Lithium therapy improves neurological function and hippocampal dendritic arborization in a spinocerebellar ataxia type 1 mouse model

Abstract

Background: Spinocerebellar ataxia type 1 (SCA1) is a dominantly inherited neurodegenerative disorder characterized by progressive motor and cognitive dysfunction. Caused by an expanded polyglutamine tract in ataxin 1 (ATXN1), SCA1 pathogenesis involves a multifactorial process that likely begins with misfolding of ATXN1, which has functional consequences on its interactions, leading to transcriptional dysregulation. Because lithium has been shown to exert neuroprotective effects in a variety of conditions, possibly by affecting gene expression, we tested the efficacy of lithium treatment in a knock-in mouse model of SCA1 (Sca1(154Q/2Q) mice) that replicates many features of the human disease.

Methods and findings: Sca1(154Q/2Q) mice and their wild-type littermates were fed either regular chow or chow that contained 0.2% lithium carbonate. Dietary lithium carbonate supplementation resulted in improvement of motor coordination, learning, and memory in Sca1(154Q/2Q) mice. Importantly, motor improvement was seen when treatment was initiated both presymptomatically and after symptom onset. Neuropathologically, lithium treatment attenuated the reduction of dendritic branching in mutant hippocampal pyramidal neurons. We also report that lithium treatment restored the levels of isoprenylcysteine carboxyl methyltransferase (Icmt; alternatively, Pccmt), down-regulation of which is an early marker of mutant ATXN1 toxicity.

Conclusions: The effect of lithium on a marker altered early in the course of SCA1 pathogenesis, coupled with its positive effect on multiple behavioral measures and hippocampal neuropathology in an authentic disease model, make it an excellent candidate treatment for human SCA1 patients.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. Lithium Treatment Improves Motor Coordination in Sca1154Q/2Q Mice

(A) Body weights of Sca1154Q/2Q mice on a diet with (Li KI) or without (Cont KI) 0.2% lithium carbonate supplementation. *p < 0.02 by Student t-test. (B–D) Rotarod analysis. Ten-week-old naïve mice (B and D) and 13 week-old naïve Sca1154Q/2Q (C) mice were trained on the accelerating Rotarod in four trials per day (T1–T4) for 4 d (D1–D4). Lithium treatment was started either at the time of weaning (B and C) or at five weeks of age (D), when the Sca1154Q/2Q mice already show motor impairments. In both (B) and (D), repeated measures ANOVA revealed a significant time by day interaction for each of the four groups (unpublished data). Two-way repeated measures ANOVA indicated there were main effects of genotype (p < 0.001 for [B]; p = 0.002 for [D]) and treatment (p = 0.037 for [B] and p = 0.005 for [D]), but their interaction was not significant in (B) (p = 0.491) or (D) (p = 0.115). Cont KI, Sca1154Q/2Q mice on control diet; Li KI, lithium-treated Sca1154Q/2Q mice on lithium-supplemented diet; Li WT, wild-type mice on lithium-supplemented diet; Cont WT, wild-type mice on control diet. All data are shown as mean ± SEM.

Figure 2. Sca1154Q/2Q Mice Reared on Lithium-Containing Chow Have Improved Learning and Memory Functions

(A and B) Performance of nine- to 11-week old mice on the Morris hidden-platform water maze. Sca1154Q/2Q mice on the control diet (Cont KI) learned more slowly than those given lithium (Li KI). In a 60 s probe trial (B), Cont KI spent significantly less time in the training quadrant than Li KI (p = 0.012 by multiple comparisons using Scheffe's post hoc test), whereas Li KI spent similar amounts of time in the training quadrant compared to wild-type mice given lithium (Li WT) (p = 0.770) or wild-type mice on the control diet (Cont WT) (p = 0.847). (C and D) Pavlovian conditioned fear for Sca1154Q/2Q (KI) and wild-type (WT) mice treated with lithium (Li) or fed control chow (Cont). Percentage of time spent in freezing behavior during the contextual test (C) and the cued test (D) is shown. (E) Open-field activity for Sca1154Q/2Q mice. Locomotor activity of lithium-treated and untreated mutant mice was measured two weeks after the conditioned fear test. Each panel indicates total distance traveled (left graph) and time spent moving (right graph). All data are shown as mean ± SEM.

