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1.
EMBO Mol Med ; 13(11): e14095, 2021 11 08.
Article in English | MEDLINE | ID: mdl-34632710

ABSTRACT

Spinocerebellar ataxia type 8 (SCA8), a dominantly inherited neurodegenerative disorder caused by a CTG•CAG expansion, is unusual because most individuals that carry the mutation do not develop ataxia. To understand the variable penetrance of SCA8, we studied the molecular differences between highly penetrant families and more common sporadic cases (82%) using a large cohort of SCA8 families (n = 77). We show that repeat expansion mutations from individuals with multiple affected family members have CCG•CGG interruptions at a higher frequency than sporadic SCA8 cases and that the number of CCG•CGG interruptions correlates with age at onset. At the molecular level, CCG•CGG interruptions increase RNA hairpin stability, and in cell culture experiments, increase p-eIF2α and polyAla and polySer RAN protein levels. Additionally, CCG•CGG interruptions, which encode arginine interruptions in the polyGln frame, increase toxicity of the resulting proteins. In summary, SCA8 CCG•CGG interruptions increase polyAla and polySer RAN protein levels, polyGln protein toxicity, and disease penetrance and provide novel insight into the molecular differences between SCA8 families with high vs. low disease penetrance.


Subject(s)
Spinocerebellar Degenerations , Trinucleotide Repeat Expansion , Ataxia , Humans , Nerve Tissue Proteins/genetics , Penetrance , Proteins , RNA, Long Noncoding/genetics , Spinocerebellar Degenerations/genetics
2.
Hum Mol Genet ; 29(24): 3900-3918, 2021 02 25.
Article in English | MEDLINE | ID: mdl-33378537

ABSTRACT

C9orf72 ALS/FTD patients show remarkable clinical heterogeneity, but the complex biology of the repeat expansion mutation has limited our understanding of the disease. BAC transgenic mice were used to better understand the molecular mechanisms and repeat length effects of C9orf72 ALS/FTD. Genetic analyses of these mice demonstrate that the BAC transgene and not integration site effects cause ALS/FTD phenotypes. Transcriptomic changes in cell proliferation, inflammation and neuronal pathways are found late in disease and alternative splicing changes provide early molecular markers that worsen with disease progression. Isogenic sublines of mice with 800, 500 or 50 G4C2 repeats generated from the single-copy C9-500 line show longer repeats result in earlier onset, increased disease penetrance and increased levels of RNA foci and dipeptide RAN protein aggregates. These data demonstrate G4C2 repeat length is an important driver of disease and identify alternative splicing changes as early biomarkers of C9orf72 ALS/FTD.


Subject(s)
Alternative Splicing , Amyotrophic Lateral Sclerosis/pathology , C9orf72 Protein/metabolism , DNA Repeat Expansion , Disease Models, Animal , Frontotemporal Dementia/pathology , Penetrance , Amyotrophic Lateral Sclerosis/etiology , Amyotrophic Lateral Sclerosis/metabolism , Animals , C9orf72 Protein/genetics , Frontotemporal Dementia/etiology , Frontotemporal Dementia/metabolism , Humans , Mice , Mice, Transgenic , Mutation , Phenotype
3.
Neuron ; 108(4): 784-796.e3, 2020 11 25.
Article in English | MEDLINE | ID: mdl-33022226

ABSTRACT

Mordes et al. (2020) did not detect the survival or motor phenotypes in C9orf72 BAC transgenic mice originally described by Liu et al. (2016). We discuss methodological differences between the Mordes and Liu studies, several additional studies in which survival and motor phenotypes were found, and possible environmental and genetic effects. First, Nguyen et al. (2020) showed robust ALS/FTD phenotypes in C9-BAC versus non-transgenic (NT) mice and that α-GA1 treatment improved survival, behavior, and neurodegeneration. The groups of Gelbard and Saxena also show decreased survival of C9-BAC versus NT mice and neuropathological and behavioral deficits similar to those shown by Liu et al. (2016). Although FVB/N mice can have seizures, increases in seizure severity and death of C9 and NT animals, which may mask C9 disease phenotypes, have been observed in recent C9-500 FVB/NJ-bred cohorts. In summary, we provide an update on phenotypes seen in FVB C9-BAC mice and additional details to successfully use this model. This Matters Arising Response paper addresses the Mordes et al. (2020) Matters Arising paper, published concurrently in Neuron.


