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Rinnakkaistallenteet Terveystieteiden tiedekunta
2018
Interrelationship between the Levels of C9orf72 and Amyloid-ß Protein
Precursor and Amyloid-ß in Human Cells and Brain Samples
Leskelä, S
IOS Press
Tieteelliset aikakauslehtiartikkelit
© IOS Press and the authors All rights reserved
http://dx.doi.org/10.3233/JAD-170362
https://erepo.uef.fi/handle/123456789/6602
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Interrelationship between the levels of C9orf72and amyloid- precursor protein and -amyloid in human cells and brain samples
Stina Leskeläa,¶, Mari Takaloa,b,¶, Mikael Marttinenb, Nadine Hubera, Jussi Paananenb, Vikram Mitrah, Tuomas Rauramaac,e, Petra Mäkinenb, Ville Leinoneng, Hilkka Soininend,f, Ian Pikeh, Anne M. Remesd,f, Mikko Hiltunenb,d,f, and Annakaisa Haapasaloa,f,*
aA.I. Virtanen Institute for Molecular Sciences, Neulaniementie 2, bInstitute of Biomedicine, Yliopistonranta 1E, cInstitute of Clinical Medicine – Pathology Yliopistonranta 1C, and
dInstitute of Clinical Medicine – Neurology, Yliopistonranta 1C, University of Eastern Finland, 70211 Kuopio, Finland; eDepartment of Pathology, Kuopio University Hospital, Puijonlaaksontie 2, 70210 Kuopio, Finland; fNeuroCenter, Neurology, and gNeurosurgery of NeuroCenter, Kuopio University Hospital and University of Eastern Finland, P.O. Box 100, 70029, Kuopio, Finland; and hProteome Sciences plc, Coveham House, Downside Bridge Road, Cobham, Surrey KT11 3EP, United Kingdom
¶These authors contributed equally to this work
*Corresponding author:
Annakaisa Haapasalo
A. I. Virtanen Institute for Molecular Sciences University of Eastern Finland (UEF)
P. O. Box 1627 (Neulaniementie 2) 70211 Kuopio
Finland
Tel: +358 40 355 2768 Fax: +358 17 163 025
Email: annakaisa.haapasalo@uef.fi
Running title: Interrelationship between the levels ofC9orf72, A PP, and A
Abstract
A subset of C9orf72 repeat expansion-carrying frontotemporal dementia patients display an Alzheimer-like decrease in cerebrospinal fluid amyloid- (A ) biomarker levels. We report that downregulation ofC9orf72 in non-neuronal human cells overexpressing amyloid- precursor protein (A PP) resulted in increased levels of secreted A PP fragments and A , while levels of PP or its C-terminal fragments (CTFs) remained unchanged. In neuronal cells, A PP and C83 CTF levels were decreased upon C9orf72 knockdown, but those of secreted A PP fragments or A remained unchanged. C9orf72 protein levels significantly increased in human brain with advancing neurofibrillary pathology and positively correlated with brain A 42 levels. Our data suggest that alteredC9orf72levels may lead to cell-type specific alterations in
PP processing, but warrant further studies to clarify the underlying mechanisms.
Key words: Alzheimer’s disease; amyloid- ; amyloid- precursor protein; C9orf72;
frontotemporal dementia
Introduction
The GGGGCC hexanucleotide repeat expansion in C9orf72 gene is a major genetic cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) [1,2]. The frequency of theC9orf72 repeat expansion varies in different countries. For example, 2-19% of sporadic and 14-48% familial FTD cases, with Finland showing the highest frequency, are expansion carriers [3]. The suggested pathogenic mechanisms in the carriers include haploinsufficiency and formation of RNA foci and dipeptide-repeat (DPR)-containing protein inclusions in cells [1,4-8]. In humans,C9orf72 encodes three mRNA variants, which yield two protein isoforms, the long isoform a (~50 Da) and the short isoform b (~25 kDa). Isoform a is implicated in vesicular trafficking in the endosomal and autophagosomal/lysosomal pathways [9-11] and isoform b in nucleocytoplasmic trafficking [12].
