Striatal Postsynaptic Ultrastructural Alterations Following ...

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Ann. N.Y. Acad. Sci. 965: 381–398 (2002). © 2002 New York Academy of Sciences.

Striatal Postsynaptic Ultrastructural Alterations Following Methylenedioxymethamphetamine Administration

F. FORNAI,a,b M. GESI,a P. LENZI,a M. FERRUCCI,a A. PELLEGRINI,a

S. RUGGIERI,b A. CASINI,c AND A. PAPARELLIa

aDepartment of Human Morphology and Applied Biology, University of Pisa,Pisa, ItalybI.R.C.C.S. I.N.M. Neuromed, Pozzilli, ItalycDepartment of Experimental Pathology, University of Pisa, Pisa, Italy

ABSTRACT: Amphetamine derivatives, such as methamphetamine (METH) and3,4-methylenedioxymethamphetamine (MDMA), act as monoaminergic neuro-toxins in the central nervous system. Although there are slight differences intheir mechanism of action, these compounds share a final common pathway,which involves dopamine release and oxidative stress. Apart from striatal tox-icity involving monoamine axons, no previous report evidenced any alterationat the striatal level concerning postsynaptic sites. Given the potential toxicityfor extracellular dopamine at the striatal level, and the hypothesis for neuro-toxic effects of dopamine on striatal medium-sized neurons in Huntington’sdisease, we evaluated at an ultrastructural level the effects of MDMA on intrin-sic striatal neurons of the mouse. In this study, administering MDMA, we notedultrastructural alterations of striatal postsynaptic GABAergic cells consistingof neuronal inclusions shaped as whorls of concentric membranes. Thesewhorls stained for ubiquitin but not for synuclein and represent the first mor-phologic correlate of striatal postsynaptic effects induced by MDMA.

KEYWORDS: dopamine; ecstasy; MDMA; whorls; ubiquitin

INTRODUCTION

Amphetamine derivatives, such as methamphetamine (METH) and 3,4-methyl-enedioxymethamphetamine (MDMA), act as monoaminergic neurotoxins in the cen-tral nervous system (CNS).1 Although there are slight differences in theirmechanism of action, these compounds share a final common neurotoxic mecha-nism, which involves oxidative stress.2

Address for correspondence: Francesco Fornai, Department of Human Morphology andApplied Biology, University of Pisa 56100, Pisa, Italy. Voice: +39-050-835927; fax: +39-050-835925.

[email protected]

382 ANNALS NEW YORK ACADEMY OF SCIENCES

In particular, METH and MDMA administration, apart from releasing serotonin(5HT), produces a marked release of dopamine (DA) in several brain areas, thestriatum3 being the main target region. Such DA release represents the triggeringmechanism that induces oxidative stress.

In fact, although there are species differences in the neuronal system selectivelyinvolved in amphetamine-induced neurotoxicity,4 DA plays a major role in thepathogenesis of the lesions.5,6 In keeping with this, enhancement of DA metabolism,by producing toxic quinones and cysteinyl derivatives, is thought to be responsiblefor the rapid increase in cerebral content of free radicals and reactive oxygen species(ROS),2,7 leading to damage of striatal DA terminals arising from the substantia ni-gra pars compacta (SNpc) and 5HT striatal terminals arising from the dorsal raphenucleus. Potential neurotoxicity of cerebral DA extends not only to DA neurons, butalso to the surrounding 5HT axons8 and nonmonoaminergic neurons.9 Recent stud-ies lend substance to these effects, showing that endogenous DA might trigger thedegeneration of striatal medium-sized γ-aminobutyric acid (GABA) neurons in Hun-tington’s disease.10

The main mechanism responsible for DA-mediated neurotoxicity consists of thehigh production of free radicals and ROS. Indeed, neurons are particularly suscepti-ble to the toxic effects of ROS, as compared with other cell types;11 on the otherhand, brain metabolism requires great amounts of molecular oxygen, resulting in thegeneration of high levels of oxygen-free radicals, also in basal conditions.11 There-fore, the role of antioxidant systems in the CNS is critical to preventing cell deathduring both acute injuries and chronic degenerative disorders.11

Therefore, it is likely that oxidative stress might arise not only from the enhance-ment of free radical production, but also from the depletion of physiologic antioxi-dant mechanisms.

