Cannabis use and measurement of cannabinoids in plasma and breast milk of breastfeeding mothers.

BACKGROUND: Information on cannabinoids in breast milk and maternal cannabis use is limited. We quantified cannabinoids in plasma and breast milk of breastfeeding mothers and assessed cannabis use patterns. METHODS: This is a prospective study at a university hospital in a state with legal medical and recreational cannabis. Breast milk and plasma samples along with survey data were collected from volunteers using cannabis in the last 48 h at 2 weeks and 2 months postpartum. RESULTS: Twenty subjects were enrolled. Median age (IQR) was 27 (24-34) years. Median (IQR) instances of cannabis use in the last 7 days were visit 1: 17 (6-29) and visit 2: 23 (15-45). Median (IQR) tetrahydrocannabinol (THC) concentrations were: plasma 3.7 ng/ml (0.8-56.8) and breast milk 27.5 ng/ml (0.8-190.5). Median (IQR) cannabidiol (CBD) concentrations were: plasma 0.6 ng/ml (0.5-6.4) and breast milk 1.2 ng/ml (0.5-17.0). Median (IQR) THC M/P: 7.0 (1.8-34.6) and CBD M/P: 2.6. Median breast milk THC concentration increased from visit 1 to visit 2 by 30.2 ng/ml (95% CI 3.05-69.3 ng/ml). CONCLUSIONS: THC and CBD accumulate in breast milk. Breastfeeding mothers used cannabis frequently and increased use in the early postpartum period. Research on the effects of infant exposure to cannabinoids in breast milk is urgently needed. IMPACT: Cannabis use is increasing in the general population and many nursing mothers use cannabis. THC has been previously detected in breast milk but little is known on how it concentrates relative to plasma. Data on cannabinoids other than THC, reasons for cannabis use, and patterns of use in breastfeeding women are also limited. We detected THC and CBD in breast milk. Both concentrate in breast milk relative to plasma. We showed that breastfeeding mothers increased cannabis use in the weeks after childbirth. Further research is needed to evaluate infant exposure to cannabinoids via breast milk and effects on infant health.

Consensus clinical management guideline for beta-propeller protein-associated neurodegeneration.

This review provides recommendations for the evaluation and management of individuals with beta-propeller protein-associated neurodegeneration (BPAN). BPAN is one of several neurodegenerative disorders with brain iron accumulation along with pantothenate kinase-associated neurodegeneration, PLA2G6-associated neurodegeneration, mitochondrial membrane protein-associated neurodegeneration, fatty acid hydroxylase-associated neurodegeneration, and COASY protein-associated neurodegeneration. BPAN typically presents with global developmental delay and epilepsy in childhood, which is followed by the onset of dystonia and parkinsonism in mid-adolescence or adulthood. BPAN is an X-linked dominant disorder caused by pathogenic variants in WDR45, resulting in a broad clinical phenotype and imaging spectrum. This review, informed by an evaluation of the literature and expert opinion, discusses the clinical phenotype and progression of the disease, imaging findings, epilepsy features, and genetics, and proposes an approach to the initial evaluation and management of disease manifestations across the life span in individuals with BPAN. What this paper adds The complex epilepsy profile of beta-propeller protein-associated neurodegeneration (BPAN) often resolves in adolescence. The treatment for an individual with BPAN is supportive, with attention to sleep disorders, complex epilepsy, and behavioral problems. Individuals with BPAN have shifting needs throughout their life span requiring multidisciplinary care.

Surgical Resection of Focal Cortical Dysplasia in a Neonate with Novel TSC1 Mutation Leading to Resolution of Refractory Seizures: Case Report.

We describe a neonate presenting on first day of life with refractory seizures secondary to a single, large area of focal cortical dysplasia (FCD) who underwent surgical resection at age 3 weeks leading to resolution of seizure activity and dramatic improvement in developmental trajectory. Surgical intervention for epilepsy is infrequently offered for neonates, often reserved only for those with catastrophic presentations. This case demonstrates that surgical intervention can be safe and efficacious in neonates for pharmaco-resistant seizures associated with a focal lesion. Rapid whole exome sequencing in this case yielded a germline novel de novo TSC1 mutation, leading to a genetic diagnosis of tuberous sclerosis complex (TSC). Our patient demonstrates an atypical neonatal presentation of TSC. Limited data is available for those with isolated FCD in TSC; this is the first reported case in a neonate.

