EunRan Suh

AN INTESTINE-SPECIFIC HOMEOBOX GENE REGULATES PROLIFERATION AND DIFFERENTIATION

Precise regulation of cellular proliferation, differentiation, and senescence results in the continuous renewal of the intestinal epithelium with maintenance of a highly ordered tissue architecture. Here we show that an intestine-specific homeobox gene, Cdx2, is a transcription factor that regulates both proliferation and differentiation in intestinal epithelial cells. Conditional expression of Cdx2 in IEC-6 cells, an undifferentiated intestinal cell line, led to arrest of proliferation for several days followed by a period of growth resulting in multicellular structures containing a well-formed columnar layer of cells. The columnar cells had multiple morphological characteristics of intestinal epithelial cells. Enterocyte-like cells were polarized with tight junctions, lateral membrane interdigitations, and well-organized microvilli with associated glycocalyx located at the apical pole. Remarkably, there were also cells with a goblet cell-like ultrastructure, suggesting that two of the four intestinal epithelial cell lineages may arise from IEC-6 cells. Molecular evidence for differentiation was shown by demonstrating that cells expressing high levels of Cdx2 expressed sucrase-isomaltase, an enterocyte-specific gene which is a well-defined target for the Cdx2 protein. Taken together, our data suggest that Cdx2 may play a role in directing early processes in intestinal cell morphogenesis and in the maintenance of the differentiated phenotype by supporting transcription of differentiated gene products. We propose that Cdx2 is part of a regulatory network that orchestrates a developmental program of proliferation, morphogenesis, and gene expression in the intestinal epithelium.

A homeodomain protein related to caudal regulates intestine-specific gene transcription.

The continually renewing epithelium of the intestinal tract arises from the visceral endoderm by a series of complex developmental transitions. The mechanisms that establish and maintain the processes of cellular renewal, cell lineage allocation, and tissue restriction and spatial assignment of gene expression in this epithelium are unknown. An understanding of the regulation of intestine-specific gene regulation may provide information on the molecular mechanisms that direct these processes. In this regard, we show that intestine-specific transcription of sucrase-isomaltase, a gene that is expressed exclusively in differentiated enterocytes, is dependent on binding of a tissue-specific homeodomain protein (mouse Cdx-2) to an evolutionarily conserved promoter element in the sucrase-isomaltase gene. This protein is a member of the caudal family of homeodomain genes which appear to function in early developmental events in Drosophila melanogaster, during gastrulation in many species, and in intestinal endoderm. Unique for this homeodomain gene family, we show that mouse Cdx-2 binds as a dimer to its regulatory element and that dimerization in vitro is dependent on redox potential. These characteristics of the interaction of Cdx-2 with its regulatory element provide for a number of potential mechanisms for transcriptional regulation. Taken together, these findings suggest that members of the Cdx gene family play a fundamental role both in the establishment of the intestinal phenotype during development and in maintenance of this phenotype via transcriptional activation of differentiated intestinal genes.

Stages of pTDP-43 pathology in amyotrophic lateral sclerosis

To see whether the distribution patterns of phosphorylated 43kDa TAR DNA‐binding protein (pTDP‐43) intraneuronal inclusions in amyotrophic lateral sclerosis (ALS) permit recognition of neuropathological stages.

C9orf72 hypermethylation protects against repeat expansion-associated pathology in ALS/FTD

Abstract Hexanucleotide repeat expansions of C9orf72 are the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal degeneration. The mutation is associated with reduced C9orf72 expression and the accumulation of potentially toxic RNA and protein aggregates. CpG methylation is known to protect the genome against unstable DNA elements and to stably silence inappropriate gene expression. Using bisulfite cloning and restriction enzyme-based methylation assays on DNA from human brain and peripheral blood, we observed CpG hypermethylation involving the C9orf72 promoter in cis to the repeat expansion mutation in approximately one-third of C9orf72 repeat expansion mutation carriers. Promoter hypermethylation of mutant C9orf72 was associated with transcriptional silencing of C9orf72 in patient-derived lymphoblast cell lines, resulting in reduced accumulation of intronic C9orf72 RNA and reduced numbers of RNA foci. Furthermore, demethylation of mutant C9orf72 with 5-aza-deoxycytidine resulted in increased vulnerability of mutant cells to oxidative and autophagic stress. Promoter hypermethylation of repeat expansion carriers was also associated with reduced accumulation of RNA foci and dipeptide repeat protein aggregates in human brains. These results indicate that C9orf72 promoter hypermethylation prevents downstream molecular aberrations associated with the hexanucleotide repeat expansion, suggesting that epigenetic silencing of the mutant C9orf72 allele may represent a protective counter-regulatory response to hexanucleotide repeat expansion.

