Biology subjects

Munoz-Sanjuan, I.

Publications and source records attributed to Munoz-Sanjuan, I..

8 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

Mapping brain volume changes in the zQ175DN mouse model of Huntington's disease: a longitudinal MRI study

Huntingtons disease (HD) is a progressive neurodegenerative disease affecting motor and cognitive abilities, as well as exhibiting psychiatric manifestations. Studies in people with HD (PwHD) consistently report atrophy of the caudate and putamen as an early pathological event and is therefore considered an early biomarker. Investigating whether similar phenotypic features are apparent in rodent HD models is important since it could have translational potential in evaluating the efficacy of novel therapeutic strategies. We used high-resolution anatomical images to longitudinally investigate brain morphology in the zQ175DN heterozygous mouse model (HET) and wildtype littermates (WT) at 3, 6, and 10 months of age (M), which reflect different stages of phenotypic progression. We investigated volumetric alterations using semi-automatic segmentations of HD on relevant regions-of-interest (striatum, cerebellum, corpus callosum, cerebral cortex, ventricles, and total brain volume) and whole brain voxel-wise Tensor Based Morphometry (TBM) analysis. The striatum showed the earliest progressive lower absolute volume in HET mice compared to WT, starting from 3M, followed by lower absolute volume of cortex and corpus callosum concomitantly at 6 and 10M. TBM highlighted lower relative local volume in the rostral-medial striatum at all ages, and in cerebral cortex in HET mice at 6 and 10M. A bigger relative local volume in the cerebellum was observed at all ages in HET mice, and in the globus pallidus, substantia nigra, at 6 and 10M. Overall, this study revealed key structural abnormalities that resemble the natural history of PwHD. Hence, analysis of brain structure through MRI in the zQ175DN heterozygous mouse model holds potential for testing disease-modifying treatments expected to slow down or prevent structural degeneration. HighlightsO_LIThe striatum of zQ175DN heterozygous mice shows volume decrease at 3 months compared to WT mice C_LIO_LIWidespread volume reductions are observed from 6 months in zQ175DN heterozygous mice C_LIO_LITensor Based Morphometry highlights vulnerability of the dorsomedial striatum in the zQ175DN heterozygous mouse model C_LI

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↗

bioRxiv2024

Preclinical evaluation of the novel CHDI-650 PET ligand for non-invasive quantification of mutant huntingtin aggregates in Huntington's disease

PurposePositron emission tomography (PET) imaging of mutant huntingtin (mHTT) aggregates is a potential tool to monitor disease progression as well as the efficacy of candidate therapeutic interventions for Huntingtons disease (HD). To date, the focus has been mainly on the investigation of 11C radioligands; however, favourable 18F radiotracers will facilitate future clinical translation. This work aimed at characterising the novel [18F]CHDI-650 PET radiotracer using a combination of in vivo and in vitro approaches in a mouse model of HD. MethodsAfter characterising [18F]CHDI-650 using in vitro autoradiography, we assessed in vivo plasma and brain radiotracer stability as well as kinetics through dynamic PET imaging in the heterozygous (HET) zQ175DN mouse model of HD and wild-type (WT) littermates at 9 months of age. Additionally, we performed a head-to-head comparison study at 3 months with the previously published [11C]CHDI-180R radioligand. ResultsPlasma and brain radiometabolite profiles indicated a suitable metabolic profile for in vivo imaging of [18F]CHDI-650. Both in vitro autoradiography and in vivo [18F]CHDI-650 PET imaging at 9 months of age demonstrated a significant genotype effect (p<0.0001) despite the poor test-retest reliability. [18F]CHDI-650 PET imaging at 3 months of age displayed higher differentiation between genotypes when compared to [11C]CHDI-180R. ConclusionOverall, [18F]CHDI-650 allows for discrimination between HET and WT zQ175DN mice at 9 and 3 months of age. [18F]CHDI-650 represents the first suitable 18F radioligand to image mHTT aggregates in mice and its clinical evaluation is underway.

neuroscience↗

bioRxiv2022

Resting-State fMRI reveals Longitudinal Alterations in Brain Network Connectivity in a Mouse Model of Huntington's Disease

