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Published as doi: 10.1096/fj.07-8386com.
 (The FASEB Journal. 2007;21:3835-3844.)
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Prevention and restoration of lactacystin-induced nigrostriatal dopamine neuron degeneration by novel brain-permeable iron chelators

Wen Zhu*,{dagger}, Wenjie Xie*, Tianhong Pan*, Pingyi Xu{dagger}, Mati Fridkin{ddagger}, Hailin Zheng{ddagger}, Joseph Jankovic*, Moussa B. H. Youdim§,1 and Weidong Le*,1

* Department of Neurology, Baylor College of Medicine, Houston, Texas, USA;

{dagger} Department of Neurology, The 1st Affiliated Hospital of Sun Yat-sen University, Guangzhou, China;

{ddagger} The Weizmann Institute of Science, Department of Chemistry, Rehovot, Israel; and

§ Technion-Rappaport Family Faculty of Medicine, Haifa, Israel

1Correspondence: W.L., Department of Neurology, NB 205, Baylor College of Medicine, Houston, TX 77030, USA. E-mail: weidongl{at}bcm.tmc.edu; or M.B.H.Y., Technion-Rappaport Family Faculty of Medicine, Efron St., P.O. Box 9697, Haifa 31096, Israel. E-mail: youdim{at}tx.technion.ac.il


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Dysfunction of the ubiquitin-proteasome system (UPS) and accumulation of iron in substantia nigra (SN) are implicated in the pathogenesis of Parkinson’s disease (PD). UPS dysfunction and iron misregulation may reinforce each other’s contribution to the degeneration of dopamine (DA) neurons. In the present study, we use a new brain-permeable iron chelator, VK-28 [5-(4-(2-hydroxyethyl) piperazin-1-yl (methyl)-8-hydroxyquinoline], and its derivative M30 [5-(N-methyl-N-propargyaminomethyl)-8-hydroxyquinoline] in vivo to test their neuroprotective and neurorestorative properties against proteasome inhibitor (lactacystin) -induced nigrostriatal degeneration. Bilateral microinjections of lactacystin (1.25 µg/side) into the mouse medial forebrain bundle were performed. Administration of VK-28 (5 mg/kg, once a day) or M30 (5 mg/kg, once a day) was applied intraperitoneally 7 days before or after the lactacystin microinjection until the mice were sacrificed 28 days after microinjection. We found that VK-28 and M30 both significantly improved behavioral performances and attenuated lactacystin-induced DA neuron loss, proteasomal inhibition, iron accumulation, and microglial activation in SN. In addition, M30 restored the Bcl-2 level, which was suppressed after lactacystin injection. These findings suggest that brain-permeable iron chelators can improve DA neuron survival under UPS impairment. Furthermore, M30, a derivative of VK-28 and neuroprotective agent rasagiline, may serve as a better neuroprotective therapy for PD.—Zhu, W., Xie, W., Pan, T., Xu, P., Fridkin, M., Zheng, H., Jankovic, J., Youdim, M. B. H., Le, W. Prevention and restoration of lactacystin-induced nigrostriatal dopamine neuron degeneration by novel brain-permeable iron chelators.


Key Words: ubiquitin-proteasome system • Parkinson’s disease • VK-28 • M30


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
PARKINSON’S DISEASE (PD) IS DEFINED pathologically by degeneration of dopamine (DA) neurons in the substantia nigra (SN) and by the intracytoplasmic accumulation of proteinaceous inclusions, known as Lewy bodies (1) . In PD, 50–70% of the DA neurons had already been lost at the time of diagnosis (2) . Thus, a neuroprotective therapy that slows, stops, or reverses disease progression is the major unmet medical need in PD.

A pivotal role of iron in the pathogenesis of PD has been hypothesized since numerous studies have shown a progressive accumulation of iron and ferritin in PD patients, specifically in the SN pars compacta (3 , 4) . The major mechanism of iron-mediated damage in nigrostriatal neurons is related to the fact that iron is a significant generator of reactive oxygen species (ROS). Accessible ferrous iron (Fe2+) can react with hydrogen peroxide (H2O2) produced during oxidative deamination of DA to generate hydroxyl radicals (·OH) that can damage proteins, nucleic acids, and membrane phospholipids, leading to cellular degeneration (5) . Recently, iron chelation was shown to protect neurodegeneration in PD animal models induced by N-methyl-4-pheny-1, 2, 3, 6-tetrahydropyridine (MPTP). Chelation of iron either by the oral administration of iron chelators such as R-apomorphine (6) or green tea (–)-epigallocatechin-3-gallate (EGCG) (7) , clioquinol (8) , M30 (9) , or overexpression of the heavy subunit of the iron binding protein ferritin (8) protected DA neurons, maintained DA production, and prevented motor deficits associated with MPTP administration. More recently, iron has been found in the rim of Lewy bodies where {alpha}-synuclein, ubiquitin, and tyrosine hydroxylase (TH) were also present (10) . Furthermore, there is also evidence that iron overload facilitates fibrillation of human {alpha}-synuclein and impairs the proteasomal activity (11 , 12) . Accordingly, pretreatment of R-apomorphine and EGCG, which possess iron-chelating properties, has been shown to prevent the increase of {alpha}-synuclein protein in SN (13) . The above findings suggest that the neuroprotective mechanism of iron chelators may be via their ability to interfere with impairment of ubiquitin-proteasome system (UPS) and aggregation of abnormal protein.

