5176 • The Journal of Neuroscience, April 14, 2010 • 30(15):5176–5188

Development/Plasticity/Repair Metabotropic 1 Mediates the Electrophysiological and Toxic Actions of the Cycad Derivative ␤-N-Methylamino-L- on Substantia Nigra Pars Compacta DAergic Neurons

Maria Letizia Cucchiaroni,1 Maria Teresa Viscomi,1 Giorgio Bernardi,1,2 Marco Molinari,1 Ezia Guatteo,1* and Nicola B. Mercuri1,2* 1Santa Lucia Foundation Istituto di Ricovero e Cura a Carattere Scientifico, 00143 Rome, Italy, and 2Department of Neuroscience, University of Rome “Tor Vergata,” 00133 Rome, Italy

Amyotrophic lateral sclerosis–Parkinson dementia complex (ALS-PDC) is a neurodegenerative disease with ALS, parkinsonism, and Alzheimer’ssymptomsthatisprevalentintheGuampopulation.␤-N-Methylaminoalanine(BMAA)hasbeenproposedasthetoxicagent damaging several neuronal types in ALS-PDC, including substantia nigra pars compacta dopaminergic (SNpc DAergic) neurons. BMAA is a mixed glutamate receptor agonist, but the specific pathways activated in DAergic neurons are not yet known. We combined electro- physiology, microfluorometry, and confocal microscopy analysis to monitor membrane potential/current, cytosolic calcium concentra- 2ϩ tion ([Ca ]i ) changes, cytochrome-c (cyt-c) immunoreactivity, and reactive oxygen species (ROS) production induced by BMAA. Rapid 2ϩ toxin applications caused reversible membrane depolarization/inward current and increase of firing rate and [Ca ]i in DAergic neu- rons. The inward current (IBMAA ) was mainly mediated by activation of metabotropic glutamate receptor 1 (mGluR1), coupled to transient receptor potential (TRP) channels, and to a lesser extent, AMPA receptors. Indeed, mGluR1 (CPCCOEt) and TRP channels (SKF

96365; Ruthenium Red) antagonists reduced IBMAA , and a small component of IBMAA was reduced by the AMPA CNQX. Calcium accumulation was mediated by mGluR1 but not by AMPA receptors. Application of a low concentration of NMDA potentiated the BMAA-mediated calcium increase. Prolonged exposure to BMAA caused significant modifications of membrane prop- erties, calcium overload, cell shrinkage, massive cyt-c release into the cytosol and ROS production. In SNpc GABAergic neurons, BMAA activated only AMPA receptors. Our study identifies the mGluR1-activated mechanism induced by BMAA that may cause the neuronal degenerationandparkinsoniansymptomsseeninALS-PDC.Moreover,environmentalexposuretoBMAAmightpossiblyalsocontribute to idiopathic PD.

Introduction water bodies (Metcalf et al., 2008), in South Africa freshwater The amyotrophic lateral sclerosis–Parkinson dementia complex impoundments (Esterhuizen and Downing, 2008) and in Peru- (ALS-PDC) is a rare form of neuronal degeneration, occurring vian highland lakes (Johnson et al., 2008), suggesting that it may among the Chamorro people of Guam (Reed and Brody, 1975; be of concern not only for specific populations of Western Pacific Garruto and Yase, 1986; Spencer et al., 1986). It has been pro- Islands but also for the world-wide population. Moreover, posed to be caused by exposure to the non-protein amino acid BMAA can accumulate in bacteria, plant, animal, and human ␤-N-methylamino-L-alanine (BMAA) (Spencer et al., 1986, tissues as free amino acid as well as bound within proteins 1987). The recent discovery that BMAA can be produced by cya- (Murch et al., 2004). This creates an endogenous reservoir of nobacteria throughout the world, including symbionts and free- neurotoxin within the human body, capable of slow release into living cyanobacteria (Cox et al., 2003; Ince and Codd, 2005), has cerebral and other tissues during protein catabolism for years or increased the interest for this toxin involvement in other neuro- decades after its consumption. In vivo (Spencer et al., 1987; Smith degenerative diseases. Indeed, BMAA has been found in British and Meldrum, 1990; Rakonczay et al., 1991; Chang et al., 1993; Santucci et al., 2009) and in vitro studies have reported that BMAA acts as a glutamate agonist onto spinal motoneurons (Rao Received Oct. 29, 2009; revised Dec. 22, 2009; accepted Jan. 8, 2010. The study was supported by grants from the Ministero della Salute (to N.B.M.). We are grateful to Dr. Peter S. et al., 2006) and cortical (Lobner et al., 2007) and cerebellar Freestone and Prof. Janusz Lipski for their comments on the manuscript. (Staton and Bristow, 1997) neurons. However, a detailed descrip- *E.G. and N.B.M. contributed equally to this work. tion of its actions on substantia nigra pars compacta (SNpc) do- Correspondence should be addressed to Prof. Nicola B. Mercuri, Fondazione Santa Lucia, Via del Fosso di Fiorano paminergic cells (DAergic) is still lacking. This would be of 64, 00143 Rome, Italy. E-mail: [email protected]. DOI:10.1523/JNEUROSCI.5351-09.2010 particular interest, as it would help to understand mechanisms of Copyright © 2010 the authors 0270-6474/10/305176-13$15.00/0 neuronal degeneration involved not only in ALS-PDC, but also in Cucchiaroni et al. • Actions of BMAA onto SNpc DAergic Neurons J. Neurosci., April 14, 2010 • 30(15):5176–5188 • 5177

sporadic Parkinson’s disease (PD) (Hanna and Bhatia, 1997; CaCl2 (0.1), MgCl2 (2), KCl (10), EGTA (0.75), and 0.25 fura-2 pentapotas- Beal, 1998; Ince and Codd, 2005). To this regard, the neuronal sium salt (Invitrogen), pH 7.3, osmolarity 280 mOsm. loss within the SNpc that characterizes PD may be due either to Whole-cell voltage-clamp or current-clamp experiments were per- genetic (Klein and Schlossmacher, 2007; Bogaerts et al., 2008) formed with an Axopatch 200 B amplifier (Molecular Devices), filtered at 1 kHz, and digitized at 10 kHz. In voltage clamp, holding potential and/or environmental factors (Le Couteur et al., 2002; Di Monte, Ϫ (Vhold) was 60 mV. No hyperpolarizing holding currents were applied 2003; Greenamyre et al., 2003). There is evidence that in PD, in current-clamp recordings except, in the prolonged treatment experi- glutamate may cooperate to induce nigral excitotoxicity (Rodriguez ments, for membrane resistance (Rm) and number of evoked spikes mea- et al., 1998) and, in addition to NMDA receptors, metabotropic Ϫ surements, in which the cells were held at 50 mV. Rm changes were receptors (mGluRs) possibly mediate neuronal death (Vernon et calculated at the end of the hyperpolarizing potentials produced by in- al., 2005, 2007). Indeed, mGluR1 activation promotes numerous jecting a negative current pulse (Ϫ30 pA, 700 ms duration), whereas the processes, such as activation of inward cationic currents (Guatteo number of spikes were calculated in response to a depolarizing current et al., 1999; Tozzi et al., 2003), inhibition of potassium channels, step (ϩ100 pA, 700 ms). ␮ and direct influx of calcium ions, which leads to inappropriate BMAA (3 mM,or1mM in Fig. 9) and glutamate (300 M), dissolved in activation of kinases, proteases, and phospholipases and finally to ACSF, were pressure applied (10 psi, 0.5–1.0 s), through a patch electrode connected to a Pneumatic Pico-pump PV 800 (WPI). The puffing elec- energy deficits (Salin´ ska et al., 2005). trode was positioned above the slice in close proximity to the recorded Therefore, the aim of the present study was to identify the neuron and the agonist-mediated inward currents (IBMAA; IGLU) were BMAA-induced electrophysiological changes, intracellular calcium elicited every 2 min. In experiments illustrated in Figures 1A–F and 7, modifications, and toxic effects on SNpc neurons. We found that BMAA was bath-applied (0.1–10 mM). The current/voltage (I–V) rela-

