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

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.

Direct Neuronal Reprogramming for Disease Modeling Studies Using Patient-Derived Neurons: What Have We Learned?

“…Disease-associated features are not always unique to the neurons and as a result, several of these phenotypes can be observed in both iNs and fibroblasts (Santambrogio et al, 2015 ; Lim et al, 2016a ). However, as iNs adopt a neuronal-like morphology and at least some functional properties of neurons, they provide an opportunity to study diseases in the cell type primarily clinically affected.…”

“…However, as iNs adopt a neuronal-like morphology and at least some functional properties of neurons, they provide an opportunity to study diseases in the cell type primarily clinically affected. For example, iNs derived from adult-onset Krabbe disease had the same lysosomal storage defects as the starting fibroblasts, but unique to the iNs was the abnormal neuronal branching, which may be more relevant to the clinical expression of this disorder (Lim et al, 2016a ). In fact, a few studies have now reported that disease-associated features could only be seen in iNs.…”

“…To date, the predominant method by which to isolate/identify the iN population to assess disease relevant phenotypes in patient derived iNs, has involved either an antibiotic selection to remove cells that are not expressing the reprogramming construct(s) (Liu et al, 2014 ; Su et al, 2014 ; Bavamian et al, 2015 ; Lim et al, 2016a ) or based on the expression of neuronal markers such as TUJ1 (βIII-Tubulin) (Iovino et al, 2014 ; Liu et al, 2014 ; Fiesel et al, 2015 ; Lim et al, 2016a , b ; Puschmann et al, 2017 ). However, direct neuronal reprogramming studies that have used antibiotic selection to purify the neuronal culture have consistently reported that a significant percentage of cells do not convert even though the reprogramming constructs are expressed (Victor et al, 2014 ; Mertens et al, 2015a ; Huh et al, 2016 ; Liu et al, 2016 ; Xue et al, 2016 ), which presents the need to identify the iN population even after antibiotic selection.…”

GALC mutations in Chinese patients with late-onset Krabbe disease: a case report

“…Brain MRI and GLAC activity assay are essential for patients manifesting chronic progressive corticospinal tract impaired. However, the relationship between the deficiencies in the lysosomal enzyme activities and the degree of clinical severity appears obscure [24,25] . Both the two mutations are reported in the gnomad dataset (http://gnomad.broadinstitute.org).…”

Percutaneous Radiofrequency Ablation Combined With Transarterial Chemoembolization Plus Sorafenib for Large Hepatocellular Carcinoma Invading the Portal Venous System: A Prospective Randomized Study

“…Although technology was improved thereby promoting the development of HCC therapy, the treatment outcomes of HCC with PVTT remains poor, as expected. Currently, sorafenib or lenvatinib, transarterial chemoembolization (TACE), TACE plus sorafenib, percutaneous radiofrequency ablation (RFA), and radiotherapy, among others, are used in the clinical treatment of PVTT, but these treatment options remain unsatisfactory (8)(9)(10). Therefore, further research is urgently needed to investigate the optimal therapeutic strategy for HCC with PVTT.…”