Alterations of Calcium Channels in a Mouse Model of Huntington’s Disease and Neuroprotection by Blockage of CaV1 Channels

Huntington’s disease (HD) is a neurodegenerative autosomal dominant disorder, characterized by symptoms of involuntary movement of the body, loss of cognitive function, psychiatric disorder, leading inevitably to death. It has been previously described that higher levels of brain expression of Cav1 channels are involved in major neurodegenerative disorders, such as Alzheimer’s disease and Parkinson’s disease. Our results demonstrate that a bacterial artificial chromosome (BAC)-mediated transgenic mouse model (BACHD mice) at the age of 3 and 12 months exhibits significantly increased Cav1.2 protein levels in the cortex, as compared with wild-type littermates. Importantly, electrophysiological analyses confirm a significant increase in L-type Ca2+ currents and total Ca2+ current density in cortical neurons from BACHD mice. By using an in vitro assay to measure neuronal cell death, we were able to observe neuronal protection against glutamate toxicity after treatment with Cav1 blockers, in wild-type and, more importantly, in BACHD neurons. According to our data, Cav1 blockers may offer an interesting strategy for the treatment of HD. Altogether, our results show that mutant huntingtin (mHtt) expression may cause a dysregulation of Cav1.2 channels and we hypothesize that this contributes to neurodegeneration during HD.


Introduction
Huntington's disease (HD) is a progressive neurodegenerative disorder characterized by symptoms that include involuntary movement of the body, loss of cognitive function, psychiatric disorders, and inevitable death (Ehrnhoefer et al., 2009, Labbadia andMorimoto, 2013). HD is an autosomal dominant disease caused by poly-glutamine expansion in a protein named huntingtin (Htt), leading to aggregate formation, as in a typical case of protein misfolding (Li and Li, 2004). However, the molecular mechanisms linking Htt mutation and neuronal cell death have not yet been fully elucidated (Davies et al., 1997, Tobin and Signer, 2000, Labbadia and Morimoto, 2013. The development of HD is determined by the progressive neuronal cell death occurring in the neocortex and striatum of affected patients (DiFiglia, 1990). There are some data showing that the neuronal cell loss that takes place in HD is due to glutamatergic excitotoxicity, which is mediated by excessive influx of intracellular Ca 2þ (DiFiglia, 1990, Calabresi et al., 1999. Several studies indicate that mHtt promotes Ca 2þ signaling alterations, which might be closely associated with the death of striatal neurons (Oliveira et al., 2006, Rosenstock et al., 2010, Wu et al., 2016, Raymond, 2017. It has been described that high levels of intracellular Ca 2þ produce abnormalities in the central nervous system that might be associated with voltage-gated Ca 2þ channels (Sattler and Tymianski, 2000, Cano-Abad et al., 2001, Luo et al., 2005, Swayne et al., 2005, Kim et al., 2008, Min et al., 2013. Alterations of voltage-gated channels may play an important part in disorders such as Alzheimer's disease (AD), Parkinson's disease (PD), epilepsy, and ischemia (Kim and Rhim, 2004, Pierrot et al., 2004, Luo et al., 2005, Swayne et al., 2005. Furthermore, dysfunction of L-type Ca 2þ channels has been implicated in some of the most prevalent neurodegenerative diseases, including AD and PD (Hurley et al., 2013, Min et al., 2013. Notably, previous studies have demonstrated that high levels of Ca v 1.2 channel expression in the brain are involved in AD (Kim and Rhim, 2011), and additional data showed that isradipine, an L-type Ca 2þ channel blocker, offers neuroprotection in a mouse model of PD (Ilijic et al., 2011). Therefore, we attempted to investigate whether Ca v 1 channels played a role on HD using a bacterial artificial chromosome (BAC)-mediated transgenic mouse model, BACHD (Gray et al., 2008). BACHD mice express full-length human Htt, exhibit progressive motor deficits, and lateonset selective neurodegeneration in the cortex and striatum. Accordingly, this mouse model is well suited for therapeutic trials (Ehrnhoefer et al., 2009).
Our results show that Ca v 1.2 protein levels are increased in the cortex of BACHD mice at 3 and 12 months of age. On the other hand, BACHD mice at 12 months did not show any alterations of Ca v 1.3 protein levels in the cortex, hippocampus, and striatum tissue. Interestingly, Ca v 1.2 mRNA levels were not different when comparing WT and BACHD mice, indicating that changes in protein levels are not due to gene expression alterations. Furthermore, whole-cell electrophysiology recordings from cortical BACHD neurons show an increase in L-type Ca 2þ currents and also in Ca 2þ current density, as compared with WT. In addition, we performed an assay using nifedipine and isradipine, which are L-type Ca 2þ channel blockers, to assess the neuroprotective effect of these drugs following glutamate insult. Our data showed that after applying the tested Ca v 1.2 channel blockers, there was a decrease in neuronal cell death in both WT and BACHD cultured neurons, even though cell death levels were higher in BACHD neurons. Notably, our results indicate the set point theory that calcium homeostatic mechanisms, including Ca v 1.2 channelsmediated influx, regulate the intracellular Ca 2þ levels at or near an optimal set point. Thus, different concentrations of Ca v 1.2 channel blockers appeared to stabilize the free cytosolic Ca 2þ concentration at optimal levels, even if they incompletely suppressed Ca 2þ influx. Altogether, these data suggest a previously unrecognized mechanism, where an increase in protein levels and function of Ca v 1.2 channels may lead to enhanced levels of intracellular Ca 2þ , which ultimately could damage irreversibly the neurons and contribute to HD pathogenesis.

