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1.
J Neurosci ; 43(10): 1692-1713, 2023 03 08.
Artículo en Inglés | MEDLINE | ID: mdl-36717230

RESUMEN

The brain µ-opioid receptor (MOR) is critical for the analgesic, rewarding, and addictive effects of opioid drugs. However, in rat models of opioid-related behaviors, the circuit mechanisms of MOR-expressing cells are less known because of a lack of genetic tools to selectively manipulate them. We introduce a CRISPR-based Oprm1-Cre knock-in transgenic rat that provides cell type-specific genetic access to MOR-expressing cells. After performing anatomic and behavioral validation experiments, we used the Oprm1-Cre knock-in rats to study the involvement of NAc MOR-expressing cells in heroin self-administration in male and female rats. Using RNAscope, autoradiography, and FISH chain reaction (HCR-FISH), we found no differences in Oprm1 expression in NAc, dorsal striatum, and dorsal hippocampus, or MOR receptor density (except dorsal striatum) or function between Oprm1-Cre knock-in rats and wildtype littermates. HCR-FISH assay showed that iCre is highly coexpressed with Oprm1 (95%-98%). There were no genotype differences in pain responses, morphine analgesia and tolerance, heroin self-administration, and relapse-related behaviors. We used the Cre-dependent vector AAV1-EF1a-Flex-taCasp3-TEVP to lesion NAc MOR-expressing cells. We found that the lesions decreased acquisition of heroin self-administration in male Oprm1-Cre rats and had a stronger inhibitory effect on the effort to self-administer heroin in female Oprm1-Cre rats. The validation of an Oprm1-Cre knock-in rat enables new strategies for understanding the role of MOR-expressing cells in rat models of opioid addiction, pain-related behaviors, and other opioid-mediated functions. Our initial mechanistic study indicates that lesioning NAc MOR-expressing cells had different effects on heroin self-administration in male and female rats.SIGNIFICANCE STATEMENT The brain µ-opioid receptor (MOR) is critical for the analgesic, rewarding, and addictive effects of opioid drugs. However, in rat models of opioid-related behaviors, the circuit mechanisms of MOR-expressing cells are less known because of a lack of genetic tools to selectively manipulate them. We introduce a CRISPR-based Oprm1-Cre knock-in transgenic rat that provides cell type-specific genetic access to brain MOR-expressing cells. After performing anatomical and behavioral validation experiments, we used the Oprm1-Cre knock-in rats to show that lesioning NAc MOR-expressing cells had different effects on heroin self-administration in males and females. The new Oprm1-Cre rats can be used to study the role of brain MOR-expressing cells in animal models of opioid addiction, pain-related behaviors, and other opioid-mediated functions.


Asunto(s)
Dependencia de Heroína , Heroína , Ratas , Masculino , Femenino , Animales , Heroína/farmacología , Analgésicos Opioides/farmacología , Núcleo Accumbens , Receptores Opioides/metabolismo , Ratas Transgénicas , Receptores Opioides mu/genética , Receptores Opioides mu/metabolismo , Dolor/metabolismo
2.
J Neurochem ; 2024 Mar 15.
Artículo en Inglés | MEDLINE | ID: mdl-38491746

RESUMEN

Dysregulation of synaptic glutamate levels can lead to excitotoxicity such as that observed in stroke, traumatic brain injury, and epilepsy. The role of increased intracellular calcium (Ca2+ ) in the development of excitotoxicity is well established. However, less is known regarding the impact of glutamate on endoplasmic reticulum (ER)-Ca2+ -mediated processes such as proteostasis. To investigate this, we expressed a secreted ER Ca2+ modulated protein (SERCaMP) in primary cortical neurons to monitor exodosis, a phenomenon whereby ER calcium depletion causes the secretion of ER-resident proteins that perform essential functions to the ER and the cell. Activation of glutamatergic receptors (GluRs) led to an increase in SERCaMP secretion indicating that normally ER-resident proteins are being secreted in a manner consistent with ER Ca2+ depletion. Antagonism of ER Ca2+ channels attenuated the effects of glutamate and GluR agonists on SERCaMP release. We also demonstrate that endogenous proteins containing an ER retention/retrieval sequence (ERS) are secreted in response to GluR activation supporting that neuronal activation by glutamate promotes ER exodosis. Ectopic expression of KDEL receptors attenuated the secretion of ERS-containing proteins caused by GluR agonists. Taken together, our data indicate that excessive GluR activation causes disruption of neuronal proteostasis by triggering the secretion of ER-resident proteins through ER Ca2+ depletion and describes a new facet of excitotoxicity.

