The capsaicin receptor TRPV1 has been widely characterized in the sensory system as a key component of pain and inflammation. A large amount of evidence shows that TRPV1 is also functional in the brain although its role is still debated. Here we report that TRPV1 is highly expressed in microglial cells rather than neurons of the anterior cingulate cortex and other brain areas. We found that stimulation of microglial TRPV1 controls cortical microglia activation per se and indirectly enhances glutamatergic transmission in neurons by promoting extracellular microglial microvesicles shedding. Conversely, in the cortex of mice suffering from neuropathic pain, TRPV1 is also present in neurons affecting their intrinsic electrical properties and synaptic strength. Altogether, these findings identify brain TRPV1 as potential detector of harmful stimuli and a key player of microglia to neuron communication.

Endocannabinoids are neuroprotective in vivo and in vitro, but the mechanisms by which they act are largely unknown. The present study addressed the role of cannabinoid receptors during remote cell death of central neurons in a model that is based on cerebellar lesions. A lesion in one cerebellar hemisphere induced remote cell death and type 2 cannabinoid receptor (CB2R) expression in contralateral precerebellar neurons. Of the selective agonists and antagonists that modulated cannabinoid receptor activity, we found that the CB2R agonist JWH-015 reduced neuronal loss and cytochrome-c release, leading to neurological recovery; these effects were reversed by the selective CB2R antagonist SR144528. Analysis of CB2R-triggered signal transduction demonstrated that in axotomized neurons, CB2R regulated Akt and JNK phosphorylation through a PI3K-dependent pathway, whereas other major signaling routes that are dependent on CB2R, such as ERK1/2 and p38, were not involved. This result was corroborated by the observation that the selective PI3K inhibitor LY294002 blocked the CB2R stimulation effects on neuronal survival as well as Akt and JNK phosphorylation levels. Together, these data demonstrate that axonal damage induces CB2R expression in central neurons and that stimulation of this receptor has a neuroprotective effect that is achieved through PI3K/Akt signaling.

The molecular basis for the control of energy balance by the endocannabinoid anandamide (AEA) is still unclear. Here, we show that murine 3T3-L1 fibroblasts have the machinery to bind, synthesize and degrade AEA, and that their differentiation into adipocytes increases by approximately twofold the binding efficiency of cannabinoid receptors (CBR), and by approximately twofold and approximately threefold, respectively, the catalytic efficiency of the AEA transporter and AEA hydrolase. In contrast, the activity of the AEA synthetase and the binding efficiency of vanilloid receptor were not affected by the differentiation process. In addition, we demonstrate that AEA increases by approximately twofold insulin-stimulated glucose uptake in differentiated adipocytes, according to a CB1R-dependent mechanism that involves nitric oxide synthase, but not lipoxygenase or cyclooxygenase. We also show that AEA binding to peroxisome proliferator-activated receptor-gamma, known to induce differentiation of 3T3-L1 fibroblasts into adipocytes, is not involved in the stimulation of glucose uptake.

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Transglutaminases (TGases) are seven enzymes, cross-linking proteins by ␥-glutamil-⑀-lysine bonds, four of which are expressed in the skin. A new member of the TGase family, TGase 5, has been identified recently, and in the present study we evaluated its role in keratinocyte differentiation in vitro. In addition to the previously described isoforms, full-length TGase 5 and ⌬3 (deletion of exon 3), we identified two new splicing variants, ⌬11 and ⌬3⌬11 (deletion of exons 11 or 3, 11). We expressed full-length TGase 5, ⌬3, ⌬11, and ⌬3⌬11 isoforms in the keratinocyte and baculovirus systems. The results indicate that both full-length TGase 5 and ⌬11 are active, whereas ⌬3 and ⌬3⌬11 have very low activity. Expression studies show that full-length TGase 5 is induced during the early stages of keratinocyte differentiation and is differently regulated in comparison with the other epidermal TGases. Kinetic and in vitro crosslinking experiments indicate that full-length TGase 5 is very efficient in using specific epidermal substrates (loricrin, involucrin, and SPR3). In keratinocyte expression system, TGase 5 isoforms are retained in an intermediate filament-enriched fraction, suggesting its association with insoluble proteins. Indeed, TGase 5 co-localize with vimentin and it is able to cross-link vimentin in vitro.

