New anti-huntingtin monoclonal antibodies: implications for huntingtin conformation and its binding proteins (2023)

Huntington’s disease is caused by a polyglutamine (polyQ) expansion in the huntingtin protein. Huntingtin exon 1 (Httex1), as well as other naturally occurring N-terminal huntingtin fragments with expanded polyQ are prone to aggregation, forming potentially cytotoxic oligomers and fibrils. Antibodies and other N-terminal huntingtin binders are widely explored as biomarkers and possible aggregation-inhibiting therapeutics. A monoclonal antibody, MW1, is known to preferentially bind to huntingtin fragments with expanded polyQ lengths, but the molecular basis of the polyQ length specificity remains poorly understood. Using solution NMR, electron paramagnetic resonance, and other biophysical methods, we investigated the structural features of the Httex1–MW1 interaction. Rather than recognizing residual α-helical structure, which is promoted by expanded Q-lengths, MW1 caused the formation of a new, non-native, conformation in which the entire polyQ is largely extended. This non-native polyQ structure allowed the formation of large mixed Httex1-MW1 multimers (600–2900 kD), when Httex1 with pathogenic Q-length (Q46) was used. We propose that these multivalent, entropically favored interactions, are available only to proteins with longer Q-lengths and represent a major factor governing the Q-length preference of MW1. The present study reveals that it is possible to target proteins with longer Q-lengths without having to stabilize a natively favored conformation. Such mechanisms could be exploited in the design of other Q-length specific binders.

Huntington's disease (HD) is the most common monogenic neurodegenerative disease and is fatal. CAG repeat expansions in mutant Huntingtin (mHTT) exon 1encode for polyglutamine (polyQ) stretches and influence age of onset and disease severity, depending on their length. mHTT is more structured compared to wild-type (wt) HTT, resulting in a decreased N-terminal conformational flexibility. mHTT inflexibility may contribute to both gain of function toxicity, due to increased mHTT aggregation propensity, but also to loss of function phenotypes, due to decreased interactions with binding partners. High-throughput-screening techniques to identify mHTT flexibility states and potential flexibility modifying small molecules are currently lacking. Here, we propose a novel approach for identifying small molecules that restore mHTT's conformational flexibility in human patient fibroblasts. We have applied a well-established antibody-based time-resolved Förster resonance energy transfer (TR-FRET) immunoassay, which measures endogenous HTT flexibility using two validated HTT-specific antibodies, to a high-throughput screening platform. By performing a small-scale compound screen, we identified several small molecules that can partially rescue mHTT inflexibility, presumably by altering HTT post-translational modifications. Thus, we demonstrated that the HTT TR-FRET immunoassay can be miniaturized and applied to a compound screening workflow in patient cells. This automated assay can now be used in large screening campaigns to identify previously unknown HD drugs and drug targets.

Huntington’s disease is a heritable neurodegenerative disease that is caused by a CAG expansion in the first exon of the huntingtin gene. This expansion results in an elongated polyglutamine domain that increases the propensity of huntingtin exon-1 to form cross-β fibrils. Although the polyglutamine domain is important for fibril formation, the dynamic, C-terminal proline-rich domain (PRD) of huntingtin exon-1 makes up a large fraction of the fibril surface. Because potential fibril toxicity has to be mediated by interactions of the fibril surface with its cellular environment, we wanted to model the conformational space adopted by the PRD. We ran 800-ns long molecular dynamics simulations of the PRD using an explicit water model optimized for intrinsically disordered proteins. These simulations accurately predicted our previous solid-state NMR data and newly acquired electron paramagnetic resonance double electron-electron resonance distances, lending confidence in their accuracy. The simulations show that the PRD generally forms an imperfect polyproline (polyP) II helical conformation. The two polyP regions within the PRD stay in a polyP II helix for most of the simulation, whereas occasional kinks in the proline-rich linker region cause an overall bend in the PRD structure. The dihedral angles of the glycine at the end of the second polyP region are very variable, effectively decoupling the highly dynamic 12 C-terminal residues from the rest of the PRD.

Intrabodies (both single-chain Fv and single-domain VH, VHH, and VL nanobodies) offer unique solutions to some of the challenges of delivery and target engagement posed by immunotherapeutics for the brain and other areas of the nervous system. The specificity, which includes the recognition of post-translational modifications, and capacity for engineering that characterize these antibody fragments can be especially well-focused when the genes encoding only the binding sites of the antibody are expressed intracellularly. Multifunctional constructs use fusions with peptides that can re-target antigen-antibody complexes to enhance both pharmacodynamic activity and intracellular solubility simultaneously. Fusions with proteolytic targeting signals, such as the PEST degron, greatly enhance potency in some cases. Stem cell transplants can be protected from exogenous misfolded proteins by stable transfection with intrabodies. Tandem expression to target two or more misfolding proteins in one treatment may be especially valuable for proteostatic disruptions due to genetic, aging, or toxic triggers. Advances in bioinformatics, screening protocols, and especially gene therapy are showing great promise for intrabody/ nanobody treatments of a full range of neurological disorders, including Alzheimer's disease and related tau dementias, Parkinson's disease and Lewy body diseases, Huntington's disease, amyotrophic lateral sclerosis, and prion diseases, among others.

