Neuroprotective effects of lithium on medium-size lamina VII neurons. (a) Shows representative micrographs of those H&E-stained lamina VII neurons that were selected for the count based on size specificity (diameter ranging from 10 to 20 μm). (b) Graph indicates the severe loss of these neurons in G93A mice, which exceeded the loss of MN. Remarkably, the G93A mice treated with lithium showed a much higher number of lamina VII medium-size neurons even compared with saline-treated WT mice. (c–e) These results were confirmed by gephyrin immunostaining, as shown here and by all of the staining procedures summarized in SI Fig. 16. Counts represent the mean ± SEM of 62,000 cells per group (3,100 per mouse in groups of 20 mice). Comparison among groups was made by using one-way ANOVA. *, P ≤ 0.05 compared with WT saline-treated group. #, P ≤ 0.001 compared with G93A saline-treated groups. (Scale bars, 17 μm.)

Effects of lithium on autophagy in vivo and in vitro. (a–d) MDC-positive small vacuoles in the lumbar spinal cord of WT (a and c, arrows) and G93A mice (b and d, arrows). (e) Representative picture of beclin immunostained vacuoles in the cytoplasm of alpha MN from a G93A lithium-treated mouse. The ImmunoGold particles (20 nm) are localized on both the membrane (that surround the core) and the electrondense core (arrows). (f) LC3 immunostaining is present on a larger membranous structure; the ImmunoGold particles (20 nm) are randomly localized (arrows). (g) The count of beclin immunostained structures shows a marked effect of lithium in MN both WT and G93A mice. (h) Likewise, LC3 immunopositive vacuoles increase significantly in G93A and WT mice administered lithium. (i–l) Phase-contrast microscopic images of lithium-induced accumulation of vacuoles in SH-SY5Y cells exposed or not for 72 h to 1 mM lithium (j), or lithium plus 50 mM asparagine (Asn) (a slight autophagy blocker acting downstream of lithium) (k), or 400 nM rapamycin (Rap) (l). Arrows point to cytoplasmic vacuoles that accumulate in cells treated with lithium or Rap (a known autophagy inducer). No vacuolization was observed in control (i) or lithium plus Asn-treated cells. (m–p) A parallel experiment was performed with transfected SH-SY5Y cells stably expressing the GFP-LC3 chimeric fluorescent protein. The images clearly show that both lithium (n) and rapamycin (p) change the cytoplasmic diffuse fluorescence pattern of GFP-LC3 to a punctuated pattern indicative of autophagosome formation. Asparagine (o) inhibited the effect of lithium on GFP-LC3 localization. *, P < 0.05 compared with saline. (Scale bars: a–d, 14 μm; e, 0.1 μm; f, 0.08 μm; i–l, 20 μm; m–p, 50 μm.)

Effects of lithium treatment on the lifespan and neurological symptoms of G93A mice. (a) Survival curve for saline- and lithium-treated G93A mice. Lithium carbonate (1 mEq/kg, daily, i.p.) treatment significantly increased the survival time of G93A mice compared with saline-treated mice. (b) Effects of lithium on specific symptoms, such as hind limb adduction, gait impairment, and the onset of severe paralysis. (c) Symptomatic effects and prolongation of the life span induced by lithium. Values represent the mean ± SEM of 10 mice per group in two different experiments (total N per group = 20). Comparison was made by using ANOVA with Sheffe's post hoc analysis. *, P ≤ 0.05 compared with G93A mice administered saline. **, P ≤ 0.001 compared with G93A mice administered saline.

Effects of lithium administration on motor neurons mitochondria in vivo. (a–h) Representative pictures of mitochondria (arrows) in MN from the spinal cord of WT mice treated with saline (a and b) or lithium (c and d) and from G93A mice treated with saline (e and g) or lithium (f and h). (g) In G93A mice treated with saline, TEM shows mitochondrial vacuolization (arrowheads). (f and h) This vacuolization is consistently absent in mitochondria of G93A mice treated with lithium. (d and h–j) Lithium decreases the size of mitochondria both in WT and G93A mice (d and h, respectively) both in the cervical (i) and lumbar (j) spinal cord. (k and l) Lithium increases the number of mitochondria both in cervical (k) and lumbar (l) MN both in WT and G93A. Values are the mean ± SEM. Comparison between groups was made by using one-way ANOVA. *, P ≤ 0.05 compared with saline-treated mice. **, P ≤ 0.01 compared with saline treated mice. (Scale bars: a, c, e, and g, 1.8 μm; b, d, f, and h, 0.25 μm.)

Effects of lithium treatment on disease symptom progression and survival in patients with ALS. (a, b, d, and e) Symptoms progression (evaluated every 3 months) in controls (riluzole-treated) and treated patients (lithium plus riluzole-treated patients) expressed as raw data, using the Norris (a) and ALSFRS-R (b) rating scales and FVC (d) and MRC (e). There was no significant progression in the lithium-treated patients at any time interval apart from the last two evaluations using the MRC scale. In the riluzole-treated patients, symptoms progressed significantly starting at 3 or 6 months (depending on the scale). (c) Survival curve as normalized data shows the percentage of patients surviving over the 15 months of treatment in the riluzole and lithium groups. None of the patients died in the lithium-treated group; however, in the control group, although the patients had comparable disease severity at enrollment, ≈30% died. Intra- and intergroup analyses were performed by unpaired t test or ANOVA followed by the Bonferroni post hoc test. (f) Shows the breakdown between treated and control patients for FVC. (g) Shows the breakdown between groups for FVC (calculated at 1 month before death) in those patients affected by the bulbar form. *, P < 0.05, **, P < 0.01 compared with baseline value. #, P < 0.05; ##, P < 0.01 compared with control patients.