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National Institutes of Health

Eunice Kennedy Shriver National Institute of Child Health and Human Development

2022 Annual Report of the Division of Intramural Research

Cellular Stress in Development and Disease

An Dang Do
  • An N. Dang Do, MD, PhD, Head, Unit on Cellular Stress in Development and Diseases
  • Danielle O'Mard, BS, MPH, Laboratory Technician
  • Ewelina Dwojak, BS, MS, Postbaccalaureate IRTA

The overarching goal of the Unit is to build a foundation for a translational research program. The basic science component of the program will focus on investigating the regulation of the cellular integrated stress response (ISR) as (1) a mechanism for variable phenotypic expressions and (2) a potential therapeutic approach to relevant human diseases. The clinical component will focus on using lysosomal diseases, such as CLN3 (Batten disease), as models for the pursuits of investigator-initiated and sponsored interventional trials.

ISR is an evolutionarily conserved process capable of inducing pro-survival or pro-apoptotic status in cells experiencing endoplasmic-reticulum (ER) and other stresses by activating either autophagy or apoptosis. Cellular stresses, such as unfolded protein accumulation, trigger the ISR through one of the four known eIF2α kinases. The integration occurs as Ser51-phosphorylated eIF2α inhibits the guanine nucleotide exchange activity of eIF2B, halts the formation of the translation initiation ternary complex, and effectively attenuates global mRNA translation. The reduced ternary complex formation paradoxically allows for increased translation of selected mRNAs, many of whose protein products are involved in determining cell fate and may be specific in the response to the instigating stress (Sonenberg N, Hinnebusch AG. Cell 2009;136:731). As an instigator or sequelae, aberrant ISR is implicated in human disorders of metabolism (diabetes), growth (skeletal dysplasia, cancer) and neurologic processes (MEHMO, Down syndrome, Alzheimer's disease), amongst many others. Regulation of the ISR could provide therapeutic benefit.

CLN3 is a rare, fatal, pediatric, neurodegenerative, lysosomal disorder with no currently approved treatment. Syndromic CLN3 presentation includes vision loss, neurodevelopmental plateauing and decline, behavioral inflexibility and emotional lability, seizures, and motor dysfunction, as described previously (OMIM 204200) and also observed in a cohort of natural history study (NCT03307304) participants at the NIH. Accumulations of lipopigment consisting of carbohydrates, lipids, metal ions, and proteins (particularly mitochondrial ATP synthase subunit C [SCMAS]) form intracellular and lysosomal deposits that autofluorescence under UV light and present as fingerprint patterns under electron microscopy. The affected CLN3 gene encodes a 468–amino acid, transmembrane, ubiquitously expressed protein implicated in many cellular pathways, whose exact function remains to be elucidated.

Findings from cells and flies implicate perturbed ISR following stress induction in the CLN3–deficient compared with wild-type (WT) controls. Reduced expression of WT CLN3 lowered SH-SY5Y cell viability following treatment with tunicamycin (Wu D et al. Biochem Biophys Res Commun 2014;447:115), and reduced Drosophila survival following exposure to chemical inducers of oxidative stress (Tuxworth RI et al. Hum Mol Genet 2011;20:2037). Higher eIF2α phosphorylation and BiP/Grp78 expression in fibroblast cell lines from individuals with CLN3 disease than in WT controls followed treatment with NH4Cl (Wei H et al. Hum Mol Genet. 2008;17:469). Disturbances in the lysosome autophagic pathway may also interconnect with the ISR through the transcription factor EB (TFEB) (Martina JA et al. EMBO J 2016;35:479; Burton TD et al. J Biol Chem 2020;295:7418). TFEB is a widely studied autophagy regulator and a treatment target of interest for conditions involving lysosomes and neurodegeneration (Cortes CJ, La Spada AR. Neurobiol Dis 2019;122:83). Cln3 mutant mice treated with trehalose, a disaccharide activator of TFEB, had improved intracellular lipopigment accumulation, brain weight, pain sensitivity, and survival (Palmieri M et al. Nat Commun 2017;8:14338). Thus, further understanding of the ISR in CLN3 disease models would provide insight into the underlying pathophysiology and inform novel therapeutic approaches.

Development of a human sample biorepository and experimental models

The Unit on Cellular Stress in Development and Diseases was established in October 2021, and completed recruitment of laboratory personnel in April 2022. We are developing laboratory reagents, experimental models, and collaborations to address the overall objective of understanding the role of the integrated stress response in human diseases such as CLN3.

We continue to conduct extensive characterization of the phenotype of individuals with variants in CLN3, and establish a biorepository of corresponding biosamples (CSF, blood, urine). To date, our study contains the largest cohort of individuals with CLN3–related disorders extensively characterized and prospectively followed, with corresponding biosamples collected. This (1) permits the development and assessment of outcome measures applicable for therapeutic trials (NCT03307304); (2) builds the infrastructures for engagement in a sponsored Phase 1/2 study of miglustat therapy trial (NCT05174039), and in future investigator-initiated and sponsored interventional trials; and (3) provides the means for further CLN3 research through identification of disease-reflective biomarkers.