3.
Proc Natl Acad Sci U S A ; 117(14): 8126-8134, 2020 04 07.
Artículo en Inglés | MEDLINE | ID: mdl-32205443

RESUMEN

We recently reported that social choice-induced voluntary abstinence prevents incubation of methamphetamine craving in rats. This inhibitory effect was associated with activation of protein kinase-Cδ (PKCδ)-expressing neurons in central amygdala lateral division (CeL). In contrast, incubation of craving after forced abstinence was associated with activation of CeL-expressing somatostatin (SOM) neurons. Here we determined the causal role of CeL PKCδ and SOM in incubation using short-hairpin RNAs against PKCδ or SOM that we developed and validated. We injected two groups with shPKCδ or shCtrlPKCδ into CeL and trained them to lever press for social interaction (6 d) and then for methamphetamine infusions (12 d). We injected two other groups with shSOM or shCtrlSOM into CeL and trained them to lever press for methamphetamine infusions (12 d). We then assessed relapse to methamphetamine seeking after 1 and 15 abstinence days. Between tests, the rats underwent either social choice-induced abstinence (shPKCδ groups) or homecage forced abstinence (shSOM groups). After test day 15, we assessed PKCδ and SOM, Fos, and double-labeled expression in CeL and central amygdala medial division (CeM). shPKCδ CeL injections decreased Fos in CeL PKCδ-expressing neurons, increased Fos in CeM output neurons, and reversed the inhibitory effect of social choice-induced abstinence on incubated drug seeking on day 15. In contrast, shSOM CeL injections decreased Fos in CeL SOM-expressing neurons, decreased Fos in CeM output neurons, and decreased incubated drug seeking after 15 forced abstinence days. Our results identify dissociable central amygdala mechanisms of abstinence-dependent expression or inhibition of incubation of craving.


Asunto(s)
Núcleo Amigdalino Central/fisiología , Ansia/fisiología , Comportamiento de Búsqueda de Drogas/fisiología , Relaciones Interpersonales , Animales , Conducta Animal , Modelos Animales de Enfermedad , Humanos , Masculino , Metanfetamina/administración & dosificación , Metanfetamina/efectos adversos , Neuronas/metabolismo , Proteína Quinasa C-delta/genética , Proteína Quinasa C-delta/metabolismo , ARN Interferente Pequeño/administración & dosificación , Ratas , Ratas Sprague-Dawley , Autoadministración , Somatostatina/genética , Somatostatina/metabolismo
4.
J Neurosci ; 40(44): 8463-8477, 2020 10 28.
Artículo en Inglés | MEDLINE | ID: mdl-33051346

RESUMEN

Relapse to drug use can be initiated by drug-associated cues. The intensity of cue-induced drug seeking in rodent models correlates with the induction of transient synaptic potentiation (t-SP) at glutamatergic synapses in the nucleus accumbens core (NAcore). Matrix metalloproteinases (MMPs) are inducible endopeptidases that degrade extracellular matrix (ECM) proteins, and reveal tripeptide Arginine-Glycine-Aspartate (RGD) domains that bind and signal through integrins. Integrins are heterodimeric receptors composed of αß subunits, and a primary signaling kinase is focal adhesion kinase (FAK). We previously showed that MMP activation is necessary for and potentiates cued reinstatement of cocaine seeking, and MMP-induced catalysis stimulates ß3-integrins to induce t-SP. Here, we determined whether ß3-integrin signaling through FAK and cofilin (actin depolymerization factor) is necessary to promote synaptic growth during t-SP. Using a small molecule inhibitor to prevent FAK activation, we blocked cued-induced cocaine reinstatement and increased spine head diameter (dh). Immunohistochemistry on NAcore labeled spines with ChR2-EYFP virus, showed increased immunoreactivity of phosphorylation of FAK (p-FAK) and p-cofilin in dendrites of reinstated animals compared with extinguished and yoked saline, and the p-FAK and cofilin depended on ß3-integrin signaling. Next, male and female transgenic rats were used to selectively label D1 or D2 neurons with ChR2-mCherry. We found that p-FAK was increased during drug seeking in both D1 and D2-medium spiny neurons (MSNs), but increased p-cofilin was observed only in D1-MSNs. These data indicate that ß3-integrin, FAK and cofilin constitute a signaling pathway downstream of MMP activation that is involved in promoting the transient synaptic enlargement in D1-MSNs induced during reinstated cocaine by drug-paired cues.SIGNIFICANCE STATEMENT Drug-associated cues precipitate relapse, which is correlated with transient synaptic enlargement in the accumbens core. We showed that cocaine cue-induced synaptic enlargement depends on matrix metalloprotease signaling in the extracellular matrix (ECM) through ß3-integrin to activate focal adhesion kinase (FAK) and phosphorylate the actin binding protein cofilin. The nucleus accumbens core (NAcore) contains two predominate neuronal subtypes selectively expressing either D1-dopamine or D2-dopamine receptors. We used transgenic rats to study each cell type and found that cue-induced signaling through cofilin phosphorylation occurred only in D1-expressing neurons. Thus, cocaine-paired cues initiate cocaine reinstatement and synaptic enlargement through a signaling cascade selectively in D1-expressing neurons requiring ECM stimulation of ß3-integrin-mediated phosphorylation of FAK (p-FAK) and cofilin.


