Yamini Achari

Ting Lu

David A Hart
email: hartd@ucalgary.ca

Dr David A Hart Mccaig

Polymorphisms in the promoter regions for human MMP-1 and MMP-13 lead to differential responses to the alpha and beta isoforms of estrogen receptor and their ligand in vitro

Keywords: Polymorphism, MMP-13, ER-α, ER-β, 1G, 2G, Gene regulation

Estrogen receptors (ER) are present in connective tissues and therefore it is possible that the loss of estrogen after menopause influences the integrity of these tissues, contributing to development of degenerative conditions such as osteoporosis and osteoarthritis in a subset of women. Aberrant expression of matrix metalloproteinases (e.g. MMP-1 & MMP-13) has been implicated in the progression of these diseases. The present study investigated potential molecular mechanisms involved in the regulation of expression of MMP-1 and MMP-13 promoter variants by ER-α and ER-β (+/estrogen) in a transient transfection system. The results demonstrate that the activity of human MMP-1 and MMP-13 polymorphic variants are elevated in the presence of ER-α and ERβ, and the single nucleotide polymorphisms present in the promoters of MMP-1 and MMP-13 variants lead to differential activities in response to the ER isoforms. Furthermore, the influence of 17-β estradiol also varies depending upon whether the alpha or the beta isoform of ER is the modulator of these polymorphic variants. These findings support the conclusion that ER isoforms may be contributing to disease development and/or progression in genetically distinct subsets of women following menopause, and provide mechanistic insights into how such contributions are manifested.

Please cite this article as: Yamini Achari, Ting Lu, David A. Hart, Polymorphisms in the promoter regions for human MMP-1 and MMP-13 lead to differential responses to the alpha and beta isoforms of estrogen receptor and their ligand in vitro, BBA -Molecular Basis of Disease (2008), doi: 10.1016/j.bbadis.2008.02.009 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

A C C E P T E D M A N U S C R I P T A C C E P T E D M A N U S C R I P T Introduction

The incidence of osteoarthritis (OA) and osteoporosis (OP) is more prevalent in females than in males, particularly after menopause when estrogen levels in the body decline. Degradation of articular cartilage is a feature of OA and degradation of bone matrix a feature of OP. In both conditions aberrant expression of matrix metalloproteinases (MMPs) has been implicated (reviewed in 1; 2, 3). In addition, depletion of estrogen as a result of ovariectomy has been observed to lead to increased cartilage degradation in a rodent model [START_REF] Christgau | Suppression of elevated cartilage turnover in postmenopausal women and in ovariectomized rats by estrogen and a selective estrogen-receptor modulator (SERM)[END_REF][START_REF] Høegh-Andersen | Ovariectomized rats as a model of postmenopausal osteoarthritis: validation and application[END_REF]. Furthermore, in vivo animal studies on ovariectomized sheep [START_REF] Turner | Biochemical effects of estrogen on articular cartilage in ovariectomized sheep[END_REF] and monkeys [START_REF] Ham | Effects of long-term estrogen replacement therapy on osteoarthritis severity in cynomolgus monkeys[END_REF] also suggest that estrogen replacement therapy may have a protective effect against OA as it reverses the damage to biomechanical properties of cartilage and/or decreases the severity of cartilage degradation. In vitro studies, which have investigated the effects of 17-β estradiol on IL-1 beta induced MMP expression, suggest that high concentration of estrogen can lead to increased IL-1 beta stimulated cartilage degradation [START_REF] Richette | Dual effects of 17β-oestradiol on interleukin 1b-induced proteoglycan degradation in chondrocytes[END_REF]. Destruction of cartilage has also been associated to production of catabolic factors such as NO by chondrocytes [START_REF] Pelletier | The increased synthesis of inducible nitric oxide inhibits IL-1ra synthesis by human articular chondrocytes: possible role in osteoarthritic cartilage degradation[END_REF].

It is well known that the major actions of the hormone estrogen are generally mediated through two intracellular receptors, ER-alpha and ER-beta (discussed in [START_REF] Lu | Estrogen receptor alpha regulates matrix metalloproteinase-13 promoter activity primarily through the AP-1 transcriptional regulatory site[END_REF][START_REF] Lu | Estrogen receptor beta +/-ligands can modulate MMP-13 promoter activity by HIG-82 cells in vitro: Evidence that ER-beta enhancement of activity can be modulated by estrogen and involves specific sites on the promoter[END_REF].

Previous studies have shown that both ER-α and ER-β can elevate the activity of the promoter for the rabbit MMP-13, a member of the family of MMPs. Furthermore, those in vitro studies have also shown that in the presence of the estrogenic ligand 17-β estradiol, the enhancement of rabbit MMP-13 promoter activity by ER is depressed (10.11). Thus, it would appear that the receptors for estrogen have potential to exhibit a modulatory role on MMP-13 expression, and it is governed in part by the presence and absence of the hormone estrogen.

Studies in the progression of atherosclerosis lesions in the abdominal aorta led to the discovery of two polymorphic variants in the promoter of the MMP-13 gene [START_REF] Yoon | MMP13 promoter polymorphism is associated with atherosclerosis in the abdominal aorta of young black males[END_REF].

Sequence analysis of the MMP-13 promoter revealed an insertion of an adenine residue at position -291 leading to a stretch of 12A residues in one variant form. Alternately, in the other MMP-13 allele there is an absence of the adenine residue at -nt 291 leading to a stretch of only 11 adenine residues. Furthermore, an A to G transition at nt -77 changes the PEA-3 transcriptional regulatory site from AGGAAG to GGGAAG, thus leading to the formation of another polymorphic mutation. Increased development of atherosclerosis lesions in the young black males have been attributed, in part, to the polymorphisms observed in the MMP-13 promoter [START_REF] Yoon | MMP13 promoter polymorphism is associated with atherosclerosis in the abdominal aorta of young black males[END_REF]. Thus, far it is not known whether these MMP-13 polymorphic promoter variants are influenced differently by the alpha and beta isoforms of ER in vitro.

Some sequence variations in the promoters of other members of the metalloproteinase family have also been detected. In particular, the 1G and 2 G variants are two well characterized polymorphic variations in the promoter for MMP-1 [START_REF] Rutter | A single nucleotide polymorphism in the matrix metalloproteinase-1 promoter creates an Ets binding site and augments transcription[END_REF]. The insertion of a guanine residue at position -1607 leads to the formation of an Ets transcription factor binding site which along with the AP-1 site at nt -1602 leads to increased transcription of the 2G variant [START_REF] Rutter | Cell-type specific regulation of human interstitial collagenase-1 gene expression by interleukin-1 beta (IL-1 beta) in human fibroblasts and BC-8701 breast cancer cells[END_REF][START_REF] Rutter | A single nucleotide polymorphism in the matrix metalloproteinase-1 promoter creates an Ets binding site and augments transcription[END_REF]. In contrast, the 1G variant lacks this insertion of a guanine residue in its promoter and consequently is reported to exhibit lower basal transcriptional levels [START_REF] Rutter | Cell-type specific regulation of human interstitial collagenase-1 gene expression by interleukin-1 beta (IL-1 beta) in human fibroblasts and BC-8701 breast cancer cells[END_REF][START_REF] Rutter | A single nucleotide polymorphism in the matrix metalloproteinase-1 promoter creates an Ets binding site and augments transcription[END_REF]. It is reported that MMP-1 is a proteinase produced by multiple cells in the joint inclusive of synovial cells, and it is believed to influence the onset and progression of osteoarthritis by promoting degradation of collagen fibrils [START_REF] Burrage | Matrix metalloproteinases: role in arthritis[END_REF]. Besides arthritis, MMPs are also postulated to play a role in the metastasis of the tumors. Previous studies have indicated that the hormone estrogen has an inhibitory effect on the activities of MMPs in breast cancer cells [START_REF] Philips | Inhibition of transforming growth factor-beta and matrix-metalloproteinases by estrogen and prolactin in breast cancer cells[END_REF].

Interestingly, ovariectomy in rats leads to a marked elevation in the expression of the rodent form of MMP-13 in bone [START_REF] Li | Effects of 17 beta-estradiol on the expression of interstitial collagenases-8 and -13 (MMP-8 and MMP-13) and tissue inhibitor of metalloproteinase-1 (TIMP-1) in ovariectomized rat osteoblastic cells[END_REF].

Joint tissues are recognized as estrogen responsive tissues as several studies have identified estrogen receptors in several connective tissues [START_REF] Sciore | Identification of sex hormone receptors in human and rabbit ligaments of the knee by reverse transcription-polymerase chain reaction: evidence that receptors are present in tissue from both male and female subjects[END_REF][START_REF] Liu | Estrogen affects the cellular metabolism of the anterior cruciate ligament. A potential explanation for female athletic injury[END_REF]. Different tissues express different levels of ER-α and ER-β, and furthermore these isoforms are influenced differently by estrogen agonists and antagonists. Therefore, the aim of the present study was to understand the influence of ER-α and ER-β on several of the known polymorphic variants of the human MMP-13 and MMP-1 promoters, and to decipher the potential role of estrogen in such modulation. Polymorphisms found in the MMP promoters and their response to the changes in hormonal levels may provide a partial explanation for why only subsets of women develop osteoarthritis or osteoporosis with different clinical phenotypes following menopause.

