Aims

Altered mitochondrial homeostasis occurs early in Alzheimer’s disease (AD) and has been consistently linked to Amyloid beta (Aβ). We investigated the contribution of the amyloid precursor protein C-terminal fragments (APP-CTFs) to mitochondrial defects and examined the therapeutic potential of the AMP-activated protein kinase (AMPK) signaling cascade.

Methods

We studied mitochondria structure, function and mitophagy in cells and mice models mimicking familial forms of AD (FAD). We investigated the contribution of APP-CTFs by modulating β- and γ-secretases activities, expressing APP-CTFs in cells and mice, using a transgenic mouse model accumulating APP-CTFs in the absence of Aβ, and by validating our data in human sporadic AD (SAD) samples. We pharmacologically and genetically modulated AMPK in AD models.

Results

We demonstrated the localization and the accumulation of APP-CTFs in mitochondria of both FAD models and SAD samples [1-3]. We reported that APP-CTFs accumulation triggers, independently from Aβ, mitochondrial structure and function alterations and leads to mitophagic failure phenotype [3]. We reported a repressed AMPK cascade in AD and showed that this contributes to APP-CTFs accumulation, mitochondrial dysfunction and mitophagy. Inversely, activation of AMPK cascade showed several beneficial effects including mitochondria structure, dendritic spines maturation, learning capacity and neuroinflammation.

Conclusions

We unravelled the toxicity of APP-CTFs towards mitochondria, and highlighted a pathogenic role of AMPK cascade repression in AD. Our data pointed-out AMPK cascade as a potential AD therapeutic approach.

1.Del Prete, D., et al. J Alzheimers Dis, 2017.

2.Eysert, F., et al. Int J Mol Sci, 2020.

3.Vaillant-Beuchot, L., et al. Acta Neuropathol, 2021.

Aims

The significant increase in life expectancy in the last 60 years urges the handling of age-associated diseases. Alzheimer's disease (AD) is currently considered the major cause of dementia. However, despite being one of the most studied neurodegenerative diseases, little therapeutic progress was achieved in the last decades. Familial Alzheimer’s disease comprises of 5% of the cases and display an early onset, making it a valuable model for the study of the disease. One of the hallmarks of AD is its mitochondrial metabolism disruption. Here, we aim to investigate the metabolic mechanisms which are altered in AD.

Methods

We successfully generated and characterized neuroprogenitor cells (NPCs) from familial AD, their respective isogenic controls, and healthy subjects-derived iPSCs. Our differentiation protocol makes use of a 15 days 3D embryoid body formation step, followed by FACS sorting of the NPC population (CD271-, CD44-, CD184+, CD24+, CD15-/+) which renders a population of more than 90% NPC. The overall expression of metabolic pathways proteins was determined by proteomics. Metabolic activity was assessed by measurements of mitochondrial respiration, glycolytic rate, and metabolic substrate usage.

Results

We report an overall decrease in glycolytic proteins expression and substrate usage, OCR and ECAR parameters.

Conclusions

Our results indicate that metabolic alterations are already perceivable in AD-derived NPCs, which can be a target for early intervention. References: 1. N. Gunhanlar et al., Mol Psychiatry 23, 1336-1344 (2018); 2. M. Oksanen et al., Stem Cell Reports 9, 1885-1897 (2017); 3. M. Trombetta-Lima et al., Cell Calcium 94:102362 (2021).

Aims

Basal forebrain cholinergic neuron (BFCN) degeneration is a hallmark of aging and Alzheimer’s disease (AD). The reasons for this degeneration are unclear. BFCNs depend on retrograde axonal transport of neurotrophins like pro nerve growth factor (proNGF) for survival and function. ProNGF transport is reduced in aging and AD and coincides with loss of its receptor, tropomyosin-related kinase A (TrkA), while pan-neurotrophin receptor (p75NTR) levels remain unchanged. We sought to determine whether oxidative stress accounts for these reductions.

Methods

Embryonic rat BFCNs cultured in microfluidic chambers were incubated in antioxidant-poor medium to induce oxidative stress. Thioredoxin-1 and thioredoxin reductase or protein tyrosine phosphatase 1B (PTP1B) siRNA or antagonist TCS401 were applied exclusively to the cell bodies. Retrograde transport was assayed by adding quantum dot-labelled cleavage-resistant proNGF or proNGFs that bind only to TrkA (proNGF-KKE) or to p75NTR (proNGF-9/13) to the axon terminals.

Results

ProNGF retrograde transport depended upon TrkA but not p75NTR. Antioxidant deprivation reduced TrkA immunoreactivity and proNGF retrograde transport without affecting p75NTR levels. Oxidative stress triggered axonal degeneration in the presence of proNGF or proNGF-9/13 but not proNGF-KKE. Knockdown or inhibition of PTP1B, which regulates TrkA trafficking, reduced TrkA levels, decreased proNGF retrograde transport and lowered axonal uptake of proNGF-KKE. Treatment of antioxidant-deprived BFCNs with thioredoxin-1, which reactivates oxidized PTP1B, increased TrkA levels and rescued proNGF transport and axonal degeneration.

Conclusions

Oxidative stress reduces TrkA levels and proNGF transport through a PTP1B-dependent mechanism. Impaired proNGF retrograde transport due to oxidative PTP1B-mediated TrkA loss may contribute to BFCN degeneration in aging and AD.

Aims

RNA modifications offer a new and emerging field of cellular regulation and are involved in several human diseases. However, each RNA modification must be considered individually, because depending on localization and modification level, its effects may be different. Therefore we aim to unravel the function and impact of specific RNA modifications in the mitochondrial epitranscriptome and observe their involvement in mitochondrial dysfunction during Alzheimer’s Disease.

