Cytochrome P450 2D18 in the mammalian brain: Recombinant expression, purification, and characterization of xenobiotic and endobiotic metabolism

The cytochromes P450 (P450) comprise a superfamily of hemoproteins that function in concert with NADPH-cytochrome P450 reductase (P450-reductase) to metabolize both endogenous and exogenous compounds. Many pharmacological agents undergo phase I metabolism by this P450 and P450-reductase monooxygenase system. Phase I metabolism ensures that these highly hydrophobic xenobiotics are made more hydrophilic, and hence easier to extrude from the body. While the majority of phase I metabolism occurs in the liver, metabolism in extrahepatic organ-systems like the intestine, kidney, and brain can have important roles in drug metabolism and/or efficacy. ^ While P450-mediated phase I metabolism has been well studied, investigators have only recently begun to elucidate what physiological roles P450 may have. One way to approach this question is to study P450s that are highly or specifically expressed in extrahepatic tissues. In this project I have studied the role of a recently cloned P450 family member, P450 2D18, that was previously shown to be expressed in the rat brain and kidney, but not in the liver. To this end, I have used the baculovirus expression system to over-express recombinant P450 2D18 and purified the functional enzyme using nickel and hydroxylapatite chromatography. SDS-PAGE analysis indicated that the enzyme was purified to electrophoretic homogeneity and Western analysis showed cross-reactivity with rabbit anti-human P450 2D6. Carbon monoxide difference spectra indicated that the purified protein contained no denatured P450 enzyme; this allowed for further characterization of the substrates and metabolites formed by P450 2D18-mediated metabolism. ^ Because P450 2D18 is expressed in brain, we characterized the activity toward several psychoactive drugs including the antidepressants imipramine and desipramine, and the anti-psychotic drugs chlorpromazine and haloperidol. P450 2D18 preferentially catalyzed the N-demethylation of imipramine, desipramine, and chlorpromazine. This is interesting given the fact that other P450 isoforms form multiple metabolites from such compounds. This limited metabolic profile might suggest that P450 2D18 has some unique function, or perhaps a role in endobiotic metabolism. ^ Further analysis of possible endogenous substrates for P450 2D18 led to the identification of dopamine and arachidonic acid as substrates. It was shown that P450 2D18 catalyzes the oxidation of dopamine to aminochrome, and that the enzyme binds dopamine with an apparent KS value of 678 μM, a value well within reported dopamine concentration in brain dopaminergic systems. Further, it was shown that P450 2D18 binds arachidonic acid with an apparent KS value of 148 μM, and catalyzes both the ω-hydroxylation and epoxygenation of arachidonic acid to metabolites that have been shown to have vasoactive properties in brain, kidney, and heart tissues. These data provide clues for endogenous roles of P450 within the brain, and possible involvement in the pathogenesis of Parkinson's disease. ^

