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AGel amyloidosis, formerly known as familial amyloidosis of the Finnish-type, is caused by pathological aggregation of proteolytic fragments of plasma gelsolin. So far, four mutations in the gelsolin gene have been reported as responsible for the disease. Although D187N is the first identified variant and the best characterized, its structure has been hitherto elusive. Exploiting a recently-developed nanobody targeting gelsolin, we were able to stabilize the G2 domain of the D187N protein and obtained, for the first time, its high-resolution crystal structure. In the nanobody-stabilized conformation, the main effect of the D187N substitution is the impairment of the calcium binding capability, leading to a destabilization of the C-terminal tail of G2. However, molecular dynamics simulations show that in the absence of the nanobody, D187N-mutated G2 further misfolds, ultimately exposing its hydrophobic core and the furin cleavage site. The nanobody's protective effect is based on the enhancement of the thermodynamic stability of different G2 mutants (D187N, G167R and N184K). In particular, the nanobody reduces the flexibility of dynamic stretches, and most notably decreases the conformational entropy of the C-terminal tail, otherwise stabilized by the presence of the Ca 2+ ion. A Caenorhabditis elegans-based assay was also applied to quantify the proteotoxic potential of the mutants and determine whether nanobody stabilization translates into a biologically relevant effect. Successful protection from G2 toxicity in vivo points to the use of C. elegans as a tool for investigating the mechanisms underlying AGel amyloidosis and rapidly screen new therapeutics., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Purpose: Gelsolin amyloidosis (AGel), also known as familial amyloidosis, Finnish type (FAF), is an autosomal, dominant, incurable disease caused by a point mutation (G654A/T) in the gelsolin (GSN) gene. The mutation results in loss of a Ca 2+ -binding site in the second gelsolin domain. Subsequent incorrect folding exposes a cryptic furin cleavage site, leading to the formation of a 68-kDa C-terminal cleavage product (C68) in the trans-Golgi network. This C68 fragment is cleaved by membrane type 1-matrix metalloproteinase (MT1-MMP) during secretion into the extracellular environment, releasing 8- and 5-kDa amyloidogenic peptides. These peptides aggregate and cause disease-associated symptoms. We set out to investigate whether AGel-specific nanobodies could be used to monitor amyloidogenic gelsolin buildup., Procedures: Three nanobodies (FAF Nb1-3) raised against the 8-kDa fragment were screened as AGel amyloid imaging agents in WT and AGel mice using 99m Tc-based single-photon emission computed tomography (SPECT)/X-ray tomography (CT), biodistribution analysis, and immunofluorescence (IF). The quantitative characteristics were analyzed in a follow-up study with a Nb11-expressing mouse model., Results: All three nanobodies possess the characteristics desired for a  99m Tc-based SPECT/CT imaging agent, high specificity and a low background signal. FAF Nb1 was identified as the most potent, based on its superior signal-to-noise ratio and signal specificity. As a proof of concept, we implemented 99m Tc-FAF Nb1 in a follow-up study of the Nb11-expressing AGel mouse model. Using biodistribution analysis and immunofluorescence, we demonstrated the validity of the data acquired via 99m Tc-FAF Nb1 SPECT/CT., Conclusion: These findings demonstrate the potential of this nanobody as a non-invasive tool to image amyloidogenic gelsolin deposition and assess the therapeutic capacity of AGel therapeutics currently under development. We propose that this approach can be extended to other amyloid diseases, thereby contributing to the development of specific therapies.

