Breuer Annika, Kamalizade Delara, Baumgartner Tobias, Berns Juliane L, Opitz Thoralf, Thaler Franziska S, Schoch Susanne, Komorowski Lars, Helmstaedter Christoph, Surges Rainer, Becker Albert J, Pitsch Julika
Department of Epileptology, Medical Faculty, University of Bonn, Venusberg-Campus 1, D-53127, Bonn, Germany.
Inst. of Experimental Epileptology and Cognition Research, University Hospital Bonn, Bonn, Germany.
J Neuroinflammation. 2025 Jul 26;22(1):194. doi: 10.1186/s12974-025-03521-4.
Autoantibodies targeting the intracellular 65-kDa isoform of glutamic acid decarboxylase (anti-GAD65) have been associated with a variety of autoimmune-related syndromes involving a spectrum of difficult-to-treat neurological disorders. However, the pathophysiological role of anti-GAD65 in neuroinflammation remains vague. Its understanding may be complicated by the possible pathogenic interaction between anti-GAD65 and potentially coexisting autoantibodies.
We combined a broad spectrum of approaches ranging from antibody-antigen identification, immunoblotting, immunoprecipitation, mass-spectrometry, cell-based assays, subcellular binding pattern analysis in primary neuronal cultures, and immunohistochemistry to in vitro assays of neuronal uptake, viability, and multi-electrode arrays.
In anti-GAD65-positive neurological patients, mass-spectrometric analysis revealed cytosolic 5'-nucleotidase 1 A (CN1A syn. NT5C1A) as the most abundant antigen. Subsequent screening of 118 anti-GAD65-positive patients revealed that 32 of them had additional autoantibodies targeting CN1A, which were also present in all available corresponding CSF samples. Limbic encephalitis was more often diagnosed in anti-CN1A/anti-GAD65-positive compared to the anti-GAD65-positive patients. Functionally, incubation of primary hippocampal neurons with anti-GAD65, but not with anti-CN1A, resulted in uptake into GABAergic neurons, neuronal cell death, and increased neuronal network activity. Moreover, simultaneous incubation with both antibodies (anti-CN1A/anti-GAD65) resulted in concomitant intraneuronal uptake in a concentration-dependent manner, which correlated with enhanced autophagy followed by massive neuronal death.
GAD65 antibodies directly affect neuronal viability and network activity. Co-existing autoantibodies against CN1A, present in anti-GAD65-positive patients, enhance autophagy and subsequent neuronal death in vitro. Clinically, anti-GAD65-positive patients should be screened for anti-CN1A-associated diseases, and evaluation of anti-CN1A in anti-GAD65-related autoimmune conditions may clarify links between systemic autoimmunity and epilepsy.
靶向谷氨酸脱羧酶细胞内65-kDa异构体的自身抗体（抗GAD65）与多种自身免疫相关综合征有关，这些综合征涉及一系列难以治疗的神经系统疾病。然而，抗GAD65在神经炎症中的病理生理作用仍不明确。抗GAD65与潜在共存的自身抗体之间可能存在的致病相互作用可能会使对其的理解变得复杂。
我们结合了多种方法，包括抗体-抗原鉴定、免疫印迹、免疫沉淀、质谱分析、基于细胞的检测、原代神经元培养中的亚细胞结合模式分析以及免疫组化，还进行了神经元摄取、活力和多电极阵列的体外检测。
在抗GAD65阳性的神经系统疾病患者中，质谱分析显示胞质5'-核苷酸酶1A（CN1A，同义词NT5C1A）是最丰富的抗原。随后对118例抗GAD65阳性患者进行筛查，发现其中32例还存在靶向CN1A的其他自身抗体，这些抗体也存在于所有可用的相应脑脊液样本中。与抗GAD65阳性患者相比，抗CN1A/抗GAD65阳性患者更常被诊断为边缘叶脑炎。在功能上，用抗GAD65而非抗CN1A孵育原代海马神经元，会导致其被GABA能神经元摄取、神经元细胞死亡以及神经元网络活动增加。此外，同时用两种抗体（抗CN1A/抗GAD65）孵育会导致神经元内同时摄取，且呈浓度依赖性，这与自噬增强继而大量神经元死亡相关。
GAD65抗体直接影响神经元活力和网络活动。抗GAD65阳性患者中存在的针对CN1A的共存自身抗体在体外会增强自噬并导致随后的神经元死亡。临床上，应针对抗CN1A相关疾病对抗GAD65阳性患者进行筛查，在抗GAD65相关自身免疫性疾病中对抗CN1A的评估可能会阐明全身自身免疫与癫痫之间的联系。
