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Background: The expansion of GGC repeats (typically exceeding 60 repeats) in the 5' untranslated region (UTR) of the NOTCH2NLC gene (N2C) is linked to N2C-related repeat expansion disorders (NREDs), such as neuronal intranuclear inclusion disease (NIID), frontotemporal dementia (FTD), essential tremor (ET), and Parkinson's disease (PD). These disorders share common clinical manifestations, including parkinsonism, dementia, seizures, and muscle weakness. Intermediate repeat sizes ranging from 40 to 60 GGC repeats, particularly those with AGC-encoded serine insertions, have been reported to be associated with PD; however, the functional implications of these intermediate repeats with serine insertion remain unexplored. Methods: Here, we utilized cellular models harbouring different sizes of N2C variant 2 (N2C2) GGC repeat expansion and CRISPR-Cas9 engineered transgenic mouse models carrying N2C2 GGC intermediate repeats with and without serine insertion to elucidate the underlying pathophysiology associated with N2C intermediate repeat with serine insertion in NREDs. Results: Our findings revealed that the N2C2 GGC intermediate repeat with serine insertion (32G13S) led to mitochondrial dysfunction and cell death in vitro. The neurotoxicity was influenced by the length of the repeat and was exacerbated by the presence of the serine insertion. In 12-month-old transgenic mice, 32G13S intensified intranuclear aggregation and exhibited early PD-like characteristics, including the formation of α-synuclein fibers in the midbrain and the loss of tyrosine hydroxylase (TH)-positive neurons in both the cortex and striatum. Additionally, 32G13S induced neuronal hyperexcitability and caused locomotor behavioural impairments. Transcriptomic analysis of the mouse cortex indicated dysregulation in calcium signaling and MAPK signaling pathways, both of which are critical for mitochondrial function. Notably, genes associated with myelin sheath components, including MBP and MOG, were dysregulated in the 32G13S mouse. Further investigations using immunostaining and transmission electron microscopy revealed that the N2C intermediate repeat with serine induced mitochondrial dysfunction-related hypermyelination in the cortex. Conclusions: Our in vitro and in vivo investigations provide the first evidence that the N2C-GGC intermediate repeat with serine promotes intranuclear aggregation of N2C, leading to mitochondrial dysfunction-associated hypermyelination and neuronal hyperexcitability. These changes contribute to motor deficits in early PD-like neurodegeneration in NREDs. [ABSTRACT FROM AUTHOR]

Abstract Background The expansion of GGC repeats (typically exceeding 60 repeats) in the 5’ untranslated region (UTR) of the NOTCH2NLC gene (N2C) is linked to N2C-related repeat expansion disorders (NREDs), such as neuronal intranuclear inclusion disease (NIID), frontotemporal dementia (FTD), essential tremor (ET), and Parkinson’s disease (PD). These disorders share common clinical manifestations, including parkinsonism, dementia, seizures, and muscle weakness. Intermediate repeat sizes ranging from 40 to 60 GGC repeats, particularly those with AGC-encoded serine insertions, have been reported to be associated with PD; however, the functional implications of these intermediate repeats with serine insertion remain unexplored. Methods Here, we utilized cellular models harbouring different sizes of N2C variant 2 (N2C2) GGC repeat expansion and CRISPR-Cas9 engineered transgenic mouse models carrying N2C2 GGC intermediate repeats with and without serine insertion to elucidate the underlying pathophysiology associated with N2C intermediate repeat with serine insertion in NREDs. Results Our findings revealed that the N2C2 GGC intermediate repeat with serine insertion (32G13S) led to mitochondrial dysfunction and cell death in vitro. The neurotoxicity was influenced by the length of the repeat and was exacerbated by the presence of the serine insertion. In 12-month-old transgenic mice, 32G13S intensified intranuclear aggregation and exhibited early PD-like characteristics, including the formation of α-synuclein fibers in the midbrain and the loss of tyrosine hydroxylase (TH)-positive neurons in both the cortex and striatum. Additionally, 32G13S induced neuronal hyperexcitability and caused locomotor behavioural impairments. Transcriptomic analysis of the mouse cortex indicated dysregulation in calcium signaling and MAPK signaling pathways, both of which are critical for mitochondrial function. Notably, genes associated with myelin sheath components, including MBP and MOG, were dysregulated in the 32G13S mouse. Further investigations using immunostaining and transmission electron microscopy revealed that the N2C intermediate repeat with serine induced mitochondrial dysfunction-related hypermyelination in the cortex. Conclusions Our in vitro and in vivo investigations provide the first evidence that the N2C-GGC intermediate repeat with serine promotes intranuclear aggregation of N2C, leading to mitochondrial dysfunction-associated hypermyelination and neuronal hyperexcitability. These changes contribute to motor deficits in early PD-like neurodegeneration in NREDs.