Manuel Guzmán’s research while affiliated with Instituto de Salud Carlos III and other places

The E3 ligase substrate adapter cereblon (CRBN), the primary target of clinical agents thalidomide and lenalidomide, recognizes endogenous substrates bearing the C-terminal cyclic imide modification. Although C-terminal cyclic imides can form spontaneously, an enzyme that regulates the formation of these modifications and thereby promotes a biological pathway connecting substrates to CRBN is unknown. Here, we report that protein carboxymethyltransferase (PCMT1) promotes formation of the C-terminal cyclic imide on C-terminal asparagine residues of CRBN substrates. PCMT1 and CRBN co-regulate the levels of metabolic enzymes glutamine synthetase (GLUL) and inorganic pyrophosphatase 1 (PPA1) in vitro, in cells, and in vivo, and this regulation is associated with the proepileptic phenotype of CRBN knockout mouse models. The discovery of an enzyme that regulates CRBN substrates through the C-terminal cyclic imide modification reveals a previously unknown biological pathway that is perturbed by thalidomide derivatives and provides a biochemical basis for the connection between multiple biological processes and CRBN.

Background Although the formal diagnosis of HD is mainly based on the presence of motor abnormalities, most HD patients develop a decline in cognitive abilities that often precedes motor symptoms. Cognitive decline (CD) molecular mechanisms are still undetermined, and the hippocampus remains an underexplored region. The scarcity of knowledge arises the need for innovative molecular targets and emerge in the absence of CD treatments. Several evidences emphasized hippocampal CB1R (hCB1R) importance in cognitive functions, mostly in GABAergic and glutamatergic neurons. Interestingly, CB1R alteration has been revealed in several neurodegenerative disorders. Consequently, this project proposes CB1R as altered factor leading to CD in HD. Aims In HD male and female mice, we aim to:1) characterize hCB1R expression and function, 2) manipulate hCB1R in GABAergic/glutamatergic populations,3) explore the benefits of CB1R agonist WIN 55,212-2(WIN) for CD. Methods. hCB1R was assessed from pre to post-symptomatic stages in GABAergic/glutatamergic cells. Cognitive performances were evaluated after viral increase of hCB1R in GABAergic/glutamatergic cells. WIN benefits were proved on behavior and synaptic plasticity measuring GABA/glutamatergic transmission and CB1R density. Results hCB1R decreases in HD mice, specifically in GABAergic neurons. GABAergic transmission is altered and viral hCB1R increase in GABAergic cells ameliorate CD in HD mice. WIN treatment rescues CD, ameliorates CB1R levels, dendritic spines and GABAergic transmission in HD mice hippocampus (Figure 1). Conclusions Results showed alteration in the inhibitory/excitatory balance of HD mice and confirm hCB1R as crucial target for CD in HD. CB1R functional activation in GABAergic cells rescues CD in mice and cannabinoid compound assumes benefic role on CD. • Download figure • Open in new tab • Download powerpoint Abstract I028 Figure 1 CB1R agonist WIN rescues normal memory in HD mice

Growth-associated protein of 43 kDa (GAP43) is a key cytoskeleton-associated component of the presynaptic terminal that facilitates neuroplasticity. Downregulation of GAP43 expression has been associated to various psychiatric conditions in humans and evokes hippocampus-dependent memory impairments in mice. Despite the extensive studies conducted on hippocampal GAP43 in past decades, however, very little is known about its roles in modulating the excitatory vs. inhibitory balance in other brain regions. We recently generated conditional knockout mice in which the Gap43 gene was selectively inactivated in either telencephalic glutamatergic neurons (Gap43fl/fl ;Nex1Cre mice, hereafter Glu-GAP43-/- mice) or forebrain GABAergic neurons (Gap43fl/fl ;Dlx5/6Cre mice, hereafter GABA-GAP43-/- mice). Here, we show that Glu-GAP43-/- but not GABA-GAP43-/- mice of either sex show a striking hyperactive phenotype when exposed to a novel environment. This behavioral alteration of Glu-GAP43-/- mice was linked to a selective activation of dorsal-striatum neurons, as well as to an enhanced corticostriatal glutamatergic transmission and an abrogation of corticostriatal endocannabinoid-mediated long-term depression. In line with these observations, GAP43 was abundantly expressed in corticostriatal glutamatergic terminals of wild-type mice. The novelty-induced hyperactive phenotype of Glu-GAP43-/- mice was abrogated by chemogenetically inhibiting corticostriatal afferences with a Gi-coupled "designer receptor exclusively activated by designer drugs" (DREADD), thus further supporting that novelty-induced activity is controlled by GAP43 at corticostriatal excitatory projections. Taken together, these findings show an unprecedented regulatory role of GAP43 in the corticostriatal circuitry and provide a new mouse model with a delimited neuronal-circuit alteration for studying novelty-induced hyperactivity, a phenotypic shortfall that occurs in diverse psychiatric diseases.Significance statement Psychiatric alterations such as attention deficit/hyperactivity disorder, schizophrenia and bipolar disorder pose a significant health and socioeconomic burden to our society. Animal models that recapitulate precise phenotypical traits of those diseases are therefore warranted for developing new therapeutic interventions. Here, we found that mice lacking the protein GAP43 selectively in telencephalic glutamatergic neurons show a robust novelty-induced hyperactive phenotype, a behavioral deficit often associated to psychiatric diseases. These mice exhibit profound alterations in corticostriatal excitatory plasticity and a selective overactivation of dorsal-striatum neurons in response to a novel environment. Our findings thus unveil an important role of GAP43 in corticostriatal function and provide a new animal model with a delimited neuronal-circuit alteration for studying novelty-induced hyperactivity in psychiatric disorders.

