Trpm2 Ablation Accelerates Protein Aggregation by Impaired ADPR and Autophagic Clearance in the Brain
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TRPM2 a cation channel is also known to work as an enzyme that hydrolyzes highly reactive, neurotoxic ADP-ribose (ADPR). Although ADPR is hydrolyzed by NUT9 pyrophosphatase in major organs, the enzyme is defective in the brain. The present study questions the role of TRPM2 in the catabolism of ADPR in the brain. Genetic ablation of Trpm2 results in the disruption of ADPR catabolism that leads to the accumulation of ADPR and reduction in AMP. Trpm2−/− mice elicit the reduction in autophagosome formation in the hippocampus. Trpm2−/− mice also show aggregations of proteins in the hippocampus, aberrant structural changes and neuronal connections in synapses, and neuronal degeneration. Trpm2−/− mice exhibit learning and memory impairment, enhanced neuronal intrinsic excitability, and imbalanced synaptic transmission. These results respond to long-unanswered questions regarding the potential role of the enzymatic function of TRPM2 in the brain, whose dysfunction evokes protein aggregation. In addition, the present finding answers to the conflicting reports such as neuroprotective or neurodegenerative phenotypes observed in Trpm2−/− mice.
KeywordsADPR AMP Autophagy Protein aggregation TRPM2
Transient receptor potential ion channel subtype M2 (TRPM2, formerly named LTRPC2 or TRPC7) is a multifunctional, nonselective, Ca2+ permeable, cation channel [1, 2]. TRPM2 is ubiquitous in the brain, where it is involved in neurite growth during fetal neurodevelopment and functions as an oxidative sensor in neurons [3, 4, 5]. Moreover, its genetic or pathogenic dysfunction is associated with bipolar disorder and neurodegenerative diseases, including Parkinson’s and Alzheimer’s [6, 7, 8, 9]. TRPM2 is activated by ADP-ribose (ADPR), nicotinamide adenine dinucleotide (NAD+), mild heat, or H2O2 [1, 2, 10]. Being a Ca2+-permeable cation channel, when activated by reactive oxygen species (ROS), TRPM2 induces ROS-mediated neuronal degeneration and chemokine production in the macrophages [11, 12]. Thus, genetic ablation of trpm2 leads to neuroprotective effects in β-amyloid- or ischemia-induced brain [7, 13, 14]. On the contrary, some reports show neurodegenerative phenotypes in Trpm2−/− mice [8, 15]. However, the reason for the conflicting results is not known.
Unlike other TRP channels, only TRPM2 possesses a pore region and an enzymatic domain, NudT9-H, in its C-terminus region. The NudT9-H region shares significant homology with NudT9 ADPR pyrophosphatase (NudT9) that hydrolyzes ADPR into adenosine monophosphate (AMP) and a ribose [1, 2, 16]. Thus, it is often referred to as a chanzyme. Indeed, TRPM2 can hydrolyze ADPR weakly in vitro . However, it has still been veiled on in vivo hydrolase activity of TRPM2 in the brain.
ADPR is a metabolic product of pyridine nucleotides NAD (H) or NADP (H) and reacts readily with various proteins due to the nature of a reactive nucleotide-sugar, inducing protein aggregation [17, 18]. Therefore, its degradation is essential for normal cellular functions [19, 20]. Cellular ADPRs are catabolized by an ADPR pyrophosphatase, a member of the Nudix gene family . Nudix hydrolases remove the deleterious metabolite, ADPR, from biochemical pathways, preventing the excessive accumulation of ADPR in cells [19, 20]. If allowed to accumulate, ADPR tends to react with the lysine and arginine residues of proteins due to the activity of ADP-ribosyltransferase or non-enzymatic way, leading to an excessive mass of protein aggregates [17, 18, 21]. Protein oxidation by oxidative stress in aging and neurodegenerative brains induces oxidized and cross-linked proteins, which further accelerates the formation of protein aggregates [22, 23]. The damaged proteins and lipids are rapidly recycled by the autophagy pathway. Autophagy is a lysosomal proteolytic pathway that is widely involved in the degradation of damaged or defective cellular proteins and organelles . However, the weak proteolytic efficiency of autophagy during aging is now known to induce the accumulation of intracellular waste products, which make individuals susceptible to age-related neurodegenerative diseases, such as Alzheimer’s and Parkinson’s . Thus, ADPR accumulation along with weak autophagic activity would lead to deleterious protein aggregations.
