Cross-Talk Between Amyloidogenic Proteins in Type-2 Diabetes and Parkinson’S Disease

Cross-talk between amyloidogenic proteins in type-2 diabetes and Parkinson’s disease

Istvan Horvatha and Pernilla Wittung-Stafshedea,1

aDepartment of Biology and Biological Engineering, Chalmers University of Technology, 412 96 Gothenburg, Sweden

Edited by Alan R. Fersht, Medical Research Council Laboratory of Molecular Biology, Cambridge, United Kingdom, and approved September 23, 2016 (received for review June 29, 2016) In type-2 diabetes (T2D) and Parkinson’s disease (PD), polypeptide mechanistic level, impairment of prohormone processing has assembly into amyloid fibers plays central roles: in PD, α-synuclein been thought to play a role in initiation and progression of T2D (aS) forms amyloids and in T2D, amylin [islet amyloid polypeptide (22, 23). Insulin and pro-IAPP (22, 24–26), but not proinsulin, (IAPP)] forms amyloids. Using a combination of biophysical meth- can inhibit IAPP amyloid formation in vitro and in mice, sug- ods in vitro we have investigated whether aS, IAPP, and unpro- gesting that accumulation of unprocessed proinsulin may processed IAPP, pro-IAPP, polypeptides can cross-react. Whereas IAPP mote IAPP amyloid formation (22, 24). Insulin-degrading forms amyloids within minutes, aS takes many hours to assemble enzyme (IDE) is a conserved metallopeptidase that can degrade into amyloids and pro-IAPP aggregates even slower under the insulin and a variety of other small peptides including IAPP in same conditions. We discovered that preformed amyloids of pro- the pancreas (27, 28). Genome-wide association studies have IAPP inhibit, whereas IAPP amyloids promote, aS amyloid forma- linked IDE to T2D (29, 30) and Ide mutant mice were found to tion. Amyloids of aS promote pro-IAPP amyloid formation, have impaired glucose-stimulated insulin secretion as well as whereas they inhibit IAPP amyloid formation. In contrast, mixing increased levels of IAPP, insulin, and, surprisingly, aS in pan- of IAPP and aS monomers results in coaggregation that is faster creatic islets (31, 32). Here, aS may be associated with insulin than either protein alone; moreover, pro-IAPP can incorporate aS biogenesis and exocytic release, as it was found to localize with monomers into its amyloid fibers. From this intricate network of insulin-secretory granules in pancreatic β-cells (33). We recently cross-reactivity, it is clear that the presence of IAPP can accelerate demonstrated in vitro that IDE readily inhibits aS amyloid for- aS amyloid formation. This observation may explain why T2D pa- mation via C-terminal binding and, in parallel, IDE activity to- tients are susceptible to developing PD. ward insulin and other small substrates increases (34, 35). Together, the key role of aS in PD and the inverse correlation α-synuclein | fluorescence | amyloid | atomic force microscopy | amylin of impaired insulin secretion and increased aS levels in the pancreatic β-cells, imply that PD and T2D may be connected. In arkinson’s disease (PD) is the second most common neuro- support, reports have suggested that patients with T2D are Plogical disorder and the most common movement disorder. It predisposed toward PD (36, 37). For Alzheimer’s disease (AD),

