The fate of neuronal synapse homeostasis in aging, infection, and inflammation.

Neuroplasticity is the brain's ability to reorganize and modify its neuronal connections in response to environmental stimuli, experiences, learning, and disease processes. This encompasses a variety of mechanisms, including changes in synaptic strength and connectivity, the formation of new synapses, alterations in neuronal structure and function, and the generation of new neurons. Proper functioning of synapses, which facilitate neuron-to-neuron communication, is crucial for brain activity. Neuronal synapse homeostasis, which involves regulating and maintaining synaptic strength and function in the central nervous system (CNS), is vital for this process. Disruptions in synaptic balance, due to factors like inflammation, aging, or infection, can lead to impaired brain function. This review highlights the main aspects and mechanisms underlying synaptic homeostasis, particularly in the context of aging, infection, and inflammation.

Monitoring regional astrocyte diversity by cell type-specific proteomic labeling in vivo.

Astrocytes exhibit regional heterogeneity in morphology, function and molecular composition to support and modulate neuronal function and signaling in a region-specific manner. To characterize regional heterogeneity of astrocytic proteomes of different brain regions we established an inducible Aldh1l1-methionyl-tRNA-synthetaseL274G (MetRSL274G ) mouse line that allows astrocyte-specific metabolic labeling of newly synthesized proteins by azidonorleucine (ANL) in vivo and subsequent isolation of tagged proteins by click chemistry. We analyzed astrocytic proteins from four different brain regions by mass spectrometry. The induced expression of MetRSL274G is restricted to astrocytes and identified proteins show a high overlap with proteins compiled in "AstroProt," a newly established database for astrocytic proteins. Gene enrichment analysis reveals a high similarity among brain regions with subtle differences in enriched biological processes and in abundances of key astrocytic proteins for hippocampus, cortex and striatum. However, the cerebellar proteome stands out with proteins being highly associated with the calcium signaling pathway or with bipolar disorder. Subregional analysis of single astrocyte TAMRA intensities in hippocampal layers indicates distinct subregional heterogeneity of astrocytes and highlights the applicability of our toolbox to study differences of astrocytic proteomes in vivo.

A role for TASK2 channels in the human immunological synapse.

The immunological synapse is a transient junction that occurs when the plasma membrane of a T cell comes in close contact with an APC after recognizing a peptide from the antigen-MHC. The interaction starts when CRAC channels embedded in the T cell membrane open, flowing calcium ions into the cell. To counterbalance the ion influx and subsequent depolarization, Kv 1.3 and KCa3.1 channels are recruited to the immunological synapse, increasing the extracellular K+ concentration. These processes are crucial as they initiate gene expression that drives T cell activation and proliferation. The T cell-specific function of the K2P channel family member TASK2 channels and their role in autoimmune processes remains unclear. Using mass spectrometry analysis together with epifluorescence and super-resolution single-molecule localization microscopy, we identified TASK2 channels as novel players recruited to the immunological synapse upon stimulation. TASK2 localizes at the immunological synapse, upon stimulation with CD3 antibodies, likely interacting with these molecules. Our findings suggest that, together with Kv 1.3 and KCa3.1 channels, TASK2 channels contribute to the proper functioning of the immunological synapse, and represent an interesting treatment target for T cell-mediated autoimmune disorders.

14-3-3 Proteins regulate K2P 5.1 surface expression on T lymphocytes.

K2P 5.1 channels (also called TASK-2 or Kcnk5) have already been shown to be relevant in the pathophysiology of autoimmune disease because they are known to be upregulated on peripheral and central T lymphocytes of multiple sclerosis (MS) patients. Moreover, overexpression of K2P 5.1 channels in vitro provokes enhanced T-cell effector functions. However, the molecular mechanisms regulating intracellular K2P 5.1 channel trafficking are unknown so far. Thus, the aim of the study is to elucidate the trafficking of K2P 5.1 channels on T lymphocytes. Using mass spectrometry analysis, we have identified 14-3-3 proteins as novel binding partners of K2P 5.1 channels. We show that a non-classical 14-3-3 consensus motif (R-X-X-pT/S-x) at the channel's C-terminus allows the binding between K2P 5.1 and 14-3-3. The mutant K2P 5.1/S266A diminishes the protein-protein interaction and reduces the amplitude of membrane currents. Application of a non-peptidic 14-3-3 inhibitor (BV02) significantly reduces the number of wild-type channels in the plasma membrane, whereas the drug has no effect on the trafficking of the mutated channel. Furthermore, blocker application reduces T-cell effector functions. Taken together, we demonstrate that 14-3-3 interacts with K2P 5.1 and plays an important role in channel trafficking.