Materials and Methods

Cell culture ---The rabbit synoviocyte cell line HIG-82 was obtained from the American Type Culture Collection (Rockville, Md). Cells were maintained at 37C under 5% CO2 in Ham's F-12 Nutrient Media (Life technologies) supplemented with 10% fetal calf serum (Invitrogen Canada Inc.) and 1% Penicillin/Streptomycin. Cells were sub-cultured 1:4 after trypsinization with 0.25% trypsin. This cell line lacks endogenous ER and has been used in previous studies [START_REF] Lu | Estrogen receptor alpha regulates matrix metalloproteinase-13 promoter activity primarily through the AP-1 transcriptional regulatory site[END_REF][START_REF] Lu | Estrogen receptor beta +/-ligands can modulate MMP-13 promoter activity by HIG-82 cells in vitro: Evidence that ER-beta enhancement of activity can be modulated by estrogen and involves specific sites on the promoter[END_REF].

A C C E P T E D M A N U S C R I P T

Isolation and cloning of the human MMP-13 promoters --

The MMP-13 promoter construct (12 AA) was isolated from the total DNA obtained from human HL-60 cells by amplification with PCR using specific primers generated from published sequences [START_REF] Tardif | Cloning, sequencing and characterization of the 5′-flanking region of the human collagenase-3 gene[END_REF].

This was labeled as the primary PCR reaction. The sequences of the primers for the human MMP-13 gene used in this primary PCR reaction were:

Forward 5'-CTTCTGCAGGGGACCAAAGATG-3' (nt -1174 to -1153)

Reverse 5'-CTCAAGAAGAGGAAGGCAGCCA-3' (nt 60 to 39)

The secondary or nested PCR reaction used 1ul of the primary PCR reaction as a template. The sequences of the secondary primers were:

Forward 5'-CTGAGAATGGAAAGGGTGGAGGTC-3' (nt -939 to -916)

Reverse 5'-TCTTGAATGGTGATGCCTGGG-3' (nt 23 to 3)

The 939 bp PCR fragment obtained from the secondary reaction was cloned directly into the PCR 2.1-TOPO vector (Invitrogen Inc.). Following this, the 939 bp fragment was released from the PCR 2.1-TOPO vector by digestion with KpnI / XhoI, and subcloned into the KpnI/XhoI site of the promoterless pGL2 Basic Luciferase reporter vector. The resulting plasmids were transformed into One shot E. coli and purified on the QI-A spin Miniprep Kit ( Qiagen, Mississauga, ON), according to the manufacturer's instructions and subjected to sequencing analysis to verify the orientation and accuracy of each construct. Two polymorphic MMP-13 constructs were identified, one which had a stretch of 12 adenine residues and one with a stretch of 11 adenine residues. The PEA-3 site began with an adenine residue -AGGAAG in these constructs and hence they were given the label of 12AA and 11AA. Two more polymorphic constructs 12AG and 11AG

were generated with a guanine residue at the PEA-3 site (GGGAAG) replacing the adenine residue. This was done using the Quick Change Site-Directed Mutagenesis kit (Stratagene Inc).

Human MMP-1 promoter constructs:

The human 1G and 2G MMP-1 promoter constructs were a generous gift from Dr. C.E Brinckerhoff (Dartmouth Medical School, NH, USA). Both the 1G and 2G constructs were subcloned into the pGL-3 luciferase expression vector. The deletion fragments -1458 bp, -572 bp, -143 bp and -73 bp were generated by RT-PCR using a 5' forward primer containing the MMP-1 sequence (underlined) along with a MluI site (ACGCGT).

-1458 5'CGACGCGTCCATAAGGTGCTGAAGTG3' (nt-1458 to -1441)

-572 5'CGACGCGTATGGCCTGGCTCTGAGTAA-3' (nt -572 to -555)

-143 5'CGACGCGTCTGCTAGGAGTCACCAT-3' (nt -143 to -126)

-72 5'CGACGCGTTGAGTCAGACACCTCTGG-3' (nt -72 to -55)

The reverse primer with the Hind III restriction enzyme site was common for all the RT-PCRs.

5' CCAAGCTTCAGTGCAAGGTAAGTG -3' (nt -51 to -36)

The PCR products were purified and digested with MluI/HindIII. The final products were then ligated into MluI/HindIII digested pGL3 basic Vector.

The PEA-3 site consensus sequence AGGAAG at nt -1385 was altered to AGCGAG using the Quick Change Site-Directed Mutagenetic kit (Stratagene, La Jolla, CA USA)

and the nt -1458 construct. The mutation was verified by sequencing. The orientation and integrity of all reporter constructs were assessed by DNA sequencing.

Additional Control and Expression Plasmids --

The internal control plasmid for the dual luciferase assays, pRLSV40, which constitutively expresses the renilla form of luciferase under the strong SV40 promoter, was obtained from Promega (Madison, WI).

The expression vectors for ER-α (pKCR2-ERα) and ER-β (pKCRE-ER-β) were a generous gift from Dr. Pierre Chambon (Strasbourg, France) and Dr. Koen Dechering (Organon, The Netherlands), respectively. Since the ER-α and ER-β constructs were obtained in different vectors, the inserts were cleaved from their original vectors and subcloned into the pSG5 vector. The orientation and integrity of the inserts were confirmed by sequencing.

Transient Transfection and Luciferase assay --

The rabbit synoviocyte cell line, HIG-82, was transfected using the FuGene6 Transfection reagent (Roche Molecular, Indianapolis. IN) as directed by the manufacturer and as described previously [START_REF] Kydd | The healing rabbit medial collateral ligament of the knee responds to systemically administered glucocorticoids differently than the uninjured tissues of the same joint or the uninjured MCL: a paradoxical shift in impact on specific mRNA levels and MMP-13 protein expression in injured tissues[END_REF][START_REF] Lu | Estrogen receptor alpha regulates matrix metalloproteinase-13 promoter activity primarily through the AP-1 transcriptional regulatory site[END_REF].

Previous analysis has revealed that this cell line is devoid of detectable ERs [START_REF] Lu | Estrogen receptor alpha regulates matrix metalloproteinase-13 promoter activity primarily through the AP-1 transcriptional regulatory site[END_REF][START_REF] Lu | Estrogen receptor beta +/-ligands can modulate MMP-13 promoter activity by HIG-82 cells in vitro: Evidence that ER-beta enhancement of activity can be modulated by estrogen and involves specific sites on the promoter[END_REF].

Briefly, HIG-82 cells were seeded on 12-well plates on the day before transfection at a density of 1X10 5 cells/well. The cells were washed with PBS prior to transfection and then transfected with a constant amount (1µg) of reporter plasmids (shown to be optimal in preliminary studies; data not shown), pRLSV40 (50 ng) and pSG5 vector alone (0. Twenty four hours after transfection and (+/-) ligand treatment, the medium was removed from the cells and the cells were washed once with phosphate buffered saline (PBS).

The cells were then lysed directly in the wells with 1X Passive Lysis Buffer (Promega Corp.). Luciferase activity was determined on cell lysates using a Turner TD-20

illuminometer and the Dual Luciferase TM Reporter Assay kit (Promega Madison, WI). All transfection experiments were performed in triplicate and repeated three or more times with results very similar to those reported obtained.

Western Blot Analysis:

The HIG-82 cells were either left un-transfected (Control) or were transiently transfected with ER-α (pSG5-ERα) or ER-β (pSG5-ERβ) for 24 hr. At that time, the cells were washed and then lysed in Mammalian Cell Lysis Kit buffer (Sigma Chemical Co). The cellular extracts from control and transfected cells were separated on a 10% SDS-PAGE gel and then transferred to a nitrocellulose membrane.

Western blotting was performed using a mouse monoclonal to ERβ (ab1103) (obtained from Ab Cam. Inc.) or a rabbit polyclonal antibody for ER-α: ER-α (HC-20) ( Santa Cruz Inc.).

Statistical Analysis:

Statistical analysis of the data was performed using ANOVA, STDEVA, STEYX in the Excel 5.0 software.

Results

Influence of increasing concentrations of ER-β and ER-α on MMP-1 promoter variant activity.

The effect of increasing concentrations of ER-β and ER-α on MMP-1 promoter activity was examined in HIG-82 cells. Two well characterized MMP-1 promoter variants 1G and 2G were used in this study. HIG-82 cells were transiently co-transfected with increasing concentrations (0.5, 1 and 2 μg) of pSG5ER-β or pSG5ER-α DNA, simultaneously with either 1G or 2G constructs (1.0 μg) and the internal control plasmid, pRL-SV40 (0.05 μg). After 24 hours, the luciferase activity of the cell lysates was determined. In the absence of exogenous ER-α or ER-β, 1G and 2G activity were both detectable in the HIG-82 cells. Furthermore, the 2G variant consistently exhibited an ~ 2.5 fold higher activity than the 1G variant. The results presented in Figure 1A suggest that co- Western blot analysis conducted on transfected and un-transfected cells indicated that un-transfected HIG-82 cells did not exhibit the presence of either ER-α or ER-β proteins (Figure 1C, Lane 1), and furthermore the cells transfected with pSG5-ERβ or pSG5-ERα for 24 hrs exhibited the presence of these proteins at nearly identical levels (Figure 1C, Lanes 2 and 3). These results support the conclusion that the differences in activity levels detected above were not due to differences in the translation of the ER constructs.

transfection

Influence of 17-β estradiol on the modulating effects of ER isoforms on the activity of MMP-1 promoter variants.

Next, the effect of the estrogenic ligand, 17-β estradiol was investigated on the ER-α and ER-β mediated augmentation of 1G and 2G variant activities (see Figure 2A). once again did not exert any detectable impact on the activity of the 2G construct. These findings support the concept that effects of estrogen modulation on the ER isoforms may be different, and additionally these also influence the polymorphic variants differently.

Functional characterization of the promoter elements of the MMP-1 promoter involved in mediating the influences of ER-α & ER-β (+/-) ligands.