Asunto(s)
Factores Despolimerizantes de la Actina/metabolismo , Trastornos Relacionados con Cocaína/fisiopatología , Neuronas Dopaminérgicas/metabolismo , Quinasa 1 de Adhesión Focal/metabolismo , Integrina beta3/metabolismo , Receptores de Dopamina D1/metabolismo , Animales , Trastornos Relacionados con Cocaína/psicología , Señales (Psicología) , Espinas Dendríticas/efectos de los fármacos , Espinas Dendríticas/ultraestructura , Comportamiento de Búsqueda de Drogas , Activación Enzimática , Humanos , Masculino , Ratas , Ratas Long-Evans , Ratas Sprague-Dawley , Ratas Transgénicas , Recurrencia , Sinapsis
5.
J Neurosci ; 39(11): 2041-2051, 2019 03 13.
Artículo en Inglés | MEDLINE | ID: mdl-30622165

RESUMEN

Outputs from the nucleus accumbens (NAc) include projections to the ventral pallidum and the ventral tegmental area and subtantia nigra in the ventral mesencephalon. The medium spiny neurons (MSN) that give rise to these pathways are GABAergic and consist of two populations of equal number that are segregated by differentially expressed proteins, including D1- and D2-dopamine receptors. Afferents to the ventral pallidum arise from both D1- and D2-MSNs, whereas the ventral mesencephalon is selectively innervated by D1-MSN. To determine the extent of collateralization of D1-MSN to these axon terminal fields we used retrograde labeling in transgenic mice expressing tdTomato selectively in D1-MSN, and found that a large majority of D1-MSN in either the shell or core subcompartments of the accumbens collateralized to both output structures. Approximately 70% of D1-MSNs projecting to the ventral pallidum collateralized to the ventral mesencephalon, whereas >90% of mesencephalic D1-MSN afferents collateralized to the ventral pallidum. In contrast, <10% of dorsal striatal D1-MSNs collateralized to both the globus pallidus and ventral mesencephalon. D1-MSN activation is required for conditioned cues to induce cocaine seeking. To determine which D1-MSN projection mediates cued cocaine seeking, we selectively transfected D1-MSNs in transgenic rats with an inhibitory Gi-coupled DREADD. Activation of the transfected Gi-DREADD with clozapine-N-oxide administered into the ventral pallidum, but not into the ventral mesencephalon, blocked cue-induced cocaine seeking. These data show that, although accumbens D1-MSNs largely collateralize to both the ventral pallidum and ventral mesencephalon, only D1-MSN innervation of the ventral pallidum is necessary for cue-induced cocaine seeking.SIGNIFICANCE STATEMENT Activity in D1 dopamine receptor-expressing neurons in the NAc is required for rodents to respond to cocaine-conditioned cues and relapse to drug seeking behaviors. The D1-expressing neurons project to both the ventral pallidum and ventral mesencephalon, and we found that a majority of the neurons that innervate the ventral pallidum also collateralize to the ventral mesencephalon. However, despite innervating both structures, only D1 innervation of the ventral pallidum mediates cue-induced cocaine seeking.


Asunto(s)
Prosencéfalo Basal/fisiología , Cocaína/administración & dosificación , Comportamiento de Búsqueda de Drogas/fisiología , Neuronas/fisiología , Núcleo Accumbens/fisiología , Receptores de Dopamina D1/fisiología , Animales , Prosencéfalo Basal/citología , Condicionamiento Clásico , Femenino , Masculino , Ratones Endogámicos C57BL , Ratones Transgénicos , Vías Nerviosas/citología , Vías Nerviosas/fisiología , Neuronas/citología , Núcleo Accumbens/citología , Ratas Long-Evans , Ratas Transgénicas
6.
Mol Ther ; 27(1): 151-163, 2019 01 02.
Artículo en Inglés | MEDLINE | ID: mdl-30389355

RESUMEN

Investigators have utilized the CRISPR/Cas9 gene-editing system to specifically target well-conserved regions of HIV, leading to decreased infectivity and pathogenesis in vitro and ex vivo. We utilized a specialized extracellular vesicle termed a "gesicle" to efficiently, yet transiently, deliver Cas9 in a ribonucleoprotein form targeting the HIV long terminal repeat (LTR). Gesicles are produced through expression of vesicular stomatitis virus glycoprotein and package protein as their cargo, thus bypassing the need for transgene delivery, and allowing finer control of Cas9 expression. Using both NanoSight particle and western blot analysis, we verified production of Cas9-containing gesicles by HEK293FT cells. Application of gesicles to CHME-5 microglia resulted in rapid but transient transfer of Cas9 by western blot, which is present at 1 hr, but is undetectable by 24 hr post-treatment. Gesicle delivery of Cas9 protein preloaded with guide RNA targeting the HIV LTR to HIV-NanoLuc CHME-5 cells generated mutations within the LTR region and copy number loss. Finally, we demonstrated that this treatment resulted in reduced proviral activity under basal conditions and after stimulation with pro-inflammatory factors lipopolysaccharide (LPS) or tumor necrosis factor alpha (TNF-α). These data suggest that gesicles are a viable alternative approach to deliver CRISPR/Cas9 technology.