Magnetic resonance imaging-guided laser-induced thermal therapy for functional hemispherotomy in a child with refractory epilepsy and multiple medical comorbidities.

Magnetic resonance imaging-guided laser-induced thermal therapy (MRgLITT) is a minimally invasive surgical approach increasingly employed for precise targeted ablation of epileptogenic brain foci. Recent reports have described corpus callosotomy using MRgLITT, though its application in more extensive functional disconnections has not been documented. Here, the authors detail its use in achieving a palliative hemispherotomy in a 5-year-old with medically refractory hemiclonic seizures following a hemispheric infarction, highlighting a novel use of this surgical technique. In this particular case, open craniotomy was deemed high risk given the multiple medical comorbidities including congenital cardiac disease and end-stage renal failure. MRgLITT was considered an alternative approach with a lower risk for periprocedural hemodynamic perturbations. The patient tolerated the procedure well, attaining an Engel class IB outcome at 16 months' follow-up. This suggests that MRgLITT may be an alternative approach to an open hemispherectomy, particularly in cases in which multiple comorbidities pose significant risks and preclude an open procedure.

Systems approach to explore components and interactions in the presynapse.

The application of proteomic techniques to neuroscientific research provides an opportunity for a greater understanding of nervous system structure and function. As increasing amounts of neuroproteomic data become available, it is necessary to formulate methods to integrate these data in a meaningful way to obtain a more comprehensive picture of neuronal subcompartments. Furthermore, computational methods can be used to make biologically relevant predictions from large proteomic data sets. Here, we applied an integrated proteomics and systems biology approach to characterize the presynaptic (PRE) nerve terminal. For this, we carried out proteomic analyses of presynaptically enriched fractions, and generated a PRE literature-based protein-protein interaction network. We combined these with other proteomic analyses to generate a core list of 117 PRE proteins, and used graph theory-inspired algorithms to predict 92 additional components and a PRE complex containing 17 proteins. Some of these predictions were validated experimentally, indicating that the computational analyses can identify novel proteins and complexes in a subcellular compartment. We conclude that the combination of techniques (proteomics, data integration, and computational analyses) used in this study are useful in obtaining a comprehensive understanding of functional components, especially low-abundance entities and/or interactions in the PRE nerve terminal.

Clathrin assembly protein AP180 and CALM differentially control axogenesis and dendrite outgrowth in embryonic hippocampal neurons.

Emerging data suggest that, much like epithelial cells, the polarized growth of neurons requires both the secretory and endocytic pathways. The clathrin assembly proteins AP180 and CALM (clathrin assembly lymphoid myeloid protein) are known to be involved in clathrin-mediated endocytosis, but their roles in mammalian neurons and, in particular, in developmental processes before synaptogenesis are unknown. Here we provide evidence that AP180 and CALM play critical roles in establishing the polarity and controlling the growth of axons and dendrites in embryonic hippocampal neurons. Knockdown of AP180 primarily impairs axonal development, whereas reducing CALM levels results in dendritic dystrophy. Conversely, neurons that overexpress AP180 or CALM generate multiple axons. Ultrastructural analysis shows that CALM affiliates with a wider range of intracellular trafficking organelles than does AP180. Functional analysis shows that endocytosis is reduced in both AP180-deficient and CALM-deficient neurons. Additionally, CALM-deficient neurons show disrupted secretory transport. Our data demonstrate previously unknown functions for AP180 and CALM in intracellular trafficking that are essential in the growth of neurons.

Partially overlapping distribution of epsin1 and HIP1 at the synapse: analysis by immunoelectron microscopy.

