Send Orders for Reprints to [email protected] 478 Current Neurovascular Research, 2019, 16, 478-490 RESEARCH ARTICLE Combined Transcriptomic and Proteomic Analyses of Cerebral Frontal Lobe Tissue Identified RNA Metabolism Dysregulation as One Potential Pathogenic Mechanism in Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy (CADASIL) Marie-Françoise Ritz1,*, Paul Jenoe2, Leo Bonati3, Stefan Engelter3,4, Philippe Lyrer3 and Nils Peters3,4 1Department of Biomedicine, Brain Tumor Biology Laboratory, University of Basel, and University Hospital of Basel, Hebelstrasse 20, 4031 Basel, Switzerland; 2Proteomics Core Facility, Biocenter, University of Basel, Klingelbergstrasse 50/70, 4056 Basel, Switzerland; 3Department of Neurology and Stroke Center, University Hospital Basel and University of Basel, Petersgraben 4, 4031 Basel, Switzerland; 4Neurorehabilitation Unit, University of Basel and University Center for Medicine of Aging, Felix Platter Hospital, Burgfelderstrasse 101, 4055 Basel, Switzerland Abstract: Background: Cerebral small vessel disease (SVD) is an important cause of stroke and vascular cognitive impairment (VCI), leading to subcortical ischemic vascular dementia. As a he- reditary form of SVD with early onset, cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) represents a pure form of SVD and may thus serve as a model disease for SVD. To date, underlying molecular mechanisms linking vascular pathol- ogy and subsequent neuronal damage in SVD are incompletely understood. A R T I C L E H I S T O R Y Objective: We performed comparative transcriptional profiling microarray and proteomic analyses Received: October 01, 2019 on post-mortem frontal lobe specimen from 2 CADASIL patients and 5 non neurologically dis- Revised: October 11, 2019 Accepted: October 15, 2019 eased controls in order to identify dysregulated pathways potentially involved in the development DOI: of tissue damage in CADASIL. 10.2174/1567202616666191023111059 Methods: Transcriptional microarray analysis of material extracted from frontal grey and white matter (WM) identified subsets of up- or down-regulated genes enriched into biological pathways mostly in WM areas. Proteomic analysis of these regions also highlighted cellular processes identi- fied by dysregulated proteins. Results: Discrepancies between proteomic and transcriptomic data were observed, but a number of pathways were commonly associated with genes and corresponding proteins, such as: “ribosome” identified by upregulated genes and proteins in frontal cortex or “spliceosome” associated with down-regulated genes and proteins in frontal WM. Conclusion: This latter finding suggests that defective expression of spliceosomal components may alter widespread splicing profile, potentially inducing expression abnormalities that could contribute to cerebral WM damage in CADASIL. Keywords: CADASIL, transcriptomic, proteomic, pathomechanisms, spliceosome, ribosome. 1. INTRODUCTION dominant arteriopathy with subcortical infarcts and leukoen- cephalopathy (CADASIL) is the most common hereditary Small vessel disease (SVD) of the brain is a common form of SVD [2], caused by mutations in NOTCH3 gene on condition responsible for approximately 25% of all strokes chromosome 19q12, which encodes a transmembrane recep- and 40% of vascular dementia [1]. Cerebral autosomal tor playing a crucial role within vascular smooth muscle and pericyte signaling pathway [3]. Clinical manifestations ap- pearing in mid adulthood (between 30 and 40 years of age) *Address correspondence to this author at the University of Basel and comprise migraine with aura, lacunar stroke and vascular University Hospital of Basel, Department of Biomedicine, Brain Tumor cognitive impairment. Upon MR-imaging, the disease is Biology Laboratory, Hebelstrasse 20, 4031 Basel, Switzerland; E-mail: [email protected] characterized by typical SVD related lesions including dif- 1567-2026/19 $58.00+.00 © 2019 Bentham Science Publishers Transcriptomic and Proteomic Analyses of CADASIL Brains Current Neurovascular Research, 2019, Vol. 16, No. 5 479 fuse ischemic white matter lesions (WML), lacunar infarcts logical disease - in particular sporadic SVD, CADASIL or and microbleeds and eventually brain atrophy [4-6]. neurodegenerative diseases - were obtained from the Insti- tute of Pathology, University Hospital Basel and were used The extracellular domain of the mutated Notch3 receptor as healthy controls (72 ± 15 years, M/F: 4/1). The use of accumulates in the membrane of blood vessels, most likely postmortem material from deceased human subjects as con- leading to a progressive loss of vascular small muscle cells trols was approved by the local Ethics committee (EKBB, (VSMCs), thickening