In line with this, it has been shown that MDMA produces a significant reductionin the concentrations of antioxidant molecules, such as vitamin E and ascorbic acid(AA) in the brain. This latter effect, in addition to MDMA-induced oxidative stress,can contribute to enhancing the toxicity of this neurotoxin.12 Ascorbic acid has greatimportance in the CNS, where it has different effects. Recently, the key role of AAas a brain antioxidant was pointed out as consisting of scavenging aqueous free rad-icals and ROS and decreasing quinone formation.13

Accordingly, pretreatment with AA can prevent MDMA-induced hydroxyl radi-cal formation.12 A new enzyme, a glutathione (GSH)-dependent dehydroascorbate(DHA) reductase, plays a pivotal role in regenerating AA from its oxidation product,DHA.14 In previous studies we localized DHA reductase within the CNS, where itwas found to be particularly abundant in certain brain areas (i.e., cerebellum, stria-tum, rhinencephalon, brain stem, and substantia nigra). At subcellular levels, DHAreductase was found in the cytosol, in the nucleus, associated with the chromatin fil-aments, and spared in the axoplasm.15 In the present study we evaluated at ultra-structural levels the occurrence of this enzyme in those subcellular compartmentsthat appear to be affected by the administration of amphetamine derivatives.

Another system that appears crucial to counteract degenerative processes selec-tively involving the SNpc DA neurons is the locus coeruleus (LC), the most rostralnoradrenaline (NA) complex, representing the major source of NA in the CNS. Infact, recent studies suggest that impairment of LC can increase the nigrostriatal DAtoxicity produced in various experimental models.16,17

383FORNAI et al.: METHYLENEDIOXYMETHAMPHETAMINE

In the present study, therefore, we evaluated at an ultrastructural level the modu-lation of the NA system on subcellular alterations induced by amphetamines. Be-sides oxidative stress, the striatum is extremely sensitive to different types of injurysuch as cerebral ischemia, virus infections, and specific neuronal genetic alterations,which provoke cell suffering or degeneration.18–20 The latter conditions involve stri-atal intrinsic neurons. By contrast, amphetamine derivatives have been extensivelystudied for their effects on striatal nerve endings arising from brain stem mono-aminergic nuclei.

Therefore, apart from the well characterized methamphetamine-induced striataltoxicity involving monoamine axons, no previous report evidenced any alteration atthe striatal level concerning postsynaptic sites. This refers mainly to biochemicaland immunohistochemical studies. Therefore, given the potential toxicity for extra-cellular DA at the striatal level, as just reviewed, and the hypothesis for neurotoxiceffects of DA on striatal medium-sized neurons in Huntington’s disease,10 we decid-ed to evaluate at an ultrastructural level the effects of amphetamines on the striatalneurons of the mouse.

In addition, since, as reported above, previous studies indicated a crucial role forthe endogenous NA system in conditioning the toxicity of amphetamines on DA neu-rons, we investigated the importance of LC integrity on amphetamine-induced ultra-structural changes in striatal neurons. For this purpose, we administered theneurotoxin N-(-2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4), which selec-tively destroys NA axons arising from LC neurons and spares extra-coeruleus NAterminals.21

MATERIAL AND METHODS

Animals

Male C57 Black mice (C57BL/6J) 9–10 weeks old were obtained from Harlan In-dustries (San Pietro al Natisone, Italy). Mice were kept under controlled conditions(12-hour light/dark cycle with lights on between 07:00 and 19:00) and were fed andallowed to drink water ad libitum. As the toxicity of amphetamine derivatives ishighly variable, critically depending on room temperature and number of animalsper cage,8 we carefully kept a constant room temperature (21°C); 24 hours beforetreatment, we housed animals one per cage (size of the cage: 11 × 10 cm wide and15 cm high), allowing the mouse to move freely inside. Animals were handled in ac-cordance with the Guidelines for Animal Care and Use developed by the NationalInstitutes of Health. All appropriate efforts were made to minimize animal sufferingand to reduce the number of animals used.