THE EFFECT OF GABAMIMETICS ON NEUROTRANSMITTER RECEPTOR BINDING AND FUNCTION IN RAT BRAIN

Gamma-aminobutyric acid (GABA) is a major inhibitory neurotransmitter in the central nervous system and alterations in central GABAergic transmission may contribute to the symptoms of a number of neurological and psychiatric disorders. Because of this relationship, numerous laboratories are attempting to develop agents which will selectively enhance GABA neurotransmission in brain. Due to these efforts, several promising compounds have recently been discovered. Should these drugs prove to be clinically effective, they will be used to treat chronic neuropsychiatric disabilities and, therefore, will be administered for long periods of time. Accordingly, the present investigation was undertaken to determine the neurochemical consequences of chronic activation of brain GABA systems in order to better define the therapeutic potential and possible side-effect liability of GABAmimetic compounds.^ Chronic (15 day) administration to rats of low doses of amino-oxyacetic acid (AOAA, 10 mg/kg, once daily), isonicotinic acid hydrazide (20 mg/kg, b.i.d.), two non-specific inhibitors of GABA-T, the enzyme which catabolizes GABA in brain, or (gamma)-acetylenic GABA (10 mg/kg, b.i.d.) a catalytic inhibitor of this enzyme, resulted in a significant elevation of brain and CSF GABA content throughout the course of treatment. In addition, chronic administration of these drugs, as well as the direct acting GABA receptor agonists THIP (8 mg/kg, b.i.d.) or kojic amine (18 mg/kg, b.i.d.) resulted in a significant increase in dopamine receptor number and a significant decrease in GABA receptor number in the corpus striatum of treated animals as determined by standard in vitro receptor binding techniques. Changes in the GABA receptor were limited to the corpus striatum and occurred more rapidly than did alterations in the dopamine receptor. The finding that dopamine-mediated stereotypic behavior was enhanced in animals treated chronically with AOAA suggested that the receptor binding changes noted in vitro have some functional consequence in vitro.^ Coadministration of atropine (a muscarinic cholinergic receptor antagonist) blocked the GABA-T inhibitor-induced increase in striatal dopamine receptors but was without effect on receptor alterations seen following chronic administration of direct acting GABA receptor agonists. Atropine administration failed to influence the drug-induced decreases in striatal GABA receptors.^ Other findings included the discovery that synaptosomal high affinity ('3)H-choline uptake, an index of cholinergic neuronal activity, was significantly increased in the corpus striatum of animals treated acutely, but not chronically, with GABAmimetics.^ It is suggested that the dopamine receptor supersensitivity observed in the corpus striatum of animals following long-term treatment with GABAmimetics is a result of the chronic inhibition of the nigrostriatal dopamine system by these drugs. Changes in the GABA receptor, on the other hand, are more likely due to a homospecific regulation of these receptors. An hypothesis based on the different sites of action of GABA-T inhibitors vis-a-vis the direct acting GABA receptor agonists is proposed to account for the differential effect of atropine on the response to these drugs.^ The results of this investigation provide new insights into the functional interrelationships that exist in the basal ganglia and suggest that chronic treatment with GABAmimetics may produce extrapyramidal side-effects in man. In addition, the constellation of neurochemical changes observed following administration of these drugs may be a useful guide for determining the GABAmimetic properties of neuropharmacological agents. ^

Actigraphic assessment of periodic leg movements in patients with restless legs syndrome.

The diagnosis of restless legs syndrome (RLS) relies upon diagnostic criteria which are based on history only, and dopaminergic treatment is not normally the first choice of treatment for all patients. It would be worthwhile to identify patients non-responsive to dopaminergic treatment beforehand, because they may suffer from a restless legs-like syndrome and may require alternative treatment. We included retrospectively 24 adult patients fulfilling the four essential criteria for restless legs and 12 age-matched healthy controls. They were investigated by ambulatory actigraphy from both legs over three nights, and patients started treatment with dopamine agonists after this diagnostic work-up. We examined 12 responders to dopaminergic treatment and 12 non-responders and studied the association between response to dopaminergic treatment and the periodic limb movement index (PLMI) as assessed with actigraphy. Demographic characteristics, excessive daytime sleepiness and fatigue at baseline were similar in all three groups. Baseline RLS severity was similar between responders and non-responders [International Restless Legs Severity Scale (IRLS): 25 ± 9 and 24 ± 8]. Group comparisons of PLMI before treatment initiation showed significant differences between the three groups. Post-hoc pairwise comparisons revealed that healthy controls had significantly lower PLMI (4.9 ± 4.5) than responders (29.3 ± 22.7) and non-responders (13.3 ± 11.2). Similarly, the PLMI in responders was lower than in non-responders. PLMI day-to-day variability did not differ between responders and non-responders and there was no correlation between treatment effect, as assessed by the decrease of the IRLS and baseline PLMI. Our retrospective study indicates that actigraphy to assess periodic limb movements may contribute to a better diagnosis of dopamine-responsive restless legs syndrome.

