Lysosomal storage, impaired autophagy and innate immunity in Gaucher and Parkinson's diseases: insights for drug discovery.

Impairment of autophagic-lysosomal pathways is increasingly being implicated in Parkinson's disease (PD). GBA1 mutations cause the lysosomal storage disorder Gaucher disease (GD) and are the commonest known genetic risk factor for PD. GBA1 mutations have been shown to cause autophagic-lysosomal impairment. Defective autophagic degradation of unwanted cellular constituents is associated with several pathologies, including loss of normal protein homeostasis, particularly of α-synuclein, and innate immune dysfunction. The latter is observed both peripherally and centrally in PD and GD. Here, we will discuss the mechanistic links between autophagy and immune dysregulation, and the possible role of these pathologies in communication between the gut and brain in these disorders. Recent work in a fly model of neuronopathic GD (nGD) revealed intestinal autophagic defects leading to gastrointestinal dysfunction and immune activation. Rapamycin treatment partially reversed the autophagic block and reduced immune activity, in association with increased survival and improved locomotor performance. Alterations in the gut microbiome are a critical driver of neuroinflammation, and studies have revealed that eradication of the microbiome in nGD fly and mouse models of PD ameliorate brain inflammation. Following these observations, lysosomal-autophagic pathways, innate immune signalling and microbiome dysbiosis are discussed as potential therapeutic targets in PD and GD. This article is part of a discussion meeting issue 'Understanding the endo-lysosomal network in neurodegeneration'.

Autophagic dysfunction and gut microbiota dysbiosis cause chronic immune activation in a Drosophila model of Gaucher disease.

Mutations in the GBA1 gene cause the lysosomal storage disorder Gaucher disease (GD) and are the greatest known genetic risk factors for Parkinson's disease (PD). Communication between the gut and brain and immune dysregulation are increasingly being implicated in neurodegenerative disorders such as PD. Here, we show that flies lacking the Gba1b gene, the main fly orthologue of GBA1, display widespread NF-kB signalling activation, including gut inflammation, and brain glial activation. We also demonstrate intestinal autophagic defects, gut dysfunction, and microbiome dysbiosis. Remarkably, modulating the microbiome of Gba1b knockout flies, by raising them under germ-free conditions, partially ameliorates lifespan, locomotor and immune phenotypes. Moreover, we show that modulation of the immune deficiency (IMD) pathway is detrimental to the survival of Gba1 deficient flies. We also reveal that direct stimulation of autophagy by rapamycin treatment achieves similar benefits to germ-free conditions independent of gut bacterial load. Consistent with this, we show that pharmacologically blocking autophagosomal-lysosomal fusion, mimicking the autophagy defects of Gba1 depleted cells, is sufficient to stimulate intestinal immune activation. Overall, our data elucidate a mechanism whereby an altered microbiome, coupled with defects in autophagy, drive chronic activation of NF-kB signaling in a Gba1 loss-of-function model. It also highlights that elimination of the microbiota or stimulation of autophagy to remove immune mediators, rather than prolonged immunosuppression, may represent effective therapeutic avenues for GBA1-associated disorders.

Fine-tuning autophagy maximises lifespan and is associated with changes in mitochondrial gene expression in Drosophila.

Increased cellular degradation by autophagy is a feature of many interventions that delay ageing. We report here that increased autophagy is necessary for reduced insulin-like signalling (IIS) to extend lifespan in Drosophila and is sufficient on its own to increase lifespan. We first established that the well-characterised lifespan extension associated with deletion of the insulin receptor substrate chico was completely abrogated by downregulation of the essential autophagy gene Atg5. We next directly induced autophagy by over-expressing the major autophagy kinase Atg1 and found that a mild increase in autophagy extended lifespan. Interestingly, strong Atg1 up-regulation was detrimental to lifespan. Transcriptomic and metabolomic approaches identified specific signatures mediated by varying levels of autophagy in flies. Transcriptional upregulation of mitochondrial-related genes was the signature most specifically associated with mild Atg1 upregulation and extended lifespan, whereas short-lived flies, possessing strong Atg1 overexpression, showed reduced mitochondrial metabolism and up-regulated immune system pathways. Increased proteasomal activity and reduced triacylglycerol levels were features shared by both moderate and high Atg1 overexpression conditions. These contrasting effects of autophagy on ageing and differential metabolic profiles highlight the importance of fine-tuning autophagy levels to achieve optimal healthspan and disease prevention.

