Generation and characterization of six human induced pluripotent stem cell lines (hiPSCs) from three individuals with SSADH Deficiency and CRISPR-corrected isogenic controls.

Succinic Semialdehyde Dehydrogenase Deficiency (SSADHD) is an ultra-rare autosomal recessive neurometabolic disorder caused by ALDH5A1 mutations presenting with autism and epilepsy. Here, we report the generation and characterization of human induced pluripotent stem cells (hiPSCs) derived from fibroblasts of three unrelated SSADHD patients - one female and two males with the CRISPR-corrected isogenic controls. These individuals are clinically diagnosed and are being followed in a longitudinal clinical study.

Combining Off-flow, a Nextflow-coded program, and whole genome sequencing reveals unintended genetic variation in CRISPR/Cas-edited iPSCs.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas nucleases and human induced pluripotent stem cell (iPSC) technology can reveal deep insight into the genetic and molecular bases of human biology and disease. Undesired editing outcomes, both on-target (at the edited locus) and off-target (at other genomic loci) hinder the application of CRISPR-Cas nucleases. We developed Off-flow, a Nextflow-coded bioinformatic workflow that takes a specific guide sequence and Cas protein input to call four separate off-target prediction programs (CHOPCHOP, Cas-Offinder, CRISPRitz, CRISPR-Offinder) to output a comprehensive list of predicted off-target sites. We applied it to whole genome sequencing (WGS) data to investigate the occurrence of unintended effects in human iPSCs that underwent repair or insertion of disease-related variants by homology-directed repair. Off-flow identified a 3-base-pair-substitution and a mono-allelic genomic deletion at the target loci, KCNQ2, in 2 clones. Unbiased WGS analysis further identified off-target missense variants and a mono-allelic genomic deletion at the targeted locus, GNAQ, in 10 clones. On-target substitution and deletions had escaped standard PCR and Sanger sequencing analysis, while missense variants at other genomic loci were not detected by Off-flow. We used these results to filter out iPSC clones for subsequent functional experiments. Off-flow, which we make publicly available, works for human and mouse genomes currently and can be adapted for other genomes. Off-flow and WGS analysis can improve the integrity of studies using CRISPR/Cas-edited cells and animal models.

ALDH5A1-deficient iPSC-derived excitatory and inhibitory neurons display cell type specific alterations.

Succinic semialdehyde dehydrogenase deficiency (SSADHD) is a neurometabolic disorder caused by ALDH5A1 mutations presenting with autism and epilepsy. SSADHD leads to impaired GABA metabolism and results in accumulation of GABA and γ-hydroxybutyrate (GHB), which alter neurotransmission and are thought to lead to neurobehavioral symptoms. However, why increased inhibitory neurotransmitters lead to seizures remains unclear. We used induced pluripotent stem cells from SSADHD patients (one female and two male) and differentiated them into GABAergic and glutamatergic neurons. SSADHD iGABA neurons show altered GABA metabolism and concomitant changes in expression of genes associated with inhibitory neurotransmission. In contrast, glutamatergic neurons display increased spontaneous activity and upregulation of mitochondrial genes. CRISPR correction of the pathogenic variants or SSADHD mRNA expression rescue various metabolic and functional abnormalities in human neurons. Our findings uncover a previously unknown role for SSADHD in excitatory human neurons and provide unique insights into the cellular and molecular basis of SSADHD and potential therapeutic interventions.

Increased degradation of FMRP contributes to neuronal hyperexcitability in tuberous sclerosis complex.

Autism spectrum disorder (ASD) is a highly prevalent neurodevelopmental disorder, but new therapies have been impeded by a lack of understanding of the pathological mechanisms. Tuberous sclerosis complex (TSC) and fragile X syndrome are associated with alterations in the mechanistic target of rapamycin (mTOR) and fragile X messenger ribonucleoprotein 1 (FMRP), which have been implicated in the development of ASD. Previously, we observed that transcripts associated with FMRP were down-regulated in TSC2-deficient neurons. In this study, we find that FMRP turnover is dysregulated in TSC2-deficient rodent primary neurons and human induced pluripotent stem cell (iPSC)-derived neurons and is dependent on the E3 ubiquitin ligase anaphase-promoting complex. We also demonstrate that overexpression of FMRP can partially rescue hyperexcitability in TSC2-deficient iPSC-derived neurons. These data indicate that FMRP dysregulation represents an important pathological mechanism in the development of abnormal neuronal activity in TSC and illustrate a molecular convergence between these two neurogenetic disorders.

