Inhibition of Store-Operated Calcium Entry Attenuates MPP+-Induced Oxidative Stress via Preservation of Mitochondrial Function in PC12 Cells: Involvement of Homer1a

The process of store-operated calcium entry (SOCE), whereby the release of intracellular Ca²⁺ from endoplasmic reticulum (ER) activates Ca²⁺ influx channels in the plasma membrane, has been demonstrated to impact a diverse range of cell functions. In the present study, we investigated the potential protective effect of SOCE inhibition against 1-methyl-4-phenylpyridinium (MPP⁺) injury by using pharmacological antagonists or specific small interfering RNA (siRNA) in PC12 cells. The results showed that both antagonists (15 μM MRS-1845 and 50 μM ML-9) and stromal interacting molecule-1 (STIM1) targeted siRNA (Si-STIM1) significantly increased cell viability, decreased apoptotic cell death and reduced intracellular reactive oxygen species (ROS) production and lipid peroxidation in MPP⁺ injured PC12 cells. SOCE inhibition also prevented MPP⁺ induced mitochondrial dysfunction and activation of mitochondrial related apoptotic factors, while had no effect on mitochondrial biogenesis. Moreover, inhibition of SOCE by antagonists and siRNA increased the expression levels of Homer1a mRNA and protein, and knockdown of Homer1a expression by specific siRNA partly reversed the protective effects induced by SOCE inhibition in PC12 cells. All these results indicated that SOCE inhibition protected PC12 cells against MPP⁺ insult through upregulation of Homer1a expression, and SOCE might be an ideal target for investigating therapeutic strategy against neuronal injury in PD patients.     

Protective effect of Homer 1a against hydrogen peroxide-induced oxidative stress in PC12 cells

Oxidative stress-induced cell damage is involved in many neurological diseases. Homer protein, as an important scaffold protein at postsynaptic density, regulates synaptic structure and function. Here, we reported that hydrogen peroxide (H(2)O(2)) induced the expression of Homer 1a. Down-regulation of Homer 1a with a specific small interfering RNA (siRNA) exacerbated H(2)O(2)-induced cell injury. Up-regulation of Homer 1a by lentivirus transfection did not affect the anti-oxidant activity, but significantly reduced the reactive oxygen species (ROS) production and lipid peroxidation after H(2)O(2)-induced oxidative stress. Overexpression of Homer 1a attenuated the loss of mitochondrial membrane potential (MMP) and ATP production induced by H(2)O(2), and subsequently inhibited mitochondrial dysfunction-induced cytochrome c release, increase of Bax/Bcl-2 ratio and caspase-9/caspase-3 activity. Furthermore, in the presence of BAPTA-AM, an intracellular free-calcium (Ca(2+)) chelator, overexpression of Homer 1a had no significant effects on H(2)O(2)-induced oxidative stress. These results suggest that Homer 1a has protective effects against H(2)O(2)-induced oxidative stress by reducing ROS accumulation and activation of mitochondrial apoptotic pathway, and these protective effects are dependent on the regulation of intracellular Ca(2+) homeostasis.     

Homer1 knockdown protects dopamine neurons through regulating calcium homeostasis in an in vitro model of Parkinson's disease

Homer1 protein is an important scaffold protein at postsynaptic density and has been demonstrated to play a central role in calcium signaling in the central nervous system. The aim of this study was to investigate the effects of Homer1 knockdown on MPP⁺ induced neuronal injury in cultured dopamine (DA) neurons. We found that down-regulating Homer1 expression with specific small interfering RNA (siRNA) significantly suppressed LDH release, reduced Propidium iodide (PI) or Hoechst staining, increased the number of tyrosine hydroxylase (TH) positive cells and DA uptake, and attenuated apoptotic and necrotic cell death after MPP⁺ injury. Homer1 knockdown decreased intracellular reactive oxygen species (ROS) generation through inhibition of intracellular calcium overload, but did not affect the endogenous antioxidant enzyme activities. Calcium imaging was used to examine the changes of intracellular Ca^2 + concentration ([Ca^2 +]_cyt) and Ca^2 + in endoplasmic reticulum (ER) ([Ca^2 +]_ER), and the results showed that Homer1 siRNA transfection attenuated ER Ca^2 + release up to 120 min after MPP⁺ injury. Furthermore, decrease of [Ca^2 +]_cyt induced by Homer1 knockdown in MPP⁺ treated neurons was further enhanced by NMDA receptor antagonists MK-801 and AP-5, but not canonical transient receptor potential (TRPC) channel antagonist SKF-96365. l-type calcium antagonist isradipine but not nimodipine further inhibited intracellular calcium overload after MPP⁺ insult in Homer1 down-regulated neurons. These results suggest that Homer1 knockdown has protective effects against neuronal injury in in vitro PD model by reducing calcium overload mediated ROS generation, and this protection may be dependent at least in part on the regulatory effects on the function of calcium channels in both plasma membrane and ER.     