Deficiency of the immunoproteasome subunit ß5i/LMP7 supports the anxiogenic effects of mild stress and facilitates cued fear memory in mice.

Proteolysis as mediated by one of the major cellular protein degradation pathways, the ubiquitin-proteasome system (UPS), plays an essential role in learning and memory formation. However, the functional relevance of immunoproteasomes in the healthy brain and especially their impact on normal brain function including processes of learning and memory has not been investigated so far. In the present study, we analyzed the phenotypic effects of an impaired immunoproteasome formation using a ß5i/LMP7-deficient mouse model in different behavioral paradigms focusing on locomotor activity, exploratory behavior, innate anxiety, startle response, prepulse inhibition, as well as fear and safety conditioning. Overall, our results demonstrate no strong effects of constitutive ß5i/LMP7-deficiency on gross locomotor abilities and anxiety-related behavior in general. However, ß5i/LMP7-deficient mice expressed more anxiety after mild stress and increased cued fear after fear conditioning. These findings indicate that the basal proper formation of immunoproteasomes and/or at least the expression of ß5i/LMP7 in healthy mice seem to be involved in the regulation of anxiety and cued fear levels.

Proteomics of the Synapse--A Quantitative Approach to Neuronal Plasticity.

The advances in mass spectrometry based proteomics in the past 15 years have contributed to a deeper appreciation of protein networks and the composition of functional synaptic protein complexes. However, research on protein dynamics underlying core mechanisms of synaptic plasticity in brain lag far behind. In this review, we provide a synopsis on proteomic research addressing various aspects of synaptic function. We discuss the major topics in the study of protein dynamics of the chemical synapse and the limitations of current methodology. We highlight recent developments and the future importance of multidimensional proteomics and metabolic labeling. Finally, emphasis is given on the conceptual framework of modern proteomics and its current shortcomings in the quest to gain a deeper understanding of synaptic plasticity.

Proteome rearrangements after auditory learning: high-resolution profiling of synapse-enriched protein fractions from mouse brain.

Learning and memory processes are accompanied by rearrangements of synaptic protein networks. While various studies have demonstrated the regulation of individual synaptic proteins during these processes, much less is known about the complex regulation of synaptic proteomes. Recently, we reported that auditory discrimination learning in mice is associated with a relative down-regulation of proteins involved in the structural organization of synapses in various brain regions. Aiming at the identification of biological processes and signaling pathways involved in auditory memory formation, here, a label-free quantification approach was utilized to identify regulated synaptic junctional proteins and phosphoproteins in the auditory cortex, frontal cortex, hippocampus, and striatum of mice 24 h after the learning experiment. Twenty proteins, including postsynaptic scaffolds, actin-remodeling proteins, and RNA-binding proteins, were regulated in at least three brain regions pointing to common, cross-regional mechanisms. Most of the detected synaptic proteome changes were, however, restricted to individual brain regions. For example, several members of the Septin family of cytoskeletal proteins were up-regulated only in the hippocampus, while Septin-9 was down-regulated in the hippocampus, the frontal cortex, and the striatum. Meta analyses utilizing several databases were employed to identify underlying cellular functions and biological pathways. Data are available via ProteomeExchange with identifier PXD003089. How does the protein composition of synapses change in different brain areas upon auditory learning? We unravel discrete proteome changes in mouse auditory cortex, frontal cortex, hippocampus, and striatum functionally implicated in the learning process. We identify not only common but also area-specific biological pathways and cellular processes modulated 24 h after training, indicating individual contributions of the regions to memory processing.

QuaNCAT: quantitating proteome dynamics in primary cells.

