Rethinking Alzheimer's Disease Therapy: Are Mitochondria the Key?

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Mitochondrial hypermetabolism precedes impaired autophagy and synaptic disorganization in App knock-in Alzheimer mouse models.

Accumulation of amyloid β-peptide (Aβ) is a driver of Alzheimer's disease (AD). Amyloid precursor protein (App) knock-in mouse models recapitulate AD-associated Aβ pathology, allowing elucidation of downstream effects of Aβ accumulation and their temporal appearance upon disease progression. Here we have investigated the sequential onset of AD-like pathologies in AppNL-F and AppNL-G-F knock-in mice by time-course transcriptome analysis of hippocampus, a region severely affected in AD. Strikingly, energy metabolism emerged as one of the most significantly altered pathways already at an early stage of pathology. Functional experiments in isolated mitochondria from hippocampus of both AppNL-F and AppNL-G-F mice confirmed an upregulation of oxidative phosphorylation driven by the activity of mitochondrial complexes I, IV and V, associated with higher susceptibility to oxidative damage and Ca2+-overload. Upon increasing pathologies, the brain shifts to a state of hypometabolism with reduced abundancy of mitochondria in presynaptic terminals. These late-stage mice also displayed enlarged presynaptic areas associated with abnormal accumulation of synaptic vesicles and autophagosomes, the latter ultimately leading to local autophagy impairment in the synapses. In summary, we report that Aβ-induced pathways in App knock-in mouse models recapitulate key pathologies observed in AD brain, and our data herein adds a comprehensive understanding of the pathologies including dysregulated metabolism and synapses and their timewise appearance to find new therapeutic approaches for AD

Amyloid Β-Peptide Increases Mitochondria-Endoplasmic Reticulum Contact Altering Mitochondrial Function and Autophagosome Formation in Alzheimer's Disease-Related Models.

Recent findings have shown that the connectivity and crosstalk between mitochondria and the endoplasmic reticulum (ER) at mitochondria-ER contact sites (MERCS) are altered in Alzheimer's disease (AD) and in AD-related models. MERCS have been related to the initial steps of autophagosome formation as well as regulation of mitochondrial function. Here, the interplay between MERCS, mitochondria ultrastructure and function and autophagy were evaluated in different AD animal models with increased levels of Aβ as well as in primary neurons derived from these animals. We start by showing that the levels of Mitofusin 1, Mitofusin 2 and mitochondrial import receptor subunit TOM70 are decreased in post-mortem brain tissue derived from familial AD. We also show that Aβ increases the juxtaposition between ER and mitochondria both in adult brain of different AD mouse models as well as in primary cultures derived from these animals. In addition, the connectivity between ER and mitochondria are also increased in wild-type neurons exposed to Aβ. This alteration in MERCS affects autophagosome formation, mitochondrial function and ATP formation during starvation. Interestingly, the increment in ER-mitochondria connectivity occurs simultaneously with an increase in mitochondrial activity and is followed by upregulation of autophagosome formation in a clear chronological sequence of events. In summary, we report that Aβ can affect cell homeostasis by modulating MERCS and, consequently, altering mitochondrial activity and autophagosome formation. Our data suggests that MERCS is a potential target for drug discovery in AD

Essential versus accessory aspects of cell death: recommendations of the NCCD 2015

Cells exposed to extreme physicochemical or mechanical stimuli die in an uncontrollable manner, as a result of their immediate structural breakdown. Such an unavoidable variant of cellular demise is generally referred to as ‘accidental cell death’ (ACD). In most settings, however, cell death is initiated by a genetically encoded apparatus, correlating with the fact that its course can be altered by pharmacologic or genetic interventions. ‘Regulated cell death’ (RCD) can occur as part of physiologic programs or can be activated once adaptive responses to perturbations of the extracellular or intracellular microenvironment fail. The biochemical phenomena that accompany RCD may be harnessed to classify it into a few subtypes, which often (but not always) exhibit stereotyped morphologic features. Nonetheless, efficiently inhibiting the processes that are commonly thought to cause RCD, such as the activation of executioner caspases in the course of apoptosis, does not exert true cytoprotective effects in the mammalian system, but simply alters the kinetics of cellular demise as it shifts its morphologic and biochemical correlates. Conversely, bona fide cytoprotection can be achieved by inhibiting the transduction of lethal signals in the early phases of the process, when adaptive responses are still operational. Thus, the mechanisms that truly execute RCD may be less understood, less inhibitable and perhaps more homogeneous than previously thought. Here, the Nomenclature Committee on Cell Death formulates a set of recommendations to help scientists and researchers to discriminate between essential and accessory aspects of cell death

Neuronal cell-based high-throughput screen for enhancers of mitochondrial function reveals luteolin as a modulator of mitochondria-endoplasmic reticulum coupling

