Mis-spliced transcripts generate de novo proteins in TDP-43-related ALS/FTD.

Functional loss of TDP-43, an RNA binding protein genetically and pathologically linked to amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), leads to the inclusion of cryptic exons in hundreds of transcripts during disease. Cryptic exons can promote the degradation of affected transcripts, deleteriously altering cellular function through loss-of-function mechanisms. Here, we show that mRNA transcripts harboring cryptic exons generated de novo proteins in TDP-43-depleted human iPSC-derived neurons in vitro, and de novo peptides were found in cerebrospinal fluid (CSF) samples from patients with ALS or FTD. Using coordinated transcriptomic and proteomic studies of TDP-43-depleted human iPSC-derived neurons, we identified 65 peptides that mapped to 12 cryptic exons. Cryptic exons identified in TDP-43-depleted human iPSC-derived neurons were predictive of cryptic exons expressed in postmortem brain tissue from patients with TDP-43 proteinopathy. These cryptic exons produced transcript variants that generated de novo proteins. We found that the inclusion of cryptic peptide sequences in proteins altered their interactions with other proteins, thereby likely altering their function. Last, we showed that 18 de novo peptides across 13 genes were present in CSF samples from patients with ALS/FTD spectrum disorders. The demonstration of cryptic exon translation suggests new mechanisms for ALS/FTD pathophysiology downstream of TDP-43 dysfunction and may provide a potential strategy to assay TDP-43 function in patient CSF.

An ANXA11 P93S variant dysregulates TDP-43 and causes corticobasal syndrome.

As genetic testing has become more accessible and affordable, variants of uncertain significance (VUS) are increasingly identified, and determining whether these variants play causal roles in disease is a major challenge. The known disease-associated Annexin A11 (ANXA11) mutations result in ANXA11 aggregation, alterations in lysosomal-RNA granule co-trafficking, and TDP-43 mis-localization and present as amyotrophic lateral sclerosis or frontotemporal dementia. We identified a novel VUS in ANXA11 (P93S) in a kindred with corticobasal syndrome and unique radiographic features that segregated with disease. We then queried neurodegenerative disorder clinic databases to identify the phenotypic spread of ANXA11 mutations. Multi-modal computational analysis of this variant was performed and the effect of this VUS on ANXA11 function and TDP-43 biology was characterized in iPSC-derived neurons. Single-cell sequencing and proteomic analysis of iPSC-derived neurons and microglia were used to determine the multiomic signature of this VUS. Mutations in ANXA11 were found in association with clinically diagnosed corticobasal syndrome, thereby establishing corticobasal syndrome as part of ANXA11 clinical spectrum. In iPSC-derived neurons expressing mutant ANXA11, we found decreased colocalization of lysosomes and decreased neuritic RNA as well as decreased nuclear TDP-43 and increased formation of cryptic exons compared to controls. Multiomic assessment of the P93S variant in iPSC-derived neurons and microglia indicates that the pathogenic omic signature in neurons is modest compared to microglia. Additionally, omic studies reveal that immune dysregulation and interferon signaling pathways in microglia are central to disease. Collectively, these findings identify a new pathogenic variant in ANXA11, expand the range of clinical syndromes caused by ANXA11 mutations, and implicate both neuronal and microglia dysfunction in ANXA11 pathophysiology. This work illustrates the potential for iPSC-derived cellular models to revolutionize the variant annotation process and provides a generalizable approach to determining causality of novel variants across genes.

Application of Aligned-UMAP to longitudinal biomedical studies.

High-dimensional data analysis starts with projecting the data to low dimensions to visualize and understand the underlying data structure. Several methods have been developed for dimensionality reduction, but they are limited to cross-sectional datasets. The recently proposed Aligned-UMAP, an extension of the uniform manifold approximation and projection (UMAP) algorithm, can visualize high-dimensional longitudinal datasets. We demonstrated its utility for researchers to identify exciting patterns and trajectories within enormous datasets in biological sciences. We found that the algorithm parameters also play a crucial role and must be tuned carefully to utilize the algorithm's potential fully. We also discussed key points to remember and directions for future extensions of Aligned-UMAP. Further, we made our code open source to enhance the reproducibility and applicability of our work. We believe our benchmarking study becomes more important as more and more high-dimensional longitudinal data in biomedical research become available.

