Cytosolic aggregation of mitochondrial proteins disrupts cellular homeostasis by stimulating other proteins aggregation

TL;DR: In this article, it was shown that upon mitochondrial protein import impairment, high-risk precursor and immature forms of mitochondrial proteins form aberrant deposits in the cytosol, and these deposits cause further cytosolic accumulation of other mitochondrial and disease-related proteins, including α-synuclein and amyloid β.

Abstract: Mitochondria are organelles with their own genomes but rely on the import of nuclear-encoded proteins synthesized by cytosolic ribosomes. Therefore, it is important to understand whether failures in the mitochondrial uptake of these nuclear-encoded proteins may cause proteotoxic stress, and to identify which response mechanisms may be in place to respond to it. Here, we report that upon mitochondrial protein import impairment, high-risk precursor and immature forms of mitochondrial proteins form aberrant deposits in the cytosol. In turn, these deposits cause further cytosolic accumulation of other mitochondrial and disease-related proteins, including α-synuclein and amyloid β. This aberrant accumulation triggers a cytosolic protein homeostasis imbalance that is accompanied by specific molecular chaperone responses, both at the transcriptomic and protein levels. Our results provide evidence that mitochondrial dysfunction, and specifically protein import defects, can contribute to protein homeostasis impairment, thus revealing a possible molecular mechanism for mitochondrial involvement in neurodegenerative diseases.

Summary

Introduction

• Only ~1% of them are synthesized inside this organelle.
• The reverse aspect of the way in which mitochondrial dysfunction, including mitochondrial import defects, contributes to the progression of neurodegenerative diseases remains elusive.
• These findings raise the issue of whether the accumulation of mistargeted mitochondrial proteins contributes to the progression of neurodegenerative diseases.
• The authors results showed that when some of these mitochondrial proteins remain in the cytosol because of mitochondrial protein import insufficiency, they formed insoluble aggregates that disrupted protein homeostasis.
• These proteins triggered a prompt specific molecular chaperone response that aimed to minimize the consequences of protein aggregation.

Metastable mitochondrial precursor proteins can aggregate in the cytosol

• The analysis of a transcriptomic signature of Alzheimer's disease identified oxidative phosphorylation as a pathway that is metastable and downregulated in the human central nervous system (Ciryam et al., 2016; Kundra et al., 2017) .
• The authors followed a FLAG peptide signal to determine whether the protein was present in the soluble (S125k) or insoluble (P125k) fraction.
• Only subunit VIb of cytochrome c oxidase (Cox12) and subunit 6 of ubiquinol cytochrome c reductase complex (Qcr6) were mainly present in the soluble fraction .
• Next, an aggregation assay was performed to assess the fate of precursor mitochondrial proteins under conditions of chemical impairments in mitochondrial import.
• PRip1 and pSod2 accumulated and consequently formed insoluble aggregates under these conditions .

Mitochondrial protein aggregation stimulates a cytosolic molecular chaperone response

• To further investigate the cellular response to the accumulation of mitochondrial precursor forms in the cytosol, the authors investigated global transcriptomic changes that were triggered by pRip1 overproduction and pam16-3 mutation .
• To determine the function of genes whose expression changed in response to the temperature shift, the authors performed KEGG enrichment analysis for WT and the pam16-3 mutant.
• With regard to mitochondrial defects, the pam16-3 mutation had a stronger effect on the transcriptome than the effect of pRip1 .
• The authors analyzed all genes that passed a 5% false discovery rate (FDR) cut-off and were non-mitochondrial.
• The authors observed the upregulation of HSP82 and SSA4, which were also upregulated in the case of pRip1 overexpression.

Effects of the Hsp42 and Hsp104 chaperone response to mitochondrial import failure at the protein level

• Next, the authors assessed whether the observed molecular chaperone upregulation would also be observed at the protein level.
• The authors used CCCP to stimulate mitochondrial dysfunction to avoid the temperature factor at initial screening.
• After identifying Hsp42 and Hsp104 as two molecular chaperones that change significantly when mitochondrial import is impaired, the authors further examined their expression levels when metastable mitochondrial proteins were overproduced.
• The Δhsp42 strain exhibited the significant upregulation of Hsp104, suggesting a compensatory mechanism that diminishes negative consequences of Hsp42 deletion .

