Cells of the Neuronal Lineage Play a Major Role in the Generation of Amyloid Precursor Fragments in Gelsolin-related Amyloidosis*

Gelsolin-related amyloidosis or familial amyloidosis, Finnish type (FAF) (OMIM No105120) is a hereditary amyloid disease caused by a mutation in a precursor protein for amyloid (gelsolin) and characterized by corneal dystrophy and polyneuropathy.In vitro expression of the FAF-mutant (Asp187→ Asn/Tyr) secretory gelsolin in COS cells leads to generation of an aberrant polypeptide presumably representing the precursor for tissue amyloid. Here, we provide evidence that this abnormal processing results from defective initial folding of the secreted FAF gelsolin due to the lack of the Cys188-Cys201 disulfide bond, normally formed next to the FAF mutation site. We compared cells of different tissue origin and discovered a dramatic difference between the amount of cleavage of FAF gelsolin to the amyloid precursor in neuronal and non-neuronal cells. More than half of the mutant gelsolin was cleaved in PC12 and in vitro differentiated human neuronal progenitor cells. In contrast, human fibroblasts and Schwannoma cell cultures showed only a limited capacity to cleave FAF gelsolin, although the cleavage mechanism per se seems to be similar in the various cell types. The present findings of processing and distribution of secreted FAF gelsolin in the neuronal cells emphasize the role of neurons in the tissue pathogenesis of this amyloid polyneuropathy.


Gelsolin-related amyloidosis or familial amyloidosis, Finnish type (FAF) (OMIM No105120) is a hereditary amyloid disease caused by a mutation in a precursor protein for amyloid (gelsolin) and characterized by corneal dystrophy and polyneuropathy. In vitro expression of the FAF-mutant (Asp
3 Asn/Tyr) secretory gelsolin in COS cells leads to generation of an aberrant polypeptide presumably representing the precursor for tissue amyloid. Here, we provide evidence that this abnormal processing results from defective initial folding of the secreted FAF gelsolin due to the lack of the Cys 188 -Cys 201 disulfide bond, normally formed next to the FAF mutation site. We compared cells of different tissue origin and discovered a dramatic difference between the amount of cleavage of FAF gelsolin to the amyloid precursor in neuronal and non-neuronal cells. More than half of the mutant gelsolin was cleaved in PC12 and in vitro differentiated human neuronal progenitor cells. In contrast, human fibroblasts and Schwannoma cell cultures showed only a limited capacity to cleave FAF gelsolin, although the cleavage mechanism per se seems to be similar in the various cell types. The present findings of processing and distribution of secreted FAF gelsolin in the neuronal cells emphasize the role of neurons in the tissue pathogenesis of this amyloid polyneuropathy.
Amyloidosis represents a group of diseases in which abnormal fibrillar protein deposits, derived from biochemically distinct proteins are found in the extracellular space of patients' tissues (1). Gelsolin-related amyloidosis or familial amyloidosis of the Finnish type (FAF), 1 is a representative example of a hereditary amyloid polyneuropathy. The main symptoms in FAF include corneal lattice dystrophy (type II) and progressive cranial and peripheral neuropathy (2,3). The cell type responsible for amyloid deposits found in organs such as cornea, nerves, skin and kidney (3)(4)(5)(6)(7)(8)(9) has been a topic of debate. FAF is caused by the Asp 187 3 Asn or Asp 187 3 Tyr mutations of gelsolin (7,10,11), an actin-modulating protein. Intracellular and secreted forms of gelsolin differ by the presence of a disulfide bond between amino acids 188 and 201, a signal sequence, and a short amino-terminal extension in the secreted form (12,13). In addition, a novel gelsolin isoform of intracellular gelsolin, gelsolin-3, has been recently described in oligodendrocytes (14).
