Biology subjects

Wiest, A.

Publications and source records attributed to Wiest, A..

4 recordsLinked to original sources

bioRxiv2026

Gene Supplementation of MYO7A or activation of Myo7b for treatment of Usher syndrome 1B

Mutations in MYO7A result in the most severe subtype of Usher syndrome, the leading genetic cause of deafblindness. The large size of MYO7A requires dual adeno-associated virus (AAV) vectors for gene transfer or alternative methods to treat retinal defects. Here, we evaluated two treatment approaches: i) Supplementation of the human MYO7A gene via dual mRNA trans-splicing AAVs, and ii) CRISPR/Cas-mediated activation of the related murine Myo7b gene. Upon MYO7A supplementation, the transgenic MYO7A transcript and protein were expressed and correctly localized in retinal pigment epithelial (RPE) and photoreceptors of mice, pigs, and human retinal organoids. In RPE-and photoreceptor-specific Myo7a knockout mice, we could restore MYO7A expression and localization of melanosomes in RPE cells to wild-type levels. Myo7b activation led to partial restoration of melanosome localization, and the localization of MYO7B protein was largely comparable to MYO7A. These findings indicate that both approaches are in principle suitable for the therapy of Usher syndrome.

molecular biology↗

bioRxiv2024

A hypothalamic circuit mechanism underlying the impact of stress on memory and sleep

Stress profoundly affects sleep and memory processes. Stress impairs memory consolidation, and similarly, disruptions in sleep compromise memory functions. Yet, the neural circuits underlying stress-induced sleep and memory disturbances are still not fully understood. Here, we show that activation of CRHPVN neurons, similar to acute restraint stress, decreases sleep and impairs memory in a spatial object recognition task. Conversely, inhibiting CRHPVN neurons during stress reverses stress-induced memory deficits while slightly increasing the amount of sleep. We found that both stress and stimulation of CRHPVN neurons activate neurons in the lateral hypothalamus (LH), and that their projections to the LH are critical for mediating stress-induced memory deficits and sleep disruptions. Our results suggest a pivotal role for CRHPVN neuronal pathways in regulating the adverse effects of stress on memory and sleep, an important step towards improving sleep and ameliorating the cognitive deficits that occur in stress-related disorders.

neuroscience↗

bioRxiv2023

Circuit mechanism underlying fragmented sleep and memory deficits in 16p11.2 deletion mouse model of autism

Sleep disturbances are prevalent in children with autism spectrum disorder (ASD) and have a major impact on the quality of life. Strikingly, sleep problems are positively correlated with the severity of ASD symptoms, such as memory impairment. However, the neural mechanisms underlying sleep disturbances and cognitive deficits in ASD are largely unexplored. Here, we show that non-rapid eye movement sleep (NREMs) is highly fragmented in the 16p11.2 deletion mouse model of ASD. The degree of sleep fragmentation is reflected in an increased number of calcium transients in the activity of locus coeruleus noradrenergic (LC-NE) neurons during NREMs. Exposure to a novel environment further exacerbates sleep disturbances in 16p11.2 deletion mice by fragmenting NREMs and decreasing rapid eye movement sleep (REMs). In contrast, optogenetic inhibition of LC-NE neurons and pharmacological blockade of noradrenergic transmission using clonidine reverse sleep fragmentation. Furthermore, inhibiting LC-NE neurons restores memory. Rabies-mediated unbiased screening of presynaptic neurons reveals altered connectivity of LC-NE neurons with sleep- and memory regulatory brain regions in 16p11.2 deletion mice. Our findings demonstrate that heightened activity of LC-NE neurons and altered brain-wide connectivity underlies sleep fragmentation in 16p11.2 deletion mice and identify a crucial role of the LC-NE system in regulating sleep stability and memory in ASD.

neuroscience↗

bioRxiv2022

Transforming chemical proteomics enrichment into high throughput method using SP2E workflow.

Protein post-translational modifications (PTMs) play a critical role in the regulation of protein catalytic activity, localization and protein-protein interactions. Attachment of PTMs onto proteins significantly diversifies their structure and function resulting in so-called proteoforms. However, the sole identification of post-translationally modified proteins, which are often cell type and disease specific, is still a highly challenging task. Sub-stoichiometric amounts and modification of low abundant proteins necessitate purification or enrichment of the modified proteins. Although the introduction of the mass spectrometry-based chemical proteomic strategies have enabled to screen protein PTMs with increased throughput, sample preparation has remained highly time consuming and tedious. Here, we report an optimized workflow for enrichment of PTM proteins in 96-well plate format which can be possible extended to robotic automatization. This platform allows to significantly lower the input of total protein, which opens up the opportunity to screen specialized and difficult to culture cell lines in high-throughput manner. The presented SP2E protocol is robust, time- and cost-effective as well as suitable for large-scale screening of proteoforms. Application of the SP2E protocol will thus enable the characterization of proteoforms in various processes such as neurodevelopment, neurodegeneration and cancer and may contribute to an overall acceleration of the recently launched Human Proteoform Project.

biochemistry↗