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Investigations of Cellular Stress in Development and Diseases

• An N. Dang Do, MD, PhD, Head, Unit on Cellular Stress in Development and Diseases
• Kavitha Brunner, BS, 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 rare, pediatric, neurogenetic conditions as models for the pursuits of investigator-initiated and sponsored interventional trials.

The integrated stress response (ISR)

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 sequela, 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 disease

CLN3 disease (also known as Batten disease or juvenile neuronal ceroid lipofuscinosis) is a rare, fatal, pediatric, neurodegenerative, lysosomal disorder with no currently approved treatment. Syndromic CLN3 disease 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 in electron microscopy. The affected CLN3 gene encodes a 468–amino acid, transmembrane, ubiquitously expressed protein implicated in many cellular pathways, whose exact function is being defined [References 2, 3].

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 (in vitro models of neurological function) viability following treatment with the antibiotic 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 expression of the ER chaperone BiP/Grp78 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].

MEHMO syndrome (mental retardation, epileptic seizures, hypogonadism/hypogenitalism, microcephaly, obesity)

MEHMO syndrome is a very rare, X-linked recessive, pediatric, multisystemic, life-limiting condition with fewer than 100 affected males reported in the literature (OMIM 300148). Life expectancy in affected individuals ranges from infancy to early adulthood. The disease locus is located on the Xp21.1-p22.13 chromosomal band. Exome sequencing identified disease-associated variants in EIF2S3, the gene encoding the gamma subunit of the translation initiation factor eIF2, which holds a central role in the ISR, as demonstrated by pathologies and diseases associated with defective phosphorylation of the alpha subunit of eIF2 (eIF2α). Survival of affected individuals ranges from infancy to late teens. Diagnostic testing and disease-modifying therapies for MEHMO are not currently available.

Further understanding of the ISR in CLN3 disease and MEHMO syndrome models would provide insight into the underlying pathophysiology and inform novel therapeutic approaches.

The role of the integrated stress response in CLN3 (Batten disease) and the MEHMO syndrome

We continue to develop laboratory reagents, experimental models, and collaborations to address the overall objective of understanding the role of the integrated stress response in the human diseases CLN3 and MEHMO syndrome/eIF2-pathway related conditions. We are characterizing the cellular pathophysiology of the disease models, and their responses following exposure to stress conditions.

The CLN3 natural history study and biosample collection is ongoing. To date, our study contains the largest cohort of individuals with CLN3–related disorders extensively characterized and prospectively followed, with corresponding biosamples (cerebrospinal fluid [CSF], blood, urine, fibroblasts) 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. Measurement of CSF and blood neurofilament light chain level, found to be elevated in individuals with CLN3 [Reference 1], is being applied in an ongoing Phase 1/2 study of miglustat. Additional candidate fluid [Reference 5] and imaging [Dang Do AN et al. Mol Genet Metab 2023;139:107584] markers may further the progress of CLN3 translational research.

Applying experiences from CLN3 translational research, we initiated a natural history study for individuals with MEHMO syndrome or eIF2 pathway–related disorders (NCT06019182) to characterize these conditions systematically and to establish concurrent collections of phenotypic data and biomaterials. The IRB–approved study is open for enrollment.

Additional Funding

• 2022 NICHD Early Career Award
• 2023 NICHD Scientific Director's Award

Publications

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.
5. Dang Do AN, Sleat DE, Campbell K, Johnson NL, Zheng H, Wassif CA, Dale RK, Porter FD. Cerebrospinal Fluid Protein Biomarker Discovery in CLN3. J Proteome Res 2023 22:2493–2508.
6. Luckett A, Yousef M, Tifft C, Jenkins K, Smith A, Munoz A, Quimby R, Porter FD, Dang Do AN. Anesthesia outcomes in lysosomal disorders: CLN3 and GM1 gangliosidosis. Am J Med Genet A 2023 191:711–717.

Collaborators

• 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
• Quynh Chu-LaGraff, PhD, Union College, Schenectady, NY
• Susan Cotman, PhD, Massachusetts General Hospital, Boston, MA
• Ryan K. Dale, MS, 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
• Kye-yoon Park, PhD, Stem Cell Characterization Facility Center, NINDS, 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

Contact

For more information, contact an.dangdo@nih.gov.

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