CRB1-Related Retinal Dystrophies in a Cohort of 50 Patients: A Reappraisal in the Light of Specific Müller Cell and Photoreceptor CRB1 Isoforms

Pathogenic variants in CRB1 lead to diverse recessive retinal disorders from severe Leber congenital amaurosis to isolated macular dystrophy. Until recently, no clear phenotype-genotype correlation and no appropriate mouse models existed. Herein, we reappraise the phenotype-genotype correlation of 50 patients with regards to the recently identified CRB1 isoforms: a canonical long isoform A localized in Müller cells (12 exons) and a short isoform B predominant in photoreceptors (7 exons). Twenty-eight patients with early onset retinal dystrophy (EORD) consistently had a severe Müller impairment, with variable impact on the photoreceptors, regardless of isoform B expression. Among them, two patients expressing wild type isoform B carried one variant in exon 12, which specifically damaged intracellular protein interactions in Müller cells. Thirteen retinitis pigmentosa patients had mainly missense variants in laminin G-like domains and expressed at least 50% of isoform A. Eight patients with the c.498_506del variant had macular dystrophy. In one family homozygous for the c.1562C>T variant, the brother had EORD and the sister macular dystrophy. In contrast with the mouse model, these data highlight the key role of Müller cells in the severity of CRB1-related dystrophies in humans, which should be taken into consideration for future clinical trials.

An autoregulation loop in fust-1 for circular RNA regulation in Caenorhabditis elegans

Many circular RNAs (circRNAs) are differentially expressed in different tissues or cell types, suggestive of specific factors that regulate their biogenesis. Here, taking advantage of available mutation strains of RNA-binding proteins (RBPs) in Caenorhabditis elegans, I performed a screening of circRNA regulation in 13 conserved RBPs. Among them, loss of FUST-1, the homolog of Fused in Sarcoma (FUS), caused downregulation of multiple circRNAs. By rescue experiments, I confirmed FUST-1 as a circRNA regulator. Through RNA sequencing using circRNA-enriched samples, circRNAs targets regulated by FUST-1 were identified globally, with hundreds of them significantly altered. Furthermore, I showed that FUST-1 regulates circRNA formation with only small to little effect on the cognate linear mRNAs. When recognizing circRNA pre-mRNAs, FUST-1 can affect both exon-skipping and circRNA in the same genes. Moreover, I identified an autoregulation loop in fust-1, where FUST-1, isoform a (FUST-1A) promotes the skipping of exon 5 of its own pre-mRNA, which produces FUST-1, isoform b (FUST-1B) with different N-terminal sequences. FUST-1A is the functional isoform in circRNA regulation. Although FUST-1B has the same functional domains as FUST-1A, it cannot regulate either exon-skipping or circRNA formation. This study provided an in vivo investigation of circRNA regulation, which will be helpful to understand the mechanisms that govern circRNA formation.

DNAJB6b is Downregulated in Synucleinopathies

Background: α-synuclein (α-syn) aggregation contributes to the progression of multiple neurodegenerative diseases. We recently found that the isoform b of the co-chaperone DNAJB6 is a strong suppressor of a-syn aggregation in vivo and in vitro. However, nothing is known about the role of the endogenous isoform b of DNAJB6 (DNAJB6b) in health and disease, due to lack of specific antibodies. Objective: Here we generated a novel anti-DNAJB6b antibody to analyze the localization and expression this isoform in cells, in tissue and in clinical material. Methods: To address this we used immunocytochemistry, immunohistochemistry, as well as a novel quantitative DNAJB6 specific ELISA method. Results: The endogenous protein is mainly expressed in the cytoplasm and in neurites in vitro, where it is found more in dendrites than in axons. We further verified in vivo that DNAJB6b is expressed in the dopaminergic neurons of the substantia nigra pars compacta (SNpc), which is a neuronal subpopulation highly sensitive to α-syn aggregation, that degenerate to a large extend in patients with Parkinson’s disease (PD) and multiple system atrophy (MSA). When we analyzed the expression levels of DNAJB6b in brain material from PD and MSA patients, we found a downregulation of DNAJB6b by use of ELISA based quantification. Interestingly, this was also true when analyzing tissue from patients with progressive supranuclear palsy, a taupathic atypical parkinsonian disorder. However, the total level of DNAJB6 was upregulated in these three diseases, which may indicate an upregulation of the other major isoform of DNAJB6, DNAJB6a. Conclusion: This study shows that DNAJB6b is downregulated in several different neurodegenerative diseases, which makes it an interesting target to further investigate in relation to amyloid protein aggregation and disease progression.

