Akt and AMPK activators rescue hyperexcitability in neurons from patients with bipolar disorder.

BACKGROUND: Bipolar disorder (BD) is a multifactorial psychiatric illness affecting ∼1% of the global adult population. Lithium (Li), is the most effective mood stabilizer for BD but works only for a subset of patients and its mechanism of action remains largely elusive. METHODS: In the present study, we used iPSC-derived neurons from patients with BD who are responsive (LR) or not (LNR) to lithium. Combined electrophysiology, calcium imaging, biochemistry, transcriptomics, and phosphoproteomics were employed to provide mechanistic insights into neuronal hyperactivity in BD, investigate Li's mode of action, and identify alternative treatment strategies. FINDINGS: We show a selective rescue of the neuronal hyperactivity phenotype by Li in LR neurons, correlated with changes to Na+ conductance. Whole transcriptome sequencing in BD neurons revealed altered gene expression pathways related to glutamate transmission, alterations in cell signalling and ion transport/channel activity. We found altered Akt signalling as a potential therapeutic effect of Li in LR neurons from patients with BD, and that Akt activation mimics Li effect in LR neurons. Furthermore, the increased neural network activity observed in both LR & LNR neurons from patients with BD were reversed by AMP-activated protein kinase (AMPK) activation. INTERPRETATION: These results suggest potential for new treatment strategies in BD, such as Akt activators in LR cases, and the use of AMPK activators for LNR patients with BD. FUNDING: Supported by funding from ERA PerMed, Bell Brain Canada Mental Research Program and Brain & Behavior Research Foundation.

A pilot study examining the impact of lithium treatment and responsiveness on mnemonic discrimination in bipolar disorder.

INTRODUCTION: Mnemonic discrimination (MD), the ability to discriminate new stimuli from similar memories, putatively involves dentate gyrus pattern separation. Since lithium may normalize dentate gyrus functioning in lithium-responsive bipolar disorder (BD), we hypothesized that lithium treatment would be associated with better MD in lithium-responsive BD patients. METHODS: BD patients (N = 69; NResponders = 16 [23 %]) performed the Continuous Visual Memory Test (CVMT), which requires discriminating between novel and previously seen images. Before testing, all patients had prophylactic lithium responsiveness assessed over ≥1 year of therapy (with the Alda Score), although only thirty-eight patients were actively prescribed lithium at time of testing (55 %; 12/16 responders, 26/53 nonresponders). We then used computational modelling to extract patient-specific MD indices. Linear models were used to test how (A) lithium treatment, (B) lithium responsiveness via the continuous Alda score, and (C) their interaction, affected MD. RESULTS: Superior MD performance was associated with lithium treatment exclusively in lithium-responsive patients (Lithium x AldaScore ß = 0.257 [SE 0.078], p = 0.002). Consistent with prior literature, increased age was associated with worse MD (ß = -0.03 [SE 0.01], p = 0.005). LIMITATIONS: Secondary pilot analysis of retrospectively collected data in a cross-sectional design limits generalizability. CONCLUSION: Our study is the first to examine MD performance in BD. Lithium is associated with better MD performance only in lithium responders, potentially due to lithium's effects on dentate gyrus granule cell excitability. Our results may influence the development of behavioural probes for dentate gyrus neuronal hyperexcitability in BD.

USP19 deubiquitinase inactivation regulates α-synuclein ubiquitination and inhibits accumulation of Lewy body-like aggregates in mice.