Synapses of neurons use clathrin-mediated endocytic pathways for recycling of synaptic vesicles and trafficking of neurotransmitter receptors. Epsin 1 and huntingtin-interacting protein 1 (HIP1) are endocytic accessory proteins. Both proteins interact with clathrin and the AP2 adaptor complex and also bind to the phosphoinositide-containing plasma membrane via an epsin/AP180 N-terminal homology (ENTH/ANTH) domain. Epsin1 and HIP1 are found in neurons; however, their precise roles in synapses remain largely unknown. Using immunogold electron microscopy, we examine and compare the synaptic distribution of epsin1 and HIP1 in rat CA1 hippocampal synapse. We find that epsin1 is located across both sides of the synapse, whereas HIP1 displays a preference for the postsynaptic compartment. Within the synaptic compartments, espin1 is distributed similarly throughout, whereas postsynaptic HIP1 is concentrated near the plasma membrane. Our results suggest a dual role for epsin1 and HIP1 in the synapse: as broadly required factors for promoting clathrin assembly and as adaptors for specific endocytic pathways.

Synaptic distribution of the endocytic accessory proteins AP180 and CALM.

Clathrin-coated vesicles mediate a variety of endocytosis pathways in cells, including endocytic events at synapses. AP180 and clathrin assembly lymphoid myeloid leukemia protein (CALM) are clathrin accessory proteins that promote the formation of clathrin-coated vesicles. Both proteins bind to membrane lipids through their epsin N-terminal homology domains and interact with clathrin and related protein components through their carboxyl-terminal peptide motifs. We examine their neuronal expression and synaptic distribution. We show that both proteins are detected in synapses but demonstrate different distribution patterns. AP180 is located predominantly in presynaptic profiles, whereas CALM is found nonselectively in pre- and postsynaptic profiles and also in perisynaptic processes. These observations reveal an unexpected relationship between AP180 and the presumed non-neuronal homologue CALM. We propose that both AP180 and CALM function as endocytic accessory proteins at synapses, but each may regulate distinct clathrin pathways.

Antibodies to probe endogenous G protein-coupled receptor heteromer expression, regulation, and function.

Over the last decade an increasing number of studies have focused on the ability of G protein-coupled receptors to form heteromers and explored how receptor heteromerization modulates the binding, signaling and trafficking properties of individual receptors. Most of these studies were carried out in heterologous cells expressing epitope tagged receptors. Very little information is available about the in vivo physiological role of G protein-coupled receptor heteromers due to a lack of tools to detect their presence in endogenous tissue. Recent advances such as the generation of mouse models expressing fluorescently labeled receptors, of TAT based peptides that can disrupt a given heteromer pair, or of heteromer-selective antibodies that recognize the heteromer in endogenous tissue have begun to elucidate the physiological and pathological roles of receptor heteromers. In this review we have focused on heteromer-selective antibodies and describe how a subtractive immunization strategy can be successfully used to generate antibodies that selectively recognize a desired heteromer pair. We also describe the uses of these antibodies to detect the presence of heteromers, to study their properties in endogenous tissues, and to monitor changes in heteromer levels under pathological conditions. Together, these findings suggest that G protein-coupled receptor heteromers represent unique targets for the development of drugs with reduced side-effects.

Reduction of AP180 and CALM produces defects in synaptic vesicle size and density.

Clathrin assembly proteins AP180 and CALM regulate the assembly of clathrin-coated vesicles (CCVs), which mediate diverse intracellular trafficking processes, including synaptic vesicle (SV) recycling at the synapse. Although studies using several invertebrate model systems have indicated a role for AP180 in SV recycling, less is known about AP180's or CALM's function in the synapse of mammalian neurons. In this study, we examined synapses of rat hippocampal neurons in which the level of AP180 or CALM had been reduced by RNA interference (RNAi). Using light microscopy, we visualized synaptic puncta in these AP180- or CALM-reduced neurons by co-expressing Synaptophysin::EGFP (Syp::EGFP). We found that neurons with reduced AP180 or reduced CALM had smaller Syp::EGFP-illuminated puncta. Using electron microscopy, we further examined the ultrastructure of the AP180- or CALM-reduced presynaptic terminals. We found that SVs became variably enlarged in both the AP180-reduced and CALM-reduced presynaptic terminals. Lower AP180 and CALM also reduced the density of SVs and the size of SV clusters. Our findings demonstrate that in the presynaptic terminals of hippocampal neurons, AP180 and CALM have a similar role in regulating synaptic vesicles. This overlapping activity may be necessary for high-precision and high-efficacy SV formation during endocytosis.

Dimerization with cannabinoid receptors allosterically modulates delta opioid receptor activity during neuropathic pain.

