Aims

Several lines of recent evidence indicate that the amyloid precursor protein-derived C-terminal fragments (APP-CTFs) are etiological triggers of Alzheimer’s disease (AD) pathology[1]. Altered mitochondrial homeostasis is also considered as an early event in AD development. Strikingly, we and others demonstrated the localization of APP-derived fragments in mitochondria-associated membranes[2]. However, the specific contribution of APP-CTFs to mitochondrial structure, function, and mitophagy defects remains to be established.

Methods

We used human neuroblastoma SH-SY5Y cells expressing the familial APPswe mutation or C99 fragment. We studied 2xTgAD, 3xTgAD mice and adeno-associated-virus (AAV)-C99 injected mice[3]. To discriminate between APP-CTFs and Abeta, we modulated pharmacologically secretases activity. Finally, we analyzed human post-mortem sporadic AD brains.

Results

We demonstrated in cells, that APP-CTFs fragments induce mitochondrial fragmentation, cristae disorganization, mitochondrial hyperpolarization, and a higher production of mitochondrial ROS, independently of Abeta. Moreover, APP-CTFs trigger mitophagic failure characterized by the activation and accumulation of autophagic markers and mitochondrial proteins. We confirmed the contribution of APP-CTFs accumulation to morphological mitochondria alteration and impaired basal mitophagy in vivo. Importantly, we showed that APP-CTFs accumulation correlates with mitophagy failure in AD brains[4].

Conclusions

This study unravels the toxicity of APP-CTFs, independently of Abeta, towards mitochondria dysfunctions and mitophagy in AD. Potential pharmacological approaches should focus on mitophagy activation to force the elimination and/or renewal of harmful mitochondria.

[1] Lauritzen I, et al. Acta Neuropathol 2016.

[2] Del Prete D. et al. J Alzheimers Dis 2017.

[3] Bourgeois A, et al. Neurobiol Aging 2018.

[4] Vaillant-Beuchot L.^#, Mary A.^# (co-authors), Acta Neuropathol 2020, under revision.

Aims

Besides the two main Alzheimer’s disease (AD) hallmarks (neurofibrillary tangles and senile plaques), the alteration of the structure and function of mitochondria was shown to be linked to the toxicity of Aβ peptides, in particular through the increase of reactive oxygen species (ROS) production. Furthermore, we have recently shown that APP C-terminal fragments (C83 and C99) localize to mitochondria-associated membranes (1) and trigger mitochondrial dysfunctions, overproduction of ROS and blockade of the mitophagy process (2).

Aims : We assessed mitochondrial structure and function and analyzed mitophagy markers in a cohort of fibroblasts isolated from sporadic AD patients.

Methods

We used fibroblasts from control, mild-cognitive impairment (MCI), or AD (AD) patients. Mitochondrial cristae organization as well as area, perimeter and number were quantified by electron microscopy. Measurements of mitochondrial ROS production and mitochondrial potential were analyzed by flow cytometry. The expression of proteins involved in mitophagy and mitochondrial dynamics were evaluated by SDS-PAGE.

Results

Fibroblasts from AD patients exhibit disorganization of mitochondria cristae ultrastructure with an increase of size in comparison of control and MCI cells. Electronic microscopy analyses also reveal an enlargement of endosomes in AD fibroblasts. Furthermore, mitochondrial function is also disrupted with an alteration of the mitochondrial membrane potential and modifications of expression of mitophagy markers.

Conclusions

Altogether, our data highlight mitochondria impairment in peripheral cells of AD patients and emphasize their potential use in AD diagnosis and personalized medicine.

(1) Del Prete D, et al. JAD 2017.

(2) Vaillant-Beuchot L^#, Mary A^#, et al. Under revision, Acta Neuropathologica.

Aims

Mitochondria structure and function alterations are major features of AD (1). The AMP-activated protein kinase (AMPK) promotes mitochondrial health, and multiple AMPK targets are involved in various aspects of mitochondrial homeostasis, mitophagy, and inflammatory response. We investigate the impact of AMPK signaling cascade on mitochondrial dysfunctions, mitophagy and neuroinflammation in AD.

