RESUMO
Antisense oligonucleotides (ASOs) are promising therapeutics for treating various neurological disorders. However, ASOs are unable to readily cross the mammalian blood-brain barrier (BBB) and therefore need to be delivered intrathecally to the central nervous system (CNS). Here, we engineered a human transferrin receptor 1 (TfR1) binding molecule, the oligonucleotide transport vehicle (OTV), to transport a tool ASO across the BBB in human TfR knockin (TfRmu/hu KI) mice and nonhuman primates. Intravenous injection and systemic delivery of OTV to TfRmu/hu KI mice resulted in sustained knockdown of the ASO target RNA, Malat1, across multiple mouse CNS regions and cell types, including endothelial cells, neurons, astrocytes, microglia, and oligodendrocytes. In addition, systemic delivery of OTV enabled Malat1 RNA knockdown in mouse quadriceps and cardiac muscles, which are difficult to target with oligonucleotides alone. Systemically delivered OTV enabled a more uniform ASO biodistribution profile in the CNS of TfRmu/hu KI mice and greater knockdown of Malat1 RNA compared with a bivalent, high-affinity TfR antibody. In cynomolgus macaques, an OTV directed against MALAT1 displayed robust ASO delivery to the primate CNS and enabled more uniform biodistribution and RNA target knockdown compared with intrathecal dosing of the same unconjugated ASO. Our data support systemically delivered OTV as a potential platform for delivering therapeutic ASOs across the BBB.
Assuntos
Barreira Hematoencefálica , Oligonucleotídeos Antissenso , RNA Longo não Codificante , Receptores da Transferrina , Animais , Oligonucleotídeos Antissenso/farmacocinética , Oligonucleotídeos Antissenso/administração & dosagem , Barreira Hematoencefálica/metabolismo , Receptores da Transferrina/metabolismo , Humanos , RNA Longo não Codificante/metabolismo , RNA Longo não Codificante/genética , Camundongos , Transporte Biológico , Macaca fascicularis , Técnicas de Silenciamento de Genes , Distribuição TecidualRESUMO
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
BACKGROUND: Genetic mutations underlying familial Alzheimer's disease (AD) were identified decades ago, but the field is still in search of transformative therapies for patients. While mouse models based on overexpression of mutated transgenes have yielded key insights in mechanisms of disease, those models are subject to artifacts, including random genetic integration of the transgene, ectopic expression and non-physiological protein levels. The genetic engineering of novel mouse models using knock-in approaches addresses some of those limitations. With mounting evidence of the role played by microglia in AD, high-dimensional approaches to phenotype microglia in those models are critical to refine our understanding of the immune response in the brain. METHODS: We engineered a novel App knock-in mouse model (AppSAA) using homologous recombination to introduce three disease-causing coding mutations (Swedish, Arctic and Austrian) to the mouse App gene. Amyloid-ß pathology, neurodegeneration, glial responses, brain metabolism and behavioral phenotypes were characterized in heterozygous and homozygous AppSAA mice at different ages in brain and/ or biofluids. Wild type littermate mice were used as experimental controls. We used in situ imaging technologies to define the whole-brain distribution of amyloid plaques and compare it to other AD mouse models and human brain pathology. To further explore the microglial response to AD relevant pathology, we isolated microglia with fibrillar Aß content from the brain and performed transcriptomics and metabolomics analyses and in vivo brain imaging to measure energy metabolism and microglial response. Finally, we also characterized the mice in various behavioral assays. RESULTS: Leveraging multi-omics approaches, we discovered profound alteration of diverse lipids and metabolites as well as an exacerbated disease-associated transcriptomic response in microglia with high intracellular Aß content. The AppSAA knock-in mouse model recapitulates key pathological features of AD such as a progressive accumulation of parenchymal amyloid plaques and vascular amyloid deposits, altered astroglial and microglial responses and elevation of CSF markers of neurodegeneration. Those observations were associated with increased TSPO and FDG-PET brain signals and a hyperactivity phenotype as the animals aged. DISCUSSION: Our findings demonstrate that fibrillar Aß in microglia is associated with lipid dyshomeostasis consistent with lysosomal dysfunction and foam cell phenotypes as well as profound immuno-metabolic perturbations, opening new avenues to further investigate metabolic pathways at play in microglia responding to AD-relevant pathogenesis. The in-depth characterization of pathological hallmarks of AD in this novel and open-access mouse model should serve as a resource for the scientific community to investigate disease-relevant biology.
