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3' untranslated region (3'UTR) variants are strongly associated with human traits and diseases, yet few have been causally identified. We developed the massively parallel reporter assay for 3'UTRs (MPRAu) to sensitively assay 12,173 3'UTR variants. We applied MPRAu to six human cell lines, focusing on genetic variants associated with genome-wide association studies (GWAS) and human evolutionary adaptation. MPRAu expands our understanding of 3'UTR function, suggesting that simple sequences predominately explain 3'UTR regulatory activity. We adapt MPRAu to uncover diverse molecular mechanisms at base pair resolution, including an adenylate-uridylate (AU)-rich element of LEPR linked to potential metabolic evolutionary adaptations in East Asians. We nominate hundreds of 3'UTR causal variants with genetically fine-mapped phenotype associations. Using endogenous allelic replacements, we characterize one variant that disrupts a miRNA site regulating the viral defense gene TRIM14 and one that alters PILRB abundance, nominating a causal variant underlying transcriptional changes in age-related macular degeneration., Competing Interests: Declaration of interests P.C.S. is a co-founder of and consultant to Sherlock Biosciences and Board Member of Danaher Corporation. She is a shareholder in both companies., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Key Points: The lateral superior olive (LSO), a brainstem hub involved in sound localization, integrates excitatory and inhibitory inputs from the ipsilateral and the contralateral ear, respectively. In gerbils and rats, inhibition to the LSO reportedly shifts from GABAergic to glycinergic within the first three postnatal weeks. Surprisingly, we found no evidence for synaptic GABA signalling during this time window in mouse LSO principal neurons. However, we found that presynaptic GABA B Rs modulate Ca 2+ influx into medial nucleus of the trapezoid body axon terminals, resulting in reduced synaptic strength. Moreover, GABA elicited strong responses in LSO neurons that were mediated by extrasynaptic GABA A Rs. RNA sequencing revealed highly abundant δ subunits, which are characteristic of extrasynaptic receptors. Whereas GABA increased the excitability of neonatal LSO neurons, it reduced the excitability around hearing onset. Collectively, GABA appears to control the excitability of mouse LSO neurons via extrasynaptic and presynaptic signalling. Thus, GABA acts as a modulator, rather than as a classical transmitter., Abstract: GABA and glycine mediate fast inhibitory neurotransmission and are coreleased at several synapse types. Here we assessed the contribution of GABA and glycine in synaptic transmission between the medial nucleus of the trapezoid body (MNTB) and the lateral superior olive (LSO), two nuclei involved in sound localization. Whole-cell patch-clamp experiments in acute mouse brainstem slices at postnatal days (P) 4 and 11 during pharmacological blockade of GABA A receptors (GABA A Rs) and/or glycine receptors demonstrated no GABAergic synaptic component on LSO principal neurons. A GABAergic component was absent in evoked inhibitory postsynaptic currents and miniature events. Coimmunofluorescence experiments revealed no codistribution of the presynaptic GABAergic marker GAD65/67 with gephyrin, a postsynaptic marker for GABA A Rs, corroborating the conclusion that GABA does not act synaptically in the mouse LSO. Imaging experiments revealed reduced Ca 2+ influx into MNTB axon terminals following activation of presynaptic GABA B Rs. GABA B R activation reduced the synaptic strength at P4 and P11. GABA appears to act on extrasynaptic GABA A Rs as demonstrated by application of 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol, a δ-subunit-specific GABA A R agonist. RNA sequencing showed high mRNA levels for the δ-subunit in the LSO. Moreover, GABA transporters GAT-1 and GAT-3 appear to control extracellular GABA. Finally, we show an age-dependent effect of GABA on the excitability of LSO neurons. Whereas tonic GABA increased the excitability at P4, leading to spike facilitation, it decreased the excitability at P11 via shunting inhibition through extrasynaptic GABA A Rs. Taken together, we demonstrate a modulatory role of GABA in the murine LSO, rather than a function as a classical synaptic transmitter., (© 2019 The Authors. The Journal of Physiology © 2019 The Physiological Society.)

