Protective effects of s propargylcysteine (SPRC) on in vitro neuronal damage induced by amyloid beta (25 35 6 7

CHAPTER 6: FIBRILLAR Aβ 25-35PRE-TREATMENT WITH SPRC REDUCED DAMAGE INDUCED BY Aβ 25-35 FIBRILS 6.1 Results 6.1.1 Effects of incubation time on cell viability Figure 42: Effects of d

CHAPTER 6: FIBRILLAR Aβ 25-35

PRE-TREATMENT WITH SPRC REDUCED DAMAGE INDUCED BY Aβ 25-35 FIBRILS

6.1 Results

6.1.1 Effects of incubation time on cell viability

Figure 42: Effects of different times of incubation with aggregated Aβ25-35 on cell viability, expressed as percentage of cell viability compared to the untreated control ± S.E.M N= 3, *:

p<0.05 and **: p<0.01 when compared to the untreated control

To test the cytotoxicity of aggregated Aβ25-35 incubated at 37°C for 24 hours, the Aβ25-35

was fixed at a dose of 1 µM and incubated with the cells for different lengths of time (Figure 42) The cell viability of C6 cells decreased with increasing length of incubation At 3 hours, 1 µM

Aβ25-35 could already significantly reduce cell viability to 83 ± 3.5% (p<0.05) By 16 hours of incubation, the cell viability was decreased to 61 ± 0.7% (p<0.01) After 24 hours of incubation,

0 0.2 0.4 0.6 0.8 1 1.2

6.1.2 Effects of drugs on Aβ25-35 -induced cytotoxicity

6.1.2.1 SPRC on cell viability

Figure 43: Effects of SPRC treatment on Aβ-induced cytotoxicity (a) 24-hour SPRC treatment on different incubation times of Aβ1 µM (b) 24-hour pre-treatment of 0.1 µM -1 mM SPRC restored Aβ-induced cytotoxicity significantly, but did not result in any toxicity in

pre-untreated cells The values are percentages of cell viability compared to the pre-untreated control ±

S.E.M N=6, #: p<0.05 when compared to the untreated control; **: p<0.01 when compared with

the Aβ-only control

Since treatment with 1 µM aggregated Aβ25-35 resulted in comparable cytotoxicity, both time points were used to investigate the effects of SPRC on cell viability Different doses of SPRC were pre-treated to C6 cells for 24 hours before treating with 1 µM aggregated Aβ25-35 for another 3 or 16 hours (Figure 43a) Cells pre-treated with only SFM for 24 hours followed by

Aβ25-35 treatment for 3 or 16 hours resulted in 83 ± 2.6% (p<0.01) and 61 ± 0.2% (p<0.01) cell

viability respectively The longer length of incubation with Aβ25-35 resulted in lower cell viability expectedly, accounted for by the increased toxicity Pre-treatment with SPRC slightly, but not significantly, increased the cell viability in cells treated with Aβ25-35 for 3 hours (F (6,27)= 5.556;

p<0.01) However, pre-treatment with SPRC significantly increased the cell viability in cells treated with Aβ25-35 for 16 hours (F (6, 41) = 60.668; p<0.01) The dose-dependent increase in cell

viability was steady from 0.1 µM to 1 mM Cells pre-treated with 10 µM SPRC was effective

with Aβ 3 h or 16 h, resulting in 88 ± 1.0% and 93 ± 0.1% (p<0.01) of viable cells respectively

SPRC 1 µM

The effects of the SPRC pre-treatment were hence only visible upon a longer incubation time with Aβ These doses were not toxic to the cells (Figure 43b), although very slight variations in cell viability following SPRC treatment could be observed at 10 µM and 100 µM Both doses resulted in 102 ± 1.2% and 101 ± 4.1% cell viability respectively

Hence, 24-hour pre-treatment with SPRC followed by 16-hour incubation with 1 µM aggregated Aβ25-35 was selected as the experimental model Also, the dose of interest for SPRC was fixed at 10 µM for subsequent studies

6.1.2.2 SAC and NaHS on cell viability

Figure 44: Effects of SAC and NaHS pre-treatment on Aβ-induced cytotoxicity, expressed as percentage of cell viability compared to the untreated control ± S.E.M 24-hour 0.1 µM -1mM of

both drugs pre-treatment resulted in significant restoration of cell viability N≥5, #: p<0.05 when compared to the untreated control; *: p<0.05; **: p<0.01 when compared with the Aβ-only

and the highest restoration was 94 ± 1.1% of control (p<0.01), observed at 100 µM Even the lowest dose of 0.1 µM resulted in a viability of 87 ± 1.2% of control (p<0.01), significantly

higher than that in the Aβ-only group

Likewise, pre-treatment with 0.1 µM -1 mM NaHS increased cell viability significantly across all doses (F (7, 47) = 11.02; p<0.01) However, the extent of restoration was lower than that

observed in either SPRC- or SAC-treated cells, in which generally cell viability hovered around

82% Cells were most significantly increased to 85 ± 1.4% of control (p<0.01) after

pre-treatment with 100 µM NaHS

6.1.2.3 Comparisons with equimolar concentrations of drugs

Figure 45: Comparison between equimolar concentrations of drugs on Aβ-induced cytotoxicity, expressed as percentage of cell viability compared to the untreated control ± S.E.M Pre-treating

cells with 10 µM of SPRC, SAC or NaHS restored viability significantly N=6, #: p<0.05 when compared to the untreated control; **: p<0.01 when compared with the Aβ control