Transient expression of the wild-type and mutant gelsolin in COS-1 cells results in the abnormal cleavage of the secretory form of FAF-mutant gelsolin. Consequently, secretion of both the full-length (83 kDa) and the 68-kDa carboxyl-terminal fragment (GSN-c68) of gelsolin consisting of amino acids 173-755 is observed (15,16). Interestingly, GSN-c68 has been found also in the cerebrospinal fluid (CSF) and plasma of FAF patients (16 -18). The second putative cleavage of this fragment at amino acid position 244 is likely to generate the FAF amyloid (amino acids 173-243) (19,20) and, consequently, a 60-kDa carboxyl-terminal fragment also observed in the serum of the patients and CSF (17, 21) (see Fig. 1). These in vitro and in vivo findings have led to the conclusion that GSN-c68 represents the immediate precursor for FAF amyloid, and thus, generation of this polypeptide seems to be the first crucial event in the molecular pathogenesis of FAF.
This study was carried out to further clarify the molecular mechanisms underlying FAF with particular emphasis on (i) the impact of the FAF mutation on the initial folding of gelsolin and (ii) on the role of the processing of FAF gelsolin by different cell types. By providing evidence for the specific role of neurons in the generation of precursor protein for FAF amyloid, our data give interesting insight into the vigorously studied issue of the cellular pathogenesis of an amyloidosis affecting the nervous system.

EXPERIMENTAL PROCEDURES
Cell Lines and Cultures-COS-1and Hep2C cells as well as cultured primary fibroblasts from a control individual and from a 55-year-old male FAF patient with the G654A mutation of gelsolin (22) were cultured in Dulbecco's modified Eagle's medium and A549 and Madin-Darby canine kidney (MDCK) cells in modified Eagle's medium supplemented with 10 or 5% fetal calf serum (FCS). Undifferentiated PC12 cells were cultured on plates coated with a 1:40 dilution of Matrigel * This study was supported by the The Academy of Finland and by the Maud Kuistila and Emil Aaltonen Foundations. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ These authors contributed equally to this work. ‡ ‡ To whom correspondence should be addressed. (Becton Dickinson Labware, MA) and in RPM1 1640 medium supplemented with 10% normal horse serum (HS) and 5% FCS. For differentiation, a medium with 1% HS and 50 ng/ml nerve growth factor (NGF) (Alomone Labs, Israel) was used.
Human macrophages and monocytes were obtained as described previously (23). Primary human Schwannoma cell cultures were derived from the trigeminal Schwannoma of a 15-year-old male and from the vestibular Schwannoma of a 59-year-old female with histopathological diagnosis of neurilemmoma atypicum and neurilemmoma as published earlier (24). Staining with S-100 antibody, a marker for Schwann cells, was performed to confirm the phenotype of the cultured cells. Human neuronal telencephalic progenitor cells (HNP) were obtained after the legal abortion of an 8-week old fetus and cultivated as earlier described (25) with the addition of dibutyryl cyclic AMP (1 mM) (Sigma) to the culture medium. This medium allowed differentiation of the progenitors into cells of neuronal lineage, as was confirmed by the staining of most of the cells with antibodies against the microtubuleassociated protein, MAP-2.
Construction of the Expression Plasmids and Recombinant Adenoviruses-Site-directed mutagenesis changing nucleotide G658 to C (Cys 188 3 Ser) was performed with a Chameleon TM double-stranded, site-directed mutagenesis kit (Stratagene) on pcDX-X expression vectors containing either the wild-type (Asp 187 ) or FAF-mutant (Asn 187 ) secretory gelsolin cDNA (15). The mutant clones (p-sGSNSer 188 and p-sGSNAsn 187 Ser 188 ) were identified using solid-phase minisequencing.
cDNAs encoding for the wild-type and the FAF-mutant gelsolin were ligated to a shuttle vector between the long terminal repeat from Rous sarcoma virus and the SV40 polyadenylation signal sequence, flanked by regions derived from the adenoviral genome. Linearized shuttle plasmid and the large ClaI fragment of Ad-5 DNA with deletion in E3 regions were co-transfected to the 293 cell line. After homologous recombination, the recombinant E1/E3 deleted adenoviruses were plaquepurified and expanded in 293 cells (26). The correct structures were verified by restriction enzyme digestion and Southern blotting as well as by a minisequencing test for gelsolin (22).
