BACKGROUND:Differences in immune responses between women and men are leading to a strong sex bias in the incidence of autoimmune diseases that predominantly affect women, such as multiple sclerosis (MS). MS manifests in more than twice as many women, making sex one of the most important risk factor. However, it is incompletely understood which genes contribute to sex differences in autoimmune incidence. To address that, we conducted a gene expression analysis in female and male human spleen and identified the transmembrane protein CD99 as one of the most significantly differentially expressed genes with marked increase in men. CD99 has been reported to participate in immune cell transmigration and T cell regulation, but sex-specific implications have not been comprehensively investigated. METHODS:In this study, we conducted a gene expression analysis in female and male human spleen using the Genotype-Tissue Expression (GTEx) project dataset to identify differentially expressed genes between women and men. After successful validation on protein level of human immune cell subsets, we assessed hormonal regulation of CD99 as well as its implication on T cell regulation in primary human T cells and Jurkat T cells. In addition, we performed in vivo assays in wildtype mice and in Cd99-deficient mice to further analyze functional consequences of differential CD99 expression. RESULTS:Here, we found higher CD99 gene expression in male human spleens compared to females and confirmed this expression difference on protein level on the surface of T cells and pDCs. Androgens are likely dispensable as the cause shown by in vitro assays and ex vivo analysis of trans men samples. In cerebrospinal fluid, CD99 was higher on T cells compared to blood. Of note, male MS patients had lower CD99 levels on CD4+ T cells in the CSF, unlike controls. By contrast, both sexes had similar CD99 expression in mice and Cd99-deficient mice showed equal susceptibility to experimental autoimmune encephalomyelitis compared to wildtypes. Functionally, CD99 increased upon human T cell activation and inhibited T cell proliferation after blockade. Accordingly, CD99-deficient Jurkat T cells showed decreased cell proliferation and cluster formation, rescued by CD99 reintroduction. CONCLUSIONS:Our results demonstrate that CD99 is sex-specifically regulated in healthy individuals and MS patients and that it is involved in T cell costimulation in humans but not in mice. CD99 could potentially contribute to MS incidence and susceptibility in a sex-specific manner.

A disturbed balance between excitation and inhibition (E/I balance) is increasingly recognized as a key driver of neurodegeneration in multiple sclerosis (MS), a chronic inflammatory disease of the central nervous system. To understand how chronic hyperexcitability contributes to neuronal loss in MS, we transcriptionally profiled neurons from mice lacking inhibitory metabotropic glutamate signaling with shifted E/I balance and increased vulnerability to inflammation-induced neurodegeneration. This revealed a prominent induction of the nuclear receptor NR4A2 in neurons. Mechanistically, NR4A2 increased susceptibility to excitotoxicity by stimulating continuous VGF secretion leading to glycolysis-dependent neuronal cell death. Extending these findings to people with MS (pwMS), we observed increased VGF levels in serum and brain biopsies. Notably, neuron-specific deletion of Vgf in a mouse model of MS ameliorated neurodegeneration. These findings underscore the detrimental effect of a persistent metabolic shift driven by excitatory activity as a fundamental mechanism in inflammation-induced neurodegeneration.

Inflammation-induced neurodegeneration is a defining feature of multiple sclerosis (MS), yet the underlying mechanisms remain unclear. By dissecting the neuronal inflammatory stress response, we discovered that neurons in MS and its mouse model induce the stimulator of interferon genes (STING). However, activation of neuronal STING requires its detachment from the stromal interaction molecule 1 (STIM1), a process triggered by glutamate excitotoxicity. This detachment initiates non-canonical STING signaling, which leads to autophagic degradation of glutathione peroxidase 4 (GPX4), essential for neuronal redox homeostasis and thereby inducing ferroptosis. Both genetic and pharmacological interventions that target STING in neurons protect against inflammation-induced neurodegeneration. Our findings position STING as a central regulator of the detrimental neuronal inflammatory stress response, integrating inflammation with glutamate signaling to cause neuronal cell death, and present it as a tractable target for treating neurodegeneration in MS.

