Page 1 of 23 Movement Disorders

Page 1 of 23 Movement Disorders

Protective Effects ofUridine Plus Docosahexaenoic Acid in a Rat Model of

Parkinson's Disease

Mehmet Cansev\,2, Ismail H. Ulusl,2,Lei Wang\, Timothy J. Maherl,3, Richard J.

Wurtman\'*

IMassachusetts Institute of Technology, Department of Brain and Cognitive Sciences,

Cambridge MA, 02139, USA

2Uludag University School of Medicine, Department of Pharmacology and Clinical

Pharmacology, Bursa 16059, Turkey

3Massachusetts College of Pharmacy and Health Sciences, Department of Pharmaceutical

Sciences, Boston MA, 02115, USA

Word Count: 4108

*Corresponding Author:

Richard J. Wurtman, MD

MIT, 43 Vassar St., Building 46, Room 5023B

Cambridge MA, 02139

Phone: 617-253-6731

Fax: 617-253-6882

E-mail: [email protected]

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Movement Disorders Page 2 of 23

Abstract

Parkinson's disease (PD) causes degeneration of midbrain dopaminergic neurons

and diminishes striatal dopaminergic transmission. We previously showed in normal-<.

animals that administering the phosphatide precursors uridine (as uridine-5'-

monophosphate [UMPD and docosahexaenoic acid (DHA) can increase the quantity of

synaptic membrane per brain cell, and that uridine can enhance dopamine release. We

have now tested their effects on rotational behavior and brain composition in a rat model

of PD. Rats started receiving a control or UMP-supplemented (0.5%) diet, and/or, by

gavage, DHA (300 mg/kg) on the first day of the study; they were injected with 6-

hydroxydopamine (6-0HDA) into the right striatum on day 4; tested for d-amphetamine-

induced rotational behavior on day 25; and sacrificed on day 29. Giving UMP, DHA, or

both reduced d-amphetamine-induced ipsilateral rotations by 48%,47%, or 57% three

weeks following 6-0HDA injections. The combination also increased dopamine levels,

tyrosine hydroxylase (TOH) activity, TOH protein levels, and phosphatide levels in

lesioned striata, and dopamine and phosphatide levels in intact striata. Synapsin-l levels,

reduced in lesioned striata of control rats, were restored following all three treatments.

These data indicate that administering the phosphatide precursors uridine and DHA can

ameliorate behavioral and biochemical defects in a rat model of PD.

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Parkinson's disease (PD) is characterized by the progressive degeneration of

dopamine-containing neurons in the midbrain, particularly in the pars compacta of the

substantia nigral.2, and by a reduction in dopamine (DA) levels and release in the basal

ganglia. Moreover, the striatal DA depletion is associated with a decrease in dendritic

spine density on striatal medium spiny neurons3, observed both in postmortem studies4.5

and in animal models ofPD3.6. This impairment in dopaminergic neurotransmission leads

to characteristic motor symptoms including akinesia, rigidity, and resting tremor7.

Current treatment ofPD can be surgical, ie., pallidotomy, deep brain stimulation,

or foetal graft implantation8, or, more commonly, medical, using drugs to increase

synaptic DA (e.g. Levodopa [L-dopa]), or various anticholinergic or antiglutamatergic

agents9. Various allegedly neuroprotective agents have been proposed as treatments, for

example riluzolelO, coenzyme Q 1011,glial-derived neurotrophic factor (GDNF)12 and

vitamin El3. However, compelling supporting data are lacking for clinical use of any of

these compounds9. No treatment is available that has been shown to increase the numbers

or sizes of dopaminergic nigrostriatal terminals in Parkinsonian brains.

We previously observed that chronic oral administration of two circulating

phosphatide precursors, uridine (as uridine-5' -monophosphate [UMP]) and

docosahexaenoic acid (DHA), to gerbils increases the amounts of brain phosphatides and

specific pre- and postsynaptic proteins per brain cellI4,15,and that chronic administration

ofuridine to rats enhances potassium-evoked striatal dopamine releasel6. Chronic

administration ofUMP and DHA also increases the number of hippocampal dendritic

spines in gerbil brainsl7. We have now tested the effects ofUMP and/or DHA on

chemical and behavioral aspects of impaired DA neurotransmission in a rat model of PD.

