Sigma-1 receptors regulate hippocampal dendritic spine formation via a free radical-sensitive mechanism involving Rac1 GTP pathway
Shang-Yi Tsaia, Teruo Hayashia, Brandon K. Harveyb, Yun Wangb, Wells W. Wuc, Rong-Fong Shenc, Yongqing Zhangd, Kevin G. Beckerd, Barry J. Hoffere, and Tsung-Ping Sua,1
aCellular Pathobiology Section, Cellular Neurobiology Research Branch, and bNeural Protection and Regeneration Section, Molecular Neuropsychiatry Branch, Intramural Research Program, National Institute on Drug Abuse, 333 Cassell Drive, Baltimore, MD 21224; cProteomics and Analytical Biochemistry Unit and dGene Expression and Genomics Unit, Research Resources Branch, Intramural Research Program, National Institute on Aging, Biomedical Research Center, 251 Bayview Boulevard, National Institutes of Health, United States Department of Health and Human Services, Baltimore, MD 21224; and eCellular Neurophysiology Section, Cellular Neurobiology Research Branch, Intramural Research Program, National Institute on Drug Abuse, 333 Cassell Drive, Baltimore, MD 21224
Edited by Solomon Snyder, Johns Hopkins University School of Medicine, Baltimore, MD, and approved October 16, 2009 (received for review August 12, 2009) Sigma-1 receptors (Sig-1Rs) are endoplasmic reticulum (ER)-resident specific GEF TIAM1 regulate the late stage of neural morphogenesis proteins known to be involved in learning and memory. Dendritic by targeting themselves to dendritic spines and controlling the forma- spines in hippocampal neurons play important roles in neuroplasticity tion of mature forms of spines (8–10). TIAM1-Rac1 GTP signaling is and learning and memory. This study tested the hypothesis that also important for dendrite branching. Thus, Tiam1-Rac1 GTP signal- Sig-1Rs might regulate denritic spine formation in hippocampal neu- ing is critical for the morphogenesis of dendritic spines especially in the rons and examined potential mechanisms therein. In rat hippocampal later stages of neuronal maturation. primary neurons, the knockdown of Sig-1Rs by siRNAs causes a deficit Originally mistaken as a subtype of opioid receptors (11), the in the formation of dendritic spines that is unrelated to ER Ca2؉ Sigma-1 receptor (Sig-1R) (12–15) is now known to be a nonopioid signaling or apoptosis, but correlates with the mitochondrial perme- endoplasmic reticulum (ER)-resident protein (16–19). Sig-1Rs are ability transition and cytochrome c release, followed by caspase-3 postulated to be involved in neuropsychiatric diseases including activation, Tiam1 cleavage, and a reduction in Rac1 GTP. Sig-1R- amnesia and addiction (13, 20, 21). By using CHO cells, we recently knockdown neurons contain higher levels of free radicals when (17) identified the Sig-1R as a ligand-operated ER receptor chap- compared to control neurons. The activation of superoxide dismutase erone that regulates Ca2ϩ signaling specifically by chaperoning type or the application of the hydroxyl-free radical scavenger N-acetyl 3IP3 receptors. Importantly, Sig-1Rs regulate neuritogenesis in cysteine (NAC) to the Sig-1R-knockdown neurons rescues dendritic PC12 cells (22) and enhance cell differentiation in rat oligoden- spines and mitochondria from the deficits caused by Sig-1R siRNA. drocytes (23). Sig-1Rs also promote reconstitution of lipid rafts in Further, the caspase-3-resistant TIAM1 construct C1199DN, a stable plasma membranes (22). Taken together, these results suggest the guanine exchange factor able to constitutively activate Rac1 in the possibility that Sig-1Rs might affect the morphogenesis of neurons form of Rac1 GTP, also reverses the siRNA-induced dendritic spine in the CNS. However, this possibility has never been examined. deficits. In addition, constitutively active Rac1 GTP reverses this def- We tested if the ER Sig-1R might regulate neuronal morphology icit. These results implicate Sig-1Rs as endogenous regulators of in this study, specifically dendritic spine formation in rat primary hippopcampal dendritic spine formation and suggest a free radical- hippocampal neurons. We also examined potential underlying sensitive ER-mitochondrion-Rac1 GTP pathway in the regulation of mechanisms of such effect, if present. We report here that Sig-1Rs dendritic spine formation in the hippocampus. regulate dendritic spine formation via a potential ER-mitochon- drion-Rac GTP pathway, apparently due to this receptor’s ability to mitochondria ͉ ROS ͉ N-acetyl cyteine ͉ learning and memory ͉ caspase-3 regulate the redox state of neurons.
