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1.
iScience ; 27(4): 109512, 2024 Apr 19.
Artículo en Inglés | MEDLINE | ID: mdl-38715938

RESUMEN

LMTK3 is a brain-specific transmembrane serine/threonine protein kinase that acts as a scaffold for protein phosphatase-1 (PP1). Although LMKT3 has been identified as a risk factor for autism and epilepsy, its physiological significance is unknown. Here, we demonstrate that LMTK3 copurifies and binds to KCC2, a neuron-specific K+/Cl- transporter. KCC2 activity is essential for Cl--mediated hyperpolarizing GABAAR receptor currents, the unitary events that underpin fast synaptic inhibition. LMTK3 acts to promote the association of KCC2 with PP1 to promote the dephosphorylation of S940 within its C-terminal cytoplasmic domain, a process the diminishes KCC2 activity. Accordingly, acute inhibition of LMTK3 increases KCC2 activity dependent upon S940 and increases neuronal Cl- extrusion. Consistent with this, LMTK3 inhibition reduced intrinsic neuronal excitability and the severity of seizure-like events in vitro. Thus, LMTK3 may have profound effects on neuronal excitability as an endogenous modulator of KCC2 activity.

2.
Commun Biol ; 6(1): 11, 2023 01 05.
Artículo en Inglés | MEDLINE | ID: mdl-36604600

RESUMEN

Fast synaptic inhibition is dependent on targeting specific GABAAR subtypes to dendritic and axon initial segment (AIS) synapses. Synaptic GABAARs are typically assembled from α1-3, ß and γ subunits. Here, we isolate distinct GABAARs from the brain and interrogate their composition using quantitative proteomics. We show that α2-containing receptors co-assemble with α1 subunits, whereas α1 receptors can form GABAARs with α1 as the sole α subunit. We demonstrate that α1 and α2 subunit-containing receptors co-purify with distinct spectrin isoforms; cytoskeletal proteins that link transmembrane proteins to the cytoskeleton. ß2-spectrin was preferentially associated with α1-containing GABAARs at dendritic synapses, while ß4-spectrin was associated with α2-containing GABAARs at AIS synapses. Ablating ß2-spectrin expression reduced dendritic and AIS synapses containing α1 but increased the number of synapses containing α2, which altered phasic inhibition. Thus, we demonstrate a role for spectrins in the synapse-specific targeting of GABAARs, determining the efficacy of fast neuronal inhibition.


Asunto(s)
Receptores de GABA-A , Espectrina , Receptores de GABA-A/metabolismo , Espectrina/metabolismo , Sinapsis/metabolismo , Proteínas de la Membrana/metabolismo , Ácido gamma-Aminobutírico/metabolismo
3.
Brain Sci ; 11(2)2021 Jan 26.
Artículo en Inglés | MEDLINE | ID: mdl-33530492

RESUMEN

Traumatic brain injury (TBI) is a well-established risk factor for several neurodegenerative disorders including Alzheimer's disease and Parkinson's disease, however, a link between TBI and amyotrophic lateral sclerosis (ALS) has not been clearly elucidated. Using the SOD1G93A rat model known to recapitulate the human ALS condition, we found that exposure to mild, repetitive TBI lead ALS rats to experience earlier disease onset and shortened survival relative to their sham counterparts. Importantly, increased severity of early injury symptoms prior to the onset of ALS disease symptoms was linked to poor health of corticospinal motor neurons and predicted worsened outcome later in life. Whereas ALS rats with only mild behavioral injury deficits exhibited no observable changes in corticospinal motor neuron health and did not present with early onset or shortened survival, those with more severe injury-related deficits exhibited alterations in corticospinal motor neuron health and presented with significantly earlier onset and shortened lifespan. While these studies do not imply that TBI causes ALS, we provide experimental evidence that head injury is a risk factor for earlier disease onset in a genetically predisposed ALS population and is associated with poor health of corticospinal motor neurons.