The role of learning-related dopamine signals in addiction vulnerability

Dopaminergic signals play a mathematically precise role in reward-related learning, and variations in dopaminergic signaling have been implicated in vulnerability to addiction. Here, we provide a detailed overview of the relationship between theoretical, mathematical, and experimental accounts of phasic dopamine signaling, with implications for the role of learning-related dopamine signaling in addiction and related disorders. We describe the theoretical and behavioral characteristics of model-free learning based on errors in the prediction of reward, including step-by-step explanations of the underlying equations. We then use recent insights from an animal model that highlights individual variation in learning during a Pavlovian conditioning paradigm to describe overlapping aspects of incentive salience attribution and model-free learning. We argue that this provides a computationally coherent account of some features of addiction.

The Role of BDNF, Leptin and Catecholamines in Reward Learning in Bulimia Nervosa

Background: A relationship between bulimia nervosa (BN) and reward-related behavior is supported by several lines of evidence. The dopaminergic dysfunctions in the processing of reward-related stimuli have been shown to be modulated by the neurotrophin brain derived neurotrophic factor (BDNF) and the hormone leptin. Methods: Using a randomized, double-blind, placebo-controlled, crossover design, a reward learning task was applied to study the behavior of 20 female subjects with remitted BN (rBN) and 27 female healthy controls under placebo and catecholamine depletion with alpha-methyl-para-tyrosine (AMPT). The plasma levels of BDNF and leptin were measured twice during the placebo and the AMPT condition, immediately before and 1 h after a standardized breakfast. Results: AMPT-induced differences in plasma BDNF levels were positively correlated with the AMPT-induced differences in reward learning in the whole sample (p = 0.05). Across conditions, plasma BDNF levels were higher in rBN subjects compared to controls (diagnosis effect; p = 0.001). Plasma BDNF and leptin levels were higher in the morning before compared to after a standardized breakfast across groups and conditions (time effect; p < 0.0001). The plasma leptin levels were higher under catecholamine depletion compared to placebo in the whole sample (treatment effect; p = 0.0004). Conclusions: This study reports on preliminary findings that suggest a catecholamine-dependent association of plasma BDNF and reward learning in subjects with rBN and controls. A role of leptin in reward learning is not supported by this study. However, leptin levels were sensitive to a depletion of catecholamine stores in both rBN and controls.

Dopamine receptor D4 polymorphism predicts the effect of L-DOPA on gambling behavior

BACKGROUND There is ample evidence that a subgroup of Parkinson's disease patients who are treated with dopaminergic drugs develop certain behavioral addictions such as pathological gambling. The fact that only a subgroup of these patients develops pathological gambling suggests an interaction between dopaminergic drug treatment and individual susceptibility factors. These are potentially of genetic origin, since research in healthy subjects suggests that vulnerability for pathological gambling may be linked to variation in the dopamine receptor D4 (DRD4) gene. Using a pharmacogenetic approach, we investigated how variation in this gene modulates the impact of dopaminergic stimulation on gambling behavior in healthy subjects. METHODS We administered 300 mg of L-dihydroxyphenylalanine (L-DOPA) or placebo to 200 healthy male subjects who were all genotyped for their DRD4 polymorphism. Subjects played a gambling task 60 minutes after L-DOPA administration. RESULTS Without considering genetic information, L-DOPA administration did not lead to an increase in gambling propensity compared with placebo. As expected, however, an individual's DRD4 polymorphism accounted for variation in gambling behavior after the administration of L-DOPA. Subjects who carry at least one copy of the 7-repeat allele showed an increased gambling propensity after dopaminergic stimulation. CONCLUSIONS These findings demonstrate that genetic variation in the DRD4 gene determines an individual's gambling behavior in response to a dopaminergic drug challenge. They may have implications for the treatment of Parkinson's disease patients by offering a genotype approach for determining individual susceptibilities for pathological gambling and may also afford insights into the vulnerability mechanisms underlying addictive behavior.

