Alzheimer's disease transcriptional landscape in ex-vivo human microglia.

Microglia are resident immune cells of the brain and are implicated in the etiology of Alzheimer's Disease (AD) and other diseases. Yet the cellular and molecular processes regulating their function throughout the course of the disease are poorly understood. Here, we present the transcriptional landscape of primary microglia from 189 human postmortem brains, including 58 healthy aging individuals and 131 with a range of disease phenotypes, including 63 patients representing the full spectrum of clinical and pathological severity of AD. We identified transcriptional changes associated with multiple AD phenotypes, capturing the severity of dementia and neuropathological lesions. Transcript-level analyses identified additional genes with heterogeneous isoform usage and AD phenotypes. We identified changes in gene-gene coordination in AD, dysregulation of co-expression modules, and disease subtypes with distinct gene expression. Taken together, these data further our understanding of the key role of microglia in AD biology and nominate candidates for therapeutic intervention.

Long-read RNA-seq atlas of novel microglia isoforms elucidates disease-associated genetic regulation of splicing.

Microglia, the innate immune cells of the central nervous system, have been genetically implicated in multiple neurodegenerative diseases. We previously mapped the genetic regulation of gene expression and mRNA splicing in human microglia, identifying several loci where common genetic variants in microglia-specific regulatory elements explain disease risk loci identified by GWAS. However, identifying genetic effects on splicing has been challenging due to the use of short sequencing reads to identify causal isoforms. Here we present the isoform-centric microglia genomic atlas (isoMiGA) which leverages the power of long-read RNA-seq to identify 35,879 novel microglia isoforms. We show that the novel microglia isoforms are involved in stimulation response and brain region specificity. We then quantified the expression of both known and novel isoforms in a multi-ethnic meta-analysis of 555 human microglia short-read RNA-seq samples from 391 donors, the largest to date, and found associations with genetic risk loci in Alzheimer's disease and Parkinson's disease. We nominate several loci that may act through complex changes in isoform and splice site usage.

Lineage specific 3D genome structure in the adult human brain and neurodevelopmental changes in the chromatin interactome.

The human brain is a complex organ comprised of distinct cell types, and the contribution of the 3D genome to lineage specific gene expression remains poorly understood. To decipher cell type specific genome architecture, and characterize fine scale changes in the chromatin interactome across neural development, we compared the 3D genome of the human fetal cortical plate to that of neurons and glia isolated from the adult prefrontal cortex. We found that neurons have weaker genome compartmentalization compared to glia, but stronger TADs, which emerge during fetal development. Furthermore, relative to glia, the neuronal genome shifts more strongly towards repressive compartments. Neurons have differential TAD boundaries that are proximal to active promoters involved in neurodevelopmental processes. CRISPRi on CNTNAP2 in hIPSC-derived neurons reveals that transcriptional inactivation correlates with loss of insulation at the differential boundary. Finally, re-wiring of chromatin loops during neural development is associated with transcriptional and functional changes. Importantly, differential loops in the fetal cortex are associated with autism GWAS loci, suggesting a neuropsychiatric disease mechanism affecting the chromatin interactome. Furthermore, neural development involves gaining enhancer-promoter loops that upregulate genes that control synaptic activity. Altogether, our study provides multi-scale insights on the 3D genome in the human brain.

Multi-omic atlas of the parahippocampal gyrus in Alzheimer's disease.

Alzheimer's disease (AD) is the most common form of dementia worldwide, with a projection of 151 million cases by 2050. Previous genetic studies have identified three main genes associated with early-onset familial Alzheimer's disease, however this subtype accounts for less than 5% of total cases. Next-generation sequencing has been well established and holds great promise to assist in the development of novel therapeutics as well as biomarkers to prevent or slow the progression of this devastating disease. Here we present a public resource of functional genomic data from the parahippocampal gyrus of 201 postmortem control, mild cognitively impaired (MCI) and AD individuals from the Mount Sinai brain bank, of which whole-genome sequencing (WGS), and bulk RNA sequencing (RNA-seq) were previously published. The genomic data include bulk proteomics and DNA methylation, as well as cell-type-specific RNA-seq and assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) data. We have performed extensive preprocessing and quality control, allowing the research community to access and utilize this public resource available on the Synapse platform at https://doi.org/10.7303/syn51180043.2 .

Efficient differential expression analysis of large-scale single cell transcriptomics data using dreamlet.