is characterized by widespread degeneration of subcortical a direct link with T2D was found (15, 38). Amyloid fiber seeds of BIOPHYSICS AND structures of the brain, especially dopaminergic neurons in the the AD peptide, amyloid-β, were shown to efficiently accelerate COMPUTATIONAL BIOLOGY substantia nigra. These changes are coupled with bradykinesia, amyloid formation of IAPP in vitro (39, 40) and IAPP was part of rigidity, and tremor, resulting in difficulties in walking and ab- amyloid-β plaque found in mice brains (41). To address the normal gait in patients (1). The assembly process of the in- unexplored question of cross-reactivity between the amyloido- trinsically unstructured 140-residue protein α-synuclein (aS) into genic peptides in PD and T2D, we here investigated cross- amyloid fibers has been linked to the molecular basis of PD. aS is reactivity among aS, IAPP, and pro-IAPP using biophysical a major component of amyloid aggregates found in Lewy body methods in vitro. inclusions, which are the pathological hallmark of PD, and du- plications, triplications, and point mutations in the aS gene are Significance related to familial PD cases (2, 3). The exact function of aS is unknown, but it is suggested to be involved in synaptic vesicle Protein assembly into ordered so-called amyloid fibers is a release and trafficking, regulation of enzymes and transporters, process that promotes several neurodegenerative disorders, and control of the neuronal apoptotic response (4, 5). aS is such as Alzheimer’s and Parkinson’s disease (PD). Also type-2 – present at presynaptic nerve terminals (6 8) and, intriguingly, diabetes (T2D) is a disease involving amyloid formation, al- also in many cells outside the brain (e.g., red blood cells and though it occurs in the pancreas. Since the protein that forms β pancreatic -cells). aS can assemble via oligomeric intermediates amyloids in PD, α-synuclein (aS), is also expressed in the pan- to amyloid fibrils under pathological conditions (9). Although creas, we investigated whether it could affect aggregation of soluble aS oligomers have been proposed to be toxic (10, 11), the peptide involved in T2D, and vice versa. Using in vitro work with preformed aS fibrils has demonstrated that the amy- methods and purified proteins, we here demonstrate that the loid fibrils themselves are toxic and can be transmitted from cell two proteins cross-react and, importantly, the T2D amyloid to cell and are also able to cross the blood–brain barrier (12–14). protein (both as monomer and amyloid seed) can accelerate aS Type-2 diabetes (T2D) is another disease involving amyloid aggregation. This result provides a possible explanation for formation. Here, the primary pathological characteristic is islet why patients with T2D are more prone to getting PD. amyloid of the hormone amylin, also known as islet amyloid polypeptide (IAPP), in pancreatic β-cells (15–18). The process of Author contributions: I.H. and P.W.-S. designed research; I.H. performed research; I.H. and islet amyloid formation (19–21) leads to pancreatic β-cell dys- P.W.-S. analyzed data; and P.W.-S. wrote the paper. function, cell death, and development of diabetes. IAPP (37 The authors declare no conflict of interest. residues, natively unfolded) is cosecreted with insulin after en- This article is a PNAS Direct Submission. zymatic maturation of prohormones pro-IAPP (67 residues) and Freely available online through the PNAS open access option. proinsulin in secretory granules. IAPP and insulin play roles in 1To whom correspondence should be addressed. Email: [email protected]. controlling gastric emptying, glucose homeostasis, and in the This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. suppression of glucagon release. Although not understood on a 1073/pnas.1610371113/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1610371113 PNAS | November 1, 2016 | vol. 113 | no. 44 | 12473–12477 Downloaded by guest on September 27, 2021 about 3–4 nm for IAPP and pro-IAPP, but 7–9 nm for aS (Fig. 1 A 1,0 D D–F and Table 1). Reported amyloid fiber dimensions for IAPP ∼ – 0,8 are 5 nm for single fibers (42 44). For aS, the just-released solid-state NMR structure of full-length aS amyloid has a core 0,6 dimension of ∼4.5 nm, surprisingly, with its β-strands arranged in a Greek Key motif (45). This information implies that our larger 0,4 aS amyloids may be assemblies of more than one aS proto-fibril. We found that in all three cases (aS, IAPP, and pro-IAPP), 0,2 preformed amyloid fiber seeds, speed up aggregation of mono-

