Mitochondria-endoplasmic reticulum contacts in neuronal cells : from physiology to therapeutics

Mitochondria and the endoplasmic reticulum (ER) are intracellular organelles that play vital physiological functions. Mitochondria are key players in energy production through adenosine triphosphate (ATP) production and calcium (Ca2+) buffering, while the ER is involved in protein and lipid synthesis along with Ca2+ signalling in the cell. In the last 10 years scientists have realised the importance of intracellular organelle communication as a pivotal process for physiological functions. Among these interactions, mitochondria and ER functionally and structurally interact with each other forming mitochondria-ER contact sites (MERCS). Importantly, these structures oversee a variety of pathways including intracellular Ca2+ signalling. Indeed, ER to mitochondria Ca2+ shuttling has been shown to impact on mitochondrial respiration and bioenergetics. On the other hand, sustained increase in Ca2+ signalling between these two organelles can cause activation of apoptosis mediators leading to cell death. In Alzheimer´s disease (AD), cerebral hypometabolism, mitochondrial dysfunction, and functional and structural upregulation of ER to mitochondria apposition appear as early events in disease pathogenesis. Despite over 30 years of studies, the causes of AD are essentially unknown and only two symptomatic drugs have been approved for treatment, which means that AD leads to decline of quality of life and ultimately death. In this thesis, using human brain biopsies from idiopathic normal pressure hydrocephalus (iNPH) patients, mouse models of AD and cellular models, we investigated the role of mitochondria and MERCS in synapses and exocytotic mechanism and their role in the development of pathology in AD. Additionally, we have set up a high throughput screen (HTS) to find potential modulators of mitochondrial function with the overarching aim to find drugs to target neurodegeneration. In PAPER I, for the first time we have shown the presence of several organelle contact sites in human brain material and we have confirmed the presence of MERCS in human synapses. In this study we have also shown that patients suffering from dementia have more MERCS compared to non-demented patients. Furthermore, we have shown correlation of soluble Aβ levels, thought to be one of the initiators of AD, and MERCS number in iNPH patients. In PAPER II, through knockdown of Mitofusin 2 (Mfn2) in SH-SY5Y cells, a negative regulator of MERCS, we have detected substantial increased juxtaposition between ER and mitochondria. Upon Mfn2 knockdown, we have observed decreased levels of cytoplasmic vesicle and increased vesicle release upon cellular depolarization. Furthermore, we have shown that this mechanism was dependent on IP3Rs activity, an important channel for Ca2+ transfer from ER to mitochondria. In PAPER III we have characterised in vitro a novel knock-in model of AD, the AppNL-F model, which overcomes the problem of overexpressing amyloid precursor protein (APP). We have shown that embryonic cells derived from AppNL-F mice are capable of secreting levels of Aβ similar to adult brains, causing bioenergetics impairments, movement abnormalities along neurites and increased MERCS functions. Furthermore, these cells seem to be more susceptible to cell death upon inhibition of mitochondrial respiration compared to WT cells. In PAPER IV, we have assessed whether the other pathological protein in AD, tau, impacts on mitochondrial function and MERCS using the pure tauopathy model P301s. We detected that before tau pathology onset, at 22 days post-natal, animals displayed mitochondrial respiration dysfunctions and increase in MERCS. This pathology was sustained throughout mice life up to 10 months of age. In PAPER V, setting up a HTS platform evaluating mitochondrial enhancers, we have found luteolin, a natural compound from the flavonoid family, to be capable of increasing ATP production in vitro in SH-SY5Y cells and primary cortical neurons, and ex vivo in isolated mitochondria and synaptosomes. The ATP increase shown was due to increased ER to mitochondria juxtaposition and Ca2+ transfer. We have further tested luteolin in Huntington’s disease mutations bearing primary cortical neurons and C.elegans, showing improvement in respiration in vitro and recovery in movement in vivo. In conclusion, this thesis has contributed to expand the knowledge on the role of mitochondria and MERCS in synapses and in exocytotic mechanisms. We have further shown that MERCS and bioenergetics dysfunction occur early during the pathogenic development of disease in tau and amyloid AD models. We have also provided a platform for the study of drugs in neuronal cells, revealing luteolin as a promising enhancer of mitochondrial function

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

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

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 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

Upregulation of calpain activity precedes tau phosphorylation and loss of synaptic proteins in Alzheimer’s disease brain

Summary of postmortem brain characteristics (DOC 28 kb

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

Alterations in mitochondria-endoplasmic reticulum connectivity in human brain biopsies from idiopathic normal pressure hydrocephalus patients

Abstract Idiopathic normal pressure hydrocephalus (iNPH) is a neuropathology with unknown cause characterised by gait impairment, cognitive decline and ventriculomegaly. These patients often present comorbidity with Alzheimer’s disease (AD), including AD pathological hallmarks such as amyloid plaques mainly consisting of amyloid β-peptide and neurofibrillary tangles consisting of hyperphosphorylated tau protein. Even though some of the molecular mechanisms behind AD are well described, little is known about iNPH. Several studies have reported that mitochondria-endoplasmic reticulum contact sites (MERCS) regulate amyloid β-peptide metabolism and conversely that amyloid β-peptide can influence the number of MERCS. MERCS have also been shown to be dysregulated in several neurological pathologies including AD. In this study we have used transmission electron microscopy and show, for the first time, several mitochondria contact sites including MERCS in human brain biopsies. These unique human brain samples were obtained during neurosurgery from 14 patients that suffer from iNPH. Three of these 14 patients presented comorbidities with other dementias: one patient with AD, one with AD and vascular dementia and one patient with Lewy body dementia. Furthermore, we report that the numbers of MERCS are increased in biopsies obtained from patients diagnosed with dementia. Moreover, the presence of both amyloid plaques and neurofibrillary tangles correlates with decreased contact length between endoplasmic reticulum and mitochondria, while amyloid plaques alone do not seem to affect endoplasmic reticulum-mitochondria apposition. Interestingly, we report a significant positive correlation between the number of MERCS and ventricular cerebrospinal fluid amyloid β-peptide levels, as well as with increasing age of iNPH patients