Background: Mitochondrial dysfunction is a common feature of aging, neurodegeneration, and metabolic diseases. Hence, mitotherapeutics may be valuable disease modifiers for a large number of conditions. In this study, we have set up a large-scale screening platform for mitochondrial-based modulators with promising therapeutic potential. Results: Using differentiated human neuroblastoma cells, we screened 1200 FDA-approved compounds and identified 61 molecules that significantly increased cellular ATP without any cytotoxic effect. Following dose response curve-dependent selection, we identified the flavonoid luteolin as a primary hit. Further validation in neuronal models indicated that luteolin increased mitochondrial respiration in primary neurons, despite not affecting mitochondrial mass, structure, or mitochondria-derived reactive oxygen species. However, we found that luteolin increased contacts between mitochondria and endoplasmic reticulum (ER), contributing to increased mitochondrial calcium (Ca2+) and Ca2+-dependent pyruvate dehydrogenase activity. This signaling pathway likely contributed to the observed effect of luteolin on enhanced mitochondrial complexes I and II activities. Importantly, we observed that increased mitochondrial functions were dependent on the activity of ER Ca2+-releasing channels inositol 1,4,5-trisphosphate receptors (IP3Rs) both in neurons and in isolated synaptosomes. Additionally, luteolin treatment improved mitochondrial and locomotory activities in primary neurons and Caenorhabditis elegans expressing an expanded polyglutamine tract of the huntingtin protein. Conclusion: We provide a new screening platform for drug discovery validated in vitro and ex vivo. In addition, we describe a novel mechanism through which luteolin modulates mitochondrial activity in neuronal models with potential therapeutic validity for treatment of a variety of human diseases

The modular systems biology approach to investigate the control of apoptosis in Alzheimer's disease neurodegeneration

Apoptosis is a programmed cell death that plays a critical role during the development of the nervous system and in many chronic neurodegenerative diseases, including Alzheimer's disease (AD). This pathology, characterized by a progressive degeneration of cholinergic function resulting in a remarkable cognitive decline, is the most common form of dementia with high social and economic impact. Current therapies of AD are only symptomatic, therefore the need to elucidate the mechanisms underlying the onset and progression of the disease is surely needed in order to develop effective pharmacological therapies. Because of its pivotal role in neuronal cell death, apoptosis has been considered one of the most appealing therapeutic targets, however, due to the complexity of the molecular mechanisms involving the various triggering events and the many signaling cascades leading to cell death, a comprehensive understanding of this process is still lacking. Modular systems biology is a very effective strategy in organizing information about complex biological processes and deriving modular and mathematical models that greatly simplify the identification of key steps of a given process. This review aims at describing the main steps underlying the strategy of modular systems biology and briefly summarizes how this approach has been successfully applied for cell cycle studies. Moreover, after giving an overview of the many molecular mechanisms underlying apoptosis in AD, we present both a modular and a molecular model of neuronal apoptosis that suggest new insights on neuroprotection for this disease

Mechanisms of apoptosis in secretory and neuronal cells : role of oxidative stress and calcium overload

Apoptosis is a widespread physiological mechanism to regulate tissue homeostasis bothduring development and in adult organs. However, the cell deletion program can beinappropriately activated or suppressed under pathological conditions. The present project wasdesigned to study the role of apoptosis in the toxicity caused by oxidative stress and calciumoverload. The effects of the free radical nitric oxide (NO ) were studied in a pancreatic beta-cell line(RlNm5F). The NO-releasing compound sodiumnitroprusside or interleukin-lbeta (IL-Ibeta)induced endogenous NO-production, stimulated inhibitory auto-ADP-ribosylation of theglycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). At later time-pointsRlNm5F cells exposed to NO underwent apoptosis. Inhibition of the nitric oxide synthaseactivity by NG-monomethyl-L-arginine prevented IL-1beta-induced NO generation and apoptoticcell killing. DNA-damage, caused by irradiation or free radicals, can stimulate expression of thetumor suppressor gene p53. The p53 protein is believed to produce growth arrest in G1-S whichallows DNA-repair. However, such conditions have also been shown to favor the occurrence ofapoptosis. We detected accumulation of the p53 protein prior to onset of apoptosis both inRINm5F cells and RAW 264.7 macrophages. To further characterize the mechanisms involved in the deletion of pancreatic cellsduring oxidative stress we used 2,3-dimethoxy-1,4-naphthoquinone (DMNQ). We found thatDMNQ, depending on the dose, induced cell proliferation, apoptosis or necrosis. Cellproliferation was associated with induction of enzymes involved in intracellular polyaminesynthesis (i.e. ornithine decarboxylase, ODC and S-adenosyl-L-methionine decarboxylase,SAMDC). Conversely, prior to the onset of apoptosis, ODC- and SAMDC activities decreasedand cells were rapidly depleted of polyamines. Interestingly, cells were protected from apoptosiswhen incubated with the phorbol ester TPA to induce ODC and SAMDC or by supplementingspermine. Oxidative stress, calcium overload and NO-production have also been implicated inneuronal damage following ischemia. Thus, primary cultures of cerebellar granule cells (CGC)were used to further study the mechanisms of glutamate-induced cell killing. Part of the CGCpopulation exposed to glutamate rapidly lost their mitochondrial membrane potential and diedby necrosis. The surviving population recovered their mitochondrial membrane potential butlater underwent apoptosis. Nuclear lamins were degraded prior to DNA-fragmentation,indicating an early activation of proteases in cells triggered to undergo apoptosis. Furtherstudies suggested that protection from loss of mitochondrial membrane potential as well asinhibition of the phosphatase calcineurin play important roles in both necrotic and apoptoticneuronal death. In conclusion, this thesis presents evidence that apoptosis is an important determinant ofcell death both in pancreatic and neuronal cells exposed to oxidative stress or calcium overload.Onset of apoptosis was under different conditions associated with depletion of intracellularpolyamines, accumulation of p53 or degradation of nuclear larnins. Depending on the dose andduration of exposure as well as cell sensitivity, cells died by apoptosis or necrosis in bothsystems used. Neuronal apoptosis was shown to be dependent on intact mitochondria supplyingenergy, while neurons dying by necrosis rapidly lost their mitochondrial membrane potentialand were depleted of energy.Doctoral Thesis 1996 Maria AnkarcronaISBN 91-628-1867-

