Restorative Effects of Uridine Plus Docosahexaenoic Acid in a Rat Model of Parkinson’s Disease

Summary

Administering uridine-5’-monophosphate (UMP) and docosahexaenoic acid (DHA) increases synaptic membranes (as characterized by pre-and post-synaptic proteins) and dendritic spines in rodents. We examined their effects on rotational behavior and dopaminergic markers in rats with partial unilateral 6-hydroxydopamine (6-OHDA)-induced striatal lesions. Rats receiving UMP, DHA, both, or neither, daily, and intrastriatal 6-OHDA 3 days after treatment onset, were tested for d-amphetamine-induced rotational behavior and dopaminergic markers after 24 and 28 days, respectively. UMP/DHA treatment reduced ipsilateral rotations by 57% and significantly elevated striatal dopamine, tyrosine hydroxylase (TH) activity, TH protein and Synapsin-1 on the lesioned side. Hence, giving uridine and DHA may partially restore dopaminergic neurotransmission in this model of Parkinson’s Disease.

Parkinson’s disease (PD) is characterized by the progressive degeneration of dopaminergic nigrostriatal neurons, and reductions in striatal dopamine (DA) levels, dopaminergic synapses, and the density of dendritic spines on striatal medium spiny neurons. No treatment for PD currently available is thought to restore the numbers of dopaminergic nigrostriatal terminals or striatal dendritic spines.

We previously observed that chronic oral administration of two circulating phosphatide precursors, uridine (as UMP) and the omega-3 fatty acid DHA, along with dietary choline, can increase neuronal levels of the phosphatides and of specific proteins that characterize synaptic membranes (Wurtman et al., 2006), as well as the numbers of dendritic spines, in rodent brain (Sakamoto et al., 2007). The present report examines the effects of giving these precursors in a rat model of PD with unilateral neurotoxin-induced nigrostriatal damage and impaired DA neurotransmission. In this model 6-hydroxydopamine (6-OHDA), injected into one corpus striatum, causes ipsilateral decreases in DA synthesis and release, and characteristic turning behavior.

Male Sprague-Dawley rats (200–250 g) consumed the control diet (containing 16% protein and 0.1% choline) fortified with UMP (0.5%) and received by daily gavage (1 ml/kg) 300 mg/kg of DHA in 5% gum arabic solution; control rats were gavaged with DHA’s vehicle. Three days after the start of UMP/DHA administration, rats were injected with 6-OHDA (8 µg in 2 µl of 0.3% ascorbic acid/0.9% saline) into two different sites within their right striata (Kirik et al., 1998). In preliminary experiments that used DA levels, TH levels, and TH activity as markers, we found that pretreatment with UMP and DHA did not diminish the initial toxic responses to the 6-OHDA. Hence, subsequent studies used animals killed 28 days after starting UMP/DHA, a period previously shown to reliably increase membrane phosphatides, synaptic proteins (Wurtman et al., 2006) and dendritic spines (Sakamoto, et al., 2007).

Rotational behavior was induced by intraperitoneal injection of d-amphetamine (5 mg/kg) 3 weeks after animals received the 6-OHDA treatment (day 25), and ipsilateral rotations by the rats, videotaped between 15 and 45 minutes following the d-amphetamine injection, were counted by two blind observers. Animals were sacrificed 3 days after testing for rotational behavior. Striatal DA was measured using an HPLC assay (Wang et al., 2005); and TH activity was determined by a radiometric method (Ulus and Wurtman, 1976). Striatal phospholipids were extracted and individual phospholipid classes were separated and quantified by measuring their phosphorus contents (Cansev and Wurtman, 2007). TH, Synapsin-1 and β-tubulin proteins were analyzed by Western blot (Cansev and Wurtman, 2007).

Data were analyzed using one-way analysis of variance (ANOVA) followed by post hoc Tukey tests. Comparisons between values obtained from intact and lesioned striata were made using Student’s t-test. Data are presented as mean±SEM; P less than 0.05 was considered significant.

Animals that received UMP, DHA, or UMP plus DHA for 24 days exhibited significant reductions in the numbers of d-amphetamine-induced rotations, compared with those in control rats, by 48%, 47%, or 57% (all P<0.05), respectively (Table 1). The numbers of rotations exhibited by individual animals in all of the experimental groups were inversely correlated with the dopamine contents (r=−0.447;P<0.05) and TH activities (r=−0.546;P<0.01) in the lesioned striata.

Table 1.

Rotational Behavior

Treatment	Ipsilateral rotations/30 min
Control diet + Vehicle	151 ± 21
UMP diet + Vehicle	79 ± 22*
Control diet + DHA	81 ± 12*
UMP diet + DHA	65 ± 18**

Rats receiving UMP, DHA, both, or neither, daily, and intrastriatal 6-OHDA 3 days after treatment onset, were tested for d-amphetamine-induced rotational behavior after 24 days. Ipsilateral rotations were recorded for 30 minutes, between 15 and 45 min, after i.p. injection of 5 mg/kg of d-amphetamine. N = 6–9 in each group.

