Global Protein Profiling in Processed Immunohistochemistry Tissue Sections.

Tissue sections, which are widely used in research and diagnostic laboratories and have already been examined by immunohistochemistry (IHC), may subsequently provide a resource for proteomic studies, even though only small amount of protein is available. Therefore, we established a workflow for tandem mass spectrometry-based protein profiling of IHC specimens and characterized defined brain area sections. We investigated the CA1 region of the hippocampus dissected from brain slices of adult C57BL/6J mice. The workflow contains detailed information on sample preparation from brain slices, including removal of antibodies and cover matrices, dissection of region(s) of interest, protein extraction and digestion, mass spectrometry measurement, and data analysis. The Gene Ontology (GO) knowledge base was used for further annotation. Literature searches and Gene Ontology annotation of the detected proteins verify the applicability of this method for global protein profiling using formalin-fixed and embedded material and previously used IHC slides.

BDNF effects on dendritic spine morphology and hippocampal function.

Neurotrophins, including brain-derived neurotrophic factor (BDNF), are expressed in the hippocampus, as well as their precursors, the pro-neurotrophins. The neurotrophins signal through specific tyrosine kinase receptors and the low affinity receptor p75NTR. Moreover, the pro-neurotrophins are considered to be biologically active by signaling through specific receptors. The neurotrophins, especially BDNF, are involved in processes related to learning and memory. Furthermore, it is thought that BDNF also plays a crucial role in major depression. This points to a role of BDNF as a central regulator of neuronal plasticity within the postnatal hippocampus. Morphological correlates of neuronal plasticity are changes on the level of the dendritic spines and, at least in the dentate gyrus of the hippocampus, on the level of adult neurogenesis. Specific changes in dendritic spines as well as in adult hippocampal neurogenesis can be seen in the context of several forms of learning and memory, and it is known that depression is accompanied by declines in the rate of adult neurogenesis and in spine densities. The possible roles of BDNF in neuronal plasticity within the hippocampus are highlighted in this review by focusing on the morphological components of neuronal plasticity.

Deletion of p75NTR enhances the cholinergic innervation pattern of the visual cortex.

The cholinergic system is involved in cortical plasticity, attention, and learning. Within the visual cortex the cholinergic system seems to play a role in visual perception. The cholinergic neurons which project into the visual cortex are located in the basal forebrain. It has been shown that mice deficient for the low-affinity neurotrophin receptor p75NTR display increased numbers of cholinergic neurons in the basal forebrain and a denser cholinergic innervation of the hippocampus. This prompted us to analyze whether the cholinergic system is altered in adult p75NTR deficient mice. By analyzing the densities of cholinergic fibers within layer IV as well as within layer V of the visual cortex, we found that adult p75NTR deficient mice display increased cholinergic fiber densities. However, this increase was not accompanied by an increase in the density of local cholinergic neurons within the visual cortex. This indicates that the enhanced cholinergic innervation of the visual cortex is due to alteration of the cholinergic neurons located in the basal forebrain, projecting to the visual cortex. The increased cholinergic innervation of the visual cortex makes the p75NTR deficient mice an attractive model to study the necessity of the cholinergic system for the visual cortex.

Morphological and behavioral analysis of Slc35f1-deficient mice revealed no neurodevelopmental phenotype.

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.

Deficiency in FTSJ1 Affects Neuronal Plasticity in the Hippocampal Formation of Mice.

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.

Localization and expression of the Mas-related G-protein coupled receptor member D (MrgD) in the mouse brain.

Numerous studies in the last decades have provided evidence for the existence of a local renin-angiotensin system (RAS) in the central nervous system (CNS). Widespread distribution of the different RAS components in the brain demonstrates the pleiotropic role of this system in the structure and function of CNS. With the advent of new molecular techniques, a novel receptor has been identified within the beneficial arm of the RAS, the Mas-related G-protein coupled receptor D (MrgD), which can be stimulated by two heptapeptides, Ala1-(Ang-(1-7), also named alamandine, and Ang-(1-7). However, the biological and physiological relevance of this interaction remains obscure. Since several recent studies hinted at a role of MrgD in the CNS, we determined the distribution pattern of MrgD receptors in the adult mouse brain by using a genetic mouse model with tracers of MrgD expression. MrgD-positive cells could be identified in some forebrain areas, including cortex, hippocampus, amygdala, hypothalamus, habenular nuclei, striatum and pallidum, as well as in some mid-brain nuclei in a region-specific manner. The specific localization of MrgD in the reward- and limbic-related areas can hint at a role of MrgD in processes such as pain perception/modulation, synaptic plasticity, learning, memory and cognition.

A mouse model for intellectual disability caused by mutations in the X-linked 2'­O­methyltransferase Ftsj1 gene.