In mammals, ADPR is hydrolyzed by NudT-5 and NudT-9 in the major organs, but not in the brain [19, 20]. As a result, the enzyme responsible for ADPR degradation in the brain has come under question. Given the abundance of TRPM2 in the brain, it is possible that TRPM2 may hydrolyze the reactive ADPR. The present study was thus undertaken to determine the role of TRPM2 in hydrolyzing ADPR in the brain and the possible pathology when this function is chronically impaired.
Materials and Methods
Animal care and handling were carried out according to guidelines issued by the Institutional Animal Care and Use Committee at the Korea Advanced Institute of Science and Technology (KAIST) and Seoul National University.
The generation of mice lacking in TRPM2 for the study is described in the previous report . TRPM2 heterozygous knockout (Trpm2± ) mice were backcrossed into the C57BL/6J inbred background over 10 generations. Male TRPM2 wild-type (WT, Trpm2+/+) and TRPM2-deficient (Trpm2−/−) mice with a C57BL/6J background were used for the analysis. All of the experiments were performed on 8- to 12-week-old mice unless otherwise indicated. The animals were provided with free access to food and water under a 12:12-h light:dark cycle.
Extracellular Recordings from Brain Slices
Recordings were obtained of prepared hippocampal (300–330 μm-thick), as described in our previous report . Brain slices were prepared in oxygenated (95% O2, 5% CO2), cold, artificial cerebrospinal fluid (in mM, 124 of NaCl, 3.5 of KCl, 1.25 of NaH2PO4, 2.5 of CaCl2, 1.3 of MgSO4, 26 of NaHCO3, and 10 of glucose, at pH 7.4). After allowing 1 h for recovery, the slices were incubated in artificial cerebrospinal fluid, and whole-cell recordings were obtained for the hippocampal CA1 neurons at 32 °C using glass pipette electrodes (3–5 MΩ). To measure the action potentials and miniature excitatory postsynaptic currents (mEPSCs), glass pipettes were filled with a solution, containing (in mM) 135 of K-gluconate, 5 of KCl, 10 of HEPES, 2 of MgCl2, 0.3 of Na-GTP, 5 of Mg-ATP, 0.5 of CaCl2, and 5 of EGTA (pH 7.3). APs were triggered by injecting currents, ranging from – 150 to + 150 pA, in 30 pA steps using the current clamp mode. The number of spikes that were evoked by the currents injected in the hippocampal neurons isolated from WT or Trpm2−/− mice was compared. The mEPSC experiment was performed in the presence of 1 μM of tetrodotoxin, 100 μM of picrotoxin (a GABAA receptor antagonist), and 5 μM of CGP 55845 (a GABAB receptor antagonist). For miniature inhibitory postsynaptic current (mIPSC) measurements, the internal pipette solution contained (in mM) 140 of KCl, 0.5 of CaCl2, 2 of MgCl2, 5.8 of EGTA, 10 of HEPES, 5 of Mg-ATP, and 0.3 of Na-GTP (pH 7.3). The mIPSC experiment was performed in the presence of 10 μM of DNQX (an AMPA receptor blocker) and 50 μM of D-AP5 (an NMDA receptor antagonist) in the voltage clamp mode. For mEPSC and mIPSC measurements, neurons were voltage-clamped at − 60 mV. Patch-clamp recordings were also performed using a Multiclamp 700B amplifier. Data were filtered at 1 kHz and sampled at 10 kHz using a Digidata 1440A (Axon Instruments). The acquired data were analyzed using a pCLAMP version 10.2 (Axon Instruments) and the Mini Analysis Program (Synaptosoft). For the LTP experiment, field excitatory postsynaptic potentials (fEPSPs) were recorded at hippocampal CA3-CA1 synapses. A bipolar stimulating electrode was placed in the stratum radiatum in the CA1 region, and extracellular field potentials were also recorded in the stratum radiatum using a glass microelectrode (borosilicate glass, 3–5 MΩ, filled with 3 M of NaCl). A baseline stimulation was delivered at an intensity eliciting 40% of the maximum evoked response. A theta-burst LTP was induced by the theta-burst stimulation protocol, consisting of four trains with 10-s intervals between each. Each of the trains was comprised of 10 bursts separated by 200 ms. Each burst included four pulses delivered at 100 Hz.
