Gremlin1 Regulates Epileptogenisis By Mediating Astrocytic Potassium Buffering

Part I Expression and localization of Gremlin1 in epilepsyObjectives:1.To determine the expression and cellular localization of Gremlin1 in the brain tissue of TLE patients and control patients.2.To determine the temporal and spatial expression patterns of Gremlin1 in the hippocampus of KA epilepsy mice.Methods:1.Collect brain tissues from TLE patients and traumatic brain injury patients as control group.2.Construct mouse epilepsy model by dorsal hippocampus KA injection.3.Gremlin1 m RNA and protein expression were detected in brain tissues of TLE and control patients as well as hippocampus of control mice and epilepsy mice at different time points after KA using RT-q PCR and Western blot.4.Detecting the cellular localization of Gremlin1 in the brain of TLE and control patients and KA epileptic mice using immunofluorescent double-labeling techniques.5.Mouse primary neurons and primary astrocytes were cultured and were constructed to epilepsy model,respectively.The m RNA and protein expression of Gremlin1 in primary neurons and astrocytes epilepsy models were detected using RT-q PCR and western blot.Results:1.Gremlin1 protein was significantly upregulated in brain tissues of TLE patients compared with controls,and Gremlin1 was expressed in neurons and astrocytes in control and TLE patient brain tissues.2.Gremlin1 m RNA and protein levels were elevated in KA epileptic mice starting at 2 days post-KA,peaking at 7 days post-KA and remaining until 60 days post-KA.Gremlin1 expression was elevated on GFAP⁺astrocytes at 7 days post-KA in CA1 region,but was not significantly altered in Neu N⁺neurons.3.Neither m RNA nor protein levels of Gremlin1 were not significantly altered in the Mg²⁺-free neuronal epilepsy model compared with controls.Both m RNA and protein levels of Gremlin1 were significantly increased in astrocytes treated with conditioned Mg²⁺-free neuronal medium.Conclusions:1.Gremlin1 expression was elevated in brain tissue of TLE patients.2.Gremlin1 was upregulated in KA epileptic mice and this elevation was mainly attributed to astrocytes at 7 days post-KA.Part ? Gremlin1 regulates epilepsy susceptibilityObjective:To determine the role of Gremlin1 on epilepsy.Methods:1.Constructed of lentiviral vectors with Gremlin1 overexpression or knockdown.2.Mice received LV-Gremlin1 overexpression or knockdown were underwent KA modeled and subjected to 24-hour continuous video monitoring.3.Local field potential monitoring was performed in all mice.Results:1.Lentiviruses of Gremlin1 overexpression and knockdown were successfully constructed and injected into the hippocampus of mice.2.Gremlin1 overexpression prolonged the latency of epilepsy,reduced the number of SRSs episodes and the proportion of grade 4-5 SRSs episodes,while Gremlin1 knockdown shortened the latency of epilepsy and increased the number of SRSs episodes and the proportion of grade 4-5 SRSs episodes.3.Gremlin1 overexpression decreased the number and duration of SLEs discharges during the monitoring period of LFPs,and Gremlin1knockdown increased the number and duration of SLEs discharges during the monitoring period of LFPs.Conclusion:Gremlin1 overexpression attenuated seizure susceptibility and Gremlin1 knockdown increased seizure susceptibility.Part ? Gremlin1 regulated reactive astrogliosis and astrocytic potassium buffering dysfunction in KA epilepsy miceObjective:To investigate the role of Gremlin1 on reactive astrogliosis and astrocytic dysfunction in KA epilepsy miceMethods:1.Detection the GFAP expression in KA epilepsy mice using immunofluorescence and Western blot techniques.2.Detection of MMP2/3/9 m RNA levels in KA epilepsy mice using RT-q PCR technique.3.Detection of changes in extracellular potassium levels following excitation of pyramidal neurons in the CA1 region in KA epilepsy mice using potassium-selective microelectrodes.4.Detection of astrocytic Kir currents and gap junction coupling of CA1 region in KA epilepsy mice after using a patch-clamp technique.5.Detection of Kir4.1,Connexin 43,GLT1 and GLAST protein expression in KA epilepsy mice using Western blot.Results:1.Overexpression of Gremlin1 attenuated reactive astrogliosis and decreased GFAP expression,and knockdown of Gremlin1 aggravated reactive astrogliosis and increased GFAP expression in KA epilepsy mice.2.Neither overexpression nor knockdown of Gremlin1 affected the m RNA levels of MMP2/3/9 in KA epilepsy mice.3.Overexpression of Gremlin1 decreased the magnitude of the increase in[K⁺]_o levels due to neuronal excitation in the CA1 region,and knockdown of Gremlin1 decreased the magnitude of the increase in[K⁺]_olevels due to neuronal excitation in KA epilepsy mice.4.Overexpression of Gremlin1 increased Kir currents and gap junctional coupling in astrocytes of CA1 region in KA epileptic mice,and knockdown of Gremlin1 decreased Kir currents and gap junctional coupling in astrocytes of CA1 region in KA epileptic mice.5.Overexpression of Gremlin1 increased Kir 4.1 and Connexin 