To determine the interaction between iron accumulation and UPS impairment, we coinjected brain-impermeable iron chelator deferoxamine with proteasome inhibitor lactacystin locally into the medial forebrain bundle (MFB) in mouse and showed for the first time that deferoxamine significantly protected against the DA neuron degeneration induced by lactacystin in vivo (14) . This result implies that iron chelation may act through UPS to protect DA neurons. Two newly developed brain-permeable iron chelators, VK-28 [5-(4-(2-hydroxyethyl) piperazin-1-yl (methyl)-8-hydroxyquinoline] and M30 [5-(N-methyl-N-propargyaminomethyl)-8-hydroxyquinoline], recently showed neuroprotection against PD animal models induced by either 6-hydroxydopamine or MPTP (9 , 15) . In the present study, we evaluated not only neuroprotective but also neurorestorative properties of these two iron chelators, VK-28 and M30, in vivo, against nigrostriatal degeneration induced by lactacystin.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals and treatments
The proposed animal study was approved by the Baylor College of Medicine Animal Use and Care Committee and was conducted in accordance with the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the National Institutes of Health. Male C57BL/6 mice aged 12 wk were randomly signed into seven groups: control, Lac 7d, Lac 28d, Pre-VK-28, Pre-M30, Post-VK-28, and Post-M30. They were housed five animals per cage in a colony room maintained at constant temperature and humidity, with a 12 h light/dark cycle, and allowed at least 7 days to acclimatize before any treatment. Intraperitoneal (i.p.) administration of VK-28 (5 mg/kg, once a day) or M30 (5 mg/kg, once a day) started 7 days before or after microinjection with lactacystin and continued until the mice were sacrificed. Administration of the same volume of saline served as control. For stereotactic injection of lactacystin, mice were deeply anesthetized and placed in a Kopf stereotactic frame (Kopf Instruments, Tujunga, CA, USA). An injection cannula was inserted through a hole drilled in the skull into the MFB using the following coordinates (in mm): 1.34 posterior, ±1.17 lateral, and 5.1 ventral from bregma of each mouse. Two microliters of phosphate-buffered saline (PBS, 0.1M) as control or lactacystin (1.25 µg; A.G. Scientific, San Diego, CA, USA) in PBS was injected into the MFB of each mouse. Five mice injected with lactacystin only (group Lac 7d) were sacrificed 7 days after microinjection. Other mice were sacrificed at the end of the study, 28 days after microinjection of lactacystin. The mice were sacrificed by terminal anesthesia, followed by transcardial perfusion with ice-cold PBS. The mice were decapitated and the brains were immediately removed, placed on ice, and transected coronally at the infundibular stem. The midbrain blocks were fixed with 4% paraformaldehyde in PBS for 2 days and cryoprotected in 30% sucrose for 2 days at 4°C, followed by histological analysis. Striatal tissues and ventral midbrains were rapidly dissected out and stored at –80°C until analysis. The samples were divided to perform different sets of experiments.

Locomotive activities and rotarod performance
Locomotive activities and rotarod performance were tested 1 day before and 7 and 28 days after microinjection of lactacystin. Locomotive activities were monitored by the AccuScan Digiscan system (AccuScan Instruments, Inc., Columbus, OH, USA). Data collected by computer included total distance traveled (cm/60 min) and moving time (s/60 min). The measurements were carried out from 9 AM to 11 AM in a dark room. Each mouse was placed in the testing chamber for 30 min for adaptation, followed by a 60 min recording by the computer-generated automatic analysis system. Motor coordination was determined with an accelerating rotarod treadmill (Columbus Instruments, Columbus, OH, USA). Initially, each mouse was required to perch on the stationary rod for 30 s to accustom itself to the environment. Then the animals were trained at a constant speed of 5 rpm for 90 s. After this pretraining, the mice were tested three times at 1 h intervals on 3 consecutive days for a total of nine tests. During each test, the rotarod was set at a starting speed of 5 rpm for 30 s and the speed was increased by 0.1 revolution per second. All animals were tested three times for each experiment, and the means of the test results underwent statistical analysis.