The USP19 deubiquitinase is found in a locus associated with Parkinson's Disease (PD), interacts with chaperonins, and promotes secretion of α-synuclein (α-syn) through the misfolding-associated protein secretion (MAPS) pathway. Since these processes might modulate the processing of α-syn aggregates in PD, we inactivated USP19 (KO) in mice expressing the A53T mutation of α-syn and in whom α-syn preformed fibrils (PFF) had been injected in the striatum. Compared to WT, KO brains showed decreased accumulation of phospho-synuclein (pSyn) positive aggregates. This improvement was associated with less activation of microglia and improved performance in a tail-suspension test. Exposure of primary neurons from WT and KO mice to PFF in vitro also led to decreased accumulation of pSyn aggregates. KO did not affect uptake of PFF nor propagation of aggregates in the cultured neurons. We conclude that USP19 instead modulates intracellular dynamics of aggregates. At an early time following PFF injection when the number of pSyn-positive neurons were similar in WT and KO brains, the KO neurons contained less aggregates. KO brain aggregates stained more intensely with anti-ubiquitin antibodies. Immunoprecipitation of soluble proteins from WT and KO brains with antibodies to pSyn showed higher levels of ubiquitinated oligomeric species in the KO samples. We propose that the improved pathology in USP19 KO brains may arise from decreased formation or enhanced clearance of the more ubiquitinated aggregates and/or enhanced disassembly towards more soluble oligomeric species. USP19 inhibition may represent a novel therapeutic approach that targets the intracellular dynamics of α-syn complexes.

Transcriptomic effects of propranolol and primidone converge on molecular pathways relevant to essential tremor.

Essential tremor (ET) is one of the most common movement disorders, affecting nearly 5% of individuals over 65 years old. Despite this, few genetic risk loci for ET have been identified. Recent advances in pharmacogenomics have previously been useful to identify disease related molecular targets. Notably, gene expression has proven to be quite successful for the inference of drug response in cell models. We sought to leverage this approach in the context of ET where many patients are responsive to two drugs: propranolol and primidone. In this study, cerebellar DAOY and neural progenitor cells were treated for 5 days with clinical concentrations of propranolol and primidone, after which RNA-sequencing was used to identify convergent differentially expressed genes across treatments. Propranolol was found to affect the expression of genes previously associated with ET and other movement disorders such as TRAPPC11. Pathway enrichment analysis of these convergent drug-targeted genes identified multiple terms related to calcium signaling, endosomal sorting, axon guidance, and neuronal morphology. Furthermore, genes targeted by ET drugs were enriched within cell types having high expression of ET-related genes in both cortical and cerebellar tissues. Altogether, our results highlight potential cellular and molecular mechanisms associated with tremor reduction and identify relevant genetic biomarkers for drug-responsiveness in ET.

Dendritic Polyglycerol Amine: An Enhanced Substrate to Support Long-Term Neural Cell Culture.

Long-term stable cell culture is a critical tool to better understand cell function. Most adherent cell culture models require a polymer substrate coating of poly-lysine or poly-ornithine for the cells to adhere and survive. However, polypeptide-based substrates are degraded by proteolysis and it remains a challenge to maintain healthy cell cultures for extended periods of time. Here, we report the development of an enhanced cell culture substrate based on a coating of dendritic polyglycerol amine (dPGA), a non-protein macromolecular biomimetic of poly-lysine, to promote the adhesion and survival of neurons in cell culture. We show that this new polymer coating provides enhanced survival, differentiation and long-term stability for cultures of primary neurons or neurons derived from human induced pluripotent stem cells (hiPSCs). Atomic force microscopy analysis provides evidence that greater nanoscale roughness contributes to the enhanced capacity of dPGA-coated surfaces to support cells in culture. We conclude that dPGA is a cytocompatible, functionally superior, easy to use, low cost and highly stable alternative to poly-cationic polymer cell culture substrate coatings such as poly-lysine and poly-ornithine. Summary statementHere, we describe a novel dendritic polyglycerol amine-based substrate coating, demonstrating superior performance compared to current polymer coatings for long-term culture of primary neurons and neurons derived from induced pluripotent stem cells.

Chronic lithium treatment alters the excitatory/ inhibitory balance of synaptic networks and reduces mGluR5-PKC signalling in mouse cortical neurons.

