The mitochondrion is an essential cytoplasmic organelle that provides most of the energy
necessary for eukaryotic cell physiology. Mitochondrial structure and functions are
maintained by proteins of both mitochondrial and nuclear origin. These organelles are
organized in an extended network that dynamically fuses and divides. Mitochondrial
morphology results from the equilibrium between fusion and fission processes, controlled by
a family of “mitochondria-shaping” proteins. It is becoming clear that defects in
mitochondrial dynamics can impair mitochondrial respiration, morphology and motility,
leading to apoptotic cell death in vitro and more or less severe neurodegenerative disorders in
vivo in humans.
Mutations in OPA1, a nuclear encoded mitochondrial protein, cause autosomal Dominant
Optic Atrophy (DOA), a heterogeneous blinding disease characterized by retinal ganglion cell
degeneration leading to optic neuropathy (Delettre et al., 2000; Alexander et al., 2000). OPA1
is a mitochondrial dynamin-related guanosine triphosphatase (GTPase) protein involved in
mitochondrial network dynamics, cytochrome c storage and apoptosis. This protein is
anchored or associated on the inner mitochondrial membrane facing the intermembrane space.
Eight OPA1 isoforms resulting from alternative splicing combinations of exon 4, 4b and 5b
have been described (Delettre et al., 2001). These variants greatly vary among diverse organs
and the presence of specific isoforms has been associated with various mitochondrial
functions. The different spliced exons encode domains included in the amino-terminal region
and contribute to determine OPA1 functions (Olichon et al., 2006). It has been shown that
exon 4, that is conserved throughout evolution, confers functions to OPA1 involved in
maintenance of the mitochondrial membrane potential and in the fusion of the network.
Conversely, exon 4b and exon 5b, which are vertebrate specific, are involved in regulation of
cytochrome c release from mitochondria, and activation of apoptosis, a process restricted to
vertebrates (Olichon et al., 2007).
While Mgm1p has been identified thanks to its role in mtDNA maintenance, it is only recently
that OPA1 has been linked to mtDNA stability. Missense mutations in OPA1 cause
accumulation of multiple deletions in skeletal muscle. The syndrome associated to these
mutations (DOA-1 plus) is complex, consisting of a combination of dominant optic atrophy,
progressive external ophtalmoplegia, peripheral neuropathy, ataxia and deafness (Amati-
Bonneau et al., 2008; Hudson et al., 2008). OPA1 is the fifth gene associated with mtDNA
“breakage syndrome” together with ANT1, PolG1-2 and TYMP (Spinazzola et al., 2009).
In this thesis we show for the first time that specific OPA1 isoforms associated to exon 4b are
important for mtDNA stability, by anchoring the nucleoids to the inner mitochondrial membrane.
Our results clearly demonstrate that OPA1 isoforms including exon 4b are intimately
associated to the maintenance of the mitochondrial genome, as their silencing leads to
mtDNA depletion. The mechanism leading to mtDNA loss is associated with replication
inhibition in cells where exon 4b containing isoforms were down-regulated. Furthermore
silencing of exon 4b associated isoforms is responsible for alteration in mtDNA-nucleoids
distribution in the mitochondrial network.
In this study it was evidenced that OPA1 exon 4b isoform is cleaved to provide a 10kd peptide
embedded in the inner membrane by a second transmembrane domain, that seems to be
crucial for mitochondrial genome maintenance and does correspond to the second
transmembrane domain of the yeasts orthologue encoded by MGM1 or Msp1, which is also
mandatory for this process (Diot et al., 2009; Herlan et al., 2003). Furthermore in this thesis
we show that the NT-OPA1-exon 4b peptide co-immuno-precipitates with mtDNA and
specifically interacts with two major components of the mitochondrial nucleoids: the
polymerase gamma and Tfam. Thus, from these experiments the conclusion is that NT-OPA1-
exon 4b peptide contributes to the nucleoid anchoring in the inner mitochondrial membrane, a
process that is required for the initiation of mtDNA replication and for the distribution of
nucleoids along the network.