Two isogenic hiPSC lines, ZIPi013-B-1 and ZIPi013-B-2, were generated by CRISPR/Cas9-mediated indels in the TRPM4 gene of the previously published ZIPi013-B. TRPM4 belongs to the evolutionarily conserved family of transient receptor potential (TRP) channels. It is expressed ubiquitously and its activity is regulated by intracellular calcium binding, changes in membrane potential, phosphoinositide lipids in the plasma membrane and the local concentration of cytoplasmic ATP and ADP. TRPM4 has been implicated in various diseases, including neurological and immune system disorders, cardiac diseases and cancer. Both new cell lines offer the opportunity to model human diseases and test therapeutic modalities addressing these.

Migratory dendritic cells (migDCs) continuously patrol tissues and are activated by injury and inflammation. Extracellular adenosine triphosphate (ATP) is released by damaged cells or actively secreted during inflammation and increases migDC motility. However, the underlying molecular mechanisms by which ATP accelerates migDC migration is not understood. Here, we show that migDCs can be distinguished from other DC subsets and immune cells by their expression of the voltage-gated calcium channel subunit β3 (Cavβ3; CACNB3), which exclusively facilitates ATP-dependent migration in vitro and during tissue damage in vivo. By contrast, CACNB3 does not regulate lipopolysaccharide-dependent migration. Mechanistically, CACNB3 regulates ATP-dependent inositol 1,4,5-trisphophate receptor–controlled calcium release from the endoplasmic reticulum. This, in turn, is required for ATP-mediated suppression of adhesion molecules, their detachment, and initiation of migDC migration. Thus, Cacnb3 -deficient migDCs have an impaired migration after ATP exposure. In summary, we identified CACNB3 as a master regulator of ATP-dependent migDC migration that controls tissue-specific immunological responses during injury and inflammation.

Neuroinflammation leads to neuronal stress responses that contribute to neuronal dysfunction and loss. However, treatments that stabilize neurons and prevent their destruction are still lacking. Here, we identify the histone methyltransferase G9a as a druggable epigenetic regulator of neuronal vulnerability to inflammation. In murine experimental autoimmune encephalomyelitis (EAE) and human multiple sclerosis (MS), we found that the G9a-catalyzed repressive epigenetic mark H3K9me2 was robustly induced by neuroinflammation. G9a activity repressed anti-ferroptotic genes, diminished intracellular glutathione levels, and triggered the iron-dependent programmed cell death pathway ferroptosis. Conversely, pharmacological treatment of EAE mice with a G9a inhibitor restored anti-ferroptotic gene expression, reduced inflammation-induced neuronal loss, and improved clinical outcome. Similarly, neuronal anti-ferroptotic gene expression was reduced in MS brain tissue and was boosted by G9a inhibition in human neuronal cultures. This study identifies G9a as a critical transcriptional enhancer of neuronal ferroptosis and potential therapeutic target to counteract inflammation-induced neurodegeneration.

The transient receptor potential melastatin 4 (TRPM4) channel contributes to disease severity in the murine experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis and to neuronal cell death in models of excitotoxicity and traumatic brain injury. As TRPM4 is activated by intracellular calcium and conducts monovalent cations, we hypothesized that TRPM4 may contribute to and boost excitatory synaptic transmission in CA1 pyramidal neurons of the hippocampus. Using single-spine calcium imaging and electrophysiology, we found no effect of the TRPM4 antagonists 9-phenanthrol and glibenclamide on synaptic transmission in hippocampal slices from healthy mice. In contrast, glibenclamide but not 9-phenanthrol reduced excitatory synaptic potentials in slices from EAE mice, an effect that was absent in slices from EAE mice lacking TRPM4. We conclude that TRPM4 plays little role in basal hippocampal synaptic transmission, but a glibenclamide-sensitive TRPM4-mediated contribution to excitatory postsynaptic responses is upregulated at the acute phase of EAE.