10 µM was used as a dose for comparison between the drugs in all ensuing studies as this

is the effective dose that resulted in restoration of cell viability following SPRC pre-treatment (Figure 45) While pre-treatment with SPRC, SAC or NaHS resulted in significant differences in viability (F (4, 29) = 28.070; p<0.01), SPRC pre-treatment could best restore Aβ-induced

cytotoxicity to 93 ± 0.6% of control (p<0.01), a large increase in viable cells SAC pre-treatment

NaHS 10 µM

restored the cell viability slightly lower compared to the SPRC-treated cells, to 91 ± 1.1% of

control (p<0.01) Pre-treatment with 10 µM NaHS also significantly increased cell viability;

however, the extent of restoration was the weakest amongst all three drugs, about 84 ± 2.0% of

6.1.3.1 Effects on H 2 S content in cell medium

Figure 46: Effects of pre-treatment of drugs on H2S concentrations in cell medium The values are expressed as fold increase in concentration compared to the untreated control ± S.E.M N=3,

#: p<0.05 compared to untreated control; *: p<0.05 compared to Aβ-only group

The H2S concentration in the cell culture media changed significantly following Aβ treatment (F (4, 34) = 5.498; p<0.01) (Figure 46) The Aβ treatment reduced the H2S concentration

0 0.2 0.4 0.6 0.8 1 1.2

µM

SAC 10 µM

NaHS 10 µM

pre-treatment of SAC did not change the H2S concentration in the culture media, maintaining the

H2S content at 0.79 ± 0.04- fold

6.1.3.2 Effects on CBS expression

Figure 47: Effects of pre-treatment of drugs on CBS expression in cell lysates (a) Representative blots for CBS expression in treated cells N=3 (b) Percentage fold difference in CBS expression

± S.E.M N=3, #: p<0.05 compared to untreated control

The Aβ25-35 treatment significantly reduced the CBS expression (Figure 47) to 86 ± 0.4%, from 100% in the untreated control cells and similar to that in Part II: Oligomeric Aβ Pre-

treatment of 10 µM SPRC and 10 µM NaHS both restored the CBS expression to 92 ± 2.5% and

95 ± 4.3% respectively SAC pre-treatment however, did not result in any observable change in the CBS expression, remaining at 86 ± 1.8%

NaHS 10 µM

1µM Aβ 25-35

(a)

(b)

6.1.3.3 Effects of CBS inhibitor on cell viability

Figure 48: Effects of the CBS inhibitor AOAA on cell viability (a) Dose dependence of 24h treatment of AOAA with and without 1µM Aβ25-35 treatment (b) Pre-treatment of 0.1-100 µM SPRC restored the aggravated cytoxicity by AOAA on Aβ injury significantly The values are

pre-percentages of cell viability compared to the untreated control ± S.E.M N>6, #: p<0.05 when compared to the untreated control; &: p<0.05 when compared to the SFM + Aβ control; *:

p<0.05 when compared with the Aβ + 100 µM AOAA control

Different doses of the CBS inhibitor aminoxyacetic acid (AOAA) were pre-treated to the cells for 24 hours before the additional Aβ25-35 insult for 16 hours (Figure 48a) Similar to Part II: Oligomeric Aβ, there was a visible, significant dose-dependent aggravation in the cell viability with increasing doses of AOAA The Aβ-only control group resulted in cell viability of 64 ± 4.6%

(p<0.01) but this was aggravated to 50 ± 2.6% in the group with additional 100 µM AOAA

(p<0.05) While increasing doses of AOAA in normal cells without Aβ25-35 did not significantly decline in cell viability, there was a slight cytotoxicity at 100 µM AOAA with 87 ± 2.2%

(µM) - - - 0.1 1 10 100

was a gradual, dose-dependent increase in cell viability following SPRC treatment (Figure 48b), although the restoration in cell viability was not to a great extent as in Part II: Oligomeric Aβ The maximum restoration was observed in the pre-treatment from 10 µM SPRC onwards where the Aβ-induced cytotoxicity was significantly increased from 50 ± 3.0% in the Aβ + AOAA-

control to 59 ± 7.6% (p<0.05) Likewise, 100 µM SPRC also increased the cell viability to 60 ± 7.2% (p<0.05) significantly Even with SPRC pre-treatment, the cell viability could not be

restored to the levels of the Aβ-only control

6.1.4 Effects on oxidative stress

6.1.4.1 Production of DCF-DA

Figure 49: Effects of pre-treatment of drugs on DCF production, presented as percentage control

± S.E.M N=5, #: p<0.05 compared to untreated control; *: p<0.05, **: p<0.01 compared to

Aβ-only group

Aβ treatment drastically increased the oxidative stress seen in the increased DCF

produced (Figure 49) to 214 ± 15% compared to the 100% in the untreated control (p<0.01)

This is similar to Part II: Oligomeric Aβ and the oxidative stress was also significantly altered (F