Cereblon/CRBN is a substrate-recognition component of the Cullin4A-DDB1-Roc1 E3 ubiquitin ligase complex. Destabilizing mutations in the human CRBN gene cause a form of autosomal recessive non-syndromic intellectual disability (ARNSID) that is modelled by knocking-out the mouse Crbn gene. A reduction in excitatory neurotransmission has been proposed as an underlying mechanism of the disease. However, the precise factors eliciting this impairment remain mostly unknown. Here we report that CRBN molecules selectively located on glutamatergic neurons are necessary for proper memory function. Combining various in vivo approaches, we show that the cannabinoid CB 1 receptor (CB 1 R), a key suppressor of synaptic transmission, is overactivated in CRBN deficiency-linked ARNSID mouse models, and that the memory deficits observed in these animals can be rescued by acute CB 1 R-selective pharmacological antagonism. Molecular studies demonstrated that CRBN interacts physically with CB 1 R and impairs the CB 1 R-G i/o -cAMP-PKA pathway in a ubiquitin ligase-independent manner. Taken together, these findings unveil that CB 1 R overactivation is a driving mechanism of CRBN deficiency-linked ARNSID and anticipate that the antagonism of CB 1 R could constitute a new therapy for this orphan disease.

Mammary gland development occurs primarily in adulthood, undergoing extensive expansion during puberty followed by cycles of functional specialization and regression with every round of pregnancy/lactation/involution. This process is ultimately driven by the coordinated proliferation and differentiation of mammary epithelial cells. However, the endogenous molecular factors regulating these developmental dynamics are still poorly defined. Endocannabinoid signaling is known to determine cell fate-related events during the development of different organs in the central nervous system and the periphery. Here, we report that the endocannabinoid-degrading enzyme fatty acid amide hydrolase (FAAH) plays a pivotal role in adult mammary gland development. Specifically, it is required for luminal lineage specification in the mammary gland, and it promotes hormone-driven secretory differentiation of mammary epithelial cells by controlling the endogenous levels of anandamide and the subsequent activation of cannabinoid CB 1 receptors. Together, our findings shed light on the role of the endocannabinoid system in breast development and point to FAAH as a therapeutic target in milk-production deficits.

Cannabinoids exert pleiotropic effects on the brain by engaging the cannabinoid CB1 receptor (CB1R), a presynaptic metabotropic receptor that regulates key neuronal functions in a highly context-dependent manner. We have previously shown that CB1R interacts with growth-associated protein of 43 kDa (GAP43) and that this interaction inhibits CB1R function on hippocampal excitatory synaptic transmission, thereby impairing the therapeutic effect of cannabinoids on epileptic seizures in vivo. However, the underlying molecular features of this interaction remain unexplored. Here, we conducted mechanistic experiments on HEK293T cells co-expressing CB1R and GAP43 and show that GAP43 modulates CB1R signalling in a strikingly selective manner. Specifically, GAP43 did not affect the archetypical agonist-evoked (i) CB1R/Gi/o protein-coupled signalling pathways, such as cAMP/PKA and ERK, or (ii) CB1R internalization and intracellular trafficking. In contrast, GAP43 blocked an alternative agonist-evoked CB1R-mediated activation of the cytoskeletal-associated ROCK signalling pathway, which relied on the GAP43-mediated impairment of CB1R/Gq/11 protein coupling. GAP43 also abrogated CB1R-mediated ROCK activation in mouse hippocampal neurons, and this process led in turn to a blockade of cannabinoid-evoked neurite collapse. An NMR-based characterization of the CB1R-GAP43 interaction supported that GAP43 binds directly and specifically through multiple amino acid stretches to the C-terminal domain of the receptor. Taken together, our findings unveil a CB1R-Gq/11-ROCK signalling axis that is selectively impaired by GAP43 and may ultimately control neurite outgrowth.