Vitamin D Affects Neuronal Peptides in Neurodegenerative Disease: Differences of V-D2 and V-D3 for Affinity to Amyloid-β and Scrapie Prion Protein In Vitro

The misfolding of neuronal peptides such as Aβ40/42 in Alzheimer’s disease and cellular prion protein in scrapie induce abnormal aggregation of the peptides in the brain. The seeding of peptides’ oligomers from monomers is the initial step to form molten-globule states before abnormal aggregation. Therefore, compounds targeting the step are useful to clarify the mechanisms underlying aggregation of the proteins and Vitamin D derivatives, which can interact with both Aβ40 and cellular prion protein; however they show different effects in the oligomerization step of the proteins. We discuss the different effects of Vitamin D2 and Vitamin D3 in the interaction with these peptides in brain.


Introduction
Recently, involvement of Vitamin D (V-D) in cognitive impairment is reported.
V-D is a secosteroid and occurs in two distinctive major forms: Vitamin D 2 (V-D 2 ) and Vitamin D 3 (V-D 3 ). V-D 3 is a 27-carbon derivative of cholesterol, and V-D 2 is a 28-carbon derivative from plant ergosterol. The structure of V-D 2 differs from V-D 3 by containing an extra methyl group and a double bond between carbon 22 and 23 (Figure 1). Both V-D derivatives appear to have similar biological effects in humans [1,2]. V-D 3 is about four times as potent as V-D 2 [3]. Interestingly, V-D 2 is a naturally occurring V-D form derived from a fat extract of yeast by the exposure to UV light, and the metabolites were not detectable in the blood of vertebrates such as humans, unless administered from an external source [3,4]. Thus, V-D 2 is not synthesized in vivo and is regarded as a supplement. The metabolites derived from V-D 2 are not equivalent to those for V-D 3 [5]. In contrast to V-D 2 , V-D 3 is the naturally synthesized within the skin and oils of fur. Although both microsomal and mitochondrial 25-hydroxylases act on V-D 3 , they do not act on V-D 2 [4,6,7], and furthermore the V-D binding protein shows lower affinity for V-D 2 than V-D 3 and its metabolites [8]. Currently, clinical applications of V-D for immunosuppression and reduction of pro-inflammatory immune pathways demonstrate that V-D is a prosteroid hormone rather than a vitamin [9,10]. V-D cross blood-brain barrier by passive diffusion and enter the cerebrospinal fluid and brain. The beneficial effects in reducing the relapse risk in multiple sclerosis through its immune-regulatory effects were reported [11]. Recent epidemiologic studies report V-D 3 deficiency as a risk factor of cardiovascular disease including cardiac hypertrophy, myocardial remodeling developed to heart failure (HF) [12,13] and some prospective studies report the relationship between hypovitaminosis-D and an increased risk of cognitive decline in elderly population [14] and suggested that supplementation of V-D could prevent the cognitive disorders [15][16][17], and its effects for the clearance of aggregated amyloid-β (Aβ) in AD brain [18].
In this chapter, we present the different binding affinity of V-D 2 and V-D 3 to amyloidogenic protein in brain: Aβ and prion protein.

Amyloid-β protein in Alzheimer's disease and scrapie prion protein in prion disease
AD and prion diseases are neurodegenerative diseases in brain and cause dementia. AD is the most common case of senile dementia and the number of AD patients is increasing and recent study shows that 46.8 million of AD patients live in the world and it is estimated to reach 131.5 million by 2050 [19]. It is featured by memory loss, deterioration of cognitive and behavioral process, and diminished social life. These symptoms do not improve and progress with life time.
The main pathological hallmark with AD patients is the senile plaque in the brain [20]. Extracellular accumulation of insoluble Aβ protein is the main component of the plaque that induces synaptic dysfunction and neuronal loss resulting in progressive dementia [21]. The Aβ is composed of 39-43 amino acids, naturally produced by proteolytic cleavage of integral membrane protein, 100-135 kDa amyloid precursor protein [22]. The majority of the secreted Aβ alloform includes the C-terminal Aβ40 and Aβ42. Quantitative analyses have shown that, on average, 60% of all plaques contain Aβ42 and 31% contain Aβ40 [23]. The misfolding and aggregation of Aβ and tau proteins are two principal aggregating proteins in AD brain [24,25]. Growth of the fibrils occurs by assembly of the Aβ seeds into intermediate protofibrils, and self-associates to form mature fibers [26]. This multistep process may be influenced at various stages by factors that promote Aβ fiber formation and aggregation, and the seeding of Aβ40 oligomers is the initial step of the process [27,28].
The emergence of a prion disease in cattle is known as bovine spongiform encephalopathy (BSE) and a possible transmission to humans by the exposure to BSE has been suggested [29,30]. Gerstmann-Straussler-Scheinker disease and Creutzfeldt-Jacob are well-known naturally occurring prion diseases in human and they are transmissible and fatal. The main event contributing to the pathogenesis of prion disease is the conversion of the cellular prion protein (PrP c ) into scrapie prion protein (PrP sc ), which is a protease-resistant, insoluble protein [31,32]. PrP c is predominantly expressed in neurons, and attached to extracellular space of plasma membrane through a glycophosphatidylinositol. It is a sialoglycoprotein with a molecular weight of approximately 33-35 kDa [33,34]. Studies have shown that PrP c (90-231), which is N-terminal truncated fragments of PrP c and corresponds to the core of the protease K (PK) resistant prion protein, preserve the pathogenic features of PrP sc [35,36]. Gerstmann-Straussler-Scheinker disease and Creutzfeldt-Jacob disease are caused by mutations in the PrP gene [37] and the mutations directly link to conformational conversion from PrP c to PrP sc and amplification of PrP sc without exogenous PrP sc [38,39], and the infectivity can be explained by the direct PrP sc -PrP c interaction [40]. In vitro generation of infectious PrP sc has demonstrated the protein-only hypothesis of prion propagation [41,42]. Many reports have suggested that the multistep process of conversion from PrP c into PrP sc includes an oligomerization/polymerization step [43,44]. The oligomerization or molten-globule state is a preliminary step required for the formation of insoluble protein in the brain like that of Aβ aggregates in AD brain, and soluble oligomers appear to be more cytotoxic than mature aggregates [45].

