Mitochondrial ubiquitin ligase alleviates Alzheimer's disease pathology via blocking the toxic amyloid-β oligomer generation

Alzheimer's disease (AD) is a prevalent neurodegenerative disorder characterized by cognitive decline and memory impairment. Amyloid-beta (Aβ) aggregation in the brain is a crucial early step in AD pathogenesis. While Aβ plaques (fibrils and prefibrils) are a hallmark of AD, growing evidence suggests that soluble Aβ oligomers are also highly pathogenic. Familial AD mutations can lead to oligomer accumulation without plaque formation, and in sporadic AD, soluble Aβ oligomer levels correlate with disease severity more strongly than insoluble Aβ. However, the in vivo mechanisms determining whether Aβ forms oligomers or plaques remain unclear. Mitochondrial dysfunction is another common feature of AD, suggesting a role for mitochondrial homeostasis in disease development. Mitochondria undergo constant fusion and fission, processes crucial for maintaining mitochondrial quality. AD pathology, including Aβ, disrupts mitochondrial dynamics. MITOL/MARCH5, a mitochondrial E3 ubiquitin ligase, is a key regulator of mitochondrial morphology and ER tethering, modulating substrates involved in fusion and fission. Neuron-specific MITOL knockout mice show mitochondrial structural abnormalities, and Alzbase databases suggest reduced MITOL expression in AD patients. This study investigates the role of MITOL in Aβ pathology and mitochondrial dysfunction in AD.

Extensive research has established the central role of amyloid-beta (Aβ) peptide aggregation in Alzheimer's disease (AD) pathogenesis. While the presence of senile plaques, composed of aggregated Aβ fibrils, is a hallmark of the disease, increasing evidence points to the significant contribution of soluble Aβ oligomers as the primary toxic species driving neuronal dysfunction and cognitive impairment. Studies have demonstrated that soluble oligomers disrupt synaptic plasticity, induce oxidative stress, and trigger neuroinflammation, all contributing to the progression of AD. Moreover, the relationship between Aβ oligomer accumulation and mitochondrial dysfunction in AD is becoming increasingly recognized. Mitochondria play a crucial role in cellular energy production, calcium homeostasis, and apoptosis regulation. Impairments in mitochondrial function, such as reduced oxidative phosphorylation and increased oxidative stress, have been consistently observed in AD brains, linking mitochondrial damage to the neurodegenerative process. The dynamic interplay between Aβ aggregation and mitochondrial dysfunction remains an area of active research, with investigations focusing on the mechanisms by which Aβ oligomers directly interact with mitochondria to disrupt their function and the role of mitochondrial dysfunction in influencing Aβ aggregation pathways.

The study employed both in vivo and in vitro approaches. In vivo, the researchers used APP/PS1 transgenic mice, a well-established model for AD-related Aβ pathology. Cerebral cortex- and hippocampus-specific MITOL knockout (KO) mice were generated by crossing MITOLF/F mice with Emx1-Cre transgenic APP/PS1 mice. The researchers analyzed mitochondrial morphology using transmission electron microscopy (TEM), mitochondrial respiratory activity via COX staining, and ATP production. Behavioral tests, including the novel object recognition test, Y-maze test, and Barnes maze test, assessed cognitive function. Immunohistochemistry and immunoblotting analyzed neuronal morphology, synaptic markers (synaptophysin and PSD-95), microglial activation (Iba1 and CD68), and pro-inflammatory cytokines (TNFα, IL-1β, and IL-6). Aβ metabolism was characterized by analyzing APP processing products, Aβ species (Aβ38, Aβ40, Aβ42, Aβ43), and Aβ-degrading enzyme expression. The seeding activity of Aβ fibrils was assessed using a previously described method involving purification of Aβ fibrils from the brain and analyzing their ability to catalyze the aggregation of free Aβ monomers using ThT fluorescence. Aβ oligomer levels were measured by ELISA and dot blot analysis using Aβ oligomer-specific antibodies. In vitro studies used SH-SY5Y cells stably expressing APPswe, with siRNA targeting PS1 to mimic Aβ pathology. Oxygen consumption rates (OCRs) were measured using a Seahorse XFp Analyzer. Cell death assays (LDH release and caspase-3/7 activity) were performed to evaluate the neurotoxicity of Aβ fibrils and oligomers. Rifampicin (RFP), an antibiotic shown to inhibit Aβ oligomerization, was used to test the role of Aβ oligomers in the observed phenotypes. Statistical analysis included Student's t-tests and one-way ANOVA with post hoc tests.

