Molecular Mechanisms of Lithium Action: Switching the Light on Multiple Targets for Dementia Using Animal Models

Lithium has long been used for the treatment of psychiatric disorders, due to its robust beneficial effect as a mood stabilizing drug. Lithium’s effectiveness for improving neurological function is therefore well-described, stimulating the investigation of its potential use in several neurodegenerative conditions including Alzheimer’s (AD), Parkinson’s (PD) and Huntington’s (HD) diseases. A narrow therapeutic window for these effects, however, has led to concerted efforts to understand the molecular mechanisms of lithium action in the brain, in order to develop more selective treatments that harness its neuroprotective potential whilst limiting contraindications. Animal models have proven pivotal in these studies, with lithium displaying advantageous effects on behavior across species, including worms (C. elegans), zebrafish (Danio rerio), fruit flies (Drosophila melanogaster) and rodents. Due to their susceptibility to genetic manipulation, functional genomic analyses in these model organisms have provided evidence for the main molecular determinants of lithium action, including inhibition of inositol monophosphatase (IMPA) and glycogen synthase kinase-3 (GSK-3). Accumulating pre-clinical evidence has indeed provided a basis for research into the therapeutic use of lithium for the treatment of dementia, an area of medical priority due to its increasing global impact and lack of disease-modifying drugs. Although lithium has been extensively described to prevent AD-associated amyloid and tau pathologies, this review article will focus on generic mechanisms by which lithium preserves neuronal function and improves memory in animal models of dementia. Of these, evidence from worms, flies and mice points to GSK-3 as the most robust mediator of lithium’s neuro-protective effect, but it’s interaction with downstream pathways, including Wnt/β-catenin, CREB/brain-derived neurotrophic factor (BDNF), nuclear factor (erythroid-derived 2)-like 2 (Nrf2) and toll-like receptor 4 (TLR4)/nuclear factor-κB (NFκB), have identified multiple targets for development of drugs which harness lithium’s neurogenic, cytoprotective, synaptic maintenance, anti-oxidant, anti-inflammatory and protein homeostasis properties, in addition to more potent and selective GSK-3 inhibitors. Lithium, therefore, has advantages as a multi-functional therapy to combat the complex molecular pathology of dementia. Animal studies will be vital, however, for comparative analyses to determine which of these defense mechanisms are most required to slow-down cognitive decline in dementia, and whether combination therapies can synergize systems to exploit lithium’s neuro-protective power while avoiding deleterious toxicity

pGluAβ increases accumulation of Aβ in vivo and exacerbates its toxicity

Several species of β-amyloid peptides (Aβ) exist as a result of differential cleavage from amyloid precursor protein (APP) to yield various C-terminal Aβ peptides. Several N-terminal modified Aβ peptides have also been identified in Alzheimer’s disease (AD) brains, the most common of which is pyroglutamate-modified Aβ (AβpE3-42). AβpE3-42 peptide has an increased propensity to aggregate, appears to accumulate in the brain before the appearance of clinical symptoms of AD, and precedes Aβ1-42 deposition. Moreover, in vitro studies have shown that AβpE3-42 can act as a seed for full length Aβ1-42. In this study, we characterized the Drosophila model of AβpE3-42 toxicity by expressing the peptide in specific sets of neurons using the GAL4-UAS system, and measuring different phenotypic outcomes. We found that AβpE3-42 peptide had an increased propensity to aggregate. Expression of AβpE3-42 in the neurons of adult flies led to behavioural dysfunction and shortened lifespan. Expression of AβpE3-42 constitutively in the eyes led to disorganised ommatidia, and activation of the c-Jun N-terminal kinase (JNK) signaling pathway. The eye disruption was almost completely rescued by co-expressing a candidate Aβ degrading enzyme, neprilysin2. Furthermore, we found that neprilysin2 was capable of degrading AβpE3-42. Also, we tested the seeding hypothesis for AβpE3-42 in vivo, and measured its effect on Aβ1-42 levels. We found that Aβ1-42 levels were significantly increased when Aβ1-42 and AβpE3-42 peptides were co-expressed. Furthermore, we found that AβpE3-42 enhanced Aβ1-42 toxicity in vivo. Our findings implicate AβpE3-42 as an important source of toxicity in AD, and suggest that its specific degradation could be therapeuti

Lithium suppresses Aβ pathology by inhibiting translation in an adult Drosophila model of Alzheimer's disease