Methods

We used SH-SY5Y cells overexpressing the AD Swedish mutation (APPswe), which accumulate Aβ and APP C-terminal fragments (APP-CTFs), ex vivo in hippocampal organotypic slices transduced with APPswe lentiviruses, and in 3xTgAD mice. We studied mitochondrial functions and mitophagy using biochemical, and imagery approaches. We analyzed neuroinflammation in mice using immunohistochemistry, RT-qPCR, and flux cytometry analyses. APP processing was modulated using γ-secretase inhibitor, or harboring the APPswe expression. We used pharmacological and genetic tools to modulate AMPK expression and activity.

Results

Cells expressing APPswe display a repressed AMPK cascade. Inversely, γ-secretase-mediated blockade of Aβ production and accumulation of APP-CTFs enhance AMPK activity, mitochondrial dysfunctions, and trigger mitophagy blockade. Interestingly, repressing AMPK cascade amplify APP-CTFs accumulation, impair mitochondrial functions, activate mitophagy and trigger dendrite shape alterations. Interestingly, activation of AMPK shows beneficial effects by increasing the number of mature dendritic spines and rescue the learning capacity of 3xTgAD mice. Studies investigating the impact of AMPK modulation on neuroinflammation in 3xTgAD mice are ongoing.

Conclusions

Our data highlight a pathogenic role of AMPK cascade repression in AD and question the potential of AMPK stimulation as a novel track for AD therapy.

(1) Vaillant-Beuchot, L^#., Mary, A^#. et al. ^#Co authors. Acta Neuropathologica (under revision).

Aims

Mitochondrial dysfunctions have been described in neurodegenerative diseases including Alzheimer’s disease (AD). Age-related accumulation of mitochondrial DNA (mtDNA) mutations and altered mtDNA copy number (mtDNAcn) can both disrupt mitochondrial energetics. Here, we disentangle how mtDNA quality and quantity are related to pathologies in different brain regions, and whether mtDNA alterations affect cognition independent of pathologies and neuronal loss.

Methods

We used whole genome sequencing data to estimate mtDNAcn and detect mtDNA heteroplasmies in n=1361 samples from 5 brain regions. Cognitive scores, (semi-)quantitative measurements of 10 neuropathologies, and neuronal proportions were available for n=454 dorsolateral prefrontal cortex (DLPFC) samples and n=242 cerebellum samples.

Results

The mtDNAcn was consistently decreased in AD in all four cortical regions but not in the cerebellum. In a multivariable regression with 10 different neuropathologies, only tau (p=0.004) remained significantly negatively associated with mtDNAcn in the DLPFC. Tau also remained significant when adjusting for neuronal proportion, which was positively associated with mtDNAcn (p=5.5×10^-5). Age and gender were not associated with mtDNAcn. Interestingly, mtDNAcn had a significant effect on cognition (p=0.034) after adjustment for pathologies and neuronal proportion. In cortical regions, mtDNA heteroplasmies were more frequent than in the cerebellum and increased by 1.64% per year (p=2.8×10^-5), but were not associated with pathologies or cognition.

Conclusions

MtDNA heteroplasmies in the cortex accumulate with age, but this process is unrelated to brain pathologies in our data. In contrast, mtDNAcn reduction is associated with tau burden and independently affects cognitive performance, indicating that maintaining mtDNAcn levels may attenuate tau-driven cognitive decline.

Aims

Mitochondrial damage and oxidative stress are well-established players in the pathophysiology of several neurodegenerative diseases, including Parkinson’s disease, Alzheimer’s disease, and Down syndrome (DS). Using a novel high-resolution density step-gradient to isolate and fractionate subpopulations of extracellular vesicles (EVs) from the brain parenchyma, we investigated the effect of mitochondrial dysfunction on the number and content of EVs in DS brains.

Methods

We isolated EVs from murine and human DS and diploid control post-mortem brains or from cell media. EVs were analyzed by nanoparticle tracking analysis, cryogenic electron microscopy, Western blotting, mass spectrometry, and qPCR.

Results

We found that the extracellular matrix of the brain contains a newly identified population of metabolically active, double-membrane, electron-dense EVs of mitochondrial origin that we have named ‘mitovesicles’. In vitro study revealed that oxidative stress enhances mitovesicle release in a mitophagy-independent fashion. In wild-type brains, we revealed that mitovesicles are low in number and encapsulate a specialized subset of mitochondrial constituents that reflects only partially the composition of intracellular mitochondria. Conversely, in human and murine DS brains, mitovesicle number is higher when compared to controls. The content is also modified, as the amount of the pro-inflammatory mitochondrial DNA in mitovesicles was higher in DS compared to controls.