Mitochondrial protein import failure impairs cellular protein homeostasis

• The authors next investigated whether the presence of metastable and aggregation-prone mitochondrial precursors initiates the accumulation of p forms of other mitochondrial proteins.
• Nevertheless, Rip1, Sod2, and Mdh1 co-aggregated with Atp2 and Cox8 metastable proteins in the insoluble fraction, based on the aggregation assay analysis .
• Thus, the greater abundance of aggregation-prone mitochondrial precursors resulted in the progression of mitochondrial protein import defect and consequently the larger cytosolic aggregation of mitochondrial precursors.
• Here, mitochondrial defects were stimulated by the pam16-3 mutant.
• The A53T mutation of α-Syn resulted in larger aggregates compared with α-Syn WT (Outeiro & Lindquist, 2003) .

Mitochondrial dysfunction results in protein aggregation in Caenorhabditis elegans

• To assess whether mitochondrial precursor aggregation that is caused by mitochondrial protein import deficiency compromises protein homeostasis at the organismal level, the authors used Caenorhabditis elegans as a model system.
• The authors first monitored the aggregation of two model proteins in cytosol, red fluorescent protein (RFP) and GFP, in the transgenic strain that expressed RFP and GFP in the body wall muscle to assess whether the RNAi silencing of dnj-21 in early adulthood in C. elegans is sufficient to stimulate their aggregation .
• Therefore, the authors next tested whether changes in cellular homeostasis that are attributable to mitochondrial defects affect the health of C. elegans when α-Syn is produced.
• The silencing of dnj-21, accompanied by α-Syn expression, decreased worm fitness, manifested by a slower speed and fewer bends compared with the effect of dnj-21 silencing alone .
• The authors used worms that carried Aβ peptides in body wall muscles and exhibited paralysis in adults when the temperature shifted to 25C (Sorrentino et al., 2017) .

Discussion

• The present study showed that a group of mitochondrial proteins that are downregulated in Alzheimer's disease (i.e., Rip1, Atp2, Cox8, and Atp20) can aggregate in the cytosol and that the overexpression of these proteins upregulates Hsp42 and Hsp104, two molecular chaperones that are associated with inclusion bodies (Balchin et al., 2016; Mogk et al., 2015; Mogk et al., 2019) .
• Unknown, however, is whether they act independently or whether concurrent actions of all of them are required to secure balanced cellular protein homeostasis.
• The cytosolic responses, which are aiming to clear clogged translocase of the outer membrane (TOM) and to clear the precursor proteins prior to their import through proteasomal activity, are accompanied by the aggregate-specific molecular chaperone response identified in the present study.
• Defects in mitochondrial function, which are commonly observed during ageing and in neurodegeneration, trigger a vicious cycle of protein aggregation.

Materials and methods

• No statistical methods were used to predetermine sample size.
• The yeast culture was performed at 28°C to the early logarithmic growth phase and further induced with 100 μM CuSO4 for 4 h unless otherwise indicated.
• After 30 min of incubation on ice, the samples were centrifuged at 20,000  g for 15 min at 4°C, washed with ice-cold acetone, and centrifuged again.
• The resulting raw reads were assessed for quality, adapter content, and duplication rates with FastQC (Andrews, 2010) .
• Expression matrices for the pam16-3 and pRip1 strains are available in Figure2-Source Data 3 and Figure2-Source Data 4, respectively.

KEGG enrichment analysis.

• The KEGG enrichment analysis was performed for RNA-seq using in-house developed methods based on KEGG.db (v. 3.2.3) and org.
• Pathways and genes that were selected in each developmental stage were filtered after Benjamini-Hochberg correction for an adjusted p < 0.05.
• Because of the small sizes of the aggregates that were at the resolution limit of the confocal microscope and the large diversity of their intensity in each cell, an automatic particle analysis based on image thresholding was not possible.
• Therefore, the authors analyzed the average size of the aggregates using a manual approach of defining aggregate boundaries and measuring the aggregate size with ImageJ software, with n = 10 analyzed for each condition.