In addition to FTD or ALS, C9orf72 repeat expansion has been identified in some patients with other neurodegenerative disorders, including Alzheimer’s disease (AD). However, the significance of the repeat expansion beyond disorders of the FTD-ALS spectrum is not clear [13-15]. AD is the most common cause of dementia and its central neuropathological hallmarks include intraneuronal neurofibrillary tangles, composed of hyperphosphorylated tau protein, and amyloid- (A ) plaques in the brain parenchyma [16]. Increased levels of total and phosphorylated tau and decreased levels of A 42 peptides in the cerebrospinal fluid (CSF) are used as diagnostic biomarkers for AD [17,18]. We have previously reported that 25% of Finnish C9orf72 repeat expansion-carrying FTD patients show decreased CSF A 1-42 levels [19]. The patients with such altered biomarker status fulfilled the clinical criteria of the behavioral variant FTD with no clinical signs of AD. In this study, neuropathological confirmation was available only for one patient, showing TAR DNA-binding protein 43 (TDP- 43) pathology, but no A or tau pathology [19]. Albeit the reason for the decrease in the CSF 1-42 levels remains thus far unknown, these results suggest that decreased CSF A 1-42
levels may not excludeC9orf72 repeat expansion-carrying FTD patients in clinical diagnostics [19]. Given this AD-like CSF A finding in a subset of C9orf72 expansion-carrying FTD patients, we asked here if modifying the levels of C9orf72 affects A PP processing and generation of A peptides.
Materials and methods
siRNAs and cDNA constructs
Dharmacon (ON-TARGETplus, L-013341-01-005; siRNA 1) or Santa Cruz (sc-92761; siRNA 2) C9orf72 siRNA pools were used forC9orf72 knockdown. Silencer Negative control siRNA (Ambion, AM4611) was used as a control. Expression cDNA constructs encoding C9orf72 isoform a containing a C-terminal green fluorescent protein (GFP) tag (isoform a-GFP) [11]
and C9orf72 isoform b containing a C-terminal myc-DDK tag (isoform b-myc-DDK) were purchased from Origene. Empty plasmid (pcDNA3.1, Invitrogen) was used as a control in the cDNA transfections.
Cell culture, transfection, and treatments
Human embryonic kidney HEK293-AP-A PP cells overexpressing A PP751 with a thermostable N-terminal alkaline phosphatase (AP) tag were cultured as previously [20,21].
Human H4 neuroglioma cells overexpressing A PP751 (H4-A PP751) were cultured as in [22]. Twenty-five nM of each siRNA or 4 µg of plasmid DNA and 10 l Lipofectamine 2000 reagent (Invitrogen) were used per transfection according to manufacturer’s instructions to knockdownC9orf72 or overexpress C9orf72 protein isoforms. Fresh media was added 6 hours post transfection. A subset of HEK293-AP-A PP or H4-A PP751 cells were treated with 100 ng/ml phorbol myristate acetate (PMA) and 250 nM -secretase inhibitor N-[N-(3,5-
generation of A PP C-terminal fragments (CTFs) C83 and C99. These samples were used as positive controls for validation of the correct C83 and C99 bands in the Western blot analyses.
Cells and conditioned media were collected 48 (overexpression) or 96 hours (knockdown) post transfection for analyses.
Protein extraction from cells and Western blotting
Protein extraction and Western blotting were performed as in [21]. Following primary antibodies were used: anti-C9orf72 (1:1000; Santa Cruz, sc-13876); anti-A PP C-terminus (1:2000; Sigma, A8717); anti-A PP N-terminus [1:1000; Millipore, MAB348; used to detect total amount of sA PP fragments (sA PPtot = sA PP + sA PP )]; anti- -amyloid (1:1000;
BioSite, 6E10; used to specifically detect sA PP forms); anti-sA PP (1:500, Covance; to specifically detect A PP forms); and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 1:5000, Abcam, ab8245). Levels of the investigated proteins were quantified using Quantity One or Image Lab (BioRad) and normalized to those of GAPDH in the same samples.