Asunto(s)
Proteína 9 Asociada a CRISPR/metabolismo , Sistemas CRISPR-Cas/fisiología , Edición Génica/métodos , Ribonucleoproteínas Nucleares Heterogéneas/metabolismo , Proteína 9 Asociada a CRISPR/genética , Sistemas CRISPR-Cas/efectos de los fármacos , Sistemas CRISPR-Cas/genética , Células HEK293 , Duplicado del Terminal Largo de VIH/genética , Duplicado del Terminal Largo de VIH/fisiología , Ribonucleoproteínas Nucleares Heterogéneas/genética , Humanos , Lipopolisacáridos/farmacología , Mutación/genética , Provirus/genética , Provirus/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Vesiculovirus/genética , Vesiculovirus/metabolismo
7.
Eur J Neurosci ; 50(5): 2801-2813, 2019 09.
Artículo en Inglés | MEDLINE | ID: mdl-31063250

RESUMEN

Designer receptors exclusively activated by designer drugs (DREADDs) are extensively used to modulate neuronal activity in rodents, but their use in primates remains limited. An essential need that remains is the demonstration that DREADDs are efficiently expressed on the plasma membrane of primate neurons. To address this issue, electron microscopy immunogold was used to determine the subcellular localization of the AAV vector-induced DREADDs hM4Di and hM3Dq fused to different tags in various brain areas of rhesus monkeys and mice. When hM4Di was fused to mCherry, the immunogold labelling was mostly confined to the intracellular space, and poorly expressed at the plasma membrane in monkey dendrites. In contrast, the hM4Di-mCherry labelling was mostly localized to the dendritic plasma membrane in mouse neurons, suggesting species differences in the plasma membrane expression of these exogenous proteins. The lack of hM4Di plasma membrane expression may limit the functional effects of systemic administration of DREADD-actuators in monkey neurons. Removing the mCherry and fusing of hM4Di with the haemagglutinin (HA) tag resulted in strong neuronal plasma membrane immunogold labelling in both monkeys and mice neurons. Finally, hM3Dq-mCherry was expressed mostly at the plasma membrane in monkey neurons, indicating that the fusion of mCherry with hM3Dq does not hamper membrane incorporation of this specific DREADD. Our results suggest that the pattern of ultrastructural expression of DREADDs in monkey neurons depends on the DREADD/tag combination. Therefore, a preliminary characterization of plasma membrane expression of specific DREADD/tag combinations is recommended when using chemogenetic approaches in primates.


Asunto(s)
Encéfalo/metabolismo , Membrana Celular/metabolismo , Neuronas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animales , Dendritas/metabolismo , Femenino , Macaca mulatta , Masculino , Ratones
8.
Biomarkers ; 23(8): 756-765, 2018 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-30095301

RESUMEN

CONTEXT: Endoplasmic reticulum (ER) calcium depletion is associated with diverse diseases, including cardiac, hepatic, and neurologic diseases. OBJECTIVE: The aim of the present study was to identify and characterize an endogenous protein that could be used to monitor ER calcium depletion comparably to a previously described exogenous reporter protein. MATERIALS AND METHODS: The use of a selective esterase-fluorescein diester pair allowed for carboxylesterase activity in extracellular fluid to be measured using a fluorescent readout. Cell culture media from three different cell lines, rat plasma, and human serum all possess quantifiable amounts of esterase activity. RESULTS: Fluorescence produced by the interaction of carboxylesterases with a fluorescein diester substrate tracks with pharmacological and physiological inducers of ER calcium depletion. The fluorescence measured for in vitro and in vivo samples were consistent with ER calcium depletion being the trigger for increased esterase activity. DISCUSSION: Decreased luminal ER calcium causes ER resident esterases to be released from the cell, and, when assessed concurrently with other disease biomarkers, these esterases may provide insight into the role of ER calcium homeostasis in human diseases. CONCLUSION: Our results indicate that carboxylesterases are putative markers of ER calcium dysfunction.


Asunto(s)
Calcio/deficiencia , Hidrolasas de Éster Carboxílico/análisis , Medios de Cultivo Condicionados/química , Retículo Endoplásmico/química , Animales , Línea Celular , Esterasas/análisis , Colorantes Fluorescentes , Fluorometría/métodos , Humanos , Ratas
9.
Neurobiol Dis ; 97(Pt B): 189-200, 2017 01.
Artículo en Inglés | MEDLINE | ID: mdl-27189755

RESUMEN

Drug addiction is a chronic brain disease and drugs of abuse cause long lasting neuroadaptations. Addiction is characterized by the loss of control over drug use despite harmful consequences, and high rates of relapse even after long periods of abstinence. Neurotrophic factors (NTFs) are well known for their actions on neuronal survival in the peripheral nervous system. Moreover, NTFs have been shown to be involved in synaptic plasticity in the brain. Brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF) are two of the most studied NTFs and both of them have been reported to increase craving when administered into the mesocorticolimbic dopaminergic system after drug self-administration. Here we review recent data on BDNF and GDNF functions in addiction-related behavior and discuss them in relation to previous findings. Finally, we give an insight into how new technologies could aid in further elucidating the role of these factors in drug addiction.