The greatest risk factor for Alzheimer's disease (AD) is age, and changes in the ageing nervous system are likely contributors to AD pathology. Amyloid beta (Aβ) accumulation, which occurs as a result of the amyloidogenic processing of amyloid precursor protein (APP), is thought to initiate the pathogenesis of AD, eventually leading to neuronal cell death. Previously, we developed an adult-onset Drosophila model of AD. Mutant Aβ42 accumulation led to increased mortality and neuronal dysfunction in the adult flies. Furthermore, we showed that lithium reduced Aβ42 protein, but not mRNA, and was able to rescue Aβ42-induced toxicity. In the current study, we investigated the mechanism/s by which lithium modulates Aβ42 protein levels and Aβ42 induced toxicity in the fly model. We found that lithium caused a reduction in protein synthesis in Drosophila and hence the level of Aβ42. At both the low and high doses tested, lithium rescued the locomotory defects induced by Aβ42, but it rescued lifespan only at lower doses, suggesting that long-term, high-dose lithium treatment may have induced toxicity. Lithium also down-regulated translation in the fission yeast Schizosaccharomyces pombe associated with increased chronological lifespan. Our data highlight a role for lithium and reduced protein synthesis as potential therapeutic targets for AD pathogenesis

Additional file 5: Figure S5. of pGluAβ increases accumulation of Aβ in vivo and exacerbates its toxicity

pGluAβ increases accumulation of Aβ1-42 in vivo. (A). Flies co-expressing Aβ1-42 and AβpE3-42 peptide, had substantially more Aβ1-42 levels than flies expressing Aβ1-42 alone. Aβ was not detected in flies expressing a single copy of Aβ1-42 because it does not accumulate enough protein. However, Aβ1-42 was detected in flies expressing 2 copies of Aβ1-42 (B), confirming protein expression in these flies with the antibody. Furthermore, to validate specificity, Aβ1-42 was not detected in flies expressing either a single copy or double copy of AβpE3-42. GMR-GAL4 was used to drive expression of Aβ1-42 and AβQ3-42 transgenic flies. (PDF 10727 kb

Additional file 4: Figure S4. of pGluAβ increases accumulation of Aβ in vivo and exacerbates its toxicity

pGluAβ increases accumulation of Aβ in vivo. Flies co-expressing Aβ1-42 and AβpE3-42 peptide, had significantly higher Aβ levels than flies co-expressing Aβ1-42 and Aβ1-42. Data are presented as means ± SEM and were analysed by student’s t-test, P < 0.01. GMR-GAL4 was used to drive expression of Aβ1-42 and AβQ3-42 transgenic flies. (PDF 417 kb

Additional file 2: Figure S2. of pGluAβ increases accumulation of Aβ in vivo and exacerbates its toxicity

Expression of AβpE3-42 causes locomotor dysfunction. Climbing ability of elavGS/UAS-AβQ3-42and elavGS flies on + RU486 SY medium was assessed at the indicated time-points (see Materials & Methods). Expression of AβpE3-42 in adult neurons reduced climbing ability of the flies in comparison to control elavGS driver flies. Data are presented as the average performance index (PI) ± SEM and were compared using 2-way ANOVA (number of independent tests (n) = 3 P < 0.01. (PDF 306 kb

Additional file 3: Figure S3. of pGluAβ increases accumulation of Aβ in vivo and exacerbates its toxicity

(A). Confirming the expression of Neprilysin 2 in the EP(3)3549 fly strain. There was a significant increase in nep2 transcript levels in the flies expressing the EP(3)3549 EP element, in comparison to the control fly lines expressing LACZ. Data are presented as means ± SEM and were analysed by student’s t-test, P < 0.001. (B) Neprilysin2 significantly reduces Aβ protein levels, *P < 0.001 . AβX-42 levels were quantified by ELISA. Data are presented as means ± SEM and were analysed by Student t test. (C) Neprilysin2 does not reduce AβQ3-42 RNA levels. There was a significant increase in AβQ3-42 RNA levels in flies co-expressing AβQ3-42 and NEP2 in comparison to flies expressing AβQ3-42 and EGFP, by quantitative RTPCR, P < 0.01, student’s t-test. GMR-GAL4 was used to drive expression of AβQ3-42 transgenic flies. (PDF 369 kb

Additional file 1: Figure S1. of pGluAβ increases accumulation of Aβ in vivo and exacerbates its toxicity

Generation of AβpE3-42. The proenkephalin signaling peptide upstream of AβQ3-42 is cleaved by prohormone convertases, AβQ3-42 is then released, and glutaminyl cyclase catalyses its conversion to AβpE3-42. (PDF 271 kb