Downregualtion of dynamin-related protein 1 attenuates glutamate-induced excitotoxicity via regulating mitochondrial function in a calcium dependent manner in HT22 cells

Glutamate-mediated excitotoxicity is involved in many acute and chronic brain diseases. Dynamin related protein 1 (Drp-1), one of the GTPase family of proteins that regulate mitochondrial fission and fusion balance, is associated with apoptotic cell death in cancer and neurodegenerative diseases. Here we investigated the effect of downregulating Drp-1 on glutamate excitotoxicity-induced neuronal injury in HT22 cells. We found that downregulation of Drp-1 with specific small interfering RNA (siRNA) increased cell viability and inhibited lactate dehydrogenase (LDH) release after glutamate treatment. Downregulation of Drp-1 also inhibited an increase in the Bax/Bcl-2 ratio and cleavage of caspase-9 and caspase-3. Drp-1 siRNA transfection preserved the mitochondrial membrane potential (MMP), reduced cytochrome c release, enhanced ATP production, and partly prevented mitochondrial swelling. In addition, Drp-1 knockdown attenuated glutamate-induced increases of cytoplasmic and mitochondrial Ca²⁺, and preserved the mitochondrial Ca²⁺ buffering capacity after excitotoxicity. Taken together, these results suggest that downregulation of Drp-1 protects HT22 cells against glutamate-induced excitatory damage, and this neuroprotection may be dependent at least in part on the preservation of mitochondrial function through regulating intracellular calcium homeostasis.     

Knockdown of STIM1 inhibits 6-hydroxydopamine-induced oxidative stress through attenuating calcium-dependent ER stress and mitochondrial dysfunction in undifferentiated PC12 cells

Abstract

Stromal interaction molecule (STIM) proteins are parts of elaborate eukaryotic Ca²⁺ signaling systems and are considered to be important players in regulating neuronal Ca²⁺ homeostasis under normal ageing and pathological conditions. Here, we investigated the potential role of STIM1 in 6-hydroxydopamine (6-OHDA)-induced toxicity in undifferentiated PC12 cell lines. Cells exposed to 6-OHDA demonstrated alterations in the generation of reactive oxygen species (ROS) in a Ca²⁺-dependent manner. Downregulation of STIM1 expression by specific small interfering RNA (siRNA) attenuated apoptotic cell death, reduced intracellular ROS production, and partially prevented the impaired endogenous antioxidant enzyme activities after 6-OHDA treatment. Furthermore, STIM1 knockdown significantly attenuated 6-OHDA-induced intracellular Ca²⁺ overload by inhibiting endogenous store-operated calcium entry (SOCE). The effect of STIM1 siNRA on SOCE was related to orai1 and L-type Ca²⁺ channels, but not to transient receptor potential canonical type 1 (TRPC1) channel. In addition, silencing of STIM1 increased the Ca²⁺ buffering capacity of the endoplasmic reticulum (ER) in 6-OHDA-injured cells. ER vacuoles formed from the destruction of ER structural integrity and activation of ER-related apoptotic factors (CHOP and Caspase-12) were partially prevented by STIM1 knockdown. Moreover, STIM1 knockdown attenuated 6-OHDA-induced mitochondrial Ca²⁺ uptake and mitochondrial dysfunction, including the collapse of mitochondrial membrane potential (MMP) and the decrease of ATP generation. Taken together, our data provide the first evidence that inhibition of STIM1-meditated intracellular Ca²⁺ dyshomeostasis protects undifferentiated PC12 cells against 6-OHDA toxicity and indicate that STIM1 may be responsible for neuronal oxidative stress induced by ER stress and mitochondrial dysfunction in PD.     