Shifting equilibriums in Alzheimer's disease: the complex roles of microglia in neuroinflammation, neuronal survival and neurogenesis.

Alzheimer's disease is the leading cause of dementia. Its increased prevalence in developed countries, due to the sharp rise in ageing populations, presents one of the costliest challenges to modern medicine. In order to find disease-modifying therapies to confront this challenge, a more complete understanding of the pathogenesis of Alzheimer's disease is necessary. Recent studies have revealed increasing evidence for the roles played by microglia, the resident innate immune system cells of the brain. Reflecting the well-established roles of microglia in reacting to pathogens and inflammatory stimuli, there is now a growing literature describing both protective and detrimental effects for individual cytokines and chemokines produced by microglia in Alzheimer's disease. A smaller but increasing number of studies have also addressed the divergent roles played by microglial neurotrophic and neurogenic factors, and how their perturbation may play a key role in the pathogenesis of Alzheimer's disease. Here we review recent findings on the roles played by microglia in neuroinflammation, neuronal survival and neurogenesis in Alzheimer's disease. In each case, landmark studies have provided evidence for the divergent ways in which microglia can either promote neuronal function and survival, or perturb neuronal function, leading to cell death. In many cases, the secreted molecules of microglia can lead to divergent effects depending on the magnitude and context of microglial activation. This suggests that microglial functions must be maintained in a fine equilibrium, in order to support healthy neuronal function, and that the cellular microenvironment in the Alzheimer's disease brain disrupts this fine balance, leading to neurodegeneration. Thus, an understanding of microglial homeostasis, both in health and across the trajectory of the disease state, will improve our understanding of the pathogenic mechanisms underlying Alzheimer's disease, and will hopefully lead to the development of microglial-based therapeutic strategies to restore equilibrium in the Alzheimer's disease brain.

Agephagy - Adapting Autophagy for Health During Aging.

Autophagy is a major cellular recycling process that delivers cellular material and entire organelles to lysosomes for degradation, in a selective or non-selective manner. This process is essential for the maintenance of cellular energy levels, components, and metabolites, as well as the elimination of cellular molecular damage, thereby playing an important role in numerous cellular activities. An important function of autophagy is to enable survival under starvation conditions and other stresses. The majority of factors implicated in aging are modifiable through the process of autophagy, including the accumulation of oxidative damage and loss of proteostasis, genomic instability and epigenetic alteration. These primary causes of damage could lead to mitochondrial dysfunction, deregulation of nutrient sensing pathways and cellular senescence, finally causing a variety of aging phenotypes. Remarkably, advances in the biology of aging have revealed that aging is a malleable process: a mild decrease in signaling through nutrient-sensing pathways can improve health and extend lifespan in all model organisms tested. Consequently, autophagy is implicated in both aging and age-related disease. Enhancement of the autophagy process is a common characteristic of all principal, evolutionary conserved anti-aging interventions, including dietary restriction, as well as inhibition of target of rapamycin (TOR) and insulin/IGF-1 signaling (IIS). As an emerging and critical process in aging, this review will highlight how autophagy can be modulated for health improvement.

A triple drug combination targeting components of the nutrient-sensing network maximizes longevity.

Increasing life expectancy is causing the prevalence of age-related diseases to rise, and there is an urgent need for new strategies to improve health at older ages. Reduced activity of insulin/insulin-like growth factor signaling (IIS) and mechanistic target of rapamycin (mTOR) nutrient-sensing signaling network can extend lifespan and improve health during aging in diverse organisms. However, the extensive feedback in this network and adverse side effects of inhibition imply that simultaneous targeting of specific effectors in the network may most effectively combat the effects of aging. We show that the mitogen-activated protein kinase kinase (MEK) inhibitor trametinib, the mTOR complex 1 (mTORC1) inhibitor rapamycin, and the glycogen synthase kinase-3 (GSK-3) inhibitor lithium act additively to increase longevity in Drosophila Remarkably, the triple drug combination increased lifespan by 48%. Furthermore, the combination of lithium with rapamycin cancelled the latter's effects on lipid metabolism. In conclusion, a polypharmacology approach of combining established, prolongevity drug inhibitors of specific nodes may be the most effective way to target the nutrient-sensing network to improve late-life health.

