Sigma-1 receptors regulate hippocampal dendritic spine formation via a free radical-sensitive mechanism involving Rac1⅐GTP 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-Rac1⅐GTP signaling is and learning and memory. This study tested the hypothesis that also important for dendrite branching. Thus, Tiam1-Rac1⅐GTP 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 Rac1⅐GTP. 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 Rac1⅐GTP, also reverses the siRNA-induced dendritic spine in the CNS. However, this possibility has never been examined. deficits. In addition, constitutively active Rac1⅐GTP 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-Rac1⅐GTP 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-Rac⅐GTP 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 Ͻ2␮m 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.