Reevaluating the cause of laminopathy in Alzheimer’s disease

Alzheimer’s disease (AD) is the most common form of dementia and is diagnosed clinically by cognitive deficits, and anatomically by accumulation of extracellular amyloid plaques and intracellular neurofibrillary tangles (NFT) containing hyper-phosphorylated Tau. Over the past three decades of AD research, the “amyloid hypothesis” gained the most attention as the main player in neurodegeneration. This was mostly based on specific hereditary forms of AD and patients with Down syndrome that linked the level of amyloid-beta (Aβ) directly to the risk of developing these diseases. However, in non-hereditary forms of AD, little correlation exists between Aβ levels or its location with the clinical stages of the disease. While still a hotly debated topic, a large body of literature based on years of preclinical models, suggests that Aβ deposition alone is not sufficient to induce AD, rather it is a pre-requisite for formation of NFT by promoting Tau hyper-phosphorylation. Neuropathology and imaging studies also suggest that hyper-phosphorylation of Tau and its spreading through the brain, better conform to the clinical stages of the disease (Long and Holtzman, 2019). Accumulation of Aβ and NFT are observed in normal aging, which indicates the perturbation of neuronal homeostatic systems including the ubiquitin proteasomal system and lysosomal autophagy. These systems are sensitive to reactive oxygen species-mediated damage and are specifically augmented in patients with AD. Experimental conditions that can reactivate cellular antioxidative capacity have been shown to improve clinical outcomes in animal models of the disease. Nuclear factor erythroid 2-related factor 2 is a major transcriptional regulator of cellular antioxidants and is reported to decrease in AD, explaining the excessive oxidative damage in this disease. DNA hypermethylation is suggested to be responsible for nuclear factor erythroid 2-related factor 2 downregulation and its pharmacological reversal is shown to be protective as it positively alleviates the increased β-amyloid precursor protein cleaving enzyme 1 activity, the enzyme responsible for increased Aβ-deposition and cognitive loss in animal models (Bahn et al., 2019). Excessive β-amyloid precursor protein cleaving enzyme 1 activation is also associated with epigenetic changes including DNA hypomethylation in autopsy samples from human patients (Do Carmo et al., 2016). These data collectively indicate that chromatin remodeling is an important underlying mechanism in the pathophysiology of AD and that Aβ plaques and NFTs might be the consequence of nuclear events and abnormal gene expression and not the primary cause of neuronal death. Therefore, attempts for removing Aβ and Tau toxicity, even if successful cannot cure the disease or reverse the cognition loss. A major question remaining in the pathophysiology of AD is the cause of epigenetic changes and chromatin remodeling. Evidence of oxidative stress involvement in the induction of nuclear changes and downstream reshaping of chromatin as well as aberrant gene regulation was originally shown in a model of the neurodegeneration in Drosophila. The group showed that increased expression of pathological Tau was associated with nuclear chromatin relaxation and cell death. Oxidative stress was also sufficient to enhance euchromatin abundance. These degenerative changes were rescued after increasing the heterochromatin/euchromatin ratio (Frost et al., 2014). Chromatin rearrangement can be caused by damage/changes in the nuclear envelope. The envelope is connected to the cytoskeleton on its outer side and the chromatin on the inner side, by a thin protein layer, known as the nuclear lamina. The major constituents of the nuclear lamina are a class of detergent-resistant proteins with a molecular mass of 65–70 kDa, known as nuclear lamins. The lamina is a complex array of interconnecting intermediate filaments, with an overall diameter of 15–20 nm, and is composed of two major classes of lamins: A-type lamin (lamin A, C, lamin C2, and ΔA10) and B-type lamin (lamin B1, B2, and lamin B3). Lamin A and lamin C are the most common forms of the A-type lamins and are encoded by a single gene LMNA, and B-type lamins are products of two separate genes, LMNB1 and LMNB2. Lamin A, B1, and B2 contain a common