In order to characterize the promoter elements of the human MMP-1 promoter for their potential role in mediating the effects of the ER isoforms, a series of 5' deletion constructs, ranging in length from 1458 bp to 73 bp of the MMP-1 promoter were generated (Fig. 3 panel A). The 1458 bp MMP-1 construct lacks the important Ets and AP-1 binding sites present at position -1607 and -1602, respectively. Previous studies have shown these sites to be of importance in the activity of MMP-1 promoter (13).

However, this construct still has several other transcriptional regulatory sites present such as the TATA box at -30 bp and AP-1 sites at -70, -181, -429 and -556 nt, and the PEA-3 sites at -88 and -1385 nt. which are proximal to the transcriptional start site.

These constructs were used in co-transfection studies in HIG-82 cells along with ER-α or ER-β and the pRL-SV40, followed by luciferase assays to determine activity. The results presented in Figure 3B demonstrate that the activity of the -1458 construct was 75% lower than that of the intact 1G promoter in the presence of ER-α. In the presence of ERβ, a mean 70% decrease was observed in the activity of the -1458 construct when compared to the intact 1G construct (see Figure 3C). Interestingly, the mean percent decreases in activity observed for the -1458 construct with ER-α and ER-β were not significantly different. These results suggest that the AP-1 site present at position -1602 plays a primary role in elevating the activity of the MMP-1 promoter in response to ER-α and ER-β. Additionally, the ETS binding site present at -1607 bp in the 2G construct likely also contributes to the elevation in activity observed with the ER isoforms.

Next, the 572 bp fragment of the MMP-1 promoter was examined +/-ER-α or ER-β. In contrast to the significant decreases in activity seen for the 1458 bp fragment with ER-α and ER-β, this shorter fragment of MMP-1 demonstrated an 88% increase in the activity with ER-α and a 49% increase in activity in the presence of ER-β (Figure 3B & C Bar 4). When compared to the promoter of the -1458 bp fragment, this -572 bp fragment is missing the PEA-3 site at -1385 position. These results imply that the promoter activity is regulated positively and negatively by the various transcriptional regulatory sites of MMP-1 in this cell line, and suggest a negative regulatory role for the PEA-3 site at the -1385 location. To confirm this suggestion, the PEA-3 site at -1385 in the -1458 construct was mutated to yield a construct -1458M with this PEA-3 site inactivated. As shown in Figure 3D, in the presence of ER-alpha the activity of the mutated -1458 construct was actually significantly elevated compared to intact 1G. In contrast, in the presence of ER-beta, the activity was partially restored compared to that with the 1G construct but the activity with the mutant was still less than that with the 1G variant.

Furthermore, a MMP-1 promoter fragment which is only -143 bp in length, was also assessed. A ~68% increase in the promoter activity as compared to intact 1G promoter activity was observed with this construct when ER-α was the modulator.

Alternately, when ER-β was the modulator, the activity of this -143 bp MMP-1 construct decreased moderately (~5%) with respect to the intact 1G promoter construct. While overall both ER isoforms elevated the activity of MMP-1 promoter, subtle differences in the response of these transcriptional regulatory sites of the MMP-1 promoter to these ER isoforms were detected. These observations are further corroborated by the response of the -73 bp fragment of the MMP-1 promoter to the ER isoforms. The -70 bp construct containing only the AP-1 site was tested along with ER-α and a mean 24% decrease in the activity was observed compared to the intact 1G variant. When ER-β was the modulator, the -73 bp construct exhibited a mean 68% decrease in activity when compared to the 1G promoter (Figure 3 B & C, Bar 5).

Comparison of the effects of ER-α and ER-β on the human MMP-13 polymorphic promoter variants.

In this study, polymorphic promoter variants of a second matrix metalloproteinase, MMP-13, were also analyzed. As shown in Figure 4B, no activity was observed for all four human MMP-13 constructs in the absence of either ER-α or ER-β. Both pSG5-ERα and pSG5-ERβ induced expression of the human MMP-13 polymorphic variants. Interestingly, the induction of 11AA and 11AG MMP-13 promoter activity by the ER-β construct was consistently approximately 4.7 times higher than that observed for an equivalent amount of the ER-α construct (Figure 4B). The polymorphic variants 12 AA and 12 AG showed an elevation of approximately ~7.5 fold in activity in the presence of ER-β when compared to the influence of ER-α on these constructs. Furthermore, the activity of 12AA was 1.6 fold higher than that of 11AA suggesting that the presence of an extra adenine residue exerted a positive influence in elevating the activity of the 12AA

A C C E P T E D M A N U S C R I P T

construct. Similarly, a 1.6 fold elevation in activity was observed when comparisons were made between the 11AG and 12AG constructs (Figure 4B). These results suggest that ER-β is a stronger modulator than ER-α of the human MMP-13 DNA constructs. The number (11 or 12) of adenine residues also appear to have an impact on the response of these constructs to ER-B with constructs with 12 adenine residues showing higher activity levels. Interestingly, this impact was observed only with ER-β and not with ER-α.

Polymorphisms in the human MMP-13 promoter lead to differential responses to ER isoforms (+/-17-β estradiol).

In this aspect of the studies, polymorphic variants of the MMP-13 promoter were also analyzed for their response to the alpha and beta isoforms of ER in the presence and absence of the estrogenic ligand 17-β estradiol. The HIG-82 cells were cotransfected with the human MMP-13 polymorphic variants along with ER-α or ER-β and the internal control plasmid, pRL-SV40 in the presence and absence of 17-β estradiol.

After 24 hours, luciferase assays were used to analyze the cell lysates.

The results presented in Figure 5A show the influence of ER-α on the human MMP-13 constructs. The activity exhibited by the 11AA MMP-13 promoter was used as the positive control and values obtained in the presence of ER-α were set to 100%. The activity of all of the human MMP-13 variants were high in the presence of ER-α.

Interestingly, the activity of the human MMP-13 variants remained unaffected in the presence of estrogenic ligand, when ER-α was the modulator (Figure 5A).

Similar analyses with ER-β are shown in Figure 5B. The activities exhibited by the human MMP-13 promoter variants were again significantly increased by the addition of ER-β. Furthermore, all of the human MMP-13 constructs displayed an ~ 80% decrease in their activity in the presence of 17-β estradiol when ER-β was the modulator. Thus, some ER isoform-specific influences on the MMP-13 promoter activity may be operative in this system.

Discussion

The results of the present study suggest that genetic polymorphisms in the promoter of the human MMP-1 and MMP-13 genes can influence the response to the modulating influences of the ER isoforms. The activities of two well characterized polymorphic promoter variants of the MMP-1 gene, 1G and 2G were found to increase with increasing concentrations of ER-α and ER-β. Furthermore, the activity of the 2G variant remained consistently higher than that of 1G in the absence or presence of ER isoforms. Previous studies have indicated that the presence of the Ets binding site led to

A C C E P T E D M A N U S C R I P T

increased transcription of the 2G variant in both normal fibroblasts and cancer cells [START_REF] Rutter | Cell-type specific regulation of human interstitial collagenase-1 gene expression by interleukin-1 beta (IL-1 beta) in human fibroblasts and BC-8701 breast cancer cells[END_REF][START_REF] Kanamori | Correlation between expression of the matrix metalloproteinase-1 gene in ovarian cancers and an insertion/deletion polymorphism in its promoter region[END_REF][START_REF] Chambers | Changing views of the role of matrix metalloproteinases in metastasis[END_REF]. Additionally, the proportional increase in activity for 2G was less than the increase in activity for the 1G variant at different concentrations of ER-β (see Figure 1).

Moreover, the ratio of activity of 2G vs. 1G also decreased with increasing concentrations of ER-β.

When the estrogenic ligand, 17-β estradiol was present at saturating levels along with ER-β, a suppression of 1G and 2G promoter activity was observed. Interestingly, repression of 1G activity when 17-β estradiol was present was insignificant (p > 0.05)

when ER-α was the modulator. In contrast, the activity of the 2G variant was repressed by the ligand when ER-α was the modulator but not to the same extent as was observed when ER-β was the modulator. These results indicate that ER-β is likely a stronger modulator of MMP-1 variants than is ER-α. Several independent reports suggest that the two isoforms of the estrogen receptor have differential influences on different MMPs.

Previous studies on rabbit MMP-13 regulation have also demonstrated ER-β to be a stronger modulator of this promoter than ER-α [START_REF] Lu | Estrogen receptor beta +/-ligands can modulate MMP-13 promoter activity by HIG-82 cells in vitro: Evidence that ER-beta enhancement of activity can be modulated by estrogen and involves specific sites on the promoter[END_REF].

Abberant overexpression of MMP-1 protein has been correlated with the degradation of stromal layers leading to metastasis of primary tumor in cancer and also in progression of endometriosis [START_REF] Chambers | Changing views of the role of matrix metalloproteinases in metastasis[END_REF][START_REF] Gottschalk | Matrix metalloproteinases and TACE play a role in the pathogenesis of endometriosis[END_REF]. One study has reported that MMP-1 and ER-β expression are elevated, but ER-α expression is down-regulated in the endometriotic lesions, suggesting that estrogenic effects on MMP-1 expression are modulated primarily through ER-β [START_REF] Hudelist | Estrogen receptor beta and matrix metalloproteinase 1 are coexpressed in uterine endometrium and endometriotic lesions of patients with endometriosis[END_REF]. Interestingly, bimodal effects of estrogen have been observed in a study on human retinal pigment epithelium. A very high level of 17-β estradiol (10 -8 M) was found to suppress ER and MMP-2 expression whereas opposite effects were observed on both the proteins at a more physiological concentration of 10 -10 M (26).