Background: Bipolar disorder is characterized by cyclical alternation between mania and depression, often comorbid with psychosis and suicide. Compared with other medications, the mood stabilizer lithium is the most effective treatment for the prevention of manic and depressive episodes. However, the pathophysiology of bipolar disorder and lithium's mode of action are yet to be fully understood. Evidence suggests a change in the balance of excitatory and inhibitory activity, favouring excitation in bipolar disorder. In the present study, we sought to establish a holistic understanding of the neuronal consequences of lithium exposure in mouse cortical neurons, and to identify underlying mechanisms of action. Methods: We used a range of technical approaches to determine the effects of acute and chronic lithium treatment on mature mouse cortical neurons. We combined RNA screening and biochemical and electrophysiological approaches with confocal immunofluorescence and live-cell calcium imaging. Results: We found that only chronic lithium treatment significantly reduced intracellular calcium flux, specifically by activating metabotropic glutamatergic receptor 5. This was associated with altered phosphorylation of protein kinase C and glycogen synthase kinase 3, reduced neuronal excitability and several alterations to synapse function. Consequently, lithium treatment shifts the excitatory­inhibitory balance toward inhibition. Limitations: The mechanisms we identified should be validated in future by similar experiments in whole animals and human neurons. Conclusion: Together, the results revealed how lithium dampens neuronal excitability and the activity of the glutamatergic network, both of which are predicted to be overactive in the manic phase of bipolar disorder. Our working model of lithium action enables the development of targeted strategies to restore the balance of overactive networks, mimicking the therapeutic benefits of lithium but with reduced toxicity.

Missense mutation of Fmr1 results in impaired AMPAR-mediated plasticity and socio-cognitive deficits in mice.

Fragile X syndrome (FXS) is the most frequent form of inherited intellectual disability and the best-described monogenic cause of autism. CGG-repeat expansion in the FMR1 gene leads to FMR1 silencing, loss-of-expression of the Fragile X Mental Retardation Protein (FMRP), and is a common cause of FXS. Missense mutations in the FMR1 gene were also identified in FXS patients, including the recurrent FMRP-R138Q mutation. To investigate the mechanisms underlying FXS caused by this mutation, we generated a knock-in mouse model (Fmr1R138Q) expressing the FMRP-R138Q protein. We demonstrate that, in the hippocampus of the Fmr1R138Q mice, neurons show an increased spine density associated with synaptic ultrastructural defects and increased AMPA receptor-surface expression. Combining biochemical assays, high-resolution imaging, electrophysiological recordings, and behavioural testing, we also show that the R138Q mutation results in impaired hippocampal long-term potentiation and socio-cognitive deficits in mice. These findings reveal the functional impact of the FMRP-R138Q mutation in a mouse model of FXS.

Sushi domain-containing protein 4 controls synaptic plasticity and motor learning.

Fine control of protein stoichiometry at synapses underlies brain function and plasticity. How proteostasis is controlled independently for each type of synaptic protein in a synapse-specific and activity-dependent manner remains unclear. Here, we show that Susd4, a gene coding for a complement-related transmembrane protein, is expressed by many neuronal populations starting at the time of synapse formation. Constitutive loss-of-function of Susd4 in the mouse impairs motor coordination adaptation and learning, prevents long-term depression at cerebellar synapses, and leads to misregulation of activity-dependent AMPA receptor subunit GluA2 degradation. We identified several proteins with known roles in the regulation of AMPA receptor turnover, in particular ubiquitin ligases of the NEDD4 subfamily, as SUSD4 binding partners. Our findings shed light on the potential role of SUSD4 mutations in neurodevelopmental diseases.

Epigenetics in bipolar disorder: a critical review of the literature.

INTRODUCTION: Bipolar disorder (BD) is a chronic, disabling disease characterised by alternate mood episodes, switching through depressive and manic/hypomanic phases. Mood stabilizers, in particular lithium salts, constitute the cornerstone of the treatment in the acute phase as well as for the prevention of recurrences. The pathophysiology of BD and the mechanisms of action of mood stabilizers remain largely unknown but several pieces of evidence point to gene x environment interactions. Epigenetics, defined as the regulation of gene expression without genetic changes, could be the molecular substrate of these interactions. In this literature review, we summarize the main epigenetic findings associated with BD and response to mood stabilizers. METHODS: We searched PubMed, and Embase databases and classified the articles depending on the epigenetic mechanisms (DNA methylation, histone modifications and non-coding RNAs). RESULTS: We present the different epigenetic modifications associated with BD or with mood-stabilizers. The major reported mechanisms were DNA methylation, histone methylation and acetylation, and non-coding RNAs. Overall, the assessments are poorly harmonized and the results are more limited than in other psychiatric disorders (e.g. schizophrenia). However, the nature of BD and its treatment offer excellent opportunities for epigenetic research: clear impact of environmental factors, clinical variation between manic or depressive episodes resulting in possible identification of state and traits biomarkers, documented impact of mood-stabilizers on the epigenome. CONCLUSION: Epigenetic is a growing and promising field in BD that may shed light on its pathophysiology or be useful as biomarkers of response to mood-stabilizer.