Protein levels in conditioned media were normalized to total protein lysate concentrations in each sample. Data are shown as % of the level compared to that with control siRNA or plasmid- transfected cells (set to 100%).
Soluble A PP measurement using alkaline phosphatase assay
Soluble A PP fragments containing the N-terminal AP tag (AP-sA PP) and released into the conditioned media were measured as previously by alkaline phosphatase assay [20].
ELISA
x-40 and A x-42 levels in conditioned media were measured using Human/Rat Amyloid 40 (Cat no 294-64701) and Human/Rat Amyloid 42 (Cat no 292-64501) ELISA Kits (Wako)
according to kit instructions as previously [20]. A concentrations were normalized to total protein lysate concentrations in each sample.
Human brain sample cohort
The human brain samples from the inferior temporal cortex used in the analyses have been previously described [23]. The samples were classified into groups according to Braak staging (0-VI) depending on the extent of neurofibrillary pathology [24]. The Ethics Committee of the Kuopio University Hospital has approved the study.
Extraction and analysis of C9orf72 RNA and protein from the frozen brain tissue samples RNA and protein from 71 human brain tissue samples were extracted as described previously [25,26]. Global expression ofC9orf72 from 60 RNA samples from the inferior temporal cortex was investigated by using exon array probe FILL_23_P405873 (NM_145005). Assessment was implemented using Agilent One-Color Microarray-Based Exon Analysis as described in [23].
Global expression of C9orf72 was investigated from 36 protein samples from the inferior temporal cortex using SysQuant™ global proteomics (Proteome Sciences) as described in [26].
In total, 25 human brain tissue samples underwent both exon array and proteomic analysis.
Statistical analyses
Cell-based data are shown as mean % ± standard error of mean (SEM) of control plasmid- or siRNA-transfected cells. Statistical analyses were performed using GraphPad Prism5 or R.
One-way analysis of variance (ANOVA), followed by Newman-Keuls or Fisher’s least significant difference post-hoc test or Mann-Whitney U test, was used to test statistical significance between sample groups. Correlation between variables was assessed using Spearman’s rank-order correlation. The level of statistical significance was defined as p < 0.05.
Results
To find out if decreased levels ofC9orf72result in altered A PP processing and A generation, we first used siRNA-mediated knockdown of C9orf72 in non-neuronal HEK293-AP-A PP cells that have been widely used to screen for novel substrates for the AD-associated secretases and factors influencing A PP processing [20,27]. Knockdown of C9orf72 with two different siRNA pools (C9orf72 siRNA 1 and 2) led to significantly decreased levels of the C9orf72 protein isoform a by 78.4 % and 73.4 %, respectively, as assessed by Western blotting (Figure 1A and C). Endogenous expression of isoform b was not detectable. We observed a moderate, but statistically not significant, increase in the levels of full-length AP-A PP and C83 A PP C- terminal fragment (CTF) in C9orf72 siRNA-transfected cells as compared to control cells (Figure 1A and B). The -secretase-cleaved C99 CTFs were not detectable. As a positive control to verify the correct sizes of the observed A PP CTFs, a subset of cells was treated with PMA and the -secretase inhibitor DAPT. Under these conditions, increase in both C83 and C99 levels could be observed (Figure 1A). Western blotting of conditioned media from the same cells showed a significant increase in the levels of soluble AP-A PP (AP-sA PP ) and total soluble AP-A PP (AP-sA PPtot = AP-sA PP + AP-sA PP ) fragments uponC9orf72 knockdown. The levels of sA PP , as assessed by an antibody specifically recognizing this soluble fragment in the media, were not detectable (Figure 1D and E). An alkaline phosphatase- based assay [20], which detects the levels of AP-sA PPtot, also indicated a significant increase in the conditioned media of C9orf72 siRNA-transfected cells as compared to control cells (Figure 1 F), confirming the result from Western blot analysis. There were no statistically significant alterations in the levels of endogenous sA PP or sA PPtot, even though there was a small trend towards increased sA PPtot levels (Figure 1D and E). A significant increase in soluble A 40 and A 42 levels in the conditioned media of C9orf72 siRNA-transfected cells
was evident compared to control cells. There were no differences in the A 42/A 40 ratio (Figure 1G).
