Biomimetic Inorganic Nanovectors as Tumor-Targeting Theranostic Platform against Triple-Negative Breast Cancer.

Mesoporous silicon nanoparticles (PSi NPs) are promising platforms of nanomedicine because of their good compatibility, high payload capacities of anticancer drugs, and easy chemical modification. Here, PSi surfaces were functionalized with bisphosphonates (BP) for radiolabeling, loaded with doxorubicin (DOX) for chemotherapy, and the NPs were coated with cancer cell membrane (CCm) for homotypic cancer targeting. To enhance the CCm coating, the NP surfaces were covered with polyethylene glycol prior to the CCm coating. The effects of the BP amount and pH conditions on the radiolabeling efficacy were studied. The maximum BP was (2.27 wt%) on the PSi surfaces, and higher radiochemical yields were obtained for 99mTc (97% ± 2%) and 68Ga (94.6% ± 0.2%) under optimized pH conditions (pH = 5). The biomimetic NPs exhibited a good radiochemical and colloidal stability in phosphate-buffered saline and cell medium. In vitro studies demonstrated that the biomimetic NPs exhibited an enhanced cellular uptake and increased delivery of DOX to cancer cells, resulting in better chemotherapy than free DOX or pure NPs. Altogether, these findings indicate the potential of the developed platform for cancer treatment and diagnosis.

PET imaging studies to investigate functional expression of mGluR2 using [11C]mG2P001.

Metabotropic glutamate receptor 2 (mGluR2) has been extensively studied for the treatment of various neurological and psychiatric disorders. Understanding of the mGluR2 function is pivotal in supporting the drug discovery targeting mGluR2. Herein, the positive allosteric modulation of mGluR2 was investigated via the in vivo positron emission tomography (PET) imaging using 2-((4-(2-[11C]methoxy-4-(trifluoromethyl)phenyl)piperidin-1-yl)methyl)-1-methyl-1H-imidazo[4,5-b]pyridine ([11C]mG2P001). Distinct from the orthosteric compounds, pretreatment with the unlabeled mG2P001, a potent mGluR2 positive allosteric modulator (PAM), resulted in a significant increase instead of decrease of the [11C]mG2P001 accumulation in rat brain detected by PET imaging. Subsequent in vitro studies with [3H]mG2P001 revealed the cooperative binding mechanism of mG2P001 with glutamate and its pharmacological effect that contributed to the enhanced binding of [3H]mG2P001 in transfected CHO cells expressing mGluR2. The in vivo PET imaging and quantitative analysis of [11C]mG2P001 in non-human primates (NHPs) further validated the characteristics of [11C]mG2P001 as an imaging ligand for mGluR2. Self-blocking studies in primates enhanced accumulation of [11C]mG2P001. Altogether, these studies show that [11C]mG2P001 is a sensitive biomarker for mGluR2 expression and the binding is affected by the tissue glutamate concentration.

A robust approach to make inorganic nanovectors biotraceable.

Nuclear medicine imaging plays an important role in nanomedicine. However, it is still challenging to develop a versatile platform to make the nonviral nanovectors used in cancer therapy biotraceable. In the present study, a robust approach to radiolabel inorganic nanovectors for SPECT and PET imaging was developed. The approach was based on the bisphosphonates (BP) conjugated on the nanovector, mesoporous silicon (PSi) nanoparticles. BP served as an efficient chelator for various radionuclides. For both of the 99mTc and 68Ga radionuclides utilized, the radiochemical purity and radiochemical yield were ∼99% and ∼90%, respectively. Because of the short decay time of the radionuclides, an easy, fast and effective PEGylation method was developed to improve the residence time in systemic circulation. Both PEG-99mTc-BP-PSi and PEG-68Ga-BP-PSi NPs, where PEGylation was performed after the labeling, had excellent colloidal and radiochemical stability in vitro. The plain particles without PEGylation accumulated fast in the reticuloendothelial system organs upon intravenous administration, while PEGylation prolonged the residence time of the particles in systemic circulation. Overall, the developed approach proved to be applicable for labeling nonviral nanovectors with various radionuclides easily and robustly. Considering the nature of mesoporous nanoparticles, the approach does not hamper the addition of other functionalities on the vector, nor its capability to carry high payloads.

