Endocannabinoids play central roles in retrograde signaling at a wide variety of synapses throughout the CNS. Although several molecular components of the endocannabinoid system have been identified recently, their precise location and contribution to retrograde synaptic signaling is essentially unknown. Here we show, by using two independent riboprobes, that principal cell populations of the hippocampus express high levels of diacylglycerol lipase ␣ (DGL-␣), the enzyme involved in generation of the endocannabinoid 2-arachidonoyl-glycerol (2-AG). Immunostaining with two independent antibodies against DGL-␣ revealed that this lipase was concentrated in heads of dendritic spines throughout the hippocampal formation. Furthermore, quantification of high-resolution immunoelectron microscopic data showed that this enzyme was highly compartmentalized into a wide perisynaptic annulus around the postsynaptic density of axospinous contacts but did not occur intrasynaptically. On the opposite side of the synapse, the axon terminals forming these excitatory contacts were found to be equipped with presynaptic CB 1 cannabinoid receptors. This precise anatomical positioning suggests that 2-AG produced by DGL-␣ on spine heads may be involved in retrograde synaptic signaling at glutamatergic synapses, whereas CB 1 receptors located on the afferent terminals are in an ideal position to bind 2-AG and thereby adjust presynaptic glutamate release as a function of postsynaptic activity. We propose that this molecular composition of the endocannabinoid system may be a general feature of most glutamatergic synapses throughout the brain and may contribute to homosynaptic plasticity of excitatory synapses and to heterosynaptic plasticity between excitatory and inhibitory contacts.

Fragile X syndrome, the most commonly known genetic cause of autism, is due to loss of the fragile X mental retardation protein, which regulates signal transduction at metabotropic glutamate receptor-5 in the brain. Fragile X mental retardation protein deletion in mice enhances metabotropic glutamate receptor-5-dependent long-term depression in the hippocampus and cerebellum. Here we show that a distinct type of metabotropic glutamate receptor-5-dependent long-term depression at excitatory synapses of the ventral striatum and prefrontal cortex, which is mediated by the endocannabinoid 2-arachidonoyl-sn-glycerol, is absent in fragile X mental retardation protein-null mice. In these mutants, the macromolecular complex that links metabotropic glutamate receptor-5 to the 2-arachidonoyl-sn-glycerol-producing enzyme, diacylglycerol lipase-α (endocannabinoid signalosome), is disrupted and metabotropic glutamate receptor-5-dependent 2-arachidonoyl-sn-glycerol formation is compromised. These changes are accompanied by impaired endocannabinoid-dependent long-term depression. Pharmacological enhancement of 2-arachidonoyl-sn-glycerol signalling normalizes this synaptic defect and corrects behavioural abnormalities in fragile X mental retardation protein-deficient mice. The results identify the endocannabinoid signalosome as a molecular substrate for fragile X syndrome, which might be targeted by therapy.

An unusual protease ␥-secretase requires functional presenilins and cleaves substrates (e.g. amyloid ␤-protein precursor and Notch) with very loose amino acid sequence specificity within the transmembrane region. Here we report that ErbB4, a tyrosine kinase receptor for neuregulins, is a substrate for presenilin-dependent ␥-secretase. Our studies show that constitutive ectodomain shedding of full-length ErbB4 yields the ϳ80-kDa membrane-associated C-terminal fragment (B4-CTF). Subsequent intramembrane cleavage of the B4-CTF was inhibited in the cells devoid of functional presenilins or by treatment of cells with a ␥-secretase inhibitor, leading to enhanced accumulation of B4-CTF. Furthermore, an in vitro ␥-secretase assay demonstrated that the intracellular domain of ErbB4 (B4-ICD) was produced and subsequently released into the soluble fraction in a presenilin-dependent manner. We have also shown that ectopically expressed B4-ICD is localized to the nucleus, suggesting that the presenilin-dependent cleavage of ErbB4 generates the soluble B4-ICD that functions in the nucleus presumably at transcriptional level. Our study indicates that ErbB4 represents a first receptor tyrosine kinase that undergoes intramembrane proteolysis and may mediate a novel signaling function independent of its canonical role as a receptor tyrosine kinase. Our studies also support the idea that presenilins play a generic role in intramembrane cleavage of selected type I membrane proteins.ErbB4 is a type I membrane receptor tyrosine kinase, which belongs to the epidermal growth receptor family and mediates response to multiple growth factors, including neuregulins (reviewed in Refs. 1-3). ErbB4 has been implicated in many important biological and pathological processes, such as cardiovascular, mammary gland, and neural development, as well as malignancy and heart disease (1-3).Presenilins (PS1 and PS2), 1 gene products of the major earlyonset familial Alzheimer's disease genes (reviewed in Refs. 4 -6), are required for the activity of ␥-secretase, an unusual aspartyl protease that cleaves substrates within the predicted transmembrane region (7; reviewed in Refs. 8 -10). Two of the characteristics of ␥-secretase include a lack of requirement for specific amino acid target sequences within the transmembrane domain and a requirement for ectodomain shedding to produce membrane-anchored truncated C-terminal derivatives (10, 11). These observations imply that the presenilins may also be involved in the intramembrane cleavage of other type I membrane proteins. The ␥-secretase cleavage of amyloid ␤-protein precursor (APP) is a critical rate-limiting step toward the production of amyloid ␤-peptide (A␤) in Alzheimer's disease (6). In addition to APP, transmembrane cleavage of Notch, which releases the Lin-12/Notch intracellular domain, plays a pivotal role in cell fate determination (12, 13). Ectopically expressed intracellular domains of APP (AICD) and Notch (NICD) appear to be localized in the nucleus and participate in gene transcription (12-20)...