Several studies suggest that the synergistic interaction between the transcription factors binding to the closely spaced ETS and AP-1 transcription regulatory sites are essential for the basal activation of the 2G variant of MMP-1 [START_REF] Westermarck | Differential regulation of interstitial collagenase (MMP-1) gene expression by ETS transcription factors[END_REF][START_REF] Westermarck | Regulation of matrix metalloproteinase expression in tumor invasion[END_REF]. In general, Ets binding proteins have been shown to bind to both c-fos and c-jun proteins, proteins which also bind to AP-1 sites (29), and furthermore it has been reported that Ets-1 and c-jun are the likely candidates to interact and influence the MMP-1 promoter activity [START_REF] Rutter | A single nucleotide polymorphism in the matrix metalloproteinase-1 promoter creates an Ets binding site and augments transcription[END_REF].

The present study also demonstrated that the Ets and AP-1 site at locus -1602 and -1607 to be one of the primary regulatory sites involved in the regulation of the MMP-1 activity in response to ER isoforms. Although, MMP-1 has several proximal promoter elements, these two distal transcriptional regulatory elements appear to play an important role in the stimulation of MMP-1 promoter activity. In addition, a separate study which focused on the role of reactive oxygen species on the elevation of MMP-1 promoter activity also established that site-directed mutations of these Ets and AP-1 sites at nt -1607 and -1602 led to significant decreases in the activity of the MMP-1 promoter [START_REF] Nelson | Early post-traumatic osteoarthritis-like changes in human articular cartilage following rupture of the anterior cruciate ligament[END_REF].

Deletion of the (-1607 bp) Ets and (-1602 bp) AP-1 sites in the -1458 construct led to a significant reduction in the activity of MMP-1 (see Figure 3), findings consistent with the results of Nelson et al. [START_REF] Nelson | Early post-traumatic osteoarthritis-like changes in human articular cartilage following rupture of the anterior cruciate ligament[END_REF]. The presence of multiple positive and negative cisacting transcriptional regulatory elements have been reported in the promoter of the MMP-1 gene [START_REF] Rutter | Cell-type specific regulation of human interstitial collagenase-1 gene expression by interleukin-1 beta (IL-1 beta) in human fibroblasts and BC-8701 breast cancer cells[END_REF]. Moreover the same study also reported the presence of several putative silencer elements located between -572 bp and -1772 bp in the MMP-1

promoter. Additionally, a number of silencer/antisilencer motifs have been described in the region between -1653 bp and -2672 bp of the MMP-1 gene and it is suggested that the anti-silencer motifs override regulation by the silencer motifs, similar to that seen in the regulation of the vimentin gene [START_REF] Rutter | Cell-type specific regulation of human interstitial collagenase-1 gene expression by interleukin-1 beta (IL-1 beta) in human fibroblasts and BC-8701 breast cancer cells[END_REF][START_REF] Stover | A negative regulatory factor is missing in a human metastatic breast cancer cell line[END_REF]. Sequence analysis of the -1458 bp construct used in the present study revealed the presence of one silencer element and no antisilencer elements. This may provide an explanation for the observed decrease in the transcriptional activity of this construct in the presence of ER-α and ER-β. It has also been suggested that more than one silencer element may be required for the repression of the MMP-1 gene (13), a possibility which may further explain the partial (~70%) depression observed in the activity of the -1458 bp construct compared to the intact 1G construct. Mutation of the PEA-3 site at the -1385 bp position led to increases in the activity of the -p1485M construct in the presence of ER-α and ER-β. These results support the conclusion that the intact PEA-3 site at the -1385 bp position has a negative regulatory role on the activity of the 1G construct. Furthermore, as the elevation in activity with -p1458M was less in the presence of ER-beta than in the presence of ER-α, there may be some ER isoform-specific influences on the impact of this negative regulatory site.

Interestingly, elevations in the activity of MMP-1 promoter activity compared to the 1G construct were observed when shorter constructs (-572 and -143 nt) of the promoter were used. The activity of these two constructs were elevated to somewhat similar levels (p = 0.5 when the activity of the -572 bp construct was compared to the activity of the -143 bp construct) when ER-α was the modulator. However, when ER-β was the modulator, the activity of the -572 bp fragment was elevated more than that observed for the -143 bp construct. Thus, these findings in the present study suggest that ER-α and ER-β may use different regions of the MMP-1 promoter to mediate their influence. Furthermore, based on the studies discussed above, both of these short constructs lack the putative silencer elements which might contribute to their higher activity levels in the presence of ER-α and ER-β. In contrast to the elevated activity levels observed for the -572 bp promoter construct of MMP-1, the activity of the -73 bp fragment of the MMP-1 promoter demonstrated decreases in activity of ~24% and ~68%

with ER-α or ER-β, respectively. Although the -73 bp construct lacks the putative negative regulatory element, the present findings are in agreement with the conclusions of others [START_REF] Angel | Specific members of the Jun protein family regulate collagenase expression in response to various extracellular stimuli[END_REF][START_REF] Doyle | Transcriptional induction of collagenase-1 in differentiated monocyte-like (U937) cells is regulated by AP-1 and an upstream C/EBPbeta site[END_REF], which indicated that the presence of only the proximal AP-1 element (at -73 bp) is not sufficient to confer maximal transcriptional activation of the MMP-1

promoter. Previous studies have reported that phorbol 12-myristate 13-acetate (PMA)mediated transcriptional elevation of MMP-1 requires the proximal AP-1 site, as well as the distal transcriptional regulatory sites [START_REF] Doyle | Transcriptional induction of collagenase-1 in differentiated monocyte-like (U937) cells is regulated by AP-1 and an upstream C/EBPbeta site[END_REF]. Moreover, studies with the rabbit collagenase-1(MMP-1) promoter have indicated that mutation of the AP-1 site at the -77 bp position led to a reduction in the basal transcription of MMP-1 [START_REF] White | Two activator protein-1 elements in the matrix metalloproteinase-1 promoter have different effects on transcription and bind Jun D, c-Fos, and Fra-2[END_REF].

Previous studies have indicated that remodeling of connective tissues occurs in human atherosclerotic lesions and MMPs have been implicated in this process [START_REF] Henney | Localization of stromelysin gene expression in atherosclerotic plaques by in situ hybridization[END_REF][START_REF] Sukhova | Evidence for increased collagenolysis by interstitial collagenases-1 and -3 in vulnerable human atheromatous plaques[END_REF][START_REF] Yoon | MMP13 promoter polymorphism is associated with atherosclerosis in the abdominal aorta of young black males[END_REF]. Furthermore, genetic polymorphisms in the MMP-13 promoter have been linked to increased fibrous plaque observed in the abdominal aorta of young black males [START_REF] Yoon | MMP13 promoter polymorphism is associated with atherosclerosis in the abdominal aorta of young black males[END_REF].

The present study also evaluated the response of MMP-13 polymorphic mutants to the modulatory influences of the ER-α and ER-β. The presence of ER-α elevated the activity of MMP-1 promoter variants (12AA, 12AG, 11AA and 11AG) and addition of the estrogenic ligand, 17-β estradiol, at saturating concentrations (10 -8 M) did not influence the, MMP-13 activity. However, when ER-β was the modulator, the human MMP-13

promoter variants did show an increase in the absence of estrogenic ligand and a decrease in the presence of 10 -8 M 17-β estradiol. Similar to the observations with the human MMP-13 promoter variants, previous studies have demonstrated an elevation in the rabbit MMP-13 promoter with both ER-α and ER-β. In contrast to the human MMP-13 promoter variants, these elevations in rabbit MMP-13 promoter activity by both ER isoforms is subject to repression in the presence of 10 -8 M estradiol [START_REF] Lu | Estrogen receptor alpha regulates matrix metalloproteinase-13 promoter activity primarily through the AP-1 transcriptional regulatory site[END_REF][START_REF] Lu | Estrogen receptor beta +/-ligands can modulate MMP-13 promoter activity by HIG-82 cells in vitro: Evidence that ER-beta enhancement of activity can be modulated by estrogen and involves specific sites on the promoter[END_REF].

Interestingly, previous studies have reported a higher sequence homology between the rabbit and human MMP-13 promoter than between human and mouse MMP-13 promoters (37). However, the differences observed in the regulation of the rabbit and human MMP-13 genes by ER-α may be attributed in part, to cell-specific and species specific behavior of ER isoforms on the regulation of this gene.

It is well known that binding of steroid hormones to their specific intracellular receptors can modulate gene expression within hormone-responsive tissues. The results from the present studies suggest that MMP-1 and MMP-13 promoter variants are influenced differently by ER-α and ER-β depending upon the presence or absence of the hormone estrogen. These findings may have relevance to the functioning of knee

Figure 4: Comparison of the influence of ER-α and ER-β on the activity of polymorphic variants of the MMP-13 promoter.