Normalized fluorescence 300nm 0,0 mers of the same protein (Fig. 1 B and C; not shown for IAPP as 010203040 the seeded reaction is too fast to capture by manual mixing). time (h) B 1,0 E Cross-Seeding of Amyloid Formation. Next we tested the possibility 0,8 of cross-seeding of one protein’s amyloid formation reaction with preformed amyloid seeds of another protein. We found that 0,6 preformed IAPP amyloid seeds speed up aS amyloid formation (Fig. 2A), and preformed aS amyloid seeds speed up pro-IAPP 0,4 amyloid formation (Fig. 2B), whereas preformed aS amyloid seeds inhibit IAPP amyloid formation (Fig. 2 C and G), and 0,2 300nm preformed pro-IAPP amyloid seeds inhibit aS amyloid formation Normalized fluorescence 0,0 (Fig. 2 D and H). The resulting amyloid fibers (from reactions in 0 10203040506070 Fig. 2 A and B) have the dimensions matching that of the ag- C time (h) F gregating protein when aggregating alone (Table 1 and Fig. 2 E 1,0 and F). This implies that added amyloid fiber seeds (when con-

0,8 taining a protein that has an intrinsically faster amyloid formation reaction than the monomeric protein it is mixed with) 0,6 trigger the “normal” aggregation reaction. In contrast, when amyloid fiber seeds of a protein that forms amyloid fibers slower 0,4 than the protein it is mixed with (i.e., aS amyloid fiber seeds

0,2 added to IAPP monomers, and pro-IAPP amyloid fiber seeds added to aS monomers; Fig. 2 C and D) are added, the outcome

Normalized fluorescence 300nm 0,0 is instead reduction of amyloid formation. 0,0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 Time (h) Coaggregation of aS and Pro-IAPP. When monomers of pro-IAPP Fig. 1. Amyloid fiber formation of 70 μM aS alone (black) and in the are added to aS aggregation reactions, the pro-IAPP monomers presence of 0.7 μM preformed amyloid fiber seeds (red) (A), 140 μM pro-IAPP inhibit aS aggregation in a concentration-dependent manner alone (black) and with 7 μM preformed amyloid fiber seeds (red) (B), and (Fig. 3A). Notably, under these conditions, pro-IAPP itself does IAPP at 5-μM (black) and 10-μM concentration (red) (C) as probed by ThT not form amyloid fibers. Thus, like the pro-IAPP seeds (Fig. 2D), emission. AFM images of resulting amyloid fibers are shown for aS (D), pro- pro-IAPP monomers block aS from aggregation and the atomic IAPP (E), and IAPP (F). force microscopy (AFM) images lack amyloids or oligomers (Fig. 3D). In contrast, if aS monomers are added to high con- Results and Discussion centrations of pro-IAPP (concentration that aggregates with lag time of 30 h), the resulting thioflavin T (ThT) emission becomes Individual Protein Aggregation Reactions. IAPP, pro-IAPP, and aS significantly increased but the lag time remains the same (Fig. 3B). all form amyloid fibers in vitro upon incubation at pH 7, 37 °C. This finding implies that aS monomers become incorporated into μ With agitation, aS (70 M) aggregates with a lag time of about the pro-IAPP amyloids without effect on formation speed but A μ 20 h (Fig. 1 ), whereas pro-IAPP (140 M) has a longer lag time allowing for increased ThT emission. The increased ThT emission of about 30 h (Fig. 1B). For IAPP, amyloid formation is much may be due to better ThT binding to resulting mixed amyloid fi- faster, with lag times of 5–10 min for 5 μM and 1–2 min for bers or simply more amyloids are made. Notably, as aS alone (at 10 μM IAPP (Fig. 1C). One may speculate that variation in the 70 μM) would have a lag time of 20 h, and such a process is lacking intrinsic amyloid formation speed is dependent on the ratio of in Fig. 3B, interactions of aS with pro-IAPP (monomers, oligo- amyloid-forming segment size over total length of the peptide mers, and protofibrils) must overrule independent aS amyloid (which will be highest for IAPP), as fluctuating peptides formation. This conclusion correlates with the finding that pro- extending from the amyloid fiber core may slow down secondary IAPP monomers (at concentrations not readily assembling into nucleation. The resulting amyloid fibers have cross-sections of fibers) added to aggregating aS monomers, result in aS amyloid

Table 1. Summary of key properties of amyloids formed in the different aS/IAPP/pro-IAPP mixtures based on AFM analysis IAPP seeds aS seeds to IAPP + Pro-IAPP + Amyloid fiber aS Pro-IAPP IAPP to aS pro-IAPP aS monomers aS monomers

Fiber height (nm) 6–10 2.5–4.5 2.5–4.5 7–10 4–62–38–10 Morphology Long >1 μm Short <1 μm Short Long Short Long needle- Short clustering clustering clustering like soft clustering

Amyloid fiber height is a rough estimate that is based on Z measurements of 15–20 individual fibers in each case (distributions shown in Fig. S3). Morphology is based on visual inspection of a number of AFM images for each sample and reports the most common features observed throughout.

