Biology subjects

Elgersma, Y.

Publications and source records attributed to Elgersma, Y..

9 recordsLinked to original sources

bioRxiv2024

Simultaneous loss of CAMK2A and CAMK2B reveals endogenous in vivo substrates

Ca2+/calmodulin-dependent protein kinase 2 (CAMK2) plays a critical role in calcium signaling. Recent gene knockout studies show that CAMK2A and CAMK2B can have distinct roles yet also partially compensate for each other in yet unknown brain functions. In order to provide insight into potential novel CAMK2 functions, we performed parallel phosphoproteomic analyses on non-stimulated cortex tissue from inducible Camk2a and Camk2b double knockout (Camk2af/f;Camk2bf/f;CAG-CreESR) mice and from wild type mice. A total of 5622 phosphorylated peptides derived from 2080 proteins were identified. Phosphorylation at serine/threonine residues in 130 proteins were downregulated in the double knockout mice, including residues in 113 proteins that have not previously been identified as potential CAMK2 substrates. Comparison of amino acid sequences surrounding the downregulated phosphorylation residues provided new insights into the CAMK2-substrate consensus sequences in vivo. This dataset provides an important resource for future studies examining novel roles for CAMK2 in the brain.

neuroscience↗

bioRxiv2024

UBE3A reinstatement restores behavior and proteome in an Angelman Syndrome mouse model of Imprinting Defects

Angelman Syndrome (AS) is a severe neurodevelopmental disorder wionly symptomatic treatment currently available. Besides mutations within the UBE3A gene, AS is caused by deletions, imprinting center defects (mICD) or uniparental disomy of chromosome 15 (UPD). Current mouse models are Ube3a-centric and do not address expression changes of other 15q11-q13 genes on AS pathophysiology. Here, we studied a mouse line that harbors a mutation affecting the AS-PWS imprinting center, hence modeling mICD/UPD AS subtypes. mICD mice showed significant reduction in UBE3A protein, bi-allelic expression of Ube3a-ATS and Mkrn3-Snord115 gene cluster, leading to robust AS behavioral deficits and proteome alterations similar to Ube3aKO mice. Genetic UBE3A overexpression in mICD mice, mimicking therapeutic strategies that effectively activate the biallelic silenced Ube3a gene, resulted in a complete rescue of all behavioral and proteome alterations. Subsequently, treatment with an antisense oligonucleotide (ASO) to directly activate the biallelic silenced Ube3a gene in mICD mice also resulted in efficient reinstatement of UBE3A, alongside a partial rescue of behavioral phenotypes. Taken together, these findings demonstrate that UBE3A loss is the primary factor underlying AS phenotypes in the mICD/UPD mouse model, and also corroborate that UBE3A reinstatement is an attractive therapeutic strategy for mICD/UPD AS individuals.

neuroscience↗

bioRxiv2024

Modeling mTORopathy-related epilepsy in cultured murine hippocampal neurons using the multi-electrode array

