Susanna Campesan

Glutathione peroxidase activity is neuroprotective in models of Huntington's disease

Huntingtons disease is a fatal neurodegenerative disorder caused by a CAG repeat expansion encoding a polyglutamine tract in the huntingtin (Htt) protein. Here we report a genome-wide overexpression suppressor screen in which we identified 317 ORFs that ameliorate the toxicity of a mutant Htt fragment in yeast and that have roles in diverse cellular processes, including mitochondrial import and copper metabolism. Two of these suppressors encode glutathione peroxidases (GPxs), which are conserved antioxidant enzymes that catalyze the reduction of hydrogen peroxide and lipid hydroperoxides. Using genetic and pharmacological approaches in yeast, mammalian cells and Drosophila, we found that GPx activity robustly ameliorates Huntingtons disease–relevant metrics and is more protective than other antioxidant approaches tested here. Notably, we found that GPx activity, unlike many antioxidant treatments, does not inhibit autophagy, which is an important mechanism for clearing mutant Htt. Because previous clinical trials have indicated that GPx mimetics are well tolerated in humans, this study may have important implications for treating Huntingtons disease.

Dendritic spine loss and neurodegeneration is rescued by Rab11 in models of Huntington's disease

Huntingtons disease (HD) is a fatal neurodegenerative disorder caused by expansion of a polyglutamine tract in the huntingtin protein (htt) that mediates formation of intracellular protein aggregates. In the brains of HD patients and HD transgenic mice, accumulation of protein aggregates has been causally linked to lesions in axo-dendritic and synaptic compartments. Here we show that dendritic spines – sites of synaptogenesis – are lost in the proximity of htt aggregates because of functional defects in local endosomal recycling mediated by the Rab11 protein. Impaired exit from recycling endosomes (RE) and association of endocytosed protein with intracellular structures containing htt aggregates was demonstrated in cultured hippocampal neurons cells expressing a mutant htt fragment. Dendrites in hippocampal neurons became dystrophic around enlarged amphisome-like structures positive for Rab11, LC3 and mutant htt aggregates. Furthermore, Rab11 overexpression rescues neurodegeneration and dramatically extends lifespan in a Drosophila model of HD. Our findings are consistent with the model that mutant htt aggregation increases local autophagic activity, thereby sequestering Rab11 and diverting spine-forming cargo from RE into enlarged amphisomes. This mechanism may contribute to the toxicity caused by protein misfolding found in a number of neurodegenerative diseases.

Rab11 rescues synaptic dysfunction and behavioural deficits in a Drosophila model of Huntington's disease

Synapse abnormalities in Huntingtons disease (HD) patients can precede clinical diagnosis and neuron loss by decades. The polyglutamine expansion in the huntingtin (htt) protein that underlies this disorder leads to perturbations in many cellular pathways, including the disruption of Rab11-dependent endosomal recycling. Impairment of the small GTPase Rab11 leads to the defective formation of vesicles in HD models and may thus contribute to the early stages of the synaptic dysfunction in this disorder. Here, we employ transgenic Drosophila melanogaster models of HD to investigate anomalies at the synapse and the role of Rab11 in this pathology. We find that the expression of mutant htt in the larval neuromuscular junction decreases the presynaptic vesicle size, reduces quantal amplitudes and evoked synaptic transmission and alters larval crawling behaviour. Furthermore, these indicators of early synaptic dysfunction are reversed by the overexpression of Rab11. This work highlights a potential novel HD therapeutic strategy for early intervention, prior to neuronal loss and clinical manifestation of disease.

