The Analysis of Embryoid Body Formation and Its Role in Retinal Organoid Development.

Within the last decade, a wide variety of protocols have emerged for the generation of retinal organoids. A subset of studies have compared protocols based on stem cell source, the physical features of the microenvironment, and both internal and external signals, all features that influence embryoid body and retinal organoid formation. Most of these comparisons have focused on the effect of signaling pathways on retinal organoid development. In this study, our aim is to understand whether starting cell conditions, specifically those involved in embryoid body formation, affect the development of retinal organoids in terms of differentiation capacity and reproducibility. To investigate this, we used the popular 3D floating culture method to generate retinal organoids from stem cells. This method starts with either small clumps of stem cells generated from larger clones (clumps protocol, CP) or with an aggregation of single cells (single cells protocol, SCP). Using histological analysis and gene-expression comparison, we found a retention of the pluripotency capacity on embryoid bodies generated through the SCP compared to the CP. Nonetheless, these early developmental differences seem not to impact the final retinal organoid formation, suggesting a potential compensatory mechanism during the neurosphere stage. This study not only facilitates an in-depth exploration of embryoid body development but also provides valuable insights for the selection of the most suitable protocol in order to study retinal development and to model inherited retinal disorders in vitro.

The Road towards Gene Therapy for X-Linked Juvenile Retinoschisis: A Systematic Review of Preclinical Gene Therapy in Cell-Based and Rodent Models of XLRS.

X-linked juvenile retinoschisis (XLRS) is an early-onset progressive inherited retinopathy affecting males. It is characterized by abnormalities in the macula, with formation of cystoid retinal cavities, frequently accompanied by splitting of the retinal layers, impaired synaptic transmission of visual signals, and associated loss of visual acuity. XLRS is caused by loss-of-function mutations in the retinoschisin gene located on the X chromosome (RS1, MIM 30083). While proof-of-concept studies for gene augmentation therapy have been promising in in vitro and rodent models, clinical trials in XLRS patients have not been successful thus far. We performed a systematic literature investigation using search strings related to XLRS and gene therapy in in vivo and in vitro models. Three rounds of screening (title/abstract, full text and qualitative) were performed by two independent reviewers until consensus was reached. Characteristics related to study design and intervention were extracted from all studies. Results were divided into studies using (1) viral and (2) non-viral therapies. All in vivo rodent studies that used viral vectors were assessed for quality and risk of bias using the SYRCLE's risk-of-bias tool. Studies using alternative and non-viral delivery techniques, either in vivo or in vitro, were extracted and reviewed qualitatively, given the diverse and dispersed nature of the information. For in-depth analysis of in vivo studies using viral vectors, outcome data for optical coherence tomography (OCT), immunohistopathology and electroretinography (ERG) were extracted. Meta-analyses were performed on the effect of recombinant adeno-associated viral vector (AAV)-mediated gene augmentation therapies on a- and b-wave amplitude as well as the ratio between b- and a-wave amplitudes (b/a-ratio) extracted from ERG data. Subgroup analyses and meta-regression were performed for model, dose, age at injection, follow-up time point and delivery method. Between-study heterogeneity was assessed with a Chi-square test of homogeneity (I2). We identified 25 studies that target RS1 and met our search string. A total of 19 of these studies reported rodent viral methods in vivo. Six of the 25 studies used non-viral or alternative delivery methods, either in vitro or in vivo. Of these, five studies described non-viral methods and one study described an alternative delivery method. The 19 aforementioned in vivo studies were assessed for risk of bias and quality assessments and showed inconsistency in reporting. This resulted in an unclear risk of bias in most included studies. All 19 studies used AAVs to deliver intact human or murine RS1 in rodent models for XLRS. Meta-analyses of a-wave amplitude, b-wave amplitude, and b/a-ratio showed that, overall, AAV-mediated gene augmentation therapy significantly ameliorated the disease phenotype on these parameters. Subgroup analyses and meta-regression showed significant correlations between b-wave amplitude effect size and dose, although between-study heterogeneity was high. This systematic review reiterates the high potential for gene therapy in XLRS, while highlighting the importance of careful preclinical study design and reporting. The establishment of a systematic approach in these studies is essential to effectively translate this knowledge into novel and improved treatment alternatives.