C-terminal CaaX motif, in which C = Cys, a = aliphatic amino acids, and X can be any other amino acids, usually represented by Methionine, and is the site of post-translational modifications including farnesylation, phosphorylation, methylation. During the last few years, we have learned that lamins’ involvement in cellular biology is beyond the originally defined structural role, and these proteins play important roles in developmental stages and disease progression. Our knowledge of nuclear lamina role in health and disease has expanded vastly because of a group of diseases known as laminopathy that are caused by structural and functional abnormalities in nuclear lamins. A few hundred of mutations in LMNA have been identified that are linked to different disease conditions. Initial studies have shown that lamin A is not expressed in all cell types, including embryonic stem cells, intestinal epithelial cells, and connective tissues. The expression of lamin A is only detectable in postnatal heart and skeletal muscle cells. B-type lamins are expressed in all cell types, indicating their importance for all nucleated cells. Lamin B1 level plays a determining role in brain development, and its deletion is shown to decrease neurogenesis and promote astrocyte formation in mouse embryonic stem cells (Mahajani et al., 2017). In contrast to the widespread involvement of LMNA mutations in laminopathies, there is no evidence of LMNB mutations in any diseases. However, duplication of the LMNB1 gene has been shown in autosomal dominant leukodystrophy, a central nervous system disease diagnosed with degeneration of myelin in white matter. Evidence of lamin B1 involvement in neurodegenerative diseases is increasingly reported. In Huntington’s disease, an increase of lamin B1 protein was shown in CNS neurons with a deformed nuclear morphology that was associated with altered nucleocytoplasmic transport in the R6/1 mouse model of Huntington’s disease. The mice also showed changes in lamin B1 in the hippocampal neurons with induced LAD-associated chromatin modifications and transcriptional dysregulation of some genes including LAMP5, PRX, NEUROD6, CAMK2A, etc. (Alcala-Vida et al., 2021). Loss of lamin B1 has also been reported in Parkinson’s disease models that are induced by the administration of α-synuclein preformed fibrils or 1-methyl-4-phenylpyridinium (MPP+) (Verma et al., 2021). Depletion of lamin B1 in neurons resulted in progressive loss of dopaminergic neurons in the Protocerebral Anterior Medial cluster in Drosophila. This effect was more prevalent in aged Drosophila indicating that lamin B1 level is crucial for normal aging of the brain (Oyston et al., 2018). This is further supported by the aging-associated loss of lamin B1 in granular and molecular cell layers of the hippocampal region and astrocytes as well as in a cellular model of aging (Matias et al., 2022). Research from our group has predominantly focused on finding the underpinning mechanism/s or molecular players that mediate nuclear lamina damage under oxidative stress. Lamin B1 is the main component of the nuclear lamina in neural cells and is critically involved in the regulation of cellular resistance to oxidative stress. Using SH-SY5Y neuroblastoma cell lines, we have shown that stress such as serum deprivation is associated with nuclear damage as detected by nuclear lamina invagination and mild degradation of lamin B1. This coincided with the spontaneous inactivation of the thioredoxin antioxidant system. Thioredoxin-1 (Trx1) is a cytoplasmic thiol with the highest reducing capacity amongst the other cellular thiols for oxidized proteins. This is mediated through electron donation from two cysteine thiols at its active site. While the target protein is reduced, Trx1 is oxidized and must be reduced by Trx-reductase-1 (Trx-R1). The involvement of the Trx1 system in nuclear integrity in our in vitro model of oxidative stress was further confirmed by pharmacologic and genetic inhibition of Trx1 or Trx-R1. We showed that inhibition of Trx but not glutathione system severely enhanced nuclear lamina invagination and lamin B1 degradation. We identified that elevated caspase-6 was responsible for the catalytic degradation of lamin B1. Previous studies have linked the upregulation of caspase-6 to mild cognitive impairment which was attenuated by the inhibition of