Results endritic spines in the CNS are important for many functions. DDendritic atrophy in the neocortical region is related to Sig-1Rs Regulate the Morphogenesis of Hippocampal Primary Neu- aging-induced amnesia, and its reversal improves memory retention rons. We examined the roles of Sig-1Rs in the morphogenesis of (1). Similarly, the loss of dendritic spine-related synapses is cur- hippocampal primary neurons in vitro by silencing Sig-1R expres- rently a strong pathologic correlate of cognitive decline, and sion using siRNAs. Small hairpin RNAs, constructed in the pSI- synaptic dysfunction is evident long before synapses and neurons REN vector, were transfected into primary neurons by using the are lost (2). On the other hand, exposure to drugs of abuse including liposome-delivering system in this initial portion of the study. Rat cocaine, nicotine, or morphine produces persistent changes, usually hippocampal primary neurons were thus transfected with siRNAs in the form of increased dendritic spines and arborizations, in cells (17, 23) together with pEGFP-N1 vectors on DIV 7. Morphologies in brain regions involved in incentive motivation and reward (3). were observed on DIV 14 or 21. Control neurons receiving These persistent changes are thought to represent the neuronal transfection of control siRNAs (siCon-tf) on DIV 14 showed a reorganization that contributes to some of the persistent sequelae associated with drug use, including the establishment of motiva- Author contributions: S.-Y.T., T.H., B.K.H., and T.-P.S. designed research; S.-Y.T., and T.H. tional conditioning and learning (3). performed research; B.K.H. and Y.W. contributed new reagents/analytic tools; S.-Y.T., T.H., The morphology of dendritic spines and axons is determined by the B.K.H., Y.W., W.W.W., R.-F.S., Y.Z., K.G.B., B.J.H., and T.-P.S. analyzed data; and S.-Y.T., T.H., dynamic cytoskeleton protein actin. Rho family small GTPases includ- B.J.H., and T.-P.S. wrote the paper. ing Rho, Cdc42, and Rac1 regulate the dynamics of actin and are critical The authors declare no conflict of interest. for neuronal polarization and morphogenesis (4–6). Rho proteins are This article is a PNAS Direct Submission. regulated by guanine nucleotide exchange factors (GEFs). In early Freely available online through the PNAS open access option. stages of neural morphogenesis, the activation of Cdc42 promotes the 1To whom correspondence should be addressed. E-mail: [email protected]. formation of filopodia, the long thin protrusions serving as primary This article contains supporting information online at www.pnas.org/cgi/content/full/ precursors of axons and dendritic spines (7). However, Rac1 and its 0909089106/DCSupplemental.
22468–22473 ͉ PNAS ͉ December 29, 2009 ͉ vol. 106 ͉ no. 52 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0909089106 Downloaded by guest on September 27, 2021 Fig. 1. Inhibition of dendritic morphogenesis in Sig-1R-knockdown hippocampal neurons. (A–F) Effects of siSig-1R-tf on dendrite formation. Neurons were transfected with siRNAs on DIV 7 and were stained on 14 or 22 DIV with anti-MAP-2B antibodies. The number of established primary dendrites and branches were counted. Primary dendrites are defined as neurites originating from the neuronal soma and are at least longer than two times the diameter of the cell body. (Scale bar, 100 m.) ***P Ͻ 0.001; n ϭ 3, five to 10 neurons were quantified in each individual experiment. (G–J) Effect of siSig-1R-tf on spine formation. Neurons were transfected on DIV 7 with siCon (G and I) or siSig-1R (Hand J) vectors in combination with EGFP. Neurons were stained with rhodamin phalloidin for F-actin. Note elongated filopodia in si-Sig-1R-tf neurons. (Kand L) Quantitative assessments were made of the effects of siSig-1Rs on protrusion length, dendrite branching, and filopodium formation. The control neurons at DIV 22 had protrusions mostly Ͻ2m in length and mushroom shaped, which is characteristic of dendritic spines, whereas the siRNA neurons failed to retract protrusions (i.e., exhibited elongated filopodia). Protrusions equals dendritic spines plus filopodia. (Scale bar, 50 m.) ***P ϭ 0.0002; n ϭ 2, at least 20 neurons were captured in each experiment, and two to three dendrites were randomly selected from each neuron for counting of protrusions. (M) Synaptophysin immunostaining. (N) Effects of siSig-1Rs on synaptic activity. FM4–64 (red)-labeled neurons were depolarized with KCl for measurement of synaptic activity. (Scale bars, M 20 m and N 5 m.)