The WT and Trpm2−/− mice (8–12 weeks) were deeply anesthetized with 15% chloral hydrate. They were transcardially perfused with 100 ml of normal saline, followed by 500 ml of a freshly prepared fixative that contained 2% paraformaldehyde and 2.5% glutaraldehyde in a 0.1 M phosphate buffer (pH 7.4). The brains were removed and sectioned with a Vibratome at 500 μm. The sections, including the hippocampus sections, were post-fixed in the same fixative for 3 h, washed in phosphate buffer containing 4.5% sucrose for 15 min (3 × 5 min), and then post-fixed in a 1% OsO4 in phosphate buffer for 1 h. Each section was then rewashed in a phosphate buffer containing 4.5% sucrose and dehydrated in a graded alcohol series. During dehydration, the sections were stained en bloc with 1% uranyl acetate in 70% alcohol for 1 h, transferred to propylene oxide, flat-embedded in Epon 812, and cured at 60 °C for 3 days. Small pieces containing the CA1 pyramidal cell layer and stratum radiatum were then cut out and attached to an Epon support for further ultrathin sectioning (Reichert-Jung, Nuβloch, Germany). Ultrathin sections (70–90 nm thick) were collected on 1-hole grids coated with Formvar and examined under an electron microscope (Jeol 1200EX, Tokyo, Japan). Randomly selected neuropil areas, within 70–100 μm from the cell bodies, were photomicrographed at a 40,000× and used for quantification. Three electron micrographs representing 159.9 μm2 neuropil regions were taken per mouse. Digital images were captured with a CCD camera (SC1000 Orius; Gatan Inc., Pleasanton, CA) and saved as TIFF files. Image brightness and contrast were adjusted using Adobe Photoshop (Adobe Systems, San Jose, CA).
After being anesthetized, the WT and Trpm2−/− mice (8–12 weeks) were perfused transcardially with saline, followed by 4% paraformaldehyde. Their brains were excised and placed in 4% paraformaldehyde for 24 h. Fixed tissues were then embedded in an OCT compound (Sakura Finetechnical Company, Chuo-Ku, Japan) and placed on slides. Immunohistochemical staining was performed using antibodies to NeuN (Millipore). The brain tissues were first incubated for 1 h in a solution containing 4% bovine serum albumin and 0.05% Tween 20, and then incubated overnight at 4 °C in a solution containing primary antibodies. Finally, sections were incubated with Alexa Fluor 488-conjugated donkey anti-mouse IgG (Invitrogen) and Hoechst 33342 (Invitrogen) for 1 h at room temperature to perform nuclear staining.
All behavioral procedures were video-recorded and an experimenter who was unaware of the genotypes analyzed the recorded data.
Classical Fear Conditioning Test
As previously described , the mice were first habituated in a fear-conditioning apparatus chamber for 5 min and then subjected to a 28-s acoustic conditioned stimulus (CS). A 0.7-mA shock (unconditioned stimulus) was applied to the floor grid for 2 s immediately afterward (Panlab, S.L.U.). Conditioned stimulus-unconditioned stimulus coupling was carried out three times at 60-s intervals. To assess contextual memory, the animals were placed back into the training context 24 h after they received their training. The duration of their fear response (freezing behavior) was measured for 4 min. To assess the cued memory, the animals were placed in a different context (a novel chamber) 24 h after training and their behavior was monitored for 5 min. During the last 3 min of this test, the animals were exposed to the CS. The duration of their freezing behavior was measured throughout the 3-min test. To evaluate the foot-shock intensity, naive animals were placed into the fear-conditioning apparatus chamber and subjected to a series of foot shocks lasting 1 s. The foot shocks gradually increased in amperage (intensity) (0.02, 0.06, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, and 1.0 mA) at 30-s intervals. The shock intensities that evoked initial sensation responses (flinching and abrupt walking), running, vocalization, and jumping were recorded for each mouse.