caspase-6 (Zhou et al., 2019). These observations highlight the role of Trx1 in the pathophysiology of AD, as it is decreased in autopsy samples from AD patients’ brains. Additionally, our study provided a mechanistic link between oxidative stress and the reported caspase-6 activity in the early stages of AD (Islam et al., 2019). Despite the in vitro and in vivo data from our animal studies highlighting the Trx1-caspase-6-lamin B1 axis in AD, examination of AD patients’ brain tissue did not provide evidence of caspase-6 activation, despite the presence of neuronal nuclear lamina invagination and proteolytic cleavage of lamin B1. Considering that caspase-6 activation is regarded as an early biomarker of AD, we asked whether another system might be contributing to nuclear damage in the later stages of the disease. Using the amyloid beta 42 (Aβ42) toxicity model in primary mouse and rat hippocampal and cortical neurons we detected cathepsin L as another protease responsible for the cleavage of lamin B1, though in a different fashion from caspase-6. Our cellular model showed that cathepsin L-mediated cleavage of lamin B1 can induce nuclear lamina invagination and histone modifications marked by acetylation and methylation at lysine9. These results were further corroborated in postmortem human AD patients’ brain tissue (Islam et al., 2022). These observations link chromatin remodeling to decreased cellular antioxidants and are in agreement with a recent report by another group (Moore et al., 2019) that showed abnormal chromatin relaxation in oxidative stress conditions. The implication of two different proteases, lysosomal/nuclear and cytoplasmic, remains to be investigated. Caspase-6 is a unique member of apoptosis machinery that is involved in a variety of cellular defense systems including inflammasome activation and execution of cell death. Evidence of simultaneous activation of both enzymes for degradation of nuclear lamina was detectable in western blotting from hippocampal homogenates in 3xTg mice model of AD, However, we did not detect any evidence of caspase-6 activation in the post-mortem AD hippocampus or in our cellular model of Aβ42 toxicity. The cathepsin L-mediated lamin B1 cleavage was also observed in human postmortem AD tissue and after Aβ42 toxicity in cells. Since caspase-6 is enzymatically degraded by cathepsin L in Aβ42 treated cells and in in vitro enzymatic assays (Islam et al., 2022), we suggest a temporal pattern in the degradation of nuclear lamina by caspase-6 in the early stages of AD followed by lysosomal dysfunction and activation of cathepsin L as the executioner of cell death in later stages of the disease. Other plausible explanation could be that the two different proteolytic events may occur simultaneously in different cell types which cannot be properly examined by western blotting. Moreover, the use of cell lines, primary neurons, and variance in autopsy brain tissue might not reflect the exact etiology of neurodegeneration, which could be overcome by using a large number of human tissue and more animal models of AD. Overall, our research has illuminated the molecular machinery that is involved in nuclear laminopathy (Figure 1). We linked the oxidative stress and lysosomal dysfunction to the induction of nuclear lamina damage and invagination that results in epigenetic changes as we showed by changes in histone modification. These studies suggest that neuronal nucleus might be a new frontier for fighting neurodegeneration. Our work also provides experimental therapeutic targets that can be further pursued for finding treatments for AD.Figure 1: Schematic illustration of laminopathy and histone modifications induced by Aβ42 and Trx1 oxidation.(1, 2, 3) Aβ42 induces endoplasmic reticulum stress and lysosomal membrane permeabilization activating CTSL which cleaves LB1 and induces histone modifications. The gene expression pattern resulted from histone modifications requires further studies. CTSL inhibits caspase-6 mediated LB1 cleavage by degrading caspase-6. Oxidation/inhibition of Trx1 activates caspase-6 and initiates LB1 cleavage and downstream changes. Aβ42: Amyloid-beta 42; CTSL: cathepsin L; ER: endoplasmic reticulum; LB1: lamin B1; Trx1: thioredoxin-1. Created with BioRender.com.C-Editors: Zhao M, Liu WJ, Qiu Y; T-Editor: Jia Y

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