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Background The increasing age of global populations highlights the urgent need to understand the biological underpinnings of ageing. To this end, inhibition of the insulin/insulin-like signalling (IIS) pathway can extend healthy lifespan in diverse animal species, but with trade-offs including delayed development. It is possible that distinct cell types underlie effects on development and ageing; cell-type-specific strategies could therefore potentially avoid negative trade-offs when targeting diseases of ageing, including prevalent neurodegenerative diseases. The highly conserved diversity of neuronal and non-neuronal (glial) cell types in the Drosophila nervous system makes it an attractive system to address this possibility. We have thus investigated whether IIS in distinct glial cell populations differentially modulates development and lifespan in Drosophila. Results We report here that glia-specific IIS inhibition, using several genetic means, delays development while extending healthy lifespan. The effects on lifespan can be recapitulated by adult-onset IIS inhibition, whereas developmental IIS inhibition is dispensable for modulation of lifespan. Notably, the effects we observe on both lifespan and development act through the PI3K branch of the IIS pathway and are dependent on the transcription factor FOXO. Finally, IIS inhibition in several glial subtypes can delay development without extending lifespan, whereas the same manipulations in astrocyte-like glia alone are sufficient to extend lifespan without altering developmental timing. Conclusions These findings reveal a role for distinct glial subpopulations in the organism-wide modulation of development and lifespan, with IIS in astrocyte-like glia contributing to lifespan modulation but not to developmental timing. Our results enable a more complete picture of the cell-type-specific effects of the IIS network, a pathway whose evolutionary conservation in humans make it tractable for therapeutic interventions. Our findings therefore underscore the necessity for cell-type-specific strategies to optimise interventions for the diseases of ageing.

Frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) are neurodegenerative disorders characterized by declining motor and cognitive functions. Even though these diseases present with distinct sets of symptoms, FTD and ALS are two extremes of the same disease spectrum, as they show considerable overlap in genetic, clinical and neuropathological features. Among these overlapping features, mitochondrial dysfunction is associated with both FTD and ALS. Recent studies have shown that cells derived from patients’ induced pluripotent stem cells (iPSC)s display mitochondrial abnormalities, and similar abnormalities have been observed in a number of animal disease models. Drosophila models have been widely used to study FTD and ALS because of their rapid generation time and extensive set of genetic tools. A wide array of fly models have been developed to elucidate the molecular mechanisms of toxicity for mutations associated with FTD/ALS. Fly models have been often instrumental in understanding the role of disease associated mutations in mitochondria biology. In this review, we discuss how mutations associated with FTD/ALS disrupt mitochondrial function, and we review how the use of Drosophila models has been pivotal to our current knowledge in this field.

Accumulation of Aβ in the brain is one of the hallmarks of Alzheimer’s disease (AD). In the adult Drosophila brain, human Aβ over-expression is toxic and leads to deterioration of climbing ability and shortened lifespan. However, it remains unknown if Aβ is inherently toxic or if it triggers toxic downstream pathways that lead to neurodegeneration. Here, we describe a novel, and previously unidentified, protective role of intracellular laminin chain accumulation. Despite high Aβ levels, over-expression of the extracellular matrix protein subunit Laminin B1 (LanB1) resulted in a robust rescue of toxicity, highlighting a potential protective mechanism of resistance to Aβ. Over-expression of other Laminin subunits and a Collagen IV subunit also significantly rescued Aβ toxicity, while combining LanB1 with these subunits led to an even larger rescue. Imaging revealed that LanB1 was retained in the ER but had no effect on the secretion of Aβ into the extracellular milieu. LanB1 rescued toxicity independently of the IRE1α/XBP1-mediated branch of the ER stress response. Interestingly, over-expression of ER-targeted GFP also rescued Aβ toxicity, indicating a potentially broader benefit of ER protein retention. Finally, in proof-of-principle lentiviral transduction experiments using murine organotypic hippocampal slice cultures, over-expression of mouse Lamb1 resulted in ER-retention in transduced cells, highlighting a conserved mechanism. Typically, retention of proteins in the ER is detrimental to cellular health, but in the context of neuronal Aβ toxicity it may prove to be beneficial and a new therapeutic avenue for AD.

