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Embryonic development is a complex and dynamic process that unfolds over time and involves the production and diversification of increasing numbers of cells. The impact of developmental time on the formation of the central nervous system is well documented, with evidence showing that time plays a crucial role in establishing the identity of neuronal subtypes. However, the study of how time translates into genetic instructions driving cell fate is limited by the scarcity of suitable experimental tools. We introduce BirthSeq, a new method for isolating and analyzing cells based on their birth date. This innovative technique allows for in vivo labeling of cells, isolation via fluorescence-activated cell sorting, and analysis using high-throughput techniques. We calibrated the BirthSeq method for developmental organs across three vertebrate species (mouse, chick and gecko), and utilized it for single-cell RNA sequencing and novel spatially resolved transcriptomic approaches in mouse and chick, respectively. Overall, BirthSeq provides a versatile tool for studying virtually any tissue in different vertebrate organisms, aiding developmental biology research by targeting cells and their temporal cues.
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Análise de Célula Única , Animais , Camundongos , Análise de Célula Única/métodos , Embrião de Galinha , Lagartos/genética , Lagartos/embriologia , Desenvolvimento Embrionário/genética , Transcriptoma/genética , Citometria de Fluxo/métodos , Vertebrados/genética , Separação Celular/métodos , Galinhas , Análise de Sequência de RNA/métodosRESUMO
Alzheimer's disease (AD) is an age-related neurodegenerative condition and the most common type of dementia, characterised by pathological accumulation of extracellular plaques and intracellular neurofibrillary tangles that mainly consist of amyloid-ß (Aß) and hyperphosphorylated tau aggregates, respectively. Previous studies in mouse models with a targeted knock-out of the microtubule-associated protein tau (Mapt) gene demonstrated that Aß-driven toxicity is tau-dependent. However, human cellular models with chronic tau lowering remain unexplored. In this study, we generated stable tau-depleted human induced pluripotent stem cell (iPSC) isogenic panels from two healthy individuals using CRISPR-Cas9 technology. We then differentiated these iPSCs into cortical neurons in vitro in co-culture with primary rat cortical astrocytes before conducting electrophysiological and imaging experiments for a wide range of disease-relevant phenotypes. Both AD brain derived and recombinant Aß were used in this study to elicit toxic responses from the iPSC-derived cortical neurons. We showed that tau depletion in human iPSC-derived cortical neurons caused considerable reductions in neuronal activity without affecting synaptic density. We also observed neurite outgrowth impairments in two of the tau-depleted lines used. Finally, tau depletion protected neurons from adverse effects by mitigating the impact of exogenous Aß-induced hyperactivity, deficits in retrograde axonal transport of mitochondria, and neurodegeneration. Our study established stable human iPSC isogenic panels with chronic tau depletion from two healthy individuals. Cortical neurons derived from these iPSC lines showed that tau is essential in Aß-driven hyperactivity, axonal transport deficits, and neurodegeneration, consistent with studies conducted in Mapt-/- mouse models. These findings highlight the protective effects of chronic tau lowering strategies in AD pathogenesis and reinforce the potential in clinical settings. The tau-depleted human iPSC models can now be applied at scale to investigate the involvement of tau in disease-relevant pathways and cell types.
