Curr Alzheimer Res. 2014;11(8):745-54.

Different effect of vitamin D2 and vitamin D3 on amyloid-β40 aggregation in vitro.

Suenaga M, Takahashi H, Imagawa H, Wagatsuma M, Ouma S, Tsuboi Y, Furuta A, Matsunaga Y1.

Department of Medical Pharmacology, Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Yamashiro-cho, Tokushima 770-8514, Japan. matsunaga@ph.bunri-u.ac.jp.

 

Abstract

The seeding of amyloid-β 40 (Aβ40) oligomers from monomers is the initial step of Aβ aggregation, and many reports have suggested that cholesterol enhances this step. We studied the potential of secosteroid vitamin D derivatives for Aβ40 aggregation in vitro. The quartz-crystal microbalance technique demonstrated that vitamin D3 does not show any effect on Aβ40 aggregation while vitamin D2 promoted it and docking simulation but that vitamin D2 has high potential in this regard. Thus, stacking of the Phe19 benzene ring in Aβ40 and the C22-C23 double bond in vitamin D2 may alter the energy of these molecules. Electron microscopy revealed the potential of vitamin D2 to increase Aβ40 aggregation. Thioflavin-T assays indicated that Vitamin D2 induced increased fluorescence at 490 nm, as typically observed for amyloid fibrils but also for protofibrils; in both cases this reflects of the increase of β-sheet contents. Aβ40 aggregation was further confirmed in ELISA, SDS-PAGE and dot blot analysis which revealed changes in protease K resistance. These results suggest a possible mechanism, of how vitamin D2 could increase Aβ40 aggregation and the docking simulation explains, why the same is not observed with vitamin D3.

PMID: 25212913

 

Supplement

This is an extension of the previous study of V-D derivatives for prion protein [1].

Prion disease and Alzheimer’s disease (AD) are neurodegenerative disorder based on the conformational transition of the normal protein in the brain and we have previously reported that vitamin D2 (VD2) can bind directly to PrP and break PrPc oligomerization in vitro. This time, we studied the effect of vitamin D (VD) derivatives on Aβ40 aggregation in vitro.

Recently, many clinical reports suggest that VD3 deficiency may increase the risk of cognitive decline [2], and a prospective study exhibited the low levels of serum 25(OH)VD3 were associated with an increased risk of substantial cognitive impairment in the elderly population and also suggested the importance of 25(OH)VD3 raise for the prevention and treatments of AD [3]. The 1,25(OH)2VD3 is the most active form of VD3 metabolites in vivo and 25(OH)VD3 have been detected in CSF, and this could cross the blood-brain barrier [4]. The beneficial effects of 1, 25(OH)2VD3 is also demonstrated for AD patient and VD3 derivatives are used as medicine or supplements.

Though, the VD2 and VD3 appear to have similar biological effects in humans [5] , V-D2 is regarded as a drug and not a physiological product and our results indicate that VD2 is more potent than VD3 in Aβ40 aggregation and suggest that VD3 administration do not lead to amyloid aggregation in the brain. In the present study, both concentrations of Aβ40 and VD derivatives are hundreds to thousands of times higher relevant to our understanding of the naturally occurring concentrations in the human brain [4] and the observations do not reflect the in vivo states and real AD, however our results may give an insight to the different potentials between VD2 and VD3 for the interaction with Aβ40. We suggest the use of VD3 for medical purposes to avoid the risk of Aβ aggregation by VD2 in the brain.

The only structural difference between VD2 and VD3 is the presence of the C24 methyl group and C22–C23 double bond in the side chains (Fig 1). In general, the presence of a double bond in a linear structure influences the conformational flexibility of the molecule through allylic strain and rigidity of the double bond against rotation. [6]

 

Figure 1-1

Figure 1. Structural differences between Vitamin D2, Vitamin D3

Figure 3

Figure 3. Electron microscopic observation for Aβ40 without or with VD2 or VD3.

Aβ40 in the artificial CSF without VD as a control, Aβ40 with VD2 and Aβ40 with VD3. Bar in the photo indicates 100 nm.

 

 

Figure 2

Figure 2. Quartz-crystal microbalance pattern for Aβ40 aggregation with VD derivatives.