A review of treatment modalities in gyrate atrophy of the choroid and retina (GACR).

Gyrate atrophy of the choroid and retina (GACR) is a rare inborn error of amino acid metabolism caused by bi-allelic variations in OAT. GACR is characterised by vision decline in early life eventually leading to complete blindness, and high plasma ornithine levels. There is no curative treatment for GACR, although several therapeutic modalities aim to slow progression of the disease by targeting different steps within the ornithine pathway. No international treatment protocol is available. We systematically collected all international literature on therapeutic interventions in GACR to provide an overview of published treatment effects. METHODS: Following the PRISMA guidelines, we conducted a systematic review of the English literature until December 22nd 2020. PubMed and Embase databases were searched for studies related to therapeutic interventions in patients with GACR. RESULTS: A total of 33 studies (n = 107 patients) met the inclusion criteria. Most studies were designed as case reports (n = 27) or case series (n = 4). No randomised controlled trials or large cohort studies were found. Treatments applied were protein-restricted diets, pyridoxine supplementation, creatine or creatine precursor supplementation, l-lysine supplementation, and proline supplementation. Protein-restricted diets lowered ornithine levels ranging from 16.0-91.2%. Pyridoxine responsiveness was reported in 30% of included mutations. Lysine supplementation decreased ornithine levels with 21-34%. Quality assessment showed low to moderate quality of the articles. CONCLUSIONS: Based primarily on case reports ornithine levels can be reduced by using a protein restricted diet, pyridoxine supplementation (variation-dependent) and/or lysine supplementation. The lack of pre-defined clinical outcome measures and structural follow-up in all included studies impeded conclusions on clinical effectiveness. Future research should be aimed at 1) Unravelling the OAT biochemical pathway to identify other possible pathologic metabolites besides ornithine, 2) Pre-defining GACR specific clinical outcome measures, and 3) Establishing an international historical cohort.

The Role of Small Molecules and Their Effect on the Molecular Mechanisms of Early Retinal Organoid Development.

Early in vivo embryonic retinal development is a well-documented and evolutionary conserved process. The specification towards eye development is temporally controlled by consecutive activation or inhibition of multiple key signaling pathways, such as the Wnt and hedgehog signaling pathways. Recently, with the use of retinal organoids, researchers aim to manipulate these pathways to achieve better human representative models for retinal development and disease. To achieve this, a plethora of different small molecules and signaling factors have been used at various time points and concentrations in retinal organoid differentiations, with varying success. Additions differ from protocol to protocol, but their usefulness or efficiency has not yet been systematically reviewed. Interestingly, many of these small molecules affect the same and/or multiple pathways, leading to reduced reproducibility and high variability between studies. In this review, we make an inventory of the key signaling pathways involved in early retinogenesis and their effect on the development of the early retina in vitro. Further, we provide a comprehensive overview of the small molecules and signaling factors that are added to retinal organoid differentiation protocols, documenting the molecular and functional effects of these additions. Lastly, we comparatively evaluate several of these factors using our established retinal organoid methodology.

An alternative approach to produce versatile retinal organoids with accelerated ganglion cell development.

Genetically complex ocular neuropathies, such as glaucoma, are a major cause of visual impairment worldwide. There is a growing need to generate suitable human representative in vitro and in vivo models, as there is no effective treatment available once damage has occured. Retinal organoids are increasingly being used for experimental gene therapy, stem cell replacement therapy and small molecule therapy. There are multiple protocols for the development of retinal organoids available, however, one potential drawback of the current methods is that the organoids can take between 6 weeks and 12 months on average to develop and mature, depending on the specific cell type wanted. Here, we describe and characterise a protocol focused on the generation of retinal ganglion cells within an accelerated four week timeframe without any external small molecules or growth factors. Subsequent long term cultures yield fully differentiated organoids displaying all major retinal cell types. RPE, Horizontal, Amacrine and Photoreceptors cells were generated using external factors to maintain lamination.

The circadian clock regulates RPE-mediated lactate transport via SLC16A1 (MCT1).

