Ku70 alleviates neurodegeneration in Drosophila models of Huntington's disease

DNA damage accumulates in genome DNA during the long life of neurons, thus DNA damage repair is indispensable to keep normal functions of neurons. We previously reported that Ku70, a critical molecule for DNA double strand break (DSB) repair, is involved in the pathology of Huntington's disease (HD). Mutant huntingtin (Htt) impaired Ku70 function via direct interaction, and Ku70 supplementation recovered phenotypes of a mouse HD model. In this study, we generate multiple Drosophila HD models that express mutant huntingtin (Htt) in eye or motor neuron by different drivers and show various phenotypes. In such fly models, Ku70 co-expression recovers lifespan, locomotive activity and eye degeneration. In contrast, Ku70 reduction by heterozygous null mutation or siRNA-mediated knock down accelerates lifespan shortening and locomotion disability. These results collectively support that Ku70 is a critical mediator of the HD pathology and a candidate therapeutic target in HD.     

Brain-derived neurotrophic factor modulates dopaminergic deficits in a transgenic mouse model of Huntington's disease

Dysfunction of dopaminergic neurons may contribute to motor impairment in Huntington's disease. Here, we study the role of brain-derived neurotrophic factor (BDNF) in alterations of the nigrostriatal system associated with transgenics carrying mutant huntingtin. Using huntingtin-BDNF+/- double-mutant mice, we analyzed the effects of reducing the levels of BDNF expression in a model of Huntington's disease (R6/1). When compared with R6/1 mice, these mice exhibit an increased number of aggregates in the substantia nigra pars compacta. In addition, reduction of BDNF expression exacerbates the dopaminergic neuronal dysfunction seen in mutant huntingtin mice, such as the decrease in retrograde labelling of dopaminergic neurons and striatal dopamine content. However, mutant huntingtin mice with normal or lowered BDNF expression show the same decrease in the anterograde transport, number of dopaminergic neurons and nigral volume. In addition, reduced BDNF expression causes decreased dopamine receptor expression in mutant huntingtin mice. Examination of changes in locomotor activity induced by dopamine receptor agonists revealed that, in comparison with R6/1 mice, the double mutant mice exhibit lower activity in response to amphetamine, but not to apomorphine. In conclusion, these findings demonstrate that the decreased BDNF expression observed in Huntington's disease exacerbates dopaminergic neuronal dysfunction, which may participate in the motor disturbances associated with this neurodegenerative disorder.     

Huntingtin aggregation kinetics and their pathological role in a Drosophila Huntington's disease model

Huntington's disease is a neurodegenerative disorder resulting from expansion of a polyglutamine tract in the Huntingtin protein. Mutant Huntingtin forms intracellular aggregates within neurons, although it is unclear whether aggregates or more soluble forms of the protein represent the pathogenic species. To examine the link between aggregation and neurodegeneration, we generated Drosophila melanogaster transgenic strains expressing fluorescently tagged human huntingtin encoding pathogenic (Q138) or nonpathogenic (Q15) proteins, allowing in vivo imaging of Huntingtin expression and aggregation in live animals. Neuronal expression of pathogenic Huntingtin leads to pharate adult lethality, accompanied by formation of large aggregates within the cytoplasm of neuronal cell bodies and neurites. Live imaging and Fluorescence Recovery After Photobleaching (FRAP) analysis of pathogenic Huntingtin demonstrated that new aggregates can form in neurons within 12 hr, while preexisting aggregates rapidly accumulate new Huntingtin protein within minutes. To examine the role of aggregates in pathology, we conducted haplo-insufficiency suppressor screens for Huntingtin-Q138 aggregation or Huntingtin-Q138-induced lethality, using deficiencies covering ~80% of the Drosophila genome. We identified two classes of interacting suppressors in our screen: those that rescue viability while decreasing Huntingtin expression and aggregation and those that rescue viability without disrupting Huntingtin aggregation. The most robust suppressors reduced both soluble and aggregated Huntingtin levels, suggesting toxicity is likely to be associated with both forms of the mutant protein in Huntington's disease.     

