Although it is well-known that synaptosomal proteins are deranged in neurodegenerative disorders, no information is available at the protein-chemical level as mainly immunochemical or immunohistochemical data were reported previously. We therefore investigated synaptosomal proteins in brain specimens from patients with Down syndrome (DS) and Alzheimer’s disease (AD) to challenge the DS synaptic pathology as well as the relevance of DS to AD in synaptic pathology. For the aim of this study, we employed two-dimensional electrophoresis and matrix-associated laser desorption ionization mass spectroscopy and determined β-soluble N-ethylmaleimide-sensitive factor attachment protein (β-SNAP), γ-SNAP and synaptotagmin I (SYT I) in 7 individual brain regions of controls and patients with DS and AD. In DS brain, β-SNAP was significantly reduced in temporal cortex (p < 0.01). SYT I (p65) and SYT I (pI 7.0) were significantly reduced in thalamus (p < 0.01 and p < 0.05, respectively). In AD brain, β-SNAP was significantly decreased in temporal cortex (p < 0.05). SYT I (p65) was significantly reduced in cerebellum (p < 0.05), and temporal (p < 0.001) and parietal cortex (p < 0.01). SYT I (pI 7.0) was significantly reduced in temporal (p < 0.001) and parietal cortex (p < 0.01) and thalamus (p < 0.01). γ-SNAP did not show any change in both DS and AD. The findings may explain impaired synaptogenesis in DS and AD brain, which is well documented in DS brain already early in life, and/or synaptosomal loss secondary to neuronal loss observed in both neurodegenerative disorders. It may also represent, reflect or account for the impaired neuronal transmission in DS and AD, caused by deterioration of the exocytic machinery. Here, we provide evidence for several deranged synaptosomal proteins in several brain regions at the protein level indicating deficient synaptosomal wiring of the brain in DS and AD.