Immunohistochemistry
Midbrain blocks were cut into 30 µm sections and systematically picked at 150 µm intervals. Free-floating sections were incubated successively for 15 min with 0.05% H2O2 in 0.1 mol/L PBS to remove endogenous peroxidase activity for 1 h with 2% goat serum/0.1% Triton X-100 in 0.1 mol/L PBS to block nonspecific binding sites and for 24 h at 4°C with primary antibodies, rabbit antityrosine hydroxylase (TH, 1:1500; Protos Biotech, New York, NY, USA) and mouse anti-TH (1:500; Sigma-Aldrich, St. Louis, MO, USA), to detect DA neurons; mouse antiglial fibrillary acidic protein (GFAP, 1:500; Chemicon International Inc., Temecula, CA, USA) to label astrocytes; rat anti-CD11b (against MAC1, 1:50; Chemicon International Inc.) to detect microglia; and mouse anti-{alpha}-synuclein (1:1000; BD Transduction Laboratories, San Jose, CA, USA) and rabbit antiubiquitin (1:500; Chemicon International Inc.) to detect protein accumulations. Sections were then incubated for 2 h at room temperature with the appropriate biotinylated secondary antibody (anti-rabbit or anti-rat IgG, 1:200; Vector Laboratories Inc., Burlingame, CA, USA). The avidin-biotin method was used to amplify the signal (ABC Kit; Vector Laboratories Inc., Burlingame, CA, USA) and 3, 3'-diaminobenzidine tetrachloride (DAB) was used to visualize bound antibodies. For double-immunofluorescent staining, Alex Fluor 546 goat anti-rabbit IgG and 488 goat anti-mouse IgG (1:200; Molecular Probes, Eugene, OR, USA) were used. The DA neurons (TH-positive cells) and microglia in the SN were counted as described (16) .

Determination of striatal DA and its metabolites
The concentrations of DA, 4-dihydroxy-phenylacetic acid (DOPAC), homovanillic acid (HVA), serotonin (5-HT), and 5-hydroxyindolacetic acid (5-HIAA) were quantified in striatal tissues by HPLC. Briefly, striatal tissues were homogenized (10% w/v) by sonication in ice-cold 0.1M perchloric acid. Homogenates were centrifuged at 10,000 g for 10 min at 4°C; the supernatants were collected and filtered through acro-disc filters (0.25 µm, Fisher Scientific) and subjected to HPLC (HTEC-500; Eicom, Kyoto, Japan) with the column (EICOMPAK SC-3ODS; Eicom, Kyoto, Japan) and detected by an electrochemical detector (AD Instruments Pty Ltd., Castle Hill, NSW, Australia). The mobile phase consisted of 0.1 mM citric acid, 0.1M sodium acetate, 220 mg/L octane sulfate sodium, 5 mg/L EDTA, and 20% methanol (pH=3.5).

Proteasome activity assay
Ventral midbrains were placed on ice and homogenized in lysis buffer (50 mM HEPES, pH 7.5, 5 mM EDTA, 150 mM NaCl, 0.5 mM ATP, and 1%Triton X-100). The lysates were centrifuged at 14,000 g at 4°C for 20 min. The resulting supernatants were placed on ice and assayed for protein concentrations by the Bradford’s method (Bio-Rad, Hercules, CA, USA). The 20S Proteasome Activity kit (Chemicon International Inc.) was used to measure chymotrypsin-like activity. Assays were carried out with 50 µg of midbrain lysates and appropriate substrate for 90 min incubation at 37°C. Activity was measured by detection of the fluorophore 7-amido-4-methylcoumarin (AMC) after cleavage from the synthetic fluorogenic peptide Leu-Leu-Val-Tyr-AMC, using a spectrofluorimeter (Cytofluor II; PerSeptive Biosystems, Framingham, MA, USA) at excitation/emission wavelengths of 380/460nm. Results are expressed as fluorescence units/mg protein.

Iron measurement in the ventral midbrain
Ventral midbrains were weighed and digested in concentrated hydrochloric acid. Tissue iron concentrations (nmol/g wet tissue) were determined spectrophotometrically by using the kit from DCL (Diagnostic Chemicals Ltd., Oxford, CT, USA) in a modified microtiter plate assay.

Bcl-2 immunoblot
Tissues of mice were extracted with mammalian tissue lysis/extraction reagent (Sigma-Aldrich) supplemented with complete protease inhibitor cocktail (Sigma-Aldrich). Equal amounts of lysate protein were denatured in sodium dodecyl sulfate (SDS) sample buffer applied onto a 12% SDS-polyacrylamide gel and transferred onto a polyvinyl difluoride membrane. After being blocked in 6% nonfat milk, membranes were incubated in the presence of primary antibodies Bcl-2 (1:1000; Chemicon International Inc.) or β-actin (1:5,000; Sigma-Aldrich), followed by incubation with horseradish peroxidase-labeled secondary antibodies (1:2000; Chemicon International Inc.); signals were detected using ECL (Amersham, Arlington Heights, IL, USA).