The emerging role of autophagic-lysosomal dysfunction in Gaucher disease and Parkinson's disease.

Gaucher disease (GD), the commonest lysosomal storage disorder, results from the lack or functional deficiency of glucocerebrosidase (GCase) secondary to mutations in the GBA1 gene. There is an established association between GBA1 mutations and Parkinson's disease (PD), and indeed GBA1 mutations are now considered to be the greatest genetic risk factor for PD. Impaired lysosomal-autophagic degradation of cellular proteins, including α-synuclein (α-syn), is implicated in the pathogenesis of PD, and there is increasing evidence for this also in GD and GBA1-PD. Indeed we have recently shown in a Drosophila model lacking neuronal GCase, that there are clear lysosomal-autophagic defects in association with synaptic loss and neurodegeneration. In addition, we demonstrated alterations in mechanistic target of rapamycin complex 1 (mTORC1) signaling and functional rescue of the lifespan, locomotor defects and hypersensitivity to oxidative stress on treatment of GCase-deficient flies with the mTOR inhibitor rapamycin. Moreover, a number of other recent studies have shown autophagy-lysosomal system (ALS) dysfunction, with specific defects in both chaperone-mediated autophagy (CMA), as well as macroautophagy, in GD and GBA1-PD model systems. Lastly we discuss the possible therapeutic benefits of inhibiting mTOR using drugs such as rapamycin to reverse the autophagy defects in GD and PD.

A Drosophila Model of Neuronopathic Gaucher Disease Demonstrates Lysosomal-Autophagic Defects and Altered mTOR Signalling and Is Functionally Rescued by Rapamycin.

Glucocerebrosidase (GBA1) mutations are associated with Gaucher disease (GD), an autosomal recessive disorder caused by functional deficiency of glucocerebrosidase (GBA), a lysosomal enzyme that hydrolyzes glucosylceramide to ceramide and glucose. Neuronopathic forms of GD can be associated with rapid neurological decline (Type II) or manifest as a chronic form (Type III) with a wide spectrum of neurological signs. Furthermore, there is now a well-established link between GBA1 mutations and Parkinson's disease (PD), with heterozygote mutations in GBA1 considered the commonest genetic defect in PD. Here we describe a novel Drosophila model of GD that lacks the two fly GBA1 orthologs. This knock-out model recapitulates the main features of GD at the cellular level with severe lysosomal defects and accumulation of glucosylceramide in the fly brain. We also demonstrate a block in autophagy flux in association with reduced lifespan, age-dependent locomotor deficits and accumulation of autophagy substrates in dGBA-deficient fly brains. Furthermore, mechanistic target of rapamycin (mTOR) signaling is downregulated in dGBA knock-out flies, with a concomitant upregulation of Mitf gene expression, the fly ortholog of mammalian TFEB, likely as a compensatory response to the autophagy block. Moreover, the mTOR inhibitor rapamycin is able to partially ameliorate the lifespan, locomotor, and oxidative stress phenotypes. Together, our results demonstrate that this dGBA1-deficient fly model is a useful platform for the further study of the role of lysosomal-autophagic impairment and the potential therapeutic benefits of rapamycin in neuronopathic GD. These results also have important implications for the role of autophagy and mTOR signaling in GBA1-associated PD SIGNIFICANCE STATEMENT: We developed a Drosophila model of neuronopathic GD by knocking-out the fly orthologs of the GBA1 gene, demonstrating abnormal lysosomal pathology in the fly brain. Functioning lysosomes are required for autophagosome-lysosomal fusion in the autophagy pathway. We show in vivo that autophagy is impaired in dGBA-deficient fly brains. In response, mechanistic target of rapamycin (mTOR) activity is downregulated in dGBA-deficient flies and rapamycin ameliorates the lifespan, locomotor, and oxidative stress phenotypes. dGBA knock-out flies also display an upregulation of the Drosophila ortholog of mammalian TFEB, Mitf, a response that is unable to overcome the autophagy block. Together, our results suggest that rapamycin may have potential benefits in the treatment of GD, as well as PD linked to GBA1 mutations.