International validation of a behavioral scale in Parkinson's disease without dementia.

The "Ardouin Scale of Behavior in Parkinson's Disease" is a new instrument specifically designed for assessing mood and behavior with a view to quantifying changes related to Parkinson's disease, to dopaminergic medication, and to non-motor fluctuations. This study was aimed at analyzing the psychometric attributes of this scale in patients with Parkinson's disease without dementia. In addition to this scale, the following measures were applied: the Unified Parkinson's Disease Rating Scale, the Montgomery and Asberg Depression Rating Scale, the Lille Apathy Rating Scale, the Bech and Rafaelsen Mania Scale, the Positive and Negative Syndrome Scale, the MacElroy Criteria, the Patrick Carnes criteria, the Hospital Anxiety and Depression Scale, and the Mini-International Neuropsychiatric Interview. Patients (n = 260) were recruited at 13 centers across four countries (France, Spain, United Kingdom, and United States). Cronbach's alpha coefficient for domains ranged from 0.69 to 0.78. Regarding test-retest reliability, the kappa coefficient for items was higher than 0.4. For inter-rater reliability, the kappa values were 0.29 to 0.81. Furthermore, most of the items from the Ardouin Scale of Behavior in Parkinson's Disease correlated with the corresponding items of the other scales, depressed mood with the Montgomery and Asberg Depression Rating Scale (ρ = 0.82); anxiety with the Hospital Anxiety and Depression Scale-anxiety (ρ = 0.56); apathy with the Lille Apathy Rating Scale (ρ = 0.60). The Ardouin Scale of Behavior in Parkinson's disease is an acceptable, reproducible, valid, and precise assessment for evaluating changes in behavior in patients with Parkinson's disease without dementia. © 2015 International Parkinson and Movement Disorder Society.

Localization and production of peptide endocannabinoids in the rodent CNS and adrenal medulla.

The endocannabinoid system (ECS) comprises the cannabinoid receptors CB1 and CB2 and their endogenous arachidonic acid-derived agonists 2-arachidonoyl glycerol and anandamide, which play important neuromodulatory roles. Recently, a novel class of negative allosteric CB1 receptor peptide ligands, hemopressin-like peptides derived from alpha hemoglobin, has been described, with yet unknown origin and function in the CNS. Using monoclonal antibodies we now identified the localization of RVD-hemopressin (pepcan-12) and N-terminally extended peptide endocannabinoids (pepcans) in the CNS and determined their neuronal origin. Immunohistochemical analyses in rodents revealed distinctive and specific staining in major groups of noradrenergic neurons, including the locus coeruleus (LC), A1, A5 and A7 neurons, which appear to be major sites of production/release in the CNS. No staining was detected in dopaminergic neurons. Peptidergic axons were seen throughout the brain (notably hippocampus and cerebral cortex) and spinal cord, indicative of anterograde axonal transport of pepcans. Intriguingly, the chromaffin cells in the adrenal medulla were also strongly stained for pepcans. We found specific co-expression of pepcans with galanin, both in the LC and adrenal gland. Using LC-MS/MS, pepcan-12 was only detected in non-perfused brain (∼40 pmol/g), suggesting that in the CNS it is secreted and present in extracellular compartments. In adrenal glands, significantly more pepcan-12 (400-700 pmol/g) was measured in both non-perfused and perfused tissue. Thus, chromaffin cells may be a major production site of pepcan-12 found in blood. These data uncover important areas of peptide endocannabinoid occurrence with exclusive noradrenergic immunohistochemical staining, opening new doors to investigate their potential physiological function in the ECS. This article is part of a Special Issue entitled 'Fluorescent Neuro-Ligands'.

Is Parkinson's disease of early onset a separate disease entity?