Figure 3. Lithium Treatment Does Not Affect NI Formation in Sca1154Q/2Q Mice

The percentage of NI-harboring neurons is shown for 30- to 36-week-old mutant mice. Numbers of animals used for counting hippocampal neurons were six for lithium-treated Sca1154Q/2Q mice (Li KI) and four for Sca1154Q/2Q mice on the control diet (Cont KI); numbers of animals used for counting Purkinje cells were three for Li KI and three for Cont KI. Error bars indicate SEM.

Figure 4. Lithium Treatment Partially Rescues Dendritic Pathology in Hippocampal CA3 Pyramidal Neurons of Sca1154Q/2Q Mice

(A–D) Camera lucida drawings of representative pyramidal cell neurons from each experimental group. Shown are wild-type, control diet (Cont WT; n = 5 mice, 50 neurons) (A); wild-type, lithium diet (Li WT; n = 3 mice, 30 neurons) (B); Sca1154Q/2Q knock-in, control diet (Cont KI; n = 3, 30 neurons) (C); and (D) Sca1154Q/2Q knock-in, lithium diet (Li KI; n = 6, 60 neurons). (E and F) The effects of lithium treatment on the branching pattern of basilar (E) and apical (F) dendrites at increasing distances from the soma are shown. Genotype and lithium treatment significantly altered both basilar (E) and apical dendritic intersections (F) (genotype effect: p < 0.001, for basilar and apical; lithium treatment effect: p = 0.025 for basilar, p = 0.018 for apical). Sholl analysis additionally indicated a significant interaction between lithium treatment and genotype (p < 0.001 for basilar, p = 0.053 for apical). Values represent mean ± SEM.

Figure 5. Lithium Treatment Increases Icmt/Pccmt mRNA Levels in Sca1154Q/2Q Cerebellum

(A) Temporal profile of Icmt/Pccmt mRNA down-regulation in the Sca1154Q/2Q cerebellum. Cerebellar expression levels of Icmt/Pccmt mRNA in Sca1154Q/2Q mice and wild-type littermates were compared by Northern analysis. Ethidium bromide-stained RNA gel images are shown as a loading control, which was confirmed by hybridization with Gapdh. 25 μg of total RNA obtained from one cerebellum was loaded in each lane. Densitometric analysis using Gapdh as a control revealed a relative abundance of 0.465 for the 25 week-old Sca1154Q/2Q cerebellum compared to the respective wild-type littermate cerebella. (B) Northern blots of cerebellar RNA (25 μg) from two Sca1154Q/2Q mice and two wild-type littermates treated with lithium (+) or control diet (−) at ten weeks of age (blots on left) were probed with Icmt/Pccmt and Gapdh cDNAs. Quantitative analysis from four independent experiments (bar graph on right), in which total cerebellar RNAs were obtained from one or two Sca1154Q/2Q mice and one or two wild-type littermates for each experimental group (in total, six Sca1154Q/2Q mice on lithium diet, four Sca1154Q/2Q mice on control diet, six wild-type mice on lithium diet, and six wild-type mice on control diet), indicates Icmt/Pccmt expression was significantly increased in lithium-treated Sca1154Q/2Q mice (bar graph on right). Error bars indicate mean ± SEM.

Figure 6. Phospho-Ser-9-GSK3β, but not Phospho-Akt, was Up-Regulated in Lithium-Treated Mouse Brains

(A) Phospho-Ser-9-GSK3β, but not phospho-Ser-473-Akt, was up-regulated in lithium-treated mouse brains. Shown are blots of hippocampal (left) and cerebellar (right) extracts from Sca1154Q/2Q mice (KI) and wild-type littermates (WT), with (+) or without (−) lithium treatment, immunolabeled with phospho-Ser9-GSK3β, phospho-Ser473-Akt, and GAPDH antibodies. Each number indicates relative intensity of the band compared to the intensity of wild-type mice without the treatment. (B) Phospho-Thr-308-Akt was not up-regulated in the lithium treated mouse brains. Shown are blots of hippocampal (left) and cerebellar (right) extracts from Sca1154Q/2Q mice (KI) and wild-type littermates (WT), with (+) or without (−) lithium treatment, immunolabeled with phospho-Thr308-Akt, total Akt, and GAPDH antibodies. Similar results were obtained from the extracts from the rest of the brains. Each number indicates relative intensity of the band compared to the intensity of wild-type mice without the treatment. On each immunoblot, individual lanes were loaded with a protein extract prepared from one animal and similar results were reproduced with one to three more independent experiments (two to four animals in total were used).