Biology subjects

Verpeut, J. L.

Publications and source records attributed to Verpeut, J. L..

6 recordsLinked to original sources

bioRxiv2022

The Alphaherpesvirus Latency Associated Promoter 2 (LAP2) Drives Strong Transgene Expression in Peripheral Tissue Depending on Administration Route and AAV Serotype

Adeno-associated virus (AAV) has shown great translational potential in treating a variety of diseases often requiring strong and ubiquitous transgene expression. However, the genetic payload of AAV vectors is limited to <4.9 kb and some commonly used gene promoters are large in sizeable and susceptible to transcriptional silencing. We validated a short (404 bp), strong and persistent promoter obtained from the genome of pseudorabies virus (PRV) called alphaherpesvirus latency-associated promoter 2 (LAP2). We evaluated the biodistribution and potency of transgene expression in mouse peripheral tissue and organs when using AAV8-LAP2 and AAV9-LAP2, both of which achieved transgene expression like that of the ubiquitous promoter, EF1. LAP2 drives potent transgene expression in liver and kidney after systemic retro-orbital administration and in skeletal muscle after intramuscular delivery. Additionally, we observed broad transduction throughout the lung albeit at lower levels than other tissues. Notably, in skeletal muscle LAP2 resulted in preferential transduction of myofiber types 2. A direct side-by-side comparison between LAP2 and EF1, demonstrates that regardless of the AAV serotype and route of administration, LAP2 is as powerful and persistent as EF1 promoter despite being 66% smaller in size, thus allowing for larger therapeutic payloads.

cell biology↗

bioRxiv2021

Cerebellar contributions to a brainwide network for flexible behavior

The cerebellum regulates nonmotor behavior, but the routes by which it exerts its influence are not well characterized. Here we report a necessary role for the posterior cerebellum in guiding a reversal learning task, acting through a network of diencephalic and neocortical structures. After chemogenetic inhibition of Purkinje cells in lobule VI or crus I, mice could learn a water Y-maze task but were impaired in their ability to reverse their initial choice. To map targets of perturbation, we imaged c-Fos activation in cleared whole brains using light-sheet microscopy. Reversal learning activated diencephalic regions and associative neocortical regions. Distinctive subsets of structures were altered by perturbation of lobule VI (including thalamus and habenula) and crus I (including hypothalamus and prelimbic/orbital cortex), and both perturbations influenced anterior cingulate and infralimbic cortex. To identify functional networks, we used correlated variation in c-Fos activation within each test group. Lobule VI inactivation weakened within-thalamus correlations, while crus I inactivation divided neocortical activity into well-separated sensorimotor and associative subnetworks. In both groups, high-throughput automated analysis of complex whole-body movement revealed deficiencies in across-day adaptation to an open field environment. Neither perturbation affected gait, within-day open-field adaptation, or location preference. Taken together, these experiments reveal brainwide systems for cerebellar influence that can affect multiple flexible responses. Significance statementThe cerebellum, a part of all vertebrate brains, provides feedback to the rest of the brain on a split-second basis in response to unexpected events. The consequent changes can range from minute adjustments in movement to broad changes in behavior. In mice, we investigated cerebellar regions that help guide flexible behavior. Inactivation of these regions prevented mice from responding flexibly to a changing Y-shaped maze. We used light-sheet microscopy of c-fos protein expression to map brainwide effects of different stages of learning and cerebellar inactivation. Inactivation caused changes in thalamocortical activity that were similar in pattern, but opposite in sign, from normal learning. Lobule VI inactivation weakened thalamic functional networks, while crus I inactivation divided sensorimotor from associative neocortical networks. The same inactivation also impaired multiday adaptation to an open arena, as tracked using machine vision. Our work shows how the cerebellums influence over flexible response can be mediated by a widespread brain network.

neuroscience↗

bioRxiv2021

Adeno-associated virus (AAV) reduces cortical dendritic complexity in a TLR9-dependent manner

Recombinant adeno-associated viruses (AAVs) allow rapid and efficient gene delivery in the nervous system. AAVs are widely used in research and are the basis of multiple FDA-approved gene therapies. Here, we find that the immune response to AAVs genome reduces dendritic complexity in mammalian cortex. Dendritic loss associated with AAV-mediated gene delivery occurs at experimentally-relevant titers, cannot be explained by responses to transgene expression or surgery, and is not restricted to a particular capsid serotype, encoded transgene, promoter, or production facility. AAV-associated dendritic loss is accompanied by a decrease in the frequency and amplitude of miniature excitatory postsynaptic currents (mEPSCs) and upregulation of immune molecules that can limit dendritic complexity and synaptic transmission. Blocking detection of unmethylated CpG-rich DNA via Toll-like receptor 9 (TLR9) protects dendritic complexity, suggesting that immunodetection of a core feature of the AAV genome triggers dendritic loss. These results reveal previously unsuspected impacts of AAV on neuronal structure and function and identify TLR9 inhibitors as important tools to improve the safety and efficacy of AAV-mediated gene delivery in the nervous system.