The diversity of receptor signaling is increased by receptor heteromerization leading to dynamic regulation of receptor function. While a number of studies have demonstrated that family A G-protein-coupled receptors are capable of forming heteromers in vitro, the role of these heteromers in normal physiology and disease has been poorly explored. In this study, direct interactions between CB(1) cannabinoid and delta opioid receptors in the brain were examined. Additionally, regulation of heteromer levels and signaling in a rodent model of neuropathic pain was explored. First we examined changes in the expression, function and interaction of these receptors in the cerebral cortex of rats with a peripheral nerve lesion that resulted in neuropathic pain. We found that, following the peripheral nerve lesion, the expression of both cannabinoid type 1 receptor (CB(1)R) and the delta opioid receptor (DOR) are increased in select brain regions. Concomitantly, an increase in CB(1)R activity and decrease in DOR activity was observed. We hypothesize that this decrease in DOR activity could be due to heteromeric interactions between these two receptors. Using a CB(1)R-DOR heteromer-specific antibody, we found increased levels of CB(1)R-DOR heteromer protein in the cortex of neuropathic animals. We subsequently examined the functionality of these heteromers by testing whether low, non-signaling doses of CB(1)R ligands influenced DOR signaling in the cortex. We found that, in cortical membranes from animals that experienced neuropathic pain, non-signaling doses of CB(1)R ligands significantly enhanced DOR activity. Moreover, this activity is selectively blocked by a heteromer-specific antibody. Together, these results demonstrate an important role for CB(1)R-DOR heteromers in altered cortical function of DOR during neuropathic pain. Moreover, they suggest the possibility that a novel heteromer-directed therapeutic strategy for enhancing DOR activity, could potentially be employed to reduce anxiety associated with chronic pain.

Receptor heteromerization expands the repertoire of cannabinoid signaling in rodent neurons.

A fundamental question in G protein coupled receptor biology is how a single ligand acting at a specific receptor is able to induce a range of signaling that results in a variety of physiological responses. We focused on Type 1 cannabinoid receptor (CB1R) as a model GPCR involved in a variety of processes spanning from analgesia and euphoria to neuronal development, survival and differentiation. We examined receptor dimerization as a possible mechanism underlying expanded signaling responses by a single ligand and focused on interactions between CB1R and delta opioid receptor (DOR). Using co-immunoprecipitation assays as well as analysis of changes in receptor subcellular localization upon co-expression, we show that CB1R and DOR form receptor heteromers. We find that heteromerization affects receptor signaling since the potency of the CB1R ligand to stimulate G-protein activity is increased in the absence of DOR, suggesting that the decrease in CB1R activity in the presence of DOR could, at least in part, be due to heteromerization. We also find that the decrease in activity is associated with enhanced PLC-dependent recruitment of arrestin3 to the CB1R-DOR complex, suggesting that interaction with DOR enhances arrestin-mediated CB1R desensitization. Additionally, presence of DOR facilitates signaling via a new CB1R-mediated anti-apoptotic pathway leading to enhanced neuronal survival. Taken together, these results support a role for CB1R-DOR heteromerization in diversification of endocannabinoid signaling and highlight the importance of heteromer-directed signal trafficking in enhancing the repertoire of GPCR signaling.

Cannabinoid-opioid interactions during neuropathic pain and analgesia.

Opiates and exogenous cannabinoids, both potent analgesics used for the treatment of patients with neuropathic pain, bind to and activate class A G-protein-coupled receptors (GPCRs). Several lines of evidence have recently suggested that opioid and cannabinoid receptors can functionally interact in the central nervous system (CNS). These interactions may be direct, such as through receptor heteromerization, or indirect, such as through signaling cross-talk that includes agonist-mediated release and/or synthesis of endogenous ligands that can activate downstream receptors. Interactions between opioid and cannabinoid receptors may mediate many of the behavioral phenomena associated with the use of these drugs, including the production of acute antinociception and the development of tolerance and cross-tolerance to the antinociceptive effects of opioid and cannabinoid-specific ligands. This review summarizes behavioral, anatomical, and molecular data characterizing these interactions during the development of neuropathic pain and during antinociceptive treatment with these drugs alone or in combination. These studies are critical for understanding how the receptor systems involved in pain relief are altered during acute or chronic pain, and for designing better antinociceptive drug therapies, such as the combined use of opioid and cannabinoid receptor agonists or selective activation of receptor heteromers, that directly target the altered neurophysiology of patients experiencing pain.