Advances in single-cell and -nucleus transcriptomics have enabled generation of increasingly large-scale datasets from hundreds of subjects and millions of cells. These studies promise to give unprecedented insight into the cell type specific biology of human disease. Yet performing differential expression analyses across subjects remains difficult due to challenges in statistical modeling of these complex studies and scaling analyses to large datasets. Our open-source R package dreamlet (DiseaseNeurogenomics.github.io/dreamlet) uses a pseudobulk approach based on precision-weighted linear mixed models to identify genes differentially expressed with traits across subjects for each cell cluster. Designed for data from large cohorts, dreamlet is substantially faster and uses less memory than existing workflows, while supporting complex statistical models and controlling the false positive rate. We demonstrate computational and statistical performance on published datasets, and a novel dataset of 1.4M single nuclei from postmortem brains of 150 Alzheimer's disease cases and 149 controls.

Carbon footprint analysis and comprehensive evaluation of municipal wastewater treatment plants under different typical upgrading and reconstruction modes.

The issue of greenhouse gas (GHG) emissions resulting from the upgrading and reconstruction of municipal wastewater treatment plants (MWWTPs) along with improved water quality is receiving attention and research. There is an urgent need to explore the impact of upgrading and reconstruction on carbon footprint (CF) in order to address concerns that the upgrading and reconstruction will increase GHG emissions while improving water quality. Here we accounted for the CF of five MWWTPs in Zhejiang Province, China, before and after three different upgrading and reconstruction models - "Improving quality and efficiency" ("Mode I"), "Upgrading and renovation" ("Mode U") and "Improving quality and efficiency plus Upgrading and renovation" ("Mode I plus U"). The upgrading and reconstruction was found to not necessarily result in more GHG emissions. In contrast, the "Mode I" had a more significant advantage in terms of CF reduction (1.82-12.6 % reduction in CF). Overall, the ratio of indirect emissions to direct emissions (indirect emissions/direct emissions) and the amount of GHG emitted per unit of pollutant removed (CFCOD、CFTN、CFTP) decreased, while both the carbon and energy neutral rates increased significantly (up to 33.29 % and 79.36 % respectively) after all three upgrading and reconstruction modes. In addition, the wastewater treatment efficiency and capacity are the main factors that affect the level of carbon emission. The results of this study can provide a calculation model that can be used for other similar MWWTPs during the upgrading and reconstruction processes. More importantly, it can provide a new research perspective as well as valuable information to revisit the impact of upgrading and reconstruction in MWWTPs on GHG emissions.

Efficient differential expression analysis of large-scale single cell transcriptomics data using dreamlet.

Advances in single-cell and -nucleus transcriptomics have enabled generation of increasingly large-scale datasets from hundreds of subjects and millions of cells. These studies promise to give unprecedented insight into the cell type specific biology of human disease. Yet performing differential expression analyses across subjects remains difficult due to challenges in statistical modeling of these complex studies and scaling analyses to large datasets. Our open-source R package dreamlet (DiseaseNeurogenomics.github.io/dreamlet) uses a pseudobulk approach based on precision-weighted linear mixed models to identify genes differentially expressed with traits across subjects for each cell cluster. Designed for data from large cohorts, dreamlet is substantially faster and uses less memory than existing workflows, while supporting complex statistical models and controlling the false positive rate. We demonstrate computational and statistical performance on published datasets, and a novel dataset of 1.4M single nuclei from postmortem brains of 150 Alzheimer's disease cases and 149 controls.

The fate of phthalate acid esters in wastewater treatment plants and their impact on receiving waters.

Phthalates (PAEs) are gaining attention and being researched as an endocrine disruptor as global plastic use surge. There is an urgent need to explore the key factors affecting the removal of PAEs from wastewater and the impact of wastewater effluent on receiving water. Here we investigated the levels and distribution patterns of 16 typical PAEs in surface water and five wastewater treatment plants (WWTPs) along the Dongyang River from Yiwu, China, collecting 42 surface water and 31 wastewater samples. We found that influent PAEs concentration and treatment process were the key factors affecting the degradation efficiency of PAEs in primary and secondary treatment, respectively. In primary treatment, long-chain PAEs were more easily removed (and sometimes less likely to accumulate) than short-chain PAEs, regardless of the influent PAEs concentration (a key factor in primary treatment), while in secondary treatment, short-chain PAEs were easily removed regardless of the treatment process (a factor in secondary treatment). This was not the case for long-chain PAEs, which were only more readily removed in the A/A/O process. In addition, by comparing the significant differences between wastewater and surface water, we found that the total PAEs in the treated effluent were significantly lower than in surface water upstream and in built-up urban areas, indicating that wastewater discharges in the study area did not increase PAEs in the receiving water. Finally, river in the city center and artificial treatment facilities in the study area were identified as requiring priority attention. The results of this study can serve as a model for controlling PAEs in other similar developing cities in China and provide valuable information on the fate of endocrine disruptor from wastewater treatment in China and their impact on surface water.

