Reduced Levels of Brain-Derived Neurotrophic Factor Affect Body Weight, Brain Weight and Behavior.

Neurotrophins, which belong to the family of growth factors, not only play crucial roles during development but are also involved in many processes in the postnatal brain. One representative of neurotrophins is brain-derived neurotrophic factor (BDNF). BDNF plays a role in the regulation of body weight and neuronal plasticity and is, therefore, also involved in processes associated with learning and memory formation. Many of the studies on BDNF have been carried out using BDNF-deficient mice. Unfortunately, homozygous deletion of BDNF is lethal in the early postnatal stage, so heterozygous BDNF-deficient mice are often studied. Another possibility is the use of conditional BDNF-deficient mice in which the expression of BDNF is strongly downregulated in some brain cells, for example, in the neurons of the central nervous system, but the expression of BDNF in other cells in the brain is unchanged. To further reduce BDNF expression, we crossed heterozygous BDNF-deficient mice with mice carrying a deletion of BDNF in neurofilament L-positive neurons. These offspring are viable, and the animals with a strong reduction in BDNF in the brain show a strongly increased body weight, which is accompanied by a reduction in brain weight. In addition, these animals show behavioral abnormalities, particularly with regard to locomotion.

Introduction: What Are Dendritic Spines?

Dendritic spines are cellular specializations that greatly increase the connectivity of neurons and modulate the "weight" of most postsynaptic excitatory potentials. Spines are found in very diverse animal species providing neural networks with a high integrative and computational possibility and plasticity, enabling the perception of sensorial stimuli and the elaboration of a myriad of behavioral displays, including emotional processing, memory, and learning. Humans have trillions of spines in the cerebral cortex, and these spines in a continuum of shapes and sizes can integrate the features that differ our brain from other species. In this chapter, we describe (1) the discovery of these small neuronal protrusions and the search for the biological meaning of dendritic spines; (2) the heterogeneity of shapes and sizes of spines, whose structure and composition are associated with the fine-tuning of synaptic processing in each nervous area, as well as the findings that support the role of dendritic spines in increasing the wiring of neural circuits and their functions; and (3) within the intraspine microenvironment, the integration and activation of signaling biochemical pathways, the compartmentalization of molecules or their spreading outside the spine, and the biophysical properties that can affect parent dendrites. We also provide (4) examples of plasticity involving dendritic spines and neural circuits relevant to species survival and comment on (5) current research advancements and challenges in this exciting research field.

Neurotrophic Factors and Dendritic Spines.

Dendritic spines are highly dynamic structures that play important roles in neuronal plasticity. The morphologies and the numbers of dendritic spines are highly variable, and this diversity is correlated with the different morphological and physiological features of this neuronal compartment. Dendritic spines can change their morphology and number rapidly, allowing them to adapt to plastic changes. Neurotrophic factors play important roles in the brain during development. However, these factors are also necessary for a variety of processes in the postnatal brain. Neurotrophic factors, especially members of the neurotrophin family and the ephrin family, are involved in the modulation of long-lasting effects induced by neuronal plasticity by acting on dendritic spines, either directly or indirectly. Thereby, the neurotrophic factors play important roles in processes attributed, for example, to learning and memory.

Glial Cell Modulation of Dendritic Spine Structure and Synaptic Function.

Glia comprise a heterogeneous group of cells involved in the structure and function of the central and peripheral nervous system. Glial cells are found from invertebrates to humans with morphological specializations related to the neural circuits in which they are embedded. Glial cells modulate neuronal functions, brain wiring and myelination, and information processing. For example, astrocytes send processes to the synaptic cleft, actively participate in the metabolism of neurotransmitters, and release gliotransmitters, whose multiple effects depend on the targeting cells. Human astrocytes are larger and more complex than their mice and rats counterparts. Astrocytes and microglia participate in the development and plasticity of neural circuits by modulating dendritic spines. Spines enhance neuronal connectivity, integrate most postsynaptic excitatory potentials, and balance the strength of each input. Not all central synapses are engulfed by astrocytic processes. When that relationship occurs, a different pattern for thin and large spines reflects an activity-dependent remodeling of motile astrocytic processes around presynaptic and postsynaptic elements. Microglia are equally relevant for synaptic processing, and both glial cells modulate the switch of neuroendocrine secretion and behavioral display needed for reproduction. In this chapter, we provide an overview of the structure, function, and plasticity of glial cells and relate them to synaptic maturation and modulation, also involving neurotrophic factors. Together, neurons and glia coordinate synaptic transmission in both normal and abnormal conditions. Neglected over decades, this exciting research field can unravel the complexity of species-specific neural cytoarchitecture as well as the dynamic region-specific functional interactions between diverse neurons and glial subtypes.

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.

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.

Psilocybin targets a common molecular mechanism for cognitive impairment and increased craving in alcoholism.