Transfection and Transduction-Transfection with the plasmid constructs as well as collection of the transfected cells and their media were performed as described earlier (15,16). Cells were infected for 1-2 h in serum-free medium using the recombinant adenoviruses at different amounts of multiples of infectivity (1-100) in order to avoid a plausible artifact caused by the varying capability of the vectors to transduce different types of cells. After infection, medium supplemented with 2% FCS or 1% FCS and 1% HS was added to all cell types except for the HNP cells, the medium of which contained no FCS. In protease inhibition assays, EDTA (1.2 or 10 mg/ml) was added to serum-free medium, and the cells were incubated for 4 h. Similar efficiency of transduction in COS-1 and PC12 cell cultures was confirmed by immunocytochemical staining of gelsolin in the transduced cells.
Western blotting and detection of proteins as well as the metabolic labeling of PC12 cells were prepared as described in (15,16). The optical densities of the gelsolin-specific bands were determined by a BioImage Analyzer (Milligen, Bioresearch) on autoradiographs. Immunoprecipitations were performed as published in Kangas et al. (16) or with a mixture of AM904 and COOH961 antibodies at a 1:100 dilution using protein A-Sepharose (Pharmacia, Sweden). The lysed cells were incubated first with the antibody followed by incubation with protein A-Sepharose, both at 4°C for 1 h. In temperature-shift experiments, the cells were labeled for 2 h at 19.5°C (16). The cells and media were harvested after a 1-h chase and immediately cooled on ice. In some experiments, the cells were transferred to 37°C for 1 h after the pulse.
Immunofluorescence staining of PC12 cells was performed as described previously for COS-1 cells (16), and immunocytochemical stainings were performed with standard techniques using the Vectastain ABC-standard kit (Vector Laboratories, CA). Double labeling with two monoclonal antibodies involved the secondary antibody with a peroxidase-conjugated anti-mouse antibody, and the staining and development reactions were carried out separately. The coverslips were mounted in GelMount (Biomeda Corp., Foster City, CA), Vectashield mounting medium (Vector Laboratories), or glycerol gelatin (Sigma) and viewed with a Leica confocal microscope or a Zeiss Axiophot light microscope using a ϫ 40 or ϫ 63 objective.

RESULTS
The Disulfide Bond between Cys 188 and Cys 201 Is Crucial for the Normal Processing of Secreted Gelsolin-We wanted to determine whether the abnormal cleavage of FAF secretory gelsolin to GSN-c68 fragment results from disruption of the Cys 188 -Cys 201 bond formed in the immediate vicinity of the FAF-causing mutation in secretory gelsolin (13). A Cys 188 3 Ser (G658C) mutation was introduced into expression plasmids coding for both wild-type and FAF-mutant gelsolin. The plasmids were expressed in COS-1 cells, and the cells and media were analyzed by Western blotting using antibodies recognizing different epitopes of gelsolin (Fig. 1).
Cells transfected with either of the two plasmids containing the cysteine mutation secreted not only the full-length gelsolin but also the truncated polypeptide of the expected size and immunoreactivity as that of the GSN-c68 gelsolin fragment (Fig. 2). Based on densitometric scanning, approximately 30% of the mutant gelsolin was processed to the abnormal 68-kDa form (GSN-c68). The cell fraction contained only the full-length gelsolin, and the aberrant fragmentation could be inhibited by EDTA (Fig. 2). Thus the results obtained for the secreted gelsolin with the disrupted cysteine bridge Cys 188 -201 were identical to those we had obtained earlier for gelsolin carrying the FAF mutation (15).