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MATERIALS AND METHODS

Animals

Male Sprague Dawley rats (200-250 g BW; Charles River, Wilmington MA) were

housed at room temperature, under 12-h Iight/12-h dark conditions with ad libitum access

to food and water. Experiments were conducted according to the National Institutes of

Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80-23)

revised 1996, and formal approval to conduct them was obtained from MIT's Committee

on Animal Care. All efforts were made to minimize the number of animals used and their

suffering.

Surgery

Rats anesthetized intraperitoneally with ketamine and xylasine (80 and 10 mglkg

BW, respectively) received unilateral injections of 6-hydroxydopamine (6-0HDA;

Sigma, St. Louis, MO), each containing 8 Ilg dissolved in 2 IIIof 0.3% L-ascorbic

acidlO.9%saline, into their right striata. Two small burr holes were drilled using a bone

drill (Ideal micro-drill; CellPoint Scientific, Gaithersburg, MD) on the right side ofthe

skull; injections were made into each using a 10-1l1microsyringe (Hamilton Company,

Reno, NY) fitted with a 26-gauge steel cannulal8. Coordinates for the injection sites

were: (first injection site) anterior-posterior (AP) = +0.5 mm relative to bregma, medial-

\t. lateral (ML) = -2.4 mm fromthe mid-line,dorsal-ventral(DV)= -5.0 mm from dura;

(second injection site) AP =-0.5 mm relative to bregma, ML = -4.2 from the mid-line and

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DV = -5.0 mm from dura, with the tooth bar set at 0 mm. The 6-0HDA was injected at a

rate of 1 ~Vmin using a microinfusion pump (CMAIlOO;Bioanalytical Systems, W.

Lafayette, IN).

Treatments

In one set of experiments, control rats were given access to standard 0.1%

choline-containing rodent diet (Teklad Global 16% protein rodent diet, Harlan-Teklad,

Madison, WI); a second group received this diet supplemented with uridine

monophosphate (UMP; 0.5%); a third group of rats received the control

(unsupplemented) diet plus, each day, 300 mglkg of docosahexaenoic acid (by gavage inL

1 ml/kg of 5% Arabic gum solution dissolved in deionized water); and a fourth group

received the 0.5% UMP supplemented diet plus, by gavage, 300 mglkg ofDHA. All

treatments were given for 4 weeks starting 3 days prior to 6-0HDA injections. Animals

not receiving DHA were gavaged daily for 4 weeks with its vehicle, 5% Arabic gum

solution.

In the other set of experiments, the same protocol was followed except that a

lower dose (l00 mglkg) ofDHA, in combination with UMP-supplemented diet was also

tested.

Assessment of Rotational Behavior

Each animal was systematically handled on a regular basis throughout the study.

Drug-induced (d-amphetamine; 5 mg/kg, Lp.) rotational behavior was tested 3 weeks

following 6-0HDA treatment. After receiving the d-amphetamine, animals were placed

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into metal cylinders (35 cm diameter, 37 cm height) and their movements recorded

between 15 and 45 minutes after injection, using a camcorder placed 1.5 meters above the

cylinders. Ipsilateral (towards the lesioned side) rotations by each rat were counted by a

blind observer. Rats were allowed a drug washout period of three days before sacrifice.

Biochemical Assays

Treatment with phosphatide precursors was terminated on the 28th day (24 days

after lesioning). The following day rats were sacrificed and their brains were removed;

striata were dissected, transferred into pre-weighed eppendorf tubes, and frozen

immediately on dry ice. Samples were subsequently assayed for DA, tyrosine

hydroxylase (TOH) activity, TOH and Synapsin-I protein levels, and phospholipid

composition.