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Alpha-synuclein pathology is associated with dopaminergic neuronal loss in the substantia nigra (SN) of Parkinson's patients. Working across human and mouse models, we investigated mechanisms by which the accumulation of soluble α-synuclein oligomers leads to neurodegeneration. Biochemical analysis of the midbrain of α-synuclein overexpressing BAC-transgenic male and female mice revealed age- and region-dependent mitochondrial dysfunction and accumulation of damaged proteins downstream of the RE1 Silencing Transcription Factor (REST). Vulnerable SN dopaminergic neurons displayed low REST levels compared with neighboring protected SN GABAergic neurons, which correlated with the accumulation of α-synuclein oligomers and disrupted mitochondrial morphology. Consistent with a protective role, REST levels were reduced in patient induced pluripotent stem cell-derived dopaminergic neurons carrying the SNCA-Triplication mutation, which accumulated α-synuclein oligomers and mitochondrial damage, and displayed REST target gene dysregulation. Furthermore, CRISPR-mediated REST KO induced mitochondrial dysfunction and impaired mitophagy in vitro Conversely, REST overexpression attenuated mitochondrial toxicity and mitochondrial morphology disruption through the transcription factor PGC-1α. Finally, decreased α-synuclein oligomer accumulation and mitochondrial dysfunction in mice correlated with nuclear REST and PGC-1α in protected SN GABAergic neurons compared with vulnerable dopaminergic neurons. Our findings show that increased levels of α-synuclein oligomers cause dopaminergic neuronal-specific dysfunction through mitochondrial toxicity, which can be attenuated by REST in an early model of Parkinsonian pathology. These findings highlight REST as a mediator of dopaminergic vulnerability in PD.SIGNIFICANCE STATEMENT Understanding early Parkinsonian pathophysiology through studies of advanced preclinical models is fundamental to the translation of disease-modifying therapies. Here we show disease-relevant levels of α-synuclein expression in mice leads to accumulation of α-synuclein oligomers in the absence of overt aggregation, and mitochondrial dysfunction in dopaminergic neurons lacking the RE1 Silencing Transcription Factor. Our findings identify the mechanism of action of RE1 Silencing Transcription Factor and PGC-1α as mediators of dopaminergic vulnerability in α-synuclein BAC-transgenic mice and induced pluripotent stem cell-derived dopaminergic cultures, highlighting their potential as therapeutic targets.
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Neurônios Dopaminérgicos/patologia , Mitocôndrias/patologia , Proteínas Repressoras/genética , Sinucleinopatias/genética , Sinucleinopatias/patologia , alfa-Sinucleína/genética , Animais , Sistemas CRISPR-Cas , Cromossomos Artificiais Bacterianos , Feminino , Neurônios GABAérgicos/patologia , Regulação da Expressão Gênica , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Estresse Oxidativo , Doença de Parkinson/patologia , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genéticaRESUMO
Degeneration of locus ceruleus (LC) neurons and dysregulation of noradrenergic signaling are ubiquitous features of Parkinson's disease (PD). The LC is among the first brain regions affected by α-synuclein (asyn) pathology, yet how asyn affects these neurons remains unclear. LC-derived norepinephrine (NE) can stimulate neuroprotective mechanisms and modulate immune cells, while dysregulation of NE neurotransmission may exacerbate disease progression, particularly nonmotor symptoms, and contribute to the chronic neuroinflammation associated with PD pathology. Although transgenic mice overexpressing asyn have previously been developed, transgene expression is usually driven by pan-neuronal promoters and thus has not been selectively targeted to LC neurons. Here we report a novel transgenic mouse expressing human wild-type asyn under control of the noradrenergic-specific dopamine ß-hydroxylase promoter (DBH-hSNCA). These mice developed oligomeric and conformation-specific asyn in LC neurons, alterations in hippocampal and LC microglial abundance, upregulated GFAP expression, degeneration of LC fibers, decreased striatal DA metabolism, and age-dependent behaviors reminiscent of nonmotor symptoms of PD that were rescued by adrenergic receptor antagonists. These mice provide novel insights into how asyn pathology affects LC neurons and how central noradrenergic dysfunction may contribute to early PD pathophysiology.SIGNIFICANCE STATEMENT É-Synuclein (asyn) pathology and loss of neurons in the locus ceruleus (LC) are two of the most ubiquitous neuropathologic features of Parkinson's disease (PD). Dysregulated norepinephrine (NE) neurotransmission is associated with the nonmotor symptoms of PD, including sleep disturbances, emotional changes such as anxiety and depression, and cognitive decline. Importantly, the loss of central NE may contribute to the chronic inflammation in, and progression of, PD. We have generated a novel transgenic mouse expressing human asyn in LC neurons to investigate how increased asyn expression affects the function of the central noradrenergic transmission and associated behaviors. We report cytotoxic effects of oligomeric and conformation-specific asyn, astrogliosis, LC fiber degeneration, disruptions in striatal dopamine metabolism, and age-dependent alterations in nonmotor behaviors without inclusions.
