Esophageal epithelial Ikkß deletion promotes eosinophilic esophagitis in experimental allergy mouse model.

Background: Eosinophilic esophagitis (EoE) is a chronic T helper type 2 (Th2)-associated inflammatory disorder triggered by food allergens, resulting in esophageal dysfunction through edema, fibrosis, and tissue remodeling. The role of epithelial remodeling in EoE pathogenesis is critical but not fully understood. Objective: To investigate the role of epithelial IKKß/NFκB signaling in EoE pathogenesis using a mouse model with conditional Ikk ß knockout in esophageal epithelial cells ( Ikk ß EEC-KO ). Methods: EoE was induced in Ikkß EEC-KO mice through skin sensitization with MC903/Ovalbumin (OVA) followed by intraesophageal OVA challenge. Histological and transcriptional analyses were performed to assess EoE features. Single-cell RNA sequencing (scRNA-seq) was used to profile esophageal mucosal cell populations and gene expression changes. Results: Ikkß EEC-KO /EoE mice exhibited hallmark EoE features, including eosinophil infiltration, intraepithelial eosinophils, microabscesses, basal cell hyperplasia, and lamina propria remodeling. RNA-seq revealed significant alterations in IKKß/NFκB signaling pathways, with decreased expression of RELA and increased expression of IKKß negative regulators. scRNA- seq analyses identified disrupted epithelial differentiation and barrier integrity, alongside increased type 2 immune responses and peptidase activity. Conclusion: Our study demonstrates that loss of epithelial IKKß signaling exacerbates EoE pathogenesis, highlighting the critical role of this pathway in maintaining epithelial homeostasis and preventing allergic inflammation. The Ikkß EEC-KO /EoE mouse model closely mirrors human EoE, providing a valuable tool for investigating disease mechanisms and therapeutic targets. This model can facilitate the development of strategies to prevent chronic inflammation and tissue remodeling in EoE. Key Messages: Critical Role of Epithelial IKKß/NFκB Signaling: Loss of this signaling exacerbates EoE, causing eosinophil infiltration, basal cell hyperplasia, and tissue remodeling, highlighting its importance in esophageal health.Molecular Insights and Therapeutic Targets: scRNA-seq identified disrupted epithelial differentiation, barrier integrity, and enhanced type 2 immune responses, suggesting potential therapeutic targets for EoE. Relevance of the Ikkß EEC-KO /EoE Mouse Model: This model replicates human EoE features, making it a valuable tool for studying EoE mechanisms and testing treatments, which can drive the development of effective therapies. Capsule Summary: This study reveals the crucial role of epithelial IKKß/NFκB signaling in EoE, providing insights into disease mechanisms and potential therapeutic targets, highly relevant for advancing clinical management of EoE.

The Transcription Factor, α1ACT, Acts Through a MicroRNA Network to Regulate Neurogenesis and Cell Death During Neonatal Cerebellar Development.

MicroRNAs, a class of small RNA regulators, function throughout neurodevelopment, from neural stem cell neurogenesis to neuronal maturation, synaptic formation, and plasticity. α1ACT, a transcription factor (TF), plays a critical role in neonatal cerebellar development by regulating an ensemble of genes. Of these, ChIP-seq analysis matched near 50% genes directly regulated by α1ACT. Yet, more than half the regulated transcripts lacked direct interaction with α1ACT. To investigate whether α1ACT acts through a microRNA network, we studied α1ACT-associated simultaneous miRNA:mRNA transcriptome profiles, using miRNA-seq paired with RNA-seq. Thirty-one differentially expressed miRNAs (DEMs) associated with α1ACT-regulated differentially expressed genes (DEGs) were profiled in α1ACT-overexpressing PC12 cells and were further validated in neonatal transgenic mouse cerebellum overexpressing α1ACT in a context-dependent manner. Here, we also demonstrated that α1ACT facilitates neurogenesis and development of dendritic synapses and is partially a result of the downregulation of the miR-99 cluster, miR-143, miR-23, miR-146, miR-363, and miR-484. On the other hand, the miR-181, miR-125, and miR-708 clusters were upregulated by α1ACT, which inhibit MAPK signaling and cell death pathways by targeting Ask1, Odc1, Atf4, and Nuf2 for decreased expression. MiR-181a-5p was verified as the most abundant DEM in neonatal cerebellum, which was further induced by α1ACT. Overall, under α1ACT modulation, up-/downregulated miRNA clusters with their paired target genes may form a regulatory network controlling the balance between the neuronal proliferation, differentiation, and cell death in the cerebellum to promote neonatal development. Our findings concerning the α1ACT-related miRNA/mRNA expression profiles in neonatal cerebellum may inform future investigations for cerebellar development.

Sensory Over-responsivity and Aberrant Plasticity in Cerebellar Cortex in a Mouse Model of Syndromic Autism.

