John Pablo

Cyanobacterial neurotoxin BMAA in ALS and Alzheimer's disease.

Objective –  The aim of this study was to screen for and quantify the neurotoxic amino acid β‐N‐methylamino‐l‐alanine (BMAA) in a cohort of autopsy specimens taken from Alzheimer’s disease (AD), amyotrophic lateral sclerosis (ALS), Huntington’s disease (HD), and non‐neurological controls. BMAA is produced by cyanobacteria found in a variety of freshwater, marine, and terrestrial habitats. The possibility of geographically broad human exposure to BMAA had been suggested by the discovery of BMAA in brain tissues of Chamorro patients with ALS/Parkinsonism dementia complex from Guam and more recently in AD patients from North America. These observations warranted an independent study of possible BMAA exposures outside of the Guam ecosystem.

Dopamine transport function is elevated in cocaine users.

Dopaminergic transmission has been suggested to be a primary mechanism mediating reinforcement, withdrawal and craving associated with psychostimulant addiction. Pyschostimulants attenuate dopamine transporter (DAT) clearance efficiency, resulting in a net increase in synaptic dopamine levels. Re‐uptake rate is determined by the number of functional DAT molecules at the membrane surface. Previous in vivo imaging studies in humans and in vitro studies in post‐mortem human brain have demonstrated that chronic cocaine abuse results in a neuroadaptive increase in DAT‐binding site density in the limbic striatum. Whether this increase in DAT availability represents an increase in the functional activity of the transporter is unknown. Here, we present evidence that DAT function is elevated by chronic cocaine abuse. The effect of increasing post‐mortem interval on the functional viability of synaptosomes was modeled in the baboon brain. Baboon brains sampled under conditions similar to human brain autopsies yielded synaptosomal preparations that were viable up to 24 h post‐mortem. Dopamine (DA) uptake was elevated twofold in the ventral striatum from cocaine users as compared to age‐matched drug‐free control subjects. The levels of [3H]DA uptake were not elevated in victims of excited cocaine delirium, who experienced paranoia and marked agitation prior to death. In keeping with the increase in DAT function, [3H]WIN 35,428 binding was increased in the cocaine users, but not in the victims of excited delirium. These results demonstrate that DA uptake function assayed in cryopreserved human brain synaptosomes is a suitable approach for testing hypotheses of the mechanisms underlying human brain disorders and for studying the actions of addictive drugs in man.

Characterization and Distribution of Transferrin Receptors in the Rat Brain

Abstract: Transferrin receptors were characterized with 125Iferrotransferrin on membrane fractions prepared from the rodent forebrain. The distribution of transferrin receptors in the rat brain was investigated further by in vitro autoradiography. Saturation binding analysis revealed an apparent single class of sites with a dissociation constant of 2 nM and a binding site density of 15 pmol/g. The Hill coefficient derived from these data was 1.05. indicating the absence of cooperativity and that 125I‐ferrotransferrin binds to a single class of sites. Estimates of the kinetically determined Kd for forebrain membranes were within the 2–4 nM range, in agreement with the equilibrium measurements. Apotransferrin and ferrotransferrin competitively displaced the binding of 125I‐ferrotransferrin, while ferritin, albumin, and cytochrome c failed to compete for the binding site. Ceruloplasmin, the copper transport protein, was a weak inhibitor of 125I‐ferrotransferrin binding. Autoradiographic localization studies demonstrate a heterogeneous distribution of transferrin receptors in the rat brain. Transferrin receptor densities were markedly elevated over the cerebral cortex and the hippocampus. Moderate to high 125I‐ferrotransferrin binding was also apparent throughout areas involved in motor functions, including the caudate‐putamen, the nucleus accumbens, the substantia nigra, the red nucleus, and the cerebellum.

Gene expression in human hippocampus from cocaine abusers identifies genes which regulate extracellular matrix remodeling.

The chronic effects of cocaine abuse on brain structure and function are blamed for the inability of most addicts to remain abstinent. Part of the difficulty in preventing relapse is the persisting memory of the intense euphoria or cocaine “rush”. Most abused drugs and alcohol induce neuroplastic changes in brain pathways subserving emotion and cognition. Such changes may account for the consolidation and structural reconfiguration of synaptic connections with exposure to cocaine. Adaptive hippocampal plasticity could be related to specific patterns of gene expression with chronic cocaine abuse. Here, we compare gene expression profiles in the human hippocampus from cocaine addicts and age-matched drug-free control subjects. Cocaine abusers had 151 gene transcripts upregulated, while 91 gene transcripts were downregulated. Topping the list of cocaine-regulated transcripts was RECK in the human hippocampus (FC = 2.0; p<0.05). RECK is a membrane-anchored MMP inhibitor that is implicated in the coordinated regulation of extracellular matrix integrity and angiogenesis. In keeping with elevated RECK expression, active MMP9 protein levels were decreased in the hippocampus from cocaine abusers. Pathway analysis identified other genes regulated by cocaine that code for proteins involved in the remodeling of the cytomatrix and synaptic connections and the inhibition of blood vessel proliferation (PCDH8, LAMB1, ITGB6, CTGF and EphB4). The observed microarray phenotype in the human hippocampus identified RECK and other region-specific genes that may promote long-lasting structural changes with repeated cocaine abuse. Extracellular matrix remodeling in the hippocampus may be a persisting effect of chronic abuse that contributes to the compulsive and relapsing nature of cocaine addiction.