DA was assayed using an ESA Coulochem II 51OOAdetector (E 1= -175 mV; E2=

325 mY; Eguard= 350 mY) with an ESA Microdialysis Cell (modeI5014B, ESA, North

Chelmsford, MA). The flow rate of the mobile phase (MD-TM, ESA) was 0.4 mUmin.

The column (ESA MD 150, 3 x 150 mm, 311m) was kept at 40°C. Samples were injected

and analyzed by Alltech AllChromsystem (Alltech, Deerfield, IL) as described previously

[16].

TOH activity was measured according to a previously-described protocol19.The

rate ofl4C02 formation from 1-[carboxy-14C]tyrosine(Perkin-Elmer, Waltham, MA) was

measured, and TOH activity was expressed as nmol DOPA formed per hour per mg

protein tissue.

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The levels ofTOH and Synapsin-l proteins were determined by Western blot as

described previously15.The following primary antibodies were used: mouse anti-

Synapsin-l (Calbiochem, EMD Chemicals, San Diego, CA), and rabbit anti-tyrosine

hydroxylase (Abeam, Cambridge, MA).

Striatal phospholipids were extracted and measured as described previously15.

Statistics

Data were analyzed using one-way analysis of variance (ANOYA) followed by

post hoc Tukey test. Student's t-test was applied as appropriate. Yalues ofP less than

0.05 were considered to be significant. Data are presented as the mean I S.E.M.

RESULTS

As expected20, intrastriatal injection of6-0HDA, a neurotoxin that destroys

dopaminergic nerve terminals, caused 64% and 65% decreases in striatal DA levels and

TOH activity, respectively. There was a 35% loss in TOH protein and a 15% loss in

Synapsin-l, while phospholipid levels did not change in the lesioned striatum compared

with the intact striatum.

Rotational Behavior

Intraperitoneal injection of d-amphetamine (5 mg/kg) 3 weeks following 6-

OHDA injections into right striata induced ipsilateral rotations in all rats. Compared with

control rats (receiving the unsupplemented diet and DHA's vehicle by gavage), oral

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administration for 24 days ofthe phosphatide precursors UMP, DHA, or UMP plus DHA

significantly reduced the number of rotations by 48%,47%, or 57%, respectively (all

P<0.05) (Table I). In a separate experiment, the effect ofDHA on rotational behavior

was confirmed with the 300 mg/kg dose, but not with a 100 mglkg dose (data not shown).

Striatal DA Levels

DA levels in the lesioned (right) striata were 36% of those in the intact (left)

striata of control rats (0.25 :i: 0.02 vs 0.70 :i: 0.02 nmol/mg protein) (Table 2). Chronic

administration of the UMP-supplemented diet alone increased DA levels in the lesioned

but not the unlesioned striata (by 41%; P<O.OI)(Table 2). Combining UMP and DHA

increased DA levels in both the lesioned (by 37%; P<0.05) and the intact (18%; P<O.OI)

striata (Table 2). Supplementation ofDHA alone tended to increase DA levels in lesioned

striata, but not significantly (Table 2).

TOH Activity and TOH Protein Levels

TOH activity in the lesioned (right) striata was 35% ofthat in intact striata (1.40 :i:

0.06 vs 3.98:i: 0.26 nmol DOPA formed/h/mg protein) (Table 3A). UMP administration,

alone or with DHA, increased TOH activity in lesioned striata by 53% or 52%,

respectively (Table 3A), but had no effect on TOH activity in intact striata.

DHA supplementation alone, and when given with UMP, increased TOH protein

levels in the lesioned striata, by 21% and 22%, respectively (Table 3B); UMP failed to

produce this significant increase. TOH protein levels in lesioned striata were reduced by

about 35% compared with those in intact striata of control rats (data not shown).

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... - -. ... .. .. . ......


Synapsin-l Levels

Levels of Synapsin-l, a specific presynaptic protein located on vesicular

membranes, were reduced in the lesioned striata by 15% (P<O.OO1), and restored by all

treatments (Figure 1).

