α-Lipoic Acid Has the Potential to Normalize Copper Metabolism, Which Is Dysregulated in Alzheimer's Disease.

BACKGROUND: Alzheimer's disease (AD) is an age-dependent progressive neurodegenerative disorder and the most common cause of dementia. The treatment and prevention of AD present immense yet unmet needs. One of the hallmarks of AD is the formation of extracellular amyloid plaques in the brain, composed of amyloid-ß (Aß) peptides. Besides major amyloid-targeting approach there is the necessity to focus also on alternative therapeutic strategies. One factor contributing to the development of AD is dysregulated copper metabolism, reflected in the intracellular copper deficit and excess of extracellular copper. OBJECTIVE: In the current study, we follow the widely accepted hypothesis that the normalization of copper metabolism leads to the prevention or slowing of the disease and search for new copper-regulating ligands. METHODS: We used cell culture, ICP MS, and Drosophila melanogaster models of AD. RESULTS: We demonstrate that the natural intracellular copper chelator, α-lipoic acid (LA) translocates copper from extracellular to intracellular space in an SH-SY5Y-based neuronal cell model and is thus suitable to alleviate the intracellular copper deficit characteristic of AD neurons. Furthermore, we show that supplementation with LA protects the Drosophila melanogaster models of AD from developing AD phenotype by improving locomotor activity of fruit fly with overexpression of human Aß with Iowa mutation in the fly brain. In addition, LA slightly weakens copper-induced smooth eye phenotype when amyloid-ß protein precursor (AßPP) and beta-site AßPP cleaving enzyme 1 (BACE1) are overexpressed in eye photoreceptor cells. CONCLUSION: Collectively, these results provide evidence that LA has the potential to normalize copper metabolism in AD.

Daughterless, the Drosophila orthologue of TCF4, is required for associative learning and maintenance of the synaptic proteome.

Mammalian transcription factor 4 (TCF4) has been linked to schizophrenia and intellectual disabilities, such as Pitt-Hopkins syndrome (PTHS). Here, we show that similarly to mammalian TCF4, fruit fly orthologue Daughterless (Da) is expressed widely in the Drosophila brain. Furthermore, silencing of da, using several central nervous system-specific Gal4 driver lines, impairs appetitive associative learning of the larvae and leads to decreased levels of the synaptic proteins Synapsin (Syn) and Discs large 1 (Dlg1), suggesting the involvement of Da in memory formation. Here, we demonstrate that Syn and dlg1 are direct target genes of Da in adult Drosophila heads, as Da binds to the regulatory regions of these genes and the modulation of Da levels alter the levels of Syn and dlg1 mRNA. Silencing of da also affects negative geotaxis of the adult flies, suggesting the impairment of locomotor function. Overall, our findings suggest that Da regulates Drosophila larval memory and adult negative geotaxis, possibly via its synaptic target genes Syn and dlg1 These behavioural phenotypes can be further used as a PTHS model to screen for therapeutics.This article has an associated First Person interview with the first author of the paper.

In vivo screening reveals interactions between Drosophila Manf and genes involved in the mitochondria and the ubiquinone synthesis pathway.

BACKGROUND: Mesencephalic Astrocyte-derived Neurotrophic Factor (MANF) and Cerebral Dopamine Neurotrophic Factor (CDNF) form an evolutionarily conserved family of neurotrophic factors. Orthologues for MANF/CDNF are the only neurotrophic factors as yet identified in invertebrates with conserved amino acid sequence. Previous studies indicate that mammalian MANF and CDNF support and protect brain dopaminergic system in non-cell-autonomous manner. However, MANF has also been shown to function intracellularly in the endoplasmic reticulum. To date, the knowledge on the interacting partners of MANF/CDNF and signaling pathways they activate is rudimentary. Here, we have employed the Drosophila genetics to screen for potential interaction partners of Drosophila Manf (DmManf) in vivo. RESULTS: We first show that DmManf plays a role in the development of Drosophila wing. We exploited this function by using Drosophila UAS-RNAi lines and discovered novel genetic interactions of DmManf with genes known to function in the mitochondria. We also found evidence of an interaction between DmManf and the Drosophila homologue encoding Ku70, the closest structural homologue of SAP domain of mammalian MANF. CONCLUSIONS: In addition to the previously known functions of MANF/CDNF protein family, DmManf also interacts with mitochondria-related genes. Our data supports the functional importance of these evolutionarily significant proteins and provides new insights for the future studies.

Exploring the Conserved Role of MANF in the Unfolded Protein Response in Drosophila melanogaster.

Disturbances in the homeostasis of endoplasmic reticulum (ER) referred to as ER stress is involved in a variety of human diseases. ER stress activates unfolded protein response (UPR), a cellular mechanism the purpose of which is to restore ER homeostasis. Previous studies show that Mesencephalic Astrocyte-derived Neurotrophic Factor (MANF) is an important novel component in the regulation of UPR. In vertebrates, MANF is upregulated by ER stress and protects cells against ER stress-induced cell death. Biochemical studies have revealed an interaction between mammalian MANF and GRP78, the major ER chaperone promoting protein folding. In this study we discovered that the upregulation of MANF expression in response to drug-induced ER stress is conserved between Drosophila and mammals. Additionally, by using a genetic in vivo approach we found genetic interactions between Drosophila Manf and genes encoding for Drosophila homologues of GRP78, PERK and XBP1, the key components of UPR. Our data suggest a role for Manf in the regulation of Drosophila UPR.

