Locus Coeruleus Optogenetic Light Activation Induces Long-Term Potentiation of Perforant Path Population Spike Amplitude in Rat Dentate Gyrus

Norepinephrine (NE) in dentate gyrus (DG) produces NE-dependent long-term potentiation (NE-LTP) of the perforant path-evoked potential population spike both in vitro and in vivo. Chemical activators infused near locus coeruleus (LC), the source of DG NE, produce a NE-LTP that is associative, i.e., requires concurrent pairing with perforant path (PP) input. Here, we ask if LC optogenetic stimulation that allows us to activate only LC neurons can induce NE-LTP in DG. We use an adeno-associated viral vector containing a depolarizing channel (AAV8-Ef1a-DIO-eChR2(h134r)-EYFP-WPRE) infused stereotaxically into the LC of TH:Cre rats to produce light-sensitive LC neurons. A co-localization of ~62% in LC neurons was observed for these channels. Under urethane anesthesia, we demonstrated that 5–10 s 10 Hz trains of 30 ms light pulses in LC reliably activated neurons near an LC optoprobe. Ten minutes of the same train paired with 0.1 Hz PP electrical stimulation produced a delayed NE-LTP of population spike amplitude, but not EPSP slope. A leftward shift in the population spike input/output curve at the end of the experiment was also consistent with long-term population spike potentiation. LC neuron activity during the 10 min light train was unexpectedly transient. Increased LC neuronal firing was seen only for the first 2 min of the light train. NE-LTP was more delayed and less robust than reported with LC chemo-activation. Previous estimates of LC axonal conduction times suggest acute release of NE occurs 40–70 ms after an LC neuron action potential. We used single LC light pulses to examine acute effects of NE release and found potentiated population spike amplitude when a light pulse in LC occurred 40–50 ms, but not 20–30 ms, prior to a PP pulse, consistent with conduction estimates. These effects of LC optogenetic activation reinforce evidence for a continuum of NE potentiation effects in DG. The single pulse effects mirror an earlier report using LC electrical stimulation. These acute effects support an attentional role of LC activation. The LTP of PP responses induced by optogenetic LC activation is consistent with the role of LC in long-term learning and memory

ChlP-Seq reveals that QsMYB1 directly targets genes involved in lignin and suberin biosynthesis pathways in cork oak (Quercus suber)

Research ArticleBackground: Gene activity is largely controlled by transcriptional regulation through the action of transcription factors and other regulators. QsMYB1 is a member of the R2R3-MYB transcription factor family related to secondary growth, and in particular, with the cork development process. In order to identify the putative gene targets of QsMYB1 across the cork oak genome we developed a ChIP-Seq strategy. Results: Results provide direct evidence that QsMY1B targets genes encoding for enzymes involved in the lignin and suberin pathways as well as gene encoding for ABCG transporters and LTPs implicated in the transport of monomeric suberin units across the cellular membrane. These results highlight the role of QsMYB1 as a regulator of lignin and suberin biosynthesis, transport and assembly. Conclusion: To our knowledge, this work constitutes the first ChIP-Seq experiment performed in cork oak, a non-model plant species with a long-life cycle, and these results will contribute to deepen the knowledge about the molecular mechanisms of cork formation and differentiationinfo:eu-repo/semantics/publishedVersio

HNRNPC haploinsufficiency affects alternative splicing of intellectual disability-associated genes and causes a neurodevelopmental disorder

