A compact beta model of huntingtin toxicity

Huntington disease results from an expanded polyglutamine region in the N terminus of the huntingtin protein. HD pathology is characterized by neuronal degeneration and protein inclusions containing N-terminal fragments of mutant huntingtin. Structural information is minimal, though it is believed that mutant huntingtin polyglutamine adopts β structure upon conversion to a toxic form. To this end, we designed mammalian cell expression constructs encoding compact β variants of Htt exon 1 N-terminal fragment and tested their ability to aggregate and induce toxicity in cultured neuronal cells. In parallel, we performed molecular dynamics simulations, which indicate that constructs with expanded polyglutamine β-strands are stabilized by main-chain hydrogen bonding. Finally, we found a correlation between the reactivity to 3B5H10, an expanded polyglutamine antibody that recognizes a compact β rich hairpin structure, and the ability to induce cell toxicity. These data are consistent with an important role for a compact β structure in mutant huntingtin-induced cell toxicity.     

A screen for enhancers of clearance identifies huntingtin as a heat shock protein 90 (Hsp90) client protein

Mechanisms to reduce the cellular levels of mutant huntingtin (mHtt) provide promising strategies for treating Huntington disease (HD). To identify compounds enhancing the degradation of mHtt, we performed a high throughput screen using a hippocampal HN10 cell line expressing a 573-amino acid mHtt fragment. Several hit structures were identified as heat shock protein 90 (Hsp90) inhibitors. Cell treatment with these compounds reduced levels of mHtt without overt toxic effects as measured by time-resolved Förster resonance energy transfer assays and Western blots. To characterize the mechanism of mHtt degradation, we used the potent and selective Hsp90 inhibitor NVP-AUY922. In HdhQ150 embryonic stem (ES) cells and in ES cell-derived neurons, NVP-AUY922 treatment substantially reduced soluble full-length mHtt levels. In HN10 cells, Hsp90 inhibition by NVP-AUY922 enhanced mHtt clearance in the absence of any detectable Hsp70 induction. Furthermore, inhibition of protein synthesis with cycloheximide or overexpression of dominant negative heat shock factor 1 (Hsf1) in HdhQ150 ES cells attenuated Hsp70 induction but did not affect NVP-AUY922-mediated mHtt clearance. Together, these data provided evidence that direct inhibition of Hsp90 chaperone function was crucial for mHtt degradation rather than heat shock response induction and Hsp70 up-regulation. Co-immunoprecipitation experiments revealed a physical interaction of mutant and wild-type Htt with the Hsp90 chaperone. Hsp90 inhibition disrupted the interaction and induced clearance of Htt through the ubiquitin-proteasome system. Our data suggest that Htt is an Hsp90 client protein and that Hsp90 inhibition may provide a means to reduce mHtt in HD.     

Dithiol-based compounds maintain expression of antioxidant protein peroxiredoxin 1 that counteracts toxicity of mutant huntingtin

Mitochondrial dysfunction and elevated reactive oxygen species are strongly implicated in both aging and various neurodegenerative disorders, including Huntington disease (HD). Because reactive oxygen species can promote the selective oxidation of protein cysteine sulfhydryl groups to disulfide bonds we examined the spectrum of disulfide-bonded proteins that were specifically altered in a HD context. Protein extracts from PC12 cells overexpressing the amino-terminal fragment of the Huntingtin (Htt) protein with either a nonpathogenic or pathogenic polyglutamine repeat (Htt-103Q) were resolved by redox two-dimensional PAGE followed by mass spectrometry analysis. Several antioxidant proteins were identified that exhibited changes in disulfide bonding unique to Htt-103Q expressing cells. In particular, the antioxidant protein peroxiredoxin 1 (Prx1) exhibited both decreased expression and hyperoxidation in response to mutant Htt expressed in either PC12 cells or immortalized striatal cells exposed to 3-nitropropionic acid. Ectopic expression of Prx1 in PC12 cells attenuated mutant Htt-induced toxicity. In contrast, short hairpin RNA-mediated knockdown of Prx1 potentiated mHtt toxicity. Furthermore, treatment with the dithiol-based compounds dimercaptopropanol and dimercaptosuccinic acid suppressed toxicity in both HD cell models, whereas monothiol compounds were relatively ineffective. Dimercaptopropanol treatment also prevented mutant Htt-induced loss of Prx1 expression in both cell models. Our studies reveal for the first time that pathogenic Htt can affect the expression and redox state of antioxidant proteins; an event countered by specific dithiol-based compounds. These findings should provide a catalyst to explore the use of dithiol-based drugs for the treatment of neurodegenerative diseases.     

