Biology subjects

Zhang, V.

Publications and source records attributed to Zhang, V..

12 recordsLinked to original sources

bioRxiv2026

Sequence and structure of protein binding sites in RNA impact biomolecular condensates

Biomolecular condensates are central to subcellular compartmentalization. Although many condensates contain and regulate RNA, research has primarily focused on protein interactions. Here, we investigate RNA-protein interactions underlying cell cycle-regulating condensates in the multinucleate fungus Ashbya gossypii. These condensates form through interactions between G1 cyclin mRNA CLN3 and RNA-binding protein Whi3, which was predicted by homology to recognize a five-nucleotide motif repeated within the transcript. Natural variation in motif number in Ashbya strains led us to hypothesize that binding site valence may influence condensate properties. Using unbiased binding assays, we determined the preference of Ashbya Whi3 protein for specific primary RNA sequence and mutated individual Whi3-binding sites within CLN3 mRNA. Mutants exhibited distinct condensate properties despite having the same valence in terms of binding site number. Mutations altered the saturation concentration (Csat) and dense phase concentration of RNA and protein in cell-free reconstitution experiments. A subset of mutants, showed reduced number of condensates and deregulation of the cell cycle in cells. We also find that enhanced availability of single-stranded RNA can compensate for loss of binding sites. Together, these data indicate that differences in RNA protein binding-site context and not simply valence plays a critical role in determining condensate properties.

cell biology↗

bioRxiv2024

Biomolecular condensates control and are defined by RNA-RNA interactions that arise in viral replication

Cells must limit RNA-RNA interactions to avoid irreversible RNA entanglement. Cells may prevent deleterious RNA-RNA interactions by genome organization to avoid complementarity however, RNA viruses generate long, perfectly complementary antisense RNA during replication. How do viral RNAs avoid irreversible entanglement? One possibility is RNA sequestration into biomolecular condensates. To test this, we reconstituted critical SARS-CoV-2 RNA-RNA interactions in Nucleocapsid condensates. We observed that RNAs with low propensity RNA-RNA interactions resulted in more round, liquid-like condensates while those with high sequence complementarity resulted in more heterogeneous networked morphology independent of RNA structure stability. Residue-resolution molecular simulations and direct sequencing-based detection of RNA-RNA interactions support that these properties arise from degree of trans RNA contacts. We propose that extensive RNA-RNA interactions in cell and viral replication are controlled via a combination of genome organization, timing, RNA sequence content, RNA production ratios, and emergent biomolecular condensate material properties. Graphical Abstract: SARS-CoV-2 replication cycle employs weak and strong RNA-RNA interactions O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=102 SRC="FIGDIR/small/630161v2_ufig1.gif" ALT="Figure 1000"> View larger version (32K): org.highwire.dtl.DTLVardef@1e75ad7org.highwire.dtl.DTLVardef@1fcda3aorg.highwire.dtl.DTLVardef@15b556eorg.highwire.dtl.DTLVardef@145b3b6_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2024

RNA encodes physical information

Most amino acids are encoded by multiple codons, making the genetic code degenerate. Synonymous mutations affect protein translation and folding, but their impact on RNA itself is often neglected. We developed a genetic algorithm that introduces synonymous mutations to control the diversity of structures sampled by an mRNA. The behavior of the designed mRNAs reveals a physical code layered in the genetic code. We find that mRNA conformational heterogeneity directs physical properties and functional outputs of RNA-protein complexes and biomolecular condensates. The role of structure and disorder of proteins in biomolecular condensates is well appreciated, but we find that RNA conformational heterogeneity is equally important. This feature of RNA enables both evolution and engineers to build cellular structures with specific material and responsive properties.