Reactive oxygen species-mediated endoplasmic reticulum stress contributes to aldosterone-induced apoptosis in tubular epithelial cells

Apoptosis contributes to tubular epithelial cell death and atrophy in aldosterone (Aldo)-induced renal injury. This study aimed to determine mechanisms underlying Aldo-induced reactive oxygen species (ROS) and endoplasmic reticulum (ER) stress in tubular epithelial cells. Intracellular ROS generation was evaluated by 2',7'-dichlorofluorescin diacetate fluorescence. Apoptosis was detected by annexin V/propidium iodide staining and flow cytometry. ER stress induced protein and mRNA were evaluated by Western blot and real-time PCR, respectively. Aldo promoted tubular epithelial cell apoptosis, increased intracellular ROS production and induced ER stress, as evidenced by increased expression of glucose-regulated protein 78 (GRP78) and CAAT/enhancer-binding protein homologous protein (CHOP) in a dose- and time-dependent manner. Additionally, siRNA knockdown of CHOP and antioxidant N-acetyl-l-cysteine (NAC) attenuated ER stress-mediated apoptosis. NAC also could inhibit Aldo-induced expression of GRP78 and CHOP. Altogether, these observations suggest that Aldo induces apoptosis via ROS-mediated, CHOP-dependent activation in renal tubular epithelial cells.     

Homer1b/c在缺氧复氧损伤神经元中的保护作用研究

目的:探讨Homer1b/c在缺氧复氧损伤神经元中的作用。方法:构建pc DNA3.1-Homer1b/c质粒和Homer1b/c si RNA,过表达及si RNA干扰Neuro-2a细胞中Homer1b/c的水平,western blot分析观察上调及下调Homer1b/c后Homer1b/c的蛋白表达变化,再予细胞缺氧复氧损伤处理,观察细胞生存率、乳酸脱氢酶(Lactic dehydrogenase,LDH)释放量及caspase-3活性的变化。结果:过表达及si RNA干扰Homer1b/c后,Homer1b/c的蛋白水平分别较正常组明显增高及降低(P值分别0.01),提示过表达及干扰Homer1b/c的效果均明显,分别能明显提高及降低Homer1b/c的水平。在缺氧复氧损伤条件下,Homer1b/c过表达组较空质粒转染组,其细胞生存率明显增加,LDH释放量、caspase-3活性明显降低(P值分别0.05);相反,下调Homer1b/c的水平后,与control si RNA转染组相比,细胞生存率明显降低,而LDH释放量和caspase-3活性明显增加(P值分别0.05)。结论:Homer1b/c在缺氧复氧损伤神经元中具有神经保护作用。     

Protective effect of Homer 1a against hydrogen peroxide-induced oxidative stress in PC12 cells

Abstract

Oxidative stress-induced cell damage is involved in many neurological diseases. Homer protein, as an important scaffold protein at postsynaptic density, regulates synaptic structure and function. Here, we reported that hydrogen peroxide (H₂O₂) induced the expression of Homer 1a. Down-regulation of Homer 1a with a specific small interfering RNA (siRNA) exacerbated H₂O₂-induced cell injury. Up-regulation of Homer 1a by lentivirus transfection did not affect the anti-oxidant activity, but significantly reduced the reactive oxygen species (ROS) production and lipid peroxidation after H₂O₂-induced oxidative stress. Overexpression of Homer 1a attenuated the loss of mitochondrial membrane potential (MMP) and ATP production induced by H₂O₂, and subsequently inhibited mitochondrial dysfunction-induced cytochrome c release, increase of Bax/Bcl-2 ratio and caspase-9/caspase-3 activity. Furthermore, in the presence of BAPTA-AM, an intracellular free-calcium (Ca^{2 +}) chelator, overexpression of Homer 1a had no significant effects on H₂O₂-induced oxidative stress. These results suggest that Homer 1a has protective effects against H₂O₂-induced oxidative stress by reducing ROS accumulation and activation of mitochondrial apoptotic pathway, and these protective effects are dependent on the regulation of intracellular Ca^{2 +} homeostasis.     