Dynamic 3D Combinatorial Generation of hPSC-Derived Neuromesodermal Organoids With Diverse Regional and Cellular Identities.

Neuromesodermal progenitors represent a unique, bipotent population of progenitors residing in the tail bud of the developing embryo, which give rise to the caudal spinal cord cell types of neuroectodermal lineage as well as the adjacent paraxial somite cell types of mesodermal origin. With the advent of stem cell technologies, including induced pluripotent stem cells (iPSCs), the modeling of rare genetic disorders can be accomplished in vitro to interrogate cell-type specific pathological mechanisms in human patient conditions. Stem cell-derived models of neuromesodermal progenitors have been accomplished by several developmental biology groups; however, most employ a 2D monolayer format that does not fully reflect the complexity of cellular differentiation in the developing embryo. This article presents a dynamic 3D combinatorial method to generate robust populations of human pluripotent stem cell-derived neuromesodermal organoids with multi-cellular fates and regional identities. By utilizing a dynamic 3D suspension format for the differentiation process, the organoids differentiated by following this protocol display a hallmark of embryonic development that involves a morphological elongation known as axial extension. Furthermore, by employing a combinatorial screening assay, we dissect essential pathways for optimally directing the patterning of pluripotent stem cells into neuromesodermal organoids. This protocol highlights the influence of timing, duration, and concentration of WNT and fibroblast growth factor (FGF) signaling pathways on enhancing early neuromesodermal identity, and later, downstream cell fate specification through combined synergies of retinoid signaling and sonic hedgehog activation. Finally, through robust inhibition of the Notch signaling pathway, this protocol accelerates the acquisition of terminal cell identities. This enhanced organoid model can serve as a powerful tool for studying normal developmental processes as well as investigating complex neurodevelopmental disorders, such as neural tube defects. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Robust generation of 3D hPSC-derived spheroid populations in dynamic motion settings Support Protocol 1: Pluronic F-127 reagent preparation and coating to generate low-attachment suspension culture dishes Basic Protocol 2: Enhanced specification of hPSCs into NMP organoids Support Protocol 2: Combinatorial pathway assay for NMP organoid protocol optimization Basic Protocol 3: Differentiation of NMP organoids along diverse cellular trajectories and accelerated terminal fate specification into neurons, neural crest, and sclerotome derivatives.

Enhanced prime editing systems by manipulating cellular determinants of editing outcomes.

While prime editing enables precise sequence changes in DNA, cellular determinants of prime editing remain poorly understood. Using pooled CRISPRi screens, we discovered that DNA mismatch repair (MMR) impedes prime editing and promotes undesired indel byproducts. We developed PE4 and PE5 prime editing systems in which transient expression of an engineered MMR-inhibiting protein enhances the efficiency of substitution, small insertion, and small deletion prime edits by an average 7.7-fold and 2.0-fold compared to PE2 and PE3 systems, respectively, while improving edit/indel ratios by 3.4-fold in MMR-proficient cell types. Strategic installation of silent mutations near the intended edit can enhance prime editing outcomes by evading MMR. Prime editor protein optimization resulted in a PEmax architecture that enhances editing efficacy by 2.8-fold on average in HeLa cells. These findings enrich our understanding of prime editing and establish prime editing systems that show substantial improvement across 191 edits in seven mammalian cell types.

miR-142-3p regulates cortical oligodendrocyte gene co-expression networks associated with tauopathy.

