RESULTADOS PRIMER PISO DE INDUSTRIAL 1 Resultados de la sala de red 1.

nmol/min/mg II+II nmol/min/mg IV k/min/mg CS nmol/min/mg con (+) 14.5 + 2.0 15.2 + 0.7 2.0+ 0.0 196.9+19.2 LPS/IFN 15.6 + 3.3 12.8+1.4 1.8+ 0.1 190.7 + 21.2 BSO 15.7 + 2.1 13.1+2.3 1.9+ 0.3 227.1 +28.1 LPS/IFN/BSO 19.2+1.3 10.8 + 0.4* 1.5 ± 0 .1 * ' 188.3 + 26.6

Table 6.1. ETC complex activity in neurones cocultured with astrocytes

Neurones were incubated with untreated astrocytes (con (+)), activated astrocytes (LPS/IFN), GSH depleted astrocytes (BSO), or activated astrocytes depleted of GSH (LPS/IFN/BSO) for 24 hours, and ETC complex activity determined in neuronal homogenates. Complex 11+111 was significantly lower in neurones cocultured with activated astrocytes depleted of GSH compared to neurones cocultured with untreated astrocytes, while complex IV activity was significantly lower in neurones cocultured with activated astrocytes depleted of GSH compared to neurones cocultured with untreated or activated astrocytes. Values are mean ± SEM (n=4-6 independent cell preparations). Statistical significance was determined by one-way ANOVA followed by a least significant difference test. * p < 0.05 compared to con (+); ^ p < 0.05 compared to LPS/IFN.

6 4.2.2. ETC complex activities in neurones cocultured with astrocytes

The ETC complex activities were determined in neuronal homogenates cocultured with astrocytes (Table 6.1) A significant 29% loss of Complex II+III activity was observed in neurones cocultured with activated astrocytes depleted of GSH, compared to neurones cocultured with untreated astrocytes. Complex IV activity was significantly reduced by 24% and 17% in neurones cocultured with activated astrocytes depleted of GSH, compared to neurones cocultured with untreated or activated astrocytes respectively. No loss of complex I or citrate synthase activity was observed following incubation with activated astrocytes depleted of GSH.

No increase in LDH activity was observed in neurones cocultured with activated astrocytes (4.2 ±1. 1 %LDH release), GSH depleted astrocytes (4.0 ± 2.2 %LDH release), or activated astrocytes depleted of GSH (4.4 ± 1 . 5 %LDH release), compared to neurones cultured with untreated astrocytes (2.2 ± 0.4 %LDH release; n=4 independent cell preparations).

6.5. Discussion

The increased GSH levels observed in neurones cocultured with untreated or activated astrocytes, compared to neurones cultured alone, are consistent with previous studies (Sagara et al., 1993; Bolanos et al., 1996; Dringen et al., 1999a). The results in this study suggest that the elevation of GSH levels in cocultured neurones is dependent on GSH release by astrocytes, since GSH levels were not elevated in neurones when they were cocultured with GSH depleted astrocytes that released very little GSH. The elevation of GSH levels in cocultured neurones was not concomitant with an increase in neuronal GCL activity, indicating that the release of GSH by astrocytes alone maybe sufficient to increase neuronal GSH levels. The lack of GSH released by astrocytes will significantly lower the amount of CysGly available to neurones, and may explain why neurones cannot increase GSH levels under these conditions. Dringen et al (1999a) have reported than inhibition of y-GT, the ectoenzyme that metabolises GSH to CysGly, abolishes the elevation of neuronal GSH levels when cocultured with astrocytes, further supporting the hypothesis that CysGly is necessary for elevated neuronal GSH

levels. However, it should be noted that incubation of neurones with 100 p.M CysGly for 24 hours in this chapter did not result in an increase in neuronal GSH levels. An explanation for this maybe that an increase in neuronal GSH levels may occur at an earlier time point, and that by 24 hours, all the CysGly has been exhausted {i.e., taken up by neurones, oxidised to CysGly disulphide), and GSH levels have returned to basal levels. GSH levels in neurones need to be determined at earlier time points to see if this is the case. Neuronal GSH levels have been reported to double following incubation with 100 pM CysGly for 4 hours (Dringen et al., 1999a).