The mechanistic target of rapamycin complex 1 (mTORC1) signaling pathway is a ubiquitous cellular pathway. mTORopathies, a group of disorders characterized by hyperactivity of the mTORC1 pathway, illustrate the prominent role of the mTOR pathway in disease pathology, often profoundly affecting the central nervous system. One of the most debilitating symptoms of mTORopathies is drug-resistant epilepsy, emphasizing the urgent need for a deeper understanding of disease mechanisms to develop novel anti-epileptic drugs. In this study, we explored the multiwell Multi-electrode array (MEA) system as a tool to identify robust network activity parameters in an approach to model mTORopathy-related epilepsy in vitro. To this extent, we cultured mouse primary hippocampal neurons on the multiwell MEA to identify robust network activity phenotypes in mTORC1-hyperactive neuronal networks. mTOR-hyperactivity was induced either through deletion of Tsc1 or overexpression of a constitutively active RHEB variant identified in patients, RHEBp.P37L. mTORC1 dependency of the phenotypes was assessed using rapamycin, and vigabatrin was applied to treat epilepsy-like phenotypes. We show that hyperactivity of the mTORC1 pathway leads to aberrant network activity. In both the Tsc1-KO and RHEB-p.P37L models, we identified changes in network synchronicity, rhythmicity, and burst characteristics. The presence of these phenotypes is prevented upon early treatment with the mTORC1-inhibitor rapamycin. Application of rapamycin in mature neuronal cultures could only partially rescue the network activity phenotypes. Additionally, treatment with the anti-epileptic drug vigabatrin reduced network activity and restored burst characteristics. Taken together, we showed that mTORC1-hyperactive neuronal cultures on the multiwell MEA system present reliable network activity phenotypes that can be used as an assay to explore the potency of new drug treatments targeting epilepsy in mTORopathy patients and may give more insights into the pathophysiological mechanisms underlying epilepsy in these patients. ABBREVIATIONS AED, anti-epileptic drug, CoVNIBI, coefficient of variance of NIBI, CTR, control transduced cultures, DIV, days in vitro, DMEM, Dulbeccos modified Eagle medium, DMSO, dimethyl sulfoxide, GABA, gamma-aminobutyric acidergic, GAPDH, Glyceraldehyde-3-Phosphate Dehydrogenase, iPSC, induced pluripotent stem cell, KO, knock-out, LV, lentivirus, MEA, multi-electrode array, MFR, mean firing rate, mTORC1, mechanistic target of rapamycin complex 1, NB, network burst, NBC, network burst composition, NBD, network burst duration, NBM, neurobasal medium, NBR, network burst rate, NIBI, network interburst interval, NT, non-transduced, RHEB, Ras-homolog enriched in brain, %RS, percentage of random spikes, TBS, tris buffered saline, TSC, Tuberous sclerosis complex, WT, wildtype

neuroscience↗

bioRxiv2024

Localization of Human UBE3A Isoform 3 is Highly Sensitive to Amino Acid Substitutions at p.Met21 Position

UBE3A encodes three isoforms of Ubiquitin E3 ligase A, which differ in their N-terminal sequence, abundance, and localization. Recently, three individuals diagnosed with Angelman Syndrome have been described who carry a variant that abrogates the start codon of the predominant nuclear isoform 1 (hUBE3A-Iso1p.Met1Thr) and concomitantly results in a missense variant in isoform 3 (hUBE3A-Iso1p.Met21Thr), which we previously reported to be nuclear enriched as well. Here, we studied the effect of the p.Met21Thr variant on hUBE3A-Iso3 localization. Recombinant expression of hUBE3A-Iso3p.Met21Thr in U2-OS and mouse neurons revealed similar UBE3A labelling in the nucleus and cytosol, indicating hUBE3A-Iso3 localization is sensitive to amino acid changes at this position. This finding prompted us to revisit hUBE3A-Iso3 localization, since we previously introduced a p.Met21Ala/p.Met22Ala amino acid substitution in hUBE3A-Iso3 and its mouse orthologue mUBE3A-Iso2 to prevent translation of the shorter hUBE3A-Iso1 and mUBE3A-Iso3 nuclear isoforms. Introduction of silent mutations to disfavour translation of the short UBE3A isoforms enabled us to determine the localization hUBE3A-Iso3 and mUBE3A-Iso2 in the absence of amino acid changes at the p.Met21/p.Met22 position, respectively. Surprisingly, hUBE3A-Iso3 localization shifted from predominant nuclear localization for hUBE3A-Iso3p.Met21Ala to a predominant cytosolic localization of the Kozak optimized hUBE3A-Iso3KOZAK, while their mouse orthologues mUBE3A-Iso2p.Met22Ala and mUBE3A-Iso2KOZAK both localized predominantly to the cytosol. Taken together, these experiments indicate that the localization of human UBE3A-Iso3 is highly sensitive to amino acid substitutions at the p.Met21 position and that variants at this position not only abrogate the translation of hUBE3A-Iso1, but can also change the localization of hUBE3A-Iso3.

