Our results show that increasing striatal GLT1 expression attenuates the neurological signs of HD in R6/2 mice. The GLT1 increase, moreover, enhances glutamate uptake, suggesting that a dysregulation of striatal glutamate transmission plays a key role in HD. It also is interesting that, although glutamate uptake is attenuated in symptomatic R6/2 mice relative to wild-type, there is no difference in the expression level of GLT1. Thus, the GLT1 deficit appears to represent a deficiency in function rather than level of protein. The increase in GLT1 expression after ceftriaxone treatment, however, is accompanied by an increase in uptake, suggesting that the newly expressed protein is functional. Our results, therefore, pave the way for assessment of the potential therapeutic benefits of ceftriaxone and other compounds that may increase GLT1 expression in HD.
We assessed several behavioral signs of HD in R6/2 mice, and in each case ceftriaxone treatment led to a significant improvement relative to vehicle. Clasping or dyskinesia of the limbs when R6/2 mice are suspended by the tail is one of the first noticeable signs of the HD phenotype (Mangiarini et al., 1996
). A ceftriaxone-induced improvement was evident by the third treatment day and persisted for each treatment day thereafter as well as the first post-treatment test day. Although the loss of this effect one week after treatment argues against a long-term change by ceftriaxone, our results in the plus-maze and open-field tests suggest a more complex picture. For example, on post-treatment day 1, R6/2 mice treated with ceftriaxone matched WT performance in both the plus maze and climbing in the open-field. Their performance, moreover, was not different than WT or saline-treated R6/2 mice on post-treatment day 7. In addition, R6/2 open-field twitching, which did not improve on the first post-treatment day in ceftriaxone-treated animals, showed a significant improvement over saline one week later. In fact, twitching had declined so dramatically by post-treatment day 7 that ceftriaxone-treated R6/2 mice were not significantly different from WT. Conceivably, different neural circuits changing at different time courses may explain these results, a conclusion supported by evidence of multiple behavioral pathways within striatal circuitry (Alexander et al., 1986
). Other interpretations, however, are equally likely, including a complex interaction among the behaviors themselves such that a change in one could, over time, force a change in another. Thus, although it may be difficult to link a specific mechanism to a specific behavioral change, our results reveal significant improvement in the HD phenotype with ceftriaxone.
Another interesting observation is that ceftriaxone failed to alter the behavioral response of WT mice. None of our behavioral tests revealed a difference between WT animals treated with saline or ceftriaxone. Even open-field activity, which includes multiple movements and motor sequences (Dorner et al., 2007
), was unaffected in WT mice. Although we cannot rule out some subtle behavioral effects of ceftriaxone not detected by our measurements, it appears that the drug does not alter common WT behavioral responses.
The behavioral effects of ceftriaxone in R6/2 mice cannot be explained by an antibiotic action since there is no indication of sepsis at this stage of HD. In fact, we focused on animals at 7-8 weeks of age because of the clear HD behavioral signs without the complications (e.g., diabetes, weight loss) that emerge at later ages. It also is unlikely that ceftriaxone exerted a general sedative or muscle-relaxing effect on behavior since the improvements with ceftriaxone include increases in turning and climbing behavior. We also saw none of the side effects characteristic of high ceftriaxone doses (e.g., diarrhea and nausea), ruling out a non-selective dose effect on behavior. It is also relevant that 3.5 μmol/L ceftriaxone, the EC50 required to increase GLT1 expression (Rothstein et al., 2005
), is comparable to levels of ceftriaxone found in the central nervous system of patients undergoing therapy for meningitis (0.3 to 6 μmol/L) (Nau et al., 1993
). A related issue is that the polyglutamine repeat length in R6/2 mice may vary among litters between 110-150 repeats. Although repeat length influences onset and progression of the HD phenotype (Brandt et al., 1996
), all our R6/2 mice had a quantitatively reproducible phenotype with little variation between animals. This result is consistent with our repeat-length analysis, which also showed little variation (see Experimental Procedures).
We chose our ceftriaxone dose and treatment schedule because of our interest in GLT1. At 200 mg/kg for 5 consecutive days, ceftriaxone is known to elicit a maximal increase in GLT1 expression without causing neurotoxic effects (Rothstein et al., 2005
; Chu et al., 2007
). Our immunohistochemistry and Western blot data not only confirm these results, but also indicate that HD itself is not an impediment to increasing GLT1 expression. Thus, additional compounds believed to increase glutamate transport (e.g., the synthetic neuroimmunophilin GPI-1046) (Ganel et al., 2006
) also may effectively reverse HD signs in transgenic models. Moreover, further testing of ceftriaxone (e.g., lower doses, longer treatment duration) in models showing a relatively long pre-symptomatic period can be used to address the possibility that increasing GLT1 expression even before symptom onset could significantly delay and perhaps minimize HD progression.
