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Simple Summary
Neuronal plasticity refers to the brain’s ability to adapt in response to activity-dependent changes. This process, among others, allows the brain to acquire memory or to compensate for a neurocognitive deficit. We analyzed adult FTSJ1-deficient mice in order to gain insight into the role of FTSJ1 in neuronal plasticity. These mice displayed alterations in the hippocampus (a brain structure that is involved in memory and learning, among other functions) e.g., in the form of changes in dendritic spines. Changes in dendritic spines are considered to represent a morphological hallmark of altered neuronal plasticity, and thus FTSJ1 deficiency might have a direct effect upon the capacity of the brain to adapt to plastic changes. Long-term potentiation (LTP) is an electrophysiological correlate of neuronal plasticity, and is related to learning and to processes attributed to memory. Here we show that LTP in FTSJ1-deficient mice is reduced, hinting at disturbed neuronal plasticity. These findings suggest that FTSJ1 deficiency has an impact on neuronal plasticity not only morphologically but also on the physiological level.
Abstract
The role of the tRNA methyltransferase FTSJ1 in the brain is largely unknown. We analyzed whether FTSJ1-deficient mice (KO) displayed altered neuronal plasticity. We explored open field behavior (10 KO mice (aged 22–25 weeks)) and 11 age-matched control littermates (WT) and examined mean layer thickness (7 KO; 6 WT) and dendritic spines (5 KO; 5 WT) in the hippocampal area CA1 and the dentate gyrus. Furthermore, long-term potentiation (LTP) within area CA1 was investigated (5 KO; 5 WT), and mass spectrometry (MS) using CA1 tissue (2 each) was performed. Compared to controls, KO mice showed a significant reduction in the mean thickness of apical CA1 layers. Dendritic spine densities were also altered in KO mice. Stable LTP could be induced in the CA1 area of KO mice and remained stable at for at least 1 h, although at a lower level as compared to WTs, while MS data indicated differential abundance of several proteins, which play a role in neuronal plasticity. FTSJ1 has an impact on neuronal plasticity in the murine hippocampal area CA1 at the morphological and physiological levels, which, in conjunction with comparable changes in other cortical areas, might accumulate in disturbed learning and memory functions.
Until today, more than 17% of the population in Mecklenburg Western-Pomerania suffer from chronic kidney disease (CKD) which was revealed by the SHIP study (Study of Health in Pomerania). 20% of CKD cases can be traced back to glomerulopathies. One common characteristic of glomerulopathies is the morphologic change of the glomerular filtration barrier which consists of endothelial cells, the glomerular basement membrane and podocytes. Under healthy conditions, the foot processes of the podocytes interdigitate with the foot processes of the neighboring podocytes with a filtration slit in between. Apart from the slit membrane protein nephrin, typical adherens junction proteins like occludin or JAM-A are also expressed at this cell-cell junction. This junction is therefore considered to be a specialized type of adherens junction, necessary to maintain the size-selectivity of the filtration barrier. During podocyte injury, podocyte foot processes lose their characteristic morphology and the typical meandering filtration slit becomes linearized, a process which is described as foot process effacement.
Since morphological change is directly linked to change or loss of function, ultrastructural analysis of the foot processes is necessary for diagnostics and research. By using 3D-structured illumination microscopy (3D-SIM), we quantified these morphological changes as well as studied a possible biomarker, the tight junction protein claudin 5 (CLDN5). Our study showed a spatially restricted up-regulation of CLDN5 in effaced filtration slit areas in biopsies of patients suffering from minimal change disease (MCD), focal and segmental glomerulosclerosis (FSGS) as well as in mice after NTS injection and in the uninephrectomy DOCA-salt mouse model. CLDN5/nephrin ratios of biopsies from patients with glomerulopathies and of tissue received from NTS-treated mice were significantly higher compared to controls. We found that in patients the CLDN5/nephrin ratios were negatively correlated with the filtration slit density. Since CLDN5 up-regulation was observed in several areas of high filtration slit density, we hypothesized that CDLN5 upregulation preceded visible foot process effacement. Taken together, we suggest that CLDN5 could be a helpful biomarker to identify an early change of the foot process morphology in addition to filtration slit density measurement. Additionally, correlation analysis of foot process effacement with patient data showed a significant negative correlation of the filtration slit density with proteinuria in MCD patients.