Quantitative Profiling of Synuclein Species: Application to Transgenic Mouse Models of Parkinson's Disease.

INTRODUCTION: Improved analytical tools for detailed characterization of synucleins in pre-clinical models of Parkinson's disease (PD) and related synucleinopathies are needed. OBJECTIVE: Develop a multiple reaction monitoring (MRM) liquid chromatography tandem mass spectrometry (LC-MS/MS) assay to quantify species-specific sequences and structural heterogeneity in soluble α- and ß-synucleins in brain tissue. METHODS: Using a proteolytic digestion workflow, the MRM LC-MS/MS method assayed six proteotypic peptides from the α-synuclein sequence; three unique to mouse or human α-synuclein and three conserved in α- and ß-synuclein. For quantification, we used labeled α-synuclein as the internal standard and an external calibration curve. As proof of concept, the synuclein LC-MS/MS method was applied to brain tissue specimens from M83 transgenic PD mice, which overexpresses human α-synuclein, relative to wild-type littermate controls. RESULTS: The synuclein MRM assay was linear over a wide concentration range (at least one order of magnitude). The assay had several advantages over ligand binding analytical methods, such as western blotting and enzyme-linked immunosorbent assays. These advantages included the ability to: quantify 1) total α-synuclein, 2) combined α- and ß-synucleins, 3) species-specific contributions to total α-synuclein (e.g., in mice expressing both mouse and human α-synuclein), and 4) identify peptide-specific profile differences that may reflect post-translational modifications, all within a single analysis. CONCLUSION: With improved and expanded analytical characteristics coupled with a streamlined sample preparation workflow, the quantitative synuclein profiling LC-MS/MS assay provides a versatile and efficient platform to characterize synuclein biology in pre-clinical models and the potential for application to human tissues and fluids.

The synaptic balance between sumoylation and desumoylation is maintained by the activation of metabotropic mGlu5 receptors.

Sumoylation is a reversible post-translational modification essential to the modulation of neuronal function, including neurotransmitter release and synaptic plasticity. A tightly regulated equilibrium between the sumoylation and desumoylation processes is critical to the brain function and its disruption has been associated with several neurological disorders. This sumoylation/desumoylation balance is governed by the activity of the sole SUMO-conjugating enzyme Ubc9 and a group of desumoylases called SENPs, respectively. We previously demonstrated that the activation of type 5 metabotropic glutamate receptors (mGlu5R) triggers the transient trapping of Ubc9 in dendritic spines, leading to a rapid increase in the overall synaptic sumoylation. However, the mechanisms balancing this increased synaptic sumoylation are still not known. Here, we examined the diffusion properties of the SENP1 enzyme using a combination of advanced biochemical approaches and restricted photobleaching/photoconversion of individual hippocampal spines. We demonstrated that the activation of mGlu5R leads to a time-dependent decrease in the exit rate of SENP1 from dendritic spines. The resulting post-synaptic accumulation of SENP1 restores synaptic sumoylation to initial levels. Altogether, our findings reveal the mGlu5R system as a central activity-dependent mechanism to maintaining the homeostasis of sumoylation at the mammalian synapse.

Involvement of Phosphodiesterase 2A Activity in the Pathophysiology of Fragile X Syndrome.

