Combined Transcriptomics and Proteomics in Frontal Cortex Area 8 in Frontotemporal Lobar Degeneration Linked to C9ORF72 Expansion

22 Background: Frontotemporal lobar degeneration with TDP-43 immunoreactive inclusions (FTLD-TDP) may appear as sporadic (sFTLD-TDP) or linked to mutations in various genes including expansions of the non-coding region of C9ORF72 (c9FTLD). 23

C9ORF72 expansions produce haploinsufficiency of C9orf72 protein, RNA foci sequestering of various RNA-binding proteins, dipeptide repeat protein inclusions probably sequestering additional proteins, abnormal protein binding, TDP-43 aggregation and loss of function, nucleocytoplasmic transport defects, RNA mis-splicing, DNA damage, abnormal stress granule dynamics, and altered autophagy, among others [8,.
Complementary information has been gained from the application of transcriptomics and proteomics in FTLD-TDP and ALS [42][43][44][45] but studies including FTLD linked to C9ORF72 expansion (c9FTLD) are scarce [46].The present study was designed to gain understanding about c9FTLD pathogenesis by using vascular diseases, neoplastic disorders affecting the nervous system, metabolic syndrome, hypoxia, and prolonged axonal states such as those occurring in intensive care units, were excluded.The whole series included 19 familial cases of fFTLD associated with C9ORF72 mutation, henceforth named c9FTLD for practical purposes (mean age 70 years; 10 men and 9 women), and 14 cases (mean age 67 years; 8 men and 6 women) not suffering from neurologic or psychiatric diseases, and without abnormalities in the neuropathologic examination, which were assessed in parallel as age-matched controls (Table 1).Details of the clinical symptoms were very brief in the accompanying data provided with the brain samples used for study.Apathy, loss of empathy, disinhibition, executive dysfunction, memory loss, hallucinations, and delusions were common, often accompanied by motor neuron disease; parkinsonism and progressive aphasia were reported in some cases.

RNA purification
RNA from frozen frontal cortex area 8 was extracted following the instructions of the supplier (RNeasy Mini Kit, Qiagen® GmbH, Hilden, Germany).RNA integrity and 28S/18S ratios were determined with the Agilent Bioanalyzer (Agilent Technologies Inc, Santa Clara, CA, USA) to assess RNA quality combined with DNase digestion to avoid extraction and later amplification of genomic DNA, and the RNA concentration was evaluated using a NanoDrop™ Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA).RIN values are shown in Table 1.Special care was taken to assess premortem and postmortem factors which may interfere with RNA processing [49].

Microarray hybridization
Samples were analyzed by microarray hybridization with Human Clariom™ D Assay kit and GeneChip WT Plus Reagent Kit, and microarray 7000G platform from Affymetrix® (Affymetrix, Santa Clara, CA, USA) with a capacity to detect more than 540,000 transcripts.Preprocessing of raw data and statistical analyses were performed using bioconductor packages in an R programming environment for genes [50] which enabled data preprocessing for differential gene expression analysis and enrichment analysis.Gene selection was based upon their values using a test for differential expression between two classes (Student's t-test).Genes differentially  is represented in a heat-map (Fig. 1A).Forty-eight 351 genes were differentially expressed in frontal cortex 352 area 8 of c9FTLD when compared with controls (11 upregulated, 37 downregulated).Using GO database, we identified clusters of deregulated genes related to DNA recombination, RNA splicing regulation, RNA polymerase transcription, and centriole (Fig. 1B).

Gene validation
Aberrant accumulation of hyper-phosphorylated TDP43 and toxic aggregates of C9orf72 are common hallmarks in c9FTLD.mRNA levels of TARDBP and C9ORF72 were evaluated with RT-qPCR.

