Comparative analysis of hippocampal extracellular space uncovers widely altered peptidome upon epileptic seizure in urethane-anaesthetized rats.

BACKGROUND: The brain extracellular fluid (ECF), composed of secreted neurotransmitters, metabolites, peptides, and proteins, may reflect brain processes. Analysis of brain ECF may provide new potential markers for synaptic activity or brain damage and reveal additional information on pathological alterations. Epileptic seizure induction is an acute and harsh intervention in brain functions, and it can activate extra- and intracellular proteases, which implies an altered brain secretome. Thus, we applied a 4-aminopyridine (4-AP) epilepsy model to study the hippocampal ECF peptidome alterations upon treatment in rats. METHODS: We performed in vivo microdialysis in the hippocampus for 3-3 h of control and 4-AP treatment phase in parallel with electrophysiology measurement. Then, we analyzed the microdialysate peptidome of control and treated samples from the same subject by liquid chromatography-coupled tandem mass spectrometry. We analyzed electrophysiological and peptidomic alterations upon epileptic seizure induction by two-tailed, paired t-test. RESULTS: We detected 2540 peptides in microdialysate samples by mass spectrometry analysis; and 866 peptides-derived from 229 proteins-were found in more than half of the samples. In addition, the abundance of 322 peptides significantly altered upon epileptic seizure induction. Several proteins of significantly altered peptides are neuropeptides (Chgb) or have synapse- or brain-related functions such as the regulation of synaptic vesicle cycle (Atp6v1a, Napa), astrocyte morphology (Vim), and glutamate homeostasis (Slc3a2). CONCLUSIONS: We have detected several consequences of epileptic seizures at the peptidomic level, as altered peptide abundances of proteins that regulate epilepsy-related cellular processes. Thus, our results indicate that analyzing brain ECF by in vivo microdialysis and omics techniques is useful for monitoring brain processes, and it can be an alternative method in the discovery and analysis of CNS disease markers besides peripheral fluid analysis.

Inflammatory processes in the prefrontal cortex induced by systemic immune challenge: Focusing on neurons.

Peripheral immune challenge induces neurobiological alterations in the brain and related neuropsychiatric symptoms both in humans and other mammals. One of the best known physiological effects of systemic inflammation is sickness behavior. However, in addition to this depression-like state, there are other cognitive outcomes of peripherally induced neuroinflammation that can be linked to the dysfunction of higher-order cortical areas, such as the prefrontal cortex (PFC). As the physiological activity of the PFC is largely based on the balanced interplay of excitatory pyramidal cells and inhibitory interneurons, it may be hypothesized that neuroinflammatory processes result in a shift of excitatory/inhibitory balance, which is a common hallmark of several neuropsychiatric conditions. Indeed, many data suggest that peripherally induced neuroinflammation is strongly associated with molecular and functional changes in PFC neurons leading to disturbances in their synaptic networks. Different experimental approaches may cause some incongruence in the reviewed data. However, it is commonly agreed that acute systemic inflammation leads to changes in the excitatory/inhibitory balance in the PFC by proinflammatory signaling at the brain borders and in the brain parenchyma. These cellular changes result in altered local and brain-wide network activity inducing disturbances in the top-down control of goal-directed behavior and cognition regulated by the PFC. Lipopolysaccharide (LPS)-treated rodents are the most widely used experimental models of peripherally induced neuroinflammation, so the majority of the reviewed data come from studies utilizing the LPS model. This may limit their general interpretation regarding the neuronal effects of peripheral immune activation. In addition, several biological variables (e.g., sex, age) can influence the PFC effects of systemic immune challenge, not only the nature and severity of immune activation. Therefore, it would be desirable to investigate inflammation-related neuronal changes in the PFC using other models of systemic inflammation as well, and to focus on the targeted fine-tuning of the affected cell types via common molecular mechanisms of the immune and nervous systems.

Chronic Cerebral Hypoperfusion-Induced Disturbed Proteostasis of Mitochondria and MAM Is Reflected in the CSF of Rats by Proteomic Analysis.

Declining cerebral blood flow leads to chronic cerebral hypoperfusion which can induce neurodegenerative disorders, such as vascular dementia. The reduced energy supply of the brain impairs mitochondrial functions that could trigger further damaging cellular processes. We carried out stepwise bilateral common carotid occlusions on rats and investigated long-term mitochondrial, mitochondria-associated membrane (MAM), and cerebrospinal fluid (CSF) proteome changes. Samples were studied by gel-based and mass spectrometry-based proteomic analyses. We found 19, 35, and 12 significantly altered proteins in the mitochondria, MAM, and CSF, respectively. Most of the changed proteins were involved in protein turnover and import in all three sample types. We confirmed decreased levels of proteins involved in protein folding and amino acid catabolism, such as P4hb and Hibadh in the mitochondria by western blot. We detected reduced levels of several components of protein synthesis and degradation in the CSF as well as in the subcellular fractions, implying that hypoperfusion-induced altered protein turnover of brain tissue can be detected in the CSF by proteomic analysis.

