Gene choice in cancer cells is exclusive in ion transport but concurrent in DNA replication.

Cancers share common cellular and physiological features. Little is known about whether distinctive gene expression patterns can be displayed at the single-cell level by gene families in cancer cells. The expression of gene homologs within a family can exhibit concurrence and exclusivity. Concurrence can promote all-or-none expression patterns of related genes and underlie alternative physiological states. Conversely, exclusive gene families express the same or similar number of homologs in each cell, allowing a broad repertoire of cell identities to be generated. We show that gene families involved in the cell-cycle and antigen presentation are expressed concurrently. Concurrence in the DNA replication complex MCM reflects the replicative status of cells, including cell lines and cancer-derived organoids. Exclusive expression requires precise regulatory mechanism, but cancer cells retain this form of control for ion homeostasis and extend it to gene families involved in cell migration. Thus, the cell adhesion-based identity of healthy cells is transformed to an identity based on migration in the population of cancer cells, reminiscent of epithelial-mesenchymal transition.

Reactive astrocytes promote tumor progression by up-regulating tumor protocadherin 1 expression in lung cancer brain metastasis.

Brain metastasis (BM) is one of the main causes of death in patients with non-small cell lung carcinoma. The specific pathological processes of BM, which are inextricably linked to the brain tumor microenvironment, such as the abundance of astrocytes, lead to limited treatment options and poor prognosis. Reactive astrocytes are acquired in the BM; however, the underlying mechanisms remain unclear. This study aimed to explore the mechanisms by which astrocytes promote BM development. We determined the crucial role of reactive astrocytes in promoting the proliferation and migration of brain metastatic lung tumor cells by upregulating protocadherin 1 (PCDH1) expression in an in vitro co-culture model. The overexpression of PCDH1 was confirmed in clinical BM samples using immunohistochemical staining. Survival analysis indicated that high-PCDH1 expression was associated with poor survival in patients with lung adenocarcinoma. In vivo assays further showed that silence of PCDH1 effectively inhibited the tumor progression of brain metastases and prolonged the survival of animals. RNA sequencing has revealed that PCDH1 plays an important role in cell proliferation and adhesion. In conclusion, the present study revealed the promoting role of astrocytes in enhancing the aggressive phenotype of brain metastatic tumor cells by regulating the expression of PCDH1, which might be a biomarker for BM diagnosis and prognosis, suggesting the potential efficacy of targeting important astrocyte-tumor interactions in the treatment of patients with non-small cell lung carcinoma with BM.

Multicenter retrospective study of patients with PCDH19-related epilepsy: The first Hungarian cohort.

OBJECTIVE: PCDH19-related epilepsy occurs predominantly in girls and is caused by pathogenic variant of the protocadherin-19 gene. The initial seizures usually develop in association with fever, begin on average at 15 months of age, and often occur in clusters. Autistic symptoms, intellectual disability, and sleep disturbance are often associated. METHODS: In our retrospective, multicenter study, we reviewed clinical data of nine children with epilepsy genetically confirmed to be associated with PCDH19. RESULTS: In the Hungarian patient population aged 0-18 years, the prevalence of PCDH19-related epilepsy was found to be lower (1/100000 live births in females) than the reported international data (4-5/100000 live births in females). Four of our nine patients had positive family history of epilepsy (cousins, sister, and mother). We assessed brain anomalies in three patients (in one patient focal cortical dysplasia and left anterior cingulate dysgenesis, and in two children right or left hippocampal sclerosis) and in another three cases incidentally identified benign alterations on brain MRI were found. The first seizure presented as a cluster in seven out of nine children. In seven out of nine cases occurred status epilepticus. Six out of nine children had autistic symptoms and only one child had normal intellectual development. Seven of our patients were seizure free with combined antiseizure medication (ASM). The most effective ASMs were levetiracetam, valproate, and clobazam. SIGNIFICANCE: The prevalence of PCDH19-related epilepsy is presumably underestimated because of the lack of widely performed molecular genetic evaluations. Molecular genetic testing including PCDH19 pathogenic variants is recommended for female patients with an onset of seizures before the age of 3 years.

NGS-Based Identification of Two Novel PCDH19 Mutations in Female Patients with Early-Onset Epilepsy.

