Biology subjects

Palczewski, K.

Publications and source records attributed to Palczewski, K..

7 recordsLinked to original sources

bioRxiv2026

Engineered mRNA-packaging virus-like particles enable efficient and precise mitochondrial base editing in vitro and in vivo

Mitochondrial DNA (mtDNA) base editors (mtBEs) are promising tools for studying and correcting pathogenic mtDNA mutations. However, the application of mtBEs is often hindered by substantial mtDNA-wide off-target editing that accumulates with prolonged mtBE expression resulting from plasmid DNA or viral delivery. To overcome this limitation, we developed mtBE-VLPs, a virus-like-particle (VLP) platform for transiently delivering mtBEs in vitro and in vivo with minimal off-target effects. While canonical protein-packaging VLPs yielded undetectable mtDNA base editing, engineered mRNA-packaging VLPs enabled efficient but transient mtBE cargo delivery. By iteratively engineering mRNA-packaging VLP compositions, we improved mtBE-VLP-mediated base editing efficiencies from less than 1% to greater than 50% across multiple target mtDNA loci in bulk populations of treated cultured cells. To further improve the packaging and delivery of certain mtBEs, we developed a strategy to selectively reduce mtBE cargo expression in VLP producer cells but not transduced cells, which increased VLP-mediated base editing efficiencies for certain mtBEs by 7.1-fold. Compared to plasmid DNA delivery in vitro, mtBE-VLPs exhibited equivalent on-target editing but 23- to 41-fold lower mtDNA-wide off-target editing, highlighting the improved editing specificity offered by transient mtBE delivery. mtBE-VLPs efficiently installed a pathogenic mtDNA mutation in mouse embryonic fibroblasts (88% efficiency) and corrected a pathogenic mtDNA mutation in a human patient-derived cell line (41% efficiency). Additionally, a single subretinal injection of mtBE-VLPs yielded 6-20% (average 12%) mtDNA base editing in the mouse retinal pigment epithelium with minimal mtDNA-wide off-target editing. These findings establish mRNA-packaging mtBE-VLPs as promising vehicles for efficient and precise mitochondrial base editing via transient mtBE delivery.

bioengineering↗

bioRxiv2023

Identification of conserved gene expression programs activated in multiple modes of torpor across vertebrate clades.

Torpor encompasses adaptations to diverse extreme environmental stressors such as hibernation, aestivation, brumation, and daily torpor. Here we introduce StrokeofGenus, an analytic pipeline that identifies distinct transcriptomic states and conservation of gene expression patterns across studies, tissues, and species. We use StrokeofGenus to study multiple and diverse forms of torpor from publicly available RNA-seq datasets that span eight species and two classes. We identify three transcriptionally distinct states during the cycle of heterothermia: euthermia, torpor, and interbout arousal. We also identify torpor-specific gene expression patterns that are conserved both across tissues and between species with over three hundred million years of evolutionary divergence. We further demonstrate the general conservation of gene expression patterns in multiple forms of torpor, implying a common evolutionary origin for this process. Although here we apply StrokeofGenus to analysis of torpor, it can be used to interrogate any other complex physiological processes defined by transient transcriptomic states. HIGHLIGHTSO_LIStrokeofGenus integrates orthologue annotation, non-negative matrix factorization, and transfer learning for cross-species analysis. C_LIO_LIStrokeofGenus identifies conserved topor-related gene expression patterns across divergent species and cell types. C_LIO_LIHeterothermia has three distinct transcriptomic states. C_LIO_LITorpor-specific gene expression patterns are conserved between species and forms of torpor. C_LI

physiology↗

bioRxiv2022

Visual system hyperexcitability and compromised V1 receptive field properties in early-stage retinitis pigmentosa in mice

Inherited retinal degenerative diseases are a prominent cause of blindness. Even though mutations causing death of photoreceptors are mostly known, the pathophysiology downstream in the inner retina and along the visual pathway is incompletely characterized in the earliest disease stages. Here we investigated retinal, midbrain and cortical visual function using electroretinography (ERG), the optomotor response (OMR), visual evoked potentials (VEPs), respectively, and single unit electrophysiology at the primary visual cortex (V1) in light-adapted juvenile ([~] 1-month-old) and young adult (3-month-old) RhoP23H/WT mice, representative of early-stage retinitis pigmentosa (RP). Photopic ERG revealed up to [~] 30 % hypersensitivity to light in RhoP23H/WT mice, as measured by the light intensity required to generate half-maximal b-wave (I50 parameter). RhoP23H/WT mice also showed increased optomotor responses towards low spatial frequency drifting gratings, indicative of visual overexcitation at the midbrain level. At the V1 level, VEPs and single-cell recordings revealed prominent hyperexcitability in the juvenile RhoP23H/WT mice. Mean VEP amplitudes for light ON stimuli were nearly doubled in 1-month-old RhoP23H/WT mice compared to controls, and more than doubled for light OFF. Single-cell recordings showed a significantly increased spontaneous V1 neuron firing in the RhoP23H/WT mice, and persistent contrast and temporal sensitivities. In contrast, direction selectivity was severely compromised. Our data suggest that during early RP, the visual pathway becomes hyperexcited. This could have both compensatory and deleterious consequences for visual behavior. Further studies on the mechanisms of hyperexcitability are warranted as this could lead to therapeutic interventions for RP. Significance statementLost retinal function in many blinding retinal degenerative disorders could soon be alleviated by advanced therapies that restore photoreception. However, it is unknown whether a visual system rewired downstream of the photoreceptors can process signals adequately. We studied the functional consequences of early rod death along the visual pathway in young retinitis pigmentosa (RP) mice. Photopic inner retina responses were moderately hypersensitized in the electroretinograms of RP mice. Reflex-based visual behavior and visual cortex electrophysiology showed hyperexcitability. Some aspects of complex visual processing were remarkably resistant to degeneration, whereas others were severely impacted. We conclude that the visual system adapts to lost photoreception by increasing sensitivity, but simultaneously becomes detrimentally hyperexcited. Mechanistic understanding could lead to therapeutic preservation and restoration of vision.