LPS-induced acute neuroinflammation, involving interleukin-1 beta signaling, leads to proteomic, cellular, and network-level changes in the prefrontal cortex of mice.

Neuroinflammation induced by peripheral infections leads to various neuropsychiatric symptoms both in humans and laboratory animals, e.g., to the manifestation of sickness behavior that resembles some features of clinical depression. However, in addition to depression-like behavior, there are other symptoms of acute systemic inflammation that can be associated with the impairment of prefrontal cortex (PFC)-regulated cognitive functions. Thus, we investigated the electrophysiological and proteomic alterations of the PFC using brain slices and the lipopolysaccharide (LPS) model of acute peripheral infection in male mice. Based on the gene expression differences of the coreceptor (Il1rap) of interleukin-1 beta (IL-1ß) between neuron types in our previous single-cell sequencing dataset, we first compared the electrophysiological effects of IL-1ß on PFC pyramidal cells and interneurons. We found that pyramidal cells are more responsive to IL-1ß, as could be presumed from our transcriptomic data. To examine the possible circuit-level correlates of the cellular changes, frontal electroencephalographic (EEG) activity and fronto-occipital functional connectivity were analyzed in LPS-treated mice and significant changes were found in the fronto-occipital EEG correlation and coherence in the delta and high-gamma frequency bands. The upregulation of the prefrontal IL-1 system (IL-1ß and its receptor) after LPS treatment was revealed by immunoassays simultaneously with the observed EEG changes. Furthermore, we investigated the LPS-induced alterations of the synaptic proteome in the PFC using 2-D differential gel electrophoresis and mass spectrometry and found 48 altered proteins mainly related to cellular signaling, cytoskeletal organization, and carbohydrate/energy metabolism. Thus, our results indicate remarkable electrophysiological and molecular changes in the PFC related to acute systemic inflammation that may explain some of the concomitant behavioral and physiological symptoms.

Neuronal-specific septin-3 binds Atg8/LC3B, accumulates and localizes to autophagosomes during induced autophagy.

In synapses that show signs of local apoptosis and mitochondrial stress and undergo neuro-immunological synapse pruning, an increase in the levels of the presynaptic protein, neuronal-specific septin-3 can be observed. Septin-3 is a member of the septin GTPase family with the ability to form multimers and contribute to the cytoskeleton. However, the function of septin-3 remains elusive. Here, we provide evidence that septin-3 is capable of binding the most-studied autophagy protein Atg8 homolog microtubule-associated protein 1 light chain 3B (LC3B), besides another homolog, GABA receptor-associated protein-like 2 (GABARAPL2). Moreover, we demonstrate that colocalization of septin-3 and LC3B increases upon chemical autophagy induction in primary neuronal cells. Septin-3 is accumulated in primary neurons upon autophagy enhancement or blockade, similar to autophagy proteins. Using electron microscopy, we also show that septin-3 localizes to LC3B positive membranes and can be found at mitochondria. However, colocalization results of septin-3 and the early mitophagy marker PTEN-induced kinase 1 (PINK1) do not support that binding of septin-3 to mitochondria is mitophagy related. We conclude that septin-3 correlates with synaptic/neuronal autophagy, binds Atg8 and localizes to autophagic membranes that can be enhanced with chemical autophagy induction. Based on our results, elevated septin-3 levels might indicate enhanced or impeded autophagy in neurons.

Designed Peptide Inhibitors of STEP Phosphatase-GluA2 AMPA Receptor Interaction Enhance the Cognitive Performance in Rats.

Cognitive impairment and learning ability of the brain are directly linked to synaptic plasticity as measured in changes of long-term potentiation (LTP) and long-term depression (LTD) in animal models of brain diseases. LTD reflects a sustained reduction of the synaptic AMPA receptor content based on targeted clathrin-mediated endocytosis. AMPA receptor endocytosis is initiated by dephosphorylation of Tyr876 on the C-terminus of the AMPAR subunit GluA2. The brain-specific striatal-enriched protein tyrosine phosphatase (STEP) is responsible for this process. To identify new, highly effective inhibitors of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) internalization, we performed structure-based design of peptides able to inhibit STEP-GluA2-CT complex formation. Two short peptide derivatives were found as efficient in vitro inhibitors. Our in vivo experiments evidenced that both peptides restore the memory deficits and display anxiolytic and antidepressant effects in a scopolamine-treated rat model. The interference peptides identified and characterized here represent promising lead compounds for novel cognitive enhancers and/or behavioral modulators.