To study the effects of C9orf72 knockdown in neuronal cells, H4-A PP751 cells were transfected with C9orf72 siRNA1. Similar to HEK293-AP-A PP cells, a significant decrease in C9orf72 levels by 73.2 % was observed (Figure 2C). Opposite to HEK293-AP-A PP cells, the levels of mature and immature forms of A PP and C83 CTF were observed to decrease uponC9orf72 knockdown in H4-A PP751 cells. The decrease in the levels of immature A PP was statistically significant. C99 levels were not detectable in the H4-A PP751 cells even after PMA and DAPT treatment (Figure 2A and B). Also in contrast to the HEK293-AP-A PP cells, the levels of the soluble sA PP (sA PPtot or sA PP ) fragments remained unchanged in C9orf72 siRNA-transfected H4-A PP751 cells as compared to control cells. sA PP levels were undetectable in H4-A PP751 cells (Figure 2D and E). There were no differences in the 40 and A 42 levels or A 42/A 40 ratio in the conditioned media between C9orf72 siRNA- transfected and control cells (Figure 2F). Similar results related to A PP processing upon C9orf72 knockdown were also obtained in H4 cells without A PP overexpression (data not shown).
To test whether overexpression of the individual C9orf72 protein isoforms affected A PP processing, transfection of HEK293-AP-A PP cells with C9orf72 isoform a-GFP [11] or isoform b-myc-DDK was performed. Western blot analysis of cell lysates from isoform a-GFP- or isoform b-myc-DDK-transfected cells did not indicate differences in full-length AP-A PP or C83A PP CTF levels between cells overexpressing the two isoforms or the control cells (Supplementary Figure 1A and B). Western blot analysis of conditioned media from the same cells did not reveal statistically significant differences in AP-sA PPtot or AP-sA PP levels (Supplementary Figure 1C and D). Levels of the endogenously expressed sA PP or sA PPtot did not show alterations by Western blotting (Supplementary Figure 1C and D) or AP-based
assay (Supplementary Figure 1E). The levels of secreted A 40 or A 42 or A 40/A 42 ratio remained unchanged in the conditioned media of cells transfected with either of the C9orf72 isoforms compared to control cells (Supplementary Figure 1F).
Finally, the levels of C9orf72 mRNA and protein in an existing human brain sample cohort from temporal cortex subcategorized according to advancing neurofibrillary pathology (Braak staging 0-VI) were examined. We have previously used this cohort to assess AD-related changes in gene expression [23,27]. We observed a small decrease inC9orf72 mRNA levels at Braak stage IV as compared to Braak stage 0, but overall the mRNA levels ofC9orf72 did not show major alterations at different Braak stages (Figure 3A). However, the C9orf72 protein levels showed a clear increase along with increasing neurofibrillary pathology and these increases were statistically significant at Braak stages II-VI as compared to Braak stage 0 (Figure 3B). Correlation analyses indicated a highly significant positive correlation between C9orf72and PP mRNA levels (Figure 3C). A similar mild, but not statistically significant, trend in the correlation between C9orf72 and A PP protein levels could also be observed (Figure 3D). The C9orf72 protein, but not mRNA, levels showed a statistically significant positive correlation with brain A 42 levels (Figure 3E and F).
Discussion
TheC9orf72hexanucleotide repeat expansion is suggested to lead to FTD or ALS pathogenesis through haploinsufficiency, resulting in decreased levels ofC9orf72 mRNA and protein [1,28], and formation of nuclear RNA foci and cytoplasmic inclusions containing dipeptide repeat (DPR)-containing proteins [1,4-8]. Also, some patients with other neurodegenerative disorders, including AD, have been reported to carryC9orf72repeat expansion, suggesting its potential involvement in neurodegeneration beyond the FTD/ALS spectrum [13-15]. On the other hand,
in our previous study, we found that 25% of Finnish C9orf72repeat expansion-carrying FTD patients display decreased CSF A 1-42 biomarker levels similarly to AD patients [19].