SorLA is a member of the Vps10p-domain (Vps10p-D) receptor family of type-I transmembrane proteins conveying neuronal endosomal sorting. The extracellular/luminal moiety of SorLA has a unique mosaic domain composition and interacts with a large number of different and partially unrelated ligands, including the amyloid precursor protein as well as amyloid-β. Several studies support a strong association of SorLA with sporadic and familial forms of Alzheimer's disease (AD). Although SorLA seems to be an important factor in AD, the large number of different ligands suggests a role as a neuronal multifunctional receptor with additional intracellular sorting capacities. Therefore, understanding the determinants of SorLA's subcellular targeting might be pertinent for understanding neuronal endosomal sorting mechanisms in general. A number of cytosolic adaptor proteins have already been demonstrated to determine intracellular trafficking of SorLA. Most of these adaptors and several ligands of the extracellular/luminal moiety are shared with the Vps10p-D receptor Sortilin. Although SorLA and Sortilin show both a predominant intracellular and endosomal localization, they are targeted to different endosomal compartments. Thus, independent adaptor proteins may convey their differential endosomal targeting. Here, we hypothesized that Sortilin and SorLA interact with the cytosolic adaptors PSD95 and PICK1 which have been shown to bind the Vps10p-D receptor SorCS3. We observed only an interaction for SorLA and PICK1 in mammalian-two-hybrid, pull-down and cellular recruitment experiments. We demonstrate by mutational analysis that the C-terminal minimal PDZ domain binding motif VIA of SorLA mediates the interaction. Moreover, we show co-localization of SorLA and PICK1 at vesicular structures in primary neurons. Although the physiological role of the interaction between PICK1 and SorLA remains unsolved, our study suggests that PICK1 partakes in regulating SorLA's intracellular itinerary.

PSENEN/PEN2 is the smallest subunit of the γ-secretase complex, an intramembrane protease that cleaves proteins within their transmembrane domains. Mutations in components of the γ-secretase underlie familial Alzheimer disease. In addition to its proteolytic activity, supplementary, γ-secretase independent, functions in the macroautophagy/autophagy-lysosome system have been proposed. Here, we screened for PSENEN-interacting proteins and identified CLN3. Mutations in CLN3 are causative for juvenile neuronal ceroid lipofuscinosis, a rare lysosomal storage disorder considered the most common neurodegenerative disease in children. As mutations in the PSENEN and CLN3 genes cause different neurodegenerative diseases, understanding shared cellular functions of both proteins might be pertinent for understanding general cellular mechanisms underlying neurodegeneration. We hypothesized that CLN3 modulates γ-secretase activity and that PSENEN and CLN3 play associated roles in the autophagy-lysosome system. We applied CRISPR gene-editing and obtained independent isogenic HeLa knockout cell lines for PSENEN and CLN3. Following previous studies, we demonstrate that PSENEN is essential for forming a functional γ-secretase complex and is indispensable for γ-secretase activity. In contrast, CLN3 does not modulate γ-secretase activity to a significant degree. We observed in PSENEN- and CLN3-knockout cells corresponding alterations in the autophagy-lysosome system. These include reduced activity of lysosomal enzymes and lysosome number, an increased number of autophagosomes, increased lysosome-autophagosome fusion, and elevated levels of TFEB (transcription factor EB). Our study strongly suggests converging roles of PSENEN and CLN3 in the autophagy-lysosome system in a γ-secretase activity-independent manner, supporting the idea of common cytopathological processes underlying different neurodegenerative diseases.Abbreviations: Aβ, amyloid-beta; AD, Alzheimer disease; APP, amyloid precursor protein; ATP5MC, ATP synthase membrane subunit c; DQ-BSA, dye-quenched bovine serum albumin; ER, endoplasmic reticulum; GFP, green fluorescent protein; ICC, immunocytochemistry; ICD, intracellular domain; JNCL, juvenile neuronal ceroid lipofuscinosis; KO, knockout; LC3, microtubule associated protein 1 light chain 3; NCL, neuronal ceroid lipofuscinoses; PSEN, presenilin; PSENEN/PEN2: presenilin enhancer, gamma-secretase subunit; TAP, tandem affinity purification; TEV, tobacco etch virus; TF, transferrin; WB, Western blot; WT, wild type.

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system with continuous neuronal loss. Treatment of clinical progression remains challenging due to lack of insights into inflammation-induced neurodegenerative pathways. Here, we show that an imbalance in the neuronal receptor inter actome is driving glutamate excitotoxicity in neurons of MS patients and identify the MS risk-associated metabotropic glutamate receptor 8 (GRM8) as a decisive modulator. Mechanistically, GRM8 activation counteracted neuronal cAMP accumulation, thereby directly desensitizing the inositol 1,4,5-trisphosphate receptor (IP3R). This profoundly limited glutamate-induced calcium release from the endoplasmic reticulum and subsequent cell death. Notably, we found Grm8-deficient neurons to be more prone to glutamate excitotoxicity, whereas pharmacological activation of GRM8 augmented neuroprotection in mouse and human neurons as well as in a preclinical mouse model of MS. Thus, we demonstrate that GRM8 conveys neuronal resilience to CNS inflammation and is a promising neuroprotective target with broad therapeutic implications.