Phospholipid Levels

As expectedfromthe neurochemicalspecificityof its toxicity, injectionof 6-

OHDA into right striata did not change phospholipid levels significantly (i.e., total

phospholipids in lesioned striata and intact striata of control rats were 376 ~ 11 and 377 ~

16 nmol/mg protein, respectively) (Table 4). Administration of both UMP and DHA

increased total phospholipid levels by 15% (P<0.05) and 21 % (P<O.OO1) in lesioned and

the intact striata, respectively (Table 4). Levels of individual phospholipids such as

phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS) and

sphingomyelin (SM) were also significantly increased in lesioned and intact striata, by

18-60% and 19-45% following UMP plus DHA treatment, respectively (Table 4).

Phosphatidylinositol (PI) levels in intact striata were increased by 2-fold and 2.7-fold

following DHA supplementation alone and in combination with UMP, respectively,

while PI increased by 50% in lesioned striata with either treatment (Table 4). DHA

supplementation also alone increased striatal PE and PS, levels by 19% and 27% in the

intact striata, respectively (Table 4).

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DISCUSSION

These data show that chronic oral administration of the phosphatide precursors

uridine (as UMP) and/or DHA significantly reduces the number of d-amphetamine-

induced ipsilateral rotations in rats with unilateral striatal lesions caused by 6-0HDA

(Table I). UMP alone or in combination with DHA significantly increased DA levels

(Table 2) and TOH activity (Table 3A) in lesioned striata, while UMP plus DHA

increased DA levels significantly in intact striata (Table 2). DHA alone enhanced TOH

protein levels (Table 3B) and brain PI levels (Table 4) significantly, while its

combination with UMP likewise enhanced levels ofTOH protein (Table 3B) and all

phosphatide classes investigated (i.e., PC, PE, PS, SM, and PI) in lesioned striata as well

as in intact striata (Table 4). Moreover, levels ofSynapsin-l, a presynaptic vesicular

protein21,which were reduced in lesioned striata of control rats, were restored following

UMP, DHA or UMP plus DHA treatment (Figure I).

In the present study, the injection of 6-0HDA (8 ~g) into two different regions of

the right striatum caused about a 65% decrease in TOH activity in the lesioned striata,

compared with that in the intact (left) striata of control rats (1.40 :f: 0.06 vs 3.98 :f: 0.26

nmol DOPA formed/h/mg protein) (Table 3A). Similarly, DA levels in the lesioned

striata decreased by about 64% compared with those in the intact (left) striata of control

rats (0.25:f: 0.02 vs 0.70:f: 0.02 nmol/mg protein) (Table 2). These observations are in

good accord with previous studies which reported a 50-80% reduction in TOH-

immunoreactive cells in rat striatum following two injections of6-0HDA (each 10 ~g)18.

It has long been known that peripheral administration of d-amphetamine to rats with

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unilateral striatal lesions induced by 6-0HDA causes ipsilateral circling by acting

presynaptically, presumably by releasing DA trom nigrostriatal terminals which are more

abundant on the intact side22.Consistently, rats in our study exhibited ipsilateral rotations

following intraperitoneal injection of d-amphetamine 3 weeks after their right striata were

lesioned by 6-0HDA. Chronic oral supplementation with the phosphatide precursors

uridine and/or DHA ameliorated this rotational behavior: Giving UMP alone, DHA

alone, or a combination ofUMP and DHA reduced d-amphetamine-induced ipsilateral

rotations by 48%, 47%, or 57%, respectively (Table I). These reductions were

accompanied by recoveries in biochemical parameters, discussed below.