12474 | www.pnas.org/cgi/doi/10.1073/pnas.1610371113 Horvath and Wittung-Stafshede Downloaded by guest on September 27, 2021 A B C D 1,0 1,0 150 50000

0,8 0,8 40000

100 0,6 0,6 30000

0,4 0,4 20000 50

0,2 0,2 10000 Tht fluorescence (a.u.) Normalized fluorescence Tht Fluorescence (a.u.) Normalized fluorescence

0,0 0,0 0 0 02040600 10203040506070 0,0 0,1 0,2 0,3 0,4 0,5 0,6 010203040 Time (h) Time (h) Time (h) Time (h) E FG H

500nm 300nm 500nm 2.0µm

Fig. 2. Cross-seeding of amyloid fiber formation of aS by IAPP amyloid seeds: 70 μM aS alone (black), with 3.5 μM (red) and 7 μM (blue) IAPP amyloid seeds added (A) and of pro-IAPP by aS amyloid seeds: 140 μM pro-IAPP alone (black) and with 7 μM (red) aS amyloid seeds added (B). Inhibition of amyloid fiber formation of IAPP by aS amyloid seeds: 5 μM IAPP alone (black) and with 1.4 μM aS amyloid seeds (red) (C) and of aS by pro-IAPP amyloid seeds: 70 μMaS alone (black), with 7 μM (red) and 14 μM (blue) pro-IAPP fibrils (D). AFM images of the resulting amyloid fibers in A and B (shown in E and F) and dem- onstration of inhibitory effects in C and D (shown in G and H). In G, the large structures observed are not amyloids, but likely aggregates of protein.

inhibition (Fig. 3A). In essence, therefore, aS amyloid formation reaction kinetics, strongly argue for the formation of coassembled becomes slowed down when monomers are added to high con- amyloids of aS and IAPP. centrations of pro-IAPP and instead pro-IAPP is capable of Addition of aS monomers to aggregating IAPP after the ThT recruiting the aS monomers into its amyloid fibers. AFM images emission plateau was reached (i.e., at 20 min; IAPP amyloids al- of these mixtures imply that thicker fibrils are formed (8–10 nm) ready formed) did not result in further ThT increase within the compared with the fibrils of pro-IAPP alone (Fig. 3E and Table 1). time frame of hours (Fig. S2). This result demonstrates that the BIOPHYSICS AND