Statistical analysis
All results of the study were from groups of 3–10 mice. Intergroup differences between the various dependent variables were assessed using 1-way ANOVA, followed by the Dunnett post hoc multiple comparisons test. P values lower than 0.05 were considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
VK-28 and M30 improved behavioral performance in lactacystin-lesioned mice
Both locomotive activities and rotarod performance were performed 1 day before and 7 and 28 days after the microinjection of lactacystin. The day of microinjection with lactacystin was set to be day 0. Compared with the control, locomotive activities (total distance traveled and moving time) were significantly decreased by 67.9% and 71.6%, respectively (Fig. 1 and Fig. 2 a, b), and the rotarod performance (the time remaining on the rod) was reduced by 70.0% (Fig. 2c ) in the mice injected with lactacystin on day 7. Locomotive tests and rotarod performance remained unchanged by the end of the study (28 days after lactacystin microinjection). Pretreatment of VK-28 and M30 significantly attenuated behavioral impairment by 86.6% and 97.2% in total distance traveled, by 78.4% and 85.6% in moving time, and by 69.1% and 72.2% in rotarod time, respectively, on day 7 (Fig. 2) . Recovery was seen on the mice posttreated with VK-28 (73.4% of control in total distance traveled, 87.4% of control in moving time, and 71.5% of control in rotarod time), an even better recovery was documented on the mice posttreated with M30 (87.2% of control in moving distance, 87.4% of control in moving time, and 81.7% of control in rotarod time) in the behavioral performances (Fig. 2) .


Figure 1
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  		Figure 1. The structures of prototype iron chelator VK-28 [5-(4-(2-hydroxyethyl) piperazin-1-yl (methyl)-8-hydroxyquinoline] and its neuroprotective propargylamine possessing derivative, M30 [5-(N-methyl-N-propargyaminomethyl)-8-hydroxyquinoline].


Figure 2
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  		Figure 2. VK-28 and M30 improved behavioral performance in lactacystin-lesioned mice. Two microliters of phosphate-buffered saline (PBS, 0.1 M) as control or lactacystin (1.25 µg) in PBS was injected into the MFB of each mouse. Intraperitoneal (i.p.) administration of VK-28 (5 mg/kg, once a day) or M30 (5 mg/kg, once a day) started 7 days before (pretreatment) or after (posttreatment) microinjection with lactacystin, while administration of the same volume saline served as control. Locomotive activities and rotarod performance and were performed 1 day before and 7 and 28 days after microinjection of lactacystin. The day of microinjection with lactacystin was set to be day 0. Changes in locomotive activity are shown by total distance traveled (a) and moving time (b). The alteration of rotarod performance was recorded by time staying on the rod (c). Data were expressed as means ± SE (n=7). *P < 0.05 vs. control and #P < 0.05 vs. Lac 28d.

VK-28 and M30 reduced lactacystin-induced loss of DA neurons in SN
Compared with the control, a loss of 22.5% DA neurons was traced in the SN 7 days after injection with lactacystin; a loss of 67.0% DA neurons was evidenced on day 28, indicating that the neurodegeneration induced by lactacystin is a progressive process (Fig. 3 ). Pretreatment of either VK-28 or M30 showed marked protective effects on DA neurons against lactacystin-induced injury at the end of the study, with a 73.8% and 86.3% reduction in DA neuron loss, respectively (Fig. 3) . The ability of VK-28 or M30 to restore the DA neuron loss was determined after a 21 day posttreatment that started 7 days after the lactacystin injection. Our results showed that both VK-28 and M30 significantly attenuated the lactacystin-induced loss of DA neurons in SN by 63.2% and 82.2%, respectively (Fig. 3) . Furthermore, compared with VK-28 (5 mg/kg/day), M30 (5 mg/kg/day) was more potent in protecting or restoring DA neurons (Fig. 3) .


Figure 3
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  		Figure 3. VK-28 and M30 reduced lactacystin-induced loss of DA neurons in SN. Five mice injected with lactacystin only were sacrificed 7 days after microinjection with lactacystin. Other mice were sacrificed at the end of the study (day 28). a) Representative photomicrographs of SN with TH immunohistochemistry (10x). A–G) Control, Lac 7d, Lac 28d, Pre-VK-28, Pre-M30, Post-VK-28, and Post-M30 groups, respectively. b) Quantitative analysis of TH immunopositive neurons in the SN. Each value was presented by the mean ± SE based on the number of TH immunopositive neurons in right hemispheric nigral slices (n=8). *P < 0.01 and **P < 0.001 vs. control, #P < 0.01 vs. Lac 28d and $P < 0.05 vs. VK-28.