BMAA preferentially activates an mGluR1-mediated pathway on tionship of BMAA-induced current (IBMAA) was measured using a ramp SNpc DAergic neurons and that its prolonged application causes (1.5 s, from Ϫ65 mV to ϩ70 mV) executed before (control ramp) and at significant functional alterations inducing also massive cyt-c release the peak of IBMAA (BMAA ramp, 3 mM, 2 s). The net IBMAA was calculated from mitochondria and reactive oxygen species (ROS) production. as digital subtraction between the control and the BMAA ramps. For Indeed, these excitotoxic effects were mitigated by mGluR1 antago- these experiments, K-gluconate in the pipette solution was replaced with Cs-methanesulfonate (pH 7.3 adjusted with CsOH) and QX-314 (5 mM) nists, the mitochondrial transition pore inhibitor cyclosporine A, was added. The extracellular solution was initially as follows (in mM): and the antioxidant 1,4-dithiothreitol. NaCl, 141; KCl, 2.5; MgCl2, 1.2; CaCl2, 2.4; NaOH, 9; glucose, 10; and ϩ ϩ 2ϩ HEPES, 20. Before applying ramps, we blocked Na ,K , Ih,Ca , AMPA, and GABA conductances by replacing this solution with one Materials and Methods A containing the following (in mM): NaCl, 126; 4-aminopyridine, 5; KCl, Tissue preparation. All procedures were approved by the Animal Ethics 2.5; MgCl2, 1.2; CsCl, 2; CaCl2, 1.4; CoCl2, 1; HEPES, 20; NaOH, 5; Committee of the University of Rome Tor Vergata. Preparation of rat glucose, 10; tetrodotoxin, 0.001; picrotoxin, 0.1; nifedipine, 0.01; CNQX, midbrain slices was performed as described previously (Lacey et al., 1989; 0.01; and CdCl , 0.1. Both the HEPES-buffered solutions were equili- Mercuri et al., 1995). Briefly, Wistar rats (P18–P22) (Charles River Lab- 2 brated with 100% O2. oratories) were deeply anesthetized with and decapitated. The Data were acquired using Clampex/AxoScope softwares (version 9, brain was rapidly removed and horizontal slices (thickness 250 ␮m), Molecular Devices). Measurements (every 2 min) of Rm were performed containing the substantia nigra pars compacta (SNpc) and the ventral in voltage clamp using Ϫ5 mV command pulses (20–30 ms). Off-line tegmental area (VTA), were cut with a vibratome (VT 1000s, Leica) in data analysis was performed using Origin (OriginLab) and Clampfit ice-cold modified artificial CSF (ACSF). This solution contained the fol- (Molecular Devices) softwares. lowing (in mM): 126 NaCl, 2.5 KCl, 1.2 MgCl2, 1.2 NaH2PO4, 2.4 CaCl2, Ϫ Microfluorometry. Neurons were filled with a solution containing the 1 2ϩ 10 glucose, and 24 NaHCO3, 290 mOsm , and was gassed with 95% Ca -sensitive dye fura-2 pentapotassium salt (Invitrogen) by diffusion O2–5% CO2, pH 7.4. After 30 min recovery in 95% O2–5% CO2-gassed from the patch pipette. UV excitation light (340 and 380 nm) was pro- ACSF at 34°C, a single slice was transferred to a recording chamber vided by the monochromator (Till Photonics). Emitted light passed a (volume 0.6 ml) and completely submerged with continuously flowing barrier filter (440 nm), was detected by the CCD camera (Till Photonics) Ϫ1 (2.5 ml ⅐ min ) ACSF at 34–35°C. and images were acquired at 2 or 5 s intervals using TillVision software. Patch-clamp recordings. The recording chamber was mounted on the Fluorescence changes expressed as “ratio” (r) were calculated from the stage of an upright microscope (Olympus BX51WI) equipped with a ϭ Ϫ Ϫ formula r (F340soma F340bg)/(F380soma F380bg), where F340 and F380 CCD camera (Till Photonics) and monochromator (Polychrome IV, Till were the fluorescence emitted at the excitation wavelengths 340 and 380 Photonics) for microfluorometry. Individual neurons were visualized nm, respectively, for the soma and background (bg), measured from a through a 40ϫ water-immersion objective (Olympus) using an infrared region Ͼ100 ␮m away from the soma (Guatteo et al., 1998). Ratio values differential interference contrast (IR-DIC) system. Whole-cell patch- were converted into ion concentrations using the method of Grynkiewicz

clamp recordings, combined with microfluorometry, were performed on et al. (1985). The rmin and rmax values (Grynkiewicz et al., 1985) were 2ϩ SNpc neurons localized in a region of tightly packed neurons adjacent to obtained in situ by exposing perforated cells (1 ␮M ionomycin) to Ca - 2ϩ the medial terminal nucleus of the accessory optic tract. DAergic neurons free (0 mM calcium, 1 mM EGTA) and 1 mM Ca -containing solutions. were electrophysiologically identified based on a slow and regular spon- Confocal microscopy. Slice preparation was identical to that described taneous firing (1–5 Hz), hyperpolarizing/outward response to dopamine for electrophysiological recordings. After cutting, slices were transferred ␮ (30 M), prominent Ih in response to hyperpolarizing voltage steps to a holding chamber and left to recover for 30 min (33°C) in standard

(Mercuri et al., 1995) and, in current-clamp mode, a depolarizing sag ACSF medium saturated with an O2/CO2 gas mixture. Slices were then when negative currents were applied (Grillner and Mercuri, 2002). The placed in seven chambers containing standard saturated ACSF. In the GABAergic interneurons in the SNpc showed a high regular spontaneous first chamber (CTRL), no extra compounds were added; in the second firing at rest (25 Hz) (Lacey et al., 1989; Berretta et al., 2000; Gernert et al., (BMAA), we added BMAA (3 mM), and in the others, we added BMAA ␮ ␮ ␮ 2004), no response to dopamine application, modest Ih and sag potential plus CPCCOEt (100 M), CNQX (10 M), JNJ (1 M), a mixture of JNJ in response to hyperpolarizing voltage and current pulses, respectively. plus CNQX, or cyclosporine A (CsA, 10 ␮M). After 1 h treatment, slices Patch pipettes (2–5 M⍀), pulled with a PP 83 Narishige puller, were from all the groups were left to recover (5 h) in separate chambers filled

filled with a solution containing the following (in mM): K-gluconate (115), with standard saturated ACSF gassed with 95%O2,5%CO2. For ROS CaCl2 (4), MgCl2 (2), KCl (10), EGTA (10), HEPES (10), Mg2-ATP (2), and detection experiments (supplemental Fig. S3, available at www.jneurosci. Na3-GTP (0.3), pH 7.3, osmolarity 280 mOsm. For microfluorometry, we org as supplemental material), we divided slices into 3 groups: CTRL, used a pipette solution containing the following (in mM): K-gluconate (135), BMAA, and BMAA plus 1,4-dithiothreitol (DTT; 500 ␮M). After 1 h 5178 • J. Neurosci., April 14, 2010 • 30(15):5176–5188 Cucchiaroni et al. • Actions of BMAA onto SNpc DAergic Neurons

