RESUMO
Eukaryotic translation initiation factor 2B (eIF2B) is a key component of the integrated stress response (ISR), which regulates protein synthesis and stress granule formation in response to cellular insult. Modulation of the ISR has been proposed as a therapeutic strategy for treatment of neurodegenerative diseases such as vanishing white matter (VWM) disease and amyotrophic lateral sclerosis (ALS) based on its ability to improve cellular homeostasis and prevent neuronal degeneration. Herein, we report the small-molecule discovery campaign that identified potent, selective, and CNS-penetrant eIF2B activators using both structure- and ligand-based drug design. These discovery efforts culminated in the identification of DNL343, which demonstrated a desirable preclinical drug profile, including a long half-life and high oral bioavailability across preclinical species. DNL343 was progressed into clinical studies and is currently undergoing evaluation in late-stage clinical trials for ALS.
Assuntos
Esclerose Lateral Amiotrófica , Leucoencefalopatias , Doenças Neurodegenerativas , Humanos , Doenças Neurodegenerativas/tratamento farmacológico , Doenças Neurodegenerativas/metabolismo , Esclerose Lateral Amiotrófica/tratamento farmacológico , Esclerose Lateral Amiotrófica/metabolismo , Mutação , Fator de Iniciação 2B em Eucariotos/genética , Fator de Iniciação 2B em Eucariotos/metabolismo , Encéfalo/metabolismo , Leucoencefalopatias/metabolismoRESUMO
Dual leucine zipper kinase (DLK) and leucine zipper-bearing kinase (LZK) are regulators of neuronal degeneration and axon growth. Therefore, there is a considerable interest in developing DLK/LZK inhibitors for neurodegenerative diseases. Herein, we use ligand- and structure-based drug design approaches for identifying novel amino-pyrazine inhibitors of DLK/LZK. DN-1289 (14), a potent and selective dual DLK/LZK inhibitor, demonstrated excellent in vivo plasma half-life across species and is anticipated to freely penetrate the central nervous system with no brain impairment based on in vivo rodent pharmacokinetic studies and human in vitro transporter data. Proximal target engagement and disease relevant pathway biomarkers were also favorably regulated in an in vivo model of amyotrophic lateral sclerosis.
Assuntos
Esclerose Lateral Amiotrófica , Doenças Neurodegenerativas , Humanos , Esclerose Lateral Amiotrófica/tratamento farmacológico , Esclerose Lateral Amiotrófica/metabolismo , Zíper de Leucina , MAP Quinase Quinase Quinases , Sistema Nervoso Central/metabolismo , Encéfalo/metabolismo , Doenças Neurodegenerativas/metabolismoRESUMO
Loss-of-function (LOF) variants of TREM2, an immune receptor expressed in microglia, increase Alzheimer's disease risk. TREM2 senses lipids and mediates myelin phagocytosis, but its role in microglial lipid metabolism is unknown. Combining chronic demyelination paradigms and cell sorting with RNA sequencing and lipidomics, we find that wild-type microglia acquire a disease-associated transcriptional state, while TREM2-deficient microglia remain largely homeostatic, leading to neuronal damage. TREM2-deficient microglia phagocytose myelin debris but fail to clear myelin cholesterol, resulting in cholesteryl ester (CE) accumulation. CE increase is also observed in APOE-deficient glial cells, reflecting impaired brain cholesterol transport. This finding replicates in myelin-treated TREM2-deficient murine macrophages and human iPSC-derived microglia, where it is rescued by an ACAT1 inhibitor and LXR agonist. Our studies identify TREM2 as a key transcriptional regulator of cholesterol transport and metabolism under conditions of chronic myelin phagocytic activity, as TREM2 LOF causes pathogenic lipid accumulation in microglia.