The present results do not indicate whether the supply of cysteine to neurones in coculture is due to the reduction of cystine by GSH released from astrocytes (Figure 1.7, route 1; Wang & Cynader et ah, 2000), or metabolism of extracellular GSH by y-GT to supply CysGly (Figure 1.7, route 2; Dringen et al.,

1999a). However, it should be noted that the experiments investigating the conversion of cystine to cysteine by extracellular GSH were performed in culture media containing 33 mM glucose (Wang & Cynader, 2000), which is far greater than the physiological concentration of approximately 5 mM. Furthermore, the rate of GSH release from these cultured rat cortical astrocytes was at least 5-fold lower than reported by others (Sagara et al., 1996; Dringen et al., 1997a; Stone et al., 1999). The observed loss of cystine from the cell culture media by Wang & Cynader (2000) could be accounted for by astrocytic uptake, rather than conversion to cysteine, while the increased cysteine levels could be due to hydrolysis of CysGly. No experiments were performed in the presence o f acivicin, an inhibitor of y-GT, to see if this had any effect on extracellular GSH and cysteine levels.

GCL activity in neurones was unchanged when incubated with 100 pM CysGly, or cocultured with either untreated or activated astrocytes. However, neuronal GCL activity was increased by more than 2-fold when cocultured with either GSH depleted astrocytes or activated astrocytes depleted of GSH. Since GCL activity was increased by a similar amount in both cases, it would suggest that this observation is a result of incubation with astrocytes depleted of GSH, rather than

exposure to NO. Indeed, the previous chapter of this thesis (chapter 5) has indicated that neurones exposed to the NO donor DETA-NO do not increase GCL activity.

The results also suggest that the elevation in GCL activity maybe due to a signal released from the GSH depleted astrocytes, rather than just a response to a lack of GSH or GSH precursors {e.g., CysGly), since GCL activity in neurones cultured alone, which in theory are exposed to very little GSH or CysGly, had similar GCL activity to that of neurones cocultured with untreated astrocytes or activated astrocytes. It is unclear whether the increased GCL activity observed in neurones cocultured with GSH depleted astrocytes is due to increased expression o f the enzyme or a posttranslational modification of the enzyme. Determination of GCL activity at earlier time points {e.g., 1 hour) and measurement of GCL mRNA levels at several time points may indicate the probable reason for the increase in enzyme activity.

Astrocytes release a variety of signalling molecules such as cytokines {e.g.,

interleukin Ip), tumour necrosis factor (TNF), and neurotrophins {e.g., brain derived neurotrophic factor (BDNF), glial derived neurotrophic factor (GDNF); Mollace et al., 1998; McNaught & Jenner, 2000b). Interleukin Ip, TNF, and certain neurotrophic factors (e.g., nerve growth factor) have been reported to increase expression of GCL (Pan & Perez-Polo, 1993; Ikegami et al., 2000; Soltaninassab et al., 2000). Therefore, the depletion o f GSH in astrocytes may prevent or induce the release of a particular signalling molecule. Indeed, astrocytes depleted of GSH have been reported to lower the release of BDNF and GDNF (McNaught & Jenner, 2000b). Furthermore, neurones incubated in astrocyte-conditioned media, but not neuronal media, are able to increase GSH levels and GCL expression upon exposure to hydrogen peroxide (Iwata-Ichikawa

et al., 1999).

Alternatively, the depletion of GSH in astrocytes may increase the amount of reactive oxygen species within the cell, which may be released, and act as a signal to increase GCL activity in the cocultured neurones.

Although GCL activity is increased in neurones cocultured with astrocytes depleted o f GSH, no increase in neuronal GSH levels was observed. This in part can be explained by the lack of GSH released by astrocytes. However, it also may suggest that the amount of cysteine (or cysteine containing molecules) available to neurones in cell culture neuronal media maybe limiting the amount of de novo

neuronal GSH synthesis. This could he due to low concentrations o f cysteine following 24 hours of uptake by neurones and astrocytes. Supplementation of neuronal media with cysteine or CysGly following coculture for 24 hours with GSH depleted astrocytes, or a change of media at this point, followed by incubation for a short period {i.e., 4 hours) prior to determination of GSH levels, may indicate whether a lack of cysteine in neuronal medium is preventing an elevation o f GSH levels in neurones with increased GCL activity.