Tissue kallikrein protects neurons from hypoxia/reoxygenation-induced cell injury through Homer1b/c

Previous studies have demonstrated that human tissue kallikrein (TK) gene delivery protects against mouse cerebral ischemia/reperfusion (I/R) injury through bradykinin B2 receptor (B2R) activation. We have also reported that exogenous TK administration can suppress glutamate- or acidosis-induced neurotoxicity through the extracellular signal-regulated kinase1/2 (ERK1/2) pathway. To further explore the neuroprotection mechanisms of TK, in the present study we performed immunoprecipitation analysis and identified a scaffolding protein Homer1b/c using MALDI-TOF MS analysis. Here, we tested the hypothesis that TK reduces cell injury induced by oxygen and glucose deprivation/reoxygenation (OGD/R) through activating Homer1b/c. We found that TK increased the expression of Homer1b/c in a concentration- and time-dependent manner. Moreover, TK facilitated the translocation of Homer1b/c to the plasma membrane under OGD/R condition by confocal microscope assays. We also observed that overexpression of Homer1b/c showed the neuroprotection against OGD/R-induced cell injury by enhancing cell survival, reducing LDH release, caspase-3 activity and cell apoptosis. However, the knockdown of Homer1b/c by small interfering RNA showed the opposite effects, indicating that Homer1b/c had protective effects against OGD/R-induced neuronal injury. More interestingly, TK exerted its much more significantly neuroprotective effects after Homer1b/c overexpression, whereas it exerted its reduced effects after Homer1b/c knockdown. In addition, TK pretreatment increased the phosphorylation of the ERK1/2 and Akt-GSK3β through Homer1b/c activation. The beneficial effects of Homer1b/c were abolished by the ERK1/2 or PI3K antagonist. Therefore, we propose novel signaling mechanisms involved in the anti-hypoxic function of TK through activation of Homer1b/c-ERK1/2 and Homer1b/c-PI3K-Akt signaling pathways.     

Endoplasmic reticulum stress plays a role in the advanced glycation end product-induced inflammatory response in endothelial cells

Aims

Both advanced glycation end products (AGEs) and endoplasmic reticulum (ER) stress play important roles in the development of various diseases. This study aimed to clarify the consequence of AGE-induced ER stress and its underlying mechanisms in human umbilical venous endothelial cells (HUVECs).

Main methods

AGE-induced ER stress was assessed by the increased expression and activation of the ER stress marker proteins GRP78, IRE1α and JNK, which were detected using Western blot. NF-κB translocation was revealed using Western blot and immunofluorescent staining in IRE1α-knockdown HUVECs. The mechanism of AGE-induced ER stress was also explored by inhibiting the effect of reactive oxygen species (ROS) using NADPH oxidase 4 (Nox4) siRNA and the antioxidant reduced glutathione (GSH). The cellular ROS level was measured using flow cytometry.

Key findings

AGEs time- and dose-dependently enhanced the expression of GRP78 and increased the phosphorylation of IRE1α and its downstream signal JNK in HUVECs. siRNA-induced IRE1α down-regulation suppressed AGE-induced NF-κB p65 nuclear translocation. Inhibiting the ROS production using Nox4 siRNA or antagonizing ROS using GSH reduced cellular ROS level and attenuated AGE-induced GRP78 expression and IRE1α and JNK activation.

Significance

This study confirms that AGE-induced ER stress in HUVECs focuses on the ER stress-enhanced inflammatory response through JNK and NF-κB activation. It further reveals the involvement of ROS in the AGE-induced ER stress mechanism.     

An N-terminal sequence specific for a novel Homer1 isoform controls trafficking of group I metabotropic glutamate receptor in mammalian cells

Homer proteins bind to a proline-rich region of the group I metabotropic glutamate receptors (mGluRs) and control their expression and localization at the excitatory postsynaptic density. We isolated a novel isoform of Homer1, Homer1d, from a mouse heart cDNA library. Its N-terminal end of 18 amino acids was unique among Homer1 variants (Homer1a-d), while the remainder of Homer1d was identical to that of Homer1b. To clarify the function of its N-terminus, we expressed Homer1b and 1d in the presence and absence of mGluR5b in HEK293T cells. When expressed alone, both Homer proteins were distributed diffusely in the cytoplasm and mGluR5b was on the plasma membrane (PM). When co-expressed, Homer1d and mGluR5b were co-localized on the PM, while Homer1b and mGluR5b were retained in the endoplasmic reticulum (ER). Both Homer proteins bound to mGluR5b in vitro. Therefore, the N-terminal portion of Homer1d may facilitate trafficking of Homer1-mGluR5 complex from the ER to the PM.     