Genetics of the human microglia regulome refines Alzheimer's disease risk loci.

Microglia are brain myeloid cells that play a critical role in neuroimmunity and the etiology of Alzheimer's disease (AD), yet our understanding of how the genetic regulatory landscape controls microglial function and contributes to AD is limited. Here, we performed transcriptome and chromatin accessibility profiling in primary human microglia from 150 donors to identify genetically driven variation and cell-specific enhancer-promoter (E-P) interactions. Integrative fine-mapping analysis identified putative regulatory mechanisms for 21 AD risk loci, of which 18 were refined to a single gene, including 3 new candidate risk genes (KCNN4, FIBP and LRRC25). Transcription factor regulatory networks captured AD risk variation and identified SPI1 as a key putative regulator of microglia expression and AD risk. This comprehensive resource capturing variation in the human microglia regulome provides insights into the etiology of neurodegenerative disease.

A complete temporal transcription factor series in the fly visual system.

The brain consists of thousands of neuronal types that are generated by stem cells producing different neuronal types as they age. In Drosophila, this temporal patterning is driven by the successive expression of temporal transcription factors (tTFs)1-6. Here we used single-cell mRNA sequencing to identify the complete series of tTFs that specify most Drosophila optic lobe neurons. We verify that tTFs regulate the progression of the series by activating the next tTF(s) and repressing the previous one(s), and also identify more complex mechanisms of regulation. Moreover, we establish the temporal window of origin and birth order of each neuronal type in the medulla and provide evidence that these tTFs are sufficient to explain the generation of all of the neuronal diversity in this brain region. Finally, we describe the first steps of neuronal differentiation and show that these steps are conserved in humans. We find that terminal differentiation genes, such as neurotransmitter-related genes, are present as transcripts, but not as proteins, in immature larval neurons. This comprehensive analysis of a temporal series of tTFs in the optic lobe offers mechanistic insights into how tTF series are regulated, and how they can lead to the generation of a complete set of neurons.

A bidirectional competitive interaction between circHomer1 and Homer1b within the orbitofrontal cortex regulates reversal learning.

Although circular RNAs (circRNAs) are enriched in the brain, their relevance for brain function and psychiatric disorders is poorly understood. Here, we show that circHomer1 is inversely associated with relative HOMER1B mRNA isoform levels in both the orbitofrontal cortex (OFC) and stem-cell-derived neuronal cultures of subjects with psychiatric disorders. We further demonstrate that in vivo circHomer1 knockdown (KD) within the OFC can inhibit the synaptic expression of Homer1b mRNA. Furthermore, we show that circHomer1 directly binds to Homer1b mRNA and that Homer1b-specific KD increases synaptic circHomer1 levels and improves OFC-mediated behavioral flexibility. Importantly, double circHomer1 and Homer1b in vivo co-KD results in a complete rescue in circHomer1-associated alterations in both chance reversal learning and synaptic gene expression. Lastly, we uncover an RNA-binding protein that can directly bind to circHomer1 and promote its biogenesis. Taken together, our data provide mechanistic insights into the importance of circRNAs in brain function and disease.

Single-nucleus transcriptome analysis of human brain immune response in patients with severe COVID-19.

BACKGROUND: Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, has been associated with neurological and neuropsychiatric illness in many individuals. We sought to further our understanding of the relationship between brain tropism, neuro-inflammation, and host immune response in acute COVID-19 cases. METHODS: Three brain regions (dorsolateral prefrontal cortex, medulla oblongata, and choroid plexus) from 5 patients with severe COVID-19 and 4 controls were examined. The presence of the virus was assessed by western blot against viral spike protein, as well as viral transcriptome analysis covering > 99% of SARS-CoV-2 genome and all potential serotypes. Droplet-based single-nucleus RNA sequencing (snRNA-seq) was performed in the same samples to examine the impact of COVID-19 on transcription in individual cells of the brain. RESULTS: Quantification of viral spike S1 protein and viral transcripts did not detect SARS-CoV-2 in the postmortem brain tissue. However, analysis of 68,557 single-nucleus transcriptomes from three distinct regions of the brain identified an increased proportion of stromal cells, monocytes, and macrophages in the choroid plexus of COVID-19 patients. Furthermore, differential gene expression, pseudo-temporal trajectory, and gene regulatory network analyses revealed transcriptional changes in the cortical microglia associated with a range of biological processes, including cellular activation, mobility, and phagocytosis. CONCLUSIONS: Despite the absence of detectable SARS-CoV-2 in the brain at the time of death, the findings suggest significant and persistent neuroinflammation in patients with acute COVID-19.