An autoregulation loop in fust-1 for circular RNA regulation in Caenorhabditis elegans

Circular RNAs (circRNAs) are always expressed tissue-specifically, suggestive of specific factors that regulate their biogenesis. Here, taking advantage of available mutation strains of RNA binding proteins (RBPs) in Caenorhabditis elegans, I performed a screening of circRNA regulation in thirteen conserved RBPs. Among them, loss of FUST-1, the homolog of FUS (Fused in Sarcoma), caused downregulation of multiple circRNAs. By rescue experiments, I confirmed FUST-1 as a circRNA regulator. Further, I showed that FUST-1 regulates circRNA formation without affecting the levels of the cognate linear mRNAs. When recognizing circRNA pre-mRNAs, FUST-1 can affect both exon-skipping and circRNA in the same genes. Moreover, I identified an autoregulation loop in fust-1, where FUST-1, isoform a promotes the skipping of exon 5 of its own pre-mRNA, which produces FUST-1, isoform b with different N-terminal sequences. FUST-1, isoform a is the functional isoform in circRNA regulation. Although FUST-1, isoform b has the same functional domains as isoform a, it cannot regulate either exon-skipping or circRNA formation.

RHAMMB-mediated bifunctional nanotherapy targeting Bcl-xL and mitochondria for pancreatic neuroendocrine tumor treatment

The incidence of pancreatic neuroendocrine tumor (PNET) has continued to rise. Due to their indolent feature, PNET patients often present with incurable, metastatic diseases. Novel therapies are urgently needed. We have previously shown that Receptor for Hyaluronic Acid-Mediated Motility isoform B (RHAMMB) and Bcl-xL are upregulated in PNETs and both of them promote PNET metastasis. Because RHAMM protein is undetectable in most adult tissues, we hypothesized that RHAMMB could be a gateway for nanomedicine delivery into PNETs. To test this, we developed RHAMMB-targeting nanoparticle. Inside this nanoparticle, we assembled siRNA against Bcl-xL (siBcl-xL) and mitochondria-fusing peptide KLA. We demonstratsed that RHAMMB-positive PNETs picked up the RHAMMB-targeting nanoparticles. siBcl-xL or KLA alone only killed 30% of PNET cells. In contrast, a synergistic killing effect was achieved with the co-delivery of siBcl-xL and KLA peptide in vitro. Unexpectedly, siBcl-xL induced cell death before reducing Bcl-xL protein levels. The systemically-injected RHAMMB-targeting nanoparticles carrying siBcl-xL and KLA peptide significantly reduced tumor burden in mice bearing RHAMMB-positive PNETs. Together, these findings indicate that the RHAMMB-targeting nanotherapy serves as a promising drug delivery system for PNET and possibly other malignancies upregulating RHAMMB. The combination of siBcl-xL and KLA peptide can be a therapy for PNET treatment.

P2X7 Variants in Oncogenesis

The P2X7 receptor for extracellular ATP is a well-established mediator of tumoral development and progression both in solid cancers and hematological malignancies. The human P2X7 gene is highly polymorphic, and several splice variants of the receptor have been identified in time. P2X7 single-nucleotide polymorphisms (SNPs) have been broadly analyzed by studies relating them to pathologies as different as infectious, inflammatory, nervous, and bone diseases, among which cancer is included. Moreover, in the last years, an increasing number of reports concentrated on P2X7 splice variants’ different roles and their implications in pathological conditions, including oncogenesis. Here, we give an overview of established and recent literature demonstrating a role for human P2X7 gene products in oncological conditions, mainly focusing on current data emerging on P2X7 isoform B and nfP2X7. We explored the role of these and other genetic variants of P2X7 in cancer insurgence, dissemination, and progression, as well as the effect of chemotherapy on isoforms expression. The described literature strongly suggests that P2X7 variants are potential new biomarkers and therapeutical targets in oncological conditions and that their study in carcinogenesis deserves to be further pursued.

