Protective effects of s propargylcysteine (SPRC) on in vitro neuronal damage induced by amyloid beta (25 35 4

4.1.2 Effects of Aβ25-35 aggregation with temperature Figure 12: Aggregation of Aβ25-35 with increasing temperature for 96 hours.. 4.1.3 Effects of SPRC on Aβ25-35 aggregationFigure 13

CHAPTER 4: PART I - IN VITRO STUDY

INVESTIGATING THE ANTI-AMYLOIDOGENIC AND FREE RADICAL

Representative gel photo of protein bands incubated at 37°C for different lengths of time using Coomassie blue staining N=3 (c) Representative gel photo of Aβ oligomers left in wells of the stacking gel after 0h and 96h incubation at 37°C respectively

The shift in thioflavin T fluorescence was employed to track the self-aggregation of Aβ

25-35 at 37°C over 96 hours There was a significant increase (F (4,194) = 60.131; p<0.01) in the

aggregation of 50 µM Aβ25-35 from 0-96 hours, presented as relative fluorescence units per 24

hours 50 µM Aβ25-35 aggregated fairly rapidly and significantly to about 120% of the initial

fluorescence at 0 hour in the first 24 hours (Figure 11a) and gradually slowed down to a constant

rate to 96 hours At the end of 96 hours, the final products produced a shift of about 1.4 RFU, or

40% higher than the initial fluorescence Correspondingly, there was a noticeable decrease in the

Coomassie blue staining of monomers in a time-dependent manner (Figure 11b) The stained

bands decreased in size and density as the incubation time increased, resulting in the smallest

stained band after 96 h incubation These bands were concluded to be the monomers as the

aggregation produced fibrils which could not enter the acrylamide gel and were left in the

stacking gel as observed in Figure 11c After 96 h incubation at 37°C, there was an obvious

darker band left in the well of the stacking gel which was not observed with the freshly prepared

sample The band stained rapidly in Coomassie blue and is believed to be large molecular weight

aggregates unable to enter the 4% stacking gel and could not be visualized in the resolving gel

4.1.2 Effects of Aβ25-35 aggregation with temperature

Figure 12: Aggregation of Aβ25-35 with increasing temperature for 96 hours (a) Average

fluorescence change over incubation time measured by shift in Thio-T fluorescence N≥6 (b) Representative gel photo of protein bands incubated at different temperatures for 96 hours using

Coomassie blue staining N=3, p<0.05

The aggregation of Aβ25-35 was investigated by incubating 50 µM Aβ25-35 at increasing

temperature for 96 hours Thio-T fluorescence was significantly different upon increasing

incubation temperatures (F (2,23)= 3.674; p<0.05) (Figure 12a) However, there was no

significance observed between incubation at 4°C and 25°C, or between 25°C and 37°C There

was only significant increase (p<0.05) when comparing between the 4°C and 37°C incubations

Similarly, there were obvious decreases in stained monomers between samples incubated at

different temperatures (Figure 12b) Aβ25-35 incubated at 4°C showed a dark band which became

less dense than that incubated at 25°C and 37°C The monomer band for Aβ incubated at 37°C

was the weakest when compared between incubations at different temperatures

4.1.3 Effects of SPRC on Aβ25-35 aggregation

Figure 13: Effects of different concentrations of SPRC on Aβ25-35 aggregation (a) Change in Thio-T fluorescence tracked over 96 h when 50 µM Aβ25-35 was incubated with different

concentrations of SPRC at 37°C (b) Thio-T fluorescence of different concentrations of SPRC at

0 h, 24 h, 48 h, 72 h and 96 h Co-incubation with SPRC showed significant decreases as the

concentrations and incubation times increased N=12, *: p<0.05, **: p<0.01 when compared to

the control group (c) Rates of change (slope) for the 96-hour incubation decreased with

increasing concentrations of SPRC N=12, **: p<0.01 when compared to the control group (d)

