Reading Frame Repair of TTN Truncation Variants Restores Titin Quantity and Functions

Background: Titin truncation variants (TTNtvs) are the most common inheritable risk factor for dilated cardiomyopathy (DCM), a disease with high morbidity and mortality. The pathogenicity of TTNtvs has been associated with structural localization as A-band variants overlapping myosin heavy chain-binding domains are more pathogenic than I-band variants by incompletely understood mechanisms. Demonstrating why A-band variants are highly pathogenic for DCM could reveal new insights into DCM pathogenesis, TTN functions and therapeutic targets. Methods: We constructed human cardiomyocyte models harboring DCM-associated TTNtvs within A-band and I-band structural domains using induced pluripotent stem cell and CRISPR technologies. We characterized normal TTN isoforms and variant-specific truncation peptides by their expression levels and cardiomyocyte localization using TTN protein gel electrophoresis and immunofluorescence, respectively. Using CRISPR to ablate A-band variant-specific truncation peptides through introduction of a proximal I-band TTNtv, we studied genetic mechanisms in single cardiomyocyte and 3-dimensional, biomimetic cardiac microtissue functional assays. Finally, we engineered a full-length TTN protein reporter assay and utilized next-generation sequencing assays to develop a CRISPR therapeutic for somatic cell genome editing TTNtvs. Results: An A-band TTNtv dose-dependently impaired cardiac microtissue twitch force, reduced full-length TTN levels, and produced abundant TTN truncation peptides. TTN truncation peptides integrated into nascent myofibril-like structures and impaired myofibrillogenesis. CRISPR-ablation of TTN truncation peptides using a proximal I-band TTNtv partially restored cardiac microtissue twitch force deficits. Cardiomyocyte genome-editing using SpCas9 and a TTNtv-specific guide RNA restored TTN protein reading frame, which increased full length TTN protein levels, reduced TTN truncation peptides, and increased sarcomere function in cardiac microtissue assays. Conclusions: An A-band TTNtv diminished sarcomere function greater than an I-band TTNtv in proportion to estimated DCM pathogenicity. While both TTNtvs resulted in full-length TTN haploinsufficiency, only the A-band TTNtv produced TTN truncation peptides that impaired myofibrillogenesis and sarcomere function. CRISPR-mediated reading frame repair of the A-band TTNtv restored functional deficits, and could be adapted as a "one-and-done" genome editing strategy to target ∼30% of DCM-associated TTNtvs.

CACNA1A Mutations Causing Early Onset Ataxia: Profiling Clinical, Dysmorphic and Structural-Functional Findings

The CACNA1A gene encodes the pore-forming α1A subunit of the voltage-gated CaV2.1 Ca2+ channel, essential in neurotransmission, especially in Purkinje cells. Mutations in CACNA1A result in great clinical heterogeneity with progressive symptoms, paroxysmal events or both. During infancy, clinical and neuroimaging findings may be unspecific, and no dysmorphic features have been reported. We present the clinical, radiological and evolutionary features of three patients with congenital ataxia, one of them carrying a new variant. We report the structural localization of variants and their expected functional consequences. There was an improvement in cerebellar syndrome over time despite a cerebellar atrophy progression, inconsistent response to acetazolamide and positive response to methylphenidate. The patients shared distinctive facial gestalt: oval face, prominent forehead, hypertelorism, downslanting palpebral fissures and narrow nasal bridge. The two α1A affected residues are fully conserved throughout evolution and among the whole human CaV channel family. They contribute to the channel pore and the voltage sensor segment. According to structural data analysis and available functional characterization, they are expected to exert gain- (F1394L) and loss-of-function (R1664Q/R1669Q) effect, respectively. Among the CACNA1A-related phenotypes, our results suggest that non-progressive congenital ataxia is associated with developmental delay and dysmorphic features, constituting a recognizable syndromic neurodevelopmental disorder.