Mis-spliced transcripts generate de novo proteins in TDP-43-related ALS/FTD.

Functional loss of TDP-43, an RNA-binding protein genetically and pathologically linked to ALS and FTD, leads to inclusion of cryptic exons in hundreds of transcripts during disease. Cryptic exons can promote degradation of affected transcripts, deleteriously altering cellular function through loss-of-function mechanisms. However, the possibility of de novo protein synthesis from cryptic exon transcripts has not been explored. Here, we show that mRNA transcripts harboring cryptic exons generate de novo proteins both in TDP-43 deficient cellular models and in disease. Using coordinated transcriptomic and proteomic studies of TDP-43 depleted iPSC-derived neurons, we identified numerous peptides that mapped to cryptic exons. Cryptic exons identified in iPSC models were highly predictive of cryptic exons expressed in brains of patients with TDP-43 proteinopathy, including cryptic transcripts that generated de novo proteins. We discovered that inclusion of cryptic peptide sequences in proteins altered their interactions with other proteins, thereby likely altering their function. Finally, we showed that these de novo peptides were present in CSF from patients with ALS. The demonstration of cryptic exon translation suggests new mechanisms for ALS pathophysiology downstream of TDP-43 dysfunction and may provide a strategy for novel biomarker development.

Maximizing CRISPRi efficacy and accessibility with dual-sgRNA libraries and optimal effectors.

CRISPR interference (CRISPRi) enables programmable, reversible, and titratable repression of gene expression (knockdown) in mammalian cells. Initial CRISPRi-mediated genetic screens have showcased the potential to address basic questions in cell biology, genetics, and biotechnology, but wider deployment of CRISPRi screening has been constrained by the large size of single guide RNA (sgRNA) libraries and challenges in generating cell models with consistent CRISPRi-mediated knockdown. Here, we present next-generation CRISPRi sgRNA libraries and effector expression constructs that enable strong and consistent knockdown across mammalian cell models. First, we combine empirical sgRNA selection with a dual-sgRNA library design to generate an ultra-compact (1-3 elements per gene), highly active CRISPRi sgRNA library. Next, we compare CRISPRi effectors to show that the recently published Zim3-dCas9 provides an excellent balance between strong on-target knockdown and minimal non-specific effects on cell growth or the transcriptome. Finally, we engineer a suite of cell lines with stable expression of Zim3-dCas9 and robust on-target knockdown. Our results and publicly available reagents establish best practices for CRISPRi genetic screening.

A reference human induced pluripotent stem cell line for large-scale collaborative studies.

Human induced pluripotent stem cell (iPSC) lines are a powerful tool for studying development and disease, but the considerable phenotypic variation between lines makes it challenging to replicate key findings and integrate data across research groups. To address this issue, we sub-cloned candidate human iPSC lines and deeply characterized their genetic properties using whole genome sequencing, their genomic stability upon CRISPR-Cas9-based gene editing, and their phenotypic properties including differentiation to commonly used cell types. These studies identified KOLF2.1J as an all-around well-performing iPSC line. We then shared KOLF2.1J with groups around the world who tested its performance in head-to-head comparisons with their own preferred iPSC lines across a diverse range of differentiation protocols and functional assays. On the strength of these findings, we have made KOLF2.1J and its gene-edited derivative clones readily accessible to promote the standardization required for large-scale collaborative science in the stem cell field.

Tackling neurodegenerative diseases with genomic engineering: A new stem cell initiative from the NIH.

The iPSC Neurodegenerative Disease Initiative (iNDI) is the largest-ever iPSC genome engineering project. iNDI will model more than 100 mutations associated with Alzheimer's disease and related dementias (ADRD) in isogenic iPSC lines. Resulting cell lines and phenotypic datasets will be broadly shared.

Impaired prenatal motor axon development necessitates early therapeutic intervention in severe SMA.