Multiple retinal cells harbor a circadian oscillator, including retinal pigment epithelial cells (RPE). However, little is known about the functions that are regulated by the RPE clock. The aim of this study was to investigate whether the circadian clock in the RPE regulates the transport of glucose and its glycolytic metabolic by-product - lactate. To that end, we first characterized the mRNA expression profile of glucose and monocarboxylate transporters in ARPE-19 cells. We found that SLC2A1 and SLC16A1 were, respectively, the most abundantly expressed glucose and lactate (monocarboxylate) transporters. We further observed that the protein products of SLC2A1 (encoding GLUT1) and SLC16A1 (encoding MCT1) localize on the apical membrane of ARPE-19 monolayers. In a subsequent time-course experiment, we found that SLC2A1 and SLC16A1 mRNA oscillated in ARPE-19 monolayers, but not in dispersed cells, suggesting that monolayer cellular organization is necessary for rhythmic regulation of these transporters. In these monolayers, we found that MCT1 proteins varied over time, in contrast to GLUT1 proteins which did not vary over time. Spectrophotometric measurements of supernatants sampled from ARPE-19 monolayer cultures revealed that glucose concentrations did not significantly differ between apical (Api) supernatants and basolateral (BL) ones. In addition, we did not find rhythms in Api or BL glucose concentrations. Conversely, we found higher lactate concentrations in Api supernatants than BL ones. Further, we found that Api lactate concentrations were rhythmic. Pearson's r revealed that the concentration gradients (Api - BL) of glucose and lactate correlated with the gene expression of respective SLC2A1 and SLC16A1 transporters. Incubation with photoreceptor outer segments (POS) affected the mRNA expression of SLC16A1 and SLC2A1 in ARPE-19 monolayers in a time-dependent manner, thus suggesting that the retina might modulate the RPE clock-controlled expression of transporters via interactions with POS. In conclusion, this work provides evidence that the transport of lactate is regulated by the circadian clock in the RPE.

Does the circadian clock make RPE-mediated ion transport "tick" via SLC12A2 (NKCC1)?

The presence of a circadian clock in the retinal pigment epithelium (RPE) was discovered recently. However, little is known about mechanisms or processes regulated by the RPE clock. We cultured ARPE-19 monolayers in a transwell culture system, and we found rhythmic mRNA expression of the sodium-potassium-chloride co-transporter SLC12A2. We localized the corresponding protein product, NKCC1, on the apical membrane of ARPE-19 cells. We found that concentrations of sodium, potassium, and chloride oscillated in apical supernatants. The ion concentration gradients between supernatants strongly correlated with SLC12A2 mRNA expression. Our results suggest that the circadian clock regulates ion transport by the RPE via NKCC1 expression.

ALS-associated missense and nonsense TBK1 mutations can both cause loss of kinase function.

Mutations in TANK binding kinase 1 (TBK1) have been linked to amyotrophic lateral sclerosis. Some TBK1 variants are nonsense and are predicted to cause disease through haploinsufficiency; however, many other mutations are missense with unknown functional effects. We exome sequenced 699 familial amyotrophic lateral sclerosis patients and identified 16 TBK1 novel or extremely rare protein-changing variants. We characterized a subset of these: p.G217R, p.R357X, and p.C471Y. Here, we show that the p.R357X and p.G217R both abolish the ability of TBK1 to phosphorylate 2 of its kinase targets, IRF3 and optineurin, and to undergo phosphorylation. They both inhibit binding to optineurin and the p.G217R, within the TBK1 kinase domain, reduces homodimerization, essential for TBK1 activation and function. Finally, we show that the proportion of TBK1 that is active (phosphorylated) is reduced in 5 lymphoblastoid cell lines derived from patients harboring heterozygous missense or in-frame deletion TBK1 mutations. We conclude that missense mutations in functional domains of TBK1 impair the binding and phosphorylation of its normal targets, implicating a common loss of function mechanism, analogous to truncation mutations.