The kynurenine pathway of tryptophan metabolism detected in the nervous system of Drosophila melanogaster

Isolated thoracic ganglia were incubated in physiological solution containing 14C-tryptophan. After this procedure, they were homogenized in a 1 per cent solution of HCL in methanol, and supernatant was subjected to two-dimensional thin layer chromatography in the presence of tryptophan, kynurenine, 3-hydroxy kynurenine, as well as kynurenic, anthranilic and xanthurenic acids. The spots were cut out and counted by liquid scintillation technique. Except tryptophan, only kynurenine and 3-hydroxy kynurenine spots contained notable radioactivity. Therefore, at least the initial stages of kynurenine pathway operate in the nervous system of Drosophila melanogaster. This finding is in accordance with observations of the effects of kynurenines on insect behaviour.     

From neuronal inclusions to neurodegeneration: neuropathological investigation of a transgenic mouse model of Huntington's disease.

Huntington's disease (HD) is an inherited progressive neurodegenerative disease caused by the expansion of a polyglutamine repeat sequence within a novel protein. Recent work has shown that abnormal intranuclear inclusions of aggregated mutant protein within neurons is a characteristic feature shared by HD and several other diseases involving glutamine repeat expansion. This suggests that in each of the these disorders the affected nerve cells degenerate as a result of these abnormal inclusions. A transgenic mouse model of HD has been generated by introducing exon 1 of the HD gene containing a highly expanded CAG sequence into the mouse germline. These mice develop widespread neuronal intranuclear inclusions and neurodegeneration specifically within those areas of the brain known to degenerate in HD. We have investigated the sequence of pathological changes that occur after the formation of nuclear inclusions and that precede neuronal cell death in these cells. Although the relation between inclusion formation and neurodegeneration has recently been questioned, a full characterization of the pathways linking protein aggregation and cell death will resolve some of these controversies and will additionally provide new targets for potential therapies.     

RACK1 modulates polyglutamine-induced neurodegeneration by promoting ERK degradation in Drosophila

Polyglutamine diseases are neurodegenerative diseases caused by the expansion of polyglutamine (polyQ) tracts within different proteins. Although multiple pathways have been found to modulate aggregation of the expanded polyQ proteins, the mechanisms by which polyQ tracts induced neuronal cell death remain unknown. We conducted a genome-wide genetic screen to identify genes that suppress polyQ-induced neurodegeneration when mutated. Loss of the scaffold protein RACK1 alleviated cell death associated with the expression of polyQ tracts alone, as well as in models of Machado-Joseph disease (MJD) and Huntington’s disease (HD), without affecting proteostasis of polyQ proteins. A genome-wide RNAi screen for modifiers of this rack1 suppression phenotype revealed that knockdown of the E3 ubiquitin ligase, POE (Purity of essence), further suppressed polyQ-induced cell death, resulting in nearly wild-type looking eyes. Biochemical analyses demonstrated that RACK1 interacts with POE and ERK to promote ERK degradation. These results suggest that RACK1 plays a key role in polyQ pathogenesis by promoting POE-dependent degradation of ERK, and implicate RACK1/POE/ERK as potent drug targets for treatment of polyQ diseases.     

TOR-mediated cell-cycle activation causes neurodegeneration in a Drosophila tauopathy model

BACKGROUND: Previous studies have demonstrated reexpression of cell-cycle markers within postmitotic neurons in neurodegenerative tauopathies, including Alzheimer's disease (AD). However, the critical questions of whether cell-cycle activation is causal or epiphenomenal to tau-induced neurodegeneration and which signaling pathways mediate cell-cycle activation in tauopathy remain unresolved. RESULTS: Cell-cycle activation accompanies wild-type and mutant tau-induced neurodegeneration in Drosophila, and genetically interfering with cell-cycle progression substantially reduces neurodegeneration. Our data support a role for cell-cycle activation downstream of tau phosphorylation, directly preceding apoptosis. We accordingly show that ectopic cell-cycle activation leads to apoptosis of postmitotic neurons in vivo. As in AD, TOR (target of rapamycin kinase) activity is increased in our model and is required for neurodegeneration. TOR activation enhances tau-induced neurodegeneration in a cell cycle-dependent manner and, when ectopically activated, drives cell-cycle activation and apoptosis in postmitotic neurons. CONCLUSIONS: TOR-mediated cell-cycle activation causes neurodegeneration in a Drosophila tauopathy model, identifying TOR and the cell cycle as potential therapeutic targets in tauopathies and AD.     

