Biology subjects

Cariulo, C.

Publications and source records attributed to Cariulo, C..

4 recordsLinked to original sources

bioRxiv2026

Development and validation of highly selective monoclonal antibodies for the detection of huntingtin neoepitopes

Huntington's disease is caused by a CAG repeat tract expansion in the huntingtin gene, resulting in production of pathogenic N-terminal huntingtin protein fragments associated with disease pathology. Despite their central role, detection of these fragments has relied on a limited antibody repertoire with reproducibility concerns. Here, we describe the generation and characterization of recombinant rabbit monoclonal antibodies targeting two reciprocal neoepitopes flanking the huntingtin exon 1/exon 2 junction corresponding to amino acids P90 and K91. The P90 antibodies (clones 1B12, 11G2) demonstrate fragment-length-selective recognition of the C-terminal HTTexon1 P90 neoepitope with no detectable binding to full length huntingtin. A side-by-side comparison of the widely used monoclonal antibody MW8 from two different sources revealed measurable lot-to-lot drift in its fragment selectivity, whereas the recombinant P90 antibodies, expressed from a defined, sequenced clone, maintained consistent specificity, addressing this source-dependent variability. Whereas P90-positive fragments can arise through alternative splicing of the HTT1a transcript, generation of the reciprocal K91 N-terminal HTTexon2 neoepitope would require site-specific proteolytic cleavage, a mechanism that has not yet been directly tested for lack of a suitable reagent. The K91 antibody (clone 7G10) binds the N-terminal K91 neoepitope with high affinity and specificity over full length huntingtin and provides, for the first time, a tool capable of directly interrogating whether such cleavage occurs. Neoepitope specificity of these antibodies was orthogonally confirmed by protease digestion (Lys-N and Arg-C) coupled with intact mass spectrometry. As an additional outcome of the immunization and selection strategy, we discovered human-mouse cross-reactive antibodies (clones 27F5, 31C10) targeting the proline-rich domain of huntingtin that will facilitate mouse-human translational studies. All antibodies are recombinant, ensuring long-term reproducibility, and are being made available, along with their sequences, to the research community.

neuroscience↗

bioRxiv2025

Modulation of huntingtin S421 phosphorylation in a Huntington's disease mouse model and its detection in nonhuman primate cerebrospinal fluid

Huntingtons disease (HD) is a progressive neurodegenerative disease caused by the pathologic expansion of a CAG repeat in the first exon of the huntingtin (HTT) gene, resulting in a huntingtin (HTT) protein with an expanded polyglutamine (polyQ) tract. Phosphorylation at residue S421 (pS421) is one of the post-translational modifications proposed to influence the biology of wild-type and mutant (m)HTT, such as HTT stability and clearance, HTT subcellular localization, mHTT toxicity, and regulation of HTT function in axonal transport. However, the detection and quantification of S421-HTT phosphorylation in relevant biological contexts have remained challenging and the consequences of pS421 in HD pathogenesis remains unclear. Here we report the development of a novel ultrasensitive immunoassay enabling the specific and sensitive detection of pS421-HTT in a variety of biologically relevant contexts. With this assay we conducted a longitudinal assessment of pS421 levels in tissues from a mouse model of HD to investigate the relationship between S421 phosphorylation and phenotypic progression. We also identified PRKACA, the cAMP-regulated catalytic subunit of PKA, as a kinase capable of phosphorylating S421-HTT, demonstrating its ability to regulate endogenous pS421 in human cells. Finally, we exploited the sensitivity of the assay to detect endogenous pS421-HTT in cerebrospinal fluid (CSF) from nonhuman primates, showing for the first time that phosphorylation at S421-HTT can be detected in this bio-fluid. These reagents and assay will enable investigation of the biological consequence and the relevance of pS421 in the natural history of HD.

neuroscience↗

bioRxiv2022

IKBKB reduces huntingtin aggregation by phosphorylating Serine 13 via a non-canonical IKK pathway

N-terminal phosphorylation at residues T3 and S13 is believed to have important beneficial implications for the biological and pathological properties of mutant huntingtin, where IKBKB was identified as a candidate regulator of huntingtin N-terminal phosphorylation. The paucity of mechanistic information on IKK pathways, together with the lack of sensitive methods to quantify endogenous huntingtin phosphorylation, prevented detailed study of the role of IKBKB in Huntingtons disease. Using novel ultrasensitive assays, we demonstrate that IKBKB can regulate endogenous S13 huntingtin phosphorylation in a manner dependent on its kinase activity and known regulators. We found that the ability of IKBKB to phosphorylate endogenous huntingtin S13 is mediated through a non-canonical IRF3-mediated IKK-pathway, distinct from the established involvement of IKBKB in mutant huntingtins pathological mechanisms mediated via the canonical pathway. Furthermore, increased huntingtin S13 phosphorylation by IKBKB resulted in decreased aggregation of mutant huntingtin in cells, again dependent on its kinase activity. These findings point to a non-canonical IKK-pathway linking S13 huntingtin phosphorylation to the pathological properties of mutant huntingtin aggregation, thought to be significant to Huntingtons disease.

neuroscience↗

bioRxiv2020

Dose-dependent and disease-modifying effects of striatal infusion of cholesterol in Huntington's disease

A variety of pathophysiological mechanisms are implicated in Huntingtons disease (HD). Among them, reduced cholesterol biosynthesis has been detected in the HD mouse brain from pre-symptomatic stages, leading to diminished cholesterol synthesis, particularly in the striatum. In addition, systemic injection of cholesterol-loaded brain-permeable nanoparticles ameliorates synaptic and cognitive function in a transgenic mouse model of HD. To identify an appropriate treatment regimen and gain mechanistic insights into the beneficial activity of exogenous cholesterol in the HD brain, we employed osmotic mini-pumps to infuse three escalating doses of cholesterol directly into the striatum of HD mice in a continuous and rate-controlled manner. All tested doses prevented cognitive decline, while amelioration of disease-related motor defects was dose-dependent. In parallel, we found morphological and functional recovery of synaptic transmission involving both excitatory and inhibitory synapses of striatal medium spiny neurons. The treatment also enhanced endogenous cholesterol biosynthesis and clearance of mutant Huntingtin aggregates. These results indicate that cholesterol infusion to the striatum can exert a dose-dependent, disease-modifying effect and may be therapeutically relevant in HD.

neuroscience↗