Alzheimer's disease: insights from Drosophila melanogaster models.

The power of fruit fly genetics is being deployed against some of the most intractable and economically significant problems in modern medicine, the neurodegenerative diseases. Fly models of Alzheimer's disease can be exposed to the rich diversity of biological techniques that are available to the community and are providing new insights into disease mechanisms, and assisting in the identification of novel targets for therapy. Similar approaches might also help us to interpret the results of genome-wide association studies of human neurodegenerative diseases by allowing us to triage gene "hits" according to whether a candidate risk factor gene has a modifying effect on the disease phenotypes in fly model systems.

ALS mutations in FUS cause neuronal dysfunction and death in Caenorhabditis elegans by a dominant gain-of-function mechanism.

It is unclear whether mutations in fused in sarcoma (FUS) cause familial amyotrophic lateral sclerosis via a loss-of-function effect due to titrating FUS from the nucleus or a gain-of-function effect from cytoplasmic overabundance. To investigate this question, we generated a series of independent Caenorhabditis elegans lines expressing mutant or wild-type (WT) human FUS. We show that mutant FUS, but not WT-FUS, causes cytoplasmic mislocalization associated with progressive motor dysfunction and reduced lifespan. The severity of the mutant phenotype in C. elegans was directly correlated with the severity of the illness caused by the same mutation in humans, arguing that this model closely replicates key features of the human illness. Importantly, the mutant phenotype could not be rescued by overexpression of WT-FUS, even though WT-FUS had physiological intracellular localization, and was not recruited to the cytoplasmic mutant FUS aggregates. Our data suggest that FUS mutants cause neuronal dysfunction by a dominant gain-of-function effect related either to neurotoxic aggregates of mutant FUS in the cytoplasm or to dysfunction in its RNA-binding functions.

Iron promotes the toxicity of amyloid beta peptide by impeding its ordered aggregation.

We have previously shown that overexpressing subunits of the iron-binding protein ferritin can rescue the toxicity of the amyloid ß (Aß) peptide in our Drosophila model system. These data point to an important pathogenic role for iron in Alzheimer disease. In this study, we have used an iron-selective chelating compound and RNAi-mediated knockdown of endogenous ferritin to further manipulate iron in the brain. We confirm that chelation of iron protects the fly from the harmful effects of Aß. To understand the pathogenic mechanisms, we have used biophysical techniques to see how iron affects Aß aggregation. We find that iron slows the progression of the Aß peptide from an unstructured conformation to the ordered cross-ß fibrils that are characteristic of amyloid. Finally, using mammalian cell culture systems, we have shown that iron specifically enhances Aß toxicity but only if the metal is present throughout the aggregation process. These data support the hypothesis that iron delays the formation of well ordered aggregates of Aß and so promotes its toxicity in Alzheimer disease.

Impaired tissue growth is mediated by checkpoint kinase 1 (CHK1) in the integrated stress response.

The integrated stress response (ISR) protects cells from numerous forms of stress and is involved in the growth of solid tumours; however, it is unclear how the ISR acts on cellular proliferation. We have developed a model of ISR signalling with which to study its effects on tissue growth. Overexpression of the ISR kinase PERK resulted in a striking atrophic eye phenotype in Drosophila melanogaster that could be rescued by co-expressing the eIF2alpha phosphatase GADD34. A genetic screen of 3000 transposon insertions identified grapes, the gene that encodes the Drosophila orthologue of checkpoint kinase 1 (CHK1). Knockdown of grapes by RNAi rescued eye development despite ongoing PERK activation. In mammalian cells, CHK1 was activated by agents that induce ER stress, which resulted in a G2 cell cycle delay. PERK was both necessary and sufficient for CHK1 activation. These findings indicate that non-genotoxic misfolded protein stress accesses DNA-damage-induced cell cycle checkpoints to couple the ISR to cell cycle arrest.

The TRiC/CCT chaperone is implicated in Alzheimer's disease based on patient GWAS and an RNAi screen in Aß-expressing Caenorhabditis elegans.

The human Aß peptide causes progressive paralysis when expressed in the muscles of the nematode worm, C. elegans. We have exploited this model of Aß toxicity by carrying out an RNAi screen to identify genes whose reduced expression modifies the severity of this locomotor phenotype. Our initial finding was that none of the human orthologues of these worm genes is identical with the genome-wide significant GWAS genes reported to date (the "white zone"); moreover there was no identity between worm screen hits and the longer list of GWAS genes which included those with borderline levels of significance (the "grey zone"). This indicates that Aß toxicity should not be considered as equivalent to sporadic AD. To increase the sensitivity of our analysis, we then considered the physical interactors (+1 interactome) of the products of the genes in both the worm and the white+grey zone lists. When we consider these worm and GWAS gene lists we find that 4 of the 60 worm genes have a +1 interactome overlap that is larger than expected by chance. Two of these genes form a chaperonin complex, the third is closely associated with this complex and the fourth gene codes for actin, the major substrate of the same chaperonin.