Developmental and epileptic encephalopathy-9 (DEE9) is characterized by seizure onset in infancy, mild to severe intellectual impairment, and psychiatric features and is caused by a mutation in the PCDH19 gene on chromosome Xq22. The rare, unusual X-linked type of disorder affects heterozygous females and mosaic males; transmitting males are unaffected. In our study, 165 patients with epilepsy were tested by Next Generation Sequencing (NGS)-based panel and exome sequencing using Illumina technology. PCDH19 screening identified three point mutations, one indel, and one 29 bp-long deletion in five unrelated female probands. Two novel mutations, c.1152_1180del (p.Gln385Serfs*6) and c.830_831delinsAA (p.Phe277*), were identified and found to be de novo pathogenic. Moreover, among the three inherited mutations, two originated from asymptomatic mothers and one from an affected father. The PCDH19 c.1682C>T and c.1711G>T mutations were present in the DNA samples of asymptomatic mothers. After targeted parental testing, X chromosome inactivation tests and Sanger sequencing were carried out for mosaicism examination on maternal saliva samples in the two asymptomatic PCDH19 mutation carrier subjects. Tissue mosaicism and X-inactivation tests were negative. Our results support the opportunity for reduced penetrance in DEE9 and contribute to expanding the genotype-phenotype spectrum of PCDH19-related epilepsy.

Altered socio-affective communication and amygdala development in mice with protocadherin10-deficient interneurons.

Autism spectrum disorder (ASD) is a group of neurodevelopmental conditions associated with deficits in social interaction and communication, together with repetitive behaviours. The cell adhesion molecule protocadherin10 (PCDH10) is linked to ASD in humans. Pcdh10 is expressed in the nervous system during embryonic and early postnatal development and is important for neural circuit formation. In mice, strong expression of Pcdh10 in the ganglionic eminences and in the basolateral complex (BLC) of the amygdala was observed at mid and late embryonic stages, respectively. Both inhibitory and excitatory neurons expressed Pcdh10 in the BLC at perinatal stages and vocalization-related genes were enriched in Pcdh10-expressing neurons in adult mice. An epitope-tagged Pcdh10-HAV5 mouse line revealed endogenous interactions of PCDH10 with synaptic proteins in the young postnatal telencephalon. Nuanced socio-affective communication changes in call emission rates, acoustic features and call subtype clustering were primarily observed in heterozygous pups of a conditional knockout (cKO) with selective deletion of Pcdh10 in Gsh2-lineage interneurons. These changes were less prominent in heterozygous ubiquitous Pcdh10 KO pups, suggesting that altered anxiety levels associated with Gsh2-lineage interneuron functioning might drive the behavioural effects. Together, loss of Pcdh10 specifically in interneurons contributes to behavioural alterations in socio-affective communication with relevance to ASD.

Mapping combinatorial expression of non-clustered protocadherins in the developing brain identifies novel PCDH19-mediated cell adhesion properties.

Non-clustered protocadherins (ncPcdhs) are adhesive molecules with spatio-temporally regulated overlapping expression in the developing nervous system. Although their unique role in neurogenesis has been widely studied, their combinatorial role in brain physiology and pathology is poorly understood. Using probabilistic cell typing by in situ sequencing, we demonstrate combinatorial inter- and intra-familial expression of ncPcdhs in the developing mouse cortex and hippocampus, at single-cell resolution. We discovered the combinatorial expression of Protocadherin-19 (Pcdh19), a protein involved in PCDH19-clustering epilepsy, with Pcdh1, Pcdh9 or Cadherin 13 (Cdh13) in excitatory neurons. Using aggregation assays, we demonstrate a code-specific adhesion function of PCDH19; mosaic PCDH19 absence in PCDH19+9 and PCDH19 + CDH13, but not in PCDH19+1 codes, alters cell-cell interaction. Interestingly, we found that PCDH19 as a dominant protein in two heterophilic adhesion codes could promote trans-interaction between them. In addition, we discovered increased CDH13-mediated cell adhesion in the presence of PCDH19, suggesting a potential role of PCDH19 as an adhesion mediator of CDH13. Finally, we demonstrated novel cis-interactions between PCDH19 and PCDH1, PCDH9 and CDH13. These observations suggest that there is a unique combinatorial code with a cell- and region-specific characteristic where a single molecule defines the heterophilic cell-cell adhesion properties of each code.

Abnormal cell sorting and altered early neurogenesis in a human cortical organoid model of Protocadherin-19 clustering epilepsy.