^*P<0.05; and

^**P<0.025 compared with Control diet + Vehicle group using One Way ANOVA followed by post hoc Tukey test.

Among control rats DA levels and TH activity (Table 2), and TH protein (Figure 1A) in the lesioned striata were 64%, 65%, and 35% lower (all P<0.001) than those in the intact striata, respectively, and levels of the two DA metabolites dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) were reduced by about 50%. UMP administration, alone or with DHA, restored DA levels [by 41% (P<0.01) or by 37% (P<0.05)], as well as TH activity [by 53% (P<0.05) or 52% (P<0.05)] in lesioned striata (Table 2). Reductions in TH protein levels in lesioned striata were partially restored, increasing by 21% (P<0.01) or 22% (P<0.01), following DHA supplementation alone or after UMP plus DHA (Figure 1A). UMP plus DHA treatment also increased DOPAC levels from 0.18±0.01 to 0.22±0.01 nmol/mg protein (P<0.05) and HVA levels from 0.15±0.01 to 0.19±0.04 nmol/mg protein (P<0.05) in lesioned striata.

Table 2.

Dopamine (DA) Levels and Tyrosine Hydroxylase (TH) Activity

Table 2A	Dopamine Levels (nmol/mg protein)
Treatments	Left (Intact) Striatum	Right (Lesioned) Striatum
Control diet + Vehicle	0.704 ± 0.024	0.252 ± 0.018a
UMP diet + Vehicle	0.749 ± 0.022	0.355 ± 0.025**
Control diet + DHA	0.745 ± 0.021	0.311 ± 0.016
UMP diet + DHA	0.830 ± 0.022**	0.345 ± 0.017*

Table 2B	TH Activity (nmol DOPA formed/h/mg protein)
Treatments	Left (Intact) Striatum	Right (Lesioned) Striatum
Control diet + Vehicle	3.983 ± 0.26	1.405 ± 0.06a
UMP diet + Vehicle	3.591 ± 0.20	2.144 ± 0.19*
Control diet + DHA	4.014 ± 0.12	1.906 ± 0.17
UMP diet + DHA	4.189 ± 0.24	2.131 ± 0.17*

Rats receiving the treatments for 28 days were sacrificed on day 29. Striata were assayed for DA levels (A) and TH activity (B). N = 6–9 in each group.

^*P<0.05; and

^**P<0.01 compared with Control diet + Vehicle group within the same localization using One Way ANOVA followed by post hoc Tukey test.

^aP<0.001 compared with values from left striata of animals that received the Control diet + Vehicle using Student’s t test.

Figure 1. TH Protein and Synapsin-1 Levels.

Homogenates of the lesioned striata were analyzed for TH protein (A) and Synapsin-1 (B). Values obtained from rats treated with UMP, DHA, and UMP+DHA were expressed by reference to those (normalized to 100) obtained from rats that received the control diet+vehicle (control group). β -tubulin was used as the loading control. N=6 per group. *P<0.05 and **P<0.01 compared with control group using One Way ANOVA.

Levels of Synapsin-1, reduced in lesioned striata by 15% (P<0.001) (Figure 1B), were increased significantly by UMP, DHA, or UMP plus DHA (by 17%, 16%, or 25% respectively (Figure 1B). In contrast, levels of β-tubulin, our loading control, were unaffected by either 6-OHDA injection or dietary treatments (Figure 1). Since intrastriatal 6-OHDA is selectively neurotoxic to DA terminals, and since these terminals comprise only a small proportion of striatal structures, its administration failed to affect striatal phospholipid levels. Administration of UMP plus DHA did increase these levels (Table 3), as expected (Wurtman et al., 2006) in both the lesioned and the control sides, those of each of the individual phosphatides [i.e., phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS) phosphatidylinositol (PI) and sphingomyelin (SM)] also increasing significantly (Table 3). This treatment is known to increase phosphatide levels significantly throughout the brain (Cansev and Wurtman, 2007), and probably affects most or all types of brain cells since all, apparently, utilize the same substrate-unsaturated Kennedy Cycle enzymes (Wurtman et al., In Press) for phosphatide synthesis.

Table 3.

Phospholipid Levels

Left (Intact) Striatum	Phospholipid Levels (nmol/mg protein)
Treatments	Total PL	PC	PE	PS	SM	PI
Control diet + Vehicle	377 ± 16	128 ± 5	125 ± 8	22 ± 1	16 ± 1	10 ± 1
UMP diet + Vehicle	394 ± 15	126 ± 4	139 ± 5	25 ± 1	16 ± 1	11 ± 1
Control diet + DHA	402 ± 9	142 ± 3	149 ± 4*	28 ± 1**	16 ± 1	20 ± 1**
UMP diet + DHA	455 ± 9***	152 ± 4**	158 ± 7**	32 ± 1***	21 ± 2*	27 ± 3***
Right (Lesioned) Striatum	Phospholipid Levels (nmol/mg protein)
Treatments	Total PL	PC	PE	PS	SM	PI
Control diet + Vehicle	376 ± 11	130 ± 4	127 ± 6	22 ± 1	15 ± 1	10 ± 1
UMP diet + Vehicle	424 ± 14	142 ± 5	138 ± 3	25 ± 1	19 ± 1	14 ± 1
Control diet + DHA	419 ± 13	145 ± 5	144 ± 7	25 ± 1	16 ± 1	15 ± 2*
UMP diet + DHA	433 ± 14*	153 ± 4**	150 ± 3*	29 ± 1***	24 ± 4*	15 ± 1*

Rats receiving the treatments for 28 days were sacrificed on day 29. N = 6–9 in each group.

