The mechanism of polyQ-expanded Htt induced toxicity is believed to involve a conformational change in mutant Htt protein or its N-terminal proteolytic fragments, which leads to abnormal protein interactions, resulting in cellular dysfunction and death. Previous studies of the Htt interactome, based on yeast two-hybrid screens and affinity pull-downs followed by MS, were not designed to compare normal and expanded Htt protein interactions. Here, we used, for the first time, quantitative proteomics to assess and quantify the changes in Htt interactome induced by polyQ expansion in striatal neuronal precursor cell line, the most relevant for HD cell type. Our findings support the hypothesis that polyQ expansion induces abnormal interactions of Htt, which may disrupt key cellular functions and networks. We showed that among the most altered are Htt interactions with several mitochondrial proteins, including AIFM1, consistent with a role for mitochondrial dysfunction in HD pathogenesis. Furthermore, our data suggest a novel role of mutant Htt in RNA processing and regulation of translation via its interaction and co-localization with SG-associated RNA-binding proteins Caprin-1 and G3BP1 in striatal cells under the ER-stress conditions.
The N-terminal segment of Htt possesses a mix of hydrophobic and hydrophilic amino acids and is predicted to have both significant α helix potential and a tendency toward a compact structure in the presence of a binding partner.40,41
CD spectra and molecular dynamics simulations42
suggest that the N-terminal sequence by itself has a tendency to take on some α-helical secondary structure, while expanded polyQ was shown to aquire a β-sheet conformation.43
Formation of amyloid-like fibrils with β-sheet structure is observed with many protein sequences and is linked to many neurodegenerative conditions, including Parkinson, Huntington and Alzheimer diseases. If the underlying mechanism is mediated by aberrant interactions of aggregates with other cellular proteins, resulting in their sequestration and functional impairment, are there specific sequence structural features which make cellular proteins especially prone to such sequestration? In a recent report, the authors used quantitative proteomics to study interactions of artificial β-sheet proteins forming amyloid-like fibrils.26
They found that these interactors are particularly enriched in intrinsically disordered (unstructured) regions, a feature linked to multifunctionality. Consistent with these data, we found that preferential interactors of expanded Htt-N586 were also enriched in such metastable disordered regions, which further supports previously suggested role of intrinsically disordered proteins in neurodegenerative disorders.26,27
The hypothesis that a structural conformational change in expanded Htt leads to abnormal protein interactions was recently challenged in a study designed to assess the influence of Htt aggregation on its interactions.44
The authors found that in the absence of aggregation, normal and expanded Htt interacts with endophillin-3 with similar affinity, as measured by surface plasmon resonance, suggesting that expanded polyQ per se does not alter interactions. The authors demonstrate that Htt aggregates strongly affect the outcome of the pull-down experiments and propose that aggregates are forming molecular platforms that influence the Htt-interacting network. Although we did not specifically control for the absence of aggregates in our experimental system, we observed neither visible aggregate formation in striatal cells transfected with expanded Htt, nor accumulation of SDS-insoluble material on the top of the gel. However, we have observed high molecular weight Htt species formed in cells transfected with Htt constructs ( and , middle parts). Although these species were more evident after immunoprecipitation, we often observed trace amonts of similar material also in the analysis of inputs. We think that these may represent soluble oligomeric species of expanded Htt formed in cells, thus presenting efficient molecular platforms that perturb Htt interactions,44
and, consequently, our results may partially reflect binding to these oligomers. Whether abnormal interactions of expanded Htt are mediated by expanded polyQ, per se, or by certain aggregated or soluble oligomeric forms of expanded Htt, the understanding of such changes in Htt interactome helps to illuminate the pathways and molecular targets affected, with a potential to develop new HD therapies.