Chronic hypometabolism in striatum and hippocampal network after traumatic brain injury and their relation with memory impairment - [18F]-FDG-PET and MRI 4 months after fluid percussion injury in rat.

Hippocampal and thalamo-cortico-striatal networks are critical for memory function as well as execution of a variety of learning strategies. In subjects with memory impairment as a sequel of traumatic brain injury (TBI), the contribution of late metabolic depression across these networks to memory deficit is poorly understood. We used [18F]-FDG-PET to measure chronic post-TBI glucose uptake in the striatum and connected brain areas (septal and temporal hippocampus, thalamus, entorhinal cortex, frontoparietal cortex and amygdala) in rats with lateral fluid-percussion injury (LFPI). Then we assessed a link between network hypometabolism and memory impairment. At 4 months post TBI, glucose uptake was decreased in ipsilateral striatum (10%, p = 0.027), frontoparietal cortex (17%, p = 0.00009), and hippocampus (22%, p = 0.027) as compared to sham operated controls. Thalamic uptake was 6% lower ipsilaterally than contralaterally, p = 0.00004). At 5 months, Morris water maze (MWM) showed memory impairment in 83% of the rats with TBI. The lower the hippocampal or striatal [18F]-FDG uptake, the poorer the MWM performance (hippocampus: r = -0.471, p < 0.05; striatum: r = -0.696, p < 0.001). Striatal [18F]-FDG-PET identified the injured animals with memory impairment with 100% specificity and sensitivity (AUC = 1.000, p = 0.009). Interestingly, the low striatal glucose uptake was a better diagnostic biomarker for memory impairment than the reduced hippocampal (AUC = 0.806, p = 0.112) or entorhinal (AUC = 0.528, p = 0.885) glucose uptake. The volumetric atrophy assessed in T2 weighted MRI or the gliotic area in Nissl staining did not correlate with glucose uptake. Arterial spin labeling did not indicate any reduction in the striatal blood flow. Our study suggests that TBI-induced chronic hypometabolism in striatum contributes to the cognitive deficits.

AAV2-VEGF-B gene therapy failed to induce angiogenesis in ischemic porcine myocardium due to inflammatory responses.

Therapeutic angiogenesis induced by gene therapy is a promising approach to treat patients suffering from severe coronary artery disease. In small experimental animals, adeno-associated viruses (AAVs) have shown good transduction efficacy and long-term transgene expression in heart muscle and other tissues. However, it has been difficult to achieve cardiac-specific angiogenic effects with AAV vectors. We tested the hypothesis whether AAV2 gene transfer (1 × 1013 vg) of vascular endothelial growth factor B (VEGF-B186) together with immunosuppressive corticosteroid treatment can induce long-term cardiac-specific therapeutic effects in the porcine ischemic heart. Gene transfers were delivered percutaneously using direct intramyocardial injections, improving targeting and avoiding direct contact with blood, thus reducing the likelihood of immediate immune reactions. After 1- and 6-month time points, the capillary area was analyzed, myocardial perfusion reserve (MPR) was measured with radiowater positron emission tomography ([15O]H2O-PET), and fluorodeoxyglucose ([18F]FDG) uptake was used to evaluate myocardial viability. Clinical chemistry and immune responses were analyzed using standard methods. After 1- and 6-month follow-up, AAV2-VEGF-B186 gene transfer failed to induce angiogenesis and improve myocardial perfusion and viability. Here, we show that inflammatory responses attenuated the therapeutic effect of AAV2 gene transfer by significantly reducing successful transduction and long-term gene expression despite the efforts to reduce the likelihood of immune reactions and the use of targeted local gene transfer methods.

Adenoviral VEGF-B186R127S gene transfer induces angiogenesis and improves perfusion in ischemic heart.

