Kininogen Enhances Seizure Susceptibility Possibly through Bradykinin-induced Modulation of Calcium Transients in Glutamatergic and GABAergic Neurons

Arijit Ghosh¹Jiwen Huang¹Qian Wang¹Yanling Gong¹Srishti Jain²Abhimanyu Thakur³Amiyangshu De²Meixi Gong¹Yoshie Kiyohara¹Jingtai Hu⁴Lingyan Jin⁵Sharba Bandyopadhyay²Fang Chen^6*Shengtian Li¹

• ¹Shanghai Jiao Tong University, Shanghai, Shanghai Municipality, China
• ²Indian Institute of Technology Kharagpur, Kharagpur, West Bengal, India
• ³Delhi Institute of Pharmaceutical Sciences and Research, University of Delhi, New Delhi, India
• ⁴East China Normal University, Shanghai, Shanghai Municipality, China
• ⁵Fudan University, Shanghai, Shanghai Municipality, China
• ⁶First Affiliated Hospital of Xiamen University, Xiamen, China

Previously we showed that elevated cerebrospinal fluid (CSF) levels of kininogen during the acute phase of encephalitis are associated with symptomatic epilepsy. However, the functional role of kininogen in epileptogenesis remains unexplored. This study investigated the brain expression of kininogen and its influence on seizure susceptibility. Additionally, we examined the effects of bradykinin, a nonapeptide derived from kininogen, as a downstream mediator of kininogen’s effect on seizure susceptibility and the underlying cellular mechanisms. We analyzed brain mRNA expression of kininogen using publicly available single-cell RNA-sequencing (scRNA-seq) datasets and assessed protein expression through immunofluorescence in various brain regions. Immunoblot was conducted following pilocarpine-induced status epilepticus (Pilo-SE) to understand post-seizure kininogen dynamics. Next, to understand its functional role, kininogen was overexpressed in the hippocampal CA1 area via its AAV-mediated gene delivery, and bradykinin was administered through the fourth ventricle in mice. The effects on seizure susceptibility were evaluated using a pentylenetetrazole-induced seizure susceptibility test. Furthermore, two-photon in vivo calcium imaging of cortical layer 2/3 glutamatergic neurons and GABAergic parvalbumin-positive neurons was performed to explore the potential circuit mechanisms. While scRNA-seq analyses found kininogen gene expressions in various cell types across the brain, immunofluorescence revealed its preferential localization in neurons but not in glia. Pilo-SE decreased intact kininogen levels in the hippocampus and increased cleaved to intact kininogen (cHK / iHK) ratio. Overexpression of kininogen and exogenous bradykinin administration significantly increased pentylenetetrazole-induced seizure susceptibility in mice. Mechanistically, bradykinin was found to enhance calcium activities in cortical glutamatergic excitatory neurons in Thy1-GCaMP mice when these mice were treated with a subthreshold dose of pentylenetetrazole. In contrast, calcium activities in GABAergic parvalbumin-positive inhibitory neurons were reduced by bradykinin in PVCre-GCaMP mice, suggesting potential cellular mechanisms by which kininogen may render increased seizure susceptibility. Our findings thus reveal a novel pathological role of kininogen in seizure occurrence, explaining why kininogen might be elevated in the CSF of epilepsy-susceptible patients and suggest its potential mechanisms where it might regulate the activities of glutamatergic and GABAergic neurons through the downstream release of bradykinin. Altogether, we propose kininogen as a potential target for developing therapeutics for epilepsy intervention.