Monitoring the frontal cortical expression of specific protein mediators in c9FTLD
In order to partially validate the results obtained by LC-MS/MS, western-blotting was used as an orthogonal technique.To complement our study, total TDP43 protein levels were increased in c9FTLD when compared with controls (p = 0.02).
C9orf72 long isoform was significantly reduced (p = 0.04) whereas C9orf72 short isoform was significantly increased (p = 0.000) in c9FTLD (Fig. 5).In     in motor neurons has shown that the long iso-   (Fig. 6A).However, a protein interactome map con-   Commonalities and discrepancies are also seen when comparing the transcriptome of the frontal cortex in sALS with no mutations and ALS linked to C9orf72 mutations (c9ALS) [83].The number and type of deregulated genes in c9ALS was approximately double that seen in sALS.For example, alteration of the unfolded protein response and intracellular protein transport were identified from genes differentially upregulated in c9ALS but not in sALS, whereas alterations in oxidative phosphorylation, cytoskeleton, and synaptic transmission were predominant in frontal cortex in sALS [83].These data in frontal cortex in sALS roughly correlate with our observations in the sALS/sFTLD-TDP spectrum [45,51] although the lack of information regarding the cognitive status of patients in the c9ALS/sALS comparative study does not permit further analogies between the separate series.That study was performed using RNA-sequencing methods [83], which uses high-throughput sequencing to document all transcripts in contrast to microarrays which quantify a set of predetermined sequences.Therefore, RNAseq is presumably more robust than our microarrays approach, and may account for differences in the results obtained by these different strategies.However, focusing on c9ALS and present observations in c9FTLD, altered RNA splicing and ubiquitinproteasome system are identified in both studies.
Comparisons with other studies performed in different types of FTLD-TDP show disparate results.One gene expression analysis in fFTLD linked to GRN mutations identified abnormal regulated processes associated with lipid metabolism, MAPK signaling pathway, and transport [43], while lysosomal dysfunction was identified in another [44].Another study recognized synapse-, cytoskeletal/filament-, microtubule/axon-, and proteasome-related pathways in FTLD-TDP when compared with controls, and cytoskeletal protein-, mitochondria/energy-, synapse-, microtubule/axon-, and ubiquitin-proteasome-associated deregulation when comparing FTLD-TDP with FTLD associated with motor neuron disease [42].Common mechanisms occur within the FTLD-ALS spectrum [84].However, weighted co-expression network proteomic analysis has recently revealed 15 modules of co-expressed proteins, eight of which differed significantly across the ALS-FTD disease spectrum [46].Interestingly, a module enriched with astrocyte and microglia proteins was significantly increased in the frontal cortex in ALS cases carrying the C9orf72 mutation compared to sporadic ALS cases, suggesting that the genetic expansion is associated with inflammation in the brain [46].Increased levels of proteins linked to inflammation are also identified in the frontal cortex in C9orf72 in our study.The present dual-omic approach, like the majority of molecular studies carried out in the postmortem human brain, is based on the relative abundance of particular mRNAs and proteins or ratios of two absolute concentrations (fold-change) representing concentrations relative to reference samples.Moreover, the agonal state and postmortem delay may differentially interfere with transcription and protein synthesis/degradation.Therefore, when analyzing postmortem brain samples, a non-steady-state condition is always the real scenario.This statement is important when assessing transcripts and proteins separately, but it is especially crucial when analyzing RNA and protein correlations of particular genes in human postmortem brain [85][86][87].Moreover, distinct cell populations are usually mingled, and rates and scales of RNA and corresponding encoded proteins may be cell type-dependent.These facts, together with particular characteristics of samples, and differences in the procedures and methods, may

Fig. 1 .
Fig. 1.Microarray analysis.A) Hierarchical clustering heat-map of expression intensities of mRNA array transcripts in frontal cortex area 8 in c9FTLD compared with controls.B) GO database identifies clusters of deregulated genes in c9FTLD.Genes differentially expressed show an absolute fold change >2.0 in combination with a p-value ≤ 0.05.

Fig. 2 .
Fig. 2. mRNA expression of selected genes in c9FTLD.A) Expression levels of genes C9ORF72 and TARDBP.B) Relative expression levels of genes linked to DNA/RNA regulation mechanisms.C) Expression levels of genes coding for myelin and oligodendrocyte proteins.The significance level is set at * p < 0.05.

Fig. 3 .
Fig. 3. Differentially expressed proteins in frontal cortex area 8 in c9FTLD.A) Heat map representing the fold-change of identified proteins with associated p-values from the pair-wise quantitative comparisons with controls.Significantly upregulated proteins in c9FTLD between pair-wise comparisons are labelled in green, and significantly downregulated proteins are labelled in red.B) Volcano plot of differentially expressed proteins.