Subject(s)
Amyotrophic Lateral Sclerosis , Frontotemporal Dementia , Amyotrophic Lateral Sclerosis/genetics , Animals , C9orf72 Protein/genetics , DNA Repeat Expansion , Disease Models, Animal , Frontotemporal Dementia/genetics , Mice , Mice, Transgenic , Phenotype
4.
J Exp Zool B Mol Dev Evol ; 334(7-8): 423-437, 2020 11.
Article in English | MEDLINE | ID: mdl-32614138

ABSTRACT

Evolution in similar environments often leads to convergence of behavioral and anatomical traits. A classic example of convergent trait evolution is the reduced traits that characterize many cave animals: reduction or loss of pigmentation and eyes. While these traits have evolved many times, relatively little is known about whether these traits repeatedly evolve through the same or different molecular and developmental mechanisms. The small freshwater fish, Astyanax mexicanus, provides an opportunity to investigate the repeated evolution of cave traits. A. mexicanus exists as two forms, a sighted, surface-dwelling form and at least 29 populations of a blind, cave-dwelling form that initially develops eyes that subsequently degenerate. We compared eye morphology and the expression of eye regulatory genes in developing surface fish and two independently evolved cavefish populations, Pachón and Molino. We found that many of the previously described molecular and morphological alterations that occur during eye development in Pachón cavefish are also found in Molino cavefish. However, for many of these traits, the Molino cavefish have a less severe phenotype than Pachón cavefish. Further, cave-cave hybrid fish have larger eyes and lenses during early development compared with fish from either parental population, suggesting that some different changes underlie eye loss in these two populations. Together, these data support the hypothesis that these two cavefish populations evolved eye loss independently, yet through some of the same developmental and molecular mechanisms.


Subject(s)
Anophthalmos/veterinary , Biological Evolution , Characidae/growth & development , Animals , Caves , Characidae/abnormalities , Characidae/genetics , Eye/growth & development , In Situ Hybridization
5.
Neuron ; 105(4): 645-662.e11, 2020 02 19.
Article in English | MEDLINE | ID: mdl-31831332

ABSTRACT

The intronic C9orf72 G4C2 expansion, the most common genetic cause of ALS and FTD, produces sense- and antisense-expansion RNAs and six dipeptide repeat-associated, non-ATG (RAN) proteins, but their roles in disease are unclear. We generated high-affinity human antibodies targeting GA or GP RAN proteins. These antibodies cross the blood-brain barrier and co-localize with intracellular RAN aggregates in C9-ALS/FTD BAC mice. In cells, α-GA1 interacts with TRIM21, and α-GA1 treatment reduced GA levels, increased GA turnover, and decreased RAN toxicity and co-aggregation of proteasome and autophagy proteins to GA aggregates. In C9-BAC mice, α-GA1 reduced GA as well as GP and GR proteins, improved behavioral deficits, decreased neuroinflammation and neurodegeneration, and increased survival. Glycosylation of the Fc region of α-GA1 is important for cell entry and efficacy. These data demonstrate that RAN proteins drive C9-ALS/FTD in C9-BAC transgenic mice and establish a novel therapeutic approach for C9orf72 ALS/FTD and other RAN-protein diseases.


Subject(s)
Amyotrophic Lateral Sclerosis/genetics , Antibodies, Monoclonal/genetics , C9orf72 Protein/genetics , Frontotemporal Dementia/genetics , Genetic Therapy/methods , ran GTP-Binding Protein/metabolism , Aged , Amyotrophic Lateral Sclerosis/metabolism , Animals , Antibodies, Monoclonal/administration & dosage , Antibodies, Monoclonal/metabolism , Brain/metabolism , C9orf72 Protein/metabolism , Cell Line, Tumor , Disease Models, Animal , Female , Frontotemporal Dementia/metabolism , Gene Targeting/methods , HEK293 Cells , Humans , Mice , Mice, Inbred C57BL , Mice, Transgenic , Phenotype , Random Allocation , Recombinant Proteins/administration & dosage , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , ran GTP-Binding Protein/antagonists & inhibitors
6.
Sci Rep ; 9(1): 15778, 2019 10 31.
Article in English | MEDLINE | ID: mdl-31673015

ABSTRACT

Retinal ganglion cells can be classified into more than 40 distinct subtypes, whether by functional classification or transcriptomics. The examination of these subtypes in relation to their physiology, projection patterns, and circuitry would be greatly facilitated through the identification of specific molecular identifiers for the generation of transgenic mice. Advances in single cell transcriptomic profiling have enabled the identification of molecular signatures for cellular subtypes that are only rarely found. Therefore, we used single cell profiling combined with hierarchical clustering and correlate analyses to identify genes expressed in distinct populations of Parvalbumin-expressing cells and functionally classified RGCs. RGCs were manually isolated based either upon fluorescence or physiological distinction through cell-attached recordings. Microarray hybridization and RNA-Sequencing were employed for the characterization of transcriptomes and in situ hybridization was utilized to further characterize gene candidate expression. Gene candidates were identified based upon cluster correlation, as well as expression specificity within physiologically distinct classes of RGCs. Further, we identified Prph, Ctxn3, and Prkcq as potential candidates for ipRGC classification in the murine retina. The use of these genes, or one of the other newly identified subset markers, for the generation of a transgenic mouse would enable future studies of RGC-subtype specific function, wiring, and projection.


