Junqing Zhu

Design, Synthesis and Evaluation of Coumarin-based Molecular Probes for Imaging of Myelination

Myelination represents one of the most fundamental biological processes in the vertebrate nervous system. Abnormalities and changes in myelination in the central nervous system (CNS) are seen in many neurodegenerative disorders, such as multiple sclerosis (MS). A long-standing goal has been to directly detect and quantify myelin content in order to facilitate diagnosis and therapeutic treatments of myelin-related diseases. In the course of our studies, we have developed a series of small-molecule probes (SMP) as myelin-imaging agents. Among them are coumarin derivatives, which exhibit promising brain permeability and myelin-binding properties. Herein we report a full account of the design and synthesis of coumarin-based SMPs as myelin-imaging agents. Systematic evaluation of these SMPs in both the CNS and peripheral nervous system (PNS) allowed us to identify some lead agents for potential use as fluorescent dyes for intraoperative nerve mapping in surgical operations or as radiotracers for positron emission tomography (PET) imaging of myelination.

Kruppel-like factor 2 protects against ischemic stroke by regulating endothelial blood brain barrier function

During an ischemic stroke normal brain endothelial function is perturbed, resulting in blood brain barrier (BBB) breakdown with subsequent infiltration of activated inflammatory blood cells, ultimately leading to neuronal cell death. Kruppel-like factor 2 (KLF2) is regulated by flow, is highly expressed in vascular endothelial cells (ECs), and serves as a key molecular switch regulating endothelial function and promoting vascular health. In this study we sought to determine the role of KLF2 in cerebrovascular function and the pathogenesis of ischemic stroke. Transient middle cerebral artery occlusion was performed in KLF2-deficient (KLF2(-/-)), KLF2 overexpressing (KLF2(tg)), and control mice, and stroke volume was analyzed. BBB function was assessed in vivo by real-time neuroimaging using positron emission tomography and Evans blue dye assay. KLF2(-/-) mice exhibited significantly larger strokes and impairment in BBB function. In contrast, KLF2(tg) mice were protected against ischemic stroke and demonstrated preserved BBB function. In concordance, gain- and loss-of-function studies in primary brain microvascular ECs using transwell assays revealed KLF2 to be BBB protective. Mechanistically, KLF2 was demonstrated, both in vitro and in vivo, to regulate the critical BBB tight junction factor occludin. These data are first to identify endothelial KLF2 as a key regulator of the BBB and a novel neuroprotective factor in ischemic stroke.

Longitudinal near-infrared imaging of myelination.

Myelination is one of the fundamental biological processes in the development of vertebrate nervous system. Disturbance of myelination is found to be associated with progression in many neurological diseases such as multiple sclerosis. Tremendous efforts have been made to develop novel therapeutic agents that prevent demyelination and/or promote remyelination. These efforts need to be accompanied by the development of imaging tools that permit direct quantification of myelination in vivo. In this work, we describe a novel near-infrared fluorescence imaging technique that is capable of direct quantification of myelination in vivo. This technique is developed based on a near-infrared fluorescent probe, 3,3′-diethylthiatricarbocyanine iodide (DBT) that readily enters the brain and specifically binds to myelinated fibers. In vivo imaging studies were first conducted in two animal models of hypermyelination and hypomyelination followed by longitudinal studies in the cuprizone-induced demyelination/remyelination mouse model. Quantitative analysis suggests that DBT is a sensitive and specific imaging probe of myelination, which complements other current myelin-imaging modalities and is of low cost.

Longitudinal positron emission tomography imaging for monitoring myelin repair in the spinal cord

Novel therapeutic interventions aimed at myelin repair are now under development for neuroprotection as well as functional recovery of patients with multiple sclerosis. However, development of myelin repair therapy necessitates a noninvasive approach for measuring changes in myelin content in vivo in a quantitative fashion not yet possible using magnetic resonance imaging. For this reason, we developed a novel positron emission tomography (PET) probe, termed [11C]MeDAS, that is capable of longitudinally imaging central nervous system myelin content.

In Situ Fluorescence Imaging of Myelination

We describe a novel fluorescent dye, 3-(4-aminophenyl)-2H-chromen-2-one (termed case myelin compound or CMC), that can be used for in situ fluorescent imaging of myelin in the vertebrate nervous system. When administered via intravenous injection into the tail vein, CMC selectively stained large bundles of myelinated fibers in both the central nervous system (CNS) and the peripheral nervous system (PNS). In the CNS, CMC readily entered the brain and selectively localized in myelinated regions such as the corpus callosum and cerebellum. CMC also selectively stained myelinated nerves in the PNS. The staining patterns of CMC in a hypermyelinated mouse model were consistent with immunohistochemical staining. Similar to immunohistochemical staining, CMC selectively bound to myelin sheaths present in the white matter tracts. Unlike CMC, conventional antibody staining for myelin basic protein also stained oligodendrocyte cytoplasm in the striatum as well as granule layers in the cerebellum. In vivo application of CMC was also demonstrated by fluorescence imaging of myelinated nerves in the PNS.

