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Radioligands for Tropomyosin Receptor Kinase (Trk) Positron Emission Tomography Imaging
摘要： The tropomyosin receptor kinases family (TrkA, TrkB, and TrkC) supports neuronal growth, survival, and differentiation during development, adult life, and aging. TrkA/B/C downregulation is a prominent hallmark of various neurological disorders including Alzheimer’s disease (AD). Abnormally expressed or overexpressed full-length or oncogenic fusion TrkA/B/C proteins were shown to drive tumorigenesis in a variety of neurogenic and non-neurogenic human cancers and are currently the focus of intensive clinical research. Neurologic and oncologic studies of the spatiotemporal alterations in TrkA/B/C expression and density and the determination of target engagement of emerging antineoplastic clinical inhibitors in normal and diseased tissue are crucially needed but have remained largely unexplored due to the lack of suitable non-invasive probes. Here, we review the recent development of carbon-11- and fluorine-18-labeled positron emission tomography (PET) radioligands based on specifically designed small molecule kinase catalytic domain-binding inhibitors of TrkA/B/C. Basic developments in medicinal chemistry, radiolabeling and translational PET imaging in multiple species including humans are highlighted.
关键词： oncogenic fusions,neurodegeneration,positron emission tomography,tropomyosin receptor kinase
更新于2025-09-23 15:22:29
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A UV cross-linking method combined with infrared imaging to analyse RNA–protein interactions
摘要： Photo cross-linking of proteins with short RNA oligomers is a classical method to study RNA–protein interactions that are implicated in many aspects of RNA metabolism and function. Most commonly, this involves the use of [c-32P]-labeled RNA probes. Although very sensitive, these procedures are complicated by the safety issues associated with the use of radioisotopes. Here, we describe a modi?ed UV cross-linking method using oligonucleotide probes end labelled with the infrared dye IRDyeVR 800. After UV cross-linking, proteins are separated by SDS-PAGE and cross-linked products are visualized with the OdysseyVR Infrared Imaging system. This end labelling approach provides a streamlined alternative to random labelling which reduces the ef?ciency of in-vitro transcription. End labelling is also independent of the length of the probe, thus facili- tating quantitative comparisons. To validate the method, we have con?rmed the binding of HuD to the 30-UTR of the mRNA for the microtubule-associated protein tau, implicated in the pathogenesis of Alzheimer’s disease. UV cross-linking of HuD with a labeled 21-mer probe was successfully performed using a recombinant puri?ed glutathione-S-transferase–HuD fu- sion protein as well as with lysates from CHO cells transfected with HuD cDNA. UV cross-linking combined with infrared imaging offers a convenient and robust strategy to analyse RNA–protein interactions and their emerging importance in disease.
关键词： Tau,RNA-binding protein,UV cross-linking,neurodegeneration,HuD,OdysseyVR
更新于2025-09-23 15:19:57
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Monitoring Neuronal Survival via Longitudinal Fluorescence Microscopy
摘要： Standard cytotoxicity assays, which require the collection of lysates or fixed cells at multiple time points, have limited sensitivity and capacity to assess factors that influence neuronal fate. These assays require the observation of separate populations of cells at discrete time points. As a result, individual cells cannot be followed prospectively over time, severely limiting the ability to discriminate whether subcellular events, such as puncta formation or protein mislocalization, are pathogenic drivers of disease, homeostatic responses, or merely coincidental phenomena. Single-cell longitudinal microscopy overcomes these limitations, allowing the researcher to determine differences in survival between populations and draw causal relationships with enhanced sensitivity. This video guide will outline a representative workflow for experiments measuring single-cell survival of rat primary cortical neurons expressing a fluorescent protein marker. The viewer will learn how to achieve high-efficiency transfections, collect and process images enabling the prospective tracking of individual cells, and compare the relative survival of neuronal populations using Cox proportional hazards analysis.
关键词： fluorescence microscopy,automation,Cell death,transfection,neurodegeneration,survival analysis
更新于2025-09-19 17:15:36
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Quantification of Oxygen Consumption in Retina Ex Vivo Demonstrates Limited Reserve Capacity of Photoreceptor Mitochondria
摘要： PURPOSE. Cell death in neurodegeneration occurs at the convergence of diverse metabolic pathways. In the retina, a common underlying mechanism involves mitochondrial dysfunction since photoreceptor homeostasis and survival are highly susceptible to altered aerobic energy metabolism. We sought to develop an assay to directly measure oxygen consumption in intact retina with the goal of identifying alterations in respiration during photoreceptor dysfunction and degeneration. METHODS. Circular punches of freshly isolated mouse retina, adjacent to the optic nerve head, were used in the microplate-based Seahorse Extracellular Flux Analyzer to measure oxygen consumption. Tissue integrity was evaluated by propidium iodide staining and live imaging. Different substrates were tested for mitochondrial respiration. Basal and maximal respiration were expressed as oxygen consumption rate (OCR) and respectively measured in Ames’ medium before and after the addition of mitochondrial uncoupler, BAM15. RESULTS. We show that glucose is an essential substrate for retinal mitochondria. At baseline, mitochondria respiration in the intact wild-type retina was close to maximal, with limited reserve capacity. Similar OCR and limited mitochondrial reserve capacity was also observed in cone-only Nrl(cid:2)/(cid:2) retina. However, the retina of Pde6brd1/rd1, Cep290rd16/rd16 and Rpgrip1(cid:2)/(cid:2) mice, all with dysfunctional or no photoreceptors, had reduced OCR and higher mitochondrial reserve capacity. CONCLUSIONS. We have optimized a method to directly measure oxygen consumption in acutely isolated, ex vivo mouse retina and demonstrate that photoreceptors have low mitochondrial reserve capacity. Our data provide a plausible explanation for the high vulnerability of photoreceptors to altered energy homeostasis caused by mutations or metabolic challenges.