Mitochondrial dysfunction has been previously implicated in HD pathogenesis (reviewed in ref. 45
), although the mechanism of Htt involvement is still elusive. We found that expanded Htt complexes were particularly enriched in proteins assigned (by IPA) mitochondrial function and localization, while protein network analysis revealed potential disruption of processes related to energy production and oxidative phosphorylation. The biochemical interaction of N-terminal Htt fragments with mitochondria was previously demonstrated in HD knock-in mouse brain46
and mutant Htt oligomers co-localize with mitochondrial proteins COX1 and cytochrome C
in brain sections of HD patients.47
However, it is not clear whether Htt or its fragments can enter mitochondria. Our cell fractionation experiments demonstrated that that normal (Q7) and polyQ-expanded (Q111) Htt were present in OMM and within mitochondrial fraction, which included intermembrane space, inner membrane and matrix; however, the N-terminal fragments of expanded Htt were only detected in OMM (). We further demonstrated that expanded Htt interacted and co-localized with mitochondrial protein AIFM1. AIFM1 is attached to the inner mitochondrial membrane, where it exerts NADH oxidase activity,48
while mutations in this gene cause oxidative phosphorylation deficiency.49-51
Our biochemical fractionation confirmed the exclusive mitochondrial localization of AIFM1 in our experimental conditions. Subcellular co-localization of Htt and AIFM1 was also associated with mitochondria, with both proteins co-localizing with mitochondrial marker MnSOD (), further supporting the notion that mitochondria may be the predominant site of Htt/AIFM1 interaction. We found only the full-length Htt, but not its cleavage fragments, in the mitochondria, indicating specificity and suggesting that full-length Htt may be selectively transported into mitochondria, which may warrant further study.
AIFM1 plays a critical role in mitochondria-mediated cell death in certain cell types, including neurons.37
Induction of apoptosis results in the translocation of AIFM1 to the nucleus, where it binds DNA and affects chromosome condensation and fragmentation, although the precise mechanism of AIFM1 nuclear function is unknown.48,52-55
We showed that AIFM1 knockdown in striatal cells attenuated toxicity of expanded Htt-N585-82Q fragment (). However, we found no evidence of substantial nuclear co-localization of normal or expanded Htt with AIFM1 in our experimental conditions. To further clarify the possible role of Htt/AIFM1 interaction in mitochondria-mediated cell death, we examined whether this interaction is altered when mitochondrial death is suppressed. Bcl-2 is a multifunctional inhibitor of apoptosis that has been suggested to guard mitochondrial integrity and to control the release of mitochondrial proteins into the cytoplasm.56
Mitochondrial release of AIFM1 was also shown to be blocked by Bcl-2 at the cleavage step in the IMS.49
Our results indicate that Htt/AIFM1 interaction is preserved when mitochondrial death (and possibly the release of AIFM1 to the cytoplasm) is suppressed by Bcl2 overexpression. This is consistent with the predominant Htt/AIFM1 interaction within mitochondria. Taken together, these results suggest that AIFM1-mediated cell death pathway is activated in HD models, and that Htt and AIFM1 interaction within or in association with mitochondria, in part mediates mutant Htt toxicity.
Multiple transcript variants of AIFM1 arise from alternative splicing. Existence of a brain-specific isoform, AIF2, utilizing an alternative exon 2b, was demonstrated recently in reference 55
. AIF2 has a different IMM sorting signal that results in a stronger membrane attachment, thus AIF2 is less likely (than AIFM1) to translocate to the nucleus and mediate cell death. Since AIF2 dimerizes with AIFM1, potentially also preventing a release of AIFM1 from mitochondria, AIF2 may have a neuroprotective function. Thus it is possible that the loss of the brain-specific isoform, AIF2 (but not AIFM1), mediates CNS defects in AIF-difficient models. This hypothesis may also explain why only neurons are affected in Harlequin (Hq) mice,49
expressing less than 20% of normal levels of both AIFM1 and AIF2.55
These mice develop blindness, ataxia and neurodegeneration.49
Expression of AIF2 increases dramatically as neuronal precursor cells differentiate. Furthermore, AIF2 expression is restricted to developing and adult brain, with AIF2/AIFM1 ratio particularly increased in caudate nucleus and nucleus accumbens.55
These findings are of particular interest, since striatum is the most affected brain region in HD, and this may signify a possible interplay between brain-specific isoforms of AIF and mutant Htt.
In our iTRAQ experiments we identified two unique peptides (corresponding to AIFM), both downstream of exon 2, which is alternatively utilized in AIFM1/AIF2 isoforms. AIFM1 siRNA used in this study did not specifically target either splice variant. Thus both interaction/co-localization with Htt and attenuation of mutant Htt toxicity may be attributed to either or both AIFM1 and AIF2 isoforms. Future studies utilizing AIF2-specific reagents (antibodies and exon 2b-specific siRNAs) will elucidate possible roles of AIF isoforms in mutant Htt pathogenesis.
