Petra Bergström

Association of Nrf2-encoding NFE2L2 haplotypes with Parkinson's disease

BackgroundOxidative stress is heavily implicated in the pathogenic process of Parkinsons disease. Varying capacity to detoxify radical oxygen species through induction of phase II antioxidant enzymes in substantia nigra may influence disease risk. Here, we hypothesize that variation in NFE2L2 and KEAP1, the genes encoding the two major regulators of the phase II response, may affect the risk of Parkinsons disease.MethodsThe study included a Swedish discovery case-control material (165 cases and 190 controls) and a Polish replication case-control material (192 cases and 192 controls). Eight tag single nucleotide polymorphisms representing the variation in NFE2L2 and three representing the variation in KEAP1 were chosen using HapMap data and were genotyped using TaqMan Allelic Discrimination.ResultsWe identified a protective NFE2L2 haplotype in both of our European case-control materials. Each haplotype allele was associated with five years later age at onset of the disease (p = 0.001) in the Swedish material, and decreased risk of PD (p = 2 × 10-6), with an odds ratio of 0.4 (95% CI 0.3-0.6) for heterozygous and 0.2 (95% CI 0.1-0.4) for homozygous carriers, in the Polish material. The identified haplotype includes a functional promoter haplotype previously associated with high transcriptional activity. Genetic variation in KEAP1 did not show any associations.ConclusionThese data suggest that variation in NFE2L2 modifies the Parkinsons disease process and provide another link between oxidative stress and neurodegeneration.

Nrf2-encoding NFE2L2 haplotypes influence disease progression but not risk in Alzheimer's disease and age-related cataract.

Alzheimers disease (AD) and age-related cataract, disorders characterized by protein aggregation causing late-onset disease, both involve oxidative stress. We hypothesize that common variants of NFE2L2 and KEAP1, the genes encoding the main regulators of the Nrf2 system, an important defence system against oxidative stress, may influence risk of AD and/or age-related cataract. This case-control study combines an AD material (725 cases and 845 controls), and a cataract material (489 cases and 182 controls). Genetic variation in NFE2L2 and KEAP1 was tagged by eight and three tag single nucleotide polymorphisms (SNPs), respectively. Single SNPs and haplotypes were analyzed for associations with disease risk, age parameters, MMSE and AD cerebrospinal fluid biomarkers. NFE2L2 and KEAP1 were not associated with risk of AD or cataract. However, one haplotype allele of NFE2L2 was associated with 2 years earlier age at AD onset (p(c)=0.013) and 4 years earlier age at surgery for posterior subcapsular cataract (p(c)=0.019). Another haplotype of NFE2L2 was associated with 4 years later age at surgery for cortical cataract (p(c)=0.009). Our findings do not support NFE2L2 or KEAP1 as susceptibility genes for AD or cataract. However, common variants of the NFE2L2 gene may affect disease progression, potentially altering clinically recognized disease onset.

Repeated transient sulforaphane stimulation in astrocytes leads to prolonged Nrf2-mediated gene expression and protection from superoxide-induced damage.

Oxidative stress is a major contributor to slowly developing diseases like Parkinsons disease, Alzheimers disease and cancer and one of the main causes of tissue damage following ischemic insults in the brain. Nrf2 is a transcription factor responsible for much of the inducible cellular defense against oxidative stress. Nrf2 can also be activated by xenobiotics like sulforaphane, a component highly enriched in cruciferous vegetables such as broccoli. Ingestion of broccoli or sulforaphane results in long-term protection against radical damage, although absorbed sulforaphane is cleared from the body within a few hours. Here we have examined whether the prolonged protection induced by sulforaphane is explained by a slow down regulation of the Nrf2 response. Furthermore, to simulate daily ingestion of sulforaphane, we examined the hypothesis that repeated transient sulforaphane stimulation results in an accumulation of Nrf2-mediated gene expression and an increased protection against oxidative damage. The kinetics of sulforaphane-induced Nrf2 response was studied in astrocytes, a cell type known to be highly involved in the defense against oxidative stress in the brain. Sulforaphane stimulation for 4 h induced an Nrf2-dependent increase of Nqo1 and Hmox1 mRNA that remained elevated for 24 h, and the corresponding proteins remained elevated for over 48 h. In addition, peroxide-clearing activity and the levels of glutathione were elevated for more than 20 h after stimulation for 4 h with sulforaphane, resulting in an increased resistance to superoxide-induced cell damage. Repeated sulforaphane stimulation resulted in an accumulation of mRNA and protein levels of Nqo1 and a persistent cell protection against oxidative damage. These findings indicate that brief stimulation of the Nrf2 pathway by sulforaphane results in long-lasting elevation of endogenous antioxidants in astrocytes. The findings also demonstrate that part of this response can be built up by repeated transient stimulation, possibly explaining how intermittent intake of sulforaphane can result in long-term protection from radical-induced disease.