Typical real-time monitoring of Aβ40 (12mM) aggregation with VD2 at the concentrations of 0,0.1,0.5 and 1mM (a) or with VD3 at the concentrations of 0,0.5 and 1mM (b) in quartz-crystal microbalance measurements. The changes of frequency of Aβ40 with VD2 or VD3 for 60 min are shown. The data are representative of three experiments. Total amount of Aβ40 aggregates with VD2.(a) VD2 induced potential dose-dependent Aβ40 aggregates. Total amount of Aβ40 aggregates with VD3. (b) VD3 did not induce Aβ40 aggregation after 60 min.

Therefore, we hypothesize that conformational restriction by the double bond in the VD2 side chain facilitated binding of VD2 to the recognition site of Aβ40.

We used Quartz-Crystal Microbalance (QCM) FINIX Q8 [7], which is a highly sensitive mass-measuring apparatus to detect the promoted Aβ40 aggregates by VD derivatives . Sauerbrey’s equation [8] was obtained for the QCM in the air phase:

DF= – 2F0 2Dm / A √rqmq

where DF, measured frequency change (Hz); Dm, mass change; F0, fundamental frequency of the quartz crystal (AT cut 27 MHz); A, electrode area (0.049 cm2); rQ, density of the quartz crystal (2.648 g cm-3); mq, shear modulus of the quartz crystal (2.947 × 1013 gm-1s-2). The equation shows that a 0.61 ng/cm2 increase in mass results in a -1 Hz decrease in frequency in air.

Table 1. Measurement of frequency decrease in Aβ40 aggregation by VD2 on performing QCM 60 min later

tab1

*Direct binding of Aβ40 peptide to the Au electrode without V-D2

a: A -1 Hz decrease in frequency results in a 0.61 ng/cm2 increase in mass

b: Calculation using a molecular weight of 4331 for Aβ40

Data are presented as mean ± SD values for three independent experiments

A typical pattern of real-time monitoring of Aβ40 aggregation with VD2 or VD3 by QCM measurements are shown in Fig 2. A significant decrease in frequency began 15 min after addition of Aβ40 to each concentration of VD2 solution; the frequency reduction depend on the VD2 concentration and incubation time, as shown in Fig 2(a). In case of VD3, however no considerable decrease in frequency was observed upon Aβ40 addition, as shown in Fig 2(b). The amount of Aβ40 aggregation with VD2 was determined from the changes in frequency caused by changes in mass on the electrode (Table 1).

Electron microscopic observation revealed potential Aβ40 aggregation with VD2 , but not with VD3 in artificial cerebrospinal fluid. There was no differences in Aβ40 aggregation between the control and Aβ40 incubated with VD3 at 100 mM; however VD2 at 100 mM strongly enhanced Aβ40 aggregation in the artificial cerebrospinal fluid (Figure 3).

 

 

References

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[3] Oudshoorn C, Mattace-Roso FU, van der Velde N, Colin EM, van der Cammen TJ. High serum vitamin D3 levels are associated with better cognitive test performance in patients with Alzheimer’s disease. Dement Geriatr Cogn Disord 25: 539-43 (2008).

[4] Balabanova S Richter HP, Antoniadis G, Homoki J, Kremmer N, Hanle J, Teller WM. 25-Hydoroxyvitamin D, 24, 25-dihydroxyvitamin D and 1,25-dihydroxyvitamin D in human cerebrospinal fluid. Klin Wochenschr 62: 1086-90 (1984).

[5] Hunt RD, Garcia FG, Hegsted DM, Kaplinsky N. Vitamin D2 and D3 in new world primates: influence on calcium absorption. Science 157: 943-5 (1967).

[6] Hoffmann RW. Allylic 1,3-strain as a controlling factor in stereoselective transformations. Chem Rev 89: 1841-60 (1989).

[7] Ebara Y, Itakura K, Okahata Y. Kinetic studies of molecular recognition based on hydrogen bounding at the air-water interface by using a highly sensitive quartz-crystal microbalance. Langmuir 12: 5165-70 (1996).

[8] Sauerbrey G. Verwendrug von Schwingquarzen zur Wagung dunner Schichten, und zur Mikrowagun. Z Phys 155: 206-22 (1959).

 

 

 

 

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