Gene replacement and pre-mRNA splicing modifier therapies represent breakthrough gene targeting treatments for the neuromuscular disease spinal muscular atrophy (SMA), but mechanisms underlying variable efficacy of treatment are incompletely understood. Our examination of severe infantile onset human SMA tissues obtained at expedited autopsy revealed persistence of developmentally immature motor neuron axons, many of which are actively degenerating. We identified similar features in a mouse model of severe SMA, in which impaired radial growth and Schwann cell ensheathment of motor axons began during embryogenesis and resulted in reduced acquisition of myelinated axons that impeded motor axon function neonatally. Axons that failed to ensheath degenerated rapidly postnatally, specifically releasing neurofilament light chain protein into the blood. Genetic restoration of survival motor neuron protein (SMN) expression in mouse motor neurons, but not in Schwann cells or muscle, improved SMA motor axon development and maintenance. Treatment with small-molecule SMN2 splice modifiers beginning immediately after birth in mice increased radial growth of the already myelinated axons, but in utero treatment was required to restore axonal growth and associated maturation, prevent subsequent neonatal axon degeneration, and enhance motor axon function. Together, these data reveal a cellular basis for the fulminant neonatal worsening of patients with infantile onset SMA and identify a temporal window for more effective treatment. These findings suggest that minimizing treatment delay is critical to achieve optimal therapeutic efficacy.

The GENDULF algorithm: mining transcriptomics to uncover modifier genes for monogenic diseases.

Modifier genes are believed to account for the clinical variability observed in many Mendelian disorders, but their identification remains challenging due to the limited availability of genomics data from large patient cohorts. Here, we present GENDULF (GENetic moDULators identiFication), one of the first methods to facilitate prediction of disease modifiers using healthy and diseased tissue gene expression data. GENDULF is designed for monogenic diseases in which the mechanism is loss of function leading to reduced expression of the mutated gene. When applied to cystic fibrosis, GENDULF successfully identifies multiple, previously established disease modifiers, including EHF, SLC6A14, and CLCA1. It is then utilized in spinal muscular atrophy (SMA) and predicts U2AF1 as a modifier whose low expression correlates with higher SMN2 pre-mRNA exon 7 retention. Indeed, knockdown of U2AF1 in SMA patient-derived cells leads to increased full-length SMN2 transcript and SMN protein expression. Taking advantage of the increasing availability of transcriptomic data, GENDULF is a novel addition to existing strategies for prediction of genetic disease modifiers, providing insights into disease pathogenesis and uncovering novel therapeutic targets.

Age-dependent SMN expression in disease-relevant tissue and implications for SMA treatment.

BACKGROUNDSpinal muscular atrophy (SMA) is caused by deficient expression of survival motor neuron (SMN) protein. New SMN-enhancing therapeutics are associated with variable clinical benefits. Limited knowledge of baseline and drug-induced SMN levels in disease-relevant tissues hinders efforts to optimize these treatments.METHODSSMN mRNA and protein levels were quantified in human tissues isolated during expedited autopsies.RESULTSSMN protein expression varied broadly among prenatal control spinal cord samples, but was restricted at relatively low levels in controls and SMA patients after 3 months of life. A 2.3-fold perinatal decrease in median SMN protein levels was not paralleled by comparable changes in SMN mRNA. In tissues isolated from nusinersen-treated SMA patients, antisense oligonucleotide (ASO) concentration and full-length (exon 7 including) SMN2 (SMN2-FL) mRNA level increases were highest in lumbar and thoracic spinal cord. An increased number of cells showed SMN immunolabeling in spinal cord of treated patients, but was not associated with an increase in whole-tissue SMN protein levels.CONCLUSIONSA normally occurring perinatal decrease in whole-tissue SMN protein levels supports efforts to initiate SMN-inducing therapies as soon after birth as possible. Limited ASO distribution to rostral spinal and brain regions in some patients likely limits clinical response of motor units in these regions for those patients. These results have important implications for optimizing treatment of SMA patients and warrant further investigations to enhance bioavailability of intrathecally administered ASOs.FUNDINGSMA Foundation, SMART, NIH (R01-NS096770, R01-NS062869), Ionis Pharmaceuticals, and PTC Therapeutics. Biogen provided support for absolute real-time RT-PCR.

αvß6-Fyn Kinase Promotes Epithelial Phenotype in SYF Cells.