Conditions associated with ER dysfunction activate homer 1a expression

Homer proteins physically link metabotropic glutamate receptors with IP3 receptors located at the endoplasmic reticulum (ER) and thereby modulate receptor-activated calcium signaling. Homer 1a, the short form of constitutively expressed homer 1 proteins, exerts dominant negative activity with respect to homer 1 proteins by interfering with the formation of multiprotein complexes. Homer 1a is an immediate early gene, the expression of which is activated by various stimuli including glutamate receptor activation. The mechanisms underlying activation of homer 1a expression are however, not fully understood. Here, we show that homer 1a expression is induced in neuronal cell cultures under experimental conditions associated with ER dysfunction. Increased homer 1a mRNA levels were found in 2 sets of cultures: in those exposed to thapsigargin, a specific inhibitor of ER Ca2+-ATPase, after a transient depletion of ER calcium stores through exposure to calcium-free medium supplemented with EGTA, and in those exposed to a proteasome inhibitor known to induce ER dysfunction. Thus, homer 1a expression may be activated by impairment of ER functioning just as it is by glutamate receptor activation.     

Homer regulates gain of ryanodine receptor type 1 channel complex

Homer proteins form an adapter system that regulates coupling of group 1 metabotropic glutamate receptors with intracellular inositol trisphosphate receptors and is modified by neuronal activity. Here, we demonstrate that Homer proteins also physically associate with ryanodine receptors type 1 (RyR1) and regulate gating responses to Ca(2+), depolarization, and caffeine. In contrast to the prevailing notion of Homer function, Homer1c (long form) and Homer1-EVH1 (short form) evoke similar changes in RyR activity. The EVH1 domain mediates these actions of Homer and is selectively blocked by a peptide that mimics the Homer ligand. 1B5 dyspedic myotubes expressing RyR1 with a point mutation of a putative Homer-binding domain exhibit significantly reduced (approximately 33%) amplitude in their responses to K(+) depolarization compared with cells expressing wild type protein. These results reveal that in addition to its known role as an adapter protein, Homer is a direct modulator of Ca(2+) release gain. Homer is the first example of an "adapter" that also modifies signaling properties of its target protein. The present work reveals a novel mechanism by which Homer directly modulates the function of its target protein RyR1 and excitation-contraction coupling in skeletal myotubes. This form of regulation may be important in other cell types that express Homer and RyR1.     

Serum deprivation-induced reactive oxygen species production is mediated by Romo1

Serum deprivation-triggered increases in reactive oxygen species (ROS) are known to induce apoptotic cell death. However, the mechanism by which serum deprivation causes ROS production is not known. Since mitochondria are the main source of ROS and since mitochondrial ROS modulator 1 (Romo1) is involved in ROS production, we sought to determine if serum deprivation triggered ROS production through Romo1. To examine the relationship between Romo1 and the serum deprivation-triggered increase in ROS, we transfected Romo1 siRNA into various cell lines and looked for inhibition of mitochondrial ROS generation. Romo1 knockdown by Romo1 siRNA blocked the mitochondrial ROS production caused by serum deprivation, which originates in the mitochondrial electron transport chain. We also found that Romo1 knockdown inhibited serum deprivation-induced apoptosis. These findings suggest that Romo1-derived ROS play an important role in apoptotic cell death triggered by withdrawal of cell survival factors.     

Activation of the UPR Protects against Cigarette Smoke-induced RPE Apoptosis through Up-Regulation of Nrf2

