We previously introduced intracellular patch electrochemistry (IPE) to study the regulation of cytosolic catecholamine homeostasis in cultured primary murine adrenal chromaffin cells (Mosharov et al., 2003
) and the PC12 cell line (Mosharov et al., 2006
). To extend IPE measurements to DA neurons, we employed ventral midbrain (VM) cultures from mice that express green fluorescent protein under the control of the tyrosine hydroxylase (TH) promoter (TH-GFP, Figure S1
) (Sawamoto et al., 2001
). Immunolabeling of fixed two-week-old cultures of VM neurons for TH showed that approximately 97% of GFP+
cells were TH+
(185 of 191 cells).
Dependence of DAcyt on extracellular L-DOPA
IPE measurements in a cyclic voltammetric mode that detects DA preferentially over other intracellular metabolites (including L-DOPA and DOPAC) revealed that DAcyt
in untreated GFP+
neurons was below the detection limits of the technique (< 0.1 μM). This was similar to DAcyt
levels in PC12 cells (Mosharov et al., 2006
), but differed from the 10-20 μM cytosolic catecholamine concentrations found in untreated chromaffin cells (Mosharov et al., 2003
). We previously demonstrated that 1 h pre-treatment with 100 μM L-DOPA produces a 2-3-fold increase of cytosolic catecholamine concentration in chromaffin cells (Mosharov et al., 2003
). The same dose of L-DOPA increased DAcyt
neurons to 17.4 ± 1.7 μM (mean ± SEM; n = 74 cells).
To determine the kinetics of DAcyt changes after L-DOPA treatment, we performed IPE at 1, 8, 15 and 24 h after L-DOPA addition to the media. After 1 h of 100 μM L-DOPA exposure, DA in the cytosol reached a steady state level that was maintained for ~8 h, followed by a decline to control levels over the succeeding 24 h of drug treatment (). Interestingly, 500 μM L-DOPA increased DAcyt to the same maximum level, but the elevated steady state was maintained longer, and 24 h after L-DOPA treatment, DAcyt was still higher than in untreated cells. To study the dependence of DAcyt on extracellular L-DOPA, neuronal cultures were treated with a range of L-DOPA concentrations for 1 h at 37°C, followed by IPE measurements in the presence of the same L-DOPA concentrations in the bath and in the pipette solutions within the following 30 min at room temperature. The drug response curve was hyperbolic () with an apparent K0.5 of ~10 μM L-DOPA.
L-DOPA metabolic consumption by VM neurons
As the steady state DA concentration in neuronal cytosol is regulated by multiple enzymes and transporters, we attempted to determine which metabolic step limited DAcyt
accumulation in L-DOPA-treated cells. HPLC-EC measurements of the total intracellular DA (the majority of which represents vesicular DA (Chien et al., 1990
; Mosharov et al., 2003
)) and DOPAC contents showed that these metabolites were elevated to the same degree in neurons exposed to 100 and 500 μM L-DOPA (), indicating that there was no difference in the rates of DA oxidation by MAO and vesicular uptake by VMAT2. To establish whether L-DOPA transport into the cell or its conversion to DA by AADC limited the L-DOPA metabolic consumption, we measured total intracellular L-DOPA levels in VM cultures after 1 h treatment with 100 and 500 μM L-DOPA concentrations in the presence of AADC inhibitor benserazide to block DA synthesis (). The initial rate of L-DOPA accumulation into the cells was not saturated under these conditions, consistent with previously published data (Sampaio-Maia et al., 2001
) on the kinetics of L-DOPA uptake by cell lines derived from astrocytes and neurons (Km
= 50 - 100 μM). Overall, these data suggest that the activity of AADC limits the steady state DAcyt
concentration following L-DOPA treatment.