Ethics Statement
All procedures used in this study were approved and strictly followed the ethical principles of animal experimentation adopted by the Ethic Committee on Animal Use of Federal University of Minas Gerais and institutionally approved under protocol number 139/2013.

Animals
Mice were housed in an animal care facility at 23 C on a 12-h light/12-h dark cycle with food and water provided ad libitum. C57/BL6 mice (25-30 g) were purchased from the animal facility (CEBIO) located at the Universidade Federal de Minas Gerais. FVB/NJ wild-type (WT) and FVB/N-Tg (Htt*97Q) IXwy/J (BACHD) (Gray et al., 2008) were purchased from The Jackson Laboratory (Bar Harbor, ME, USA). BACHD mice is a transgenic mouse model expressing full-length human Htt, exhibiting progressive motor deficits and late-onset selective neurodegeneration in the cortex and striatum. genders, at the embryonic Day 15 (E15), as described previously (Doria et al., 2013). After dissection, cortical tissue was submitted to trypsin digestion followed by cell dissociation using a fire-polished Pasteur pipette. Cells were plated on poly-L-ornithine coated dishes in Neurobasal medium supplemented with N2 and B27 supplements, 2 mM GlutaMAX, 50 mg/ml penicillin, and 50 mg/ml streptomycin. Cells were incubated at 37 C and 5% CO 2 in a humidified incubator and cultured for 8 to 12 days in vitro with medium replenishment every 4 days. Previous data showed that our cultures are almost pure neuronal cultures, as 99.5% of the cells are neurons, 0.5% are microglia, and 0% are astrocytes.

Cell Death Assay
Neurons were incubated for 20 h in the presence or absence of L-type Ca 2þ channel blockers or 50 lM glutamate, as indicated in the Table 1. Cell death was determined by live or dead viability assay, as described previously (Doria et al., 2013). Briefly, neurons were stained with 2 mM calcein acetoxymethyl ester (AM) and 2 mM ethidium homodimer-1 for 15 min and the fractions of live (calcein AM positive) and dead (ethidium homodimer-1 positive) cells were determined. Neurons were visualized by fluorescence microscopy using a Floid Microscope (Life Technologies). Cells were analyzed per well in triplicate using ImageJ TM software. Dead cells were expressed as a percentage of the total number of cells.

Immunoblotting
The cortex, hippocampus, and striatum of BACHD and WT mice were dissected and lysed in RIPA buffer containing protease inhibitors. A total of 100 mg of total cellular protein for each sample was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by electroblotting onto nitrocellulose membranes. Membranes were blocked with 10% skim milk in wash buffer (150 mM NaCl, 10 mM Tris-HCl, and 0.075% Triton X 100, pH 7.4) for 1 h and then incubated with rabbit anti-Ca v 1.2 (1:200), anti-Ca v 1.3 (1:200), or mouse anti-b-actin (1:1.000) antibodies in wash buffer containing 3% skim milk overnight at 4 C. Membranes were rinsed 3 times for 5 minutes with wash buffer and then incubated with either secondary horseradish peroxidase-conjugated goat anti-rabbit IgG (1:5.000) or secondary horseradish peroxidase-conjugated goat anti-mouse IgG (1:5.000) in wash buffer containing 3% skim milk for 1 h at room temperature. Membranes were rinsed 3 times for 10 minutes with wash buffer and incubated with ECL luminol Prime. Nonsaturated, immunoreactive bands were quantified by scanning densitometry using Image Quant LAS software (GE Healthcare). Immunoband intensity was obtained by ImageJ TM software. Ca v 1.2 levels were normalized to actin levels.