BMAA treatment, hydroethidine (Invitrogen; 315 nM) was added for 30 nium oxychloride [Ruthenium Red (RR)], cyclopiazonic acid (CPA), min. For hypoxia experiments, after 30 min of recovery, slices were di- 1-[(5-chloro-1H-benzimidazol-2-yl)carbonyl]-4-methylpiperazine vided into two groups: the first (CTRL) consisted of slices kept for 30 min maleate (JNJ 16259685), and picrotoxin were obtained from Tocris in the standard ACSF gassed with 95%O2,5%CO2; the second (hypoxia) Cookson. Tetrodotoxin was from Alomone Laboratories. Dopamine hy- ␤ was placed in a chamber containing the ACSF saturated with a 95% N2 drochloride (DA), -N-methylamino-L-alanine (BMAA), glutamic and 5% CO2 for 30 min. acid, cyclosporine A (CsA), nifedipine, 4-aminopyridine, cesium To perform post hoc labeling of the recorded cells, biocytin (5 mM, free chloride, cobalt chloride, and cadmium chloride were from Sigma- base; Sigma) was added in the pipette solution. Aldrich. 1,4-Dithiothreitol (DTT) was from Boehringer Mannheim Slices from the different experimental settings were fixed overnight in and hydroethidine from Invitrogen. 4% paraformaldehyde and, after 3 washing in phosphate buffer (PB), transferred to 30% sucrose/PB at 4°C for 48 h. They were then cut into Results ␮ 2؉ 35- m-thick horizontal sections with a freezing microtome and col- Actions of BMAA on the membrane properties and [Ca ]i in lected in PB. The sections with biocytin-loaded cells were incubated for SNpc neurons 1 h at room temperature (RT) in PB-containing Cy2-conjugated Strepta- In the view that BMAA could be the toxic agent causing damage vidin (1:200; Jackson ImmunoResearch Laboratories) and 0.3% Triton and eventually cell death of DAergic and other neuronal types in X-100. The following primary antibodies were then used: goat anti- ALS/PDC as well as in sporadic PD, we studied its effects in single tyrosine hydroxylase (TH; 1:500; Santa Cruz Biotechnology), mouse SNpc DAergic neurons. We first investigated the effects of BMAA anti-cyt-c (1:100; Promega), rabbit anti-STIM1, or rabbit anti-Orai1 (1: 400; ProSci). Following overnight incubation with primary antibodies, on neuronal firing activity (Fig. 1A–D) in current-clamp mode, sections were incubated for2hatRTinamixture of secondary antibodies to detect their typical spontaneous tonic action potential discharge including the following: Cy5-conjugated donkey anti-goat IgG, Cy3- (Grillner and Mercuri, 2002). Short-lasting bath application (1.5–2 conjugated donkey anti-rabbit IgG, or Cy2-conjugated donkey anti- min) of BMAA (0.1–10 mM) increased the spontaneous firing mouse IgG (1:100; all from Jackson ImmunoResearch Laboratories). frequency in a dose-dependent manner (Fig. 1A–C) and caused a Sections were air dried and coverslipped with Gel/Mount (Biomeda). rapid and reversible membrane depolarization (Fig. 1A,C,D). Images were acquired through a confocal laser scanning microscope To determine whether continuous presence of BMAA in the (Leica SP5, Leica Microsystems) providing four excitations: 405, 488, extracellular fluid, in low concentration, induces progressive al- 543, and 633 nm. Plates were generated adjusting the contrast and bright- teration of neuronal activity, we applied 100 ␮M BMAA for 30 ness of digital images (Corel Draw, 9). Qualitative and/or quantitative min. In this experimental condition, the drug produced signifi- observations were limited to SNpc neurons, identified by shape, cellular cantly larger depolarization of the membrane potential and in- density and proximity to the medial terminal nucleus of the accessory optic tract. Analysis was conducted in five subsections obtained from crease in firing frequency than during short-lasting application Ͻ each 250-␮m-thick slice (5 slices/group). (Fig. 1B,D, white bars) ( p 0.05). Electrophysiological and microfluorometry data analysis. Data were an- Voltage-clamp recordings revealed that BMAA-induced alyzed with Origin (version 6, OriginLab), expressed as mean Ϯ SEM, membrane depolarization was due to an inward current (IBMAA), and compared using one- or two-population Student’s t test. Changes in whose mean amplitude ranged from 20.8 Ϯ 3.9 pA to 1004 Ϯ firing frequency and cell membrane depolarization were calculated as the 160.3 pA (Fig. 1F), in response to increasing BMAA concentra- difference between the peak and baseline levels. Baseline levels were mea- tions (0.1–10 mM) (Fig. 1E,F). The inward current caused by sured 10–20 s before BMAA application, and peak levels during 10–20 s high dosages (3 and 10 mM) of BMAA was often followed by an just before washout of the drug. Current areas (see Fig. 5D, inset) were outward current (Fig. 1E). quantified using an integral function (OriginLab) of the area between the Measures of I and the associated [Ca 2ϩ] dynamics were current trace and the baseline. In all measurements (including densitom- BMAA i etry and cell size measurements), significance levels were indicated as performed by applying BMAA to the recorded neuron by pres- *p Ͻ 0.05; **p Ͻ 0.01; #p Ͻ 0.0001. sure ejection, at 2 min interval (3 mM) (see Figs. 1G,H,2–6,9). Densitometric analysis of fluorescence images. Quantification of fluores- Using this experimental approach, we obtained fast transient Ϯ ϭ cence (cyt-c immunoreactivity and ROS generation) was performed in IBMAAs of mean amplitude 454.5 34.6 pA (n 73), which were 2ϩ TH-immunoreactive cells by densitometric analysis. After background accompanied by reversible and transient [Ca ]i increases of subtraction, cell-associated signals were quantified by manually outlin- 139.3 Ϯ 23.7 nM (n ϭ 13). Calcium concentration changes orig- ing individual neurons and measuring the mean pixel density (mean inated from a resting level of 87.1 Ϯ 5.2 nM, peaked at 226.4 Ϯ fluorescence) with the ImageJ software (NIH; http://rsb.info.nih.gov/ij/). 23.3 nM ( p Ͻ 0.0001, paired t test) (Fig. 1H)(n ϭ 13) and rapidly Mean fluorescence of individual cells (F) was normalized to the total returned to the baseline ( p ϭ 0.3). cellular surface (A). Quantification was done on 200 cells (5 slices/ group). Statistical analysis was performed by means of one-way ANOVA, Ͻ Glutamate receptors activated by BMAA followed by Bonferroni post hoc test with p 0.05 as minimal level for ␤ significance. Based on the evidence that the -carbamate form of BMAA, pro- Neuron size measurement. Digital images of the TH-immunoreactive duced in bicarbonate-containing medium, has a chemical struc- SNpc neurons from 5 sections were captured using a 63ϫ objective and ture similar to glutamate (Myers and Nelson, 1990; Brownson et 0.7 zoom factor. Neurons with clearly distinguishable nucleus, nucleolus, al., 2002), we aimed to determine which glutamate receptors ac- and cytoplasmic borders were sampled. Cell surface analysis was done by tivated IBMAA by using selective glutamate receptors antagonists outlining the soma borders, excluding dendrites and axon. Approxi- (Fig. 2A,B). We compared the relative contribution of different mately 200 cells were measured in each experimental group (5 slices/ receptors to the total current activated by BMAA (3 mM) with the group). The areas were calculated using ImageJ. Statistical analysis was one induced by glutamate (300 ␮M) (Fig. 2C,D). We assessed performed as described above. glutamate concentration, puff duration and electrode position to Drugs. 6-Cyano-7-nitroquinoxaline-2,3-dione (CNQX), D-2-amino- obtain similar IBMAA and IGLU amplitudes. In the presence of the 5-phosphono-pentanoic acid (APV), 7(hydroxyimino)cyclopropal[b] ␮ ␤ selective mGluR1 antagonist CPCCOEt (100 M), IBMAA was re- chromen-1a-carboxylate (CPCCOEt), DL-threo- -benzyloxyaspartic acid Ϯ Ͻ ϭ (DL-TBOA), (5S,10R)-(ϩ)-5-methyl-10,11-dihydro-5H-dibenzo[a,d] duced to 41.6 3.6% of control (Fig. 2A1,B)(p 0.0001, n cyclohepten-5,10-imine maleate (MK801), 2-methyl-6-(phenylethynyl) 24). We also tested the effect of another selective and potent pyridine hydrochloride (MPEP), 1-[2-(4-methoxyphenyl)propoxy] mGluR1 antagonist, JNJ 16259685 (1 ␮M) (Lavreysen et al., Ϯ ϭ ethyl-1H-imidazole hydrochloride (SKF 96365), ammoniated ruthe- 2004), which reduced IBMAA to 3 3.2% of control (n 3, data Cucchiaroni et al. • Actions of BMAA onto SNpc DAergic Neurons J. Neurosci., April 14, 2010 • 30(15):5176–5188 • 5179

IBMAA is activated mainly by mGluR1 and, to a lesser extent, by AMPA receptors. We refer to this mGluR1-dependent response of BMAA as “classical.” We then compared the effects of gluta- mate and BMAA on SNpc DAergic cells.

Differently from IBMAA, IGLU was reduced by either ionotropic or metabotropic type

1 receptor antagonists (Fig. 2C,D1,D2). In ␮ particular, CNQX (10 M) reduced IGLU to 42.5 Ϯ 4.2% of control ( p Ͻ 0.0001, ϭ n 10) (Fig. 2C1,D1), and APV and Ϯ Ͻ MK801 reduced IGLU to 75.5 2.8% ( p 0.01, n ϭ 6) and 70.4 Ϯ 1.1% of control ( p Ͻ 0.0001, n ϭ 5), respectively (Fig.