Drug Administration

Intraperitoneal injection of neurotoxin DSP-4 hydrochloride (RBI, Natick, MA)was carried out at a dose of 50 mg/kg, which produces the maximal NA lesions indifferent brain areas.8 DSP-4 solution was freshly prepared in 0.9% NaCl.

Two days after DSP-4 injection, MDMA hydrochloride (Sigma Chemical Co., St.Louis, MO) was administered at a dose of 30 mg/kg × 3, 2 hours apart, which has been


shown to produce depletion of striatal DA in mice.8 MDMA solutions were freshlyprepared in distilled water. Each treatment group was composed of 10 animals.

Biochemical Procedures and HPLC Analysis

One week after treatment, the animals were killed by decapitation, and theirbrains were removed and dissected in order to obtain the striatum. Tissue sampleswere homogenized in ice-cold 0.6 mL of perchloric acid (0.1 M), and an aliquot ofhomogenate (50 µL) was assayed for protein content.8 After centrifugation at8,000 g for 10 minutes, 20 µL of the clear supernatant was injected into an HPLCsystem to measure levels of NA, DA, 5HT, and metabolites, as previouslydescribed.8

Morphological Procedures

Light Microscopy and Immunohistochemistry

Under i.p. chloral hydrate anesthesia, mice were sacrificed, and the brain was re-moved and frozen at −80°C until sectioning. Twenty-micrometer thick cryostate sec-tions were mounted on slides and maintained at 37°C for 15 minutes. Slicesprocessed for tyrosine hydroxylase (TH) were fixed in cold methanol, whereas slicesfor DA transporter (DAT) and glial fibrillar acidic protein (GFAP) immunohis-tochemistry were first fixed in 4% paraformaldehyde and then permeabilized inphosphate-buffered saline (PBS) solution with 0.1% Triton 100 SX; after washing,endogenous peroxidase activity was removed by a 30-minute wash in methanol with0.3% hydrogen peroxide and pre-blocked with normal goat serum (Vector Laborato-ries, Burlingame, CA) for 2 hours. They were then incubated for 24 hours with pri-mary antibodies (Ab-I) IgG in PBS solution containing normal goat serum. We usedmouse Ab-I anti-TH IgG (Sigma Chemical Co., St. Louis, MO) diluted 1:2000; ratAb-I anti-DAT IgG (Chemicon International, Temecula, CA) diluted 1:1000; andrabbit Ab-I anti-GFAP IgG (Chemicon International, Temecula, CA) diluted 1:400.

After careful washing, sections incubated with anti-TH and anti-DAT Ab-I wereincubated with byotinylated secondary antibodies (Vector Laboratories, Burlin-game, CA), washed and revealed by using the ABC kit (Vector Laboratories, Burl-ingame, CA), followed by diaminobenzidine as chromogen (Vector Laboratories,Burlingame, CA). Sections incubated with anti-GFAP Ab-I were then processedwith a secondary fluorescein-conjugated antibody (Vector Laboratories, Burlin-game, CA).

Electron Microscopy

Perfusion-Fixation. Mice were anesthetized with chloral hydrate (4 mL/kg), tho-racotomized, and perfused transcardially. In a pilot series of experiments, we empir-ically established the optimal fixing solution: 2.0% paraformaldehyde/0.1%glutaraldehyde in 0.1 M PBS, pH 7.4.

After perfusion, brains were maintained in situ overnight at 4°C. They were thenremoved from the skull, and the caudate-putamen was dissected from a thick coronalsection of brain by a clean cut, removing the surrounding white matter and cortex.