Assuntos
Doença de Alzheimer , Precursor de Proteína beta-Amiloide , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Amiloidose/metabolismo , Animais , Encéfalo/metabolismo , Modelos Animais de Doenças , Camundongos , Camundongos Transgênicos , Microglia/metabolismo , Placa Amiloide/patologia , Receptores de GABA/metabolismoRESUMO
Delivery of biotherapeutics across the blood-brain barrier (BBB) is a challenge. Many approaches fuse biotherapeutics to platforms that bind the transferrin receptor (TfR), a brain endothelial cell target, to facilitate receptor-mediated transcytosis across the BBB. Here, we characterized the pharmacological behavior of two distinct TfR-targeted platforms fused to iduronate 2-sulfatase (IDS), a lysosomal enzyme deficient in mucopolysaccharidosis type II (MPS II), and compared the relative brain exposures and functional activities of both approaches in mouse models. IDS fused to a moderate-affinity, monovalent TfR-binding enzyme transport vehicle (ETV:IDS) resulted in widespread brain exposure, internalization by parenchymal cells, and significant substrate reduction in the CNS of an MPS II mouse model. In contrast, IDS fused to a standard high-affinity bivalent antibody (IgG:IDS) resulted in lower brain uptake, limited biodistribution beyond brain endothelial cells, and reduced brain substrate reduction. These results highlight important features likely to impact the clinical development of TfR-targeting platforms in MPS II and potentially other CNS diseases.
Assuntos
Iduronato Sulfatase , Mucopolissacaridose II , Receptores da Transferrina , Proteínas Recombinantes de Fusão , Animais , Barreira Hematoencefálica/metabolismo , Encéfalo/metabolismo , Modelos Animais de Doenças , Células Endoteliais/metabolismo , Iduronato Sulfatase/metabolismo , Iduronato Sulfatase/farmacologia , Lisossomos/metabolismo , Camundongos , Mucopolissacaridose II/metabolismo , Receptores da Transferrina/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/farmacologia , Distribuição TecidualRESUMO
Mucopolysaccharidosis type II (MPS II) is a lysosomal storage disorder caused by deficiency of the iduronate-2-sulfatase (IDS) enzyme, resulting in cellular accumulation of glycosaminoglycans (GAGs) throughout the body. Treatment of MPS II remains a considerable challenge as current enzyme replacement therapies do not adequately control many aspects of the disease, including skeletal and neurological manifestations. We developed an IDS transport vehicle (ETV:IDS) that is engineered to bind to the transferrin receptor; this design facilitates receptor-mediated transcytosis of IDS across the blood-brain barrier and improves its distribution into the brain while maintaining distribution to peripheral tissues. Here we show that chronic systemic administration of ETV:IDS in a mouse model of MPS II reduced levels of peripheral and central nervous system GAGs, microgliosis, and neurofilament light chain, a biomarker of neuronal injury. Additionally, ETV:IDS rescued auricular and skeletal abnormalities when introduced in adult MPS II mice. These effects were accompanied by improvements in several neurobehavioral domains, including motor skills, sensorimotor gating, and learning and memory. Together, these results highlight the therapeutic potential of ETV:IDS for treating peripheral and central abnormalities in MPS II. DNL310, an investigational ETV:IDS molecule, is currently in clinical trials as a potential treatment for patients with MPS II.
