Mapping the lifetime of PSD95 at single-synapse resolution across the mouse brain in health and disease

Abstract

A continuous renewal of synaptic proteins is required for healthy functioning and activity-dependent synapse adaptations in the brain. Disruptions to protein turnover lead to impairments in learning and memory and contribute to agerelated cognitive decline and dementia. Protein turnover is thought to be tightly regulated in space and time, however, no technology to date has been able to visualise protein turnover and lifetime at individual synapses across the brain. In this thesis, we present insights into the architecture and temporal changes to the lifetime of an abundant post-synaptic density scaffolding protein PSD95 at single-synapse resolution using a newly developed method.

Transgenic mice carrying a HaloTag domain fused to the endogenous PSD95 protein were intravenously injected with a fluorescent ligand that covalently binds the HaloTag domain. Fluorescent protein levels were visualized in brain sections using confocal microscopy, with loss of fluorescence over time used for estimating PSD95 protein decay. We employed Synaptome mapping pipeline to quantify the changes in numbers and morphological parameters of synaptic puncta. We performed a detailed examination of: (a) the lifetime of PSD95 in synapses of 110 brain regions, (b) the changes in protein lifetime with age of an animal, and (c) the effects of disease-relevant mutations in PSD95-interacting synaptic proteins on the turnover and lifetime of PSD95.

Strikingly, a vast majority of PSD95 protein in synapses is replaced within two weeks and the synapses with longer lived protein reside in cortical and hippocampal brain regions, areas involved with long-term memory storage. PSD95-positive puncta half-life ranges more than 6-fold between brain regions in adult mice, from ~1-2 days in olfactory bulb and thalamic nuclei to ~10-12 days in the superficial layers of the cortex.

PSD95 lifetime increases with age across the brain and the protein lasts at least twice as long in synapses of ageing 18-month-old animals compared to synapses in developing 3-week-old mice. Between developing and adult brain, the most striking changes in turnover are observed in the cortical brain areas which are known to undergo extensive synaptic pruning and remodelling during the first month of life in mice. With ageing, the largest increase in protein lifetime is detected in cerebellar areas.

Disease-relevant mutations in post-synaptic density proteins interacting with PSD95 showed differing effects on PSD95 lifetime. The mice carrying a schizophrenia- and autism-relevant deletion of a gene that codes for PSD93 protein showed a substantially increased lifetime of PSD95 compared to wildtype animals, with a gene dose-dependent effect. In contrast, mice carrying an autism and intellectual disability-relevant mutation in a gene coding for SynGAP protein showed minor changes to PSD95 turnover displaying the sensitivity of the method to detect disease-specific effects on protein turnover and lifetime.

In summary, synapse protein dynamics show a conserved spatial architecture with longest protein lifetime observed in regions involved with memory storage. Age-related increase in protein lifetime may contribute to memory impairments and dementia risk. The atlas of synaptic protein lifetimes will serve as a novel resource with applications in molecular neuroscience and brain disease research.