A. Schematic representation of the polymorphic variations present in the promoter of human MMP-13 DNA. The construct 11AA has a stretch of 11 adenine residues. The PEA-3 site (AGGAAG) in this construct starts with an A residue. The polymorphic variant 11AG has the same stretch of 11 adenine bases but the PEA-3 site starts with a Guanine residue (GGGAAG). Both the constructs 12AA and 12AG have a stretch of 12 adenine residues but differ in the presence of adenine or guanine residues at the PEA-3 site as indicated in the figure above. All four constructs contain all the known key elements of rabbit MMP-13 promoter region such as Runx domain (RD), p53, PEA-3 and AP-1 binding sites. All the promoter fragments were cloned into the vector, pGL2-Basic, which contains the promoterless luciferase gene. B. HIG-82 cells were transiently co-transfected with 0.5 µg of pSG5-ERβ or pSG5-ERα along with the human MMP-13 DNA constructs (11AA, 11AG, 12AA and 12AG), promoter (1 µg) and the internal control plasmid, pRL-SV40 (0.05 µg). Luciferase activity was expressed as the ratio of firefly luciferase activity and the Renilla luciferase reporter activity and values represent the mean +/-S.E. for three determinations. The activity of 11AA transfected with pSG5ER-α was set at 100%. The activity of the rest of the human MMP-13 constructs (+/-ER-α or ERβ) was expressed as percentage of the activity of 11AA (+ ER-α). ♣ P > 0.05 Not Significant

1 - 2

 12 μg), pSG5-ERβ (0.5 µg -4 μg) or pSG5-ERα (0.5 µg) by means of the Fugene 6 Transfection Reagent. Transfection was performed in Ham's F-12 Nutrient Medium in the absence of serum. Immediately, after transfection, 17-ß-estradiol (E2) (Sigma, Oakville, ON) was added to the medium in appropriate wells of specific experiments.

  with 0.5 μg of ER-β had a significant impact on activity levels of both the 1G and 2G MMP-1 promoter constructs. The activity of the 1G variant in the presence of ER-β was elevated approximately ~4 fold with respect to 1G activity in the absence of ER-β. The activity of the 2G variant in the presence of 0.5 μg of ER-β was elevated approximately ~3.7 fold when compared to the 2G activity in the absence of exogenously added ER-β. The activity of the 1G construct increased ~7.6 fold and ~13 fold when compared to 1G activity (-ER-β) in the presence of 1 and 2 μg of ER-β respectively. Similarly, the 2G variant also exhibited a ~4.6 fold and ~7.3 increase in activity in the presence of 1 and 2 μg of ER-β respectively. Although the activity of the 2G variant was always higher than that of the 1G variant in the absence or presence of ER-β, the proportional increase in activity for 2G was consistently less than the increase in activity for 1G at different concentrations of ER-β. In addition, the ratio of activity of 2G vs. 1G also decreased with increasing concentrations of ER-β.The results presented in Figure1B, show evidence of the effect of the alpha isoform of ER on the MMP-1 polymorphic constructs. The activity of the 1G variant increased 2.2 fold, 2.6 fold and 3.6 fold with concentrations of 0.5, 1.0 and 2 μg of pSG5ER-α DNA, respectively. The activity of the 2G variant showed a modest ~1.2 fold increase at 0.5 and 1 μg of ER-α DNA and detected activity increased approximately 2fold with 2 μg of ER-β DNA when compared to 2G activity in the absence of ER-β.However, the 2G variant always exhibited higher activity as compared to the 1G variant in the absence or presence of the ER-α construct. These results indicate a strong influence of ER-α and ER-β on the activity of MMP-1 promoter variants in this in vitro model system.

  HIG-82 cells were co-transfected with 1μg of pSG5ER-α or pSG5ER-β and the 1G variant along with 0.05 μg of pRL-SV40 DNA. The activity of the 1G variant was found to be elevated approximately 2-fold in the presence of ER-α and ~3 fold in the presence of ER-β. When 10 -8 M 17-β estradiol was added to the transfection medium, the elevation in 1G activity levels induced by ER-β was found to decrease by approximately 75%.Interestingly, the elevation in activity with the 1G variant in the presence of ER-α remained uninhibited (P > 0.05, NS) in the presence of same concentration of 17-β estradiol (Figure2A, lane 3). When the ligand, 17-β estradiol, was used at a concentration of 10-12 M with ER-α or ER-β, no notable decreases in the activity of the 1G variant were again observed (data not shown). Figure2Bshows the response of the 2G construct with ER-α or ER-β (-/+) 17-β estradiol. The activity of the 2G variant was elevated modestly in the presence of ER-α and no decreases in activity levels were again observed in the presence of 10 -12 M 17-β estradiol (data not shown). However, the activity of the 2G variant decreased by ~30% when saturating amounts of 17-β estradiol (10 -8 M) were used (see Figure2B lane 3). The activity of the 2G variant was elevated approximately 3-fold when ER-β alone was present, and this elevation was decreased ~70% in the presence of 10 -8 M 17-β estradiol. At a concentration of 10-12 M, the ligand

  Figure 4A illustrates the four polymorphic constructs of the human MMP-13 promoter used in this study. The variant 12AA contains a stretch of 12 A residues and the PEA-3 site has the consensus sequence AGGAAG. The 12 AG variant contains stretch of 12 A residues, but the PEA-3 site begins with a G residue. The 11AA and 11 AG constructs contain the stretch of 11 A residues and the PEA-3 site has either an A or a G residue, respectively. The HIG-82 cells were co-transfected with the individual human MMP-13 promoter variants, along with ER-α or ER-β and the internal control plasmid, pRL-SV40. After 24 hours, luciferase assays were used to analyze the cell lysates.

Figure 2 :

 2 Figure 2: Influence of ligand, 17-β estradiol on the modulating effects of ER-α and ER-β on MMP-1 promoter polymorphic variants. A & B.Rabbit synoviocytes (HIG-82) were transiently co-transfected with the human MMP-1 promoter constructs (1G or 2G), ER-α (pSG5ER-α) or ER-β (pSG5-ERβ), and the internal control plasmid, pRL-SV40. One set of transfected cells was treated with 10 -8 M 17-β estradiol. Cells transfected with 1G or 2G alone were used as controls in Figure2Aor 2B respectively. Luciferase activity was expressed as the ratio of 1G or 2G firefly luciferase activity and the Renilla luciferase reporter activity. Values represent the mean +/-S.E. for three determinations.

Figure 5 :

 5 Figure5: Polymorphisms in the MMP-13 promoter influence the response of the variants to the modulatory effects of ER isoforms. Rabbit Synoviocytes, HIG-82, were transiently co-transfected with either the polymorphic variants of the human MMP-13 promoter (12AA, 12AG, 11AA and 11AG along with 0.5 µg of ER-α (Figure5A) or ER-β ((Figure5B) and pRL-SV40. Cells were treated with 10 -8 M 17-β Estradiol, for a period of 24 hours post transfection. Luciferase activity was expressed as the ratio of firefly luciferase activity and the renilla luciferase reporter activity. Values represent the mean +/-S.E. for three determinations and are expressed as percentage of 11AA luciferase activity without treatment with any ligand.

Acknowledgements:

The authors thank Drs. Brinckerhoff, Chambon and Dechring for their generous gifts of constructs. These studies were supported by grants from the Institute for Gender and Health of CIHR, and The Arthritis Society. DAH is the Calgary Foundation -Grace Glaum Professor in Arthritis Research.

MMP-1 Activity (% of 1G)

A.

A C C E P T E D M A N U S C R I P T

Φ

p < 0.001 comparing the activity of 1G and 2G in the absence of exogenously added ER-β p < 0.001 comparing the activity of 1G and 2G in the presence of exogenously added ER-β B. Rabbit synoviocytes (HIG-82) were transiently co-transfected with the human MMP-1 promoter constructs (1G or 2G), ER-α (pSG5-ERα), and the internal control plasmid, pRL-SV40. Increasing concentrations of pSG5-ERα ranging from (0.5 to 2 μg) were used in this experiment. Cells transfected with 1G or 2G alone were used as controls. Luciferase activity was expressed as the ratio of 1G firefly luciferase activity and the Renilla luciferase reporter activity. Values represent the mean +/-S.E. for three determinations. † p < 0.001 as compared with 1G activity in the absence of ER-α. † p < 0.05 as compared with 2G activity in the absence of ER-α. • p > 0.05 Not Significant as compared with 2G activity in the absence of ER-α. ♣ p < 0.005 as compared with 1G activity in the absence of ER-α.

Φ

p < 0.001 comparing the activity of 1G and 2G in the absence of exogenously added ER-α p < 0.005 comparing the activity of 1G and 2G in the presence of exogenously added ER-α C. Rabbit synoviocytes (HIG-82) were either left un-transfected or were transiently transfected with either ER-α (pSG5-ERα) or ER-β (pSG5-ERβ). The protein extracts from transfected and un-transfected cells were separated on a 10% SDS-PAGE gel and then transferred onto a nitrocellulose membrane. Western blot analysis was conducted using the Mouse monoclonal to ERβ (ab1103) (obtained from Ab Cam. Inc.) and rabbit polyclonal antibody for ER-α: ER-α( HC-20) ( Santa Cruz Inc.). Lane 1 represents the un-transfected HIG-82 cells (Control); Lane 2. HIG-82 cells transfected with pSG5-ERβ. Lane 3. HIG-82 cells transfected with pSG5-ERα.


