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.
List of scientific papers
I. Nuno Santos Leal, Giacomo Dentoni, Bernadette Schreiner, Olli-Pekka Kämäräinen, Nelli Partanen, Sanna-Kaisa Herukka, Anne M Koivisto, Mikko Hiltunen, Tuomas Rauramaa, Ville Leinonen, Maria Ankarcrona. Alterations in mitochondria-endoplasmic reticulum connectivity in human brain biopsies from idiopathic normal pressure hydrocephalus patients. Acta Neuropathologica Communications. (2018) 6:102.
https://doi.org/10.1186/s40478-018-0605-2
II. Giacomo Dentoni, Luana Naia, Maria Ankarcrona. Increased mitochondria-endoplasmic reticulum contacts result in elevated exocytosis depending on inositol 1,4,5-trisphosphate receptor function in human neuroblastoma cells. [Manuscript]
III. Giacomo Dentoni*, Luana Naia*, Nuno Santos Leal, Per Nilsson, Maria Ankarcrona. In vitro characterization of mitochondrial function and mitochondria-ER contact sites in primary cortical neurons derived from the AppNL-F mouse model. *Contributed equally. [Manuscript]
IV. Giacomo Dentoni*, Ania Goncalves*, Stephane Duvezin-Caubet, Nuno Santos Leal, Severine Deforges, Anne Devin, Maria Ankarcrona, Sandrine Pouvreau. Early mitochondrial and mitochondria-endoplasmic reticulum contact sites dysfunction in the P301s tauopathy model. *Contributed equally. [Manuscript]
V. Luana Naia*, Catarina M. Pinho*, Giacomo Dentoni, Jianping Liu, Nuno Santos Leal, Duarte M. S. Ferreira, Bernadette Schreiner, Riccardo Filadi, Lígia Fão, Niamh M. C. Connolly, Pontus Forsell, Gunnar Nordvall, Makoto Shimozawa, Elisa Greotti, Emy Basso, Pierre Theurey, Anna Gioran, Alvin Joselin, Marie Arsenian-Henriksson, Per Nilsson, A. Cristina Rego, Jorge L. Ruas, David Park, Daniele Bano, Paola Pizzo, Jochen H. M. Prehn, Maria Ankarcrona. Neuronal cell-based high-throughput screen for enhancers of mitochondrial function reveals luteolin as a modulator of mitochondria-endoplasmic reticulum coupling. BMC Biology. (2021) 19:57. *Contributed equally.
https://doi.org/10.1186/s12915-021-00979-5
History
Defence date
2021-06-09Department
- Department of Neurobiology, Care Sciences and Society
Publisher/Institution
Karolinska InstitutetMain supervisor
Ankarcrona, MariaCo-supervisors
Lindskog, Mia; Pouvreau, SandrinePublication year
2021Thesis type
- Doctoral thesis
ISBN
978-91-8016-258-6Number of supporting papers
5Language
- eng