4.
J Trauma Acute Care Surg ; 89(5): 955-961, 2020 11.
Artículo en Inglés | MEDLINE | ID: mdl-32472900

RESUMEN

BACKGROUND: How recurrent traumatic brain injury (rTBI) alters brain function years after insult is largely unknown. This study aims to characterize the mechanistic cause for long-term brain deterioration following rTBI using a rat model. METHODS: Eighteen Sprague-Dawley wild-type rats underwent bilateral rTBI using a direct skull impact device or sham treatment, once per week for 5 weeks, and were euthanized 56 weeks after the first injury. Weekly rotarod performance measured motor deficits. Beam walk and grip strength were also assessed. Brain tissue were stained and volume was computed using Stereo Investigator's Cavalieri Estimator. The L5 cortical layer proximal to the injury site was microdissected and submitted for sequencing with count analyzed using R "DESeq2" and "GOStats." Brain-derived neurotrophic factor (BDNF) levels were determined using enzyme-linked immunosorbent assay. RESULTS: Rotarod data demonstrated permanent deficits 1 year after rTBI. Decreased beam walk performance and grip strength was noted among rTBI rodents. Recurrent traumatic brain injury led to thinner cortex and thinner corpus callosum, enlarged ventricles, and differential expression of 72 genes (25 upregulated, 47 downregulated) including dysregulation of those associated with TBI (BDNF, NR4A1/2/3, Arc, and Egr) and downregulation in pathways associated with neuroprotection and neuroplasticity. Over the course of the study, BDNF levels decreased in both rTBI and sham rodents, and at each time point, the decrease in BDNF was more pronounced after rTBI. CONCLUSION: Recurrent traumatic brain injury causes significant long-term alteration in brain health leading to permanent motor deficits, cortical and corpus callosum thinning, and expansion of the lateral ventricles. Gene expression and BDNF analysis suggest a significant drop in pathways associated with neuroplasticity and neuroprotection. Although rTBI may not cause immediate neurological abnormalities, continued brain deterioration occurs after the initial trauma in part due to a decline in genes associated with neuroplasticity and neuroprotection.


Asunto(s)
Lesiones Traumáticas del Encéfalo/complicaciones , Factor Neurotrófico Derivado del Encéfalo/sangre , Encéfalo/patología , Disfunción Cognitiva/etiología , Animales , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Disfunción Cognitiva/sangre , Disfunción Cognitiva/patología , Modelos Animales de Enfermedad , Regulación hacia Abajo , Humanos , Ratas , Recurrencia , Factores de Tiempo
5.
Stem Cell Reports ; 10(6): 1696-1704, 2018 06 05.
Artículo en Inglés | MEDLINE | ID: mdl-29706501

RESUMEN

Trophic factor delivery to the brain using stem cell-derived neural progenitors is a powerful way to bypass the blood-brain barrier. Protection of diseased neurons using this technology is a promising therapy for neurodegenerative diseases. Glial cell line-derived neurotrophic factor (GDNF) has provided benefits to Parkinsonian patients and is being used in a clinical trial for amyotrophic lateral sclerosis. However, chronic trophic factor delivery prohibits dose adjustment or cessation if side effects develop. To address this, we engineered a doxycycline-regulated vector, allowing inducible and reversible expression of a therapeutic molecule. Human induced pluripotent stem cell (iPSC)-derived neural progenitors were stably transfected with the vector and transplanted into the adult mouse brain. Doxycycline can penetrate the graft, with addition and withdrawal providing inducible and reversible GDNF expression in vivo, over multiple cycles. Our findings provide proof of concept for combining gene and stem cell therapy for effective modulation of ectopic protein expression in transplanted cells.


Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Factor Neurotrófico Derivado de la Línea Celular Glial/genética , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Células-Madre Neurales/citología , Células-Madre Neurales/metabolismo , Trasplante de Células Madre , Tratamiento Basado en Trasplante de Células y Tejidos , Expresión Génica , Genes Reporteros , Terapia Genética , Vectores Genéticos/genética , Factor Neurotrófico Derivado de la Línea Celular Glial/metabolismo , Humanos , Plantas Modificadas Genéticamente , Trasplante de Células Madre/métodos , Transducción Genética , Transgenes
6.
Stem Cells ; 36(7): 1122-1131, 2018 07.
Artículo en Inglés | MEDLINE | ID: mdl-29656478