Massachusetts) at room temperature (22 C-25 C).
Current recordings were filtered at 2.9 kHz and digitally sampled at 10 kHz. Patch pipette resistance was 2.5 to 3.0 MX and was filled with an internal solution composed of (120 mM CsCl, 1 mM MgCl 2 .6H 2 O, 10 mM HEPES, 10 mM EGTA, 2 mM Mg-ATP, pH 7.2). Cs þ was used in the pipette solution to block voltage-dependent K þ channels, as well as to increase membrane resistance (typically above 100MX), improving voltage-clamp quality. Ba 2þ was used as charge carrier in all experiments. Primary cultured cortical neurons with series resistance over 8.0 MX were excluded from the analysis. During whole-cell experiments, neurons were bathed in Tyrode solution (140 mM NaCl, 5.4 mM KCl, 0.5 mM MgCl 2 , 0.33 mM NaH 2 PO 4 , 1.8 mM CaCl 2 , 5mM HEPES, 11 mM glucose, pH 7.4). After the establishment of the whole-cell configuration, the plates containing cells were perfused with control barium solution (130 mM TEA-Cl, 2 mM MgCl 2 .6H 2 O, 10 mM BaCl 2 , 10mM HEPES, 10 mM Glucose, pH 7.2) for 5 min followed by 5 min exposure to nifedipine 5 lM added to the same solution. Cortical neurons were hyperpolarized to À100 mV for 50 ms from a holding potential of À80 mV, followed by a ramp protocol from À100 mV to þ50 mV at a rate of 1.5 V/s, with a frequency of 0.1 Hz. L-type Ca 2þ currents were determined by digital subtraction between Ca 2þ currents before and after the effects of nifedipine. We observed a significant rundown in four WT and three BACHD recorded neurons. Extent of rundown magnitude varied between 30% and 80% of total peak initial current, measured 300s after beginning of stimulation protocol. Also, the electrophysiological experiments were always age matched.

Statistical Analyses
Data were tested for normality using Shapiro-Wilk Test and the results indicated normal distribution of data.
Means AE standard error of the mean (SEM) are shown for the number of independent experiments indicated in Figure 1, 2, 3 and 4. GraphPad Prism TM software was used to analyze data for statistical significance determined by either unpaired t test (for comparing two groups) or two-way analysis of variance testing followed by Bonferroni post hoc multiple comparison testing.

BACHD Mice Exhibit Increased Cortical Levels of Ca v 1.2 Protein
It has been demonstrated that Ca v 1 protein expression is increased in some neurodegenerative disorders, including AD and PD (Ueda et al., 1997, Hurley et al., 2013, 2015. Thus, we investigated whether Ca v 1.2 or Ca v 1.3 protein levels are altered in BACHD mice. For that, we performed western blotting experiments to measure Ca v 1.2 or Ca v 1.3 levels in total cell lysates (Figure 1(a) to (c)).
Our results showed a significant increase in Ca v 1.2 levels in the cortex, but not in the hippocampus or striatum, of 3-month-old BACHD, as compared with WT littermates (t 4 ¼3.393 p ¼ .0146; Figure 1(d)). Moreover, Ca v 1.2 levels in 12-month-old BACHD mice were significantly increased in the cortex, but not in the hippocampus or striatum, as compared with WT mice (t 4 ¼6.297, p ¼ .0007; Figure 1(e)). Importantly, our immunoblotting analyses showed that Ca v 1.3 protein levels were not altered in the cortex, hippocampus, or striatum of 12-month-old BACHD mice, as compared with WT littermates (t 4 ¼ 4.175, p ¼ .4103; Figure 1(f)). This increase in Ca v 1.2 protein levels in the cortex could be due to either increased gene expression or decreased protein degradation. To test whether Ca v 1.2 expression was increased in BACHD mice, we performed RT-qPCR to measure Ca v 1.2 mRNA levels. Interestingly, Ca v 1.2 (Figure 2(a)), as well as Ca v 1.3 (Figure 2(b)) mRNA levels were not different when comparing WT and BACHD mice at 2 and 12 months of age. Therefore, it is possible that other posttranslational alterations, such as cell trafficking and protein degradation, could be responsible for Ca v 1.2 increased protein levels.