1. MITOL expression was significantly downregulated in the cerebral cortex of 15-month-old APP/PS1 mice compared to non-transgenic controls, confirming observations from Alzbase database. This downregulation was also observed in a cellular model expressing APPswe and siPS1. 2. MITOL deletion in APP/PS1 mice led to increased numbers of smaller mitochondria with disrupted inner membranes, reduced mitochondrial respiratory activity (COX staining), and decreased ATP production, indicating accelerated mitochondrial dysfunction. 3. MITOL-deficient APP/PS1 mice exhibited more severe cognitive impairments in behavioral tests (object recognition, Y-maze, and Barnes maze) compared to APP/PS1 mice with normal MITOL levels. 4. MITOL deletion worsened neuronal atrophy, reduced synaptic markers (synaptophysin, PSD-95), and increased microglial activation and pro-inflammatory cytokine expression. 5. Despite exacerbating Aβ toxicity, MITOL deletion did not affect the total number or overall size of Aβ plaques. However, it significantly increased the size of the fibrillized core within the plaques, suggesting enhanced fibrillization within existing plaques. This effect was corroborated by the finding that MITOL deletion selectively increased the amount of Aβ43. 6. Aβ fibrils purified from MITOL-deficient APP/PS1 brains displayed significantly higher seeding activity than those from APP/PS1 mice, shortening the lag time in the aggregation of free Aβ monomers. 7. MITOL deletion dramatically increased the levels of soluble Aβ oligomers in APP/PS1 mice, particularly off-pathway oligomers, indicating that the enhancement of Aβ secondary assembly plays a role in the generation of Aβ oligomers. 8. Aβ fibrils from MITOL-deficient APP/PS1 mice induced greater cell death and caspase-3/7 activation in SH-SY5Y cells, particularly in the presence of free Aβ monomers, highlighting their enhanced neurotoxicity. 9. Treatment with rifampicin (RFP), an inhibitor of Aβ oligomerization, rescued the synaptic loss, microglial activation, and cognitive deficits in MITOL-deficient APP/PS1 mice, confirming the central role of Aβ oligomers in the observed pathology.

This study provides compelling evidence that MITOL, a regulator of mitochondrial dynamics, plays a crucial protective role against Aβ-induced neurotoxicity in AD. The findings demonstrate that MITOL downregulation, observed in AD patients, leads to mitochondrial dysfunction, which in turn exacerbates Aβ pathology primarily by increasing the production of toxic Aβ oligomers. This effect is not due to increased Aβ production or impaired clearance, but rather to enhanced seeding of Aβ fibrils, leading to accelerated secondary aggregation of free Aβ monomers and a disproportionate accumulation of oligomers. The study highlights the importance of considering the balance between spontaneous Aβ self-assembly and fibril-catalyzed secondary assembly in AD pathogenesis. The results further underscore the importance of Aβ oligomers as key mediators of AD-related neurotoxicity and suggest a potential therapeutic strategy targeting mitochondrial function or Aβ oligomer formation to mitigate AD pathology. The rescue of cognitive impairments by RFP strongly supports the conclusion that Aβ oligomers are the main drivers of the observed pathology in this model.

This research reveals a novel mechanism linking mitochondrial dysfunction to Aβ pathology in Alzheimer's disease. MITOL, a crucial regulator of mitochondrial morphology, prevents the excessive generation of toxic Aβ oligomers by inhibiting the seeding effect of Aβ fibrils. MITOL deficiency exacerbates AD pathology through the preferential accumulation of toxic Aβ oligomers, leading to severe cognitive deficits. The study's findings provide strong support for further investigation into therapeutic strategies targeting mitochondrial function and Aβ oligomer formation as potential treatments for Alzheimer's disease. Future research could focus on the specific molecular mechanisms by which MITOL regulates Aβ aggregation and explore the therapeutic potential of targeting MITOL or its associated pathways.

The study primarily utilized a mouse model of AD, which may not fully recapitulate the complexity of human AD. The mechanisms linking mitochondrial dysfunction to enhanced Aβ fibril seeding remain to be fully elucidated. While the study provides strong evidence for the involvement of Aβ oligomers, it does not exclude the possibility that other factors contribute to the observed pathology. The impact of MITOL levels on other aspects of AD pathogenesis beyond Aβ aggregation requires further investigation.