Defective Mitochondrial Disulfide Relay System, Altered

Human Molecular Genetics, 2013, Vol. 22, No. 5 989–1004 doi:10.1093/hmg/dds503 Advance Access published on November 29, 2012 Defective mitochondrial disulfide relay system, altered mitochondrial morphology and function in Huntington’s disease Eleonora Napoli, Sarah Wong, Connie Hung, Catherine Ross-Inta, Prithvi Bomdica and Cecilia Giulivi∗ Department of Molecular Biosciences, University of California Davis, Davis, CA 95616, USA Downloaded from Received October 5, 2012; Revised November 7, 2012; Accepted November 26, 2012 A number of studies have been conducted that link mitochondrial dysfunction (MD) to Huntington’s disease (HD); however, contradicting results had resulted in a lack of a clear mechanism that links expression of mutant Huntingtin protein and MD. Mouse homozygous (HM) and heterozygous (HT) mutant striatal cells http://hmg.oxfordjournals.org/ with two or one allele encoding for a mutant huntingtin protein with 111 polyGln repeats showed a significant impairment of the mitochondrial disulfide relay system (MDRS). This system (consisting of two proteins, Gfer and Mia40) is involved in the mitochondrial import of Cys-rich proteins. The Gfer-to-Mia40 ratio was significantly altered in HM cells compared with controls, along with the expression of mitochondrial proteins considered substrates of the MDRS. In progenitors and differentiated neuron-like HM cells, impairment of MDRS were ac- companied by deficient oxidative phosphorylation, Complex I, IV and V activities, decreased mtDNA copy number and transcripts, accumulation of mtDNA deletions and changes in mitochondrial morphology, consist- ent with other MDRS-deficient biological models, thus providing a framework for the energy deficits observed at University of California, Davis on February 19, 2013 in this HD model. The majority (>90%) of the mitochondrial outcomes exhibited a gene–dose dependency with the expression of mutant Htt. Finally, decreases in the mtDNA copy number, along with the accumulation of mtDNA deletions, provide a mechanism for the progressive neurodegeneration observed in HD patients. INTRODUCTION The human HD gene (also called IT15) contains 67 exons spanning .200 kb. The translated wild-type (WT) huntingtin Huntington’s disease (HD) is a fatal neurodegenerative dis- (Htt) protein is a 350 kDa protein containing a polymorphic order characterized by psychiatric, cognitive and motor disor- stretch of between 6 and 35 Gln residues in its N-terminal ders (1). The neuropathology of HD involves the selective domain. When the number of Gln-encoding repeats exceeds dysfunction and death of specific neuronal subpopulations 36, the gene encodes a version of huntingtin or mutant huntigtin within the central nervous system, in which the most affected (mutHtt) that leads to disease (5,6). The mutant protein with the cells are the gamma-aminobutyric acid-releasing spiny pro- polyGln expansion induces the formation of neuritic, cytoplas- jecting neurons of the striatum (subcortical brain structure mic and nuclear inclusions, leading to dysfunction and finally that controls body movement) (2). Disease onset is generally death of these neurons. Analysis of postmortem brains from marked by involuntary movements of the face, fingers, feet patients at the early stages of HD revealed the presence of dys- or thorax (3). Psychiatric symptoms (depression, anxiety, trophic neurites before cell death (7). Early neuropathology has apathy and irritability) are more heterogeneous (4) and can also been detected in mouse models of HD such as electro- occur up to 20 years before the onset of the choreiform move- physiological and mitochondrial abnormalities and the presence ments. Patients usually die 10–20 years after the first symp- of neuropil aggregates in axons and axon terminals (8). toms appear, as currently there is no effective treatment to There is significant evidence that energy production is prevent or delay disease progression. impaired in HD (9). Impaired glucose metabolism has been ∗To whom correspondence should be addressed at: Department of Molecular Biosciences, University of California, One Shields Ave., 1120 Haring Hall, Davis, CA 95616, USA. Email: [email protected] # The Author 2012. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected] 990 Human Molecular Genetics, 2013, Vol. 22, No. 5 Downloaded from http://hmg.oxfordjournals.org/ at University of California, Davis on February 19, 2013 Figure 1. Evaluation of the MDRS proteins expression in striatal cells. (A). Schematic representation of the MDRS. Mia40 oxidizes the proteins that are imported into the mitochondria trapping them into the intermembrane space, and in turn it gets re-oxidized by Gfer. The transfer of electrons to cytochrome c finalizes the restoration of the oxidized form of Gfer. (B). Western blots for the indicated proteins were performed on total cell lysates as described under Materials and Methods and Supplementary Material, Table S1. Twenty-five micrograms of proteins were loaded in each lane. Actin was used as a loading control. (C). Densitometry of Gfer, Mia40, cytochrome c and Cox17, in striatal cells. Data are shown as mean + SEM. The P-values shown are relative to the statistical comparison (ANOVA followed by Bonferroni’s post-test) of HT and HM with WT. ∗ P , 0.05, ∗∗ P , 0.01. observed in striatum of HD patients at early stages of the respiratory-chain subunits, oxidative phosphorylation assembly disease (10–12), striatal hypometabolism has been observed factors, proteins involved in mitochondrial DNA (mtDNA) in presymptomatic HD cases (13) and increased lactate maintenance, factors related to mitochondrial protein synthesis, levels have been observed in the cortex and basal ganglia biosynthetic enzymes and proteins promoting mitochondrial from HD patients (5). Others have identified defects in mito- biogenesis (29). While many of these proteins are synthesized chondrial respiratory Complexes II, III and IV in postmortem in the cytosol in the form of precursor proteins and post- brain tissues from HD cases with substantial neuronal loss translationally transported to the mitochondria in an unfolded (14–17). However, others indicated that the presence of state, other low molecular weight Cys-containing proteins mutHtt did not impair the activity of respiratory complexes localized at the IMS require the sequential cooperation of the (18–20): Complex activities were not significantly different translocase of the mitochondrial outer membrane (TOM) between mouse striatal neurons with two alleles encoding complex, followed by the mitochondrial disulfide relay for a mutHtt with 111 Gln repeats compared with control system (MDRS) for intramitochondrial import (30) utilizing cells (18), in the striatum of presymptomatic or lower grade cytochrome c as the terminal electron acceptor (31). HD cases with minimal neuronal loss (19), or in several trans- The MDRS is constituted by two proteins (Fig. 1A), the genic HD mouse models (19,20). Other studies reported mito- mitochondrial intermembrane space import and assembly chondrial dysfunction (MD) in brains of transgenic mice protein 40 (Mia40 also known as coiled-coil-helix-coiled-coil- (21,22), changes in mitochondrial calcium homeostasis in helix domain-containing protein 4 or CHCHD4) and Gfer HD mouse models and in patients’ lymphoblasts (23–26), oc- (also known as Alr, Erv1, Herv1, HPO). Physiological sub- currence of a feedback loop between Complex III and the pro- strates of the MDRS include many proteins relevant to COX teasome (27), and deficiency in PGC1-a, possibly leading to biogenesis (Cox12, Cox17, Cox19, Cox23 (30,32)), several an altered mitochondria biogenesis (28). small Tim chaperones (Tim8, Tim9, Tim10 and Tim13 Mitochondrial and nuclear DNA mutations resulting in mito- (33,34) and the copper chaperone for superoxide dismutase chondrial diseases have been described in genes encoding for or Ccs1 (32), in addition to a proposed role in Fe/S traffic in Human Molecular Genetics, 2013, Vol. 22, No. 5 991 the cytosolic compartment (35); therefore, a defect in this in HM were found to be 1.3-fold of controls, maybe as a com- pathway is likely to result in multiple effects due to a defective pensation mechanism to overcome the deficiency in the import of proteins relevant to Complex IV biogenesis as well MDRS. Following the trend of Mia40, the protein level of as to a number of yet uncharacterized mitochondrial proteins. Cox17 was 72 and 46% of control values in HT and HM, re- Defects in the ERV1 gene were initially identified by a spectively (Fig. 1B and C). Other subunits of Complex IV temperature-sensitive yeast mutant with a severe defect in oxi- which do not require the participation of MDRS, such as the dative phosphorylation and reduced levels of mitochondrial mtDNA-encoded subunit III (COXIII), were found not differ- transcripts (36). In further studies, it was shown that this ent in HT versus controls but significantly lower in HM (see gene was essential for the maintenance of intact mtDNA below), suggesting a disrupted assembly/folding of Complex (37) as well as mitochondria morphology and distribution IV subunits. (38). Thus, considering that mutHtt is primarily found in the Although some substrates for MDRS have been identified, cytoplasm and the initial events leading to the disease may others still remain unknown. To this end, we screened the start in this subcellular compartment, we reasoned that an 40+ subunits of Complex I that fulfilled the requirement for

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