The Y-maze apparatus consisted of three identical arms. Each arm was 25-cm long, 5-cm wide, and 14-cm high. One of the arms of the maze was briefly closed, and the mice were placed randomly into one of the other arms (start arm) and allowed to explore the maze for 10 min (training session). After 1 h, the mice were replaced in the start arm and allowed to freely explore all three arms for 5 min (test session). The retention times (duration) in each arm were used to assess the spatial memory of the mice. The results are presented as ratios of the amount of time spent in each arm, over the total time spent in all three arms.
Novel Object Recognition Memory Test
As previously described , the mice were individually habituated to an open-field box (40 × 40 × 40 cm) for 15 min per day for 3 days. During the training trial, two objects were placed in the box, and the mice were allowed to explore the objects for 10 min. A mouse was considered to be exploring an object when its head was less than 1 in. away from the object and facing it. Twenty-four hours later, the mice were returned to the box with the two objects in the same locations, but with one novel object replacing one of the familiar objects. The mice were then allowed to explore the two objects for 10 min. Preference percentages (defined as the time spent exploring an object expressed as a percentage of the total time spent exploring both objects) were used to assess recognition memory.
Quantification of Metabolites by Triple Quadrupole Mass Spectrophotometer
As previously described, tissue extracts were analyzed by LC-MS/MS . To determine the concentrations of ADPR and AMP in the tissue extracts, hippocampi from WT and Trpm2−/− mice were briefly treated with 10% (v/v) trifluoroacetic acid and sonicated. These tissue extracts were centrifuged at 13000 rpm for 20 min, and the supernatants were diluted for analysis. The diluted supernatants were separated using a BEH Amide column (Waters ACQUITY UPLC BEH Amide, 130 Å, 1.7 μm, 2.1 mm × 50 mm). All chromatographic separations were performed at a flow rate of 0.2 mL/min. The column was equilibrated with a 100% buffer B (99.9% acetonitrile/0.1% formic acid), and the tissue extractor was eluted in a 5-min gradient to a 100% buffer A (10 mM of ammonium formate in water). The column was then rinsed with an ammonium formate buffer and re-equilibrated with the acetonitrile/formic acid buffer before the next injection. The following optimal conditions were used for the MS analysis of ADPR, NAD, and AMP: cone gas 150 L/h, nebulizer 7 Bar, and desolvation temperature at 350 °C. The ion transitions used for quantification were m/z 558.17 → 346.01 for ADPR, 662.27 → 540.14 for NAD, and 348.09 → 136.06 for AMP.
ADPR Hydrolase Activity
The hippocampus tissues from WT and Trpm2−/− KO mice were suspended in a 1 ml ice-cold lysis buffer, containing 30 mM Tris-HCl (pH 7.0), 150 mM NaCl, and a protease inhibitor cocktail. The suspensions were sonicated for 30 s and centrifuged at 12,000g for 10 min at 4 °C. The supernatants were harvested and the pellets were resuspended in the same buffer. The sonication and centrifugation of the insoluble pellets were repeated five times. The supernatants and finally re-suspended pellets were all ultra-centrifugated at 100,000g for 1 h at 4 °C. The supernatant was discarded, and the insoluble pellets were twice washed and solubilized by the addition of a lysis buffer, including 1% Triton X-100. Following solubilization, the samples were centrifuged at 120,000g for 1 h to obtain soluble fractions. The Nudix activity of lysate from each hippocampus was determined by measuring the conversion of substrate ADPR to AMP using LC-MS/MS. The reaction mixture (260 ul) consisted of 50 mM of Tris-HCl (pH 6.8), 16 mM of MgCl2, 40 μM of ADPR, and 40 μg of tissue lysate. After incubation for 1 h at 37 °C, the reaction was stopped by adding EDTA. The protein was removed by Vivaspin (3000 MWCO, Sartorius, Goettingen, Germany) and the product, AMP, was measured, as described in the Methods section above. The ms/ms response of the detected total AMP was removed with those of the sample blank and contaminated with AMP at the standard ADPR.