Increasing the information depth of single kidney biopsies can improve diagnostic precision, personalized medicine and accelerate basic kidney research. Until now, information on mRNA abundance and morphologic analysis has been obtained from different samples, missing out on the spatial context and single-cell correlation of findings. Herein, we present scoMorphoFISH, a modular toolbox to obtain spatial single-cell single-mRNA expression data from routinely generated kidney biopsies. Deep learning was used to virtually dissect tissue sections in tissue compartments and cell types to which single-cell expression data were assigned. Furthermore, we show correlative and spatial single-cell expression quantification with super-resolved podocyte foot process morphometry. In contrast to bulk analysis methods, this approach will help to identify local transcription changes even in less frequent kidney cell types on a spatial single-cell level with single-mRNA resolution. Using this method, we demonstrate that ACE2 can be locally upregulated in podocytes upon injury. In a patient suffering from COVID-19-associated collapsing FSGS, ACE2 expression levels were correlated with intracellular SARS-CoV-2 abundance. As this method performs well with standard formalin-fixed paraffin-embedded samples and we provide pretrained deep learning networks embedded in a comprehensive image analysis workflow, this method can be applied immediately in a variety of settings.
Together with endothelial cells and the glomerular basement membrane, podocytes form the size-specific filtration barrier of the glomerulus with their interdigitating foot processes. Since glomerulopathies are associated with so-called foot process effacement—a severe change of well-formed foot processes into flat and broadened processes—visualization of the three-dimensional podocyte morphology is a crucial part for diagnosis of nephrotic diseases. However, interdigitating podocyte foot processes are too narrow to be resolved by classic light microscopy due to Ernst Abbe's law making electron microscopy necessary. Although three dimensional electron microscopy approaches like serial block face and focused ion beam scanning electron microscopy and electron tomography allow volumetric reconstruction of podocytes, these techniques are very time-consuming and too specialized for routine use or screening purposes. During the last few years, different super-resolution microscopic techniques were developed to overcome the optical resolution limit enabling new insights into podocyte morphology. Super-resolution microscopy approaches like three dimensional structured illumination microscopy (3D-SIM), stimulated emission depletion microscopy (STED) and localization microscopy [stochastic optical reconstruction microscopy (STORM), photoactivated localization microscopy (PALM)] reach resolutions down to 80–20 nm and can be used to image and further quantify podocyte foot process morphology. Furthermore, in vivo imaging of podocytes is essential to study the behavior of these cells in situ. Therefore, multiphoton laser microscopy was a breakthrough for in vivo studies of podocytes in transgenic animal models like rodents and zebrafish larvae because it allows imaging structures up to several hundred micrometer in depth within the tissue. Additionally, along with multiphoton microscopy, lightsheet microscopy is currently used to visualize larger tissue volumes and therefore image complete glomeruli in their native tissue context. Alongside plain visualization of cellular structures, atomic force microscopy has been used to study the change of mechanical properties of podocytes in diseased states which has been shown to be a culprit in podocyte maintenance. This review discusses recent advances in the field of microscopic imaging and demonstrates their currently used and other possible applications for podocyte research.