Vascular endothelial growth factor B (VEGF-B) is an interesting therapeutic candidate for coronary artery disease. However, it can also cause ventricular arrhythmias, potentially preventing its use in clinics. We cloned VEGF-B isoforms with different receptor binding profiles to clarify the roles of VEGFR-1 and Nrp-1 in angiogenesis and to see if angiogenic properties can be maintained while avoiding side effects. VEGF-B constructs were studied in vivo using adenovirus (Ad)-mediated intramyocardial gene transfers into the normoxic and ischemic porcine heart (n = 51). It was found that the unprocessed isoform VEGF-B186R127S is as efficient angiogenic growth factor as the native VEGF-B186 in normoxic and ischemic heart. In addition, AdVEGF-B186R127S increased myocardial perfusion reserve by 22% in ischemic heart without any side effects. AdVEGF-B127 (VEGFR-1 and Nrp-1 ligand) and AdVEGF-B109 (VEGFR-1 ligand) did not induce angiogenesis. Thus, VEGF-B186 is angiogenic only before its proteolytic processing to VEGF-B127. Only the VEGF-B186 C-terminal fragment was associated with arrhythmias.

Severe Obstructive Sleep Apnea and Increased Cortical Amyloid-ß Deposition.

BACKGROUND: The suggested association between severe obstructive sleep apnea (OSA) and risk of Alzheimer's disease (AD) needs further study. Only few recent reports exist on associations between brain amyloid-ß (Aß) burden and severe OSA in middle-aged patients. OBJECTIVE: Examine the possible presence of cortical Aß accumulation in middle-aged patients with severe OSA. METHODS: We performed detailed multimodal neuroimaging in 19 cognitive intact patients (mean 44.2 years) with severe OSA (Apnea-Hypopnea Index >30 h-1). Known etiological factors for possible Aß accumulation were used as exclusion criteria. Aß uptake was studied with [11C]-PiB-PET, glucose metabolism with [18F]-FDG-PET, and structural imaging with 3.0T MRI. RESULTS: When analyzed individually, in [11C]-PiB-PET a substantial number (∼32%) of the patients exhibited statistically significant evidence of increased cortical Aß uptake based on elevated regional Z-score values, mostly seen bilaterally in the precuneus and posterior cingulum regions. Cortical glucose hypometabolism in [18F]-FDG-PET was seen in two patients. MRI did not show structural changes suggestive of AD-related pathology. CONCLUSION: Increased [11C]-PiB uptake was seen in middle-aged cognitively intact patients with severe OSA. These findings are similar to those described in cognitive unimpaired older OSA patients. The changes in cortical Aß uptake suggest that severe OSA itself may predispose to alterations related to AD already in middle-age. Aß clearance may be compromised without simultaneous evidence of metabolic or structural alterations. The results emphasize the importance of early diagnostics and proper treatment of severe OSA in cognitively intact middle-aged subjects, possibly diminishing the individual risk for later cognitive dysfunction.

Design, Synthesis, and Characterization of Benzimidazole Derivatives as Positron Emission Tomography Imaging Ligands for Metabotropic Glutamate Receptor 2.

Three benzimidazole derivatives (13-15) have been synthetized as potential positron emission tomography (PET) imaging ligands for mGluR2 in the brain. Of these compounds, 13 exhibits potent binding affinity (IC50 = 7.6 ± 0.9 nM), positive allosteric modulator (PAM) activity (EC50 = 51.2 nM), and excellent selectivity against other mGluR subtypes (>100-fold). [11C]13 was synthesized via O-[11C]methylation of its phenol precursor 25 with [11C]methyl iodide. The achieved radiochemical yield was 20 ± 2% (n = 10, decay-corrected) based on [11C]CO2 with a radiochemical purity of >98% and molar activity of 98 ± 30 GBq/µmol EOS. Ex vivo biodistribution studies revealed reversible accumulation of [11C]13 and hepatobiliary and urinary excretions. PET imaging studies in rats demonstrated that [11C]13 accumulated in the mGluR2-rich brain regions. Pre-administration of mGluR2-selective PAM, 17 reduced the brain uptake of [11C]13, indicating a selective binding. Therefore, [11C]13 is a potential PET imaging ligand for mGluR2 in different central nervous system-related conditions.

The Alzheimer's disease-associated protective Plcγ2-P522R variant promotes immune functions.