Assuntos
Encéfalo/metabolismo , Colesterol/metabolismo , Macrófagos/metabolismo , Glicoproteínas de Membrana/genética , Microglia/metabolismo , Bainha de Mielina/metabolismo , Fagocitose/genética , Receptores Imunológicos/genética , Acetil-CoA C-Acetiltransferase/antagonistas & inibidores , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Animais , Ésteres do Colesterol/metabolismo , Modelos Animais de Doenças , Citometria de Fluxo , Humanos , Células-Tronco Pluripotentes Induzidas , Metabolismo dos Lipídeos/genética , Lipidômica , Receptores X do Fígado/agonistas , Camundongos , Camundongos Knockout , Camundongos Knockout para ApoE , RNA-SeqRESUMO
Receptor-interacting serine/threonine-protein kinase 1 (RIPK1) regulates inflammation, cytokine release, and necroptotic cell death and is implicated in pathogenic cellular pathways in amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), and multiple sclerosis. Inhibition of RIPK1 activity protects against inflammation and cell death in multiple animal models. DNL104 is a selective, brain-penetrant inhibitor of RIPK1 phosphorylation in clinical development for AD and ALS. DNL104 was tested in 68 healthy volunteers to investigate safety and tolerability, pharmacokinetic profile in plasma and cerebrospinal fluid, and pharmacodynamic effects of RIPK1 inhibition in peripheral blood mononuclear cells in a first-in-human, placebo-controlled, double-blind, randomized single-ascending dose (SAD) and multiple-ascending dose (MAD) study. DNL104 was well-tolerated in the SAD group and during the dosing period of the MAD group. However, postdose liver toxicity in 37.5% of subjects was observed in the MAD, and assessed to be drug related. We demonstrate that DNL104 leads to RIP1 kinase inhibition, and this is not associated with central nervous system (CNS) toxicities, supporting future development of CNS penetrant RIPK1 inhibitors.
Assuntos
Proteína Serina-Treonina Quinases de Interação com Receptores/antagonistas & inibidores , Adolescente , Adulto , Área Sob a Curva , Relação Dose-Resposta a Droga , Método Duplo-Cego , Feminino , Meia-Vida , Humanos , Leucócitos Mononucleares/efeitos dos fármacos , Masculino , Taxa de Depuração Metabólica , Pessoa de Meia-Idade , Fosforilação/efeitos dos fármacos , Adulto JovemRESUMO
Microglia, the CNS-resident immune cells, play important roles in disease, but the spectrum of their possible activation states is not well understood. We derived co-regulated gene modules from transcriptional profiles of CNS myeloid cells of diverse mouse models, including new tauopathy model datasets. Using these modules to interpret single-cell data from an Alzheimer's disease (AD) model, we identified microglial subsets-distinct from previously reported "disease-associated microglia"-expressing interferon-related or proliferation modules. We then analyzed whole-tissue RNA profiles from human neurodegenerative diseases, including a new AD dataset. Correcting for altered cellular composition of AD tissue, we observed elevated expression of the neurodegeneration-related modules, but also modules not implicated using expression profiles from mouse models alone. We provide a searchable, interactive database for exploring gene expression in all these datasets (http://research-pub.gene.com/BrainMyeloidLandscape). Understanding the dimensions of CNS myeloid cell activation in human disease may reveal opportunities for therapeutic intervention.
Assuntos
Doença de Alzheimer/genética , Encéfalo/metabolismo , Microglia/metabolismo , Doença de Alzheimer/metabolismo , Animais , Modelos Animais de Doenças , Humanos , CamundongosRESUMO
The common p.D358A variant (rs2228145) in IL-6R is associated with risk for multiple diseases and with increased levels of soluble IL-6R in the periphery and central nervous system (CNS). Here, we show that the p.D358A allele leads to increased proteolysis of membrane bound IL-6R and demonstrate that IL-6R peptides with A358 are more susceptible to cleavage by ADAM10 and ADAM17. IL-6 responsive genes were identified in primary astrocytes and microglia and an IL-6 gene signature was increased in the CNS of late onset Alzheimer's disease subjects in an IL6R allele dependent manner. We conducted a screen to identify variants associated with the age of onset of Alzheimer's disease in APOE É4 carriers. Across five datasets, p.D358A had a meta Pâ=â3 ×10-4 and an odds ratioâ=â1.3, 95% confidence interval 1.12 -1.48. Our study suggests that a common coding region variant of the IL-6 receptor results in neuroinflammatory changes that may influence the age of onset of Alzheimer's disease in APOE É4 carriers.