Assuntos
Barreira Hematoencefálica/metabolismo , Terapia de Reposição de Enzimas/métodos , Iduronato Sulfatase/administração & dosagem , Mucopolissacaridose II/tratamento farmacológico , Receptores da Transferrina/metabolismo , Vesículas Transportadoras/metabolismo , Animais , Comportamento Animal/efeitos dos fármacos , Modelos Animais de Doenças , Glicosaminoglicanos/metabolismo , Iduronato Sulfatase/genética , Memória/efeitos dos fármacos , Camundongos , Camundongos Knockout , Destreza Motora/efeitos dos fármacos , Mucopolissacaridose II/genética , Mucopolissacaridose II/metabolismo , Mucopolissacaridose II/fisiopatologia , Fenótipo , Filtro Sensorial/efeitos dos fármacos , Esqueleto/efeitos dos fármacos , Aprendizagem Espacial/efeitos dos fármacos , TranscitoseRESUMO
GRN mutations cause frontotemporal dementia (GRN-FTD) due to deficiency in progranulin (PGRN), a lysosomal and secreted protein with unclear function. Here, we found that Grn-/- mice exhibit a global deficiency in bis(monoacylglycero)phosphate (BMP), an endolysosomal phospholipid we identified as a pH-dependent PGRN interactor as well as a redox-sensitive enhancer of lysosomal proteolysis and lipolysis. Grn-/- brains also showed an age-dependent, secondary storage of glucocerebrosidase substrate glucosylsphingosine. We investigated a protein replacement strategy by engineering protein transport vehicle (PTV):PGRN-a recombinant protein linking PGRN to a modified Fc domain that binds human transferrin receptor for enhanced CNS biodistribution. PTV:PGRN rescued various Grn-/- phenotypes in primary murine macrophages and human iPSC-derived microglia, including oxidative stress, lysosomal dysfunction, and endomembrane damage. Peripherally delivered PTV:PGRN corrected levels of BMP, glucosylsphingosine, and disease pathology in Grn-/- CNS, including microgliosis, lipofuscinosis, and neuronal damage. PTV:PGRN thus represents a potential biotherapeutic for GRN-FTD.
Assuntos
Produtos Biológicos/uso terapêutico , Encéfalo/metabolismo , Doenças por Armazenamento dos Lisossomos/terapia , Progranulinas/uso terapêutico , Animais , Proteínas Morfogenéticas Ósseas/metabolismo , Endossomos/metabolismo , Feminino , Demência Frontotemporal/sangue , Demência Frontotemporal/líquido cefalorraquidiano , Gliose/complicações , Gliose/patologia , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Inflamação/patologia , Metabolismo dos Lipídeos , Lipofuscina/metabolismo , Lisossomos/metabolismo , Macrófagos/metabolismo , Masculino , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microglia/metabolismo , Degeneração Neural/patologia , Fenótipo , Progranulinas/deficiência , Progranulinas/metabolismo , Receptores Imunológicos/metabolismo , Receptores da Transferrina/metabolismo , Distribuição TecidualRESUMO
BACKGROUND: Alzheimer's disease (AD) is the most frequent and costly neurodegenerative disorder. Although diverse lines of evidence suggest that the amyloid precursor protein (APP) is involved in its causation, the precise mechanisms remain unknown and no treatments are available to prevent or halt the disease. A favorite hypothesis has been that APP contributes to AD pathogenesis through the cerebral accumulation of the amyloid-ß peptide (Aß), which is derived from APP through sequential proteolytic cleavage by BACE1 and γ-secretase. However, inhibitors of these enzymes have failed in clinical trials despite clear evidence for target engagement. METHODS: To further elucidate the roles of APP and its metabolites in AD pathogenesis, we analyzed transgenic mice overexpressing wildtype human APP (hAPP) or hAPP carrying mutations that cause autosomal dominant familial AD (FAD), as well as App knock-in mice that do not overexpress hAPP but have two mouse App alleles with FAD mutations and a humanized Aß sequence. RESULTS: Although these lines of mice had marked differences in cortical and hippocampal levels of APP, APP C-terminal fragments, soluble Aß, Aß oligomers and age-dependent amyloid deposition, they all developed cognitive deficits as well as non-convulsive epileptiform activity, a type of network dysfunction that also occurs in a substantive proportion of humans with AD. Pharmacological inhibition of BACE1 effectively reduced levels of amyloidogenic APP C-terminal fragments (C99), soluble Aß, Aß oligomers, and amyloid deposits in transgenic mice expressing FAD-mutant hAPP, but did not improve their network dysfunction and behavioral abnormalities, even when initiated at early stages before amyloid deposits were detectable. CONCLUSIONS: hAPP transgenic and App knock-in mice develop similar pathophysiological alterations. APP and its metabolites contribute to AD-related functional alterations through complex combinatorial mechanisms that may be difficult to block with BACE inhibitors and, possibly, also with other anti-Aß treatments.