Introduction: Protocadherin-19 (PCDH19)-Clustering Epilepsy (PCE) is a developmental and epileptic encephalopathy caused by loss-of-function variants of the PCDH19 gene on the X-chromosome. PCE affects females and mosaic males while male carriers are largely spared. Mosaic expression of the cell adhesion molecule PCDH19 due to random X-chromosome inactivation is thought to impair cell-cell interactions between mutant and wild type PCDH19-expressing cells to produce the disease. Progress has been made in understanding PCE using rodent models or patient induced pluripotent stem cells (iPSCs). However, rodents do not faithfully model key aspects of human brain development, and patient iPSC models are limited by issues with random X-chromosome inactivation. Methods: To overcome these challenges and model mosaic PCDH19 expression in vitro, we generated isogenic female human embryonic stem cells with either HA-FLAG-tagged PCDH19 (WT) or homozygous PCDH19 knockout (KO) using genome editing. We then mixed GFP-labeled WT and RFP-labeled KO cells and generated human cortical organoids (hCOs). Results: We found that PCDH19 is highly expressed in early (days 20-35) WT neural rosettes where it co-localizes with N-Cadherin in ventricular zone (VZ)-like regions. Mosaic PCE hCOs displayed abnormal cell sorting in the VZ with KO and WT cells completely segregated. This segregation remained robust when WT:KO cells were mixed at 2:1 or 1:2 ratios. PCE hCOs also exhibited altered expression of PCDH19 (in WT cells) and N-Cadherin, and abnormal deep layer neurogenesis. None of these abnormalities were observed in hCOs generated by mixing only WT or only KO (modeling male carrier) cells. Discussion: Our results using the mosaic PCE hCO model suggest that PCDH19 plays a critical role in human VZ radial glial organization and early cortical development. This model should offer a key platform for exploring mechanisms underlying PCE-related cortical hyperexcitability and testing of potential precision therapies.

DNA methylation-altered genes in the rat hippocampal neurogenic niche after continuous exposure to amorphous curcumin.

Rat offspring who are exposed to an amorphous formula of curcumin (CUR) from the embryonic stage have anti-anxiety-like behaviors, enhanced fear extinction learning, and increased synaptic plasticity in the hippocampal dentate gyrus (DG). In the present study, we investigated the links between genes with altered methylation status in the neurogenic niche and enhanced neural functions after CUR exposure. We conducted methylation and RNA sequencing analyses of the DG of CUR-exposed rat offspring on day 77 after delivery. Methylation status and transcript levels of candidate genes were validated using methylation-sensitive high-resolution melting and real-time reverse-transcription PCR, respectively. In the CUR group, we confirmed the hypermethylation and downregulation of Gpr150, Mmp23, Rprml, and Pcdh8 as well as the hypomethylation and upregulation of Ppm1j, Fam222a, and Opn3. Immunohistochemically, reprimo-like+ hilar cells and protocadherin-8+ granule cells were decreased and opsin-3+ hilar cells were increased by CUR exposure. Both reprimo-like and opsin-3 were partially expressed on subpopulations of glutamic acid decarboxylase 67+ γ-aminobutyric acid-ergic interneurons. Furthermore, the transcript levels of genes involved in protocadherin-8-mediated N-cadherin endocytosis were altered with CUR exposure; this was accompanied by Ctnnb1 and Syp upregulation and Mapk14, Map2k3, and Grip1 downregulation, suggesting that CUR-induced enhanced synaptic plasticity is associated with cell adhesion. Together, our results indicate that functionally different genes have altered methylation and expression in different neuronal populations of the hippocampal neurogenic niche, thus enhancing synaptic plasticity after CUR exposure.

PCDH19-clustering epilepsy, pathophysiology and clinical significance.

PCDH19 clustering epilepsy (PCDH19-CE) is an X-linked epilepsy disorder associated with intellectual disability (ID) and behavioral disturbances, which is caused by PCDH19 gene variants. PCDH19 pathogenic variant leads to epilepsy in heterozygous females, not in hemizygous males and the inheritance pattern is unusual. The hypothesis of cellular interference was described as a key pathogenic mechanism. According to that, males do not develop the disease because of the uniform expression of PCDH19 (variant or wild type) unless they have a somatic variation. We conducted a literature review on PCDH19-CE pathophysiology and concluded that other significant mechanisms could contribute to pathogenesis including: asymmetric cell division and heterochrony, female-related allopregnanolone deficiency, altered steroid gene expression, decreased Gamma-aminobutyric acid receptor A (GABAA) function, and blood-brain barrier (BBB) dysfunction. Being aware of these mechanisms helps us when we should decide which therapeutic option is more suitable for which patient.

