Biology subjects

Prado, M. A. M.

Publications and source records attributed to Prado, M. A. M..

10 recordsLinked to original sources

bioRxiv2026

Phosphorylated -Synuclein shapes changes in brain structure, task-related activity and functional connectivity in M83 mice

Phosphorylated -synuclein at S129 (pS129) is the predominant form of -synuclein in the aggregates known as Lewy bodies (LB) and Lewy neurites (LN), the pathological hallmarks of Parkinson's disease (PD) and dementia with Lewy bodies (DLB), collectively known as synucleinopathies. The toxic nature of these inclusions remains contentious. To improve our understanding of these aggregates and their role in the structural and functional changes observed in synucleinopathies, we combined non-invasive structural and functional MRI in awake head-fixed M83 mice with whole-brain light-sheet fluorescence microscopy (LSFM) in the same animals following a unilateral injection of either human preformed fibrils of -synuclein (PFF) or PBS into the dorsal striatum. We developed a multimodal framework integrating the non-invasive, mesoscopic nature of MRI with the cellular-level details of LSFM to further our understanding of the underlying pathology of synucleinopathies. Using this framework, we show that four months after injection, animals injected with PFFs showed ubiquitous spread of pS129 pathology throughout the whole brain and spinal cord consistent with anatomical connectivity from the seeding region, with some structures such as the midbrain more susceptible to pS129 accumulation. Compared to controls, PFF-injected animals showed widespread atrophy as well as a decrease in functional connectivity, while evoked activity in the somatosensory/somatomotor areas in response to spontaneous paw movements showed significant hypoactivation. These cellular, structural and functional changes were significantly associated with the proportion of pS129 accumulation determined in the same animals. Our results demonstrate the deleterious effects of pS129 accumulation and argue in favor of a toxic role played by these aggregates.

neuroscience↗

bioRxiv2026

Striatal acetylcholine enables latent-state creation during reversal learning

Like dopamine, acetylcholine is modulated in the striatum by reward-predicting cues and outcomes, yet its computational role remains unclear. Here, we manipulated dorsomedial striatal acetylcholine in mice performing a reversal learning task using genetic and physiologically guided optogenetic approaches. Inhibiting phasic acetylcholine modulation impaired reversal learning, while enhancing modulation during non-rewarded trials facilitated reversal learning. Trial-by-trial acetylcholine dynamics were best explained by a reinforcement learning model in which new latent states are created when experience is poorly explained by existing states. A circuit-constrained model further suggested that acetylcholine promotes plasticity when reward-omission is ambiguous, leading to a surprising prediction: making reward omission explicit should reduce the need for acetylcholine. We confirmed this in a new experiment in which an auditory omission cue substantially reduced the reversal-learning deficit caused by genetic acetylcholine knockdown. These findings suggest that striatal acetylcholine supports reversal learning by promoting state construction in reinforcement learning.

neuroscience↗

bioRxiv2026

TPR Domains Drive the Functional Phase Separation of HOP and its Regulation by Hsp90 and Hsp70

HOP is a cochaperone that facilitates client transfer between two major chaperones, Hsp90 and Hsp70. Emerging evidence, however, suggests that HOP plays additional roles in coordinating complex proteostasis networks. Upon exposure to proteostatic stress, HOP rapidly sequesters soluble misfolded proteins into cytoplasmic foci in a Hsp90 independent manner, thereby facilitating their clearance through the ubiquitin proteasome system. We demonstrate here that stress-dependent HOP foci are biomolecular condensates formed by liquid-liquid phase separation. Purified HOP forms protein droplets that closely resemble the foci observed in cells. Our biophysical analyses show that the phase separation of HOP is driven by electrostatic interactions between its tandem TPR domains, with a critical role of its TPR2A domain. Of note, Hsp90 and Hsp70 regulate the extent of HOP phase separation, with Hsp70 driving HOP droplet formation and Hsp90 reversing it. Finally, we find that the Y354E phosphomimetic variant of HOP impairs phase separation and sensitizes cells to acute misfolding stress, suggesting a key role of HOP condensation in mitigating protein misfolding stress. Our work thus identifies a new mechanism by which HOP phase separation mitigates protein misfolding stress in eukaryotic cells and is regulated by Hsp70 and Hsp90.

biochemistry↗

bioRxiv2024

Dissociable patterns of dopamine dynamics and causal contributions to stimulus-response behaviors across striatal subregions.