Asunto(s)
Factores de Crecimiento Nervioso/metabolismo , Trastornos Relacionados con Sustancias/metabolismo , Animales , Humanos
10.
Transgenic Res ; 26(4): 477-489, 2017 08.
Artículo en Inglés | MEDLINE | ID: mdl-28608322

RESUMEN

Long Evans rat strains are applied as research models in a broad spectrum of biomedical fields (>15,800 citations, NCBI PubMed). Here, we report an approach to genetically modify the Long Evans rat germline in donor spermatogonial stem cells. Long Evans rat spermatogonial lines were derived from freshly isolated laminin-binding spermatogonia. Laminin-binding spermatogonia were cultured over multiple passages on fibroblast feeder layers in serum-free culture medium containing GDNF and FGF2. Long Evans rat spermatogonial lines were genetically modified by transposon transduction to express a germline, tdTomato reporter gene. Donor rat spermatogonial lines robustly regenerated spermatogenesis after transplantation into testes of busulfan-treated, allogenic, Long Evans rats. Donor-derived spermatogenesis largely restored testis size in the chemically sterilized, recipient Long Evans rats. Recipient Long Evans rats stably transmitted the tdTomato germline marker to subsequent generations. Overall, Long Evans rat spermatogonial lines provided effective donor germline vectors for genetically modifying Long Evans rats.


Asunto(s)
Ratas Transgénicas/genética , Espermatogénesis/genética , Células Madre/citología , Testículo/crecimiento & desarrollo , Animales , Elementos Transponibles de ADN/genética , Genes Reporteros/genética , Células Germinativas/crecimiento & desarrollo , Laminina/genética , Solanum lycopersicum/genética , Masculino , Ratas , Ratas Long-Evans/genética , Ratas Transgénicas/crecimiento & desarrollo , Espermatogonias/crecimiento & desarrollo , Testículo/citología
11.
Proc Natl Acad Sci U S A ; 111(32): 11876-81, 2014 Aug 12.
Artículo en Inglés | MEDLINE | ID: mdl-25071172

RESUMEN

Prolyl endopeptidase (PREP) has been implicated in neuronal functions. Here we report that hypothalamic PREP is predominantly expressed in the ventromedial nucleus (VMH), where it regulates glucose-induced neuronal activation. PREP knockdown mice (Prep(gt/gt)) exhibited glucose intolerance, decreased fasting insulin, increased fasting glucagon levels, and reduced glucose-induced insulin secretion compared with wild-type controls. Consistent with this, central infusion of a specific PREP inhibitor, S17092, impaired glucose tolerance and decreased insulin levels in wild-type mice. Arguing further for a central mode of action of PREP, isolated pancreatic islets showed no difference in glucose-induced insulin release between Prep(gt/gt) and wild-type mice. Furthermore, hyperinsulinemic euglycemic clamp studies showed no difference between Prep(gt/gt) and wild-type control mice. Central PREP regulation of insulin and glucagon secretion appears to be mediated by the autonomic nervous system because Prep(gt/gt) mice have elevated sympathetic outflow and norepinephrine levels in the pancreas, and propranolol treatment reversed glucose intolerance in these mice. Finally, re-expression of PREP by bilateral VMH injection of adeno-associated virus-PREP reversed the glucose-intolerant phenotype of the Prep(gt/gt) mice. Taken together, our results unmask a previously unknown player in central regulation of glucose metabolism and pancreatic function.


Asunto(s)
Glucagón/metabolismo , Hipotálamo/enzimología , Insulina/metabolismo , Serina Endopeptidasas/metabolismo , Animales , Glucemia/metabolismo , Expresión Génica , Técnicas de Silenciamiento del Gen , Técnica de Clampeo de la Glucosa , Intolerancia a la Glucosa/enzimología , Intolerancia a la Glucosa/etiología , Hipotálamo/fisiología , Indoles/farmacología , Secreción de Insulina , Canales Iónicos/genética , Masculino , Ratones , Ratones Transgénicos , Proteínas Mitocondriales/genética , Páncreas/metabolismo , Fosforilación , Prolil Oligopeptidasas , Receptor de Insulina/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Serina Endopeptidasas/deficiencia , Serina Endopeptidasas/genética , Inhibidores de Serina Proteinasa/farmacología , Tiazolidinas/farmacología , Proteína Desacopladora 1 , Núcleo Hipotalámico Ventromedial/enzimología , Núcleo Hipotalámico Ventromedial/fisiología
12.
Addict Biol ; 21(2): 255-66, 2016 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-25377775

RESUMEN

In this study, methamphetamine (Meth)- and glutamate (Glu)-mediated intracellular Ca(++) (Ca(++) i) signals were examined in real time in primary cortical neurons overexpressing an intracellular Ca(++) probe, GCaMP5, by adeno-associated viral (AAV) serotype 1. Binding of Ca(++) to GCaMP increased green fluorescence intensity in cells. Both Meth and Glu induced a rapid increase in Ca(++) i, which was blocked by MK801, suggesting that Meth enhanced Ca(++) i through Glu receptor in neurons. The Meth-mediated Ca(++) signal was also blocked by Mg(++) , low Ca(++) or the L-type Ca(++) channel inhibitor nifedipine. The ryanodine receptor inhibitor dantrolene did not alter the initial Ca(++) influx but partially reduced the peak of Ca(++) i. These data suggest that Meth enhanced Ca(++) influx through membrane Ca(++) channels, which then triggered the release of Ca(++) from the endoplasmic reticulum in the cytosol. AAV-GCaMP5 was also injected to the parietal cortex of adult rats. Administration of Meth enhanced fluorescence in the ipsilateral cortex. Using immunohistochemistry, Meth-induced green fluorescence was found in the NeuN-containing cells in the cortex, suggesting that Meth increased Ca(++) in neurons in vivo. In conclusion, we have used in vitro and in vivo techniques to demonstrate a rapid increase of Ca(++) i by Meth in cortical neurons through overexpression of GCaMP5. As Meth induces behavioral responses and neurotoxicity through Ca(++) i, modulation of Ca(++) i may be useful to reduce Meth-related reactions.