Introducing Pitt-Hopkins syndrome-associated mutations of TCF4 to Drosophila daughterless.

Pitt-Hopkins syndrome (PTHS) is caused by haploinsufficiency of Transcription factor 4 (TCF4), one of the three human class I basic helix-loop-helix transcription factors called E-proteins. Drosophila has a single E-protein, Daughterless (Da), homologous to all three mammalian counterparts. Here we show that human TCF4 can rescue Da deficiency during fruit fly nervous system development. Overexpression of Da or TCF4 specifically in adult flies significantly decreases their survival rates, indicating that these factors are crucial even after development has been completed. We generated da transgenic fruit fly strains with corresponding missense mutations R578H, R580W, R582P and A614V found in TCF4 of PTHS patients and studied the impact of these mutations in vivo. Overexpression of wild type Da as well as human TCF4 in progenitor tissues induced ectopic sensory bristles and the rough eye phenotype. By contrast, overexpression of Da(R580W) and Da(R582P) that disrupt DNA binding reduced the number of bristles and induced the rough eye phenotype with partial lack of pigmentation, indicating that these act dominant negatively. Compared to the wild type, Da(R578H) and Da(A614V) were less potent in induction of ectopic bristles and the rough eye phenotype, respectively, suggesting that these are hypomorphic. All studied PTHS-associated mutations that we introduced into Da led to similar effects in vivo as the same mutations in TCF4 in vitro. Consequently, our Drosophila models of PTHS are applicable for further studies aiming to unravel the molecular mechanisms of this disorder.

Gene expression analysis of Drosophilaa Manf mutants reveals perturbations in membrane traffic and major metabolic changes.

BACKGROUND: MANF and CDNF are evolutionarily conserved neurotrophic factors that specifically support dopaminergic neurons. To date, the receptors and signalling pathways of this novel MANF/CDNF family have remained unknown. Independent studies have showed upregulation of MANF by unfolded protein response (UPR). To enlighten the role of MANF in multicellular organism development we carried out a microarray-based analysis of the transcriptional changes induced by the loss and overexpression of Drosophila Manf. RESULTS: The most dramatic change of expression was observed with genes coding membrane transport proteins and genes related to metabolism. When evaluating in parallel the ultrastructural data and transcriptome changes of maternal/zygotic and only zygotic Manf mutants, the endoplasmic reticulum (ER) stress and membrane traffic alterations were evident. In Drosophila Manf mutants the expression of several genes involved in Parkinson's disease (PD) was altered as well. CONCLUSIONS: We conclude that besides a neurotrophic factor, Manf is an important cellular survival factor needed to overcome the UPR especially in tissues with high secretory function. In the absence of Manf, the expression of genes involved in membrane transport, particularly exocytosis and endosomal recycling pathway was altered. In neurodegenerative diseases, such as PD, correct protein folding and proteasome function as well as neurotransmitter synthesis and uptake are crucial for the survival of neurons. The degeneration of dopaminergic neurons is the hallmark for PD and our work provides a clue on the mechanisms by which the novel neurotrophic factor MANF protects these neurons.

Evidence that DmMANF is an invertebrate neurotrophic factor supporting dopaminergic neurons.

In vertebrates the development and function of the nervous system is regulated by neurotrophic factors (NTFs). Despite extensive searches no neurotrophic factors have been found in invertebrates. However, cell ablation studies in Drosophila suggest trophic interaction between neurons and glia. Here we report the invertebrate neurotrophic factor in Drosophila, DmMANF, homologous to mammalian MANF and CDNF. DmMANF is expressed in glia and essential for maintenance of dopamine positive neurites and dopamine levels. The abolishment of both maternal and zygotic DmMANF leads to the degeneration of axonal bundles in the embryonic central nervous system and subsequent nonapoptotic cell death. The rescue experiments confirm DmMANF as a functional ortholog of the human MANF gene thus opening the window for comparative studies of this protein family with potential for the treatment of Parkinson's disease.

GABA receptor rho subunit expression in the developing rat brain.

Ionotropic GABA(C) receptors are composed of rho1, rho2 and rho3 subunits. Although the distribution of rho subunit mRNAs in the adult brain has been studied, information on the developmental regulation of different rho subunits in the brain is scattered and incomplete. Here, GABA(C) receptor rho subunit expression was studied in the developing rat brain. In situ hybridization on postnatal brain slices showed rho2 mRNA expression from newborn in superficial gray layer (SGL) of superior colliculus (SuC), and from the first postnatal week in the hippocampal CA1 region and pretectal nucleus of the optic tract. rho2 mRNA was also expressed in the adult dorsal lateral geniculate nucleus. Quantitative RT-PCR revealed expression of all three rho subunits in the hippocampus and superior colliculus from the first postnatal day. In the hippocampus, rho2 mRNA expression clearly dominated over rho1 and rho3, whereas in the superior colliculus, rho1 mRNA expression levels were similar to rho2. In both areas, a clear up-modulation of rho2 and rho3 mRNA during the first postnatal week was detected. GABA(C) receptor protein expression was confirmed in adult hippocampus, superior colliculus and dorsal lateral geniculate nucleus by immunohistochemistry. Our results demonstrate for the first time the expression of all three rho subunit mRNAs in several regions of the developing and adult rat brain. Our quantitative data allows assessment of putative subunit combinations in the superior colliculus and hippocampus. From the selective distribution of rho subunits, it may be hypothesized that GABA(C) receptors are specifically involved in aspects of visual image motion processing in the rat brain.