Recent studies have revealed a role of endoplasmic reticulum (ER) stress-induced unfolded protein response (UPR) in the regulation of RPE cell activity and survival. Herein, we examined the mechanisms by which the UPR modulates apoptotic signaling in human RPE cells challenged with cigarette smoking extract (CSE). Our results show that CSE exposure induced a dose- and time-dependent increase in ER stress markers, enhanced reactive oxygen species (ROS), mitochondrial fragmentation, and apoptosis of RPE cells. These changes were prevented by the anti-oxidant NAC or chemical chaperone TMAO, suggesting a close interaction between oxidative and ER stress in CSE-induced apoptosis. To decipher the role of the UPR, overexpression or down-regulation of XBP1 and CHOP genes was manipulated by adenovirus or siRNA. Overexpressing XBP1 protected against CSE-induced apoptosis by reducing CHOP, p-p38, and caspase-3 activation. In contrast, XBP1 knockdown sensitized the cells to CSE-induced apoptosis, which is likely through a CHOP-independent pathway. Surprisingly, knockdown of CHOP reduced p-eIF2α and Nrf2 resulting in a marked increase in caspase-3 activation and apoptosis. Furthermore, Nrf2 inhibition increased ER stress and exacerbated cell apoptosis, while Nrf2 overexpression reduced CHOP and protected RPE cells. Our data suggest that although CHOP may function as a pro-apoptotic gene during ER stress, it is also required for Nrf2 up-regulation and RPE cell survival. In addition, enhancing Nrf2 and XBP1 activity may help reduce oxidative and ER stress and protect RPE cells from cigarette smoke-induced damage.     

The Homer-1 protein Ania-3 interacts with the plasma membrane calcium pump

The Homer family of scaffold proteins couples NMDA receptors to metabotropic glutamate receptors and links extracellular signals to calcium release from intracellular stores. Ania-3 is a member of the Homer family and is rapidly inducible in brain in response to diverse stimuli. Here, we report the identification of the plasma membrane Ca2+ ATPase (PMCA) as a novel Ania-3/Homer-associated protein. Ania-3/Homer interacts with the b-splice forms of all PMCAs (PMCA1b, 2b, 3b, and 4b) via their PDZ domain-binding COOH-terminal tail. Ectopically expressed Ania-3 colocalized with the PMCA at the plasma membrane of polarized MDCK epithelial cells, and endogenous Ania-3/Homer and PMCA2 are co-expressed in the soma and dendrites of primary rat hippocampal neurons. The interaction between Ania-3/Homer and PMCAs may represent a novel mechanism by which local calcium signaling and hence synaptic function can be modulated in neurons.     

Scaffolding protein Homer1a protects against NMDA-induced neuronal injury

Excessive N-methyl-D-aspartate receptor (NMDAR) activation and the resulting activation of neuronal nitric oxide synthase (nNOS) cause neuronal injury. Homer1b/c facilitates NMDAR-PSD95-nNOS complex interactions, and Homer1a is a negative competitor of Homer1b/c. We report that Homer1a was both upregulated by and protected against NMDA-induced neuronal injury in vitro and in vivo. The neuroprotective activity of Homer1a was associated with NMDA-induced Ca²⁺ influx, oxidative stress and the resultant downstream signaling activation. Additionally, we found that Homer1a functionally regulated NMDAR channel properties in neurons, but did not regulate recombinant NR1/NR2B receptors in HEK293 cells. Furthermore, we found that Homer1a detached the physical links among NR2B, PSD95 and nNOS and reduced the membrane distribution of NMDAR. NMDA-induced neuronal injury was more severe in Homer1a homozygous knockout mice (KO, Homer1a^−/−) when compared with NMDA-induced neuronal injury in wild-type mice (WT, Homer1a^+/+). Additionally, Homer1a overexpression in the cortex of Homer1a^−/− mice alleviated NMDA-induced neuronal injury. These findings suggest that Homer1a may be a key neuroprotective endogenous molecule that protects against NMDA-induced neuronal injury by disassembling NR2B-PSD95-nNOS complexes and reducing the membrane distribution of NMDARs.Glutamate (Glu) acts on glutamate receptors, such as the N-methyl-D-aspartate receptor (NMDAR), and leads to neuronal hyper-excitability and death in a dose-dependent manner.¹ NMDAR activation induces Ca²⁺ influx and specifically activates neuronal nitric oxide synthase (nNOS) and downstream signaling pathways.^{2, 3, 4} Ca²⁺ influx is involved in glutamate-induced apoptosis caused by the activation of apoptosis-related signaling pathways, mitochondrial dysfunction and ROS induction.^{3, 4} Additionally, nNOS has been reported to contribute to NMDA-induced excitotoxicity.^{5, 6} Considering that direct NMDAR inhibition has not yet demonstrated favorable efficacy in most clinic trails and further considering the remarkable role of nNOS in NMDA-induced neuronal death,⁷ measures that can effectively protect neurons from NMDA-induced neuronal injury are urgently needed and represent a worthwhile research goal.Homer proteins belong to the postsynaptic density (PSD) family and consist of two major groups: the short-form Homer proteins (Homer1a and Ania3) and the long-form Homer proteins (Homer1b/c, Homer2 and Homer3).⁸ Homer1b/c has a conserved N-terminal Ena/VASP homology 1 domain and binds to group I metabotropic glutamate receptors (mGluRs), inositol triphosphate receptors and Shank family proteins.^{9, 10, 11, 12} Homer1b/c regulates surface receptor expression,^{13, 14} clustering,¹⁵ transient receptor potential family channels and mGluRs coupled to ion channels.^{10, 16, 17, 18, 19} Additionally, because of its C-terminal coiled-coil (CC) domains, Homer1b/c can self-multimerize, form multiprotein complexes and facilitate signal transduction to downstream pathways. Homer1a, which lacks the CC domain, is believed to compete with constitutive Homer1b/c and disrupt the association of multiple Homer1b/c complexes.Notably, Homer1b/c can interact with the Glu-induced Ca²⁺ influx pathway by binding to Shank, a NMDAR complex adaptor protein (NMDAR-PSD95-GKAP-Shank-Homer1b/c).^{12, 20} Furthermore, Homer1a also interacts with Shank, NMDA, nNOS and other Homer1b/c target proteins. Homer1a has a negative regulatory role by physically replacing certain target proteins, and is involved in the regulation of a variety of cellular and molecular functions in neurological diseases.^{21, 22, 23, 24, 25} Nevertheless, the mechanisms of action and associations between Homer1a and NMDA-induced neuronal injury have not yet been studied. Here, we aimed to investigate the possible neuroprotective effects of Homer1a and explore the mechanisms underlying Homer1a activity in NMDA-induced neuronal injury.     