The transient receptor potential melastatin 4 (TRPM4) channel contributes to disease severity in the murine experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis and to neuronal cell death in models of excitotoxicity and traumatic brain injury. As TRPM4 is activated by intracellular calcium and conducts monovalent cations, we hypothesized that TRPM4 may contribute to and boost excitatory synaptic transmission in CA1 pyramidal neurons of the hippocampus. Using single-spine calcium imaging and electrophysiology, we found no effect of the TRPM4 antagonists 9-phenanthrol and glibenclamide on synaptic transmission in hippocampal slices from healthy mice. In contrast, glibenclamide but not 9-phenanthrol reduced excitatory synaptic potentials in slices from EAE mice, an effect that was absent in slices from EAE mice lacking TRPM4. We conclude that TRPM4 plays little role in basal hippocampal synaptic transmission but a glibenclamide-sensitive TRPM4-mediated contribution to excitatory postsynaptic responses is upregulated at the acute phase of EAE.

The voltage-gated proton channel Hv1 regulates proton fluxes across membranes, thereby influencing pH-dependent processes. Plasmacytoid dendritic cells (pDCs) require a particularly tight regulation of endosomal pH to ensure strong type I IFN secretion exclusively during infection, avoiding autoimmunity. However, whether Hv1 is important for pH control in pDCs is presently unknown. In this study, we show that mouse pDCs require Hv1 to achieve potent type I IFN responses after the recognition of foreign DNA by endosomal TLR9. Genetic disruption of Hvcn1, which encodes Hv1, impaired mouse pDC activation by CpG oligonucleotides in vitro and in vivo, reducing IFN-α secretion and the induction of IFN-stimulated genes. Mechanistically, Hvcn1 deficiency delayed endosomal acidification and enhanced intracellular reactive oxygen species production, consequently limiting protease activity and TLR9 signaling. Our study reveals a critical role of Hv1 during innate immune responses and places this channel as a key modulator of type I IFN production, the hallmark function of pDCs, commending Hv1 as an attractive target for modulating type I IFN-driven autoimmunity.

Significance Spatial learning and memory are hippocampal functions that emerge and mature during early postnatal development. The molecular mechanisms which shape this process are largely unknown. Here, we present evidence that the activity-regulated gene Arc/Arg3.1 is transiently up-regulated in the hippocampus of neonatal mice where it is required for establishing appropriate hippocampal network activity essential for spatial learning. Once established, network activity supports normal spatial learning in the absence of Arc/Arg3.1 while long-term memory storage continues to rely on Arc/Arg3.1 expression throughout life. These results demonstrate that hippocampal networks undergo a critical period of development mediated by Arc/Arg3.1 and open opportunities to investigate normal and pathological neurodevelopment of higher brain functions.

