Alterations in striatal dopamine catabolism precede loss of substantia nigra neurons in a mouse model of juvenile neuronal ceroid lipofuscinosis

Abstract

Batten disease, or juvenile neuronal ceroid lipofuscinosis (JNCL), results from mutations in the CLN3 gene. This disorder presents clinically around the age of 5 years with visual deficits progressing to include seizures, cognitive impairment, motor deterioration, hallucinations, and premature death by the third to fourth decade of life. The motor deficits include coordination and gait abnormalities, myoclonic jerks, inability to initiate movements, and spasticity. Previous work from our laboratory has identified an early reduction in catechol-O-methyltransferase (COMT), an enzyme responsible for the efficient degradation of dopamine. Alterations in the kinetics of dopamine metabolism could cause the accumulation of undegraded or unsequestered dopamine leading to the formation of toxic dopamine intermediates. We report an imbalance in the catabolism of dopamine in 3 month Cln3(-/-) mice persisting through 9 months of age that may be causal to oxidative damage within the striatum at 9 months of age. Combined with the previously reported inflammatory changes and loss of post-synaptic D1alpha receptors, this could facilitate cell loss in striatal projection regions and underlie a general locomotion deficit that becomes apparent at 12 months of age in Cln3(-/-) mice. This study provides evidence for early changes in the kinetics of COMT in the Cln3(-/-) mouse striatum, affecting the turnover of dopamine, likely leading to neuron loss and motor deficits. These data provide novel insights into the basis of motor deficits in JNCL and how alterations in dopamine catabolism may result in oxidative damage and localized neuronal loss in this disorder.

Figure 1. Deficits genes involved in catecholamine metabolism is restricted to a decreased in COMT in the Cln3^-/- mice

Evaluation of several published microarray data sets performed on the Cln3^-/-mice showed a selective down regulation in the expression of Comt (3.56 fold decrease, 10-week whole brain microarray) with a sparing of all other enzymes involved in catecholamine catabolism (Dopamine, A & Norepinephrine, B) (Brooks et al., 2003; Elshatory et al., 2003). Synthesis of catecholamine is achieved through conversion of tyrosine to DOPA, followed by the subsequent selective conversion to dopamine, norepinephrine, and epinephrine. Of the enzymes involved in the production of catecholamines, specifically tyrosine hydroxylase, DOPA decarboxylase, dopamine β-hydroxylase, and norephinephrine N-methyltransferase (Pnmt) none were altered in the Cln3^-/- mice aside from a slight, yet significant, decrease in the level of Pnmt, suggesting disruption in the enzyme involved in conversion of norepinephrine to epinephrine [less than 1.5 fold change (p≤0.05, Student's t-test), 10-week whole brain microarray (Brooks et al., 2003)] (C).

Figure 2. Decreased COMT protein and activity levels in 3 and 9 month Cln3^-/- mice cortex and striatum

Immunoblotting for catechol-O-methyl transferase (COMT) revealed changes in both the membrane bound (MB) and soluble (S) form of the protein in brain samples from Cln3^-/-mice compared to wild-type mice. Samples were prepared from cortex (A) and striatum (B) of both three and nine month old male mice. Top panels show representative level of COMT from the indicated samples (13% SDS-PAGE) while the lower band shows actin immunoblotting. Histograms in the lower panel depict relative levels of MB-COMT and S-COMT in wild-type and Cln3^-/- mice. Densitometry of MB-COMT and S-COMT bands were standardized to actin densities, with background subtraction being performed for both COMT and actin bands. (Mean relative intensity ± SEM). O-methylating activity of COMT was assayed by enriching for the S- or MB-COMT fractions by centrifugation at 100,000×g (confirmed by western blot), mixing samples with the co-factor S-adenosylmethionine (SAM), followed by reaction with dihydroxybenzoic acid. O-methylation of dihydroxybenzoic acid (DHBAc) to vanillic acid was assessed after 60 minute incubation times by HPLC (A, B, lower row). Samples were prepared from cortex (A) and striatum (B) of both three (left column) and nine month (right column) old male Cln3^-/- and 129/SvJ wild-type mice. Decreases in the activity of S-COMT were seen at both time points in cortex and striatum (A, B, lower row). Significant decreases in activity of the MB-COMT were seen only at nine months in the striatum (B) Activity is represented as mean pg vanillic acid/mg protein/minute ± SEM; *P≤0.05, ** P≤0.01, *** P≤0.001 (2-way ANOVA).