Mutations in the vesicular trafficking protein annexin A11 are associated with amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder. We screened 751 familial ALS patient whole-exome sequences and identified six mutations including p.D40G in the ANXA11 gene in 13 individuals. The p.D40G mutation was absent from 70,000 control whole-exome sequences. This mutation segregated with disease in two kindreds and was present in another two unrelated cases (P = 0.0102), and all mutation carriers shared a common founder haplotype. Annexin A11-positive protein aggregates were abundant in spinal cord motor neurons and hippocampal neuronal axons in an ALS patient carrying the p.D40G mutation. Transfected human embryonic kidney cells expressing ANXA11 with the p.D40G mutation and other N-terminal mutations showed altered binding to calcyclin, and the p.R235Q mutant protein formed insoluble aggregates. We conclude that mutations in ANXA11 are associated with ALS and implicate defective intracellular protein trafficking in disease pathogenesis.

Oncogenic Properties of Candidate Oncogenes in Chromosome Region 17p11.2p12 in Human Osteosarcoma.

Osteosarcomas are primary tumors of bone that most often develop in adolescents. They are characterized by complex genomic changes including amplifications, deletions, and translocations. The chromosome region 17p11.2p12 is frequently amplified in human high grade osteosarcomas (25% of cases), suggesting the presence of one or more oncogenes. In previous studies, we identified 9 candidate oncogenes in this region (GID4, ARGHAP44, LRRC75A-AS1, TOP3A, COPS3, SHMT1, PRPSAP2, PMP22, and RASD1). The aim of the present study was to determine their oncogenic properties. Therefore, we generated osteosarcoma cell lines overexpressing these genes, except for LRRC75A-AS1 and PRPSAP2, and subjected these to functional oncogenic assays. We found that TOP3A, SHMT1, and RASD1 overexpression provided increased proliferation and that ARGHAP44, COPS3, and PMP22 overexpression had a stimulatory effect on migration and invasion of the cells. COPS3 and PMP22 overexpression additionally improved the ability of the cells to form new colonies. No oncogenic effect could be demonstrated for GID4 overexpression. We conclude that the concerted amplification-mediated overexpression of these genes in 17p11.2p12 may contribute to the oncogenic process in malignant osteosarcoma.

NEK1 variants confer susceptibility to amyotrophic lateral sclerosis.

To identify genetic factors contributing to amyotrophic lateral sclerosis (ALS), we conducted whole-exome analyses of 1,022 index familial ALS (FALS) cases and 7,315 controls. In a new screening strategy, we performed gene-burden analyses trained with established ALS genes and identified a significant association between loss-of-function (LOF) NEK1 variants and FALS risk. Independently, autozygosity mapping for an isolated community in the Netherlands identified a NEK1 p.Arg261His variant as a candidate risk factor. Replication analyses of sporadic ALS (SALS) cases and independent control cohorts confirmed significant disease association for both p.Arg261His (10,589 samples analyzed) and NEK1 LOF variants (3,362 samples analyzed). In total, we observed NEK1 risk variants in nearly 3% of ALS cases. NEK1 has been linked to several cellular functions, including cilia formation, DNA-damage response, microtubule stability, neuronal morphology and axonal polarity. Our results provide new and important insights into ALS etiopathogenesis and genetic etiology.

Molecular classification of amyotrophic lateral sclerosis by unsupervised clustering of gene expression in motor cortex.

Amyotrophic lateral sclerosis (ALS) is a rapidly progressive and ultimately fatal neurodegenerative disease, caused by the loss of motor neurons in the brain and spinal cord. Although 10% of ALS cases are familial (FALS), the majority are sporadic (SALS) and probably associated to a multifactorial etiology. Currently there is no cure or prevention for ALS. A prerequisite to formulating therapeutic strategies is gaining understanding of its etio-pathogenic mechanisms. In this study we analyzed whole-genome expression profiles of 41 motor cortex samples of control (10) and sporadic ALS (31) patients. Unsupervised hierarchical clustering was able to separate control from SALS patients. In addition, SALS patients were subdivided in two different groups that were associated to different deregulated pathways and genes, some of which were previously associated to familiar ALS. These experiments are the first to highlight the genomic heterogeneity of sporadic ALS and reveal new clues to its pathogenesis and potential therapeutic targets.

Exome-wide rare variant analysis identifies TUBA4A mutations associated with familial ALS.