Consistent with our previous studiesl6, chronic oral supplementation with uridine

enhanced by 41% the reduced DA levels in lesioned striata following 6-0HDA injections

(Table 2). Moreover, a combination of uridine and DHA also increased striatal DA levels

(by 37%) on the lesioned side (Table 2). TOH activity which was reduced by 65% in the

lesioned striata likewise was partially restored by 53% and 52%, following treatment

with UMP alone or with UMP plus DHA, respectively (Table 3A). TOH protein levels,

which were reduced by 35% in the lesioned striata were enhanced significantly by 21%

and 22% following DHA, or UMP plus DHA administrations (Table 3B). Decreases (by

15%) in levels of the presynaptic vesicular protein Synapsin-121in lesioned striata were

totally restored by all three treatments (Figure I). Levels of striatal phosphatides did not

differ in lesioned vs intact striata (Table 4). UMP plus DHA treatment increased the

amounts oftotal phosphatides, as well as of individual phosphatide classes, in both

lesioned and intact striata (Table 4). DHA alone also significantly increased PI levels in

the lesioned, and PE, PS and PI levels in the intact striata (Table 4). That the amounts of

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phosphatides did not differ significantly, and those of Synapsin-l were only 15% lower in

lesioned vs intact striata was probably due to the fact that dopaminergic terminals

constitute only a small fraction of all neuronal structures in the striatum. These data

confirm our previous results which showed enhanced amounts of synaptic membranesl4.1S

and numbers of hippocampal dendritic spines17following chronic oral administration of

the phosphatide precursors uridine and DHA.

Synthesis of brain PC, the most abundant membrane phosphatide, via the

predominant Kennedy pathway3 utilizes various circulating compounds, two of which

are a pyrimidine (e.g., uridine) and a PUFA (e.g., DHA). Uridine, on entering the brain

cells by the high-affinity CNT2 transporter located at the blood-brain barrier24,is

phosphorylated to UTP by uridine/cytidine kinase (UCK) and then coverted to CTP, the

compound that usually rate-limits PC synthesis, by CTP-synthase. DHA, on entering the

brain2s,26,can be activated to docosahexaenoyl-CoA and acylated to the sn-2 position of

DAG27to form DAG species rich in DHA28.Each step in the incorporation of uridine or

DHA into brain phosphat ides is catalyzed by a relatively low-affinity enzyme; this

characteristic allows the administration of each precursor to affect the rate of phosphatide

synthesis14.29.

In conclusion, these data show that, chronic oral administration of the phosphatide

precursors uridine and DHA, probably by enhancing the amount of synaptic membranes,

can ameliorate the loss of dopaminergic terminals in 6-0HDA-lesioned striata.

Administering uridine plus DHA to patients could possibly enhance the efficacy of

current treatments for PO, by partially restoring dopaminergic nigrostriatal transmission.

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Page 13 of 23

t:

Movement Disorders

Acknowledgements

This work was supported by grants from the National Institutions of Health (Grant

MH-28783), the Center for Brain Sciences and Metabolism Charitable Trust and the

Turkish Academy of Sciences (IH Dlus).

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REFERENCES

1. Fearnley JM, Lees AJ. Ageing and Parkinson's disease: substantia nigra regional

selectivity. Brain 1991;114:2283-2301.

2. Damier P, Hirsch EC, Agid Y, Graybiel AM. The substantia nigra of the human brain.

II. Patterns of loss of dopamine-containing neurons in Parkinson's disease. Brain

1999; 122: 1437-1448.

3. Ingham CA, Hood SH, Arbuthnott GW. Spine density on neostriatal neurones changes

)with 6-hydroxydopamine lesions and with age. Brain Res 1989;503:334-338.

4. McNeill TH, Brown SA, Rafols JA, Shoulson I. Atrophy of medium spiny I striatal

dendrites in advanced Parkinson's disease. Brain Res 1988;455:148-152.

5. Stephens B, Mueller AJ, Shering AF, et al. Evidence of a breakdown of corticostriatal

connections in Parkinson's disease. Neuroscience 2005;132:741-754.

6. Ingham CA, Hood SH, van Maldegem B, Weenink A, Arbuthnott GW. Morphological

changes in the rat neostriatum after unilateral 6-hydroxydopamine injections into the

nigrostriatal pathway. Exp Brain Res 1993;93:17-27.

