Altered dynamics of glymphatic flow in a mature-onset Tet-off APP mouse model of amyloidosis.

BACKGROUND: Alzheimer's disease (AD) is an incurable neurodegenerative disorder characterised by the progressive buildup of toxic amyloid-beta (Aß) and tau protein aggregates eventually leading to cognitive decline. Recent lines of evidence suggest that an impairment of the glymphatic system (GS), a brain waste clearance pathway, plays a key role in the pathology of AD. Moreover, a relationship between GS function and neuronal network integrity has been strongly implicated. Here, we sought to assess the efficacy of the GS in a transgenic Tet-Off APP mouse model of amyloidosis, in which the expression of mutant APP was delayed until maturity, mimicking features of late-onset AD-the most common form of dementia in humans. METHODS: To evaluate GS function, we used dynamic contrast-enhanced MRI (DCE-MRI) in 14-month-old Tet-Off APP (AD) mice and aged-matched littermate controls. Brain-wide transport of the Gd-DOTA contrast agent was monitored over time after cisterna magna injection. Region-of-interest analysis and computational modelling were used to assess GS dynamics while characterisation of brain tissue abnormalities at the microscale was performed ex vivo by immunohistochemistry. RESULTS: We observed reduced rostral glymphatic flow and higher accumulation of the contrast agent in areas proximal to the injection side in the AD group. Clustering and subsequent computational modelling of voxel time courses revealed significantly lower influx time constants in AD relative to the controls. Ex vivo evaluation showed abundant amyloid plaque burden in the AD group coinciding with extensive astrogliosis and microgliosis. The neuroinflammatory responses were also found in plaque-devoid regions, potentially impacting brain-fluid circulation. CONCLUSIONS: In a context resembling late-onset AD in humans, we demonstrate the disruption of glymphatic function and particularly a reduction in brain-fluid influx in the AD group. We conjecture that the hindered circulation of cerebrospinal fluid is potentially caused by wide-spread astrogliosis and amyloid-related obstruction of the normal routes of glymphatic flow resulting in redirection towards caudal regions. In sum, our study highlights the translational potential of alternative approaches, such as targeting brain-fluid circulation as potential therapeutic strategies for AD.

Combining designer receptors exclusively activated by designer drugs and neuroimaging in experimental models: A powerful approach towards neurotheranostic applications.

The combination of chemogenetics targeting specific brain cell populations with in vivo imaging techniques provides scientists with a powerful new tool to study functional neural networks at the whole-brain scale. A number of recent studies indicate the potential of this approach to increase our understanding of brain function in health and disease. In this review, we discuss the employment of a specific chemogenetic tool, designer receptors exclusively activated by designer drugs, in conjunction with non-invasive neuroimaging techniques such as PET and MRI. We highlight the utility of using this multiscale approach in longitudinal studies and its ability to identify novel brain circuits relevant to behaviour that can be monitored in parallel. In addition, some identified shortcomings in this technique and more recent efforts to overcome them are also presented. Finally, we discuss the translational potential of designer receptors exclusively activated by designer drugs in neuroimaging and the promise it holds for future neurotheranostic applications.

Increased soluble amyloid-beta causes early aberrant brain network hypersynchronisation in a mature-onset mouse model of amyloidosis.

