Aβ-driven nuclear pore complex dysfunction alters activation of necroptosis proteins in a mouse model of Alzheimer’s Disease

Reviewer #1 (Public Review):

Summary:
This manuscript describes a deficiency in nuclear pore complexes (NPCs) to maintain proper compartmentalization between the nucleus and cytoplasm in a mouse model of AD-related Aβ pathology. Experiments demonstrate NPC dysfunction in cultured neurons and mouse tissue as a result of intracellular Aβ, which may cause reduced levels of certain nucleoporins, leading to a reduced number of NPCs, and their dysfunction in nuclear protein import and maintaining nucleocytoplasmic compartmentalization. In addition, the authors also report a potential mechanism for how NPC dysfunction may result in increased vulnerability to inflammation-induced necroptosis, where core components are reportedly activated via phosphorylation through nucleocytoplasmic shutting. Overall, the study is interesting and well conducted and reveals striking NCT defects in a Aβ pathology disease model that may have important implications for our understanding of AD pathology.

Strengths:
Previous studies have found nucleocytoplasmic transport (NCT) defects in other models of age-related neurodegenerative diseases, including Huntington's disease, tauopathy, C9orf72-linked frontotemporal dementia / amyotrophic lateral sclerosis (FTD/ALS), and TDP-43 proteinopathy in FTD/ALS. Typically, NCT defects have been linked mechanistically to aberrant co-aggregation of nucleoporins with e.g. TDP-43 and tau found in disease models and sometimes also human autopsy tissue. This study is novel, in that it describes NCT defects that are caused by Alzheimer's disease (AD) related Aβ pathology, using a human APP knock-in mouse model (AppNL-G-F/NL-G-F) that exhibits robust Aβ pathology in the CNS. The main focus of this study is on the barrier dysfunction of the NPCs leading to compartmentalization defects, while previous publications in the field have focused more on active protein import and RNA export defects. This is of considerable interest since an age-dependent decline in NPC barrier function has been observed in transdifferentiated neurons derived from normal-aged fibroblasts (Mertens et al., 2015). The potential link of NPC dysfunction to an increased vulnerability to inflammation-induced necroptosis may also be relevant to other neurodegenerative disorders with NCT dysfunction. Experiments are largely focused on either dissociated neuronal cultures, or studies using mouse tissue at different stages of disease progression. Experiments are mostly based on immunocytochemistry (ICC) and histochemistry (IHC) of nucleoporins to show morphological NPC defects and fluorescent reporter constructs and dyes of defined MW to show NPC dysfunction. The experiments using an anti-nuclear pore O-linked glycoprotein antibody [RL1], which recognizes multiple metazoan nucleoporins that are modified via post-translational O-GlcNAcylation, show a very striking reduction in staining intensity that is also replicated with antibodies specific for the FG-motif rich Nup98 and the very stable and essential NPC component Nup107. Taken together, the fluorescence microscopy studies convincingly support the claim of NPC dysfunction leading to defective compartmentalization between the nucleus and cytoplasm.

Weaknesses:
However, the molecular mechanisms leading to NPC dysfunction and the cellular consequences of resulting compartmentalization defects are not as thoroughly explored. Results from complementary key experiments using western blot analysis are less impressive than microscopy data and do not show the same level of reduction. The antibodies recognizing multiple nucleoporins (RL1 and Mab414) could have been used to identify specific nucleoporins that are most affected, while the selection of Nup98 and Nup107 is not well explained. There is also no clear hypothesis on how Aβ pathology may affect nucleoporin levels and NPC function. All functional NCT experiments are based on reporters or dyes, although one would expect widespread mislocalization of endogenous proteins, likely affecting many cellular pathways. The second part of this manuscript reports that in App KI neurons, disruption in the permeability barrier and nucleocytoplasmic transport may enhance activation of key components of the necrosome complex that include receptor-interacting kinase 3 (RIPK3) and mixed lineage kinase domain1 like (MLKL) protein, resulting in an increase in TNFα-induced necroptosis. While this is of potential interest, it is not well integrated in the study. This potential disease pathway is not shown in the very simple schematic (Fig. 8) and is barely mentioned in the Discussion section, although it would deserve a more thorough examination.

Reviewer #2 (Public Review):

Summary:
The authors try to establish that there is an Abeta-dependent loss of nuclear pores early in Alzheimer's disease. To do so the authors compared different NUP proteins and assessed their function by analyzing nuclear leakage and resistance to induction of nuclear damage and the associated necroptosis. The authors use a mouse knockin for hAPP with familial Alzheimer's mutations to model amyloidosis related to Alzheimer's disease. Treatment with an inhibitor of beta-amyloid production partially rescued the loss of nuclear pore proteins in young KI neurons, implicating beta-amyloid in Nuclear Pore dysfunction, a mechanism already described in other neurodegenerative diseases but not in Alzheimer's disease.