The global prevalence of kidney diseases has been steadily rising over the last decades. Today, around 10% of the world population suffers from relevant chronic kidney disease. Podocytes are highly specialized and terminally differentiated cells residing in the filtering units of the kidneys, the so-called glomeruli. With their interdigitating foot-processes, these cells are a crucial part of the renal filtration barrier. As podocytes are post-mitotic, injury or loss of these cells results in an impairment of the filtration barrier with subsequent loss of global kidney function. Therefore, the question whether a relevant amount of podocytes can be regenerated and if this regeneration can be influenced is crucial for future therapeutic developments. As in vivo microscopic imaging of podocytes in higher animals like mice or rats is rather challenging, larval zebrafish have been applied as an animal model for podocyte development and kidney filtration. 48 hours post fertilization, zebrafish larvae develop a single filtering glomerulus with a similar morphology and molecular construction to that in mammals. For evaluation of podocyte morphology and filtration, we used transgenic zebrafish strains in which podocytes were labeled with fluorescence proteins. Additionally, podocytes expressed the bacterial enzyme nitroreductase fused to the fluorescence protein mCherry. In this model, application of the antibiotic metronidazole leads to podocyte-specific cell death. Through cross-breeding we established strains that additionally express an eGFP-labeled protein in the blood plasma. Using in vivo two-photon microscopy, we could image podocyte-loss induced impairments of the glomerular filtration barrier. Additionally, we tracked characteristic morphological changes of podocyte morphology including podocyte foot process effacement, development of sub-podocyte pseudocysts and finally detachment of whole cells from the glomerular basement membrane. These changes have been before described histologically in different animal models as well as in patient biopsies. Using the in vivo microscopy approach, we could clearly describe the temporal sequence of these alterations. Finally, we also tracked individual, non-detached podocytes over up to 24 hours, and found that these cells were non-migratory. These results show that early podocyte-regeneration through immigration of intra- or extraglomerular cells is unlikely within the first 24 hours of acute glomerular injury.
The dentate gyrus (DG) of the hippocampus is one of the stem cell housing niches in the adult mammalian brain. Canonical Wingless-type (Wnt) signals provided by the microenvironment are one of the major niche factors that regulate the differentiation of adult neural stem cells (aNSCs) towards the neuronal lineage. Wnts are part of a complex and diverse set of signaling pathways with a wide range of possible interactions. It remains unknown whether different canonical and non-canonical Wnt signals act in a stage-specific manner to regulate distinctive steps of adult hippocampal neurogenesis. Using in vitro assays on adult hippocampal NSCs, we identified an attenuation of canonical Wnt/ß-Catenin signaling responsiveness in the course of neuronal differentiation, while non-canonical Wnt/Planar Cell Polarity (PCP) signaling events progressively increased. Single-cell genetic manipulations were performed by using retroviral vectors to target dividing progenitor cells in the murine hippocampus. Retrovirus-mediated knockdown of ATP6AP2, a recently discovered core protein involved in both Wnt signaling pathways, revealed that the dual role of this adaptor protein is dependent on the signaling context that is present. We were able to confirm its dual role in neurogenic Wnt signaling in cultured adult hippocampal progenitors (AHPs) for both canonical Wnt signaling in proliferating AHPs and non-canonical Wnt signaling in differentiating AHPs. Specific knockdown of ATP6AP2 in neural progenitor cells in vivo resulted in a decreased induction of neuronal cell fate and severe morphological defects of newborn neurons, likely via altering both canonical and non-canonical Wnt signaling. Furthermore, in vivo knockdown of PCP core proteins CELSR1-3 and FZD3 mimicked the maturational defects of ATP6AP2-deficient neuroblasts but did not affect granule cell fate. In summary, the data presented here characterize a transition of Wnt signaling responsiveness from Wnt/ß-Catenin signaling to non-canonical Wnt/PCP signaling in the course of granule cell fate that was confirmed in a human pluripotent stem cell (hPSC)-based model of dentate granule neurogenesis. Our findings suggest that these pathways show stage-dependent activities and regulate distinct steps of adult dentate granule cell neurogenesis. Conclusively, we provide evidence for a stage-specific regulation of fate determination through the Wnt/ß-Catenin pathway and granule cell morphogenesis through the Wnt/PCP signaling pathway, including the FZD3-CELSR1-3 system. Additionally, the Wnt adaptor protein ATP6AP2 is involved in earlier and later stages of adult neurogenesis and its knockdown in vivo resembles all phenotypic features of both canonical and Wnt/PCP signaling mutants.