The fragile X mental retardation protein (FMRP) is an RNA-binding protein involved in translational regulation of mRNAs that play key roles in synaptic morphology and plasticity. The functional absence of FMRP causes the fragile X syndrome (FXS), the most common form of inherited intellectual disability and the most common monogenic cause of autism. No effective treatment is available for FXS. We recently identified the Phosphodiesterase 2A (Pde2a) mRNA as a prominent target of FMRP. PDE2A enzymatic activity is increased in the brain of Fmr1-KO mice, a recognized model of FXS, leading to decreased levels of cAMP and cGMP. Here, we pharmacologically inhibited PDE2A in Fmr1-KO mice and observed a rescue both of the maturity of dendritic spines and of the exaggerated hippocampal mGluR-dependent long-term depression. Remarkably, PDE2A blockade rescued the social and communicative deficits of both mouse and rat Fmr1-KO animals. Importantly, chronic inhibition of PDE2A in newborn Fmr1-KO mice followed by a washout interval, resulted in the rescue of the altered social behavior observed in adolescent mice. Altogether, these results reveal the key role of PDE2A in the physiopathology of FXS and suggest that its pharmacological inhibition represents a novel therapeutic approach for FXS.

Sumoylation regulates FMRP-mediated dendritic spine elimination and maturation.

Fragile X syndrome (FXS) is the most frequent inherited cause of intellectual disability and the best-studied monogenic cause of autism. FXS results from the functional absence of the fragile X mental retardation protein (FMRP) leading to abnormal pruning and consequently to synaptic communication defects. Here we show that FMRP is a substrate of the small ubiquitin-like modifier (SUMO) pathway in the brain and identify its active SUMO sites. We unravel the functional consequences of FMRP sumoylation in neurons by combining molecular replacement strategy, biochemical reconstitution assays with advanced live-cell imaging. We first demonstrate that FMRP sumoylation is promoted by activation of metabotropic glutamate receptors. We then show that this increase in sumoylation controls the homomerization of FMRP within dendritic mRNA granules which, in turn, regulates spine elimination and maturation. Altogether, our findings reveal the sumoylation of FMRP as a critical activity-dependent regulatory mechanism of FMRP-mediated neuronal function.

mGlu5 receptors regulate synaptic sumoylation via a transient PKC-dependent diffusional trapping of Ubc9 into spines.

Sumoylation plays important roles in the modulation of protein function, neurotransmission and plasticity, but the mechanisms regulating this post-translational system in neurons remain largely unknown. Here we demonstrate that the synaptic diffusion of Ubc9, the sole conjugating enzyme of the sumoylation pathway, is regulated by synaptic activity. We use restricted photobleaching/photoconversion of individual hippocampal spines to measure the diffusion properties of Ubc9 and show that it is regulated through an mGlu5R-dependent signalling pathway. Increasing synaptic activity with a GABAA receptor antagonist or directly activating mGlu5R increases the synaptic residency time of Ubc9 via a Gαq/PLC/Ca(2+)/PKC cascade. This activation promotes a transient synaptic trapping of Ubc9 through a PKC phosphorylation-dependent increase of Ubc9 recognition to phosphorylated substrates and consequently leads to the modulation of synaptic sumoylation. Our data demonstrate that Ubc9 diffusion is subject to activity-dependent regulatory processes and provide a mechanism for the dynamic changes in sumoylation occurring during synaptic transmission.

Activity-dependent regulation of the sumoylation machinery in rat hippocampal neurons.

BACKGROUND INFORMATION: Sumoylation is a key post-translational modification by which the Small Ubiquitin-like MOdifier (SUMO) polypeptide is covalently attached to specific lysine residues of substrate proteins through a specific enzymatic pathway. Although sumoylation participates in the regulation of nuclear homeostasis, the sumoylation machinery is also expressed outside of the nucleus where little is still known regarding its non-nuclear functions, particularly in the Central Nervous System (CNS). We recently reported that the sumoylation process is developmentally regulated in the rat CNS. RESULTS: Here, we demonstrate that there is an activity-dependent redistribution of endogenous sumoylation enzymes in hippocampal neurons. By performing biochemical and immunocytochemical experiments on primary cultures of rat hippocampal neurons, we show that sumoylation and desumoylation enzymes are differentially redistributed in and out of synapses upon neuronal stimulation. This enzymatic redistribution in response to a neuronal depolarisation results in the transient decrease of sumoylated protein substrates at synapses. CONCLUSIONS: Taken together, our data identify an activity-dependent regulation of the sumoylation machinery in neurons that directly impacts on synaptic sumoylation levels. This process may provide a mechanism for neurons to adapt their physiological responses to changes occurring during neuronal activation.