Immunocytochemical Methods. Dihydroxynonenal, α/β-synuclein, and DHA re-ductase immunocytochemistry were carried out using post-embedding immunogold;


ubiquitin immunocytochemistry was carried out by both immunoperoxidase pre-em-bedding and immunogold post-embedding.

For pre-embedding experiments, after perfusion, thin slices (approximately200 µm) were washed in PBS, and endogenous peroxidase activity was blocked us-ing 0.3% hydrogen peroxide. Specimens were preblocked using 10% normal goat se-rum and 0.2% saponine (Sigma Chemical Co., St. Louis, MO) in PBS for 20 minutesand then incubated with rabbit primary antibodies against ubiquitin (Chemicon In-ternational, Temecula, CA) in PBS containing 0.2% saponine and 1% normal goatserum for 24 hours at 4°C. Samples were further rinsed in PBS and incubated withsecondary anti-rabbit peroxidase-labeled IgG (Sigma Chemical Co., St. Louis, MO)for 60 minutes at room temperature, washed carefully, and revealed with diami-nobenzidine (Vector Laboratories, Burlingame, CA) for 10 minutes as a chromogen.After immunocytochemistry, samples were post-fixed in 1% OsO4, dehydrated inethanol, and embedded in an epon-araldite mixture.

For post-embedding experiments, we used only immunogold staining. Briefly,specimens were post-fixed in 1% OsO4 in buffered solution, dehydrated in ethanol,and embedded in epon-araldite mixture. Thin sections, collected on nickel grids,were deosmicated in Na-metaperiodate solutions for 30 minutes at room tempera-ture, washed in PBS (15 minutes), incubated for 24 hours with primary antibodiesagainst ubiquitin (Chemicon International, Temecula, CA), dihydroxynonenal (kindgift from Prof. Alfonso Pompella, University of Pisa), α/β-synuclein (kind gift fromOliver Schlueter, Max Planck Institute, Goettingen), and DHA reductase, obtainedas described in a previous report,14 and finally processed as described for pre-em-bedding, using secondary gold conjugate antibodies (diameter 10 nm) (SigmaChemical Co., St. Louis, MO). Control sections were incubated only with secondaryantibody, and they never showed any detectable levels of immunostaining. All thinsections were contrasted with uranyl acetate and lead citrate and examined in a JeolJEM SX 100 transmission electron microscope (TEM).

Statistical Analysis of Striatal Whorls

Striatal whorls were measured by counting the number of either cytosolic or nu-clear whorls out of the total number of neurons under observation. In particular, foreach animal from each different group we randomly chose two tissue blocks dissect-ed from the dorsolateral striatum. From each block we got 10 grids, every grid con-taining several nonserial slices to observe 10 different neurons and count a finalnumber of 2,000 cells.

Data concerning the percentage of whorls in the cytosol or the nucleus of striatalcells were compared using ANOVA with Sheffè post hoc analysis. Null hypothesis(H0) was rejected when p < 0.05.

RESULTS

Striatal Monoamine Levels

In line with previous reports,8 administration of MDMA to intact mice caused asignificant decrease in both striatal DA and DOPAC levels as compared with con-trols. By contrast, striatal DA and DOPAC levels were not affected by DSP-4 treat-


ment. Administration of MDMA to mice that had undergone a previous NA lossinduced by the neurotoxin DSP-4 showed enhancement of MDMA-induced striatalDA and DOPAC loss (TABLE 1), but no decrease in 5HT levels (data not shown).

Striatal Immunohistochemistry

Control mice had marked immunostaining for TH, and a similar positivity waspresent in the striatum after DSP-4 administration (FIG. 1A and 1B). FollowingMDMA administration, the striatum exhibited less intense TH immunostaining (FIG.1C), which was further reduced in NA-depleted mice receiving MDMA (FIG. 1D).Similar results were obtained with striatal DAT immunohistochemistry (data notshown). In line with that, GFAP immunohistochemistry was more intense after com-bined treatment (data not shown).