Association of NFE2L2 and KEAP1 haplotypes with amyotrophic lateral sclerosis

Abstract Amyotrophic lateral sclerosis (ALS) is a degenerative motor neuron syndrome influenced by oxidative stress. The transcription factor Nrf2 and its repressor Keap1 constitute an important defence system in cellular protection against oxidative stress. Here we hypothesize that common genetic variations in the genes NFE2L2 and KEAP1, encoding Nrf2 and Keap1, may influence the risk and phenotype of ALS. Five hundred and twenty-two Swedish patients with sporadic ALS (SALS) and 564 Swedish control subjects were studied. Eight tag SNPs in NFE2L2 and three tag SNPs in KEAP1 were genotyped by allelic discrimination and three functional NFE2L2 promoter SNPs were genotyped by sequencing. One NFE2L2 haplotype (GGGAC) was associated with decreased risk of SALS (OR = 0.62 per allele, p = 0.003) and one haplotype in KEAP1 (CGG) was associated with later SALS onset (+3.4 years per allele, p = 0.015). When stratified by subgroup, one haplotype in NFE2L2, GAGCAGA including three functional promoter SNPs associated with high Nrf2 protein expression, was associated with 4.0 years later disease onset per allele in subgroup ALS (p = 0.008). In conclusion, these results suggest that variations in NFE2L2 and KEAP1, encoding two central proteins in cellular oxidative stress defence, may influence SALS pathogenesis.

Amyloid precursor protein expression and processing are differentially regulated during cortical neuron differentiation

Amyloid precursor protein (APP) and its cleavage product amyloid β (Aβ) have been thoroughly studied in Alzheimer’s disease. However, APP also appears to be important for neuronal development. Differentiation of induced pluripotent stem cells (iPSCs) towards cortical neurons enables in vitro mechanistic studies on human neuronal development. Here, we investigated expression and proteolytic processing of APP during differentiation of human iPSCs towards cortical neurons over a 100-day period. APP expression remained stable during neuronal differentiation, whereas APP processing changed. α-Cleaved soluble APP (sAPPα) was secreted early during differentiation, from neuronal progenitors, while β-cleaved soluble APP (sAPPβ) was first secreted after deep-layer neurons had formed. Short Aβ peptides, including Aβ1-15/16, peaked during the progenitor stage, while processing shifted towards longer peptides, such as Aβ1-40/42, when post-mitotic neurons appeared. This indicates that APP processing is regulated throughout differentiation of cortical neurons and that amyloidogenic APP processing, as reflected by Aβ1-40/42, is associated with mature neuronal phenotypes.

Low-dose γ-secretase inhibition increases secretion of Aβ peptides and intracellular oligomeric Aβ

ABSTRACT &ggr;‐Secretase inhibitors have been considered promising drug candidates against Alzheimers disease (AD) due to their ability to reduce amyloid‐&bgr; (A&bgr;) production. However, clinical trials have been halted due to lack of clinical efficacy and/or side effects. Recent in vitro studies suggest that low doses of &ggr;‐secretase inhibitors may instead increase A&bgr; production. Using a stem cell‐derived human model of cortical neurons and low doses of the &ggr;‐secretase inhibitor DAPT, the effects on a variety of A&bgr; peptides were studied using mass spectrometry. One major focus was to develop a novel method for specific detection of oligomeric A&bgr; (oA&bgr;), and this was used to study the effects of low‐dose &ggr;‐secretase inhibitor treatment on intracellular oA&bgr; accumulation. Low‐dose treatment (2 and 20 nM) with DAPT increased the secretion of several A&bgr; peptides, especially A&bgr;x‐42. Furthermore, using the novel method for oA&bgr; detection, we found that 2 nM DAPT treatment of cortical neurons resulted in increased oA&bgr; accumulation. Thus, low dose‐treatment with DAPT causes both increased production of long, aggregation‐prone A&bgr; peptides and accumulation of intracellular A&bgr; oligomers, both believed to contribute to AD pathology. HighlightsLow‐dose DAPT treatment of human cortical neurons increases secretion of long A&bgr; peptides.Higher DAPT doses decreases longer A&bgr; peptides, whereas shorter ones are increased.This paper describes a novel sensitive method for detection of oligomeric A&bgr;.Low‐dose DAPT treatment increases intracellular accumulation of oligomeric A&bgr;.

Expression and secretion of synaptic proteins during stem cell differentiation to cortical neurons

