In our kinetic and pharmacological characterization of primary astrocyte cultures, we observed increased EAAT activity in cells from PrPKO mice compared to PrP expressing WT mice. This finding was observed in astrocytes cultured both in the absence and presence of dbcAMP. Furthermore, increased transport by PrPKO astrocytes was shown to be functionally significant as PrPKO astrocytes protected both PrPKO and WT neurons from L-glutamate-mediated excitotoxicity to a greater extent than did WT astrocytes. Levels of L-glutamate are believed to reach millimolar levels in the synaptic cleft during excitatory transmission (Clements et al. 1992
) and are rapidly cleared to markedly lower (0.1–1μM) homeostatic levels (Herman and Jahr 2007). Therefore, the differences in Vmax
observed between PrPKO and WT astrocytes could have a marked influence on transmitter clearance and glutamatergic signaling.
Kinetic and physiological data showed that PrPKO astrocytes exhibit greater L-glutamate transport than WT astrocytes, although the underlying mechanisms responsible remain unclear. The increased EAAT activity in PrPKO versus WT astrocytes was accompanied by only a modest elevation in EAAT1 expression in PrPKO astrocytes, suggesting the alterations could be due to indirect mechanisms that effect surface localization and/or changes in intrinsic activity. L-Glutamate transporters are subject to interactions with modulating proteins (Jackson et al. 2001
) and to post-translational modifications that can result in altered transporter function (Adolph et al. 2007
; Duan et al. 1999
; Lin et al. 2001
; Munir et al. 2000
, Schlag et al. 1998
). The localization of PrP and EAATs to lipid rafts may provide an environment conducive to direct and/or indirect interactions between these proteins (Butchbach et al. 2004; Naslavsky et al. 1997).
In our in vitro cultured astrocytes the effect of the PrPKO genotype on glutamate transport activity appeared to be mediated primarily through an action on EAAT1. Even in astrocyte-neuron co-cultures where EAAT2 expression was highest, mRNA expression of EAAT1 was still approximately 4-fold higher than EAAT2 (), and transport experiments in the presence of EAAT inhibitors could not clearly demonstrate a significant functional contribution by EAAT2 () (Schlag et al., 1998
). Further studies will be required to determine whether PrP can also influence expression of EAAT2 in various situations in vivo. However, even if the effect of PrP expression is limited solely to EAAT1, this influence is of significant interest because this subtype has important roles in certain areas including cerebellum, inner ear, circumventricular organs and retina (for reviews see Danbolt 2001
and Rauen et al. 1999
). For example, lack of EAAT1 in knockout mice has been found to be associated with reduced motor coordination and increased sensitivity to cerebellar traumatic injury (Watase et al. 1998
) as well as increased susceptibility to noise-induced injury (Hakuba et al. 2000
). Furthermore, human patients with mutant EAAT1 were found to have clinical deficits including ataxia, hemiplegia, and seizures (Jen et al. 2005
In contrast to our findings, a previous study reported that PrPKO astrocytes transported less L-glutamate than did WT astrocytes (Brown and Mohn 1999). However, this study used 3H-L-glutamate as a substrate, rather than 3H-D-aspartate, and the observed decrease was based on determinations at a single substrate concentration (100μM), making direct comparisons difficult. The most notable difference between the two studies, however, concerns the genetics of the mouse strains used to generate the cell cultures. In the Brown and Mohn study, the PrP WT mice were (129/SvEv x C57BL/6J) F1 hybrids, while PrPKO mice were homozygous for a random mixture of 129/SvEv and C57BL/6J genes. Thus, the background genes of these PrPKO mice differed completely from the WT mice used. In contrast, in our experiments the PrPKO mice were backcrossed nine times to C57BL/10SnJ mice, the WT strain in this study. Therefore, in our PrPKO mice, only the PrP gene (Prnp) itself and areas immediately adjacent to this gene were still of strain 129/Ola origin. It is possible that these adjacent genes might also contribute to the differences observed in our experiments.