Normal Ultrastructural Features of Striatal Neurons

The neuronal population of the striatum consists of different pharmacological andanatomical types; correspondingly, striatal neurons exhibit a certain degree of mor-phological variability. Nonetheless, the most frequent cell type (>90%) is represent-ed by medium-sized neurons, which possess a large and round nucleus, with anevident nucleolus and abundant cytosol containing cisterns of rough endoplasmicreticulum (RER) and Golgi apparatus, mitochondria, and particles of glycogen(FIG. 2A).

Striatal Ultrastructural Features after MDMA Treatment

Methylenedioxymethamphetamine administration produced morphologicalchanges within the striatal neurons. These alterations consisted of various slight al-terations in the striatal ultrastructure, which were evident in some cells as enlargedcistern clusters in the Golgi apparatus (FIG. 2B). Nonetheless, typical features ap-pearing as intracellular concentric and multilamellar bodies known as whorls werepresent both in the cytosol and within the nucleus of the striatal cells, mainly in thetypical medium-sized neurons (FIG. 2C and 2D). The whorls, with a diameter rang-ing from 0.1–2.4 µm, exhibited concentric single membranes surrounding an amor-

TABLE 1. Effect of DSP-4 on MDMA-induced striatal DA and DOPAC loss

CONTROL DSP-4 MDMA

DSP-4+

MDMA

DA 120.48 ± 7.15 128.07 ± 5.14 78.34 ± 7.06* 31.11 ± 4.89**

DOPAC 8.61 ± 0.22 6.07 ± 0.36 5.41 ± 0.17* 3.10 ± 0.11**

NOTE: Striatal dopamine (DA) and DOPAC levels are expressed as ng/mg protein. Mice weresacrificed after saline (controls) or after various treatments (DSP-4, MDMA, and combinedDSP-4 + MDMA administration). MDMA produces a significant striatal DA loss that is exacer-bated in mice carrying a previous lesion of LC neurons induced by DSP-4. DSP-4 (50 mg/kg)administration is carried out 3 days before MDMA (30 mg/kg × 3, 2 hours apart). Values areexpressed as the percentage of the means ± SEM of 10 animals per group.

*p < 0.05 compared with controls; **p < 0.05 compared with MDMA.


phous material (FIG. 2E and 2F). Apart from the different localization, no structuraldifferences were detected between cytosolic and nuclear whorls (FIG. 2E and 2G).After MDMA administration 12.13 ± 1.69% of striatal cells showed cytosolic whorlsthat were often associated with smooth endoplasmic reticulum and/or the outer nu-clear membrane (FIG. 2H); on the other hand, 29.02 ± 2.04% of striatal cells pos-sessed nuclear whorls (FIG. 2G). Both cytoplasmic and nuclear whorls were absentin striatal cells of control mice.

Striatal Ultrastructural Features after DSP-4 Treatment

DSP-4 treatment induced ultrastructural modifications in striatal neurons similarto those observed after MDMA administration. Indeed, membranous whorls wereobserved at both cytosolic (15.8 ± 1.3%) and nuclear (25.38 ± 1.7%) levels.

FIGURE 1. Effects of DSP-4 and/or MDMA administration on striatal TH immuno-hystochemistry. (A) Section from control mouse in which intense TH immunoreactivity isspecifically located in the striatum. (B) A similar pattern occurs in section from DSP-4–treated mice. (C) After MDMA administration TH immunostaining is attenuated. (D) Theimmunostaining appears further reduced following DSP-4 and MDMA treatment. (X 2,9).


FIGURE 2. Effects of MDMA administration on the ultrastructure of striatal medium-sized neurons. (A) Medium-sized striatal neuron from control mouse shows round nucleusand cytoplasm rich of ribosomes and glycogen particles; cisterns of rough endoplasmicreticulum (arrow) and mitochondria are evident (× 7000). (B) After MDMA administration,Golgi apparatus shows enlargement of cisterns (× 110,000).