Methods

To address specific sites in mitochondrial RNA, a sequencing based approach is used, coupled with a reverse transcription. RNA modifications are evoking specific patterns of truncation, mismatch and jumps in the complementary strand during reverse transcription. Therefore modification levels can be estimated after Illumina sequencing and a bioinformatics analysis. Writer enzymes can be characterized in different models via Western Blot or evaluation of human databases.

Results

We found that the RNA modification N1-Methyladenosine (m¹A) is elevated on a specific mitochondrial mRNA site. Besides protein levels of the corresponding writer enzyme are increased in an AD cell and animal model and mRNA levels are increased in excitatory and inhibitory neurons of human AD patients. Our results suggest, that modified mRNA inhibits protein translation, probably provoking mitochondrial dysfunction.

Conclusions

Modifications of mitochondrial mRNA sites might interfere with translation and therefore impair the synthesis of mitochondrial proteins. Moreover, we demonstrate that there is a correlation of mRNA modification content, writer enzyme level and the associated synthesized protein.

Aims

To investigate the role of NRF2 in synaptic plasticity and mitochondrial function in neurons isolated from the A53T alpha Synuclein (A53TSyn) mouse model of alpha synuclein accumulation. Our lab has demonstrated that loss of NRF2 results in diminished synaptic density and impaired mitochondrial function in healthy neurons and that its activation improves those endpoints. Here we explore the role NRF2 in a pathological context using A53TSyn neurons.

Methods

Hippocampal neurons from A53TSyn embryos or their wild-type (WT) littermates were grown in co-culture with glial cells for 4 weeks. In the final week neurons were exposed to either the NRF2 activating compound dimethyl fumarate (DMF) or the NRF2 inhibitor ML385. Dendritic arborization and spine density were quantified. Gene expression, mitochondrial function and levels of reactive oxygen species (ROS) were also analyzed in hippocampal A53TSyn and WT neurons treated with DMF or ML385.

Results

A53TSyn neurons displayed a progressive reduction in dendritic complexity over time in culture relative to WT neurons. This was accompanied by a decrease in spine density, diminished mitochondrial function and increased levels of ROS. DMF reversed all of these changes in the A53Tsyn neurons whereas ML385 exacerbated them. Similar effects of ML385 were observed in WT neurons although DMF treatment did elicit any effects.

Conclusions

These data show that NRF2 activation can attenuate alpha synuclein-related synaptic and mitochondrial dysfunction. Because of the strong correlation between synaptic density and cognitive function, these data suggest that NRF2 may be an important target to improve cognitive function in Parkinson’s disease.

Aims

Mitochondria and mitochondrial-related pathways have become an attractive target for disease-modifying strategies, as mitochondrial dysfunction prior to clinical onset has been widely described in Alzheimer’s Disease (AD) patients and AD animal models. We aim to provide an updated overview of the mechanisms that regulate mitochondrial function and to survey mitochondrial-related pathways in publicly available findings and datasets that study AD among other neurodegenerative diseases.

Methods

Methods: Selected transcriptomic study data was parallelly screened for mitochondrial-related pathways and analyzed for gene-set enrichment considering sex-, cell-, and stage-specific changes in AD.

Results

Mitochondrial- and nuclear-encoded genes of which expression impacts mitochondrial function (e.g. oxidative phosphorylation, autophagy, mitophagy, citric acid cycle) are differentially expressed in AD in a sex-specific manner. In a stage-dependent manner, neuronal cell types are affected earlier than glial cells such as oligodendrocytes and astrocytes in AD. Transcriptomic responses of microglia against amyloid-β or tau protein yields on differential expression patterns of metabolic pathways of interest such as glycolysis, fatty acid oxidation, HIF-1α, and calcium signaling. Amyloid-β induces a transcriptomic signature similar to inflammatory microglia, and tau protein similar to homeostatic microglia.

Conclusions

Several studies have placed mitochondrial dysfunction as a process that contributes to neuronal cell loss, microglial activation and the onset of AD. Whether the dysregulated pathways we observed in AD are not only biomarkers but represent therapeutic targets remains to be investigated. OMICs technologies represent a novel avenue to delineate disease mechanisms not only in AD but in other neurodegenerative diseases.

Aims

To date, there are no effective treatments for amyotrophic lateral sclerosis (ALS), highlighting the importance of unraveling the mechanisms leading to motor neuron loss. One candidate is the WW domain-containing oxidoreductase (WWOX), widely involved in neurodegeneration.

Methods

Western blots were used to assess the levels of WWOX, and protein involved in the mitochondrial electron transport chain in ALS and control post-mortem motor cortex (mCTX). Project MinE data was assessed to identify genetic variants in WWOX. Cell viability, ATP and reactive oxygen species (ROS) were assessed in SH-SY5Y cells following WWOX knock down using small interfering RNA (siWWOX) or treatment with wild-type and mutant recombinant WWOX proteins. Furthermore, siWWOX was used in a fly model and alterations in behavior were assessed.

Results

WWOX levels were decreased in ALS mCTX and we identified several rare and ALS specific variants in WWOX. Among these variants, the stop codon mutation at amino acid 261 decreased cell viability, reduced ATP levels, and increased ROS in vitro, consistent with decreases in the mitochondrial membrane ATP synthase of complex V and the cytochrome c oxidase of complex IV in ALS mCTX. Furthermore, siWWOX decreased ROS levels in SH-SY5Y cells, suggesting a link between loss of WWOX and increases in oxidative stress in ALS. Similarly, knocking down WWOX in a fly model reduced sleep supporting a pathogenic role for loss of WWOX.

Conclusions

Together, our findings suggest that loss of WWOX or mutations in the gene that lead to a truncated protein exacerbate mitochondrial dysfunction in ALS.