442
teins linked with the calcium/calmodulin-dependent 443 (CaM) kinase cascade were selected for valida-444 tion.SNAP25 was significantly decreased (p = 0.02), 445 and CaMKII and CaMKIV protein levels were 446 significantly reduced, in c9FTLD when compared 447 with controls (p = 0.05 and p = 0.03, respectively) differential mRNA and protein 451 expression in frontal cortex area 8 in 19 fFTLD cases 452 linked to C9ORF72 expansion (c9FTLD) and 14 age-and gender-matched controls using microarray hybridization and 7000G platform technology from 455 Affymetrix® and quantitative proteomics using LC-456 MS/MS, respectively.mRNA expression for selected genes was validated with RT-qPCR, and protein levels with gel electrophoresis and western blotting.Regarding TDP-43 and C9orf72, major pathologic components in c9FTLD, TARDBP mRNA expression was preserved but total TDP-43 protein showed increased levels in c9FTLD when compared with controls.This is in accordance with the abnormal deposition of this protein in intracellular inclusions and threads characteristic of this disease.In contrast, C9ORF72 mRNA expression was significantly decreased in c9FTLD.However, the C9orf72 long isoform was significantly reduced and the C9orf72 short isoform significantly increased in c9FTLD.Reduced C9ORF72 protein levels were found in previous reports [55-58].The functional implications of the reduced levels of the long C9orf72 isoform are not known, but quantitative mass spectrometrybased proteomics used to identify interacting proteins

Fig. 4 .
Fig. 4. High-scoring protein interactome map of differentially expressed proteins in the frontal cortex in c9FTLD.Visual representation of the relationships between differentially expressed proteins and functional interactors.Downregulated proteins are highlighted in red and upregulated proteins in green.Continuous and discontinuous lines represent direct and indirect interactions, respectively.

476
form complex stabilizes SMCR8, a protein which 477 acts as an autophagic regulator[59, 60].Therefore, reduced levels of the long isoform may interfere 479 with normal autophagy and lysosomal processing 480 [60].C9orf72 also binds to several proteins including 481 members of the Rab family, endoplasmic reticulum 482 and synapses; nuclear and cytoplasmic transport, 483 endoplasmic reticulum stress, and altered synaptic 484 function have all been reported in association with 485 pathogenic C9ORF72 expansions [21, 22, 26, 28, 35, 39, 61].The present study reveals altered gene transcription related to DNA recombination, RNA splicing regulation, RNA polymerase transcription, myelin synthesis, calcium regulation, and ubiquitin-proteasome system in c9FTLD.Proteomics performed in the same tissue samples identifies altered protein expression linked to apoptosis, inflammation, metabolism of amino acids, metabolism

Fig. 5 .
Fig. 5. Gel electrophoresis and western blotting to C9orf72 isoforms, and total TDP-43, CAMKIIa, and CAMKIV in frontal cortex area 8 in c9FTLD and controls.The protein expression of C9orf72 long isoform (54 kDa) is reduced in parallel with the increased protein level of the C9orf72 short isoform (25 kDa).Total protein levels of TDP-43 are significantly increased in c9FTLD.CAMKII and CAMKIV protein levels are significantly decreased.* p < 0.05 and * * * p < 0.001.

Fig. 6 .
Fig.6.A) Venn's diagram comparing transcriptomics and proteomics profile in c9FTLD based on the present observations.B-D) Overlap of the transcriptomic profiles obtained in the frontal cortex area 8 in sALS, sFTLD-TDP, and c9FTLD based on the present observations and our previous studies cited in[45] and[51].B) Venn's diagram of downregulated genes in sALS, sFTLD, and c9FTLD.C) Venn's diagram of upregulated genes in sALS, sFTLD, and c9FTLD.D) Venn's diagram of total deregulated genes in sALS, sFTLD, and c9FTLD.

504
structed using the IPA software shows deregulation 505 of cross-linkers between DNA/RNA regulation sys-506 tems and ubiquitin-proteasome systems, suggesting 507 an imbalance in cellular transcription processes and 508 protein degradation mechanisms, which makes sense 509 in light of gene transcription observations of the main 510 contributors to the pathogenesis of neurodegenerative 511 diseases with abnormal protein aggregates, and it is 512 particularly in line with the function of TDP-43 and C9orf72.