Astrocytic EAATs mediate the fine balance between having sufficient L-glutamate in the synapse for neuronal signaling without exceeding the threshold that would trigger excitotoxic pathology. To test if the increased transport exhibited by PrPKO astrocytes was physiologically relevant, we studied the toxicity of L-glutamate on mixed cortical cultures, where differences in transport capacity have previously been shown to modulate susceptibility to excitotoxicity (Robinson et al. 1993
; Rosenberg and Aizenman 1989
; Rothstein et al. 1996
). The decreased neuronal damage observed within a narrow range of L-glutamate concentrations in cultures containing PrPKO astrocytes () suggested that enhanced glutamate transport by PrPKO astrocytes lessened the excitotoxic insult.
However, this protection from L-glutamate-mediated excitotoxicity was distinct from the increased neuronal vulnerability of PrPKO neurons to NMDA-mediated excitotoxicity. As observed previously by others (Khosravani et al. 2008
), we found that PrPKO neurons were intrinsically more vulnerable to NMDA-mediated excitotoxic injury than WT neurons. The difference between the two insults lies not with the action of L-glutamate and NMDA as NMDA receptor agonists, but with the fact that the EAATs do not transport NMDA. Thus, when NMDA was added to the mixed neuron-astrocyte co-cultures (), the experiment reflected the direct effect of NMDA on PrPKO and WT neurons. In contrast, when L-glutamate was added (), astrocytic transport of L-glutamate was able to reduce the excitotoxic challenge. Thus, increased transport of L-glutamate by PrPKO astrocytes appeared to be effective in protecting PrPKO neurons from death despite an increased vulnerability to excitotoxic injury.
Increased clearance of L-glutamate by astrocytes in vivo
may contribute to the neurophysiological abnormalities observed previously in PrPKO mice. For example, reduced excitatory post-synaptic potentials (Carleton et al. 2001
), impaired formation of long-term potentiation (Collinge et al. 1994
; Criado et al. 2005
; Manson et al. 1995), reductions in after-hyperpolarization potentials (Mallucci et al. 2002
, Ratte et al. 2002) and abnormal responses to NMDA antagonist MK-801 (Coitinho et al. 2002
) observed in PrPKO mice all suggest attenuation of L-glutamate-mediated signaling. Many of these alterations would be consistent with the premature termination of the L-glutamate signal and/or the excessive clearance of L-glutamate from the extracellular space surrounding EAA receptors. Consistent with such a conclusion, the EAATs have been shown to regulate the extracellular levels of L-glutamate available to activate synaptic and extrasynaptic receptors in specific excitatory circuits (Diamond 2005
; Dzubay and Otis 2002
; Huang et al. 2004
; Turecek and Trussell 2000
). Thus, the impact of alterations in EAAT activity may be greatest in those synaptic connections ensheathed by astrocytes, where the presence and positioning of the transporters has been shown to modulate glutamatergic neurotransmission (Anderson and Swanson 2000
; Bridges and Esslinger 2005
; Eulenburg and Gomeza 2010
Low EAAT activity has been reported in a number of neurodegenerative diseases, including amyotrophic lateral sclerosis (Rothstein et al. 1992
), HIV-associated dementia (Sardar et al. 1999
), and Alzheimer’s disease (Masliah et al. 1996
). Increased levels of glutamate in the synaptic cleft may lead to neuronal death through excitotoxicity (Beart and O’Shea 2007
). Accordingly, enhancement of L-glutamate transport has been regarded as a potential therapeutic goal. However, hyperactive EAAT activity and consequent reduced NMDA receptor signaling is not without its own complications, as has been suggested to be the case in schizophrenia (Miyamoto et al. 2005
). Future therapeutic approaches to modulating EAAT activity in any disease will require targeting a level of transporter activity that will maintain an optimal level of L-glutamate in the synaptic cleft, balancing physiological and pathological signaling during all phases of neuronal activity.