Ibogaine: Complex Pharmacokinetics, Concerns for Safety, and Preliminary Efficacy Measures

Ibogaine is an indole alkaloid found in the roots of Tabernanthe Iboga (Apocynaceae family), a rain forest shrub that is native to western Africa. Ibogaine is used by indigenous peoples in low doses to combat fatigue, hunger and thirst, and in higher doses as a sacrament in religious rituals. Members of American and European addict self‐help groups have claimed that ibogaine promotes long‐term drug abstinence from addictive substances, including psychostimulants and opiates. Anecdotal reports attest that a single dose of ibogaine eliminates opiate withdrawal symptoms and reduces drug craving for extended periods of time. The purported efficacy of ibogaine for the treatment of drug dependence may be due in part to an active metabolite. The majority of ibogaine biotransformation proceeds via CYP2D6, including the O‐demethylation of ibogaine to 12‐hydroxyibogamine (noribogaine). Blood concentration‐time effect profiles of ibogaine and noribogaine obtained for individual subjects after single oral dose administrations demonstrate complex pharmacokinetic profiles. Ibogaine has shown preliminary efficacy for opiate detoxification and for short‐term stabilization of drug‐dependent persons as they prepare to enter substance abuse treatment. We report here that ibogaine significantly decreased craving for cocaine and heroin during inpatient detoxification. Self‐reports of depressive symptoms were also significantly lower after ibogaine treatment and at 30 days after program discharge. Because ibogaine is cleared rapidly from the blood, the beneficial aftereffects of the drug on craving and depressed mood may be related to the effects of noribogaine on the central nervous system.

Cyanobacterial Neurotoxin β-N-Methylamino-L-alanine (BMAA) in Shark Fins

Sharks are among the most threatened groups of marine species. Populations are declining globally to support the growing demand for shark fin soup. Sharks are known to bioaccumulate toxins that may pose health risks to consumers of shark products. The feeding habits of sharks are varied, including fish, mammals, crustaceans and plankton. The cyanobacterial neurotoxin β-N-methylamino-L-alanine (BMAA) has been detected in species of free-living marine cyanobacteria and may bioaccumulate in the marine food web. In this study, we sampled fin clips from seven different species of sharks in South Florida to survey the occurrence of BMAA using HPLC-FD and Triple Quadrupole LC/MS/MS methods. BMAA was detected in the fins of all species examined with concentrations ranging from 144 to 1836 ng/mg wet weight. Since BMAA has been linked to neurodegenerative diseases, these results may have important relevance to human health. We suggest that consumption of shark fins may increase the risk for human exposure to the cyanobacterial neurotoxin BMAA.

Chapter 8 Ibogaine in the treatment of heroin withdrawal

Publisher Summary The chapter presents a study on the role of ibogaine in the treatment of heroin withdrawal. Pharmacological treatments for heroin addiction currently employ two treatment strategies: detoxification followed by drug-free abstinence or maintenance treatment with an opioid agonist. Because agonist maintenance with methadone usually has the goal of eventual detoxification to a drug-free state, the use of medications to facilitate this transition is a clinically important treatment strategy. Anecdotal reports suggest that ibogaine has promise as an alternative medication approach for making this transition. Ibogaine has an added benefit to other detoxification strategies in that the treatment experience seems to bolster the patients own motivational resources for change. Ibogaine is a drug with complex pharmacokinetics and an uncertain mechanism of action with regards to its alleged efficacy for the treatment of opiate dependence. Ibogaine is metabolized to noribogaine, which has a pharmacological profile that is different from that of the parent drug. The chapter presents that ibogaine is effective in blocking opiate withdrawal, providing an alternative approach for opiate-dependent patients who have failed other conventional treatments. Identifying noribogaines mechanism of action may explain the way ibogaine promotes rapid detoxification from opiates after only a single dose. Ibogaine, like most central nervous system (CNS) drugs, is highly lipophilic and is subject to extensive biotransformation. The Opiate-Symptom Checklist (OP-SCL) was developed for the present study as a subtle assessment of withdrawal symptoms.