Assuntos
Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Secretases da Proteína Precursora do Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Ácido Aspártico Endopeptidases/metabolismo , Precursor de Proteína beta-Amiloide/genética , Animais , Comportamento Animal/fisiologia , Encéfalo/metabolismo , Modelos Animais de Doenças , Técnicas de Introdução de Genes , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação , Rede Nervosa/metabolismo , Rede Nervosa/patologiaRESUMO
A large number of mouse models have been engineered, characterized and used to advance biomedical research in Alzheimer disease (AD). Early models simply damaged the rodent brain through toxins or lesions. Later, the spread of genetic engineering technology enabled investigators to develop models of familial AD by overexpressing human genes such as those encoding amyloid precursor protein (APP) or presenilins (PSEN1 or PSEN2) carrying mutations linked to early-onset AD. Recently, more complex models have sought to explore the impact of multiple genetic risk factors in the context of different biological challenges. Although none of these models has proven to be a fully faithful reproduction of the human disease, models remain essential as tools to improve our understanding of AD biology, conduct thorough pharmacokinetic and pharmacodynamic analyses, discover translatable biomarkers and evaluate specific therapeutic approaches. To realize the full potential of animal models as new technologies and knowledge become available, it is critical to define an optimal strategy for their use. Here, we review progress and challenges in the use of AD mouse models, highlight emerging scientific innovations in model development, and introduce a conceptual framework for use of preclinical models for therapeutic development.
Assuntos
Doença de Alzheimer/tratamento farmacológico , Modelos Animais de Doenças , Desenvolvimento de Medicamentos/métodos , Doença de Alzheimer/genética , Doença de Alzheimer/fisiopatologia , Animais , Biomarcadores/metabolismo , Avaliação Pré-Clínica de Medicamentos/métodos , Predisposição Genética para Doença , Humanos , Camundongos , Mutação , Fatores de RiscoRESUMO
Most lysosomal storage diseases (LSDs) involve progressive central nervous system (CNS) impairment, resulting from deficiency of a lysosomal enzyme. Treatment of neuronopathic LSDs remains a considerable challenge, as approved intravenously administered enzyme therapies are ineffective in modifying CNS disease because they do not effectively cross the blood-brain barrier (BBB). We describe a therapeutic platform for increasing the brain exposure of enzyme replacement therapies. The enzyme transport vehicle (ETV) is a lysosomal enzyme fused to an Fc domain that has been engineered to bind to the transferrin receptor, which facilitates receptor-mediated transcytosis across the BBB. We demonstrate that ETV fusions containing iduronate 2-sulfatase (ETV:IDS), the lysosomal enzyme deficient in mucopolysaccharidosis type II, exhibited high intrinsic activity and degraded accumulated substrates in both IDS-deficient cell and in vivo models. ETV substantially improved brain delivery of IDS in a preclinical model of disease, enabling enhanced cellular distribution to neurons, astrocytes, and microglia throughout the brain. Improved brain exposure for ETV:IDS translated to a reduction in accumulated substrates in these CNS cell types and peripheral tissues and resulted in a complete correction of downstream disease-relevant pathologies in the brain, including secondary accumulation of lysosomal lipids, perturbed gene expression, neuroinflammation, and neuroaxonal damage. These data highlight the therapeutic potential of the ETV platform for LSDs and provide preclinical proof of concept for TV-enabled therapeutics to treat CNS diseases more broadly.
