Removal of a partial genomic duplication restores synaptic transmission and behavior in the MyosinVA mutant mouse Flailer.

BACKGROUND: Copy number variations, and particularly duplications of genomic regions, have been strongly associated with various neurodegenerative conditions including autism spectrum disorder (ASD). These genetic variations have been found to have a significant impact on brain development and function, which can lead to the emergence of neurological and behavioral symptoms. Developing strategies to target these genomic duplications has been challenging, as the presence of endogenous copies of the duplicate genes often complicates the editing strategies. RESULTS: Using the ASD and anxiety mouse model Flailer, which contains a partial genomic duplication working as a dominant negative for MyoVa, we demonstrate the use of DN-CRISPRs to remove a 700 bp genomic region in vitro and in vivo. Importantly, DN-CRISPRs have not been used to remove genomic regions using sgRNA with an offset greater than 300 bp. We found that editing the flailer gene in primary cortical neurons reverts synaptic transport and transmission defects. Moreover, long-term depression (LTD), disrupted in Flailer animals, is recovered after gene editing. Delivery of DN-CRISPRs in vivo shows that local delivery to the ventral hippocampus can rescue some of the mutant behaviors, while intracerebroventricular delivery, completely recovers the Flailer animal phenotype associated to anxiety and ASD. CONCLUSIONS: Our results demonstrate the potential of DN-CRISPR to efficiently remove larger genomic duplications, working as a new gene therapy approach for treating neurodegenerative diseases.

A Myosin Va mutant mouse with disruptions in glutamate synaptic development and mature plasticity in visual cortex.

Myosin Va (MyoVa) mediates F-actin-based vesicular transport toward the plasma membrane and is found at neuronal postsynaptic densities (PSDs), but the role of MyoVa in synaptic development and function is largely unknown. Here, in studies using the dominant-negative MyoVa neurological mutant mouse Flailer, we find that MyoVa plays an essential role in activity-dependent delivery of PSD-95 and other critical PSD molecules to synapses and in endocytosis of AMPA-type glutamate receptors (AMPAR) in the dendrites of CNS neurons. MyoVa is known to carry a complex containing the major scaffolding proteins of the mature PSD, PSD-95, SAPAP1/GKAP, Shank, and Homer to dendritic spine synapses. In Flailer, neurons show abnormal dendritic shaft localization of PSD-95, stargazin, dynamin3, AMPARs and abnormal spine morphology. Flailer neurons also have abnormally high AMPAR miniature current frequencies and spontaneous AMPAR currents that are more frequent and larger than in wild-type while numbers of NMDAR containing synapses remain normal. The AMPAR abnormalities are consistent with a severely disrupted developmental regulation of long-term depression that we find in cortical Flailer neurons. Thus MyoVa plays a fundamentally important role both in localizing mature glutamate synapses to spines and in organizing the synapse for normal function. For this reason Flailer mice will be valuable in further dissecting the role of MyoVa in normal synaptic and circuit refinement and also in studies of neurological and neuropsychiatric diseases where disruptions of normal glutamate synapses are frequently observed.

Redefining venereology practice in Tamil Nadu, South India - Nakshatra Health - A networking model.