Podocytes are highly specialized kidney cells that are attached to the outer aspect of the glomerular capillaries and are damaged in more than 75% of patients with an impaired renal function. This specific cell type is characterized by a complex 3D morphology which is essential for proper filtration of the blood. Any changes of this unique morphology are directly associated with a deterioration of the size-selectivity of the filtration barrier. Since podocytes are postmitotic, there is no regenerative potential and the loss of these cells is permanent. Therefore, identification of small molecules that are able to protect podocytes is highly important. The aim of this work was to establish an in vivo high-content drug screening in zebrafish larvae. At first, we looked for a reliable podocyte injury model which is fast, reproducible and easy to induce. Since adriamycin is commonly used in rodents to damage podocytes, we administered it to the larvae and analyzed the phenotype by in vivo microscopy, (immuno-) histology and RT-(q)PCR. However, adriamycin did not result in a podocyte-specific injury in zebrafish larvae. Subsequently, we decided to use a genetic ablation model which specifically damages podocytes in zebrafish larvae. Treatment of transgenic zebrafish larvae with 80 µM metronidazole for 48 hours generated an injury resembling focal and segmental glomerulosclerosis which is characterized by podocyte foot process effacement, cell depletion and proteinuria. Following this, we established an in vivo high-content screening system by the use of a specific screening zebrafish strain. This screening strain expresses a circulating 78 kDa eGFP-labeled Vitamin D-binding fusion protein, which passes the filtration barrier only after glomerular injury. Therefore, we had an excellent readout to follow podocyte injury in vivo. We generated a custom image analysis software that measures the fluorescence intensity of podocytes and the vasculature automatically on a large scale. Furthermore, we screened a specific drug library consisting of 138 compounds for protective effects on larval podocytes using this in vivo high-content system. The analysis identified several initial hits and the subsequent validation experiments identified belinostat as a reliable and significant protective agent for podocytes. These results led to a patent request and belinostat is a promising candidate for a clinical use and will be tested in mammalian podocyte injury models.
Study of the effect of the podocyte-specific palladin knockout in mice with a 129 genetic background
(2023)
Worldwide, chronic kidney disease is one of the leading public health problems. Podocytes, highly specialized postmitotic cells in the filtration unit of the kidney glomerulus, are essential for the size selectivity of the filtration barrier. Loss of the complex 3D morphology of their interdigitating foot processes, effacement and detachment of the cells from the capillaries lead to proteinuria and often loss of kidney function.
Since the morphology of podocyte foot processes is highly dependent on an intact actin cytoskeleton and actin-binding proteins, we investigated the role of the actin-binding protein palladin in podocytes from mice with a 129 genetic background, that is more susceptible to kidney injury. PodoPalld129-/- mice were examined at 6 and 12 months of age using immunofluorescence staining, electron and 3D super-resolution microscopy as well as qRT-PCR.
Our analysis of PodoPalld129-/- mice at 6 and 12 months of age showed that podocyte- specific knockout of palladin results in dilation of the capillary tuft accompanied by loss of mesangial cells, indicating the influence of palladin on glomerular tuft formation. Besides, we observed morphological abnormalities such as an enlarged sub-podocyte space, cyst formations and an increased number of cell-cell contacts between podocytes and parietal epithelial cells in PodoPalld129-/- mice compared to controls. Moreover, palladin knockout resulted in downregulation of the slit diaphragm protein nephrin as well as an age-dependent significant increase in podocyte foot process effacement. Although there was a significant change in foot process morphology, we did not detect albuminuria in PodoPalld129-/- mice of both age groups. However, we found an increase of trefoil factor 1 (Tff1) in the urine of the mice, indicating an altered, more permeable filtration barrier.
Considering that palladin has several binding sites for important actin-binding and regulatory proteins, we studied the expression of Lasp-1, Pdlim2, VASP and Klotho in dependence on palladin. We found a remarkable reduction in, for example, phosphorylated Lasp-1 as well as Klotho, which could influence the morphology of podocyte foot processes.