Rationale: Midbrain dopamine (DA) neurons project principally towards the striatum, serving as key regulators of movement, motivation, and cognition. Different striatal dopaminergic (DA) pathways may regulate different aspects of cognition. Objectives: We investigated how different striatal DA pathways contribute to a known function of the striatum, visuomotor conditional learning. Methods: Using fiber photometry, we recorded DA transients in these regions as mice learned the touchscreen Visuo-Motor Conditional Learning task. Results: DA transients in all regions dynamically tracked task events, but differed in the timing of peak responses and ramp-like activity preceding a choice, indicating region-specific temporal dynamics across learning. Manipulations of reward probability revealed DA transients in all regions during reward delivery and omission that are consistent with an interpretation in terms of reward prediction error. Thus, DA dynamics in all regions could indicate involvement in visuomotor conditional learning. Therefore, to determine whether nigrostriatal or mesolimbic DA is necessary for learning, we chemogenetically inhibited DA striatal afferents, revealing that only DLS-projecting nigrostriatal DA, and not NAc-projecting mesolimbic striatal DA, was necessary for learning the task. Conclusion: These findings demonstrate functional heterogeneity of aspects of striatal DA signaling, and selective causal roles in the learning of visuomotor conditional learning.

animal behavior and cognition↗

bioRxiv2024

Sex-focused analyses of M83 A53T hemizygous mouse model with recombinant human alpha-synuclein preformed fibril injection identifies female resilience to disease progression: A combined magnetic resonance imaging and behavioural study.

Alpha-synuclein (aSyn) pathology has been extensively studied in mouse models harbouring human mutations. In spite of the known sex differences in age of onset, prevalence and disease presentation in human synucleinopathies, the impact of sex on aSyn propagation has received very little attention. To address this need, we examined sex differences in whole brain signatures of neurodegeneration due to aSyn toxicity in the M83 mouse model using longitudinal magnetic resonance imaging (MRI; T1-weighted; 100 m3 isotropic voxel; acquired -7, 30, 90 and 120 days post-injection [dpi]; n[≥]8 mice/group/sex/time point). To initiate aSyn spreading, M83 mice were inoculated with recombinant human aSyn preformed fibrils (Hu-PFF) or phosphate buffered saline (PBS) injected in the right dorsal striatum. We observed more aggressive neurodegenerative profiles over time for male M83 Hu-PFF-injected mice when examining voxel-wise trajectories. However, at 90 dpi, we observed widespread patterns of neurodegeneration in the female Hu-PFF-injected mice. These differences were not accompanied with any differences in motor symptom onset between the male and female Hu-PFF-injected mice. However, male Hu-PFF-injected mice reached their humane endpoint sooner. These findings suggest that post-motor symptom onset, even though more accelerated disease trajectories were observed for male Hu-PFF-injected mice, neurodegeneration may appear sooner in female Hu-PFF-injected mice (prior to motor symptomatology). These findings suggest that sex-specific synucleinopathy phenotypes urgently need to be considered to improve our understanding of neuroprotective and neurodegenerative mechanisms.

neuroscience↗

bioRxiv2022

Presymptomatic neuroanatomical and cognitive biomarkers of alpha-synuclein propagation in a mouse model of synucleinopathy

There is significant evidence suggesting aggregated misfolded alpha-synuclein, a major component of Lewy bodies, propagates in a prion-like manner contributing to disease progression in Parkinsons disease (PD) and other synucleinopathies. Animal models are essential for understanding and developing treatments for these diseases. However, despite modelling human pathology, most endpoints studied in mice do not translate to humans. Furthermore, the progression by which alpha-synuclein misfolding affects human-relevant measures such as brain volume and underlying subtle, high-level cognitive deficits is poorly understood. Here we used a mouse model of synucleinopathy; hemizygous M83 human A53T alpha-synuclein transgenic mice inoculated with recombinant human alpha-synuclein preformed fibrils (PFF) injected in the right striatum to initiate alpha-synuclein misfolding and aggregation. We examined alpha-synuclein-induced atrophy at 90 days post-injection using ex vivo magnetic resonance imaging as well as high-level cognition and motor function, as biomarkers of alpha-synuclein toxicity. We observed widespread atrophy in bilateral regions that project to or receive input from the injection site, highlighting a network of regions that are consistent with structural changes observed in humans with PD. Moreover, we detected early deficits in reversal learning with touchscreen testing in PFF-injected mice prior to motor dysfunction, consistent with the pathology observed in cortical-striatal and thalamic loops. We show, using translational approaches in mice, that progression of prion-like spreading of alpha-synuclein causes selective atrophy via connected brain regions leading to high-level cognitive deficits. We propose that precise imaging and cognitive biomarkers can provide a more direct and human-relevant measurement of alpha-synuclein-induced toxicity in pre-clinical testing. Significance StatementThe work described in this manuscript showcases the utility of state-of-the-art methodologies (magnetic resonance imaging and touchscreen behavioural tasks) to examine endophenotypes, both in terms of symptomatology and neuroanatomy, of alpha-synuclein propagation in a mouse model of synucleinopathy. Our work further validates the M83-Hu-PFF mouse model of synucleinopathy-associated pathogenesis of neurodegenerative diseases while highlighting precise imaging and cognitive biomarkers of protein misfolding toxicity. Specifically, we identified rapid and translational biomarkers that can serve as a proxy for the direct examination of cellular levels for pathology. We anticipate that these biomarkers can measure progression of toxicity, specifically in the early phases, and may be more reliable than end stage pathology and more useful as endpoints in the examination of novel therapeutics.