The 5-year survival rate for patients with oral cancer remains at 50%, in large part due the high rate of post-treatment recurrence. In this study, we transfected epithelial-specific integrin αvß6 and Fyn-kinase, a member of the Src-family kinases, into embryonic murine fibroblasts. In oral cancer, expression of αvß6 is neo-expressed. Using a variety of in vitro assays, including cell migration and multicellular spheroid formation, we determined that these embryonic fibroblasts expressing αvß6 and Fyn-kinase were able to acquire an epithelial phenotype. This is in direct contrast to human oral SCC, where expression of αvß6 with Fyn-kinase promotes epithelial to mesenchymal transition. This demonstrates that signaling pathways can be species-specific.

The Antisense Transcript SMN-AS1 Regulates SMN Expression and Is a Novel Therapeutic Target for Spinal Muscular Atrophy.

The neuromuscular disorder spinal muscular atrophy (SMA), the most common inherited killer of infants, is caused by insufficient expression of survival motor neuron (SMN) protein. SMA therapeutics development efforts have focused on identifying strategies to increase SMN expression. We identified a long non-coding RNA (lncRNA) that arises from the antisense strand of SMN, SMN-AS1, which is enriched in neurons and transcriptionally represses SMN expression by recruiting the epigenetic Polycomb repressive complex-2. Targeted degradation of SMN-AS1 with antisense oligonucleotides (ASOs) increases SMN expression in patient-derived cells, cultured neurons, and the mouse central nervous system. SMN-AS1 ASOs delivered together with SMN2 splice-switching oligonucleotides additively increase SMN expression and improve survival of severe SMA mice. This study is the first proof of concept that targeting a lncRNA to transcriptionally activate SMN2 can be combined with SMN2 splicing modification to ameliorate SMA and demonstrates the promise of combinatorial ASOs for the treatment of neurogenetic disorders.

Multicellular Spheroids as a Model for Hypoxia-induced EMT.

Oral cancer is aggressive and invasive. The 5-year survival rate is around 50% and has not improved in over 50 years. One-third of oral cancer patients develop local and/or regional tumor recurrence following treatment. We continue to use our multicellular spheroid (MCS) model to better understand how the extracellular matrix contributes to epithelial to mesenchymal transition and how hypoxia contributes to the progression of oral squamous cell carcinoma (SCC).

Regulation of Multicellular Spheroids by MAPK and FYN Kinase.

Understanding of the biology of oral squamous cell carcinoma (SCC) has not progressed significantly in the past 60 years, with 5-year survival remaining at approximately 50%. The epidemic of Human Papilloma Virus and its associated SCC warrants a renewed emphasis on fully understanding this disease. We previously used the 3-dimensional multicellular spheroid (MCS) model system to evaluate SCC behavior more accurately. In this study, we determined that SCC growth in MCS approximates epithelial to mesenchymal transition. Organization of an MCS requires the full-length ß6 integrin subunit and its maintenance requires mitogen-activated protein kinase (MAPK). Limiting FYN kinase activation results in the down-regulation of E-cadherin, ß-catenin and an increase in expression of N-cadherin and SNAIL. These results indicate that the microenvironment and growth patterns in an MCS are complex and require MAPK and FYN kinase.

Gingival pustules and sterile diffuse sclerosing osteomyelitis as a feature of synovitis, acne, pustulosis, hyperostosis, osteitis (SAPHO) syndrome.

Synovitis, acne, pustulosis, hyperostosis, osteitis (SAPHO) syndrome represents the rare co-occurrence of sterile inflammatory osteoarticular disease in association with a variety of cutaneous manifestations. Oral involvement is uncommon. The etiology of SAPHO is complex and is likely the combined result of infectious, genetic, and immunologic factors. Due to diverse clinical presentations, SAPHO is difficult to diagnose. Here, we describe the case of a 74-year-old man, who had a history of SAPHO syndrome and presented with gingival pustules and sterile diffuse sclerosing osteomyelitis of the mandible. This is the first case report describing neutrophilic mucositis as a feature of SAPHO.

Differential diagnosis and management of oral ulcers.

The diagnosis and treatment of oral lesions is often challenging due to the clinician's limited exposure to the conditions that may cause the lesions and their similar appearances. While many oral ulcers are the result of chronic trauma, some may indicate an underlying systemic condition such as a gastrointestinal dysfunction, malignancy, immunologic abnormality, or cutaneous disease. Correctly establishing a definitive diagnosis is of major importance to clinicians who manage patients with oral mucosal disease. Some of these diseases are infectious; however, most are chronic, symptomatic, and desquamative. Treatment and management requires an understanding of the immunopathologic nature of the lesion. This review will address how to differentiate and diagnose varying types of oral ulcers and provide a treatment strategy.