One of the pathological hallmarks of Huntington disease (HD) is accumulation of the disease-causing mutant huntingtin (mHTT), which leads to the disruption of a variety of cellular functions, ultimately resulting in cell death. Induction of autophagy, for example by the inhibition of mechanistic target of rapamycin (mTOR) signaling, has been shown to reduce HTT levels and aggregates. While rapalogs like rapamycin allosterically inhibit the mTOR complex 1 (TORC1), ATP-competitive mTOR inhibitors suppress activities of TORC1 and TORC2 and have been shown to be more efficient in inducing autophagy and reducing protein levels and aggregates than rapalogs. The ability to cross the blood-brain barrier of first generation catalytic mTOR inhibitors has so far been limited, and therefore sufficient target coverage in the brain could not be reached. Two novel, brain penetrant compounds – the mTORC1/2 inhibitor PQR620, and the dual pan-phosphoinositide 3-kinase (PI3K) and mTORC1/2 kinase inhibitor PQR530 - were evaluated by assessing their potential to induce autophagy and reducing mHTT levels. For this purpose, expression levels of autophagic markers and well-defined mTOR targets were analyzed in STHdh cells and HEK293T cells and in mouse brains. Both compounds potently inhibited mTOR signaling in cell models as well as in mouse brain. As proof of principle, reduction of aggregates and levels of soluble mHTT were demonstrated upon treatment with both compounds. Originally developed for cancer treatment, these second generation mTORC1/2 and PI3K/mTOR inhibitors show brain penetrance and efficacy in cell models of HD, making them candidate molecules for further investigations in HD.

Experimental Cell Research, Volume 340, Issue 2, 2016, pp. 227-237

Human induced pluripotent stem cells (hiPSCs) are capable of unlimited self-renewal and can generate nearly all cells in the body. Changes induced by different LSD1 activities on the regulation of hiPSC self-renewal and differentiation and the mechanism underlying such changes were determined. We used two different LSD1 inhibitors (phenelzine sulfate and tranylcypromine) and RNAi technique to inhibit LSD1 activity, and we obtained hiPSCs showing 71.3%, 53.28%, and 31.33% of the LSD1 activity in normal hiPSCs. The cells still maintained satisfactory self-renewal capacity when LSD1 activity was at 71.3%. The growth rate of hiPSCs decreased and cells differentiated when LSD1 activity was at approximately 53.28%. The hiPSCs were mainly arrested in the G0/G1 phase and simultaneously differentiated into endodermal tissue when LSD1 activity was at 31.33%. Teratoma experiments showed that the downregulation of LSD1 resulted in low teratoma volume. When LSD1 activity was below 50%, pluripotency of hiPSCs was impaired, and the teratomas mainly comprised endodermal and mesodermal tissues. This phenomenon was achieved by regulating the critical balance between histone methylation and demethylation at regulatory regions of several key pluripotent and developmental genes.

The American Journal of Human Genetics, Volume 104, Issue 6, 2019, pp. 1116-1126

Huntington disease (HD) is caused by a CAG repeat expansion in the huntingtin (HTT) gene. Although the length of this repeat is inversely correlated with age of onset (AOO), it does not fully explain the variability in AOO. We assessed the sequence downstream of the CAG repeat in HTT [reference: (CAG)n-CAA-CAG], since variants within this region have been previously described, but no study of AOO has been performed. These analyses identified a variant that results in complete loss of interrupting (LOI) adenine nucleotides in this region [(CAG)n-CAG-CAG]. Analysis of multiple HD pedigrees showed that this LOI variant is associated with dramatically earlier AOO (average of 25 years) despite the same polyglutamine length as in individuals with the interrupting penultimate CAA codon. This LOI allele is particularly frequent in persons with reduced penetrance alleles who manifest with HD and increases the likelihood of presenting clinically with HD with a CAG of 36–39 repeats. Further, we show that the LOI variant is associated with increased somatic repeat instability, highlighting this as a significant driver of this effect. These findings indicate that the number of uninterrupted CAG repeats, which is lengthened by the LOI, is the most significant contributor to AOO of HD and is more significant than polyglutamine length, which is not altered in these individuals. In addition, we identified another variant in this region, where the CAA-CAG sequence is duplicated, which was associated with later AOO. Identification of these cis-acting modifiers have potentially important implications for genetic counselling in HD-affected families.

