Effect of fluoxetine on disease progression in a mouse model of ALS

Abstract

Selective serotonin reuptake inhibitors (SSRIs) and other antidepressants are often prescribed to amyotrophic lateral sclerosis (ALS) patients; however, the impact of these prescriptions on ALS disease progression has not been systematically tested. To determine whether SSRIs impact disease progression, fluoxetine (Prozac, 5 or 10 mg/kg) was administered to mutant superoxide dismutase 1 (SOD1) mice during one of three age ranges: neonatal [postnatal day (P)5-11], adult presymptomatic (P30 to end stage), and adult symptomatic (P70 to end stage). Long-term adult fluoxetine treatment (started at either P30 or P70 and continuing until end stage) had no significant effect on disease progression. In contrast, neonatal fluoxetine treatment (P5-11) had two effects. First, all animals (mutant SOD1(G93A) and control: nontransgenic and SOD1(WT)) receiving the highest dose (10 mg/kg) had a sustained decrease in weight from P30 onward. Second, the high-dose SOD1(G93A) mice reached end stage ∼8 days (∼6% decrease in life span) sooner than vehicle and low-dose animals because of an increased rate of motor impairment. Fluoxetine increases synaptic serotonin (5-HT) levels, which is known to increase spinal motoneuron excitability. We confirmed that 5-HT increases spinal motoneuron excitability during this neonatal time period and therefore hypothesized that antagonizing 5-HT receptors during the same time period would improve disease outcome. However, cyproheptadine (1 or 5 mg/kg), a 5-HT receptor antagonist, had no effect on disease progression. These results show that a brief period of antidepressant treatment during a critical time window (the transition from neonatal to juvenile states) can be detrimental in ALS mouse models.

Keywords: Prozac; amyotrophic lateral sclerosis; antidepressant; fluoxetine; motoneuron excitability.

Fig. 1.

Fluoxetine postnatal day (P)70 to end stage. A: rotarod: fluoxetine dose had no significant effect on rotarod performance. B: weight: fluoxetine dose had no significant effect on weight. C: % survival: fluoxetine dose had no significant effect on survival. D: fluid intake: fluoxetine dose had no effect on fluid intake. The gray horizontal bar at P70 signifies the onset of fluoxetine treatment. n = 20 per group for controls; n = 14 per group for SOD1^G93A. Drug concentration: vehicle (light gray), 5 mg/kg (dark gray), 10 mg/kg (black); Genotype: controls (dashed lines) and SOD1^G93A (solid lines). dpn, Postnatal day. Means ± SE are displayed for each treatment group at each time point in A, B, and D.

Fig. 2.

Fluoxetine P30 to end stage. A: rotarod: fluoxetine dose had no significant effect on rotarod performance. B: weight: fluoxetine dose had no significant effect on weight. C: % survival: fluoxetine dose had no significant effect on survival. n = 20 per group for controls; n = 14 per group for SOD1^G93A. Drug concentration: vehicle (light gray), 5 mg/kg (dark gray), 10 mg/kg (black). Genotype: controls (dashed lines) and SOD1^G93A (solid lines). Means ± SE are displayed for each treatment group at each time point in A and B.

Fig. 3.

Neonatal fluoxetine P5 to P11. A: rotarod: rotarod performance was maintained in all control animals. Rotarod performance declined with age in SOD1^G93A mice; however, this decline was dependent on fluoxetine dose [repeated-measures (RM) ANOVA: Age × Genotype × Drug level, F(5,322) = 2.61, P < 0.5]. Post hoc tests with Bonferroni correction indicated that the high-dose SOD1^G93A group (10 mg/kg, solid black trace) had worse rotarod performance than both vehicle (solid light gray trace) (P < 0.05) and low-dose (solid dark gray trace) (P < 0.05) groups and that these differences occurred in the age range between P125 and P135 as indicated on the graph. B: % survival: all control animals survived regardless of fluoxetine dose. SOD1^G93A mice receiving the high fluoxetine dose reached end stage sooner than both vehicle [Kaplan Meier (K-M) log rank: χ² = 7.4, P ≤ 0.01] and low-dose (K-M log rank: χ² = 11.4; P ≤ 0.001) groups. n = 20 per group for controls and SOD1^G93A. Drug concentration: vehicle (light gray), 5 mg/kg (dark gray), 10 mg/kg (black). Means ± SE are displayed for each treatment group at each time point in A. Significance values indicate a significant difference in Drug level at individual time points: *P < 0.05, **P < 0.01.