biophysics↗

bioRxiv2024

A protective role for the cerebellum in cognitive aging

BackgroundBrain reserve -- the brains resilience to age-related change or damage -- provides protection against cognitive decline. The cerebellum is relatively unstudied as a contributor to brain reserve. This study investigates cerebellar brain reserve in the largest cohort to date. MethodsWe used data from the Human Connectome Project (n=708, 36-100yrs), UK Biobank (n=45,013, 44-81yrs), and ADNI (n=1,423, 56-95yrs). ADNI participants were cognitively normal or had a diagnosis of mild cognitive impairment or Alzheimers disease (AD) dementia. We examined associations between cerebellar tissue volume, age, Montreal Cognitive Assessment (MoCA) scores, global PET amyloid burden, and APOE genotype. FindingsHCP-Aging data revealed heterogenous aging-associated changes in cerebellar volume, with the greatest effects in posterior hemispheric regions (crus I) (Bonferroni-corrected, p<0{middle dot}05). MoCA scores were associated with higher tissue density in the cerebellum (p<0{middle dot}0001) to the same extent as neocortex, and MoCA scores coupled most strongly with posterior cerebellar cortex. Strikingly, greater volume in MoCA visuospatial-related cerebellar cortex protected against aging-related cognitive decline (p=0{middle dot}0001). We replicated tissue aging results in the UK Biobank with the greatest aging-related effects in posterior cerebellum (p<0{middle dot}0001), and an association of greater cerebellar volumes with less cognitive decline (Trails Making-B: p<0{middle dot}00001; Digit Symbol Substitution: p=0{middle dot}034). AD patients with low amyloid-beta burden (A{beta}-) exhibited the strongest cerebellar association with MoCA (volume x group, A{beta}- AD: p=0{middle dot}0001). In A{beta}- individuals, APOE {varepsilon}4/{varepsilon}4 carriers showed the greatest effect with MoCA (volume x APOE, {varepsilon}4/{varepsilon}4: p=0{middle dot}017). InterpretationOur large-scale study demonstrates a potentially strong role for the cerebellum in mitigating cognitive decline. The persistence of this protection in APOE {varepsilon}4/{varepsilon}4 carriers reshapes our understanding of reserve and AD risk. Our findings open the cerebellum as a novel target for future clinical research on brain reserve in aging populations. FundingNational Science Foundation, National Academies of Sciences, Engineering and Medicine, National Institutes of Health. Research in ContextO_ST_ABSEvidence before this studyC_ST_ABSWe searched PubMed and GoogleScholar between August 12, 2023, and September 25, 2024 for articles irrespective of language or date of publication, relating to measures of cortical and cerebellar aging. Search terms included "cerebellum", "cognitive aging", "cerebellar reserve", "Alzheimers disease", "covariance", "retrogenesis", and "connectivity". Prior research demonstrated that the cerebellum may play a role in cognitive function. Studies have shown that cerebellar development is spatially heterogenous, with posterior regions undergoing the most protracted change in structure and function. Brain reserve has been predominantly studied in the neocortex, with some studies suggesting the cerebellum may contribute to cognitive changes in diseases like Alzheimers disease (AD) dementia. Cerebellar volume differences between young and older adults have been noted, as well as between healthy adults and neurodegenerative conditions like Parkinsons disease. However, the cerebellums contribution to cognitive reserve, particularly in healthy aging and individuals with a clinical diagnosis of mild cognitive impairment or AD dementia, had not been explored especially in large population-based datasets. Added value of this studyTo our knowledge, this study is the largest to date looking into how cerebellar structures contribute to cognitive outcomes in both healthy older adults, and those with mild cognitive impairment or AD dementia. We leverage three large neuroimaging datasets -- HCP-Aging, UK Biobank, and ADNI -- and demonstrate that cerebellar aging is spatially heterogeneous, with posterior regions showing the greatest age-related decline. We further establish a significant link between larger cerebellar volumes and better cognitive outcomes, suggesting that the cerebellum plays a role in cognitive resilience. Additionally, we identify how cerebellar structures predict cognitive performance and interact with amyloid-beta brain pathology and APOE genotype, particularly in those with low amyloid brain burden and those at greatest risk of AD such as in homozygous APOE e4 allele carriers. Implications of all the available evidenceThe aging of the global population raises the challenge of maintaining cognitive health in older age. Our research focuses on the cerebellum as a novel mediator of preserved cognitive function in old age and clinical dementia. Our findings have profound implications, given that individuals with robust cerebellar structures may be missed during cognitive and clinical screenings. Greater cerebellum volume seems to be most advantageous in those at higher risk for Alzheimers disease based on APOE4 status and those who have yet to accumulate substantial amyloid-beta brain pathology. Our study underscores the importance of the cerebellum as a novel brain reserve mechanism in aging populations.

neuroscience↗

bioRxiv2024

Lack of circadian entrainment and limited nocturnal plasticity in response to a cyclic aversive stimulus in a diurnal rodent, the antelope ground squirrel (Ammospermophilus leucurus)