typical morphology of a stage V neuron, having several long the longer the culture time, the more pronounced the abnormal dendrites with tapering and branching characteristics (Fig. 1 A–F). phenotype was seen, such as increased numbers of elongated The Sig-1R-siRNA-transfected (siSig-1R-tf) neurons, however, filopodia-like protrusions lacking head structures. In general, si- showed a reduction of extension and branching of dendrites. Long lencing of Sig-1R expression increased the length of protrusions by filopodia-like structures protruding from soma were often observed 59% on DIV 16 and 159% on DIV 22, respectively. These results in siSig-1R-tf neurons. Similar effects were seen on DIV 21 in suggest a dysregulation of stage-dependent signalings for actin siSig-1R-tf neurons. Sig-1Rs also affected the formation and mat- compartmentalization/polymerization in Sig-1R-deficient neurons. uration of dendritic spines. Results are shown in Fig. 1 G–L.On
DIV 16, control neurons formed filopodia-like long protrusions on Sig-1R-Knockdown Inhibits Synapse Formation. Active synapses ex- NEUROSCIENCE dendrites, with majority of them (Ͼ60%) showing F-actin-enriched press specific proteins and receptors for proper function. We next asked head structures at the tip. On DIV 22, control neurons formed if the knockdown of Sig-1Rs might affect the formation of synapses. In clusters of stubby mushroom-shaped spine heads and possessed no control neurons, staining for NR-1, GluR2/3, and PSD-95 showed filopodia, indicating the maturation of spines. In contrast, siSig- puncta of strong immunoreactivities on dendritic spines. However, no 1R-tf neurons formed long and thin protrusions, and only few such puncta were observed in the long filopodia-like protrusions in possessed spine heads at the tip. Further, F-actins were seen siSig-1R-tf neurons; these proteins remained in the dendritic shafts (Fig. diffusing over the whole cytoplasm in the filopodia of siSig-1R-tf S1 in SI Appendix). Axon terminals in siSig-1R-tf neurons also exhibited neurons, whereas F-actins discretely accumulated at the tip of significantly less immunoreactivities for synaptophysin (Fig. 1M). Syn- filopodia in control neurons (Fig. 1 G–J). For siSig-1R-tf neurons, aptic activities assessed by the depolarization-induced uptake of
Tsai et al. PNAS ͉ December 29, 2009 ͉ vol. 106 ͉ no. 52 ͉ 22469 Downloaded by guest on September 27, 2021 measured by a pull-down assay, the Rac1 GTP in the raft fractions (fractions 4 and 5) was reduced in AAV-siSig-1R-td neurons (Fig. 2 F and G). The Sig-1R-knockdown also caused a decrease of intact TIAM1 in the raft fractions. The cleaved TIAMs, which were increased in AAV-si-Sig-1R-td neurons, were present only in nonraft fractions (Fig. 2 H and I). These data suggest that: (i) The active full-length TIAM1 preferentially exists at the raft where TIAM1 serves to activate Rac1 and (ii) the knockdown of Sig-1Rs decreases the amount of full-length TIAM1 at rafts, leading to a reduction of Rac1 activation. How the knockdown of Sig-1Rs might reduce the full-length TIAM1 is examined in the next section.