Role of antibodies in developing drugs that target G-protein-coupled receptor dimers.

G-protein-coupled receptors are important molecular targets in drug discovery. These receptors play a pivotal role in physiological signaling pathways and are targeted by nearly 50% of currently available drugs. Mounting evidence suggests that G-protein-coupled receptors form dimers, and various studies have shown that dimerization is necessary for receptor maturation, signaling, and trafficking. However, the physiological implications of dimerization in vivo have not been well explored because detection of GPCR dimers in endogenous systems has been a challenging task. One exciting new approach to this challenge is the generation of antibodies against specific G-protein-coupled receptor dimers. Such antibodies could be used as tools for characterization of heteromer-specific function; as reagents for their purification, tissue localization, and regulation in vivo; and as probes for mapping their functional domains. In addition, such antibodies could serve as alternative ligands for G-protein-coupled receptor heteromers. Thus, heteromer-specific antibodies represent novel tools for the exploration and manipulation of G-protein-coupled receptor-dimer pharmacology.

Quantitative neuroproteomics of the synapse.

An emerging way to study neuropsychiatric or neurodegenerative diseases is by performing proteomic analyses of brain tissues. Here, we describe methods used to isolate and identify the proteins associated with a sample of interest, such as the synapse, as well as to compare the levels of proteins in the sample under different conditions. These techniques, involving subcellular fractionation and modern quantitative proteomics using isotopic labels, can be used to understand the organization of neuronal compartments and the regulation of synaptic function under various conditions.

Increased abundance of opioid receptor heteromers after chronic morphine administration.

The mu and delta types of opioid receptors form heteromers that exhibit pharmacological and functional properties distinct from those of homomeric receptors. To characterize these complexes in the brain, we generated antibodies that selectively recognize the mu-delta heteromer and blocked its in vitro signaling. With these antibodies, we showed that chronic, but not acute, morphine treatment caused an increase in the abundance of mu-delta heteromers in key areas of the central nervous system that are implicated in pain processing. Because of its distinct signaling properties, the mu-delta heteromer could be a therapeutic target in the treatment of chronic pain and addiction.

Plasma membrane ion permeability induced by mutant alpha-synuclein contributes to the degeneration of neural cells.

Mutations in alpha-synuclein cause some cases of familial Parkinson's disease (PD), but the mechanism by which alpha-synuclein promotes degeneration of dopamine-producing neurons is unknown. We report that human neural cells expressing mutant alpha-synuclein (A30P and A53T) have higher plasma membrane ion permeability. The higher ion permeability caused by mutant alpha-synuclein would be because of relatively large pores through which most cations can pass non-selectively. Both the basal level of [Ca2+]i and the Ca2+ response to membrane depolarization are greater in cells expressing mutant alpha-synuclein. The membrane permeable Ca2+ chelator BAPTA-AM significantly protected the cells against oxidative stress, whereas neither L-type (nifedipine) nor N-type (omega-conotoxin-GVIA) Ca2+ channel blockers protected the cells. These findings suggest that the high membrane ion permeability caused by mutant alpha-synuclein may contribute to the degeneration of neurons in PD.

Preserved synaptic vesicle recycling in hippocampal neurons in a mouse Alzheimer's disease model.

A recently described triple-transgenic mouse model (3xTg, PS1(M146V), APP(Swe), and tau(P301L)) develops a neuropathology similar to the brains of Alzheimer's disease patients including progressive deposits of plaques and tangles [Neuron 39 (2003) 409]. These mice also show age-related deficits in hippocampal synaptic plasticity that occurs before the development of plaques and tangles. Here we report unchanged synaptic vesicle recycling, as measured by FM1-43 release, in the hippocampal neurons of the 3xTg mice. Expression levels of presynaptic protein synaptophysin and of proteins involved in synaptic vesicle recycling including AP180, dynamin I, and synaptotagmin I also remain unaffected. These data suggest that the synaptic deficits observed in the 3xTg neurons may not arise from the preserved synaptic vesicle recycling.