Die Konsolidierung des Gedachtnisses hin zu einer langanhaltenden Form, ist von der Induktion Aktivitats-regulierter Gene und deren Einfluss auf neuronale Verbindungen abhangig. Eines dieser Gene ist das immediate early gene Arc/Arg3.1, dessen mRNA und Protein in Abhangigkeit Plastizitats-induzierender Stimuli in die Dendriten der aktivierten Neurone transportiert wird. Mause mit einer genetischen Deletion von Arc/Arg3.1 zeigen schwere Storungen in der Konsolidierung der synaptischer Plastizitat und des Langzeitgedachtnisses, wahrend die grundlegende synaptische Transmission und das Kurzzeitgedachtnis intakt sind. Arc/Arg3.1 interagiert mit Komponenten der Clathrin-abhangigen Endozytose-Maschinerie und beeinflusst die Oberflachenexpression von ionotropen Glutamat-Rezeptoren vom Typ AMPA (α-amino-5-hydroxy-3-methyl-4-isoxazole propionic acid). Die Anderung der Komposition und Menge an synaptischen AMPA-Rezeptoren wird als zentraler Mechanismus der synaptischen Plastizitat angesehen. Der Einfluss von Arc/Arg3.1 auf diesen Mechanismus und die zu Grunde liegenden molekularen Wechselwirkungen sind weitgehend unverstanden. Neben einer Verbindung zur Endozytose weist die Interaktion von Arc/Arg3.1 mit der γ-Secretase auf eine Funktion in der endosomalen Sortierung von Membranproteinen hin. Die Prozessierung der Membranproteine APP (Amyloid Precurser Protein) und Notch1 durch die γ-Secretase wird entscheidend durch die Aktivitats-regulierte Expression von Arc/Arg3.1 verandert. Es ist jedoch unklar wie Arc/Arg3.1 die endosomale Sortierung der γ-Secretase und moglicherweise auch von AMPA-Rezeptoren beeinflussen kann. Zur Beantwortung dieser Frage und zum besseren Verstandnis der molekularen Funktion von Arc/Arg3.1 haben wir im Hefe-Zwei-Hybrid-System nach neuen Arc/Arg3.1-Bindungspartnern gesucht. Mehrere der neu identifizierten Bindungspartner sind mit dem endosomalen System der Zelle assoziiert. In dieser Arbeit habe ich drei der Proteine auf deren Kolokalisation und Bindungseigenschaften mit Arc/Arg3.1 hin untersucht. Sorting Nexin-7 (SNX7), ein noch nicht charakterisiertes Mitglied der Sorting-Nexin-Familie hat sich als robuster Bindungspartner von Arc/Arg3.1 erwiesen. Sorting Nexine sind durch ihre Phosphatidyl-Inositolphosphat-bindende PX-Domane charakterisiert, welche eine Bindung an spezifische Membrankompartimente vermittelt. SNX7 besitzt zudem eine BAR-(Bin-Amphiphysin-Rvs)-Domane und zahlt daher zu der Subfamilie der SNX-BAR-Proteine. SNX-BAR-Proteine sind ein zentraler Bestandteil der endosomalen, tubularen Sortierung-Maschinerie fur Membranproteine. Durch die in silico Modellierung der SNX7-BAR-Domane und der Analyse von Substitutionsmutanten war es mir moglich, die Bindungsstelle von Arc/Arg3.1 innerhalb eines von Leucinen dominierten, hydrophoben Bereiches der SNX7-BAR-Domane zu identifizieren. Die untersuchten Substitutionsmutanten beeintrachtigen nicht die Formierung des SNX7/4-Heterodimer, an welches auch Arc/Arg3.1 bindet und welches als die funktionelle Einheit in der endosomalen, tubularen Sortierung betrachtet wird. Alle drei Proteine kolokalisieren an Fruhen Endosomen und in dendritischen Spines von Neuronen. Die Verteilung der Proteine in Spines weist auf eine Subpopulation von Arc/Arg3.1 hin, welche auch dort mit SNX7/4-positiven Endosomen assoziiert ist. Daruber hinaus zeigen meine Analysen, dass SNX7 mit CPG2 (candidate plasticity gene 2) assoziiert ist, welches Teil der Endozytischen Zone in Spines und bedeutend fur die Endozytose von AMPA-Rezeptoren ist. SNX7 und Arc/Arg3.1 zeigen beide eine Assoziation mit der Clathrin-abhangigen Endozytose-Maschinerie und verstarken die Internalisierung von Transferrin. Ein weiterer neu identifizierter Bindungspartner von Arc/Arg3.1 ist AMPH2 (Amphiphysin-2), welches ebenfalls Bestandteil der Endozytose-Maschinerie ist. Der kooperative Effekt von AMPH2 auf die Arc/Arg3.1-vermittelte Erhohung der Internalisierung von Transferrin, zeigt eine funktionelle Verbindung beider Proteine in der Clathrin-abhangigen Endozytose. Meine Untersuchungen der SNX7-Expression im Mausgehirn und die Generierung von KO-Mausen zeigen zudem, dass SNX7 insbesondere in Arealen, welche mit Lern- und Gedachtnisprozessen in Verbindung gebracht werde, exprimiert wird. Die Ergebnisse meiner Arbeit zeigen zum ersten