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Neurônios Adrenérgicos/metabolismo , Gliose/genética , Locus Cerúleo/metabolismo , Doença de Parkinson/genética , alfa-Sinucleína/metabolismo , Neurônios Adrenérgicos/patologia , Animais , Ritmo Circadiano , Feminino , Gliose/patologia , Hipocampo/metabolismo , Hipocampo/patologia , Humanos , Locus Cerúleo/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microglia/metabolismo , Microglia/patologia , Movimento , Doença de Parkinson/patologia , Doença de Parkinson/fisiopatologia , alfa-Sinucleína/genéticaRESUMO
Parkinson's disease (PD) is the second most common neurodegenerative disorder and a central role for α-synuclein (αSyn; SNCA) in disease aetiology has been proposed based on genetics and neuropathology. To better understand the pathological mechanisms of αSyn, we generated induced pluripotent stem cells (iPSCs) from healthy individuals and PD patients carrying the A53T SNCA mutation or a triplication of the SNCA locus and differentiated them into dopaminergic neurons (DAns). iPSC-derived DAn from PD patients carrying either mutation showed increased intracellular αSyn accumulation, and DAns from patients carrying the SNCA triplication displayed oligomeric αSyn pathology and elevated αSyn extracellular release. Transcriptomic analysis of purified DAns revealed perturbations in expression of genes linked to mitochondrial function, consistent with observed reduction in mitochondrial respiration, impairment in mitochondrial membrane potential, aberrant mitochondrial morphology and decreased levels of phosphorylated DRP1Ser616. Parkinson's iPSC-derived DAns showed increased endoplasmic reticulum stress and impairments in cholesterol and lipid homeostasis. Together, these data show a correlation between αSyn cellular pathology and deficits in metabolic and cellular bioenergetics in the pathology of PD.
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Neurônios Dopaminérgicos/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Mitocôndrias/metabolismo , Doença de Parkinson/genética , alfa-Sinucleína/genética , Diferenciação Celular , Dinaminas/metabolismo , Estresse do Retículo Endoplasmático/genética , Metabolismo Energético/genética , Humanos , Metabolismo dos Lipídeos/genética , Potencial da Membrana Mitocondrial , Mitocôndrias/ultraestrutura , Mutação , Doença de Parkinson/metabolismo , RNA-Seq , Sinucleinopatias/metabolismo , alfa-Sinucleína/metabolismoRESUMO
Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy are neurodegenerative disorders resulting in progressive motor/cognitive deficits among other symptoms. They are characterised by stereotypical brain cell loss accompanied by the formation of proteinaceous aggregations of the protein α-synuclein (α-syn), being, therefore, termed α-synucleinopathies. Although the presence of α-syn inclusions is a common hallmark of these disorders, the exact nature of the deposited protein is specific to each disease. Different neuroanatomical regions and cellular populations manifest a differential vulnerability to the appearance of protein deposits, cell dysfunction, and cell death, leading to phenotypic diversity. The present review describes the multiple factors that contribute to the selective vulnerability in α-synucleinopathies. We explore the intrinsic cellular properties in the affected regions, including the physiological and pathophysiological roles of endogenous α-syn, the metabolic and genetic build-up of the cells and their connectivity. These factors converge with the variability of the α-syn conformational strains and their spreading capacity to dictate the phenotypic diversity and regional vulnerability of each disease. Finally, we describe the exogenous and environmental factors that potentially contribute by igniting and modulating the differential pathology in α-synucleinopathies. In conclusion, we think that it is the confluence of this disruption of the cellular metabolic state and α-syn structural equilibrium through the anatomical connectivity which appears to initiate cascades of pathological processes triggered by genetic, environmental, or stochastic events that result in the "death by a thousand cuts" profile of α-synucleinopathies.