process in Fig. 3C involves coaggregation of monomers, or early COMPUTATIONAL BIOLOGY Coaggregation of aS and IAPP. Similar to the mixing of aS and pro- assemblies of the two proteins, and is not due to initial formation of IAPP monomers, we found increased ThT emission for IAPP IAPP amyloids that subsequently template aS aggregation. Label- amyloid formation reactions when monomeric aS was added ing experiments will address the arrangement of IAPP and aS (Fig. 3C). Notably, because IAPP amyloid formation occurs monomers within the heterologous amyloids (work in progress). within 10–20 min, the added aS will not aggregate at all by itself under these conditions. Intriguingly, not only did the ThT Conclusions emission increase when aS monomers were added to IAPP Although most studies of amyloid formation have focused on monomers, also at a ratio of aS to IAPP of 2:1 (5 μM IAPP), but individual disease-specific peptides, interactions of peptides as- the lag time was also shortened to a few minutes (similar to what sociated with different amyloid diseases may modulate amyloid β was observed when IAPP concentration was increased, compare formation pathways and pathogenicity (38). IAPP and amyloid- Fig. 1C with Fig. 3C). The increased ThT emission and reduced share 50% sequence similarity and were found to cross-react lag time strongly argue that IAPP and aS readily coaggregate and both in vitro (40) and in vivo (41). Also peptides structurally and physiologically unrelated, such as IAPP and the amyloidogenic that such reactions are favorable with respect to both speed and determinant of the prion protein (PrP), can cross-react into amount. In control experiments of IAPP and aS monomers morphologically distinct amyloid aggregates (46) and amyloid-β mixed in the plate reader, we found the endpoint ThT signal fibers can seed aS amyloid formation (47). aS is present in the (after 30 min) to follow the same trend as observed for the same pancreas (33), its levels are inversely correlated with that of IDE mixtures in kinetic cuvette experiments (Fig. S1). (31), and IDE inhibits aS amyloid formation in vitro (34, 35). AFM images of these mixtures suggest that the resulting am- ∼ Despite these facts, putative cross-talk between aS and processed yloid fibers have a thickness of only 2 nm, which is thinner than and/or unprocessed IAPP has not been addressed previously. what we observe for the two homoprotein amyloids (Table 1). The importance of possible cross-reactivity between these pro- – The resulting amyloid fibers in the aS IAPP mixture appear teins is also underscored by recent reports demonstrating that needle-like with rough, but sharp, edges according to thorough IAPP is present in brain cells and may form amyloidogenic de- AFM image inspection (Fig. 3F; we note that AFM quantifica- posits there, although the IAPP peptide appeared to be synthe- tion, apart from the Z height, is uncertain). In contrast to the sized elsewhere (41, 48–50). typical amyloids formed by IAPP and aS individually, the amy- In addition to the intriguing coaggregation detected among loid fibers formed in mixtures of the two monomers are very monomers, readily forming heterologous amyloid fibers, a trend we fragile: the AFM tip easily destroys these fibers. Therefore, the discovered was that preformed amyloid fiber seeds from a faster/ tapping mode imaging was possible only at instrument settings slower aggregating protein will accelerate/inhibit aggregation of an that corresponded to the exposure to weak forces. The differ- intrinsically slower/faster aggregating protein (summarized in ences in amyloid fiber morphology, together with the altered Fig. 4). This trend may be explained by a built-in “memory” in the

Horvath and Wittung-Stafshede PNAS | November 1, 2016 | vol. 113 | no. 44 | 12475 Downloaded by guest on September 27, 2021 fiber seeds FASTER A D fiber seeds fiber seeds 12000 INHIBITION FASTER fiber seeds 10000 INHIBITION

8000 IAPP amyloid αS amyloid pro-IAPP amyloid formaon formaon formaon 6000 5-10 min ~20 h 30-40 h monomers 4000 INHIBITION monomers no eﬀect 2000 mix of monomers on speed

Tht fluorescence (a.u.) 1.0µm FASTER FASTER CO-AGGREGATION 0 0 10203040506070 Time (h) Fig. 4. Summarizing scheme of the discovered effects on amyloid forma- B 35000 E tion reactions among IAPP, aS, and pro-IAPP monomers and amyloid seeds. 30000 The statement “faster makes faster and slower makes slower” is a common 25000 conclusion among the amyloid seeding reactions.

20000 15000 Materials and Methods 10000 Proteins. aS was purified as described previously (52). The protein was freeze dried and dissolved in the appropriate buffer before use, followed by fil- Tht fluorescence (a.u.) 5000 1.0µm tration through a 0.2-μm filter. Pro-IAPP was purchased from Severn Biotech 0 102030405060 (product no. SBP0022). IAPP was obtained from Calbiochem (product no. 05– Time (h) C 250 F 23-2540). Both pro-IAPP and IAPP were treated with hexafluoroisopropanol (105228; Sigma-Aldrich) before use to dissolve any preexisting aggregates. 200 Amyloid seeds of each protein were prepared by incubating aggregated protein samples (i.e., the products of a normal amyloid formation reaction) 150 for 10 s in a sonicating bath. The incubation time has to be optimized for different sonicators because the power output can vary depending on the 100 manufacturer (extended sonication can completely destroy fibrils). Amyloid

50 seeds were characterized by AFM before use. Tht fluorescence (a.u.)