molecular biology↗

bioRxiv2022

Adult Camk2a gene reinstatement restores the learning and plasticity deficits of Camk2a knockout mice

With the recent findings that mutations in the gene encoding the -subunit of calcium/calmodulin-dependent protein kinase II (CAMK2A) causes neurodevelopmental disorder (NDD), it is of great therapeutic relevance to know if there a critical developmental time window in which CAMK2A needs to be expressed for normal brain development, or whether expression of the protein at later stages is still beneficial to restore normal functioning. To answer this question, we generated an inducible Camk2a mouse model, which allows us to express CAMK2A at any desired time. Here, we show that adult expression of CAMK2A rescues the behavioural and electrophysiological phenotypes seen in the Camk2a knock-out mice, including spatial and conditional learning and synaptic plasticity. These results suggest that CAMK2A does not play a critical irreversible role in neurodevelopment, which is of importance for future therapies to treat CAMK2A-dependent disorders.

neuroscience↗

bioRxiv2022

Bidirectional changes in excitability upon loss of both CAMK2A and CAMK2B.

The mammalian Ca2+/calmodulin-dependent protein kinase II (CAMK2) family consists of 4 different CAMK2 genes, encoding CAMK2A, CAMK2B, CAMK2D and CAMK2G, which have high structural homology. CAMK2A and CAMK2B are abundantly expressed in the brain; they play a unique role in proper neuronal functioning, since both CAMK2A and CAMK2B knockout mice show several behavioural and cellular phenotypes. However, our recent finding that deletion of both CAMK2A and CAMK2B is lethal indicates that they show redundancy and that the full spectrum of CAMK2 function in neurons remains to be uncovered. For example, it still remains unclear which overlapping functions are present at a single cell level in neuronal transmission and excitability. In order to get more insight into the full spectrum of CAMK2 functions in neurons, we performed whole-cell patch clamp experiments in inducible Camk2a/Camk2b double knockout mice, as well as the CAMK2A and CAMK2B knockout mice. We found that whereas deletion of only CAMK2A or CAMK2B did not change excitability, simultaneous deletion of CAMK2A and CAMK2B resulted in a decrease in excitability 10 days after deletion in CA1 pyramidal neurons, which reversed to increased excitability 21 days after deletion. Additionally, loss of both CAMK2A and CAMK2B resulted in a decreased frequency of both miniature excitatory and inhibitory postsynaptic currents (mEPSC and mIPSC) 21 days after deletion, but not 10 days after deletion, an effect not seen in the single mutants. Our results indicate that CAMK2 is critically important to maintain normal excitability of hippocampal CA1 pyramidal cells, as well as normal inhibitory and excitatory synaptic transmission. Together, these results lead to new insights in how CAMK2 regulates normal neuronal function and highlight the importance of having both CAMK2A and CAMK2B expressed in high levels in the brain.

neuroscience↗

bioRxiv2021

A novel automated approach for improving standardization of the marble burying test enables quantification of burying bouts and activity characteristics