The data in also provide information about the rate of L-DOPA turnover by the cells. The combined rate of DA and DOPAC synthesis during 1 h of L-DOPA exposure was ~40 fmol/μg of total protein. As each culture dish typically contained 50 - 100 μg of total protein, the rate of L-DOPA consumption during 10 h of incubation was 20-40 pmol per culture. This rate, however, is too low to account for the decline in the DAcyt
observed after 10 h of cell incubation with L-DOPA (), as the total amount of available L-DOPA was >3 orders of magnitude higher (200 nmol in 2 ml of 100 μM solution). We therefore examined the availability of extracellular L-DOPA, which is known to auto-oxidize to DOPA-semiquinone and DOPA-quinone derivatives (Sulzer and Zecca, 2000
). HPLC-EC measurements of L-DOPA concentration in cell-free media showed that the drug disappeared with first order kinetics and a half-life of 4.7 h (). This suggests that after 100 or 500 μM L-DOPA initial doses, its concentration in the media would reach the K0.5
levels in 16 h or 26 h, respectively (), which is in close agreement with the kinetics of DAcyt
changes, as a significant decrease in its initial steady state was observed at approximately these time points (). Together, these data allow us to predict the time dependence of DAcyt
changes based on the concentration of L-DOPA added to the media and the initial increase in DAcyt
determined by IPE (see Discussion).
We next investigated whether exposure to various L-DOPA concentrations, and thereby different durations of sustained elevated DAcyt, correlated with neurotoxicity. We identified an exponential dependence of neurotoxicity on the extracellular L-DOPA concentration (), whereas the time dependence for cell survival in cultures treated with 250 μM L-DOPA demonstrated that the number of TH+ neurons declined linearly after drug exposure, reaching ~50% of the control levels after 4 days, with no subsequent neuronal loss (). As L-DOPA at this concentration is cleared within ~22 h, the data indicate a lag between the end of L-DOPA/DA-mediated stress and the completed course of the catechol-induced toxicity. For further experiments we exposed cultures to 250 μM L-DOPA, which consistently produced ~50% loss of VM dopaminergic neurons 4 days following drug addition.
L-DOPA-induced neurotoxicity in DA and non-DA neurons
Extracellular DA released by neuronal activity did not seem to play a significant role in the L-DOPA-induced cell death, as neuronal viability was not affected by Na+ channel blocker tetrodotoxin (TTX, 1 μM), which inhibits stimulation-dependent transmitter release (data not shown). To further determine which pool of DA was responsible for the observed cell damage, we employed postnatal cultures of cortical and striatal neurons, which do not express TH, DAT or VMAT. Following L-DOPA treatment of cortical neurons, we detected small amounts of DA measured by HPLC-EC () and a substantial elevation of DAcyt measured by IPE (). This was accompanied by >60% loss of cells immunoreactive to microtubule-associated protein 2 (MAP2), which was used as a neuronal marker (). L-DOPA neurotoxicity was NSD-1015-sensitive, confirming AADC-mediated DA synthesis in cortical neurons (data not shown). In contrast, almost no DAcyt was detected in L-DOPA-treated striatal neurons and they were spared following L-DOPA challenge (). Together, our data suggest DAcyt as the primary source of the L-DOPA-induced neurotoxicity.
Pharmacological manipulation of DAcyt and neurotoxicity
AADC, MAO and VMAT inhibitors
To study the contribution of individual metabolic pathways in the maintenance of DAcyt steady state and to determine if changes in DA level correlate with neurotoxicity, we performed metabolite measurements and toxicity studies on L-DOPA - treated neurons that were pre-incubated with specific inhibitors of AADC, MAO and VMAT. Inhibition of the DA synthesizing enzyme AADC with NSD-1015 () or benserazide (not shown) blocked L-DOPA-induced elevation of whole-cell intracellular DA and DOPAC. Benserazide also inhibited the buildup of DAcyt following L-DOPA treatment (), consistent with the idea that the IPE oxidation signal comes from DA synthesized by AADC in the cytosol (note that NSD-1015 alters IPE sensitivity for DA, and therefore was not examined: see Methods). Moreover, the blockade of L-DOPA decarboxylation completely prevented drug-induced cell death (), indicating that an L-DOPA metabolite, but not L-DOPA itself, was responsible for the toxicity.
Effect of pharmacological inhibitors on DAcyt and neurotoxicity
The contribution of DA catabolism was investigated by treating cells with pargyline, an inhibitor of MAO that almost completely abolished DOPAC synthesis (). Pargyline also produced a several-fold increase in both the total amount of intracellular DA () and DAcyt () in L-DOPA-treated neurons, which further correlated with a significantly greater neuronal loss ().
The blockade of VMAT-mediated DA uptake into the vesicles depletes vesicular catecholamine storage, as observed by a reduction in the exocytotic quantal size (Colliver et al., 2000
). Consistently, pre-treatment of VM neurons with reserpine decreased the amount of total intracellular DA synthesized from L-DOPA by ~2-fold both with and without pargyline (). We observed, however, no effect of reserpine, or another VMAT inhibitor, tetrabenazine (10 μM; data not shown) on either DAcyt
or the number of surviving dopaminergic neurons ().
