The type 1 cannabinoid receptor (CB1R) is one of the most abundant G-protein-coupled receptors (GPCRs) in the brain, predominantly localized to axons of GABAergic neurons. Like several other neuronal GPCRs, CB1R displays significant in vitro constitutive activity (i.e., spontaneous activation in the absence of ligand). However, a clear biological role for constitutive GPCR activity is still lacking. This question was addressed by studying the consequences of constitutive activation on the intracellular trafficking of endogenous or transfected CB1Rs in cultured hippocampal neurons using optical and electron microscopy. We found that constitutive activity results in a permanent cycle of endocytosis and recycling, which is restricted to the somatodendritic compartment. Thus, CB1Rs are continuously removed by endocytosis from the plasma membrane in the somatodendritic compartment but not in axons, where CB1Rs accumulate on surface. Blocking constitutive activity by short-term incubation with inverse agonist 1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-4-morpholinyl-1H-pyrazole-3-carboxamide (AM281) results in sequestration of recycled CB1Rs on the somatodendritic plasma membrane. Long-term inhibition of endocytosis by cotransfection of dominant-negative proteins results in impaired axonal polarization of surface-bound CB1Rs. Kinetic analysis shows that the majority of newly synthesized CB1Rs arrive first to the somatodendritic plasma membrane, from where they are rapidly removed by AM281-sensitive constitutive endocytosis before being delivered to axons. Thus, constitutive-activity driven somatodendritic endocytosis is required for the proper axonal targeting of CB1R, representing a novel, conformation-dependent targeting mechanism for axonal GPCRs.

Aggregation of neurotransmitter receptors at pre- and postsynaptic structures is crucial for efficient neuronal communication. In contrast to the wealth of information about postsynaptic specializations, little is known about the molecular organization of presynaptic membrane proteins. We show here that the metabotropic glutamate receptor mGluR7a, which localizes specifically to presynaptic active zones, interacts in vitro and in vivo with PICK1. Coexpression in heterologous systems induces coclustering dependent upon the extreme C terminus of mGluR7a and the PDZ domain of PICK1. mGluR7a and PICK1 localize to excitatory synapses in hippocampal neurons. Furthermore, whereas transfected mGluR7a clusters at presynaptic sites, mGluR7adelta3 lacking the PICK1 binding site targets to axons but does not cluster. These results suggest that PICK1 is a component of the presynaptic machinery involved in mGluR7a aggregation and in modulation of glutamate neurotransmission.

Heterozygous mutations in the GRN gene lead to progranulin (PGRN) haploinsufficiency and cause frontotemporal dementia (FTD), a neurodegenerative syndrome of older adults. Homozygous GRN mutations, on the other hand, lead to complete PGRN loss and cause neuronal ceroid lipofuscinosis (NCL), a lysosomal storage disease usually seen in children. Given that the predominant clinical and pathological features of FTD and NCL are distinct, it is controversial whether the disease mechanisms associated with complete and partial PGRN loss are similar or distinct. We show that PGRN haploinsufficiency leads to NCL-like features in humans, some occurring before dementia onset. Noninvasive retinal imaging revealed preclinical retinal lipofuscinosis in heterozygous GRN mutation carriers. Increased lipofuscinosis and intracellular NCL-like storage material also occurred in postmortem cortex of heterozygous GRN mutation carriers. Lymphoblasts from heterozygous GRN mutation carriers accumulated prominent NCL-like storage material, which could be rescued by normalizing PGRN expression. Fibroblasts from heterozygous GRN mutation carriers showed impaired lysosomal protease activity. Our findings indicate that progranulin haploinsufficiency caused accumulation of NCL-like storage material and early retinal abnormalities in humans and implicate lysosomal dysfunction as a central disease process in GRN-associated FTD and GRN-associated NCL.