VK-28 and M30 restored lactacystin-induced depletion of DA and its metabolites
Accompanied by the loss of DA neurons, microinjection of lactacystin also induced a progressive depletion of DA and its metabolites, DOPAC and HVA. Compared with the control, DA, DOPAC, and HVA levels were decreased by 15.5%, 18.6%, and 14.6% on day 7, respectively, and reductions in DA, DOPAC and HVA were greater on day 28 (i.e., 66.4%, 63.4%, and 46.8%, respectively; Fig. 4 a, b). However, the levels of striatal 5-HT and its metabolite 5-HIAA were not significantly affected (Fig. 4c ), indicating that the toxicity of lactacystin might be relatively selective to the nigrostriatal dopaminergic pathway. At the end of the study, we found that pretreatment of VK-28 and M30 significantly attenuated the lactacystin-induced reduction of striatal DA by 65.5% and 91.8%, respectively. Meanwhile, posttreatment of VK-28 and M30 restored up to 68.5% and 90.8% of control striatal DA level, respectively (Fig. 4a, b ). Compared with VK-28, M30 was superior in reversing the reduction of DA and HVA (Fig. 4a, b ).


Figure 4
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  		Figure 4. VK-28 and M30 restored lactacystin-induced depletion of DA and its metabolites. The effect of coadministration with iron chelator VK-28 or M30 on lactacystin-induced changes of striatal DA (a) and its metabolites (b), DOPAC and HVA, as well as changes in striatal 5-HT and 5-HIAA (c). The results are expressed as means ± SE (n=7). *P < 0.05 and **P < 0.01 vs. control; #P < 0.05 and ##P < 0.01 vs. Lac 28d; $P < 0.05 vs. VK-28.

VK-28 and M30 alleviated lactacystin-induced proteasomal inhibition
As a proteasome inhibitor, lactacystin caused a 40.6% inhibition of the chymotrypsin-like proteasomal activity in the ventral midbrain 7 days after microinjection of lactacystin, and the proteasomal activity remained inhibited even after 28 days of lactacystin treatment (Fig. 5 ). VK-28 and M30 at 5 mg/kg/day were used in lactacystin-injected mice to observe the reverse effects on proteasomal activity. It was shown that pretreatment of VK-28 and M30 notably attenuated lactacystin-induced proteasomal inhibition by 38.7% and 74.7%, respectively. Furthermore, posttreatment with VK-28 and M30 also managed to restore proteasomal activity by 35.3% and 68.7%, respectively (Fig. 5) . Although both VK-28 and M30 could alleviate the proteasomal inhibition, the capability of M30 was almost twice that of VK-28 (Fig. 5) .


Figure 5
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  		Figure 5. VK-28 and M30 alleviated lactacystin-induced proteasomal inhibition. The effect of iron chelator VK-28 or M30 is indicated by changes in chymotrypsin-like activity induced in ventral midbrain. Results are expressed as means ± SE (n=5). *P < 0.01 vs. control; #P < 0.05 and ##P < 0.01 vs. Lac 28d; $P < 0.05 vs. VK-28.

VK-28 and M30 attenuated lactacystin-induced iron accumulation
Iron concentration in ventral midbrain was progressively elevated after microinjection of lactacystin. Seven days after lactacystin microinjcetion, there was only a mild increase in iron concentration while a 32.7% increase of iron concentration in ventral midbrain was found on day 28 (Fig. 6 ). The ability of VK-28 and M30 to chelate brain iron was tested in the lactacystin-injected mice. We found that systemic application of VK-28 or M30 significantly attenuated iron overload in the ventral midbrain (Fig. 6) . However, the iron chelating effect of VK-28 seemed more potent than M30 when administrated at the same dosage. Regardless of application before or after the lactacystin microinjection, administration of VK-28 resulted in a full inhibition of iron overload of up to 140%, and M30 alleviated iron accumulation by up to 80% (Fig. 6) .


Figure 6
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  		Figure 6. VK-28 and M30 attenuated lactacystin-induced iron accumulation. The ability of iron chelator VK-28 and M30 in passing BBB to act on lactacystin-induced iron accumulation are documented by changes in iron concentration in the ventral midbrain. Results are expressed as means ± SE (n=5). *P < 0.01 vs. control; #P < 0.05 and ##P < 0.01 vs. Lac 28d; $P < 0.05 vs. VK-28.

VK-28 and M30 decreased microglial activation in lactacystin-injected mice
Glial activation and possible inflammation in the SN were studied by immunohistochemistry. Microglia was detected by CD11b staining and morphological characterization (16) . Compared with vehicle control, an increase in microglial profile was evident in SN in mice injected with lactacystin beginning 7 days after the microinjection of lactacystin (Fig. 7 a). A dense deposition of hypertrophic microglia was seen on day 7. At the end of the study, both VK-28 and M30 managed to inhibit the microglial activation (Fig. 7a ). Compared with microinjection of lactacystin alone, pretreatment with VK-28 or M30 inhibited activation of microglia by 72.1% and 89.8%, respectively. Posttreatment with VK-28 or M30 reduced microglial activation by 60.1% and 85.3%, respectively (Fig. 7b ). GFAP staining was applied to detect astrocytes (Fig. 8 ). After the microinjection of lactacystin, a progressive increase of GFAP-positive cells was detectable in the SN compared with the vehicle control (Fig. 8) . However, neither pretreatment nor posttreatment with VK-28 interfered with the astrocytic activation in SN, whereas both pretreatment and posttreatment with M30 resulted in a decrease of astrocytes on day 28 (Fig. 8) .


