Nuclear alpha-synuclein is present in the human brain and is modified in dementia with Lewy bodies

Abstract

Background Dementia with Lewy bodies (DLB) is pathologically-defined by the cytoplasmic accumulation of alpha-synuclein (aSyn) within neuronal cells in the brain. aSyn is predominately pre-synaptic, but has been reported present in various subcellular compartments in cell and animal models. In particular, nuclear aSyn (aSynNuc) is evident in-vitro and in disease models and has been associated with altered DNA integrity, gene transcription, nuclear homeostasis. However, owing to various factors, the presence of aSynNuc in the human brain remains controversial, as does its role in synucleinopathies. Methods Here, we close this gap and provide a unique demonstration confirming the presence of aSynNuc in control (Con) and in DLB post-mortem brain tissue via immunohistochemistry, immunoblot, and label-free mass-spectrometry (MS). Results Discrete intra-nuclear aSyn puncta reactive against phosphorylated serine 129-aSyn (pS129-aSyn) and pan-aSyn antibodies were observed in cortical neurons and non-neuronal cells in fixed brain sections and isolated nuclear preparations from Con and DLB cases. Subsequent biochemical analysis of subcellular fractionated tissue confirmed aSynNuc as present at levels ~10-fold lower than in the cytoplasm. Critically, however, an increase in monomeric pS129-aSyn was observed in DLB cases alongside higher molecular weight pan- and pS129-reactive aSyn species, consistent with the formation of intranuclear phosphorylated aSynNuc oligomers. Furthermore, the occurrence of aSynNuc was confirmed via MS, with 6 unique aSyn derived peptide sequences identified in nuclear fractions (71.4% aSyn sequence coverage). Conclusions Collectively, our data confirm the presence of aSynNuc in human brain tissue and describe DLB-associated nuclear pathology. These findings address a major controversy in the synucleinopathy field by confirming the presence of aSynNuc in autoptic human brain tissue and, for the first time, identify that aSyn is aggregated into novel and potentially pathological assemblies in the nucleus as part of the DLB disease process and thus may contribute to the DLB phenotype.

Neurons with Cat’s Eyes: A Synthetic Strain of α-Synuclein Fibrils Seeding Neuronal Intranuclear Inclusions

“…Work is also in progress to further decipher the atomic organization of the 1B fibrils and to identify the cascade of molecular events prevailing to the intranuclear seeding of NIIs. Due to their small size (14 kDa), α-Syn monomers and small oligomers (<4 mer) can passively diffuse through the nuclear pore sieve and gain access the nucleoplasm [80,81]. However, regarding amyloid fibril seeds, specific transport mechanisms relying on karyopherins [82,83] are in all likelihood involved.…”

Neighbouring modifications interfere with the detection of phosphorylated alpha-synuclein at Serine 129: Revisiting the specificity of pS129 antibodies

“…We speculated that this could be due to the presence of predominantly truncated and phosphorylated C-terminal fragments of aSyn in the nucleus. A recent study by Koss et al reported strong pS129 staining in neuronal cells in the brain of healthy and DLB individuals 55 . Despite the strong signals detected by ICC, WB analysis showed that the levels of pS129 are minimal in the nuclear fraction (< 5%) relative to the total aSyn protein level 55 .…”

“…A recent study by Koss et al reported strong pS129 staining in neuronal cells in the brain of healthy and DLB individuals 55 . Despite the strong signals detected by ICC, WB analysis showed that the levels of pS129 are minimal in the nuclear fraction (< 5%) relative to the total aSyn protein level 55 . The discrepancy between the ICC signal and levels of pS129 detected by WB suggests that the pS129 antibodies could potentially be cross-reacting with other nuclear proteins.…”

“…We speculated that this could be due to the presence of predominantly truncated and phosphorylated C-terminal fragments of aSyn in the nucleus. A recent study by Koss et al reported strong pS129 staining in neuronal cells in the brain of healthy and DLB individuals 55 .…”

Aggregated Alpha-Synuclein Inclusions within the Nucleus Predict Impending Neuronal Cell Death in a Mouse Model of Parkinsonism

“…Despite early suggestions of its localization to the nucleus, the presence of nuclear aSyn has been controversial due to the existence of nonspecific staining when certain antibodies are used [ 1 , 12 ]. Recently, however, multiple different groups and approaches have confirmed that aSyn can localize to the nucleus and its function there is still being unraveled [ 13 , 14 , 15 , 16 ]. Evidence suggests that nuclear aSyn can have normal physiologic functions, including regulating gene transcription [ 15 ], participating in nucleocytoplasmic transport [ 17 ], and facilitating DNA double-strand break repair [ 13 ].…”