Two groups of patients suffering from Parkinson's disease were studied. The first group consisted of 23 patients with an onset age before 40 years; in the second group of 21 patients the onset was after age 50. The clinical findings and the course of the disease were very similar in each group. In spite of a longer disease duration in the patients with early onset of the disease there was no difference in motor impairment; the younger patients did better in mental testing and they were taking less dopaminergic medication. These differences are thought to be due to the age difference rather than to the existence of different disease entities. In the early onset group more familial cases (mostly affecting siblings) were found than in the older ones. The points in favour of there being a hereditary subgroup of early onset Parkinson's disease or of environmental factors causing the disease are reviewed.

Distinct roles of cortical and pallidal β and γ frequencies in hemiparkinsonian and dyskinetic rats.

Enhanced β band (βB) activity, which is suppressed by levodopa (LD) treatment, has been demonstrated within the basal ganglia (BG) of Parkinson's disease (PD) patients. However, some data suggest that Parkinsonian symptoms are not directly related to this brain frequency and therefore, its causative role remains questionable. A less explored phenomenon is the link between the γ band (γB) and PD phenomenology. Here, we monitored the development of the oscillatory activity during chronic LD depletion and LD treatment in Parkinsonian and levodopa-induced dyskinesia (LID) in rats. We found a significant and bilateral power increase in the high βB frequencies (20-30Hz) within the first 10days after 6-hydroxydopamine (6-OHDA) lesion, which was in accordance with a significant depletion of dopaminergic fibers in the striatum. We also observed a clear-cut γB increase during LD treatment. The development of LID was characterized by a slight increase in the cumulative power of βB accompanied by a large augmentation in the γB frequency (60-80Hz). This latter effect reached a plateau in the frontal cortex bilaterally and the left globus pallidus after the second week of LD treatment. Our data suggest that the βB parallels the emergence of Parkinsonian signs and can be taken as a predictive sign of DA depletion, matching TH-staining reduction. On the other hand, the γB is strictly correlated to the development of LID. LD treatment had an opposite effect on βB and γB, respectively.

Characterization of fetal antigen 1/delta-like 1 homologue expressing cells in the rat nigrostriatal system: effects of a unilateral 6-hydroxydopamine lesion.

Fetal antigen 1/delta-like 1 homologue (FA1/dlk1) belongs to the epidermal growth factor superfamily and is considered to be a non-canonical ligand for the Notch receptor. Interactions between Notch and its ligands are crucial for the development of various tissues. Moreover, FA1/dlk1 has been suggested as a potential supplementary marker of dopaminergic neurons. The present study aimed at investigating the distribution of FA1/dlk1-immunoreactive (-ir) cells in the early postnatal and adult midbrain as well as in the nigrostriatal system of 6-hydroxydopamine (6-OHDA)-lesioned hemiparkinsonian adult rats. FA1/dlk1-ir cells were predominantly distributed in the substantia nigra (SN) pars compacta (SNc) and in the ventral tegmental area. Interestingly, the expression of FA1/dlk1 significantly increased in tyrosine hydroxylase (TH)-ir cells during early postnatal development. Co-localization and tracing studies demonstrated that FA1/dlk1-ir cells in the SNc were nigrostriatal dopaminergic neurons, and unilateral 6-OHDA lesions resulted in loss of both FA1/dlk1-ir and TH-ir cells in the SNc. Surprisingly, increased numbers of FA1/dlk1-ir cells (by 70%) were detected in dopamine-depleted striata as compared to unlesioned controls. The higher number of FA1/dlk1-ir cells was likely not due to neurogenesis as colocalization studies for proliferation markers were negative. This suggests that FA1/dlk1 was up-regulated in intrinsic cells in response to the 6-OHDA-mediated loss of FA1/dlk1-expressing SNc dopaminergic neurons and/or due to the stab wound. Our findings hint to a significant role of FA1/dlk1 in the SNc during early postnatal development. The differential expression of FA1/dlk1 in the SNc and the striatum of dopamine-depleted rats could indicate a potential involvement of FA1/dlk1 in the cellular response to the degenerative processes.