Rescue of cone and rod photoreceptor function in a CDHR1-model of age-related retinal degeneration.

Age-related macular degeneration (AMD) is the most common cause of untreatable blindness in the developed world. Recently, CDHR1 has been identified as the cause of a subset of AMD that has the appearance of the "dry" form, or geographic atrophy. Biallelic variants in CDHR1-a specialized protocadherin highly expressed in cone and rod photoreceptors-result in blindness from shortened photoreceptor outer segments and progressive photoreceptor cell death. Here we demonstrate long-term morphological, ultrastructural, functional, and behavioral rescue following CDHR1 gene therapy in a relevant murine model, sustained to 23-months after injection. This represents the first demonstration of rescue of a monogenic cadherinopathy in vivo. Moreover, the durability of CDHR1 gene therapy seems to be near complete-with morphological findings of the rescued retina not obviously different from wildtype throughout the lifespan of the mouse model. A follow-on clinical trial in patients with CDHR1-associated retinal degeneration is warranted. Hypomorphic CDHR1 variants may mimic advanced dry AMD. Accurate clinical classification is now critical, as their pathogenesis and treatment are distinct.

Synaptic adhesion molecule protocadherin-γC5 mediates ß-amyloid-induced neuronal hyperactivity and cognitive deficits in Alzheimer's disease.

Neuronal hyperactivity induced by ß-amyloid (Aß) is an early pathological feature in Alzheimer's disease (AD) and contributes to cognitive decline in AD progression. However, the underlying mechanisms are still unclear. Here, we revealed that Aß increased the expression level of synaptic adhesion molecule protocadherin-γC5 (Pcdh-γC5) in a Ca2+-dependent manner, associated with aberrant elevation of synapses in both Aß-treated neurons in vitro and the cortex of APP/PS1 mice in vivo. By using Pcdhgc5 gene knockout mice, we demonstrated the critical function of Pcdh-γC5 in regulating neuronal synapse formation, synaptic transmission, and cognition. To further investigate the role of Pcdh-γC5 in AD pathogenesis, the aberrantly enhanced expression of Pcdh-γC5 in the brain of APP/PS1 mice was knocked down by shRNA. Downregulation of Pcdh-γC5 efficiently rescued neuronal hyperactivity and impaired cognition in APP/PS1 mice. Our findings revealed the pathophysiological role of Pcdh-γC5 in mediating Aß-induced neuronal hyperactivity and cognitive deficits in AD and identified a novel mechanism underlying AD pathogenesis.

Gamma protocadherins in vascular endothelial cells inhibit Klf2/4 to promote atherosclerosis.

Atherosclerotic cardiovascular disease (ASCVD) is the leading cause of mortality worldwide1. Laminar shear stress (LSS) from blood flow in straight regions of arteries protects against ASCVD by upregulating the Klf2/4 anti-inflammatory program in endothelial cells (ECs)2-8. Conversely, disturbed shear stress (DSS) at curves or branches predisposes these regions to plaque formation9,10. We previously reported a whole genome CRISPR knockout screen11 that identified novel inducers of Klf2/4. Here we report suppressors of Klf2/4 and characterize one candidate, protocadherin gamma A9 (Pcdhga9), a member of the clustered protocadherin gene family12. Pcdhg deletion increases Klf2/4 levels in vitro and in vivo and suppresses inflammatory activation of ECs. Pcdhg suppresses Klf2/4 by inhibiting the Notch pathway via physical interaction of cleaved Notch1 intracellular domain (NICD Val1744) with nuclear Pcdhg C-terminal constant domain (CCD). Pcdhg inhibition by EC knockout (KO) or blocking antibody protects from atherosclerosis. Pcdhg is elevated in the arteries of human atherosclerosis. This study identifies a novel fundamental mechanism of EC resilience and therapeutic target for treating inflammatory vascular disease.

In pursuit of a minimal CHO genome: Establishment of large-scale genome deletions.