Analyzing Brain Morphology in Alzheimer's Disease Using Discriminative and Generative Spiral Networks

Several patterns of atrophy have been identified and strongly related to Alzheimer's disease (AD) pathology and its progression. Morphological changes in brain shape have been identified up to ten years before clinical diagnoses of AD, making its early diagnosis more desirable. We propose novel geometric deep learning frameworks for the analysis of brain shape in the context of neurodegeneration caused by AD. Our deep neural networks learn low-dimensional shape descriptors of multiple neuroanatomical structures, instead of handcrafted features for each structure. A discriminative network using spiral convolution on 3D meshes is constructed for the in-vivo binary classification of AD from healthy controls (HCs) using a fast and efficient "spiral" convolution operator on 3D triangular mesh surfaces of human brain subcortical structures extracted from T1-weighted magnetic resonance imaging (MRI). Our network architecture consists of modular learning blocks using residual connections to improve overall classifier performance. In this work: (1) a discriminative network is used to analyze the efficacy of disease classification using input data from multiple brain structures and compared to using a single hemisphere or a single structure. It also outperforms prior work using spectral graph convolution on the same the same tasks, as well as alternative methods that operate on intermediate point cloud representations of 3D shapes. (2) Additionally, visual interpretations for regions on the surface of brain structures that are associated to true positive AD predictions are generated and fall in accordance with the current reports on the structural localization of pathological changes associated to AD. (3) A conditional generative network is also implemented to analyze the effects of phenotypic priors given to the model (i.e. AD diagnosis) in generating subcortical structures. The generated surface meshes by our model indicate learned morphological differences in the presence of AD that agrees with the current literature on patterns of atrophy associated to the disease. In particular, our inference results demonstrate an overall reduction in subcortical mesh volume and surface area in the presence of AD, especially in the hippocampus. The low-dimensional shape descriptors obtained by our generative model are also evaluated in our discriminative baseline comparisons versus our discriminative network and the alternative shape-based approaches.

Cell Surface Processing of CD109 by Meprin β Leads to the Release of Soluble Fragments and Reduced Expression on Extracellular Vesicles

Cluster of differentiation 109 (CD109) is a glycosylphosphatidylinositol (GPI)-anchored protein expressed on primitive hematopoietic stem cells, activated platelets, CD4+ and CD8+ T cells, and keratinocytes. In recent years, CD109 was also associated with different tumor entities and identified as a possible future diagnostic marker linked to reduced patient survival. Also, different cell signaling pathways were proposed as targets for CD109 interference including the TGFβ, JAK-STAT3, YAP/TAZ, and EGFR/AKT/mTOR pathways. Here, we identify the metalloproteinase meprin β to cleave CD109 at the cell surface and thereby induce the release of cleavage fragments of different size. Major cleavage was identified within the bait region of CD109 residing in the middle of the protein. To identify the structural localization of the bait region, homology modeling and single-particle analysis were applied, resulting in a molecular model of membrane-associated CD109, which allows for the localization of the newly identified cleavage sites for meprin β and the previously published cleavage sites for the metalloproteinase bone morphogenetic protein-1 (BMP-1). Full-length CD109 localized on extracellular vesicles (EVs) was also identified as a release mechanism, and we can show that proteolytic cleavage of CD109 at the cell surface reduces the amount of CD109 sorted to EVs. In summary, we identified meprin β as the first membrane-bound protease to cleave CD109 within the bait region, provide a first structural model for CD109, and show that cell surface proteolysis correlates negatively with CD109 released on EVs.