Loss of Nogo-A-expressing neurons in a rat model of Parkinson's disease

The myelin-associated protein Nogo-A is among the most potent neurite growth inhibitors in the adult CNS. Recently, Nogo-A expression was demonstrated in a number of neuronal subpopulations of the adult and developing CNS but at present, little is known about the expression of Nogo-A in the nigrostriatal system, a brain structure severely affected in Parkinson's disease (PD). The present study sought to characterize the expression pattern of Nogo-A immunoreactive (ir) cells in the adult ventral mesencephalon of control rats and in the 6-hydroxydopamine (6-OHDA) rat model of PD. Immunohistochemical analyses of normal adult rat brain showed a distinct expression of Nogo-A in the ventral mesencephalon, with the highest level in the substantia nigra pars compacta (SNc) where it co-localized with dopaminergic neurons. Analyses conducted 1week and 1 month after unilateral striatal injections of 6-OHDA disclosed a severe loss of the number of Nogo-A-ir cells in the SNc. Notably, at 1week after treatment, more dopaminergic neurons expressing Nogo-A were affected by the 6-OHDA toxicity than Nogo-A-negative dopaminergic neurons. However, at later time points more of the surviving dopaminergic neurons expressed Nogo-A. In the striatum, both small and large Nogo-A-positive cells were detected. The large cells were identified as cholinergic interneurons. Our results suggest yet unidentified functions of Nogo-A in the CNS beyond the inhibition of axonal regeneration and plasticity, and may indicate a role for Nogo-A in PD.

TIME DEPENDENT ALTERATIONS IN CENTRAL NERVOUS SYSTEM DOPAMINE RECEPTORS INDUCED BY DOPAMINE AGONIST TREATMENT

Levodopa, the precursor of dopamine, is currently the drug of choice in the treatment of Parkinson's disease. Recently, two direct dopamine agonists, bromocriptine and pergolide, have been tested for the treatment of Parkinson's disease because of reduced side effects compared to levodopa. Few studies have evaluated the effects of long-term treatment of dopamine agonists on dopamine receptor regulation in the central nervous system. Thus, the purpose of this study was to determine whether chronic dopamine agonist treatment produces a down-regulation of striatal dopamine receptor function and to compare the results of the two classes of dopaminergic drugs.^ Levodopa with carbidopa, a peripheral decarboxylase inhibitor, was administered orally to rats whereas bromocriptine and pergolide were injected intraperitoneally once daily. Several neurochemical parameters were examined from 1 to 28 days.^ Levodopa minimally decreased striatal D-1 receptor activity but increased the number of striatal D-2 binding sites. Levodopa increased the V(,max) of tyrosine hydroxylase (TH) in all brain regions tested. Protein blot analysis of striatal TH indicated a significant increase in the amount of TH present. Dopamine-beta-hydroxylase (DBH) activity was markedly decreased in all brain regions studied and mixing experiments of control and drug-treated cortices did not show the presence of an increased level of endogenous inhibitors.^ Bromocriptine treatment decreased the number of D-2 binding sites. Striatal TH activity was decreased and protein blot analysis indicated no change in TH quantity. The specificity of bromocriptine for striatal TH suggested that bromocriptine preferentially interacts with dopamine autoreceptors.^ Combination levodopa-bromocriptine was administered for 12 days. There was a decrease in both D-1 receptor activity and D-2 binding sites, and a decrease in brain HVA levels suggesting a postsynaptic receptor action. Pergolide produced identical results to the combination levodopa-bromocriptine studies.^ In conclusion, combination levodopa-bromocriptine and pergolide treatments exhibited the expected down-regulation of dopamine receptor activity. In contrast, levodopa appeared to up-regulate dopamine receptor activity. Thus, these data may help to explain, on a biochemical basis, the decrease in the levodopa-induced side effects noted with combination levodopa-bromocriptine or pergolide therapies in the treatment of Parkinson's disease. ^