The Single-Cell Transcriptomic Analysis of Prefrontal Pyramidal Cells and Interneurons Reveals the Neuronal Expression of Genes Encoding Antimicrobial Peptides and Immune Proteins.

The investigation of the molecular background of direct communication of neurons and immune cells in the brain is an important issue for understanding physiological and pathological processes in the nervous system. Direct contacts between brain-infiltrating immune cells and neurons, and the neuromodulatory effect of immune cell-derived regulatory peptides are well established. Several aspects of the role of immune and glial cells in the direct neuro-immune communication are also well known; however, there remain many questions regarding the molecular details of signaling from neurons to immune cells. Thus, we report here on the neuronal expression of genes encoding antimicrobial and immunomodulatory peptides, as well as proteins of immune cell-specific activation and communication mechanisms. In the present study, we analyzed the single-cell sequencing data of our previous transcriptomic work, obtained from electrophysiologically identified pyramidal cells and interneurons of the murine prefrontal cortex. We filtered out the genes that may be associated with the direct communication between immune cells and neurons and examined their expression pattern in the neuronal transcriptome. The expression of some of these genes by cortical neurons has not yet been reported. The vast majority of antimicrobial (~53%) and immune cell protein (~94%) transcripts was identified in the transcriptome of the 84 cells, owing to the high sensitivity of ultra-deep sequencing. Several of the antimicrobial and immune process-related protein transcripts showed cell type-specific or enriched expression. Individual neurons transcribed only a fraction of the investigated genes with low copy numbers probably due to the bursting kinetics of gene expression; however, the comparison of our data with available transcriptomic datasets from immune cells and neurons suggests the functional relevance of the reported findings. Accordingly, we propose further experimental and in silico studies on the neuronal expression of immune system-related genes and the potential role of the encoded proteins in neuroimmunological processes.

Cell Surface Protein mRNAs Show Differential Transcription in Pyramidal and Fast-Spiking Cells as Revealed by Single-Cell Sequencing.

The prefrontal cortex (PFC) plays a key role in higher order cognitive functions and psychiatric disorders such as autism, schizophrenia, and depression. In the PFC, the two major classes of neurons are the glutamatergic pyramidal (Pyr) cells and the GABAergic interneurons such as fast-spiking (FS) cells. Despite extensive electrophysiological, morphological, and pharmacological studies of the PFC, the therapeutically utilized drug targets are restricted to dopaminergic, glutamatergic, and GABAergic receptors. To expand the pharmacological possibilities as well as to better understand the cellular and network effects of clinically used drugs, it is important to identify cell-type-selective, druggable cell surface proteins and to link developed drug candidates to Pyr or FS cell targets. To identify the mRNAs of such cell-specific/enriched proteins, we performed ultra-deep single-cell mRNA sequencing (19 685 transcripts in total) on electrophysiologically characterized intact PFC neurons harvested from acute brain slices of mice. Several selectively expressed transcripts were identified with some of the genes that have already been associated with cellular mechanisms of psychiatric diseases, which we can now assign to Pyr (e.g., Kcnn2, Gria3) or FS (e.g., Kcnk2, Kcnmb1) cells. The earlier classification of PFC neurons was also confirmed at mRNA level, and additional markers have been provided.

Chronic stepwise cerebral hypoperfusion differentially induces synaptic proteome changes in the frontal cortex, occipital cortex, and hippocampus in rats.

During chronic cerebral hypoperfusion (CCH), the cerebral blood flow gradually decreases, leading to cognitive impairments and neurodegenerative disorders, such as vascular dementia. The reduced oxygenation, energy supply induced metabolic changes, and insufficient neuroplasticity could be reflected in the synaptic proteome. We performed stepwise bilateral common carotid occlusions on rats and studied the synaptic proteome changes of the hippocampus, occipital and frontal cortices. Samples were prepared and separated by 2-D DIGE and significantly altered protein spots were identified by HPLC-MS/MS. We revealed an outstanding amount of protein changes in the occipital cortex compared to the frontal cortex and the hippocampus with 94, 33, and 17 proteins, respectively. The high alterations in the occipital cortex are probably due to the hypoxia-induced retrograde degeneration of the primary visual cortex, which was demonstrated by electrophysiological experiments. Altered proteins have functions related to cytoskeletal organization and energy metabolism. As CCH could also be an important risk factor for Alzheimer's disease (AD), we investigated whether our altered proteins overlap with AD protein databases. We revealed a significant amount of altered proteins associated with AD in the two neocortical areas, suggesting a prominent overlap with the AD pathomechanism.