BACHD Neurons Display Increased L-Type Ca 2þ Currents
Whole-cell patch-clamp experiments were conducted in primary cultured cortical neurons to evaluate whether enhanced Ca v 1.2 protein levels would reflect in an increase in L-type Ca 2þ current density. By digital subtraction of the total Ca 2þ current before and after exposure to 5 lM of nifedipine, we assessed L-type Ca 2þ current (nifedipine-sensitive current). We used 5 lM nifedipine to access the full effect on Ca v 1.2 channels. Figure 3(a) displays the ramp protocol used in patchclamp experiments. Representative records of the current-voltage relationship of WT (left panel) and BACHD (right panel) cortical neurons before and after the exposure to 5 lM nifedipine are displayed in Figure 3 (b). It is evident that both total Ca 2þ peak current density (À31.74 AE 5.4 pA/pF vs. À54.44 AE 8.3 pA/pF; WT vs. BACHD; Figure 3(c)) and L-type Ca 2þ peak current density (À10.05 AE 1.7 pA/pF vs. À20.8 AE 2.9 pA/pF; WT vs. BACHD; Figure 3(d)) were increased in BACHD cortical neurons, as compared with WT cortical neurons.
Nifedipine was able to decrease Ca 2þ peak current from both WT and BACHD neurons; however, nifedipine-resistant Ca 2þ current was still higher in WT compared with BACHD, suggesting that a different voltage-gated Ca 2þ channel may contribute to the putative increase in Ca 2þ current density observed in cortical neurons from BACHD mice (Figure 3(e)). Cell capacitance was averaged at 36.9 AE 3.6 pF, n ¼ 8 for WT and 37.3 AE 4.1 pF n ¼ 7 for BACHD and did not differ from each other. Membrane resistance was estimated for each cell and multiplied by its respective capacitance to obtain s values estimation (2.34 AE 0.44 ms for WT vs. 2.99 AE 0.62 ms for BACHD), which did not differ from each other. Altogether, these data strongly indicate that there is more Ca v 1.2 activity in BACHD cortical neurons, which per se could underlie increased intracellular Ca 2þ levels and, consequently, cell death. Indeed, our group have previously assessed the nifedipineresistant Ca 2þ current presented in Figure 3(e).
Importantly, we observed a twofold increase in Ca v 2.2 currents from BACHD-derived neurons that match the fraction of residual increase in total Ca 2þ current apart from the Ca v 1.2-dependent increase demonstrated in this article (Silva et al., 2017).