Background: Patients with autism spectrum disorder often show altered responses to sensory stimuli as well as motor deficits, including an impairment of delay eyeblink conditioning, which involves integration of sensory signals in the cerebellum. Here, we identify abnormalities in parallel fiber (PF) and climbing fiber (CF) signaling in the mouse cerebellar cortex that may contribute to these pathologies. Methods: We used a mouse model for the human 15q11-13 duplication (patDp/+) and studied responses to sensory stimuli in Purkinje cells from awake mice using two-photon imaging of GCaMP6f signals. Moreover, we examined synaptic transmission and plasticity using in vitro electrophysiological, immunohistochemical, and confocal microscopic techniques. Results: We found that spontaneous and sensory-evoked CF-calcium transients are enhanced in patDp/+ Purkinje cells, and aversive movements are more severe across sensory modalities. We observed increased expression of the synaptic organizer NRXN1 at CF synapses and ectopic spread of these synapses to fine dendrites. CF-excitatory postsynaptic currents recorded from Purkinje cells are enlarged in patDp/+ mice, while responses to PF stimulation are reduced. Confocal measurements show reduced PF+CF-evoked spine calcium transients, a key trigger for PF long-term depression, one of several plasticity types required for eyeblink conditioning learning. Long-term depression is impaired in patDp/+ mice but is rescued on pharmacological enhancement of calcium signaling. Conclusions: Our findings suggest that this genetic abnormality causes a pathological inflation of CF signaling, possibly resulting from enhanced NRXN1 expression, with consequences for the representation of sensory stimuli by the CF input and for PF synaptic organization and plasticity.

HER2 c-Terminal Fragments Are Expressed via Internal Translation of the HER2 mRNA.

The HER2/neu signaling pathway is one of the most frequently mutated in human cancer. Although therapeutics targeting this pathway have good efficacy, cancer cells frequently develop resistance. The HER2 gene encodes the full-length HER2 protein, as well as smaller c-terminal fragments (CTFs), which have been shown to be a cause of resistance. Here, we show that HER2 CTFs, exclusive from the full-length HER2 protein, are generated via internal translation of the full-length HER2 mRNA and identify regions which are required for this mechanism to occur. These regions of the HER2 mRNA may present novel sites for therapeutic intervention via small molecules or antisense oligonucleotides (ASOs).

Loss-of-function BK channel mutation causes impaired mitochondria and progressive cerebellar ataxia.

Despite a growing number of ion channel genes implicated in hereditary ataxia, it remains unclear how ion channel mutations lead to loss-of-function or death of cerebellar neurons. Mutations in the gene KCNMA1, encoding the α-subunit of the BK channel have emerged as responsible for a variety of neurological phenotypes. We describe a mutation (BKG354S) in KCNMA1, in a child with congenital and progressive cerebellar ataxia with cognitive impairment. The mutation in the BK channel selectivity filter dramatically reduced single-channel conductance and ion selectivity. The BKG354S channel trafficked normally to plasma, nuclear, and mitochondrial membranes, but caused reduced neurite outgrowth, cell viability, and mitochondrial content. Small interfering RNA (siRNA) knockdown of endogenous BK channels had similar effects. The BK activator, NS1619, rescued BKG354S cells but not siRNA-treated cells, by selectively blocking the mutant channels. When expressed in cerebellum via adenoassociated virus (AAV) viral transfection in mice, the mutant BKG354S channel, but not the BKWT channel, caused progressive impairment of several gait parameters consistent with cerebellar dysfunction from 40- to 80-d-old mice. Finally, treatment of the patient with chlorzoxazone, a BK/SK channel activator, partially improved motor function, but ataxia continued to progress. These studies indicate that a loss-of-function BK channel mutation causes ataxia and acts by reducing mitochondrial and subsequently cellular viability.

α1ACT Is Essential for Survival and Early Cerebellar Programming in a Critical Neonatal Window.

Postnatal cerebellar development is a precisely regulated process involving well-orchestrated expression of neural genes. Neurological phenotypes associated with CACNA1A gene defects have been increasingly recognized, yet the molecular principles underlying this association remain elusive. By characterizing a dose-dependent CACNA1A gene deficiency mouse model, we discovered that α1ACT, as a transcription factor and secondary protein of CACNA1A mRNA, drives dynamic gene expression networks within cerebellar Purkinje cells and is indispensable for neonatal survival. Perinatal loss of α1ACT leads to motor dysfunction through disruption of neurogenesis and synaptic regulatory networks. However, its elimination in adulthood has minimal effect on the cerebellum. These findings shed light on the critical role of α1ACT in facilitating neuronal development in both mice and humans and support a rationale for gene therapies for calcium-channel-associated cerebellar disorders. Finally, we show that bicistronic expression may be common to the voltage-gated calcium channel (VGCC) gene family and may help explain complex genetic syndromes.