BACKGROUND: Microglia-specific genetic variants are enriched in several neurodegenerative diseases, including Alzheimer's disease (AD), implicating a central role for alterations of the innate immune system in the disease etiology. A rare coding variant in the PLCG2 gene (rs72824905, p.P522R) expressed in myeloid lineage cells was recently identified and shown to reduce the risk for AD. METHODS: To assess the role of the protective variant in the context of immune cell functions, we generated a Plcγ2-P522R knock-in (KI) mouse model using CRISPR/Cas9 gene editing. RESULTS: Functional analyses of macrophages derived from homozygous KI mice and wild type (WT) littermates revealed that the P522R variant potentiates the primary function of Plcγ2 as a Pip2-metabolizing enzyme. This was associated with improved survival and increased acute inflammatory response of the KI macrophages. Enhanced phagocytosis was observed in mouse BV2 microglia-like cells overexpressing human PLCγ2-P522R, but not in PLCγ2-WT expressing cells. Immunohistochemical analyses did not reveal changes in the number or morphology of microglia in the cortex of Plcγ2-P522R KI mice. However, the brain mRNA signature together with microglia-related PET imaging suggested enhanced microglial functions in Plcγ2-P522R KI mice. CONCLUSION: The AD-associated protective Plcγ2-P522R variant promotes protective functions associated with TREM2 signaling. Our findings provide further support for the idea that pharmacological modulation of microglia via TREM2-PLCγ2 pathway-dependent stimulation may be a novel therapeutic option for the treatment of AD.

A multimodal approach to identify clinically relevant biomarkers to comprehensively monitor disease progression in a mouse model of pediatric neurodegenerative disease.

While research has accelerated the development of new treatments for pediatric neurodegenerative disorders, the ability to demonstrate the long-term efficacy of these therapies has been hindered by the lack of convincing, noninvasive methods for tracking disease progression both in animal models and in human clinical trials. Here, we unveil a new translational platform for tracking disease progression in an animal model of a pediatric neurodegenerative disorder, CLN6-Batten disease. Instead of looking at a handful of parameters or a single "needle in a haystack", we embrace the idea that disease progression, in mice and patients alike, is a diverse phenomenon best characterized by a combination of relevant biomarkers. Thus, we employed a multi-modal quantitative approach where 144 parameters were longitudinally monitored to allow for individual variability. We use a range of noninvasive neuroimaging modalities and kinematic gait analysis, all methods that parallel those commonly used in the clinic, followed by a powerful statistical platform to identify key progressive anatomical and metabolic changes that correlate strongly with the progression of pathological and behavioral deficits. This innovative, highly sensitive platform can be used as a powerful tool for preclinical studies on neurodegenerative diseases, and provides proof-of-principle for use as a potentially translatable tool for clinicians in the future.

Synthesis and Characterization of Fluorine-18-Labeled N-(4-Chloro-3-((fluoromethyl-d2)thio)phenyl)picolinamide for Imaging of mGluR4 in Brain.

We have synthesized and characterized [18F]-N-(4-chloro-3-((fluoromethyl-d2)thio)phenyl)-picolinamide ([18F]15) as a potential ligand for the positron emission tomography (PET) imaging of mGluR4 in the brain. Radioligand [18F]15 displays central nervous system drug-like properties, including mGluR4 affinity, potent mGluR4 PAM activity, and selectivity against other mGluRs, as well as sufficient metabolic stability. Radiosynthesis was carried out in two steps. The radiochemical yield of [18F]15 was 11.6 ± 2.9% (n = 7, decay corrected) with a purity of 99% and a molar activity of 84.1 ± 11.8 GBq/µmol. Ex vivo biodistribution studies showed reversible binding of [18F]15 in all investigated tissues including the brain, liver, heart, lungs, and kidneys. PET imaging studies in male Sprague Dawley rats showed that [18F]15 accumulates in the brain regions known to express mGluR4. Pretreatment with the unlabeled mGluR4 PAM compounds 13 (methylthio analogue) and 15 showed significant dose-dependent blocking effects. These results suggest that [18F]15 is a promising radioligand for PET imaging mGluR4 in the brain.

