Examination of healthy and diseased human brain is essential to translational neuroscience. Protein–protein interactions play a pivotal role in physiological and pathological processes, but their detection is difficult, especially in aged and fixed human brain tissue. We used the in-situ proximity ligation assay (PLA) to broaden the range of molecular interactions assessable in-situ in the human neuropathology. We adapted fluorescent in-situ PLA to detect ubiquitin-modified proteins in human brains with Alzheimer’s disease (AD), including approaches for the management of autofluorescence and quantification using a high-content image analysis system. We confirmed that phosphorylated microtubule-associated protein tau (Serine202, Threonine205) aggregates were modified by ubiquitin and that phospho-tau-ubiquitin complexes were increased in hippocampal and frontal cortex regions in AD compared to non-AD brains. Overall, we refined PLA for use in human neuropathology, which has revealed a profound change in the distribution of ubiquitin in AD brain and its association with characteristic tau pathologies.

Examination of healthy and diseased human brain is essential to translational neuroscience. Protein-protein interactions play a pivotal role in physiological and pathological processes, but their detection is difficult, especially in aged and fixed human brain tissue. We used the proximity ligation assay (PLA) to broaden the range of molecular interactions assessable in-situ in human neuropathology. We adapted fluorescent in-situ PLA to detect ubiquitin-modified proteins in human brains with Alzheimer’s disease (AD), including approaches for the management of autofluorescence and quantification using a high-content image analysis system. We confirmed that hyperphosphorylated microtubule-associated protein tau (Serine202, Threonine205) aggregates were modified by ubiquitin and that phospho-tau-ubiquitin complexes were increased in hippocampal and frontal cortex regions in AD compared to non-AD brains. Overall, we refined PLA for use in human neuropathology, which has revealed a profound change in the distribution of ubiquitin in AD brain and its association with characteristic tau pathologies.

Background: Proteinopathy is a common feature of multiple neurodegenerative diseases. Recent studies have demonstrated lysosomal dysfunction is closely linked to neurodegenerative proteinopathies. In the context of Alzheimer's disease (AD), the lysosomal protein phospholipase D3 (PLD3) has been identified as a potential molecular player, but PLD3's distinct role in lysosome function is not completely understood. Method:We used immunohistochemistry, western blot, and proximity ligation assays to determine PLD3's localization, expression level, post-translational modification and protein-protein interaction profile. Experiments were performed using iPSC-neuron cultures and human brain tissue obtained from patients with AD and neurological controls through the Vanderbilt Brain and Biospecimen Bank.Result: PLD3 is neuronal widely distributed throughout brain regions relevant to memory and cognition and PLD3 levels were significantly lower in AD brains compared to the controls. We discovered that PLD3 ubiquitination was significantly increased in AD brains relative to the controls, providing a possible explanation for the PLD3 reduction and localization into hallmark pathological structures of the AD brain. Furthermore, PLD3-ubiquitin complexes were enriched in parenchymal β-amyloid deposits and intraneuronal tau pathologies, both neuropil threads and neurofibrillary tangles.We provide the first experimental evidence for the physical interaction between PLD3 and Cathepsin D (CTD), both in human brain tissue and iPSC neurons. The interaction between PLD3/CTD was promoted by exposure of iPSC-neurons to human Aβ seeds isolated from post-mortem brain, suggesting this protein-protein interaction is relevant to Aβ pathology. We found this heterodimer was dramatically reduced in AD brain, especially in dystrophic neurites. The loss of this interaction in dystrophic neurites is notable because even though PLD3 levels are reduced in the AD brain, both PLD3 and CTD are present in abundance around β-amyloid plaques. The loss of PLD3/CTD interaction observed in AD brain is only partly explained by reduced expression of