Early Presymptomatic Changes in the Proteome of Mitochondria-Associated Membrane in the APP/PS1 Mouse Model of Alzheimer's Disease.

Intracellular ß-amyloid (Aß) accumulation is an early event in Alzheimer's disease (AD) progression. Recently, it has been uncovered that presenilins (PSs), the key components of the amyloid precursor protein (APP) processing and the ß-amyloid producing γ-secretase complex, are highly enriched in a special sub-compartment of the endoplasmic reticulum (ER) functionally connected to mitochondria, called mitochondria-associated ER membrane (MAM). A current hypothesis of pathogenesis of Alzheimer's diseases (AD) suggests that MAM is involved in the initial phase of AD. Since MAM supplies mitochondria with essential proteins, the increasing level of PSs and ß-amyloid could lead to metabolic dysfunction because of the impairment of ER-mitochondrion crosstalk. To reveal the early molecular changes of this subcellular compartment in AD development MAM fraction was isolated from the cerebral cortex of 3 months old APP/PS1 mouse model of AD and age-matched C57BL/6 control mice, then mass spectrometry-based quantitative proteome analysis was performed. The enrichment and purity of MAM preparations were validated with EM, LC-MS/MS and protein enrichment analysis. Label-free LC-MS/MS was used to reveal the differences between the proteome of the transgenic and control mice. We obtained 77 increased and 49 decreased protein level changes in the range of - 6.365 to + 2.988, which have mitochondrial, ER or ribosomal localization according to Gene Ontology database. The highest degree of difference between the two groups was shown by the ATP-binding cassette G1 (Abcg1) which plays a crucial role in cholesterol metabolism and suppresses Aß accumulation. Most of the other protein changes were associated with increased protein synthesis, endoplasmic-reticulum-associated protein degradation (ERAD), oxidative stress response, decreased mitochondrial protein transport and ATP production. The interaction network analysis revealed a strong relationship between the detected MAM protein changes and AD. Moreover, it explored several MAM proteins with hub position suggesting their importance in Aß induced early MAM dysregulation. Our identified MAM protein changes precede the onset of dementia-like symptoms in the APP/PS1 model, suggesting their importance in the development of AD.

Chronic Cerebral Hypoperfusion Induced Synaptic Proteome Changes in the rat Cerebral Cortex.

Chronic cerebral hypoperfusion (CCH) evokes mild cognitive impairment (MCI) and contributes to the progression of vascular dementia and Alzheimer's disease (AD). How CCH induces these neurodegenerative processes that may spread along the synaptic network and whether they are detectable at the synaptic proteome level of the cerebral cortex remains to be established. In the present study, we report the synaptic protein changes in the cerebral cortex after stepwise bilateral common carotid artery occlusion (BCCAO) induced CCH in the rat. The occlusions were confirmed with magnetic resonance angiography 5 weeks after the surgery. Synaptosome fractions were prepared using sucrose gradient centrifugation from cerebral cortex dissected 7 weeks after the occlusion. The synaptic protein differences between the sham operated and CCH groups were analyzed with label-free nanoUHPLC-MS/MS. We identified 46 proteins showing altered abundance due to CCH. In particular, synaptic protein and lipid metabolism, as well as GABA shunt-related proteins showed increased while neurotransmission and synaptic assembly-related proteins showed decreased protein level changes in CCH rats. Protein network analysis of CCH-induced protein alterations suggested the importance of increased synaptic apolipoprotein E (APOE) level as a consequence of CCH. Therefore, the change in APOE level was confirmed with Western blotting. The identified synaptic protein changes would precede the onset of dementia-like symptoms in the CCH model, suggesting their importance in the development of vascular dementia.

Mitochondrial Proteome Changes Correlating with ß-Amyloid Accumulation.