Defects in IGF-1 receptor, insulin receptor and IRS-1/2 in Alzheimer's disease indicate possible resistance to IGF-1 and insulin signalling.

Insulin like growth factor-1 receptor (IGF-1R) and insulin receptor (IR) signalling control vital growth, survival and metabolic functions in the brain. Here we describe specific and significant alterations in IGF-1R, IR, and their key substrate adaptor proteins IRS-1 and IRS-2 in Alzheimer's disease (AD). Western immunoblot analysis detected increased IGF-1R levels, and decreased levels of IGF-1-binding protein-2 (IGFBP-2), a major IGF-1-binding protein, in AD temporal cortex. Increased IGF-1R was observed surrounding and within amyloid-beta (Abeta)-containing plaques, also evident in an animal model of AD, and in astrocytes in AD. However, despite the overall increase in IGF-1R levels, a significantly lower number of neurons expressed IGF-1R in AD, and IGF-1R was aberrantly distributed in AD neurons especially evident in those with neurofibrillary tangles (NFTs). IR protein levels were similar in AD and control cases, however, the IR was concentrated intracellularly in AD neurons, unlike its distribution throughout the neuronal cell soma and in dendrites in control brain. Significant decreases in IRS-1 and IRS-2 levels were identified in AD neurons, in association with increased levels of inactivated phospho(Ser312)IRS-1 and phospho(Ser616)IRS-1, where increased levels of these phosphoserine epitopes colocalised strongly with NFTs. Our results show that IGF-1R and IR signalling is compromised in AD neurons and suggest that neurons that degenerate in AD may be resistant to IGF-1R/IR signalling.

Akt signal transduction dysfunction in Parkinson's disease.

Significant attention has been drawn to the potential role of defective PI3-kinase-Akt (PKB) signalling in Parkinson's disease (PD) neurodegeneration and to the possibility that activation of Akt may provide neuroprotection in PD. However, little knowledge exists on the integrity of the Akt system in PD. Results of the present study show diminished levels of both total and active phospho(Ser473)-Akt in the brain in PD. This was evident by western blot analysis of midbrain fractions from PD compared to non-PD control brain, but more specifically by immunofluorescence microscopy of the substantia nigra pars compacta (SNpc). Here, double immunofluorescence microscopy found Akt and phospho(Ser473)-Akt to be expressed at high levels in tyrosine hydroxylase (TH) immunopositive dopaminergic neurons in control human brain. Selective loss of these neurons was accompanied by a marked decrease of Akt and phospho(Ser473)-Akt expression in the PD brain, however Akt and active phospho(Ser473)-Akt are still evident in degenerating dopaminergic neurons in the disease. This suggests that it may be possible to target neuronal Akt in advanced PD. Converse to the marked loss of neuronal Akt in PD, increased Akt and phospho(Ser473)-Akt levels were observed in small non-TH positive cells in PD SNpc, whose increased number and small nuclear size indicate they are glia. These findings implicate defective Akt as a putative signalling pathway linked to loss of dopaminergic neurons in PD.

Activation of Akt/PKB, increased phosphorylation of Akt substrates and loss and altered distribution of Akt and PTEN are features of Alzheimer's disease pathology.

Studies suggest that activation of phosphoinositide 3-kinase-Akt may protect against neuronal cell death in Alzheimer's disease (AD). Here, however, we provide evidence of increased Akt activation, and hyperphosphorylation of critical Akt substrates in AD brain, which link to AD pathogenesis, suggesting that treatments aiming to activate the pathway in AD need to be considered carefully. A different distribution of Akt and phospho-Akt was detected in AD temporal cortex neurons compared with control neurons, with increased levels of active phosphorylated-Akt in particulate fractions, and significant decreases in Akt levels in AD cytosolic fractions, causing increased activation of Akt (phosphorylated-Akt/total Akt ratio) in AD. In concordance, significant increases in the levels of phosphorylation of total Akt substrates, including: GSK3beta(Ser9), tau(Ser214), mTOR(Ser2448), and decreased levels of the Akt target, p27(kip1), were found in AD temporal cortex compared with controls. A significant loss and altered distribution of the major negative regulator of Akt, PTEN (phosphatase and tensin homologue deleted on chromosome 10), was also detected in AD neurons. Loss of phosphorylated-Akt and PTEN-containing neurons were found in hippocampal CA1 at end stages of AD. Taken together, these results support a potential role for aberrant control of Akt and PTEN signalling in AD.