Mitochondria-Endoplasmic Reticulum Interplay Regulates Exo-Cytosis in Human Neuroblastoma Cells

Mitochondria–endoplasmic reticulum (ER) contact sites (MERCS) have been emerging as a multifaceted subcellular region of the cell which affects several physiological and pathological mechanisms. A thus far underexplored aspect of MERCS is their contribution to exocytosis. Here, we set out to understand the role of these contacts in exocytosis and find potential mechanisms linking these structures to vesicle release in human neuroblastoma SH-SY5Y cells. We show that increased mitochondria to ER juxtaposition through Mitofusin 2 (Mfn2) knock-down resulted in a substantial upregulation of the number of MERCS, confirming the role of Mfn2 as a negative regulator of these structures. Furthermore, we report that both vesicle numbers and vesicle protein levels were decreased, while a considerable upregulation in exocytotic events upon cellular depolarization was detected. Interestingly, in Mfn2 knock-down cells, the inhibition of the inositol 1,4,5-trisphosphate receptor (IP3R) and the mitochondrial calcium (Ca2+) uniporter (MCU) restored vesicle protein content and attenuated exocytosis. We thus suggest that MERCS could be targeted to prevent increased exocytosis in conditions in which ER to mitochondria proximity is upregulated

Mitochondrial accumulation of APP and Abeta:significance for Alzheimer disease pathogenesis

Accumulating evidence suggest that alterations in energy metabolism are among the earliest events that occur in the Alzheimer disease (AD) affected brain. Energy consumption is drastically decreased in the AD-affected regions of cerebral cortex and hippocampus pointing towards compromised mitochondrial function of neurons within specific brain regions. This is accompanied by an elevated production of reactive oxygen species contributing to increased rates of neuronal loss in the AD-affected brain regions. In this review, we will discuss the role of mitochondrial function and dysfunction in AD. We will focus on the consequences of amyloid precursor protein and amyloid-β peptide accumulation in mitochondria and their involvement in AD pathogenesis

Mitochondrial Alterations in Neurons Derived from the Murine AppNL-F Knock-In Model of Alzheimer's Disease

Background: Alzheimer’s disease (AD) research has relied on mouse models overexpressing human mutant A βPP; however, newer generation knock-in models allow for physiological expression of amyloid-β protein precursor (AβPP) containing familial AD mutations where murine AβPP is edited with a humanized amyloid-β (Aβ) sequence. The AppNL-F mouse model has shown substantial similarities to AD brains developing late onset cognitive impairment. Objective: In this study, we aimed to characterize mature primary cortical neurons derived from homozygous AppNL-F embryos, especially to identify early mitochondrial alterations in this model. Methods: Primary cultures of AppNL-F neurons kept in culture for 12–15 days were used to measure Aβ levels, secretase activity, mitochondrial functions, mitochondrial-ER contacts, synaptic function, and cell death. Results: We detected higher levels of Aβ42 released from AppNL-F neurons as compared to wild-type neurons. AppNL-F neurons, also displayed an increased Aβ42/Aβ40 ratio, similar to adult AppNL-F mouse brain. Interestingly, we found an upregulation in mitochondrial oxygen consumption with concomitant downregulation in glycolytic reserve. Furthermore, AppNL-F neurons were more susceptible to cell death triggered by mitochondrial electron transport chain inhibition. Juxtaposition between ER and mitochondria was found to be substantially upregulated, which may account for upregulated mitochondrial-derived ATP production. However, anterograde mitochondrial movement was severely impaired in this model along with loss in synaptic vesicle protein and impairment in pre- and post-synaptic function. Conclusion: We show that widespread mitochondrial alterations can be detected in AppNL-F neurons in vitro, where amyloid plaque deposition does not occur, suggesting soluble and oligomeric Aβ-species being responsible for these alterations