Figure 3. Alterations in level of Dopamine, DOPAC, and H VA in midbrain and cortex wild-type and Cln3^-/- mice

Deproteinized homogenates were sonicated and catecholamine metabolites were fractionated on an ESA column (70-0636). Dopamine, HVA and DOPAC came off at 3.33, 5.65, and 2.73 minutes, and showed maximum responses at channels set to 25, 250 and 100mV, respectively. The levels of dopamine, HVA, and DOPAC were assayed three and nine month old Cln3-/- and 129/SvJ wild-type mice in cortex and midbrain and presented as level of metabolite in ng normalized to the total amount of protein in μg. This analysis revealed global disruption in the levels of these metabolites in Cln3-/- mice. In both the cerebral cortex (A) and the striatum (B) there was a significant decrease in the levels of dopamine and DOPAC, although HVA levels remained unaffected. Data are presented as mean ng metabolite/μg total protein ± SEM, *P≤0.05, ** P≤0.01, *** P≤0.001 (2-way ANOVA).

Figure 4. Elevated oxidative striatal stress and cellular atrophy in the substantia nigra pars reticulata of Cln3^-/- mice

Proteins from three and nine month old male Cln3^-/- and 129/SvJ cortex (A, upper row) and striatum (A, lower row) were dot blotted onto PVDF and carbonyls were derivativitzed with DNPH to form a stable product that was detected by immunoblotting. This revealed an increase in the level of oxidative stress at nine months of age in the striatum of Cln3^-/- mice (A, lower right panel). Histograms depict densitometry of derivatized carbonyls standardized to total protein, determined by staining of the PVDF with Sypro Ruby Red (Mean relative intensity ± SEM). Unbiased Cavalieri estimates of regional volume were measured on Nissl stained brain sections. Regional volumes were expressed in μm³ and the mean volume of each region calculated for control and homozygous Cln3^-/- mice. There was a significant regional atrophy was seen in Cln3^-/- vs. 129/SvJ (+/+) at six months of age within the substantia nigra pars reticulata (SN-pr) (B, left panel), although no difference was observed in the volume of the substantia nigra pars compacta (B, right panel). This cell loss was specific to the parvalbumin positive cells of the pars reticulata and was progressive as the animals aged. Optical fractionator cell counts of the substantia nigra pars reticulata and pars compacta were performed to determine absolute numbers of cells within each region. To distinguish cells types, cells of the pars reticulata were immuno-labeled with parvalbumin (PV) and the cells of the pars compacta were labeled with tyrosine hydroxylase at three, six, and nine months of age. A significant decrease was seen in the number of PV positive pars reticulata cells, progressing to near 35% cell loss by nine months of age (129/SvJ: 1788±21.3, Cln3^-/-: 1214±135.87) although no change was seen at the early time points (3 month: 129/SvJ: 1766±166.89, Cln3^-/-: 1916±228.27; 6 month: 129/SvJ: 1785±87.79, Cln3^-/-: 1501±72.68) (C). No decrease within the pars compacta was noted at any of the time points (3 month: 129/SvJ: 2906±287.23, Cln3^-/-: 2023±329.48; 6 month: 129/SvJ: 3100±193.69, Cln3^-/-: 3650±109.83; 9 month: 129/SvJ: 3859±74.71, Cln3^-/-: 3324±153.05) (D). Data are presented as mean ± SEM, *p≤0.05, ** p≤0.01 (Student's t-test).

Figure 5. Degenerative changes in motor activity of Cln3^-/- mice

Locomotor testing was performed on 129/SvJ and Cln3^-/- mice at three different time points in an open field activity chamber. Animals were habituated for two days for 30 minutes followed by testing trial of 30 minutes. Separate groups of mice were tested at three, nine, and twelve months of age. The total distance each animal traveled (A, C, E) and the amount of time spent moving (B, D, F) was calculated. No differences in total distance traveled or time spent ambulating was detected at three or nine months of age. By twelve months of age, there was a significant decrease in distance traveled and amount of time spent ambulating in Cln3^-/- mice (E-F). Mean time or distance traveled ± SEM, two way ANOVA, F(1,23)=2.421, P=0.004 (time) and (F(1,23)=2.560, P=0.001 (distance).

Figure 6. Decreased D1α receptor expression at nine month Cln3^-/- mice striatum

Immunoblotting for the dopamine 1α (D1α) receptor revealed a decrease in the expression level of the protein in striatum brain samples from nine month old Cln3^-/- mice compared to 129/SvJ wild-type mice. Samples were prepared from cortex (A) and striatum (B) of both three and nine month old male mice. Top panels show level of D1α receptor from the indicated samples while the lower band shows actin immunoblotting. Histograms in the lower panel depict relative levels of D1α receptor in wild-type and Cln3^-/- mice. Densitometry of D1α receptor bands were standardized to actin densities, with background subtraction being performed for both receptor and actin bands. Data are presented as mean ± SEM, *p≤0.05 (Student's t-test).

Figure 7. Summary of dopamine metabolism and handling

1. Decrease in both COMT levels and activity

2. Decrease in the metabolites dopamine and DOPAC

3. Decrease in the postsynaptic D_1α receptor

4. No difference in the expression of DAT, MAO-B, VMAT, or NET