To assess the potential link betweenC9orf72 and altered A PP processing and A production, we modulated expression of C9orf72 by RNA interference-mediated knockdown or overexpression of the individual C9orf72 protein isoforms a and b in non-neuronal HEK293- AP-A PP or neuronal H4-A PP751 cells. The knockdown approach was used to model the suggested haploinsufficiency and the overexpression to allow investigation of the effects of individual C9orf72 protein isoforms. Overexpression of either C9orf72 protein isoform did not significantly affect A PP processing in HEK293-AP-A PP cells. However, a moderate increase in the levels of full-length A PP or its C83 CTF upon C9orf72 knockdown was observed. In addition, the levels of secreted sA PP fragments and A 40 and A 42 were significantly increased in HEK293-AP-A PP cells with decreased C9orf72 expression. In contrast to these non-neuronal cells, knockdown ofC9orf72in the neuronal H4-A PP751 cells overexpressing A PP, or H4 cells expressing endogenous A PP, led to decreased levels of full- length A PP and C83 CTF, but did not influence the levels of the secreted sA PP fragments or peptides. These results altogether indicate that alterations inC9orf72 expression may have an impact on A PP levels and processing, but such effects may be cell type-specific in non- neuronal and neuronal cells.
C9orf72 proteins, especially the isoform a, are implicated in protein trafficking in the endosomal-lysosomal and autophagosomal pathways and suggested to function as GDP-GTP exchange factors for Rab proteins, key regulators of vesicular trafficking [9,11]. It is well established that A PP trafficking and subcellular localization determine whether A PP goes through amyloidogenic or non-amyloidogenic processing. A PP undergoes maturation by glycosylation when trafficking on the secretory pathway from ER to Golgi [29]. The mature, N- and O-glycosylated A PP then traffics to plasma membrane and is cleaved by -secretases.
This cleavage prevents A generation and yields sA PP potentially having neuroprotective properties [30,31]. Alternatively, A PP is internalized from the plasma membrane and either recycled via the early endosomal compartment or targeted to later endosomal compartments and lysosomes. Endosomes are the major sites of A PP amyloidogenic processing by - and - secretase and A production [32,33]. Our results suggest that decreased or increased levels of C9orf72 might not directly affect A PP cleavage by the secretases, because we did not observe major differences in the levels of -secretase- or -secretase-cleaved A PP CTFs. Moreover, the levels of C83 A PP CTF normalized to those of full-length A PP were not altered, also further inferring that the -secretase-mediated cleavage of A PP was not affected. The fact that C9orf72 knockdown did not result in increased levels of sA PP or C99 A PP CTF argues against an enhanced -secretase-mediated A PP cleavage. On the other hand, investigations in both non-neuronal and neuronal cells suggested that C9orf72 knockdown may lead to altered levels of full-length A PP, but whether the A PP levels increase or decrease, appears to depend on the cell type. Increased levels of both secreted sA PP and A in HEK293-AP-A PP cells uponC9orf72knockdown imply that decreased levels ofC9orf72 may lead to alterations in the secretion of these proteins at least in these non-neuronal cells. Alternatively, these increases might result from the initially slightly increased full-length A PP levels.
Our findings in human brain samples suggest that the expression levels ofC9orf72 and A PP positively correlate with each other. This correlation was statistically significant at the mRNA level and there was a trend towards a positive correlation also at the protein level. These data are in line with the observed decrease of A PP levels in the neuronal H4 cells either overexpressing or endogenously expressing A PP upon C9orf72 downregulation. Also, the levels of C9orf72 protein with the brain A 42 levels displayed a statistically significant positive correlation. These results together point out that the hexanucleotide repeat expansion-associated
haploinsufficiency leading to decreased levels of C9orf72 may have an impact on A PP expression or the levels of A in human brain.