Generation of a new form of human PrP(Sc) in vitro by interspecies transmission from cervid prions

Prion diseases are infectious neurodegenerative disorders that affect humans and animals and that result from the conversion of normal prion protein (PrP(C)) into the misfolded prion protein (PrP(Sc)). Chronic wasting disease (CWD) is a prion disorder of increasing prevalence within the United States that affects a large population of wild and captive deer and elk. Determining the risk of transmission of CWD to humans is of utmost importance, considering that people can be infected by animal prions, resulting in new fatal diseases. To study the possibility that human PrP(C) can be converted into the misfolded form by CWD PrP(Sc), we performed experiments using the protein misfolding cyclic amplification technique, which mimics in vitro the process of prion replication. Our results show that cervid PrP(Sc) can induce the conversion of human PrP(C) but only after the CWD prion strain has been stabilized by successive passages in vitro or in vivo. Interestingly, the newly generated human PrP(Sc) exhibits a distinct biochemical pattern that differs from that of any of the currently known forms of human PrP(Sc). Our results also have profound implications for understanding the mechanisms of the prion species barrier and indicate that the transmission barrier is a dynamic process that depends on the strain and moreover the degree of adaptation of the strain. If our findings are corroborated by infectivity assays, they will imply that CWD prions have the potential to infect humans and that this ability progressively increases with CWD spreading.     

Dynamic imaging by fluorescence correlation spectroscopy identifies diverse populations of polyglutamine oligomers formed in vivo

Protein misfolding and aggregation are exacerbated by aging and diseases of protein conformation including neurodegeneration, metabolic diseases, and cancer. In the cellular environment, aggregates can exist as discrete entities, or heterogeneous complexes of diverse solubility and conformational state. In this study, we have examined the in vivo dynamics of aggregation using imaging methods including fluorescence microscopy, fluorescence recovery after photobleaching (FRAP), and fluorescence correlation spectroscopy (FCS), to monitor the diverse biophysical states of expanded polyglutamine (polyQ) proteins expressed in Caenorhabditis elegans. We show that monomers, oligomers and aggregates co-exist at different concentrations in young and aged animals expressing different polyQ-lengths. During aging, when aggregation and toxicity are exacerbated, FCS-based burst analysis and purified single molecule FCS detected a populational shift toward an increase in the frequency of brighter and larger oligomeric species. Regardless of age or polyQ-length, oligomers were maintained in a heterogeneous distribution that spans multiple orders of magnitude in brightness. We employed genetic suppressors that prevent polyQ aggregation and observed a reduction in visible immobile species with the persistence of heterogeneous oligomers, yet our analysis did not detect the appearance of any discrete oligomeric states associated with toxicity. These studies reveal that the reversible transition from monomers to immobile aggregates is not represented by discrete oligomeric states, but rather suggests that the process of aggregation involves a more complex pattern of molecular interactions of diverse intermediate species that can appear in vivo and contribute to aggregate formation and toxicity.     

Proteolysis of Mutant Huntingtin Produces an Exon 1 Fragment That Accumulates as an Aggregated Protein in Neuronal Nuclei in Huntington Disease

Huntingtin proteolysis has been implicated in the molecular pathogenesis of Huntington disease (HD). Despite an intense effort, the identity of the pathogenic smallest N-terminal fragment has not been determined. Using a panel of anti-huntingtin antibodies, we employed an unbiased approach to generate proteolytic cleavage maps of mutant and wild-type huntingtin in the HdhQ150 knock-in mouse model of HD. We identified 14 prominent N-terminal fragments, which, in addition to the full-length protein, can be readily detected in cytoplasmic but not nuclear fractions. These fragments were detected at all ages and are not a consequence of the pathogenic process. We demonstrated that the smallest fragment is an exon 1 huntingtin protein, known to contain a potent nuclear export signal. Prior to the onset of behavioral phenotypes, the exon 1 protein, and possibly other small fragments, accumulate in neuronal nuclei in the form of a detergent insoluble complex, visualized as diffuse granular nuclear staining in tissue sections. This methodology can be used to validate the inhibition of specific proteases as therapeutic targets for HD by pharmacological or genetic approaches.     

CX3CR1 protein signaling modulates microglial activation and protects against plaque-independent cognitive deficits in a mouse model of Alzheimer disease

Aberrant microglial activation has been proposed to contribute to the cognitive decline in Alzheimer disease (AD), but the underlying molecular mechanisms remain enigmatic. Fractalkine signaling, a pathway mediating the communication between microglia and neurons, is deficient in AD brains and down-regulated by amyloid-β. Although fractalkine receptor (CX3CR1) on microglia was found to regulate plaque load, no functional effects have been reported. Our study demonstrates that CX3CR1 deficiency worsens the AD-related neuronal and behavioral deficits. The effects were associated with cytokine production but not with plaque deposition. Ablation of CX3CR1 in mice overexpressing human amyloid precursor protein enhanced Tau pathology and exacerbated the depletion of calbindin in the dentate gyrus. The levels of calbindin in the dentate gyrus correlated negatively with those of tumor necrosis factor α and interleukin 6, suggesting neurotoxic effects of inflammatory factors. Functionally, removing CX3CR1 in human amyloid precursor protein mice worsened the memory retention in passive avoidance and novel object recognition tests, and their memory loss in the novel object recognition test is associated with high levels of interleukin 6. Our findings identify CX3CR1 as a key microglial pathway in protecting against AD-related cognitive deficits that are associated with aberrant microglial activation and elevated inflammatory cytokines.     