V-D-induced Aβ40 oligomerization
Quartz-crystal microbalance (QCM) measurement is a highly sensitive mass-measuring system [46,47] We applied Sauerbrey's equation for the QCM in the air phase: Vitamin D Affects Neuronal Peptides in Neurodegenerative Disease: Differences of V-D2 and V-D3 for Affinity... http://dx.doi.org/10.5772/64508 where ΔF is measured frequency change (Hz), Δm is mass change, F 0 is fundamental frequency of the quartz-crystal, A is an electrode area, ρ q is density of quartz-crystal, and μ q is the shear modulus of quartz-crystal.
The equation indicates that a 0.61 ng/cm 2 increase in mass means a −1 Hz decrease in frequency. The change of frequency is proportional to that of mass.
The change of mass in Aβ40 with V-D 2 or V-D 3 was determined [49].  Table 1). In case of V-D 3 , however, no considerable decrease in frequency upon Aβ40 addition was observed (Figure 2b). These results show a different potential of V-D 2 and V-D 3 for Aβ40 oligomerization in vitro.

Electron microscopic observation exhibited V-D 2 -induced Aβ40 oligomerization
Aβ40 in artificial cerebrospinal fluid without V-D as a control induced weak self-oligomerization and V-D 3 induced no enhancement to the control, however V-D 2 enhanced potent oligomerization for Aβ40 as Figure 3(a-c) [49]. Vitamin D Affects Neuronal Peptides in Neurodegenerative Disease: Differences of V-D2 and V-D3 for Affinity... http://dx.doi.org/10.5772/64508

Thioflavin-T assay revealed β-sheet formation of Aβ40 with V-D 2
Amyloid fibers are ordered β-sheet-rich proteins. Benzothiazole dye, Thioflavin-T (Th-T) is used to probe amyloid fibril formation due to specific noncovalent interactions that yield strong fluorescence upon binding [50]. Aβ40 showed a peak at 490 nm, indicating β-sheet formation [51]. V-D 2 increased peak intensity at 490 nm dose-dependently, indicating that V-D 2 facilitates β-sheet formation in Aβ40. The V-D 3 do not increase peak fluorescent intensity at 490 nm, indicating that V-D 3 facilitate no β-sheet formation in Aβ40 peptide (Figure 4) [49].

Docking simulation between Aβ40 and V-D 2 or V-D 3
In silico docking analysis at the tertiary structure level by Molecular Operating Environment   Vitamin D Affects Neuronal Peptides in Neurodegenerative Disease: Differences of V-D2 and V-D3 for Affinity... http://dx.doi.org/10.5772/64508

Reactivity of 3F4 antibody with Hu-rPrP c (90-231) bound to V-D 2 , as monitored by ELISA
The responsible fragment within Hu-rPrP c (90-231) that was affected by V-D 2 was determined by ELISA. The reactivity of the 3F4 antibody to PrP c (90-231) that was incubated with V-D 2 showed decreasing signals toward PrP c (90-231) bound with V-D 2 in a dose-dependent manner (Figure 7) [52]. These results confirm the observation by Biacore assay (Figure 6).

Structural difference of V-D 2 and V-D 3 could explain different potential for the affinity to Aβ40 and PrP c (90-231)
The C22-C23 double bond contained in V-D 2 structure may influence the conformational flexibility of the molecule through allylic strain and rigidity of the double bond against rotation [57,58]. Therefore, we hypothesize that conformational restriction by the double bond in the V-D 2 side chain facilitated binding of V-D 2 to the recognition site of Aβ40 and PrP c (90-231).

Conclusion
We detected V-D 2 -induced Aβ40 oligomerization by QCM, and electron microscopic observation demonstrated the potential of V-D 2 for Aβ40 oligomerization through β-sheet formation as revealed by Th-T study. V-D 2 -mediated Aβ40 oligomerization occurs through interaction between the Phe19 benzene ring of Aβ40 and the C22-C23 double bond of V-D 2 . In case of prion, the fragment of V-D 2 binding to PrP c (90-231) is around 3F4 epitope, 109-112 amino acid in PrP c (90-231). These fragments are involved in the sensitive fragments to pH shifts and thermal stress. The binding of V-D 2 to amyloidogenic peptides in brain might give some insights to oligomerization of these peptides in the brain.