Subject(s)
Eye Proteins/biosynthesis , Gene Expression Profiling , Gene Expression Regulation , Oligonucleotide Array Sequence Analysis , Retinal Ganglion Cells/metabolism , Single-Cell Analysis , Animals , Eye Proteins/genetics , Mice , Mice, Transgenic , Retinal Ganglion Cells/cytology
7.
PLoS One ; 13(9): e0202867, 2018.
Article in English | MEDLINE | ID: mdl-30208054

ABSTRACT

To better understand the mechanisms that govern the development of retinal neurons, it is critical to gain additional insight into the specific intrinsic factors that control cell fate decisions and neuronal maturation. In the developing mouse retina, Atoh7, a highly conserved transcription factor, is essential for retinal ganglion cell development. Moreover, Atoh7 expression in the developing retina occurs during a critical time period when progenitor cells are in the process of making cell fate decisions. We performed transcriptome profiling of Atoh7+ individual cells isolated from mouse retina. One of the genes that we found significantly correlated with Atoh7 in our transcriptomic data was the E3 ubiquitin ligase, Trim9. The correlation between Trim9 and Atoh7 coupled with the expression of Trim9 in the early mouse retina led us to hypothesize that this gene may play a role in the process of cell fate determination. To address the role of Trim9 in retinal development, we performed a functional analysis of Trim9 in the mouse and did not detect any morphological changes in the retina in the absence of Trim9. Thus, Trim9 alone does not appear to be involved in cell fate determination or early ganglion cell development in the mouse retina. We further hypothesize that the reason for this lack of phenotype may be compensation by one of the many additional TRIM family members we find expressed in the developing retina.


Subject(s)
Retina/metabolism , Tripartite Motif Proteins/metabolism , Animals , Basic Helix-Loop-Helix Transcription Factors/metabolism , Carrier Proteins/genetics , Carrier Proteins/metabolism , Embryo, Mammalian/metabolism , Female , Genotype , In Situ Hybridization , Mice , Mice, Knockout , Nerve Tissue Proteins/deficiency , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Pregnancy , Retina/pathology , Retinal Neurons/metabolism , Retinal Neurons/pathology , Transcriptome , Tripartite Motif Proteins/genetics , Ubiquitin-Protein Ligases
8.
Gene Expr Patterns ; 30: 14-31, 2018 12.
Article in English | MEDLINE | ID: mdl-30165106

ABSTRACT

Amyotrophic lateral sclerosis (ALS) is characterized by progressive muscle atrophy resulting from the deterioration of motor neurons in the central nervous system (CNS). Recent genome-wide association studies have revealed several genes linked to ALS, further demonstrating the complexity of the disease. The zebrafish (Danio rerio) is an attractive model organism to study the function of the rapidly expanding number of ALS-associated genes, in part, due to the development of genome editing techniques that have facilitated specific gene targeting. Before investing in the manipulation and phenotypic examination of these genes, however, it is important to ascertain the localization of expression in this organism. We performed an expression analysis of 29 total ALS-linked genes in the developing zebrafish, specifically focusing on those genes that displayed robust and reproducible expression at multiple different timepoints. First, we classified a subset of the most robustly expressed genes into three distinct groups: head-only expression, head and weak trunk expression, and head and robust trunk expression. Then, we defined the characteristic pattern of each gene at 2, 3, and 4 days post fertilization. This analysis will facilitate improved mutant phenotype assessment in zebrafish by focusing researchers on the areas of expression.