Our no-net-flux microdialysis data reveal for the first time that striatal glutamate uptake in symptomatic R6/2 mice is, in fact, impaired. This finding in saline-treated animals not only confirms the importance of a glutamate dysregulation in HD, but also that the uptake problem occurs when animals are fully awake and behaving. Interestingly, however, a decline in glutamate uptake was not reflected in an increase in extracellular glutamate. Thus, glutamate transmission in these mice may adapt to the loss of uptake with a compensatory decrease in glutamate release. Although this hypothesis is difficult to confirm in that most extracellular glutamate sampled by microdialysis appears to originate from a non-synaptic source (Timmerman and Westerink, 1997
), previous data based on microdialysis done in conjunction with CE-LIF technology show that basal glutamate levels in striatum are sensitive to both electrical stimulation of corticostriatal neurons and application of tetrodotoxin, suggesting that the source of glutamate is, at least in part, synaptic (Lada et al., 1998
, Rebec et al., 2005
). Furthermore, it has been well established that the no-net-flux curves represent in vivo
recovery of the probes, such that increases in cellular uptake increase recovery as well as the slope of the nonet-flux linear regression (Bungay et al., 1990
; Parsons and Justice, 1992
; Smith and Justice, 1994
; Melendez et al., 2005
). Finally, to confirm that recovery represents cellular uptake, we applied PDC, a glutamate uptake inhibitor, through the microdialysis probe and showed a marked increase in basal glutamate concomitant with decreased recovery of the probe.
Treatment with ceftriaxone restored glutamate uptake in R6/2 mice to saline-treated WT levels. Immunohistochemistry and Western blot confirmed that this effect occurred in conjunction with up-regulation of GLT1 expression. Unlike our behavioral results, however, the increase in GLT1 appeared in both WT and R6/2 mice. In WT animals, therefore, an increase in GLT1 and a concomitant reduction in glutamate have little, if any, behavioral impact, perhaps owing to the non-synaptic nature of glutamate in extracellular fluid (see above). Even if this is the case, however, it is clear that the change in uptake and extracellular glutamate induced by ceftriaxone has a significant effect on HD mice. This effect may be due to other HD-related changes in glutamate transmission such as a change in the sensitivity of glutamate receptors (see Zeron et al., 2002
). A related point is that we found no difference in GLT1 expression between WT and R6/2 mice regardless of treatment. Thus, the uptake deficit in R6/2 mice appears to be due to dysfunctional rather than missing GLT1. If so, then ceftriaxone not only increases the amount of GLT1 but the increase is also functional. Interestingly, Kalivas and colleagues have also reported deficits in glutamate uptake that were not accompanied by decreased glutamate transporter expression in rats that were exposed to repeated ethanol injections (Melendez et al., 2005
). It is also relevant that deficient glutamate uptake was observed in postmortem brain tissue taken from HD patients, while no changes were found in glutamate transporter levels (Hassel et al., 2007
Previous studies using cultured striatal tissue from relatively old (12-14 weeks of age) and presumably severely symptomatic R6/2 mice, have reported decreased GLT1 expression along with deficient glutamate uptake (Lievens et al., 2001
; Behrens et al., 2002
; Shin et al., 2005
). The inconsistency with our data may be related to the preparation since our results are based on intact, behaving animals, and extracellular glutamate is sensitive to the level of behavioral activation (Sandstrom and Rebec, 2007
), most likely due to changes in cortical input (Lada et al, 1998
). It is also relevant that R6/2 mice >12 weeks of age are often severely symptomatic (Carter et al., 1999
). Our evidence of a glutamate uptake deficit at a relatively early stage of symptom development is important because such a deficit could be precursory to other pathogenic mechanisms in HD.
The ability of ceftriaxone to reverse the glutamate uptake deficit indicates that the increase in GLT1 expression can overcome dysfunctional GLT1 in HD. Although we cannot rule out other central mechanisms of action of ceftriaxone in our HD animals, it is unlikely that other glutamate transporters (e.g., GLAST and EAAC1) can account for the change in uptake since ceftriaxone acts selectively on GLT1 (Rothstein et al., 2005
). In addition, GLAST and EAAC1 are not differentially expressed in HD mouse models compared to WT (Lievens et al., 2001
; Behrens et al., 2002
Several mechanisms, acting alone or in combination, may contribute to GLT1 dysfunction in HD. One comes from evidence that operation of GLT1 is sensitive to oxidative damage (Trotti et al., 1998
), which is common in HD pathology (Browne and Beal, 2006
). That R6/2 mice have low striatal extracellular ascorbate (Rebec et al., 2002
), an antioxidant vitamin linked to glutamate uptake (Rebec and Pierce, 1994
), is consistent with this view. Inhibition of glutamate transport, moreover, creates sufficient oxidative stress to overwhelm antioxidant protection (Nagatomo et al., 2007
). It also is noteworthy that treatment of R6/2 mice with ascorbate, which restores striatal ascorbate to WT levels, attenuates the abnormally high firing rate of R6/2 striatal neurons (Rebec et al., 2006
) and attenuates the HD phenotype (Rebec et al., 2003
), both of which could be explained by improved GLT1 function. Another mechanism relates to mutant huntingtin, which accumulates in astrocytes as early as ~4 weeks of age (Shin et al., 2005
). The resulting disruption of intracellular protein trafficking (DiFiglia et al., 1995
; Gunawarden and Goldstein, 2005
) may cause improper localization or insertion of GLT1 into the membrane. Finally, medium spiny neurons in R6/2 mice exhibit abnormal dendritic morphology (Klapstein et al., 2001
), which may cause altered coupling of astrocytic processes to the synapse, leading to inefficient glutamate uptake and spillover into the extracellular space. Our results suggest a need to investigate the mechanisms by which GLT1 becomes dysfunctional in HD.