43expression in KA epileptic mice and knockdown of Gremlin1 decreased Kir 4.1 and Connexin 43 expression in KA epilepsy mice.6.Neither overexpression nor knockdown of Gremlin1 affected GLT1or GLAST protein expression in KA epilepsy mice.Conclusions:1.Gremlin1 regulated reactive astrogliosis in KA epilepsy mice.2.Gremlin1 did not regulate ECM remodeling or astrocyte glutamate transporter expression in KA epilepsy mice.3.Gremlin1 regulated astrocyte potassium buffering by upregulated expression of Kir 4.1 and Connexin 43 then increased Kir currents and gap junction coupling in KA epilepsy mice.Part ? Gremlin1 in primary astrocytes improves neuronal activity by downregulating GFAP expression and increasing potassium bufferingObjectives:1.To explore the effect of Gremlin1 on potassium buffering in primary astrocytes.2.To investigate the role of Gremlin1 in astrocytes on the neuronal activity in epilepsy.Methods:1.Primary astrocytes were treated with recombinant mouse Gremlin1protein(Rm Gremlin1),then detect GFAP expression using Western blot,immunofluorescence,and proliferation of astrocytes using CCK8.2.Detecting the effects of Gremlin1 on astrocytic potassium buffering,Kir currents,intercellular gap junction communication,Kir4.1 and Connexin 43 expression using patch-clamp technique,Western blot and Lucifer Yellow diffusion assay.3.Examining the effect of Gremlin1 in astrocytes on the neuronal activity of epilepsy models using astrocyte-neuron co-culture,membrane clamp technique.Results:1.Gremlin1 downregulated GFAP expression in a dose-dependent manner but did not affect astrocyte proliferation.2.Gremlin1 increased potassium buffering in primary astrocytes,increased Kir currents and intercellular gap junctional communication,and increased Kir 4.1 and Connexin 43 expression.3.Rm Gremlin1-treated astrocytes hyperpolarized epileptic neurons and reduced the frequency of spontaneous AP in epileptic neurons but did not affect their evoked AP properties.Conclusions:1.Gremlin1 downregulated GFAP expression in primary astrocytes.2.Gremlin1 increased potassium buffering in primary astrocytes.3.Gremlin1 in astrocytes improved epileptic neuronal activity.Part ? Mechanisms of Gremlin1 in regulating astrocytic potassium bufferingObjective:To explore the molecular mechanisms by which Gremlin1regulates reactive astrogliosis and potassium buffering in KA epilepsy mice.Methods:1.Recombinant mouse BMP4 protein(Rm BMP4)or VEGFR2inhibitor SU5416 were co-incubation with Rm Gremlin1 in Primary astrocytes,and the expression GFAP,Kir 4.1 Connexin 43,p.Smad1/5/9and Smad1/5/9 were detected using Western blot.2.Expression and localization of BMP4/Smad signaling in KA epilepsy mice were detected using Western blot and immunofluorescence double-labeling technique.3.Mice received BMP4/Smad inhibitor LDN193189,then were underwent KA modeled and subjected to 24-hour continuous video monitoring.4.Detection of Smad1/5/9 phosphorylation in Gremlin1overexpressing or knockdown mice using Western blot.5.Knockdown of Gremlin1 along with inhibition of BMP4/Smad signaling using LDN193189 and then observe the epileptic behavior tests,LFPs,reactive astrogliosis and astrocytic potassium buffering in KA epilepsy mice.Results:1.In primary astrocytes,Rm BMP4 reversed the regulation of GFAP,Kir 4.1 and Connexin 43 proteins by Rm Gremlin1,while co-treated of SU5416 with Rm Gremlin1 did not affect the expression of these proteins.2.Rm BMP4 increased phosphorylation of Smad1/5/9,and Rm Gremlin1 decreased phosphorylation of Smad1/5/9 in primary astrocyte.3.BMP4 and Smad1/5/9 phosphorylation were increased in KA epilepsy mice,and BMP4 expression was increased in astrocytes in the hippocampal CA1 region of epilepsy mice.4.Treatment with LDN193189 significantly prolonged the latency period,reduced the number of seizures per week in SRSs and the proportion of grade 4-5 SRSs in KA epilepsy mice,and reduced the number of seizures and total duration of seizures of LFPs.5.Gremlin1 overexpression decreased phosphorylation of Smad1/5/9and Gremlin1 knockdown increased phosphorylation of Smad1/5/9 in KA epilepsy mice.6.Gremlin1 knockdown+LDN193189 treatment significantly prolonged seizure latency,reduced the number of seizures per week in SRSs and the proportion of grade 4-5 SRSs seizures,and reduced the number of seizures and total duration of seizures in SLEs during LFPs testing.7.Gremlin1 knockdown+LDN193189 treatment significantly attenuated reactive astrocyte proliferation and increased Kir4.1 and Connexin 43 protein expression.Conclusions:1.In primary astrocytes,Gremlin1 regulated the expression of GFAP,Kir 4.1 and Connexin 43 by inhibiting BMP4/Smad1/5/9 signaling.2.In epilepsy mice 7 days post-KA,BMP4/Smad signaling was significantly increased in astrocytes in the CA1 region of hippocampus.3.Inhibition of BMP4/Smad signaling suppressed seizure susceptibility in KA epileptic mice.4.Gremlin1 regulates epilepsy susceptibility and reactive astrogliosis and astrocytic potassium buffering through inhibition of BMP4/Smad signaling in KA epilepsy mice.