Figure 7
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  		Figure 7. VK-28 and M30 decreased microglial activation induced by lactacystin. a) The changes of microglial activation in SN are demonstrated by microglia with CD11b immunohistochemistry (40x). A–G) Control, Lac 7d, Lac 28d, Pre-VK-28, Pre-M30, Post-VK-28, and Post-M30 groups, respectively. b) Quantification of CD11b-positive microglia in the right SN. The results were expressed as means ± SE (n=5). *P < 0.01 vs. control and #P < 0.01 vs. Lac 28d.


Figure 8
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  		Figure 8. Effects of representative VK-28 and M30 on astrocytic activation after lactacystin injection. An increased amount of GFAP immunoreactivity is detectable in the ipsilateral SN after lactacystin injection (10x). A–G) Control, Lac 7d, Lac 28d, Pre-VK-28, Pre-M30, Post-VK-28, and Post-M30 groups, respectively.

M30 reversed the reduction in Bcl-2 protein level caused by proteasome inhibition
Compared with vehicle control, a significant reduction of Bcl-2 was observed in the ventral midbrain 7 and 28 days after lactacystin microinjection (Fig. 9 ). These findings may suggest that lactacystin induces cell death related at least in part to the decrease of Bcl-2. It is interesting to find that pretreatment and posttreatment with M30 but not VK-28 significantly elevated Bcl-2 (105.6% of control), which may contribute to its neuroprotective and neurorestorative properties (Fig. 9) .


Figure 9
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  		Figure 9. M30 reversed the reduction in Bcl-2 protein level caused by proteasome inhibition. Protein level of Bcl-2 in the ventral midbrain was analyzed by Western blot at the end of the study. a) Lanes A to G represent the control, Lac 7d, Lac 28d, Pre-VK-28, Pre-M30, Post-VK-28, and Post-M30 groups, respectively. Loading of the lanes was normalized to levels of β-actin and the experiment is representative of 3 independent experiments. b) The calculated densitometry intensities of the respective bands were presented as % of control. The results are expressed as means ± SE (n=3). *P < 0.01 vs. control, #P < 0.01 vs. Lac 28d and $P < 0.01 vs. VK-28.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study we found that bilateral stereotactic injection with lactacystin into the MFB of mice induces behavioral deficits and nigrostriatal degeneration. We also demonstrated that focal injection of lactacystin can result in protein aggregation positively immunostained with {alpha}-synuclein and ubiquitin (for details, see Supplemental Fig. 1 and legend). These findings resemble some core features of PD. Furthermore, stereotactic injection with lactacystin also triggers proteasome activity reduction, iron overload, glial activation, and Bcl-2 suppression, which may account for the underlying mechanisms of nigral neurodegeneration. Using this mouse model, we have identified the potential neuroprotective and neurorestorative properties of iron chelators VK-28 and M30 against nigrostriatal degeneration induced by UPS impairment. Both VK-28 (15) and M30 (9) are newly developed iron chelators with a potency equivalent to that of desferrioxamine (17) , which has been shown to possess neuroprotective properties against lactacystin-induced nigrostriatal neurodegeneration (14) . Unlike desferrioxamine, both VK-28 and M30 can pass the blood-brain barrier (BBB) to target the brain when given orally or intraperitoneally, which makes them applicable for clinical use; thus, testing their ability to protect or restore would be meaningful. In our study, we have demonstrated that pretreatment of VK-28 or M30 can protect against the lactacystin-induced DA neuron degeneration in the SN. More importantly, we also documented that both VK-28 and M30 have neurorestorative effects against neurodegeneration induced by lactacystin when given after lactacystin lesions. Administration of VK-28 or M30 for 21 days after microinjection of lactacystin managed to restore the severe reduction in DA neuron number and striatal DA level, and also attenuated proteasomal inhibition, iron accumulation, and microglial activation. Furthermore, M30 is capable of reversing the reduction of Bcl-2 protein level caused by lactacystin. These findings all suggest that VK-28 and M30 may be promising disease-modifying agents that warrant further clinical investigation for PD.