Mutations in the X chromosomal tRNA 2'­O­methyltransferase FTSJ1 cause intellectual disability (ID). Although the gene is ubiquitously expressed affected individuals present no consistent clinical features beyond ID. In order to study the pathological mechanism involved in the aetiology of FTSJ1 deficiency-related cognitive impairment, we generated and characterized an Ftsj1 deficient mouse line based on the gene trapped stem cell line RRD143. Apart from an impaired learning capacity these mice presented with several statistically significantly altered features related to behaviour, pain sensing, bone and energy metabolism, the immune and the hormone system as well as gene expression. These findings show that Ftsj1 deficiency in mammals is not phenotypically restricted to the brain but affects various organ systems. Re-examination of ID patients with FTSJ1 mutations from two previously reported families showed that several features observed in the mouse model were recapitulated in some of the patients. Though the clinical spectrum related to Ftsj1 deficiency in mouse and man is variable, we suggest that an increased pain threshold may be more common in patients with FTSJ1 deficiency. Our findings demonstrate novel roles for Ftsj1 in maintaining proper cellular and tissue functions in a mammalian organism.

A patient-specific induced pluripotent stem cell model for West syndrome caused by ST3GAL3 deficiency.

ST3GAL3 encodes the Golgi enzyme beta-galactoside-alpha-2,3-sialyltransferase-III that in humans forms, among others, the sialyl Lewis a (sLea) epitope on proteins. Functionally deleterious variants in this gene were previously identified in patients with either non-syndromic or syndromic intellectual disability such as West syndrome, an age-dependent epileptic encephalopathic syndrome associated with developmental arrest or regression. The aim of this study was to further elucidate the molecular and cellular mechanisms causing West syndrome by lack of ST3GAL3 function. For this purpose we generated induced pluripotent stem cell (iPSC) lines from fibroblasts obtained from a patient with West syndrome, carrying a variant in exon 12 (c.958G>C, p.(Ala320Pro)) of ST3GAL3, and a healthy sibling, using lentiviral reprogramming. iPSCs and cortical neurons derived thereof were analysed by lectin blots, mRNA sequencing, adherence assays, and FACS. While no significant difference was observed at stem cell or fibroblast level between patient and control cells, patient-derived cortical neurons displayed an altered lectin blot staining pattern, enhanced adherence to a poly-L-ornithine/laminin-coated surface and decreased levels of neurons expressing T-box transcription factor brain 1. Our results suggest that changes in the sialylation pattern on the surface of specific neuronal cell types affect adhesive interactions during development, which in turn may cause subtle changes in tissue composition that could result in the occurrence of epilepsy and might impair neural development to an extent that is detrimental to the development and maintenance of normal cognitive functions.

Impact of a deletion of the full-length and short isoform of p75NTR on cholinergic innervation and the population of postmitotic doublecortin positive cells in the dentate gyrus.

Analyses of mice carrying a deletion of the pan-neurotrophin receptor p75NTR have allowed identifying p75NTR as an important structural regulator of the hippocampus. Most of the previous analyses were done using p75NTR (ExIII) knockout mice which still express the short isoform of p75NTR. To scrutinize the role of p75NTR in the hippocampus, we analyzed adult and aged p75NTR (ExIV) knockout mice, in which both, the short and the full-length isoform are deleted. Deletion of these isoforms induced morphological alterations in the adult dentate gyrus (DG), leading to an increase in the thickness of the molecular and granular layer. Based on these observations, we next determined the morphological substrates that might contribute to this phenotype. The cholinergic innervation of the molecular and granular layer of the DG was found to be significantly increased in the knockout mice. Furthermore, adult neurogenesis in the DG was found to be significantly altered with increased numbers of doublecortin (DCX) positive cells and reduced numbers of apoptotic cells in p75NTR (ExIV) knockout mice. However, cell proliferation as measured by phosphohiston H3 (PH3) positive cell numbers was not affected. These morphological alterations (number of DCX-positive cells and increased cholinergic fiber densities) as well as reduced cell death in the DG are likely to contribute to the observed thickening of the granular layer in p75NTR (ExIV) knockout mice. In addition, Sholl-analysis of DCX-positive neurons revealed a higher dendritic complexity and could thus be a possible morphological correlate for the increased thickness of the molecular layer in p75NTR deficient animals. Our data clearly demonstrate that deletion of both, the short and the full-length isoform of p75NTR affects DG morphology, due to alterations of the cholinergic system and an imbalance between neurogenesis and programmed cell death within the subgranular zone.

Distribution of PCP4 protein in the forebrain of adult mice.

Purkinje-cell protein 4 (PCP4) is a small calmodulin (CaM)-binding protein that has been discovered to be selectively expressed by cerebellar Purkinje cells in the adult rodent brain. In addition, expression of PCP4 mRNA has been detected in the hippocampus and in the cortex. In order to determine the expression of PCP4 protein in the brain, we performed an immunohistochemical analysis using adult mice. We could demonstrate that PCP4 is expressed in neocortical structures, especially in the deep layers, as well as in other cortical structures and parts of the hippocampal formation. Moreover, PCP4 protein is highly expressed in the olfactory bulb and caudate putamen. PCP4 positive cells were also detected in specific areas of the amygdala, thalamus (especially dorsal lateral geniculate nucleus) and hypothalamus. By performing double-labeling experiments together with NeuN (a neuronal marker), we could demonstrate that PCP4 expressing cells in the brain are of neuronal origin.