(4, 24) = 10.34; p<0.01) After pre-treatment with the various drugs, the oxidative stress induced

by Aβ25-35 in the cells was generally reduced Pre-treatment with 10 µM SPRC significantly

NaHS 10 µM

reduced the DCF produced to 128 ± 12% (p<0.01) Likewise, the pre-treatment with 10 µM SAC significantly reduced the DCF produced to 112 ± 13% (p<0.01) Pre-treatment with NaHS also significantly reduced the production of DCF to 137 ± 12% (p<0.05)

Figure 50: Effects of pre-treatment of drugs on DHE fluorescence (a) Representative photos of DHE-stained cells under 20X magnification with a scale bar of 100 µm indicated at the lower left

of each panel I: SFM-only control; II: 1 µM Aβ-only control; III: 10 µM SPRC + 1 µM Aβ; IV:

10 µM SAC + 1 µM Aβ; V: 10 µM NaHS + 1 µM Aβ (b) Fold difference of fluorescence units

as calculated from the DHE staining ± S.E.M N=4, #: p<0.05 compared to untreated control

The free radicals levels visualized in cells were decidedly different following Aβ

treatment (F (4, 19) = 3.810; p<0.05) (Figure 50a) Aβ25-35 typically increases the intracellular free radical levels that contribute to increased oxidative stress, similarly observed using the dye DCF-

DA presented in Part 6.1.4.1 As such, the panels treated with Aβ25-35 (Figure 50a, Panels II-V) showed stronger red fluorescence compared to the untreated control (Figure 50a, Panel I), where weak red fluorescence represented the background free radical levels The Aβ-only group

recorded a significantly higher 1.26 ± 0.07- fold of red fluorescence (p<0.05) that were reversed

by pre-treatment of any drugs (Figure 50b) While all drugs slightly reduced the fold differences, none were significant statistically SPRC pre-treatment resulted in a 1.18 ± 0.06-fold, SAC pre-

treatment resulted in a 1.18 ± 0.05-fold (p=N.S) and NaHS pre-treatment resulted in a 1.22 ± 0.05-fold (p=N.S)

0 0.2 0.4 0.6 0.8 1 1.2 1.4

µM

SAC 10 µM

NaHS 10 µM

6.1.4.3 Expression of antioxidant enzymes

Figure 51: Effects of pre-treatment of drugs on SOD-1 expression in cell lysates (a)

Representative blots for SOD-1 expression in treated cells (b) Percentage fold difference in SOD-1 expression ± S.E.M N=6 (c) Effects of pre-treatment of drugs on total SOD activity The values are expressed as a percentage of the activity in the untreated cells ± S.E.M N=5, #:

p<0.05 compared to untreated control; *: p<0.05, **: p<0.01 compared to Aβ-only group

The SOD-1 expression in the cells changed significantly after the various drug treatments (F (4, 20) = 23.336; p<0.05), suggesting an alteration to the amounts of SOD-1 in the cell lysates

(Figure 51a) Aβ-only treatment significantly reduced the SOD-1 expression to 0.80 ± 0.02-fold

compared to the untreated control (p<0.01) (Figure 51b), accounting for the oxidative stress

observed The pre-treatment of all three drugs positively up-regulated the SOD-1 expression; and

oxidative stress observed The total SOD activity changed significantly after the various drug treatments as calculated from one-way ANOVA (F(4, 24) =5.677; p<0.01) Pre-treatment with all

three drugs significantly restored the total SOD activity comparable to the untreated control, and the effects were equal amongst the drugs This trend was similar to that observed in the SOD protein expressions SPRC pre-treatment significantly restored the SOD activity to 91 ± 3.4%

(p<0.05) while SAC pre-treatment significantly restored the activity to 92 ± 6.7% (p<0.05) NaHS pre-treatment also restored the activity to 93 ± 2.2% (p<0.05)

Figure 52: Effects of pre-treatment of drugs on catalase expression in cell lysates (a)

Representative blots for catalase expression in treated cells (b) Percentage fold difference in catalase expression ± S.E.M N=6 (c) Effects of pre-treatment of drugs on catalase activity The

values are expressed as units of activity/µg protein ± S.E.M N≥3 #: p<0.05 compared to

untreated control; *: p<0.05 compared to Aβ-only group; **: p<0.01 compared to Aβ-only

group

The catalase expression following Aβ25-35 treatment was significantly altered within the different drug groups (F (4, 17) = 7.76; p<0.01) (Figure 52a) Catalase is slightly but significantly up-regulated after the Aβ-only treatment to 1.07 ± 0.01-fold (p<0.05) (Figure 52b) Pre-

10 µM

SAC 10 µM

treatment of 10µM SPRC significantly reversed this Aβ-induced up-regulation to 0.84 ±

0.004-fold (p<0.01) While the same trend was observed following SAC pre-treatment, the decrease to 0.85 ± 0.12- fold was not statistically significant (p=N.S) In contrast, NaHS pre-treatment resulted in even higher expression of catalase (1.27 ± 0.04- fold; p<0.01) that suggested a

different mode of regulation on this antioxidant enzyme compared to other drugs of interest Catalase activities were measured spectrophotometrically and were significantly increased after all drug treatments (F (4, 16) = 8.572; p<0.01) (Figure 52c) There was significant increase after