Asunto(s)
Calcio/metabolismo , Dopaminérgicos/farmacología , Ácido Glutámico/farmacología , Metanfetamina/farmacocinética , Neuronas/metabolismo , Análisis de Varianza , Animales , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio/efectos de los fármacos , Proteínas de Unión al Calcio/metabolismo , Calmodulina/metabolismo , Células Cultivadas , Corteza Cerebral/efectos de los fármacos , Dantroleno/farmacología , Maleato de Dizocilpina/farmacología , Antagonistas de Aminoácidos Excitadores/farmacología , Femenino , Proteínas Fluorescentes Verdes/metabolismo , Indicadores y Reactivos/farmacología , Masculino , Relajantes Musculares Centrales/farmacología , Nifedipino/farmacología , Ratas Sprague-Dawley , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal/efectos de los fármacos
13.
J Biol Chem ; 288(6): 4209-25, 2013 Feb 08.
Artículo en Inglés | MEDLINE | ID: mdl-23255601

RESUMEN

Mesencephalic astrocyte-derived neurotrophic factor (MANF) is an endoplasmic reticulum (ER) stress-responsive protein with neuroprotective effects in animal models of neurodegeneration, but the underlying mechanism is not understood. We constructed a set of lentiviral vectors that contain or lack the highly conserved final four amino acids of MANF ("RTDL"), which resemble the canonical ER retention signal ("KDEL"), to study MANF regulation in neuroblastoma cells and rat primary cortical neurons. The RTDL sequence was required for both ER retention and secretory response to ER stress. Overexpression of KDEL receptor paralogs (KDELRs) differentially reduced MANF secretion but had no effect on MANF lacking RTDL. MANF binding to the plasma membrane also required the RTDL sequence and was inhibited with a peptide known to interact with KDELRs, suggesting MANF binds KDELRs at the surface. We detected surface localization of FLAG-tagged KDELRs, with levels increasing following ER stress. Our study provides new insight into the regulation of MANF trafficking and has implications for other secreted proteins containing a KDEL-like retention signal.


Asunto(s)
Membrana Celular/metabolismo , Estrés del Retículo Endoplásmico , Retículo Endoplásmico/metabolismo , Factores de Crecimiento Nervioso/metabolismo , Receptores de Péptidos/metabolismo , Animales , Línea Celular Tumoral , Membrana Celular/genética , Retículo Endoplásmico/genética , Humanos , Factores de Crecimiento Nervioso/genética , Enfermedades Neurodegenerativas/genética , Enfermedades Neurodegenerativas/metabolismo , Unión Proteica , Señales de Clasificación de Proteína/genética , Transporte de Proteínas/genética , Ratas , Receptores de Péptidos/genética
14.
Neurobiol Dis ; 68: 1-15, 2014 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-24746855

RESUMEN

The misfolding and aggregation of α-synuclein (aSyn) eventually lead to an accumulation of toxic forms that disturb normal neuronal function and result in cell death. aSyn rich inclusions are seen in Parkinson's disease, dementia with Lewy bodies and other synucleinopathies. Prolyl oligopeptidase (PREP) can accelerate the aggregation process of aSyn and the inhibition of PREP leads to a decreased amount of aggregated aSyn in cell models and in aSyn transgenic mice. In this study, we investigated the effect of 5- and 28-day PREP inhibitor (KYP-2047) treatments on a mouse strain carrying a point mutation in the aSyn coding gene. Following PREP inhibition, we found a decrease in high molecular-weight oligomeric aSyn and a concomitant increase in the amount of the autophagosome marker, LC3BII, suggesting enhanced macroautophagy (autophagy) and aSyn clearance by KYP-2047. Moreover, 28-day treatment with KYP-2047 caused significant increases in striatal dopamine levels. In cell culture, overexpression of PREP reduced the autophagy. Furthermore, the inhibition of PREP normalized the changes on autophagy markers (LC3BII and p62) caused by an autophagy inhibition or aSyn overexpression, and induced the expression of beclin 1, a positive regulator of autophagy. Taken together, our results suggest that PREP inhibition accelerates the clearance of protein aggregates via increased autophagy and thus normalizes the cell functions in vivo and in vitro. Therefore, PREP inhibition may have future potential in the treatment of synucleinopathies.