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Encéfalo/patologia , Atrofia de Múltiplos Sistemas/patologia , Doença de Parkinson/patologia , Sinucleinopatias/patologia , Animais , Humanos , Corpos de Lewy/patologia , Doença por Corpos de Lewy/patologiaRESUMO
Abnormal alpha-synuclein (α-synuclein) expression and aggregation is a key characteristic of Parkinson's disease (PD). However, the exact mechanism(s) linking α-synuclein to the other central feature of PD, dopaminergic neuron loss, remains unclear. Therefore, improved cell and in vivo models are needed to investigate the role of α-synuclein in dopaminergic neuron loss. MicroRNA-7 (miR-7) regulates α-synuclein expression by binding to the 3' UTR of the Synuclein Alpha Non A4 Component of Amyloid Precursor (SNCA) gene and inhibiting its translation. We show that miR-7 is decreased in the substantia nigra of patients with PD and, therefore, may play an essential role in the regulation of α-synuclein expression. Furthermore, we have found that lentiviral-mediated expression of miR-7 complementary binding sites to stably induce a loss of miR-7 function results in an increase in α-synuclein expression in vitro and in vivo. We have also shown that depletion of miR-7 using a miR-decoy produces a loss of nigral dopaminergic neurons accompanied by a reduction of striatal dopamine content. These data suggest that miR-7 has an important role in the regulation of α-synuclein and dopamine physiology and may provide a new paradigm to study the pathology of PD.
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Neurônios Dopaminérgicos/metabolismo , MicroRNAs/metabolismo , Substância Negra/metabolismo , alfa-Sinucleína/metabolismo , Animais , Humanos , Lentivirus/genética , Locomoção/genética , Locomoção/fisiologia , Masculino , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Doença de Parkinson/genética , Doença de Parkinson/metabolismo , alfa-Sinucleína/genéticaRESUMO
Alpha-synuclein is a protein implicated in Parkinson's disease and thought to be one of the main pathological drivers in the disease, although it remains unclear how this protein elicits its neurotoxic effects. Recent findings indicate that the assembly of toxic oligomeric species of alpha-synuclein may be one of the key processes for the pathology and spread of the disease. The absence of a sensitive in situ detection method has hindered the study of these oligomeric species and the role they play in the human brain until recently. In this review, we assess the evidence for the toxicity and prion-like activity of oligomeric forms of alpha-synuclein and discuss the advances in our understanding of the role of alpha-synuclein in Parkinson's disease that may be brought about by the specific and sensitive detection of distinct oligomeric species in post-mortem patient brain. Finally, we discuss current approaches being taken to therapeutically target alpha-synuclein oligomers and their implications.
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Antiparkinsonianos/uso terapêutico , Doença de Parkinson/metabolismo , Doença de Parkinson/terapia , alfa-Sinucleína/metabolismo , Animais , Antiparkinsonianos/farmacologia , Biomarcadores/metabolismo , Humanos , Agregação Patológica de Proteínas/metabolismo , Agregação Patológica de Proteínas/terapia , alfa-Sinucleína/toxicidadeRESUMO
Dickkopf-3 (Dkk-3) is a secreted protein whose expression is downregulated in many types of cancer. Endogenous Dkk-3 is required for formation of acini in 3D cultures of prostate epithelial cells, where it inhibits transforming growth factor (TGF)-ß/Smad signaling. Here, we examined the effects of Dkk-3 on the expression and activity of matrix metalloproteases (MMPs), which mediate the effects of TGF-ß on extracellular matrix disassembly during tissue morphogenesis and promote invasion of tumor cells. Silencing of Dkk-3 in prostate epithelial cells resulted in increased expression and enzyme activity of MMP-2 and MMP-9. Inhibition of MMP-9 partially restored normal acinar morphogenesis in Dkk-3-silenced RWPE-1 prostate epithelial cells. In PC3 prostate cancer cells, Dkk-3 inhibited TGF-ß-dependent migration and invasion. Inhibition was mediated by the Dkk-3 C-terminal cysteine-rich domain (Cys2), which also inhibited TGF-ß-induced expression of MMP9 and MMP13. In contrast, Dkk-3, but not Cys2, increased formation of normal acini in Dkk-3-silenced prostate epithelial cells. These observations highlight a role for Dkk-3 in modulating TGF-ß/MMP signals in the prostate, and suggest that the Dkk-3 Cys2 domain can be used as a basis for therapies that target the tumor promoting effects of TGF-ß signaling in advanced prostate cancer.