Here we demonstrate quantitation of stimuli-induced proteome dynamics in primary cells by combining the power of bio-orthogonal noncanonical amino acid tagging (BONCAT) and stable-isotope labeling of amino acids in cell culture (SILAC). In conjunction with nanoscale liquid chromatography-tandem mass spectrometry (nanoLC-MS/MS), quantitative noncanonical amino acid tagging (QuaNCAT) allowed us to monitor the early expression changes of >600 proteins in primary resting T cells subjected to activation stimuli.

NMDAR-dependent proteasome activity in the gustatory cortex is necessary for conditioned taste aversion.

Taste information is processed in different brain structures in the mammalian brain, including the gustatory cortex (GC), which resides within the insular cortex. N-methyl-d-aspartate receptor (NMDAR) activity in the GC is necessary for the acquisition of conditioned taste aversion (CTA) but not positive novel taste learning. Previous studies have shown that taste memory consolidation requires intact protein synthesis in the GC. In addition, the direct involvement of translation initiation and elongation factors was documented in the GC during taste learning. However, protein expression is defined by protein synthesis, degradation, and localization. Protein degradation is critical for the consolidation and reconsolidation of other forms of learning, such as fear learning and addiction behavior, but its role in cortical-dependent learning is not clear. Here, we show for the first time that proteasome activity is specifically increased in the GC 4h following experiencing of a novel taste. This increase in proteasome activity was abolished by local administration to the GC of the NMDA antagonist, APV, as well as a CaMKII inhibitor, at the time of acquisition. In addition, local application of lactacystin, a proteasome inhibitor, resulted in impaired CTA, but not novel taste learning. These results suggest that NMDAR-dependent proteasome activity in the GC participates in the association process between novel taste experience and negative visceral sensation.

Differential effects of dopamine signalling on long-term memory formation and consolidation in rodent brain.

BACKGROUND: Using auditory discrimination learning in gerbils, we have previously shown that activation of auditory-cortical D1/D5 dopamine receptors facilitates mTOR-mediated, protein synthesis-dependent mechanisms of memory consolidation and anterograde memory formation. To understand molecular mechanisms of this facilitatory effect, we tested the impact of local pharmacological activation of different D1/D5 dopamine receptor signalling modes in the auditory cortex. To this end, protein patterns in soluble and synaptic protein-enriched fractions from cortical, hippocampal and striatal brain regions of ligand- and vehicle-treated gerbils were analysed by 2D gel electrophoresis and mass spectrometry 24 h after intervention. RESULTS: After auditory-cortical injection of SKF38393 - a D1/D5 dopamine receptor-selective agonist reported to activate the downstream effectors adenylyl cyclase and phospholipase C - prominent proteomic alterations compared to vehicle-treated controls appeared in the auditory cortex, striatum, and hippocampus, whereas only minor changes were detectable in the frontal cortex. In contrast, auditory-cortical injection of SKF83959 - a D1/D5 agonist reported to preferentially stimulate phospholipase C - induced pronounced changes in the frontal cortex. At the molecular level, we detected altered regulation of cytoskeletal and scaffolding proteins, changes in proteins with functions in energy metabolism, local protein synthesis, and synaptic signalling. Interestingly, abundance and/or subcellular localisation of the predominantly presynaptic protein α-synuclein displayed dopaminergic regulation. To assess the role of α-synuclein for dopaminergic mechanisms of memory modulation, we tested the impact of post-conditioning systemic pharmacological activation of different D1/D5 dopamine receptor signalling modes on auditory discrimination learning in α-synuclein-mutant mice. In C57BL/6JOlaHsd mice, bearing a spontaneous deletion of the α-synuclein-encoding gene, but not in the related substrains C57BL/6JCrl and C57BL/6JRccHsd, adenylyl cyclase-mediated signalling affected acquisition rates over future learning episodes, whereas phospholipase C-mediated signalling affected final memory performance. CONCLUSIONS: Dopamine signalling modes via D1/D5 receptors in the auditory cortex differentially impact protein profiles related to rearrangement of cytomatrices, energy metabolism, and synaptic neurotransmission in cortical, hippocampal, and basal brain structures. Altered dopamine neurotransmission in α-synuclein-deficient mice revealed that distinct D1/D5 receptor signalling modes may control different aspects of memory consolidation.

Tff3 is expressed in neurons and microglial cells.