Oligodendrocytes exist in a heterogenous state and are implicated in multiple neuropsychiatric diseases including dementia. Cortical oligodendrocytes are a glial population uniquely positioned to play a key role in neurodegeneration by synchronizing circuit connectivity but molecular pathways specific to this role are lacking. We utilized oligodendrocyte-specific translating ribosome affinity purification and RNA-seq (TRAP-seq) to transcriptionally profile adult mature oligodendrocytes from different regions of the central nervous system. Weighted gene co-expression network analysis reveals distinct region-specific gene networks. Two of these mature myelinating oligodendrocyte gene networks uniquely define cortical oligodendrocytes and differentially regulate cortical myelination (M8) and synaptic signaling (M4). These two cortical oligodendrocyte gene networks are enriched for genes associated with dementia including MAPT and include multiple gene targets of the regulatory microRNA, miR-142-3p. Using a combination of TRAP-qPCR, miR-142-3p overexpression in vitro, and miR-142-null mice, we show that miR-142-3p negatively regulates cortical myelination. In rTg4510 tau-overexpressing mice, cortical myelination is compromised, and tau-mediated neurodegeneration is associated with gene co-expression networks that recapitulate both the M8 and M4 cortical oligodendrocyte gene networks identified from normal cortex. We further demonstrate overlapping gene networks in mature oligodendrocytes present in normal cortex, rTg4510 and miR-142-null mice, and existing datasets from human tauopathies to provide evidence for a critical role of miR-142-3p-regulated cortical myelination and oligodendrocyte-mediated synaptic signaling in neurodegeneration.

Klotho, PTSD, and advanced epigenetic age in cortical tissue.

This study examined the klotho (KL) longevity gene polymorphism rs9315202 and psychopathology, including posttraumatic stress disorder (PTSD), depression, and alcohol-use disorders, in association with advanced epigenetic age in three postmortem cortical tissue regions: dorsolateral and ventromedial prefrontal cortices and motor cortex. Using data from the VA National PTSD Brain Bank (n = 117), we found that rs9315202 interacted with PTSD to predict advanced epigenetic age in motor cortex among the subset of relatively older (>=45 years), white non-Hispanic decedents (corrected p = 0.014, n = 42). An evaluation of 211 additional common KL variants revealed that only variants in linkage disequilibrium with rs9315202 showed similarly high levels of significance. Alcohol abuse was nominally associated with advanced epigenetic age in motor cortex (p = 0.039, n = 114). The rs9315202 SNP interacted with PTSD to predict decreased KL expression via DNAm age residuals in motor cortex among older white non-Hispanics decedents (indirect ß = -0.198, p = 0.027). Finally, in dual-luciferase enhancer reporter system experiments, we found that inserting the minor allele of rs9315202 in a human kidney cell line HK-2 genomic DNA resulted in a change in KL transcriptional activities, likely operating via long noncoding RNA in this region. This was the first study to examine multiple forms of psychopathology in association with advanced DNA methylation age across several brain regions, to extend work concerning the association between rs9315202 and advanced epigenetic to brain tissue, and to identify the effects of rs9315202 on KL gene expression. KL augmentation holds promise as a therapeutic intervention to slow the pace of cellular aging, disease onset, and neuropathology, particularly in older, stressed populations.

PTSD and the klotho longevity gene: Evaluation of longitudinal effects on inflammation via DNA methylation.

BACKGROUND: Longevity gene klotho (KL) is associated with age-related phenotypes including lifespan, cardiometabolic disorders, cognition, and brain morphology, in part, by conferring protection against inflammation. We hypothesized that the KL/inflammation association might be altered in the presence of psychiatric stress and operate via epigenetic pathways. We examined KL polymorphisms, and their interaction with posttraumatic stress disorder (PTSD) symptoms, in association with KL DNA methylation in blood. We further examined KL DNA methylation as a predictor of longitudinal changes in a peripheral biomarker of inflammation (C-reactive protein; CRP). METHODS: The sample comprised 309 white non-Hispanic military veterans (93.5 % male; mean age: 32 years, range: 19-65; 30 % PTSD per structured diagnostic interview); 111 were reassessed approximately two years later. RESULTS: Analyses revealed a methylation quantitative trait locus at rs9527025 (C370S, previously implicated in numerous studies of aging) in association with a Cytosine-phosphate-Guanine site (cg00129557; B = -.65, p = 1.29 X 10-20), located within a DNase hypersensitivity site in the body of KL. There was also a rs9527025 x PTSD severity interaction (B = .004, p = .035) on methylation at this locus such that the minor allele was associated with reduced cg00129557 methylation in individuals with few or no PTSD symptoms while this effect was attenuated in those with elevated levels of PTSD. Path models revealed that methylation at cg00129557 was inversely associated with CRP over time (B = -.14, p = .005), controlling for baseline CRP. There was also an indirect effect of rs9527025 X PTSD on subsequent CRP via cg00129557 methylation (indirect B = -.002, p = .033). CONCLUSIONS: Results contribute to our understanding of the epigenetic correlates of inflammation in PTSD and suggest that KL methylation may be a mechanism by which KL genotype confers risk vs. resilience to accelerated aging in those experiencing traumatic stress.