2C2,D1). To isolate a pure mGluR-media- ted current, we blocked all ionotropic components by applying CNQX (10 ␮M) ␮ with MK801 (10 M) (Fig. 2C3,C4,D2). In Ϯ this condition IGLU was reduced to 29.6 7.3% of control ( p Ͻ 0.0001, n ϭ 7) (Fig.

2D1). The mGluR1 and mGluR5 antag- onists CPCCOEt (100 ␮M) and MPEP (10 ␮M) reduced the remaining current to 77 Ϯ 2.7% ( p Ͻ 0.01, n ϭ 7) and 88.7 Ϯ 2.8% ( p ϭ 0.06, n ϭ 3) of con- trol, respectively. In a small population of neurons (15/ 88), BMAA caused an inward current that was mostly insensitive to the mGluR1 an-

tagonist CPCCOEt. In these cells, IBMAA was mediated by AMPA receptors, as it was strongly reduced by CNQX (10 ␮M, data not shown) but was not by the kai- nate receptor antagonist GYKI (300 ␮M). Interestingly, the electrophysiological properties of these neurons (firing fre- quency, sensitivity to DA, and presence of a time- and voltage-dependent inward

current, Ih) fulfilled the criteria typical of DAergic neurons (data not shown). The following focuses on the classical 2ϩ BMAA-induced responses. Figure 1. BMAA-induced firing frequency increase, membrane depolarization, inward current, and [Ca ]i modification in DAergic neurons. A, C, Example traces showing transient firing rate increase in response to 0.1, 0.3, and 1 mM BMAA bath applications (1.5–2 min). B, D, Plots showing mean changes of firing frequency (FF) and membrane potential, respectively, in TRPC-like channels mediate the responsetoshort(1.5–2min,0.1–10mM,blackbars)andlong(30min,0.1mM,whitebars)BMAAbathapplications.Theseeffects BMAA-induced inward current ϭϪ Ϯ were due to activation of an inward current (IBMAA) as revealed by voltage-clamp recordings (Vhold 60 mV) (E, F). Means It has been previously shown in DAergic SEMoftheplotsB,D,andFareshowninsupplementalTable1(availableatwww.jneurosci.orgassupplementalmaterial).G,Fast neurons that mGluR1s couple, in a BMAA application(1–1.5 s, arrow) induced an inward current (top) that was associated with calcium transients (bottom). H, Top, G-protein-dependent manner, to tran- ϭ Averagedcalciumtrace(n 13)andmeancalciumvalues(bottom)measuredatthetimesindicatedbythecorrespondingletters sient receptor potential (TRP) channels, # Ͻ in the trace. p 0.0001. mostly type C1, C3, and C5 (Riccio et al., 2002; Tozzi et al., 2003). Thus, we sought to

not shown). However, the antagonistic effect of this drug did not determine whether IBMAA is also mediated by TRPC-like channels by washout within 1 h. In the presence of the selective AMPA recep- perfusing the nonselective TRP channel blockers SKF 96365 (100 ␮ ␮ ␮ tor antagonist CNQX (10 M), IBMAA was slightly but signifi- M) and RR (20 M) (Fig. 3A,B). Figure 3A shows the time Ϯ Ͻ cantly reduced to 93.1 2% of control (Fig. 2A2,B)(p 0.01, course of IBMAA amplitude, elicited every 2 min, in control (a, n ϭ 24). The NMDA receptor antagonists MK801 (10 ␮M) and c) and during SKF 96365 (b) and RR (d) bath application. Both ␮ ␮ APV (50 M) and the mGluR5 antagonist MPEP (10 M) did not TRP antagonists caused a marked reduction of IBMAA ampli- Ϯ Ͻ ϭ Ϯ significantly affect IBMAA amplitude (Fig. 2A3,A4,B). Indeed, in tude to 42.1 4.3% ( p 0.0001, n 8) and 27 8.3% of the presence of APV or MK801, the current was 98.7 Ϯ 2.2% of control ( p Ͻ 0.01, n ϭ 6), respectively. ϭ ϭ Ϯ ϭ ϭ control ( p 0.6, n 6) and 104 8.8% of control ( p 0.7, n To further test the hypothesis that IBMAA is due to the activa- Ϯ 4), respectively, and in the presence of MPEP, IBMAA was 95.4 tion of a mixed cationic current, likely TRPC channels, we mea- ϭ ϭ 4.9% of control ( p 0.4, n 7). These data demonstrate that sured the I–V relationship of IBMAAs by using voltage ramps (Fig. 5180 • J. Neurosci., April 14, 2010 • 30(15):5176–5188 Cucchiaroni et al. • Actions of BMAA onto SNpc DAergic Neurons

3C, see Materials and Methods). The cur- rent had a reversal potential of ϳ1.0 mV (n ϭ 4) (Fig. 3D), consistent with the opening of a mixed cationic channel (Okada et al., 1998; Strubing et al., 2001; Tozzi et al., 2003; Faber et al., 2006). It has been recently demonstrated that TRPC channels require STIM1 and/or Orai1 activation, to act as store-operated calcium channels (SOCs) (Kim et al., 2009; Liao et al., 2009) (for review, see Ca- halan, 2009). Therefore, we made whole- cell recordings from SNpc DAergic neurons with biocytin-containing pi- pettes and characterized IBMAA by using mGluR1, AMPA, and TRPC antagonists. We then conducted immunohistochemis- try for TH, STIM1, and Orai1 (n ϭ 20). All the biocytin-labeled cells were TH, STIM1, and Orai1 immunoreactive (Fig. 3E,F). Thus, the expression of STIM1 and Orai1 proteins in biocytin-labeled TH- immunoreactive neurons might suggest their putative involvement, together with Figure 2. A, C, IBMAA is mediated by the activation of mGluR1. Example traces of IBMAA (A) and IGLU (C) on expanded time scale, TRPC channels, in the mGluR1-mediated incontrolandinthepresenceofglutamatereceptorantagonists,werealignedontheirrisingphase.B,D,HistogramsofIBMAA and responses. However, the functional inter- IGLU amplitudes, expressed as percentage of controls, in the presence of glutamate receptor antagonists. CPCCOEt (A1) and, to a ϭ action between STIM1, TRPC, and Orai1 lesser extent, CNQX (A2) significantly reduced IBMAA (B, n 24 for both). APV, MK801 (A3), and MPEP (A4) did not modify IBMAA ϭ proteins is still questioned (DeHaven et (B, n 6, 4, and 7, respectively). IGLU was significantly affected by all ionotropic (CNQX, MK801, APV, and CNQX plus MK801) ϭ receptor antagonists (C1, C2, D1, n 10, 5, 6, 5, respectively). The IGLU sensitivity to mGluR antagonists was assessed in the al., 2009) and needs further investigation. ϭ presence of CNQX plus MK801 (D2). CPCCOEt (C3) significantly reduced IGLU (D2, n 7), whereas MPEP (C4) left IGLU unaffected (D , n ϭ 3). **p Ͻ 0.01, #p Ͻ 0.0001. Intracellular calcium changes in 2 response to BMAA application ϩ sarcoplasmic reticulum Ca 2 -ATPase, BMAA-induced calcium As reported in Figure 1G, IBMAA is also associated with transient ϩ ϩ was reduced to 12 Ϯ 5.7% of control ( p ϭ 0.001, n ϭ 5, data not [Ca 2 ] elevation. We then explored whether the [Ca 2 ] rise was i i shown). dependent on activation of membrane receptors as mGluR1 and To verify that BMAA does not produce any additional effects AMPA, and whether the opening of TRPC-like channels is re- other than those due to mGluR1-TRPC and AMPA receptor ac- quired (Fig. 4). Therefore, we recorded I (upper traces) and BMAA tivation, we measured I and calcium levels in the presence of calcium changes (bottom traces) in fura-2-loaded DAergic neu- BMAA mGluR1, TRPC, and AMPA receptor antagonists (CPCCOEt, rons before, during, and after the application of CPCCOEt (100 SKF 96365, and CNQX, respectively). In the presence of this ␮M) (Fig. 4A), CNQX (10 ␮M) (Fig. 4B), and SKF 96365 (100 mixture (supplemental Fig. S1A,B, available at www.jneurosci. ␮M) (Fig. 4C). I was reduced by all drugs to 21.1 Ϯ 4.2% BMAA org as supplemental material), BMAA responses were abolished, ( p Ͻ 0.0001, n ϭ 7), 75 Ϯ 7% ( p Ͻ 0.05, n ϭ 5), and 54.1 Ϯ 4.1% ϩ excluding the possibility that other mechanisms might be in- of control ( p Ͻ 0.0001, n ϭ 7), respectively. In contrast, [Ca 2 ] i volved (current: 10 Ϯ 1% of control; calcium: 7.5 Ϯ 2.5% of was not affected by CNQX (93.3 Ϯ 13.2% of control, p ϭ 0.63, control). These data suggest that BMAA activates mGluR1-TRPC n ϭ 6) (Fig. 4B), suggesting that while the contribution of AMPA pathway as previously described for the selective mGluR1 agonist receptors to IBMAA is small but significant, the AMPA-mediated 2ϩ (S)-3,5- (DHPG) (Tozzi et al., 2003). In- changes in [Ca ]i are negligible. Interestingly, the BMAA- ϩ deed, direct activation of mGluR1 with DHPG (100 ␮M) com- induced [Ca 2 ] increase was largely dependent on both mGluR1 i pletely occluded BMAA effects (supplemental Fig. S2, available at and TRPC-like channel activation, since their antagonists strongly www.jneurosci.org as supplemental material) (current: 31 Ϯ reduced calcium signals (CPCCOEt: 26.6 Ϯ 6.6% of control, p Ͻ 9.9% of control; calcium: 2 Ϯ 0.5% of control). 0.0001, n ϭ 7; SKF 96365: 38 Ϯ 10.3% of control, p Ͻ 0.01, n ϭ 7, respectively) (Fig. 4A,C). All drugs washed out upon re- moval from bathing solution. Involvement of EAA transporters in BMAA-induced currents Coapplication of CPCCOEt and SKF 96365 did not cause ad- We evaluated the possibility that excitatory amino acid trans- 2ϩ Ϯ ditive effect on IBMAA and [Ca ]i decrease [current: 48. 7 5.8% porters (EAATs) could contribute to the clearance of BMAA of control (supplemental Fig. S1A, available at www.jneurosci. from the extracellular medium affecting amplitude and/or dura- Ϯ org as supplemental material); calcium: 27 3.8% of control tion of IBMAA in dopamine neurons. Therefore, we studied the (supplemental Fig. S1B, available at www.jneurosci.org as sup- effect of the potent inhibitor of EAATs, DL-TBOA (100 ␮M) plemental material)], and the values were not significantly differ- (Shimamoto et al., 1998) on IBMAA amplitude and area (see Ma- ent from those obtained in the presence of the two compounds terials and Methods) (Fig. 5A). First, we assessed the effect of separately. Thus, mGluR1 activation and TRPC-like channel DL-TBOA on glutamate-mediated inward currents (IGLU, 300 opening are two subsequent steps in the same pathway. More- ␮M) (Fig. 5B), which are known to be highly modulated by over, in the presence of CPA (30 ␮M), a reversible inhibitor of EAATs (Tong and Jahr, 1994). As expected, DL-TBOA signifi- Cucchiaroni et al. • Actions of BMAA onto SNpc DAergic Neurons J. Neurosci., April 14, 2010 • 30(15):5176–5188 • 5181