7. Agid Y. Parkinson's disease: pathophysiology. Lancet 1991;337:1321-1324.

8. Horstink M, Tolosa E, Bonuccelli U, et al. Review of the therapeutic management of

Parkinson's disease. Report of ajoint task force of the European Federation of

Neurological Societies (EFNS) and the Movement Disorder Society-European Section

(MDS-ES). Part II: late (complicated) Parkinson's disease. Eur J Neurosci 2006;13:1186-

1202.

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9. Horstink M, Tolosa E, Bonuccelli D, et al. Review of the therapeutic management of

Parkinson's disease. Report of ajoint task force of the European Federation of

Neurological Societies and the Movement Disorder Society-European Section. Part I:

early (uncomplicated) Parkinson's disease. Eur J Neurosci 2006; 13:1170-1185.

10. Jankovic H, Hunter C. A double-blind, placebo-controlled and longitudinal study of

riluzole in early Parkinson's disease. Parkinsonism Relat Disord 2002;8:271-276.

11. Shults CW, Oakes 0, Kieburtz K, et al. Effects of coenzyme Q10 in early Parkinson's

disease: evidence of slowing of the functional decline. Arch NeuroI2002;59: 1541-1550.

12. Nutt JG, Burchiel KJ, Comella CL, et al. Randomized, double-blind trial of glial cell

line-derived neurotrophic factor (GDNF) in PD. Neurology 2003;60:69-73.

13. Parkinson Study Group. Effect of deprenyl on the progression of disability in early

Parkinson's disease. New Eng J Med 1989;321:1364-1371.

14. Wurtman RJ, Dlus IH, Cansev M, Watkins CJ, Wang L, MarzloffG. Synaptic

~0proteins and phospholipids are increased in gerbil brain by administering uridine plus

docosahexaenoic acid orally. Brain Res 2006;1088:83-92.

15. Cansev M, Wurtman RJ. Chronic administration ofdocosahexaenoic acid or

eicosapentaenoic acid, but not arachidonic acid, alone or in combination with uridine,

increases brain phosphatide and synaptic protein levels in gerbils. Neuroscience In Press.

16. Wang L, Pooler AM, Albrecht MA, Wurtman RJ. Dietary uridine-5'-monophosphate

supplementation increases potassium-evoked dopamine release and promotes neurite

outgrowth in aged rats. J Mol Neurosci 2005;27:137-145.

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17. Sakamoto T, Wurtman RJ. Increased dendritic spine density in gerbil hippocampus

following oral UMP and DHA supplementation. 10thInternational Conference on

Alzheimer's Disease and Related Disorders, Madrid, Spain, 2006.

18. Kirik D, Rosenblad C, Bjorklund A. Characterization of behavioral and

neurodegenerative changes following partial lesions of the nigrostriatal dopamine system

induced by intrastriatal 6-Hydroxydopamine in the rat. Exp Neurol 1998;152:259-277.

19. Waymire IC, Bjur R, Weiner N. Assay of tyrosine hydroxylase by coupled

decarboxylation of dopa formed from 1-14C-L-tyrosine.Anal Biochem 1971;43:588-600.

20. Hefti F, Melamed E, Wurtman RJ. Partial lesions of the dopaminergic nigrostriatal

system in rat brain: biochemical characterization. Brain Res 1980;195:123-137.

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release. Cell Mol Life Sci 2002;59:589-595.

22. Ungerstedt U. Postsynaptic supersensitivity after 6-hydroxydopamine induced

degeneration of nigro-striatal dopamine system. Acta Physiol Scand Suppl1971 ;367:69-

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23. Kennedy EM, Weiss SB. The function of cytidine coenzymes in the biosynthesis of

phospholipids. J BioI Chern 1956;222:193-214.

24. Cansev M. Uridine and cytidine in the brain: Their transport and utilization. Brain Res Brain

Res Rev 2006;52:389-397.

