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

Abstract

Dementia with Lewy bodies (DLB) is pathologically defined by the cytoplasmic accumulation of alpha-synuclein (aSyn) within neurons in the brain. Predominately pre-synaptic, aSyn has been reported in various subcellular compartments in experimental models. Indeed, nuclear alpha-synuclein (aSynNuc) is evident in many models, the dysregulation of which is associated with altered DNA integrity, transcription and nuclear homeostasis. However, the presence of aSynNuc in human brain cells remains controversial, yet the determination of human brain aSynNuc and its pathological modification is essential for understanding synucleinopathies. Here, using a multi-disciplinary approach employing immunohistochemistry, immunoblot, and mass-spectrometry (MS), we confirm aSynNuc in post-mortem brain tissue obtained from DLB and control cases. Highly dependent on antigen retrieval methods, in optimal conditions, intra-nuclear pan and phospho-S129 positive aSyn puncta were observed in cortical neurons and non-neuronal cells in fixed brain sections and in isolated nuclear preparations in all cases examined. Furthermore, an increase in nuclear phospho-S129 positive aSyn immunoreactivity was apparent in DLB cases compared to controls, in both neuronal and non-neuronal cell types. Our initial histological investigations identified that aSynNuc is affected by epitope unmasking methods but present under optimal conditions, and this presence was confirmed by isolation of nuclei and a combined approach of immunoblotting and mass spectrometry, where aSynNuc was approximately tenfold less abundant in the nucleus than cytoplasm. Notably, direct comparison of DLB cases to aged controls identified increased pS129 and higher molecular weight species in the nuclei of DLB cases, suggesting putative pathogenic modifications to aSynNuc in DLB. In summary, using multiple approaches we provide several lines of evidence supporting the presence of aSynNuc in autoptic human brain tissue and, notably, that it is subject to putative pathogenic modifications in DLB that may contribute to the disease phenotype.

Post-mortem evidence for a reciprocal relationship between genomic DNA damage and alpha-synuclein pathology in dementia with Lewy bodies

“…a+b). As previously reported[34], nuclear pS129 aSyn was increased in the DLB cohort, in NeuN +ve and NeuN -ve nuclei (p<0.01, for both, Fig 3. a+c).…”

“…Building on our prior studies of the nuclear localisation of aSyn [23, 34, 53] and its pathological modification in DLB cortical tissue[34], we here demonstrate a disease dependent accumulation of DSBs as per γH2A.X in neuronal and non-neuronal cells in the temporal cortex of DLB cases. γH2A.X results from the rapid serine 139 phosphorylation of histone H2.A and is instrumental in the initiation of DSB repair, facilitating DDR molecule recruitment and is terminated upon repair completion [39].…”

“…Consistently, both pathological modification of aSyn and SNCA gene knockout in rodents have been associated with a reduced capacity for DNA damage repair, leading to elevated levels of DSBs and downstream neurodegeneration [57, 79]. Strikingly, we recently demonstrated the occurrence of nuclear aSyn in postmortem human tissue and the occurrence of disease-dependent modifications of nuclear aSyn in DLB cases [34]. Nevertheless, the consequences of nuclear aSyn pathology upon genomic stability, DNA damage and the ensuing changes in nuclear proteasome have yet to be determined.…”

“…Slides were washed in Tris-buffered Saline (TBS; 5mM Tris, 145mM NaCl, pH 7.4) for 5 mins, prior to antigen retrieval. Optimised nuclear staining as previously determined [34] was achieved via pressured heating in EDTA (10mM, pH 8) for 2mins, prior to submersion in formic acid (90%, 10mins) at RT. Slides were washed in TBS x2 and in TBS with 0.1 % Tween-20 (TBST) for 5mins, prior to being blocked in TBST containing 10% normal goat serum (NBS, Sigma) for 1hr at RT.…”

“…Nuclear isolation was performed as previously reported [34]. In Brief, ∼250mg of frozen lateral temporal cortex from Con and DLB cases (n=9) were homogenised 1:16 (W:V) in nuclear extraction buffer (0.32 mM sucrose, 5 mM CaCl 2 , 3 mM Mg(Ac) 2 , 10 mM Tris-HCl, 0.1% NP-40, pH 8) supplemented with cOmplete proteases inhibitor cocktail and phostop tablets (1 per 10 ml, Sigma) via a dounce homogeniser (50 manual strokes per sample).…”