The purpose of this statement is to provide consensus-based recommendations for optimal management and care for patients with primary mitochondrial disease. This statement is intended for physicians who are engaged in the diagnosis and management of these patients. Working group members were appointed by the Mitochondrial Medicine Society. The panel included members with several different areas of expertise. The panel members utilized surveys and the Delphi method to reach consensus. We anticipate that this statement will need to be updated as the field continues to evolve. Consensus-based recommendations are provided for the routine care and management of patients with primary genetic mitochondrial disease.

Cav1.2 and Cav1.3 are the major L-type voltage-gated Ca(2+) channels in the CNS. Yet, their individual in vivo functions are largely unknown. Both channel subunits are expressed in the auditory brainstem, where Cav1.3 is essential for proper maturation. Here, we investigated the role of Cav1.2 by targeted deletion in the mouse embryonic auditory brainstem. Similar to Cav1.3, loss of Cav1.2 resulted in a significant decrease in the volume and cell number of auditory nuclei. Contrary to the deletion of Cav1.3, the action potentials of lateral superior olive (LSO) neurons were narrower compared with controls, whereas the firing behavior and neurotransmission appeared unchanged. Furthermore, auditory brainstem responses were nearly normal in mice lacking Cav1.2. Perineuronal nets were also unaffected. The medial nucleus of the trapezoid body underwent a rapid cell loss between postnatal days P0 and P4, shortly after circuit formation. Phosphorylated cAMP response element-binding protein (CREB), nuclear NFATc4, and the expression levels of p75NTR, Fas, and FasL did not correlate with cell death. These data demonstrate for the first time that both Cav1.2 and Cav1.3 are necessary for neuronal survival but are differentially required for the biophysical properties of neurons. Thus, they perform common as well as distinct functions in the same tissue., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Short-term plasticity plays a key role in synaptic transmission and has been extensively investigated for excitatory synapses. Much less is known about inhibitory synapses. Here we analyze the performance of glycinergic connections between the medial nucleus of the trapezoid body (MNTB) and the lateral superior olive (LSO) in the auditory brainstem, where high spike rates as well as fast and precise neurotransmission are hallmarks. Analysis was performed in acute mouse slices shortly after hearing onset (postnatal day (P)11) and 8 days later (P19). Stimulation was done at 37°C with 1-400 Hz for 40 s. Moreover, in a novel approach named marathon experiments, a very prolonged stimulation protocol was employed, comprising 10 trials of 1-min challenge and 1-min recovery periods at 50 and 1 Hz, respectively, thus lasting up to 20 min and amounting to >30,000 stimulus pulses. IPSC peak amplitudes displayed short-term depression (STD) and synaptic attenuation in a frequency-dependent manner. No facilitation was observed. STD in the MNTB-LSO connections was less pronounced than reported in the upstream calyx of Held-MNTB connections. At P11, the STD level and the failure rate were slightly lower within the ms-to-s range than at P19. During prolonged stimulation periods lasting 40 s, P19 connections sustained virtually failure-free transmission up to frequencies of 100 Hz, whereas P11 connections did so only up to 50 Hz. In marathon experiments, P11 synapses recuperated reproducibly from synaptic attenuation during all recovery periods, demonstrating a robust synaptic machinery at hearing onset. At 26°C, transmission was severely impaired and comprised abnormally high amplitudes after minutes of silence, indicative of imprecisely regulated vesicle pools. Our study takes a fresh look at synaptic plasticity and stability by extending conventional stimulus periods in the ms-to-s range to minutes. It also provides a framework for future analyses of synaptic plasticity.

Although several hundred regions of the human genome harbor signals of positive natural selection, few of the relevant adaptive traits and variants have been elucidated. Using full-genome sequence variation from the 1000 Genomes (1000G) Project and the composite of multiple signals (CMS) test, we investigated 412 candidate signals and leveraged functional annotation, protein structure modeling, epigenetics, and association studies to identify and extensively annotate candidate causal variants. The resulting catalog provides a tractable list for experimental follow-up; it includes 35 high-scoring nonsynonymous variants, 59 variants associated with expression levels of a nearby coding gene or lincRNA, and numerous variants associated with susceptibility to infectious disease and other phenotypes. We experimentally characterized one candidate nonsynonymous variant in Toll-like receptor 5 (TLR5) and show that it leads to altered NF-κB signaling in response to bacterial flagellin. PAPERFLICK:, (Copyright © 2013 Elsevier Inc. All rights reserved.)