Satyakam Bhagavati
email: satyakamb@hotmail.com

S A Shafiq

Weimin Xu

whether they are published or not. The documents may come

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

  Corrigendum to "(CTG) repeats markedly inhibit differentiation of the C2C12 myoblast cell line: Implications for congenital myotonic dystrophy" [Biochim. Biophys. Acta 1453 (1999) 221-229] Satyakam Bhagavati, S.A. Shafiq, Weimin Xu To cite this version: Satyakam Bhagavati, S.A. Shafiq, Weimin Xu. Corrigendum to "(CTG) repeats markedly inhibit differentiation of the C2C12 myoblast cell line: Implications for congenital myotonic dystrophy" [Biochim. Biophys. Acta 1453 (1999) 221-229]. Biochimica et Biophysica Acta -Molecular Basis of Disease, 2008, 1782 (5), pp.362. 10.1016/j.bbadis.2008.03.005. hal-00562833 ÔØ Å ÒÙ× Ö ÔØ Corrigendum to "(CTG) n repeats markedly inhibit differentiation of the C2C12 myoblast cell line: Implications for congenital myotonic dystrophy" [Biochim. Biophys. Acta 1453 (1999) 221-229] Satyakam Bhagavati, S.A. Shafiq, Weimin Xu PII: S0925-4439(08)00077-X DOI: doi: 10.1016/j.bbadis.2008.03.005 Reference: BBADIS 62806 To appear in: BBA -Molecular Basis of Disease Please cite this article as: Satyakam Bhagavati, S.A. Shafiq, Weimin Xu, Corrigendum to "(CTG) n repeats markedly inhibit differentiation of the C2C12 myoblast cell line: Implications for congenital myotonic dystrophy" [Biochim. Biophys. Acta 1453 (1999) 221-229], BBA -Molecular Basis of Disease (2008), doi: 10.1016/j.bbadis.2008.03.005


Satyakam Bhagavati
email: satyakamb@hotmail.com

Ashwini Ghatpande

Betty Leung

]

ÔØ Å ÒÙ× Ö ÔØ

Corrigendum to "Normal levels of DM RNA and myotonin protein kinase in skeletal muscle from adult myotonic dystrophy (DM) patients" [Biochim. Biophys. Acta 1317(1996) This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.


Ariane Kretlow

Qi Wang

Michael Beekes

Dieter Naumann

PhD Lisa M Miller
email: lmiller@bnl.gov

Changes in Protein Structure and Distribution Observed at Pre-Clinical Stages of Scrapie Pathogenesis

Keywords: prion, scrapie, dorsal root ganglia, protein structure, protein-folding, infrared microspectroscopy Protein Structural Changes during Scrapie Pathogenesis Transmissible Spongiform Encephalopathy (TSE), dorsal root ganglia

Scrapie is a neurodegenerative disorder that involves the misfolding, aggregation and accumulation of the prion protein (PrP). The normal cellular PrP (PrP C ) is rich in αhelical secondary structure, whereas the disease-associated pathogenic form of the protein (PrP Sc ) has an anomalously high ß-sheet content. In this study, protein structural changes were examined in situ in the dorsal root ganglia from perorally 263K scrapieinfected and mock-infected hamsters using synchrotron Fourier Transform InfraRed Microspectroscopy (FTIRM) at four time points over the course of the disease (preclinical, 100 & 130 days post-infection (dpi); first clinical signs (~145 dpi); and terminal (~170 dpi)). Results showed clear changes in the total protein content, structure, and distribution as the disease progressed. At pre-clinical time points, the scrapie-infected animals exhibited a significant increase in protein expression, but the β-sheet protein content was significantly lower than controls. Based on these findings, we suggest that the pre-clinical stages of scrapie are characterized by an overexpression of proteins low in β-sheet content. As the disease progressed, the β-sheet content increased significantly.

Immunostaining with a PrP-specific antibody, 3F4, confirmed that this increase was partly -but not solely -due to the formation of PrP Sc in the tissue and indicated that other proteins high in β-sheet were produced, either by overexpression or misfolding. Elevated β-sheet was observed near the cell membrane at pre-clinical time points and also in the cytoplasm of infected neurons at later stages of infection. At the terminal stage of the disease, the protein expression declined significantly, likely due to degeneration and death of neurons. These dramatic changes in protein content and structure, especially at pre-clinical time points, emphasize the possibility for identifying other proteins involved in early pathogenesis, which are important for further understanding the disease.

A C C E P T E D M A N U S C R I P T INTRODUCTION

A characteristic feature of scrapie, a neurodegenerative disorder naturally occurring in sheep and goats, is the accumulation of a β-sheet rich protein termed PrP Sc , or scrapie-associated prion protein, predominantly in the brain and spinal cord. First described in 1732 in sheep, scrapie belongs to the family of Transmissible Spongiform Encephalopathies (TSEs), which can also occur in cattle (bovine spongiform encephalopathy), deer (chronic wasting disease), mink (transmissible mink encephalopathy) and other animals. The Creutzfeldt-Jakob disease in humans can occur sporadically but may also have genetic and infectious origins, which is a unique feature of this group of diseases. According to the prion hypothesis, the causative agent of TSEs is believed to be a proteinaceous infectious particle ("prion") that lacks agent-specific nucleic acid and consists mainly -if not entirely -of misfolded and pathologically aggregated prion protein [START_REF] Prusiner | Prion diseases and the BSE crisis[END_REF]. Normal PrP and its pathological isoform have an identical amino acid sequence but different secondary (and tertiary) structures. PrP C , the cellular prion protein, mainly expressed by neuronal cells, is high in α-helix (42%) and consists of only 3% β-sheet, whereas the misfolded or disease-associated form PrP Sc shows a high amount of β-sheet structure (43%) and has less α-helix (30%) [START_REF] Pan | Conversion of alpha-helices into beta-sheets features in the formation of the scrapie prion proteins[END_REF].

The nervous system of hamsters orally infected with scrapie has been studied extensively for the temporal-spatial course of PrP Sc deposition (for review see: [START_REF] Beekes | The spread of prions through the body in naturally acquired transmissible spongiform encephalopathies[END_REF]). After centripetally reaching the spinal cord, subsequent centrifugal spread of PrP Sc deposition proceeds to the corresponding afferent dorsal root ganglia (DRG, nodules on a dorsal spinal root that contain cell bodies of afferent nerve fibers), where the protein can first be detected 76 days post-infection in half of the examined hamsters [START_REF] Beekes | Cerebral targeting indicates vagal spread of infection in hamsters fed with scrapie[END_REF][START_REF] Mcbride | Early spread of scrapie from the gastrointestinal tract to the central nervous system involves autonomic fibers of the splanchnic and vagus nerves[END_REF][START_REF] Mcbride | Pathological PrP is abundant in sympathetic and sensory ganglia of hamsters fed with scrapie[END_REF]. Immunostained

A C C E P T E D M A N U S C R I P T

sections have shown that PrP Sc does not accumulate in all neurons of the DRG, where only certain cells stain strongly. In prion research, conventional techniques like Western Blot analysis are sensitive for examining the total amount of PrP Sc in homogenized tissue.

In situ, PET blotting (paraffin embedded tissue blot) [START_REF] Schulz-Schaeffer | The paraffin-embedded tissue blot detects PrP(Sc) early in the incubation time in prion diseases[END_REF] labels the misfolded prion protein directly within the tissue, providing spatially-resolved information on the location of PrP Sc . However, both techniques are limited to the detection of PrP Sc , and do not provide information on other compositional changes that occur throughout the disease, especially at early time points in pathogenesis before PrP Sc shows detectable accumulation.

In contrast, Fourier Transform Infrared Microspectroscopy (FTIRM) is an in situ technique that is not restricted to the detection of PrP Sc . In fact, an infrared spectrum of a biological sample is composed of characteristic absorption bands that originate from all tissue components, e.g. proteins, lipids, nucleic acids, and carbohydrates. Since the combination of all molecular parameters (structure, composition, and/or interactions) in a specific tissue, cell type, or subcellular component is unique, FTIRM is an in situ technique for examining the molecular composition of biological materials [START_REF] Wetzel | Imaging molecular chemistry with infrared microscopy[END_REF]. No labels, stains or dyes are required for FTIR microspectroscopy and it is a non-destructive, sensitive and fast analytical tool. Thus, coupled with PrP Sc -specific imaging techniques, FTIRM can be used to determine compositional changes in prion-infected tissue associated with scrapie pathogenesis [START_REF] Kneipp | Molecular changes of preclinical scrapie can be detected by infrared spectroscopy[END_REF][START_REF] Kneipp | Detection of pathological molecular alterations in scrapie-infected hamster brain by Fourier transform infrared (FT-IR) spectroscopy[END_REF].

In this study, protein content, structure, and distribution throughout scrapie pathogenesis were investigated using synchrotron FTIRM. Syrian hamsters (Mesocricetus auratus) perorally infected with scrapie strain 263K were studied at 4

MATERIALS AND METHODS

Animal experiments and tissue preparation

All animal experiments were carried out in accordance with European, German, and USA legal and ethical regulations. 20 outbred Syrian hamsters at an age of approximately 8 weeks were fed with 1-3 x 10 7 50% intracerebral lethal doses of scrapie strain 263K as described elsewhere [START_REF] Baldauf | Evidence for an alternative direct route of access for the scrapie agent to the brain bypassing the spinal cord[END_REF]. 12 mock-infected hamsters of the same age were similarly fed with normal brain homogenate and served as controls. Five infected and three control hamsters each were euthanized by euthanasia with CO 2 , at each of four time points: 100 dpi, 130 dpi, fcs (~145 dpi), and at the terminal stage of the disease (~170 dpi). While the first two time points were fixed dates, fcs and terminal stage depend on the progression of the disease in each individual animal. Fcs is defined as a stage where the animals start to show symptoms that are specific for a 263K scrapie infection in hamsters. These are most commonly a hypersensitivity to touch and noise,

A C C E P T E D M A N U S C R I P T

where the animals often twitch, or have difficulties in maintaining balance and rising from a supine position. At the terminal stage, animals show head bobbing, ataxia of gait, and generalized tremor.

After euthanizing the animals, the dorsal root ganglia attached to the thoracic spinal cord were removed and stored at -70 o C. After embedding the samples in Jung tissue freezing medium (Leica Instruments, Germany), 10 µm thick cryo-sections were cut at a temperature of -20°C and mounted on IR transparent slides (CaF 2 , 1 mm in thickness) (Korth Kristalle GmbH, Altenholz, Germany). Adjacent sections were cut at the same thickness and mounted on standard glass microscope slides for immunostaining.