Assuntos
Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Encéfalo/metabolismo , Polimorfismo de Nucleotídeo Único , Receptores de Interleucina-6/genética , Receptores de Interleucina-6/metabolismo , Proteína ADAM10/metabolismo , Proteína ADAM17/metabolismo , Idoso , Idoso de 80 Anos ou mais , Alelos , Animais , Apolipoproteína E4/genética , Astrócitos/metabolismo , Linfócitos T CD4-Positivos/metabolismo , Técnicas de Cocultura , Estudos de Coortes , Feminino , Células HEK293 , Humanos , Interleucina-6/metabolismo , Masculino , Camundongos , Microglia/metabolismo , Proteínas Recombinantes/metabolismoRESUMO
TREM and TREM-like receptors are a structurally similar protein family encoded by genes clustered on chromosome 6p21.11. Recent studies have identified a rare coding variant (p.R47H) in TREM2 that confers a high risk for Alzheimer's disease (AD). In addition, common single nucleotide polymorphisms in this genomic region are associated with cerebrospinal fluid biomarkers for AD and a common intergenic variant found near the TREML2 gene has been identified to be protective for AD. However, little is known about the functional variant underlying the latter association or its relationship with the p.R47H. Here, we report comprehensive analyses using whole-exome sequencing data, cerebrospinal fluid biomarker analyses, meta-analyses (16,254 cases and 20,052 controls) and cell-based functional studies to support the role of the TREML2 coding missense variant p.S144G (rs3747742) as a potential driver of the meta-analysis AD-associated genome-wide association studies signal. Additionally, we demonstrate that the protective role of TREML2 in AD is independent of the role of TREM2 gene as a risk factor for AD.
Assuntos
Doença de Alzheimer/genética , Variação Genética/genética , Estudo de Associação Genômica Ampla , Mutação de Sentido Incorreto/genética , Receptores Imunológicos/genética , Doença de Alzheimer/prevenção & controle , Biomarcadores/líquido cefalorraquidiano , Cromossomos Humanos Par 6 , Humanos , Metanálise como Assunto , Polimorfismo de Nucleotídeo Único/genética , Receptores Imunológicos/fisiologia , RiscoRESUMO
The growth and guidance of many axons in the developing nervous system require Netrin-mediated activation of Deleted in Colorectal Cancer (DCC) and other still unknown signaling cues. Commissural axon guidance defects are more severe in DCC mutant mice than Netrin-1 mutant mice, suggesting additional DCC activating signals besides Netrin-1 are involved in proper axon growth. Here we report that interaction screens on extracellular protein microarrays representing over 1,000 proteins uniquely identified Cerebellin 4 (CBLN4), a member of the C1q-tumor necrosis factor (TNF) family, and Netrin-1 as extracellular DCC-binding partners. Immunofluorescence and radio-ligand binding studies demonstrate that Netrin-1 competes with CBLN4 binding at an overlapping site within the membrane-proximal fibronectin domains (FN) 4-6 of DCC and binds with â¼5-fold higher affinity. CBLN4 also binds to the DCC homolog, Neogenin-1 (NEO1), but with a lower affinity compared to DCC. CBLN4-null mice did not show a defect in commissural axons of the developing spinal cord but did display a transient increase in the number of wandering axons in the brachial plexus, consistent with a role in axon guidance. Overall, the data solidifies CBLN4 as a bona fide DCC ligand and strengthens its implication in axon guidance.