RESUMEN

Early dysfunction of cortical motor neurons may underlie the initiation of amyotrophic lateral sclerosis (ALS). As such, the cortex represents a critical area of ALS research and a promising therapeutic target. In the current study, human cortical-derived neural progenitor cells engineered to secrete glial cell line-derived neurotrophic factor (GDNF) were transplanted into the SOD1G93A ALS rat cortex, where they migrated, matured into astrocytes, and released GDNF. This protected motor neurons, delayed disease pathology and extended survival of the animals. These same cells injected into the cortex of cynomolgus macaques survived and showed robust GDNF expression without adverse effects. Together this data suggests that introducing cortical astrocytes releasing GDNF represents a novel promising approach to treating ALS. Stem Cells 2018;36:1122-1131.


Asunto(s)
Terapia Genética/métodos , Factor Neurotrófico Derivado de la Línea Celular Glial/metabolismo , Esclerosis Amiotrófica Lateral , Animales , Modelos Animales de Enfermedad , Neuronas Motoras , Ratas
7.
J Trauma Acute Care Surg ; 82(6): 1039-1048, 2017 06.
Artículo en Inglés | MEDLINE | ID: mdl-28520686

RESUMEN

INTRODUCTION: Chronic traumatic encephalopathy (CTE) is a neurodegenerative disease linked to repetitive head injuries. Chronic traumatic encephalopathy symptoms include changes in mood, behavior, cognition, and motor function; however, CTE is currently diagnosed only postmortem. Using a rat model of recurrent traumatic brain injury (TBI), we demonstrate rodent deficits that predict the severity of CTE-like brain pathology. METHODS: Bilateral, closed-skull, mild TBI was administered once per week to 35 wild-type rats; eight rats received two injuries (2×TBI), 27 rats received five injuries (5×TBI), and 13 rats were sham controls. To determine clinical correlates for CTE diagnosis, TBI rats were separated based on the severity of rotarod deficits and classified as "mild" or "severe" and further separated into "acute," "short," and "long" based on age at euthanasia (90, 144, and 235 days, respectively). Brain atrophy, phosphorylated tau, and inflammation were assessed. RESULTS: All eight 2×TBI cases had mild rotarod deficiency, 11 5×TBI cases had mild deficiency, and 16 cases had severe deficiency. In one cohort of rats, tested at approximately 235 days of age, balance, rearing, and grip strength were significantly worse in the severe group relative to both sham and mild groups. At the acute time period, cortical thinning, phosphorylated tau, and inflammation were not observed in either TBI group, whereas corpus callosum thinning was observed in both TBI groups. At later time points, atrophy, tau pathology, and inflammation were increased in mild and severe TBI groups in the cortex and corpus callosum, relative to sham controls. These injury effects were exacerbated over time in the severe TBI group in the corpus callosum. CONCLUSIONS: Our model of repeat mild TBI suggests that permanent deficits in specific motor function tests correlate with CTE-like brain pathology. Assessing balance and motor coordination over time may predict CTE diagnosis.


Asunto(s)
Conmoción Encefálica/complicaciones , Encefalopatía Traumática Crónica/diagnóstico , Animales , Atrofia , Encéfalo/patología , Conmoción Encefálica/patología , Encefalopatía Traumática Crónica/patología , Encefalopatía Traumática Crónica/fisiopatología , Cuerpo Calloso/patología , Modelos Animales de Enfermedad , Masculino , Destreza Motora , Fosforilación , Equilibrio Postural , Ratas , Ratas Sprague-Dawley , Proteínas tau/metabolismo
8.
Hepatology ; 63(6): 1783-95, 2016 06.
Artículo en Inglés | MEDLINE | ID: mdl-26638120

