Patient fibroblasts-derived induced neurons demonstrate autonomous neuronal defects in adult-onset Krabbe disease

// Su Min Lim 1,2,* , Byung-Ok Choi 3,* , Seong-il Oh 4,* , Won Jun Choi 5 , Ki-Wook Oh 2,6 , Minyeop Nahm 2 , Yuanchao Xue 7 , Jae Hyeok Choi 2 , Ji Young Choi 2 , Young-Eun Kim 8 , Ki Wha Chung 9 , Xiang-Dong Fu 10 , Chang-Seok Ki 11 and Seung Hyun Kim 2,6 1 Biomedical Research Institute, Hanyang University, Seoul, Republic of Korea 2 Cell Therapy Center, Hanyang University Hospital, Seoul, Republic of Korea 3 Department of Neurology and Neuroscience Center, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Republic of Korea 4 Department of Neurology, Busan Paik Hospital, Inje University College of Medicine, Busan, Republic of Korea 5 Department of Neurology, Sheikh Khalifa Specialty Hospital, Ras Al Khaimah, United Arab Emirates 6 Department of Neurology, College of Medicine, Hanyang University, Seoul, Republic of Korea 7 Key Laboratory of RNA Biology, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China 8 Green Cross Genome, Yongin, Republic of Korea 9 Department of Biological Sciences, Gongju National University, Gongju, Republic of Korea 10 Department of Cellular Molecular Medicine, University of California, San Diego, La Jolla, CA, USA 11 Department of Laboratory Medicine and Genetics, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Republic of Korea * These authors have contributed equally to this work Correspondence: Chang-Seok Ki, email: // Seung Hyun Kim, email: // Keywords : krabbe disease; globoid cell leukodystrophy; β-galactosylceramidase; psychosine; induced neuron; Gerotarget Received : October 17, 2015 Accepted : October 14, 2016 Published : October 21, 2016 Abstract Krabbe disease (KD) is an autosomal recessive neurodegenerative disorder caused by defective β-galactosylceramidase (GALC), a lysosomal enzyme responsible for cleavage of several key substrates including psychosine. Accumulation of psychosine to the cytotoxic levels in KD patients is thought to cause dysfunctions in myelinating glial cells based on a comprehensive study of demyelination in KD. However, recent evidence suggests myelin-independent neuronal death in the murine model of KD, thus indicating defective GALC in neurons as an autonomous mechanism for neuronal cell death in KD. These observations prompted us to generate induced neurons (iNeurons) from two adult-onset KD patients carrying compound heterozygous mutations (p.[K563*];[L634S]) and (p.[N228_S232delinsTP];[G286D]) to determine the direct contribution of autonomous neuronal toxicity to KD. Here we report that directly converted KD iNeurons showed not only diminished GALC activity and increased psychosine levels, as expected, but also neurite fragmentation and abnormal neuritic branching. The lysosomal-associated membrane proteins 1 (LAMP1) was expressed at higher levels than controls, LAMP1-positive vesicles were significantly enlarged and fragmented, and mitochondrial morphology and its function were altered in KD iNeurons. Strikingly, we demonstrated that psychosine was sufficient to induce neurite defects, mitochondrial fragmentation, and lysosomal alterations in iNeurons derived in healthy individuals, thus establishing the causal effect of the cytotoxic GALC substrate in KD and the autonomous neuronal toxicity in KD pathology.