Diagnosis of Parkinson's disease on the basis of clinical and genetic classification: a population-based modelling study

BACKGROUND Accurate diagnosis and early detection of complex diseases, such as Parkinsons disease, has the potential to be of great benefit for researchers and clinical practice. We aimed to create a non-invasive, accurate classification model for the diagnosis of Parkinsons disease, which could serve as a basis for future disease prediction studies in longitudinal cohorts. METHODS We developed a model for disease classification using data from the Parkinsons Progression Marker Initiative (PPMI) study for 367 patients with Parkinsons disease and phenotypically typical imaging data and 165 controls without neurological disease. Olfactory function, genetic risk, family history of Parkinsons disease, age, and gender were algorithmically selected by stepwise logistic regression as significant contributors to our classifying model. We then tested the model with data from 825 patients with Parkinsons disease and 261 controls from five independent cohorts with varying recruitment strategies and designs: the Parkinsons Disease Biomarkers Program (PDBP), the Parkinsons Associated Risk Study (PARS), 23andMe, the Longitudinal and Biomarker Study in PD (LABS-PD), and the Morris K Udall Parkinsons Disease Research Center of Excellence cohort (Penn-Udall). Additionally, we used our model to investigate patients who had imaging scans without evidence of dopaminergic deficit (SWEDD). FINDINGS In the population from PPMI, our initial model correctly distinguished patients with Parkinsons disease from controls at an area under the curve (AUC) of 0·923 (95% CI 0·900-0·946) with high sensitivity (0·834, 95% CI 0·711-0·883) and specificity (0·903, 95% CI 0·824-0·946) at its optimum AUC threshold (0·655). All Hosmer-Lemeshow simulations suggested that when parsed into random subgroups, the subgroup data matched that of the overall cohort. External validation showed good classification of Parkinsons disease, with AUCs of 0·894 (95% CI 0·867-0·921) in the PDBP cohort, 0·998 (0·992-1·000) in PARS, 0·955 (no 95% CI available) in 23andMe, 0·929 (0·896-0·962) in LABS-PD, and 0·939 (0·891-0·986) in the Penn-Udall cohort. Four of 17 SWEDD participants who our model classified as having Parkinsons disease converted to Parkinsons disease within 1 year, whereas only one of 38 SWEDD participants who were not classified as having Parkinsons disease underwent conversion (test of proportions, p=0·003). INTERPRETATION Our model provides a potential new approach to distinguish participants with Parkinsons disease from controls. If the model can also identify individuals with prodromal or preclinical Parkinsons disease in prospective cohorts, it could facilitate identification of biomarkers and interventions. FUNDING National Institute on Aging, National Institute of Neurological Disorders and Stroke, and the Michael J Fox Foundation.

The Caudal-related Homeodomain Protein Cdx1 Inhibits Proliferation of Intestinal Epithelial Cells by Down-regulation of D-type Cyclins

Cdx1 is a homeodomain transcription factor that regulates intestine-specific gene expression. Experimental evidence suggests that Cdx1 may be involved in cell cycle regulation, but its role is ill defined and the mechanisms have not been explored. We used stable transfection of inducible constructs and transient expression with a replication-deficient adenovirus to induce Cdx1 expression in rat IEC6 cells, a non-transformed intestinal epithelial cell line that does not express Cdx1 protein. Expression of Cdx1 markedly reduced proliferation of IEC6 cells with accumulation of cells in the G0/G1 phase of the cell cycle. Cell cycle arrest was accompanied by an increase in the hypophosphorylated forms of the retinoblastoma protein (pRb) and the pRb-related p130 protein. Protein levels of multiple cyclin-dependent kinase inhibitors were either unchanged (p16, p18, p21, p27, and p57) or were not detected (p15 and p19). Most significantly, levels of cyclins D1 and D2 were markedly diminished with Cdx1 expression, but not cyclins D3, E, or the G1 kinases. Additionally, cyclin-dependent kinase-4 activity was decreased in association with decreased cyclin D protein. We conclude that Cdx1 regulates intestinal epithelial cell proliferation by inhibiting progression through G0/G1, most likely via modulation of cyclin D1 and D2 protein levels.

A platform for discovery: The University of Pennsylvania Integrated Neurodegenerative Disease Biobank

Neurodegenerative diseases (NDs) are defined by the accumulation of abnormal protein deposits in the central nervous system (CNS), and only neuropathological examination enables a definitive diagnosis. Brain banks and their associated scientific programs have shaped the actual knowledge of NDs, identifying and characterizing the CNS deposits that define new diseases, formulating staging schemes, and establishing correlations between neuropathological changes and clinical features. However, brain banks have evolved to accommodate the banking of biofluids as well as DNA and RNA samples. Moreover, the value of biobanks is greatly enhanced if they link all the multidimensional clinical and laboratory information of each case, which is accomplished, optimally, using systematic and standardized operating procedures, and in the framework of multidisciplinary teams with the support of a flexible and user‐friendly database system that facilitates the sharing of information of all the teams in the network. We describe a biobanking system that is a platform for discovery research at the Center for Neurodegenerative Disease Research at the University of Pennsylvania.

