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

Su Min Lim,Ji Young Choi,Yuanchao Xue,Byung-Ok Choi,Ki-Wook Oh,Xiang-Dong Fu,Won Jun Choi,Minyeop Nahm,Seung Hyun Kim,Ki Wha Chung,Jae Hyeok Choi,Young-Eun Kim,Chang-Seok Ki,Seong-Il Oh

doi:10.18632/oncotarget.12812

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.

Highlights

Krabbe disease (KD) characterized by globoid cell leukodystrophy is an autosomal recessive lysosomal storage disease caused by mutations in the β-galactosylceramidase (GALC) gene that impair the enzymatic function
Laboratory studies of biochemical and cerebrospinal fluid screening indicated that plasma electrolytes, liver function, calcium, phosphate, thyroid function, full blood count, vitamin B-12 and folate, syphilis serology, and autoantibody profile, were all unremarkable
Specialized laboratory analyses further excluded some rare metabolic disorders and the level of very long chain fatty acids (VLCFA) level remained in a normal range

Summary

INTRODUCTION

Krabbe disease (KD) characterized by globoid cell leukodystrophy is an autosomal recessive lysosomal storage disease caused by mutations in the β-galactosylceramidase (GALC) gene that impair the enzymatic function. Psychosine has been found to alter the angiogenesis process in the murine model, and linked to neuronal inclusion of misfolded and aggregated ɑ-synuclein in postmortem brains from both infantile and late onset KD patients [10, 11] These studies all point to potential autonomous neuronal dysfunction independent of myelin defects in leukodystrophic pathology, which may precede myelin loss. In patient-derived iNeurons, we demonstrate a direct relationship of GALC mutation and abnormal psychosine accumulation with axonal and dendritic defects with morphological and functional impairments in lysosomes and mitochondria These myelin-independent axonal and neuronal defects strongly argue for autonomous neuronal toxicity in adultonset KD

RESULTS

DISCUSSION

MATERIALS AND METHODS