Microscopy

• Standard conditions were used for the propagation of C. elegans (Brenner, 1974) .
• The following C. elegans strains were used: Worm transformation.
• LB medium (10 g/L tryptone, 10 g/L NaCl, and 5 g/L yeast extract) was inoculated with transformed bacteria and cultured at 37C at 180 rotations per minute.
• Worm total protein isolation and Western blot.
• The pellet that contained debris was discarded.

Motility assay.

• For each experiment, at least 35 worms were analyzed.
• Automated motility assessment was performed with a tracking device that was developed and described previously (Perni et al., 2018) .
• Briefly, worms were washed off the plates with M9 buffer and spread over a 9 cm NGM plate in a final volume of 5 mL, after which their movements were recorded for 120 s. Videos were analyzed in a consistent manner to track worm motility (bends per minute) and swimming speed.
• The results show the mean ± SEM from two independent experiments.

Amyloid β (Aβ) quantification.

• Aβ aggregates were calculated by dividing the signal that was detected with anti-Aβ antibody by the protein signal that was detected with Coomassie staining.
• For each temperature condition, aggregate levels were normalized to the control.
• Overall differences between conditions were assessed using unpaired t-tests by assuming unequal variance.

Statistical analysis.

• For the statistical analysis, two-tailed, unpaired t-tests were used by assuming equal variance unless otherwise stated.
• The authors declare that data that support the findings of this study are available within the paper, supporting information, and source data.
• The gene name is displayed for each molecular chaperone if it was detected as differentially expressed (i.e., 5% FDR, log2 fold change [log2FC] ± 1). (C) (D) Analysis of changes in the expression of genes that encode molecular chaperones using pam16-3 samples that were treated as in B.
• Up-and downregulated genes (5% FDR) are shown in green and pink, respectively.
• Scores > 1 characterize highly soluble proteins and sequence residues (blue).

Full text

1
Cytosolic aggregation of mitochondrial proteins disrupts cellular homeostasis by
1
stimulating the aggregation of other proteins
2
3
4
Urszula Nowicka
1, 2, 3
, Piotr Chroscicki
2, 4, 7
, Karen Stroobants
5, 7
, Maria Sladowska
2, 4
,
5
Michal Turek
1, 2, 4
, Barbara Uszczynska-Ratajczak
2, 6
, Rishika Kundra
5
, Tomasz Goral
1,
6
2
, Michele Perni
5
, Christopher M. Dobson
5
, Michele Vendruscolo
5
, & Agnieszka
7
Chacinska
1, 2, 3,
*
8
9
1
ReMedy International Research Agenda Unit, University of Warsaw, Warsaw, Poland
10
2
Centre of New Technologies, University of Warsaw, Warsaw, Poland
11
3
IMol Polish Academy of Sciences, Warsaw, Poland
12
4
International Institute of Molecular and Cell Biology, Warsaw, Poland
13
5
Centre for Misfolding Diseases, Department of Chemistry, University of Cambridge,
14
Cambridge, United Kingdom
15
6
Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznan, Poland
16
7
These authors contributed equally to this work.
17
18
*Correspondence should be directed to a.chacinska@imol.institute
19
20
.CC-BY-NC-ND 4.0 International licensemade available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
The copyright holder for this preprintthis version posted July 9, 2021. ; https://doi.org/10.1101/2021.05.02.442342doi: bioRxiv preprint