The marble burying test is a commonly used paradigm to screen phenotypes in mouse models of neurodevelopmental and psychiatric disorders. The current methodological approach relies solely on reporting the number of buried marbles at the end of the test. By measuring the proxy of the behavior (buried marbles), rather than the behavior itself (burying bouts), many important characteristics regarding the temporal aspect of this assay are lost. Here we introduce a novel, automated method to quantify mouse behavior throughout the duration of the marble burying test with the focus on the burying bouts. Using open-source software packages, we trained a supervised machine learning algorithm (the "classifier") to distinguish burying behavior in freely moving mice. In order to confirm the classifiers accuracy and uncover the behavioral meaning of the marble burying test, we performed marble burying test in three mouse models: Ube3am-/p+ (Angelman Syndrome model), Shank2-/- (autism model), and Sapap3-/- (obsessive-compulsive disorder model) mice. The classifier scored burying behavior accurately and consistent with the literature in the Ube3am-/p+ mice, which showed decreased levels of burying compared to controls. Shank2-/- mice showed a similar pattern of decreased burying behavior, which was not found in Sapap3-/- mice. Tracking mouse behavior throughout the test enabled us to quantify activity characteristics, revealing hypoactivity in Ube3am-/p+ and hyperactivity in the Shank2-/- mice, indicating that mouse activity is unrelated to burying behavior. Together, we demonstrate that our classifier is an accurate method for the analysis of the marble burying test, providing more information than the currently used methods. Significance StatementThe marble burying test is widely used in phenotyping neurodevelopmental and neuropsychiatric disorder mouse models. Currently, its analysis consists of manually scoring the number of buried marbles upon the completion of the assay. This approach is not standardized across laboratories, and leaves out important variables such as movement characteristics and information about the burying bouts. This leads to divergent interpretations of the marble burying test, ranging from anxiety to cognitive impairment. We introduce a method that reliably tracks mouse behavior throughout the experiment, classifies the duration and number of the burying bouts, and is generalizable across laboratories. Using machine learning for measuring the actual burying behavior standardizes this method, and provides rich information about the burying characteristics.

neuroscience↗

bioRxiv2020

Mono-ubiquitination of Rabphilin 3A by UBE3A serves a non-degradative function

Angelman syndrome (AS) is a severe neurodevelopmental disorder caused by brain-specific loss of UBE3A, an E3 ubiquitin protein ligase. A substantial number of possible ubiquitination targets of UBE3A have been identified, although evidence of being direct UBE3A substrates is often lacking. Here we identified the synaptic protein Rabphilin-3a (RPH3A), an effector of the RAB3A small GTPase involved in axonal vesicle priming and docking, as a ubiquitination target of UBE3A. We found that the UBE3A and RAB3A binding sites on RPH3A partially overlap, and that RAB3A binding to RPH3A interferes with UBE3A binding. We confirmed previous observations that RPH3A levels are critically dependent on RAB3A binding but, rather surprisingly, we found that the reduced RPH3A levels in the absence of RAB3A are not mediated by UBE3A. Indeed, while we found that RPH3A is ubiquitinated in a UBE3A-dependent manner in mouse brain, UBE3A mono-ubiquitinates RPH3A and does not facilitate RPH3A degradation. Moreover, we found that an AS-linked UBE3A missense mutation in the UBE3A region that interacts with RPH3A, abrogates the interaction with RPH3A. In conclusion, our results identify RPH3A as a novel target of UBE3A and suggest that UBE3A-dependent ubiquitination of RPH3A serves a non-degradative function.

neuroscience↗

bioRxiv2018

Within-subject consistency of paired associative stimulation as assessed by linear mixed models.

ObjectivePaired associative stimulation (PAS) is a TMS paradigm used to induce long-term potentiation in the human cortex. Little is known about the within-subject consistency of PAS-induced effects. We determined PAS-induced effects and their consistency in healthy volunteers between two PAS sessions. Additionally, we assessed the benefit of applying linear mixed models (LMMs) to PAS data.\n\nMethodsThirty-eight healthy volunteers underwent two identical PAS sessions with a >1 week interval. During each session, motor evoked potentials (MEPs) were assessed once before PAS induction and 3 times after at 30 min intervals.\n\nResultsWe did not detect any significant potentiation of MEP size after PAS induction. However, MEP size during PAS induction showed significant potentiation over time in both sessions (LR(1) = 13.36, p<0.001). Nevertheless, there was poor within-subject consistency of PAS-induced effects both during (ICC=0.15) and after induction (ICC=0.03-0.08). Additionally, statistical model selection procedures demonstrate that LMMs are more appropriate than conventional longitudinal models for estimating PAS-induced effects (LR(34) = 214.73, p < 0.001).\n\nConclusionPAS-induced effects are more pronounced during than after induction, have a low within-subject consistency in any phase of the measurement, and are best estimated with LMMs. The implication of our study is that PAS is an inappropriate method to assess the evolution of brain plasticity over time periods longer than the PAS measurement itself.

neuroscience↗