physiology↗

bioRxiv2021

Determinants shaping the nanoscale architecture of the mouse rod outer segment

The unique membrane organization of the rod outer segment (ROS), the specialized sensory cilium of rod photoreceptor cells, provides the foundation for phototransduction, the initial step in vision. ROS architecture is characterized by a stack of identically shaped and tightly packed membrane disks loaded with the visual receptor rhodopsin. A wide range of genetic aberrations compromise ROS ultrastructure, impairing photoreceptor viability and function. Yet, the structural basis giving rise to the remarkable long-range order of ROS membrane stacks and the molecular mechanisms underlying genetically inherited diseases remain elusive. Here, cryo-electron tomography (cryo-ET) performed on native ROS at molecular resolution provides insights into key structural determinants of ROS membrane architecture. Our data reveal the existence of two molecular connectors/spacers which likely contribute to the nanometer-scale precise stacking of the ROS disks. We further show that the extreme radius of curvature at the disk rims is enforced by a continuous supramolecular assembly composed of peripherin-2 (PRPH2) and rod outer segment membrane protein 1 (ROM1) tetramers. We suggest that, together these molecular assemblies constitute the structural basis of the highly specialized ROS functional architecture. Cryo-ET therefore provides novel quantitative and structural information on the molecular architecture in ROS and insights into possible mechanisms underlying pathologies of certain PRPH2 mutations leading to blindness.

cell biology↗

bioRxiv2021

Dissecting lipid contents in the distinct regions of native retinal rod disk membranes

Photoreceptors rely on distinct membrane compartments to support their specialized function. Unlike protein localization, identification of critical differences in membrane content has not yet been expanded to lipids, due to the difficulty of isolating domain-specific samples. We have overcome this by using SMA to co-immunopurify membrane proteins and their native lipids from two regions of photoreceptor ROS disks. Each samples copurified lipids were subjected to untargeted lipidomic and fatty acid analysis. Extensive differences between center (rhodopsin) and rim (ABCA4 and PRPH2/ROM1) samples included a lower PC to PE ratio and increased LC- and VLC-PUFAs in the center relative to the rim region, which were enriched in shorter, saturated FAs. The comparatively few differences between the two rim samples likely reflect specific protein-lipid interactions. High-resolution profiling of the ROS disk lipid composition provides a model for future studies of other complex cellular structures, and gives new insights into how intricate membrane structure and protein activity are balanced within the ROS. SUMMARYSander et al. have parsed the lipid composition of native-source photoreceptor disks and find large differences in fatty acid unsaturation and chain length between the center and rim regions. They selectively copurify membrane proteins and lipids from each region in SMALPs using nanobodies and antibodies.

cell biology↗

bioRxiv2020

Homeostatic plasticity triggered by rod photoreceptor degenerative disease is associated with maintenance of sensitive night vision

Neuronal plasticity of the inner retina has been observed in response to photoreceptor degeneration. Typically, this phenomenon has been considered maladaptive and may preclude vision restoration in the blind. However, several recent studies utilizing triggered photoreceptor ablation have shown adaptive responses in bipolar cell dendrites expected to support normal vision. Whether such homeostatic plasticity occurs during progressive photoreceptor degenerative disease to help maintain normal visual behavior is unknown. We addressed these issues in an established mouse model of Retinitis Pigmentosa caused by the P23H mutation in rhodopsin. We show robust modulation of the retinal transcriptomic network reminiscent of the neurodevelopmental state as well as potentiation of rod - rod bipolar cell signaling following rod photoreceptor degeneration. Additionally, we found highly sensitive night vision in P23H mice even when more than half of the rod photoreceptors were lost. The results implicate retinal adaptation leading to persistent visual function during photoreceptor degenerative disease.

neuroscience↗

bioRxiv2020

Melanopsin Carboxy-terminus Phosphorylation Plasticity and Bulk Negative Charge, not Strict Site Specificity, Achieves Phototransduction Deactivation

Melanopsin is a visual pigment expressed in a small subset of ganglion cells in the mammalian retina known as intrinsically photosensitive retinal ganglion cells (ipRGCs) and is implicated in regulating non-image forming functions such as circadian photoentrainment and pupil constriction and contrast sensitivity in image formation. Mouse melanopsins Carboxy-terminus (C-terminus) possesses 38 serine and threonine residues, which can potentially serve as phosphorylation sites for a G-protein Receptor Kinase (GRK) and be involved in the deactivation of signal transduction. Previous studies suggest that S388, T389, S391, S392, S394, S395 on the proximal region of the C-terminus of mouse melanopsin are necessary for melanopsin deactivation. We expressed a series of mouse melanopsin C-terminal mutants in HEK293 cells and using calcium imaging, and we found that the necessary cluster of six serine and threonine residues, while being critical, are insufficient for proper melanopsin deactivation. Interestingly, the additional six serine and threonine residues adjacent to the required six sites, in either proximal or distal direction, are capable of restoring wild-type deactivation of melanopsin. These findings suggest an element of plasticity in the molecular basis of melanopsin phosphorylation and deactivation. In addition, C-terminal chimeric mutants and molecular modeling studies support the idea that the initial steps of deactivation and {beta}-arrestin binding are centered around these critical phosphorylation sites (S388-S395). This degree of functional versatility could help explain the diverse ipRGC light responses as well as non-image and image forming behaviors, even though all six sub types of ipRGCs express the same melanopsin gene OPN4.

neuroscience↗