Significance Aging is the main risk factor for the costliest diseases in today’s world. However, significant gaps remain in understanding how different cell types modulate this most common physiological process. Here, we use published single-cell gene expression data to map the prolongevity roles of two evolutionarily conserved Drosophila transcription factors, FKH and FOXO, onto either neuronal or glial cells. We then demonstrate that neuronal FKH preserves healthy function even under stress. Finally, we identify an autophagy-related gene as one of FKH’s downstream prolongevity effectors. Our results exemplify tapping into publicly available gene expression datasets to extract physiological insights, and highlight the need to shift away from organism-wide approaches and toward cell type-specific strategies to obtain meaningful insights in aging research., Reduced activity of insulin/insulin-like growth factor signaling (IIS) increases healthy lifespan among diverse animal species. Downstream of IIS, multiple evolutionarily conserved transcription factors (TFs) are required; however, distinct TFs are likely responsible for these effects in different tissues. Here we have asked which TFs can extend healthy lifespan within distinct cell types of the adult nervous system in Drosophila. Starting from published single-cell transcriptomic data, we report that forkhead (FKH) is endogenously expressed in neurons, whereas forkhead-box-O (FOXO) is expressed in glial cells. Accordingly, we find that neuronal FKH and glial FOXO exert independent prolongevity effects. We have further explored the role of neuronal FKH in a model of Alzheimer’s disease-associated neuronal dysfunction, where we find that increased neuronal FKH preserves behavioral function and reduces ubiquitinated protein aggregation. Finally, using transcriptomic profiling, we identify Atg17, a member of the Atg1 autophagy initiation family, as one FKH-dependent target whose neuronal overexpression is sufficient to extend healthy lifespan. Taken together, our results underscore the importance of cell type-specific mapping of TF activity to preserve healthy function with age.

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., Author summary The increasing number of people living with age-related diseases underscores the importance of ageing research to improve healthspan. Two well-studied evolutionary conserved interventions that extend lifespan and improve health are dietary restriction and down-regulation of nutrient sensing pathways, such as glucose sensing by insulin and amino acid sensing by the target-of-rapamycin signalling pathway. One common characteristic of these anti-ageing interventions is an increase in autophagy, a cellular pathway that degrades damaged proteins and organelles to supply essential building blocks and energy. To help provide a more direct link between autophagy and healthy ageing, we fine-tuned overexpression of Atg1 kinase, which is critical for autophagy induction, and measured its effect on longevity in the fruit fly Drosophila. Interestingly, we observed that a moderate increase in autophagy is beneficial in extending healthy lifespan, whereas strong autophagy up-regulation is detrimental and leads to progressive lipid loss and decreased lifespan. Moderate and stronger Atg1 overexpression displayed opposing transcriptional profiles of mitochondrial genes, being upregulated in long-lived and down-regulated in short-lived Atg1 over-expressing animals. Overall, we provide a detailed description of the phenotypes associated with varying degrees of autophagy up-regulation in vivo, demonstrating that autophagy enhancement delays ageing only when applied in moderation.