ϭ Ϯ 0.3, n 6) and IBMAA area was 101.8 7.8% of control ( p ϭ 0.8, n ϭ 6).

BMAA activates AMPA receptors in GABAergic neurons of the SNpc It has been demonstrated that BMAA ac- tivates different types of glutamate recep- tors in diverse brain areas (Spencer et al., 1987; Copani et al., 1990, 1991; Allen et al., 1993; Pai et al., 1993; Staton and Bristow, 1998; Rao et al., 2006; Lobner et al., 2007). To this regard, we aimed to ex- plore its effect onto the “secondary” cells of the SNpc, presumably GABAergic neu- rons (Fig. 6) (Lacey et al., 1989). We found that, in these cells, BMAA evoked an inward current (Fig. 6A, upper panel) (mean amplitude: 166 Ϯ 20 pA; n ϭ 9) significantly smaller than in DAergic cells. This difference was not due to cell size

since IBMAA, normalized to cell capaci- tance, was also significantly different Ϯ (IBMAA in DAergic cells 5.18 0.7 pA/pF, n ϭ 16; in GABAergic cells 2.56 Ϯ 0.5 pA/ pF, n ϭ 6, p Ͻ 0.05; data not shown).

Moreover, in GABAergic cells, IBMAA was reduced by CNQX (10 ␮M; 33.4 Ϯ 2.8% of control, p Ͻ 0.0001, n ϭ 9) but not by CPCCOEt (100 ␮M; 97.4 Ϯ 5.2% of con- trol, p ϭ 0.64, n ϭ 7) (Fig. 6C,D) and was 2ϩ not associated with [Ca ]i increase [Fig. 6A (lower panel),B](p ϭ 0.14, paired t test, n ϭ 4).

Prolonged exposure to BMAA cause significant changes in the properties of DAergic neurons Based on the evidence that BMAA can ac- cumulate within the human body (Murch et al., 2004), we sought to determine whether prolonged exposure to the toxin could alter DAergic neuron functional properties. To test this hypothesis, we monitored different cellular parameters before and after BMAA (3 mM) bath ap- plications of 12, 20, and 30 min. The eval- uations were done 20 min after BMAA ␮ Figure3. TRPCsmediateIBMAA.A,TimecourseofIBMAA amplitudeinresponsetoperfusionwithSKF96365(100 M)andRR(20 washout, and were expressed as percent- ␮ M). Top, Raw traces of IBMAA acquired at the times indicated by the corresponding letters in the plot. B, Histogram of IBMAA age of the control value before BMAA ap- amplitude, expressed as percentage of control (white bar), following exposure to SKF 96365 and RR (n ϭ 8 and 6, respectively). plication. The examined parameters ϭϪ C,IBMAA recordedfromaDAergicneuron(Vhold 65mV)inresponsetoaBMAApuff(3mM,2s,arrow).Atthepeakoftheinward included the following: number of spikes currentavoltageramp(V ,Ϫ65toϩ70mV,1.5s,seeinset)isexecuted.D,MeanI–VofthenetI obtainedfromfourcells. ramp BMAA evoked by a depolarizing current step E, F, Confocal laser scanning microscope images of SNpc DAergic neurons loaded with biocytin (green) and stained with TH 2ϩ (Fig. 7A), [Ca ]i levels (Fig. 7B), mem- (blue) antibody. E, Triple labeling with STIM1 (red) antibody. F, Triple labeling with Orai1 (red) antibody. As shown in the brane resistance R (Fig. 7C), and mem- mergeimages,biocytin-loadedTH-positiveneuronsareimmunopositiveforbothSTIM1(E)andOrai1(F ).Scalebar:6␮m. m brane potential changes V (Fig. 7D). The **p Ͻ 0.01, #p Ͻ 0.0001. m same parameters were also measured in a control population of cells recorded for 50 min, but not exposed to the toxin (no- Ϯ Ͻ cantly increased IGLU amplitude and area to 448 18.6% ( p drug group) (Fig. 7A–D, white bar). All the experiments were 0.01, n ϭ 3) and to 753 Ϯ 127% of control ( p Ͻ 0.05, n ϭ 3), performed in current-clamp mode.

respectively (Fig. 5C,D, black bars). In contrast, IBMAA was largely In the no-drug group, all the parameters remained unchanged unaffected by the EAAT inhibitor (Fig. 5C,D, white bars). Indeed, in up to 50 min of recording: spike frequency was 99.8 Ϯ 17.8% of Ϯ ϭ ϭ ϭ 2ϩ Ϯ the presence of DL-TBOA, IBMAA amplitude was 90.6 8.7% ( p control ( p 0.99, n 11); [Ca ]i was 119.9 11% of control 5182 • J. Neurosci., April 14, 2010 • 30(15):5176–5188 Cucchiaroni et al. • Actions of BMAA onto SNpc DAergic Neurons

ϭ ϭ Ϯ ( p 0.11, n 8); Rm was 86.9 7.2% of ϭ ϭ control ( p 0.11, n 8); and Vm was 101.8 Ϯ 2.2% of control ( p ϭ 0.44, n ϭ