BACKGROUND/AIMS: The trefoil factor family (TFF) peptide TFF3 is typically secreted by mucous epithelia, but is also expressed in the immune system and the brain. It was the aim of this study to determine the cerebral cell types which express Tff3. METHODS: Primary cultures from rat embryonic or neonatal cerebral cortex and hippocampus, respectively, were studied by means of RT-PCR and immunofluorescence. Moreover, Tff3 expression was localized by immunocytochemistry in sections of adult rat cerebellum. RESULTS: Tff3 transcripts were detectable in neural cultures of both the cortex and the hippocampus as well as in glial cell-enriched cultures. Tff3 peptide co-localized with Map2 indicating an expression in neurons in vitro. The neuronal expression was confirmed by immunofluorescence studies of adult rat cerebellum. Furthermore, Tff3 peptide showed also a clear co-localization with Iba-1 in vitro typical of activated microglial cells. CONCLUSION: The neuronal expression of Tff3 is in line with a function of a typical neuropeptide influencing, e.g., fear, memory, depression and motoric skills. The expression in activated microglial cells, which is demonstrated here for the first time, points towards a possible function for Tff3 in immune reactions in the CNS. This opens a plethora of additional possible functions for Tff3 including synaptic plasticity and cognition as well as during neuroinflammatory diseases and psychiatric disorders.

Orexinergic modulation of chronic jet lag-induced deficits in mouse cognitive flexibility.

Cognitive flexibility and working memory are important executive functions mediated by the prefrontal cortex and can be impaired by circadian rhythm disturbances such as chronic jet lag (CJL) or shift work. In the present study, we used mice to investigate whether (1) simulated CJL impairs cognitive flexibility, (2) the orexin system is involved in such impairment, and (3) nasal administration of orexin A is able to reverse CJL-induced deficits in cognitive flexibility and working memory. Mice were exposed to either standard light-dark conditions or simulated CJL consisting of series of advance time shifts. Experiment (1) investigated the effects of a mild CJL protocol on cognitive flexibility using the attentional set shifting task. Experiment (2) used a stronger CJL protocol and examined CJL effects on the orexin system utilizing c-Fos and orexin immunohistochemistry. Experiment (3) tested whether nasal orexin application can rescue CJL-induced deficits in cognitive flexibility and working memory, the latter by measuring spontaneous alternation in the Y-maze. The present data show that CJL (1) impairs cognitive flexibility and (2) reduces the activity of orexin neurons in the lateral hypothalamus. (3) Nasal administration of orexin A rescued CJL-induced deficits in working memory and cognitive flexibility. These findings suggest that executive function impairments by circadian rhythm disturbances such as CJL are caused by dysregulation of orexinergic input to the prefrontal cortex. Compensation of decreased orexinergic input by nasal administration of orexin A could be a potential therapy for CJL- or shift work-induced human deficits in executive functions.

Manipulation of DHPS activity affects dendritic morphology and expression of synaptic proteins in primary rat cortical neurons.

Deoxyhypusine synthase (DHPS) catalyzes the initial step of hypusine incorporation into the eukaryotic initiation factor 5A (eIF5A), leading to its activation. The activated eIF5A, in turn, plays a key role in regulating the protein translation of selected mRNAs and therefore appears to be a suitable target for therapeutic intervention strategies. In the present study, we analyzed the role of DHPS-mediated hypusination in regulating neuronal homeostasis using lentivirus-based gain and loss of function experiments in primary cortical cultures from rats. This model allows us to examine the impact of DHPS function on the composition of the dendritic and synaptic compartments, which may contribute to a better understanding of cognitive function and neurodevelopment in vivo. Our findings revealed that shRNA-mediated DHPS knockdown diminishes the amount of hypusinated eIF5A (eIF5AHyp), resulting in notable alterations in neuronal dendritic architecture. Furthermore, in neurons, the synaptic composition was also affected, showing both pre- and post-synaptic changes, while the overexpression of DHPS had only a minor impact. Therefore, we hypothesize that interfering with the eIF5A hypusination caused by reduced DHPS activity impairs neuronal and synaptic homeostasis.

Probing cognitive flexibility in Shank2-deficient mice: Effects of D-cycloserine and NMDAR signaling hub dynamics.