This study evaluates the outcome of urgent neurologic referrals. This was a retrospective review of all referrals to the Floating Hospital for Children in 1 month. The total number of patients referred to our center was 223. Amongst those, 108 were new patients and 195 were follow-up visits; 30 patients were deemed urgent, yet 6 of them did not present to their visit. Urgent and routinely scheduled patients were compared based on the need for further evaluation or medication initiation following their visit. The frequency of visit outcomes was statistically similar between urgently and nonurgently referred patients. We did observe though, that diagnostic testing and medication were initiated more frequently for the patients urgently referred for seizure compared with those routinely scheduled patients for seizure evaluation. For this reason, we suggest that pediatric neurologists preferentially should hold clinic space open for urgent referrals for patients with new-onset seizure.

Voltage-gated Ca(2+) (Ca(v))1.3 α-subunits of high voltage-activated Ca(2+) channels (HVACCs) are essential for Ca(2+) influx and transmitter release in cochlear inner hair cells and therefore for signal transmission into the central auditory pathway. Their absence leads to deafness and to striking structural changes in the auditory brain stem, particularly in the lateral superior olive (LSO). Here, we analyzed the contribution of various types of HVACCs to the total Ca(2+) current (I(Ca)) in developing mouse LSO neurons to address several questions: do LSO neurons express functional Ca(v)1.3 channels? What other types of HVACCs are expressed? Are there developmental changes? Do LSO neurons of Ca(v)1.3(-/-) mice show any compensatory responses, namely, upregulation of other HVACCs? Our electrophysiological and pharmacological results showed the presence of functional Ca(v)1.3 and Ca(v)1.2 channels at both postnatal days 4 and 12. Aside from these L-type channels, LSO neurons also expressed functional P/Q-type, N-type, and, most likely, R-type channels. The relative contribution of the four different subtypes to I(Ca) appeared to be 45%, 29%, 22%, and 4% at postnatal day 12, respectively. The physiological results were flanked and extended by quantitative RT-PCR data. Altogether, LSO neurons displayed a broad repertoire of HVACC subtypes. Genetic ablation of Ca(v)1.3 resulted in functional reorganization of some other HVACCs but did not restore normal I(Ca) properties. Together, our results suggest that several types of HVACCs are of functional relevance for the developing LSO. Whether on-site loss of Ca(v)1.3, i.e., in LSO neurons, contributes to the recently described malformation of the LSO needs to be determined by using tissue-specific Ca(v)1.3(-/-) animals.

Thapsigargin is a specific inhibitor of the sarco/endoplasmic reticulum Ca(2+) ATPase of the endoplasmic reticulum. Here, we show that stimulation of human HaCaT keratinocytes with nanomolar concentrations of thapsigargin triggers expression of activating transcription factor (ATF) 3, a basic-region leucin zipper transcription factor. ATF3 expression was also up-regulated in thapsigargin-stimulated glioma cells, hepatoma cells, retinal pigment epithelial cells, and airway epithelial cells. Thapsigargin-induced up-regulation of ATF3 expression in keratinocytes was attenuated by BAPTA-acetoxymethyl ester or by expression of the Ca(2+)-binding protein parvalbumin in the cytosol of HaCaT cells but not by a panel of pharmacological agents that chelate extracellular Ca(2+) (EGTA) or inhibit either ryanodine receptors (dantrolene) or voltage-gated Ca(2+) channels (nifedipine). Hence, elevated levels of intracellular Ca(2+), released from intracellular stores, are essential for the effect of thapsigargin on the biosynthesis of ATF3. The thapsigargin-induced signaling pathway was blocked by expression of either mitogen-activated protein kinase phosphatase-1 or -5. Experiments involving pharmacological and genetic tools revealed the importance of c-Jun N-terminal protein kinase (JNK) within the signaling cascade, whereas inhibition of extracellular signal-regulated protein kinase or p38 protein kinase did not attenuate thapsigargin-induced expression of ATF3. Functional studies showed that treatment of HaCaT keratinocytes with thapsigargin led to a 2-fold induction of caspase-3/7 activity. The up-regulation of caspase-3/7 activity in thapsigargin-stimulated HaCaT cells was attenuated by inhibition of JNK. Together, these data show that stimulation of HaCaT cells with thapsigargin induces a specific signaling pathway in keratinocytes involving activation of JNK, biosynthesis of ATF3, and up-regulation of caspase-3/7 activity.