The Processing of Mutant Secreted Gelsolin in Neuronal and Non-neuronal Cells-To monitor the processing of secreted FAF gelsolin in various cell types, we utilized recombinant adenoviruses encoding for the wild-type or FAF form of secretory gelsolin (Ad-sGSNwt and Ad-sGSNAsn 187 , respectively) and infected cells of different tissue origin with these constructs. The processing of mutant gelsolin in COS-1 cells was found to be similar both in the adenovirus-driven and in the transient expression experiments (Fig. 3, left).
MDCK cells (27% of total gelsolin) and lowest in the media of Hep2c cells (6%). In contrast, human fibroblasts transduced with Ad-sGSNAsn 187 similarly to fibroblasts derived from a FAF patient secreted only the full-length gelsolin into the culture media (Fig. 3). To ascertain whether fibroblasts have any role in the processing of exogenous FAF gelsolin, non-transduced control fibroblast cultures were incubated with culture media derived from Ad-sGSNAsn 187 -infected MDCK cells. Further cleavage of the mutant secretory gelsolin was not observed, and in particular, no FAF amyloid polypeptide was observed. Similar results were obtained using cultured monocytes instead of fibroblasts (data not shown).
Since neuropathy is a characteristic feature of FAF, we were interested in analyzing the processing of FAF gelsolin in cells of neuroectodermal origin. Schwannoma cell cultures obtained from two patients showed a limited capacity to cleave FAF gelsolin because only 0 -7% of secreted FAF gelsolin was cleaved (Fig. 4, left). However, unlike all the other cell types tested so far, a major fraction (approximately 60 -90%) of the mutant gelsolin was cleaved in both undifferentiated and NGFinduced PC12 cells (Fig. 4, middle). Furthermore, the abnormal GSN-c68 polypeptide was detected not only in the medium but also in the PC12 cell extracts (Fig. 4, middle). In order to confirm the results obtained with PC12 cells, HNP cells that had been differentiated in vitro into cells of neuronal lineage were applied for transduction analyses. Also in these cells, more than half of the secreted FAF gelsolin was cleaved to GSN-c68 (Fig. 4, right).
The cleavage of FAF gelsolin (83 kDa) into GSN-c68 should also generate an amino-terminal 15 kDa polypeptide (Fig. 1). We detected this polypeptide both in the cells and media of NGF-induced PC12 cells expressing FAF gelsolin. The polypeptide proved to be unstable because it was not found in the COS-1 cells or media and only occasionally in the media from the MDCK and A549 cells expressing FAF gelsolin (Fig. 5).
Kinetics of Processing of Mutant Gelsolin in PC12 Cells-Pulse-chase experiments revealed that the GSN-c68 polypeptide was secreted from the PC12 cells at the same rate as the full-size gelsolin while no signs of endocytosis of gelsolin polypeptides were observed (Fig. 6). This was further confirmed by the finding of only the 83-kDa gelsolin in the media and no endocytosed gelsolin polypeptides from non-transduced PC12 cells that had been incubated with medium derived from fibroblasts expressing FAF gelsolin (data not shown). Pulsechase experiments on a temperature block of 19.5°C (28) showed that secreted FAF gelsolin gets cleaved in the PC12 cells after the trans-Golgi network but prior to secretion (Fig.  6B). Finally, a high concentration (10 mg/ml) of EDTA had some inhibitory effect on the fragmentation of FAF-gelsolin (Fig. 6C). Thus the results obtained here are very similar to those we obtained earlier in the COS-cells (15), suggesting that the enzymatic process cleaving FAF gelsolin is most likely similar in the PC12 and COS cells.