Neurodevelopmental disorders such as autism spectrum disorder (ASD) have a heterogeneous etiology but are largely associated with genetic factors. Robust evidence from recent human genetic studies has linked mutations in the Shank2 gene to idiopathic ASD. Modeling these Shank2 mutations in animal models recapitulates behavioral changes, e.g. impaired social interaction and repetitive behavior of ASD patients. Shank2-deficient mice exhibit NMDA receptor (NMDAR) hypofunction and associated behavioral deficits. Of note, NMDARs are strongly implicated in cognitive flexibility. Their hypofunction, e.g. observed in schizophrenia, or their pharmacological inhibition leads to impaired cognitive flexibility. However, the association between Shank2 mutations and cognitive flexibility is poorly understood. Using Shank2-deficient mice, we explored the role of Shank2 in cognitive flexibility measured by the attentional set shifting task (ASST) and whether ASST performance in Shank2-deficient mice can be modulated by treatment with the partial NMDAR agonist D-cycloserine (DCS). Furthermore, we investigated the effects of Shank2 deficiency, ASST training, and DCS treatment on the expression level of NMDAR signaling hub components in the orbitofrontal cortex (OFC), including NMDAR subunits (GluN2A, GluN2B, GluN2C), phosphoglycerate dehydrogenase and serine racemase. Surprisingly, Shank2 deficiency did not affect ASST performance or alter the expression of the investigated NMDAR signaling hub components. Importantly, however, DCS significantly improved ASST performance, demonstrating that positive NMDAR modulation facilitates cognitive flexibility. Furthermore, DCS increased the expression of GluN2A in the OFC, but not that of other NMDAR signaling hub components. Our findings highlight the potential of DCS as a pharmacological intervention to improve cognitive flexibility impairments downstream of NMDAR modulation and substantiate the key role of NMDAR in cognitive flexibility.

Alteration of cGAS-STING signaling pathway components in the mouse cortex and hippocampus during healthy brain aging.

The cGAS-STING pathway is a pivotal element of the innate immune system, recognizing cytosolic DNA to initiate the production of type I interferons and pro-inflammatory cytokines. This study investigates the alterations of the cGAS-STING signaling components in the cortex and hippocampus of mice aged 24 and 108 weeks. In the cortex of old mice, an increase in the dsDNA sensor protein cGAS and its product 2'3'-cGAMP was observed, without corresponding activation of downstream signaling, suggesting an uncoupling of cGAS activity from STING activation. This phenomenon may be attributed to increased dsDNA concentrations in the EC neurons, potentially arising from nuclear DNA damage. Contrastingly, the hippocampus did not exhibit increased cGAS activity with aging, but there was a notable elevation in STING levels, particularly in microglia, neurons and astrocytes. This increase in STING did not correlate with enhanced IRF3 activation, indicating that brain inflammation induced by the cGAS-STING pathway may manifest extremely late in the aging process. Furthermore, we highlight the role of autophagy and its interplay with the cGAS-STING pathway, with evidence of autophagy dysfunction in aged hippocampal neurons leading to STING accumulation. These findings underscore the complexity of the cGAS-STING pathway's involvement in brain aging, with regional variations in activity and potential implications for neurodegenerative diseases.

Cognitive Flexibility in Mice: Effects of Puberty and Role of NMDA Receptor Subunits.

Cognitive flexibility refers to the ability to adapt flexibly to changing circumstances. In laboratory mice, we investigated whether cognitive flexibility is higher in pubertal mice than in adult mice, and whether this difference is related to the expression of distinct NMDA receptor subunits. Using the attentional set shifting task as a measure of cognitive flexibility, we found that cognitive flexibility was increased during puberty. This difference was more pronounced in female pubertal mice. Further, the GluN2A subunit of the NMDA receptor was more expressed during puberty than after puberty. Pharmacological blockade of GluN2A reduced the cognitive flexibility of pubertal mice to adult levels. In adult mice, the expression of GluN2A, GluN2B, and GluN2C in the orbitofrontal cortex correlated positively with performance in the attentional set shifting task, whereas in pubertal mice this was only the case for GluN2C. In conclusion, the present study confirms the observation in humans that cognitive flexibility is higher during puberty than in adulthood. Future studies should investigate whether NMDA receptor subunit-specific agonists are able to rescue deficient cognitive flexibility, and whether they have the potential to be used in human diseases with deficits in cognitive flexibility.