关键词： photoreceptor homeostasis,neurodegeneration,mitochondrial function,retinal disease,oxidative stress
更新于2025-09-10 09:29:36
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Changes in Vascular and Immune Cell Content of an LPS Treated Rat Olfactory Bulb PD Model, Measured by Fluorescence Deconvolution Microscopy
摘要： Objective: We have previously shown changes in protein, immune cell, nitric oxide a neurotrophic factor content and distribution in the olfactory bulb of an endotoxin-treated rat model, as all these factors have been shown to be involved in neurodegeneration. We expanded our studies by fluorescently imaging smooth muscle actin and endothelial nitric oxide synthase (eNOS), both markers of blood vessels, to investigate loss of vasculature content, as well as imaging locations and quantities of immune cells. The work was performed to shed further light on associations between vessel integrity and immune cell initiated endothelial disruption. Our goal was to demonstrate that cytokine production, NOS induction and immune cell increases, are likely part of the process that leads to a loss of olfaction and dopaminergic signaling and includes vascular perturbations. Methods: Rats were sacrificed following lipopolysaccharide (LPS) treatment. Olfactory bulbs were harvested, sectioned from top to bottom to include the tract and sensory neurons, and probed for markers of inflammation. Inducible nitric oxide synthase (iNOS), neuronal nitric oxide synthase (nNOS), eNOS, interleukin-1 beta (IL-1β), TNF-α, interleukin-6, glial cell derived neurotrophic factor (GDNF) and circulating nitric oxide (NO) were imaged together with tagged macrophages, T-cells, B-cells and neutrophils. Results: Serum NO levels indicated that an inflammatory episode had occurred, being significantly higher in treated animals, with tissue levels of NOS elevated for an extended period of time. Immune cell clusters were seen in a number of areas and the localization of NOS isomers suggests that they have divergent roles in neurodegeneration. For instance, eNOS was associated with blood vessels, iNOS with glial and matrix cells and nNOS with glial cells and neurons. T and B-cell numbers showed a sustained increase; neutrophil numbers rapidly increased then returned to baseline levels; macrophage numbers increased and remained high; LAMP positive cell numbers (NK-cells) increased and remained high; GDNF content increased; IL-6, TNF-α and IL-1β levels all rapidly increased, before dropping to untreated levels, while circulating, NO levels increased dramatically. Of interest, the images of vascular content, immune cell content, eNOS and smooth muscle actin, allowed us to show detrimental interactions between cells, factors and vessels. Our data show that the majority of the vessels were intact, though sections of interest were ‘extracted’ to reveal possible leaky areas. Specific sites of IL-6 positive lymphocyte clustering were noted around vessels, suggesting that interactions are occurring that lead to disruptions of blood vessel tunicae, allowing the internalization of circulating cells and subsequent cytokine-initiated endothelial cell death. Conclusion: Our findings suggest that protective GDNF and eNOS, which maintains vascular tone, are possibly synthesized too late to combat cytokine initiated neuron damage, glial activation and chronic loss of vascular integrity.
关键词： Blood-brain-barrier,Cytokines,Neurodegeneration,Nitric oxide,Olfaction
更新于2025-09-09 09:28:46
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Degeneration of Photoreceptor Cells in Arylsulfatase G-Deficient Mice
摘要： Retinal degeneration is a common feature of several lysosomal storage disorders, including the mucopolysaccharidoses, a group of metabolic disorders that is characterized by widespread accumulation of glycosaminoglycans due to lysosomal enzyme dysfunction. We used a new mouse model of mucopolysaccharidosis IIIE to study the effect of Arylsulfatase G (ARSG) deficiency on retina integrity. The retina of Arsg knockout mice aged 1 to 24 months was studied by immunohistochemistry and Western blot analysis. Electron microscopic analyses were performed on retinas from 15- and 22-month-old animals. Photoreceptor and microglia cell numbers and retina thickness were determined to quantitatively characterize retinal degeneration in ARSG-deficient mice. Arsg knockout mice showed a progressive degeneration of photoreceptor cells starting between 1 and 6 months of age, resulting in the loss of more than 50% of photoreceptor cells in 24-month-old mice. Photoreceptor loss was accompanied by reactive astrogliosis, reactive microgliosis that was evident in the outer but not inner retina, and elevated expression levels of some lysosomal proteins. Electron microscopic analyses of retinas revealed no evidence for the presence of storage vacuoles. Of note, expression of ARSG protein in wild-type mice was detectable only in the RPE which, however, appeared morphologically unaffected in knockout mice at the electron microscopic level. To our knowledge, this is the first study demonstrating that ARSG deficiency results in progressive photoreceptor degeneration and dysregulation of various lysosomal proteins.