The CaF 2 slides were kept in a dry and dark environment until the FTIR microspectroscopic measurements were carried out. One animal died before reaching the stage of fcs, resulting in a total of 19 infected and 12 mock-infected control animals included in the study.

Immunohistochemistry

Adjacent sections on glass slides were stained for PrP using the monoclonal antibody 3F4 as described previously [START_REF] Wang | In situ characterization of prion protein structure and metal accumulation in Scrapie-Infected Cells by Synchrotron Infrared and X-ray Imaging[END_REF]. In short, the slides were fixed three times: After FTIRM data collection, the ganglia on CaF 2 windows were also immunostained accordingly.

FTIRM Data collection

The FTIRM experiments were conducted at beamline U10B at the National Synchrotron Light Source, Brookhaven National Laboratory (Upton, NY). A Thermo Nicolet Magna 860 FTIR spectrometer, coupled to a Continuum FTIR microscope (ThermoNicolet, Madison, WI), was used with synchrotron light as the infrared source.

The microscope was equipped with matching 32X Schwarzschild objectives, a motorized

x-y mapping stage, an adjustable rectangular aperture, and a mercury cadmium telluride (MCT-A) detector. The aperture of the microscope was set to 10 x 10 µm. A pre-defined area of approximately 100 x 100 µm encompassing about 15 -20 cells was rasterscanned with a step size of 4 µm using Omnic software (ThermoNicolet). At each point, an absorbance spectrum was recorded in transmission mode. Each spectrum was collected in the mid-infrared spectral range (800 -4000 cm -1 ) with a spectral resolution of

Data Analysis

The FTIRM data were analyzed using Thermo Nicolet's software Omnic 7.3.

Absorbances from 1616 to 1636 cm -1 of protein infrared spectra are attributed primarily to β-sheet structures, which have been shown to increase in studies of recombinant and protease-resistant Syrian hamster PrP in vitro [START_REF] Troullier | Transient non-native secondary structures during the refolding of alpha-lactalbumin detected by infrared spectroscopy[END_REF][START_REF] Caughey | Strain-dependent differences in beta-sheet conformations of abnormal prion protein[END_REF][START_REF] Sokolowski | Formation of critical oligomers is a key event during conformational transition of recombinant syrian hamster prion protein[END_REF][START_REF] Spassov | Structural differences between TSEs strains investigated by FT-IR spectroscopy[END_REF]. Thus, to examine the relative content of β-sheet structure in the tissue, each absorbance spectrum was integrated from 1624 -1628 cm -1 , applying a linear baseline from 1480 -1715 cm -1 . To normalize for potential differences in tissue thickness among samples, the β-sheet area was divided by the area under the Amide I protein band (1600 -1700 cm -1 ), applying the same linear baseline. Accordingly, the relative α-helical content was calculated by integrating each absorbance spectrum from 1654 -1658 cm -1 and normalizing to the area under the Amide I band. For both α-helix and β-sheet content, narrow regions were integrated in order to prevent overlap with other secondary structure elements [START_REF] Fabian | Methods to study protein folding by stopped-flow FT-IR[END_REF]. Chemical images in α-helix and β-sheet content were generated using Transform (Fortner Software, USA).

To estimate the relative protein content at each pixel, the integrated area under the Amide I protein band was divided by the area under the entire infrared spectrum, in order to normalize for potential differences in tissue thickness among samples. The total integrated area was calculated as the integrated area from 2500 -3700 cm -1 (baseline from 2500 to 3700 cm -1 ) plus the integrated area from 1760 -1000 cm -1 (baseline from 1760 -1000 cm -1 ). The whole spectral region was chosen since it includes the vibrational modes of C-H, N-H and O-H bonds of all major biomolecules and therefore

A C C E P T E D M A N U S C R I P T

represents the total biomass. Thus, by dividing the total protein by the total biomass, a relative estimate of total protein content was determined.

For each animal, the α-helix, β-sheet, and total protein content were determined for every pixel in each map. These values were averaged to obtain a mean and standard deviation for each animal. Then the individual animal values were averaged at their respective time points. An unpaired, two-sided t-test (Student's t-test) was performed on all data to test for significant differences (p<0.05) between scrapie and control at each time point, and differences between time points. In the analysis, those spectra derived from areas outside the ganglion or from tissue artifacts were excluded from the calculation.

RESULTS

To examine changes in the secondary structure of proteins as a function of disease severity, the mean values ± standard deviation for β-sheet content were calculated and plotted in Figure 1A. As disease progressed, results showed that the amount of β-sheet remained relatively constant in control hamsters (gray line), but substantially increased with time in the scrapie-infected animals (black line). The most dramatic difference between the control and infected animals was observed at 100 dpi, where the β-sheet content in scrapie-infected ganglia was significantly lower than in the control ganglia (p=0.013). Over the course of the disease, we found a significant increase in β-sheet content in the infected animals of about 8% (p=0.003). In contrast, the β-sheet content in the control ganglia remained unchanged over the time course studied here.

content was unaffected by the disease (black line) or by age (control, gray line).

To determine how the changes in protein structure were related to total protein expression, the total protein content was determined as a function of disease severity (Figure 1C). At 100 dpi, the scrapie-infected animals (black line) showed a significant increase in relative protein content when compared to the control animals (p=0.020).

Moreover, the total protein content of scrapie-infected animals (black line) declined gradually during pathogenesis and reached a significantly lower level at the terminal stage of the disease (p=0.013). The control animals did not exhibit a change in total protein expression throughout the time course studied here.

In addition to measuring relative protein content and structure, the spatial distribution of β-sheet protein within the tissue was also examined. In (obtained from points in the center of the cell indicated by white asterisk in Figure 2.)

However, the area near the cell membrane (black asterisk in Figure 2) exhibited spectra with increased peak intensities at 1681 cm -1 (attributed to β-turns and loops), 1638 and 1632 cm -1 (attributed to β-sheet), and a broad shoulder at around 1648 cm -1 (attributed to unordered structures), while the intensity of the α-helical peak at around 1656 cm -1 was shown to decrease slightly [START_REF] Spassov | Structural differences between TSEs strains investigated by FT-IR spectroscopy[END_REF], indicating changes in a number of secondary structures, beyond α-helix and β-sheet.

Since the FTIRM data showed an increase in β-sheet content with disease progression, consistent with the accumulation of PrP Sc , immunostaining with the 3F4 antibody for the prion protein was performed on adjacent tissue sections (Figure 4). Due to the fact that the antibody 3F4 stains both the cellular and the misfolded form of the prion protein [START_REF] Kascsak | Mouse polyclonal and monoclonal antibody to scrapie-associated fibril proteins[END_REF], a more or less homogeneous brown background can be seen in all images, where the intensity or distribution does not significantly change between time points or infection state. However, in the infected tissue, comparative immunohistochemical and PET blot staining has shown that PrP Sc accumulates as micro disperse aggregates that present as characteristic granular deposits of immunoreactive

A C C E P T E D M A N U S C R I P T

material [START_REF] Mcbride | Early spread of scrapie from the gastrointestinal tract to the central nervous system involves autonomic fibers of the splanchnic and vagus nerves[END_REF]. Figure 4 shows a clear progression of PrP Sc deposition during pathogenesis by the increased appearance of brown, granular, immunoreactive material, indicating the presence of pathologically aggregated PrP. At 100 dpi, very few neurons and satellite cells showed such immunostaining for PrP Sc , whereas the accumulation of PrP Sc became more prominent at later stages of incubation as indicated by the increasing number of characteristic granular deposits at 130 dpi, fcs, and at the terminal stage of disease.

To directly correlate the elevated β-sheet content with the distribution of aggregated PrP Sc , the FTIRM samples were subsequently stained with the 3F4 monoclonal antibody. Figure 5 shows the results for an infected animal at 100 dpi (left column) and at first clinical signs (right column). The first row displays the unstained photomicrographs, while the second row shows the same tissue stained with the 3F4 antibody. At 100 dpi, the immunostained section shows no evidence of PrP Sc inside the neurons, but in some of the surrounding satellite cells (arrows). The third and fourth rows represent the β-sheet and α-helix distributions, respectively, as measured with FTIRM. At 100 dpi, the satellite cells that stained positive for PrP Sc also have higher β-sheet content.

Furthermore, some areas showed both elevated β-sheet and α-helical content (asterisks).

At fcs, some neurons stained positive for PrP Sc (as can be seen by granular deposits and indicated by arrows) and show a high content of β-sheet in the corresponding FTIRM map (e.g. the cell in the center of the image). In contrast, the cell shown in the upper left of the fcs photomicrograph (arrowhead) shows elevated β-sheet content but no PrP Sc staining, indicating that β-sheet rich proteins other than PrP Sc also increase during pathogenesis.

A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT

13

DISCUSSION

FTIR spectra of isolated PrP fragments (PrP 27-30 ) and protease-resistant PrP (PrP res ) from 263K scrapie infected hamster were shown to exhibit strong absorbance bands at around 1636 and 1626 cm -1 , indicating the presence of β-sheet structures [START_REF] Troullier | Transient non-native secondary structures during the refolding of alpha-lactalbumin detected by infrared spectroscopy[END_REF][START_REF] Caughey | Strain-dependent differences in beta-sheet conformations of abnormal prion protein[END_REF][START_REF] Spassov | Structural differences between TSEs strains investigated by FT-IR spectroscopy[END_REF] Furthermore, under destabilizing conditions and low pH, recombinant Syrian hamster prion protein showed dominant amide I band components at 1620 and 1691 cm -1 , which are generally assigned to intermolecularly arranged antiparallel β-sheet structures [START_REF] Sokolowski | Formation of critical oligomers is a key event during conformational transition of recombinant syrian hamster prion protein[END_REF].