Alzheimer's disease (AD) is the most common form of dementia in the elderly. According to the amyloid hypothesis, the accumulation and deposition of amyloid-beta (Aß) peptides play a key role in AD. Soluble Aß (sAß) oligomers were shown to be involved in pathological hypersynchronisation of brain resting-state networks in different transgenic developmental-onset mouse models of amyloidosis. However, the impact of protein overexpression during brain postnatal development may cause additional phenotypes unrelated to AD. To address this concern, we investigated sAß effects on functional resting-state networks in transgenic mature-onset amyloidosis Tet-Off APP (TG) mice. TG mice and control littermates were raised on doxycycline (DOX) diet from 3d up to 3 m of age to suppress transgenic Aß production. Thereafter, longitudinal resting-state functional MRI was performed on a 9.4 T MR-system starting from week 0 (3 m old mice) up to 28w post DOX treatment. Ex-vivo immunohistochemistry and ELISA analysis was performed to assess the development of amyloid pathology. Functional Connectivity (FC) analysis demonstrated early abnormal hypersynchronisation in the TG mice compared to the controls at 8w post DOX treatment, particularly across regions of the default mode-like network, known to be affected in AD. Ex-vivo analyses performed at this time point confirmed a 20-fold increase in total sAß levels preceding the apparition of Aß plaques and inflammatory responses in the TG mice compared to the controls. On the contrary at week 28, TG mice showed an overall hypoconnectivity, coinciding with a widespread deposition of Aß plaques in the brain. By preventing developmental influence of APP and/or sAß during brain postnatal development, we demonstrated FC abnormalities potentially driven by sAß neurotoxicity on resting-state neuronal networks in mature-induced TG mice. Thus, the Tet-Off APP mouse model could be a powerful tool while used as a mature-onset model to shed light into amyloidosis mechanisms in AD.

Clinical and immunological control of experimental autoimmune encephalomyelitis by tolerogenic dendritic cells loaded with MOG-encoding mRNA.

BACKGROUND: Although effective in reducing relapse rate and delaying progression, current therapies for multiple sclerosis (MS) do not completely halt disease progression. T cell autoimmunity to myelin antigens is considered one of the main mechanisms driving MS. It is characterized by autoreactivity to disease-initiating myelin antigen epitope(s), followed by a cascade of epitope spreading, which are both strongly patient-dependent. Targeting a variety of MS-associated antigens by myelin antigen-presenting tolerogenic dendritic cells (tolDC) is a promising treatment strategy to re-establish tolerance in MS. Electroporation with mRNA encoding myelin proteins is an innovative technique to load tolDC with the full spectrum of naturally processed myelin-derived epitopes. METHODS: In this study, we generated murine tolDC presenting myelin oligodendrocyte glycoprotein (MOG) using mRNA electroporation and we assessed the efficacy of MOG mRNA-electroporated tolDC to dampen pathogenic T cell responses in experimental autoimmune encephalomyelitis (EAE). For this, MOG35-55-immunized C57BL/6 mice were injected intravenously at days 13, 17, and 21 post-disease induction with 1α,25-dihydroxyvitamin D3-treated tolDC electroporated with MOG-encoding mRNA. Mice were scored daily for signs of paralysis. At day 25, myelin reactivity was evaluated following restimulation of splenocytes with myelin-derived epitopes. Ex vivo magnetic resonance imaging (MRI) was performed to assess spinal cord inflammatory lesion load. RESULTS: Treatment of MOG35-55-immunized C57BL/6 mice with MOG mRNA-electroporated or MOG35-55-pulsed tolDC led to a stabilization of the EAE clinical score from the first administration onwards, whereas it worsened in mice treated with non-antigen-loaded tolDC or with vehicle only. In addition, MOG35-55-specific pro-inflammatory pathogenic T cell responses and myelin antigen epitope spreading were inhibited in the peripheral immune system of tolDC-treated mice. Finally, magnetic resonance imaging analysis of hyperintense spots along the spinal cord was in line with the clinical score. CONCLUSIONS: Electroporation with mRNA is an efficient and versatile tool to generate myelin-presenting tolDC that are capable to stabilize the clinical score in EAE. These results pave the way for further research into mRNA-electroporated tolDC treatment as a patient-tailored therapy for MS.

Normalized averaged range (nAR), a robust quantification method for MPIO-content.