Knockdown of Sig-1Rs Activates Caspase-3 in an ER-Stress and Apo- ptosis-Independent Manner. TIAM1, by activating Rac1, is known to maintain the structural configuration of dendritic spines (8–10). TIAM1 in the motor neuron is inactivated by caspase-dependent proteolysis when cells undergo stress (25). To determine whether increased cleavage of TIAM1, as seen above in AAV-siSig-1R-td neurons, is associated with caspase activation, active forms of several caspase members were analyzed by Western blots. As shown in Fig. S4 in SI Appendix, in the absence of ER stressors like thapsigargin, AAV-siSig-1R-td neurons showed a reduction of procaspase-3 with a concomitant increase in active caspase-3, seen as the cleaved form, when compared to the control (Fig. S4A, lane 2 vs. lane 1). There was no effect on the level of ER stress-responding proteins including caspase-12, GRP94, calnexin, ATF4, and BiP (Fig. S4 B and C, lane 2 vs. lane 1 in each panel). These results suggest that Sig-1R-knockdown did not elicit ER stress and that the knockdown of Sig-1Rs might have Fig. 2. Sig-1R-knockdown causes TIAM1 GEF cleavage and the inactivation caused an increase in caspase-3 in an ER stress-independent manner. of Rac. (A and B) Immunocytochemisty for Rac and Tiam1 in AAV-siCon-td or We thus decided to use thapsigargin, a well-known ER stressor that AAV-siSig-1R-td neurons at DIV 22. (Scale bar, 5 m.) (C) TIAM1 cleavage causes an increase in many of the ER stress-responding proteins detected by Western blots. Note the decrease of intact TIAM1 (200 kDa) with detailed above. Inasmuch as the knockdown of Sig-1Rs did not cause concomitant increases of cleaved TIAM1 (120, 85, and 74 kDa) in AAV-siSig- 1R-td neurons. (D–G) Effect of AAV-siSig-1Rs on lipid raft distributions of total ER stress, we postulated that the caspase-3 increase seen in AAV- Rac (D–G)orRac GTP (F and G). *P Ͻ 0.05; n ϭ 3. (H and I) Lipid raft distributions siSig-1R-td neurons should continue to be present if neurons are of TIAM1 and its proteolytic products. challenged with thapsigargin, whereas the thapsigargin-elicited ER stress-responding proteins should not be affected, or more specifically enhanced, by the Sig-1R-knockdown. Indeed, we found that the FM4–64 showed that the siSig-1R-tf-induced protrusions did not form caspase-3 activation continued to be seen in thapsigargin-treated Sig- functional synapses (Fig. 1N). These findings suggest that the filopodia- 1R-knockdown neurons (Fig. S4A), whereas no further enhancement like long protrusions caused by siSig-1Rs failed to form functional of the ER stress-responding proteins was seen in thapsigargin-treated synapses. Sig-1R-knockdown neurons (Fig. S4C). Thus, the activation of capase-3 induced by the Sig-1R-knockdown may not involve ER stress but Sig-1R-Knockdown Inhibits Activation of Rac1 Rho GTPase. Rac1 regulates instead may involve other, as yet unidentified, pathway(s). dendritic growth and spine maturation but not filopodium formation To ascertain whether the knockdown of Sig-1Rs would cause (8–10). Since the knockdown of Sig-1Rs stunt dendritic growth and apoptosis, Hoechst 33342 staining was used to detect chromatin synapse formation but not the formation of filopodia, we hypothesized condensation in control and transfected or tranduced neurons. that the Sig-1R might regulate Rac1 signaling in neurons. Both Rac1 Staurosporine-treated (STS; 1 M for 24 h) neurons served as and TIAM1 clustered in dendritic shafts and spines of control neurons positive controls for apoptosis. In both AAV-siCon-td and AAV- (Fig. 2A and B). However, intensities of Rac1 and TIAM1 in filopo- siSig-1R-td neurons, only a very small fraction of cells underwent dium-like protrusions on the dendrites of siSig-1R-tf neurons were apoptosis. No difference