Proteasomes are multisubunit, multicatalytic proteases and are the principal enzymes responsible for the degradation of unwanted proteins in the cytoplasm, nucleus, and endoplasmic reticulum of eukaryotic cells (18) . A growing body of evidence suggests that failure of the proteasomal dysfunction might play an important role in the pathogenic process of sporadic and familial PD and may underlie protein accumulation, Lewy body formation, and neurodegeneration in the SN (19 , 20) . It has been shown that striatal microinfusions of the proteasome inhibitor lactacystin or epoxomycin can reproduce selective retrograde nigral damage in the ipsilateral SN (21) . More recently it was reported that systemic administration of proteasome inhibitors causes DA neuron death (22) , although some investigators have not been able to replicate these findings (23 24 25) . The reasons why this model is hard to replicate are complicated. One possibility is that lower doses of the proteasome inhibitors do not sufficiently inactivate proteasome function to induce the model; higher doses are insoluble and do not cross the BBB and may cause peripheral toxicity (26) . Comparatively, focal microinjection of proteasome inhibitors can directly target the nigro-striatal pathway by overcoming the BBB problem. Thus, our study has clearly shown that microinjection with lactacystin into MFB can produce profound nigral DA neuron degeneration in the mouse, which may serve as an animal model of PD.

Iron is the most abundant transition metal in the body, with high concentrations in the brain (27) . The highest iron concentrations in the brain are found in the globus pallidus, followed by the red nucleus, SN, putamen, and caudate nucleus, which are most vulnerable to PD (28) . At the cellular level, iron is stored mainly by ferritin and hemosiderin in glial cells and by neuromelanin in neurons (29) . The main route of iron uptake is through the transferrin receptor (TfR) at the cell surface (30) . The regulation of iron metabolism in mammalian cells is controlled by two cytoplasmic iron regulatory proteins (IRP): IRP-1 and IRP-2 (31) . The IRPs control ferritin and the TfR level according to the labile iron pool. When cellular iron levels are high, the IRPs are inactivated, resulting in an increased translation of ferritin heavy (H) and light (L) subunit and in destabilization of the TfR message, and vice versa. The two IRPs are functionally and structurally homologous, and both have been found to undergo proteasomal degradation (32 , 33) . Thus, in the present study the increase of iron concentration in SN after microinjection of lactacystin may be 1) caused by a dysregulation of nigral iron homeostasis as a result of DA neurons loss and iron released from neuromelanin; 2) related to the accumulation of IRPs caused by UPS impairment; 3) associated with infiltrating reactive glial cells, an important downstream cascade of proteasomal inhibition (34) ; 4) originated by a dysfunction in the BBB induced by lactacystin.

Although it is still not clear whether the increased iron concentration is a primary or secondary event in PD, a pivotal role of iron in the pathogenesis has been emphasized because of its capacity to enhance the production of oxygen radicals and accelerate neuronal degeneration (35) . More recently, iron and oxidative stress has been found in association with the UPS dysregulation and protein aggregation. Recent studies have shown that overexpression of {alpha}-synuclein can form toxic aggregates in the presence of iron (36) . Its 5'-untranslated messenger RNA contains a predicted iron-responsive element similar to ferritin, and thus it is regulated by iron (37 , 38) . This is regarded as contributing to the formation of Lewy body via oxidative stress (39) . Iron released from neuromelanin has been reported to cause mitochondrial dysfunction and to reduce proteasomal function (40) . Oxidative stress can facilitate mutant protein aggregation, mimicking proteasomal malfunction (41) . Dysregulation of UPS and/or accumulation of iron may induce a vicious circle in which UPS impairment may cause iron overload, and increased iron may further aggravate UPS dysfunction. Thus, application of iron chelators VK-28 or M30 may sequester the redundant iron to prevent the ability of iron to induce oxidative stress as a consequence of reactive hydroxyl radical generation via its interaction with hydrogen peroxide and may further stop the amplified damage triggered by UPS malfunction. These ideas were supported by the fact that administration of VK-28 or M30 significantly alleviated the neurodegeneration and iron accumulation induced by lactacystin microinjection. However, iron accumulation is not the only mechanism underlying the nigral neurodegeneration; we found that although iron chelator VK-28 was more potent in chelating brain iron than M30, it was not as good as M30 either in attenuating nigral neurodegeneration induced by lactacystin or in reducing proteasomal inhibition after lactacystin microinjection. These findings suggest that M30 is involved in neuroprotective activities other than iron chelating.

In addition to iron deposition and UPS failure, inflammatory processes have been shown to be associated with the pathogenesis of PD. Activated microglia, and to a lesser extent reactive astrocytes, have been reported in the area associated with DA neuron loss in PD (16) . Many compelling findings support the notion that microglia are responsible for the deleterious effects seen in PD by releasing proinflammatory prostaglandins or cytokines (42) . In the present study, remarkable microglial activation in the lactacystin-injected mice was observed at an early stage of the study (day 7), and remained highly activated at day 28. On the one hand, matrix metalloproteinase 3, {alpha}-synuclein, and neuromelanin released by damaged DA neurons may account for the continuous activation of microglia (43) . On the other hand, the overactivated microglia can induce more significant and highly detrimental neurotoxic effects by excess production of a large array of cytotoxic factors. In our study, administration of iron chelator VK-28 or M30 significantly inhibited the activation of microglia. Mechanisms underlying the microglial inhibition by VK-28 and M30 remain unknown. We speculate that VK-28 and M30 may directly interfere with the microglia or other inflammatory cells by blocking microglial activation signals (43) or suppress the microglial activation indirectly by inhibiting iron-mediated oxidative stress damage to DA neuron in order to reduce the release of the matrix metalloproteinase 3, {alpha}-synuclein, and neuromelanin, which can activate microglia.