There are several possible mechanisms, which might underlie the previously observed decrease in CSF A 1-42 levels in repeat expansion-carrying FTD patients, including changes in A secretion, clearance, synaptic pools, transport through the blood-brain barrier, or degradation by A -degrading enzymes. Our cell-based findings suggesting potential alterations in the expression of A PP or secretion of sA PP and A agree well with these patient data, even though uncovering the specific molecular mechanisms in different cell types still requires further investigations. Therefore, it would be important to investigate the levels of A PP, its cleavage products, or A in the cells or brain samples derived from the hexanucleotide repeat carriers. To our knowledge, we provide here for the first time information on the relationship between C9orf72 and A PP levels and processing. However, further investigations are warranted in different model systems and patient-derived cells or tissue samples with decreased C9orf72 expression to reveal the underlying molecular mechanisms leading to potentially altered A PP levels and/or processing.
Acknowledgements
The authors would like to express their gratitude to Dr. Stefan Lichtenthaler (DZNE, Munich, Germany) for generously providing the HEK293-AP-A PP cells. This study was supported by VTR grants 5772795 and 5772816 of Kuopio University Hospital, Sigrid Jusélius Foundation, and Doctoral Program of Molecular Medicine (DPMM), University of Eastern Finland.
Conflict of Interest/Disclosure Statement The authors have no conflict of interest to report.
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Figure legends
Figure 1. Knockdown of C9orf72 significantly increases the levels of soluble A PP fragments and A 40 and A 42 and mildly the levels of full-length A PP or C83 C- terminal fragment in non-neuronal HEK293-AP-A PP cells. A) Western blot of total protein lysates of HEK293-AP-A PP cells transfected with C9orf72 siRNA 1 or 2 or control siRNA showing the levels of full-length AP-A PP (AP-A PP), C83 A PP C-terminal fragment, endogenous C9orf72 isoform a, or GAPDH (loading control). Treatment with 100 ng/ml PMA and 250 nM DAPT for 3 hours (positive control for the CTFs on the blot) leads to increased levels of both C83 and C99 A PP CTFs. Endogenous C9orf72 isoform b levels were not detectable. All samples were run on the same gel. Molecular weight markers are indicated on the left as kDa. An unspecific band, detected by the C9orf72 antibody and not affected by the siRNA treatment, is indicated by an asterisk (*).B) Quantification of full-length AP-A PP and C83 levels normalized to those of GAPDH from A. C83 levels were also normalized to those of AP-A PP. Data are shown as mean % ± SEM of the protein levels compared to those in the control cells. AP-A PP n = 9; C83/GAPDH n = 6; C83/AP-A PP n = 6; One-way ANOVA, Newman-Keuls, not significant. C) Quantification of C9orf72 levels normalized to those of GAPDH from A. Data are shown as mean % ± SEM of the protein levels compared to those in the control cells. n = 9 for all transfections, One-way ANOVA, Newman-Keuls, ***p 0.001. D) Western blot of conditioned media of HEK293-AP-A PP cells transfected with C9orf72 siRNA 1 or 2 or control siRNA showing the levels of the endogenous soluble A PP (sA PP ) and soluble A PPtot (sA PPtot = sA PP + sA PP ) and soluble AP-A PP (AP- sA PP ) and AP-A PPtot (AP-sA PPtot) derived from overexpressed AP-A PP. Soluble sA PP levels remained undetectable. Molecular weight markers are indicated on the left as kDa.E) Quantification of sA PP , sA PPtot, AP-sA PP and AP-sA PPtot from D. Data are shown as mean % ± SEM of the protein levels compared to those in the control cells. n = 6 for
all transfections, One-way ANOVA, Newman-Keuls, *p 0.05, **p 0.01, ***p 0.001. F) Alkaline phosphatase assay-based determination of the soluble AP-sA PPtot levels in the conditioned media of HEK293-AP-A PP cells transfected with C9orf72 siRNA 1 or 2 or control siRNA. The assay detects both AP-sA PP and AP-sA PP forms (= AP-sA PPtot).