and stenosis of vessel walls by various Basel, Switzerland). Times to autopsy were <24 h post- types of collagens, laminins, and fibronectins, as well as de- mortem for all subjects. There was no significant difference posits of granular osmiophilic material (GOM) [7, 8] and in age between CADASIL and control groups (p=0.204, t- von Willebrand factor [9]. test). Tissue blocks dissected during autopsy were shock To date, the underlying molecular mechanisms linking frozen at -80°C until mRNA or protein extraction. vascular pathology in SVD and subsequent neuronal damage are incompletely understood. Various mechanisms have been 2.2. RNA Preparation and Microarray Processing discussed, including endothelial dysfunction, inflammatory The frozen brain samples were homogenized in Purezol processes, hemostasis, oxidative stress, disturbance of capil- solution (Bio-Rad Laboratories, Hercules, CA, USA) using a lary flow and altered function of the blood-brain-barrier [10, bead type-homogenizer (MM-301, Retsch, Haan, Germany). 11]. We recently studied the transcriptome profiles in post- Isolation of total RNA from the lysate including DNAse I mortem brains affected by sporadic SVD in comparison to treatment was carried out using the Aurum Total RNA Fatty normal brains in order to gain insight in the pathogenesis and Fibrous Tissue kit (BioRad Laboratories) according to [12]. We found regional alterations in gene expression in- the manufacturer’s protocol. The purity and integrity of the volving various pathways, such as inflammation, apoptosis, extracted total RNA were determined using the Agilent 2100 and alterations in lipid metabolism and coagulation. How- Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). ever, given the age association of sporadic SVD, there are All RNA samples had an integrity number > 7. Double potentially confounding alterations present, such as concomi- strand cDNA and labeled cRNA were prepared as previously tant neurodegeneration, having a possible influence on the published [14] and 12 µg fragmented cRNA was used for observed findings. As a hereditary condition with earlier hybridization to the Affymetrix GeneChip® Human Gene 1.0 onset, CADASIL can be regarded as a pure form of SVD, ST Arrays (Affymetrix, Santa Clara, CA, USA). Sample potentially allowing to study the underlying mechanisms of labeling, hybridization, scanning and data outputs were per- SVD in more detail. Therefore, in this study, a similar whole formed according to Affymetrix protocol. Data expression genome analysis was performed in CADASIL brains com- values were collected as individual CEL files. bined with a proteomic approach, in order to identify the correspondence of transcriptional responses to cellular pro- 2.3. Analysis of Gene Expression Data tein abundance. Indeed, proteins are effectors of biological ® function and their levels are not only dependent on mRNA Microarray data were normalized using Partek Genom- TM levels but also on host translational control and regulation ics Suite version 6.6 (Partek Inc., St. Louis, MO, USA). [13]. Thus, the proteomics should be considered as the most Data analysis including background subtraction, normaliza- relevant data set to characterize a biological system. tion, and elimination of false positives was performed using the default parameters. The principal component analysis The outcome of such an analysis is thought to offer a (PCA) was performed to assess the variation in the expres- comprehensive view of the biological roles of the selected sion of genes among the different samples. The unsupervised genes/proteins through highlighting key pathways and cellu- gene hierarchical clustering containing all the genes differ- lar processes in which they are involved. ently expressed between the two groups was used to estimate similarities in expression patterns between samples. One- Focusing on the changes of expression of genes and pro- teins in the frontal lobe, this work identified deregulation of way analysis of variance (ANOVA) was used to identify the spliceosome, which could produce splicing abnormalities genes differentially expressed between CADASIL and con- leading to generalized protein disbalance. This finding repre- trol samples for each brain region. Criteria for significant sents a yet unsuspected molecular dysfunction in cerebral changes in signal levels were: False Discovery Rate (FDR) small vessel diseases that needs to be further investigated. value < 0.05, and a fold change F (log transformed base 2.0) ≥ 1.2 in both directions. 2. MATERIALS AND METHODS 2.4. Proteomic Analysis 2.1. Human Brain Samples 2.4.1. Protein and Peptide Preparation The use of material from human patients was approved Biopsies