FIGURE 2—continued. (C and D) MDMA-induced membranous bodies in the nucleusand cytoplasm (arrows). (× 7000; × 10,000).


FIGURE 2—continued. At higher magnifications (E and F), a cytoplasmic membra-nous body shows electron-dense single membranes whorled around an amorphous material(arrows) (× 20,000; × 33,000).


FIGURE 2—continued. In the nucleus (G), a membranous whorl appears in the neigh-bor of the nucleolus (Nu) (× 40,000) or (H) at the level of the outer nuclear envelope, wheremembranes are blebbing into the cytoplasm (× 130,000). Cy, cytoplasm; M, mitochondria;N, nucleus.


DSP-4 Enhances MDMA-Induced Morphological Alterations in the Striatum

Similar ultrastructural alterations, such as membranous cytosolic and nuclearwhorls, were observed in the striatal cells after combined treatment (not shown). Acomparable number of striatal cells (15.03 ± 1.2%) exhibited cytoplasmic mem-branous whorls, whereas a significant enhancement in the number of nuclear whorls(41.22 ± 2.45%) was counted, in comparison with what occurred after DSP-4 orMDMA treatment.

Immunocytochemical Characterization of the Striatal Whorls

Immunocytochemical investigation revealed that whorls were positive for ubiq-uitin and that the immunogold reaction was specifically located on the membrane ofwhorls (FIG. 3A), whereas it was not detected in the surrounding cytosol. Similarfindings were obtained using immunoperoxidase (FIG. 3B). Membranous whorlsalso showed immunopositivity for DHA reductase (FIG. 3C), whereas they were notstained by antibodies directed against dihydroxynonenal (FIG. 3D) even when dihy-droxynonenal positivity was found scattered in the nucleoplasm (FIG. 3E). Negativeimmunolabeling was also found for the presynaptic protein α- and β-synuclein (notshown).

DISCUSSION

Even though several hypotheses suggest a potential striatal toxicity of substitutedamphetamines, to our knowledge this is the first work showing the occurrence ofconstant ultrastructural alterations of striatal postsynaptic GABAergic neurons. Al-though we did not report these data, medium-sized neurons, which have been studiedhere, positively stain for GAD 67 and GAD 65.

These inclusions appear as whorls of concentric membranes. Although thesemembranous whorls have been generally described in the brain and periphery for de-cades,22,23 they remain undefined. At first, they were considered an artifact due tofixation procedures; however, it has been established that this may occur only whenspecimens are stored for months in glutaraldehyde.24 In our experimental conditionsstorage time was limited to a few hours, and we found that these alterations occurredrarely (up to 1 in 100 cells) in control mice. In the brain, due to the abundance ofphospholipids and free fatty acids, these artifacts might occur more frequently; how-ever, it is necessary to wait for 1 month to observe an increase in whorls as a conse-quence of tissue storage in glutaraldehyde. The other variable, which might interferewith the formation of whorls, consists in fixation temperature. In particular, whorlsincrease in size as the temperature of fixation rises from 4° to 37°C.24 In our exper-imental procedures we stored the tissue at 0°–4°C, ruling out even this variable.

Positive staining for ubiquitin is accompanied by positive staining for the anti-oxidant enzyme DHA reductase, which strongly suggests that these formations rep-resent a functional appendix of the ubiquitin-proteasome pathway in conditions ofoxidative stress. This is confirmed by the localization of whorls in association withthe smooth endoplasmic reticulum and the nuclear membrane. Ascorbic acid, whichis regenerated by the enzyme DHA reductase, plays an important role in preventingbrain damage induced by lipid peroxidation;25 this might explain why membranous


FIGURE 3. Immunocytochemistry of striatal whorls. High magnifications of whorls.(A and B) Anti-ubiquitin immunogold and immunoperoxidase staining (arrows) are placedon the surface of the concentric membranes (× 100,000; × 70,000).