Assuntos
Barreira Hematoencefálica , Iduronato Sulfatase , Animais , Encéfalo , Modelos Animais de Doenças , Terapia de Reposição de Enzimas , Lisossomos , CamundongosRESUMO
Effective delivery of protein therapeutics to the central nervous system (CNS) has been greatly restricted by the blood-brain barrier (BBB). We describe the development of a BBB transport vehicle (TV) comprising an engineered Fc fragment that exploits receptor-mediated transcytosis for CNS delivery of biotherapeutics by binding a highly expressed brain endothelial cell target. TVs were engineered using directed evolution to bind the apical domain of the human transferrin receptor (hTfR) without the use of amino acid insertions, deletions, or unnatural appendages. A crystal structure of the TV-TfR complex revealed the TV binding site to be away from transferrin and FcRn binding sites, which was further confirmed experimentally in vitro and in vivo. Recombinant expression of TVs fused to anti-ß-secretase (BACE1) Fabs yielded antibody transport vehicle (ATV) molecules with native immunoglobulin G (IgG) structure and stability. Peripheral administration of anti-BACE1 ATVs to hTfR-engineered mice and cynomolgus monkeys resulted in substantially improved CNS uptake and sustained pharmacodynamic responses. The TV platform readily accommodates numerous additional configurations, including bispecific antibodies and protein fusions, yielding a highly modular CNS delivery platform.
Assuntos
Secretases da Proteína Precursora do Amiloide , Barreira Hematoencefálica , Secretases da Proteína Precursora do Amiloide/metabolismo , Animais , Ácido Aspártico Endopeptidases/metabolismo , Barreira Hematoencefálica/metabolismo , Encéfalo/metabolismo , Haplorrinos/metabolismo , Fragmentos Fc das Imunoglobulinas , Camundongos , Receptores da Transferrina/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
Analogous behavioral assays are needed across animal models and human patients to improve translational research. Here, we examined the extent to which performance in the Morris water maze - the most frequently used behavioral assay of spatial learning and memory in rodents - translates to humans. We designed a virtual version of the assay for human subjects that includes the visible-target training, hidden-target learning, and probe trials that are typically administered in the mouse version. We compared transgenic mice that express human amyloid precursor protein (hAPP) and patients with mild cognitive impairment due to Alzheimer's disease (MCI-AD) to evaluate the sensitivity of performance measures in detecting deficits. Patients performed normally during visible-target training, while hAPP mice showed procedural learning deficits. In hidden-target learning and probe trials, hAPP mice and MCI-AD patients showed similar deficits in learning and remembering the target location. In addition, we have provided recommendations for selecting performance measures and sample sizes to make these assays sensitive to learning and memory deficits in humans with MCI-AD and in mouse models. Together, our results demonstrate that with careful study design and analysis, the Morris maze is a sensitive assay for detecting AD-relevant impairments across species.
Assuntos
Doença de Alzheimer/fisiopatologia , Aprendizagem em Labirinto , Memória , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Precursor de Proteína beta-Amiloide/biossíntese , Precursor de Proteína beta-Amiloide/genética , Animais , Feminino , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Especificidade da EspécieRESUMO
Aging is the principal demographic risk factor for Alzheimer disease (AD), the most common neurodegenerative disorder. Klotho is a key modulator of the aging process and, when overexpressed, extends mammalian lifespan, increases synaptic plasticity, and enhances cognition. Whether klotho can counteract deficits related to neurodegenerative diseases, such as AD, is unknown. Here we show that elevating klotho expression decreases premature mortality and network dysfunction in human amyloid precursor protein (hAPP) transgenic mice, which simulate key aspects of AD. Increasing klotho levels prevented depletion of NMDA receptor (NMDAR) subunits in the hippocampus and enhanced spatial learning and memory in hAPP mice. Klotho elevation in hAPP mice increased the abundance of the GluN2B subunit of NMDAR in postsynaptic densities and NMDAR-dependent long-term potentiation, which is critical for learning and memory. Thus, increasing wild-type klotho levels or activities improves synaptic and cognitive functions, and may be of therapeutic benefit in AD and other cognitive disorders.