We found that Htt interacted and co-localized with SG-associated RNA-binding proteins Caprin-1 and G3BP1 in striatal cells under the ER-stress conditions. Both proteins were enriched within expanded Htt complexes, as shown by quantitative proteomics analysis. Caprin-1/G3BP1 physical and functional interaction has been shown to be involved in synaptic plasticity and neuronal network formation.57
In the brain, Caprin-1 (RNG 105) is expressed in postsynaptic granules (stress granules, SGs) in dendrites in the hippocampus and neocortex.38
It is found in messenger ribonucleoprotein particles (mRNPs) that also contain RNA-binding proteins like hnRNPK, PABP-1 Staufen, β tubulin and the motor protein dynein.58
SG formation occurs in cells exposed to environmental stress and is usually induced by stalled preinitiation complexes accumulating due to phosphorylation of eukatyotic translation initiation factor 2α (eIF2α), which blocks translation initiation. SGs contain mRNAs associated with small ribosomal subunit and certain translation factors, and SG assembly is promoted by any one of the RNA-binding proteins, many of which are able to oligomerize. Such proteins include TIA-1, FXR1, G3BP1 and others.59
Caprin-1 was shown to bind directly to mRNAs and repress translation via induction of phosphorylation of eIF2α;38,39
Caprin-1 associates and colocalizes with G3BP-1 in cytoplasmic RNA granules associated with microtubules.39
RasGAP-associated phosphorylation-dependent endoribonuclease G3BP (RasGAP SH3 domain binding protein-1) is a marker for SG and an effector of SG assembly.60,61
Caprin-1-localizing granules are specifiically associated with CaMKII, CREB, MAP2, BDNF and TrkB mRNAs.38
Thus Caprin-1/G3BP-1 complex is likely to regulate the transport and translation of mRNAs of proteins involved with synaptic plasticity in neurons, including BDNF.
BDNF has been linked to HD pathogenesis, since its levels are reduced in HD patients (reviewed in ref. 62
). The deregulation of BDNF gene transcription and defects in the microtubule-dependent transport of vesicles containing BDNF were also reported in HD neurons.63,64
A recent study from Tanese’s group demonstrates co-localization of BDNF mRNA with Htt, Ago2, CPEB and dynein in neuronal granules of cultured cortical neurons and the rat cortex.65
The authors propose that Htt may play a role in post-transcriptional transport/targeting of mRNA for BDNF, thus contributing to neuronal survival. Our new discovery that Htt associates with Caprin-1/G3BP1 complex in the SGs of striatal cells supports these ideas and suggests a new mechanism of regulation of BDNF mRNA processing and translation.
Neurons contain several types of RNA granules, which may contain specific and shared components. Mutant Htt associates with Ago2 in P bodies and contributes to RNA-mediated gene silencing.23,24
P bodies, unlike SG, are not associated with ribosome and mostly serve for storage and degradation of repressed mRNAs. P bodies contain decapping enzymes and endonucleases and may associate with components of RISC complex (argonaute, DICER). In our experiments, Htt associated with neuronal granules mostly resembling SG, since their formation was induced by ER stress, and they were positive for Caprin-1 and G3BP1, markers for SG. We also found other RNA-binding proteins associated with SG-PABP1, FXR1, Eif4G, dynein, FUS/TLS and TDP-43 among Htt-associated proteins (data not shown). In neurons, transport of translationally silenced mRNAs to dendritic synapses for translation occurs in the neuronal post-synaptic RNA granules, containing both small and large ribosomal subunits and translation initiation factors. Future studies will determine if normal or mutant Htt is also involved in these processes.
RNA metabolism has been increasingly implicated in a variety of motor neuron and neurodegenerative disorders. The mechanisms include all steps of RNA processing, such as RNA synthesis, splicing, localization, transport and translation.66-70
A major contribution to the field was a discovered link between two RNA-binding proteins, TDP-43 and FUS/TLS, and the pathogenesis of amyotrophic lateral sclerosis (ALS) and other neurodegenerative disorders. Postmortem analysis of patients with different polyQ diseases, including HD, showed the association of TDP-43 and FUS/TLS with intranuclear and cytoplasmic inclusions and co-localization with Htt (reviewed in ref. 71
). RNA processing has been previously implicated in HD based on several expression profiling studies in cells, mouse, yeast and fly models of HD.72-76
These studies demonstrate enrichment in ribosomal and RNA-processing proteins. In line with this, we showed that expanded Htt complexes were enriched in proteins related to RNA processing and regulation of translation and identified several novel Htt interactors within these pathways. It should be noted that many interactions of Htt identified in our studies may be indirect and are perhaps mediated by other proteins or even RNAs. The exact composition of Htt protein and RNA complexes will be determined by further studies.
In summary, our work presents the first systematic assessment of cellular processes that may be disrupted by abnormal interactions of expanded Htt. This information will serve as a platform for emerging studies that may pinpoint the key pathways and molecules involved, with a potential to identify new therapeutic targets for HD.