Local consequences of FMRP loss: cell type-specific mRNA and protein profiles of Fmr1-KO synapses

Brain function relies on billions of neurons interconnected by synapses with unique molecular composition specialized for cell type, brain region, and their role in circuit dynamics. Pathological synaptic dysfunctions - so-called synaptopathies - underlie many neurodevelopmental disorders (NDDs), including Fragile X Syndrome (FXS). While synaptic deficits are well documented in FXS, molecular studies have largely relied on bulk brain homogenates or synaptic fractions, which typically represent ''average'' molecular identities, potentially masking synapse type-specific alterations. To provide synapse type resolution, we use fluorescence-activated synaptosome sorting coupled to RNA-sequencing and mass spectrometry to isolate and profile cortical excitatory and inhibitory synapses of hemizygous and heterozygous Fmr1-deficient mice. We found that full or partial FMRP loss leads to cell type- and compartment-specific molecular remodeling (syndive.org), affecting mRNAs and proteins related to synaptic organization and metabolic pathways. Strikingly, only a minority of dysregulated mRNAs/proteins were shared across excitatory and inhibitory synapses - the substantial majority were uniquely regulated at one subtype. Indeed, FMRP loss often resulted in opposing protein changes in each synapse type. Changes in mRNA or protein abundance were also not well coordinated, consistent with substantial post-transcriptional regulation, including changes to the translational and ubiquitin-dependent protein degradation machinery. For example, our data suggest a disturbed, cell type-distinct coordination of protein degradation via CAND1 as one key consequence of FMRP loss. Furthermore, we observed differences in male (Fmr1-/y) and female (Fmr1-/+) transcriptomes, where, for example, FMRP dosage was coupled to reciprocal regulation of the NDD-risk gene Trpc4 in both synapse types.