25. Abumrad NA, Park JH, Park CR. Permeation of long-chain fatty acid into adipocytes.

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26. Kamp F, WesterhoffHV, Hamilton JA. Movement offatty acids, fatty acid

analogues, and bile acids across phospholipid bilayers. Biochemistry 1993;32:11074-

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27. Robinson PJ, Noronha J, DeGeorge JJ, Freed LM, Nariai T, Rapoport SL A

quantitative method for measuring regional in vivo fatty-acid incorporation into and

turnover within brain phospholipids: review and critical analysis. Brain Res Brain Res

Rev 1992; 17: 187-214.

28. Bazan NG. Supply ofn-3 polyunsaturated fatty acids and their significance in the central

nervous system. In: Wurtman RJ and Wurtman JJ, editors. Nutrition and the Brain. New York:

Raven Press; 1990. p 1-24 (Volume 8).

29. Cansev M, Watkins CJ, van der Beek EM, Wurtman RJ. Oral uridine 5' monophosphate

(UMP) increases brain COP-choline levels in gerbils. Brain Res 2005;1058:101-108.

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Movement Disorders

Figure Legend

Fieure 1. Synapsin-l Levels

Experiments were carried out as described in Table 1 legend. On the 29thday of treatment

rats were sacrificed; brains were removed, and striata were dissected and assayed for

Synapsin-llevels using Western Blotting. ***P<O.OOIcompared with left (intact)

striatum using Student's t test.

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18


Table 1. Rotational Behavior

Rats were given, daily for 28 days, either a control or a UMP-containing (0.5%) diet

(both also contained 0.1% choline), and received, by gavage, DHA (300 mg/kg; in a

vehicle of 5% Arabic gum solution) or just its vehicle. Three days after the treatment had

started (day 4),8 Jlg of 6-0HDA dissolved in 2 JlIof 0.3% L-ascorbic acidlO.9% saline

was injected into two different sites within their right striata. Three weeks following the

6-0HDA injections (day 25), ipsilateral (towards the lesioned side) rotations by each rat

were recorded for 30 minutes, between 15 and 45 min, after the i.p. injection of d-

amphetamine (5 mglkg). *P<0.05; and **P<0.025 compared with Control diet + Vehicle

group using One Way ANOVA followed by post hoc Tukey test

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Table lA

Treatment Ipsilateral rotations/30 min

Control diet + Vehicle 151 :t21

UMP diet + Vehicle 79 :t 22*

Control diet + DHA 81 :t 12*

UMP diet + DHA 65:t 18**


Table 2. Dopamine (DA)Levels

Experiments were carried out as described in Table I legend. Striata were assayed for DA

levels. *P<0.05; and **P<O.OIcompared with Control diet + Vehicle group using One

Way ANOV A followed by post hoc Tukey test.

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Dopamine Levels (nmollmg protein)

Treatments Left (Intact) Striatum Right (Lesioned) Striatum

Control diet + Vehicle 0.704:t 0.024 0.252:t 0.018

UMP diet + Vehicle 0.749:t 0.022 0.355 :t 0.025**

Control diet + DHA 0.745 :t 0.021 0.311 :t 0.016

UMP diet + DHA 0.830 :t 0.022** 0.345:t 0.017*


Table 3. Tyrosine Hydroxylase (TOH) Activity and TOH Protein Levels

Experiments were carried out as described in Table 1 legend. Striata were assayed for

TOH activity (Table 3A) using radioenzymatic method and TOH protein levels (Table

3B) using Western Blotting. *P<0.05; and **P<O.OIcompared with Control diet +

Vehicle group using One Way ANOVA followed by post hoc Tukey test.