Reactive oxygen species (ROS) are involved in many physiological and pathophysiological cellular processes. We used lymphocytes, which are exposed to highly oxidizing environments during inflammation, to study the influence of ROS on cellular function. Calcium ion (Ca(2+)) influx through Ca(2+) release-activated Ca(2+) (CRAC) channels composed of proteins of the ORAI family is essential for the activation, proliferation, and differentiation of T lymphocytes, but whether and how ROS affect ORAI channel function have been unclear. Here, we combined Ca(2+) imaging, patch-clamp recordings, and measurements of cell proliferation and cytokine secretion to determine the effects of hydrogen peroxide (H(2)O(2)) on ORAI channel activity and human T helper lymphocyte (T(H) cell) function. ORAI1, but not ORAI3, channels were inhibited by oxidation by H(2)O(2). The differential redox sensitivity of ORAI1 and ORAI3 channels depended mainly on an extracellularly located reactive cysteine, which is absent in ORAI3. T(H) cells became progressively less redox-sensitive after differentiation into effector cells, a shift that would allow them to proliferate, differentiate, and secrete cytokines in oxidizing environments. The decreased redox sensitivity of effector T(H) cells correlated with increased expression of Orai3 and increased abundance of several cytosolic antioxidants. Knockdown of ORAI3 with small-interfering RNA rendered effector T(H) cells more redox-sensitive. The differential expression of Orai isoforms between naïve and effector T(H) cells may tune cellular responses under oxidative stress.

Traumatic brain injury frequently elicits epileptic seizures hours or days after the impact. The mechanisms on cellular level are poorly understood. Because posttraumatic epilepsy appears in many cases as a temporal-lobe epilepsy which originated the hippocampus, we studied trauma-induced hyperexcitability on the cellular level in this brain area. We used the model of closed head injury to analyse the electrophysiological changes in CA1 and CA3 pyramidal cells and in interneurones of the CA1 field, which is extremely sensitive to ischemia. We found that morphologically closed head injury (CHI) led to a gradual progressive, cell type specific time course in neuronal degeneration. To analyse electrophysiological impairment we measured resting membrane potential, recorded spontaneous action potentials and induced action potentials by current pulses at different times after CHI. We found a dramatic increase in the frequency of spontaneous action potentials of CA1 but not of CA3 pyramidal cells after CHI. This hyperexcitability was maximal at 2 h (4.5-fold higher than sham), was also observed at 24 h after CHI and disappeared after 3 days. We found that CA1 interneurones responded by a much weaker increase of AP frequency after CHI. We conclude that the strong hyperexcitability after CHI is cell-type specific and transient. The understanding of the complex neuronal interactions probably offers a promising possibility for pharmacological intervention to prevent posttraumatic epilepsy.

Clonal T cell expansion through proliferation is a central process of the adaptive immune response. Apoptosis of activated T cells is required to avoid chronic inflammation. T cell proliferation and apoptosis are often analyzed with stimuli that do not induce formation of a functional immunological synapse. Here we analyze the Ca(2+) dependence of proliferation and apoptosis in primary human CD4(+) T cells following stimulation with anti-CD3/anti-CD28-coated beads, which induce a tight interaction similar to the immunological synapse. We found this focal stimulation to be much more efficient for stimulating IL-2 production and proliferation than non-focal TCR stimuli. Surprising little Ca(2+) entry through Ca(2+) channels was required for T cell proliferation. Transient free intracellular calcium concentration ([Ca(2+)](i)) elevations of up to 220 nM from a baseline level of around 40 nM were sufficient for maximal proliferation in primary human CD4(+) T cells. We also show that proliferation was very Ca(2+) sensitive in the range 90-120 nM, whereas apoptosis was basically constant for [Ca(2+)](i) levels of 90-120 nM. We conclude that very small changes in [Ca(2+)](i) can dramatically change the ratio between proliferation and apoptosis, thus keeping the balance between overshooting and inefficient immune responses.