Compared with PodoPalldBL/6-/- mice, PodoPalld129-/- mice showed stronger glomerular tuft dilation and developed podocytes with increased morphological abnormalities, underlining the importance of the genetic background.
In conclusion, these results demonstrate the essential role of palladin for podocyte morphology in mice with a 129 genetic background.
Chronic kidney disease (CKD) is a major public health burden affecting more than 500 million people worldwide. Podocytopathies are the main cause for the majority of CKD cases due to pathogenic morphological as well as molecular biological alterations of postmitotic podocytes. Podocyte de-differentiation is associated with foot process effacement subsequently leading to proteinuria. Since currently no curative drugs are available, high throughput screening methods using a small number of animals are a promising and essential tool to identify potential drugs against CKD in the near future. Our study presents the implementation of the already established mouse GlomAssay as a semi-automated high-throughput screening method—shGlomAssay—allowing the analysis of several hundreds of FDA-verified compounds in combination with downstream pathway analysis like transcriptomic and proteomic analyses from the same samples, using a small number of animals. In an initial prescreening we have identified vitamin D3 and its analog calcipotriol to be protective on podocytes. Furthermore, by using RT-qPCR, Western blot, and RNA sequencing, we found that mRNA and protein expression of nephrin, the vitamin D receptor and specific podocyte markers were significantly up-regulated due to vitamin D3- and calcipotriol-treatment. In contrast, kidney injury markers were significantly down-regulated. Additionally, we found that vitamin D3 and calcipotriol have had neither influence on the expression of the miR-21 and miR-30a nor on miR-125a/b, a miRNA described to regulate the vitamin D receptor. In summary, we advanced the established mouse GlomAssay to a semi-automated high-throughput assay and combined it with downstream analysis techniques by using only a minimum number of animals. Hereby, we identified the vitamin D signaling pathway as podocyte protective and to be counteracting their de-differentiation.
Chronic kidney disease is a major public health burden associated with a drastically reduced quality of living and life span that lacks suitable, individualized therapeutic strategies. Here we present a human induced pluripotent stem cell line (iPSC, UMGACBi001-A) reprogrammed from urine cells of an acute septic dialysis patient suffering from chronic kidney disease using non-integrating administration of RNAs. The generated iPSCs were positively characterized for typical morphology, pluripotency marker expression, directed differentiation potential, non-contamination, chromosomal consistency and donor identity. This iPSC-line can be a useful source for in vitro disease modelling and individualized therapeutic approaches.
Morphological changes of the complex 3-D architecture of podocytes as well as the loss of these post-mitotic cells often result in severe kidney disease. Since currently, there are no curative drugs, we focused on the identification of non-invasive biomarkers, allowing an early detection of the onset of such diseases. Therefore, we analyzed the cellular- and the cell-free fractions of urine samples from patients suffering from chronic kidney disease (CKD), especially for injury markers as well as for exosome-derived miRNAs.
We identified the mRNA of the neuronal protein brain-derived neurotrophic factor (BDNF) in the cellular fraction of 120 CKD patients and found that the expression was highly correlated with the mRNA expression of the kidney injury marker molecule 1 (KIM-1). Furthermore, we found that both were correlated with the mRNA expression of the podocyte-specific gene Nephrin (NPHS1), suggesting that podocytes are very likely the cellular source.
Beside this, we observed that BDNF is upregulated in biopsies of diabetic patients and seems to be involved in the differentiation of podocytes. Immunofluorescence staining clearly showed that BDNF is localized in the cell body and major processes of podocytes within the glomerulus. Knockdown experiments in zebrafish larvae, a well-established animal model to study kidney function, showed the importance of BDNF on kidney function, morphology and filtration in vivo.
Additionally, we analyzed circulating exosomal microRNAs (miRs) isolated from the cell-free urine fraction. After the optimization of a column-based isolation protocol for exosomes, we identified miR-16 from a pre-selected set of candidates as a suitable endogenous reference gene for data normalization. Subsequently, we analyzed the exosomal levels of miR-21, miR-30a-5p and miR-92a in urine samples of 41 CKD patients and 5 healthy controls. We found significantly enhanced levels of miR-21 in CKD patients that were also negatively correlated with the eGFR, suggesting a negative influence on kidney function. MiR-21 was also highly upregulated in de-differentiated glomeruli and in kidneys of nephrotoxic serum- (NTS-) treated mice as an in vivo kidney injury model.