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Table 3A TOH Activity (nmol DOPA formedlhlmg protein)


Control diet + Vehicle 3.983 :t 0.26 1.405 :t 0.06

UMP diet + Vehicle 3.591 :t 0.20 2.144:t 0.19*

Control diet + DHA 4.014:t 0.12 1.906 :t 0.17

UMP diet + DHA 4.189 :t 0.24 2.131 :t 0.17*

Table 3B TOH Protein Levels (Percent of Control)


Control diet + Vehicle 100:t3 100:t3

UMP diet + Vehicle 95 :t 3 114 :t 4

Control diet + DHA 98 :t 5 121 :t2**

UMP diet + DHA 100:t5 122 :t 6**--- -- --


Table 4. Phospholipid Levels

Experiments were carried out as described in Table 1 legend. *P<0.05; **P<O.OI;and

***P<O.OOI compared with Control Diet + Vehicle group using One Way ANOV A

followed by post hoc Tukey test. Total PL, Total Phospholipids; PC,

Phosphatidylcholine; PE, Phosphatidylethanolamine; PS, Phosphatidylserine; PI,

Phosphatidylinositol; SM, Sphingomyelin.

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Left (Intact) Striatum Phospholipid Levels (nmol/mg protein)

Treatments Total PL PC PE PS SM PI

Control diet + Vehicle 377:t 16 128 :t 5 125 :t 8 22 :t 1 16:t 1 lO:tl

UMP diet + Vehicle 394:t 15 126 :t 4 139:t5 25 :t 1 16:t 1 l1:tl

Control diet + DHA 402 :t 9 142 :t 3 149 :t 4* 28 :t 1** 16:t 1 20 :t 1**

UMP diet + DHA 455 :t 9*** 152 :t 4** 158 :t 7** 32 :t 1*** 21 :t 2* 27 :t 3***

Right (Lesioned) Striatum Phospholipid Levels (nmol/mg protein)

Treatments Total PL PC PE PS SM PI

Control diet + Vehicle 376 :t 11 130 :t 4 127 :t 6 22 :t 1 15 :t 1 lO:tl

UMP diet + Vehicle 424:t 14 142 :t 5 138:t3 25 :t 1 19:t 1 14:t 1

Control diet + DHA 419:t 13 145 :t 5 144 :t 7 25 :t 1 16:t 1 15 :t 2*

UMP diet + DHA 433 :t 14* 153 :t 4** 150 :t 3* 29 :t 1*** 24 :t 4* 15 :t 1*


UMP Diet + Vehicle

left (Intact) Righi (lesioned)Striatun.. SbiabJm

270x355mm (300 X 300 DPI)

John Wiley & 50ns

Control Diet + Vehicle140 140

120 .... 120

§ 100 *** § 10010 1;;1: 80 'C 80lii lii- -u 60 u GO«I «IB B

O O.. ..

20 20

left (Inlact) Right (lesioned)StriabJm Striatum

- --T

T

Control Diet + DHA UMP Diet + DHA140 140

120 ... ..'"1

.. ..§ 100

T T E 100 T T::r

1;; -«I

'C 80 'C 80lii liiV -

60 u 60«I «I.5 .5

o o.. ..

20 20

0 oU I L

left (Intact) Right (lesioned) left (Inlact) Right (lesioned)Striatum Striatum Striatum Striatum

RICHARDJ. WURTMAN

ITINERARY FORTUESDAY SEPTEMBER 4 - SUNDAY SEPTEMBER 9,2007

BOSTON-AMSTERDAM- NICE-LONDON- BOSTON

Note: Dr. Tremblay's mobile number: 06 07 55 94 00

DEPART ARRIVE ARRIVALDATE LEAVE FROM FL.# TIME AT TIME HOTEL PHONE & FAX

Tue9/4 Boston NW38 7:10 P Schipol 8:00 A 5th Airport Sheraton 011 31 203164300 (P)Amsterdam 011 31 203164399 (F)

Grand Softtel 31 (0) 20 555 3 111 (P)Amsterdam 31 (0) 20 555 3 222 (F)Hotel Softtel La 33(0) 49 297 7000 (P)

Wed. 9/6 Amsterdam KLM 12:10 P Nice 2:15 P Napoule 33(0) 493495150 (F)1265

Sun. 9/9 Nice BA 347 1:35 P London 2:35 P

Sun. 9/9 London AA 155 6:05 P Boston 8:20 P

Documents

Page 1 of 23 Movement Disorders