Development and Validation of Pedigree Classification Criteria for Frontotemporal Lobar Degeneration

IMPORTANCE A significant portion of frontotemporal lobar degeneration (FTLD) is due to inherited gene mutations, and we are unaware of a large sequential series that includes a recently discovered inherited cause of FTLD. There is also great need to develop clinical tools and approaches that will assist clinicians in the identification and counseling of patients with FTLD and their families regarding the likelihood of an identifiable genetic cause. OBJECTIVES To ascertain the frequency of inherited FTLD and develop validated pedigree classification criteria for FTLD that provide a standardized means to evaluate pedigree information and insight into the likelihood of mutation-positive genetic test results for C9orf72, MAPT, and GRN. DESIGN Information about pedigrees and DNA was collected from 306 serially assessed patients with a clinical diagnosis of FTLD. This information included gene test results for C9orf72, MAPT, and GRN. Pedigree classification criteria were developed based on a literature review of FTLD genetics and pedigree tools and then refined by reviewing mutation-positive and -negative pedigrees to determine differentiating characteristics. SETTING Academic medical center. PARTICIPANTS Patients with FTLD. MAIN OUTCOMES AND MEASURES Familial risk. RESULTS The rate of C9orf72, MAPT, or GRN mutation-positive FTLD in this series was 15.4%. Categories designating the risk level for hereditary cause were termed high, medium, low, apparent sporadic, and unknown significance. Thirty-nine pedigrees (12.7%) met criteria for high, 31 (10.1%) for medium, 46 (15.0%) for low, 91 (29.7%) for apparent sporadic, and 99 (32.4%) for unknown significance. The mutation-detection rates were as follows: high, 64.1%; medium, 29%; low, 10.9%; apparent sporadic, 1.1%; and unknown significance, 7.1%. Mutation-detection rates differed significantly between the high and other categories. CONCLUSIONS AND RELEVANCE Mutation rates are high in FTLD spectrum disorders, and the proposed criteria provide a validated standard for the classification of FTLD pedigrees. The combination of pedigree criteria and mutation-detection rates has important implications for genetic counseling and testing in clinical settings.

Genetic and neuroanatomic associations in sporadic frontotemporal lobar degeneration

Genome-wide association studies have identified single nucleotide polymorphisms (SNPs) that are sensitive for tau or TDP-43 pathology in frontotemporal lobar degeneration (FTLD). Neuroimaging analyses have revealed distinct distributions of disease in FTLD patients with genetic mutations. However, genetic influences on neuroanatomic structure in sporadic FTLD have not been assessed. In this report, we use novel multivariate tools, Eigenanatomy, and sparse canonical correlation analysis to identify associations between SNPs and neuroanatomic structure in sporadic FTLD. Magnetic resonance imaging analyses revealed that rs8070723 (MAPT) was associated with gray matter variance in the temporal cortex. Diffusion tensor imaging analyses revealed that rs1768208 (MOBP), rs646776 (near SORT1), and rs5848 (PGRN) were associated with white matter variance in the midbrain and superior longitudinal fasciculus. In an independent autopsy series, we observed that rs8070723 and rs1768208 conferred significant risk of tau pathology relative to TDP-43, and rs646776 conferred increased risk of TDP-43 pathology relative to tau. Identified brain regions and SNPs may help provide an in vivo screen for underlying pathology in FTLD and contribute to our understanding of sporadic FTLD.

Transcriptomic Changes Due to Cytoplasmic TDP-43 Expression Reveal Dysregulation of Histone Transcripts and Nuclear Chromatin

TAR DNA-binding protein 43 (TDP-43) is normally a nuclear RNA-binding protein that exhibits a range of functions including regulation of alternative splicing, RNA trafficking, and RNA stability. However, in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with TDP-43 inclusions (FTLD-TDP), TDP-43 is abnormally phosphorylated, ubiquitinated, and cleaved, and is mislocalized to the cytoplasm where it forms distinctive aggregates. We previously developed a mouse model expressing human TDP-43 with a mutation in its nuclear localization signal (ΔNLS-hTDP-43) so that the protein preferentially localizes to the cytoplasm. These mice did not exhibit a significant number of cytoplasmic aggregates, but did display dramatic changes in gene expression as measured by microarray, suggesting that cytoplasmic TDP-43 may be associated with a toxic gain-of-function. Here, we analyze new RNA-sequencing data from the ΔNLS-hTDP-43 mouse model, together with published RNA-sequencing data obtained previously from TDP-43 antisense oligonucleotide (ASO) knockdown mice to investigate further the dysregulation of gene expression in the ΔNLS model. This analysis reveals that the transcriptomic effects of the overexpression of the ΔNLS-hTDP-43 transgene are likely due to a gain of cytoplasmic function. Moreover, cytoplasmic TDP-43 expression alters transcripts that regulate chromatin assembly, the nucleolus, lysosomal function, and histone 3’ untranslated region (UTR) processing. These transcriptomic alterations correlate with observed histologic abnormalities in heterochromatin structure and nuclear size in transgenic mouse and human brains.