Pharmacological screen for activities of 12=hydroxyibogamine: a primary metabolite of the indole alkaloid ibogaine

The purported efficacy of ibogaine for the treatment of drug dependence may be due in part to an active metabolite. Ibogaine undergoes first pass metabolism and isO-demethylated to 12-hydroxyibogamine (12-OH ibogamine). Radioligand binding assays were conducted to identify the potency and selectivity profiles for ibogaine and 12-OH ibogamine. A comparison of 12-OH ibogamine to the primary molecular targets identified previously for ibogaine demonstrates that the metabolite has a binding profile that is similar, but not identical to the parent drug. Both ibogaine and 12-OH ibogamine demonstrated the highest potency values at the cocaine recognition site on the 5-HT transporter. The same rank order (12-OH ibogamine > ibogaine), but lower potencies were observed for the [3H]paroxetine binding sites on the 5-HT transporter. Ibogaine and 12-OH ibogamine were equipotent at vesicular monoamine and dopamine transporters. The metabolite demonstrated higher affinity at the kappa-1 receptor and lower affinity at the NMDA receptor complex compared to the parent drug. Quantitation of the regional brain levels of ibogaine and 12-OH ibogamine demonstrated micromolar concentrations of both the parent drug and metabolite in rat brain. Drug dependence results from distinct, but inter-related neurochemical adaptations, which underlie tolerance, sensitization and withdrawal. Ibogaine’s ability to alter drug-seeking behavior may be due to combined actions of the parent drug and metabolite at key pharmacological targets that modulate the activity of drug reward circuits.

Distribution and number of transferrin receptors in Parkinson's disease and in MPTP-treated mice

Transferrin is a glycoprotein that functions primarily to deliver iron to the cell. Recent studies suggest that the transferrin receptor mediates the intracellular delivery and transport of iron bound to transferrin in the CNS. Iron-catalyzed free radical generation has been proposed as a possible cause of nigral cell death in Parkinsons disease. Our hypothesis is that abnormal iron handling by the transferrin receptor may contribute to the formation of free radical species which catalyze the lipid peroxidation of nigral cell membranes. We have assessed the number of transferrin receptors on membrane fractions prepared from the human striatum from control subjects and patients with Parkinsons disease. Equilibrium-binding studies demonstrated a reversible, saturable, and high-affinity transferrin binding site (KD = 3 nM) in human brain membranes. Regional binding assays indicate that the number of transferrin receptors in the putamen was reduced significantly in Parkinsons disease. The density of transferrin receptors was unaltered in membranes prepared from the caudate nuclei and the globus pallidus. To address the possibility that transferrin receptors are located on dopaminergic terminals, we have examined the distribution and number of transferrin receptors in the striatum of MPTP-treated mice using in vitro autoradiographic methods. In these experiments, the loss of dopaminergic terminals in the striatum was visualized by differential [3H]mazindol uptake site autoradiography. A marked reduction in the density of both transferrin receptors and [3H]mazindol binding sites was observed in the mouse striatum 7 days post-MPTP treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Multiregional gene expression profiling identifies MRPS6 as a possible candidate gene for Parkinson's disease

Combining large-scale gene expression approaches and bioinformatics may provide insights into the molecular variability of biological processes underlying neurodegeneration. To identify novel candidate genes and mechanisms, we conducted a multiregional gene expression analysis in postmortem brain. Gene arrays were performed utilizing Affymetrix HG U133 Plus 2.0 gene chips. Brain specimens from 21 different brain regions were taken from Parkinsons disease (PD) (n = 22) and normal aged (n = 23) brain donors. The rationale for conducting a multiregional survey of gene expression changes was based on the assumption that if a gene is changed in more than one brain region, it may be a higher probability candidate gene compared to genes that are changed in a single region. Although no gene was significantly changed in all of the 21 brain regions surveyed, we identified 11 candidate genes whose pattern of expression was regulated in at least 18 out of 21 regions. The expression of a gene encoding the mitochondria ribosomal protein S6 (MRPS6) had the highest combined mean fold change and topped the list of regulated genes. The analysis revealed other genes related to apoptosis, cell signaling, and cell cycle that may be of importance to disease pathophysiology. High throughput gene expression is an emerging technology for molecular target discovery in neurological and psychiatric disorders. The top gene reported here is the nuclear encoded MRPS6, a building block of the human mitoribosome of the oxidative phosphorylation system (OXPHOS). Impairments in mitochondrial OXPHOS have been linked to the pathogenesis of PD.