A method to specifically activate the Klotho promoter by using zinc finger proteins constructed from modular building blocks and from naturally engineered Egr1 transcription factor backbone.

There is an unmet need for treatments for diseases associated with aging. The antiaging, life-extending, and cognition-enhancing protein Klotho is neuroprotective due to its anti-inflammatory, antioxidative, and pro-myelinating effects. In addition, Klotho is also a tumor suppressor and has beneficial roles in multiple organs. Klotho is downregulated as part of the aging process. Thus, upregulating Klotho in the brain may lead to novel therapeutics to people suffering or at risk for neurodegenerative diseases such as Alzheimer's, Parkinson's, and amyotrophic lateral sclerosis, and demyelinating diseases such as multiple sclerosis. We attempted to upregulate Klotho for its beneficial effects in the brain and elsewhere. Here, we describe a method to specifically activate Klotho gene expression. To accomplish this task, we designed zinc finger proteins (ZFPs) targeting within -300 bps of the human Klotho promoter. We designed the ZPF constructs either de novo from modular building blocks, or modified sequences from the natural endogenous Egr1 transcription factor backbone structure. Egr1 is known to upregulate Klotho expression. We tested the transcriptional activation effects of these ZFPs in a dual luciferase coincidence reporter system under the control of 4-kb promoter of human Klotho in stable HEK293 cells and in HK-2 cells that express Klotho protein endogenously. We found that the best ZFPs are the de novo designed ones targeting -250 bps of Klotho promoter and one of the Egr1-binding sites. We further enhanced Klotho's activation using p65-Rta transcriptional activation domains in addition to VP64. These upregulation approaches could be useful for studying Klotho's protective effects and designing Klotho boosting therapeutics for future in vivo experiments.

Identification of the cleavage sites leading to the shed forms of human and mouse anti-aging and cognition-enhancing protein Klotho.

Klotho is an age-extending, cognition-enhancing protein found to be down-regulated in aged mammals when age-related diseases start to appear. Low levels of Klotho occur in neurodegenerative diseases, kidney disease and many cancers. Many normal and pathologic processes involve the proteolytic shedding of membrane proteins. Transmembrane (TM) Klotho contains two homologous domains, KL1 and KL2 with homology to glycosidases. After shedding by ADAM 10 and 17, a shed Klotho isoform is released into serum and urine by the kidney, and into the CSF by the choroid plexus. We previously reported that human Klotho contains two major cleavage sites. However, the exact cleavage site responsible for the cleavage between the KL1 and KL2 domains remains unknown for the human Klotho, and both sites are unknown for mouse Klotho. In this study, we aimed to identify the cleavage sites leading to the shed forms of human and mouse Klotho. Mutations in the region close to the TM domain of mouse Klotho result in the reduced shedding of the 130 kD (KL1+KL2) and 70 kD (KL1) fragments, suggesting that the cleavage site lies within the mutated region. We further identified the cleavage sites responsible for the cleavage between KL1 and KL2 of human and mouse Klotho. Moreover, mutated Klotho proteins have similar subcellular localization patterns as wild type Klotho. Finally, in an FGF23 functional assay, all Klotho mutants with a nine amino acid deletion can also function as an FGFR1 co-receptor for FGF23 signaling, however, the signaling activity was greatly reduced. The study provides new and important information on Klotho shedding, and paves the way for studies aimed to distinguish between the distinct roles of the various isoforms of Klotho.

Klotho regulation by albuminuria is dependent on ATF3 and endoplasmic reticulum stress.