Enhanced Aggregation of Androgen Receptor in Induced Pluripotent Stem Cell-derived Neurons from Spinal and Bulbar Muscular Atrophy

Spinal and bulbar muscular atrophy (SBMA) is an X-linked motor neuron disease caused by a CAG repeat expansion in the androgen receptor (AR) gene. Ligand-dependent nuclear accumulation of mutant AR protein is a critical characteristic of the pathogenesis of SBMA. SBMA has been modeled in AR-overexpressing animals, but precisely how the polyglutamine (polyQ) expansion leads to neurodegeneration is unclear. Induced pluripotent stem cells (iPSCs) are a new technology that can be used to model human diseases, study pathogenic mechanisms, and develop novel drugs. We established SBMA patient-derived iPSCs, investigated their cellular biochemical characteristics, and found that SBMA-iPSCs can differentiate into motor neurons. The CAG repeat numbers in the AR gene of SBMA-iPSCs and also in the atrophin-1 gene of iPSCs derived from another polyQ disease, dentato-rubro-pallido-luysian atrophy (DRPLA), remain unchanged during reprogramming, long term passage, and differentiation, indicating that polyQ disease-associated CAG repeats are stable during maintenance of iPSCs. The level of AR expression is up-regulated by neuronal differentiation and treatment with the AR ligand dihydrotestosterone. Filter retardation assays indicated that aggregation of ARs following dihydrotestosterone treatment in neurons derived from SBMA-iPSCs increases significantly compared with neurological control iPSCs, easily recapitulating the pathological feature of mutant ARs in SBMA-iPSCs. This phenomenon was not observed in iPSCs and fibroblasts, thereby showing the neuron-dominant phenotype of this disease. Furthermore, the HSP90 inhibitor 17-allylaminogeldanamycin sharply decreased the level of aggregated AR in neurons derived from SBMA-iPSCs, indicating a potential for discovery and validation of candidate drugs. We found that SBMA-iPSCs possess disease-specific biochemical features and could thus open new avenues of research into not only SBMA, but also other polyglutamine diseases.     

Identification of low molecular weight pyroglutamate A{beta} oligomers in Alzheimer disease: a novel tool for therapy and diagnosis

N-terminally truncated Aβ peptides starting with pyroglutamate (AβpE3) represent a major fraction of all Aβ peptides in the brain of Alzheimer disease (AD) patients. AβpE3 has a higher aggregation propensity and stability and shows increased toxicity compared with full-length Aβ. In the present work, we generated a novel monoclonal antibody (9D5) that selectively recognizes oligomeric assemblies of AβpE3 and studied the potential involvement of oligomeric AβpE3 in vivo using transgenic mouse models as well as human brains from sporadic and familial AD cases. 9D5 showed an unusual staining pattern with almost nondetectable plaques in sporadic AD patients and non-demented controls. Interestingly, in sporadic and familial AD cases prominent intraneuronal and blood vessel staining was observed. Using a novel sandwich ELISA significantly decreased levels of oligomers in plasma samples from patients with AD compared with healthy controls were identified. Moreover, passive immunization of 5XFAD mice with 9D5 significantly reduced overall Aβ plaque load and AβpE3 levels, and normalized behavioral deficits. These data indicate that 9D5 is a therapeutically and diagnostically effective monoclonal antibody targeting low molecular weight AβpE3 oligomers.     

Defining the protein complexome of translation termination factor eRF1: Identification of four novel eRF1‐containing complexes that range from 20S to 57S in size

The eukaryotic eRF1 translation termination factor plays an important role in recognizing stop codons and initiating the end to translation. However, which exact complexes contain eRF1 and at what abundance is not clear. We have used analytical ultracentrifugation with fluorescent detection system to identify the protein complexome of eRF1 in the yeast Saccharomyces cerevisiae. In addition to eRF1 presence in translating polysomes, we found that eRF1 associated with five other macromolecular complexes: 77S, 57S, 39S, 28S, and 20S in size. Generally equal abundances of each of these complexes were found. The 77S complex primarily contained the free 80S ribosome consistent with in vitro studies and did not appear to contain significant levels of the monosomal translating complex that co‐migrates with the free 80S ribosome. The 57S and 39S complexes represented, respectively, free 60S and 40S ribosomal subunits bound to eRF1, associations not previously reported. The novel 28S and 20S complexes (containing minimal masses of 830 KDa and 500 KDa, respectively) lacked significant RNA components and appeared to be oligomeric, as eRF1 has a mass of 49 KDa. The majority of polysomal complexes containing eRF1 were both substantially deadenylated and lacking in closed‐loop factors eIF4E and eIF4G. The thirteen percent of such translating polysomes that contained poly(A) tails had equivalent levels of eIF4E and eIF4G, suggesting these complexes were in a closed‐loop structure. The identification of eRF1 in these unique and previously unrecognized complexes suggests a variety of new roles for eRF1 in the regulation of cellular processes.