Methamphetamine and cocaine
The discrepancy between the reduction of the total DA in reserpine-treated neurons and the lack of the effect of VMAT inhibition on DAcyt might be explained by the distribution of synaptic vesicles between neuronal cell bodies and the neurites. If the majority of vesicles filled with DA are located in synaptic terminals far from the cell bodies, any transmitter redistributed from them would be invisible to IPE (see Methods).
To examine the effects of other DA-releasing drugs on somatic DA concentration, we exposed L-DOPA-treated neurons to methamphetamine (METH), which disrupts the vesicular proton gradient and redistributes DA from synaptic vesicles to the cytosol (Sulzer et al., 2005
). In contrast to the increased cytosolic catecholamine levels observed in chromaffin cells acutely treated with METH (Mosharov et al., 2003
), neurons exposed to 50 μM METH showed decreased DAcyt
(); 5 μM METH produced the same decrease of DAcyt
(38 % of untreated cells; p<0.005 by t-test). METH-mediated reduction of DAcyt
was blocked by the DA uptake transporter (DAT) blocker cocaine, suggesting that the effect was due to reverse transport through DAT (Sulzer et al., 2005
). METH-mediated decrease of DAcyt
and its blockade by cocaine were also observed in L-DOPA-treated cells in the presence of PGL (data not shown). Note that DAcyt
in L-DOPA-treated neurons was unaffected by DAT inhibitors cocaine () or nomifensine (5 μM; data not shown), supporting the idea that reverse transport is not induced by the elevated DAcyt
alone, but requires additional METH-mediated effects on DAT (Kahlig et al., 2005
Consistent with the IPE results, METH protected neuronal cell bodies from L-DOPA toxicity (), despite the considerable neurite loss that occurs in METH-treated cultured DA neurons (Cubells et al., 1994
; Larsen et al., 2002
). When applied for 4 days without L-DOPA, neither METH or cocaine nor the combination of the two drugs had a significant effect on the number of surviving neurons (data not shown).
Genetic manipulation of DAcyt and neurotoxicity
We previously developed a recombinant adenovirus that overexpresses VMAT2 (rVMAT2), resulting in reduced neuronal neuromelanin content, enhanced quantal size and increased the number of evoked quantal neurotransmitter release events from the terminals of cultured VM dopaminergic neurons (Pothos et al., 2000
; Sulzer and Pothos, 2000
). To investigate whether enhanced vesicular uptake may decrease DAcyt
and rescue neurons from L-DOPA-induced toxicity, we employed an adenoviral construct that resulted in overexpression of the recombinant VMAT2 in 80-90% of both dopaminergic and non-dopaminergic cells (Figure S2
VMAT2 overexpression significantly decreased DAcyt
in L-DOPA-treated GFP+
VM neurons (from 17.4 ± 1.7 μM, n = 74 neurons to 3.1 ± 0.8 μM, n = 28; ), but had no effects on GFP-
neurons from the same culture (2.4 ± 0.8 μM, n = 33 vs. 2.3 ± 1.9 μM, n = 7). GFP+
neurons in cultures treated with a virus that did not contain rVMAT2 displayed the same DAcyt
levels after L-DOPA treatment as control cells (data not shown). Consistent with the kinetics of L-DOPA metabolic consumption (), rVMAT2 lowered DAcyt
to the same extent in cells treated with 100 μM and 500 μM L-DOPA (). Moreover, infection with rVMAT2 effectively protected TH+
neurons from the L-DOPA-mediated neurotoxicity (). It should be noted, however, that these data also suggest that somatic vesicles or other organelles that do not normally sequester substantial levels of DA can do so after transporter overexpression, as has been demonstrated for non-catecholaminergic AtT-20 cells (Pothos et al., 2000
) and hippocampal neurons (Li et al., 2005
Effect of VMAT2 overexpression and α-syn knock-out on DAcyt and toxicity
To investigate the role of α-syn in mediating DAcyt neurotoxicity, we generated α-syn deficient mice that express eGFP in dopaminergic neurons (see Methods). While VM neurons from α-syn-/- and α-syn+/- mice were more resistant to L-DOPA-induced stress than neurons from their wild-type littermates (), IPE measurements indicated no difference in the DAcyt concentration between the three groups (). These data suggest that the pathogenic effect of α-syn is downstream of DA synthesis (see Discussion), and that the presence of both elevated DAcyt and α-syn is required for L-DOPA-induced neurotoxicity.