Assuntos
Precursor de Proteína beta-Amiloide/fisiologia , Cognição/fisiologia , Glucuronidase/fisiologia , Longevidade/genética , Precursor de Proteína beta-Amiloide/genética , Animais , Comportamento Animal/fisiologia , Transtornos Cognitivos/genética , Transtornos Cognitivos/psicologia , Humanos , Proteínas Klotho , Longevidade/fisiologia , Aprendizagem em Labirinto/fisiologia , Camundongos , Camundongos Transgênicos , Rede Nervosa/patologia , Receptores de N-Metil-D-Aspartato/fisiologia , Sinapses/patologia , Proteínas tau/metabolismoRESUMO
OBJECTIVE: Dementia with Lewy bodies (DLB) is associated with the accumulation of wild-type human α-synuclein (SYN) in neurons and with prominent slowing of brain oscillations on electroencephalography (EEG). However, it remains uncertain whether the EEG abnormalities are actually caused by SYN. METHODS: To determine whether SYN can cause neural network abnormalities, we performed EEG recordings and analyzed the expression of neuronal activity-dependent gene products in SYN transgenic mice. We also carried out comparative analyses in humans with DLB. RESULTS: We demonstrate that neuronal expression of SYN in transgenic mice causes a left shift in spectral power that closely resembles the EEG slowing observed in DLB patients. Surprisingly, SYN mice also had seizures and showed molecular hippocampal alterations indicative of aberrant network excitability, including calbindin depletion in the dentate gyrus. In postmortem brain tissues from DLB patients, we found reduced levels of calbindin mRNA in the dentate gyrus. Furthermore, nearly one quarter of DLB patients showed myoclonus, a clinical sign of aberrant network excitability that was associated with an earlier age of onset of cognitive impairments. In SYN mice, partial suppression of epileptiform activity did not alter their shift in spectral power. Furthermore, epileptiform activity in human amyloid precursor protein transgenic mice was not associated with a left shift in spectral power. INTERPRETATION: We conclude that neuronal accumulation of SYN slows brain oscillations and, in parallel, causes aberrant network excitability that can escalate into seizure activity. The potential role of aberrant network excitability in DLB merits further investigation.
RESUMO
OBJECTIVE: Reducing levels of the microtubule-associated protein tau has shown promise as a potential treatment strategy for diseases with secondary epileptic features such as Alzheimer disease. We wanted to determine whether tau reduction may also be of benefit in intractable genetic epilepsies. METHODS: We studied a mouse model of Dravet syndrome, a severe childhood epilepsy caused by mutations in the human SCN1A gene encoding the voltage-gated sodium channel subunit Nav 1.1. We genetically deleted 1 or 2 Tau alleles in mice carrying an Nav 1.1 truncation mutation (R1407X) that causes Dravet syndrome in humans, and examined their survival, epileptic activity, related hippocampal alterations, and behavioral abnormalities using observation, electroencephalographic recordings, acute slice electrophysiology, immunohistochemistry, and behavioral assays. RESULTS: Tau ablation prevented the high mortality of Dravet mice and reduced the frequency of spontaneous and febrile seizures. It reduced interictal epileptic spikes in vivo and drug-induced epileptic activity in brain slices ex vivo. Tau ablation also prevented biochemical changes in the hippocampus indicative of epileptic activity and ameliorated abnormalities in learning and memory, nest building, and open field behaviors in Dravet mice. Deletion of only 1 Tau allele was sufficient to suppress epileptic activity and improve survival and nesting performance. INTERPRETATION: Tau reduction may be of therapeutic benefit in Dravet syndrome and other intractable genetic epilepsies.