Ca v 1.2 Channel Blockers Protect Against Glutamate-Induced Neuronal Cell Death in BACHD Cultured Neurons
To test whether the antagonism of Ca v 1.2 channels could be efficient to promote neuroprotection, we performed an in vitro assay to measure neuronal cell death. Primary cultured corticostriatal neurons from C57/BL6 (Figure 4(a) Figure 2. mRNA of Ca v 1.2 and Ca v 1.3 are not altered in the cortex of 2-and 12-month-old BACHD mice. Graphs show mRNA of Ca v 1.2 (A) and mRNA of Ca v 1.3 (B), in the cortex, of either WT or BACHD mice at 2 and 12 months of age. mRNA levels were assessed by quantitative RT-PCR, which was performed in triplicate and normalized to RPL32 mRNA levels and actin. Data represent the means AE SEM, n ¼ 6. * indicates significant differences (p < .05). and (b)), FVB/NJ (WT; Figure 4(c)) or BACHD mouse embryos exposed to 50 lM glutamate for 20 h exhibited high level of neuronal cell death (Figure 4(c)). To establish a concentration-response curve, C57/BL6 neurons were incubated with glutamate in the presence of increasing concentrations (0.1, 1, and 10 nM) of nifedipine (Figure 4(a)) or israpidine (Figure 4(b)), and, as observed, neuronal cell death was significantly decreased. Interestingly, nifedipine at 10 nM was not able to promote neuroprotection. In fact, 10 nM nifedipine triggered neuronal cell death even in the absence of glutamate (Figure 4(a)). In addition, the most common adverse effect associated with nifedipine is reflex tachycardia secondary to pronounced vasodilatation. Some patients may experience symptoms of hypotension and flushing, as well as some severe adverse affects, including retinal ischemia, cerebral vascular accident, and myocardial ischemia and infarction (Murakami et al., 1972). Nifedipine and isradipine are examples of calcium channel blockers with 1,4-dihydropyridine (DHP) scaffold. Even though effectively used in clinics for the treatment of hypertension, the binding mechanism to their target, the L-type Ca 2þ channel, Ca v 1.2, is not completely understood. As previously reported, dihydropyridines can bind L-type Ca 2þ channels in an isoform-selective manner, which may explain or after exposure to nifedipine 5 lM (gray). (c) Graphs show peak total Ca 2þ current-density and (d) L-type Ca 2þ current-density from WT and BACHD mice. (e) Graphs show peak total Ca 2þ current-density after exposure to nifedipine 5 lM from WT (n ¼ 8) and BACHD (n ¼ 7) mice. *indicate significant differences as compared with matched WT (p < .05).
some of the clinical differences (Sinnegger-Brauns et al., 2009). Thus, we settled to perform more experiments using isradipine that is also able to block Ca v 1.2 channels and has been described as neuroprotective (Anekonda andQuinn, 2011, Ilijic et al., 2011). To test whether this Ca v 1.2 channel blocker could also be efficient to protect neurons expressing mutant Htt, BACHD corticostriatal neurons were also tested and compared with WT neurons (Figure 4(c) and (d)). BACHD neurons exhibited increased basal neuronal cell death levels, as compared with WT neurons (Figure 4(c) and (d)). Likewise, BACHD neurons were also more susceptible to glutamate insult than WT neurons (Figure 4(c) and (d)). Notably, this excitotoxic effect of glutamate was blocked by 1 nM isradipine in both WT and BACHD neurons (Figure 4(c) and (d)). In addition, 1 nM isradipine diminished cell death induced by glutamate to the same levels as those of untreated neurons (Figure 4(c) and (d)). Overall, these data pinpoint that Ca v 1.2 channel blockers are capable of preventing the death of WT and BACHD primary cultured neurons in response to glutamate insult.

Calcium Dysfunction in Neurodegenerative Diseases
Ca 2þ fluxes across the plasma membrane and between intracellular compartments play important roles in neuronal function, including cell survival, synaptic transmission, plasticity, and gene transcription (Berridge, 2004). In most neurodegenerative disorders, Ca 2þ regulation processes are compromised leading the neuronal cells to endure synaptic dysfunction, impairment in plasticity, oxidative stress, apoptosis, and death (Hajnoczky et al., 2003, Orrenius et al., 2003. In HD, it is also observed that excitotoxicity mediated by excessive activation of glutamate receptors leading to an excessive increase in intracellular Ca 2þ concentration (DiFiglia, 1990, Chen et al., 1999. Several studies indicate that mHtt promotes Ca 2þ signaling alterations, which are closely associated with cell death of striatal neurons (Zeron et al., 2002, Ribeiro et al., 2010. For instance, it has been described that mHtt is able to sensitize NMDA receptors, increasing NMDA channel permeability to Ca 2þ in striatal neurons (Chen et al., 1999, Sun et al., 2001. Also, mHtt protein promotes destabilization of mitochondria, decreasing the ability of this organelle to regulate Ca 2þ levels (Panov et al., 2002, Choo et al., 2004. In addition, mHtt is able to sensitize the inositol trisphosphate receptor (InsP 3 R), increasing the release of Ca 2þ from intracellular stores (Tang et al., 2003, Bezprozvanny, 2011. Moreover, it has been shown that signaling through ryanodine receptor, critical for skeletal muscle excitation-contraction coupling, is altered by mHtt in HD neurons . Indeed, authors showed a significant decrease in L-type Ca 2þ channel conductance, leading to changes of Ca 2þ turnover in skeletal muscle of R6/2 mice and suggesting that changes may be associated with muscle pathology in HD (Braubach et al., 2014). Therefore, mHtt-mediated increased intracellular Ca 2þ levels play an important role in the neuronal cell death that takes place in HD. Alterations in voltage-gated Ca 2þ channels are also implicated in age-related neuronal dysfunctions (Ueda et al., 1997, Tai et al., 2011, Daschil et al., 2013, Hurley et al., 2015. Interestingly, a relationship between voltage-gated L-type Ca 2þ channels and AD has already been demonstrated, as published data indicate that b-amyloid peptide is capable of interacting with voltage-gated L-type Ca 2þ channels, altering channel activity and promoting an increase in the expression of this Ca 2þ channel at the plasma membrane (Scragg et al., 2005, Kim andRhim, 2011). Notably, L-type Ca 2þ channel blockers were also found to have neuroprotective effects against Ab-induced neuronal apoptosis in cultured rat cortical neurons (Yagami et al., 2004) and from amyloid precursor protein (APP)-induced neurotoxicity in neuroblastoma cells (Anekonda and Quinn, 2011). Despite these various studies relating voltagegated L-type Ca 2þ channel and neurodegenerative diseases, there is no evidence of whether these channels could play a role in the Ca 2þ alteration that occurs in HD patients. Our results presented here suggest that Ca v 1 channels are altered in a mouse model of HD.