Chinese hamster ovary (CHO) cells are the most commonly used mammalian cell line for the production of complex therapeutic glycoproteins. As CHO cells have evolved as part of a multicellular organism, they harbor many cellular functions irrelevant for their application as production hosts in industrial bioprocesses. Consequently, CHO cells have been the target for numerous genetic engineering efforts in the past, but a tailored host cell chassis holistically optimized for its specific task in a bioreactor is still missing. While the concept of genome reduction has already been successfully applied to bacterial production cells, attempts to create higher eukaryotic production hosts exhibiting reduced genomes have not been reported yet. Here, we present the establishment and application of a large-scale genome deletion strategy for targeted excision of large genomic regions in CHO cells. We demonstrate the feasibility of genome reduction in CHO cells using optimized CRISPR/Cas9 based experimental protocols targeting large non-essential genomic regions with high efficiency. Achieved genome deletions of non-essential genetic regions did not introduce negative effects on bioprocess relevant parameters, although we conducted the largest reported genomic excision of 864 kilobase pairs in CHO cells so far. The concept presented serves as a directive to accelerate the development of a significantly genome-reduced CHO host cell chassis paving the way for a next generation of CHO cell factories.

Concurrent glomerular PCDH7 deposits in PLA2R-associated membranous nephropathy.

The understanding of membranous nephropathy (MN) has undergone impressive advancements in the last 5 years, particularly due to identification of novel antigenic targets. M-type phospholipase A2 receptor (PLA2R) and thrombospondin type 1 domain-containing 7A (THSD7A) account for approximately 70% and 1-5% of the target antigens in primary MN, respectively. Recently, more novel/putative antigens have been identified in the remaining cases of MN that include exostosin 1/exostosin 2 (EXT1/EXT2), neural epidermal growth factor-like 1 protein (NELL-1), semaphorin 3B (SEMA3B) and protocadherin 7 (PCDH7). However, comparatively little is known about the PCDH7 among these novel antigens. As such, we herein described a unique case of positive glomerular PCDH7 deposits in PLA2R-associated MN, which may offer a deeper insight into the role of PCDH7 in MN and improve our understanding of glomerular diseases in the post-COVID era, particularly with the emerging variants.

Genome-wide identification and spatiotemporal expression analysis of cadherin superfamily members in echinoderms.

BACKGROUND: Cadherins are calcium-dependent transmembrane cell-cell adhesion proteins that are essential for metazoan development. They consist of three subfamilies: classical cadherins, which bind catenin, protocadherins, which contain 6-7 calcium-binding repeat domains, and atypical cadherins. Their functions include forming adherens junctions, establishing planar cell polarity (PCP), and regulating cell shape, proliferation, and migration. Because they are basal deuterostomes, echinoderms provide important insights into bilaterian evolution, but their only well-characterized cadherin is G-cadherin, a classical cadherin that is expressed by many embryonic epithelia. We aimed to better characterize echinoderm cadherins by conducting phylogenetic analyses and examining the spatiotemporal expression patterns of cadherin-encoding genes during Strongylocentrotus purpuratus development. RESULTS: Our phylogenetic analyses conducted on two echinoid, three asteroid, and one crinoid species identified ten echinoderm cadherins, including one deuterostome-specific ortholog, cadherin-23, and an echinoderm-specific atypical cadherin that possibly arose in an echinoid-asteroid ancestor. Catenin-binding domains in dachsous-2 orthologs were found to be a deuterostome-specific innovation that was selectively lost in mouse, while those in Fat4 orthologs appeared to be Ambulacraria-specific and were selectively lost in non-crinoid echinoderms. The identified suite of echinoderm cadherins lacks vertebrate-specific innovations but contains two proteins that are present in protostomes and absent from mouse. The spatiotemporal expression patterns of four embryonically expressed cadherins (fat atypical cadherins 1 and 4, dachsous-2, and protocadherin-9) were dynamic and mirrored the expression pattern of Frizzled 5/8, a non-canonical Wnt PCP pathway receptor protein essential for archenteron morphogenesis. CONCLUSIONS: The echinoderm cadherin toolkit is more similar to that of an ancient bilaterian predating protostomes and deuterostomes than it is to the suite of cadherins found in extant vertebrates. However, it also appears that deuterostomes underwent several cadherin-related innovations. Based on their similar spatiotemporal expression patterns and orthologous relationships to PCP-related and tumor-suppressing proteins, we hypothesize that sea urchin cadherins may play a role in regulating the shape and growth of embryonic epithelia and organs. Future experiments will examine cadherin expression in non-echinoid echinoderms and explore the functions of cadherins during echinoderm development.