13). Except for Vm (Fig. 7D), all the pa- rameters were significantly modified by prolonged BMAA exposures (12, 20, or 30 min). Particularly, spike frequency (Fig. 7A) was reduced to 70.4 Ϯ 20.2% of con- trol ( p ϭ 0.2, n ϭ 6) after 12 min, to 14.5 Ϯ 8% of control ( p Ͻ 0.0001, n ϭ 10) after 20 min, and to 0 Ϯ 0.3% of con- Ͻ ϭ trol ( p 0.0001, n 4) after 30 min. Rm (Fig. 7C) was reduced to 77.8 Ϯ 2.7% of control ( p Ͻ 0.01, n ϭ 5) after 12 min, to 80.2 Ϯ 3.9% of control ( p Ͻ 0.01, n ϭ 5) after 20 min, and to 32 Ϯ 9% of control Ͻ ϭ 2ϩ ( p 0.01, n 4) after 30 min. [Ca ]i (Fig. 7B) increased to 173.9 Ϯ 32.6% of control ( p Ͻ 0.05, n ϭ 10) after 12 min, to 192.5 Ϯ 26.6% of control ( p Ͻ 0.01, n ϭ 12) after 20 min, and to 473.5 Ϯ 109.3% of control ( p Ͻ 0.0001, n ϭ 4) after 30 min. Since all the parameters were measured 20 min after BMAA washout, we considered these changes as functional evidence of neu- ronal damage. To additionally confirm that BMAA exclusively activates mGluR1-TRPC and AMPA receptors, slices were treated for 30 min with BMAA in the presence of JNJ 16259685 and CNQX (Fig. 7A–D). In this condition, the BMAA-induced effects were prevented. Indeed, spike frequency was 134.8 Ϯ 21.7% (Fig. 7A)(p ϭ 0.3, n ϭ 2ϩ Ϯ 4), [Ca ]i was 124.6 10.8% (Fig. 7B) ϭ ϭ Ϯ ( p 0.8, n 4), Rm was 100.3 13.8% ϭ ϭ 2ϩ (Fig. 7C)(p 0.37, n 4), and Vm was Figure 4. BMAA-induced [Ca ]i increase is sensitive to mGluR and TRPC channel blockers. A–C, Simultaneous recordings of 2ϩ 99.7 Ϯ 3.2% (Fig. 7D)(p ϭ 0.66, n ϭ 4) of currents(uppertraces)and[Ca ]i (lowertraces)showpairsofBMAAresponsesincontrolsolutions,followingbathapplicationof control. The antagonists alone (without the drugs CPCCOEt (100 ␮M)(A), CNQX (10 ␮M)(B), SKF 96365 (100 ␮M)(C), and washout. Histograms show cumulative data of I and [Ca 2ϩ] in the presence of the drugs CPCCOEt (A), CNQX (B), SKF 96365 (C), and washout normalized to the control BMAA) left the parameters unchanged BMAA i response (white bar), measured in the same cell. *p Ͻ 0.05, **p Ͻ 0.01, #p Ͻ 0.0001. (Fig. 7A–D). ϩ Ͻ ϩ ϩ Ͻ BMAA induces cytochrome-c release, which is prevented by 0.0001), BMAA JNJ ( p 0.01), BMAA JNJ CNQX ( p ϩ Ͻ mGluR1 and mPTP antagonists, and activates ROS 0.0001), and BMAA CsA groups ( p 0.0001). No significant ϩ production differences were observed between the BMAA and BMAA ϩ ϩ ϩ It has been proposed that mitochondrial Ca 2 overload leads to CPCCOEt, BMAA and BMAA CNQX, or BMAA CPCCOEt ϩ Ͼ cyt-c release due to the opening of the mitochondrial permeability and BMAA CNQX groups (all p 0.05). Furthermore, signif- ϩ transition pore (mPTP) (Hajno´czky et al., 2003; Orrenius et al., icant differences were observed between BMAA CPCCOEt or ϩ ϩ ϩ ϩ ϩ 2003) and that glutamate receptor-mediated Ca 2 entry triggers BMAA CNQX and BMAA CNQX JNJ or BMAA CsA Ͻ cyt-c release from mitochondria inducing neuronal degeneration data ( p 0.01) (Fig. 8H). On the other hand, the differences (Tang et al., 2005). In view of these data, we evaluated the toxic between CTRL and BMAA ϩ CNQX ϩ JNJ or CTRL and BMAA ϩ effects of BMAA by morphological analysis and immunohisto- CsA or BMAA ϩ CNQX ϩ JNJ and BMAA ϩ CsA groups were not chemical quantification of cyt-c release in TH-positive cells. significant ( p Ͼ 0.05). These latter comparisons indicate that block- BMAA exposure (3 mM, 1 h) resulted in clear cell shrinkage in ing either mGluR1 and AMPA receptors or mPTP (through CsA) DAergic neurons. Indeed, morphological analyses showed that prevents BMAA-induced neuronal damage (Fig. 8H). TH-positive neurons were significantly smaller in the BMAA- We further evaluated the ability of different ionotropic and exposed than in the CTRL groups (Fig. 8H). One-way ANOVA metabotropic glutamate receptor antagonists to prevent BMAA- analysis, performed on DAergic neuron cell size, among CTRL, induced changes. The cyt-c immunoreactivity (IR) was almost BMAA, BMAA ϩ CPCCOEt, BMAA ϩ CNQX, BMAA ϩ JNJ, absent in the CTRL group (Fig. 8A), while after BMAA treat- BMAA ϩ JNJ ϩ CNQX, and BMAA ϩ CsA (an mPTP blocker) ment, it dramatically increased (Fig. 8B). Compared to BMAA groups showed a significant overall effect of treatment (F ϭ treatment alone, BMAA ϩ CPCCOEt and BMAA ϩ CNQX treat- 15.48; p Ͻ 0.0001). Post hoc comparison demonstrated that ments presented a small reduction of cyt-c IR intensity (Fig. BMAA group data were significantly different from CTRL ( p Ͻ 8C,D). JNJ 16259685 treatment presented a better protection to Cucchiaroni et al. • Actions of BMAA onto SNpc DAergic Neurons J. Neurosci., April 14, 2010 • 30(15):5176–5188 • 5183

Figure 5. BMAA is not a substrate of EAATs. Traces showing IBMAA (A) and IGLU (B), evoked every 2 min, during DL-TBOA bath application (100 ␮M). C, D, Histograms of mean ϭ ϭ IBMAA (white bars, n 6) and IGLU (black bars, n 3) amplitude and area, respectively, calculated as indicated in the corresponding insets. Data are expressed as percentage of controls. *p Ͻ 0.05, **p Ͻ 0.01.

Figure 7. Effects of BMAA long treatments on DAergic cell viability. Histograms show- ing the number of action potentials in response to depolarizing step (100 pA, 700 ms) (A), Figure 6. Responses of GABAergic neurons to BMAA. A, Example traces of IBMAA and 2ϩ calcium changes during BMAA puff applications (2 min interval). Note that current ampli- [Ca ]i (B), membrane resistance Rm (C), and membrane potentials Vm (D) in five exper- tude was significantly smaller than in DAergic cells ( p Ͻ 0.05) (Fig. 1G). B, Histogram imental conditions: neurons exposed to BMAA for 12, 20, and 30 min, to BMAA ϩ JNJ ϩ 2ϩ Ϯ Ϯ CNQX for 30 min, and to JNJ ϩ CNQX for 30 min. The levels of significance have been showing mean [Ca ]i before (basal) (102.9 10.6 nM), during (BMAA) (110.3 13.9 nM), and after (post puff) (104.8 Ϯ 11 nM) BMAA application (n ϭ 4). C, Bottom, Time obtained by comparing each experimental condition with no drug. Other comparisons are ␮ indicated in the figure. Each parameter was measured after 20 min of BMAA washout. course of IBMAA amplitude in response to application of CPCCOEt (100 M) and CNQX (10 ␮ *p Ͻ 0.05, **p Ͻ 0.01, #p Ͻ 0.0001. M). Top, Raw traces of IBMAA acquired at the times indicated by the corresponding letters in the plot. D, Histogram of IBMAA amplitude, expressed as percentage of control (white bar), following exposure to CPCCOEt and CNQX (n ϭ 7 and 9, respectively). #p Ͻ 0.0001. 5184 • J. Neurosci., April 14, 2010 • 30(15):5176–5188 Cucchiaroni et al. • Actions of BMAA onto SNpc DAergic Neurons