Alterations of Ca v 1 Channels in a Mouse Model of HD
Our investigation started with the observation that Ca v 1.2 total protein levels are increased in the cortex of 3-and 12-month-old BACHD mice. As Ca v 1.2 proteins are mostly located postsynaptically at somatodendritic locations, these channels regulate neuronal excitability and are also known to be involved in translating synaptic activity into alterations in gene expression and neuronal cell death (Westenbroek et al., 1990;Murphy et al., 1991;Hell et al., 1993;Obermair et al., 2004;Helton et al., 2005;Wankerl et al., 2010). Our immunoblotting analyses and also RT-qPCR results showed that neither Ca v 1.3 protein nor mRNA levels are altered. Ca v 1.3 channels activate more rapidly and at more negative voltages than Ca v 1.2, which allows them to contribute to the stabilization of upstate potentials and the control of neuronal firing (Koschak et al., 2001). Interestingly, whole-cell patch-clamp data indicated that this increase in Ca v 1.2 protein levels in BACHD mice are accompanied by enhanced L-type Ca 2þ current density. Altogether, these data may suggest that an increase in intracellular Ca 2þ observed in HD could also be related to alterations in Ca v 1.2 protein levels and activity. Thus, therapeutic agents aiming to diminish this increase in cytosolic Ca 2þ levels, by partially blocking L-type Ca 2þ currents, may play an important role in protecting corticostriatal neurons against Htt-mediated neuronal cell death. Dihydropyridines are commonly used to establish the contribution of L-type Ca 2þ currents in several neuronal processes, but the efficiency of these blockers depends on membrane potential, channel state, and channel subtype (Hess et al., 1984;Holz et al., 1988). Notably, the results with nifedipine and isradipine are very intriguing, as both drugs are able to bind to either Ca v 1.2 or Ca v 1.3 binding pockets (Takahashi and Catterall, 1987;Sinnegger-Brauns et al., 2009). However, the contribution of Ca v 1.2 and Ca v 1.3 isoforms to L-type Ca 2þ currents in different neurons is difficult to assess using pharmacological tools, as a consequence of partial selectivity of DHPs. Thus, to clarify the potential role of Ca v 1.2 and Ca v 1.3 channels in HD pathology by using electrophysiology protocols, newer selective blockers for those channels have to be developed. According to our results, the excitotoxic effect caused by high concentrations of glutamate in BACHD cultured neurons was diminished by adding 1 nM isradipine to the culture. Furthermore, 1 nM isradipine was able to reduce glutamate-induced neuronal cell death to the same levels as control. As observed in our whole-cell patch-clamp data, Ca v 1.2 blockage did not completely abolish the increase in total Ca 2þ current observed in BACHD neurons, suggesting that a different voltage-gated Ca 2þ channels may play a part in HD dysfunction. As previously mentioned, our research group observed an increase in Ca v 2.2 currents in BACHD derived neurons (Silva et al., 2017). Therefore, there are likely two components rendering the increase in total calcium current from BACHD cortical neurons compared with WT: a twofold increase in L-type Ca 2þ currents observed in this study and a twofold increase in N-type Ca 2þ currents (Silva et al., 2017). Interestingly, even though more subtypes of Ca 2þ channels seem to be affected by mutant Htt, the blockage of L-type Ca 2þ currents by isradipine (0.1, 1, and 10 nM) was sufficient for completely rescuing glutamate-induced neuronal cell death, at least an in vitro setting using primary neuronal cultures. Nifedipine was also efficient to rescue neuronal cell death at the concentrations of 0.1 and 1 nM. However, 10 nM nifedipine did not prevent glutamateinduced neuronal cell death and was also neurotoxic even in the absence of glutamate. Thus, 0.1 to 1 nM of nifedipine appeared to stabilize the free cytosolic calcium concentration at optimal levels, even if it incompletely suppressed Ca 2þ influx.
It is also important to emphasize that we used an in vitro assay to measure neuronal cell death against glutamate toxicity and neuroprotection after treatment with Ca v 1 blockers. It is well known that cultures from embryonic neurons have substantially abnormal environment (Millet and Gillette, 2012), as in this case neuronal cultures are devoid of glial cells and, therefore, are not in the presence of many factors that could alter neuronal survival or cell death (Doria et al., 2013). Furthermore, although in vitro models are successfully used in biological fields to study biological and pharmacological mechanisms, extrapolation of the results to human are not possible. Thus, additional in vivo studies are needed in order to claim that blocking Ca v 1channels could be neuroprotective.
Another key point to be raised is whether glutamate induced calcium entry through Ca v 1.2 channels may reflect what occurs in HD. We believe that Ca v 1.2 channels may play a small piece in the puzzle of increasing intracellular Ca 2þ observed in HD. Thus, we cannot discard the role of NMDA receptors, IP3 receptors, and others in inducing neuronal cell death (DiFiglia, 1990, Chen et al., 1999, Tang et al., 2003, Bezprozvanny, 2011. Maybe by blocking all other targets, we could drastically reduce neuronal cell death in BACHD neurons. However, blocking all Ca 2þ influx may also interfere with the homeostasis of neuronal cells (Hajnoczky et al., 2003). Besides, literature has identified Ca v 1.2 and Ca v 1.3 channels as specific molecular and cellular cascades that underlie mood (anxiety and depression), social behavior, and cognition in rodents (Mogilnicka et al., 1987;Bader et al., 2011;Kabir et al., 2016Kabir et al., , 2017 As well, interesting data demonstrated that the BACHD mouse recaps clinical HD with early psychiatric aspects, such as depressive and anxiety-like features (Hult Lundh et al., 2013). Likewise, more in vivo data are also required to demonstrate whether blocking Ca v 1.2 is sufficient to delay or ameliorate the onset of behavioral deficits in BACHD mice. Consequently, we hypothesize that partially blocking Ca v 1.2 channels could provide a better option for preventing neuronal cell loss, likely by modulating the overactivity of those channels ( Figure 5).