The P2X(7) receptor is an ATP-gated ionotropic receptor that is permeable for small cations including Ca(2+) ions. Using 293 cells expressing P2X(7) receptors, we show that the P2X(7) receptor-specific ligand 2',3'-O-(4-benzoyl-benzoyl)-ATP (BzATP) induces a signaling cascade leading to the biosynthesis of biologically active Egr-1, a zinc finger transcription factor. BzATP-triggered Egr-1 biosynthesis was attenuated by the mitogen-activated protein kinase kinase inhibitor PD98059, by BAPTA-AM, the acetoxymethylester of the cytosolic Ca(2+) chelator BAPTA, and by an epidermal growth factor (EGF) receptor-specific tyrosine kinase inhibitor (AG1478). These results indicate that phosphorylation and activation of extracellular signal-regulated protein kinase ERK, elevated levels of intracellular Ca(2+) and the transactivation of the EGF receptor are essential for BzATP-induced upregulation of Egr-1. The requirement of Ca(2+) within the signaling cascade was upstream of Raf kinase activation. Lentiviral-mediated expression of MAP kinase phosphatase-1 (MKP-1), a dual-specific phosphatase that dephosphorylates and inactivates ERK in the nucleus, inhibited Egr-1 biosynthesis following BzATP stimulation, indicating that MKP-1 functions as a nuclear shut-off device. Furthermore, the ternary complex factor Elk-1 was phosphorylated and the transcriptional activation potential of Elk-1 was enhanced following P2X(7) receptor stimulation. Expression of a dominant-negative mutant of Elk-1 impaired BzATP-induced upregulation of Egr-1 biosynthesis. Thus, Elk-1 connects the intracellular signaling cascade elicited by activation of P2X(7) receptors with the transcription of the Egr-1 gene., ((c) 2007 Wiley-Liss, Inc.)

Lymphocyte signaling and activation leads to the influx of extracellular Ca(2+) via the activation of Ca(2+) release activated Ca(2+) (CRAC) channels in the plasma membrane. Activation of CRAC channels occurs following emptying of the endoplasmic reticulum intracellular Ca(2+) stores. One model to explain the coupling of store-emptying to CRAC activation is the secretion-like conformational coupling model. This model proposes that store depletion increases junctions between the endoplasmic reticulum and the plasma membrane in a manner that could be regulated by the cortical actin cytoskeleton. Here, we show that stabilization or depolymerization of the actin cytoskeleton failed to affect CRAC activation. We therefore conclude that rearrangement of the actin cytoskeleton is dispensable for store-operated Ca(2+) entry in T-cells.

Activation of T-lymphocytes requires stimulation of T-cell receptors (TCR) and co-stimulatory signals. Among different signalling cascades, TCR engagement induces Ca(2+) entry through plasma membrane Ca(2+) channels, which is an indispensable step for T-cells to expand clonally and to acquire effector functions. The Ca(2+) channels are activated by depletion of Ca(2+) stores and are called Ca(2+) release-activated Ca(2+) (CRAC) channels. Ca(2+) influx through CRAC channels is also controlled, directly or indirectly, by K(+) channels, Ca(2+)-ATPases, mitochondria, endoplasmic reticulum and Ca(2+) buffers. We review the functional implications of these transporters, organelles and buffers and develop a model of Ca(2+) signal generation that depends mainly on their relative mutual localization. This model offers the possibility of controlling amplitude and kinetics of Ca(2+) signals in T-cells. Decoding of various Ca(2+) signals allows differential activation of the transcription factor families nuclear factor of activated T-cells (NFAT), nuclear factor-kappaB (NF-kappaB) and activator protein-1 (AP-1). Variation of amplitude and kinetics of Ca(2+) signals thus is an important mechanism for modulating the specificity of T-cell responses.