Mitochondrial dysfunction and defects in lipid homeostasis as therapeutic targets in neurodegeneration with brain iron accumulation.

The PLA2G6 gene encodes a group VIA calcium independent phospholipase A2 (iPLA2ß), which hydrolyses glycerophospholipids to release fatty acids and lysophospholipids. Mutations in PLA2G6 are associated with a number of neurodegenerative disorders including neurodegeneration with brain iron accumulation (NBIA), infantile neuroaxonal dystrophy (INAD), and dystonia parkinsonism, collectively known as PLA2G6-associated neurodegeneration (PLAN). Recently Kinghorn et al. demonstrated in Drosophila and PLA2G6 mutant fibroblasts that loss of normal PLA2G6 activity is associated with mitochondrial dysfunction and mitochondrial lipid peroxidation. Furthermore, they were able to show the beneficial effects of deuterated polyunsaturated fatty acids (D-PUFAs), which reduce lipid peroxidation. D-PUFAs were able to rescue the locomotor deficits of flies lacking the fly ortholog of PLA2G6 (iPLA2-VIA), as well as the mitochondrial abnormalities in PLA2G6 mutant fibroblasts. This work demonstrated that the iPLA2-VIA knockout fly is a useful organism to dissect the mechanisms of pathogenesis of PLAN, and that further investigation is required to determine the therapeutic potential of D-PUFAs in patients with PLA2G6 mutations. The fruit fly has also been used to study some of the other genetic causes of NBIA, and here we also describe what is known about the mechanisms of pathogenesis of these NBIA variants. Mitochondrial dysfunction, defects in lipid metabolism, as well as defective Coenzyme A (CoA) biosynthesis, have all been implicated in some genetic forms of NBIA, including PANK2, CoASY, C12orf19 and FA2H.

Lithium Promotes Longevity through GSK3/NRF2-Dependent Hormesis.

The quest to extend healthspan via pharmacological means is becoming increasingly urgent, both from a health and economic perspective. Here we show that lithium, a drug approved for human use, promotes longevity and healthspan. We demonstrate that lithium extends lifespan in female and male Drosophila, when administered throughout adulthood or only later in life. The life-extending mechanism involves the inhibition of glycogen synthase kinase-3 (GSK-3) and activation of the transcription factor nuclear factor erythroid 2-related factor (NRF-2). Combining genetic loss of the NRF-2 repressor Kelch-like ECH-associated protein 1 (Keap1) with lithium treatment revealed that high levels of NRF-2 activation conferred stress resistance, while low levels additionally promoted longevity. The discovery of GSK-3 as a therapeutic target for aging will likely lead to more effective treatments that can modulate mammalian aging and further improve health in later life.

Genetics and pharmacology of longevity: the road to therapeutics for healthy aging.

Aging can be defined as the progressive decline in tissue and organismal function and the ability to respond to stress that occurs in association with homeostatic failure and the accumulation of molecular damage. Aging is the biggest risk factor for human disease and results in a wide range of aging pathologies. Although we do not completely understand the underlying molecular basis that drives the aging process, we have gained exceptional insights into the plasticity of life span and healthspan from the use of model organisms such as the worm Caenorhabditis elegans and the fruit fly Drosophila melanogaster. Single-gene mutations in key cellular pathways that regulate environmental sensing, and the response to stress, have been identified that prolong life span across evolution from yeast to mammals. These genetic manipulations also correlate with a delay in the onset of tissue and organismal dysfunction. While the molecular genetics of aging will remain a prosperous and attractive area of research in biogerontology, we are moving towards an era defined by the search for therapeutic drugs that promote healthy aging. Translational biogerontology will require incorporation of both therapeutic and pharmacological concepts. The use of model organisms will remain central to the quest for drug discovery, but as we uncover molecular processes regulated by repurposed drugs and polypharmacy, studies of pharmacodynamics and pharmacokinetics, drug-drug interactions, drug toxicity, and therapeutic index will slowly become more prevalent in aging research. As we move from genetics to pharmacology and therapeutics, studies will not only require demonstration of life span extension and an underlying molecular mechanism, but also the translational relevance for human health and disease prevention.