Assuntos
Epilepsias Mioclônicas/metabolismo , Hipocampo/metabolismo , Convulsões/metabolismo , Proteínas tau/metabolismo , Alelos , Animais , Comportamento Animal/fisiologia , Modelos Animais de Doenças , Eletroencefalografia , Epilepsias Mioclônicas/fisiopatologia , Epilepsias Mioclônicas/terapia , Feminino , Hipocampo/patologia , Hipocampo/fisiopatologia , Aprendizagem/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação/genética , Canal de Sódio Disparado por Voltagem NAV1.1/genética , Convulsões/etiologia , Convulsões/fisiopatologia , Proteínas tau/genéticaRESUMO
We show that a natural behavior, exploration of a novel environment, causes DNA double-strand breaks (DSBs) in neurons of young adult wild-type mice. DSBs occurred in multiple brain regions, were most abundant in the dentate gyrus, which is involved in learning and memory, and were repaired within 24 h. Increasing neuronal activity by sensory or optogenetic stimulation increased neuronal DSBs in relevant but not irrelevant networks. Mice transgenic for human amyloid precursor protein (hAPP), which simulate key aspects of Alzheimer's disease, had increased neuronal DSBs at baseline and more severe and prolonged DSBs after exploration. Interventions that suppress aberrant neuronal activity and improve learning and memory in hAPP mice normalized their levels of DSBs. Blocking extrasynaptic NMDA-type glutamate receptors prevented amyloid-ß (Aß)-induced DSBs in neuronal cultures. Thus, transient increases in neuronal DSBs occur as a result of physiological brain activity, and Aß exacerbates DNA damage, most likely by eliciting synaptic dysfunction.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Quebras de DNA de Cadeia Dupla , Neurônios/fisiologia , Precursor de Proteína beta-Amiloide/genética , Animais , Animais Recém-Nascidos , Células Cultivadas , Córtex Cerebral/citologia , Córtex Cerebral/efeitos dos fármacos , Channelrhodopsins , Corpo Estriado/efeitos dos fármacos , Corpo Estriado/metabolismo , Corticosterona/sangue , Modelos Animais de Doenças , Antagonistas de Aminoácidos Excitatórios/farmacologia , Comportamento Exploratório/efeitos dos fármacos , Comportamento Exploratório/fisiologia , Regulação da Expressão Gênica/efeitos dos fármacos , Hipocampo/citologia , Hipocampo/efeitos dos fármacos , Histonas/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neurônios/efeitos dos fármacos , Estimulação Luminosa , Quinoxalinas/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Estresse Fisiológico/fisiologia , Valina/análogos & derivados , Valina/farmacologia , Proteínas tau/genéticaRESUMO
Environmental enrichment of laboratory animals influences brain plasticity, stimulates neurogenesis, increases neurotrophic factor expression, and protects against the effects of brain insult. However, these positive effects are not constantly observed, probably because standardized procedures of environmental enrichment are lacking. Therefore, we engineered an enriched cage (the Marlau™ cage), which offers: (1) minimally stressful social interactions; (2) increased voluntary exercise; (3) multiple entertaining activities; (4) cognitive stimulation (maze exploration), and (5) novelty (maze configuration changed three times a week). The maze, which separates food pellet and water bottle compartments, guarantees cognitive stimulation for all animals. Compared to rats raised in groups in conventional cages, rats housed in Marlau™ cages exhibited increased cortical thickness, hippocampal neurogenesis and hippocampal levels of transcripts encoding various genes involved in tissue plasticity and remodeling. In addition, rats housed in Marlau™ cages exhibited better performances in learning and memory, decreased anxiety-associated behaviors, and better recovery of basal plasma corticosterone level after acute restraint stress. Marlau™ cages also insure inter-experiment reproducibility in spatial learning and brain gene expression assays. Finally, housing rats in Marlau™ cages after severe status epilepticus at weaning prevents the cognitive impairment observed in rats subjected to the same insult and then housed in conventional cages. By providing a standardized enriched environment for rodents during housing, the Marlau™ cage should facilitate the uniformity of environmental enrichment across laboratories.
