Infantile neuronal ceroid lipofuscinosis (INCL), the most severe form of neuronal ceroid lipofuscinoses, is caused by mutations in the lysosomal enzyme palmitoyl protein thioesterase 1 (PPT1). Typical symptoms of this disease include progressive psychomotor developmental retardation, visual failure, seizures, and premature death. Here, we investigated seizure activity and relevant pathological changes in PPT1 knock-in mice (PPT1 KI). The behavior studies in this study demonstrated that PPT1 KI mice had no significant seizure activity until 7 months of age, and local field potentials also displayed epileptiform activity at the same age. The expression levels of Iba-1 and CD68 demonstrated, by Western blot analysis, the inflammatory cytokine TNF-α content measured with enzyme-linked immunosorbent assay, and the number of microglia demonstrated by immunohistochemistry (IHC) were significantly increased at age of 7 months, all of which indicate microglia activation at an age of seizure onset. The increased expression of GFAP were seen at an earlier age of 4 months, and such an increase reached its peak at age of 6 months, indicating that astrocyte activation precedes microglia. The purinergic P2X7 receptor (P2X7R) is an ATP-sensitive ionic channel that is highly expressed in microglia and is fundamental to microglial activation, proliferation, cytokines release and epilepsy. We show that the ATP concentration in hippocampal tissue in PPT1 KI mice was increased using an enhanced ATP assay kit and demonstrated that the antagonist of P2X7R, A-438079, significantly reduced seizures in PPT1 KI mice. In contrast to glial cell activation and proliferation, a significant reduction in synaptic proteins GABA_AR was seen in PPT1 KI mice. These results indicate that seizure in PPT1 KI mice may be associated with microglial activation involved in ATP-sensitive P2X7R signaling and impaired inhibitory neurotransmission.

The infantile neuronal ceroid lipofuscinosis, also called CLN1 disease, is a fatal neurodegenerative disease caused by mutations in the CLN1 gene encoding palmitoyl protein thioesterase 1 (PPT1). Identifying the depalmitoylation substrates of PPT1 is crucial for understanding CLN1 disease. In this study, we found that GABA_AR, the critical synaptic protein essential for inhibitory neurotransmission, is a substrate of PPT1. PPT1 depalmitoylates GABA_AR α1 subunit at Cystein-260, while binding to Cystein-165 and -179. Mutations of PPT1 or its GABA_AR α1 subunit binding site enhanced inhibitory synaptic transmission and strengthened oscillations powers but disrupted phase coupling in CA1 region and impaired learning and memory in 1- to 2-months-old PPT1-deficient and Gabra1^em1 mice. Our study highlights the critical role of PPT1 in maintaining GABA_AR palmitoylation homeostasis and reveals a previously unknown molecular pathway in CLN1 diseases induced by PPT1 mutations.

Palmitoylation is a reversible lipid modification, regulating protein localization and signaling in neurons. Growth-associated protein 43 (GAP43) requires palmitoylation for axonal development and synaptic plasticity; however, its depalmitoylase and regulation in neural circuits remain unknown. We investigated whether palmitoyl-protein thioesterase 1 (PPT1) is the principal depalmitoylase for GAP43 and examined how disrupted PPT1-GAP43 signaling affects neuronal morphology and circuit function. Using biochemical assays, structural modeling, CRISPR-Cas9–generated GAP43 point mutation mice (GAP43-PM), PPT1-knockout mice (PPT1-KO), electrophysiology, and behavior, we demonstrated that PPT1 interacts with GAP43 at Cys ³ and Cys ⁴ to mediate its depalmitoylation, disruption of this interaction causes GAP43 hyperpalmitoylation that drove excessive dendritic arborization and aberrant growth cone expansion, enhanced glutamatergic transmission, and hippocampal network hyperexcitability, resulting in cognitive deficits without lysosomal storage pathology. Exogenous PPT1 reduced these morphological/synaptic abnormalities. Our findings establish the PPT1-GAP43 depalmitoylation pathway as essential for neuronal circuit homeostasis; its dysfunction contributes to neurodevelopmental disorders, identifying a potential therapeutic target for palmitoylation-related neurodevelopmental disorders.

Summary: Palmitoylation is a reversible and dynamic process involving addition of palmitic acid to cysteine residues of proteins. Studies have indicated that a variety of neuronal receptors, including glutamate receptors such as AMPAR, NMDAR, and GABAAR, are palmitoylated, which contributes to the dynamic modulation of synaptic strength in response to neuronal activity. However, little is known about the depalmitoylation of these receptors. In this study, we generated PPT1-deficient mice, an animal model that closely mimics human disease of infantile neuronal ceroid lipofuscinosis (INCLs), by knocking in a CLN1 c.451C>T nonsense mutation. We identified for the first time that the GABA_ARα1 subunit rather than AMPAR is the substrate of PPT1. In PPT1-deficient mice, excessive palmitoylation and extended membrane location of GABA_AR were detected. Simultaneously, spatial learning and memory deficits with dysregulation of neuronal network γ oscillation and impairment of long-term plasticity were shown in the mice at as early as 2-month-old. Application of N-tert-butylhydroxylamine hydrochloride, a thioesterase mimetic, attenuated PPT1 mutation-induced GABA_AR hyper-palmitoylation and its membrane accumulation with improved neuronal transmission and memory functions in the mice. These data provide new insights into the mechanisms of neuronal disorder caused by depalmitoylation deficiency and offer a clue for further intervention for INCLs and other neurodegenerative diseases. Funding: This work was supported by funds from the National Natural Science Foundation (CBL, No.: 81771517) and a Xinxiang Medical University endowment (SYP, No.: 505182). Declaration of Interests: The authors declare no competing interests. Ethics Approval Statement: Animal use and procedures were performed according to the regulations and requirements of Xinxiang Medical University Animal Ethics Committee (No. XYLL 2021053).

The neuronal ceroid lipofuscinosis family of lysosomal storage diseases, also called CLN1 disease, is characterized by the deficiency of palmitoyl-protein thioesterase 1 (PPT1). In this study, we investigated the impact of PPT1 deficiency on hippocampal GABAergic interneurons (INs) and associated neural network oscillations in a PPT1-KI ( CLN1 c.451 C > T (p.R151X)) mouse model. Using a combination of in vivo electrophysiology, immunostaining, and fiber photometry, we observed that PPT1 deficiency led to the activation of caspase 3 in parvalbumin-positive (PV ⁺ ) INs, an increased activity of pyramidal neurons and theta/gamma oscillation power, and the disruption of theta-gamma cross-frequency coupling (CFC) in the early stage of the CLN1 disease model. In the late stage of the CLN1 disease model, we observed the reduced neuronal activity, extensive neuronal loss including PV ⁺ INs, and the emergence of spontaneous epileptiform discharges and the pathological ripples. Treatment with diazepam partially restored oscillatory coupling and reduced seizure-like activities. Our research indicated that PPT1 deficiency leads to early selective impairment of PV ⁺ INs, triggering overactivation of pyramidal neurons and network dysfunction, which consequently results in seizures and neurodegeneration. This research provides novel insights into the pathogenesis of CLN1 disease and potential therapeutic strategies for the intervention of CLN1 disease by improving the function of inhibitory INs via caspase inhibition.