564
and downregulated genes clustering into oligoden-565 drocyte function and myelin-related proteins [51].566 Venn's diagrams serve to illustrate downregulated 567 and upregulated genes shared in these diseases: 568 sALS, sFTLD-TDP, and c9FTLD (Fig. 6B-D).569 Curiously, some downregulated clusters related 570 mainly to synapses and neurotransmission in frontal 571 cortex in sFTLD-TDP were upregulated in frontal 572 cortex in sALS without dementia [45, 51].The 573 present observations in c9FTLD reveal some commonalities with sFTLD-TDP in altered clusters but not in particular genes.Downregulation of genes linked to oligodendrocytes and myelin in frontal cortex area 8 is shared in c9FTLD and sALS.Recent studies have shown oligodendrocytes as key players in neurodegenerative diseases with abnormal protein aggregates [76].Oligodendrogliopathy is common in sALS and FTLD-TDP.Phosphorylated-TDP-43-immunoreactive oligodendroglial inclusions are found, in addition to spinal cord motor neurons, in the motor, sensory and premotor cortex, but not in the corpus callosum, cingulum or lateral tracts of the spinal cord [77].TDP-43 oligodendroglial inclusions are common in the deep layers of the cerebral cortex and white matter in FTLD-TDP [78].The functional effects of oligodendroglial TDP-43 inclusions are not known, but present findings indicate altered oligodendroglial gene expression in the frontal cortex in c9FTLD.Particular features are linked to C9orf72 hexanucleotide repeat expansion [79, 80], and TDP-43-dependent or TDP-43-independent oligodendroglial dysfunction might be a characteristic trait linked to C9orf72 hexanucleotide repeat expansion.In contrast, although astrocytes play key pathogenic roles in ALS [81], TDP-43immunoreactive inclusions are rare in sALS and FTLD-TDP [82].

Table 1
Summary of the thirty-three cases analyzed, including frontal cortex area 8 of 14 controls and 19 fFTLD cases.c9FTLD, familial FTLD linked to C9orf72 expansion; F, female; M, male; PMD, postmortem delay (hours, minutes); RIN, RNA integrity number 172For each 10 L TaqMan reaction, 4.5 L cDNA was 173 mixed with 0.5 L 20× TaqMan Gene Expression

Table 2
Genes, gene symbols, and TaqMan probes used for the study of gene expression including probe for normalization (GUS-␤) tive features.Output data files were managed using Perseus Software for subsequent statistical analyses and representation [54].Proteins identified by site (identification based only on a modification), reverse proteins (identified by decoy database), and potential contaminants were filtered out.Proteins quantified with at least two unique peptides, a pvalue lower than 0.05 and an absolute fold change of <0.77 (downregulation) or >1.3 (upregulation) in linear scale, were considered to be significantly differentially expressed.MS raw data and search results files were deposited in the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org)via the PRIDE partner repository with the dataset identifiers PXD011713.tory/metabolic networks identified in c9FTLD were analyzed with the use of QIAGEN's Ingenuity® Pathway Analysis (IPA) (QIAGEN Redwood City, test.The non-parametric Mann-Whitney test was 333 performed to compare each group when values did not follow a normal distribution, whereas the 335 unpaired t-test was used for normal variables.Statisti-336 cal analysis and graphic design were performed with 337 GraphPad Prism version 5.01 (La Jolla, CA, USA).338 Outliers were detected using the GraphPad software 339 QuickCalcs (p < 0.05).The data were expressed as 340 mean ± SEM, and significance levels were set at 341 * p < 0.05, * * p < 0.01, * * * p < 0.001.342 RESULTS 343 Microarray analysis 344 Cofactors age, gender, RIN value, and postmortem 345 delay were not relevant for the analysis.After fil-346 tering, 4,851 genes were included in the analysis.347 Gene expression in control and c9FTLD cases, with 348 p-values equal to or lower than 0.05 and absolute 349 fold change logarithm equal to or greater than 0.5,

Table 4
Deregulated proteins in c9FTLD compared with controls