An efficient autometallography approach to localize lead at ultra-structural levels of cultured cells

Understanding the precise intracellular localization of lead (Pb) is a key in deciphering processes in Pb-induced toxicology. However, it is a great challenge to trace Pb in vitro, especially in cultured cells. We aimed to find an innovative and efficient approach to investigate distribution of Pb in cells and to validate it through determining the subcellular Pb content. We identified its ultra-structural distribution with autometallography under electron microscopy in a choroidal epithelial Z310 cell line. Electron microscopy in combination with energy-dispersive X-ray spectroscope (EDS) was employed to provide further evidence of Pb location. In addition, Pb content was determined in the cytosol, membrane/organelle, nucleus and cytoskeleton fractions with atomic absorption spectroscopy. Pb was found predominantly inside the nuclear membranes and some was distributed in the cytoplasm under low-concentration exposure. Nuclear existence of Pb was verified by EDS under electron microscopy. Once standardized for protein content, Pb percentage in the nucleus fraction reached the highest level (76%). Our results indicate that Pb is accumulated mainly in the nucleus of choroid plexus. This method is sensitive and precise in providing optimal means to study the ultra-structural localization of Pb for in vitro models. In addition, it offers the possibility of localization of other metals in cultured cells. Some procedures may also be adopted to detect target proteins via immunoreactions.

Characterization of cathepsin S exosites that govern its elastolytic activity

We have previously determined that the elastolytic activities of cathepsins (Cat) K and V require two exosites sharing the same structural localization on both enzymes. The structural features involved in the elastolytic activity of CatS have not yet been identified. We first mutated the analogous CatK and V putative exosites of CatS into the elastolytically inactive CatL counterparts. The modification of the exosite 1 did not affect the elastase activity of CatS whilst mutation of the Y118 of exosite 2 decreased the cleavage of elastin by ∼70% without affecting the degradation of other macromolecular substrates (gelatin, thyroglobulin). T06, an ectosteric inhibitor that disrupt the elastolytic activity of CatK, blocked ∼80% of the elastolytic activity of CatS without blocking the cleavage of gelatin and thyroglobulin. Docking studies showed that T06 preferentially interacts with a binding site located on the Right domain of the enzyme, outside of the active site. The structural examination of this binding site showed that the loop spanning the L174N175G176K177 residues of CatS is considerably different from that of CatL. Mutation of this loop into the CatL-like equivalent decreased elastin degradation by ∼70% and adding the Y118 mutation brought down the loss of elastolysis to ∼80%. In addition, the Y118 mutation selectively reduced the cleavage of the basement membrane component laminin by ∼50%. In summary, our data show that the degradation of elastin by CatS requires two exosites where one of them is distinct from those of CatK and V whilst the cleavage of laminin requires only one exosite.

Percutaneous Endoscopic Debridement and Drainage with Four Different Approach Methods for the Treatment of Spinal Infection

Background: The incidence of spinal infection seems to be increasing in recent years. Percutaneous endoscopic debridement and drainage (PEDD) has become an effective alternative to extensive open surgery. Objective: This study reviewed the charter of patients who received PEDD using 4 different approach methods to evaluate the clinical results. Study Design: An Institutional Review Board (IRB)-approved retrospective chart review. Setting: University hospital inpatient referred to our pain clinic. Methods: A retrospective patient chart analysis of PEDD procedures in spinal infections over a 7-year period was done for the evaluation of structural location, symptoms and signs, etiologic agents, and outcomes. Results: Seventeen patients (11 men and 6 women, mean age 70.4 ± 11.1 years) with spinal infections received PEDD. According to the structural localization of the spinal infections, 6 cases of spondylodiscitis alone, 5 cases of spondylodiscitis with a psoas abscess, one case of spondylodiscitis with an epidural abscess, 4 cases of spondylodiscitis with epidural and psoas abscesses, and one case of spondylodiscitis with a facet joint abscess were found. All patients had preoperative symptoms of unremitting backache and febrile sensation, and signs of paravertebral muscle tenderness and limitation of spine motion. The most common etiologic bacteria were Staphylococcus aureus. Most patients (14/17) improved; the 2 failed patients received a second PEDD after recurrence, and the other received open surgery without re-PEDD. Both the numeric rating scale and Oswestry disability index scores were significantly reduced after PEDD. No complications related to PEDD were found. Limitation: This study is limited by its retrospective design. Conclusions: PEDD using 4 different routes brought immediate pain relief and reduced disability in treating spinal infections, especially in elderly patients with comorbid underlying disorders.