RESUMEN

UNLABELLED: Persistent infection of hepatitis C virus (HCV) is one of the leading causes of end-stage liver disease (ESLD), such as decompensated cirrhosis and liver cancer. Of particular note, nearly half of HCV-infected people in the United States are reported to be heavy drinkers. This particular group of patients is known to rapidly progress to the ESLD. Although accelerated disease progression among alcohol abusers infected with HCV is clinically well recognized, the molecular pathophysiology behind this manifestation has not been well elucidated. Hepatocytes metabolize ethanol (EtOH) primarily through two steps of oxidative catabolism in which alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) play central roles. The ADH-ALDH pathway also governs the metabolism of retinol (vitamin A) to its transcriptionally active metabolite, retinoic acid (RA). In this study, we defined that the ADH-ALDH pathway serves as a potent antiviral host factor in hepatocytes, which regulates the expression of interferon (IFN)-stimulated genes (ISGs) by biogenesis of RA. ISGs constitute over 300 antiviral effectors, which cooperatively govern intracellular antiviral innate immunity. Our study revealed that intracellular RA levels greatly influence ISG expression under basal conditions. Moreover, RA augments ISG induction in response to viral infection or exposure to IFN in a gene-specific manner. Lastly, our results demonstrated that EtOH attenuates the antiviral function of the ADH-ALDH pathway, which suggests the possibility that EtOH-retinol metabolic competition is one of the molecular mechanisms for the synergism between HCV and alcohol abuse in liver disease progression. CONCLUSIONS: RA plays a critical role in the regulation of intracellular antiviral innate immunity in hepatocytes. (Hepatology 2016;63:1783-1795).


Asunto(s)
Regulación de la Expresión Génica , Hepatocitos/inmunología , Inmunidad Innata , Fallo Hepático/etiología , Vitamina A/metabolismo , Animales , Línea Celular , Etanol/efectos adversos , Etanol/metabolismo , Hepatitis C Crónica/complicaciones , Hepatocitos/metabolismo , Humanos , Hepatopatías Alcohólicas/complicaciones , Ratones Endogámicos C57BL
9.
Proc Natl Acad Sci U S A ; 111(5): 1909-14, 2014 Feb 04.
Artículo en Inglés | MEDLINE | ID: mdl-24449862

RESUMEN

In response to viral infection, the host induces over 300 IFN-stimulated genes (ISGs), which are the central component of intracellular antiviral innate immunity. Inefficient induction of ISGs contributes to poor control and persistence of hepatitis C virus infection. Therefore, further understanding of the hepatocytic ISG regulation machinery will guide us to an improved management strategy against hepatitis C virus infection. In this study, comprehensive genome-wide, high-throughput cDNA screening for genes regulating ISG expression identified a tyrosine kinase nonreceptor 1 (TNK1) as a unique player in the ISG induction pathway. The immune-modulatory function of TNK1 has never been studied, and this study characterizes its significance in antiviral innate immunity. TNK1 is abundantly expressed in hepatocytes and maintains basal ISG expression. More importantly, TNK1 plays a critical role in type I IFN-mediated ISG induction. We discovered that the activated IFN receptor complex recruits TNK1 from the cytoplasm. TNK1 is then phosphorylated to enhance its kinase activity. The activated TNK1 potentiates JAK-STAT signaling through dual phosphorylation of STAT1 at tyrosine 701 and serine 727 amino acid positions. Our loss-of-function approach demonstrated that TNK1 governs a cluster of ISG expression that defines the TNK1 pathway effector genes. More importantly, TNK1 abundance is inversely correlated to viral replication efficiency and is also a determinant factor for the hepatocytic response to antiviral treatment. Taken together, our studies found a critical but unidentified integrated component of the IFN-JAK-STAT signaling cascade.


Asunto(s)
Antivirales/metabolismo , Proteínas Fetales/metabolismo , Interferones/metabolismo , Fosfoserina/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Factor de Transcripción STAT1/metabolismo , Transducción de Señal , Animales , Línea Celular Tumoral , ADN Complementario/genética , Susceptibilidad a Enfermedades , Eliminación de Gen , Regulación de la Expresión Génica , Pruebas Genéticas , Genoma Humano/genética , Hepacivirus/fisiología , Hepatitis C/enzimología , Hepatitis C/genética , Hepatitis C/patología , Hepatitis C/virología , Hepatocitos/enzimología , Hepatocitos/patología , Hepatocitos/virología , Humanos , Inmunidad Innata/genética , Janus Quinasa 1/metabolismo , Hígado/metabolismo , Ratones , Ratones Endogámicos C57BL , Fosforilación
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