Synaptic vesicle endocytosis deficits underlie GBA-linked cognitive dysfunction in Parkinson's disease and Dementia with Lewy bodies
GBA mutations are major risk factors for Parkinson's disease (PD) and Dementia with Lewy Bodies (DLB), two common a-synucleinopathies associated with cognitive impairment. Here, we investigated the role of GBA mutations in cognitive decline by utilizing Gba L444P mutant mice, SNCA transgenic (tg), and Gba-SNCA double mutant mice. Notably, Gba mutant mice showed early cognitive deficits but no PD-like motor deficits up to 12 months old. Conversely, SNCA tg mice displayed age-related motor deficits but no cognitive abnormalities. Gba-SNCA mice exhibited exacerbated motor deficits and cognitive decline.  Immunohistological analysis revealed cortical phospho-a-synuclein pathology in SNCA tg mice, which was exacerbated in Gba-SNCA mice, especially in layer 5 cortical neurons. Significantly, Gba mutant mice did not show a-synuclein pathology. Single-nucleus RNA sequencing of cortices instead uncovered selective synaptic vesicle cycle defects in excitatory neurons of Gba mutant and Gba-SNCA mice, via robust downregulation in gene networks regulating synapse vesicle cycle and synapse assembly. Meanwhile SNCA tg mice displayed broader synaptic changes. Immunohistochemical and electron microscopic analyses validated these findings. Together, our results indicate that Gba mutations, while exacerbating pre-existing a-synuclein aggregation and PD-like motor deficits, contribute to cognitive deficits through a-synuclein-independent mechanisms, likely involving dysfunction in synaptic vesicle endocytosis. Additionally, Gba-SNCA mice are a valuable model for studying cognitive and motor deficits in PD and DLB Overall design: Mice: Gba mutant mice have been previously described in Mistry et al. (2010)1 and Taguchi et al. (2017)2. These mice have a copy of the Gba L444P mutant allele and a Gba KO allele, with Gba expression rescued in skin to prevent early lethality. SNCA tg mice overexpress the human a-synuclein A30P transgene (heterozygous), and have also been previously described. Gba mutant mice were crossed to SNCA tg to obtain Gba-SNCA double mutant mice. Age and sex matched WT mice were used as normal controls. We performed single nucleus RNA sequencing (snRNA-seq) on cortical brain tissue from adult mice belonging to the four genotypes (n=14) 3-4 mice per genotype were used: 4 Gba mutant mice (192, 193, 185, and 201), 3 SNCA tg mice (729, 754, 740), 3 Gba-SNCA double mutant mice (187, 191, 184) and 4 WT (normal control) mice of the B6 strain. We chose to perform this analysis on 12-month-old mice, as Gba-SNCA mice show enhanced behavioral deficits and a-synuclein pathology, while lacking gross neuronal loss or degeneration in the cortex, allowing us to investigate disease relevant mechanisms. Nuclei isolation from cerebral cortex: Fresh cortical tissue were dissected from left hemisphere of 12 month old WT, Gba, SNCA tg and Gba-SNCA mice after euthanasia. Single nuclei were isolated as previously described with modifications. All procedures were carried out on ice or at 4 degrees C. Cortical snRNA expression was analysed in mice with a pathogenic mutation - i.e., Gba mutant mice, SNCA tg mice, and Gba-SNCA double mutant mice - and compared, respectively, with WT mice cortical snRNA expression.