Drosophila: a "model" model system to study neurodegeneration

The fruit fly, Drosophila melanogaster, is a powerful model genetic organism that has been used since the turn of the previous century in the study of complex biological problems. In the last decade, numerous researchers have focused their attention on understanding neurodegenerative diseases by utilizing this model system. Numerous Drosophila mutants have been isolated that profoundly affect neural viability and integrity of the nervous system with age. Additionally, many transgenic strains have been developed as models of human disease conditions. We review the existing Drosophila neurodegenerative mutants and transgenic disease models, and discuss the role of the fruit fly in therapeutic development for neurodegenerative diseases.     

Antioxidants cannot suppress the lethal phenotype of a Drosophila melanogaster model of Huntington's disease.

Substantial evidence suggests that antioxidants may play a major role in delaying the progress of Huntington's disease (HD). Here we investigated the effects of superoxide dismutase (cytoplasmic Cu/ZnSOD and mitochondrial MnSOD) and supplementation with dietary antioxidants (alpha-tocopherol and coenzyme Q10) on survival to adulthood in a Drosophila melanogaster model of HD. Our results illustrate that neither overexpression of superoxide dismutase nor supplementation of dietary antioxidants can rescue the lethal phenotype of HD flies. We discuss these results in conjunction with other evidence that antioxidants may only avert the oxidative stress induced progression of HD.     

Glutathione s-transferase omega 1 activity is sufficient to suppress neurodegeneration in a Drosophila model of Parkinson disease

A loss-of-function mutation in the gene parkin causes a common neurodegenerative disease that may be caused by mitochondrial dysfunction. Glutathione S-transferase Omega (GSTO) is involved in cell defense mechanisms, but little is known about the role of GSTO in the progression of Parkinson disease. Here, we report that restoration of Drosophila GSTO1 (DmGSTO1), which is down-regulated in parkin mutants, alleviates some of the parkin pathogenic phenotypes and that the loss of DmGSTO1 function enhances parkin mutant phenotypes. We further identified the ATP synthase β subunit as a novel in vivo target of DmGSTO1. We found that glutathionylation of the ATP synthase β subunit is rescued by DmGSTO1 and that the expression of DmGSTO1 partially restores the activity and assembly of the mitochondrial F(1)F(0)-ATP synthase in parkin mutants. Our results suggest a novel mechanism for the protective role of DmGSTO1 in parkin mutants, through the regulation of ATP synthase activity, and provide insight into potential therapies for Parkinson disease neurodegeneration.     

Pur alpha binds to rCGG repeats and modulates repeat-mediated neurodegeneration in a Drosophila model of fragile X tremor/ataxia syndrome

Fragile X-associated tremor/ataxia syndrome (FXTAS) is a recently recognized neurodegenerative disorder in fragile X premutation carriers with FMR1 alleles containing 55-200 CGG repeats. Previously, we developed a Drosophila model of FXTAS and demonstrated that transcribed premutation repeats alone are sufficient to cause neurodegeneration, suggesting that rCGG-repeat-binding proteins (RBPs) may be sequestered from their normal function by rCGG binding. Here, we identify Pur alpha and hnRNP A2/B1 as RBPs. We show that Pur alpha and rCGG repeats interact in a sequence-specific fashion that is conserved between mammals and Drosophila. Overexpression of Pur alpha in Drosophila could suppress rCGG-mediated neurodegeneration in a dose-dependent manner. Furthermore, Pur alpha is also present in the inclusions of FXTAS patient brains. These findings support the disease mechanism of FXTAS of rCGG repeat sequestration of specific RBPs, leading to neuronal cell death, and implicate that Pur alpha plays an important role in the pathogenesis of FXTAS.     