The conclusions of this paper related to familial AD are well supported by data but are not related to an aging decline in NUP function, where it is required to extend data analysis and one additional experiment.

1. Adding statistics and comparisons between wild-type changes at different times/ages to determine if the nuclear pore changes with time in wild-type neurons. The images show differences in the Nuclear pore in neurons from the wild-type mice, with time in culture and age. However, a rigorous statistical analysis is lacking to address the impact of age/development on NUP function. Although the authors state that nuclear pore transport is reported to be altered in normal brain aging, the authors either did not design their experiments to account for the normal aging mechanisms or overlooked the analysis of their data in this light.

2. Add experiments to assess the contribution of wild-type beta-amyloid accumulation with aging. It was described in 2012 (Guix FX, Wahle T, Vennekens K, Snellinx A, Chávez-Gutiérrez L, Ill-Raga G, Ramos-Fernandez E, Guardia-Laguarta C, Lleó A, Arimon M, Berezovska O, Muñoz FJ, Dotti CG, De Strooper B. 2012. Modification of γ-secretase by nitrosative stress links neuronal ageing to sporadic Alzheimer's disease. EMBO Mol Med 4:660-673, doi:10.1002/emmm.201200243) and 2021 (Burrinha T, Martinsson I, Gomes R, Terrasso AP, Gouras GK, Almeida CG. 2021. Upregulation of APP endocytosis by neuronal aging drives amyloid-dependent synapse loss. J Cell Sci 134. doi:10.1242/jcs.255752), 28 DIV neurons are senescent and accumulate beta-amyloid42. In addition, beta-amyloid 42 accumulates normally in the human brain (Baker-Nigh A, Vahedi S, Davis EG, Weintraub S, Bigio EH, Klein WL, Geula C. 2015. Neuronal amyloid-β accumulation within cholinergic basal forebrain in ageing and Alzheimer's disease. Brain 138:1722-1737. doi:10.1093/brain/awv024), thus, it would be important to determine if it contributes to NUP dysfunction. Unfortunately, the authors tested the Abeta contribution at div14 when wild-type Abeta accumulation was undetected. It would enrich the paper and allow the authors to conclude about normal aging if additional experiments were performed, namely, treating 28Div neurons with DAPT and assessing if NUP is restored.

Reviewer #3 (Public Review):

Summary:
This manuscript reports the novel observation of alterations in the nuclear pore (NUP) components and the function of the nuclear envelope in knock-in models of APP and presenilin mutations. The data show that loss of NUP immunoreactivity (IR) and pore density are observed at times prior to plaque deposition in this model. The loss of NUP IR is correlated with an increase in intraneuronal Abeta IR with two monoclonal antibodies that react with the N-terminus of Abeta. Similar results are observed in cultured neurons from APP-KI and Wt mice where further results with cultured neurons indicate that Abeta "drives" this process: incubation of neurons with oligomeric, but not monomeric or fibrillar Abeta causes loss of NUP IR, incubation with conditioned media from KI cells but not wt cells also causes loss of NUP IR and treatment with the gamma secretase inhibitor, NAPT partially blocks the loss of NUP IR. Further data show that nuclear envelope function is altered in KI cells and KI cells are more sensitive to TNFalpha-induced necroptosis. This is potentially an important and significant report, but how this fits within the larger picture of what is known about amyloid aggregation and accumulation and pathogenesis in neurons needs to be clarified. The results from mouse brains are strong, while the results from cultured cells are in some instances are of a lower magnitude, less convincing, ambiguous, and sometimes over-interpreted.

Strengths:
1. Loss of NUP expression and activity is a novel observation.
2. Its association with intraneuronal Abeta immunoreactivity suggests an association with Alzheimer's disease.
3. The experiments generally appear to be well-controlled.
4. Multiple approaches are sometimes used to increase the robustness of the data.

Weaknesses:
1. It does not consider the relationship of the findings here to other published work on the intraneuronal perinuclear and nuclear accumulation of amyloid in other transgenic mouse models and in humans.
2. It appears to presume that soluble, secreted Abeta is responsible for the effect rather than the insoluble amyloid fibrils.
3. Most of the critical findings on the association with Abeta and the functional consequences are done in cultured neurons and not in mouse models.
4. There is no evidence from the human brain that would strengthen the significance.
5. It is not clear when the alteration in NUP expression begins in the KI mice as there is no time at which there is no difference between NUP expression in KI and Wt and the earliest time shown is 2 months. If NUP expression is decreased from the earliest times at birth, then this makes the significance of the observation of the association with amyloid pathology less clear.