ABSTRACT Synaptic function and neurotransmitter release are regulated by specific proteins. Cortical neuronal differentiation of human induced pluripotent stem cells (hiPSC) provides an experimental model to obtain more information about synaptic development and physiology in vitro. In this study, expression and secretion of the synaptic proteins, neurogranin (NRGN), growth‐associated protein‐43 (GAP‐43), synaptosomal‐associated protein‐25 (SNAP‐25) and synaptotagmin‐1 (SYT‐1) were analyzed during cortical neuronal differentiation. Protein levels were measured in cells, modeling fetal cortical development and in cell‐conditioned media which was used as a model of cerebrospinal fluid (CSF), respectively. Human iPSC‐derived cortical neurons were maintained over a period of at least 150 days, which encompasses the different stages of neuronal development. The differentiation was divided into the following stages: hiPSC, neuro‐progenitors, immature and mature cortical neurons. We show that NRGN was first expressed and secreted by neuro‐progenitors while the maximum was reached in mature cortical neurons. GAP‐43 was expressed and secreted first by neuro‐progenitors and its expression increased markedly in immature cortical neurons. SYT‐1 was expressed and secreted already by hiPSC but its expression and secretion peaked in mature neurons. SNAP‐25 was first detected in neuro‐progenitors and the expression and secretion increased gradually during neuronal stages reaching a maximum in mature neurons. The sensitive analytical techniques used to monitor the secretion of these synaptic proteins during cortical development make these data unique, since the secretion of these synaptic proteins has not been investigated before in such experimental models. The secretory profile of synaptic proteins, together with low release of intracellular content, implies that mature neurons actively secrete these synaptic proteins that previously have been associated with neurodegenerative disorders, including Alzheimers disease. These data support further studies of human neuronal and synaptic development in vitro, and would potentially shed light on the mechanisms underlying altered concentrations of the proteins in bio‐fluids in neurodegenerative diseases. HighlightsHuman iPSC‐derived cortical neurons express and secrete synaptic proteins.NRGN, GAP‐43 and SNAP‐25 are first expressed and secreted by neuro‐progenitors.SYT‐1 is expressed and secreted by stem cells.Synaptic proteins are secreted the highest when mature cortical neurons are formed.

SUBCELLULAR CO-LOCALIZATION OF APP AND APP-CLEAVING ENZYMES

AD-associated variants in SORL1 lead to detectable and modifiable endocytic phenotypes in human neurons. Methods: hiPSC-derived neurons are generated from control and patient fibroblasts. Neurons are generated from hiPSCs using established differentiation protocols. Patient hiPSC lines harbor predicted pathogenic variants in SORL1. CRISPR/Cas9 will be used to correct SORL1 variants. Analysis of endosomal network dysfunction and cellular AD phenotypes will be: Rab5 immunostaining, Transferring recycling assay, amyloid beta ELISA, phospho-Tau ELISA. Results: In hiPSC-derived neural cells expressing a SORL1 shRNA, we show increased amyloid beta peptides, reduced transferring recycling, and increased size of Rab5+ endosomes. In collaboration with the UWAlzheimer’s Disease Research Center, we have identified AD patients with SORL1 coding variants and are generating hiPSCs from these patients. We will assay hiPSC-derived neurons from SORL1 variant carriers for endosomal phenotypes and use CRISPR/Cas9 gene-editing to correct the variants to determine whether the predicted pathogenic variant leads to endocytic dysfunction in human neurons.Conclusions:This work is significant in that it will investigate a functional genotype-phenotype relationship of genetic variants in the endosomal network, which is known to be disrupted early in AD pathogenesis. Investigating this driver of disease pathogenesis and how it relates to human genetic background is critical in the development of new and precision treatments for AD.

ACUTE, TRANSIENT INCREASE IN SECRETION OF NEURODEGENERATION BIOMARKERS AFTER IRRADIATION OF HUMAN CORTICAL NEURONS

both produce and uptake beta-amyloid. AD astrocytes secrete significantly higher amounts of the 1-42 form and show an elevated 1-42/1-40 ratio as compared to controls, but respond well to treatment with gamma-secretase inhibitor DAPT. Conclusions:Our data show that pathological findings commonly seen in AD patients are recapitulated in the iPSC-derived astrocytes. Thus, these cells offer a valuable tool for studying the disease mechanisms and for drug screening and testing novel therapeutic approaches in a cell-type specific manner.

EXPRESSION AND SECRETION OF SYNAPTIC PROTEINS DURING DIFFERENTIATION OF PLURIPOTENT STEM CELLS TO CORTICAL NEURONS

P1-221 EXPRESSION AND SECRETION OF SYNAPTIC PROTEINS DURING DIFFERENTIATION OF PLURIPOTENT STEM CELLS TO CORTICAL NEURONS Faisal Hayat Nazir, Lotta Agholme, Tugce Munise Satir, Ann Brinkmalm, Annika € Ohrfelt, Kaj Blennow, Bruno Becker, Petra Bergstr€om, Henrik Zetterbeg, Institute of Neuroscience and Physiology, The Sahlgrenska Academy at University of Gothenburg, Gothenburg, Sweden; Institute of Neuroscience and Physiology, Department of Psychiatry and Neurochemistry, The Sahlgrenska Academy at the University of Gothenburg; Clinical Neurochemistry Laboratory, Sahlgrenska University Hospital, M€olndal, Sweden; Institute of Neuroscience and Physiology, The Sahlgrenska Academy at University of Gothenburg, M€olndal, Sweden; UCL Institute of Neurology, Department of Molecular Neuroscience, University College London, Queen Square, London, United Kingdom. Contact e-mail: Faisal.nazir@neuro.gu.se