Activating transcription factor 4-dependent lactate dehydrogenase activation as a protective response to amyloid beta toxicity

Accumulation of amyloid beta peptides is thought to initiate the pathogenesis of Alzheimer’s disease. However, the precise mechanisms mediating their neurotoxicity are unclear. Our microarray analyses show that, in Drosophila models of amyloid beta 42 toxicity, genes involved in the unfolded protein response and metabolic processes are upregulated in brain. Comparison with the brain transcriptome of early-stage Alzheimer’s patients revealed a common transcriptional signature, but with generally opposing directions of gene expression changes between flies and humans. Among these differentially regulated genes, lactate dehydrogenase (Ldh) was up-regulated by the greatest degree in amyloid beta 42 flies and the human orthologs (LDHA and LDHB) were down-regulated in patients. Functional analyses revealed that either over-expression or inhibition of Ldh by RNA interference (RNAi) slightly exacerbated climbing defects in both healthy and amyloid beta 42-induced Drosophila. This suggests that metabolic responses to lactate dehydrogenase must be finely-tuned, and that its observed upregulation following amyloid beta 42 production could potentially represent a compensatory protection to maintain pathway homeostasis in this model, with further manipulation leading to detrimental effects. The increased Ldh expression in amyloid beta 42 flies was regulated partially by unfolded protein response signalling, as ATF4 RNAi diminished the transcriptional response and enhanced amyloid beta 42-induced climbing phenotypes. Further functional studies are required to determine whether Ldh upregulation provides compensatory neuroprotection against amyloid beta 42-induced loss of activating transcription factor 4 activity and endoplasmatic reticulum stress.Our study thus reveals dysregulation of lactate dehydrogenase signalling in Drosophila models and patients with Alzheimer’s disease, which may lead to a detrimental loss of metabolic homeostasis. Importantly, we observed that down-regulation of ATF4-dependent endoplasmic reticulum-stress signalling in this context appears to prevent Ldh compensation and to exacerbate amyloid beta 42-dependent neuronal toxicity. Our findings therefore suggest caution in the use of therapeutic strategies focused on down-regulation of this pathway for treatment of Alzheimer’s disease, since its natural response to the toxic peptide may induce beneficial neuroprotective effects

Aβ42 inhibits cncC activity in <i>Drosophila</i>.

<p>(A) Nrf2/cncC activity was measured by crossing <i>gstD1</i>(ARE)–GFP reporter flies to UAS-attP Aβ-expressing lines, under control of the <i>Drosophila</i> necrotic signal peptide [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006593#pgen.1006593.ref036" target="_blank">36</a>], then measuring GFP expression in heads by Western blotting. (B) Quantitation of WB depicted in (A) above. A separate experiment was run for each Aβ-expressing line. GFP expression was normalized to actin, for–RU and +RU samples, then expressed as a percentage of the average–RU value for each blot to enable comparison. ArcAβ42 significantly reduced GFP expression compared to controls (** <i>p</i>< 0.01 comparing +RU to–RU). <i>p</i>>0.05 comparing +RU to–RU for WT Aβ40 and 42 lines (student’s t-test). Data are presented as means ± SEM and were analysed by student’s t-test for each genotype. N = 4 biological repeats of 10 fly heads per sample. (C) Analysis of Nrf2/cncC activity in response to high levels of WT Aβ42 expression. <i>gstD1</i>(ARE)–GFP reporter flies were crossed to flies expressing a tandem double copy of WT Aβ42 under the control of the argos signal peptide [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006593#pgen.1006593.ref037" target="_blank">37</a>]. Data were analysed as described in (B). * <i>p</i><0.05 comparing -RU to +RU (student’s t-test). N = 4 biological repeats of 10 fly heads per sample. (D) Heatmaps depicting gene ontology (GO) categories altered by ArcAβ42 expression in + RU vs–RU UAS-Arc Aβ42>elavGS flies (column Arc Aβ42) and that overlap with categories altered by cncC over-expression[<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006593#pgen.1006593.ref038" target="_blank">38</a>]; column cncC). Red indicates higher expression; blue indicates lower expression (scale = log₁₀ fold change). A random insertion UAS-ArcAβ42 line was used for these experiments[<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006593#pgen.1006593.ref039" target="_blank">39</a>]. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006593#pgen.1006593.s001" target="_blank">S1A Fig</a> for effects of ArcAβ42 induction of Nrf2 activity in heads and bodies using these flies. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006593#pgen.1006593.s001" target="_blank">S1B Fig</a> for Venn diagrams of reciprocal effects of ArcAβ42 and cncC activation on GO category expression.</p