neuroscience↗

bioRxiv2021

Deep behavioral phenotyping of mouse autism models using open-field behavior

AO_SCPLOWBSTRACTC_SCPLOWAutism is noted for both its genotypic and phenotypic diversity. Repetitive action, resistance to environmental change, and motor disruptions vary from individual to individual. In animal models, conventional behavioral phenotyping captures such fine-scale variations incompletely. Here we use advances in computer vision and deep learning to develop a framework for characterizing mouse behavior on multiple time scales using a single popular behavioral assay, the open field test. We observed male and female C57BL/6J mice to develop a dynamic baseline of adaptive behavior over multiple days. We then examined two rodent models of autism, a cerebellum-specific model, L7-Tsc1, and a whole-brain knockout model, Cntnap2. Both Cntnap2 knockout and L7-Tsc1 mutants showed forelimb lag during gait. L7-Tsc1 mutants showed complex defects in multi-day adaptation, lacking the tendency of wild-type mice to spend progressively more time in corners of the arena. In L7-Tsc1 mutant mice, failure-to-adapt took the form of maintained ambling, turning, and locomotion, and an overall decrease in grooming. Adaptation in Cntnap2 knockout mice more broadly resembled that of wild-type. L7-Tsc1 mutant and Cntnap2 knockout mouse models showed different patterns of behavioral state occupancy. Our automated pipeline for deep phenotyping successfully captures model-specific deviations in adaptation and movement as well as differences in the detailed structure of behavioral dynamics.

neuroscience↗

bioRxiv2020

Parallel organization of cerebellar pathways to sensorimotor, associative, and modulatory forebrain

Cerebellar outputs take polysynaptic routes to reach the rest of the brain, impeding conventional tracing. Here we quantify pathways between cerebellum and forebrain using transsynaptic tracing viruses and a whole-brain quantitative analysis pipeline. Retrograde tracing found a majority of descending paths originating from somatomotor cortex. Anterograde tracing of ascending paths encompassed most thalamic nuclei, especially ventral posteromedial, lateral posterior, mediodorsal, and reticular nuclei; in neocortex, sensorimotor regions contained the most labeled neurons, but higher densities were found in associative areas, including orbital, anterior cingulate, prelimbic, and infralimbic cortex. Patterns of ascending expression correlated with c-Fos expression after optogenetic inhibition of Purkinje cells. Our results reveal homologous networks linking single areas of cerebellar cortex to diverse forebrain targets. We conclude that shared areas of cerebellum are positioned to provide sensory-motor information to regions implicated in both movement and nonmotor function. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/979153v3_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@1bead0corg.highwire.dtl.DTLVardef@1531de2org.highwire.dtl.DTLVardef@1cf02f6org.highwire.dtl.DTLVardef@1cb6774_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2019

SMALL ALPHAHERPESVIRUS LATENCY-ASSOCIATED PROMOTERS DRIVE EFFICIENT AND LONG-TERM TRANSGENE EXPRESSION IN THE CENTRAL NERVOUS SYSTEM

Recombinant adeno-associated viral vectors (rAAV) are used as gene therapy vectors to treat central nervous system (CNS) diseases. Despite their safety and broad tropism, important issues need to be corrected such as the limited payload capacity and the lack of small gene promoters providing long-term, pan-neuronal transgene expression in the CNS. Commonly used gene promoters are relatively large and can be repressed a few months after CNS transduction, risking the long-term performance of single-dose gene therapy applications. We used a whole-CNS screening approach based on systemic delivery of AAV-PHP.eB, iDisco+ tissue-clearing and light-sheet microscopy, to identify three small latency-associated promoters (LAP) from the herpesvirus pseudorabies virus (PRV). These promoters are LAP1 (404bp), LAP2 (498bp) and LAP1_2 (880bp). They drive chronic transcription of the virus encoded latency-associated transcript (LAT) during productive and latent phases of PRV infection. We observed stable, pan-neuronal transgene transcription and translation from AAV-LAP in the CNS for six months post AAV transduction. In several CNS areas, the number of cells expressing the transgene was higher for LAP2 than the large conventional EF1 promoter (1264bp). Our data suggests that the LAP are suitable candidates for viral vector-based CNS gene therapies requiring chronic transgene expression after one-time viral-vector administration.

neuroscience↗