Myelin Imaging Compound (MIC) Enhanced Magnetic Resonance Imaging of Myelination

The vertebrate nervous system is characterized by myelination, a fundamental biological process that protects the axons and facilitates electric pulse transduction. Damage to myelin is considered a major effect of autoimmune diseases such as multiple sclerosis (MS). Currently, therapeutic interventions are focused on protecting myelin integrity and promoting myelin repair. These efforts need to be accompanied by an effective imaging tool that correlates the disease progression with the extent of myelination. To date, magnetic resonance imaging (MRI) is the primary imaging technique to detect brain lesions in MS. However, conventional MRI cannot differentiate demyelinated lesions from other inflammatory lesions and therefore cannot predict disease progression in MS. To address this problem, we have prepared a Gd-based contrast agent, termed MIC (myelin imaging compound), which binds to myelin with high specificity. In this work, we demonstrate that MIC exhibits a high kinetic stability toward transmetalation with promising relaxometric properties. MIC was used for in vivo imaging of myelination following intracerebroventricular infusion in the rat brain. MIC was found to distribute preferentially in highly myelinated regions and was able to detect regions of focally induced demyelination.

Synthesis and characterization of a novel gadolinium-based contrast agent for magnetic resonance imaging of myelination

Myelin is a membrane system that fosters nervous impulse conduction in the vertebrate nervous system. Myelin sheath disruption is a common characteristic of several neurodegenerative diseases such as multiple sclerosis (MS) and various leukodystrophies. To date, the diagnosis of MS is obtained using a set of criteria in which MRI observations play a central role. However, because of the lack of specificity for myelin integrity, the use of MRI as the primary diagnostic tool has not yet been accepted. In order to improve MR specificity, we began developing MR probes targeted toward myelin. In this work we describe a new myelin-targeted MR contrast agent, Gd-DODAS, based on a stilbene binding moiety and demonstrate its ability to specifically bind to myelin in vitro and in vivo. We also present evidence that Gd-DODAS generates MR contrast in vivo in T1-weighed images and in T1 maps that correlates to the myelin content.

Design, Synthesis, and Evaluation of Fluorinated Radioligands for Myelin Imaging.

Myelination is one of the fundamental processes in vertebrates. A major challenge is to quantitatively image myelin distribution in the central nervous system. For this reason, we designed and synthesized a series of fluorinated radioligands that can be radiolabeled as radiotracers for positron emission tomography (PET) imaging of myelin. These newly developed radioligands readily penetrate the blood-brain barrier and selectively bind to myelin membranes in the white matter region. Structure-activity relationship studies of such ligands suggested that optimal permeability could be achieved with calculated lipophilicty in the range of 3-4. After radiolabeling with fluorine-18, the brain uptake and retention of each radioligand were determined by microPET/CT imaging studies. These pharmacokinetic studies led us to identify a lead compound ([(18)F]FMeDAS, 32) with promising in vivo binding properties, which was subsequently validated by ex vivo autoradiography.

Discovery of 1,2,3-Triazole Derivatives for Multimodality PET/CT/Cryoimaging of Myelination in the Central Nervous System

Myelin pathology is present in many neurological conditions such as multiple sclerosis (MS) and traumatic spinal cord injury (SCI). To facilitate development of novel therapies aimed at myelin repair, we set out to develop imaging agents that permit direct quantification of myelination in vivo. In this work, we designed and synthesized a series of fluorescent fluorinated myelin imaging agents that can be used for in vivo positron emission tomography (PET) imaging combined with subsequent post-mortem fluorescent cryoimaging. Structure-activity relationship (SAR) studies of the newly developed myelin imaging agents led us to identify a lead compound (TAFDAS, 21) that readily enters the brain and spinal cord and selectively binds to myelin. By conducting sequential PET and 3D cryoimaging in an SCI rat model, we demonstrated for the first time that PET and cryoimaging can be combined as a novel technique to image the spinal cord with high sensitivity and spatial resolution.

High-resolution dynamic oxygen-17 MR imaging of mouse brain with golden-ratio–based radial sampling and k-space–weighted image reconstruction

The current study aimed to develop a three‐dimensional (3D) dynamic oxygen‐17 (17O) MR imaging method with high temporal and spatial resolution to delineate the kinetics of 17O water uptake and washout in the brains of mice with glioblastoma (GBM).