Conclusions

Brain mitovesicles are tightly regulated in normal conditions and are modified in DS, suggesting that mitovesicles are a previously unrecognized player in mitochondria quality control and may have a yet undiscovered role in the inter-cellular response to oxidative stress and neuroinflammation.

Aims

Mitochondrial dysfunction is a hallmark of Alzheimer’s disease (AD). However, how mitochondria activity varies along pathology progression is still poorly understood. Additionally, untangling the effects of elevated Aβ from those due to APP overexpression is a common challenge when interpreting mitochondrial phenotypes.

Methods

We performed several functional analyses using isolated hippocampal mitochondria at different disease stages as well as primary neurons from App^NL-F and App^NL-G-F mouse models containing a humanized Aβ region with pathogenic mutations.

Results

Hippocampal mitochondria isolated from pre-/early-symptomatic App^NL-G-F mice show upregulated mitochondrial complex I and IV activities and higher capacity to generate ATP as compared to wildtype. This results in increased basal and maximal mitochondrial respiration. Primary App^NL-F neurons follow a similar pattern, with increased mitochondrial respiration to compensate for glycolytic defects, suggesting that embryonic neurons may mimic early disease stages. Interestingly, both isolated mitochondria from 2 months-old App^NL-G-F mice and primary App^NL-F neurons show lower mitochondrial calcium handling capacity. Additionally, App^NL-F neurons show significant deficits in anterograde mitochondrial movement. These data reveal an early mitochondrial dysfunction, despite increased ATP production. With pathology progression, App^NL-G-F mitochondria show severe deficits on mitochondrial complex I-III activities, and decreased mitochondrial maximal respiration induced by the uncoupler FCCP. Moreover, synapses from both 12 months-old App^NL-F and App^NL-G-F mice show lower number of mitochondria, suggesting that mitochondrial deficits may influence synaptic integrity.

Conclusions

Overall, we show that App knock-in mice have an early upregulation of mitochondrial activity accompanied by deficits on mitochondrial calcium handling and movement, which may influence late mitochondrial activity decay.

Aims

Alzheimer’s disease (AD) is a complex multifactorial disease in which neural death occurs predominantly by apoptosis. The p53 protein functions as a key regulator of apoptosis and accumulates in brains from AD patients. This protein naturally occurs in humans in two functional polymorphic variants, resulting in Arg or Pro at residue 72, which regulates p53 apoptotic activity. Here we evaluate the relevance of the p53 Arg72Pro single nucleotide polymorphism (SNP) in Amyloid-β(Aβ)-induced neurotoxicity both in vitro and in vivo.

Methods

We used primary cortical neurons from humanized Tp53 Arg72Pro knock-in mice treated with Aβ oligomers (10 µM) and an Aβ (5 nmol) intracerebroventricular injection model in Tp53 Arg72Pro knock-in mice to assess mitochondrial function, neurodegeneration and cognitive status.

Results

We found that the Arg genotype increased neuronal susceptibility to Aβ toxicity upon p53 mitochondrial stabilization in vitro. Accordingly, Arg72 mice were more susceptible to Aβ-induced neurodegeneration and memory loss than Pro72 mice. Interestingly, treatment with PFTμ (40 mgKg^-1), a small molecule that inhibits p53 mitochondrial binding without affecting its transactivation activity, prevented Aβ-induced memory deficit in Arg72 mice.

Conclusions

Our results demonstrate that blocking the p53-mitochondria interaction prevents cognitive decline in Aβ-injected mice. Moreover, PFTμ may be useful to develop novel therapeutic approaches for AD, especially in patients carrying the Arg72 SNP variant.

Funded by Instituto de Salud Carlos III (PI18/00265; RD16/0019/0018); FEDER; European Union’s Horizon 2020 Research and Innovation Programme (Grant Agreement 686009) and Junta de Castilla y León (Escalera de Excelencia (CLU-2017-03 Cofinanciado por el P.O. FEDER de Castilla y León).