FIGURE 3—continued. (C) Anti-DHA reductase immunogold particles (arrows) aresimilarly placed on a whorl (× 50,000), whereas (D) dihydroxynonenal immunostaining (ar-row) is located out of the external membrane of the whorl (× 100,000) and (E) widespreadin the nucleoplasm (arrows) (× 80,000).


whorls, which possess a phospholipid structure,26 are formed in conditions of lipidperoxidation.

Recently, membranous whorls were described in cell cultures transfected with thegene for the mutant torsinA; these inclusions positively stain for mutated torsinA,which belongs to the wide class of heat-shock proteins, which are believed to pro-vide a protective effect against oxidative stress.27

Previous studies in which these ultrastructural membranous bodies were de-scribed may help again to understand their significance. They are structurally ar-ranged similar to multivesicular bodies contained in type II pneumocytes asprecursors of the lung surfactant. Interestingly, after exposure to high oxygen con-centrations up to pure oxygen, there is a marked increase in these multivesicular bod-ies.23 Also, following cerebral ischemia in the dorsolateral striatum, some cells showstructural abnormalities consisting of cytoplasmic membranous whorls.18

Membranous whorls are found in several different conditions characterized byfunctional alterations in neurons of specific brain areas associated with hormonaldeficits28 or genetic diseases.29 Whorls were described as a characteristic feature ofa few viral infections19 and resulted from unusual proliferation of the inner mesaxonof innumerable myelinated fibers in an experimental model of Creutzfeldt-Jacob dis-ease.30 Ultrastructural alterations consisting in whorls of membrane were found inaxonal profiles from experimental models of Alzheimer’s disease.20 Moreover, in-

FIGURE 3—continued.


tramitochondrial membranous whorls were observed in liver cells, after chloroform-induced hepatotoxicity,31 and after pharmacological therapy.32

Ubiquitin represents a selective signal for intracytosolic protein degradation,which occurs in the proteasome. The ubiquitin-proteasome pathway has a centralrole in catalyzing proteolysis, and it is involved in the selective elimination of abnor-mal proteins.33 Intracellular multiubiquitinated aggregates were found in some dis-eases such as inclusion body myositis34 and cystic fibrosis.35 Within the CNS,ubiquitin specifically marks intracytoplasmic inclusions such as Loewy bodies with-in SNpc DA neurons, LC, and other brain areas in Parkinson’s disease.36

Ubiquitin-positive whorls, which we have observed after amphetamine treatment,might represent the morphological profile of the ubiquitin-proteasome pathway, en-hanced by the neurotoxic activity of these compounds. In particular, MDMA produc-es hydroxyl radicals and peroxynitrite.2 These highly reactive species can oxidase alarge amount of intracellular proteins, altering their normal structures and makingthem ideal substrates for ubiquitin linkage and their entry into the ubiquitin-protea-some pathway.

The significance of these inclusions is still unknown. In the presence of an intactcellular machinery to recognize and degrade misfolded proteins, it is likely that theseaggregation bodies could occur when the capacity of the proteasome is exceeded ortheir activity is inhibited.

The presence of ubiquitin in MDMA-induced whorls suggests that these forma-tions contain misfolded proteins produced by neurotoxic treatment. For instance, itis established that both MDMA and METH produce marked striatal DA release6

which is enhanced in mice bearing an NA lesion.17 Interestingly, a recent study dem-onstrated that DA inhibits the proteasome pathway,37 and there is evidence for suchinhibition in Huntington’s disease38 in which DA release is supposed to play a caus-ative role.10 This might be crucial in light of new evidence showing that huntingtininhibits the proteasome,38 thereby making more critical the oxidative effects of DAon “proteasome-impaired” GABAergic cells.

We hypothesize that the observed membranous whorls might be the ultrastructur-al evidence of a functional alteration in membranes consisting of impairment of mo-lecular trafficking.

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