Improved synthesis of [18F] fallypride and characterization of a Huntington's disease mouse model, zQ175DN KI, using longitudinal PET imaging of D2/D3 receptors.

PURPOSE: Dopamine receptors are involved in pathophysiology of neuropsychiatric diseases, including Huntington's disease (HD). PET imaging of dopamine D2 receptors (D2R) in HD patients has demonstrated 40% decrease in D2R binding in striatum, and D2R could be a reliable quantitative target to monitor disease progression. A D2/3R antagonist, [18F] fallypride, is a high-affinity radioligand that has been clinically used to study receptor density and occupancy in neuropsychiatric disorders. Here we report an improved synthesis method for [18F]fallypride. In addition, high molar activity of the ligand has allowed us to apply PET imaging to characterize D2/D3 receptor density in striatum of the recently developed zQ175DN knock-in (KI) mouse model of HD. METHODS: We longitudinally characterized in vivo [18F] fallypride -PET imaging of D2/D3 receptor densities in striatum of 9 and 12 month old wild type (WT) and heterozygous (HET) zQ175DN KI mouse. Furthermore, we verified the D2/D3 receptor density in striatum with [3H] fallypride autoradiography at 12 months of age. RESULTS: We implemented an improved synthesis method for [18F] fallypride to yield high molar activity (MA, 298-360 GBq/µmol) and good reproducibility. In the HET zQ175DN KI mice, we observed a significant longitudinal decrease in binding potential (BPND) (30.2%, p < 0.001, 9 months of age and 51.6%, p < 0.001, 12 months of age) compared to WT littermates. No mass effect was observed when the MA of [18F] fallypride was > 100 GBq/µmol at the time of injection. Furthermore, the decrease of D2/D3 receptor density in striatum in HET zQ175DN KI was consistent using [3H] fallypride autoradiography. CONCLUSIONS: We observed a significant decrease in D2/D3R receptor densities in the striatum of HET zQ175DN KI mice compared to WT mice at 9 and 12 months of age. These results are in line with clinical findings in HD patients, suggesting [18F] fallypride PET imaging has potential as a quantitative translational approach to monitor disease progression in preclinical studies.

MRS Reveals Chronic Inflammation in T2w MRI-Negative Perilesional Cortex - A 6-Months Multimodal Imaging Follow-Up Study.

Sustained inflammation in the injured cortex is a promising therapeutic target for disease-modification after traumatic brain injury (TBI). However, its extent and dynamics of expansion are incompletely understood which challenges the timing and placement of therapeutics to lesioned area. Our aim was to characterize the evolution of chronic inflammation during lesion expansion in lateral fluid-percussion injury (FPI) rat model with focus on the MRI-negative perilesional cortex. T2-weighted MR imaging (T2w MRI) and localized magnetic resonance spectroscopy (MRS) were performed at 1, 3, and 6 months post-injury. End-point histology, including Nissl for neuronal death, GFAP for astrogliosis, and Prussian Blue for iron were used to assess perilesional histopathology. An additional animal cohort was imaged with a positron emission tomography (PET) using translocator protein 18 kDa (TSPO) radiotracer [18F]-FEPPA. T2w MRI assessed lesion growth and detected chronic inflammation along the lesion border while rest of the ipsilateral cortex was MRI-negative (MRI-). Instead, myo-inositol that is an inflammatory MRS marker for gliosis, glutathione for oxidative stress, and choline for membrane turnover were elevated throughout the 6-months follow-up in the MRI- perilesional cortex (all p < 0.05). MRS markers revealed chronically sustained inflammation across the ipsilateral cortex but did not indicate the upcoming lesion expansion. Instead, the rostral expansion of the cortical lesion was systematically preceded by a hyperintense band in T2w images months earlier. Histologic analysis of the hyperintensity indicated scattered astrocytes, incomplete glial scar, and intracellularly packed and free iron. Yet, the band was negative in [18F]-FEPPA-PET. [18F]-FEPPA also showed no cortical TSPO expression within the MRS voxel in MRI- perilesional cortex or anywhere along glial scar when assessed at 2 months post-injury. However, [18F]-FEPPA showed a robust signal increase, indicating reactive microgliosis in the ipsilateral thalamus at 2 months post-TBI. We present evidence that MRS reveals chronic posttraumatic inflammation in MRI-negative perilesional cortex. The mismatch in MRS, MRI, and PET measures may allow non-invasive endophenotyping of beneficial and detrimental inflammatory processes to aid targeting and timing of anti-inflammatory therapeutics.

