Biology subjects

Curti, L.

Publications and source records attributed to Curti, L..

5 recordsLinked to original sources

bioRxiv2024

Optogenetic inhibition quenches the integration of sensory input in the cortex

SignificanceWe designed a novel all-optical tool to simultaneously silence neuronal activity at arbitrary sites on the dorsal cortex, and monitor the consequences of the manipulation. Optogenetic inhibition of primary sensory regions determined short and long-term dampening of the sensory response across a distributed cortical network. IntroductionMany fundamental processes of brain computation, such as sensory perception and motor control, heavily rely on the mesoscopic dynamics of activity across the cerebral cortex. Manipulating mesoscale activity and observing its effects across multiple brain regions is crucial for understanding the causal link between cortical dynamics and behavior. ObjectiveThe goal of this study was to develop a novel all-optical system that allows inhibition of excitatory neurons while simultaneously monitoring cortical responses at arbitrary sites across the entire dorsal cortex of mice. MethodsWe combined wide-field imaging and optogenetics to create a mesoscale all-optical approach, enabling simultaneous monitoring and manipulation of cortical activity using light. Intravenous injection of two PHP.eB AAVs enabled the whole-brain co-expression of the red-shifted calcium indicator jRCaMP1b and the inhibitory actuator stGtACR2, with stable expression over several weeks. This system was calibrated, and the effects of inhibition on sensory responses were tested. ResultsIncreasing laser power progressively reduced spontaneous activity at the site of irradiation. A single 5-second pulse on the barrel field cortex significantly decreased the amplitude of sensory-evoked responses, not only in the stimulated region but across the entire stimulated hemisphere. ConclusionsThis novel all-optical system enables targeted inhibition while concurrently monitoring mesoscale cortical activity. It provides insights into the dynamics of cortical circuits and offers a milestone for investigating the causal links between neuronal activity and behavior. Future research can use this tool to address sensory responsiveness impairments in neurological and neuropsychiatric disorders.

neuroscience↗

bioRxiv2024

Age-dependent cortical overconnectivity revers under anesthesia in Shank3 mice

Growing evidence points to brain network dysfunction as a central neurobiological basis for autism spectrum disorders (ASDs). As a result, studies on Functional Connectivity (FC) have become pivotal for understanding the large-scale network alterations associated with ASD. Despite ASD being a neurodevelopmental disorder, and FC being significantly influenced by the brain state, existing FC studies in mouse models predominantly focus on adult subjects under anesthesia. The differential impact of anesthesia and age on cortical functional networks in ASD subjects remains unexplored. To fill this gap, we conducted a longitudinal evaluation of FC across three brain states and three ages in the Shank3b mouse model of autism. We utilized wide-field calcium imaging to monitor cortical activity in Shank3b+/- and Shank3b+/+ mice fromlate development (P45) through adulthood (P90), and isoflurane anesthesia to manipulate the brain state. Our findings reveal that network hyperconnectivity, emerging from the barrel-field cortices during the juvenile stage, progressively expands to encompass the entire dorsal cortex in adult Shank3b+/- mice. Notably, the severity of FC imbalance is highly dependent on the brain state: network alterations are more pronounced in the awake state and shift towards hypoconnectivity under anesthesia. These results underscore the crucial role of anesthesia in detecting autism-related FC alterations and identify a significant network of early cortical dysfunction associated with autism. This network represents a potential target for non-invasive translational treatments.

neuroscience↗

bioRxiv2024

CDK12 controls transcription at damaged genes and prevents MYC-induced transcription-replication conflicts

Oncogene-induced replicative stress is a potent tumor-suppressive mechanism that must be kept in check for cancer cells to thrive. Thus, the identification of genes and pathways involved in replicative stress is key to understand cancer evolution and to identify prospective therapeutic targets. Here, we investigated factors that modulate replicative stress upon deregulation of the MYC oncogene. We identified the cyclin-dependent kinase CDK12 as selectively required to prevent transcription-replication conflicts and the activation of a cytotoxic DNA-damage response (DDR). At the mechanistic level, CDK12 was recruited to damaged genes by PARP-dependent DDR-signaling and elongation-competent RNAPII. Once recruited, CDK12 repressed transcription by preventing the association of CDK9 with RNAPII. Either loss or chemical inhibition of CDK12 led to DDR-resistant transcription at damaged genes. Genome-wide profiling revealed that loss of CDK12 exacerbated transcription-replication conflicts in MYC-overexpressing cells and led to the accumulation of double-strand DNA breaks (DSBs), occurring preferentially between early- replicating regions and transcribed genes, organized in a co-directional head-to-tail orientation. Overall, our data demonstrate that CDK12 protects genome integrity by repressing transcription of damaged genes, which is required for proper resolution of DSBs at oncogene-induced transcription-replication conflicts. This provides a rationale that explains both how CDK12 deficiency can promote tandem duplications of early-replicated regions during tumor evolution, and how CDK12 targeting can exacerbate replicative-stress in tumors.

cancer biology↗

bioRxiv2020

Activation of pancreatic B-cell genes by multiplex epigenetic CRISPR-editing

CRISPR-based systems for epigenetic editing are promising molecular tools that could be harnessed for directed differentiation of pluripotent stem cells. We used the CRISPR/dCas9-VP160, CRISPR/dCas9-TET1 and CRISPR/dCas9-P300 systems for multiplex epigenetic editing and activation of human beta pancreatic genes (PDX1, NEUROG3, PAX4 and INS). The CRISPR/dCas9-P300 system was the most effective at activating genes with reduced number of sgRNA. Using small number of sgRNA per gene was important to induce multiplex gene activation. Combined activation of transcription factors (TFs) involved in beta cell development resulted in INS gene expression; in which sequential TFs activation was more effective than simultaneous activation. Full CRISPR RNA-based delivery system was able to activate all targeted genes. Overall, this study shows the utility of CRISPR tools for epigenetic editing and directed cellular differentiation.

molecular biology↗

bioRxiv2020

An ultrasensitive, rapid, and portable coronavirus SARS-CoV-2 sequence detection method based on CRISPR-Cas12

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has received global attention due to the recent outbreak in China. In this work, we report a CRISPR-Cas12 based diagnostic tool to detect synthetic SARS-CoV-2 RNA sequences in a proof-of-principle evaluation. The test proved to be sensitive, rapid, and potentially portable. These key traits of the CRISPR method are critical for virus detection in regions that lack resources to use the currently available methods.

molecular biology↗