Exome sequencing is an effective strategy for identifying human disease genes. However, this methodology is difficult in late-onset diseases where limited availability of DNA from informative family members prohibits comprehensive segregation analysis. To overcome this limitation, we performed an exome-wide rare variant burden analysis of 363 index cases with familial ALS (FALS). The results revealed an excess of patient variants within TUBA4A, the gene encoding the Tubulin, Alpha 4A protein. Analysis of a further 272 FALS cases and 5,510 internal controls confirmed the overrepresentation as statistically significant and replicable. Functional analyses revealed that TUBA4A mutants destabilize the microtubule network, diminishing its repolymerization capability. These results further emphasize the role of cytoskeletal defects in ALS and demonstrate the power of gene-based rare variant analyses in situations where causal genes cannot be identified through traditional segregation analysis.

Myelin and axon pathology in a long-term study of PMP22-overexpressing mice.

We analyzed clinical and pathological disease in 2 peripheral myelin protein-22 (PMP22) overexpressing mouse models for 1.5 years. C22 mice have 7 and C3-PMP mice have 3 to 4 copies of the human PMP22 gene. C3-PMP mice showed no overt clinical signs at 3 weeks and developed mild neuromuscular impairment; C22 mice showed signs at 3 weeks that progressed to severe impairment. Adult C3-PMP mice had very similar, stable, low nerve conduction velocities similar to adults with human Charcot-Marie-Tooth disease type 1A (CMT1A); velocities were much lower in C22 mice. Myelination was delayed, and normal myelination was not reached in either model but the degree of dysmyelination in C3-PMP mice was considerably less than that in C22 mice; myelination was stable in the adult mice. Numbers of myelinated, fibers were reduced at 3 weeks in both models, suggesting that normal numbers of myelinated fibers are not reached during development in the models. In adult C3-PMP and wild-type mice, there was no detectable loss of myelinated fibers,whereas there was clear loss of myelinated fibers in C22 mice.In C3-PMP mice, there is a balance between myelination status and axonal function early in life, whereas in C22 mice, early reduction of axons is more severe and there is major loss of axons in adulthood. We conclude that C3-PMP mice may be an appropriate model for most CMT1A patients, whereas C22 mice may be more relevant to severely affected patients in the CMT1 spectrum.

Mouse Schwann cells activate MHC class I and II restricted T-cell responses, but require external peptide processing for MHC class II presentation.

Schwann cells are the myelinating glia cells of the peripheral nervous system (PNS). In inflammatory neuropathies like the Guillain-Barré syndrome (GBS) Schwann cells become target of an autoimmune response, but may also modulate local inflammation. Here, we tested the functional relevance of Schwann cell derived MHC expression in an in vitro coculture system. Mouse Schwann cells activated proliferation of ovalbumin specific CD8+ T cells when ovalbumin protein or MHC class I restricted ovalbumin peptide (Ova(257-264) SIINFEKL) was added and after transfection with an ovalbumin coding vector. Schwann cells activated proliferation of ovalbumin specific CD4+ T cells in the presence of MHC class II restricted ovalbumin peptide (Ova(323-339) ISQAVHAAHAEINEAGR). CD4+ T-cell proliferation was not activated by ovalbumin protein or transfection with an ovalbumin coding vector. This indicates that Schwann cells express functionally active MHC class I and II molecules. In this study, however, Schwann cells lacked the ability to process exogenous antigen or cross-present endogenous antigen into the MHC class II presentation pathway. Thus, antigen presentation may be a pathological function of Schwann cells exacerbating nerve damage in inflammatory neuropathies.

Myelination competent conditionally immortalized mouse Schwann cells.

Numerous mouse myelin mutants are available to analyze the biology of the peripheral nervous system related to health and disease in vivo. However, robust in vitro biochemical characterizations of players in peripheral nerve processes are still not possible due to the limited growth capacities of Schwann cells. In order to generate cell lines from peripheral nerves that are amenable to experimental manipulation, we have isolated Schwann cells from transgenic mice (H-2Kb-tsA58) carrying the temperature sensitive SV40 large T oncogene under the control of the interferon gamma (IFNgamma) H-2Kb promoter. These cells are immortalized at 33 degrees C when the SV40 large T antigen has a stable conformation. At the non-permissive temperature of 37 degrees C and in the absence of IFNgamma, the growth rate of the cultures reduces and typical Schwann cell markers such as p75(NGFR) become upregulated. The conditionally immortalized Schwann cells allow genetic manipulation as demonstrated here by the generation of a stable eGFP expressing cell line. They regain their characteristic non-immortalized properties at non-permissive temperature and differentiate to myelin-forming cells when seeded on dorsal root ganglia neurons. The Schwann cell lines derived are valuable tools for in vitro studies involving demyelinating diseases.