0 Amyloid Formation Assay. aS and pro-IAPP amyloid formation reactions were 0,00 0,20 0,40 0,60 0,80 2.0µm conducted in a plate reader (Fluostar Optima; BMG Labtech) in TBS (0.05 M Tris Time (h) HCl buffer, pH 7.4 with 0.15 M NaCl, 93318; Sigma-Aldrich) buffer in the presence of 20 μM ThT (T3516; Sigma-Aldrich) at 37 °C with agitation and a 2-mm glass Fig. 3. Coaggregation upon mixing of monomers. (A) Effects on aS amyloid bead in each well. Samples were typically incubated for 60 h and fluorescence formation upon the addition of pro-IAPP monomers [70 μM aS alone (black) was measured every 20 min. Due to its faster aggregation kinetics, IAPP amyloid and with 17 μM (red) and 35 μM (blue) proIAPP added]. (B) Effects on pro- formation was monitored in a 1-cm cuvette using a Cary Eclipse spectropho- IAPP amyloid formation upon the addition of aS monomers [140 μM proIAPP tometer (with a magnetic stir bar). IAPP aggregation was initiated by pipetting a alone (black) and with 35 μM (red) or 70 μM (blue) aS]. (C) Effects on IAPP small volume of IAPP from the 2.5 mM stock (in hexafluoroisopropanol) into the amyloid formation upon the addition of aS monomers [5 μM IAPP alone reaction buffer (10 μM ThT in TBS) followed by fluorescence measurements (black) and with 10 μM aS (red)]. AFM images of the resulting amyloid fibers every 30 s. Comparison of endpoint ThT values for IAPP amyloid formation in a are shown in D (for A), E (for B), and F (for C). cuvette and in the plate reader suggested that the IAPP aggregation process was similar in both setups. For both plate reader and cuvette experiments, ThT was amyloid seed (envisioned as fine structural differences) that excited at 440 nm and emission was recorded at 480 nm. All reported ThT curves are averages of triplicate experiments, and all experiments were performed (in becomes transmitted into newly formed amyloids and thereby triplicate) three independent times. (For all of the mixture experiments of dictates the aggregation speed of the other protein. With more monomers/amyloid seeds of two proteins in various combinations, a range of structural studies of amyloids at atomic resolution, such micro- concentrations was tested. The included figures show representative data that scopic variations may eventually be revealed. illustrate the trends we discovered from a number of experimental conditions.) Finally, our findings provide a simple justification for why T2D – is a risk factor for PD (as IAPP amyloid seeds, present in T2D, AFM. Aggregated samples were diluted into Milli-Q water (10 20 times) and deposited on freshly cleaved mica. After 10 min, the mica was rinsed with fil- trigger aS amyloid formation), whereas patients with PD are tered Milli-Q water and dried under a gentle nitrogen stream. Images were not prone to getting T2D (aS amyloid seeds, present in PD, in- recorded on an NTEGRA Prima setup (NT-MDT) using a gold-coated single hibit IAPP amyloid formation) (36, 37). Because aS and IAPP crystal silicon cantilever (NSG01, spring constant of ∼5.1 N/m; NT-MDT) and a may come in contact with each other both in pancreatic β-cells resonance frequency of ∼180 kHz. The 512-pixel images were acquired with a (33) and in various brain cells (48–50), and amyloidogenic as- 0.5-Hz scan rate. Images were analyzed using the WSxM 5.0 software (53). semblies may spread from cell to cell (51), in vivo studies are de- ACKNOWLEDGMENTS. Funding is acknowledged from the Knut and Alice sired to reveal the biological consequences of the cross-reactivity Wallenberg Foundation, the Swedish Research Council, and the Chalmers detected here. Foundation.

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Horvath and Wittung-Stafshede PNAS | November 1, 2016 | vol. 113 | no. 44 | 12477 Downloaded by guest on September 27, 2021