The protein lysates from the hippocampi of WT or Trpm2−/− mice were prepared in a RIPA cell lysis buffer (GenDEPOT), containing a protease inhibitor cocktail (Roche). These lysates were then subjected to an 8% SDS-PAGE gel. The proteins were transferred to PVDF membranes and then treated for 1 h with a TBS-T solution (20 mM of Tris/HCl, 500 mM of NaCl, 0.1% Tween 20), containing 2–5% skimmed milk powder. They were incubated with primary antibodies against β-actin (Sigma), α-tubulin (Millipore), GAPDH (Santa Cruz), calnexin (Santa Cruz), EEA-1 (Abcam), LAMP-1 (Santa Cruz), or autophagy-related proteins (LC3B:#2755, mTOR: #2972, phospho-mTOR: #5536, Raptor:#2280, AMPKα: #2532, phospho-AMPKα: #2531, AMPKβ1: #12063, phospho-AMPKβ1: #4181, ULK1: #8054, phospho-ULK1 (Ser555): #5869, phospho-ULK1 (Ser757): #14202, from Cell Signaling) at 4 °C on a rotary shaker overnight. The membranes were washed three times in a TBS-T solution, incubated with a secondary antibody for 1 h, and then treated with WEST-ZOL® ECL solution (iNtRON biotech). Blots were analyzed using an ImageQuant™ LAS 4000 chemiluminescence (GE Healthcare).
Experimental Design and Statistical Analysis
All of the results are shown as means ± SEMs. The unpaired two-tailed Student’s t test was used to determine the statistical differences between the two means. A one-way or two-way analysis of variance (ANOVA) with Tukey’s post hoc analysis was used to conduct multiple comparisons of the means. Mann-Whitney or Kruskal-Wallis analysis was used to conduct multiple comparisons of the means with small number of experiments (n≤ 6). Statistical significance was accepted for p value of < 0.05.
Reduced Catabolism of ADPR in the Trpm2 −/− Mice Brains
Accumulated Aggregates in Trpm2 −/− Mice Brains
Because ADPR reacts with various proteins due to the nature of a reactive nucleotide sugar [17, 18], we assumed that the accumulation of ADPR might induce protein aggregation in Trpm2−/− mice brains. To confirm the possibility, we conducted ultrastructural analysis using electron microscopy. Strikingly, we observed the presence of multiple inclusion bodies in the dendrites of the hippocampal neurons in the brains of young adults (8~12 week old) (Fig. 1d–e) . These round and varied protein aggregates were observed throughout dendrites, including the spines. The protein aggregates appeared different to the neurofilamentary tangles of Tau . The proportion of hippocampal neurons containing inclusion bodies was about 9 times higher in Trpm2−/− mice than in the hippocampal neurons of WT mice (Fig. 1f). In every 1000 μm2, 55.6 ± 4.8 (n = 9) dendrites in the hippocampus of the Trpm2−/− mice showed inclusion bodies, whereas for WT mice, only 6.3 ± 2.9 (n = 9) dendrites in the hippocampus contained such aggregates (p < 0.001, Student’s t test).
