F. In an amphetamine hypersensitization psychosis-like model in rats, we found that subchronic intracerebroventricularly delivered HAL (0.1 mg/kg/day), but not HAL-F (0.3–1.5 mg/kg/day), attenuates psychotic-like behavior in rats. Conclusions/interpretation: These findings suggest the presynaptic accumulation of HAL may serve as an essential prerequisite for its full antipsychotic action and may explain the time course of APD action. Targeting accumulation properties of APDs may, thus, become a new strategy to improve APD action.

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