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Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Fator de Crescimento Transformador beta/metabolismo , Células Acinares/metabolismo , Células Acinares/patologia , Proteínas Adaptadoras de Transdução de Sinal , Linhagem Celular Tumoral , Quimiocinas , Ativação Enzimática , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Inativação Gênica , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Masculino , Metaloproteinase 2 da Matriz/biossíntese , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 9 da Matriz/biossíntese , Metaloproteinase 9 da Matriz/genética , Morfogênese , Invasividade Neoplásica , Neoplasias da Próstata/enzimologia , Neoplasias da Próstata/genética , Estrutura Terciária de Proteína , Transdução de SinaisRESUMO
The first mammalian Wnt to be discovered, Wnt-1, was found to be essential for the development of a large part of the mouse brain over 25 years ago. We have since learned that Wnt family secreted glycolipoproteins, of which there are nineteen, which activate a diverse network of signals that are particularly important during embryonic development and tissue regeneration. Wnt signals in the developing and adult brain can drive neural stem cell self-renewal, expansion, asymmetric cell division, maturation and differentiation. The molecular events taking place after a Wnt binds to its cell-surface receptors are complex and, at times, controversial. A deeper understanding of these events is anticipated to lead to improvements in the treatment of neurodegenerative diseases and stem cell-based replacement therapies. Here, we review the roles played by Wnts in neural stem cells in the developing mouse brain, at neurogenic sites of the adult mouse and in neural stem cell culture models.
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Encéfalo/metabolismo , Células-Tronco Neurais/metabolismo , Via de Sinalização Wnt , Animais , Encéfalo/citologia , Encéfalo/crescimento & desenvolvimento , Diferenciação Celular/fisiologia , Humanos , Camundongos , Células-Tronco Neurais/citologia , Células-Tronco Neurais/fisiologia , Neurogênese/fisiologia , Células-Tronco/citologia , Células-Tronco/fisiologia , Proteínas Wnt/genética , Proteínas Wnt/metabolismoRESUMO
Wnt/ß-catenin signaling is essential for neurogenesis but less is known about ß-catenin-independent Wnt signals. We show here that Wnt/activator protein-1 (AP-1) signaling drives differentiation of human embryonic stem cell and induced pluripotent stem cell-derived neural progenitor cells. Neuronal differentiation was accompanied by a reduction in ß-catenin/Tcf-dependent transcription and target gene expression, increased levels and/or phosphorylation of activating transcription factor 2 (ATF2), cyclic AMP response element-binding protein, and c-Jun, and increased AP-1-dependent transcription. Inhibition of Wnt secretion using the porcupine inhibitors IWP-2 and Wnt-C59 blocked neuronal differentiation, while activation or inhibition of Wnt/ß-catenin signaling had no effect. Neuronal differentiation increased expression of several Wnt genes, including WNT3A, silencing of which reduced differentiation. Addition of recombinant Wnt-3a to cells treated with IWP-2 or Wnt-C59 increased AP-1 levels and restored neuronal differentiation. The effects of Wnt-3a could not be blocked by addition of Dkk-1 or IWR-1, suggesting the involvement of noncanonical signaling. Consistent with this, restoration of neuronal differentiation by Wnt-3a was reduced by inhibition of Jun N-terminal kinase (JNK) and by gene silencing of ATF2. Together, these observations suggest that ß-catenin-independent Wnt signals promote neural stem/progenitor cell differentiation in a signaling pathway involving Wnt-3a, JNK, and ATF2.
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Diferenciação Celular/fisiologia , Células-Tronco Neurais/citologia , Neurogênese/fisiologia , Neurônios/citologia , Via de Sinalização Wnt/fisiologia , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Neurais/metabolismo , Transdução de Sinais/fisiologia , Fator de Transcrição AP-1/metabolismo , Proteínas Wnt/metabolismo , beta Catenina/metabolismoRESUMO
Understanding medium spiny neuron (MSN) physiology is essential to understand motor impairments in Parkinson's disease (PD) given the architecture of the basal ganglia. Here, we developed a custom three-chambered microfluidic platform and established a cortico-striato-nigral microcircuit partially recapitulating the striatal presynaptic landscape in vitro using induced pluripotent stem cell (iPSC)-derived neurons. We found that, cortical glutamatergic projections facilitated MSN synaptic activity, and dopaminergic transmission enhanced maturation of MSNs in vitro. Replacement of wild-type iPSC-derived dopamine neurons (iPSC-DaNs) in the striatal microcircuit with those carrying the PD-related GBA-N370S mutation led to a depolarisation of resting membrane potential and an increase in rheobase in iPSC-MSNs, as well as a reduction in both voltage-gated sodium and potassium currents. Such deficits were resolved in late microcircuit cultures, and could be reversed in younger cultures with antagonism of protein kinase A activity in iPSC-MSNs. Taken together, our results highlight the unique utility of modelling striatal neurons in a modular physiological circuit to reveal mechanistic insights into GBA1 mutations in PD.
