Mitochondrion to endoplasmic reticulum apposition length in zebrafish embryo spinal progenitors is unchanged in response to perturbations associated with Alzheimer’s disease

Abstract

Mutations in the human genes PRESENILIN1 (PSEN1), PRESENILIN2 (PSEN2) and AMYLOID BETA A4 PRECURSOR PROTEIN (APP) have been identified in familial Alzheimer’s disease (AD). The length of mitochondrion-endoplasmic reticulum (M-ER) appositions is increased in Psen1-/-/Psen2-/- double knockout murine embryonic fibroblasts and in fibroblasts from AD-affected individuals. Development of an easily accessible, genetically manipulable, in vivo system for studying M-ER appositions would be valuable so we attempted to manipulate M-ER apposition length in zebrafish (Danio rerio) embryos. We injected fertilized zebrafish eggs with antisense morpholino oligonucleotides (MOs) that inhibit expression of zebrafish familial AD gene orthologues psen1 and psen2. Furthermore, we treated zebrafish embryos with DAPT (a highly specific γ-secretase inhibitor) or with sodium azide (to mimic partially hypoxic conditions). We then analyzed M-ER apposition in an identified, presumably proliferative neural cell type using electron microscopy. Our analysis showed no significant differences in M-ER apposition lengths at 48 hours post fertilization (hpf) between psen1 & psen2 MO co-injected embryos, embryos treated with DAPT, or sodium azide, and control embryos. Instead, the distribution of M-ER apposition lengths into different length classes was close to identical. However, this indicates that it is feasible to reproducibly measure M-ER size distributions in zebrafish embryos. While our observations differ from those of murine and human studies, this may be due to differences in cellular differentiation and metabolic state, cell age, or species-specific responses. In particular, by focusing on a presumably proliferative embryonic cell type, we may have selected a cell heavily already reliant on anaerobic glycolysis and less responsive to factors affecting M-ER apposition. Future examination of more differentiated, more secretory cell types may reveal measurable responses of M-ER apposition to environmental and genetic manipulation.

Perturbed mitochondria–ER contacts in live neurons that model the amyloid pathology of Alzheimer's disease

“…Finally, the results obtained using cells lines, even though they are informative, present limitations to infer what happens in live neurons. In this regard, Newman and colleagues recently analyzed by TEM the interface length, as previously reported in fibroblast from human subjects (Area-Gomez et al, 2012), in neuronal progenitors from zebrafish embryos injected with morpholines against PSEN1and/or PSEN2, and found no significant differences in PSEN1/2 knock-down cells as compared to controls(Newman et al, 2017), reinforcing the concept that MERC function and/or activity is cell-dependent, and although neurons share many features found in other cell types, they possess some special characteristics that cannot be reproduced in un-differentiated cells. AlthoughHedskog et al (Hedskog et al, 2013) employed primary mouse hippocampal neurons, the increase in MERC number detected by confocal microscopy after acute synthetic Aβ exposure may represent an artefactual effect, since Aβ affects intracellular trafficking of Mt inducing accumulation of Mt in perinuclear regions, which are regions rich in ER(De Vos et al, 2008).…”

“…In the last few years, some studies in cell lines, fibroblasts from AD patients (Area-Gomez et al, 2012;Del Prete et al, 2017) and zebrafish neuronal progenitors (Newman et al, 2017) have reported alterations in MERC, with contradictory results. However, none of them analyzed the gap width between the organelles in live neurons and with methods to evaluate proximity with the appropriate resolution.…”

Studying zebrafish nervous system structure and function in health and disease with electron microscopy

“…Even though zebrafish can evidently be a valuable model for Alzheimer's research, one should always stay aware of the limits of any model. This was showcased by Newman and colleagues in their TEM study of zebrafish larvae (Newman et al, 2017). They used morpholinos to knock down proteins that are mutated in patients suffering from familial Alzheimer's disease.…”