Potential Mechanisms Explaining mHtt Effects on Ca v 1.2 Channels
Another remarkable question hoist by our data is how mHtt alters Ca v 1.2 channel levels. It has been shown previously by our group and others that mHtt can interact with voltage-gated Ca 2þ channels, such as Ca v 2.2 (Swayne et al., 2005, Silva et al., 2017. Also, it seems that mHtt can regulate Ca v 2.2 at many levels by altering channel levels, modifying Ca v 2.2 interaction with its protein partners and changing channel activity (Silva et al., 2017). Interestingly, although Ca v 1.2 protein levels are increased in BACHD mice, Ca v 1.2 mRNA levels are not different when comparing WT and BACHD mice. Then, we suggest that Htt mutation is not affecting Ca v 1.2 gene expression but protein trafficking or degradation. mHtt could change Ca v 1.2 cell trafficking and localization, which could prevent Ca v 1.2 protein degradation. For instance, mHtt may interact with auxiliary subunits of L-type Ca channels, altering channel trafficking. In addition, growing evidence has suggested that expanded polyglutamine repeats facilitate the interactions of mHtt protein with huntingtin-associated proteins selectively expressed in the striatum and cortex and also other proteins that are ubiquitously expressed (Martin et al., 1999). Among these proteins are calmodulin, Huntingtin-associated protein 1 (Hap1), huntingtin protein interacting proteins (Hip1 and 2), and glyceraldehyde-3-phosphate dehydrogenase (Bao et al., 1996, Burke et al., 1996, Li and Li, 2004. Particularly, Hap1 may maintain neuronal transmission and neurotrophic functions by regulating intracellular trafficking, recycling, and stabilization of receptors (Gauthier et al., 2004). Recently, Hap1 was shown to regulate the surface expression level and intracellular trafficking of Ca v 1.2 channel in INS-1 cells, a pancreatic b-cell line, therefore regulating insulin secretion (Pan et al., 2016). According to these data, impaired Hap1 function is involved in altering the distribution of Ca v 1.2 on the plasma membrane of INS-1 cells, leading to a decrease in insulin release (Pan et al., 2016). Since Hap1 is highly expressed in the brain, interaction of mHtt protein with Hap1 could induce other protein dysfunctions and consequently lead to the toxicity characteristic of HD (Li et al., 1995). In the future, it would be interesting to investigate whether impaired Hap1 function is involved in the dysregulation of Ca v 1 or Ca v 2 channels observed in our model. Thus, to elucidate the underlying mechanism of our findings, further experiments are essential to test the hypothesis that the trafficking of those channels are altered in our model, which could alter Ca v 1.2 protein degradation.
In conclusion, our results suggest that L-type Ca 2þ channels are affected in a mouse model of HD. mHtt protein might disrupt calcium homeostasis via upregulation of L-type Ca 2þ channels, triggering the production of free radicals and oxidative stress, mitochondrial dysfunction, and, eventually, cell death. Finally, in vitro concentration-response assay measuring neuronal cell death showed that L-type Ca 2þ channel blockers were able to reduce neuronal cell death of WT and BACHD cultured neurons ( Figure 5). Altogether, the data constitute a previously unrecognized mechanism that may contribute to our understanding of HD pathogenesis. In addition, Ca v 1 channel blockers should be further investigated as potential therapeutics tools.