in the percentage of apoptotic cells was markedly decreased (Fig. 2 A and B). The expression levels of these seen in AAV-siCon-td and AAV-siSig-1R-td neurons (Fig. S4D). proteins were examined by Western blotting, a technique that requires Therefore, Sig-1R-knockdown in hippocampal primary neurons more protein to be analyzed. Since gene transfer using liposomes yields did not induce apoptosis. only a few percentage of successful transfection in primary neurons, we used an AAV-based gene delivery system that yields Ͼ70% efficacy in Sig-1R-Knockdown Does Not Affect Ca2؉ Dynamics in Hippocampal gene expression. Details of vector constructions are described in SI Neurons but Nevertheless Causes Mitochondrial Dysfunction. Con- Methods and Fig. S2 in SI Appendix. Western blots showed that the sistent with other reports (26, 27), we found that type-1 IP3 knockdown of Sig-1Rs (i.e., AAV-siSig-1R transduced or abbreviated receptors, and not type-3 receptors, were the predominant IP3 as AAV-siSig-1R-td) did not affect the total level of Rac1 protein in receptors in hippocampal neurons. As Sig-1R chaperones are hippocampal neurons. However, the knockdown caused a down- known to regulate only type-3 IP3 receptors but not the type-1 regulation of full-length TIAM1, accompanied by increases in its receptors (17), we found that the basal or activated level of proteolytic products (Fig. 2C). mitochondrial or cytosolic Ca2ϩ transient/concentration in hip- Rac1 is compartmentalized in lipid rafts of postsynaptic mem- pocampal neurons was not affected by Sig-1R-knockdown (Fig. branes (24). We found (Fig. 2D) that Rac 1 existed in both raft S3 A–D in SI Appendix). We found, however, that Sig-1R- (fractions 3, 4, and 5) and nonraft fractions (fractions 6 and higher). knockdown caused a disruption of mitochondrial membrane The knockdown of Sig-1Rs did not apparently affect the distribu- potential (28) as well as an enhanced release of cytochrome c tion pattern of Rac1 in raft or nonraft fractions (Fig. 2E). However, from mitochondria (Fig. S3 E–L in SI Appendix). Thus, Sig-1R- when active forms of GTP-binding Rac1 (i.e., Rac1 GTP) were knockdown caused the activation of caspase-3 by increasing
22470 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0909089106 Tsai et al. Downloaded by guest on September 27, 2021 Fig. 3. Free radicals in Sig-1R-knockdown neurons. (A) Gen- eration of superoxide anions by GSH and sodium selenite. • Ϫ Sodium selenite was mixed with GSH at 0.2 mg/mL. O2 were monitored by lucigenin-based chemiluminescense (lucigenin • Ϫ at 50 M). (B) Effect of hippocampal neuronal lysates on O2 generated by GSH/selenite. Neurons were transduced with AAV-siCons or AAV-siSig-1Rs for 10 days before assay. (C) The AUC, calculated from assays in panel B (between the 10th and 25th intervals), seen with increasing lysates (0, 0.03, 0.1, 0.3, 1, and 3 g protein). (D) Effect of AAV-siSig-1R-td on the NADPH- • Ϫ induced generation of O2 in living hippocampal neurons. NADPH at 45 M was applied to neurons in the presence of 50 M lucigenin at the beginning of the fifth interval (AUC from 10th to 40th interval). (E) Inhibition of AAV-siSig-1R-td- induced mitochondrial permeability transition by free radical scavengers NAC and Tempol. The puncta of JC-1 positive mi- tochondria in dendrites were measured. ***P Ͻ 0.001; n ϭ 5, each group in triplicates. (F) Effects of free radical scavengers on dendritic spine formation in AAV-siSig-1R-td neurons. Neu- rons were transduced with AAV-siSig-1Rs on DIV 13 when 50 M Tempol, NAC, or NAL were also added. Protrusion lengths were measured on day 5 posttransduction. Frequencies of various protrusion lengths are shown; n ϭ 3 independent experiments. Representative morphologies are shown at the Top. (Scale bar, 5 m.)