Representative gel photo of the bands of 50 µM Aβ25-35 that were incubated with different

concentrations of SPRC, stained with Coomassie blue N=3

Incubation of various doses of SPRC with 50 µM Aβ25-35 slowed the aggregation over

96h at 37°C (Figure 13a) Co-incubation of Aβ25-35 with SPRC1 µM, SPRC 10 µM and SPRC 50

µM resulted in gradual, dose-dependent declines in the rate of aggregation which were

observable from 12 hours The shifts in fluorescence, which represent the rate of aggregation,

were significantly different from the control group after 72 h for co-incubation with smaller

doses of SPRC (Figure 13b) However, these differences were significant after only 48 h for

co-incubation with larger doses of SPRC, especially for 50 µM SPRC, in which the significance was

observed starting from 24 h and sustained till 96 h This suggested that the larger doses

decreased the rates of aggregation more than that of the smaller doses At the end of the

incubation, or after 96 h, the aggregation products were all significantly less upon co-incubation

with SPRC The final changes in fluorescence recorded for both 50 µM and 100 µM SPRC were

half of that observed in the control group The rates of change in fluorescence across 96 h were

also found using the average slopes (Figure 13c) The control group had the largest slope of

about 3.91 x 10-3 The slopes gradually decreased with increasing doses of SPRC, in which 50

µM showed the smallest slope of about 1.16 x 10-3 This suggested that SPRC can slow the rate

of aggregation of Aβ25-35 The final aggregation products of Aβ25-35 were reduced after incubating

increasing doses of SPRC with Aβ25-35 (Figure 13d) The amounts of monomers, or unaggregated

Aβ25-35, were largely decreased after a 96 h-incubation, evident from the lack of staining of the

15 kDa smear (Lane 2 vs 3) This lack of the smear was also observed when Aβ25-35 was

co-incubated with 1 µM or 10 µM SPRC (Lanes 4 and 5) The stained smears or monomers

re-appeared much darker and intense than that for 100 µM SPRC These data suggested that SPRC

could reduce aggregation, and the optimal dose was 50 µM

4.1.4 Effects of SAC on Aβ25-35 aggregation

Figure 14: Effects of different concentrations of SAC on Aβ25-35 aggregation (a) Change in Thio-T fluorescence tracked over 96 h when 50 µM Aβ25-35 was incubated with different

concentrations of SAC at 37°C (b) Thio-T fluorescence of different concentrations of SAC at 0

h, 24 h, 48 h, 72 h and 96 h Co-incubation with SAC only showed significant decreases after 72

h incubation N>12, *: p<0.05, **: p<0.01 when compared to the control group (c) Rates of

change (slope) for the 96-hour incubation showed a biphasic decrease with concentration of SAC

N>12, **: p<0.01 when compared to the control group

Incubation with increasing doses of SAC generally discourages Aβ25-35 aggregation

significantly (Figure 14a) The shifts in Thio-T fluorescence after incubating Aβ25-35 with SAC

appeared to be biphasic While 1 µM and 10 µM SAC incubations decreased the shifts in Thio-T

fluorescence dose-dependently, but 50 µM and 100 µM SAC incubations resulted in reduced

Thio-T fluorescence more than the lower doses Differences in fluorescence after incubation

with SAC were only significant after 72 h (Figure 14b); the effects of the co-incubation were

only evident after 72 h All doses except the co-incubation with 50 µM SAC resulted in

significant decreases after this period, and co-incubation with 50 µM SAC was only significantly

deviated from normal Aβ25-35 aggregation at 96 h This trend can also be seen in the rate of

change in shift of fluorescence measured (Figure 14c) Co-incubation Aβ25-35 with SAC

decreased the slope significantly at lower doses, but the addition of 50 µM SAC did not follow

the trend Rather, co-incubation with 50 µM SAC resulted in a larger slope of about 1.66 x 10-3

