Synaptophysin density underestimates synapse counts in the human brain - An anatomical study on synaptoporin protein expression.

Synaptoporin (SPO; synaptophysin II) has classically been described as a presynaptic vesicle protein enriched in the hippocampal mossy fiber pathway, distinguishing it from the ubiquitously expressed synaptophysin I (SYP). However, its distribution and synapse-type specificity in the human brain have not been studied yet. Here, we comprehensively mapped SPO protein expression across multiple human brain regions using immunofluorescence staining of post mortem tissue and biochemical analysis of brain lysates. We identified high SPO protein expression in the hippocampus, cerebral cortex, and dorsal horn of the spinal cord; moderate expression in the cerebellum and amygdala; and low levels in the putamen. The analyzed brainstem regions and the thalamus were devoid of SPO. Notably, SPO was present in distinct components of the human auditory pathway, mirroring rodent patterns. Colocalization analyses revealed largely separate distributions of SPO and SYP, challenging the concept of SYP as 'pan-synaptic' marker. In the cerebral cortex, a region with pronounced SPO expression, SYP-based synapse quantification underestimated total synapse numbers by up to 35%, a result replicated across independent brain tissue sources. SPO puncta were associated with both excitatory and inhibitory synaptic markers, reflecting the highly heterogeneous human synaptome. Collectively, these findings demonstrate that SYP-only assays overlook a substantial subset of synapses and they highlight the combinatorial complexity of vesicle protein expression in the human brain. This work establishes SPO as a critical determinant of synaptic diversity and can serve as reference to identify vulnerable synaptic subtypes in neurodegenerative diseases.