mitochondrial cytochrome c release. However, this action of drial function in AAV-siSig-1R-td neurons, we next examined if free Sig-1Rs in this regard did not involve mitochondrial Ca2ϩ. radical scavengers might block the MPT in those neurons. N-acetyl cysteine (NAC) or 4-hydroxy-2,2,6,6-tetramethylpiperidinyloxy (Tem- Reduced Radical Scavenging Activity in Sig-1R-Knockdown Neurons. pol), scavengers of hydroxyl or superoxide dismutase activator, respec- In addition to Ca2ϩ overload in mitochondria, free radical-related tively, successfully blocked the MPT caused by the knockdown of oxidative stress is also known to causes mitochondrial membrane Sig-1Rs (Fig. 3E). The nitric oxide synthase inhibitor nitro-L-arginine disruption, which leads to mitochondrial permeability transition (NAL) was, however, ineffective. Thus, siSig-1R-induced disruption of (MPT) (29–31). Thus, we next examined if free radicals are mitochondrial membrane potential is, at least, in part due to the involved in the siSig-1R-induced MPT. First, we examined if the neurons’ deficiency in scavenging free radicals. Sig-1R-knockdown affected the free radical scavenging activity of Free radical scavengers were also used to examine if they might block neurons. An in vitro test, employing the selenite-catalyzed super- the aberrant spine development in AAV-siSig-1R-td neurons. Indeed, oxide generation system (32), was used. Selenite at 0.06 mM NAC and Tempol successfully rescued spine formation in Sig-1R- effectively induces superoxide anion generation upon the addition knockdown neurons (Fig. 3F). Specifically, NAC- or Tempol-treated •Ϫ of glutathione (GSH; 0.2 mg/mL) (Fig. 3A). The superoxides (O2 ) AAV-siSig-1R-td neurons were able to retract filopodia and form thus generated were monitored by lucigenin-based chemilumines- dendritic spines. NAL was again less effective in this regard (Fig. 3F). cence. Fig. 3B shows that lysates from AAV-siSig-1R-td neurons •Ϫ were less effective in scavenging O2 when compared to control Caspase-3-Resistant TIAM1 Construct Rescues the Spine Formation in lysates. The scavenging activity was lysate protein-dose-dependent. Sig-1R-Knockdown Neurons. Our hypothesis suggests that the final Analyses of the area-under-the-curve (AUC) indicate an EC50 ϭ pathway of Sig-1R-knockdown effects on dendritic spine formation 0.0144 g protein for AAV-siCon-td controls and 0.3791 g protein is related to the inactivation of Rac1, which in turn is hinged upon for AAV-siSig-1R-td neurons (Fig. 3C). We next examined if TIAM1 cleavage by caspase-3. To provide evidence for the in- similar results might be seen if assays were done in the living system. volvement of Sig-1Rs in this pathway, we examined if the deficit of
Accordingly, NADPH was applied to living neurons to generate spine formation caused by Sig-1R-knockdown might be rescued by NEUROSCIENCE •Ϫ •Ϫ O . NADPH induced a rapid generation of O in both groups, but a caspase-3-resistant TIAM1 construct. We therefore transfected 2 •Ϫ 2 higher levels of detectable O2 were seen in AAV-siSig-1R-td siSig-1R-tf neurons with either an N-terminal-truncated TIAM1 neurons (Fig. 3D). These results with the NADPH assay suggest construct called C1199, which is more stable when compared to that either AAV-siSig-1R-td neurons are less effective in scaveng- full-length TIAM1 (10), or a construct derived from C1199, called •Ϫ •Ϫ ing O2 or more O2 are generated in those neurons. C1199DN, that is fully resistant to caspase-3 (25). Initially, we used Neuro-2a cells, in lieu of primary hippocampal neurons, to char- Free Radical Scavengers Block Mitochondrial Dysfunction and Rescue acterize and confirm the properties of C1199 and C1199DN, Dendritic Spine Formation in Sig-1R-Knockdown Neurons. To establish because of the need for the abundant samples available from a causal relation between redox state and the alteration of mitochon- Neuro-2a cells for such an extensive characterization. We specifi-
Tsai et al. PNAS ͉ December 29, 2009 ͉ vol. 106 ͉ no. 52 ͉ 22471 Downloaded by guest on September 27, 2021 following modifications in the protocol: (i) Neurons seeded at higher density (1 ϫ 105 cells/well); (ii) pretreatment with kineuric acid and MgCl2 1 h before transfection; and (iii) neurobasal medium used as a diluent of the DNA-lipofectamine complexes. This modified protocol yielded no cytotoxicity. Our results showed that the overexpression of C1199 in siSig-1R-tf neurons slightly improved the maturation of dendritic spines, but filopodium-like protrusions still dominated the phenotype (Fig. 4C). This result is consistent with another report showing that branched filopodia and lamelapodia were occasionally observed in neurons overexpressing C1199 (10). The overexpression of caspase-3-resistant C1199DN in siSig-1R-tf neurons, however, successfully rescued dendritic spine formation in those neurons notably by decreasing the number of long filopodia while concomitantly increasing mature spines with mushroom-shaped head structures (Fig. 4 C–E).