Alzheimer’s disease (AD) is the most common form of dementia and the most prevalent neurodegenerative disease. Genome-wide association studies have linked PICALM to AD risk. PICALM has been implicated in Aβ42 production and turnover, but whether it plays a direct role in modulating Aβ42 toxicity remains unclear. We found that increased expression of the Drosophila PICALM orthologue lap could rescue Aβ42 toxicity in an adult-onset model of AD, without affecting Aβ42 level. Imbalances in the glutamatergic system, leading to excessive, toxic stimulation, have been associated with AD. We found that Aβ42 caused the accumulation of presynaptic vesicular glutamate transporter (VGlut) and increased spontaneous glutamate release. Increased lap expression reversed these phenotypes back to control levels, suggesting that lap may modulate glutamatergic transmission. We also found that lap modulated the localization of amphiphysin (Amph), the homologue of another AD risk factor BIN1, and that Amph itself modulated postsynaptic glutamate receptor (GluRII) localization. We propose a model where PICALM modulates glutamatergic transmission, together with BIN1, to ameliorate synaptic dysfunction and disease progression., Graphical Abstract Graphical Abstractlap and Amph in glutamate metabolism. Aβ affects the localization of both VGlut and GluRIIA, leading to increased glutamate signalling. Lap and Amph restore glutamate signalling, potentially by promoting presynaptic endocytosis of VGlut via lap and/or Rab5/EndoA, and postsynaptic retrival of GluRIIA via Amph.

Inhibition of signalling through several receptor tyrosine kinases (RTKs), including the insulin‐like growth factor receptor and its orthologues, extends healthy lifespan in organisms from diverse evolutionary taxa. This raises the possibility that other RTKs, including those already well studied for their roles in cancer and developmental biology, could be promising targets for extending healthy lifespan. Here, we focus on anaplastic lymphoma kinase (Alk), an RTK with established roles in nervous system development and in multiple cancers, but whose effects on aging remain unclear. We find that several means of reducing Alk signalling, including mutation of its ligand jelly belly (jeb), RNAi knock‐down of Alk, or expression of dominant‐negative Alk in adult neurons, can extend healthy lifespan in female, but not male, Drosophila. Moreover, reduced Alk signalling preserves neuromuscular function with age, promotes resistance to starvation and xenobiotic stress, and improves night sleep consolidation. We find further that inhibition of Alk signalling in adult neurons modulates the expression of several insulin‐like peptides, providing a potential mechanistic link between neuronal Alk signalling and organism‐wide insulin‐like signalling. Finally, we show that TAE‐684, a small molecule inhibitor of Alk, can extend healthy lifespan in Drosophila, suggesting that the repurposing of Alk inhibitors may be a promising direction for strategies to promote healthy aging., The receptor tyrosine kinase Alk is expressed primarily in neurons, where its signalling overlaps with that of the lifespan‐modulating insulin signalling pathway. Here, we find that Alk inhibition extends lifespan, preserves locomotor function and improves stress resistance in female fruit flies, potentially via a mechanism involving insulin‐like peptide signalling from neurons to and from adipose tissues. These findings pave the way for future studies on Alk and aging in more complex animals.

Parkinson's disease patients report sleep disturbances well ahead of motor symptoms. In this issue of Neuron, Valadas et al. (2018) report that the disease genes pink1 and parkin exert novel, cell-type-specific effects to modulate ER-mitochondria contacts, neuropeptidergic transmission, and sleep patterns.

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.

The inflammatory response is a fundamental driving force in the pathogenesis of Alzheimer's disease (AD). In the setting of accumulating immunogenic As peptide assemblies, microglia, the innate immune cells of the brain, generate a non-resolving immune response and fail to adequately clear accumulating As peptides, accelerating neuronal and synaptic injury. Pathological, biomarker, and imaging studies point to a prominent role of the innate immune response in AD development, and the molecular components of this response are beginning to be unraveled. The inflammatory cyclooxygenase-PGE2 pathway is implicated in pre-clinical development of AD, both in epidemiology of normal aging populations and in transgenic mouse models of Familial AD. The cyclooxygenase-PGE2 pathway modulates the inflammatory response to accumulating As peptides through actions of specific E-prostanoid G-protein coupled receptors.