the BMAA-induced effects (Fig. 8E). Fur- thermore, the greatest protection was achieved when a combination of JNJ and CNQX was used or when BMAA was co- applied with CsA. These latter conditions almost completely prevented the BMAA effects on cyt-c IR (Fig. 8F,G). One-way ANOVA performed on the cyt-cF/A values of CTRL, BMAA, BMAA ϩ CPCCOEt, BMAA ϩ CNQX, BMAA ϩ JNJ, BMAA ϩ JNJ ϩ CNQX, and BMAA ϩ CsA showed a significant overall effect of treat- ment (F ϭ 133.84; p Ͻ 0.0001). Post hoc comparisons demonstrated that BMAA data were significantly different from those of all the other groups considered ( p Ͻ 0.0001). Notably, the entity of BMAA-induced cyt-c release (F/A ratio ϭ 0.43) was similar to that caused by hyp- oxia (F/A ratio ϭ 0.5, data not shown) in DAergic neurons of SNpc, a condition known to irreversibly impair neuronal function (Geracitano et al., 2005). The comparisons between BMAA ϩ CPCCOEt or BMAA ϩ CNQX and BMAA ϩ JNJ groups ( p Ͻ 0.0001) or between BMAA ϩ JNJ and BMAA ϩ JNJ ϩ CNQX groups ( p Ͻ 0.01) were also significant (Fig. 8I). No significant differences were ob- served between BMAA ϩ CPCCOEt and BMAA ϩ CNQX, between CTRL and BMAA ϩ CNQX ϩ JNJ, between CTRL and BMAA ϩ CsA, or between BMAA ϩ CNQX ϩ JNJ and BMAA ϩ CsA groups ( p Ͼ 0.05) (Fig. 8I). These latter data indi- cate that the combined antagonism of mGluR1 and AMPA receptors, as well as the combination of BMAA and CsA treatments, completely prevents BMAA-induced neu- ronal damage. Furthermore, to determine the in- volvement of mitochondrial stress in BMAA toxicity, slices were treated with the ROS indicator hydroethidine (Anan- tharam et al., 2007; Liu et al., 2009; Zhang et al., 2009) to detect the production of superoxide following BMAA exposure (supplemental Fig. S3, available at www. jneurosci.org as supplemental material). BMAA caused a marked increase of the hydroethidine fluorescence in TH-posi- tive cells compared to CTRL, indicating an augmentation of ROS production. In-

4

of TH-immunoreactive neurons (in square micrometers) (H) and densitometric values of cyt-c release expressed as mean fluorescenceofindividualcellsnormalizedtototalcellularsur- face (F/A)(I) in CTRL, BMAA, BMAA ϩ CPCCOEt, BMAA ϩ CNQX,BMAAϩJNJ,BMAAϩCNQXϩJNJ,andBMAAϩCsA Figure 8. Prolonged exposure to BMAA causes neuronal shrinkage and massive cyt-c release into the cytosol of SNpc DAergic groups. Data are reported as means Ϯ SEM (n ϭ 200 cells per neurons.A–G,cyt-candTHdouble-labelingconfocalimagesofcontrol(CTRL;A)andBMAA-(B),BMAAϩCPCCOEt-(C),BMAAϩ group). Statistical comparison versus BMAA group, **p Ͻ CNQX- (D), BMAA ϩ JNJ- (E), BMAA ϩ CNQX ϩ JNJ- (F), and BMAA ϩ CsA- (G) treated slices. H, I, Histograms of soma cell size 0.01, #p Ͻ 0.0001. Scale bar: 18 ␮m. Cucchiaroni et al. • Actions of BMAA onto SNpc DAergic Neurons J. Neurosci., April 14, 2010 • 30(15):5176–5188 • 5185

tions. Taking into account the findings by Duncan et al. (1991), reporting that 10–30 ␮g/g BMAA in the rat brain corre- sponds approximately to the concentra- tion of Յ0.25 mM, it can be concluded that BMAA concentration in the human brain of patients is in the low millimolar range. The deleterious mechanisms of the toxin could underlie neurological condi- tions such as ALS-PDC and, potentially, idiopathic Parkinson’s disease. Few stud-

Figure 9. Activation of NMDA receptors increases BMAA-induced calcium modifications. A, Simultaneous recordings of IBMAA ies have investigated the effects of BMAA 2ϩ ␮ (uppertraces)and[Ca ]i (lowertraces)incontrolandduringbathapplicationofNMDA(20 M).BMAAconcentrationinthepuff on nigrostriatal DAergic neurons (Lind- ϭ 2ϩ ϭ pipette was 1 mM. B, Histograms showing cumulative data of IBMAA (black bar, n 5) and [Ca ]i (hatched bar, n 5) in the strom et al., 1990; Santiago et al., 2006). presenceof20␮M NMDA,normalizedtocontrolresponsebeforeNMDAapplication,inthesamecells(dottedline).NotethatNMDA However, BMAA toxicity was evaluated ϭ 2ϩ Ͻ did not affect IBMAA ( p 0.36), but it increased the BMAA-induced [Ca ]i accumulations. *p 0.05. indirectly, by measuring the ability of these cells to uptake or release dopamine. deed, this increase was significantly reduced by treatment with In the present study, we investigated the the antioxidant 1,4-dithiothreitol (DTT, 500 ␮M). One-way direct effects of BMAA onto single DAergic neurons of the SNpc ANOVA performed on the ROS F/A index of CTRL, BMAA, and in an in vitro slice preparation. We found that brief BMAA appli- BMAA ϩ DTT showed a significant overall effect of treatment cations, at a concentration similar to that found in ALS-PDC (F ϭ 129.66; p Ͻ 0.0001). Post hoc comparisons demonstrated brains (Murch et al., 2004; Pablo et al., 2009), induced reversible that BMAA data were significantly different from CTRL ( p Ͻ and reproducible membrane depolarizations/inward currents, ϩ 0.001) and BMAA ϩ DTT groups ( p Ͻ 0.001). Furthermore, the firing increase, and Ca 2 accumulation. In contrast, prolonged comparison between BMAA ϩ DTT and CTRL was also signifi- exposures (Ͼ12 min) caused functional impairment, cellular cant ( p Ͻ 0.05) (supplemental Fig. S3, available at www. shrinkage, calcium overload, cyt-c release, and ROS production jneurosci.org as supplemental material). that did not return to control values for up to 20 min or several hours after BMAA washout. BMAA and the “multiple-hits hypothesis” In the majority of cells tested, IBMAA was principally mediated Although several gene mutations have been proposed as risk fac- by activation of mGluR1-TRPC and, to a lesser extent, AMPA ϩ tors for PD development, the low penetrance of some of them receptors. In contrast, Ca 2 accumulation was strongly depen- suggests that PD may be due to the interaction between multiple dent on mGluR1-TRPC but not AMPA receptor activation. In- ϩ factors. The “multiple hits hypothesis” (Sulzer, 2007) combines deed, BMAA-induced Ca 2 increase was strongly reduced by toxic stress, as mitochondrial dysfunction, with decrease of neu- CPA, indicating that this amino acid activates a mGluR1-induced ϩ roprotective responses and exposure to environmental neuro- Ca 2 release from endoplasmic reticulum. 2ϩ toxic agents. Moreover, BMAA-induced responses (IBMAA and [Ca ]i in- To this regard, it has been demonstrated in cortical cell cul- crease) were abolished by a mixture of mGluR1, TRPC, and tures that low concentrations of BMAA, in combination with AMPA receptor antagonists, and occluded by the selective other insults (␤-amyloid, NMDA, and MPP ϩ), induces neuronal mGluR1 agonist DHPG. Therefore, it is unlikely that BMAA ac- death (Lobner et al., 2007). To test this hypothesis in midbrain tivates additional mechanisms. The contribution of mGluR5 to slice preparation, we puffed 1 mM BMAA (instead of 3 mM) with BMAA responses is minimal or absent since the specific antago- and without bath application of 20 ␮M NMDA for 8–10 min. In nist MPEP was without effect. When comparing the pharmacol- this condition, BMAA caused a significantly larger elevation of ogy of similar IGLU and IBMAA amplitudes, we found that IGLU,in 2ϩ [Ca ]i than in control, without affecting membrane current DAergic cells, was highly sensitive to both ionotropic and 2ϩ Ϯ (Fig. 9A,B). Indeed, [Ca ]i increased to 338.4 79.6% of con- mGluR1 antagonists, further suggesting that, despite the struc- Ͻ Ϯ trol ( p 0.05), whereas IBMAA remained 92.4 7.4% of control tural similarity between BMAA and glutamate, the former is pref- ( p ϭ 0.36, n ϭ 5 for both). These results indicate that calcium erentially an mGluR1 agonist. loading induced by lower BMAA concentrations can be potenti- We have previously reported that activation of mGluR1 by ated in stressed neurons, such as during exposure to NMDA. DHPG, caused an inward current in DAergic neurons (Guatteo et al., 1999) that was mediated by TRPC-like channels (Tozzi et al.,