关键词： Sanfilippo syndrome,mucopolysaccharidosis,retinal pigment epithelium,Arylsulfatase G,retina,neurodegeneration,photoreceptor cells
更新于2025-09-09 09:28:46
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Retinal Neurodegeneration in Diabetic Patients Without Diabetic Retinopathy
摘要： PURPOSE. To compare the thickness of all retinal layers between a nondiabetic group and diabetic patients without diabetic retinopathy (DR). METHODS. Cross-sectional study, in which all subjects underwent an ophthalmic examination including optical coherence tomography. After automatic retinal segmentation, each retinal layer thickness (eight separate layers and overall thickness) was calculated in all nine Early Treatment Diabetic Retinopathy Study (ETDRS) areas. The choroidal thickness (CT) also was measured at five locations. Generalized additive regression models were used to analyze the data. RESULTS. A total of 175 patients were recruited, 50 nondiabetic subjects and 125 diabetic patients without DR, stratified into three groups according to diabetes duration: group I (<5 years, n = 55), group II (5–10 years, n = 39), and group III (>10 years, n = 31). Overall, groups I and III of diabetic patients had a decrease in the photoreceptor layer (PR) thickness, when compared with the nondiabetic subjects in six ETDRS areas (P < 0.0007). Patients with more recent diagnosis (group I) had thinner PR than those with moderate duration (group II). Interestingly, patients with longer known disease (group III) had the thinnest PR values. There were no overall differences in the remaining retinal parameters. CONCLUSIONS. Retinal thickness profile is not linear throughout disease duration. Even in the absence of funduscopic disease, PR layer in diabetic patients seems to differ from nondiabetic subjects, thus suggesting that some form of neurodegeneration may take place before clinical signs of vascular problems arise.
关键词： neurodegeneration,retinal layers,diabetic retinopathy,optical coherence tomography
更新于2025-09-09 09:28:46
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Automated Behavioral Analysis of Large <em>C. elegans</em> Populations Using a Wide Field-of-view Tracking Platform
摘要： Caenorhabditis elegans is a well-established animal model in biomedical research, widely employed in functional genomics and ageing studies. To assess the health and fitness of the animals under study, one typically relies on motility readouts, such as the measurement of the number of body bends or the speed of movement. These measurements usually involve manual counting, making it challenging to obtain good statistical significance, as time and labor constraints often limit the number of animals in each experiment to 25 or less. Since high statistical power is necessary to obtain reproducible results and limit false positive and negative results when weak phenotypic effects are investigated, efforts have recently been made to develop automated protocols focused on increasing the sensitivity of motility detection and multi-parametric behavioral profiling. In order to extend the limit of detection to the level needed to capture the small phenotypic changes that are often crucial in genetic studies and drug discovery, we describe here a technological development that enables the study of up to 5,000 individual animals simultaneously, increasing the statistical power of the measurements by about 1,000-fold compared to manual assays and about 100-fold compared to other available automated methods.
关键词： nematode library,neurodegeneration,amyloid formation,Alzheimer's disease,Drug discovery,Issue 141,phenotype-based screening,high-throughput screening,Immunology and Infection,C. elegans,large population analysis
更新于2025-09-09 09:28:46
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Multimodal Imaging of the Initial Stages of Diabetic Retinopathy. Different Disease Pathways in Different Patients
摘要： To evaluate the prevalence of different disease pathways (ischemia, neurodegeneration and edema) in the initial stages of diabetic retinopathy (DR). In this retrospective cross-sectional study, eyes were grouped by DR severity using the 7-field ETDRS protocol (levels 10-20, 35 and 43-47). Neurodegeneration was identified by thinning of the retinal nerve fiber layer (RNFL) and/or ganglion cell layer (GCL). Edema was identified by thickening of the inner nuclear layer (INL), outer plexiform layer (OPL), or full retina. Ischemia was identified by metrics of retinal vessel density. 142 eyes from 142 patients (28% women) aged 52-88 years were imaged. Vessel density (ischemia) was significantly different between ETDRS groups (p<0.020). On multivariate regression analysis, it remained significantly different between stages of the disease and showed associations with age (p<0.001), gender (p=0.028) and metabolic control (p=0.034). No significant differences between ETDRS groups were found in retinal thinning (neurodegeneration) or retinal thickness (edema). Eyes with the same ETDRS retinopathy grading from different diabetic patients show that the prevalence of different disease pathways, varies between patients even within the same severity group. Ischemia (capillary dropout) is the only disease pathway that shows correlation with retinopathy severity and metabolic control.
关键词： Retinal Edema,Ischemia,Neurodegeneration,Diabetic Retinopathy
更新于2025-09-04 15:30:14