These data provide new crucial insights in understanding the mechanism involved in
maintenance of mtDNA integrity, because they clearly demonstrate that, besides genes
implicated in mtDNA replications (i.e. polymerase gamma, Tfam, twinkle and genes involved
in the nucleotide pool metabolism), OPA1 and mitochondrial membrane dynamics play also
an important role. Noticeably, the effect on mtDNA is different depending on the specific
OPA1 isoforms down-regulated, suggesting the involvement of two different combined
mechanisms.
Over two hundred OPA1 mutations, spread throughout the coding region of the gene, have
been described to date, including substitutions, deletions or insertions. Some mutations are
predicted to generate a truncated protein inducing haploinsufficiency, whereas the missense
nucleotide substitutions result in aminoacidic changes which affect conserved positions of the
OPA1 protein. So far, the functional consequences of OPA1 mutations in cells from DOA
patients are poorly understood. Phosphorus MR spectroscopy in patients with the
c.2708delTTAG deletion revealed a defect in oxidative phosphorylation in muscles (Lodi et
al., 2004). An energetic impairment has been also show in fibroblasts with the severe OPA1
R445H mutation (Amati-Bonneau et al., 2005). It has been previously reported by our group
that OPA1 mutations leading to haploinsufficiency are associated in fibroblasts to an oxidative
phosphorylation dysfunction, mainly involving the respiratory complex I (Zanna et al., 2008).
In this study we have evaluated the energetic efficiency of a panel of skin fibroblasts derived
from DOA patients, five fibroblast cell lines with OPA1 mutations causing haploinsufficiency
(DOA-H) and two cell lines bearing mis-sense aminoacidic substitutions (DOA-AA), and
compared with control fibroblasts. Although both types of DOA fibroblasts maintained a
similar ATP content when incubated in a glucose-free medium, i.e. when forced to utilize the
oxidative phosphorylation only to produce ATP, the mitochondrial ATP synthesis through
complex I, measured in digitonin-permeabilized cells, was significantly reduced in cells with
OPA1 haploinsufficiency only, whereas it was similar to controls in cells with the missense
substitutions.
Furthermore, evaluation of the mitochondrial membrane potential (DYm) in the two fibroblast
lines DOA-AA and in two DOA-H fibroblasts, namely those bearing the c.2819-2A>C
mutation and the c.2708delTTAG microdeletion, revealed an anomalous depolarizing
response to oligomycin in DOA-H cell lines only. This finding clearly supports the
hypothesis that these mutations cause a significant alteration in the respiratory chain function,
which can be unmasked only when the operation of the ATP synthase is prevented.
Noticeably, oligomycin-induced depolarization in these cells was almost completely
prevented by preincubation with cyclosporin A, a well known inhibitor of the permeability
transition pore (PTP). This results is very important because it suggests for the first time that
the voltage threshold for PTP opening is altered in DOA-H fibroblasts. Although this issue
has not yet been addressed in the present study, several are the mechanisms that have been
proposed to lead to PTP deregulation, including in particular increased reactive oxygen
species production and alteration of Ca2+ homeostasis, whose role in DOA fibroblasts PTP
opening is currently under investigation. Identification of the mechanisms leading to altered
threshold for PTP regulation will help our understanding of the pathophysiology of DOA, but
also provide a strategy for therapeutic intervention.
| Identifer | oai:union.ndltd.org:unibo.it/oai:amsdottorato.cib.unibo.it:2815 |
| Date | 20 April 2010 |
| Creators | Vidoni, Sara <1979> |
| Contributors | Rugolo, Michela |
| Publisher | Alma Mater Studiorum - Università di Bologna |
| Source Sets | Università di Bologna |
| Language | French |
| Detected Language | English |
| Type | Doctoral Thesis, PeerReviewed |
| Format | application/pdf |
| Rights | info:eu-repo/semantics/restrictedAccess |
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