The smallest slope recorded was for the co-incubation with 10 µM SAC at 1.3 x 10-3

4.1.5 Effects of NaHS on Aβ25-35 aggregation

Figure 15: Effects of different concentrations of NaHS on Aβ25-35 aggregation (a) Change in Thio-T fluorescence tracked over 96 h when 50 µM Aβ25-35 was incubated with different

concentrations of NaHS at 37°C (b) Thio-T fluorescences of different concentrations of NaHS at

0 h, 24 h, 48 h, 72 h and 96 h Co-incubation with NaHS showed significant decreases in

fluorescence for all doses as early as 24 h N=12, *: p<0.05, **: p<0.01 when compared to the

control group (c) Rates of change (slope) for the 96-hour incubation showed a biphasic decrease

with the concentration of NaHS N=12, *: p<0.05, **: p<0.01 when compared to the control

group

Co-incubating different doses of NaHS 50 µM with Aβ25-35 declined the change in

fluorescence significantly (Figure 15a) The effects were observable as early as 4 hours and

generally slowed for all doses, most significantly seen with 100 µM NaHS There appears to be a

biphasic trend where 10 µM NaHS decreased aggregation rate more than the 50 µM group and

this decline was repeated by the higher 100 µM group When compared to the control group at

every 24-hour time point (Figure 15b), all doses of NaHS showed significant decreases in

fluorescence as early as 24 hours Interestingly, both 10 µM and 100 µM NaHS resulted in

significantly lower fluorescence at all time points when compared to the control group The rate

of change in fluorescence (Figure 15c) decreased upon co-incubation with NaHS, where the

similar biphasic trend was observed 1 µM and 50 µM resulted in slopes of about 2 x 10-3, though

10 µM and 100 µM NaHS resulted in much more significant reduction (p<0.01) in slopes to 1.32

x 10-3 and 1.35 x 10-3 respectively

4.1.6 Comparison of equimolar concentration of drugs on aggregation

Figure 16: Comparison between treatments with different drugs (a) Thio-T fluorescence for incubation with different drugs over 96 h (b) Rates of change in fluorescence after co-incubation with 50 µM of SPRC, SAC or NaHS (c) Comparison of percentage decreases in end-point fluorescence by incubation with different drugs (d) Representative gel photo of the bands of 50

µM Aβ25-35 that were incubated with 50 µM of different drugs over 96 h, stained with Coomassie

blue Values are expressed with S.E.M N=3, **: p<0.01

50 µM was chosen as the dose for comparison to investigate the effects of drug

incubation on Aβ25-35 aggregation The shifts in Thio-T fluorescence tracked over 96 h at37°C

for equimolar concentrations of different drugs were compared on the same axis (Figure 16a)

SPRC 50 µM maintained the lowest fluorescence shift consistently over 96h SAC 50 µM and

NaHS 50 µM both showed slightly higher fluorescence shifts than SPRC, although NaHS was

more effective than SAC in slowing Aβ25-35 aggregation The slopes of the Thio-T fluorescence

assay were taken as the rates of change in fluorescence (Figure 16b) While no significance was

observed between the groups, the smallest slope after incubation with SPRC 50 µM (1.29 x 10-3

± 0.21) implied the slowest rate of aggregation The incubation with 50 µM of SAC or NaHS

showed a larger rate of aggregation at 1.66 x 10-3 and 1.78 x 10-3 respectively, although the

difference in slopes is small SPRC 50 µM resulted in about 20% decrease in the end-point

fluorescence, and this was significantly lower than when incubated with SAC (p<0.01)