The generation of biologically active proteins by regulated intramembrane proteolysis is a highly conserved mechanism in cell signaling. Presenilin-dependent ␥-secretase activity is responsible for the intramembrane proteolysis of selected type I membrane proteins, including ␤-amyloid precursor protein (APP) and Notch. A small fraction of intracellular domains derived from both APP and Notch translocates to and appears to function in the nucleus, suggesting a generic role for ␥-secretase cleavage in nuclear signaling. Here we show that the p75 neurotrophin receptor (p75 NTR ) undergoes presenilin-dependent intramembrane proteolysis to yield the soluble p75-intracellular domain. The p75 NTR is a multifunctional type I membrane protein that promotes neurotrophininduced neuronal survival and differentiation by forming a heteromeric co-receptor complex with the Trk receptors. Mass spectrometric analysis revealed that ␥-secretase-mediated cleavage of p75 NTR occurs at a position located in the middle of the transmembrane (TM) domain, which is reminiscent of the amyloid ␤-peptide 40 (A␤40) cleavage of APP and is topologically distinct from the major TM cleavage site of Notch 1. Size exclusion chromatography and co-immunoprecipitation analyses revealed that TrkA forms a molecular complex together with either full-length p75 or membrane-tethered C-terminal fragments. The p75-ICD was not recruited into the TrkAcontaining high molecular weight complex, indicating that ␥-secretase-mediated removal of the p75 TM domain may perturb the interaction with TrkA. Independent of the possible nuclear function, our studies suggest that ␥-secretase-mediated p75 NTR proteolysis plays a role in the formation/disassembly of the p75-TrkA receptor complex by regulating the availability of the p75 TM domain that is required for this interaction.The p75 neurotrophin receptor (p75 NTR

Activation of group I metabotropic glutamate (mGlu) receptors recruits the endocannabinoid system to produce both shortand long-term changes in synaptic strength in many regions of the brain. Although there is evidence that the endocannabinoid 2-arachidonoylglycerol (2-AG) mediates this process, the molecular mechanism underlying 2-AG mobilization remains unclear. In the present study, we used a combination of genetic and targeted lipidomic approaches to investigate the role of the postsynaptic membrane-associated lipase, diacylglycerol lipase type-␣ (DGL-␣), in mGlu receptor-dependent 2-AG mobilization. DGL-␣ overexpression in mouse neuroblastoma Neuro-2a cells increased baseline 2-AG levels. This effect was accompanied by enhanced utilization of the 2-AG precursor 1-stearoyl,2-arachidonoyl-sn-glycerol and increased accumulation of the 2-AG breakdown product arachidonic acid. A similar, albeit less marked response was observed with other unsaturated and polyunsaturated monoacylglycerols, 1,2-diacylglycerols, and fatty acids. Silencing of DGL-␣ by RNA interference elicited lipidomic changes opposite those of DGL-␣ overexpression and abolished group I mGlu receptor-dependent 2-AG mobilization. Coimmunoprecipitation and site-directed mutagenesis experiments revealed that DGL-␣ interacts, via a PPxxF domain, with the coiled-coil (CC)-Homer proteins Homer-1b and Homer-2, two components of the molecular scaffold that enables group I mGlu signaling. DGL-␣ mutants that do not bind Homer maintained their ability to generate 2-AG in intact cells but failed to associate with the plasma membrane. The findings indicate that DGL-␣ mediates group I mGlu receptor-induced 2-AG mobilization. They further suggest that the interaction of CC-Homer with DGL-␣ is necessary for appropriate function of this lipase.