Comparison of Schwann cell and sciatic nerve transcriptomes indicates that mouse is a valid model for the human peripheral nervous system.

High-throughput gene expression analyses of murine models of the peripheral nervous system (PNS), and its cellular components, have yielded enormous amounts of expression data of the PNS in various conditions. These data provided clues for future research directions to further decipher this complex organ in relation to acquired and inherited PNS diseases. Various studies addressing the validity of mouse models for human conditions in other tissues and cell types have indicated that in many cases the mouse model only poorly represents the human situation. To determine how well the mouse can serve as model to study the biological processes occurring in the PNS, we compared the gene expression profiles that we generated for mouse and human sciatic nerve and cultured Schwann cells derived thereof. A two-way analysis based on the differentially expressed genes between the sciatic nerve and the cultured Schwann cell, and which takes into account the differential expression between mouse and man, indicates that the human PNS is well represented by that of the mouse in terms of the "biological processes" ontology.

Expression profiling of sciatic nerve in a Charcot-Marie-Tooth disease type 1a mouse model.

Expression profiling was performed on sciatic nerve of normal mice and of transgenic mice overexpressing the peripheral myelin protein 22 kDa (PMP22). These mice represent a model for the hereditary peripheral neuropathy Charcot-Marie Tooth type 1A. Comparison of the profiles reveals that the proteasomal degradation pathway and various signaling mechanisms are up-regulated in the diseased nerve. The down-regulated processes represent cell shape and adhesion as well as cellular activity and metabolism. In addition, we found that the most significantly up-regulated differences could not be mapped on known transcripts and thus might represent not identified transcripts. Our data will be helpful to direct future research aimed at deciphering the molecular pathogenesis of the most prevalent hereditary peripheral neuropathy.

In vivo tumor growth inhibition and biodistribution studies of locked nucleic acid (LNA) antisense oligonucleotides.

Locked nucleic acids (LNA) are novel high-affinity DNA analogs that can be used as genotype-specific drugs. The LNA oligonucleotides (LNA PO ODNs) are very stable in vitro and in vivo without the need for a phosphorothiolated backbone. In this study we tested the biological fate and the efficacy in tumor growth inhibition of antisense oligonucleotides directed against the gene of the large subunit of RNA polymerase II (POLR2A) that are completely synthesized as LNA containing diester backbones. These full LNA oligonucleotides strongly reduce POLR2A protein levels. Full LNA PO ODNs appeared to be very stable compounds when injected into the circulation of mice. Full LNA PO ODNs were continuously administered for 14 days to tumor-bearing nude mice. Tumor growth was inhibited sequence specifically at dosages from 1 mg/kg/day. LNA PO ODNs appeared to be non-toxic at dosages <5 mg/kg/day. Biodistribution studies showed the kidneys to have the highest uptake of LNA PO ODNs and urinary secretion as the major route of clearance. This report shows that LNA PO ODNs are potent genotype-specific drugs that can inhibit tumor growth in vivo.

Ribonuclease H1 maps to chromosome 2 and has at least three pseudogene loci in the human genome.

We have analyzed the genomic structure of ribonuclease H1 (RNase H1) loci in the human genome. Human PAC library screening combined with database searches indicated that several loci are present. The transcribed gene is localized on chromosome 2p25. This was confirmed by RNA analysis of a monochromosomal hybrid cell line that expressed human chromosome 2. These data contradict a previous report, as well as the current Human Genome Project (HGP) annotation, which had placed the gene on chromosome 17p11.2. This location represents a pseudogene. Another highly similar pseudogene is present at a separate locus located more distal on chromosome 17p, while a third pseudogene is localized on chromosome 1q.