To summarize, we identified two promising new and non-invasive biomarkers for CKD in the urine of patients which may also have a functional relevance on kidney function.
Chronic alcohol abuse is one of the most common addictions and one of the most substantial public health problems as it affects millions of people physically as well as mentally around the world. Globally more than 3 million deaths are assignable to alcohol intake each year. Chronic alcoholism is a multi-component disease and its development is associated with both environmental as well as genetic factors. However, the key mechanisms underlying an addiction, especially on a cellular and physiological basis, are still unknown. Bio-medically an influence of chronic alcohol consumption on synaptic plasticity in the brain of humans as well as rodents has been proven.
On the dendritic shaft of nervous brain cells, small membrane protrusions called dendritic spines can be found. These spines possess the capacity to change their morphology and quantity and are thought to play an important role in learning and memory forming, and seem to be impaired in multiple neurological disorders. These dynamics are called synaptic plasticity. Most of these studies however, were carried out on the cortex. These previous observations raise the question whether such alterations in synaptic plasticity can also be observed in regions of the brain that contribute to the limbic system and therefore to the processing of emotional responses, learning and decision making. The amygdala is of special interest when trying to understand the neurobiology and pathophysiology that lead to the emergence and up keeping of an alcohol addiction. In this thesis a closer look has been taken at possible alterations in synaptic plasticity within different amygdaloid nuclei by the help of a rat model. These rats were put into the so called postdependent state, one of the most common animal models to investigate excessive ethanol intake in rodents. The postdependent state is a model in which the key driving force to obtain alcohol as part of a preserved addiction cycle is based on negative affect. Studies showed differences in the behavioural outcome of those animals that were exposed to chronic intermittent alcohol consumption compared to a control group, so it was of special interest to see whether those behavioural changes also show on a cellular basis.
In the study, a morphological comparison of the spine length as well as the spine density of alcohol dependent rats with a comparable control group has been made. The medial, the central, the lateral and the basolateral amygdaloid nucleus were of special interest in this research project.
The results showed no significant difference of the spine densities in any of the four amygdaloid regions. When comparing the spine morphology within the ethanol and the control group, differences showed in the lateral amygdaloid nucleus. In this region the spines of the ethanol group were significantly smaller. This leads to the conclusion that chronic alcohol intake can have an influence on the spine morphology and hence alter anatomical brain structures.
80% of chronic kidney diseases are caused by the loss and the damage of a differentiated and postmitotic cell type, the podocytes. The size-selectivity of the blood filtration barrier is highly dependent on the complex interdigitation of the podocyte foot processes as well as of the slit membrane which is spanned in between. Changes of this specific morphology as well as a detachment of podocytes lead to the clinical hallmark of a nephrotic syndrome e.g. proteinuria and oedema formation.
Since specific drugs or therapies are usually not available, patients are often dependent on dialysis and transplantation. Therefore, intensive studies are necessary to understand the pathogenesis of glomerulopathies as well as to identify specific drugs. In the past, it was already demonstrated that the zebrafish is an ideal model to study kidney function and to screen for drugs, since the larvae quickly develop a simple glomerulus that is comparable to the glomeruli of mice, rat and human.