Recent studies have shown that cyclic aversive stimuli, such as random footshocks, act as a nonphotic zeitgeber to entrain circadian behaviors in nocturnal rodents, a pattern termed "fear entrainment". However, it remains unknown whether diurnal species exhibit similar plasticity in behavioral timing. This study aimed to determine if antelope ground squirrels (Ammospermophilus leucurus; AGS), a naturally diurnal rodent, can also shift their activity patterns to cyclic aversive stimuli. We conducted two experiments with 20 AGS housed in custom cages featuring a safe nesting area and a separate foraging area (for feeding and drinking), rendered aversive by presentation of unsignaled, time-specific footshocks. In the first experiment, animals were subjected to a 12:12 light-dark (LD) cycle. One group experienced aversion during the light phase, while the control group received the same treatment during the dark phase. In the second experiment, a 16:8 LD cycle was used, and animals were divided into three groups with the foraging area rendered aversive either during the first or second half of the light phase or during the dark phase. Following each of these treatments, animals were released into constant darkness (DD) to assess the phase and period of free-running rhythms. Contrary to previous findings in nocturnal rodents, AGS did not exhibit consistent shifts in activity to avoid footshocks. Most animals maintained their normal diurnal activity, with only minor and inconsistent phase shifts. In experiment two, animals exposed to footshocks during half of the light phase also failed to reliably shift activity to the opposite "safe" portion of the light phase. Together, these findings show AGS lack the ability to entrain to cyclic aversive stimuli or become nocturnal; this result suggests a lack of substantial plasticity in activity timing. These results highlight the importance of considering species-specific differences in nonphotic circadian entrainment and temporal niche plasticity.

animal behavior and cognition↗

bioRxiv2024

NPD1/GPR37 axis protects painful traumatic brain injury and its complications

Patients with traumatic brain injury (TBI) frequently exhibit heightened pain and associated complications such as cognitive decline, depression, and anxiety. GPR37 is widely expressed in various brain regions, but its function remains largely unclear. We recently discovered neuroprotectin D1 (NPD1) as a novel GPR37 ligand. In this study, we examined the protective role of the NPD1/GPR37 signaling pathway in TBI-induced neuropathic pain and its complications. TBI was induced by closed-head impact and resulted in transient neuropathic pain for less than two weeks, showing periorbital and cutaneous mechanical allodynia/hyperalgesia, as well as motor deficiency and cognitive impairment. We found that peri-surgical treatment with NPD1, effectively prevented TBI-induced mechanical hypersensitivity, motor deficiency, and cognitive impairment. NPD1 treatment also substantially inhibited TBI-induced microgliosis, astrogliosis (including A1 astrocyte markers), and neuroinflammation in the sensory cortex and hippocampus. RNA sequencing and GO enrichment analysis revealed downregulations of genes related to "calcium ion homeostasis," and "GPCR signaling pathway" in the TBI-affected brain. These downregulations were restored by NPD1 treatment. RNAscope in situ hybridization revealed predominant Gpr37 mRNA expression in oligodendrocytes. TBI resulted in rapid and remarkable demyelination and downregulation of Gpr37 mRNA expression in oligodendrocytes, and both were protected by NPD1 treatment. NPD1s inhibition of periorbital and cutaneous mechanical pain was abolished in Gpr37-/- mice. Moreover, TBI-induced neuropathic pain was prolonged by swimming stress, and NPD1 treatment prevented the stress-induced transition from acute to chronic pain in wild-type mice but not Gpr37-/- mice. Finally, chronic pain was associated with depression and anxiety, and NPD1 treatment mitigated these chronic pain complications through GPR37. Thus, through modulation of demyelination, diverse responses of glial cells, and neuroinflammation, the NPD1/GPR37 axis serves as a protective mechanism and a therapeutic target against painful traumatic brain injury and its complications.

animal behavior and cognition↗

bioRxiv2024

CRISPR/Cas9 gene editing in Drosophila via visual selection in a summer classroom

CRISPR/Cas9 methods are a powerful in vivo approach to edit the genome of Drosophila melanogaster. To convert existing Drosophila GAL4 lines to LexA driver lines in a secondary school classroom setting, we applied the CRISPR-based genetic approach to a collection of Gal4 driver lines. The integration of the yellow+ coat color marker into homology-assisted CRISPR knock-in (HACK) enabled visual selection of Gal4-to-LexA conversions using brightfield stereo-microscopy available in a broader set of standard classrooms. Here, we report the successful conversion of eleven Gal4 lines with expression in neuropeptide-expressing cells into corresponding, novel LexA drivers. The conversion was confirmed by LexA- and Gal4-specific GFP reporter gene expression. This curriculum was successfully implemented in a summer course running 16 hours/week for seven weeks. The modularity, flexibility, and compactness of this course should enable development of similar classes in secondary schools and undergraduate curricula, to provide opportunities for experience-based science instruction, and university-secondary school collaborations that simultaneously fulfill research needs in the community of science.

genetics↗

bioRxiv2024

Armored Bicistronic CAR T Cells with Dominant-negative TGF-β Receptor II to Overcome Resistance in Glioblastoma.