Subject(s)
Amyotrophic Lateral Sclerosis/genetics , Central Nervous System/metabolism , Zebrafish/growth & development , Zebrafish/genetics , Amyotrophic Lateral Sclerosis/pathology , Animals , Animals, Genetically Modified/genetics , Animals, Genetically Modified/growth & development , Animals, Genetically Modified/metabolism , Phenotype , Zebrafish/metabolism , Zebrafish Proteins/genetics
9.
J Vis Exp ; (123)2017 05 22.
Article in English | MEDLINE | ID: mdl-28570514

ABSTRACT

The discovery of cell type-specific markers can provide insight into cellular function and the origins of cellular heterogeneity. With a recent push for the improved understanding of neuronal diversity, it is important to identify genes whose expression defines various subpopulations of cells. The retina serves as an excellent model for the study of central nervous system diversity, as it is composed of multiple major cell types. The study of each major class of cells has yielded genetic markers that facilitate the identification of these populations. However, multiple subtypes of cells exist within each of these major retinal cell classes, and few of these subtypes have known genetic markers, although many have been characterized by morphology or function. A knowledge of genetic markers for individual retinal subtypes would allow for the study and mapping of brain targets related to specific visual functions and may also lend insight into the gene networks that maintain cellular diversity. Current avenues used to identify the genetic markers of subtypes possess drawbacks, such as the classification of cell types following sequencing. This presents a challenge for data analysis and requires rigorous validation methods to ensure that clusters contain cells of the same function. We propose a technique for identifying the morphology and functionality of a cell prior to isolation and sequencing, which will allow for the easier identification of subtype-specific markers. This technique may be extended to non-neuronal cell types, as well as to rare populations of cells with minor variations. This protocol yields excellent-quality data, as many of the libraries have provided read depths greater than 20 million reads for single cells. This methodology overcomes many of the hurdles presented by Single-cell RNA-Seq and may be suitable for researchers aiming to profile cell types in a straightforward and highly efficient manner.


Subject(s)
Retinal Ganglion Cells/cytology , Sequence Analysis, RNA , Single-Cell Analysis , Gene Regulatory Networks
10.
J Comp Neurol ; 525(12): 2735-2781, 2017 Aug 15.
Article in English | MEDLINE | ID: mdl-28510275

ABSTRACT

The vertebrate retina is a specialized photosensitive tissue comprised of six neuronal and one glial cell types, each of which develops in prescribed proportions at overlapping timepoints from a common progenitor pool. While each of these cells has a specific function contributing to proper vision in the mature animal, their differential representation in the retina as well as the presence of distinctive cellular subtypes makes identifying the transcriptomic signatures that lead to each retinal cell's fate determination and development challenging. We have analyzed transcriptomes from individual cells isolated from the chick retina throughout retinogenesis. While we focused our efforts on the retinal ganglion cells, our transcriptomes of developing chick cells also contained representation from multiple retinal cell types, including photoreceptors and interneurons at different stages of development. Most interesting was the identification of transcriptomes from individual mixed lineage progenitor cells in the chick as these cells offer a window into the cell fate decision-making process. Taken together, these data sets will enable us to uncover the most critical genes acting in the steps of cell fate determination and early differentiation of various retinal cell types.


Subject(s)
Gene Expression Regulation, Developmental/physiology , Neuroglia/metabolism , Neurons/metabolism , Organogenesis/physiology , Retina/cytology , Retina/embryology , Retinal Ganglion Cells/metabolism , Single-Cell Analysis/methods , Stem Cells/metabolism , Transcriptome/physiology , Animals , Chick Embryo , Gene Expression Profiling , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Neurons/classification , Stem Cells/cytology
11.
Bioinformatics ; 32(17): i421-i429, 2016 09 01.
Article in English | MEDLINE | ID: mdl-27587658

ABSTRACT

MOTIVATION: A central task of bioinformatics is to develop sensitive and specific means of providing medical prognoses from biomarker patterns. Common methods to predict phenotypes in RNA-Seq datasets utilize machine learning algorithms trained via gene expression. Isoforms, however, generated from alternative splicing, may provide a novel and complementary set of transcripts for phenotype prediction. In contrast to gene expression, the number of isoforms increases significantly due to numerous alternative splicing patterns, resulting in a prioritization problem for many machine learning algorithms. This study identifies the empirically optimal methods of transcript quantification, feature engineering and filtering steps using phenotype prediction accuracy as a metric. At the same time, the complementary nature of gene and isoform data is analyzed and the feasibility of identifying isoforms as biomarker candidates is examined. RESULTS: Isoform features are complementary to gene features, providing non-redundant information and enhanced predictive power when prioritized and filtered. A univariate filtering algorithm, which selects up to the N highest ranking features for phenotype prediction is described and evaluated in this study. An empirical comparison of pipelines for isoform quantification is reported by performing cross-validation prediction tests with datasets from human non-small cell lung cancer (NSCLC) patients, human patients with chronic obstructive pulmonary disease (COPD) and amyotrophic lateral sclerosis (ALS) transgenic mice, each including samples of diseased and non-diseased phenotypes. AVAILABILITY AND IMPLEMENTATION: https://github.com/clabuzze/Phenotype-Prediction-Pipeline.git CONTACT: clabuzze@iastate.edu, antoniom@bc.edu, watsondk@musc.edu, andersonpe2@cofc.edu.