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Mitochondria play a central role in the energy metabolism of cells, and their function is especially important for neurons due to their high energy demand. Therefore, mitochondrial dysfunction is a pathological hallmark of various neurological disorders, including Parkinson's disease. The shape and organization of the mitochondrial network is highly plastic, which allows the cell to respond to environmental cues and needs, and the structure of mitochondria is also tightly linked to their health. Here, we present a protocol to study mitochondrial morphology in situ based on immunostaining of the mitochondrial protein VDAC1 and subsequent image analysis. This tool could be particularly useful for the study of neurodegenerative disorders because it can detect subtle differences in mitochondrial counts and shape induced by aggregates of α-synuclein, an aggregation-prone protein heavily involved in the pathology of Parkinson's disease. This method allows one to report that substantia nigra pars compacta dopaminergic neurons harboring pS129 lesions show mitochondrial fragmentation (as suggested by their reduced Aspect Ratio, AR) compared to their healthy neighboring neurons in a pre-formed fibril intracranial injection Parkinson model.
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Mitocôndrias , Doença de Parkinson , Animais , Feminino , Masculino , Camundongos , Modelos Animais de Doenças , Mitocôndrias/patologia , Doença de Parkinson/patologia , Camundongos Endogâmicos C57BL , Linhagem CelularRESUMO
Parkinson's disease (PD) is characterized by a progressive deterioration of motor and cognitive functions. Although death of dopamine neurons is the hallmark pathology of PD, this is a late-stage disease process preceded by neuronal dysfunction. Here we describe early physiological perturbations in patient-derived induced pluripotent stem cell (iPSC)-dopamine neurons carrying the GBA-N370S mutation, a strong genetic risk factor for PD. GBA-N370S iPSC-dopamine neurons show an early and persistent calcium dysregulation notably at the mitochondria, followed by reduced mitochondrial membrane potential and oxygen consumption rate, indicating mitochondrial failure. With increased neuronal maturity, we observed decreased synaptic function in PD iPSC-dopamine neurons, consistent with the requirement for ATP and calcium to support the increase in electrophysiological activity over time. Our work demonstrates that calcium dyshomeostasis and mitochondrial failure impair the higher electrophysiological activity of mature neurons and may underlie the vulnerability of dopamine neurons in PD.
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Glycogen synthase kinase (GSK-3) is upregulated in many types of tumor, including prostate cancer. GSK-3 inhibitors reduce prostate tumor cell growth; however, it is not clear if both isoforms, GSK-3α and GSK-3ß, are involved. Here, we compared their expression in prostate tumors and used gene silencing to study their functions in 22Rv1 prostate cancer cells. Compared to normal prostate, GSK-3α and GSK-3ß were upregulated in 25/79 and 24/79 cases of prostate cancer, respectively, with GSK-3α elevated in low Gleason sum score tumors and GSK-3ß expressed in high Gleason tumors, and both isoforms correlating with high expression of the androgen receptor (AR). Gene silencing of GSK-3α and, to a lesser extent, GSK-3ß reduced AR transcriptional activity. In addition, silencing of GSK-3ß, but not GSK-3α, reduced Akt phosphorylation. Acute and chronic silencing of either isoform reduced 22Rv1 growth in colony formation assays; however, this did not correlate with effects on AR activity. The GSK-3 inhibitor CHIR99021 reduced 22Rv1 colony formation by 50% in normal growth medium and by 15% in hormone-depleted medium, suggesting that GSK-3 is required both for hormone-dependent and hormone-independent proliferation. In addition, CHIR99021 enhanced growth inhibition by the AR antagonists bicalutamide and MDV3100. Finally, expression of GSK3A and GSK3B mRNAs correlated with a gene expression signature for androgen-regulated genes. Our observations highlight the importance of the GSK-3/AR signaling axis in prostate cancer and support the case for development of isoform-specific GSK-3 inhibitors and their use, in combination with AR antagonists, to treat patients with prostate cancer.