^*P<0.05

^**P<0.01; and

^***P<0.001 compared with Control Diet + Vehicle group within the same localization using One Way ANOVA followed by post hoc Tukey test. Total PL, Total Phospholipids; PC, Phosphatidylcholine; PE, Phosphatidylethanolamine; PS, Phosphatidylserine; PI, Phosphatidylinositol; SM, Sphingomyelin.

These data show that chronic oral administration of uridine (as UMP) and/or DHA, which can stimulate brain phosphatide and synaptic protein synthesis (Wurtman et al., 2006), significantly reduces a behavioral consequence of unilateral damage to a nigrostriatal tract, i.e., amphetamine-induced ipsilateral rotational behavior, and partially restores biochemical markers of DA terminals. The observed effects of 6-OHDA on levels of DA and its metabolites, and on TH protein and activity, agree well with the previously-demonstrated 50–80% reduction in TH-immunoreactive-fiber density in rat striatum following two intrastriatal injections of 6-OHDA at doses (each 10 µg) (Kirik et al., 1998) in the range used here (8 µg). As expected (Ungerstedt, 1971; Hefti et al., 1980; Olds et al., 2006), the unilaterally-lesioned rats exhibited ipsilateral rotational behavior when given d-amphetamine 3 weeks after lesioning. If the animals also chronically received UMP alone, DHA alone, or a combination of UMP and DHA, d-amphetamine-induced ipsilateral rotations were reduced by 48%, 47%, or 57%, respectively, compatible with the partial restoration of striatal dopamine levels and, probably, release.

Giving UMP plus DHA does not protect nigrostriatal neurons from the initial toxic actions of 6-OHDA, but subsequently, it partially restores striatal DA, DOPAC and HVA levels, and TH levels and activity, possibly by increasing the number and/or size of nigrostriatal terminals and synapses. Unfortunately our laboratories were not able to perform histochemical studies that might have provided evidence of increases in the number or size of nigrostriatal synapses, so this explanation remains an hypothesis. However in support of this explanation, the treatment also elevated striatal levels of Synapsin-1, a presynaptic vesicular protein (Ferreira and Rapoport, 2002) (Figure 1B), but not of µ-tubulin (Figure 1), a ubiquitous structural protein, and in gerbil hippocampus, the treatment is known to increase the numbers of dendritic spines (Sakamoto et al., 2007) and the levels of pre- and postsynaptic proteins (e.g. PSD-95; GluR-1). That uridine can independently increase the storage and release of striatal DA in normal rats has been shown previously (Wang et al., 2005). Other investigators (Bousquet et al., 2008) have reported that supplementation with omega-3 fatty acids, including DHA, prior to administration of the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine can be beneficial in a mouse model of PD.

Besides serving as a precursor for brain cytidyl-diphosphate, the rate-limiting constituent in membrane phosphatide synthesis (Ross et al., 1997; Cansev et al., 2005) by the Kennedy cycle (Kennedy and Weiss, 1956), uridine’s phosphorylated product UTP also promotes neurite outgrowth by activating P2Y2 and other P2Y receptors, and then various intracellular signaling pathways (Pooler et al., 2005; Cansev, 2007). Conceivably, these receptors are involved in the increases in TH and Synapsin-1 proteins observed in our treated animals. Similarly, DHA, besides being a precursor preferentially incorporated into newly-synthesized brain phosphatides (DeGeorge et al., 1991), also interacts with brain receptors (e.g. syntaxin-3) to promote neuronal differentiation (Darios and Davletov, 2006). The ability of uridine and DHA to promote membrane phosphatide synthesis may also depend on the availability of a third circulating compound, choline (Wurtman et al., 2006), inasmuch as choline kinase is highly unsaturated with choline (Millington and Wurtman, 1982).

In conclusion, these data show that chronic oral administration of the phosphatide precursors uridine and DHA can ameliorate the impairment in dopaminergic terminals and transmission in 6-OHDA-lesioned rat striata, probably by increasing the amount of synaptic membrane generated by surviving striatal neurons. Since clinical manifestations of PD apparently require the loss of as many as 70–80% of nigrostriatal neurons (Bernheimer et al., 1973), a treatment like UMP plus DHA which, hypothetically, could increase nigrostriatal synapses by 15–25% (as estimated from the increases in TH and Synapsin-1 levels in rat striatum) might confer clinical benefit in patients with Parkinson’s Disease.