Optogenetic activation of cholinergic neurons in the PPT or LDT induces REM sleep.

Rapid eye movement (REM) sleep is an important component of the natural sleep/wake cycle, yet the mechanisms that regulate REM sleep remain incompletely understood. Cholinergic neurons in the mesopontine tegmentum have been implicated in REM sleep regulation, but lesions of this area have had varying effects on REM sleep. Therefore, this study aimed to clarify the role of cholinergic neurons in the pedunculopontine tegmentum (PPT) and laterodorsal tegmentum (LDT) in REM sleep generation. Selective optogenetic activation of cholinergic neurons in the PPT or LDT during non-REM (NREM) sleep increased the number of REM sleep episodes and did not change REM sleep episode duration. Activation of cholinergic neurons in the PPT or LDT during NREM sleep was sufficient to induce REM sleep.

Differential spheroid formation by oral cancer cells.

Squamous cell carcinomas (SCC) make up 96% of all oral cancers. Most laboratory SCC studies grow cells as a monolayer, which does not accurately represent the disease in vivo. We used a more relevant multicellular spheroid (MCS) model to study this disease. The SCC9ß6KDFyn cell line, which expresses full-length ß6 and a kinase dead Fyn formed the largest MCS. Cell adhesive properties are dynamic and N-cadherin was increased in the largest MCS. c-Raf mediates the survival of tumor cells and was consistently expressed both in monolayers and in the MCS by SCC9ß6D1 cells which lack the ß6 cytoplasmic tail and, do not activate Fyn. SCC9ß6KDFyn cells also express high levels of c-Raf when grown as spheroids in which Fyn suppression stimulates MCS formation. Tumor microenvironment and growth patterns modulate cell behavior and suppression of Fyn kinase may promote MCS growth.

The cytoplasmic extension of the integrin ß6 subunit regulates epithelial-to-mesenchymal transition.

Prognosis for oral cancer patients has not improved in over 60 years due to invasion and recurrence. To understand the invasive behavior of this tumor, we evaluated the role of the αvß6 integrin. Invasive oral SCC cells express the αvß6 integrin, which contains an 11-amino-acid extension on its ß-subunit unique to the integrin family. We determined that this ß6 cytoplasmic extension regulates the composition of the intermediate filament network and the organization of signaling structures called focal contacts. The auto-phosphorylation of FAK, which is localized to focal contacts, was also regulated by the ß6-cytoplasmic tail, as were the transcription factors Notch and STAT3. Lastly, we also determined that activation of MAPK required the full-length ß6 integrin. Together these results indicate that the signaling critical to epithelial-to-mesenchymal transition (EMT) is regulated by the ß6 integrin cytoplasmic domain.

Wnt signaling regulates pulp volume and dentin thickness.

Odontoblasts, cementoblasts, ameloblasts, and osteoblasts all form mineralized tissues in the craniofacial complex, and all these cell types exhibit active Wnt signaling during postnatal life. We set out to understand the functions of this Wnt signaling, by evaluating the phenotypes of mice in which the essential Wnt chaperone protein, Wntless was eliminated. The deletion of Wls was restricted to cells expressing Osteocalcin (OCN), which in addition to osteoblasts includes odontoblasts, cementoblasts, and ameloblasts. Dentin, cementum, enamel, and bone all formed in OCN-Cre;Wls(fl/fl) mice but their homeostasis was dramatically affected. The most notable feature was a significant increase in dentin volume and density. We attribute this gain in dentin volume to a Wnt-mediated misregulation of Runx2. Normally, Wnt signaling stimulates Runx2, which in turn inhibits dentin sialoprotein (DSP); this inhibition must be relieved for odontoblasts to differentiate. In OCN-Cre;Wls(fl/fl) mice, Wnt pathway activation is reduced and Runx2 levels decline. The Runx2-mediated repression of DSP is relieved and odontoblast differentiation is accordingly enhanced. This study demonstrates the importance of Wnt signaling in the homeostasis of mineralized tissues of the craniofacial complex.