Subject(s)
Algorithms , Alternative Splicing , Machine Learning , Phenotype , Amyotrophic Lateral Sclerosis , Animals , Carcinoma, Non-Small-Cell Lung , Humans , Lung Neoplasms , Mice, Transgenic , Pulmonary Disease, Chronic Obstructive
12.
Zebrafish ; 13(4): 272-80, 2016 08.
Article in English | MEDLINE | ID: mdl-26982811

ABSTRACT

During retinal development, a variety of different types of neurons are produced. Understanding how each of these types of retinal nerve cells is generated is important from a developmental biology perspective. It is equally important if one is interested in how to regenerate cells after an injury or a disease. To gain more insight into how retinal neurons develop in the zebrafish, we performed single-cell mRNA profiling and in situ hybridizations (ISHs) on retinal sections and whole-mount zebrafish. Through the series of ISHs, designed and performed solely by undergraduate students in the laboratory, we were able to retrospectively identify our single-cell mRNA profiles as most likely coming from developing amacrine cells. Further analysis of these profiles will reveal genes that can be mutated using genome editing techniques. Together these studies increase our knowledge of the genes driving development of different cell types in the zebrafish retina.


Subject(s)
Amacrine Cells/metabolism , Gene Expression Regulation, Developmental , Retina/growth & development , Retinal Ganglion Cells/metabolism , Zebrafish/genetics , Amacrine Cells/cytology , Animals , Animals, Genetically Modified/genetics , Animals, Genetically Modified/growth & development , Animals, Genetically Modified/metabolism , Gene Expression Profiling , Retina/metabolism , Retinal Ganglion Cells/cytology , Zebrafish/growth & development , Zebrafish/metabolism
13.
PLoS One ; 11(3): e0150878, 2016.
Article in English | MEDLINE | ID: mdl-26949938

ABSTRACT

During retinogenesis seven different cell types are generated in distinct yet overlapping timepoints from a population of retinal progenitor cells. Previously, we performed single cell transcriptome analyses of retinal progenitor cells to identify candidate genes that may play roles in the generation of early-born retinal neurons. Based on its expression pattern in subsets of early retinal cells, polo-like kinase 3 (Plk3) was identified as one such candidate gene. Further characterization of Plk3 expression by in situ hybridization revealed that this gene is expressed as cells exit the cell cycle. We obtained a Plk3 deficient mouse and investigated changes in the retina's morphology and transcriptome through immunohistochemistry, in situ hybridization and gene expression profiling. These experiments have been performed initially on adult mice and subsequently extended throughout retinal development. Although morphological studies revealed no consistent changes in retinogenesis upon Plk3 loss, microarray profiling revealed potential candidate genes altered in Plk3-KO mice. Further studies will be necessary to understand the connection between these changes in gene expression and the loss of a protein kinase such as Plk3.


Subject(s)
Gene Expression Regulation, Developmental , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Retina/embryology , Retina/enzymology , Animals , Gene Expression Profiling , Gene Knockout Techniques , Mice , Protein Serine-Threonine Kinases/deficiency
14.
Med Hypotheses ; 83(3): 343-5, 2014 Sep.
Article in English | MEDLINE | ID: mdl-24986645

ABSTRACT

This manuscript considers available evidence that a specific Salmonella strain could be used as an effective orally-administered option for cancer therapy involving the brain. It has been established that Salmonella preferentially colonizes neoplastic tissue and thrives as a facultative anaerobe in the intra-tumor environment. Although Salmonella accumulates in tumors by passive processes, it is still possible for lipopolysaccharide to cause sepsis and endotoxic shock during the migration of bacteria to the tumor site. An LPS-free version of a recently identified Salmonella isolate may have the capability to circumvent the blood brain barrier and provide a safer method of reaching brain tumors. This isolate merits further research as a "Trojan horse" for future oral biotherapy of brain cancer.


Subject(s)
Brain Neoplasms/microbiology , Salmonella/physiology , Animals , Antineoplastic Agents/administration & dosage , Blood-Brain Barrier , Brain/pathology , Brain Neoplasms/complications , Brain Neoplasms/therapy , Cattle , Disease Models, Animal , Humans , Hypoxia , Lipopolysaccharides/chemistry , Mutation , Neoplasms/complications , Neoplasms/microbiology , Neoplasms/therapy , Sepsis/physiopathology , Shock, Septic/physiopathology , Swine
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