SynB3 Conjugated QBP1 Passes Blood-Brain Barrier Models and Inhibits PolyQ Protein Aggregation.

BACKGROUND: Polyglutamine diseases are degenerative diseases in the central nervous system caused by CAG trinucleotide repeat expansion which encodes polyglutamine tracts, leading to the misfolding of pathological proteins. Small peptides can be designed to prevent polyglutamine diseases by inhibiting the polyglutamine protein aggregation, for example, polyglutamine binding peptide 1(QBP1). However, the transportation capability of polyglutamine binding peptide 1 across the blood-brain barrier is less efficient. We hypothesized whether its therapeutic effect could be improved by increasing the rate of membrane penetration. OBJECTIVE: The objective of the study was to explore whether polyglutamine binding peptide 1 conjugated cell-penetrating peptides could pass through the blood-brain barrier and inhibit the aggregation of polyglutamine proteins. METHODS: In order to investigate the toxic effects, we constructed a novel stable inducible PC12 cells to express Huntington protein that either has 11 glutamine repeats or 63 glutamine repeats to mimic wild type and polyglutamine expand Huntington protein, respectively. Both SynB3 and TAT conjugated polyglutamine binding peptide 1 was synthesized, respectively. We tested their capabilities to pass through a Trans-well system and subsequently studied the counteractive effects on polyglutamine protein aggregation. RESULTS: The conjugation of cell-penetrating peptides to SynB3 and TAT enhanced the transportation of polyglutamine binding peptide 1 across the mono-cell layer and ameliorated polyglutamine-- expanded Huntington protein aggregation; moreover, SynB3 showed better delivery efficiency than TAT. Interestingly, it has been observed that polyglutamine binding peptide 1 specifically inhibited polyglutamine-expanded protein aggregation rather than affected other amyloidosis proteins, for example, ß-Amyloid. CONCLUSION: Our study indicated that SynB3 could be an effective carrier for polyglutamine binding peptide 1 distribution through the blood-brain barrier model and ameliorate the formation of polyglutamine inclusions; thus SynB3 conjugated polyglutamine binding peptide 1 could be considered as a therapeutic candidate for polyglutamine diseases.

The ß-turn-supporting motif in the polyglutamine binding peptide QBP1 is essential for inhibiting huntingtin aggregation.

Aggregation of polyglutamine proteins is a hallmark of several neurodegenerative diseases. The 11-residue polyglutamine binding peptide Ac-SNWKWWPGIFD-am, known as QBP1, inhibits polyglutamine aggregation. Besides, a minimal 8-residue stretch in the QBP1 peptide (Ac-WKWWPGIF-am) is reported in the literature to retain this activity. Both peptides harbor a Pro-Gly dipeptide motif, a feature characteristic of potential ß-turn regions. Here, we investigated whether the presence of this ß-turn motif is necessary for the inhibition of huntingtin aggregation, a polyglutamine protein implicated in Huntington's disease. Using single amino acid substitutions to generate analogs that could support, introduce, or eliminate the ß-turn, we show that the turn-supporting motif is essential for QBP1-mediated inhibition of huntingtin aggregation.

Brain-Targeting Delivery of Two Peptidylic Inhibitors for Their Combination Therapy in Transgenic Polyglutamine Disease Mice via Intranasal Administration.

Polyglutamine diseases are a set of progressive neurodegenerative disorders caused by misfolding and aggregation of mutant CAG RNA and polyglutamin protein. To date, there is a lack of effective therapeutics that can counteract the polyglutamine neurotoxicity. Two peptidylic inhibitors, QBP1 and P3, targeting the protein and RNA toxicities, respectively, have been previously demonstrated by us with combinational therapeutic effects on the Drosophila polyglutamine disease model. However, their therapeutic efficacy has never been investigated in vivo in mammals. The current study aims to (a) develop a brain-targeting delivery system for both QBP1 and L1P3V8 (a lipidated variant of P3 with improved stability) and (b) evaluate their therapeutic effects on the R6/2 transgenic mouse model of polyglutamine disease. Compared with intravenous administration, intranasal administration of QBP1 significantly increased its brain-to-plasma ratio. In addition, employment of a chitosan-containing in situ gel for the intranasal administration of QBP1 notably improved its brain concentration for up to 10-fold. Further study on intranasal cotreatment with the optimized formulation of QBP1 and L1P3V8 in mice found no interference on the brain uptake of each other. Subsequent efficacy evaluation of 4-week daily QBP1 (16 µmol/kg) and L1P3V8 (6 µmol/kg) intranasal cotreatment in the R6/2 mice demonstrated a significant improvement on the motor coordination and explorative behavior of the disease mice, together with a full suppression on the RNA- and protein-toxicity markers in their brains. In summary, the current study developed an efficient intranasal cotreatment of the two peptidylic inhibitors, QBP1 and L1P3V8, for their brain-targeting, and such a novel therapeutic strategy was found to be effective on a transgenic polyglutamine disease mouse model.

Protein Misfolding and Aggregation as a Therapeutic Target for Polyglutamine Diseases.

The polyglutamine (polyQ) diseases, such as Huntington's disease and several types of spinocerebellar ataxias, are a group of inherited neurodegenerative diseases that are caused by an abnormal expansion of the polyQ tract in disease-causative proteins. Proteins with an abnormally expanded polyQ stretch undergo a conformational transition to ß-sheet rich structure, which assemble into insoluble aggregates with ß-sheet rich amyloid fibrillar structures and accumulate as inclusion bodies in neurons, eventually leading to neurodegeneration. Since misfolding and aggregation of the expanded polyQ proteins are the most upstream event in the most common pathogenic cascade of the polyQ diseases, they are proposed to be one of the most ideal targets for development of disease-modifying therapies for polyQ diseases. In this review, we summarize the current understanding of the molecular pathogenic mechanisms of the polyQ diseases, and introduce therapeutic approaches targeting misfolding and aggregation of the expanded polyQ proteins, which are not only effective on a wide spectrum of polyQ diseases, but also broadly correct the functional abnormalities of multiple downstream cellular processes affected in the aggregation process of polyQ proteins. We hope that in the near future, effective therapies are developed, to bring hope to many patients suffering from currently intractable polyQ diseases.

Assessing a peptidylic inhibitor-based therapeutic approach that simultaneously suppresses polyglutamine RNA- and protein-mediated toxicities in patient cells and Drosophila.

Polyglutamine (polyQ) diseases represent a group of progressive neurodegenerative disorders that are caused by abnormal expansion of CAG triplet nucleotides in disease genes. Recent evidence indicates that not only mutant polyQ proteins, but also their corresponding mutant RNAs, contribute to the pathogenesis of polyQ diseases. Here, we describe the identification of a 13-amino-acid peptide, P3, which binds directly and preferentially to long-CAG RNA within the pathogenic range. When administered to cell and Drosophila disease models, as well as to patient-derived fibroblasts, P3 inhibited expanded-CAG-RNA-induced nucleolar stress and suppressed neurotoxicity. We further examined the combined therapeutic effect of P3 and polyQ-binding peptide 1 (QBP1), a well-characterized polyQ protein toxicity inhibitor, on neurodegeneration. When P3 and QBP1 were co-administered to disease models, both RNA and protein toxicities were effectively mitigated, resulting in a notable improvement of neurotoxicity suppression compared with the P3 and QBP1 single-treatment controls. Our findings indicate that targeting toxic RNAs and/or simultaneous targeting of toxic RNAs and their corresponding proteins could open up a new therapeutic strategy for treating polyQ degeneration.