PCDH1, a poor prognostic biomarker and potential target for pancreatic adenocarcinoma metastatic therapy.

BACKGROUND: Pancreatic adenocarcinoma (PAAD) is an aggressive solid tumour characterised by few early symptoms, high mortality, and lack of effective treatment. Therefore, it is important to identify new potential therapeutic targets and prognostic biomarkers of PAAD. METHODS: The Cancer Genome Atlas and Genotype-Tissue Expression databases were used to identify the expression and prognostic model of protocadherin 1 (PCDH1). The prognostic performance of risk factors and diagnosis of patients with PAAD were evaluated by regression analysis, nomogram, and receiver operating characteristic curve. Paraffin sections were collected from patients for immunohistochemistry (IHC) analysis. The expression of PCDH1 in cells obtained from primary tumours or metastatic biopsies was identified using single-cell RNA sequencing (scRNA-seq). Real-time quantitative polymerase chain reaction (qPCR) and western blotting were used to verify PCDH1 expression levels and the inhibitory effects of the compounds. RESULTS: The RNA and protein levels of PCDH1 were significantly higher in PAAD cells than in normal pancreatic ductal cells, similar to those observed in tissue sections from patients with PAAD. Aberrant methylation of the CpG site cg19767205 and micro-RNA (miRNA) hsa-miR-124-1 may be important reasons for the high PCDH1 expression in PAAD. Up-regulated PCDH1 promotes pancreatic cancer cell metastasis. The RNA levels of PCDH1 were significantly down-regulated following flutamide treatment. Flutamide reduced the percentage of PCDH1 RNA level in PAAD cells Panc-0813 to < 50%. In addition, the PCDH1 protein was significantly down-regulated after Panc-0813 cells were incubated with 20 µM flutamide and proves to be a potential therapeutic intervention for PAAD. CONCLUSION: PCDH1 is a key prognostic biomarker and promoter of PAAD metastasis. Additionally, flutamide may serve as a novel compound that down-regulates PCDH1 expression as a potential treatment for combating PAAD progression and metastasis.

Reciprocal Connections between Parvalbumin-Expressing Cells and Adjacent Pyramidal Cells Are Regulated by Clustered Protocadherin γ.

Functional neural circuits in the cerebral cortex are established through specific neural connections between excitatory and various inhibitory cell types. However, the molecular mechanisms underlying synaptic partner recognition remain unclear. In this study, we examined the impact of clustered protocadherin-γ (cPcdhγ) gene deletion in parvalbumin-positive (PV+) cells on intralaminar and translaminar neural circuits formed between PV+ and pyramidal (Pyr) cells in the primary visual cortex (V1) of male and female mice. First, we used whole-cell recordings and laser-scan photostimulation with caged glutamate to map excitatory inputs from layer 2/3 to layer 6. We found that cPcdhγ-deficient PV+ cells in layer 2/3 received normal translaminar inputs from Pyr cells through layers 2/3-6. Second, to further elucidate the effect on PV+-Pyr microcircuits within intralaminar layer 2/3, we conducted multiple whole-cell recordings. While the overall connection probability of PV+-Pyr cells remained largely unchanged, the connectivity of PV+-Pyr was significantly different between control and PV+-specific cPcdhγ-conditional knock-out (PV-cKO) mice. In control mice, the number of reciprocally connected PV+ cells was significantly higher than PV+ cells connected one way to Pyr cells, a difference that was not significant in PV-cKO mice. Interestingly, the proportion of highly reciprocally connected PV+ cells to Pyr cells with large unitary IPSC (uIPSC) amplitudes was reduced in PV-cKO mice. Conversely, the proportion of middle reciprocally connected PV+ cells to Pyr cells with large uIPSC amplitudes increased compared with control mice. This study demonstrated that cPcdhγ in PV+ cells modulates their reciprocity with Pyr cells in the cortex.

Dual AAV-based PCDH15 gene therapy achieves sustained rescue of visual function in a mouse model of Usher syndrome 1F.