The Distribution of Secretory Gelsolin in the NGF-induced PC12 and Human Telencephalic Progenitor Cells-In order to examine the distribution of wild-type and mutant secretory gelsolin in neuronal cells, differentiated PC12 and HNP cells transduced either with Ad-sGSNwt or with Ad-sGSNAsn 187 were subjected to immunocytochemical stainings using antigelsolin antibody. The infected and uninfected cells were found to be morphologically similar, and no signs of induced death of cells expressing mutant gelsolin was observed. The wild-type and mutant gelsolin were identically distributed so that, in either case, immunostaining was seen both in the soma and along the processes where the staining was most prominent at the tip of the extensions (Fig. 7A, B, and D-F). Double staining of the transduced PC12 cells using anti-gelsolin antibody with antibodies against either synaptophysin, MAP-1, or MAP-2 showed similar distributions to these marker proteins. The immunostaining pattern of anti-gelsolin antibody was perhaps closest to that obtained with the synaptophysin antibody (Fig. 7C). DISCUSSION FAF is a dominantly inherited amyloidosis caused by Asp 187 3 Asn/Tyr mutations in gelsolin (7,10,11). We have shown earlier in COS cells that the abnormal cleavage of the amyloidosis-associated forms of secretory gelsolin results in the secretion of an aberrant 68-kDa carboxyl-terminal fragment of gelsolin (GSN-c68) (15), presumably representing an immediate precursor protein for FAF amyloid. Intracellular FAF gelsolin also contains the FAF mutation but is not subject to alternative cleavage in COS cells, suggesting that the secreted form of FAF gelsolin is the only source of FAF amyloid (16).
Characteristic only to the secretory gelsolin is a disulfide bond that gets formed in close vicinity to the FAF mutation, between amino acids 188 and 201 (13,29). Consequently, the aberrant processing restricted to the secretory FAF gelsolin could be related to disturbed disulfide bridge formation and abnormal primary folding of the molecule. We demonstrated here that secretory gelsolin carrying the Cys 188 3 Ser mutation was processed in a fashion similar to that of the FAF mutant protein in COS-1 cells, resulting in the secretion of a truncated carboxyl-terminal polypeptide that corresponded in size and immunoreactivity to the GSN-c68 fragment. Since the introduction of this serine mutation to FAF gelsolin did not cause any other obvious changes in the protein processing or secretion, the present data would indicate that the 188 -201 disulfide bond is not properly formed in the mutant secretory gelsolin. This would result in abnormal initial folding of the protein and aberrant proteolytic cleavage producing the FAF amyloid precursor fragment. A similar portion, about 30% of both the FAF and the Cys 188 3 Ser mutated secretory gelsolin was found to become abnormally cleaved in the expression system of COS-1 cells, most probably due to the insufficient capacity of gelsolin-cleaving enzyme(s).
Our in vitro expression data are slightly in contrast with the assumption made by Burtnick et al. (29) who suggested, based on the three-dimensional structure of wild-type horse plasma gelsolin, that the Cys 188 -Cys 201 disulfide bond might help to stabilize the FAF amyloid fragment accumulating in tissues. Although the present data strongly suggest that the formation of this bond is actually disturbed in the case of secreted FAF gelsolin, these cysteine residues might, nevertheless, play an important role in later processing steps of the GSN-c68 to FAF amyloid. Rearrangement of inter-or intramolecular disulfide bonds have been described in another protein (fibrillin) in extracellular matrix after secretion and might occur also in the case of FAF gelsolin polypeptides (30). Further, it is noteworthy that, although we demonstrated similar abnormal cleavage of FAF and cysteine-defective gelsolin in COS cells, the actual Right, after a 1-h chase, all the labeled gelsolin was secreted into the culture medium. B, left, after a 2-h incubation at 19.5°C, only the full-size gelsolin was visible in the transduced cells, indicating that the cleavage of the FAF gelsolin to GSN-c68 polypeptide occurs after the trans-Golgi network. Right, incubation for 1 h at 37°C after the 2-h pulse time allowed the normal protein transport and processing to continue, and the GSN-c68 was detected in the medium of the PC12 cells. C, left, at a concentration of 1.2 mg/ml EDTA, 94% of the mutant gelsolin in the PC12 cell extracts was in the cleaved form. At the higher concentration (10 mg/ml), only 58% of the mutant gelsolin was cleaved. Right, only the high concentration of EDTA was capable of inhibiting the secretion of the GSN-c68 polypeptide into the PC12 cell medium. p ϭ pulse; c ϭ chase; 0 ϭ background of PC12 cells.
amyloid formation might still be influenced by additional factors in vivo (31).