Data are shown as mean % ± SEM of the protein levels compared to those in the control cells.
n = 6 for all transfections, One-way ANOVA, Newman-Keuls, ***p 0.001.G) The levels of 40 or A 42, and A 40/A 42 ratio in the conditioned media from HEK293-AP-A PP cells transfected with C9orf72 siRNA 1 or 2 or control siRNA. Data are shown as mean % ± SEM of the A levels compared to those in control cells. n = 6 for all transfections, One-way ANOVA, Newman-Keuls, **p 0.01, ***p 0.001.
Figure 2. Knockdown of C9orf72 decreases the levels of mature and immature forms of full-length A PP and C83 C-terminal fragment, but does not lead to changes in the levels of soluble sA PP fragments or A in neuronal H4-A PP751 cells.A) Western blot of total protein lysates of H4-A PP751 cells transfected with C9orf72 siRNA 1 or control siRNA showing the levels of mature and immature forms of A PP, C83 A PP C-terminal fragment, endogenous C9orf72 isoform a, or GAPDH (loading control). Treatment with 100 ng/ml PMA and 250 nM DAPT for 3 hours (positive control for the CTFs on the blot) leads to increased levels of C83 A PP CTFs. Endogenous C9orf72 isoform b levels were not detectable. All samples were run on the same gel. Molecular weight markers are indicated on the left as kDa.
An unspecific band, detected by the C9orf72 antibody and not affected by the siRNA treatment, is indicated by an asterisk (*). B) Quantification of mature and immature A PP and C83 levels normalized to those of GAPDH from A. C83 levels were also normalized to those of total A PP (mature + immature A PP). Data are shown as mean % ± SEM of the protein levels compared to those in the control cells. AP-A PP n = 6; C83/GAPDH n = 6; C83/AP-A PP n = 6; One-
way ANOVA, Newman-Keuls, **p 0.01. C) Quantification of C9orf72 levels normalized to those of GAPDH from A. Data are shown as mean % ± SEM of the protein levels compared to those in the control cells. n = 6 for all transfections, Mann-Whitney U, **p 0.01. D) Western blot of conditioned media of H4-A PP751 cells transfected with C9orf72 siRNA 1 or control siRNA showing the levels of the soluble A PP (sA PP ) and soluble A PPtot (sA PPtot = sA PP + sA PP ). Soluble sA PP levels remained undetectable. Molecular weight markers are indicated on the left as kDa. E) Quantification of sA PP and sA PPtot from D. Data are shown as mean % ± SEM of the protein levels compared to those in the control cells. n = 6 for all transfections, One-way ANOVA, Newman-Keuls, not significant. F) The levels of A 40 or 42, and A 40/A 42 ratio in the conditioned media from H4-A PP751 cells transfected with C9orf72 siRNA 1 or control siRNA. Data are shown as mean % ± SEM of the A levels compared to those in control cells. n = 6 for all transfections, One-way ANOVA, Newman- Keuls, not significant.
Figure 3. The C9orf72 mRNA levels slightly decrease and protein levels show an increase in relation to increased neurofibrillary pathology in human temporal cortex. A) C9orf72 mRNA expression in the human temporal cortex samples was assessed using a FILL_23_P405873 probe, detecting global expression of all C9orf72 mRNAs. The samples were subcategorized according to the extent of neurofibrillary pathology as assessed by Braak staging. Braak 0 stage indicates no detectable neurofibrillary pathology and Braak I-VI stages indicate increasing neurofibrillary pathology (I = mildest, VI = most severe).C9orf72 mRNA levels show a slight decrease according to increasing neurofibrillary pathology and the decrease at Braak stage IV is statistically significant (*p 0.05) as compared to stage 0. Box plots show the median, 25th and 75th percentiles, error bars show 1.5 interquartile ranges. ANOVA, LSD,
*p < 0.05, n = 60. B) C9orf72 protein levels were assessed by LC-MS/MS from a subset of
patients, identifying a C9orf72-specific peptide (DVLMTF). C9orf72 protein levels are significantly increased at Braak stages II-VI according to increasing severity of the neurofibrillary pathology. Box plots show the median, 25th and 75th percentiles, error bars show 1.5 interquartile ranges. ANOVA, LSD, *p < 0.05, n = 36. C) C9orf72 mRNA levels show a significant positive correlation with PP mRNA levels in human brain. D) C9orf72 and A PP protein levels show a trend towards positive correlation in human brain. E) C9orf72 mRNA levels and A 42 levels do not correlate with each other in human brain. F) C9orf72 protein and brain A 42 levels indicate a significant positive correlation in human brain.