PS1 FAD mutants decrease ephrinB2-regulated angiogenic functions, ischemia-induced brain neovascularization and neuronal survival.

Microvascular pathology and ischemic lesions contribute substantially to neuronal dysfunction and loss that lead to Alzheimer disease (AD). To facilitate recovery, the brain stimulates neovascularization of damaged tissue via sprouting angiogenesis, a process regulated by endothelial cell (EC) sprouting and the EphB4/ephrinB2 system. Here, we show that in cultures of brain ECs, EphB4 stimulates the VE-cadherin/Rok-α angiogenic complexes known to mediate sprouting angiogenesis. Importantly, brain EC cultures expressing PS1 FAD mutants decrease the EphB4-stimulated γ-secretase cleavage of ephrinB2 and reduce production of the angiogenic peptide ephrinB2/CTF2, the VE-cadherin angiogenic complexes and EC sprouting and tube formation. These data suggest that FAD mutants may attenuate ischemia-induced brain angiogenesis. Supporting this hypothesis, ischemia-induced VE-cadherin angiogenic complexes, levels of neoangiogenesis marker Endoglin, vascular density, and cerebral blood flow recovery, are all decreased in brains of mouse models expressing PS1 FAD mutants. Ischemia-induced brain neuronal death and cognitive deficits also increase in these mice. Furthermore, a small peptide comprising the C-terminal sequence of peptide ephrinB2/CTF2 rescues angiogenic functions of brain ECs expressing PS1 FAD mutants. Together, our data show that PS1 FAD mutations impede the EphB4/ephrinB2-mediated angiogenic functions of ECs and impair brain neovascularization, neuronal survival and cognitive recovery following ischemia. Furthermore, our data reveal a novel brain angiogenic mechanism targeted by PS1 FAD mutants and a potential therapeutic target for ischemia-induced neurodegeneration. Importantly, FAD mutant effects occur in absence of neuropathological hallmarks of AD, supporting that such hallmarks may form downstream of mutant effects on neoangiogenesis and neuronal survival.

Presenilin1 familial Alzheimer disease mutants inactivate EFNB1- and BDNF-dependent neuroprotection against excitotoxicity by affecting neuroprotective complexes of N-methyl-d-aspartate receptor.

Excitotoxicity is thought to play key roles in brain neurodegeneration and stroke. Here we show that neuroprotection against excitotoxicity by trophic factors EFNB1 and brain-derived neurotrophic factor (called here factors) requires de novo formation of 'survival complexes' which are factor-stimulated complexes of N-methyl-d-aspartate receptor with factor receptor and presenilin 1. Absence of presenilin 1 reduces the formation of survival complexes and abolishes neuroprotection. EPH receptor B2- and N-methyl-d-aspartate receptor-derived peptides designed to disrupt formation of survival complexes also decrease the factor-stimulated neuroprotection. Strikingly, factor-dependent neuroprotection and levels of the de novo factor-stimulated survival complexes decrease dramatically in neurons expressing presenilin 1 familial Alzheimer disease mutants. Mouse neurons and brains expressing presenilin 1 familial Alzheimer disease mutants contain increased amounts of constitutive presenilin 1-N-methyl-d-aspartate receptor complexes unresponsive to factors. Interestingly, the stability of the familial Alzheimer disease presenilin 1-N-methyl-d-aspartate receptor complexes differs from that of wild type complexes and neurons of mutant-expressing brains are more vulnerable to cerebral ischaemia than neurons of wild type brains. Furthermore, N-methyl-d-aspartate receptor-mediated excitatory post-synaptic currents at CA1 synapses are altered by presenilin 1 familial Alzheimer disease mutants. Importantly, high levels of presenilin 1-N-methyl-d-aspartate receptor complexes are also found in post-mortem brains of Alzheimer disease patients expressing presenilin 1 familial Alzheimer disease mutants. Together, our data identify a novel presenilin 1-dependent neuroprotective mechanism against excitotoxicity and indicate a pathway by which presenilin 1 familial Alzheimer disease mutants decrease factor-depended neuroprotection against excitotoxicity and ischaemia in the absence of Alzheimer disease neuropathological hallmarks which may form downstream of neuronal damage. These findings have implications for the pathogenic effects of familial Alzheimer disease mutants and therapeutic strategies.