Proteinuria is associated with renal function decline and cardiovascular mortality. This association may be attributed in part to alterations of Klotho expression induced by albuminuria, yet the underlying mechanisms are unclear. The presence of albumin decreased Klotho expression in the POD-ATTAC mouse model of proteinuric kidney disease as well as in kidney epithelial cell lines. This downregulation was related to both decreased Klotho transcription and diminished protein half-life, whereas cleavage by ADAM proteases was not modified. The regulation was albumin specific since it was neither observed in the analbuminemic Col4α3-/- Alport mice nor induced by exposure of kidney epithelial cells to purified immunoglobulins. Albumin induced features of ER stress in renal tubular cells with ATF3/ATF4 activation. ATF3 and ATF4 induction downregulated Klotho through altered transcription mediated by their binding on the Klotho promoter. Inhibiting ER stress with 4-PBA decreased the effect of albumin on Klotho protein levels without altering mRNA levels, thus mainly abrogating the increased protein degradation. Taken together, albuminuria decreases Klotho expression through increased protein degradation and decreased transcription mediated by ER stress induction. This implies that modulating ER stress may improve proteinuria-induced alterations of Klotho expression, and hence renal and extrarenal complications associated with Klotho loss.

Klotho Is Neuroprotective in the Superoxide Dismutase (SOD1G93A) Mouse Model of ALS.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder characterized by the loss of motor neurons in the brain and spinal cord. ALS neuropathology is associated with increased oxidative stress, excitotoxicity, and inflammation. We and others reported that the anti-aging and cognition-enhancing protein Klotho is a neuroprotective, antioxidative, anti-inflammatory, and promyelinating protein. In mice, its absence leads to an extremely shortened life span and to multiple phenotypes resembling human aging, including motor and hippocampal neurodegeneration and cognitive impairment. In contrast, its overexpression extends life span, enhances cognition, and confers resistance against oxidative stress; it also reduces premature mortality and cognitive and behavioral abnormalities in an animal model for Alzheimer's disease (AD). These pleiotropic beneficial properties of Klotho suggest that Klotho could be a potent therapeutic target for preventing neurodegeneration in ALS. Klotho overexpression in the SOD1 mouse model of ALS resulted in delayed onset and progression of the disease and extended survival that was more prominent in females than in males. Klotho reduced the expression of neuroinflammatory markers and prevented neuronal loss with the more profound effect in the spinal cord than in the motor cortex. The effect of Klotho was accompanied by reduced expression of proinflammatory cytokines and enhanced the expression of antioxidative and promyelinating factors in the motor cortex and spinal cord of Klotho × SOD1 compared to SOD1 mice. Our study provides evidence that increased levels of Klotho alleviate ALS-associated pathology in the SOD1 mouse model and may serve as a basis for developing Klotho-based therapeutic strategies for ALS.

Small Molecule Amyloid-ß Protein Precursor Processing Modulators Lower Amyloid-ß Peptide Levels via cKit Signaling.

Alzheimer's disease (AD) is characterized by the accumulation of neurotoxic amyloid-ß (Aß) peptides consisting of 39-43 amino acids, proteolytically derived fragments of the amyloid-ß protein precursor (AßPP), and the accumulation of the hyperphosphorylated microtubule-associated protein tau. Inhibiting Aß production may reduce neurodegeneration and cognitive dysfunction associated with AD. We have previously used an AßPP-firefly luciferase enzyme complementation assay to conduct a high throughput screen of a compound library for inhibitors of AßPP dimerization, and identified a compound that reduces Aß levels. In the present study, we have identified an analog, compound Y10, which also reduced Aß. Initial kinase profiling assays identified the receptor tyrosine kinase cKit as a putative Y10 target. To elucidate the precise mechanism involved, AßPP phosphorylation was examined by IP-western blotting. We found that Y10 inhibits cKit phosphorylation and increases AßPP phosphorylation mainly on tyrosine residue Y743, according to AßPP751 numbering. A known cKit inhibitor and siRNA specific to cKit were also found to increase AßPP phosphorylation and lower Aß levels. We also investigated a cKit downstream signaling molecule, the Shp2 phosphatase, and found that known Shp2 inhibitors and siRNA specific to Shp2 also increase AßPP phosphorylation, suggesting that the cKit signaling pathway is also involved in AßPP phosphorylation and Aß production. We further found that inhibitors of both cKit and Shp2 enhance AßPP surface localization. Thus, regulation of AßPP phosphorylation by small molecules should be considered as a novel therapeutic intervention for AD.

Tau Phosphorylation is Impacted by Rare AKAP9 Mutations Associated with Alzheimer Disease in African Americans.

