Induced Pluripotent Stem Cells Derived from a CLN5 Patient Manifest Phenotypic Characteristics of Neuronal Ceroid Lipofuscinoses

Abstract

Neuronal ceroid lipofuscinoses (NCLs) are autosomal recessive progressive encephalopathies caused by mutations in at least 14 different genes. Despite extensive studies performed in different NCL animal models, the molecular mechanisms underlying neurodegeneration in NCLs remain poorly understood. To model NCL in human cells, we generated induced pluripotent stem cells (iPSCs) by reprogramming skin fibroblasts from a patient with CLN5 (ceroid lipofuscinosis, neuronal, 5) disease, the late infantile variant form of NCL. These CLN5 patient-derived iPSCs (CLN5Y392X iPSCs) harbouring the most common CLN5 mutation, c.1175_1176delAT (p.Tyr392X), were further differentiated into neural lineage cells, the most affected cell type in NCLs. The CLN5Y392X iPSC-derived neural lineage cells showed accumulation of autofluorescent storage material and subunit C of the mitochondrial ATP synthase, both representing the hallmarks of many forms of NCLs, including CLN5 disease. In addition, we detected abnormalities in the intracellular organelles and aberrations in neuronal sphingolipid transportation, verifying the previous findings obtained from Cln5-deficient mouse macrophages. Therefore, patient-derived iPSCs provide a suitable model to study the mechanisms of NCL diseases.

Keywords: CLN5; NCL; iPS cells; lysosomal storage disease; sphingolipid.

Figure 1

Generation and characterisation of iPS cell lines derived from the CLN5 (ceroid lipofuscinosis, neuronal, 5) patient’s fibroblasts. The CLN5 patient’s fibroblasts (A) were reprogrammed to induce pluripotent stem cells (iPSCs) using a Sendai virus-mediated delivery of the four Yamanaka factors. CLN5Y392X iPSC clones were collected on the basis of colony morphology ((B) a representative image shown); and the colonies were stained for TRA-1-60, a specific stem cell marker ((C) shown for one representative clone); RT-PCR was used to detect the expression of endogenous stem cell marker genes, NANOG, OCT3/4, REX1 and TDGF1, and the loss of virus expression confirmed by using primers specific for Sendai virus (SeV) ((D) shown for the three characterised CLN5Y392X iPSC clones (cl.) and a positive control hES cell line, H9. Sendai virus-infected fibroblasts were used as a positive control (pos.) in analysis of Sendai viral expression); The presence of the original CLN5 disease causing mutation in each analysed CLN5Y392X iPSC clone was confirmed by sequencing ((E) illustrated from one representative clone, nucleotides in the box represent second and third nucleotides of the codon TAT, and are missing in mutated DNA); Pluripotency of the three characterised CLN5Y392X iPSC lines was verified by their ability to differentiate into the three germ layers, mesoderm, ectoderm and endoderm, detected by staining with vimentin (F), β-III tubulin (G) and α-fetoprotein (AFP, (H)) antibodies, respectively (shown for one representative clone with nuclear staining shown in blue); Bar 100 µm (A,F–H), 300 µm (B) and 50 µm (C).

Figure 2

Neural differentiation of control and CLN5Y392X iPSC lines. iPS cells were differentiated into neural progenitor cells (NPCs), which grow as neurospheres in a suspension culture (A,B); Neurospheres were further differentiated on poly-l-ornithine/laminin-coated plates (C,D); At day seven, cultures were fixed and stained with the neuron-specific marker microtubule-associated protein 2 (MAP2) (green) (E,F); Bar 400 µm (A,B), 200 µm (C,D) and 50 µm (E,F).

Figure 3

Analyses of lysosomes and the endoplasmic reticulum (ER) in iPSC-derived neural lineage cells. iPSC-derived neurospheres were further differentiated in adherent cultures. After ten days in an adherent culture, the cells were processed for nuclear (Hoechst 33258, blue), and lysosomal (LAMP-1, green) or ER (PDI, green) staining. Representative ScanR microscope images of lysosomal and the ER compartments ((A,B), respectively) are shown. Quantitative image analyses show the average values (arbitrary units) of total area, the number and intensity of vesicular LAMP-1-positive compartments in control and CLN5Y392X neural lineage cells (A); the size of the ER was determined as the area of PDI-stained structures (B). All columns represent the average values of 6–8 replicates each normalised to the number of cells analysed in the replicate (average of 1231 and 1183 cells per replicate for LAMP-1 and ER analysis, respectively). The error bars represent standard deviations (stdevp). p-values, 0.0243 (*), 0.0183 (**), 0.0119 (#) and 0.0140 (##) (two-tailed Student’s t test).

Figure 4

Intracellular storage accumulation in iPSC-derived neural lineage cells. Appearance of storage material within cells analysed by measuring the amount of autofluorescence (A–D) and subunit C staining (E–I). Representative images showing autofluorescent compartments (green) and nuclei (Hoechst 33258, blue) in control (A) and CLN5Y392XiPSC-derived neural lineage cells (B) viewed using a high-content microscope; Quantitative image analysis shows the average area of autofluorescent compartments (C) as well as the average number of autofluorescent objects (D) in control (grey bars) and CLN5-deficient (black bars) cells (arbitrary units). The x axis indicates the plated cell number per 96-well (C,D). Each column in (C,D) represents the average value of four replicates per condition each normalised to the number of cells analysed in the replicate (average of 1379 cells per replicate); Representative images of control (E) and CLN5Y392XiPSC-derived neural lineage cells (F) stained with a polyclonal antibody against subunit C of the mitochondrial ATP synthase (red) and viewed using a traditional epifluorescence microscope; High-content immunofluorescence microscopy images of subunit C staining in control (G) and CLN5Y392X neural lineage cells (H); Results from the quantitative image analysis showing the average number of subunit C positive objects in control (grey bars) and CLN5-deficient cells (black bars) (I). Both columns in (I) represent the average value of 2–3 replicates each normalised to the number of cells analysed in the replicate (average of 587 cells per replicate). All error bars in the figure represent standard deviations (stdev). White arrows indicate the presence of autofluorescent/stained storage material. Bar 20 µm. p-values, 0.0024 (*), 0.0004 (**), 0.0371 (***), 0.018 (#), 0.0708 (##) and 0.0027 (****) (one-tailed Student’s t test).

Figure 5

Altered sphingolipid transport in CLN5Y392X iPSC-derived neural lineage cells. Control and CLN5Y392X cells were fed with a fluorescent glycosphingolipid analogue BODIPY-Lactosylceramide (BODIPY-LacCer). Immediately after feeding (0 min chase), BODIPY-LacCer was located at the plasma membrane (A,C,E,G); After chasing for 60 min, BODIPY LacCer displayed a typical Golgi distribution in the control cells (B,F); whereas the labelling was more dispersed in CLN5Y392X iPSC-derived neural lineage cells (D,H). Images shown in the figure are representative of experiments performed on neural lineage cells from two individual control and two CLN5-deficient hiPSC lines in two separate experiments (two different maturation time points, (A–D,E–H)). Bar 20 µm.