Hereditary gelsolin amyloidosis is an autosomal dominantly inherited amyloid disorder. A point mutation in the GSN gene (G654A being the most common one) results in disturbed calcium binding by the second gelsolin domain (G2). As a result, the folding of G2 is hampered, rendering the mutant plasma gelsolin susceptible to a proteolytic cascade. Consecutive cleavage by furin and MT1-MMP-like proteases generates 8 and 5 kDa amyloidogenic peptides that cause neurological, ophthalmological and dermatological findings. To this day, no specific treatment is available to counter the pathogenesis. Using GSN nanobody 11 as a molecular chaperone, we aimed to protect mutant plasma gelsolin from furin proteolysis in the trans-Golgi network. We report a transgenic, GSN nanobody 11 secreting mouse that was used for crossbreeding with gelsolin amyloidosis mice. Insertion of the therapeutic nanobody gene into the gelsolin amyloidosis mouse genome resulted in improved muscle contractility. X-ray crystal structure determination of the gelsolin G2:Nb11 complex revealed that Nb11 does not directly block the furin cleavage site. We conclude that nanobodies can be used to shield substrates from aberrant proteolysis and this approach might establish a novel therapeutic strategy in amyloid diseases., (© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Gelsolin amyloidosis is an autosomal dominant incurable disease caused by a point mutation in the GSN gene (G654A/T), specifically affecting secreted plasma gelsolin. Incorrect folding of the mutant (D187N/Y) second gelsolin domain leads to a pathological proteolytic cascade. D187N/Y gelsolin is first cleaved by furin in the trans-Golgi network, generating a 68 kDa fragment (C68). Upon secretion, C68 is cleaved by MT1-MMP-like proteases in the extracellular matrix, releasing 8 kDa and 5 kDa amyloidogenic peptides which aggregate in multiple tissues and cause disease-associated symptoms. We developed nanobodies that recognize the C68 fragment, but not native wild type gelsolin, and used these as molecular chaperones to mitigate gelsolin amyloid buildup in a mouse model that recapitulates the proteolytic cascade. We identified gelsolin nanobodies that potently reduce C68 proteolysis by MT1-MMP in vitro. Converting these nanobodies into an albumin-binding format drastically increased their serum half-life in mice, rendering them suitable for intraperitoneal injection. A 12-week treatment schedule of heterozygote D187N gelsolin transgenic mice with recombinant bispecific gelsolin-albumin nanobody significantly decreased gelsolin buildup in the endomysium and concomitantly improved muscle contractile properties. These findings demonstrate that nanobodies may be of considerable value in the treatment of gelsolin amyloidosis and related diseases.

Purpose: Gelsolin amyloidosis (AGel), also known as familial amyloidosis, Finnish type (FAF), is an autosomal, dominant, incurable disease caused by a point mutation (G654A/T) in the gelsolin (GSN) gene. The mutation results in loss of a Ca2+-binding site in the second gelsolin domain. Subsequent incorrect folding exposes a cryptic furin cleavage site, leading to the formation of a 68-kDa C-terminal cleavage product (C68) in the trans-Golgi network. This C68 fragment is cleaved by membrane type 1-matrix metalloproteinase (MT1-MMP) during secretion into the extracellular environment, releasing 8- and 5-kDa amyloidogenic peptides. These peptides aggregate and cause disease-associated symptoms. We set out to investigate whether AGel-specific nanobodies could be used to monitor amyloidogenic gelsolin buildup.Procedures: Three nanobodies (FAF Nb1-3) raised against the 8-kDa fragment were screened as AGel amyloid imaging agents in WT and AGel mice using 99mTc-based single-photon emission computed tomography (SPECT)/X-ray tomography (CT), biodistribution analysis, and immunofluorescence (IF). The quantitative characteristics were analyzed in a follow-up study with a Nb11-expressing mouse model.Results: All three nanobodies possess the characteristics desired for a 99mTc-based SPECT/CT imaging agent, high specificity and a low background signal. FAF Nb1 was identified as the most potent, based on its superior signal-to-noise ratio and signal specificity. As a proof of concept, we implemented 99mTc-FAF Nb1 in a follow-up study of the Nb11-expressing AGel mouse model. Using biodistribution analysis and immunofluorescence, we demonstrated the validity of the data acquired via 99mTc-FAF Nb1 SPECT/CT.Conclusion: These findings demonstrate the potential of this nanobody as a non-invasive tool to image amyloidogenic gelsolin deposition and assess the therapeutic capacity of AGel therapeutics currently under development. We propose that this approach can be extended to other amyloid diseases, thereby contributing to the development of specific therapies. [ABSTRACT FROM AUTHOR]