Site-directed spin labeling, EPR spectroscopy, and time resolved FTIR studies revealed that the conformational conversion of PrP C into PrP Sc involves major refolding of the Cterminal α-helical region of PrP C [START_REF] Cobb | Molecular architecture of human prion protein amyloid: a parallel, in-register beta-structure[END_REF][START_REF] Ollesch | Prion protein alphato-beta transition monitored by time-resolved Fourier transform infrared spectroscopy[END_REF]. But not all α-helices get converted into βsheet, as was shown by NMR [START_REF] Watzlawik | Prion protein helix1 promotes aggregation but is not converted into beta-sheet[END_REF]. However, while FTIR spectroscopy has been able to identify structural changes in PrP in vitro and ex situ, identification of structural changes within scrapie-infected tissue has been difficult.

Recently, a FTIRM study of terminally-diseased, 263K scrapie hamsters was the first to show a higher β-sheet content and a lower α-helical content in infected DRG cells [START_REF] Kneipp | In situ identification of protein structural changes in prion-infected tissue[END_REF][START_REF] Kneipp | Scrapie-infected cells, isolated prions, and recombinant prion protein: a comparative study[END_REF]. Here, this study has been extended to pre-clinical time points, and we find significant changes in protein structure, composition, and distribution throughout the course of the disease, while age-matched control animals did not exhibit any changes over the investigated time period. Specifically, we find that elevated β-sheet content was detectable earlier in the disease, i.e. with the onset of the first clinical signs. These PrP Sc accumulation as early as 76 days post infection in the 263K scrapie hamster model [START_REF] Mcbride | Early spread of scrapie from the gastrointestinal tract to the central nervous system involves autonomic fibers of the splanchnic and vagus nerves[END_REF].

A C C E P T E D M A N U S C R I P T

Over the course of the disease, results show that the increase in β-sheet was ~8%, well above the amount attributable to PrP Sc , which was shown to represent only 0.1% of all proteins in the tissue at terminal stage [START_REF] Beekes | Western blot mapping of diseasespecific amyloid in various animal species and humans with transmissible spongiform encephalopathies using a high-yield purification method[END_REF]. Furthermore, FTIRM and corresponding immunostain images revealed that not all areas with elevated β-sheet stained positive for PrP Sc , indicating that other β-sheet rich proteins also play a role in scrapie pathogenesis.

Gene profiling of 263K scrapie infected hamster identified several upregulated genes at the terminal time point [START_REF] Riemer | Identification of upregulated genes in scrapie-infected brain tissue[END_REF][START_REF] Stobart | Differential expression of interferon responsive genes in rodent models of transmissible spongiform encephalopathy disease[END_REF]. Among those were proteins with predominantly β-sheet content, such as metallothionein [START_REF] Boulanger | Model for mammalian metallothionein structure[END_REF], apolipoprotein J [START_REF] Calero | Functional and structural properties of lipid-associated apolipoprotein J (clusterin)[END_REF], IP-10 [29, 30] and β 2microglobulin, the last of which was shown to have amyloidogenic properties [START_REF] Zhang | Identification of amyloid fibril-forming segments based on structure and residue-based statistical potential[END_REF].

However, since FTIRM spectra represent the average protein structure within the volume of tissue illuminated by the infrared light, specific identification of these proteins is beyond the scope of this study.

While an increase in β-sheet content towards the terminal stage of the disease confirmed our initially proposed hypothesis, a significantly lower amount of β-sheet protein at pre-clinical time points was an unexpected finding. Since the total protein content was significantly increased in the scrapie-infected animals at 100 dpi, we suggest that the early scrapie infection is characterized by an overexpression of proteins low in βsheet content. Second derivative analysis confirmed that most spectra showed predominantly high α-helical content; however some spectra recorded near the cell's periphery exhibited an increase in random coil and β-sheet content.

Immunohistochemistry studies have previously shown that the deposition of pathological PrP Sc starts in satellite cells and in the cytoplasm and plasmalemma of neurons [START_REF] Mcbride | Pathological PrP is abundant in sympathetic and sensory ganglia of hamsters fed with scrapie[END_REF]. PrP Sc accumulation near the cell membrane has also been observed in scrapie-infected mouse

A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT 15
neuroblastoma cells [START_REF] Caughey | The scrapie-associated form of PrP is made from a cell surface precursor that is both protease-and phospholipase-sensitive[END_REF], in cultured Chinese hamster ovary cells [START_REF] Lehmann | Mutant and infectious prion proteins display common biochemical properties in cultured cells[END_REF], in the medulla oblongata, pons, and astrocytes of sheep [START_REF] Van Keulen | Immunohistochemical detection and localization of prion protein in brain tissue of sheep with natural scrapie[END_REF], and mice with natural scrapie [START_REF] Jeffrey | Murine scrapie-infected neurons in vivo release excess prion protein into the extracellular space[END_REF][START_REF] Jeffrey | Infectionspecific prion protein (PrP) accumulates on neuronal plasmalemma in scrapieinfected mice[END_REF].

At later stages of pathogenesis, the β-sheet content increased throughout the cell, which is consistent with (but not exclusively attributed to) the appearance of PrP Sc aggregates in the cytoplasm of neurons, primarily in secondary lysosomes [START_REF] Jeffrey | Sites of prion protein accumulation in scrapie-infected mouse spleen revealed by immunoelectron microscopy[END_REF],

aggrosomes [START_REF] Cohen | Scrapie-like prion protein accumulates in aggresomes of cyclosporin A-treated cells[END_REF], and in the nucleus [START_REF] Mange | Scrapie-like prion protein is translocated to the nuclei of infected cells independently of proteasome inhibition and interacts with chromatin[END_REF]. Interestingly, accumulation of PrP in the cytosol has been shown to be neurotoxic [START_REF] Ma | Neurotoxicity and neurodegeneration when PrP accumulates in the cytosol[END_REF][START_REF] Rambold | Association of Bcl-2 with misfolded prion protein is linked to the toxic potential of cytosolic PrP[END_REF].

In summary, this study showed that pronounced protein-related changes occur at the very early stages of scrapie pathogenesis, well before clinical symptoms appear.

Moreover, these changes go well beyond the transformation of PrP C to PrP Sc . In the future, efforts to identify these specific proteins that are involved at pre-clinical time points may provide an avenue for early disease detection and possible targets for treatment of scrapie infection that are not available today. and control (white lines) hamster dorsal root ganglia at the four investigated time points.

A C C E P T E D M A N U S C R I P T A C C E P T E D M A N U S C R I P T

Asterisks mark significant differences (p<0.05) between scrapie and control.

5 min 7 Kit

 57 each in 50, 100 and 50% ethanol, and then rehydrated in distilled water. The ganglia were then treated with 10% phosphate buffered formalin for 15 min, 100 mM NH 4 Cl and 0.5% Igepal in PBS (pH 7.8) for 5 min each. Sections were denatured in 3M Guanidiniumthiocyanate/10 mM TrisHCl (pH 7.8). They were blocked overnight with fetal calf serum in TBST/0.2% Tween 20 at 4 °C, incubated with antibody 3F4 (1:100) for 1 h 45 min and then with biotinylated secondary antibody Goat-anti-Mouse (Dako E 0433) (1:200) in PBS/1%BSA. Finally the sections were incubated with Vectastain ABC A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT and stained with preincubation and incubation solution containing 3,3-Diaminobenzidin (DAB) to visualize the reaction product for 10 min and 3 min, respectively. For negative controls, ganglia slices were incubated in normal mouse serum (Dako) (1:1000) instead of mAb 3F4 prior to incubation with the secondary antibody.

  8 cm -1 and 128 scans co-added. Happ-Genzel apodization and a zero-filling of level 2 were applied, resulting in approximately 1 data point per wavenumber. A background spectrum on clean substrate was also recorded by co-adding 512 scans. For each animal, one or two infrared maps were collected, yielding an average of 10500 spectra from 138A C C E P T E DM A N U S C R I P T cells (~29 cells per animal) per time point from the infected animals and 8887 spectra from 110 cells (~37 cells per animal) per time point from the controls.

Figure 2 , 3 .

 23 photomicrographs and corresponding FTIRM images of the intermolecular β-sheet

  findings are consistent with work by McBride, et al., who used immunostaining to show

Figure 1 :

 1 Figure 1: Mean values ± average deviation for the relative (A) β-sheet protein content,

Figure 2 :

 2 Figure 2: (A) Photomicrographs of unstained cryosections (1st and 3rd column) and

Figure 3 :

 3 Figure 3: Original and second derivative spectra derived from near the cell membrane

Figure 4 :

 4 Figure 4: Photomicrographs of antibody 3F4-stained adjacent sections. PrP Sc can be

Figure 5 :

 5 Figure 5: Comparison of the results from the FTIRM of an infected animal at 100 dpi

ACKNOWLEDGEMENTS

The authors like to thank Marion Joncic and Kristin Kampf (Robert Koch-Institute) for skillful technical assistance with the animal experiments. We are also grateful to the technical and safety staff at the NSLS, especially to Randy Smith, Larry Carr, and Andrew Ackerman. M. B. is grateful for ongoing support by the EU-funded Network of Excellence "NeuroPrion". This work was funded by the National Institutes of Health Grant R01-GM66873. The National Synchrotron Light Source is funded by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences, under Contract No. DE-AC02-98CH10886.