Previous experiments have suggested that the greater GSH levels in neurones cocultured with activated astrocytes resulted in the ETC being less susceptible to reactive nitrogen species, compared to neurones cultured alone exposed to the NO donor S-nitroso-N-acetylpenicillamine (Bolanos et al., 1996; Stewart et ah,

1998a). The results from this chapter showed that complexes I, II+III and IV were unaffected in neurones cocultured with activated astrocytes. These results are similar to those reported by Bolanos et al (1996), but differ from those of Stewart

et al (1998a). A 38% and 30% loss of complex II+III and IV activity was reported following exposure to activated astrocytes for 24 hours (Stewart et al., 1998a). However, the astrocytes were activated with 500 units/ml IFN-y, compared to 100 units/ml used in this chapter and Bolanos et al (1996). Therefore, the astrocytes used by Stewart et al (1998a) may have greater NOS activity, and consequently the cocultured neurones exposed to a greater concentration of NO.

When neurones were exposed to activated astrocytes depleted of GSH in this chapter, a significant 29% and 25% loss of complex II+III and IV activities respectively were observed, compared to neurones cocultured with untreated astrocytes. The activity of complex IV, but not complex II+III, in neurones cocultured with activated astrocytes depleted of GSH was also significantly lower than in neurones cocultured with activated astrocytes, which have almost twice the amount of GSH. This result supports the hypothesis that greater cellular GSH

levels protect the ETC from oxidative stress (Bolanos et al., 1995; Barker et ah,

1996). However, the damage to the ETC in these cells, compared to neurones cocultured with activated astrocytes, was perhaps less than expected. Activated astrocytes have been shown to generate a steady state NO concentration of approximately 1 pM (Brown et al., 1995) and is similar to that generated by 0.5 mM DETA-NO in the previous chapter. However the damage to neurones exposed to DETA-NO was much greater than in neurones cocultured with activated astrocytes depleted of GSH in this chapter. Perhaps other cellular antioxidant systems that may protect the ETC, in addition to the observed increase in GCL activity, could have been up regulated in these neurones. Mice over expressing CuZnSOD or Bel-2 have been reported to prevent the loss in activity of complexes I, II and IV in the brain following GSH depletion by L-BSO (Merad- Saidoune et al., 1999). Dopaminergic neurones that are induced to increase tetrahydrobiopterin levels, which can act as an antioxidant (Heales et al., 1988), also prevent the toxicity associated with depletion o f GSH (Nakamura et al.,

2000b).

As noted in the results, intracellular GSH levels were lower in both untreated astrocytes and neurones cultured alone, compared to previous chapters. These differences could be due variations between batches of animals. Variation between the batches of D- and L-valine minimal essential media or foetal bovine serum may also have affected the cells {e.g., the availability of substrates or growth factors). In the case of astrocytes, variation in media may have affected the rate of proliferation during the two weeks of culture. Decreased GSH levels have been associated with high cell density in hepatocytes (Lu & Ge, 1992), or a decrease in the proliferation rate of colon adenocarcinoma cells (Kirlin et al.,

1999).

6.6. Conclusion

Neurones in coculture require astrocytes to release GSH in order to elevate neuronal GSH levels. GCL activity was similar in either neurones cultured alone, or cocultured with untreated or activated astrocytes, implying that the supply of

GSH/CysGly by astrocytes is sufficient to increase neuronal GSH levels in coculture. Interestingly, despite GSH levels not being elevated in neurones cocultured with astrocytes depleted of GSH, GCL activity was increased in these neurones. The results suggest that the increased neuronal GCL activity is due to a signal released from astrocytes {e.g., neurotrophins, cytokines, reactive oxygen species) rather than exposure to NO, or a lack of GSH precursors supplied by astrocytes.

An increase in y-GT activity has previously been reported in the substantia nigra of PD brains at post-mortem (Sian et al., 1994b). This phenomenon has been postulated to be a protective mechanism by cells to increase the supply of GSH precursors to surviving neurones and glia. The release of signals from GSH depleted astrocytes that increase GCL activity in surviving neurones, in tandem with increased y-GT activity, could maintain or increase GSH levels in these surviving neurones, and therefore give them greater protection. The depletion of GSH in the substantia nigra is thought to be an early event in the pathogenesis of the Parkinson’s disease (PD; Dexter et al., 1994). The isolation of the putative signal that induces GCL activity in neurones could lead to treatments that prevent the loss of GSH in PD, and perhaps progression of the disease.

Chapter 7