A growing body of evidence suggests that cell death in PD occurs at least in part by signal-mediated apoptosis (44 45 46) . It was reported recently that UPS impairment may lead to nontranscriptional accumulation of p53, alterations in mitochondrial membrane permeability, and consequent apoptotic cell death (47) . In addition, the presence of elevated iron concentrations has been shown to contribute to neuronal apoptosis. Elevated iron concentrations has been found to increase cytosolic DA, leading to oxidative stress, c-Jun N-terminal kinase (JNK) pathway activation, neurite degeneration, and eventually apoptosis in vitro (47) . Redox-active ferrous iron induced apoptosis in vitro by H2O2/·OH generation, resulting in mitochondria depolarization, caspase-3 activation, and nuclear fragmentation independent of NF-{kappa}B and p53 transcription factors activation (48) . Thus, iron chelators VK-28 and M30 may attenuate the neuronal apoptosis induced by iron accumulation after lactacystin injection. Furthermore, a severe reduction of Bcl-2 in the present study appeared 7 days after lactacystin microinjection, which recovered a mere 28 days after the lesion. As overexpression of Bcl-2 and SOD in mice and PC-12 and SH-SY5Y cells has been shown to make them resistant to neurotoxin-induced neurodegeneration (49) , the reduction in Bcl-2 induced by proteasomal malfunction may contribute to the neurodegeneration. Moreover, it is reported that Bcl-2 activates the ubiquitin-proteasome system and increases all proteasome-dependent enzyme activity, with resultant inhibition of mitochondrial cytochrome c release (50 51 52) . As a derivate of rasagiline, M30 possesses the propargylamine moiety, which is believed to be neuroprotective (9) . The mechanism of neuroprotection by propargylamine has been attributed to its ability to up-regulate antiapoptotic Bcl-2 family proteins Bcl-2 and Bcl-xl while down-regulating Bad and Bax (52) . Furthermore, propargylamine and rasagiline induce mRNAs as well as proteins of glia-derived neurotrophic factor and brain-derived neurotrophic factor, which can explain their dopaminergic neurorestorative activity in post-MPTP-treated mice via tyrosine kinase pathway activation (53) . It is likely that the neurorestorative activity of M30 has a similar propargylamine-dependent mechanism, which is now being investigated. Moreover, M-30 may suppress apoptosis via multiple protection mechanisms, including inhibition of caspase 3 cleavage and prevention of Ser-139 phosphorylation of H2A.X (30) . In the present study, M30 was shown to up-regulate the level of antiapoptotic Bcl-2 protein, which may contribute to its ability to increase proteasomal activity and protect or restore DA neurons against proteasomal inhibition. We recently reported that M-30 possesses a wide range of pharmacological activities, including prosurvival neurorestorative effects and induction of neuronal differentiation in SH-SY5Y cells in culture, resulting in neurite outgrowth, regulation of the cell cycle, and an increase in GAP-43 (37) . However, VK-28 has no effect on the Bcl-2 protein level. Therefore, the differential efficacy on reversing the Bcl-2 protein level may account for the superior neuroprotection and neurorestoration effects of M30 to VK-28 in alleviating the inhibition of proteasome activity and further protecting the DA neurons in SN.

In summary, novel brain-permeable iron chelators VK-28 and M30 exert potent DA neuroprotection and neurorestoration against lactacystin-induced neurodegeneration, UPS inhibition, and iron elevation. These findings are consistent with the notion that "ironing the iron out" could be an effective approach to combat neurodegeneration in PD and other neurodegenerative disease. As brain-permeable iron chelators, VK-28 and M30 can pass the BBB to target the brain and chelate iron effectively, which makes them more applicable in the clinic to test their neuroprotection and neurorestorative effects on patients with PD. Regarding the complicated etiopathogenesis of PD, a single drug may not be adequate to induce effective neuroprotection. Thus, multifunctional drugs such as M30 may be more suited for use as disease-modifying agents.


   ACKNOWLEDGMENTS
 
This work was supported by research grants from the American Parkinson Disease Association (2005–2007) and the U.S. National Institutes of Health (NS 043567). M.B.H.Y. thanks Technion Research and Development (Haifa, Israel) and the Alzheimer’s Drug Discovery Foundation and Institute for the Study of Aging (New York, NY, USA) for their generous support.

Received for publication April 22, 2007. Accepted for publication June 21, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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