Homer 1b regulates the trafficking of group I metabotropic glutamate receptors.

The molecular basis for glutamate receptor trafficking to the plasma membrane is not understood. In the present study, we demonstrate that Homer 1b (H1b), a constitutively expressed splice form of the immediate early gene product Homer (now termed Homer 1a) regulates the trafficking and surface expression of group I metabotropic glutamate receptors. H1b inhibits surface expression of the metabotropic glutamate receptor mGluR5 in heterologous cells, causing mGluR5 to be retained in the endoplasmic reticulum (ER). In contrast, mGluR5 alone or mGluR5 coexpressed with Homer 1a successfully travels through the secretory pathway to the plasma membrane. In addition, point mutations that disrupt mGluR5 binding to H1b eliminate ER retention of mGluR5, demonstrating that H1b affects metabotropic receptor localization via a direct protein-protein interaction. Electron microscopic analysis reveals that the group I metabotropic receptor mGluR1alpha is significantly enriched in the ER of Purkinje cells, suggesting that a similar mechanism may exist in vivo. Because H1b is found in dendritic spines of neurons, local retention of metabotropic receptors within dendritic ER provides a potential mechanism for regulating synapse-specific expression of group I metabotropic glutamate receptors.     

Postsynaptic scaffold protein Homer 1a protects against traumatic brain injury via regulating group I metabotropic glutamate receptors

Traumatic brain injury (TBI) produces excessive glutamate, leading to excitotoxicity via the activation of glutamate receptors. Postsynaptic density scaffold proteins have crucial roles in mediating signal transduction from glutamate receptors to their downstream mediators. Therefore, studies on the mechanisms underlying regulation of excitotoxicity by scaffold proteins can uncover new treatments for TBI. Here, we demonstrated that the postsynaptic scaffold protein Homer 1a was neuroprotective against TBI in vitro and in vivo, and this neuroprotection was associated with its effects on group I metabotropic glutamate receptors (mGluRs). Upon further study, we found that Homer 1a mainly affected neuronal injury induced by mGluR1 activation after TBI and also influenced mGluR5 function when its activity was restored. The ability of Homer 1a to disrupt mGluR-ERK signaling contributed to its ability to regulate the functions of mGluR1 and mGluR5 after traumatic injury. Intracellular Ca²⁺ and PKC were two important factors involved in the mediation of mGluR-ERK signaling by Homer 1a. These results define Homer 1a as a novel endogenous neuroprotective agent against TBI.