Intranasal insulin activates Akt2 signaling pathway in the hippocampus of wild-type but not in APP/PS1 Alzheimer model mice.

Type 2 diabetes mellitus (T2DM) increases the risk for Alzheimer's disease (AD). Human AD brains show reduced glucose metabolism as measured by [18F]fluoro-2-deoxy-2-D-glucose positron emission tomography (FDG-PET). Here, we used 14-month-old wild-type (WT) and APPSwe/PS1dE9 (APP/PS1) transgenic mice to investigate how a single dose of intranasal insulin modulates brain glucose metabolism using FDG-PET and affects spatial learning and memory. We also assessed how insulin influences the activity of Akt1 and Akt2 kinases, the expression of glial and neuronal markers, and autophagy in the hippocampus. Intranasal insulin moderately increased glucose metabolism and specifically activated Akt2 and its downstream signaling in the hippocampus of WT, but not APP/PS1 mice. Furthermore, insulin differentially affected the expression of homeostatic microglia markers P2ry12 and Cx3cr1 and autophagy in the hippocampus of WT and APP/PS1 mice. We found no evidence that a single dose of intranasal insulin improves overnight memory. Our results suggest that intranasal insulin exerts diverse effects on Akt2 signaling, autophagy, and the homeostatic status of microglia depending on the degree of AD-related pathology.

Impaired Mitochondrial Fatty Acid Synthesis Leads to Neurodegeneration in Mice.

There has been a growing interest toward mitochondrial fatty acid synthesis (mtFAS) since the recent discovery of a neurodegenerative human disorder termed MEPAN (mitochondrial enoyl reductase protein associated neurodegeneration), which is caused by mutations in the mitochondrial enoyl-CoA/ACP (acyl carrier protein) reductase (MECR) carrying out the last step of mtFAS. We show here that MECR protein is highly expressed in mouse Purkinje cells (PCs). To elucidate mtFAS function in neural tissue, here, we generated a mouse line with a PC-specific knock-out (KO) of Mecr, leading to inactivation of mtFAS confined to this cell type. Both sexes were studied. The mitochondria in KO PCs displayed abnormal morphology, loss of protein lipoylation, and reduced respiratory chain enzymatic activities by the time these mice were 6 months of age, followed by nearly complete loss of PCs by 9 months of age. These animals exhibited balancing difficulties ∼7 months of age and ataxic symptoms were evident from 8-9 months of age on. Our data show that impairment of mtFAS results in functional and ultrastructural changes in mitochondria followed by death of PCs, mimicking aspects of the clinical phenotype. This KO mouse represents a new model for impaired mitochondrial lipid metabolism and cerebellar ataxia with a distinct and well trackable cellular phenotype. This mouse model will allow the future investigation of the feasibility of metabolite supplementation approaches toward the prevention of neurodegeneration due to dysfunctional mtFAS.SIGNIFICANCE STATEMENT We have recently reported a novel neurodegenerative disorder in humans termed MEPAN (mitochondrial enoyl reductase protein associated neurodegeneration) (Heimer et al., 2016). The cause of neuron degeneration in MEPAN patients is the dysfunction of the highly conserved mitochondrial fatty acid synthesis (mtFAS) pathway due to mutations in MECR, encoding mitochondrial 2-enoyl-CoA/ACP reductase. The report presented here describes the analysis of the first mouse model suffering from mtFAS-defect-induced neurodegenerative changes due to specific disruption of the Mecr gene in Purkinje cells. Our work sheds a light on the mechanisms of neurodegeneration caused by mtFAS deficiency and provides a test bed for future treatment approaches.

Precision Medicine in Multiple Sclerosis: Future of PET Imaging of Inflammation and Reactive Astrocytes.