Heterogeneous nuclear ribonucleoprotein C (HNRNPC) is an essential, ubiquitously abundant protein involved in mRNA processing. Genetic variants in other members of the HNRNP family have been associated with neurodevelopmental disorders. Here, we describe 13 individuals with global developmental delay, intellectual disability, behavioral abnormalities, and subtle facial dysmorphology with heterozygous HNRNPC germline variants. Five of them bear an identical in-frame deletion of nine amino acids in the extreme C terminus. To study the effect of this recurrent variant as well as HNRNPC haploinsufficiency, we used induced pluripotent stem cells (iPSCs) and fibroblasts obtained from affected individuals. While protein localization and oligomerization were unaffected by the recurrent C-terminal deletion variant, total HNRNPC levels were decreased. Previously, reduced HNRNPC levels have been associated with changes in alternative splicing. Therefore, we performed a meta-analysis on published RNA-seq datasets of three different cell lines to identify a ubiquitous HNRNPC-dependent signature of alternative spliced exons. The identified signature was not only confirmed in fibroblasts obtained from an affected individual but also showed a significant enrichment for genes associated with intellectual disability. Hence, we assessed the effect of decreased and increased levels of HNRNPC on neuronal arborization and neuronal migration and found that either condition affects neuronal function. Taken together, our data indicate that HNRNPC haploinsufficiency affects alternative splicing of multiple intellectual disability-associated genes and that the developing brain is sensitive to aberrant levels of HNRNPC. Hence, our data strongly support the inclusion of HNRNPC to the family of HNRNP-related neurodevelopmental disorders. [Display omitted] We identified genetic variants of HNRNPC in 13 individuals with intellectual disability and global developmental delay. Through a meta-analysis of multiple cell types, we found that loss of HNRNPC affects alternative splicing, in particular of intellectual disability-associated genes. In vivo assays confirmed that neurodevelopment was affected by aberrant HNRNPC levels

Dominant negative variants in IKZF2 cause ICHAD syndrome, a new disorder characterised by immunodysregulation, craniofacial anomalies, hearing impairment, athelia and developmental delay

BackgroundHelios (encoded by IKZF2), a member of the Ikaros family of transcription factors, is a zinc finger protein involved in embryogenesis and immune function. Although predominantly recognised for its role in the development and function of T lymphocytes, particularly the CD4+ regulatory T cells (Tregs), the expression and function of Helios extends beyond the immune system. During embryogenesis, Helios is expressed in a wide range of tissues, making genetic variants that disrupt the function of Helios strong candidates for causing widespread immune-related and developmental abnormalities in humans.MethodsWe performed detailed phenotypic, genomic and functional investigations on two unrelated individuals with a phenotype of immune dysregulation combined with syndromic features including craniofacial differences, sensorineural hearing loss and congenital abnormalities.ResultsGenome sequencing revealed de novo heterozygous variants that alter the critical DNA-binding zinc fingers (ZFs) of Helios. Proband 1 had a tandem duplication of ZFs 2 and 3 in the DNA-binding domain of Helios (p.Gly136_Ser191dup) and Proband 2 had a missense variant impacting one of the key residues for specific base recognition and DNA interaction in ZF2 of Helios (p.Gly153Arg). Functional studies confirmed that both these variant proteins are expressed and that they interfere with the ability of the wild-type Helios protein to perform its canonical function—repressing IL2 transcription activity—in a dominant negative manner.ConclusionThis study is the first to describe dominant negative IKZF2 variants. These variants cause a novel genetic syndrome characterised by immunodysregulation, craniofacial anomalies, hearing impairment, athelia and developmental delay

Loss-of-function in RBBP5 results in a syndromic neurodevelopmental disorder associated with microcephaly

Epigenetic dysregulation has been associated with many inherited disorders. RBBP5 (HGNC:9888) encodes a core member of the protein complex that methylates histone 3 lysine-4 (H3K4) and has not been implicated in human disease. We identify five unrelated individuals with de novo heterozygous variants in RBBP5. Three nonsense/frameshift and two missense variants were identified in probands with neurodevelopmental symptoms including global developmental delay, intellectual disability, microcephaly, and short stature. Here, we investigate the pathogenicity of the variants through protein structural analysis and transgenic Drosophila models. Both missense p.(T232I) and p.(E296D) variants affect evolutionarily conserved amino acids located at the interface between RBBP5 and the nucleosome. In Drosophila, overexpression analysis identifies partial loss-of-function mechanisms when the variants are expressed using the fly Rbbp5 or human RBBP5 cDNA. Loss of Rbbp5 leads to a reduction in brain size. The human reference or variant transgenes fail to rescue this loss and expression of either missense variant in an Rbbp5 null background results in a less severe microcephaly phenotype than the human reference, indicating both missense variants are partial loss-of-function alleles. Haploinsufficiency of RBBP5 observed through de novo null and hypomorphic loss-of-function variants is associated with a syndromic neurodevelopmental disorder. Huang et al. report the first functional validation of candidate pathological variants in RBBP5. We present three truncating p.(K244Nfs*6), p.(W254*), p.(R307*) and two missense p.(T232I), p.(E296D) variants found de novo in affected individuals sharing phenotypes including microcephaly and short stature. RBBP5 is a core member of the COMPASS complex responsible for H3 lysine 4 methylation to activate developmental target genes (COMPASS complex adapted from Namitz et al., 2023). Differentiation of neural stem cells in humans and neuroblasts in Drosophila is conserved allowing for the study of neural development in the fly model organism (neural stem cell/neuroblast differentiation diagram adapted from Kim and Hirth, 2009). We used overexpression and rescue experiments to characterize the missense variants in the fly. Neural progenitor populations were evaluated in the larval brain and tissue specific phenotypes were quantified using adult eye and wing morphology studies. We identify that the truncating and missense variants are loss-of-function alleles. As additional patients are identified, the full phenotypic spectrum of RBBP5-related disorders will be elucidated. Created with Biorender.com. [Display omitted