Micron-sized paramagnetic iron oxide particles (MPIO) are commonly used as contrast agents in magnetic resonance imaging (MRI) that produce negative contrast enhancement, i.e. darkening, on T2*-weighted images. However, estimation and quantification of MPIO in vivo is still challenging. This limitation mainly arises from smearing and displacement of the negative contrast of the MPIO, so-called blooming, potentially leading to false-positive detection. Further, the bias field induced by the MR coils also hinders visualization and quantification of the MPIO. To mitigate these drawbacks, a positive contrast image can be generated, for example by using a frequency offset technique, which can significantly improve the accuracy of quantification methods. In this research, we introduce the normalized average range (nAR) as a new way to quantify the relative MPIO content within a study. The method compares the average value of test ROIs to that of a control ROI in range filtered images. The nAR can be used on both positive and negative contrast images. The nAR was tested on agar phantoms containing various MPIO concentrations, and on a rostral migration model for MPIO labeled stem cells in mice. The amount of MPIO was quantified for biased and unbiased data, and both for positive and negative contrast images. In addition, the presence of MPIOs in the olfactory bulb was verified by histology. The results show the nAR can indicate the presence and relative content of MPIO for both negative and positive images. However, the nAR showed slightly higher sensitivity in optimized positive contrast images compared to negative contrast images. In all cases, the bias field played a minor role in the quantification, making debiasing less of a concern. The dependency of the nAR values on the MPIO content in the ROI was further validated histologically. Thus, the nAR provides a robust and reliable tool for quantification of MPIO in mice.

A smart (19) F and (1) H MRI probe with self-immolative linker as a versatile tool for detection of enzymes.

Here we report on a dual-modal (19) F and (1) H MRI paramagnetic probe with a self-immolative linker, Gd-DOMF-Gal. The enzymatic conversion of this probe by ß-galactosidase resulted in a simultaneous turning on of the fluorine signal and changed ability of the Gd(3+) complex to modulate the (1) H MR signal intensity of the surrounding water molecules. A versatile imaging platform for monitoring a variety of enzymes by (19) F and (1) H MRI using this molecular design is proposed.

Synthesis and characterization of a cell-permeable bimodal contrast agent targeting ß-galactosidase.

Noninvasive monitoring of intracellular targets such as enzymes, receptors, or mRNA by means of magnetic resonance imaging (MRI) is increasingly gaining relevance in various research areas. A vital prerequisite for their visualization is the development of cell-permeable imaging probes, which can specifically interact with the target that characterizes the cellular or molecular process of interest. Here, we describe a dual-labeled probe, Gd-DOTA-k(FR)-Gal-CPP, designed to report the presence of intracellular ß-galactosidase (ß-gal) enzyme by MRI. This conjugate consists of a galactose based core serving as cleavable spacer, incorporated between the cell-penetrating peptide D-Tat(49-57) and reporter moieties (Gd-DOTA, fluorescein (FR)). We employed a facile building block approach to obtain our bimodal probe, Gd-DOTA-k(FR)-Gal-CPP. This strategy involved the preparation of the building blocks and their subsequent assembly using Fmoc-mediated solid phase synthesis, followed by the complexation of ligand 14 with GdCl(3). Gd-DOTA-k(FR)-Gal-CPP showed a considerably higher relaxivity enhancement (16.8±0.6 mM(-1)s(-1), 123 MHz, ∼21°C) relative to the commercial Gd-DOTA (4.0±0.12 mM(-1)s(-1), 123MHz, ∼21 °C). The activation of Gd-DOTA-k(FR)-Gal-CPP was based on a cellular retention strategy that required enzymatic cleavage of the delivery vector from galactose moiety following the cell internalization to achieve a prolonged accumulation of the reporter components (Gd-DOTA/FR) in the ß-gal expressing cells. Cellular uptake of Gd-DOTA-k(FR)-Gal-CPP in ß-gal expressing C6/LacZ and enzyme deficient parental C6 rat glioma cells was confirmed by fluorescence spectroscopy, MR imaging and ICP-AES measurements. All methods showed higher accumulation of measured reporters in C6/LacZ cells compared to enzyme deficient parental C6 cells. Fluorescence microscopy of cells labeled with Gd-DOTA-k(FR)-Gal-CPP indicated a predominantly vesicular localization of the green fluorescent conjugate around cell nuclei. This cellular distribution was most likely responsible for the observed non-specific background signal in the enzyme deficient C6 cells. Even though the specific accumulation of our bimodal probe has to be further improved, it could be already used for cell imaging by MRI and optical modalities.