Assuntos
Receptores de Superfície Celular/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Axônios/metabolismo , Proteínas de Transporte , Receptor DCC , Desenvolvimento Embrionário/genética , Humanos , Cinética , Ligantes , Camundongos , Mutação , Fatores de Crescimento Neural/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Netrina-1 , Neurogênese/genética , Neurônios/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Mapeamento de Interação de Proteínas , Precursores de Proteínas/metabolismo , Receptores de Superfície Celular/química , Receptores de Superfície Celular/genética , Proteínas Supressoras de Tumor/química , Proteínas Supressoras de Tumor/genéticaRESUMO
Down syndrome cell adhesion molecule (DSCAM) has mainly been characterized for its function as an adhesion molecule in axon growth and in self-recognition between dendrites of the same neuron. Recently, it has been shown that DSCAM can bind to Netrin-1 and that downregulation of DSCAM expression by siRNAs in chick and rodent spinal cords leads to impaired growth and turning response of commissural axons to Netrin-1. To investigate the effect of complete genetic ablation of DSCAM on Netrin-1-induced axon guidance, we analyzed spinal commissural neurons in DSCAM-null mice and found that they extend axons that reach and cross the floor plate and express apparently normal levels of the Netrin receptors DCC (deleted in colorectal carcinoma) and Neogenin. In vitro, commissural neurons in dorsal spinal cord explants of DSCAM-null embryos show normal outgrowth in response to Netrin-1. We therefore conclude that DSCAM is not required for Netrin-induced commissural axon outgrowth and guidance in mice.
Assuntos
Moléculas de Adesão Celular/genética , Cones de Crescimento/metabolismo , Fatores de Crescimento Neural/fisiologia , Vias Neurais/embriologia , Medula Espinal/embriologia , Proteínas Supressoras de Tumor/fisiologia , Animais , Moléculas de Adesão Celular/deficiência , Diferenciação Celular/genética , Feminino , Cones de Crescimento/ultraestrutura , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Netrina-1 , Vias Neurais/fisiologia , Neurogênese/genética , Medula Espinal/fisiologiaRESUMO
Growing axons are guided to their targets by attractive and repulsive cues. In the developing spinal cord, Netrin-1 and Shh guide commissural axons toward the midline. However, the combined inhibition of their activity in commissural axon turning assays does not completely abrogate turning toward floor plate tissue, suggesting that additional guidance cues are present. Here we show that the prototypic angiogenic factor VEGF is secreted by the floor plate and is a chemoattractant for commissural axons in vitro and in vivo. Inactivation of Vegf in the floor plate or of its receptor Flk1 in commissural neurons causes axon guidance defects, whereas Flk1 blockade inhibits turning of axons to VEGF in vitro. Similar to Shh and Netrin-1, VEGF-mediated commissural axon guidance requires the activity of Src family kinases. Our results identify VEGF and Flk1 as a novel ligand/receptor pair controlling commissural axon guidance.
Assuntos
Axônios/fisiologia , Quimiotaxia/fisiologia , Quiasma Óptico/citologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Animais , Células Cultivadas , Quimiotaxia/genética , Embrião de Mamíferos , Ensaio de Imunoadsorção Enzimática/métodos , Glicosídeo Hidrolases/metabolismo , Cones de Crescimento/metabolismo , Proteínas Hedgehog/metabolismo , Técnicas In Vitro , Camundongos , Camundongos Transgênicos , Fatores de Crescimento Neural/metabolismo , Netrina-1 , Neurônios/citologia , Ratos , Ratos Sprague-Dawley , Ratos Wistar , Proteínas Supressoras de Tumor/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Proteína Wnt1/genéticaRESUMO
Mammalian target of rapamycin (mTOR) is implicated in synaptic plasticity and local translation in dendrites. We found that the mTOR inhibitor, rapamycin, increased the Kv1.1 voltage-gated potassium channel protein in hippocampal neurons and promoted Kv1.1 surface expression on dendrites without altering its axonal expression. Moreover, endogenous Kv1.1 mRNA was detected in dendrites. Using Kv1.1 fused to the photoconvertible fluorescence protein Kaede as a reporter for local synthesis, we observed Kv1.1 synthesis in dendrites upon inhibition of mTOR or the N-methyl-d-aspartate (NMDA) glutamate receptor. Thus, synaptic excitation may cause local suppression of dendritic Kv1 channels by reducing their local synthesis.