Non-invasive molecular imaging techniques can enhance diagnosis to achieve successful treatment, as well as reveal underlying pathogenic mechanisms in disorders such as multiple sclerosis (MS). The cooperation of advanced multimodal imaging techniques and increased knowledge of the MS disease mechanism allows both monitoring of neuronal network and therapeutic outcome as well as the tools to discover novel therapeutic targets. Diverse imaging modalities provide reliable diagnostic and prognostic platforms to better achieve precision medicine. Traditionally, magnetic resonance imaging (MRI) has been considered the golden standard in MS research and diagnosis. However, positron emission tomography (PET) imaging can provide functional information of molecular biology in detail even prior to anatomic changes, allowing close follow up of disease progression and treatment response. The recent findings support three major neuroinflammation components in MS: astrogliosis, cytokine elevation, and significant changes in specific proteins, which offer a great variety of specific targets for imaging purposes. Regardless of the fact that imaging of astrocyte function is still a young field and in need for development of suitable imaging ligands, recent studies have shown that inflammation and astrocyte activation are related to progression of MS. MS is a complex disease, which requires understanding of disease mechanisms for successful treatment. PET is a precise non-invasive imaging method for biochemical functions and has potential to enhance early and accurate diagnosis for precision therapy of MS. In this review we focus on modulation of different receptor systems and inflammatory aspect of MS, especially on activation of glial cells, and summarize the recent findings of PET imaging in MS and present the most potent targets for new biomarkers with the main focus on experimental MS research.

Synthesis and evaluation of N-(methylthiophenyl)picolinamide derivatives as PET radioligands for metabotropic glutamate receptor subtype 4.

In recent years, mGlu4 has received great research attention because of the potential benefits of mGlu4 activation in treating numerous brain disorders, such as Parkinson's disease (PD). A specific mGlu4 PET radioligand could be an important tool in understanding the role of mGlu4 in both healthy and disease conditions, and also for the development of new drugs. In this study, we synthesized four new N-(methylthiophenyl)picolinamide derivatives 11-14. Of these ligands, 11 and 14 showed high in vitro binding affinity for mGlu4 with IC50 values of 3.4nM and 3.1nM, respectively, and suitable physicochemical parameters. Compound 11 also showed enhanced metabolic stability and good selectivity to other mGluRs. [(11)C]11 and [(11)C]14 were radiolabeled using the [(11)C]methylation of the thiophenol precursors 20a and 20c with [(11)C]CH3I in 19.0% and 34.8% radiochemical yields (RCY), and their specific activities at the end of synthesis (EOS) were 496±138GBq/µmol (n=6) and 463±263GBq/µmol (n=4), respectively. The PET studies showed that [(11)C]11 accumulated fast into the brain and had higher uptake, slower washout and 25% better contrast than [(11)C]2, indicating improved imaging characteristics as PET radiotracer for mGlu4 compared to [(11)C]2. Therefore, [(11)C]11 will be a useful radioligand to investigate mGlu4 in different biological applications.

Functional modulation of G-protein coupled receptors during Parkinson disease-like neurodegeneration.

G-protein coupled dopamine and metabotropic glutamate receptors (mGlu) can modulate neurotransmission during Parkinson's disease (PD)-like neurodegeneration. PET imaging studies in a unilateral dopamine denervation model (6-OHDA) showed a significant inverse correlation of presynaptic mGlu4 and postsynaptic mGlu5 expression in the striatum and rapidly declining mGlu4 and enhanced mGlu5 expression in the hippocampus during progressive degeneration over time. Immunohistochemical studies verified the decreased mGlu4 expression in the hippocampus on the lesion side but did not show difference in mGlu5 expression between lesion and control side. Pharmacological MRI studies showed enhanced hemodynamic response in several brain areas on the lesion side compared to the control side after challenge with mGlu4 positive allosteric modulator or mGlu5 negative allosteric modulator. However, mGlu4 response was biphasic having short enhancement followed by negative response on both sides of brain. Studies in mGlu4 expressing cells demonstrated that glutamate induces cooperative increase in binding of mGlu4 ligands - especially at high glutamate levels consistent with in vivo concentration. This suggests that mGlu allosteric modulators as drug candidates will be highly sensitive to changes in glutamate concentration and hence metabolic state. These experiments demonstrate the importance of the longitudinal imaging studies to investigate temporal changes in receptor functions to obtain individual response for experimental drugs.