Spearman’s rank-order correlation coefficient, C-D; n = 55, E-F; n = 36.
Supplementary Figure 1. Overexpression of C9orf72 protein isoform a or b does not affect PP processing in HEK293-AP-A PP cells. A) Western blot showing the levels of full- length AP-A PP (AP-A PP), C83 A PP C-terminal fragment, and GAPDH (loading control) in HEK293-AP-A PP cells overexpressing C9orf72 isoform a-GFP, isoform b-myc-DDK, or empty plasmid (control). Overexpressed isoform a-GFP and isoform b-myc-DDK and endogenously expressed isoform a are indicated. Endogenous isoform b levels were not detectable. Asterisks (*) denote unspecific bands detected by the C9orf72 antibody. Molecular weight markers are indicated on the left as kDa.B) Quantification of AP-A PP and C83 levels normalized to those of GAPDH from A. Data are shown as mean % ± SEM of the protein levels compared to those in the control cells. Control n =7; isoform a-GFP n = 9; isoform b-myc-DDK n = 9. One-way ANOVA, Newman-Keuls, not significant. C)Western blot showing the levels of overexpressed AP-sA PP and total AP-sA PP (AP-sA PPtot) as well as endogenously expressed sA PP and total sA PP (sA PPtot) in the conditioned media of HEK293-AP-A PP cells overexpressing C9orf72 isoform a-GFP, isoform b-myc-DDK, or empty plasmid (control).
Molecular weight markers are indicated on the left as kDa. D) Quantification of the AP-
sA PPtot, AP-sA PP , and endogenously expressed sA PPtot, and sA PP levels from C.
Data are shown as mean % ± SEM of the protein levels compared to those in the control cells.
For AP-sA PPtot: Control n = 7; isoform a-GFP n = 8; isoform b-myc-DDK n = 9. For AP- sA PP : Control n = 7; isoform a-GFP n = 9; isoform b-myc-DDK n = 9. For endogenous sA PPtot: Control n = 5; isoform a-GFP n = 7; isoform b-myc-DDK n = 7. For endogenous sA PP : Control n = 7; isoform a-GFP n = 9; isoform b-myc-DDK n = 9. One-way ANOVA, Newman-Keuls, not significant. E) Alkaline phosphatase assay-based determination of the soluble AP-A PP (AP-sA PP) levels in the conditioned media from cells overexpressing C9orf72 isoform a-GFP, isoform b-myc-DDK, or empty plasmid (control). The assay detects both AP-sA PP and AP-sA PP forms (= AP-sA PPtot). Data are shown as mean % ± SEM of the protein levels compared to those in the control cells. Control n = 7; isoform a-GFP n = 9; isoform b-myc-DDK n = 9. One-way ANOVA, Newman-Keuls, not significant. F) The levels of A 40 or A 42, or A 40/A 42 ratio in the conditioned media from cells overexpressing C9orf72 isoform a-GFP, isoform b-myc-DDK, or empty plasmid (control). Data are shown as mean % ± SEM of the A levels compared to those in control cells. Control n = 5; isoform a- GFP n = 6; isoform b-myc-DDK n = 6. One-way ANOVA, Newman-Keuls, not significant.