Aβ treatment to 0.036 ± 0.001 U/µg protein (p<0.05), from the 0.019 ± 0.001 U/µg protein in the

untreated control group Interestingly, pre-treatments with the various drugs further increased the catalase activity, most obviously seen in the SAC pre-treated group Pre-treatment with SPRC

increased the activity to 0.043 ± 0.004 U/µg protein (p=N.S) while SAC pre-treatment increased the activity to 0.048 ± 0.005 U/µg protein (p=N.S) NaHS pre-treatment also very slightly

increased the activity to 0.038 ± 0.006 U/µg protein (p=N.S) Although no significance was

observed in all pre-treated groups, the increase in antioxidant catalase activity may be an

inherent nature of the drugs to counter the increase in oxidative stress from the Aβ treatment

Figure 53: Effects of pre-treatment of drugs on glutathione peroxidase (GPx) expression in cell lysates (a) Representative blots for GPx expression in treated cells (b) Percentage fold

difference in GPx expression ± S.E.M N=6 (c) Effects of pre-treatment of drugs on GPx

activity The values of activity are expressed as µmol/NADPH/min/mg protein ± S.E.M N=3, #:

p<0.05 compared to untreated control; *: p<0.05 compared to Aβ-only group

Drug treatments altered the expressions of GPx significantly (F (4, 18) = 6.054; p<0.01)

among the groups (Figure 53a) While the fold difference in GPx expression was significantly

lowered by Aβ-only treatment to 0.82 ± 0.05-fold (p<0.05) (Figure 53b), this decline in

expression was not restored by pre-treatment of any of the tested compounds Pre-treatment with

10 µM SPRC resulted in 0.70 ± 0.04-fold expression (p=N.S) while 10 µM SAC resulted in a comparable 0.73 ± 0.08-fold expression (p=N.S) Interestingly, 10 µM NaHS resulted in a

further dip in expression to 0.64 ± 0.09-fold (p=N.S) GPx activity changed significantly after

treatments with Aβ25-35 and the drugs of interest (F (4, 14) = 14.246; p<0.01) (Figure 53c) The

activity decreased drastically after Aβ treatment, from 3.28 ± 0.25 µmol/NADPH/min/mg

10 µM

SAC 10 µM

protein in the untreated control to 1.39 ± 0.18 µmol/NADPH/min/mg protein in the Aβ-only control There was a stark restoration in GPx activity following 10 µM SPRC pre-treatment to

3.38 ± 0.37 µmol/NADPH/min/mg protein (p<0.05) that was not observed in other groups treatment with SAC remained at 1.36 ± 0.38 µmol/NADPH/min/mg protein (p=N.S) and NaHS resulted in an activity of 1.29 ± 0.41 µmol/NADPH/min/mg protein (p=N.S)

Pre-6.1.5 Effects on inflammation

6.1.5.1 Expression of pro- inflammatory IL-1 β

Figure 54: Effects of pre-treatment of drugs on IL-1β expression (a) mRNA expression of IL-1β expressed as fold difference of the untreated control group ± S.E.M (b) Protein expression of IL-

1β expressed as concentration (pg/ml) ± S.E.M N≥3, #: p<0.05 compared to untreated control;

**: p<0.01 compared to Aβ-only group

The mRNA and protein expressions of the pro-inflammatory IL-1β altered significantly after Aβ treatment Aβ-only treatment significantly increased the mRNA expression of IL-1β to

7.08 ± 0.73-fold (p<0.01) and this increase was reversed by pre-treating the cells with 10 µM

SPRC or SAC (Figure 54a) SPRC pre-treatment reduced IL-1β mRNA expression to 3.27 ±

0.32-fold (p<0.01), and SAC pre-treatment resulted in an even lower expression of 2.43 ± fold (p<0.01) 10 µM NaHS pre-treatment slightly reduced the mRNA expression to 6.40 ± 1.01- fold (p= N.S), although it was not statistically significant

0.20-The concentration of IL-1β in the cell lysates after Aβ treatment was also significantly increased from 1689 ± 84.7 pg/ml in the untreated control to 1945 ± 44 pg/ml in the Aβ-only

control (p<0.05) (Figure 54b) Pre-treatment with the drugs significantly decreased the IL-1β

expressions in a similar manner 10 µM SPRC expressed the least pro-inflammatory IL-1β to

1484 ± 49 pg/ml (p<0.01) IL-1β expressions following SAC or NaHS treatments were

10 µM

SAC 10 µM

comparable; SAC resulted in an expression of 1519 ± 58 pg/ml (p<0.01) and NaHS resulted in

an expression of 1513 ± 71 pg/ml (p<0.01)

6.1.5.2 Expression of pro- inflammatory IL-6

Figure 55: Effects of pre-treatment of drugs on IL-6 expression (a) mRNA expression of IL-6 expressed as fold difference of the untreated control group ± S.E.M (b) Protein expression of IL-

6 expressed as concentration (pg/ml) ± S.E.M N≥3, #: p<0.05 compared to untreated control; *:

p<0.05 compared to to Aβ-only group

Both the mRNA and protein expressions of the pro-inflammatory IL-6 were up-regulated

after Aβ treatment The mRNA expression of IL-6 increased to 1.69 ± 0.11-fold (p<0.01) (Figure