Transforming growth factor-β (TGF-β) plays an important role in the development and maintenance of embryonic dopaminergic (DA) neurons in the midbrain. To study the function of TGF-β signaling in the adult nigrostriatal system, we generated transgenic mice with reduced TGF-β signaling in mature neurons. These mice display age-related motor deficits and degeneration of the nigrostriatal system. Increasing TGF-β signaling in the substantia nigra through adeno-associated virus expressing a constitutively active type I receptor significantly reduces 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced dopaminergic neurodegeneration and motor deficits. These results suggest that TGF-β signaling is critical for adult DA neuron survival and that modulating this signaling pathway has therapeutic potential in Parkinson disease.SIGNIFICANCE STATEMENTWe show that reducing Transforming growth factor-β (TGF-β) signaling promotes Parkinson disease-related pathologies and motor deficits, and increasing TGF-β signaling reduces neurotoxicity of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, a parkinsonism-inducing agent. Our results provide a rationale to pursue a means of increasing TGF-β signaling as a potential therapy for Parkinson's disease.

There is significant evidence for a central role of inflammation in the development of Alzheimer disease (AD). Epidemiological studies indicate that chronic use of nonsteroidal anti-inflammatory drugs (NSAIDs) reduces the risk of developing AD in healthy aging populations. As NSAIDs inhibit the enzymatic activity of the inflammatory cyclooxygenases COX-1 and COX-2, these findings suggest that downstream prostaglandin signaling pathways function in the preclinical development of AD. Here, we investigate the function of prostaglandin E(2) (PGE(2) ) signaling through its EP3 receptor in the neuroinflammatory response to Aβ peptide.The function of PGE(2) signaling through its EP3 receptor was examined in vivo in a model of subacute neuroinflammation induced by administration of Aβ(42) peptides. Our findings were then confirmed in young adult APPSwe-PS1ΔE9 transgenic mice.Deletion of the PGE(2) EP3 receptor in a model of Aβ(42) peptide-induced neuroinflammation reduced proinflammatory gene expression, cytokine production, and oxidative stress. In the APPSwe-PS1ΔE9 model of familial AD, deletion of the EP3 receptor blocked induction of proinflammatory gene and protein expression and lipid peroxidation. In addition, levels of Aβ peptides were significantly decreased, as were β-secretase and β C-terminal fragment levels, suggesting that generation of Aβ peptides may be increased as a result of proinflammatory EP3 signaling. Finally, deletion of EP3 receptor significantly reversed the decline in presynaptic proteins seen in APPSwe-PS1ΔE9 mice.Our findings identify the PGE(2) EP3 receptor as a novel proinflammatory, proamyloidogenic, and synaptotoxic signaling pathway, and suggest a role for COX-PGE(2) -EP3 signaling in the development of AD.

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.

Peripheral inflammation leads to immune responses in brain characterized by microglial activation, elaboration of proinflammatory cytokines and reactive oxygen species, and secondary neuronal injury. The inducible cyclooxygenase (COX), COX-2, mediates a significant component of this response in brain via downstream proinflammatory PG signaling. In this study, we investigated the function of the PGE2 E-prostanoid (EP) 4 receptor in the CNS innate immune response to the bacterial endotoxin LPS. We report that PGE2 EP4 signaling mediates an anti-inflammatory effect in brain by blocking LPS-induced proinflammatory gene expression in mice. This was associated in cultured murine microglial cells with decreased Akt and I-κB kinase phosphorylation and decreased nuclear translocation of p65 and p50 NF-κB subunits. In vivo, conditional deletion of EP4 in macrophages and microglia increased lipid peroxidation and proinflammatory gene expression in brain and in isolated adult microglia following peripheral LPS administration. Conversely, EP4 selective agonist decreased LPS-induced proinflammatory gene expression in hippocampus and in isolated adult microglia. In plasma, EP4 agonist significantly reduced levels of proinflammatory cytokines and chemokines, indicating that peripheral EP4 activation protects the brain from systemic inflammation. The innate immune response is an important component of disease progression in a number of neurodegenerative disorders, such as Alzheimer’s disease, Parkinson’s disease, and amyotrophic lateral sclerosis. In addition, recent studies demonstrated adverse vascular effects with chronic administration of COX-2 inhibitors, indicating that specific PG signaling pathways may be protective in vascular function. This study supports an analogous and beneficial effect of PGE2 EP4 receptor signaling in suppressing brain inflammation.