Presenilin1/γ-secretase protects neurons from glucose deprivation-induced death by regulating miR-212 and PEA15.

Reduced cerebral glucose utilization is found in aged individuals and often is an early sign of neurodegeneration. Here, we show that under glucose deprivation (GD) conditions, decreased expression of presenilin 1 (PS1) results in decreased neuronal survival, whereas increased PS1 increases neuronal survival. Inhibition of γ-secretase also decreases neuronal survival under GD conditions, which suggests the PS1/γ-secretase system protects neurons from GD-induced death. We also show that neuronal levels of the survival protein, phosphoprotein enriched in astrocytes at ∼15 kDa (PEA15), and its mRNA are regulated by PS1/γ-secretase. Furthermore, down-regulation of PEA15 decreases neuronal survival under reduced glucose conditions, whereas exogenous PEA15 increases neuronal survival even in the absence of PS1, which indicates that PEA15 promotes neuronal survival under GD conditions. The absence or reduction of PS1, as well as γ-secretase inhibitors, increases neuronal miR-212, which targets PEA15 mRNA. PS1/γ-secretase activates the transcription factor, cAMP response element-binding protein, regulating miR-212, which targets PEA15 mRNA. Taken together, our data show that under conditions of reduced glucose, the PS1/γ-secretase system decreases neuronal losses by suppressing miR-212 and increasing its target survival factor, PEA15. These observations have implications for mechanisms of neuronal death under conditions of reduced glucose and may provide targets for intervention in neurodegenerative disorders.-Huang, Q., Voloudakis, G., Ren, Y., Yoon, Y., Zhang, E., Kajiwara, Y., Shao, Z., Xuan, Z., Lebedev, D., Georgakopoulos, A., Robakis, N. K. Presenilin1/γ-secretase protects neurons from glucose deprivation-induced death by regulating miR-212 and PEA15.

Open chromatin profiling of human postmortem brain infers functional roles for non-coding schizophrenia loci.

Open chromatin provides access to DNA-binding proteins for the correct spatiotemporal regulation of gene expression. Mapping chromatin accessibility has been widely used to identify the location of cis regulatory elements (CREs) including promoters and enhancers. CREs show tissue- and cell-type specificity and disease-associated variants are often enriched for CREs in the tissues and cells that pertain to a given disease. To better understand the role of CREs in neuropsychiatric disorders we applied the Assay for Transposase Accessible Chromatin followed by sequencing (ATAC-seq) to neuronal and non-neuronal nuclei isolated from frozen postmortem human brain by fluorescence-activated nuclear sorting (FANS). Most of the identified open chromatin regions (OCRs) are differentially accessible between neurons and non-neurons, and show enrichment with known cell type markers, promoters and enhancers. Relative to those of non-neurons, neuronal OCRs are more evolutionarily conserved and are enriched in distal regulatory elements. Transcription factor (TF) footprinting analysis identifies differences in the regulome between neuronal and non-neuronal cells and ascribes putative functional roles to a number of non-coding schizophrenia (SCZ) risk variants. Among the identified variants is a Single Nucleotide Polymorphism (SNP) proximal to the gene encoding SNX19. In vitro experiments reveal that this SNP leads to an increase in transcriptional activity. As elevated expression of SNX19 has been associated with SCZ, our data provide evidence that the identified SNP contributes to disease. These results represent the first analysis of OCRs and TF-binding sites in distinct populations of postmortem human brain cells and further our understanding of the regulome and the impact of neuropsychiatric disease-associated genetic risk variants.

Presenilin 1 is necessary for neuronal, but not glial, EGFR expression and neuroprotection via γ-secretase-independent transcriptional mechanisms.