55a) Of note, pre-treatment with drugs enhanced the mRNA expression of IL-6, especially after SPRC pre-treatment The IL-6 mRNA expression was 3.71 ± 0.68- fold, although there was no statistical significance Likewise, SAC and NaHS pre-treatments increased the mRNA

expressions to 2.29 ± 0.12- fold (p<0.05) and 1.85 ± 0.21- fold respectively

The protein expression of IL-6 was also augmented by Aβ treatment, from 16.8 ± 4.4

pg/ml in the untreated control to 35.2 ± 4.8 pg/ml in the Aβ-only control (p<0.05) (Figure 55b)

10 µM

SAC 10 µM

respectively Though not statistically significant, SAC pre-treatment resulted in a slightly higher expression of 43.8 ± 2.5 pg/ml

6.1.5.3 Expression of pro- inflammatory TNF- α

Figure 56: Effects of pre-treatment of drugs on TNF-α expression (a) mRNA expression of TNF-α expressed as fold difference of the untreated control group ± S.E.M (b) Protein

expression of TNF-α expressed as concentration (pg/ml) ± S.E.M N≥3, #: p<0.05 compared to untreated control; *: p<0.05 compared to Aβ-only group **: p<0.01 compared to Aβ-only

group

The mRNA and protein expressions of pro-inflammatory TNF-α were significantly increased after Aβ treatment (Figure 56) The mRNA expression was increased to 2.20 ± 0.27-

fold in the Aβ-only group (p<0.05) and further increased after pre-treatment with the various

drugs (Figure 56a) Pre-treating the cells with 10µM SPRC or SAC slightly enhanced the TNF-α

mRNA expression to 2.50 ± 0.13- fold (p=N.S) and 3.10 ± 0.34- fold (p=N.S) respectively

Pre-treatment with 10 µM NaHS however, drastically increased the mRNA expression to 4.71 ±

0.29- fold (p<0.01)

Likewise, the protein expression of TNF-α in the cell lysates was increased following Aβ treatment, from 53.2 ± 7.3 pg/ml in the untreated control group to 88.9 ± 5.7 pg/ml in the Aβ-only control (Figure 56b) While both SAC and NaHS pre-treatment did not affect the protein expression, the concentration of TNF-α in the cell lysates following SPRC pre-treatment was

10 µM

SAC 10 µM

decreased to 59.6 ± 9.1 pg/ml (p<0.05) SAC and NaHS pre-treatment resulted in 102.0 ± 7.1 pg/ml expression (p=N.S) and 94.9 ± 7.6 pg/ml expression (p=N.S) respectively

6.1.5.4 Expression of anti-inflammatory IL-10

Figure 57: Effects of pre-treatment of drugs on IL-10 expression (a) mRNA expression of IL-10 expressed as fold difference of the untreated control group ± S.E.M (b) Protein expression of IL-

10 expressed as concentration (pg/ml) ± S.E.M N≥3, #: p<0.05 compared to untreated control; *:

p<0.05 compared to Aβ-only group **: p<0.01 compared to Aβ-only group

The expressions of the anti-inflammatory IL-10 were reduced drastically after Aβ

treatment The mRNA expression of IL-10 dropped significantly to 0.25 ± 0.02- fold in the

Aβ-only group (p<0.01) (Figure 57a) However, the IL-10 expression was restored after

pre-treatment with SPRC or SAC Pre-pre-treatment with SPRC increased the mRNA expression of

IL-10 to 0.69 ± 0.07- fold (p<0.05) while pre-treatment with SAC further increased the mRNA expression to 0.81 ± 0.06- fold (p<0.01) In contrast, the NaHS pre-treatment had little effect on

up-regulating IL-10 expression, with only a 0.26 ± 0.07-fold expression

IL-10 protein expression in the cell lysates significantly declined after Aβ treatment from

10 µM

SAC 10 µM

the IL-10 expression significantly to 98.7 ± 2.4 pg/ml (p<0.05) Likewise, NaHS also increased the IL-10 expression to 94.9 ± 0.5 pg/ml (p=N.S), although this was not statistically significant

6.1.6 Effects on cell death mechanisms

6.1.6.1 Cell cycle analysis

Figure 58: Effects of pre-treatment of drugs on cell cycle status (a) Pre-treatment of 10 µM of each drug altered the cell cycle profile of the glioma cells; especially obvious declines in

percentages of cells the sub-G1 phase and a slight increase in cells at S-phase (b) Percentage of cells in the sub-G1 phase for each treatment group, where pre-treatment of drugs significantly

decreased the sub-G1 cell population N=3, #: p<0.05 when compared to the untreated control; *:

p<0.05 when compared with the Aβ control; **: p<0.01 when compared with the Aβ control

The observation of changes in cell viability following drug pre-treatment suggested alterations to the cell cycle status, determining the amount of cells rescued by the pre-treatment regimes Cell cycle status was significantly affected by both pre-treatment and subsequent Aβ insult (Figure 58a), where the largest variation was observed at the R1 region (F (4, 19) = 7.782;

p<0.01) Aβ-only control resulted in about twice the percentage of cells at the sub-G1 region (Figure 58b) of 0.57 ± 0.01% of total cells, compared to 0.31 ± 0.05% in SFM-only control This increase in percentage of cells in the sub-G1 region was significantly declined by pre-treatment

of SPRC, SAC or NaHS The largest decrease was observed in the SPRC-treated cells, where

only 0.20 ± 0.01% (p<0.01) of cells were in the sub-G1 region Likewise, SAC could reverse the Aβ-induced increase to 0.33 ± 0.06% (p<0.05), and NaHS resulted in 0.27 ± 0.05% (p<0.01)