ADPR Hydrolyzing Activity Is Not Associated with its Channel Activity
We also examined whether the TRPM2 channel activity is functionally associated with the enzymatic activity of TRPM2. To determine this, we mutated Trpm2 at the pore region to block channel activity and then tested whether these mutants retain the ADPR hydrolyzing activity. We constructed two TRPM2 mutants that had mutations at C996 or C1008 in the pore region as previously reported . The application of 100 μM ADPR in the pipette solution robustly induced the currents of wild-type (WT) Trpm2, but not the two mutants, C996A and C1008A (Fig. 1g). As shown in Fig. 1h, the lysate isolated from WT Trpm2 overexpressing HEK293T cells showed significantly higher ADPR hydrolysis activity than that of mock-transfected cells (p < 0.01, Kruskal-Wallis test). More importantly, the hydrolysis activities of C996A and C1008A mutants of Trpm2 were not different from that of the WT Trpm2 overexpressing cells. Therefore, these data suggest that channel activity appears to be independent of the ADPR-hydrolysis activity of TRPM2.
Neuronal Loss in the Hippocampus of Trpm2 −/− Mice
Altered Excitability and Synaptic Transmission in Trpm2 −/− Mice
Altered Cognitive Function and Synaptic Plasticity in Trpm2 −/− Mice
Reduced Autophagy in the Trpm2 −/− Mouse Brain
Because ULK1 is known to regulate the formation of autophagosomes [24, 39], we further checked the level of microtubule-associated protein light chain 3B (LC3B), a key component required for phagophore formation. Indeed, LC3B was significantly reduced in the Trpm2−/− mice (p < 0.05, Student’s t test) (Fig. 5f), whereas the expression levels of markers for endoplasmic reticulum, early endosomes, and lysosomes were unchanged (Supplementary Fig. 2). Moreover, there was no difference in the levels of up-stream signals, including PI3 kinase, Akt, and Erk (Supplementary Fig. 3) [24, 40]. Consistently, the number of autophagosome puncta stained with LC3B antibody was reduced in the sections of the Trpm2−/− mice brains (Fig. 5g).
Reduced ADPR Hydrolase and Autophagy Activities in Hippocampal Cultures
The present study demonstrates that TRPM2 functions as a chanzyme that hydrolyzes ADPR in vivo. The genetic disruption of TRPM2 accumulates ADPR, but reduces AMP levels. This induces protein aggregation and lipid accumulation, morphological changes in the spine, and neuronal degeneration, and enhances neuronal excitability and synaptic activity. These pathologic events, in turn, become neural substrates for impaired learning and memory in Trpm2−/− mice. Thus, the present study suggests that the enzymatic activity of TRPM2 hydrolyzing ADPR is required to maintain normal brain functions. In addition, the role of TRPM2 as an enzyme to remove neurotoxic ADPR provides the possible explanation for the conflicting phenotypes, neuroprotection, or neurodegeneration, in Trmp2−/− mouse brain.
ADPR pyrophosphatase is a member of the Nudix superfamily, members of which hydrolyze substrates with a nucleoside diphosphate linked to another moiety “X” (Nudix) . Nudix hydrolases remove the deleterious metabolite, ADPR, from biochemical pathways [19, 20]. Thus, ADPR pyrophosphatases act to prevent excessive ADPR accumulation. If allowed to accumulate, ADPR, a reactive nucleotide-sugar, tends to react with the lysine and arginine residues of proteins by an ADP-ribosyltransferase or a non-enzymatic way [17, 18]. Of the known ADP-ribosylated proteins, globular actin is a known target of ADP-ribosylation in rat brains [42, 43]. The ADP-ribosylation of globular actin hinders actin polymerization, which leads to cytoskeletal disruption and, eventually, cell death . In the present study, aggregated proteins were found in the hippocampal neurons of Trpm2−/− mouse brains, which also contained elevated levels of ADPR. Thus, we believe that actin could be one of these aggregated proteins induced by increased ADPR.