Deoxynivalenol affects cell metabolism in vivo and inhibits protein synthesis in IPEC-1 cells.

Deoxynivalenol is present in forage crops in concentrations that endanger animal welfare but is also found in cereal-based food. The amphipathic nature of mycotoxins allows them to cross the cell membrane and interacts with different cell organelles such as mitochondria and ribosomes. In our study, we investigated the gene expression of several genes in vivo and in vitro that are related to the metabolism. We observed a significantly higher COX5B and MHCII expression in enterocytes of DON-fed pigs compared to CON-fed pigs and a marked increase in GAPDH and SLC7A11 in DON-fed pigs, but we could not confirm this in vitro in IPEC-1. In vitro, functional metabolic analyses were performed with a seahorse analyzer. A significant increase of non-mitochondrial respiration was observed in all DON-treatment groups (50-2000 ng/mL). The oxygen consumption of cells, which were cultured on membranes, was examined with a fiber-glass electrode. Here, we found significantly lower values for DON 200- and DON 2000-treatment group. The effect on ribosomes was investigated using biorthogonal non-canonical amino acid tagging (BONCAT) to tag newly synthesized proteins. A significantly reduced amount was found in almost all DON-treatment groups. Our findings clearly show that apical and basolateral DON-treatment of epithelial cell layer results in decreasing amounts of newly synthesized proteins. Furthermore, our study shows that DON affects enterocyte metabolism in vivo and in vitro.

Cell-selective proteomics segregates pancreatic cancer subtypes by extracellular proteins in tumors and circulation.

Cell-selective proteomics is a powerful emerging concept to study heterocellular processes in tissues. However, its high potential to identify non-cell-autonomous disease mechanisms and biomarkers has been hindered by low proteome coverage. Here, we address this limitation and devise a comprehensive azidonorleucine labeling, click chemistry enrichment, and mass spectrometry-based proteomics and secretomics strategy to dissect aberrant signals in pancreatic ductal adenocarcinoma (PDAC). Our in-depth co-culture and in vivo analyses cover more than 10,000 cancer cell-derived proteins and reveal systematic differences between molecular PDAC subtypes. Secreted proteins, such as chemokines and EMT-promoting matrisome proteins, associated with distinct macrophage polarization and tumor stromal composition, differentiate classical and mesenchymal PDAC. Intriguingly, more than 1,600 cancer cell-derived proteins including cytokines and pre-metastatic niche formation-associated factors in mouse serum reflect tumor activity in circulation. Our findings highlight how cell-selective proteomics can accelerate the discovery of diagnostic markers and therapeutic targets in cancer.

Dopaminergic modulation of the hippocampal neuropil proteome identified by bioorthogonal noncanonical amino acid tagging (BONCAT).

Local protein synthesis and its activity-dependent modulation via dopamine receptor stimulation play an important role in synaptic plasticity - allowing synapses to respond dynamically to changes in their activity patterns. We describe here the metabolic labeling, enrichment, and MS-based identification of candidate proteins specifically translated in intact hippocampal neuropil sections upon treatment with the selective D1/D5 receptor agonist SKF81297. Using the noncanonical amino acid azidohomoalanine and click chemistry, we identified over 300 newly synthesized proteins specific to dendrites and axons. Candidates specific for the SKF81297-treated samples were predominantly involved in protein synthesis and synapse-specific functions. Furthermore, we demonstrate a dendrite-specific increase in proteins synthesis upon application of SKF81297. This study provides the first snapshot in the dynamics of the dopaminergic hippocampal neuropil proteome.

Cell-selective metabolic labeling of proteins.

Metabolic labeling of proteins with the methionine surrogate azidonorleucine can be targeted exclusively to specified cells through expression of a mutant methionyl-tRNA synthetase (MetRS). In complex cellular mixtures, proteins made in cells that express the mutant synthetase can be tagged with affinity reagents (for detection or enrichment) or fluorescent dyes (for imaging). Proteins made in cells that do not express the mutant synthetase are neither labeled nor detected.