Alcohol-dependent patients commonly show impairments in executive functions that facilitate craving and can lead to relapse. However, the molecular mechanisms leading to executive dysfunction in alcoholism are poorly understood, and new effective pharmacological treatments are desired. Here, using a bidirectional neuromodulation approach, we demonstrate a causal link between reduced prefrontal mGluR2 function and both impaired executive control and alcohol craving. A neuron-specific prefrontal mGluR2 knockdown in rats generated a phenotype of reduced cognitive flexibility and excessive alcohol seeking. Conversely, virally restoring prefrontal mGluR2 levels in alcohol-dependent rats rescued these pathological behaviors. In the search for a pharmacological intervention with high translational potential, psilocybin was capable of restoring mGluR2 expression and reducing relapse behavior. Last, we propose a FDG-PET biomarker strategy to identify mGluR2 treatment-responsive individuals. In conclusion, we identified a common molecular pathological mechanism for both executive dysfunction and alcohol craving and provided a personalized mGluR2 mechanism-based intervention strategy for medication development for alcoholism.

The angiotensin converting enzyme 2 (ACE2) system in the brain: possible involvement in Neuro-Covid.

The brain has its own intrinsic renin-angiotensin system (RAS) with all its components present in the central nervous system (CNS). Recent data demonstrate that also the main components of the angiotensin concerting enzyme 2 (ACE2) system (at least ACE2 itself, as well as the biologically active angiotensin (1-7) and its cognate receptor Mas) are expressed in the brain. Aside from these members, alamadine and MrgD are discussed as further members that have neuro-active roles in the CNS. Little is known about the possible functions of MrgD within the brain. Concerning angiotensin (1-7) acting through the Mas receptor, data were accumulating that this system is involved in numerous processes contributing to neuronal plasticity and even learning and memory. Malfunctions in the brain ACE2 system are associated with disturbances in neuronal plasticity. Since SARS-CoV-2 has a high affinity towards ACE2, Neuro-Covid may directly or indirectly depend on a disturbed balance in the ACE2 derived angiotensin system in the brain. Since the ACE2 system in the brain is far from being understood, a deeper understanding of e.g. the angiotensin (1-7) / Mas system is needed, especially with regard to the roles of angiotensin (1-7) in neuronal plasticity.

Robustly High Hippocampal BDNF levels under Acute Stress in Mice Lacking the Full-length p75 Neurotrophin Receptor.

BACKGROUND: Brain-derived neurotrophic factor (BDNF) exerts its effects on neural plasticity via 2 distinct receptor types, the tyrosine kinase TrkB and the p75 neurotrophin receptor (p75NTR). The latter can promote inflammation and cell death while TrkB is critically involved in plasticity and memory, particularly in the hippocampus. Acute and chronic stress have been associated with suppression of hippocampal BDNF expression and impaired hippocampal plasticity. We hypothesized that p75NTR might be involved in the hippocampal stress response, in particular in stress-induced BDNF suppression, which might be accompanied by increased neuroinflammation. METHOD: We assessed hippocampal BDNF protein concentrations in wild-type mice compared that in mice lacking the long form of the p75NTR (p75NTRExIII-/-) with or without prior exposure to a 1-hour restraint stress challenge. Hippocampal BDNF concentrations were measured using an optimized ELISA. Furthermore, whole-brain mRNA expression of pro-inflammatory interleukin-6 (Il6) was assessed with RT-PCR. RESULTS: Deletion of full-length p75NTR was associated with higher hippocampal BDNF protein concentration in the stress condition, suggesting persistently high hippocampal BDNF levels in p75NTR-deficient mice, even under stress. Stress elicited increased whole-brain Il6 mRNA expression irrespective of genotype; however, p75NTRExIII-/- mice showed elevated baseline Il6 expression and thus a lower relative increase. CONCLUSIONS: Our results provide evidence for a role of p75NTR signaling in the regulation of hippocampal BDNF levels, particularly under stress. Furthermore, p75NTR signaling modulates baseline but not stress-related Il6 gene expression in mice. Our findings implicate p75NTR signaling as a potential pathomechanism in BDNF-dependent modulation of risk for neuropsychiatric disorders.

Structural and functional consequences in the amygdala of leptin-deficient mice.

On the one hand, the emotional state can influence food intake and on the other hand, hunger can have an impact on the emotional state. Leptin, which is encoded by the ob gene, is involved in the energy homeostasis and plays a role in development of obesity. Mice deficient for leptin (ob/ob) are obese and display several behavioral alterations. It has been shown that ob/ob mice display striking changes in neuronal plasticity within the limbic system, e.g., hippocampal formation. We focus on alterations in ob/ob mice that can be related to alter processing in another part of the limbic system, the amygdala. ob/ob mice have a higher food consumption than age-matched controls, which might have an impact on the emotional state of these mice. Since the amygdala is involved in emotional processing, we analyze whether ob/ob mice display alterations in plasticity at the electrophysiological and structural level. No changes were seen in dendritic spine densities in the basolateral and lateral (LA) nucleus of the amygdala. Interestingly and in contrast to the hippocampus (Porter et al. 2013), long-term potentiation in the LA was increased in ob/ob mice. Our results indicate that amygdalar and hippocampal synaptic plasticity are regulated in different ways by leptin deficiency in accordance with the different functions of these limbic structures in stress and anxiety.

