LATE-NC aggravates GVD-mediated necroptosis in Alzheimer's disease

Abstract

It has become evident that Alzheimer's Disease (AD) is not only linked to its hallmark lesions-amyloid plaques and neurofibrillary tangles (NFTs)-but also to other co-occurring pathologies. This may lead to synergistic effects of the respective cellular and molecular players, resulting in neuronal death. One of these co-pathologies is the accumulation of phosphorylated transactive-response DNA binding protein 43 (pTDP-43) as neuronal cytoplasmic inclusions, currently considered to represent limbic-predominant age-related TDP-43 encephalopathy neuropathological changes (LATE-NC), in up to 70% of symptomatic AD cases. Granulovacuolar degeneration (GVD) is another AD co-pathology, which also contains TDP-43 and other AD-related proteins. Recently, we found that all proteins required for necroptosis execution, a previously defined programmed form of neuronal cell death, are present in GVD, such as the phosphorylated necroptosis executioner mixed-lineage kinase domain-like protein (pMLKL). Accordingly, this protein is a reliable marker for GVD lesions, similar to other known GVD proteins. Importantly, it is not yet known whether the presence of LATE-NC in symptomatic AD cases is associated with necroptosis pathway activation, presumably contributing to neuron loss by cell death execution. In this study, we investigated the impact of LATE-NC on the severity of necroptosis-associated GVD lesions, phosphorylated tau (pTau) pathology and neuronal density. First, we used 230 human post-mortem cases, including 82 controls without AD neuropathological changes (non-ADNC), 81 non-demented cases with ADNC, i.e.: pathologically-defined preclinical AD (p-preAD) and 67 demented cases with ADNC. We found that Braak NFT stage and LATE-NC stage were good predictors for GVD expansion and neuronal loss in the hippocampal CA1 region. Further, we compared the impact of TDP-43 accumulation on hippocampal expression of pMLKL-positive GVD, pTau as well as on neuronal density in a subset of nine non-ADNC controls, ten symptomatic AD cases with (AD^TDP+) and eight without LATE-NC (AD^TDP-). Here, we observed increased levels of pMLKL-positive, GVD-exhibiting neurons in AD^TDP+ cases, compared to AD^TDP- and controls, which was accompanied by augmented pTau pathology. Neuronal loss in the CA1 region was increased in AD^TDP+ compared to AD^TDP- cases. These data suggest that co-morbid LATE-NC in AD impacts not only pTau pathology but also GVD-mediated necroptosis pathway activation, which results in an accelerated neuronal demise. This further highlights the cumulative and synergistic effects of comorbid pathologies leading to neuronal loss in AD. Accordingly, protection against necroptotic neuronal death appears to be a promising therapeutic option for AD and LATE.

Keywords: Cell death; Granulovacuolar degeneration; LATE-NC; Necroptosis; Protein aggregation; TDP-43; pMLKL; pTau.

Fig. 1

The necroptosis pathway and its presumed activation in AD. Necroptosis is a programmed form of cell death which starts with the activation of death receptors. RIPK1 is then recruited and self-phosphorylated [12]. The inhibition or activation of caspase-8 determines whether necroptosis or apoptosis takes place. If caspase-8 is inhibited, the necrosome complex is formed, which includes activated pRIPK1, pRIPK3 and pMLKL. This complex translocates to the cell membrane, resulting in membrane swelling and bursting mediated by pMLKL, the necroptosis executor [12]. This is followed by release of intracellular components, inflammatory response and ultimately neuronal death. Necroptosis activation has been previously linked to AD [12]. Here, the activated necrosome components pRIPK1, pRIPK3 and pMLKL accumulate in GVD granules presumably indicating an aberrant/delayed execution process of necroptosis in neurons with GVD [41]

Fig. 2

Tau, LATE-NC and GVD expansion are strongly associated. a Network constructed with graphical LASSO reveals that GVD is strongly associated with Braak NFT stage and mediates its relationship with LATE-NC stage (n = 221). The presence of connecting lines, i.e., edges between nodes (variables) indicates a positive and non-spurious partial correlation coefficient (with r values displayed on the edges and represented by the width of an edge). Edges representing correlation coefficients higher than 0.4 are marked in blue. b Semi-partial correlation matrix adjusted for sex shows that LATE-NC and Braak NFT stages are significantly correlated with GVD stage and CDR score, and that Braak NFT, LATE-NC and GVD stages negatively correlates with CA1-hippocampus neuronal density (n = 64). The p-values were adjusted with the Holm–Bonferroni method

Fig. 3

TDP-43 and pTau are co-expressed in necroptosis-positive neurons. Triple labeling immunofluorescence with antibodies against pTDP-43 (S409/S410), pTau (S202/T205) and pMLKL (S358) in the CA1 sub-hippocampal field of an AD^TDP+ case. Nuclei are stained with Hoechst solution. Of note, a pTDP-43 and pMLKL are co-localized in some GVD granules (arrows) and b pTDP-43 and pTau co-localize in nearby neurons (arrowheads). We exclude the signal observed being considered as unspecific lipofuscin because such signal usually shows in the blue (Hoeschst) channel as orange granules, which in this case is absent

Fig. 4

pMLKL and pTau severity as well as neuronal loss are increased in AD cases with LATE-NC. a Local accumulation of phosphorylated tau and MLKL is observed in AD^TDP+. DAB immunohistochemical staining of pTDP-43 (S409/S410), pTau (S202/T205) and pMLKL (S358) in the CA1-subiculum field of a control, AD^TDP− and AD^TDP+ case (cases 7, 16 and 21 are displayed, see Table 2). Scale bars = 50 µm. b AD^TDP+ cases display decreased neuronal density compared to controls and AD^TDP− and controls. Quantitative data representing the number of total neurons per mm² per group in the CA1 subfield of the hippocampus. Quantification of the number of CA1 positive neurons for c pTDP-43 d pMLKL and e pTau. AD^TDP+ cases display significantly higher severity of pMLKL and pTau lesions. Data are presented as mean ± SEM. N = 27 (controls = 9, AD^TDP− = 8, AD^TDP+  = 10). f Partial Spearman correlation matrix controlled for age and multiple comparisons (Holm-Bonferroni test) in this cohort confirms that the accumulation of pTDP-43, pMLKL and pTau pathologies are significantly correlated with neuronal density in the CA1, AβMTL phase and Braak NFT stage. Supporting images showing overviews of the whole hippocampus of cases 12, 16, 21 and 22 (Table 2) stained with pMLKL can be found in the public repository BioImage Archive, with the hyperlink: https://www.ebi.ac.uk/biostudies/studies/S-BIAD514?key=475a3bbe-6fc9-476e-8e45-6429422b85bf

Fig. 5

Proposed TDP-43, pTau and pMLKL interplay in AD. Aβ and TDP-43 pathologies accelerate pTau pathology in AD (black and orange arrows, respectively). We also hypothesize that TDP-43 exacerbates necrosome-positive GVD lesions through pTau (blue arrow), giving rise to neurons bearing tangles, pTDP-43 inclusions and pMLKL-positive GVD. This eventually leads to neurodegeneration and neuronal loss