Epidermal growth factor receptor (EGFR) plays pivotal roles in cell proliferation, differentiation, and tissue development, while EGFs protect neurons from toxic insults by binding EGFR and stimulating survival signaling. Furthermore, recent evidence implicates this receptor in neurometabolic disorders like Alzheimer disease and aging. Here we show that absence of presenilin 1 (PS1) results in dramatic decrease (>95%) of neuronal EGFR and that PS1-null (PS1(-/-)) brains have reduced amounts of this receptor. PS1(-/-) cortical neurons contain little EGFR and show no epidermal growth factor-induced survival signaling or protection against excitotoxicity, but exogenous EGFR rescues both functions even in absence of PS1. EGFR mRNA is greatly reduced (>95%) in PS1(-/-) neurons, and PS1(-/-) brains contain decreased amounts of this mRNA, although PS1 affects the stability of neither EGFR nor its mRNA. Exogenous PS1 increases neuronal EGFR mRNA, while down-regulation of PS1 decreases this mRNA. These effects are neuron specific, as PS1 affects the EGFR of neither glial nor fibroblast cells. In addition, PS1 controls EGFR through novel mechanisms shared with neither γ-secretase nor PS2. Our data reveal that PS1 functions as a positive transcriptional regulator of neuronal EGFR controlling its expression in a cell-specific manner. Severe downregulation of EGFR may contribute to developmental abnormalities and lethal phenotype found in PS1, but not PS2, null mice. Furthermore, PS1 may affect neuroprotection and Alzheimer disease by controlling survival signaling of neuronal EGFR.

Presenilin mediates neuroprotective functions of ephrinB and brain-derived neurotrophic factor and regulates ligand-induced internalization and metabolism of EphB2 and TrkB receptors.

Activation of EphB receptors by ephrinB (efnB) ligands on neuronal cell surface regulates important functions, including neurite outgrowth, axonal guidance, and synaptic plasticity. Here, we show that efnB rescues primary cortical neuronal cultures from necrotic cell death induced by glutamate excitotoxicity and that this function depends on EphB receptors. Importantly, the neuroprotective function of the efnB/EphB system depends on presenilin 1 (PS1), a protein that plays crucial roles in Alzheimer's disease (AD) neurodegeneration. Furthermore, absence of one PS1 allele results in significantly decreased neuroprotection, indicating that both PS1 alleles are necessary for full expression of the neuroprotective activity of the efnB/EphB system. We also show that the ability of brain-derived neurotrophic factor (BDNF) to protect neuronal cultures from glutamate-induced cell death depends on PS1. Neuroprotective functions of both efnB and BDNF, however, were independent of γ-secretase activity. Absence of PS1 decreases cell surface expression of neuronal TrkB and EphB2 without affecting total cellular levels of the receptors. Furthermore, PS1-knockout neurons show defective ligand-dependent internalization and decreased ligand-induced degradation of TrkB and Eph receptors. Our data show that PS1 mediates the neuroprotective activities of efnB and BDNF against excitotoxicity and regulates surface expression and ligand-induced metabolism of their cognate receptors. Together, our observations indicate that PS1 promotes neuronal survival by regulating neuroprotective functions of ligand-receptor systems.

Extracellular progranulin protects cortical neurons from toxic insults by activating survival signaling.

To reduce damage from toxic insults such as glutamate excitotoxicity and oxidative stresses, neurons may deploy an array of neuroprotective mechanisms. Recent reports show that progranulin (PGRN) gene null or missense mutations leading to inactive protein, are linked to frontotemporal lobar degeneration (FTLD), suggesting that survival of certain neuronal populations needs full expression of functional PGRN. Here we show that extracellular PGRN stimulates phosphorylation/activation of the neuronal MEK/extracellular regulated kinase (ERK)/p90 ribosomal S6 kinase (p90RSK) and phosphatidylinositol-3 kinase (PI3K)/Akt cell survival pathways and rescues cortical neurons from cell death induced by glutamate or oxidative stress. Pharmacological inhibition of MEK/ERK/p90RSK signaling blocks the PGRN-induced phosphorylation and neuroprotection against glutamate toxicity while inhibition of either MEK/ERK/p90RSK or PI3K/Akt blocks PGRN protection against neurotoxin MPP(+). Inhibition of both pathways had synergistic effects on PGRN-dependent neuroprotection against MPP(+) toxicity suggesting both pathways contribute to the neuroprotective activities of PGRN. Extracellular PGRN is remarkably stable in neuronal cultures indicating neuroprotective activities are associated with full-length protein. Together, our data show that extracellular PGRN acts as a neuroprotective factor and support the hypothesis that in FTLD reduction of functional brain PGRN results in reduced survival signaling and decreased neuronal protection against excitotoxicity and oxidative stress leading to accelerated neuronal cell death. That extracellular PGRN has neuroprotective functions against toxic insults suggests that in vitro preparations of this protein may be used therapeutically.