WP05: Biomarkers and modifers of CLN1

WPLeader

Anu Jalanko, PhD    
Folkhalsan

WP Summary

 

 

Objectives


  • Utilise induced pluripotent stem (iPS) cells from patients and knockout mice to model the disease and to discover modifier genes for CLN1 disease
  • Utilise patient cell lines and cultured cell models to study the cell pathology caused by Mediterranean CLN1 mutations
  • Utilise MS based imaging (MALDI-IMS) in knockout mouse and in patient brain tissue for biomarker discovery
  • Development of a new microarray setup for the analysis of differential gene expression in NCL

 


 

 

Use of induced pluripotent stem (iPS) cells from patients and knockout mice to model the disease and to discover modifier genes for CLN1 disease

 

Utilization of established Cln1 miPS cell lines in production of neuronal cell cultures was unsuccessful and therefore the study was continued with CLN1 patient-derived hiPS cell lines. In order to get a more comprehensive picture of affected pathways and pathological changes in human CLN1 disease, iPSC and iPS-derived human neuronal cell models for CLN1 disease, classic infantile and CLN1 disease, late infantile representing three disease-associated mutations in four patients of Finnish or Italian origin were established and characterized.

 

To evaluate whether the generated CLN1 iPS cell lines and CLN1 iPSC-derived cell cultures have characteristic of NCL, we analysed the presence of autofluorescent storage material by immunofluorescence microscopy. While CLN1 hiPS cell lines were not found to accumulate storage material, these compartments were clearly visible in embryoid body-derived spontaneously differentiated cultures (Fig. 4). This indicates that generated CLN1 iPS cell lines have characteristics of the disease and therefore, could be utilised as a cell model in further functional analyses.

 

  


Fig. 4. Autofluorescent storage material in CLN1 hiPSC-derived spontaneously differentiated cell cultures visualised under 488 laser line. Representative example image of CLN1 hiPS cell line generated from CLN1, late infantile patient.#


The utilisation of patient-specific iPSC cells and iPSC-derived cell cultures enables mapping of affected intracellular pathways in humans but additionally, searching for differences between phenotypically different patients. Therefore, control and CLN1 iPS cells, and iPSC-derived neurospheres and neurosphere-derived cultures were processed for whole transcriptome gene expression analyses by RNA sequencing. RNA sequencing analyses are currently in progress. Pathway analysis and verification of selected findings at protein level will be finalized post festum. These analyses may reveal further potential biomarkers for phenotypically different CLN1 disease subtypes.

 

Use of patient cell lines and cultured cell models to study the cell pathology caused by Mediterranean CLN1 mutations

 

CLN1 patients’ derived primary cell lines and cultured neuroblastoma cells have been used for the analysis of cellular biological defects in CLN1 disease. Transient transfection of SHSY5Y neuroblastoma cells with wild-type and a set of Mediterranean mutations in CLN1 could be well achieved. This has been verified by testing with qPCR, and immunocyochemistry.

 

Modelled cells have been compared with primary cell lines to investigate:

 

i) autophagic processes both morphologically and molecularly with array techniques to detect variations in protein and mRNA expression;

 

ii) mitochondrial structure and function, namely the oxidative metabolism and ATP content, by combining immunocytochemistry and ultrastructure investigations with enzymatic assays

 

iii) oxidative and endolysosomal stress, induced by appropriate changes of growth conditions and use of specific chemicals, by qualitative and quantitative analysis of morphological and biochemical methods.

 

Results of these experiments have been confirmed by direct immunodetection at the protein level and might indicate sensible biological markers of the disease to be assessed in preclinical studies.

 

Results of this part of the project have been submitted for publication:

 

Scifo E. et al. Proteomic Analysis of the Palmitoyl Protein Thioesterase 1 Interactome in SH-SY5Y Human Neuroblastoma Cells.

