Conditional deletion of Ndufs4 in dopaminergic neurons promotes Parkinson's disease-like non-motor symptoms without loss of dopamine neurons

Abstract

Reduction of mitochondrial complex I activity is one of the major hypotheses for dopaminergic neuron death in Parkinson's disease. However, reduction of complex I activity in all cells or selectively in dopaminergic neurons via conditional deletion of the Ndufs4 gene, a subunit of the mitochondrial complex I, does not cause dopaminergic neuron death or motor impairment. Here, we investigated the effect of reduced complex I activity on non-motor symptoms associated with Parkinson's disease using conditional knockout (cKO) mice in which Ndufs4 was selectively deleted in dopaminergic neurons (Ndufs4 cKO). This conditional deletion of Ndufs4, which reduces complex I activity in dopamine neurons, did not cause a significant loss of dopaminergic neurons in substantia nigra pars compacta (SNpc), and there was no loss of dopaminergic neurites in striatum or amygdala. However, Ndufs4 cKO mice had a reduced amount of dopamine in the brain compared to control mice. Furthermore, even though motor behavior were not affected, Ndufs4 cKO mice showed non-motor symptoms experienced by many Parkinson's disease patients including impaired cognitive function and increased anxiety-like behavior. These data suggest that mitochondrial complex I dysfunction in dopaminergic neurons promotes non-motor symptoms of Parkinson's disease and reduces dopamine content in the absence of dopamine neuron loss.

Figure 1. Dopaminergic neuron-specific Ndufs4 knockout does not cause dopamine neuron death.

(a) Representative photomicrographs of TH immunostaining of SNpc. cKO: conditional knockout of Ndufs4 (Dat-cre/Ndufs4^lox/lox) mice; control: Ndufs4^lox/lox littermates. Bar, 200 μm. (b) Quantification of the total number of TH⁺ neurons in the SNpc. (c) Quantification of the total number of apoptotic, TUNEL⁺ cells in the SNpc. n.s. not statistically significant. (e,f) Representative TH staining of striatal (e) and amygdalar (f) tissue from Dat-cre/Ndufs4^lox/lox (cKO) mice and Ndufs4^lox/lox (Control) mice. Bar, 200 μm. (g,h) Quantification of TH staining intensity in the striatum (g) and amygdala (h). AU, arbitrary units. n.s. not statistically significant.

Figure 2. Dopamine content, not serotonin content, is decreased in the striatum and amygdala of Ndufs4 cKO mice.

The concentrations of dopamine (a,c) and serotonin (b,d) in the striatum (a,b) and amygdala (c,d) from both Ndufs4 cKO and control mice were measured. *p < 0.05; n.s. not statistically significant.

Figure 3. Motor behavior is not affected by conditional Ndufs4 deletion.

Locomotor activity in the open field test was quantified as total distance traveled (a), and total moving time (b). n.s. not statistically significant.

Figure 4. Ndufs4 cKO mice show progressive memory loss in the novel object recognition assay.

(a–h) Ndufs4 cKO and littermate control mice were tested for the novel object recognition assay at 3 months and then again at 6 months of age. The same cohort of mice was subjected to both 1-h and 24-h memory tests on two different days using different sets of objects. (a,b)Training and 1 h memory test in 3-month-old mice. (c,d) Training and 24-h memory in 3-month-old mice. (e,f) Training and 1-h memory in 6-month-old mice. (g,h) Training and 24-h memory in 6-month-old mice. n = 7–10 mice/genotype. (i–p) Ndufs4 cKO and littermate control mice were tested for the short-term spatial memory using the novel object location test at 3 months and then again at 6 months of age. Mice were subjected to both 1-h and 24-h memory tests on two different days. (i,j) Training and 1-h test using 3-month-old mice. (k,l) Training and 24-h test using 3-month-old mice. (m,n) Training and 1-h test using 6-month-old mice. (o,p) Training and 24-h test using 6-month-old mice. n = 7–10 mice/genotype. *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 5. Ndufs4 cKO mice show impaired spatial learning and memory in the reversal Morris water maze test.

(a) Target (in quadrant 1) acquisition in the hidden platform, quantified as swim distance to platform, was similar in control and Ndufs4 cKO mice. (b) Both control and Ndufs4 cKO mice spent more time in the virtual target quadrant (quadrant 1) in the probe test conducted 24 h after training, suggesting that they retained the spatial memory. (c) Swim distance to escape in the reverse hidden platform training where the hidden platform was moved to quadrant 3. Ndufs4 cKO mice swam longer distances to locate the platform, suggesting impaired learning of newer spatial information. (d) Ndufs4 cKO mice spent less time in the virtual target quadrant (quadrant 3) than control mice during the reversal probe test. (e) The ratio of time spent in Q3 versus Q1 in the reversal probe test was significantly lower for cKO mice than control mice. (f) Control and cKO mice performed equally well in the visible platform test; they showed comparable latency to reach the platform. *p < 0.05; **p < 0.01; ***p < 0.001; n.s. not statistically significant.

Figure 6. Ndufs4 cKO mice are deficient in contextual fear memory extinction.

(a) Control and Ndufs4 cKO mice have similar levels of contextual fear memory 24 h post-training. (b) Control and Ndufs4 cKO mice showed similar cued-fear response 24 h post-training. (c) Neither control nor Ndufs4 cKO mice froze when placed in a novel context 24 h after training. (d) However, fear extinction was impaired in Ndufs4 cKO mice. (e) The freezing response that persisted in Ndufs4 cKO mice after 8-days of fear extinction training was context specific because the mice did not freeze in a novel context after fear extinction trials. **p < 0.01.

Figure 7. Anxiety-related behavior is enhanced in Ndufs4 cKO mice in the elevated plus maze test.

(a) Representative activity traces from a control and an Ndufs4 cKO mouse in the elevated plus maze test. C, closed arm; O, open arm. (b) Time spent in closed arms of an elevated plus maze. (c) Time spent in open arms of an elevated plus maze. (d) Number of entries to the open arms of an elevated plus maze of 9-month-old mice. (e) Number of entries to the open ends of an elevated plus maze of 9-month-old mice. *p < 0.05; **p < 0.01; n.s. not statistically significant.

Figure 8. Anxiety-related behavior in the open field.

(a) Representative activity traces from a control and an Ndufs4 cKO mouse in the open field test. (b) Distance traveled to the center in the open field test. (c) Total distance traveled in the entire open field. (d) Ndufs4 cKO mice show increased anxiety in a light/dark exploration test. Data shown are the number of transitions made between the light and dark chamber in a light and dark box. (e) Social interaction is reduced in Ndufs4 cKO mice. Data shown are the time spent in social interactions. *p < 0.05; **p < 0.01; n.s. not statistically significant.