PET imaging studies show enhanced expression of mGluR5 and inflammatory response during progressive degeneration in ALS mouse model expressing SOD1-G93A gene.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative motor neuron disorder. Genetic studies have linked mutation of the gene SOD1 to ALS pathology as well as several other pathological processes including modulation of glutamatergic function and inflammatory processes. Since therapeutic approaches for ALS are focused on glutamatergic function, we investigated modulation of glutamate transport based on its receptor function as well as excitotoxicity-induced inflammatory response. METHODS: In vivo positron emission tomography (PET) imaging studies of metabotropic glutamate receptor subtype 5 (mGluR5) using [(18)F]FPEB ([(18)F]3-fluoro-5-(2-pyridylethynyl)benzonitrile) and inflammatory response using [(11)C]PBR28 (peripheral benzodiazepine receptor ligand 28) were done in an early and a late phase of neurodegeneration in four ALS mice expressing SOD1-G93A gene and four control base mice (C57/BL6). Accumulation of [(18)F]FPEB and [(11)C]PBR28 were quantitated in several brain areas and spinal cord to determine degeneration-induced modulation. The studies were completed with immunohistochemical analyses of mGluR5 and inflammatory response. RESULTS: These studies showed enhanced binding potential of [(18)F]FPEB in several brain areas including striatum, hippocampus, and frontal cortex. In the whole brain, the binding potential increased 49 ± 9 % from base mice to ALS-type mice and further enhanced 23 ± 4 % during disease progression. Also, in the spinal cord 6-22 %, enhanced accumulation of [(18)F]FPEB was observed during progression of the disease. The accumulation of [(11)C]PBR28 increased by 110 ± 33 % in the whole brain during progression of the disease indicating significant inflammatory process. [(11)C]PBR28 accumulation enhanced 89-264 % in the spinal cord and 204 % in the lungs. The end point immunohistochemical analyses verified the enhanced mGluR5 expression and inflammation. CONCLUSIONS: These results confirm the role of glutamate and inflammation in ALS-type pathology. These data also support the hypothesis that excessive glutamate may contribute to inflammation in the chronic neurodegenerative processes in ALS.

Loss of Metabotropic Glutamate Receptor 5 Function on Peripheral Benzodiazepine Receptor in Mice Prenatally Exposed to LPS.

Parental microglial induced neuroinflammation, triggered by bacterial- or viral infections, can induce neuropsychiatric disorders like schizophrenia and autism to offspring in animal models. Recent investigations suggest that microglia, the resident immune cells of the brain, provides a link between neurotransmission, immune cell activation, brain inflammation and neuronal dysfunction seen with the offspring. Relatively little is known about how reduction of brain inflammation and restoration of glial function are associated with diminution of brain degeneration and behavioral deficits in offspring. Increased mGluR5 expression and the long-lasting excitotoxic effects of the neurotoxin during brain development are associated with the glial dysfunctions. We investigated the relationship of mGluR5 and PBR and how they regulate glial function and inflammatory processes in mice prenatally exposed to LPS (120µg/kg, between gestational days 15 and 17), an inflammatory model of a psychiatric disorder. Using PET imaging, we showed that pharmacological activation of mGluR5 during 5 weeks reduced expression of classic inflammation marker PBR in many brain areas and that this molecular association was not present in LPS-exposed offspring. The post-mortem analysis revealed that the down regulation of PBR was mediated through activation of mGluR5 in astrocytes. In addition, we demonstrated that this interaction is defective in a mouse model of the psychiatric deficit offering a novel insight of mGluR5 involvement to brain related disorders and PBR related imaging studies. In conclusion, mGluR5 driven glutamatergic activity regulates astrocytic functions associated with PBR (cholesterol transport, neurosteroidogenesis, glial phenotype) during maturation and could be associated with neuropsychiatric disorders in offspring.