We studied the effect of two rare mutations (rs144662445 and rs149979685) in the A-kinase anchoring protein 9 (AKAP9) gene, previously associated with Alzheimer disease (AD) in African Americans (AA), on post-translational modifications of AD-related pathogenic molecules, amyloid precursor protein (APP) and microtubule-associated protein Tau using lymphoblastoid cell lines (LCLs) from 11 AA subjects with at least one AKAP9 mutation and 17 AA subjects lacking these mutations. LCLs were transduced by viral vectors expressing causative AD mutations in APP or human full-length wild type Tau. Cell lysates were analyzed for total APP, Aß40, and total and T181 phospho-Tau (pTau). AKAP9 mutations had no effect on Aß40/APP, but significantly increased pTau/Tau ratio in LCLs treated with phosphodiesterase-4 inhibitor rolipram, which activates protein kinase A. Proteomic analysis of Tau interactome revealed enrichment of RNA binding proteins and decrease of proteasomal molecules in rolipram-treated cells with AKAP9 mutations. This study shows the impact of rare functional AKAP9 mutations on Tau, a central mechanism of AD pathogenesis, in LCLs derived from AD and control subjects.

Activation of the Anti-Aging and Cognition-Enhancing Gene Klotho by CRISPR-dCas9 Transcriptional Effector Complex.

Multiple lines of evidence show that the anti-aging and cognition-enhancing protein Klotho fosters neuronal survival, increases the anti-oxidative stress defense, and promotes remyelination of demyelinated axons. Thus, upregulation of the Klotho gene can potentially alleviate the symptoms and/or prevent the progression of age-associated neurodegenerative diseases such as Alzheimer's disease and demyelinating diseases such as multiple sclerosis. Here we used a CRISPR-dCas9 complex to investigate single-guide RNA (sgRNA) targeting the Klotho promoter region for efficient transcriptional activation of the Klotho gene. We tested the sgRNAs within the - 1 to - 300 bp of the Klotho promoter region and identified two sgRNAs that can effectively enhance Klotho gene transcription. We examined the transcriptional activation of the Klotho gene using three different systems: a Firefly luciferase (FLuc) and NanoLuc luciferase (NLuc) coincidence reporter system, a NLuc knock-in in Klotho 3'-UTR using CRISPR genomic editing, and two human cell lines: neuronal SY5Y cells and kidney HK-2 cells that express Klotho endogenously. The two sgRNAs enhanced Klotho expression at both the gene and protein levels. Our results show the feasibility of gene therapy for targeting Klotho using CRISPR technology. Enhancing Klotho levels has a therapeutic potential for increasing cognition and treating age-associated neurodegenerative, demyelinating and other diseases, such as chronic kidney disease and cancer.

The Anti-Aging Protein Klotho Enhances Remyelination Following Cuprizone-Induced Demyelination.

The current study examined whether overexpression of Klotho (KL) in transgenic mice can enhance remyelination following cuprizone-induced demyelination and improves the clinical outcome in experimental autoimmune encephalomyelitis (EAE). Demyelination was achieved by feeding transgenic mice overexpressing the transmembrane form of Klotho (KL-OE) and wild-type (WT) littermates cuprizone-containing chow for 6 weeks. The animals were then allowed to remyelinate for 3 weeks. Paraphenylenediamine staining and platelets-derived growth factor receptor α (PDGFRα) and glutathione S-transferase pi (GSTpi) immunohistochemistry were performed on corpus callosum (CC) sections for quantification of myelin and progenitor and mature oligodendrocytes, respectively. The EAE model was induced with the MOG35-55 peptide. The animals were scored daily for clinical symptoms for 30 days. Following 6 weeks of demyelination, both KL-OE mice and WT littermates demonstrated almost complete and comparable demyelination of the CC. However, the level of spontaneous remyelination was increased approximately two-fold in KL-OE mice, although no significant differences in the numbers of PDGFRα and GSTpi-positive cells were observed. Following EAE induction, Klotho overexpression did not affect the clinical scores, likely due to the different roles Klotho plays in the brain and spinal cord. Thus, increasing Klotho expression should be considered as a therapy for enhancing remyelination in the brains of individuals with multiple sclerosis.

The anti-aging and tumor suppressor protein Klotho enhances differentiation of a human oligodendrocytic hybrid cell line.