Chimeric antigen receptor (CAR) T cells have shown significant efficacy in hematological diseases. However, CAR T therapy has demonstrated limited efficacy in solid tumors, including glioblastoma (GBM). One of the most important reasons is the immunosuppressive tumor microenvironment (TME), which promotes tumor growth and suppresses immune cells to eliminate tumor cells. The human transforming growth factor-beta (TGF-{beta}) plays a crucial role in forming the suppressive GBM TME and driving the suppression of the anti-GBM response. In order to mitigate TGF-{beta} mediated suppressive activity, we combined a dominant-negative TGF-{beta} receptor II (dnTGF{beta}RII) with our previous bicistronic CART-EGFR-IL13R2 construct, currently being evaluated in a clinical trial, to generate CART-EGFR-IL13R2-dnTGF{beta}RII, a tri-modular construct we are developing for clinical application. We hypothesized that this approach would more effectively subvert resistance mechanisms observed with GBM. Our data suggests that CART-EGFR-IL13R2-dnTGF{beta}RII significantly augmented T cell proliferation and enhanced functional responses, particularly in a TGF{beta}-rich tumor environment. Additionally, in vivo studies validated the safety and efficacy of the dnTGF{beta}RII cooperating with CARs in targeting and eradicating GBM in a NSG mouse model.

cancer biology↗

bioRxiv2024

Modeling and Correction of Protein Conformational Disease in iPSC-derived Neurons through Personalized Base Editing

AbstractAltered protein conformation can cause incurable neurodegenerative disorders. Mutations in SERPINI1, the gene encoding neuroserpin, can alter protein conformation resulting in cytotoxic aggregation leading to neuronal death. Familial encephalopathy with neuroserpin inclusion bodies (FENIB) is a rare autosomal dominant progressive myoclonic epilepsy that progresses to dementia and premature death. We developed HEK293T and induced pluripotent stem cell (iPSC) models of FENIB, harboring a patient-specific pathogenic SERPINI1 variant or stably overexpressing mutant neuroserpin fused to GFP (MUT NS-GFP). Here, we utilized a personalized adenine base editor (ABE)-mediated approach to correct the pathogenic variant efficiently and precisely to restore neuronal dendritic morphology. ABE-treated MUT NS-GFP cells demonstrated reduced inclusion size and number. Using an inducible MUT NS-GFP neuron system, we identified early prevention of toxic protein expression allowed aggregate clearance, while late prevention halted further aggregation. To address several challenges for clinical applications of gene correction, we developed a neuron-specific engineered virus-like particle to optimize neuronal ABE delivery, resulting in higher correction efficiency. Our findings provide a targeted strategy which may treat FENIB and potentially other neurodegenerative diseases due to altered protein conformation such as Alzheimers and Huntingtons diseases.

molecular biology↗

bioRxiv2023

Structural covariation between cerebellum and cerebral cortex is atypically modulated by thalamus in autism spectrum disorder

Despite its strong interconnectivity with the cerebral cortex, the influence of the human cerebellum on neocortical structure and its role in the development of neuropsychiatric disorders is unclear. Because cerebellar damage in early postnatal life creates a high risk for autism spectrum disorder (ASD), we investigated inter-relationships in cerebello-cerebral morphology. Leveraging a large structural brain MRI dataset in neurotypical children (n=375) and those diagnosed with ASD (n=373), we examined whether volumetric variation in cerebellar structure across individuals was correlated with neocortical variation during development, modeling the thalamus as a moderating coupling factor. We found negative covariation between cerebellar cortical regions and thalamic/sensorimotor neocortical regions, and positive covariation between thalamic and sensorimotor neocortical regions. This pattern aligned with the major disynaptic path of cerebellar inhibition to thalamocortical excitation. Examining the dependence of this structural covariation on ASD diagnosis, we found that neurotypical and ASD children displayed inverted hemispheric biases. In ASD, the thalamus moderated structural associations between the left cerebellum and right sensorimotor cortex. For neurotypical children, right cerebellum and left sensorimotor cortex were coupled. Notably, structural coupling between cerebellum, thalamus, and neocortex was strongest in younger childhood and waned by early adolescence, a time during which behavioral differences were smallest between typically developing and autistic children. In addition to the sensorimotor cortex, cerebellar dentate nuclei in ASD displayed greater coupling to broad neocortical regions, and these nuclei related to cognitive function differently such that greater dentate nuclear volume was associated with greater behavioral impairment in ASD but not in controls. Graph analyses demonstrated that the cerebello-thalamocortical network was more densely and less prolifically interconnected in ASD than in typical development. Taken as a whole, our study reveals a developmental interplay between the cerebellum, thalamus, and neocortex which differs in ASD from neurotypical children. This pattern is consistent with a model of ASD in which early developmental influences of cerebellar output on brain maturation are specifically moderated by cerebello-thalamocortical pathways.