Loss of PLA2G6 leads to elevated mitochondrial lipid peroxidation and mitochondrial dysfunction.

The PLA2G6 gene encodes a group VIA calcium-independent phospholipase A2 beta enzyme that selectively hydrolyses glycerophospholipids to release free fatty acids. Mutations in PLA2G6 have been associated with disorders such as infantile neuroaxonal dystrophy, neurodegeneration with brain iron accumulation type II and Karak syndrome. More recently, PLA2G6 was identified as the causative gene in a subgroup of patients with autosomal recessive early-onset dystonia-parkinsonism. Neuropathological examination revealed widespread Lewy body pathology and the accumulation of hyperphosphorylated tau, supporting a link between PLA2G6 mutations and parkinsonian disorders. Here we show that knockout of the Drosophila homologue of the PLA2G6 gene, iPLA2-VIA, results in reduced survival, locomotor deficits and organismal hypersensitivity to oxidative stress. Furthermore, we demonstrate that loss of iPLA2-VIA function leads to a number of mitochondrial abnormalities, including mitochondrial respiratory chain dysfunction, reduced ATP synthesis and abnormal mitochondrial morphology. Moreover, we show that loss of iPLA2-VIA is strongly associated with increased lipid peroxidation levels. We confirmed our findings using cultured fibroblasts taken from two patients with mutations in the PLA2G6 gene. Similar abnormalities were seen including elevated mitochondrial lipid peroxidation and mitochondrial membrane defects, as well as raised levels of cytoplasmic and mitochondrial reactive oxygen species. Finally, we demonstrated that deuterated polyunsaturated fatty acids, which inhibit lipid peroxidation, were able to partially rescue the locomotor abnormalities seen in aged flies lacking iPLA2-VIA gene function, and restore mitochondrial membrane potential in fibroblasts from patients with PLA2G6 mutations. Taken together, our findings demonstrate that loss of normal PLA2G6 gene activity leads to lipid peroxidation, mitochondrial dysfunction and subsequent mitochondrial membrane abnormalities. Furthermore we show that the iPLA2-VIA knockout fly model provides a useful platform for the further study of PLA2G6-associated neurodegeneration.

[Molecular gerontology: towards healthy aging]. / Gerontología molecular: hacia un envejecimiento saludable.

For many years aging research was confined to statistics, psychology, and socioeconomic aspects of old age. However, today the study of aging is one of the most attractive and prosperous fields in biology. This change followed on from observations that single gene mutations can modulate the aging process, demonstrating the dynamic and plastic nature of the pathways involved. The field of aging is continually being fuelled by the discovery of new genes and pathways that extend lifespan when manipulated in organisms ranging from unicellular yeast to the more complex round worm C. elegans and the fruit fly Drosophila melanogaster. Such interventions have also been successful in mammals, proving the principle that discoveries in invertebrates can be evolutionarily relevant to humans. The most successful and evolutionary conserved interventions are those related to nutrient sensing pathways, the effector pathways upon which dietary restriction operates to promote health and longevity. To validate the existence of genes that modify the aging process in humans, biogerontologists have opted for a genome-wide approach to studying centenarians, those fortunate to live beyond 100 years of age. By studying these individuals, they hope to unravel the genetic signatures that promote healthy ageing and long life.

Misfolded PrP impairs the UPS by interaction with the 20S proteasome and inhibition of substrate entry.

Prion diseases are associated with the conversion of cellular prion protein (PrP(C)) to toxic ß-sheet isoforms (PrP(Sc)), which are reported to inhibit the ubiquitin-proteasome system (UPS). Accordingly, UPS substrates accumulate in prion-infected mouse brains, suggesting impairment of the 26S proteasome. A direct interaction between its 20S core particle and PrP isoforms was demonstrated by immunoprecipitation. ß-PrP aggregates associated with the 20S particle, but did not impede binding of the PA26 complex, suggesting that the aggregates do not bind to its ends. Aggregated ß-PrP reduced the 20S proteasome's basal peptidase activity, and the enhanced activity induced by C-terminal peptides from the 19S ATPases or by the 19S regulator itself, including when stimulated by polyubiquitin conjugates. However, the 20S proteasome was not inhibited when the gate in the α-ring was open due to a truncation mutation or by association with PA26/PA28. These PrP aggregates inhibit by stabilising the closed conformation of the substrate entry channel. A similar inhibition of substrate entry into the proteasome may occur in other neurodegenerative diseases where misfolded ß-sheet-rich proteins accumulate.