Mal eine direkte Verbindung von Arc/Arg3.1 mit der endosomalen Sortierung von Membranproteinen. Die Assoziation von Arc/Arg3.1 und SNX7 mit der Endozytose-Maschinerie und Endosomen in Spines weist zudem auf eine Kopplung beider Prozesse hin und eroffnet neue Erklarungsansatze fur die vielseitige Wirkung von Arc/Arg3.1 in synaptische Plastizitat Consolidation of long-term memories requires activity-dependent gene induction that is important in defining neuronal connectivity in the brain. One of these genes is the immediate early gene Arc/Arg3.1 whose mRNA and protein are rapidly distributed throughout the dendritic arbor of activated neurons. Arc/Arg3.1 KO mice show severe impairments in memory consolidation and synaptic plasticity, while basic synaptic transmission and short-term memory are unaffected. Arc/Arg3.1 binds to proteins involved in clathrin-dependent endocytosis and affects surface expression of AMPA (α-amino-5-hydroxy-3-methyl-4-isoxazole propionic acid) receptors. Changes in synaptic AMPA receptor composition and number are believed to be the basic mechanism of synaptic plasticity. How Arc/Arg3.1 controls this mechanism and the underlying molecular interactions are largely unknown. In addition, Arc/Arg3.1 interacts with Presenilin-1 a component of the γ-Secreatse complex. The processing of γ-Secreatse substrates APP and Notch1 is altered in an Arc/Arg3.1-dependend manner pointing to a function in endosomel sorting. However, it is still elusive how Arc/Arg3.1influences the endosomal sorting of Presenelin-1 and possibly AMPA receptors. To answer this question and to get deeper insights into the molecular function of Arc/Arg3.1 we searched for new Arc/Arg3.1 binding partners in a Yeast-Two-Hybrid screen. Several of the identified new binding partners are associated with the endosomal system. In this work I investigated the colocalization and binding between Arc/Arg3.1 and three? of the newly identified proteins. I found that Sorting nexin-7 (SNX7) an uncharacterized member of the Sorting nexin family was a robust binding partner of Arc/Arg3.1. Sorting nexins are characterized by their phosphatidylinositol phosphate-binding PX domain that mediates the binding to specific membrane compartments. Additionally, SNX7 belongs to the BAR (Bin-Amphiphysin-Rvs) domain containing subfamily of Sorting nexins. These SNX-BAR proteins are crucial parts of the endosomal tubular machinery that sorts membrane proteins. Based on my in silico modeling of the SNX7 BAR domain and investigation of SNX7 mutants, I identified and characterized the Arc/Arg3.1 binding site. Mutations within this binding site had no effect on the formation of a SNX7/SNX4 heterodimer but prevent binding of Arc/Arg3.1. This heteromer is believed to be the functional entity for endosomal tubular sorting and capable of Arc/Arg3.1 binding. All three proteins colocalize at early endosomes and in dendritic spines of neurons. The distribution indicates that a subpopulation of Arc/Arg3.1 is associated with SNX7/SNX4 positive endosomes within the spines. Moreover, I found an association between SNX7 and CPG2 (candidate plasticity gene 2), an endocytic zone localized protein important in AMPA receptor endocytosis. SNX7 associates with components of the clathrin-dependent endocytosis machinery and increases the amount of internalized Transferrin. Amphiphysin-2 (APMH2), a known component of the endocytic machinery was also identified as a new Arc/Arg3.1 binding partner. AMPH2 and Arc/Arg3.1 are increasing transferrin internalization cooperatively suggesting a functional connection in Clathrin-dependent endocytosis. Investigating the expression of SNX7 in mouse brains and generating SNX7 KO mice, I found expression of SNX7 particularly in areas associated with learning and memory. The results of my work show for the first time a physical link between Arc/Arg3.1 and the endosomal machinery that sorts membrane proteins. Additionally, Arc/Arg3.1 and SNX7 are both associated with Clathrin-dependent endocytosis and endosomal sorting in spines, suggesting a tight coupling of the two processes. The results of my studies lead to new insights into the molecular function of Arc/Arg3.1 and open up new explanations for the opposing effects of Arc/Arg3.1 in different forms of synaptic plasticity.