The amyloid deposits in FAF are found in particular in the cornea, nerves, skin, and kidney in addition to the walls of vessels in various organs (3)(4)(5)(6)(7)(8)(9). In addition to the high level of production of gelsolin, for example in the skin, there seem to be additional factors affecting the characteristic tissue distribution of FAF amyloid (32). One such factor could be differential processing of the mutant secretory gelsolin in different types of cells.
Adenovirus-mediated expression provided us with a convenient means to analyze the processing of FAF-mutant gelsolin in cultured cells. In cells of renal, hepatic, and lung origins, approximately 10 -30% of the mutant protein was cleaved. In contrast, fibroblasts did not process FAF gelsolin abnormally, nor did monocytes seem to degrade it, suggesting that these cells of the connective tissue and immune system do not play a major role in the generation of FAF amyloid. Interestingly, in the glial cells of the peripheral nervous system, the Schwann cells, only 0 -7% of secreted FAF gelsolin was found to be cleaved, whereas a distinct majority of the mutant protein was cleaved in PC12 cells, the classical model of neuronal lineage. Furthermore, the GSN-c68 fragment was found both in the cell culture medium of the PC12 cells and inside the cells, and the amino-terminal counterpart derived from the proteolysis of full-length FAF gelsolin was reproducibly detected only in these cells and their medium. To confirm the results obtained with PC12 cells, primary cultures of human telencephalic progenitor (HNP) cells that had been differentiated in vitro into cells of the neuronal lineage were analyzed. Also in these cells, the FAF gelsolin was extensively cleaved as compared with the non-neuronal cells. Consequently, our results emphasize the particular role of cells of neuronal origin in the abnormal proteolysis of FAF gelsolin and in production of the characteristically observed amyloid of the cranial and peripheral nerve fibers.
Since a similar level of fragmentation was observed in differentiated and undifferentiated PC12 cells, it is possible that a constantly high level or activity of gelsolin-cleaving enzyme is found in these as well as in HNP cells. Results suggesting a specific role for neuronal cells in the processing of an amyloid precursor protein have so far been obtained only for APP, the precursor protein of the ␤ amyloid found in Alzheimer's disease. For example, post-translational signaling events in APP differ between neurons and other cell types (33), and neurons also appear to generate more ␤ amyloid peptide than astrocytes or microglia (34).
Immunocytochemical analysis revealed identical distributions of wild-type and mutant secretory gelsolin in NGF-induced PC12 cells. We did not detect morphological differences between uninfected cells or cells expressing wild-type or mutant gelsolin, nor did the mutant gelsolin seem to induce increased cell death. Secretory gelsolin was localized in the cell soma, near the endoplasmic reticulum, and in Golgi-like structures as well as along the processes and nerve endings of both differentiated PC12 and HNP cells. Since the best colocalization was observed with synaptophysin, a presynaptic marker, it is likely that substantial amounts of secretory neuronal gelsolin are secreted in the presynaptic region of the neurons, although more detailed analyses including immunoelectronmicroscopy would be needed to confirm the data. Nevertheless, these observations have interesting implications when considering the deposition of FAF amyloid in those organs that are clinically perhaps most severely affected, namely the nerves and cornea, in the latter of which the characteristic lattice lines have been suggested to correspond to the trigeminal nerve endings (5,35). Thus, the present findings provide evidence for the importance of neurons in the production of precursor protein for FAF amyloid and, furthermore, in the tissue pathogenesis of an amyloid disease affecting the nervous system.