Constitutively Active Rac Rescues the Spine Formation in Sig-1R- Knockdown Neurons. To provide additional evidence for our hy- pothesis on the ER-mitochondrion- Rac1 GTP pathway, we exam- ined if constitutively active Rac1 (i.e., Q61L) might rescue the deficit of dendritic spines caused by the Sig-1R-knockdown. Our results indicate that constitutively active Rac successfully rescued the formation of dendritic spines in siSig-1R-tf neurons. The siSig-1R-tf neurons cotransfected with Q61L on DIV 18 were able to form dendritic spines on DIV 22 (Fig. 4F). Discussion In this study we have demonstrated that a potential ER- mitochondrion-Rac GTP pathway plays an important role in den- dritic spine and synapse formation in hippocampal neurons and that the redox state of neurons is an integral and critical element in this Fig. 4. Caspase-3-resistant TIAM1 construct C1199DN and its effects on pathway. Because Sig-1Rs are implicated in memory and cognition Rac GTP formation and dendritic spine maturation in Sig-1R-knockdown neu- (33, 34), our findings also provide a mechanistic insight into the rons. Effect of constitutively active Rac on dendritic spine formation in Sig- action of Sig-1Rs in neuroplasticity and cognition. 1R-knockdown neurons. (A) Characterizing the GEF activity between the ER and mitochondria have been reported to participate in full-length TIAM1, a more stable N terminus-truncated TIAM1 construct dendritic spine formation (35–39). Our results provide additional C1199, and a caspase-3-resistant TIAM1 construct C1199DN using Neuro-2a insights on how these two intracellular organelles may participate in cells. Neuro-2a cells were transfected with various TIAM1 constructs: Full- dendritic spine formation in a unique fashion. Contrary to the length (FL) Tiam1, C1199, and C1199DN. All three TIAM1s equally increased notion that ER does so by regulating Ca2ϩ efflux via ryanodine or the active form of Rac. (B) Resistance of C1199DN to proteolytic cleavage IP receptors on the ER membrane (35–37), our results indicate caused by siSig-1R-tf in Neuro-2a cells. The asterisk (*) indicates the nonspe- 3 cific band. (C–E) In primary hippocampal neurons, overexpression of C1199 that the ER might regulate dendritic spine formation in hippocam- and C1199DN rescued aberrant dendritic spine morphologies caused by siSig- pal neurons via a free radical-related mechanism that might not ϩ 1R-tf. Note the greater potency of C1199DN in rescuing the morphologies. involve Ca2 signaling. Mitochondria supply energy for cells and are ***P Ͻ 0.001 (F) The effect of constitutively active Rac1 (Q61L) on dendritic important in neural development (38, 39). Our results indicate that spine formation in siSig-1R neurons. DIV 18 neurons were cotransfected with mitochondria might participate in dendritic formation via a siRNA-Sig1R vectors together with constitutively active Rac1 or an empty TIAM1-Rac1 GTP signaling pathway. vector. The morphologies were obtained at DIV 22; n ϭ 3 experiments. At least Our results also indicate that caspase-3 plays an important role in the 5–10 neurons were counted in each experiment. Scale bar, 5 m. development of neurons. Although caspase-3 is recognized as a pro- tease that executes death signals, growing evidence suggests that cally examined the susceptibility of C1199 and C1199DN to siSig- caspase-3 may also be important in synaptic plasticity and growth cone 1R-induced proteolysis. In Neuro-2a cells, both C1199 and motility (40–43). We have thus added one more important physiolog- C1199DN were able to increase the level of Rac GTP (Fig. 4A). ical role to the action of caspase-3 and shown that it regulates dendritic spine formation in hippocampal neurons. Although Sig-1R-knockdown Both full-length TIAM1 and C1199 underwent slight cleavage in increased caspase-3 activity in primary neurons in this study, the siCon-tf Neuro-2a cells (Fig. 4B, lanes 3 and 5). In siSig-1R-tf knockdown did not cause apoptosis. As such, the increase in caspase-3 Neuro-2 cells, the cleaved form of full-length TIAM1 or C1199 activity caused by Sig-1R-knockdown might represent an early stage of increased, when compared to that seen in respective control cells denritic spine dysfunction that is before apoptosis and might still be (Fig. 4B, lanes 4 and 6 vs. lanes 3 and 5, respectively). However, the rescued by some therapeutic intervention including the use of free caspase-3-resistant construct C1199DN was resistant to