Identifying preventive targets for Alzheimer's disease is a central challenge of modern medicine. Non-steroidal anti-inflammatory drugs, which inhibit the cyclooxygenase enzymes COX-1 and COX-2, reduce the risk of developing Alzheimer's disease in normal ageing populations. This preventive effect coincides with an extended preclinical phase that spans years to decades before onset of cognitive decline. In the brain, COX-2 is induced in neurons in response to excitatory synaptic activity and in glial cells in response to inflammation. To identify mechanisms underlying prevention of cognitive decline by anti-inflammatory drugs, we first identified an early object memory deficit in APPSwe-PS1ΔE9 mice that preceded previously identified spatial memory deficits in this model. We modelled prevention of this memory deficit with ibuprofen, and found that ibuprofen prevented memory impairment without producing any measurable changes in amyloid-β accumulation or glial inflammation. Instead, ibuprofen modulated hippocampal gene expression in pathways involved in neuronal plasticity and increased levels of norepinephrine and dopamine. The gene most highly downregulated by ibuprofen was neuronal tryptophan 2,3-dioxygenase (Tdo2), which encodes an enzyme that metabolizes tryptophan to kynurenine. TDO2 expression was increased by neuronal COX-2 activity, and overexpression of hippocampal TDO2 produced behavioural deficits. Moreover, pharmacological TDO2 inhibition prevented behavioural deficits in APPSwe-PS1ΔE9 mice. Taken together, these data demonstrate broad effects of cyclooxygenase inhibition on multiple neuronal pathways that counteract the neurotoxic effects of early accumulating amyloid-β oligomers.

Microglia, the innate immune cells of the CNS, perform critical inflammatory and noninflammatory functions that maintain normal neural function. For example, microglia clear misfolded proteins, elaborate trophic factors, and regulate and terminate toxic inflammation. In Alzheimer's disease (AD), however, beneficial microglial functions become impaired, accelerating synaptic and neuronal loss. Better understanding of the molecular mechanisms that contribute to microglial dysfunction is an important objective for identifying potential strategies to delay progression to AD. The inflammatory cyclooxygenase/prostaglandin E2 (COX/PGE2) pathway has been implicated in preclinical AD development, both in human epidemiology studies and in transgenic rodent models of AD. Here, we evaluated murine models that recapitulate microglial responses to Aβ peptides and determined that microglia-specific deletion of the gene encoding the PGE2 receptor EP2 restores microglial chemotaxis and Aβ clearance, suppresses toxic inflammation, increases cytoprotective insulin-like growth factor 1 (IGF1) signaling, and prevents synaptic injury and memory deficits. Our findings indicate that EP2 signaling suppresses beneficial microglia functions that falter during AD development and suggest that inhibition of the COX/PGE2/EP2 immune pathway has potential as a strategy to restore healthy microglial function and prevent progression to AD.

Increasing survival motor neuron 2 (SMN2) gene expression may be an effective strategy for the treatment of spinal muscular atrophy (SMA). Histone deacetylase (HDAC) inhibitors have been shown to increase SMN transcript and protein levels, but the specific role of histone acetylation in regulating SMN gene expression has not been explored. Using chromatin immunopreciptation, we investigated the levels of acetylated H3 and H4 histones and HDACs associated with different regions of the human and mouse SMN genes in both cultured cells and tissues. We show that the SMN gene has a reproducible pattern of histone acetylation that is largely conserved among different tissues and species. A limited region of the promoter surrounding the transcriptional start site has relatively high levels of histone acetylation, whereas regions further upstream or downstream have lower levels. After HDAC inhibitor treatment, acetylated histone levels increased, particularly at upstream regions, correlating with a 2-fold increase in promoter activity. During development in mouse tissues, histone acetylation levels decreased and associated HDAC2 levels increased at the region closest to the transcriptional start site, correlating with a 40-60% decrease in SMN transcript and protein levels. These data indicate that histone acetylation modulates SMN gene expression and that pharmacological manipulation of this epigenetic determinant is feasible. HDAC2, in particular, may be a future therapeutic target for SMA.