Low Autophagic Formation in Trpm2 −/− Mice Brains
Autophagy is widely involved in the degradation of damaged or defective cellular proteins and organelles. The reduction in the proteolytic efficiency of autophagy during aging induces the accumulation of intracellular waste products. Thus, low autophagy activity makes individuals more susceptible to age-related neurodegenerative diseases, such as Alzheimer’s or Parkinson’s . The formation of autophagosomes is regulated by ATG, whose expression is suppressed by the mTOR complex . In addition, AMPK that is activated by AMP is an inhibitor of the mTOR complex . In the present study, elevated activity of mTOR, concomitant with low levels of AMPK and AMP, was observed in the brains of Trpm2−/− mice. Reduced autophagy formation, as shown by LC3B, was also observed in the brains of Trpm2−/− mice. Thus, the reduced ADPR hydrolyzing activity is likely responsible for the reduced autophagic activity in Trpm2−/− mice brains. Furthermore, this impaired autophagic formation may account for the protein aggregation found in Trpm2−/− brains.
Protein Accumulation in Trpm2 −/− Mice Brains
During aging, cellular oxidative stress increases significantly in the brain, which induces protein oxidation . The oxidized proteins are prone to cross-linking with each other and becoming non-functional . The accumulation of aggregated material is observed in the broad regions of the aged brain and is a major risk factor in neurodegeneration . Because TRPM2 is activated by ROS, it is considered to be an oxidative sensor responding to oxidative stress . Consistent with this, the oxidant condition regulates cell survival and death via TRPM2-dependent Ca2+ influx . TRPM2 also acts as a chanzyme to remove ADPR, a reactive metabolite induced by oxidative stress. Thus, in the brain, excessive oxidative stress activates TRPM2 and generates AMP in the cytosol, which in turn stimulates the autophagy-related signaling pathway to remove aggregated, oxidized proteins (Supplementary Fig. 4). Therefore, the function of TRPM2 as a sensor and an enzyme is required for the normal functioning of the brain.
Functional Roles of TRPM2 in Neurodegeneration
There are conflicting reports in describing the role of TRPM2 in neuroprotection or neurotoxicity. Ostapchenko and colleagues showed that TRPM2 deficiency rescues β-amyloid oligomer-mediated neurotoxicity such as synaptic loss and spatial memory deficits in double transgenic APP/PS1 mice . TRPM2 deficiency or pharmacological inhibition attenuates infarct size, neuronal loss, and memory impairment after stroke induced by transient global ischemia [13, 14, 46]. Thus, the TRPM2 loss appears to protect β-amyloid-induced or ischemia-induced neurotoxicity. On the contrary, TRPM2 deficiency leads to impaired synaptic transmission by abnormal regulation of long-term depression  and fails to reduce neurological outcomes in mice after permanent middle cerebral artery occlusion . A similar controversy over the role of TRPM2 loss on cell death extends to the heart. TRPM2 deficiency exacerbates mitochondrial dysfunction induced by ischemia-reperfusion  whereas TRPM2 deficiency attenuates infarct size in the reperfused myocardium . In the present study, we observed a neuronal and synaptic loss in the hippocampus accompanied by memory loss in Trpm2−/− mice. The two basic functions of TRPM2 may account for the conflicting results. TRPM2 is activated by ADPR or ROS thus resulting in Ca2+ influx, which leads to cell death if prolonged. On the other hand, TRPM2 is an enzyme to remove ADPR, which otherwise leads to cell death because of non-specific ribosylation of structural proteins or disruption in autophagic activity as proposed in this study. Thus, the TRPM2 deletion will result in either cell protection or cell degeneration depending on the two different functions. Experimental conditions such as types of intervention, duration of the treatment, or experimental conditions may lead to different outcomes.
We would like to thank Prof. Yasuo Mori of Kyoto University for the kind gift of TRPM2-deficient mice.
U. Oh designed and supervised the study. Y. Jang, B. Lee and S. Lee conducted the Western blots and immunohistochemistry. H. Kim mutated Trpm2 and tested the mutants for their channel activity. S. Jung performed the behavior tasks. S-Y. Lee experimented with the electrophysiological recording. J.H. Jeon and I-B. Kim performed the ultrastructural analysis. B-J. Kim, S-H. Lee, and U-H. Kim measured the ADPR metabolites. D. Jeon, Y. Lee, and S. Kim assisted with the data analysis.
Compliance with Ethical Standards
Conflict of Interest
The authors declare that they have no conflict of interest.
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