cleavage radical scavengers (see below). both in control cells and in Sig-1R-null cells (Fig. 4B, lanes 7 and One of the most important outcomes from this study is the demon- 8). These results indicated that C1199 and C1199DN would be stration that an ER protein can affect the activation of Rac1 and useful tools to test our hypothesis. associated neuroplasticity by the level of free radicals in neurons. We do Hippocampal primary neurons were subsequently transfected not know at present how Sig-1Rs modulate free radicals or reactive with a C1199 or C1199DN plasmid along with siSig-1Rs. To best oxygen species (ROS) in the neuron. Mitochondria are the main source monitor the effects of those constructs on the spine formation, of free radicals/ROS levels in the cell, but the ER can also generate free plasmids were transfected into neurons specifically on DIV 14, a radicals (44). The exact mechanism whereby free radicals are controlled time when filopodia begin to form spines in primary cultures. by the ER Sig-1Rs warrants further investigation. Gene microarray and Cytotoxicity caused by this late transfection was minimized by the proteomic studies are being planned to provide some clues in this
22472 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0909089106 Tsai et al. Downloaded by guest on September 27, 2021 regard. Nevertheless, our results with free radical scavengers being able down Sig-1Rs. Multiplicity of infection (MOI; 10,000 to Ϸ20,000) was used to rescue mitochondrial dysfunction and the aberrant morphologies of for neuronal transduction. For details, see SI Methods in SI Appendix. dendritic spines attest to the important role of redox state in the ϫ 6 Ϸ ϫ morphogenesis of neurons. Moreover, our results showing that the free Preparation of Detergent-Resistant Membrane. Approximately 5 10 1 107 cultured neurons (21 DIV) from whole brains were used. The remainder of radical scavenger NAC and the superoxide dismutase activator Tempol experimental details can be found elsewhere (22). are able to rescue the dendritic arborization also suggest the possibility that such drugs might be of therapeutic potential in treating dendritic Rac GTP Pull Down Assay. Procedures were performed according to the man- spine-related neurodegenerative diseases. Specifically, NAC is a mu- ufacturer’s instructions (Upstate Biotechnology). colytic agent used in treating respiratory conditions. Our results suggest that NAC might represent an established drug with use in treating CNS Assays for Superoxide Anions. A chemiluminescence assay was used with diseases related to dendritic spine abnormalities, especially in the modifications (32). Details of the assay are described in SI Methods in SI Appendix. hippocampus, which is well-known to play an important role in learning and memory. In fact, several reports have demonstrated that NAC is Free Radical Scavenging Experiments. Neuronal cultures were infected rou- neuroprotective and can be used to improve spatial working memory tinely on DIV 10. One hundred micromolar hydroxyl free radical scavenger in rats (45, 46). Our results provide a potential mechanism for the NAC, the superoxide dismutase activator Tempol, or the NO synthase inhibitor memory-enhancing effect of NAC. Finally, because Sig-1Rs regulate NAL were added to the cultures 24 h after infection. In ‘‘rescue’’ experiments, the ganglioside composition in lipid rafts (22), the inability of Rac GTP half of the culture medium was replaced daily with fresh medium supple- and TIAM1 to reside in the raft in Sig-1R-knockdown neurons (Fig. 2) mented with free radical scavengers. Neurons were subjected to JC-1 labeling for MPT analyses on DIV 15. suggests a dysregulation of ganglioside formation in those neurons. ACKNOWLEDGMENTS. The kindness and generosity of Dr. Margaret Chou in Materials and Methods supplying various plasmids of the TIAM1 mutants is greatly appreciated. We thank Mr. Doug Howard for his technical assistance with the AAV vectors. This For larger quantities of cell samples, Neuronal Culture and Viral Transduction. study is supported by the Intramural Research Programs of the National Institute primary neurons were prepared from whole brain in some experiments. on Drug Abuse and the National Institute on Aging, National Institutes of Health, Neurons at 10 DIV were transduced with AAV serotype 6 (AAV6) to knock Department of Health and Human Services.
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