A persistent and nonresolving inflammatory response to accumulating Aβ peptide species is a cardinal feature in the development of Alzheimer's disease (AD). In response to accumulating Aβ peptide species, microglia, the innate immune cells of the brain, generate a toxic inflammatory response that accelerates synaptic and neuronal injury. Many proinflammatory signaling pathways are linked to progression of neurodegeneration. However, endogenous anti-inflammatory pathways capable of suppressing Aβ-induced inflammation represent a relatively unexplored area. Here we report that signaling through the prostaglandin-E2(PGE2) EP4 receptor potently suppresses microglial inflammatory responses to Aβ42peptides. In cultured microglial cells, EP4 stimulation attenuated levels of Aβ42-induced inflammatory factors and potentiated phagocytosis of Aβ42. Microarray analysis demonstrated that EP4 stimulation broadly opposed Aβ42-driven gene expression changes in microglia, with enrichment for targets of IRF1, IRF7, and NF-κB transcription factors.In vivo, conditional deletion of microglial EP4 in APPSwe-PS1ΔE9(APP-PS1) mice conversely increased inflammatory gene expression, oxidative protein modification, and Aβ deposition in brain at early stages of pathology, but not at later stages, suggesting an early anti-inflammatory function of microglial EP4 signaling in the APP-PS1 model. Finally, EP4 receptor levels decreased significantly in human cortex with progression from normal to AD states, suggesting that early loss of this beneficial signaling system in preclinical AD development may contribute to subsequent progression of pathology.

Prostaglandin E2(PGE2), a potent lipid signaling molecule, modulates inflammatory responses through activation of downstream G-protein coupled EP1–4receptors. Here, we investigated the cell-specificin vivofunction of PGE2signaling through its E-prostanoid 2 (EP2) receptor in murine innate immune responses systemically and in the CNS.In vivo, systemic administration of lipopolysaccharide (LPS) resulted in a broad induction of cytokines and chemokines in plasma that was significantly attenuated in EP2-deficient mice.Ex vivostimulation of peritoneal macrophages with LPS elicited proinflammatory responses that were dependent on EP2 signaling and that overlapped within vivoplasma findings, suggesting that myeloid-lineage EP2 signaling is a major effector of innate immune responses. Conditional deletion of the EP2 receptor in myeloid lineage cells in Cd11bCre;EP2lox/loxmice attenuated plasma inflammatory responses and transmission of systemic inflammation to the brain was inhibited, with decreased hippocampal inflammatory gene expression and cerebral cortical levels of IL-6. Conditional deletion of EP2 significantly blunted microglial and astrocytic inflammatory responses to the neurotoxin MPTP and reduced striatal dopamine turnover. Suppression of microglial EP2 signaling also increased numbers of dopaminergic (DA) neurons in the substantia nigra independent of MPTP treatment, suggesting that microglial EP2 may influence development or survival of DA neurons. Unbiased microarray analysis of microglia isolated from adult Cd11bCre;EP2lox/loxand control mice demonstrated a broad downregulation of inflammatory pathways with ablation of microglial EP2 receptor. Together, these data identify a cell-specific proinflammatory role for macrophage/microglial EP2 signaling in innate immune responses systemically and in brain.