neuroscience↗

bioRxiv2022

AAV-mediated neuronal expression of a scFv antibody selective for Aβ oligomers protects synapses and rescues memory in Alzheimer models

Brain accumulation of soluble oligomers of the amyloid-{beta} peptide (A{beta}Os) has been implicated in synapse failure and memory impairment in Alzheimers disease. Here, we show that treatment with NUsc1, a single-chain variable fragment antibody (scFv) that selectively targets A{beta}Os, prevents the inhibition of long-term potentiation in hippocampal slices and memory impairment induced by A{beta}Os in mice. As a therapeutic approach for intracerebral antibody delivery, we developed an adeno-associated virus vector to drive neuronal expression of NUsc1 (AAV-NUsc1) within the brain. Transduction by AAV-NUsc1 induced NUsc1 expression and secretion in adult human brain slices, and inhibited A{beta}O binding to neurons and A{beta}O-induced loss of dendritic spine loss in primary rat hippocampal cultures. Treatment of mice with AAV-NUsc1 prevented memory impairment induced by A{beta}Os and, importantly, reversed memory deficits in aged APPswe/PS1{Delta}E9 Alzheimers disease model mice. These results support the feasibility of gene-mediated immunotherapy using single-chain antibodies as a potential therapeutic approach in Alzheimers disease.

neuroscience↗

bioRxiv2020

Chronic hM3Dq signaling in microglia ameliorates neuroinflammation in male mice

Microglia express muscarinic G protein-coupled receptors (GPCRs) that sense cholinergic activity and are activated by acetylcholine to potentially regulate microglial functions. Knowledge about how distinct types of muscarinic GPCR signaling regulate microglia function in vitro and in vivo is still poor, partly due to the fact that some of these receptors are also present in astrocytes and neurons. We generated mice expressing the hM3Dq Designer Receptor Exclusively Activated by Designer Drugs (DREADD) selectively in microglia to investigate the role of muscarinic M3Gq-linked signaling. We show that activation of hM3Dq using clozapine N-oxide (CNO) elevated intracellular calcium levels and increased phagocytosis of FluoSpheres in vitro. Acute treatment with CNO in vivo did not affect male mouse behavior, however chronic CNO treatment decreased sickness behavior triggered by lipopolysaccharide (LPS) treatment. Interestingly, whereas acute treatment with CNO increased synthesis of cytokine mRNA, chronic treatment attenuated LPS-induced cytokine mRNA changes in the brain, likely explaining the improvement in sickness behavior by chronic hM3Dq activation. No effect of CNO was observed in DREADD-negative mice. These results suggest that chronic activation of M3 muscarinic receptors (the hM3Dq progenitor) in microglia, and potentially other Gq-coupled GPCRs, preconditions microglia to decrease their response to further immunological challenges. Our results indicate that hM3Dq can be a useful tool to modulate neuroinflammation and study microglial immunological memory in vivo, which may be applicable for manipulations of neuroinflammation in neurodegenerative and psychiatric diseases. HighlightsO_LIMicroglial function was manipulated by specific activation of hM3Dq signaling. C_LIO_LIChronic hM3Dq activation prevented LPS-induced sickness behavior in mice. C_LIO_LIMicroglial hM3Dq signaling modulated expression of pro-inflammatory cytokines. C_LI

neuroscience↗

bioRxiv2019

An optimized acetylcholine sensor for monitoring in vivo cholinergic activity

The ability to directly measure acetylcholine (ACh) release is an essential first step towards understanding its physiological function. Here we optimized the GRABACh (GPCR-Activation-Based-ACh) sensor with significantly improved sensitivity and minimal downstream coupling. Using this sensor, we measured in-vivo cholinergic activity in both Drosophila and mice, revealing compartmental ACh signals in fly olfactory center and single-trial ACh dynamics in multiple regions of the mice brain under a variety of different behaviors

neuroscience↗

bioRxiv2018

A hypomorphic Stip1 allele reveals the requirement for chaperone networks in mouse development and aging

Chaperone networks are dysregulated with aging and neurodegenerative disease, but whether compromised Hsp70/Hsp90 chaperone function directly contributes to neuronal degeneration is unknown. Stress-inducible phosphoprotein-1 (STI1; STIP1; HOP) is a co-chaperone that simultaneously interacts with Hsp70 and Hsp90, but whose function in vivo remains poorly understood. To investigate the requirement of STI1-mediated regulation of the chaperone machinery in aging we combined analysis of a mouse line with a hypomorphic Stip1 allele, with a neuronal cell line lacking STI1 and in-depth analyses of chaperone genes in human datasets. Loss of STI1 function severely disturbed the Hsp70/Hsp90 machinery in vivo, and all client proteins tested and a subset of cochaperones presented decreased levels. Importantly, mice expressing a hypomorphic STI1 allele showed spontaneous age-dependent hippocampal neurodegeneration, with consequent spatial memory deficits. STI1 is a critical node for the chaperone network and it can contribute to age-dependent hippocampal neurodegeneration.

cell biology↗