Lamina propria (LP) T cells of the human intestinal mucosa usually do not develop systemic immune responses despite permanent exposure to foreign antigens. The mechanisms maintaining this hyporeactivity in the normal gut are poorly understood. It is, at present, not clear what role the microenvironment of the mucosa plays for low T cell reactivity and in the pathogenesis of mucosal inflammation. Despite the importance of cytosolic Ca(2+) signals for T lymphocyte activation, intracellular Ca(2+) concentration measurements have so far only been performed in dissociated T cells, following disruption of the microenvironment. We used two-photon technology to measure Ca(2+) signals in identified T lymphocytes within the intact mucosa to minimize impact on tissue integrity while preserving the cellular microenvironment. We show that Ca(2+) signals in LP T cells correlate with the hyporeactivity of T cells in the intestinal immune system and furthermore link Ca(2+) signals with inflammatory bowel disease. Our data implicate that Ca(2+) signals in LP T cells do not depend on the microenvironment of the intact mucosa, since they are very similar to Ca(2+) signals in dissociated LP T cells.

The contribution of ATP-sensitive potassium (K(ATP)) channels to neuronal excitability was studied in different types of pyramidal cells and interneurones in hippocampal slices prepared from 9- to 15-day-old rats. The presence of functional K(ATP) channels in the neurones was detected through the sensitivity of whole-cell currents to diazoxide, a K(ATP) channel opener, and to tolbutamide, a K(ATP) channel inhibitor. The percentages of neurones with K(ATP) channels increase in the sequence: CA1 pyramidal cells (37%)<

To better define the efficacy and tolerability of the new anticonvulsant topiramate in pediatric patients, the clinical courses of 49 children with intractable seizures were monitored during topiramate therapy. The 80% of children who had complex partial seizures experienced better seizure control with topiramate than the 20% who had generalized seizures. Efficacy was greatest with doses between 2.5 and 7.5 mg/kg/day. More than half the children on topiramate experienced adverse effects which could interfere with learning at school, but 20% demonstrated increased alertness or improved behavior. Topiramate is effective and may be considered as part of the treatment pathway for complex partial seizures in children, although careful monitoring of cognitive function is required.

Infantile spasms (IS) is an age-specific epilepsy which responds to anticonvulsant therapy but has a generally poor prognosis for normal psychomotor development. The subgroup of infants with a cryptogenic aetiology or whose therapy is initiated promptly is thought to have a more favourable prognosis. We retrospectively reviewed 28 infants with IS treated between 1990 and 1996 with adrenocorticotropic hormone (ACTH), valproic acid (VPA), or both, in order to correlate therapeutic response with long-term outcome. Mean age at onset of treatment was 6.4 months, with 57% of patients started within 1 month of IS appearance. IS was considered cryptogenic in 39%. The majority of infants responded to ACTH or VPA with a reduction in spasms of 75% or more. Total remission of seizures occurred in 52%. Death occurred in eight patients; mean duration of follow-up for survivors was 55 months. All subgroups based on age, aetiology, or treatment had poor outcomes, commonly with residual epilepsy, cerebral palsy or mental retardation. Conventional treatment for IS, even when initially successful in reducing spasms, is inadequate when viewed from a long-term developmental perspective, suggesting the need for novel innovative approaches for treating IS.

Although infantile spasms (IS) constitute a well-recognized epileptic syndrome, only recently did investigators propose that spasms be classified as a distinct seizure type, characterized by axial flexion/extension jerks in clusters. Five older children (aged 4.5-14.2 years) who underwent video-EEG monitoring in 1992 in our epilepsy program for intractable mixed seizure disorder (cryptogenic 1, symptomatic 4) demonstrated flexor and extensor spasms in clusters. Seizure onset was from birth to 1.33 years. All 5 had spasms during infancy that continued as the children aged. Ictal EEG during spasms showed a brief high-amplitude delta burst followed by diffuse background attenuation or diffuse background decrease with superimposed rhythmic beta or alpha activity. Multiple other seizure types were present. Interictal EEGs were markedly abnormal and demonstrated slowing, multifocal spikes, generalized slow spike-wave, and polyspike-wave. Two children with spasms were unsuccessfully treated with ACTH, and 3 underwent corpus callosotomy. We conclude that spasms occur in older children with intractable mixed seizure disorders and may persist beyond infancy.