Summary Statement
Our main results show that in a mouse model of Huntington's disease, Ca v 1 channels may be dysfunction. Furthermore, L-type Ca 2þ currents are enhanced in cortical neurons. By using Ca v 1 antagonists, we were able to observe neuronal protection against glutamate toxicity.

Acknowledgments
Authors thank Giovanni Gomes for his part on figure design.

Author Contributions
L. B. V. and F. M. R. designed the study. Electrophysiology recordings were performed by A. S. M. and J. S. C. P. L. C. performed RT-qPCR experiments and contributed to conception and design of the study. F. R. S. and J. M. S. conducted Western Blotting, and cell cultures experiments. I. G. O. carried out the mouse colony as well genotyping. M. V. G. made substantial contributions to conception and design of the study and revised the manuscript critically for important intellectual content. Results were analyzed by L. B. V. and F. M. R. and the Figure 5. Hypothetical mechanism of Ca v channels involved in neuronal death in a mouse model of HD. Cortical neuronal cultures obtained from BACHD mice (b) display elevated Ca v 1.2 channel levels, but not Ca v 1.3, compared with WT littermates (a). Taken together with previous observations (Silva et al. 2017), we hypothesize that (a) the disruption of mHtt interaction with Ca v 2.2 channels increase plasma membrane levels, leading to a greater Ca 2þ influx into the presynaptic compartment. This, in turn, (b) promotes a larger release of glutamate at the synaptic cleft, accentuating glutamate receptors activation, depolarizing the cell membrane, and opening Ca v 1.2 channels in the postsynaptic compartment. In addition, (c) mHtt affects Ca v 1.2 total protein levels either by altering protein trafficking or degradation; this enhancement of Ca v 1.2 channel levels trigger elevated Ca 2þ into the postsynaptic neuron and, lately neuronal death. Treatment with isradipine (b), a Ca v 1 channel blocker, is able to reduce L-type Ca 2þ currents in vitro, preventing the excitotoxic effects of glutamate insult and rescuing neuronal death in BACHD neurons to WT levels.
article was written by L. B. V. All other authors revised the data and discussed the manuscript.

Declaration of Conflicting Interests
The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding
The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This study was also supported by CNPq (