neuroscience↗

bioRxiv2022

Evaluation of an image-derived input function for kinetic modeling of nicotinic acetylcholine receptor-binding PET ligands

Development of positron emission tomography (PET) radiotracers that bind with high-affinity to 4{beta}2-type nicotinic receptors (4{beta}2Rs) allows for in vivo investigations of the mechanisms underlying nicotine addiction and smoking cessation. One challenge associated with preclinical PET imaging involves the lack of true tissue reference regions free of specific tracer binding in the rodent brain, impeding accurate quantification of the tracer binding potential. Here, we investigate the use of an image-derived arterial input function for kinetic analysis of radiotracer binding in male and female mice. Two radiotracers were explored in this study: 2-[18F]FA85380 (2-FA), which displays similar pKa and binding affinity to the smoking cessation drug varenicline (Chantix), and [18F]Nifene, which displays similar pKa and binding affinity to nicotine. For both radiotracers, time-activity curves of the left ventricle of the heart displayed similar standardized uptake values (SUVs) across wild type mice, mice lacking the {beta}2 subunit for tracer binding, and acute nicotine-treated mice, whereas typical reference tissue SUVs displayed high variation between groups. Binding potential values estimated from a two-tissue compartment model (2TCM) fit of the data with the image-derived input function were significantly higher than estimates from reference tissue-based estimations. Rate constants of radiotracer dissociation were very slow for 2-FA and very fast for Nifene, similar to the in vitro dissociation rates reported for varenicline and nicotine, respectively. We conclude that use of an image-derived input function for kinetic modeling of nicotinic PET ligands improves quantification compared to reference tissue-based methods, and that the chemical properties of 2-FA and Nifene are suitable to study receptor response to nicotine addiction and smoking cessation therapies.

neuroscience↗

bioRxiv2017

High-dimensional microbiome interactions shape host fitness

Gut bacteria can affect key aspects of host fitness, such as development, fecundity, and lifespan, while the host in turn shapes the gut microbiome. Microbiomes co-evolve with their hosts and have been implicated in host speciation. However, it is unclear to what extent individual species versus community interactions within the microbiome are linked to host fitness. Here we combinatorially dissect the natural microbiome of Drosophila melanogaster and reveal that interactions between bacteria shape host fitness through life history tradeoffs. We find that the same microbial interactions that shape host fitness also shape microbiome abundances, suggesting a potential evolutionary mechanism by which microbiome communities (rather than just individual species) may be intertwined in co-selection with their hosts. Empirically, we made germ-free flies colonized with each possible combination of the five core species of fly gut bacteria. We measured the resulting bacterial community abundances and fly fitness traits including development, reproduction, and lifespan. The fly gut promoted bacterial diversity, which in turn accelerated development, reproduction, and aging: flies that reproduced more died sooner. From these measurements we calculated the impact of bacterial interactions on fly fitness by adapting the mathematics of genetic epistasis to the microbiome. Host physiology phenotypes were highly dependent on interactions between bacterial species. Higher-order interactions (involving 3, 4, and 5 species) were widely prevalent and impacted both host physiology and the maintenance of gut diversity. The parallel impacts of bacterial interactions on the microbiome and on host fitness suggest that microbiome interactions may be key drivers of evolution.\n\nSignificanceAll animals have associated microbial communities called microbiomes that can influence the physiology and fitness of their host. It is unclear to what extent individual microbial species versus ecology of the microbiome influences fitness of the host. Here we mapped all the possible interactions between individual species of bacteria with each other and with the hosts physiology. Our approach revealed that the same bacterial interactions that shape microbiome abundances also shape host fitness traits. This relationship provides a feedback that may favor the emergence of co-evolving microbiome-host units.

microbiology↗