One-way ANOVA showed that end-point fluorescences of SPRC, SAC and NaHS differed

significantly (F (2,35)= 6.367; p,0.01) (Figure 16c) Equimolar concentrations of SAC and NaHS

resulted in about 10% and 15% decreases in end-point fluorescence respectively and no

differences were found between both groups This was observed in the decrease in density and

size of the monomer bands stained with Coomassie blue (Figure 16d) There was a significantly

lighter-stained band after 96 h incubation at 37°C which was restored to some extent after

addition of 50 µM of drugs The monomer band at 15 kDa was darkest and largest after addition

of SPRC The bands with SAC and NaHS appeared slightly less dense than the SPRC band,

although the differences were not very obvious

4.1.7 Effects on radical scavenging

Figure 17: Effects of radical scavenging abilities of various drugs (a) Dose-dependent changes

in absorbances of ABTS measured over with different drugs (b) TEAC value as compared to

Vitamin C as a control N=2; #: p<0.05 compared to 10 µM Vitamin C; *: p<0.05 compared to

50 µM Vitamin C; &: p<0.05 compared to 1000 µM Vitamin C

There were obvious dose-dependent decreases in the absorbance profiles of the various

drugs (Figure 17a) Vitamin C was used as a positive control with its known strong antioxidant

properties, and absorbance dropped starkly within the first 100 µM This trend was observed

within the first 100 µM of NaHS too SPRC and SAC showed less confident absorbance profiles,

though SPRC demonstrated a more significant dose-dependent decrease in absorbance, to about

0.3 units at 1000 µM SAC however, was less competent in decreasing the absorbance, where

absorbance remained at about 0.7 units despite increasing the dose to 1000 µM TEAC was used

to quantify the radical scavenging powers with Vitamin C as a control (100%) (Figure 17b) The

differences in groups were highly significant at 10 µM (F(3,7)= 1652.875; p<0.01), 50 µM (F(3,7)=

80248.46; p<0.01) and 1000 µM (F(3,7)= 36383.07; p<0.01) While both SPRC and SAC had

low TEAC values at lower doses (p<0.01), the TEAC value for 1000 µM SPRC was increased to

71% with 1000 µM SPRC (p<0.01) In contrast, the TEAC value for 1000 µM SAC is only 2%

The TEAC value for NaHS 10 µM was only 40% (p<0.01), but increased dramatically to close

to 100% with 50 µM and remained so for 1000 µM This showed that while NaHS is comparable

to Vitamin C as a scavenger of free radicals, SPRC is only able to do so at higher doses and SAC

is most obsolete as a free radical scavenger

4.2 Discussion

4.2.1 Aβ25-35 aggregates with increasing time and temperature

The deposition of amyloid-beta aggregates found in the Alzheimer’s disease (AD) brain

is one of the hallmarks of the disease (222) While such aggregates are not consistently

responsible for behavioural deficits in vivo, several in vitro models have shown the neurotoxicity

Of which, the truncated variant of the full-length Aβ protein, Aβ25-35, is the most toxic (15)

In this study, the temperature-dependent and time-dependent aggregation kinetics of

Aβ25-35 was investigated While much research focused on the full-length Aβ1-42 and the more

commonly detected Aβ1-40, little has been established regarding the toxic variant Aβ25-35 The

present data have established that the aggregation of Aβ25-35 follows a temperature-dependent and

time-dependent manner

Incubating the peptide at increasing temperature yielded increased aggregates evident

from the thioflavin T fluorescence and Coomassie blue staining Increasing temperature may

disrupt the thermodynamic stability of the peptide molecules and encourage bond formation The

energy barrier between that of the peptide monomers could be overcome much easier and hence

increased the possibility of interaction and aggregation This implied that the physiological

condition of 37°C was optimal for the aggregation to take place

The typical sigmoidal curve of Aβ aggregation is an expression of the

nucleation-polymerization model of assembly kinetics (223) The rate-limiting step of Aβ aggregation is the

unfavourable nucleation step to form an ordered oligomeric nucleus Subsequently in the growth

phase the nucleus grows rapidly into larger polymers There will be a steady state phase where

the ordered aggregate and monomer concentrations reach equilibrium Thioflavin T forms a