The transient receptor potential canonical (TRPC) channels are Ca2+-permeable, nonselective cation channels with different biological functions, but their roles in brain are largely unknown. Here we report that TRPC6 was localized to excitatory synapses and promoted their formation via a CaMKIV-CREB-dependent pathway. TRPC6 transgenic mice showed enhancement in spine formation, and spatial learning and memory in Morris water maze. These results reveal a previously unknown role of TRPC6 in synaptic and behavioral plasticity.

The canonical transient receptor potential channels (TRPCs) are Ca2+-permeable nonselective cation channels with various physiological functions. Here, we report that TRPC6, a member of the TRPC family, promotes hippocampal neuron dendritic growth. The peak expression of TRPC6 in rat hippocampus was between postnatal day 7 and 14, a period known to be important for maximal dendritic growth. Overexpression of TRPC6 increased phosphorylation of Ca2+/calmodulin-dependent kinase IV (CaMKIV) and cAMP-response-element binding protein (CREB) and promoted dendritic growth in hippocampal cultures. Downregulation of TRPC6 by short hairpin RNA interference against TRPC6 suppressed phosphorylation of both CaMKIV and CREB and impaired dendritic growth. Expressing a dominant-negative form of CaMKIV or CREB blocked the TRPC6-induced dendritic growth. Furthermore, inhibition of Ca2+ influx suppressed the TRPC6 effect on dendritic growth. Finally, in TRPC6 transgenic mice, the phosphorylation of CaMKIV and CREB was enhanced and the dendritic growth was also increased. In conclusion, TRPC6 promoted dendritic growth via the CaMKIV-CREB pathway. Our results thus revealed a novel role of TRPC6 during the development of the central nervous system (CNS).

Brain injury after focal cerebral ischemia, the most common cause of stroke, develops from a series of pathological processes, including excitotoxicity, inflammation, and apoptosis. While NMDA receptors have been implicated in excitotoxicity, attempts to prevent ischemic brain damage by blocking NMDA receptors have been disappointing. Disruption of neuroprotective pathways may be another avenue responsible for ischemic damage, and thus preservation of neuronal survival may be important for prevention of ischemic brain injury. Here, we report that suppression of proteolytic degradation of transient receptor potential canonical 6 (TRPC6) prevented ischemic neuronal cell death in a rat model of stroke. The TRPC6 protein level in neurons was greatly reduced in ischemia via NMDA receptor-dependent calpain proteolysis of the N-terminal domain of TRPC6 at Lys 16 . This downregulation was specific for TRPC6 and preceded neuronal death. In a rat model of ischemia, activating TRPC6 prevented neuronal death, while blocking TRPC6 increased sensitivity to ischemia. A fusion peptide derived from the calpain cleavage site in TRPC6 inhibited degradation of TRPC6, reduced infarct size, and improved behavioral performance measures via the cAMP response element-binding protein (CREB) signaling pathway. Thus, TRPC6 proteolysis contributed to ischemic neuronal cell death, and suppression of its degradation preserved neuronal survival and prevented ischemic brain damage.

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The recent Food and Drug Administration approval of immunogenic oncolytic virus (OV) has opened a new era in the treatment of advanced melanoma; however, approximately 50% of patients with melanoma develop brain metastasis, and currently there are no beneficial treatment options for such patients. To model the progression of metastases seen in patients and to overcome the hurdles of systemic delivery of OV, we developed melanoma brain metastasis models in immunocompromised and immunocompetent mice, and tested the fate and efficacy of oncolytic herpes simplex virus (oHSV)-armed mesenchymal stem cells (MSCs). Using brain-seeking patient-derived melanoma cells and real-time in vivo imaging, we show a widespread distribution of micrometastases and macrometastases in the brain, recapitulating the progression of multifoci metastases seen in patients. We armed MSCs with different oHSV variants (MSC-oHSV) and found that intracarotid administration of MSC-oHSV, but not of purified oHSV alone, effectively tracks metastatic tumor lesions and significantly prolongs the survival of brain tumor-bearing mice. In a syngeneic model of melanoma brain metastasis, a combination of MSC-oHSV and PD-L1 blockade increases IFNγ-producing CD8 + tumor-infiltrating T lymphocytes and results in a profound extension of the median survival of treated animals. This study thus demonstrates the utility of MSCs as OV carriers to disseminated brain lesions, and provides a clinically applicable therapeutic platform to target melanoma brain metastasis.stem cells | oncolytic virus | tumors | metastasis | imaging M elanoma, the most aggressive type of skin cancer, accounts for a large proportion of skin cancer-related deaths (1). Among all cancer types, melanoma has a particularly high propensity to metastasize to the brain, occurring in >50% of all patients with advanced disease. More than 90% of melanoma brain metastases lead to death, and the median survival is 17-22 wk after detection (2-4).Current therapeutic options of chemotherapy, surgery, and radiation have very limited efficacy for patients with melanoma brain metastasis (5-7). These patients either have multiple metastatic lesions or diagnostically challenging asymptomatic lesions, making surgery an inadequate therapeutic option by itself. In addition, the blood-brain barrier (BBB) limits central nervous system (CNS) penetration of systemic therapies, and the negative side effects of radiotherapy (8) pose challenges for the success of existing therapies, contributing to the failure to improve overall patient survival. As such, there is an urgent need for new therapies for melanoma brain metastasis.The development and characterization of preclinical tumor models that authentically recapitulate the clinical disease settings are critical for developing and testing new therapies. Most previous studies have used either subcutaneous (s.c.) injection or intracranial injection of established melanoma lines in mice (9-11), which do not mimic the actual clinical settings of melanoma brain metastasis,...