Notably, even though there were fluctuations in sub-G1 cell populations, the fluctuations were very small There was, however, an observable alteration of the cell population in S phase; though it was not found to be statistically different (F (4, 19) = 1.209; N.S) This showed a slight shift towards cell cycle progression after pre-treatment as there were decreases in percentage of cells in G1phase and increases in the S or G2/M phase

6.1.6.2 Apoptosis

(a)

Figure 59: Effects of pre-treatment of drugs on DNA fragmentation using TUNEL staining (a) Representative photos of drug-treated groups at 20X magnification where fragmented DNA is stained green and indicated with white arrows A scale bar of 100 µm is indicated at the lower left of each panel The nucleus is counter-stained with DAPI and presented as blue fluorescence I: Untreated control; II: Aβ-only control; III: SPRC-treated group; IV: SAC-treated group; V: NaHS-treated group (b) Proportion of green/blue

fluorescence ± S.E.M, normalized to the untreated control N=3, #: p<0.05 compared to untreated control; *: p<0.05, **: p<0.01

compared to Aβ-only group

0 0.5 1 1.5 2 2.5 3

DNA fragmentation was detected by TUNEL staining and statistically different among all treatment groups (F (4,14) = 12.448; p<0.01) The treatment period for all groups was longer

than that in Part II: Oligomeric Aβ, and that might account for a higher amount of apoptotic cells observed in the SFM-only group (Figure 59a, Panel I) Nonetheless, Aβ treatment resulted in a significantly higher proportion of apoptosis as indicated with a larger number of cells stained green (Figure 59a, Panel II) This proportion was decreased when the cells were pre-treated with drugs, where fewer cells stained green was observed (Figure 59a, Panels III- V) However, while all pre-treatments decreased the proportion of apoptosis, SPRC and SAC pre-treatments resulted

in significantly lowered proportion which was not observed in the NaHS pre-treated group, in which a proportion of 1.86 ± 0.22 was obtained (Figure 59b) The SPRC-treated group resulted

in a proportion of 1.29 ± 0.11 (p<0.01), slightly lower than the proportion of 1.49 ± 0.04 (p<0.05)

in the SAC-treated group The calculated proportions of the drug- treated groups were almost halved of that in the Aβ-only group with a proportion of 2.32 ± 0.22

Figure 60: Effects of pre-treatment of drugs on PARP and pro-caspase 3 expressions in cell lysates (a) Representative blots for PARP and pro-caspase 3 expressions in treated cells N=6 (b)

Fold difference in pro-caspase 3 expression ± S.E.M #: p<0.05 compared to untreated control; *:

p<0.05 compared to Aβ-only group

PARP cleavage was detected using Western blotting, done similarly in Part II:

Oligomeric Aβ However, the cleaved products at 87 kDa could not be visualized clearly

although the gel concentrations and antibody dilutions were kept constant (Figure 60a) Only the

116 kDa uncleaved PARP protein could be seen There was a distinctive decrease in the 116 kDa protein after Aβ treatment for 16 hours This decrease was also seen in the SAC-treated group, and to a lower extent, in the NaHS-treated group Pre-treatment with SPRC blocked this decline

as the density of the 116 kDa band remained comparable to the SFM-only control group

Changes in the pro-caspase 3 expressions were also examined (Figure 60a) Cell lysates obtained

from Aβ-treated cells showed a slight but visible decrease to 0.82 ± 0.04-fold (p<0.05) of the

control amount (Figure 60b), while all other treated groups displayed increases in the amount of pro-caspase 3 Pre-treatment with SAC significantly restored the pro-caspase 3 amounts to 1.00

± 0.03-fold (p<0.05) and pre-treatment with SPRC resulted in a significant increase to 0.97 ± 0.03-fold (p<0.05) of the control amount NaHS pre-treatment increased the protein amount the least to 0.91 ± 0.07-fold (p<N.S)

0 0.2 0.4 0.6 0.8 1 1.2

µM

SAC 10 µM

NaHS 10 µM

6.1.6.3 Acridine orange staining

(a)

V

Figure 61: Effects of pre-treatment of drugs on autophagy using acridine orange staining (a) Representative photos of drug-treated groups at 60X magnification with a scale bar of 20 µm indicated at the lower left of each panel Acridine orange enters the cell membrane to stain the cytoplasm and nucleus green, but stains the acidic autophagic vacuoles orange I: Untreated control; II: Aβ-only control; III: SPRC-treated group; IV: SAC-treated group; V: NaHS-treated

group (b) Proportion of orange/green fluorescence ± S.E.M, n=10, #: p<0.05 compared to untreated control; **: p<0.01 compared to Aβ-only group