Function of histaminergic retinopetal axons in rat and primate retinas

Mammalian retinas receive input from histaminergic neurons in the posterior hypothalamus. These neurons are most active during the waking state of the animal, but their role in retinal information processing is not known. To determine the function of these retinopetal axons, their targets in the rat and monkey retina were identified. Using antibodies to three histamine receptors, HR1, HR2, and HR3, the immunolabeling was analyzed by confocal and electron microscopy. These experiments showed that mammalian retinas possess histamine receptors. In macaques and baboons, diurnal species, HR3 receptors were found at the apex of ON-bipolar cell dendrites in cone pedicles and rod spherules, sclerad to the other neurotransmitter receptors that have been localized there. In addition, HR1 histamine receptors were localized to large puncta in the inner plexiform layer, a subset of ganglion cells and retinal blood vessels. In rats, a nocturnal species, the localization of histamine receptors in the retina was markedly different. Most HR1 receptors were localized to dopaminergic amacrine cells and on elements in the rod spherule. To determine how histaminergic retinopetal axons contribute to retinal information processing, responses of retinal ganglion cells to histamine were analyzed. The effects of histamine on the maintained and light-evoked activity of retinal ganglion cells were analyzed. In monkeys, histamine and the HR3 agonist, methylhistamine, increased or decreased the maintained activity of most ganglion cells, but a few did not respond. The responses of a subset of ganglion cells to light stimuli were decreased by histamine, a finding suggesting that histaminergic retinopetal axons contribute to light adaptation during the day. In rats, histamine nearly always increased the maintained activity and produced both increases and decreases in the light responses. The effects of histamine on maintained activity of ganglion cells in the rat can be partially attributed to HR1-mediated changes in the activity of dopaminergic amacrine cells, at night. Together, these experiments provide the first indication of the function of retinopetal axons in mammalian retinas. ^

Functional differences of beta-tubulin isotypes: A study of microtubule assembly and antimitotic drug resistance

Mammalian cells express 7 β-tubulin isotypes in a tissue specific manner. This has long fueled the speculation that different isotypes carry out different functions. To provide direct evidence for their functional significance, class III, IVa, and VI β-tubulin cDNAs were cloned into a tetracycline regulated expression vector and stably transfected Chinese hamster ovary cell lines expressing different levels of ectopic β-tubulin were compared for effects on microtubule organization, microtubule assembly and sensitivity to antimitotic drugs. It was found that all three isotypes coassembled with endogenous β-tubulin. βVI expression caused distinct microtubule rearrangements including microtubule dissociation from the centrosome and accumulation at the cell periphery; whereas expression of βIII and βVIa caused no observable changes in the interphase microtubule network. Overexpression of all 3 isotypes caused spindle malformation and mitotic defects. Both βIII and βIVa disrupted microtubule assembly in proportion to their abundance and thereby conferred supersensitivity to microtubule depolymerizing drugs. In contrast, βVI stabilized microtubules at low stoichiometry and thus conferred resistance to many microtubule destabilizing drugs but not vinblastine. The 3 isotypes caused differing responses to microtubule stabilizing drugs. Expression of βIII conferred paclitaxel resistance while βVI did not. Low expression of βIVa caused supersensitivity to paclitaxel, whereas higher expression resulted in the loss of supersensitivity. The results suggest that βIVa may possess an enhanced ability to bind paclitaxel that increases sensitivity to the drug and acts substoichiometrically. At high levels of βVIa expression, however, microtubule disruptive effects counteract the assembly promoting pressure exerted by increased paclitaxel binding, and drug supersensitivity is lost. From this study, I concluded that β-tubulin isotypes behave differently from each other in terms of microtubule organization, microtubule assembly and dynamics, and antimitotic drug sensitivity. The isotype composition of cell can impart subtle to dramatic effects on the properties of microtubules leading to potential functional consequences and opening the opportunity to exploit differences in microtubule isotype composition for therapeutic gain. ^