Klotho functions as an aging suppressor, which, in mice, extends lifespan when overexpressed and accelerates development of aging-like phenotypes when disrupted. Klotho is mainly expressed in brain and kidney and is secreted into the serum and CSF. We have previously shown that Klotho is reduced in brains of old monkeys, rats, and mice. We further reported the ability of Klotho to enhance oligodendrocyte differentiation and myelination. Here, we examined the signaling pathways induced by Klotho in MO3.13, a human oligodendrocytic hybrid cell line. We show that exogenous Klotho affects the ERK and Akt signaling pathways, decreases the proliferative abilities and enhances differentiation of MO3.13 cells. Furthermore, microarray analysis of Klotho-treated MO3.13 cells reveals a massive change in gene expression with 80 % of the differentially expressed genes being downregulated. Using gene set enrichment analysis, we predicted potential transcription factors involved in regulating Klotho-treated MO3.13 cells and found that these cells are highly enriched in the gene sets, that are similarly observed in cancer, cardiovascular disease, stress, aging, and hormone-related chemical and genetic perturbations. Since Klotho is downregulated in all brain tumors tested to date, enhancing Klotho has therapeutic potential for treating brain and other malignancies.

Identification of cleavage sites leading to the shed form of the anti-aging protein klotho.

Membrane protein shedding is a critical step in many normal and pathological processes. The anti-aging protein klotho (KL), mainly expressed in kidney and brain, is secreted into the serum and CSF, respectively. KL is proteolytically released, or shed, from the cell surface by ADAM10 and ADAM17, which are the α-secretases that also cleave the amyloid precursor protein and other proteins. The transmembrane KL is a coreceptor with the FGF receptor for FGF23, whereas the shed form acts as a circulating hormone. However, the precise cleavage sites in KL are unknown. KL contains two major cleavage sites: one close to the juxtamembrane region and another between the KL1 and KL2 domains. We identified the cleavage site involved in KL release by mutating potential sheddase(s) recognition sequences and examining the production of the KL extracellular fragments in transfected COS-7 cells. Deletion of amino acids T958 and L959 results in a 50-60% reduction in KL shedding, and an additional P954E mutation results in further reduction of KL shedding by 70-80%. Deletion of amino acids 954-962 resulted in a 94% reduction in KL shedding. This mutant also had moderately decreased cell surface expression, yet had overall similar subcellular localization as that of WT KL, as demonstrated by immunofluorescence. Cleavage-resistant mutants could function as a FGFR coreceptor for FGF23, but they lost activity as a soluble form of KL in proliferation and transcriptional reporter assays. Cleavage between the KL1 and KL2 domains is dependent on juxtamembrane cleavage. Our results shed light onto mechanisms underlying KL release from the cell membrane and provide a target for potential pharmacologic interventions aimed at regulating KL secretion.

The neuroprotective effect of Klotho is mediated via regulation of members of the redox system.

Generation of reactive oxygen species (ROS), leading to oxidative damage and neuronal cell death, plays an important role in the pathogenesis of neurodegenerative disorders, including Alzheimer disease. The present study aimed to examine the mechanism by which the anti-aging protein Klotho exerts neuroprotective effects against neuronal damage associated with neurodegeneration and oxidative stress. Pretreatment of rat primary hippocampal neurons and mouse hippocampal neuronal cell line HT22 with recombinant Klotho protected these cells from glutamate and oligomeric amyloid ß (oAß)-induced cytotoxicity. In addition, primary hippocampal neurons obtained from Klotho-overexpressing mouse embryos were more resistant to both cytotoxic insults, glutamate and oAß, compared with neurons from wild-type littermates. An antioxidative stress array analysis of neurons treated with Klotho revealed that Klotho significantly enhances the expression of the thioredoxin/peroxiredoxin (Trx/Prx) system with the greatest effect on the induction of Prx-2, an antioxidant enzyme, whose increase was confirmed at the mRNA and protein levels. Klotho-induced phosphorylation of the PI3K/Akt pathway, a pathway important in apoptosis and longevity, was associated with sustained inhibitory phosphorylation of the transcription factor forkhead box O3a (FoxO3a) and was essential for the induction of Prx-2. Down-regulation of Prx-2 expression using a lentivirus harboring shRNA almost completely abolished the ability of Klotho to rescue neurons from glutamate-induced death and significantly, but not completely, inhibited cell death mediated by oAß, suggesting that Prx-2 is a key modulator of neuroprotection. Thus, our results demonstrate, for the first time, the neuroprotective role of Klotho and reveal a novel mechanism underlying this effect.