Background: Accessing care for sexual health has always been a challenge in our Indian context. The primary reason is a lack of awareness of modes of transmission of sexually transmitted diseases (STD), appropriate testing, and treatment options. The second is taboo associated with the morality of the individual. The third is the accessibility and availability of Quality STD care by allopathic specialists in comparison to the demand. This has paved way for lots of myths and misconceptions among the general public regarding STDs and HIV disease. Compounding it is the mushrooming of nonqualified practitioners and healers who claim to cure all STDs and HIV has led to poor treatment outcomes. Several methods of partnership with qualified allopathic practitioners have been tried for the provision of quality STD care by various donor-funded, HIV and STD prevention programs in the country. The key reasons for the nonsustainability of these clinics were the lack of technical capability to handle the sexual health and STD clinical cases and the huge cost involved in the advertisement and maintenance of the clinics. Methodology: Seven clinicians from different geographical locations in Tamil Nadu, who were exclusively qualified in Venereology, conceived the idea of provision of comprehensive clinical care cum counseling and testing services through a networking model from December 01, 2020. The model comprised the following: (1) Dedicated YouTube channel (Nakshatra Health) to provide scientific information on STD and HIV, modes of transmission, clinical symptoms, lab testing, interpretation of results, clinical treatment options, and counseling on prevention, (2) Dedicated telephone helpline was established with WhatsApp to answer the queries of clients by the network venereologists, (3) Dedicated website (www.nakshatra.health) was developed to provide information on STD and HIV and options to clarify doubts and fix appointments online, (4) Clinical consultations were done in a hybrid mode with an option for direct clinic and online consultation. Prescriptions were provided using dedicated software instantly, (5) Networking was done with NABL-accredited labs and collection centers for performing STD and HIV tests with e-test request forms, (6) Networking was done with pharma companies to provide pre- and postexposure prophylaxis (PEP) services to clients through e-prescriptions. Cross referrals were made across the network members to facilitate easy access to clinical services by clients from different parts of Tamil Nadu. The entire concept was branded as "Nakshatra health" with a tagline - "Your sparkling solution for safe sexual health care." Quality STD care and ethical practice were the underlying motos of this concept. Results: During the 20 months (December 2020 to July 2022), 6442 phone calls and 9328 WhatsApp messages were received. 82.3% of the calls and messages were queries from clients related to their sexual exposures, and 16.4% were general information seekers. During this period, the Nakshatra Health YouTube channel had 1590 subscribers and nearly 2.4 lakh views of all its 24 videos. Among the viewers, 92.4% were male. 52.29% of viewers were between the ages of 25 and 34 years, 28.25% were between the ages of 18 and 24 years, and 17.25% viewers were between the ages of 35 and 44 years. 86% of the viewers were from India and 13% were Tamil-speaking viewers from Middle East, Southeast Asian countries and 1% were from European, African, and American countries. The most commonly watched videos were related to HIV symptoms and lab tests for STD and HIV. 16% of the viewers repeatedly watched the various videos in the series. The network laboratories provided testing services for 1082 clients with 2423 various STD/HIV tests. Totally 3328 clients availed of online consultation and 924 clients accessed clinic-based services across the network members. Among these cases, 18 cases of Syphilis (primary and secondary) and 12 cases of acute gonococcal urethritis, and 10 cases of genital warts were diagnosed and treated. 12 cases of phimosis and 4 cases of anal warts were surgically treated in collaboration with a surgeon. Through this initiative, PEP and PreP services were provided to 228 and 8 individuals. Conclusion: Designing a comprehensive sexual health service package is crucial to ensure the availability and ease of access to services to the general public. Provision of correct scientific information, networking and cross-referral of cases with like-minded dermatovenereologists/clinicians interested in venereology sexually transmitted infections, easily accessible clinical, laboratory, and treatment services including PreP and PEP medications, and ethical practice are the key factors for scaling this concept.

Removal of a genomic duplication by double-nicking CRISPR restores synaptic transmission and behavior in the MyosinVA mutant mouse Flailer.

Copy number variations, and particularly duplications of genomic regions, have been strongly associated with various neurodegenerative conditions including autism spectrum disorder (ASD). These genetic variations have been found to have a significant impact on brain development and function, which can lead to the emergence of neurological and behavioral symptoms. Developing strategies to target these genomic duplications has been challenging, as the presence of endogenous copies of the duplicate genes often complicates the editing strategies. Using the ASD and anxiety mouse model Flailer, that contains a duplication working as a dominant negative for MyoVa, we demonstrate the use of DN-CRISPRs to remove a 700bp genomic duplication in vitro and in vivo . Importantly, DN-CRISPRs have not been used to remove more gene regions <100bp successfully and with high efficiency. We found that editing the flailer gene in primary cortical neurons reverts synaptic transport and transmission defects. Moreover, long-term depression (LTD), disrupted in Flailer animals, is recovered after gene edition. Delivery of DN-CRISPRs in vivo shows that local delivery to the ventral hippocampus can rescues some of the mutant behaviors, while intracerebroventricular delivery, completely recovers Flailer animal phenotype associated to anxiety and ASD. Our results demonstrate the potential of DN-CRISPR to efficiently (>60% editing in vivo) remove large genomic duplications, working as a new gene therapy approach for treating neurodegenerative diseases.

Myosin Va Brain-Specific Mutation Alters Mouse Behavior and Disrupts Hippocampal Synapses.

Myosin Va (MyoVa) is a plus-end filamentous-actin motor protein that is highly and broadly expressed in the vertebrate body, including in the nervous system. In excitatory neurons, MyoVa transports cargo toward the tip of the dendritic spine, where the postsynaptic density (PSD) is formed and maintained. MyoVa mutations in humans cause neurologic dysfunction, intellectual disability, hypomelanation, and death in infancy or childhood. Here, we characterize the Flailer (Flr) mutant mouse, which is homozygous for a myo5a mutation that drives high levels of mutant MyoVa (Flr protein) specifically in the CNS. Flr protein functions as a dominant-negative MyoVa, sequestering cargo and blocking its transport to the PSD. Flr mice have early seizures and mild ataxia but mature and breed normally. Flr mice display several abnormal behaviors known to be associated with brain regions that show high expression of Flr protein. Flr mice are defective in the transport of synaptic components to the PSD and in mGluR-dependent long-term depression (LTD) and have a reduced number of mature dendritic spines. The synaptic and behavioral abnormalities of Flr mice result in anxiety and memory deficits similar to that of other mouse mutants with obsessive-compulsive disorder and autism spectrum disorder (ASD). Because of the dominant-negative nature of the Flr protein, the Flr mouse offers a powerful system for the analysis of how the disruption of synaptic transport and lack of LTD can alter synaptic function, development and wiring of the brain and result in symptoms that characterize many neuropsychiatric disorders.