Tumor cells engineered to express therapeutic agents have shown promise to treat cancer. However, their potential to target cell surface receptors specific to the tumor site and their posttreatment fate have not been explored. We created therapeutic tumor cells expressing ligands specific to primary and recurrent tumor sites (receptor self-targeted tumor cells) and extensively characterized two different approaches using (i) therapy-resistant cancer cells, engineered with secretable death receptor-targeting ligands for "off-the-shelf" therapy in primary tumor settings, and (ii) therapy-sensitive cancer cells, which were CRISPR-engineered to knock out therapy-specific cell surface receptors before engineering with receptor self-targeted ligands and reapplied in autologous models of recurrent or metastatic disease. We show that both approaches allow high expression of targeted ligands that induce tumor cell killing and translate into marked survival benefits in mouse models of multiple cancer types. Safe elimination of therapeutic cancer cells after treatment was achieved by co-engineering with a prodrug-converting suicide system, which also allowed for real-time in vivo positron emission tomography imaging of therapeutic tumor cell fate. This study demonstrates self-tumor tropism of engineered cancer cells and their therapeutic potential when engineered with receptor self-targeted molecules, and it establishes a roadmap toward a safe clinical translation for different cancer types in primary, recurrent, and metastatic settings.

Excitotoxicity induced by NMDA receptor-mediated intracellular Ca 2+ ([Ca 2+ ] i ) overload is a major cause of delayed neuronal death in cerebral ischemia. Transient receptor potential canonical (TRPC) 6 protects neurons from ischemic brain damage. However, the mechanisms by which TRPC6 protects neurons are largely unknown. Here, we reported that TRPC6 suppressed the [Ca 2+ ] i elevation induced by NMDA and protected neurons from excitotoxicity. Over-expressing or down-regulating TRPC6 suppressed or aggravated Ca 2+ overload under excitotoxicity, respectively. TRPC6 protected cultured neurons from damage caused by NMDA toxicity or oxygen glucose deprivation (OGD). Moreover, the infarct volume in TRPC6 transgenic (Tg) mice was smaller than that in wild-type (WT) littermates. The TRPC6 Tg mice had better behavior performance and lower mortality than their WT littermates. Thus, TRPC6 inhibited NMDA receptor-triggered neurotoxicity and protected neurons from ischemic brain damage. Increase in TRPC6 activity could be a potential strategy for stroke prevention and therapy. Keywords: excitotoxicity, focal ischemia, neuroprotection, transgenic mice, TRPC6. Excitotoxicity elicited by excitatory amino acid releasing during ischemia-reperfusion process is a major factor inducing ischemic insults in cerebral stroke (Dirnagl et al. 1999;Lo et al. 2003). Overactivation of NMDA receptors is a key event in excitotoxicity that acutely elevates the [Ca 2+ ] i concentration of neurons in a short time (Sattler et al. 1998;Arundine and Tymianski 2004). This sudden Ca 2+ overload in neurons activates several endonucleases, kinases, and proteases that induce severe damage to cell functions and constructions, and finally leads to cell apoptosis or necrosis (Szydlowska and Tymianski 2010). Furthermore, a wealth of evidence indicates that suppressing the Ca 2+ overload by blocking NMDA receptors can be neuroprotective in various experiment models. However, directly inhibiting NMDA receptors by antagonists for the treatment of stroke could induce severe adverse effects in the central nervous system (CNS) (Smith 2003). Finding endogenous pathways that block NMDA toxicity might provide new approaches for stroke therapy.TRPC proteins belong to the TRP superfamily. They form Ca 2+ -permeable non-selective cation channels in various cell types in mammals. TRPC family has seven members falling into four subgroups based on sequence similarity: TRPC1, TRPC2, TRPC3/6/7, and TRPC4/5. Homo-or hetero-tetramers of these subunits form functional channels (Clapham 2003;Vazquez et al. 2004). TRPC channels have high expression levels in all the regions of the CNS (Riccio et al. 2002) and have various roles, including growth cone guidance (Li et al. 2005), fear memory (Riccio et al. 2009), and neuronal development (Jia et al. 2007;Tai et al. 2008;Zhou et al. 2008). In TRPC family, TRPC6 is notable for its important roles in Received June 27, 2012; revised manuscript received September 26, 2012; accepted September 27, 2012. Address c...

Transient receptor potential canonical (TRPC) channels are Ca(2+)-permeable, nonselective cation channels formed by homomeric or heteromeric complexes of TRPC proteins that contain six transmembrane domains. These channels can be activated through a phospholipase-C-dependent mechanism, making them sensors for environmental cues. Their expression begins early in embryonic days and remains in adulthood. These channels have important roles in the processes of neuronal development, including neural stem cell proliferation, cerebellar granule cell survival, axon path finding, neuronal morphogenesis, and synaptogenesis. In this review, we will discuss functional implications of TRPC channels during brain development.