Discussion 2003). Here we found that also IBMAA is due to activation of The interest toward BMAA as putative toxic agent damaging TRPC-like channels, through mGluR1. This conclusion is sup-

DAergic cells has grown considerably due to the discovery that ported by the following evidence: (1) IBMAA was reduced by the this toxin is not only present in the Guamanian islands but ap- nonselective TRPC channels blockers SKF 96365 and RR; (2) the ϳ pears to be ubiquitous throughout the world (Cox et al., 2003; IBMAA reversal potential was 0 mV, consistent with the activa- Esterhuizen and Downing, 2008; Johnson et al., 2008; Metcalf et tion of a TRPC-mediated mixed cation current (Okada et al., al., 2008). This natural amino acid binds to proteins in human 1998; Strubing et al., 2001; Tozzi et al., 2003); (3) Orai1 and tissues and thus might represent an endogenous reservoir con- STIM1, proteins that are likely to interact with TRPC channels stantly released during protein catabolism (Murch et al., 2004). downstream of G-protein-coupled receptor activation (Wang et For this reason, only recent studies properly quantified the con- al., 2008; Kim et al., 2009; Klejman et al., 2009; Liao et al., 2009) centration of BMAA in postmortem brain tissues of patients (for review, see Cahalan, 2009), were expressed in biocytin- (Murch et al., 2004; Pablo et al., 2009), using validated methods loaded neurons; (4) CPCCOEt and SKF 96365 did not have ad- able to detect either the free or the protein-bound BMAA por- ditive effects on IBMAA, demonstrating that mGluR1 activation 5186 • J. Neurosci., April 14, 2010 • 30(15):5176–5188 Cucchiaroni et al. • Actions of BMAA onto SNpc DAergic Neurons and TRPC-like channel opening are two subsequent steps in the through the opening of TRPC-like channels (Guatteo et al., 1999; BMAA-induced pathway. Tozzi et al., 2003), and activation of voltage-dependent Na ϩ and Interestingly, GABAergic SNpc cells responded to BMAA with Ca 2ϩ channels. This prolonged intracellular ionic dysregulation an AMPA-mediated inward current that was not associated with implicates mGluRs as central players in the injury of DAergic an intracellular calcium increase. A similar drug action was also neurons (Choi, 1994; Lu et al., 1996), leading to inappropriate observed in a minority of DAergic neurons. BMAA has been activation of kinases, proteases, and phospholipases and to en- previously reported to potently activate mGluRs in hippocam- ergy deficits (Salin´ ska et al., 2005). Moreover, the excessive ele- 2ϩ pal, striatal, and cerebellar neurons (Copani et al., 1990, 1991; vation of [Ca ]i might trigger mitochondrial calcium overload Manzoni et al., 1991; Staton and Bristow, 1998; Lobner et al., (Werth and Thayer, 1994), overproduction of ROS (Lafon-Cazal 2007). Alternatively, it predominantly activates NMDA receptors et al., 1993; Dugan et al., 1995; Reynolds and Hastings, 1995; in cortical neurons (Weiss et al., 1989; Lobner et al., 2007) and Carriedo et al., 1998, 2000), and release of cyt-c from mitochon- AMPA/kainate receptors in spinal motoneurons (Rao et al., dria (Orrenius et al., 2003). In line with the idea that mGluRs 2006). Although a likely explanation for the action of BMAA on activate processes leading to excitotoxic neuronal death in the ma- different glutamate receptors in different cell types is not avail- jority of DAergic neurons, it has been recently reported that func- able yet, these results provide the idea that the responses to tional antagonism of group I mGluRs (mGluR1 and mGluR5) is BMAA may depend on isoforms of glutamate receptors, ex- neuroprotective in an animal model of PD (rats treated with pressed on each cell type, that are preferentially bound by the 6-hydroxydopamine) (Vernon et al., 2005, 2007). amino acid. The BMAA-induced cellular response could also de- It has been reported that BMAA causes neuronal death only at pend on the particular localization of mGluRs on the plasmale- high millimolar concentrations (Spencer et al., 1987; Santiago et mmal membrane (Hubert et al., 2001) that renders these al., 2006; Buenz and Howe, 2007). In line with this, we have receptors more accessible to the drug. reported that BMAA, in the low millimolar range, caused electro- Despite BMAA being a natural glutamate agonist, our exper- physiological and mitochondrial dysfunction in DAergic cells. iments using DL-TBOA suggest that it is not a substrate for EAATs Although we did not study late-onset deleterious effects of lower and thus, it does not enter DAergic neurons through these carri- BMAA concentrations (due to the limitation of an in vitro slice ers. Therefore, it is unlikely that the neurotoxic effects of BMAA preparation), we found that prolonged BMAA exposure (30 min) are either mediated by its intracellular transportation through at low concentration (100 ␮M) caused larger depolarization and EAATs or defects in their expression (Wilson and Shaw, 2007). firing frequency increases than short-lasting applications. This An important clinical implication of our results is the obser- suggests that the continuous presence of BMAA in the extracel- vation that, when the toxin was applied for Ͼ12 min, the DAergic lular fluid, even in low concentration, may induce significant neurons showed a drop in membrane resistance, reduction in the alteration of neuronal activity. 2ϩ number of evoked action potentials, and [Ca ]i increase, which This conclusion is in line with the observation that BMAA can were prevented by mGluR1 and AMPA receptor antagonists. bind to proteins in the brain and can be released back as a free Moreover, BMAA-treated TH-positive cells showed an increased amino acid during protein catabolism years after consumption ROS production, prevented by the antioxidant DTT, and a re- (Murch et al., 2004). lease of cyt-c from mitochondria. In this regard, it has been re- Moreover, according to the previous evidence on cortical neu- ported a higher frequency of somatic mutations in mitochondrial rons, that micromolar BMAA concentration is able to potentiate genome in Guam ALS and PD patients than in Guam controls, insults caused by other toxic agents (␤-amyloid, NMDA, and suggesting that mitochondria dysfunction may contribute, as ge- MPP ϩ; Lobner et al., 2007), we found that coapplication of netic susceptibility, to ALS-PDC disease (Lynch et al., 2008). In- BMAA and NMDA produced larger Ca 2ϩ accumulations than terestingly, application of the potent mGluR1 antagonist JNJ BMAA alone. This suggests a potential synergistic effect of this 16259685 considerably reduced cyt-c release, suggesting that environmental amino acid with preexisting states of neuronal mGluR1 are the principal mediators of the BMAA-induced mi- stress. Therefore, since this toxin alters cellular activity, it may tochondrial stress. An almost complete neuroprotection was cooperate in causing dysfunction and late-onset injury to meta- achieved when BMAA was applied in the presence of JNJ bolically compromised DAergic cells, such as those surviving in 16259685 and CNQX, or CsA, a potent mPTP inhibitor PD patients (“multiple hits” hypothesis) (Sulzer, 2007). It is (Ferrand-Drake et al., 2003; Precht et al., 2005). Indeed, cyt-c worth mentioning that neuropathology of ALS-PDC patients re- immunoreactivity and cellular swelling were similar to those in veals Lewy’s bodies and ␣-synuclein-positive inclusions in SNpc control cells. This supports also an additional contribution to DAergic neurons similar to those found in PD patients (Hirano et cellular damage by AMPA-like receptors. Indeed, when AMPA al., 1966). and low-potency mGluR1 antagonists (CNQX and CPCCOEt, Since we have shown that BMAA, which has a human exposure respectively) were applied separately, the BMAA-induced cyt-c more widespread than previously thought, may exacerbate physio- release was only partially prevented. These results suggest that logical events caused by excitatory inputs to DAergic neurons and AMPA receptor activation may participate in the mechanism of initiate excitotoxic pathological mechanisms by mGluRs activation, BMAA toxicity by producing neuronal depolarization (via so- these receptors could be one of the promising targets to correct neu- dium influx), which in turn promotes activation of voltage- ronal dysfunction of the DAergic system in the more common, spo- gated Na ϩ and Ca 2ϩ channels (Berman and Murray, 1996; de radic form of Parkinson’s disease. Erausquin, 2004). 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