 

Use of MS based imaging (MALDI-IMS) in knockout mouse and in patient brain tissue for biomarker discovery

 

MALDI-imaging mass spectrometry (MALDI-MSI) analyses on fresh-frozen mouse cortex tissues derived from 3(4) biological replicates at pre-symptomatic stage (1 month), symptomatic stage (3 months) and advanced stage (5 months), using 15-18 sections, 12 µm thick coronally cut sections from each brain has been completed. Proteomic profiling directly on tissues was performed by MALDI-MSI. Several dysregulated m/z in the range of 2-25 kDa were detected.

 

Laser Capture Microdissection (LCM) - based quantitative studies of the thalamus have been completed. Thalamic areas equal to ~ 500,000 μm2 (+/- 3%) and corresponding to ~ 5000 cells, were coronally cut from each specimen at the same plane (from both genotypes and all age groups) to ensure maximum reproducibility. Extraction and trypsin digestion protocols were optimized to identify and quantify nearly 400 unique proteins in each LC-MSE run. Label-free quantitation was performed in three technical replicates per sample at thresholds of p< 0.05, power >0.8 and fold change >1.2, that were used to determine a significant quantitative difference between the Ppt1  -/- and wild-type age matched control thalamic tissues. Based on the above criteria, 35 proteins (7.6% of all quantified proteins) were significantly differentially expressed at the pre-symptomatic stage, 38 (9.6%) at the symptomatic and 81 (20.6%) at the advanced stage of CLN1 disease.

 

We linked the differentially expressed proteins in the Ppt1-/- thalamus with literature knowledge using Ingenuity Pathways (IPA) algorithms at the pre-symptomatic stage. In an effort to obtain a more comprehensive picture of the disease changes at this stage, we utilized GeneMANIA to extend the network analyses by bridging human orthologs of the differentially expressed mouse proteins with proteins in the same pathways/gene ontologies and PPT1.

 

Altogether, the proteomic data confirmed the earlier studies performed in Cln1-/- mice (von Schantz et al., 2008; Blom et al., 2013) as well as the first human CLN1 iPS cell analyses performed in DEM-CHILD WP5.

 

Results of this part of the project have been submitted for publication:

 

Tikka S et al. Proteomic profiling in CLN1 disease brain: an imaging and label-free proteomics approach.

 

Development of a new microarray setup for the analysis of differential gene expression in NCL

 

Control and CLN1 patient-derived hiPS cells, hiPSC-derived neurospheres, and neurosphere-derived cultures were processed for whole transcriptome analysis carried by RNA sequencing. RNA sequencing analyses have been performed.

 

Conclusion

 

Task 1: iPS cells from CLN1 patients having different CLN1 mutations, showed autofluorescence storage material typical to NCL. Live cell imaging of iPS cell derived neurons showed defective neuronal migration and a similar defect has been previously reported in Ppt1-/- mice (von Schantz et al, 2008). These data suggest that CLN1/PPT1 has a role in neuronal extension and migration. This defect was here observed for the first time in human NCL derived cells and represents one of the earliest pathological changes in the CLN1 disease.

 

Task 2: Cultured neuroblastoma cell models overexpressing mutant CLN1 as well as CLN1 patient fibroblasts shoed enhanced apoptotic cell death after Staurosporin (STS) induction. Hyperpolarization of mitochondrial membrane was observed in both cell populations and CLN1 deficient cells showed dramatic shift to depolarized stage following STS treatment. Immunolabeling of CLN1 patient fibroblasts confirmed fragmented mitochondrial reticulum. Also, decrease of ATP production and reduction of mitochondrial mass was observed. Altogether, patient derived CLN1 fibroblasts showed a major defect in mitochondrial function.

 

Task 3: Proteomic profiling of Ppt1-/- mouse brains using MALDI-MS at presymptomatic stage thalamus revealed several changes related mostly to metabolic processes. In the symptomatic stage the changes were additionaly related to mitochondrial dysfunction, synaptosomal vesicle trafficking and signalling cascades, including RhoA signaling involved in neuronal extension and pathfinding. In the advanced stage, also strong dysregulation of myelin proteome was confirmed. Altogether, these data performed with MALDI-MS proteomics confirmed the earlier studies performed in Cln1-/- mice (von Schantz et al., 2008; Blom et al., 2013) and human CLN1 iPS cell analyses in this WP.