Asunto(s)
Autofagia/efectos de los fármacos , Encefalopatías/genética , Prolina/análogos & derivados , Serina Endopeptidasas/metabolismo , Inhibidores de Serina Proteinasa/uso terapéutico , alfa-Sinucleína/metabolismo , Alanina/genética , Animales , Autofagia/genética , Encéfalo/metabolismo , Encéfalo/patología , Encefalopatías/tratamiento farmacológico , Línea Celular Transformada , Modelos Animales de Enfermedad , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Actividad Motora/efectos de los fármacos , Actividad Motora/genética , Mutación/genética , Prolina/genética , Prolina/uso terapéutico , Prolil Oligopeptidasas , Factores de Tiempo , alfa-Sinucleína/genética
15.
Sci Rep ; 14(1): 16487, 2024 07 17.
Artículo en Inglés | MEDLINE | ID: mdl-39019902

RESUMEN

Cerebral dopamine neurotrophic factor (CDNF) and its close structural relative, mesencephalic astrocyte-derived neurotrophic factor (MANF), are proteins with neurotrophic properties. CDNF protects and restores the function of dopamine (DA) neurons in rodent and non-human primate (NHP) toxin models of Parkinson's disease (PD) and therefore shows promise as a drug candidate for disease-modifying treatment of PD. Moreover, CDNF was found to be safe and to have some therapeutic effects on PD patients in phase 1/2 clinical trials. However, the mechanism underlying the neurotrophic activity of CDNF is unknown. In this study, we delivered human CDNF (hCDNF) to the brain using an adeno-associated viral (AAV) vector and demonstrated the neurotrophic effect of AAV-hCDNF in an acute 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of PD. AAV-hCDNF resulted in the expression of hCDNF in the striatum (STR) and substantia nigra (SN), and no toxic effects on the nigrostriatal pathway were observed. Intrastriatal injection of AAV-hCDNF reduced motor impairment and partially alleviated gait dysfunction in the acute MPTP mouse model. In addition, gene therapy with AAV-hCDNF had significant neuroprotective effects on the nigrostriatal pathway and decreased the levels of interleukin 1beta (IL-1ß) and complement 3 (C3) in glial cells in the acute MPTP mouse model. Moreover, AAV-hCDNF reduced C/EBP homologous protein (CHOP) and glucose regulatory protein 78 (GRP78) expression in astroglia. These results suggest that the neuroprotective effects of CDNF may be mediated at least in part through the regulation of neuroinflammation and the UPR pathway in a mouse MPTP model of PD in vivo.


Asunto(s)
Dependovirus , Modelos Animales de Enfermedad , Neuronas Dopaminérgicas , Chaperón BiP del Retículo Endoplásmico , Estrés del Retículo Endoplásmico , Vectores Genéticos , Factores de Crecimiento Nervioso , Animales , Neuronas Dopaminérgicas/metabolismo , Dependovirus/genética , Ratones , Humanos , Factores de Crecimiento Nervioso/metabolismo , Factores de Crecimiento Nervioso/genética , Vectores Genéticos/administración & dosificación , Vectores Genéticos/genética , Técnicas de Transferencia de Gen , Masculino , Enfermedad de Parkinson/terapia , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/genética , 1-Metil-4-fenil-1,2,3,6-Tetrahidropiridina , Inflamación/metabolismo , Terapia Genética/métodos , Ratones Endogámicos C57BL , Cuerpo Estriado/metabolismo , Intoxicación por MPTP/terapia , Intoxicación por MPTP/metabolismo , Sustancia Negra/metabolismo
16.
Res Sq ; 2024 Jan 15.
Artículo en Inglés | MEDLINE | ID: mdl-38313275

RESUMEN

Classical methods of investigating protein-protein interactions (PPIs) are generally performed in non-living systems, yet in recent years new technologies utilizing proximity labeling (PL) have given researchers the tools to explore proximal PPIs in living systems. PL has distinct advantages over traditional protein interactome studies, such as the ability to identify weak and transient interactions in vitro and in vivo. Most PL studies are performed on targets within the cell or on the cell membrane. We have adapted the original PL method to investigate PPIs within the extracellular compartment, using both BioID2 and TurboID, that we term extracellular PL (ePL). To demonstrate the utility of this modified technique, we investigate the interactome of the widely expressed matrisome protein tissue inhibitor of metalloproteinases 2 (TIMP2). Tissue inhibitors of metalloproteinases (TIMPs) are a family of multi-functional proteins that were initially defined by their ability to inhibit the enzymatic activity of metalloproteinases (MPs), the major mediators of extracellular matrix (ECM) breakdown and turnover. TIMP2 exhibits a broad expression profile and is often abundant in both normal and diseased tissues. Understanding the functional transformation of matrisome regulators, like TIMP2, during the evolution of tissue microenvironments associated with disease progression is essential for the development of ECM-targeted therapeutics. Using carboxyl- and amino-terminal fusion proteins of TIMP2 with BioID2 and TurboID, we describe the TIMP2 proximal interactome. We also illustrate how the TIMP2 interactome changes in the presence of different stimuli, in different cell types, in unique culture conditions (2D vs 3D), and with different reaction kinetics (BioID2 vs. TurboID); demonstrating the power of this technique versus classical PPI methods. We propose that the screening of matrisome targets in disease models using ePL will reveal new therapeutic targets for further comprehensive studies.