Glucocerebrosidase (GBA1) mutations are associated with Gaucher disease (GD), the commonest lysosomal storage disorder, and are considered the commonest genetic defect in Parkinson9s disease (PD). GBA1 encodes a lysosomal enzyme, glucocerebrosidase (GBA), which hydrolyses glucosylceramide to ceramide and glucose. Neuronopathic GD can be associated with rapid neurological decline and death in infancy (Type II) or manifests as a chronic form (Type III) with a wide spectrum of neurological signs. Here we describe a novel Drosophila model of GBA1 loss-of-function that recapitulates the main features of GD at the cellular level with severe lysosomal defects and accumulation of the GBA substrate glucosylceramide in the fly brain. We also report the in vivo demonstration of autophagy block in the brains of GBA-deficient flies. This autophagy impairment occurs in association with reduced lifespan and age-dependent locomotor deficits and p62 and ubiquitinated protein aggregates in the fly brain. Finally, the mTOR inhibitor rapamycin is able to partially ameliorate the lifespan, starvation and oxidative stress phenotypes. Taken together our results suggest that this GBA1 knockout fly model is a useful platform for the further study of the role of lysosomal-autophagic impairment and the possible potential therapeutic benefits of rapamycin in neuronopathic GD and GBA1-associated PD.

Stroke is the third leading cause of death in the United States. Fewer than 5% of patients benefit from the only intervention approved to treat stroke. Thus, there is an enormous need to identify new therapeutic targets. The role of inducible cyclooxygenase (COX-2) activity in stroke and other neurologic diseases is complex, as both activation and sustained inhibition can engender cerebral injury. Whether COX-2 induces cerebroprotective or injurious effects is probably dependent on which downstream prostaglandin receptors are activated. Here, we investigated the function of the PGE2 receptor EP4 in a mouse model of cerebral ischemia. Systemic administration of a selective EP4 agonist after ischemia reduced infarct volume and ameliorated long-term behavioral deficits. Expression of EP4 was robust in neurons and markedly induced in endothelial cells after ischemia-reperfusion, suggesting that neuronal and/or endothelial EP4 signaling imparts cerebroprotection. Conditional genetic inactivation of neuronal EP4 worsened stroke outcome, consistent with an endogenous protective role of neuronal EP4 signaling in vivo. However, endothelial deletion of EP4 also worsened stroke injury and decreased cerebral reperfusion. Systemic administration of an EP4 agonist increased levels of activated eNOS in cerebral microvessels, an effect that was abolished with conditional deletion of endothelial EP4. Thus, our data support the concept of targeting protective prostaglandin receptors therapeutically after stroke.

We have utilized a transposable P element construct to scan the genome for modifiers of Drosophila Notch pathway phenotypes. From a collection of 2000 inserts we obtained two enhancers and one suppressor. Sequence analysis of the insertion regions demonstrated that two modifiers affect known components of the Notch pathway, whereas the third is an insert in the gene encoding ribosomal protein L13a at cytogenetic region 83B6-7. The insert in the RpL13A coding region creates a classic Minute mutation which enhances Notch pathway wing phenotypes. This report adds RpL13A to the list of Drosophila ribosomal protein genes that cause Minute phenotypes when mutated.

The Notch pathway comprises a signal transduction cascade required for the proper formation of multiple tissues during metazoan development. Originally described in Drosophila for its role in nervous system formation, the pathway has attracted much wider interest owing to its fundamental roles in a range of developmental and disease-related processes. Despite extensive analysis, Notch signaling is not completely understood and it appears that additional components of the pathway remain to be identified and characterized. Here, we describe a novel genetic strategy to screen for additional Notch pathway genes. The strategy combines partial loss of function for pathway activity with Enhancer-promoter (EP)-induced overexpression of random loci across the dorsoventral wing margin. Mastermind (Mam) is a nuclear component of the Notch signaling cascade. Using a GAL4-UAS-driven dominant-negative form of Mam, we created a genotype that exhibits a completely penetrant dominant wing-nicking phenotype. This phenotype was assayed for enhancement or suppression after outcrossing to several thousand EP lines. The screen identified known components or modifiers of Notch pathway function, as well as several potential new components. Our results suggest that a genetic screen that combines partial loss of function with random gene overexpression might be a useful strategy in the analysis of developmental pathways.