Huntingtons disease is an autosomal, dominantly inherited neurodegenerative disease caused by an expansion of the CAG repeats in exon 1 of the huntingtin gene. Neuronal degeneration and dysfunction that precedes regional atrophy result in the impairment of striatal and cortical circuits that affect the brains large-scale network functionality. However, the evolution of these disease-driven, large-scale connectivity alterations is still poorly understood. Here we used resting-state fMRI to investigate functional connectivity changes in a mouse model of Huntingtons disease in several relevant brain networks and how they are affected at different ages that follow a disease-like phenotypic progression. Towards this, we used the heterozygous (HET) form of the zQ175DN Huntingtons disease mouse model that recapitulates aspects of human disease pathology. Seed- and Region-based analyses were performed at different ages, on 3-, 6-, 10-, and 12-month-old HET and age-matched wild-type mice. Our results demonstrate decreased connectivity starting at 6 months of age, most prominently in regions such as the retrosplenial and cingulate cortices, pertaining to the default mode-like network and auditory and visual cortices, part of the associative cortical network. At 12 months, we observe a shift towards decreased connectivity in regions such as the somatosensory cortices, pertaining to the lateral cortical network, and the caudate putamen, a constituent of the subcortical network. Moreover, we assessed the impact of distinct Huntingtons Disease-like pathology of the zQ175DN HET mice on age-dependent connectivity between different brain regions and networks where we demonstrate that connectivity strength follows a nonlinear, inverted U-shape pattern, a well-known phenomenon of development and normal aging. Conversely, the neuropathologically driven alteration of connectivity, especially in the default mode and associative cortical networks, showed diminished age-dependent evolution of functional connectivity. These findings reveal that in this Huntingtons disease model, altered connectivity starts with cortical network aberrations which precede striatal connectivity changes, which appear only at a later age. Taken together, these results suggest that the age-dependent cortical network dysfunction seen in rodents could represent a relevant pathological process in Huntingtons disease progression.

neuroscience↗

bioRxiv2022

Longitudinal investigation of changes in resting-state co-activation patterns and their predictive ability in the zQ175 DN mouse model of Huntington's disease

Huntingtons disease (HD) is a neurodegenerative disorder caused by expanded ([≥]40) glutamine-encoding CAG repeats in the huntingtin gene, which leads to dysfunction and death of predominantly striatal and cortical neurons. While the genetic profile and behavioural signs of the disease are better known, changes in the functional architecture of the brain, especially before the behavioural symptoms become apparent, are not fully and consistently characterized. In this study, we sought markers at pre, early and late manifest states of phenotypic progression in the heterozygous (HET) zQ175 delta-neo (DN) mouse model, using resting-state functional magnetic resonance imaging (RS-fMRI). This mouse model shows molecular, cellular and circuitry alterations that resemble those seen in HD in humans. Specifically, we investigated, longitudinally, changes in co-activation patterns (CAPs) that are the transient states of brain activity constituting the resting-state networks (RSNs). Most robust changes in the temporal properties of CAPs occurred at the late manifest state; the durations of two anti-correlated CAPs, characterized by simultaneous co-activation of default-mode like network (DMLN) and co-deactivation of lateral-cortical network (LCN) and vice-versa, were reduced in the zQ175 DN HET animals compared to the wild-type mice. Changes in the spatial properties, measured in terms of activation levels of different brain regions, during CAPs were found at all three states and became progressively more pronounced at the manifest states. We then assessed the cross-validated predictive power of CAP metrics to distinguish HET animals from controls. Spatial properties of CAPs performed significantly better than the chance level at all three states with 80% classification accuracy at the early and late manifest states.

neuroscience↗

bioRxiv2022

Benefits of global mutant huntingtin lowering diminish over time in a Huntington's disease mouse model

We have developed a novel inducible Huntingtons disease (HD) mouse model that allows temporal control of whole-body allele-specific mutant Huntingtin (mHtt) expression. We asked whether moderate global lowering of mHtt ([~]50%) was sufficient for long-term amelioration of HD-related deficits and, if so, whether early mHtt lowering (before measurable deficits) was required. Both early and late mHtt lowering delayed behavioral dysfunction and mHTT protein aggregation, as measured biochemically. However, long-term follow up revealed that the benefits, in all mHtt lowering groups, attenuated by 12 months of age. While early mHtt lowering attenuated cortical and striatal transcriptional dysregulation evaluated at 6 months of age, the benefits diminished by 12- months of age and late mHtt lowering was unable to ameliorate striatal transcriptional dysregulation at 12 months of age. Only early mHtt lowering delayed the elevation in cerebrospinal fluid neurofilament light chain that we observed in our model starting at 9 months of age. As small-molecule HTT-lowering therapeutics progress to the clinic, our findings suggest that moderate mHtt lowering allows disease progression to continue, albeit at a slower rate, and could be relevant to the degree of mHTT lowering required to sustain long-term benefit in humans.

neuroscience↗

bioRxiv2021

A novel imaging ligand as a biomarker for mutant huntingtin-lowering in Huntington's disease

Huntingtons disease (HD) is a dominantly inherited neurodegenerative disorder caused by a CAG trinucleotide expansion in the huntingtin (HTT) gene that encodes the pathologic mutant HTT (mHTT) protein with an expanded polyglutamine (PolyQ) tract. While several therapeutic programs targeting mHTT expression have advanced to clinical evaluation, no method is currently available to visualize mHTT levels in the living brain. Here we demonstrate the development of a positron emission tomography (PET) imaging radioligand with high affinity and selectivity for mHTT aggregates. This small molecule radiolabeled with 11C ([11C]CHDI-180R) enables non-invasive monitoring of mHTT pathology in the brain and can track region-and time-dependent suppression of mHTT in response to therapeutic interventions targeting mHTT expression. We further show that therapeutic agents that lower mHTT in the striatum have a functional restorative effect that can be measured by preservation of striatal imaging markers, enabling a translational path to assess the functional effect of mHTT lowering.

neuroscience↗