The effects of autophagy on the cells are investigated using confocal microscopy and the fluorescence was presented in Figure 61 The proportion of orange/fluorescence had changed significantly after the various drug treatments (F (4,50)= 12.593; p<0.01) (Figure 61b) Unlike that

in Part II: Oligomeric Aβ, Aβ-only treatment reduced the autophagic vacuoles observed to 0.76 ±

0.02 (p<0.01), compared to 1.05 ± 0.05 in the untreated control cells The untreated control cells

demonstrated bright orange dots lining the cytoplasm while avoiding the nucleus and such

fluorescence vacuoles appeared to be diffuse but numerous (Figure 61a, Panel I) After Aβ treatment, the number of orange vacuoles reduced in both intensity and number, although some fluorescence can still be observed (Figure 61a, Panel II) Conversely, SPRC pre-treatment

restored the autophagic vacuoles in the cells that could be seen from the intense orange dots in the cell cytoplasm (Figure 61a, Panel III) The restoration was effective such that the proportion

0 0.2 0.4 0.6 0.8 1 1.2

SFM SFM SPRC 10

µM

SAC 10 µM

NaHS 10 µM

of orange/green fluorescence was 1.06 ± 0.05 (p<0.01) This restoration was not observable in SAC-treated cells (Figure 61a, Panel IV), where the proportion remained at 0.73 ± 0.05 (p= N.S)

The NaHS-treated group demonstrated some orange fluorescence, but such fluorescence

vacuoles are few in number compared to the untreated control cells (Figure 61a, Panel V) The

proportion in the NaHS-treated cells was slightly elevated at 0.83 ± 0.05 (p= N.S) but not

statistically significant

Figure 62: Effects of pre-treatment of drugs on LC3 expression in cell lysates (a) Representative blots for LC3 expression in treated cells N=6 (b) Fold difference in LC3 expression ± S.E.M *:

p<0.05 compared to Aβ-only group

The 18 kDa LC3-I expression altered significantly after drug treatment to the cells (F (4, 21)

= 21.882; p<0.01) (Figure 62a) The LC3-I expression slightly decreased after Aβ treatment - a

manifestation of the reduced orange vacuoles observed in the cells (Figure 62a, Panel II) - to

0.83 ± 0.02-fold (p<0.01) (Figure 62b) Such decrease was reversed by pre-treatment of all the

drugs of interest, most significantly in the SPRC-treated cells In accordance to the confocal microscopy results (Figure 61a, Panel III), the LC3-I expression has increased significantly to

1.30 ± 0.05-fold (p<0.01) (Figure 62b) Although there were less acridine orange fluorescence

observed in the SAC-treated cells, the LC3-I expression was increased to 1.23 ± 0.08-fold

(p<0.05) Moreover, the NaHS-treated cells also showed an elevated LC3-I expression of 1.01 ± 0.04-fold (p<0.05)

0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6

µM

SAC 10 µM

NaHS 10 µM

Figure 63: Effects of pre-treatment of drugs on the ultrastructure of the cells using transmission electron microscopy at lower

magnification Representative photos of cells from each group were compared with similar magnifications, scale bar of 5 µm indicated

although parts of the projections can be seen in the irregularly-shaped cell The plasma membrane (PM) and nuclear membrane (NM) are intact with the cytoplasm healthy There is an abundance of mitochondria (M) observed in the cell, but other organelles could not

be identified There were few whorl-like structures (W) and vacuoles (V) observed In contrast, larger vacuoles containing materials were found in the Aβ-treated cell (Figure 63, Panel II) with few membranous whorls (W) isolated Although the plasma membrane (PM) looked intact with little chromatin condensation, the relative number of mitochondria (M) decreased and part of the disrupted endoplasmic reticulum (ER) was identified There was noticeable condensation and slight invagination of the nuclear membrane (NM) with condensed chromatin found in the nucleus

SPRC pre-treatment improved the overall appearance of the cell (Figure 63, Panel III), with complete plasma (PM) and nuclear membranes (NM) Some chromatin condensation can be seen in the nucleus, but the mitochondria (M) appeared numerous and healthy

in shape Most of the mitochondria associated with small vacuoles but no internalization was observed Some vacuolization can be observed too, but such vacuoles (V) were smaller and did not contain materials compared to that in the Aβ-treated cells A few whorl-like structures can be seen too, but they were not obvious in size There were considerably fewer vacuoles (V) after SAC pre-treatment (Figure 63, Panel IV), though the whorl-like structures increased in number The nuclear membrane (NM) was slightly condensed and invaginated while part of the plasma membrane (PM) was detached with some membranous whorls However, the mitochondria remained healthy-looking and abundant in the cell NaHS pre-treatment noticeably resulted in less robust cells (Figure 63, Panel V), with one of the cells heavily vacuolized The other cell showed condensation of the nuclear membrane (NM) and both cells had intact

plasma membranes (PM) While most of the vacuoles (V) were empty, some of the detected vacuoles also contain materials (VC) in them The mitochondria (M) were drastically few in number, and few membranous whorls (W) can be detected