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Quinase 3 da Glicogênio Sintase/metabolismo , Neoplasias da Próstata/enzimologia , Antagonistas de Androgênios/farmacologia , Linhagem Celular Tumoral , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Quinase 3 da Glicogênio Sintase/genética , Glicogênio Sintase Quinase 3 beta , Humanos , Masculino , Gradação de Tumores , Fosforilação , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/patologia , RNA Mensageiro/análiseRESUMO
The septum is a key structure at the core of the forebrain that integrates inputs and relays information to other brain areas to support cognition and behaviours such as feeding and locomotion. Underlying these functions is a rich diversity of neuronal types and an intricate complexity of wiring across and within the septal region. We currently have very little understanding of how septal neuronal diversity emerges during development. Using transgenic mice expressing Cre in different subsets of telencephalic precursors we explored the origins of the three main neuronal types of the septal complex: GABAergic, cholinergic and glutamatergic neurons. We find that septal neurons originate from distinct neuroepithelial domains of the developing septum and are born at different embryonic time points. An exception to this is the GABAergic medial septal Parvalbumin-expressing population which is generated outside the septum from surrounding germinal zones. We identify the transcription factor BSX as being expressed in the developing glutamatergic neuron population. Embryonic elimination of BSX in the septum results in a reduction of septal glutamatergic cell numbers and a consequent deficit in locomotion. Further refinement of septal neuron diversity is needed to understand the multiple roles of septal neurons and their contribution to distinct behaviours.
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Neurônios , Parvalbuminas , Camundongos , Animais , Neurônios/fisiologia , Prosencéfalo , Camundongos TransgênicosRESUMO
BACKGROUND: Multimerization is a key process in prion-like disorders such as Alzheimer's disease (AD), since it is a requirement for self-templating tau and beta-amyloid amyloidogenesis. AT8-immunohistochemistry for hyperphosphorylated tau is currently used for the diagnosis and staging of tau pathology. Given that tau-tau interactions can occur in the absence of hyperphosphorylation or other post-translational modifications (PTMs), the direct visualization of tau multimerization could uncover early pathological tau multimers. METHODS: Here, we used bimolecular fluorescent complementation, rapamycin-dependent FKBP/FRB-tau interaction and transmission electron microscopy to prove the in vitro specificity of tau-proximity ligation assay (tau-PLA). We then analyzed MAPT KO and P301S transgenic mice, and human hippocampus and temporal isocortex of all Braak stages with tau-PLA and compared it with immunohistochemistry for the diagnostic antibody AT8, the early phosphorylation-dependent AT180, and the conformational-dependent antibody MC1. Finally, we performed proteinase-K treatment to infer the content of amyloidogenic beta-sheet fold. RESULTS: Our novel tau-proximity ligation assay (tau-PLA) directly visualized tau-tau interactions in situ, and exclusively recognized tau multimers but not monomers. It elicited no signal in MAPT KO mouse brains, but extensively labelled P301S transgenic mice and AD brain. Two groups of structures were detected, a previously unreported widespread small-sized diffuse pathology and large, neurofibrillary-like lesions. Tau-PLA-labelled diffuse pathology appeared from the earliest Braak stages, mostly unaccompanied by tangle-like tau-immunohistochemistry, being significantly more sensitive than any small-sized dot-/thread-like pathology labelled by AT180-, AT8- and MC1-immunohistochemistry in most regions quantified at stages 0-II. Tau-PLA-labelled diffuse pathology was extremely sensitive to Proteinase-K, in contrast to large lesions. CONCLUSIONS: Tau-PLA is the first method to directly visualize tau multimers both in vitro and in situ with high specificity. We find that tau multimerization appears extensively from the earliest presymptomatic Braak stages as a previously unreported type of diffuse pathology. Importantly, in our study multimerization is the earliest detectable molecular event of AD tau pathology. Our findings open a new window to the study of early tau pathology, with potential implications in early diagnosis and the design of therapeutic strategies.