Mutations in the PCDH15 gene, encoding protocadherin-15, are among the leading causes of Usher syndrome type 1 (USH1F), and account for up to 12% USH1 cases worldwide. A founder truncating variant of PCDH15 has a ∼2% carrier frequency in Ashkenazi Jews accounting for nearly 60% of their USH1 cases. Although cochlear implants can restore hearing perception in USH1 patients, presently there are no effective treatments for the vision loss due to retinitis pigmentosa. We established a founder allele-specific Pcdh15 knockin mouse model as a platform to ascertain therapeutic strategies. Using a dual-vector approach to circumvent the size limitation of adeno-associated virus, we observed robust expression of exogenous PCDH15 in the retinae of Pcdh15KI mice, sustained recovery of electroretinogram amplitudes and key retinoid oxime, substantially improved light-dependent translocation of phototransduction proteins, and enhanced levels of retinal pigment epithelium-derived enzymes. Thus, our data raise hope and pave the way for future gene therapy trials in USH1F subjects.

The Role of Protocadherin γ in Adult Sensory Neurons and Skin Reinnervation.

The clustered protocadherins (cPcdhs) play a critical role in the patterning of several CNS axon and dendritic arbors, through regulation of homophilic self and neighboring interactions. While not explored, primary peripheral sensory afferents that innervate the epidermis may require similar constraints to convey spatial signals with appropriate fidelity. Here, we show that members of the γ-Pcdh (Pcdhγ) family are expressed in both adult sensory neuron axons and in neighboring keratinocytes that have close interactions during skin reinnervation. Adult mice of both sexes were studied. Pcdhγ knock-down either through small interfering RNA (siRNA) transduction or AAV-Cre recombinase transfection of adult mouse primary sensory neurons from floxed Pcdhγ mice was associated with a remarkable rise in neurite outgrowth and branching. Rises in outgrowth were abrogated by Rac1 inhibition. Moreover, AAV-Cre knock-down in Pcdhγ floxed neurons generated a rise in neurite self-intersections, and a robust rise in neighbor intersections or tiling, suggesting a role in sensory axon repulsion. Interestingly, preconditioned (3-d axotomy) neurons with enhanced growth had temporary declines in Pcdhγ and lessened outgrowth from Pcdhγ siRNA. In vivo, mice with local hindpaw skin Pcdhγ knock-down by siRNA had accelerated reinnervation by new epidermal axons with greater terminal branching and reduced intra-axonal spacing. Pcdhγ knock-down also had reciprocal impacts on keratinocyte density and nuclear size. Taken together, this work provides evidence for a role of Pcdhγ in attenuating outgrowth of sensory axons and their interactions, with implications in how new reinnervating axons following injury fare amid skin keratinocytes that also express Pcdhγ.SIGNIFICANCE STATEMENT The molecular mechanisms and potential constraints that govern skin reinnervation and patterning by sensory axons are largely unexplored. Here, we show that γ-protocadherins (Pcdhγ) may help to dictate interaction not only among axons but also between axons and keratinocytes as the former re-enter the skin during reinnervation. Pcdhγ neuronal knock-down enhances outgrowth in peripheral sensory neurons, involving the growth cone protein Rac1 whereas skin Pcdhγ knock-down generates rises in terminal epidermal axon growth and branching during re-innervation. Manipulation of sensory axon regrowth within the epidermis offers an opportunity to influence regenerative outcomes following nerve injury.

Visualization of trans homophilic interaction of clustered protocadherin in neurons.

Clustered protocadherin (Pcdh) functions as a cell recognition molecule through the homophilic interaction in the central nervous system. However, its interactions have not yet been visualized in neurons. We previously reported PcdhγB2-Förster resonance energy transfer (FRET) probes to be applicable only to cell lines. Herein, we designed γB2-FRET probes by fusing FRET donor and acceptor fluorescent proteins to a single γB2 molecule and succeeded in visualizing γB2 homophilic interaction in cultured hippocampal neurons. The γB2-FRET probe localized in the soma and neurites, and FRET signals, which were observed at contact sites between neurites, eliminated by ethylene glycol tetraacetic acid (EGTA) addition. Live imaging revealed that the FRET-negative γB2 signals rapidly moved along neurites and soma, whereas the FRET-positive signals remained in place. We observed that the γB2 proteins at synapses rarely interact homophilically. The γB2-FRET probe might allow us to elucidate the function of the homophilic interaction and the cell recognition mechanism.