Biallelic variants in ribonuclease inhibitor (RNH1), an inflammasome modulator, are associated with a distinctive subtype of acute, necrotizing encephalopathy

Mendelian etiologies for acute encephalopathies in previously healthy children are poorly understood, with the exception of RAN binding protein 2 (RANBP2)–associated acute necrotizing encephalopathy subtype 1 (ANE1). We provide clinical, genetic, and neuroradiological evidence that biallelic variants in ribonuclease inhibitor (RNH1) confer susceptibility to a distinctive ANE subtype. This study aimed to evaluate clinical data, neuroradiological studies, genomic sequencing, and protein immunoblotting results in 8 children from 4 families who experienced acute febrile encephalopathy. All 8 healthy children became acutely encephalopathic during a viral/febrile illness and received a variety of immune modulation treatments. Long-term outcomes varied from death to severe neurologic deficits to normal outcomes. The neuroradiological findings overlapped with ANE but had distinguishing features. All affected children had biallelic predicted damaging variants in RNH1: a subset that was studied had undetectable RNH1 protein. Incomplete penetrance of the RNH1 variants was evident in 1 family. Biallelic variants in RNH1 confer susceptibility to a subtype of ANE (ANE2) in previously healthy children. Intensive immunological treatments may alter outcomes. Genomic sequencing in children with unexplained acute febrile encephalopathy can detect underlying genetic etiologies, such as RNH1, and improve outcomes in the probands and at-risk siblings

De novo missense variants in phosphatidylinositol kinase PIP5KIγ underlie a neurodevelopmental syndrome associated with altered phosphoinositide signaling

Phosphoinositides (PIs) are membrane phospholipids produced through the local activity of PI kinases and phosphatases that selectively add or remove phosphate groups from the inositol head group. PIs control membrane composition and play key roles in many cellular processes including actin dynamics, endosomal trafficking, autophagy, and nuclear functions. Mutations in phosphatidylinositol 4,5 bisphosphate [PI(4,5)P2] phosphatases cause a broad spectrum of neurodevelopmental disorders such as Lowe and Joubert syndromes and congenital muscular dystrophy with cataracts and intellectual disability, which are thus associated with increased levels of PI(4,5)P2. Here, we describe a neurodevelopmental disorder associated with an increase in the production of PI(4,5)P2 and with PI-signaling dysfunction. We identified three de novo heterozygous missense variants in PIP5K1C, which encodes an isoform of the phosphatidylinositol 4-phosphate 5-kinase (PIP5KIγ), in nine unrelated children exhibiting intellectual disability, developmental delay, acquired microcephaly, seizures, visual abnormalities, and dysmorphic features. We provide evidence that the PIP5K1C variants result in an increase of the endosomal PI(4,5)P2 pool, giving rise to ectopic recruitment of filamentous actin at early endosomes (EEs) that in turn causes dysfunction in EE trafficking. In addition, we generated an in vivo zebrafish model that recapitulates the disorder we describe with developmental defects affecting the forebrain, including the eyes, as well as craniofacial abnormalities, further demonstrating the pathogenic effect of the PIP5K1C variants. We describe a neurodevelopmental disorder associated with de novo gain-of-function variants in PIP5KIγ kinase. The variants cause perturbed endosomal function resulting from increased production of phosphatidylinositol 4,5 bisphosphate and enhanced association of F-actin at endosomes. Moreover, mutant zebrafish larvae recapitulate the phenotypes observed in affected individuals from our cohort