2
Abstract
21
Mitochondria are organelles with their own genomes, but they rely on the import of nuclear-
22
encoded proteins that are translated by cytosolic ribosomes. Therefore, it is important to
23
understand whether failures in the mitochondrial uptake of these nuclear-encoded proteins can
24
cause proteotoxic stress and identify response mechanisms that may counteract it. Here, we
25
report that upon impairments in mitochondrial protein import, high-risk precursor and immature
26
forms of mitochondrial proteins form aberrant deposits in the cytosol. These deposits then cause
27
further cytosolic accumulation and consequently aggregation of other mitochondrial proteins
28
and disease-related proteins, including α-synuclein and amyloid β. This aggregation triggers a
29
cytosolic protein homeostasis imbalance that is accompanied by specific molecular chaperone
30
responses at both the transcriptomic and protein levels. Altogether, our results provide evidence
31
that mitochondrial dysfunction, specifically protein import defects, contributes to impairments
32
in protein homeostasis, thus revealing a possible molecular mechanism by which mitochondria
33
are involved in neurodegenerative diseases.
34
35
36
.CC-BY-NC-ND 4.0 International licensemade available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
The copyright holder for this preprintthis version posted July 9, 2021. ; https://doi.org/10.1101/2021.05.02.442342doi: bioRxiv preprint

3
Introduction
37
Although over one thousand proteins are utilized by mitochondria to perform their
38
functions, only ~1% of them are synthesized inside this organelle. The majority of
39
mitochondrial proteins are synthesized in the cytosol and need to be actively transported to
40
mitochondria, a process that occurs via a sophisticated system that involves protein translocases
41
and sorting machineries (Calvo et al., 2016; Morgenstern et al., 2017; Neupert & Herrmann,
42
2007; Pfanner et al., 2019). The consequences of mitochondrial protein import defects on
43
cellular proteostasis can be severe and currently some response mechanisms are identified
44
(Boos et al., 2019; Izawa et al., 2017; Kim et al., 2016; Martensson et al., 2019; Priesnitz &
45
Becker, 2018; Wang & Chen, 2015; Weidberg & Amon, 2018; Wrobel et al., 2015; Wu et al.,
46
2019; Poveda-Huertes et al., 2020). Mitochondrial dysfunction is closely associated with
47
neurodegenerative disorders, and such mitochondrial defects as aberrant Ca
2+
handling,
48
increases in reactive oxygen species, electron transport chain inhibition, and impairments in
49
endoplasmic reticulum-mitochondria tethering are well described pathological markers
50
(Cabral-Costa & Kowaltowski, 2020). Still unknown, however, is whether mitochondrial
51
defects appear as a consequence of neurodegeneration, whether they contribute to it, or whether
52
both processes occur. Disease-related proteins can interfere with mitochondrial import and the
53
further processing of imported proteins within mitochondria (Cenini et al., 2016; Di Maio et
54
al., 2016; Mossmann et al., 2014; Vicario et al., 2018). Furthermore, aggregated proteins can
55
be imported into mitochondria where they can be either cleared or sequestered in specific
56
deposit sites (Bruderek et al., 2018; Ruan et al., 2017; Sorrentino et al., 2017). However, the
57
reverse aspect of the way in which mitochondrial dysfunction, including mitochondrial import
58
defects, contributes to the progression of neurodegenerative diseases remains elusive. One
59
possible mechanism may occur through alterations of cellular homeostasis, as mitochondrial
60
.CC-BY-NC-ND 4.0 International licensemade available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
The copyright holder for this preprintthis version posted July 9, 2021. ; https://doi.org/10.1101/2021.05.02.442342doi: bioRxiv preprint