DAcyt in SN and VTA neurons
To address the question of differential susceptibility of neurons from different brain regions, we prepared cultures that were enriched with cells from either SN or VTA, as previously published (Burke et al., 1998
). Immunolabel for calbindin, a protein that is expressed at higher levels in VTA than in SN (Thompson et al., 2005
), showed that in mixed VM cultures ~50% of TH+
neurons were also calbindin+
, while the proportion of TH+
cells was ~85% in VTA cultures and ~25% in SN cultures ().
Sensitivity of SN and VTA DA neurons to L-DOPA challenge
IPE measurements showed that L-DOPA treated SN neurons displayed 2-3-fold higher DAcyt than VTA neurons (). This difference in DAcyt translated into a significantly higher susceptibility of SN neurons to L-DOPA-induced toxicity, while VTA neurons were almost completely protected (). The resistance of VTA neurons to L-DOPA challenge was also seen in mixed VM cultures, where after 4 days of 250 μM L-DOPA the percentage of TH+/calbindin+ neurons increased from 52% to 88% (p<0.05 by χ2 test).
Relationship between cytoplasmic Ca2+ and DAcyt
The regulation of intracellular Ca2+
differs significantly between SN and VTA neurons (Gerfen et al., 1985
; Surmeier, 2007
; Thompson et al., 2005
). Thus, to search for the mechanism that governs the difference in DAcyt
between the two neuronal populations, we investigated whether intracellular Ca2+
had an effect on L-DOPA-induced DAcyt
Cell pretreatment with the voltage-gated calcium channel blocker CdCl2
or buffering cytoplasmic Ca2+
with membrane-permeable chelator BAPTA-AM, both significantly decreased DAcyt
in dopaminergic neurons from SN and VTA (). This suggests that the regulatory mechanism that links cytoplasmic Ca2+
exists in both cell populations, and that the difference between SN and VTA neurons might be in the steady-state Ca2+
level. This would be consistent with SN neurons relying on dihydropyridine-sensitive L-type Cav
1.3 channels for autonomous pacemaking, in contrast to VTA neurons that use HCN/Na+
channels for pacemaking. Immunostaining of VM cultures for the α1D subunit of the L-type Ca2+
channels, which is specifically present in the Cav
1.3 channels, showed that its expressed in both calbindin+
DA neurons (Figure S3
), consistent with previous reports (Rajadhyaksha et al., 2004
; Striessnig et al., 2006
). The Cav
1.3 blockers nimodipine () and nitrendipine (not shown), however, had no effect on DAcyt
in L-DOPA exposed VTA neurons, but decreased DAcyt
in SN neurons to the levels found in VTA neurons (Figure S4
). VM cultures treated with nimodipine were far more resistant to L-DOPA-induced neurodegeneration (). Pre-treatment of SN neurons for 1h with HCN channel antagonist ZD7288 (50 μM) or TTX (1 μM) had no significant effect on DAcyt
reached after L-DOPA treatment (data not shown).
Regulation of DAcyt by cytoplasmic Ca2+
To determine which enzyme or transporter is responsible for Ca2+
-induced upregulation of DA homeostasis, we first compared DAcyt
in SN and VTA neurons treated with pargyline and reserpine. DAcyt
was increased by the same percentile in both types of neurons (), suggesting that MAO and VMAT2 activities did not underlie the difference between DAcyt
handling by SN and VTA neurons and were not the regulatory targets. Next, DAcyt
in SN and VTA neuronal cultures was unaffected by the presence of DAT blocker cocaine (data not shown). Finally, the uptake of L-DOPA in VM cultures pre-treated with BAPTA-AM and benserazide was monitored by HPLC-EC and IPE in the amperometric detection mode, which in contrast to CV produces similar oxidative currents for DA and L-DOPA (Mosharov et al., 2003
). Measurements by both methods demonstrated that L-DOPA uptake was unaffected by Ca2+
buffering (). Overall, our data suggest that MAO, VMAT, DAT and the L-amino acid plasma transporter were not responsible for the difference in DAcyt
observed between SN and VTA neurons. Thus, it appears that the activity of AADC may provide the Ca2+
-sensitive metabolic step that leads to higher DAcyt
levels in SN neurons (see Discussion).