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Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Encéfalo/metabolismo , Encéfalo/patologia , Proteínas tau/metabolismo , Doença de Alzheimer/genética , Animais , Doenças Assintomáticas , Humanos , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Emaranhados Neurofibrilares/patologia , Multimerização Proteica , Proteínas tau/genéticaRESUMO
Striatal dopamine transporters (DAT) powerfully regulate dopamine signaling, and can contribute risk to degeneration in Parkinson's disease (PD). DATs can interact with the neuronal protein α-synuclein, which is associated with the etiology and molecular pathology of idiopathic and familial PD. Here, we tested whether DAT function in governing dopamine (DA) uptake and release is modified in a human-α-synuclein-overexpressing (SNCA-OVX) transgenic mouse model of early PD. Using fast-scan cyclic voltammetry (FCV) in ex vivo acute striatal slices to detect DA release, and biochemical assays, we show that several aspects of DAT function are promoted in SNCA-OVX mice. Compared to background control α-synuclein-null mice (Snca-null), the SNCA-OVX mice have elevated DA uptake rates, and more pronounced effects of DAT inhibitors on evoked extracellular DA concentrations ([DA]o) and on short-term plasticity (STP) in DA release, indicating DATs play a greater role in limiting DA release and in driving STP. We found that DAT membrane levels and radioligand binding sites correlated with α-synuclein level. Furthermore, DAT function in Snca-null and SNCA-OVX mice could also be promoted by applying cholesterol, and using Tof-SIMS we found genotype-differences in striatal lipids, with lower striatal cholesterol in SNCA-OVX mice. An inhibitor of cholesterol efflux transporter ABCA1 or a cholesterol chelator in SNCA-OVX mice reduced the effects of DAT-inhibitors on evoked [DA]o. Together these data indicate that human α-synuclein in a mouse model of PD promotes striatal DAT function, in a manner supported by extracellular cholesterol, suggesting converging biology of α-synuclein and cholesterol that regulates DAT function and could impact DA function and PD pathophysiology.
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While the initial pathology of Parkinson's disease and other α-synucleinopathies is often confined to circumscribed brain regions, it can spread and progressively affect adjacent and distant brain locales. This process may be controlled by cellular receptors of α-synuclein fibrils, one of which was proposed to be the LAG3 immune checkpoint molecule. Here, we analysed the expression pattern of LAG3 in human and mouse brains. Using a variety of methods and model systems, we found no evidence for LAG3 expression by neurons. While we confirmed that LAG3 interacts with α-synuclein fibrils, the specificity of this interaction appears limited. Moreover, overexpression of LAG3 in cultured human neural cells did not cause any worsening of α-synuclein pathology ex vivo. The overall survival of A53T α-synuclein transgenic mice was unaffected by LAG3 depletion, and the seeded induction of α-synuclein lesions in hippocampal slice cultures was unaffected by LAG3 knockout. These data suggest that the proposed role of LAG3 in the spreading of α-synucleinopathies is not universally valid.
Assuntos
Doença de Parkinson , Sinucleinopatias , Animais , Humanos , Camundongos , Camundongos Transgênicos , Neurônios , alfa-Sinucleína/genéticaRESUMO
Striatal dopamine (DA) is critical for action and learning. Recent data show that DA release is under tonic inhibition by striatal GABA. Ambient striatal GABA tone on striatal projection neurons can be determined by plasma membrane GABA uptake transporters (GATs) located on astrocytes and neurons. However, whether striatal GATs and astrocytes determine DA output are unknown. We reveal that DA release in mouse dorsolateral striatum, but not nucleus accumbens core, is governed by GAT-1 and GAT-3. These GATs are partly localized to astrocytes, and are enriched in dorsolateral striatum compared to accumbens core. In a mouse model of early parkinsonism, GATs are downregulated, tonic GABAergic inhibition of DA release augmented, and nigrostriatal GABA co-release attenuated. These data define previously unappreciated and important roles for GATs and astrocytes in supporting DA release in striatum, and reveal a maladaptive plasticity in early parkinsonism that impairs DA output in vulnerable striatal regions.