Necrosome complex detected in granulovacuolar degeneration is associated with neuronal loss in Alzheimer’s disease

Abstract

Alzheimer's disease (AD) is characterized by a specific pattern of neuropathological changes, including extracellular amyloid beta (Aβ) deposits, intracellular neurofibrillary tangles (NFTs), granulovacuolar degeneration (GVD) representing cytoplasmic vacuolar lesions, and synapse and neuronal loss. Necroptosis, a programmed form of necrosis, has recently been shown to be involved in AD. Necroptotic cell death is characterized by the assembly of the necrosome complex, consisting of phosphorylated proteins, i.e. receptor-interacting serine/threonineprotein kinase 1 and 3 (pRIPK1 and pRIPK3), and mixed lineage kinase domain-like protein (pMLKL). However, it is not yet clear whether necrosome assembly takes place in the brain regions showing AD-related neuronal loss, and whether it is associated with AD-related neuropathological changes. Here, we analyzed brains of AD, pathologically defined preclinical AD, and non-AD control cases to determine the neuropathological characteristics and distribution pattern of the necrosome components. We demonstrated that all three activated necrosome components can be detected in GVD lesions (GVDn+, i.e. GVD with activated necrosome) in neurons, and colocalized with classical GVD markers, such as pTDP-43 and CK1δ. GVDn+ neurons were inversely associated with the neuronal density in the early affected CA1 region of the hippocampus and in the late affected frontal cortex layer III. Finally, the distribution of non-phosphorylated proteins was studied. RIPK1 was mainly expressed in astrocytes and in GVD lesions, RIPK3 was detected in dystrophic neurites of neuritic plaques and in neurons. GVD lesions remained negative for non-phosphorylated RIPK3. MLKL could only be detected by western blotting showing an increase in p-preAD and AD cases. Accordingly, AD-related GVD lesions exhibited all components of the activated necrosome and were associated with reduced neuronal densities in the affected anatomical regions, and with AD-defining parameters, showing the strongest correlation and partial colocalization with NFT pathology. Therefore, we conclude that the presence of the necrosome in GVD plays a role in AD, possibly by representing an AD-specific form of necroptosis-related neuron death. Hence, necroptosis-related neuron loss could be an interesting therapeutic target for treating AD.

Untangling the origin and function of granulovacuolar degeneration bodies in neurodegenerative proteinopathies

“…Hence, GVBs and AD-related tau pathology follow a similar spatiotemporal distribution pattern in the human brain. In line with this, the Thal GVD and NFT Braak stage significantly correlate [68,132]. Moreover, GVBs and tau pathology co-localize at the single cell level in the AD brain: GVBs are typically detected in neurons that also exhibit tau pathology [5,43,72,81,90,111,122,135,146].…”

“…However, the distribution of Aβ deposits [12,13] and GVBs [132] in the AD brain follow a dissimilar pattern. In line with this, no correlation between GVBs and the occurrence of extracellular Aβ aggregates was found in AD patients [68,135] and non-demented elderly controls [125]. Furthermore, both tau pathology and GVBs are detected in non-demented controls lacking Aβ plaques [131], indicating that GVB formation occurs independently of Table 3 Box: GVB immunogenicity…”

“…GVBs have been most extensively studied in the brains of AD patients. The amount of neurons with GVBs is higher in the hippocampus of AD patients compared to age-matched controls [8,9,43,68,125,144,145]. Fig.…”

“…Alternatively, some phosphorylation-dependent antibodies may bind non-specifically to phospho-epitopes that are immunological mimics of their phosphorylated target protein. Recently, it was published that the necroptosis-related proteins RIPK1 and RIPK3 could be detected in GVBs by generic, phosphorylation-independent antibodies only after dephosphorylation of the human brain tissue [68]. This indicates that for some proteins, phosphatase treatment may be necessary to reveal their GVB localization.…”

“…However, in the human brain the distribution pattern of GVBs is similar when determining the Thal GVD stage using CK1δ, CK1ε or pTDP-43 [132]. Furthermore, no difference was found when quantifying the percentage of GVB-positive neurons in the human brain using antibodies against CK1δ or the newly discovered GVB-localizing protein pMLKL of which the staining pattern overlaps with CK1δ-and pTDP-43-positive GVBs [68]. This indicates that also pMLKL is a suitable GVB marker in the human brain, whereas its use in experimental models remains to be validated.…”