DJ-1 modulates aggregation and pathogenesis in models of Huntington's disease

The oxidation-sensitive chaperone protein DJ-1 has been implicated in several human disorders including cancer and neurodegenerative diseases. During neurodegeneration associated with protein misfolding, such as that observed in Alzheimers disease and Huntingtons disease (HD), both oxidative stress and protein chaperones have been shown to modulate disease pathways. Therefore, we set out to investigate whether DJ-1 plays a role in HD. We found that DJ-1 expression and its oxidation state are abnormally increased in the human HD brain, as well as in mouse and cell models of HD. Furthermore, overexpression of DJ-1 conferred protection in vivo against neurodegeneration in yeast and Drosophila. Importantly, the DJ-1 protein directly interacted with an expanded fragment of huntingtin Exon 1 (httEx1) in test tube experiments and in cell models and accelerated polyglutamine aggregation and toxicity in an oxidation-sensitive manner. Our findings clearly establish DJ-1 as a potential therapeutic target for HD and provide the basis for further studies into the role of DJ-1 in protein misfolding diseases.

Drosophila eye color mutants as therapeutic tools for Huntington disease

Huntington disease (HD) is a fatal inherited neurodegenerative disorder caused by a polyglutamine expansion in the huntingtin protein (htt). A pathological hallmark of the disease is the loss of a specific population of striatal neurons, and considerable attention has been paid to the role of the kynurenine pathway (KP) of tryptophan (TRP) degradation in this process. The KP contains three neuroactive metabolites: 3-hydroxykynurenine (3-HK), quinolinic acid (QUIN), and kynurenic acid (KYNA). 3-HK and QUIN are neurotoxic, and are increased in the brains of early stage HD patients, as well as in yeast and mouse models of HD. Conversely, KYNA is neuroprotective and has been shown to be decreased in HD patient brains. We recently used a Drosophila model of HD to measure the neuroprotective effect of genetic and pharmacological inhibition of kynurenine monoxygenase (KMO)—the enzyme catalyzing the formation of 3-HK at a pivotal branch point in the KP. We found that KMO inhibition in Drosophila robustly attenuated neurodegeneration, and that this neuroprotection was correlated with reduced levels of 3-HK relative to KYNA. Importantly, we showed that KP metabolites are causative in this process, as 3-HK and KYNA feeding experiments modulated neurodegeneration. We also found that genetic inhibition of the upstream KP enzyme tryptophan-2,3-dioxygenase (TDO) was neuroprotective in flies. Here, we extend these results by reporting that genetic impairment of KMO or TDO is protective against the eclosion defect in HD model fruit flies. Our results provide further support for the possibility of therapeutic KP interventions in HD.

Nitric oxide-mediated posttranslational modifications control neurotransmitter release by modulating complexin farnesylation and enhancing its clamping ability

Nitric oxide (NO) regulates neuronal function and thus is critical for tuning neuronal communication. Mechanisms by which NO modulates protein function and interaction include posttranslational modifications (PTMs) such as S-nitrosylation. Importantly, cross signaling between S-nitrosylation and prenylation can have major regulatory potential. However, the exact protein targets and resulting changes in function remain elusive. Here, we interrogated the role of NO-dependent PTMs and farnesylation in synaptic transmission. We found that NO compromises synaptic function at the Drosophila neuromuscular junction (NMJ) in a cGMP-independent manner. NO suppressed release and reduced the size of available vesicle pools, which was reversed by glutathione (GSH) and occluded by genetic up-regulation of GSH-generating and de-nitrosylating glutamate-cysteine-ligase and S-nitroso-glutathione reductase activities. Enhanced nitrergic activity led to S-nitrosylation of the fusion-clamp protein complexin (cpx) and altered its membrane association and interactions with active zone (AZ) and soluble N-ethyl-maleimide-sensitive fusion protein Attachment Protein Receptor (SNARE) proteins. Furthermore, genetic and pharmacological suppression of farnesylation and a nitrosylation mimetic mutant of cpx induced identical physiological and localization phenotypes as caused by NO. Together, our data provide evidence for a novel physiological nitrergic molecular switch involving S-nitrosylation, which reversibly suppresses farnesylation and thereby enhances the net-clamping function of cpx. These data illustrate a new mechanistic signaling pathway by which regulation of farnesylation can fine-tune synaptic release.