Using CSF samples from the natural history study, we identified the neurofilament light chain as a potential marker for disease monitoring [Reference 1], and glycerophosphodiester species as potential marker(s) for disease diagnosis [References 2&3]. Biomarker discovery efforts will continue through the use of commercial platforms (Olink® proximal extension assay), and through extramural collaborations to perform proteomic (David Sleat, NCL-Stiftung Research Award), transcriptomic (Susan Cotman), and metabolomic/lipidomic (Monther Abu-Remaileh) analyses of samples from Clinical and Basic Science projects. We also continue to work with collaborators (Jon Brudvig and Jill Weimer) on translating candidate biomarkers to screening applications

Additional Funding

  • 2022 NICHD Early Career Award


  1. Dang Do AN, Sinaii N, Masvekar RR, Baker EH, Thurm AE, Soldatos AG, Bianconi SE, Bielekova B, Porter FD. Neurofilament light chain levels correlate with clinical measures in CLN3 disease. Genet Med 2021 23(4):751–757.
  2. Brudvig JJ, Swier VJ, Johnson TB, Cain JC, Pratt M, Rechtzigel M, Leppert H, Dang Do AN, Porter FD, Weimer JM. Glycerophosphoinositol is elevated in blood samples from CLN3Δex7-8 pigs, Cln3Δex7-8 mice, and CLN3-affected individuals. Biomark Insights 2022 17:11772719221107765.
  3. Laqtom NN, Dong W, Medoh UN, Cangelosi AL, Dharamdasani V, Chan SH, Kunchok T, Lewis CA, Heinze I, Tang R, Grimm C, Dang Do AN, Porter FD, Ori A, Sabatini DM, Abu-Remaileh M. CLN3 is required for the clearance of glycerophosphodiesters from lysosomes. Nature 2022 609:1005–1011.
  4. Dang Do AN, Thurm AE, Farmer CA, Soldatos AG, Chlebowski CE, O'Reilly JK, Porter FD. Use of the Vineland-3, a measure of adaptive functioning, in CLN3. Am J Med Genet A 2022 188:1056–1064.


  • Monther Abu-Remaileh, PhD, Stanford University, Stanford, CA
  • Katharine E. Alter, MD, Functional and Applied Biomechanics Section, NIH Clinical Center, Bethesda, MD
  • Eva H. Baker, MD, PhD, Radiology and Imaging Sciences, NIH Clinical Center, Bethesda, MD
  • Brian P. Brooks, MD, PhD, Ophthalmic Genetics and Visual Function Branch, NEI, Bethesda, MD
  • Carmen C. Brewer, PhD, Audiology Unit, NIDCD, Bethesda, MD
  • Jon Brudvig, PhD, Sanford Research, Sioux Falls, SD
  • Susan Cotman, PhD, Massachusetts General Hospital, Boston, MA
  • Ryan K. Dale, PhD, Bioinformatics and Scientific Programming Core, NICHD, Bethesda, MD
  • Thomas E. Dever, PhD, Section on Protein Biosynthesis, NICHD, Bethesda, MD
  • Alex Grinberg, DVM, Mouse Transgenic Core, NICHD, Bethesda, MD
  • Hanna L. Hildenbrand, MS, OTR/L, Occupational Therapy Service, NIH Clinical Center, Bethesda, MD
  • Laryssa A. Huryn, MD, Ophthalmic Clinical Genetics Section, NEI, Bethesda, MD
  • Zou Jizhong, PhD, Induced Pluripotent Stem Cells Core, NHLBI, Bethesda, MD
  • Mark D. Levin, MD, Laboratory of Vascular & Matrix Genetics, NHLBI, Bethesda, MD
  • Jesse H. Matsubara, PT, DPT, Functional and Applied Biomechanics, NIH Clinical Center, Bethesda, MD
  • Terri D. Nguyen, MS, OTR/L, Occupational Therapy Service, NIH Clinical Center, Bethesda, MD
  • Carlo Pierpaoli, MD, PhD, Laboratory on Quantitative Medical Imaging, NIBIB, Bethesda, MD
  • Forbes D. Porter, MD, PhD, Section on Molecular Dysmorphology, NICHD, Bethesda, MD
  • David Sleat, PhD, Center for Advanced Biotechnology and Medicine, Rutgers, The State University of New Jersey, Piscataway, NJ
  • Ariane G. Soldatos, MD, MPH, Pediatric Neurology Consultation Service, NINDS, Bethesda, MD
  • Beth I. Solomon, MS, CCC-SLP, Speech Language Pathology Section Rehabilitation Medicine Department, NIH Clinical Center, Bethesda, MD
  • Audrey E. Thurm, PhD, Neurodevelopmental and Behavioral Phenotyping Service, NIMH, Bethesda, MD
  • Sara K. Young-Baird, PhD, Uniformed Services University of the Health Sciences, Bethesda, MD
  • Qingjun Wang, PhD, University of Kentucky College of Medicine, Lexington, KY
  • Jill Weimer, PhD, Sanford Research, Sioux Falls, SD
  • Cristiane Zampieri-Gallagher, PhD, Rehabilitation Medicine Department, NIH Clinical Center, Bethesda, MD
  • Wadih M. Zein, MD, Ophthalmic Genetics and Visual Function Branch, NEI, Bethesda, MD
  • Wei Zheng, PhD, Therapeutic Development Branch, NCATS, Bethesda, MD


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