In the present work, a zebrafish model was established to study a specific glomerulopathy named focal segmental glomerulosclerosis (FSGS). FSGS is mainly characterized by histology of the glomeruli which shows segmental scar formation and matrix deposition due to an activation of parietal epithelial cells (PEC) lining the Bowman’s capsule. For this purpose, we used the nitroreductase/metronidazole (NTR/MTZ) system, in which a cytotoxic agent is exclusively generated in podocytes by the enzyme NTR resulting in apoptosis of cells. Firstly, the parameters for development of an FSGS-like disease were evaluated and the glomerular response to podocyte depletion was examined during three days after the induction of podocyte damage. Using classic histological techniques, immunofluorescence staining and transmission electron microscopy, it was possible to demonstrate that zebrafish larvae phenocopy human FSGS in important characteristics after partial podocyte depletion. Secondly, by intravascular injection of fluorescence-labeled high molecular weight dextran, we found that the filtration barrier became leaky. Moreover, we identified a severe podocyte foot process effacement, formation of subpodocyte space pseudocysts and loss of the slit membrane protein podocin. Morphometrical, histological and ultrastructural analysis revealed an enlargement of the glomerulus, proliferation of cuboidal PECs and intraglomerular deposition of extracellular matrix components, all typical hallmarks of FSGS. Further, we observed adhesions between the parietal and the visceral glomerular cell layer forming sclerotic lesions. However, it remains still unclear whether an inflammatory response is involved in the development of sclerotic lesions. Our microscopic analysis provided some evidence for immigration of immunocompetent cells like neutrophils, presumably due to induction of apoptosis in our model.
Taken together, in the present work a zebrafish model was established with characteristics of mammals FSGS which will be useful for pathomechanism studies as well as for drug screening.
Niemann–Pick type C1 (NPC1) is a lysosomal storage disorder, inherited as an
autosomal-recessive trait. Mutations in the Npc1 gene result in malfunction of the NPC1 protein,
leading to an accumulation of unesterified cholesterol and glycosphingolipids. Beside visceral
symptoms like hepatosplenomegaly, severe neurological symptoms such as ataxia occur. Here,
we analyzed the sphingosine-1-phosphate (S1P)/S1P receptor (S1PR) axis in different brain regions
of Npc1−/− mice and evaluated specific effects of treatment with 2-hydroxypropyl-β-cyclodextrin
(HPβCD) together with the iminosugar miglustat. Using high-performance thin-layer chromatography
(HPTLC), mass spectrometry, quantitative real-time PCR (qRT-PCR) and western blot analyses, we
Int. J. Mol. Sci. 2020, 21, 4502; doi:10.3390/ijms21124502 www.mdpi.com/journal/ijms
Int. J. Mol. Sci. 2020, 21, 4502 2 of 31
studied lipid metabolism in an NPC1 mouse model and human skin fibroblasts. Lipid analyses
showed disrupted S1P metabolism in Npc1−/− mice in all brain regions, together with distinct changes
in S1pr3/S1PR3 and S1pr5/S1PR5 expression. Brains of Npc1−/− mice showed only weak treatment
effects. However, side effects of the treatment were observed in Npc1+/+ mice. The S1P/S1PR axis
seems to be involved in NPC1 pathology, showing only weak treatment effects in mouse brain. S1pr
expression appears to be affected in human fibroblasts, induced pluripotent stem cells (iPSCs)-derived
neural progenitor and neuronal differentiated cells. Nevertheless, treatment-induced side effects
make examination of further treatment strategies indispensable
SLC35F1 is a member of the sugar-like carrier (SLC) superfamily that is expressed in the mammalian brain. Malfunction of SLC35F1 in humans is associated with neurodevelopmental disorders. To get insight into the possible roles of Slc35f1 in the brain, we generated Slc35f1-deficient mice. The Slc35f1-deficient mice are viable and survive into adulthood, which allowed examining adult Slc35f1-deficient mice on the anatomical as well as behavioral level. In humans, mutation in the SLC35F1 gene can induce a Rett syndrome-like phenotype accompanied by intellectual disability (Fede et al. Am J Med Genet A 185:2238–2240, 2021). The Slc35f1-deficient mice, however, display only a very mild phenotype and no obvious deficits in learning and memory as, e.g., monitored with the novel object recognition test or the Morris water maze test. Moreover, neuroanatomical parameters of neuronal plasticity (as dendritic spines and adult hippocampal neurogenesis) are also unaltered. Thus, Slc35f1-deficient mice display no major alterations that resemble a neurodevelopmental phenotype.