Assuntos
Encéfalo/fisiologia , Encéfalo/fisiopatologia , Cognição , Abrigo para Animais/normas , Plasticidade Neuronal , Estado Epiléptico/patologia , Estado Epiléptico/fisiopatologia , Adaptação Psicológica/fisiologia , Animais , Ansiedade/complicações , Peso Corporal , Encéfalo/citologia , Encéfalo/patologia , Região CA1 Hipocampal/citologia , Região CA1 Hipocampal/patologia , Região CA1 Hipocampal/fisiologia , Região CA1 Hipocampal/fisiopatologia , Ingestão de Alimentos , Comportamento Exploratório/fisiologia , Saúde , Metabolismo dos Lipídeos , Masculino , Neurogênese/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Padrões de Referência , Reprodutibilidade dos Testes , Restrição Física/psicologia , Estado Epiléptico/psicologia , Estresse Psicológico/complicações , Estresse Psicológico/patologia , Estresse Psicológico/fisiopatologia , Sinapses/patologiaRESUMO
In light of the rising prevalence of Alzheimer's disease (AD), new strategies to prevent, halt, and reverse this condition are needed urgently. Perturbations of brain network activity are observed in AD patients and in conditions that increase the risk of developing AD, suggesting that aberrant network activity might contribute to AD-related cognitive decline. Human amyloid precursor protein (hAPP) transgenic mice simulate key aspects of AD, including pathologically elevated levels of amyloid-ß peptides in brain, aberrant neural network activity, remodeling of hippocampal circuits, synaptic deficits, and behavioral abnormalities. Whether these alterations are linked in a causal chain remains unknown. To explore whether hAPP/amyloid-ß-induced aberrant network activity contributes to synaptic and cognitive deficits, we treated hAPP mice with different antiepileptic drugs. Among the drugs tested, only levetiracetam (LEV) effectively reduced abnormal spike activity detected by electroencephalography. Chronic treatment with LEV also reversed hippocampal remodeling, behavioral abnormalities, synaptic dysfunction, and deficits in learning and memory in hAPP mice. Our findings support the hypothesis that aberrant network activity contributes causally to synaptic and cognitive deficits in hAPP mice. LEV might also help ameliorate related abnormalities in people who have or are at risk for AD.
Assuntos
Doença de Alzheimer/tratamento farmacológico , Anticonvulsivantes/farmacologia , Transtornos Cognitivos/tratamento farmacológico , Cognição/efeitos dos fármacos , Rede Nervosa/efeitos dos fármacos , Piracetam/análogos & derivados , Sinapses/efeitos dos fármacos , Doença de Alzheimer/complicações , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Análise de Variância , Animais , Anticonvulsivantes/sangue , Anticonvulsivantes/uso terapêutico , Western Blotting , Transtornos Cognitivos/etiologia , Eletroencefalografia , Humanos , Imuno-Histoquímica , Levetiracetam , Aprendizagem em Labirinto/efeitos dos fármacos , Camundongos , Camundongos Transgênicos , Rede Nervosa/fisiopatologia , Piracetam/sangue , Piracetam/farmacologia , Piracetam/uso terapêuticoRESUMO
Previous studies suggested that the cellular prion protein (PrP(c)) plays a critical role in the pathogenesis of Alzheimer's disease (AD). Specifically, amyloid-ß (Aß) oligomers were proposed to cause synaptic and cognitive dysfunction by binding to PrP(c). To test this hypothesis, we crossed human amyloid precursor protein (hAPP) transgenic mice from line J20 onto a PrP(c)-deficient background. Ablation of PrP(c) did not prevent the premature mortality and abnormal neural network activity typically seen in hAPPJ20 mice. Furthermore, hAPPJ20 mice with or without PrP(c) expression showed comparably robust abnormalities in learning and memory and in other behavioral domains at 6-8 months of age. Notably, these abnormalities are not refractory to therapeutic manipulations in general: they can be effectively prevented by interventions that prevent Aß-dependent neuronal dysfunction also in other lines of hAPP transgenic mice. Thus, at least in this model, PrP(c) is not an important mediator of Aß-induced neurological impairments.