17.
J Cell Sci ; 124(Pt 17): 2903-13, 2011 Sep 01.
Artículo en Inglés | MEDLINE | ID: mdl-21878498

RESUMEN

Mutations in the Caenorhabditis elegans separase gene, sep-1, are embryonic lethal. Newly fertilized mutant embryos have defects in polar body extrusion, fail to undergo cortical granule exocytosis, and subsequently fail to complete cytokinesis. Chromosome nondisjunction during the meiotic divisions is readily apparent after depletion of sep-1 by RNAi treatment, but much less so in hypomorphic mutant embryos. To identify factors that influence the activity of separase in cortical granule exocytosis and cytokinesis, we carried out a genetic suppressor screen. A mutation in the protein phosphatase 5 (pph-5) gene was identified as an extragenic suppressor of sep-1. This mutation suppressed the phenotypes of hypomorphic separase mutants but not RNAi depleted animals. Depletion of pph-5 caused no phenotypes on its own, but was effective in restoring localization of mutant separase to vesicles and suppressing cortical granule exocytosis and cytokinesis phenotypes. The identification of PPH-5 as a suppressor of separase suggests that a new phospho-regulatory pathway plays an important role in regulating anaphase functions of separase.


Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Proteínas de Ciclo Celular/metabolismo , Endopeptidasas/metabolismo , Proteínas Nucleares/metabolismo , Fosfoproteínas Fosfatasas/metabolismo , Alelos , Animales , Caenorhabditis elegans/enzimología , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Proteínas de Ciclo Celular/genética , Citocinesis/genética , Gránulos Citoplasmáticos/genética , Gránulos Citoplasmáticos/metabolismo , Endopeptidasas/genética , Exocitosis/fisiología , Mutación , Proteínas Nucleares/biosíntesis , Proteínas Nucleares/deficiencia , Proteínas Nucleares/genética , Fosfoproteínas Fosfatasas/biosíntesis , Fosfoproteínas Fosfatasas/deficiencia , Fosfoproteínas Fosfatasas/genética , Separasa
18.
bioRxiv ; 2023 Oct 09.
Artículo en Inglés | MEDLINE | ID: mdl-36798245

RESUMEN

Response to threatening environmental stimuli requires detection and encoding of important environmental features that dictate threat. Aversive events are highly salient which promotes associative learning about stimuli that signal this threat. The nucleus accumbens is uniquely positioned to process this salient, aversive information and promote motivated output, through plasticity on the major projection neurons in the brain area. We uncovered a nucleus accumbens core local circuit whereby excitatory plasticity facilitates learning and recall of discrete aversive cues. We demonstrate that putative nucleus accumbens substance P release and long-term excitatory plasticity on dopamine 2 receptor expressing projection neurons is required for cue-dependent fear learning. Additionally, we found fear learning and recall were dependent on distinct projection-neuron subtypes. Our work demonstrates a critical role for Nucleus Accumbens substance P in cue-dependent aversive learning.

20.
Sci Transl Med ; 15(706): eadd1014, 2023 07 26.
Artículo en Inglés | MEDLINE | ID: mdl-37494470

RESUMEN

Optogenetics is a widely used technology with potential for translational research. A critical component of such applications is the ability to track the location of the transduced opsin in vivo. To address this problem, we engineered an excitatory opsin, ChRERα (hChR2(134R)-V5-ERα-LBD), that could be visualized using positron emission tomography (PET) imaging in a noninvasive, longitudinal, and quantitative manner. ChRERα consists of the prototypical excitatory opsin channelrhodopsin-2 (ChR2) and the ligand-binding domain (LBD) of the human estrogen receptor α (ERα). ChRERα showed conserved ChR2 functionality and high affinity for [18F]16α-fluoroestradiol (FES), an FDA-approved PET radiopharmaceutical. Experiments in rats demonstrated that adeno-associated virus (AAV)-mediated expression of ChRERα enables neural circuit manipulation in vivo and that ChRERα expression could be monitored using FES-PET imaging. In vivo experiments in nonhuman primates (NHPs) confirmed that ChRERα expression could be monitored at the site of AAV injection in the primary motor cortex and in long-range neuronal terminals for up to 80 weeks. The anatomical connectivity map of the primary motor cortex identified by FES-PET imaging of ChRERα expression overlapped with a functional connectivity map identified using resting state fMRI in a separate cohort of NHPs. Overall, our results demonstrate that ChRERα expression can be mapped longitudinally in the mammalian brain using FES-PET imaging and can be used for neural circuit modulation in vivo.


Asunto(s)
Neoplasias de la Mama , Receptor alfa de Estrógeno , Ratas , Humanos , Animales , Femenino , Receptor alfa de Estrógeno/metabolismo , Opsinas/metabolismo , Tomografía de Emisión de Positrones , Encéfalo/diagnóstico por imagen , Encéfalo/metabolismo , Primates , Estradiol/metabolismo , Neoplasias de la Mama/metabolismo , Mamíferos/metabolismo
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