Neuroserpin binds Abeta and is a neuroprotective component of amyloid plaques in Alzheimer disease.

Alzheimer disease is characterized by extracellular plaques composed of Abeta peptides. We show here that these plaques also contain the serine protease inhibitor neuroserpin and that neuroserpin forms a 1:1 binary complex with the N-terminal or middle parts of the Abeta(1-42) peptide. This complex inactivates neuroserpin as an inhibitor of tissue plasminogen activator and blocks the loop-sheet polymerization process that is characteristic of members of the serpin superfamily. In contrast neuroserpin accelerates the aggregation of Abeta(1-42) with the resulting species having an appearance that is distinct from the mature amyloid fibril. Neuroserpin reduces the cytotoxicity of Abeta(1-42) when assessed using standard cell assays, and the interaction has been confirmed in vivo in novel Drosophila models of disease. Taken together, these data show that neuroserpin interacts with Abeta(1-42) to form off-pathway non-toxic oligomers and so protects neurons in Alzheimer disease.

Sugar and alcohol molecules provide a therapeutic strategy for the serpinopathies that cause dementia and cirrhosis.

Mutations in neuroserpin and alpha1-antitrypsin cause these proteins to form ordered polymers that are retained within the endoplasmic reticulum of neurones and hepatocytes, respectively. The resulting inclusions underlie the dementia familial encephalopathy with neuroserpin inclusion bodies (FENIB) and Z alpha1-antitrypsin-associated cirrhosis. Polymers form by a sequential linkage between the reactive centre loop of one molecule and beta-sheet A of another, and strategies that block polymer formation are likely to be successful in treating the associated disease. We show here that glycerol, the sugar alcohol erythritol, the disaccharide trehalose and its breakdown product glucose reduce the rate of polymerization of wild-type neuroserpin and the Ser49Pro mutant that causes dementia. They also attenuate the polymerization of the Z variant of alpha1-antitrypsin. The effect on polymerization was apparent even when these agents had been removed from the buffer. None of these agents had any detectable effect on the structure or inhibitory activity of neuroserpin or alpha1-antitrypsin. These data demonstrate that sugar and alcohol molecules can reduce the polymerization of serpin mutants that cause disease, possibly by binding to and stabilizing beta-sheet A.

Polymerisation underlies alpha1-antitrypsin deficiency, dementia and other serpinopathies.

We review here the molecular mechanisms that underlie alpha1-antitrypsin deficiency and show how an understanding of this mechanism has allowed us to explain the deficiency of other members of the serine proteinase inhibitor or serpin superfamily. These include the deficiency of antithrombin, C1-inhibitor and alpha1-antichymotrypsin in association with thrombosis, angio-oedema and emphysema respectively. Moreover the accumulation of mutant neuroserpin within neurones causes the novel dementia familial encephalopathy with neuroserpin inclusion bodies (FENIB). We have grouped these conditions together as the serpinopathies as recognition of their common pathophysiology provides a platform to develop strategies to treat the associated clinical syndromes.

Therapeutic targets from a Drosophila model of Alzheimer's disease.

The dominating role of the mouse in modeling Alzheimer's disease has been challenged this year by Drosophila melanogaster. Transgenic flies expressing toxic beta-amyloid peptides develop neurodegeneration. It is these peptides that accumulate in human disease and are thought to be the initiating factor in Alzheimer's disease. The flies exhibit a clear phenotype from a few days of age, including reduced locomotor function, impaired olfactory memory and shortened lifespan. Therapeutic agents that interfere with the generation of toxic aggregates of beta-amyloid peptides have been shown to rescue the flies. Several groups are now using the power and speed of genetic screens in the fly to accelerate the discovery of novel therapeutic agents.