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The endocannabinoid (eCB) system is widely expressed throughout the central nervous system (CNS) and the functionality of type-1 cannabinoid receptors in neurons is well documented. In contrast, there is little knowledge about type-2 cannabinoid receptors (CB 2 Rs) in the CNS. Here, we show that CB 2 Rs are located intracellularly in layer II/III pyramidal cells of the rodent medial prefrontal cortex (mPFC) and that their activation results in IP 3 Rdependent opening of Ca 2+ -activated Cl − channels. To investigate the functional role of CB 2 R activation, we induced neuronal firing and observed a CB 2 R-mediated reduction in firing frequency. The description of this unique CB 2 R-mediated signaling pathway, controlling neuronal excitability, broadens our knowledge of the influence of the eCB system on brain function.calcium-activated chloride current | firing rate | whole-cell current | voltage clamp | intracellular calcium stores T he endocannabinoid (eCB) system is involved in many functions of the CNS, including executive functions associated with the prefrontal cortex, such as decision-making and working memory (1). The eCB system consists of at least two G proteincoupled receptors (GPCRs), type-1 cannabinoid receptor (CB 1 R) and type-2 cannabinoid receptor (CB 2 R), lipid endogenous ligands (e.g., anandamide and 2-arachidonoylglycerol), and various enzymes responsible for the synthesis and degradation of the endogenous ligands (2-6). CB 1 Rs are among the most abundantly expressed GPCRs in the rat brain and their role, predominantly as presynaptic receptors, in modulating neurotransmission is clearly established (5,7,8). In contrast with CB 1 R, the presence and function of CB 2 R in the brain has long been a matter of debate (9). CB 2 Rs are found primarily in the immune system and were initially regarded as the "peripheral" cannabinoid receptor (10,11). This generally accepted idea is challenged by the description of CNS CB 2 R gene expression in rats and wild-type mice (12-14) and the identification of functional CB 2 Rs on glial cells and neurons (15)(16)(17)(18). In addition to the current view that supports the expression of functional CB 2 Rs in neurons upon brain stress or damage (19), it has been reported that CB 2 Rs could play a role in general CNS physiology (20)(21)(22). These developments emphasize the importance of understanding how CB 2 R activation affects neuronal functioning. To demonstrate the presence of functional CB 2 Rs in the rodent medial prefrontal cortex (mPFC) and to elucidate their functional role, we used Western blotting, a radioactive binding assay, and electrophysiological techniques (whole-cell current and voltage clamp) on layer II/III pyramidal neurons. ResultsFunctional CB 2 Rs in the mPFC. The presence of CB 2 Rs in the rat mPFC was demonstrated by a Western blot performed on homogenated mPFC samples (Fig. 1A). A band of the expected molecular weight for CB 2 R was detected, which was absent when the primary antibody was incubated with immunizing peptide (Fig. 1A)....

The cellular uptake and the intracellular synthesis/degradation of anandamide are crucial steps for controlling its extracellular level and the duration of its activity. Although the biosynthesis and breakdown of anandamide are well understood, little is known about the mechanisms underlying its intracellular transport. Here, we investigated the presence of a potential carrier-mediated trafficking of anandamide within the cytosol, using a biotinylated analog as a tool to catch by affinity chromatography anandamide-interacting proteins. The identity of two of these anandamide-binding proteins, Hsp70 and serum albumin, was determined by mass spectrometry, confirmed by western blotting and confocal microscopy, and further validated through an anandamide-binding assay. These findings suggest that the trafficking of anandamide from the plasma membrane to the internal compartments of a cell occur via a nonvesicular mechanism mediated by cytosolic carriers.