Alzheimer's disease (AD) is a multifactorial disease of wide clinical heterogenity. Overproduction of amyloid precursor protein (APP) and accumulation of ß-amyloid (Aß) and tau proteins are important hallmarks of AD. The identification of early pathomechanisms of AD is critically important for discovery of early diagnosis markers. Decreased brain metabolism is one of the earliest clinical symptoms of AD that indicate mitochondrial dysfunction in the brain. We performed the first comprehensive study integrating synaptic and non-synaptic mitochondrial proteome analysis (two-dimensional differential gel electrophoresis (2D-DIGE) and mass spectrometry) in correlation with Aß progression in APP/PS1 mice (3, 6, and 9 months of age). We identified changes of 60 mitochondrial proteins that reflect the progressive effect of APP overproduction and Aß accumulation on mitochondrial processes. Most of the significantly affected proteins play role in the mitochondrial electron transport chain, citric acid cycle, oxidative stress, or apoptosis. Altered expression levels of Htra2 and Ethe1, which showed parallel changes in different age groups, were confirmed also by Western blot. The common regulator bioinformatical analysis suggests the regulatory role of tumor necrosis factor (TNF) in Aß-mediated mitochondrial protein changes. Our results are in accordance with the previous postmortem human brain proteomic studies in AD in the case of many proteins. Our results could open a new path of research aiming early mitochondrial molecular mechanisms of Aß accumulation as a prodromal stage of human AD.

Synaptic mitochondria: a brain mitochondria cluster with a specific proteome.

The synapse is a particularly important compartment of neurons. To reveal its molecular characteristics we isolated whole brain synaptic (sMito) and non-synaptic mitochondria (nsMito) from the mouse brain with purity validated by electron microscopy and fluorescence activated cell analysis and sorting. Two-dimensional differential gel electrophoresis and mass spectrometry based proteomics revealed 22 proteins with significantly higher and 34 proteins with significantly lower levels in sMito compared to nsMito. Expression differences in some oxidative stress related proteins, such as superoxide dismutase [Mn] (Sod2) and complement component 1Q subcomponent-binding protein (C1qbp), as well as some tricarboxylic acid cycle proteins, including isocitrate dehydrogenase subunit alpha (Idh3a) and ATP-forming ß subunit of succinyl-CoA ligase (SuclA2), were verified by Western blot, the latter two also by immunohistochemistry. The data suggest altered tricarboxylic acid metabolism in energy supply of synapse while the marked differences in Sod2 and C1qbp support high sensitivity of synapses to oxidative stress. Further functional clustering demonstrated that proteins with higher synaptic levels are involved in synaptic transmission, lactate and glutathione metabolism. In contrast, mitochondrial proteins associated with glucose, lipid, ketone metabolism, signal transduction, morphogenesis, protein synthesis and transcription were enriched in nsMito. Altogether, the results suggest a specifically tuned composition of synaptic mitochondria. BIOLOGICAL SIGNIFICANCE: Neurons communicate with each other through synapse, a compartment metabolically isolated from the cell body. Mitochondria are concentrated in presynaptic terminals by active transport to provide energy supply for information transfer. Mitochondrial composition in the synapse may be different than in the cell body as some examples have demonstrated altered mitochondrial composition with cell type and cellular function in the muscle, heart and liver. Therefore, we posed the question whether protein composition of synaptic mitochondria reflects its specific functions. The determined protein difference pattern was in accordance with known functional specialties of high demand synaptic mitochondria. The data also suggest specifically tuned metabolic fluxes for energy production by means of interaction with glial cells surrounding the synapse. These findings provide possible mechanisms for dynamically adapting synaptic mitochondrial output to actual demand. In turn, an increased vulnerability of synaptic mitochondria to oxidative stress is implied by the data. This is important from theoretical but potentially also from therapeutic aspects. Mitochondria are known to be affected in some neurodegenerative and psychiatric disorders, and proteins with elevated level in synaptic mitochondria, e.g. C1qbp represent targets for future drug development, by which synaptic and non-synaptic mitochondria can be differentially affected.