Implications of the kynurenine pathway and quinolinic acid in Alzheimer's disease

Summary

Ozone, though not a free radical species, mediates its toxic effects through free radical reactions as a consequence of its high redox potential. Upon inspiration the first physical interface encountered by ozone is a thin layer of aqueous material, the epithelium lining fluid (ELF) which overlays, and is partially derived from, the underlying pulmonary epithelium. ELF is the first physical interface encountered by ozone and the majority of its primary actions are confined to this compartment. ELF contains a range of antioxidants, including the small molecular weight antioxidants: uric acid (UA), ascorbic acid (AH₂) and reduced glutathione (GSH). These compounds are present in large quantities and display high intrinsic reactivities toward ozone, consistent with their role as sacrificial substrates in this setting. In this paper we examine the concept that antioxidants, in ELF, represent the first tier of defence against the oxidizing effects of ozone. Since the concentration of these antioxidants appears to differ between individuals, we propose that these protective substances may dictate, in part, an individual's sensitivity to oxidizing air pollutants such as ozone.     

Implications of the kynurenine pathway and quinolinic acid in Alzheimer's disease

The kynurenine pathway (KP) is a major route of L-tryptophan catabolism leading to production of a number of biologically active molecules. Among them, the neurotoxin quinolinic acid (QUIN), is considered to be involved in the pathogenesis of a number of inflammatory neurological diseases. Alzheimer's disease is the major dementing disorder of the elderly that affects over 20 million peoples world-wide. Most of the approaches to explain the pathogenesis of Alzheimer's disease focus on the accumulation of amyloid beta peptide (A beta), in the form of insoluble deposits leading to formation of senile plaques, and on the formation of neurofibrillary tangles composed of hyperphosphorylated Tau protein. Accumulation of A beta is believed to be an early and critical step in the neuropathogenesis of Alzheimer's disease. There is now evidence for the KP being associated with Alzheimer's disease. Disturbances of the KP have already been described in Alzheimer's disease. Recently, we demonstrated that A beta 1-42, a cleavage product of amyloid precursor protein, induces production of QUIN, in neurotoxic concentrations, by macrophages and, more importantly, microglia. Senile plaques in Alzheimer's disease are associated with evidence of chronic local inflammation (especially activated microglia) A major aspect of QUIN toxicity is lipid peroxidation and markers of lipid peroxidation are found in Alzheimer's disease. Together, these data imply that QUIN may be one of the critical factors in the pathogenesis of neuronal damage in Alzheimer's disease. This review describes the multiple correlations between the KP and the neuropathogenesis of Alzheimer's disease and highlights more particularly the aspects of QUIN neurotoxicity, emphasizing its roles in lipid peroxidation and the amplification of the local inflammation.     

Age‐dependent alterations of the kynurenine pathway in the YAC128 mouse model of Huntington disease

Indoleamine 2,3 dioxygenase (Ido1), the first and rate‐limiting enzyme of the kynurenine pathway (KP), is a striatally enriched gene with increased expression levels in the YAC128 mouse model of Huntington disease (HD). Our objective in this study was to delineate age‐related KP alterations in this model. Three enzymes potentially catalyze the first step of the KP; Ido1 and Indoleamine 2,3 dioxygenase‐2 were highly expressed in the striatum and Tryptophan 2,3 dioxygenase (Tdo2) in the cerebellum. During development, Ido1 mRNA expression is dynamically regulated and chronically up‐regulated in YAC128 mice. Kynurenine (Kyn) to tryptophan (Trp) ratio, a measure of activity in the first step of the KP, was elevated in YAC128 striatum, but no change in Tdo2 mRNA levels or Kyn to Trp ratio was detected in the cerebellum. Ido1 induction was coincident with Trp depletion at 3 months and Kyn accumulation at 12 months of age in striatum. Changes in downstream KP metabolites of YAC128 mice generally followed a biphasic pattern with neurotoxic metabolites reduced at 3 months and increased at 12 months of age. Striatally specific induction of Ido1 and downstream KP alterations suggest involvement in HD pathogenesis, and should be taken into account in future therapeutic developments for HD.