Figure 64: Effects of pre-treatment of drugs on the ultrastructure of the cells using transmission electron microscopy at higher magnification Representative photos of cells from each group were compared with similar magnifications, scale bar of 2 µm indicated at the lower left of each photo ER: Endoplasmic reticulum; M: Mitochondria; NM: nuclear membrane; PM: plasma membrane; V: vacuole; VC: vacuole containing materials; W: whorl-like structure I: SFM-only control (9700X); II: Aβ-only control (8900X, scale bar of 0.5µm indicated); III: SPRC-treated group (9700X); IV: SAC-treated group (9700X); V: NaHS-treated group (9700X)

Larger magnification of the cell enables the comparison of the ultrastructure of the

treated groups The SFM-only group (Figure 64, Panel I) demonstrated both intact nuclear (NM) and plasma membranes (PM) There are many regularly-shaped mitochondria (M) detected in the cytoplasm with only a few vacuoles (VC) seen Vacuoles containing materials (VC) can also be

group Interestingly, some of such large vacuoles contain organelles, and even membranous whorls (W) Moreover, the presence of vacuoles containing materials was also seen in some large vacuoles The cytoplasm near the nuclear membrane (NM) showed a small degree of vacuolization, where numerous small vacuoles (V) were developing The nuclear membrane (NM) appeared quite complete with a little invagination and condensation compared to that in the untreated cells There are some slightly enlarged mitochondria (M) and even remnants of the endoplasmic reticulum (ER) that could be seen in the cell

The pre-treatment of SPRC reduced the appearance of large vacuoles (Figure 64, Panel III), instead, smaller-sized vacuoles (V) which do not contain any materials were found Slight condensation of the nuclear membrane (NM) is accompanied by some chromatin condensation The mitochondria (M) did not show any signs of swelling, but some degree of vacuolization can still be observed at the cellular region near the nucleus Some membranous whorls can also be found although such whorls are drastically fewer than in Part II: Oligomeric Aβ The SAC pre-treatment did not show as many small vacuoles (V) as observed (Figure 64, Panel IV) in the SPRC-treated group, but there were more vacuoles containing materials (VC) Several whorls (W) can also be found distributed in the cell The intact nuclear membrane (NM) and plasma membrane (PM) were accompanied by several mitochondria (M) The NaHS-treated group showed substantial vacuolization compared to the other treatment groups (Figure 64, Panel V) The vacuoles (V) observed were large in size with some containing materials (VC) The

mitochondria (M) appeared slightly swollen and the nuclear membrane (NM) was invaginated with much condensation observed in the chromatin No membranous whorls could be identified

in the cell

6.2 Discussion

6.2.1 Higher incubation temperature encourages the formation of Aβ25-35 fibrils

Part III aims to understand how increasing Aβ25-35 aggregation would affect glial cell viability The motivation for this Part IIs straightforward: incubating Aβ at 37°C mimics the actual physiological condition and produces Aβ25-35 fibrils that could give us a better insight to the behavior of the insoluble peptide on glial cells This experimental protocol also allows us to compare and contrast the neurotoxicity of the peptide with that in Part II and hopefully, predict the efficacy of drugs in different pathological situations

1µM Aβ was incubated for 24 hours in both protocols; only the temperature of incubation had been changed from 4°C to 37°C, the physiological body temperature in the latter The

concentration of Aβ incubated will affect the total concentration of fibrils formed while the incubation time can influence the fibrillization status that may eventually determine its toxicity

As such, both factors had been kept at a constant incubation time to prevent confounding the effects of changing temperature Although the temperature-dependence is assumed to follow a linear relationship that changes proportionally with incubation time, the exact relationship was not shown in this instance The change in aggregation status at 4°C cannot be tracked in a time-dependent manner similar to that in 37°C since the equipment (Varioskan Flash) used does not allow prolonged incubation at 4°C over a period of time As such, although the end-point

aggregation state of Aβ had been dramatically increased after 96-hour incubation at 37°C

However, the end-point toxic products had been shown in Part I to increase by about 20% and that gave an indication of the nature of the differently -aggregated Aβ in different

temperatures The aggregated Aβ was then applied to the cell culture studies to help identify difference in mechanisms of action of the drugs The effects of aging Aβ25-35 at different

temperatures had not been greatly investigated on glial cells and the current results may help to elucidate this knowledge gap

6.2.2 SPRC confers protection after longer pre-treatment at higher dose

The dose of fibrillar Aβ was kept at 1µM and applied to glioma cells to determine the cell viability at different time points The cell viability was significantly reduced to about 83% after just three hours of incubation By 16 hours, the Aβ -induced reduction in cell viability was 61%, compared to the 69% viability after 24 hours in Part II This implied that despite using equimolar concentrations of Aβ, the aggregation protocol in Part II This also resulted in a more toxic form

of Aβ that could reduce the cell viability faster and to a larger extent than in Part II This

augments and confirms the temperature-dependent toxicity of Aβ seen in other in vitro and in vivo models The 16-hour incubation period for Aβ was then chosen as the model of Aβ injury in Part III

Generally, pre-treating the C6 glioma cells with SPRC, SAC or NaHS could restore such

Aβ -induced cytotoxicity However, the pre-incubation period was longer than used in Part II before significant changes were visible with the treatments Both SPRC and SAC significantly restored the viability after a 24-hour pre-treatment period in a dose-dependent fashion In

contrast to the 12-hour pre-treatment conditions in Part II, the improvements in cell viability by both were not as large in Part III The best dose of SPRC was observed at 10µM, though the