4
dysfunction can affect it through multiple mechanisms (Andreasson et al., 2019; Braun &
61
Westermann, 2017; Escobar-Henriques et al., 2020).
62
Impairments in mitochondrial protein import and mitochondrial import machinery
63
overload result in the accumulation of mitochondria-targeted proteins in the cytosol and
64
stimulation of mitoprotein-induced stress (Boos et al., 2019; Wang & Chen, 2015; Wrobel et
65
al., 2015). These findings raise the issue of whether the accumulation of mistargeted
66
mitochondrial proteins contributes to the progression of neurodegenerative diseases.
67
Additionally, unknown are whether mitoprotein-induced stress is a general response to
68
precursor proteins that globally accumulate in the cytosol and whether a subset of mitochondrial
69
precursor proteins pose particularly difficult challenges to the protein homeostasis system and
70
consequently contribute to the onset and progression of neurodegenerative disorders (Boos et
71
al., 2020; Mohanraj et al., 2020).
72
The analysis of a transcriptional signature of Alzheimer’s disease supports the notion
73
that there is a subset of mitochondrial proteins that is more dangerous than others for the cell
74
(Ciryam et al., 2016; Kundra et al., 2017). These studies have shown that specific mitochondrial
75
proteins that are functionally related to oxidative phosphorylation are transcriptionally
76
downregulated in Alzheimer’s disease. In the present study, we investigated why these proteins
77
are downregulated. We hypothesized that this need arises from the potential supersaturation of
78
these proteins, which makes them prone to aggregation (Ciryam et al., 2016; Kundra et al.,
79
2017). Our results showed that when some of these mitochondrial proteins remain in the cytosol
80
because of mitochondrial protein import insufficiency, they formed insoluble aggregates that
81
disrupted protein homeostasis. These proteins triggered a prompt specific molecular chaperone
82
response that aimed to minimize the consequences of protein aggregation. However, when this
83
rescue mechanism was insufficient, these aggregates stimulated the cytosolic aggregation of
84
other mitochondrial proteins and led to the downstream aggregation of non-mitochondrial
85
.CC-BY-NC-ND 4.0 International licensemade available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
The copyright holder for this preprintthis version posted July 9, 2021. ; https://doi.org/10.1101/2021.05.02.442342doi: bioRxiv preprint

5
proteins. Our findings indicate that metastable mitochondrial proteins can be transcriptionally
86
downregulated during neurodegeneration to minimize cellular protein homeostasis imbalance
87
that is caused by their mistargeting.
88
89
Results
90
Metastable mitochondrial precursor proteins can aggregate in the cytosol
91
The analysis of a transcriptomic signature of Alzheimer’s disease identified oxidative
92
phosphorylation as a pathway that is metastable and downregulated in the human central
93
nervous system (Ciryam et al., 2016; Kundra et al., 2017). This observation suggests that a
94
group of mitochondrial proteins might be dangerous for cellular protein homeostasis because
95
of their poor supersaturation and hence solubility at cellular concentrations. From the list of
96
genes that were simultaneously downregulated and metastable in Alzheimer’s disease patients,
97
we selected all genes that encode mitochondrial proteins. Next, we identified genes that encode
98
proteins that have homologs in yeast (Figure 1–figure supplement 1). Based on the yeast
99
homolog sequence, we generated FLAG-tagged constructs that were expressed under control
100
of the copper-inducible promoter (CUP1). We then established a multi-centrifugation step assay
101
to assess whether these proteins exceed their critical concentrations and become supersaturated
102
when overproduced (Vecchi et al., 2020), thereby acquiring the ability to aggregate during their
103
trafficking to mitochondria (Figure 1–figure supplement 2A). We followed a FLAG peptide
104
signal to determine whether the protein was present in the soluble (S
125k
) or insoluble (P
125k
)
105
fraction. We found that the  and g subunits of mitochondrial F
1
F
O
adenosine triphosphate
106
(ATP) synthase (Atp2 and Atp20, respectively) were present in the insoluble fraction, indicating
107
that they formed high-molecular-weight deposits (Figure 1A and Figure 1–figure supplement
108
2B). We made a similar observation for Rieske iron-sulfur ubiquinol-cytochrome c reductase
109
(Rip1). Rip1 and subunit VIII of cytochrome c oxidase complex IV (Cox8) had entirely
110
.CC-BY-NC-ND 4.0 International licensemade available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
The copyright holder for this preprintthis version posted July 9, 2021. ; https://doi.org/10.1101/2021.05.02.442342doi: bioRxiv preprint

Frequently asked questions

Q1. What are the contributions in this paper?

In this paper, Nowicka and Chacinska proposed a method to stimulate the aggregation of other proteins.