Decoding the Role of CD271 in Melanoma

The evolution of melanoma, the most aggressive type of skin cancer, is triggered by driver mutations that are acquired in the coding regions of particularly BRAF (rat fibrosarcoma serine/threonine kinase, isoform B) or NRAS (neuroblastoma-type ras sarcoma virus) in melanocytes. Although driver mutations strongly determine tumor progression, additional factors are likely required and prerequisite for melanoma formation. Melanocytes are formed during vertebrate development in a well-controlled differentiation process of multipotent neural crest stem cells (NCSCs). However, mechanisms determining the properties of melanocytes and melanoma cells are still not well understood. The nerve growth factor receptor CD271 is likewise expressed in melanocytes, melanoma cells and NCSCs and programs the maintenance of a stem-like and migratory phenotype via a comprehensive network of associated genes. Moreover, CD271 regulates phenotype switching, a process that enables the rapid and reversible conversion of proliferative into invasive or non-stem-like states into stem-like states by yet largely unknown mechanisms. Here, we summarize current findings about CD271-associated mechanisms in melanoma cells and illustrate the role of CD271 for melanoma cell migration and metastasis, phenotype-switching, resistance to therapeutic interventions, and the maintenance of an NCSC-like state.

Purification and Characterization of Double-Stranded Nucleic Acid-Dependent ATPase Activities of Tagged Dicer-Related Helicase 1 and its Short Isoform in Caenorhabditis elegans

The Dicer-related helicases (DRHs) are members of a helicase subfamily, and mammalian DRHs such as retinoic acid-inducible gene-I (RIG-I), are involved in antiviral immunity. Caenorhabditis elegans DRH-1 and DRH-3 play crucial roles in antiviral function and chromosome segregation, respectively. Although intrinsic double-stranded RNA-dependent ATP-hydrolyzing activity has been observed in the recombinant DRH-3 protein prepared from Escherichia coli, there are no reports of biochemical studies of the nematode RIG-I homolog DRH-1. In this study, the secondary structure prediction by JPred4 revealed that DRH-1 and DRH-3 had distinct N-terminal regions and that a 200-amino acid N-terminal region of DRH-1 could form a structure very rich in α-helices. We investigated expressions and purifications of a codon-optimized DRH-1 with four different N-terminal tags, identifying poly-histidine (His)-small ubiquitin-like modifier (SUMO) as a suitable tag for DRH-1 preparation. Full-length (isoform a) and a N-terminal truncated (isoform b) of DRH-1 were purified as the His-SUMO-tagged fusion proteins. Finally, the nucleic acid-dependent ATPase activities were investigated for the two His-SUMO-tagged DRH-1 isoforms and His-tagged DRH-3. The tagged DRH-3 exhibited dsRNA-dependent ATPase activity. However, detectable dsRNA dependency of ATPase activities was not found in either isoform of tagged DRH-1 and a tag-free DRH-1 (isoform a) treated with SUMO protease. These observations suggest that DRH-1 and its short isoform have no or poor nucleic acid-dependent ATPase activity, unlike DRH-3 and mammalian DRHs.

Glycerol kinase enhances hepatic lipid metabolism by repressing nuclear receptor subfamily 4 group A1 in the nucleus

Glycerol kinase (GYK) plays a critical role in hepatic metabolism by converting glycerol to glycerol 3-phosphate in an ATP-dependent reaction. GYK isoform b is the only glycerol kinase present in whole cells, and has a non-enzymatic moonlighting function in the nucleus. GYK isoform b acts as a co-regulator of nuclear receptor subfamily 4 group A1 (NR4A1) and participates in the regulation of hepatic glucose metabolism by protein–protein interaction with NR4A1. Herein, GYK expression was found to upregulate the expression of NR4A1-mediated lipid metabolism-related genes (SREBP1C, FASN, ACACA, and GPAM) in HEK293T and L02 cells, and in mouse in vivo studies. GYK expression increased blood levels of cholesterol, triglyceride, and high-density lipoprotein cholesterol, but not low-density lipoprotein cholesterol levels. It enhanced the transcriptional activity of Nr4a1 target genes by negatively cooperating with NR4A1 and its enzymatic activity or by other undefined moonlighting functions. This enhancement was observed in both normal and diabetic mice. We also found a feed-forward regulation loop between GYK and NR4A1, serving as part of a GYK-NR4A1 regulatory mechanism in hepatic metabolism. Thus, GYK regulates the effect of NR4A1 on hepatic lipid metabolism in normal and diabetic mice, partially through the cooperation of GYK and NR4A1.