Identification of Brain-Specific Treatment Effects in NPC1 Disease by Focusing on Cellular and Molecular Changes of Sphingosine-1-Phosphate Metabolism.

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-ß-cyclodextrin (HPßCD) 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 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.

Pianp deficiency links GABAB receptor signaling and hippocampal and cerebellar neuronal cell composition to autism-like behavior.

Pianp (also known as Leda-1) is a type I transmembrane protein with preferential expression in the mammalian CNS. Its processing is characterized by proteolytic cleavage by a range of proteases including Adam10, Adam17, MMPs, and the γ-secretase complex. Pianp can interact with Pilrα and the GB1a subunit of the GABAB receptor (GBR) complex. A recent case description of a boy with global developmental delay and homozygous nonsense variant in PIANP supports the hypothesis that PIANP is involved in the control of behavioral traits in mammals. To investigate the physiological functions of Pianp, constitutive, global knockout mice were generated and comprehensively analyzed. Broad assessment did not indicate malformation or malfunction of internal organs. In the brain, however, decreased sizes and altered cellular compositions of the dentate gyrus as well as the cerebellum, including a lower number of cerebellar Purkinje cells, were identified. Functionally, loss of Pianp led to impaired presynaptic GBR-mediated inhibition of glutamate release and altered gene expression in the cortex, hippocampus, amygdala, and hypothalamus including downregulation of Erdr1, a gene linked to autism-like behavior. Behavioral phenotyping revealed that Pianp deficiency leads to context-dependent enhanced anxiety and spatial learning deficits, an altered stress response, severely impaired social interaction, and enhanced repetitive behavior, which all represent characteristic features of an autism spectrum disorder-like phenotype. Altogether, Pianp represents a novel candidate gene involved in autism-like behavior, cerebellar and hippocampal pathology, and GBR signaling.

Obesity Impairs Mobility and Adult Hippocampal Neurogenesis.

Currently, it is controversially discussed whether a relationship between obesity and cognition exists. We here analyzed a mouse model of obesity (leptin-deficient mice) to study the effects of obesity on the morphology of the hippocampus (a brain structure involved in mechanisms related to learning and memory) and on behavior. Mice aged 4 to 6 months were analyzed. At this age, the obese mice have nearly double the body weight as controls, but display smaller brains (brain volume is about 10% smaller) as control animals of the same age. Adult hippocampal neurogenesis, a process that is linked to learning and memory, might be disturbed in the obese mice and contribute to the smaller brain volume. Adult hippocampal neurogenesis was examined using specific markers for cell proliferation (phosphohistone H3), neuronal differentiation (doublecortin), and apoptosis (caspase 3). The number of phosphohistone H3 and doublecortin-positive cells was markedly reduced in leptin-deficient mice, but not the number of apoptotic cells, indicating that adult hippocampal neurogenesis on the level of cell proliferation was affected. In addition, dendritic spine densities of pyramidal neurons in the hippocampal area CA1 were analyzed using Golgi impregnation. However, no significant change in dendritic spine densities was noted in the obese mice. Moreover, the performance of the mice was analyzed in the open field as well as in the Morris water maze. In the open field test, obese mice showed reduced locomotor activity, but in the Morris water maze they showed similar performance compared with control animals.

Expression of Slc35f1 in the murine brain.

The solute carrier (SLC) group of membrane transport proteins includes about 400 members organized into more than 50 families. The SLC family that comprises nucleoside-sugar transporters is referred to as SLC35. One of the members of this family is SLC35F1. The function of SLC35F1 is still unknown; however, recent studies demonstrated that SLC35F1 mRNA is highly expressed in the brain and in the kidney. Therefore, we examine the distribution of Slc35f1 protein in the murine forebrain using immunohistochemistry. We could demonstrate that Slc35f1 is highly expressed in the adult mouse brain in a variety of different brain structures, including the cortex, hippocampus, amygdala, thalamus, basal ganglia, and hypothalamus. To examine the possible roles of Slc35f1 and its subcellular localization, we used an in vitro glioblastoma cell line expressing Slc35f1. Co-labeling experiments were performed to reveal the subcellular localization of Slc35f1. Our results indicate that Slc35f1 neither co-localizes with markers for the Golgi apparatus nor with markers for the endoplasmic reticulum. Time-lapse microscopy of living cells revealed that Slc35f1-positive structures are highly dynamic and resemble vesicles. Using super-resolution microscopy, these Slc35f1-positive spots clearly co-localize with the recycling endosome marker Rab11.

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.