Journal of Molecular Biology, Volume 427, Issue 12, 2015, pp. 2166-2178

Huntington's disease is triggered by misfolding of fragments of mutant forms of the huntingtin protein (mHTT) with aberrant polyglutamine expansions. The C4 single-chain Fv antibody (scFv) binds to the first 17 residues of huntingtin [HTT(1-17)] and generates substantial protection against multiple phenotypic pathologies in situ and in vivo. We show in this paper that C4 scFv inhibits amyloid formation by exon1 fragments of huntingtin in vitro and elucidate the structural basis for this inhibition and protection by determining the crystal structure of the complex of C4 scFv and HTT(1-17). The peptide binds with residues 3–11 forming an amphipathic helix that makes contact with the antibody fragment in such a way that the hydrophobic face of this helix is shielded from the solvent. Residues 12–17 of the peptide are in an extended conformation and interact with the same region of another C4 scFv:HTT(1-17) complex in the asymmetric unit, resulting in a β-sheet interface within a dimeric C4 scFv:HTT(1-17) complex. The nature of this scFv–peptide complex was further explored in solution by high-resolution NMR and physicochemical analysis of species in solution. The results provide insights into the manner in which C4 scFv inhibits the aggregation of HTT, and hence into its therapeutic potential, and suggests a structural basis for the initial interactions that underlie the formation of disease-associated amyloid fibrils by HTT.

Stem Cell Reports, Volume 12, Issue 2, 2019, pp. 191-200

Striatal interneurons are born in the medial and caudal ganglionic eminences (MGE and CGE) and play an important role in human striatal function and dysfunction in Huntington's disease and dystonia. MGE/CGE-like neural progenitors have been generated from human pluripotent stem cells (hPSCs) for studying cortical interneuron development and cell therapy for epilepsy and other neurodevelopmental disorders. Here, we report the capacity of hPSC-derived MGE/CGE-like progenitors to differentiate into functional striatal interneurons. Invitro, these hPSC neuronal derivatives expressed cortical and striatal interneuron markers at the mRNA and protein level and displayed maturing electrophysiological properties. Following transplantation into neonatal rat striatum, progenitors differentiated into striatal interneuron subtypes and were consistently found in the nearby septum and hippocampus. These findings highlight the potential for hPSC-derived striatal interneurons as an invaluable tool in modeling striatal development and function invitro or as a source of cells for regenerative medicine.

Handbook of Clinical Neurology, Volume 147, 2018, pp. 143-170

Throughout the genome, unstable tandem nucleotide repeats can expand to cause a variety of neurologic disorders. Expansion of a CAG triplet repeat within a coding exon gives rise to an elongated polyglutamine (polyQ) tract in the resultant protein product, and accounts for a unique category of neurodegenerative disorders, known as the CAG–polyglutamine repeat diseases. The nine members of the CAG–polyglutamine disease family include spinal and bulbar muscular atrophy (SBMA), Huntington disease, dentatorubral pallidoluysian atrophy, and six spinocerebellar ataxias (SCA 1, 2, 3, 6, 7, and 17). All CAG–polyglutamine diseases are dominantly inherited, with the exception of SBMA, which is X-linked, and many CAG–polyglutamine diseases display anticipation, which is defined as increasing disease severity in successive generations of an affected kindred. Despite widespread expression of the different polyQ-expanded disease proteins throughout the body, each CAG–polyglutamine disease strikes a particular subset of neurons, although the mechanism for this cell-type selectivity remains poorly understood. While the different genes implicated in these disorders display amino acid homology only in the repeat tract domain, certain pathologic molecular processes have been implicated in almost all of the CAG–polyglutamine repeat diseases, including protein aggregation, proteolytic cleavage, transcription dysregulation, autophagy impairment, and mitochondrial dysfunction. Here we highlight the clinical and molecular genetic features of each distinct disorder, and then discuss common themes in CAG–polyglutamine disease pathogenesis, closing with emerging advances in therapy development.

Stem Cell Reports, Volume 8, Issue 3, 2017, pp. 619-633

Huntington disease (HD) is a dominant neurodegenerative disorder caused by a CAG repeat expansion in HTT. Here we report correction of HD human induced pluripotent stem cells (hiPSCs) using a CRISPR-Cas9 and piggyBac transposon-based approach. We show that both HD and corrected isogenic hiPSCs can be differentiated into excitable, synaptically active forebrain neurons. We further demonstrate that phenotypic abnormalities in HD hiPSC-derived neural cells, including impaired neural rosette formation, increased susceptibility to growth factor withdrawal, and deficits in mitochondrial respiration, are rescued in isogenic controls. Importantly, using genome-wide expression analysis, we show that a number of apparent gene expression differences detected between HD and non-related healthy control lines are absent between HD and corrected lines, suggesting that these differences are likely related to genetic background rather than HD-specific effects. Our study demonstrates correction of HD hiPSCs and associated phenotypic abnormalities, and the importance of isogenic controls for disease modeling using hiPSCs.