Fig. 4.

Neonatal fluoxetine P5 to P11 weights. A: weight was monitored during fluoxetine injections and recorded as the average weight per pup per litter. There were no significant differences in weight during the injection period. Number of litters: n = 7 per group. Drug concentration: vehicle (light gray), 5 mg/kg (dark gray), 10 mg/kg (black). B: presymptomatic weights were significantly lower in the high-dose group (black traces) regardless of genotype [RM ANOVA: Drug level, F(2,127) = 14.03, P < 0.0001]. Post hoc tests with Bonferroni correction indicated that the high-dose group had significantly lower weights than both the vehicle (P < 0.0001) and low-dose (P < 0.0001) groups when collapsed across age and that there were significant differences at each time point as indicated on the graph. Genotypes are graphed separately for illustrative purposes only. C: postsymptomatic weight was also significantly lower in the high-dose group (black traces) regardless of genotype [RM ANOVA: Drug level, F(2,125) = 18.4, P < 0.0001]. Post hoc tests with Bonferroni correction indicated that the high-dose group had significantly lower weights than both the vehicle (P < 0.0001) and low-dose (P < 0.0001) groups when collapsed across age and that there were significant differences at each time point as indicated on the graph. Genotypes are graphed separately for illustrative purposes only. n = 20 per group for controls and SOD1^G93A. Drug concentration: vehicle (light gray), 5 mg/kg (dark gray), 10 mg/kg (black). Genotype: controls (dashed lines) and SOD1^G93A (solid lines). Means ± SE are displayed for each treatment group at each time point. Significance values indicate a significant difference in Drug level at individual time points: *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 5.

In vitro whole cell electrophysiology. Activity of typical motoneurons shown before and after bath application of a cocktail of citalopram (2–10 μM) and α-methyl 5-HT (0.1–0.3 μM). In A, B, and Cb, the motoneuron is from the same P10 nontransgenic mouse. After application of citalopram and α-methyl 5-HT, a holding current of −156 pA was needed to maintain a baseline membrane potential of −62mV for this motoneuron. A: during hyperpolarizing current steps, there is an increase in sag after application of citalopram and α-methyl 5-HT. At the offset of the current step, the voltage rebound is also increased after application of citalopram and α-methyl 5-HT. In this example, rebound action potentials are also present. B: in the same motoneuron, application of citalopram and α-methyl 5-HT decreased the current needed to elicit action potentials and increased the firing rate at a given current step. C, a: the afterhyperpolarization potential (AHP) is decreased after a single action potential in this P9 nontransgenic motoneuron. b: Expanded view from B (gray box) shows how the decrease in AHP after application of citalopram and α-methyl 5-HT affects repetitive firing. D: the persistent inward current (PIC) peak voltage is hyperpolarized (−23 mV to −40 mV in this example, black arrow) after citalopram and α-methyl 5-HT application. This example is from a P10 SOD1^G93A motoneuron. Light gray traces, control measures; black traces, after citalopram and α-methyl 5-HT application.

Fig. 6.

Neonatal cyproheptadine P5-11. A: rotarod: cyproheptadine dose had no significant effect on rotarod performance. B: weight: cyproheptadine dose had no significant effect on weight. C: % survival: cyproheptadine dose had no significant effect on survival. n = 20 per group for controls; n = 14 per group for SOD1^G93A. Drug concentration: vehicle (light gray), 1 mg/kg (dark gray), 5 mg/kg (black). Genotype: controls (dashed lines) and SOD1^G93A (solid lines). Means ± SE are displayed for each treatment group at each time point in A and B.