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Humans with monogenic inborn errors responsible for extreme disease phenotypes can reveal essential physiological pathways. We investigated germline mutations in GNAI2 , which encodes G αi2 , a key component in heterotrimeric G protein signal transduction usually thought to regulate adenylyl cyclase-mediated cyclic adenosine monophosphate (cAMP) production. Patients with activating G αi2 mutations had clinical presentations that included impaired immunity. Mutant G αi2 impaired cell migration and augmented responses to T cell receptor (TCR) stimulation. We found that mutant G αi2 influenced TCR signaling by sequestering the guanosine triphosphatase (GTPase)-activating protein RASA2, thereby promoting RAS activation and increasing downstream extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K)-AKT S6 signaling to drive cellular growth and proliferation.

Elementary emotional states and memory can be regulated by the homeostasis of the endocannabinoid system (ECS). Links between the ECS and the autophagy receptor p62 have been found at the molecular level and in animal studies. This project aimed to validate the anxiety and memory phenotype of p62 knockout (KO) animals and whether the ECS plays a role in this. We examined the behavior of p62 KO animals and analyzed whether endocannabinoid levels are altered in the responsible brain areas. We discovered in age-dependent obese p62 KO mice decreased anandamide levels in the amygdala, a brain structure important for emotional responses. Against our expectation, p62 KO animals did not exhibit an anxiety phenotype, but showed slightly increased exploratory behavior as evidenced in novel object and further tests. In addition, KO animals exhibited decreased freezing responses in the fear conditioning. Administration of the phytocannabinoid delta 9 -tetrahydrocannabinol (THC) resulted in lesser effects on locomotion but in comparable hypothermic effects in p62 KO compared with WT littermates. Our results do not confirm previously published results, as our mouse line does not exhibit a drastic behavioral phenotype. Moreover, we identified further indications of a connection to the ECS and hence offer new perspectives for future investigations.

Several studies have shown that the G-protein coupled cannabinoid receptor CB2 and its interaction partner p62 are molecularly involved in bone remodeling processes. Pharmacological activation of the CB2 receptor enhanced bone volume in postmenopausal osteoporosis and arthritis models in rodents, whereas knockout or mutation of the p62 protein in aged mice led to Paget's disease of bone-like conditions. Studies of pharmacological CB2 agonist effects on bone metabolism in p62 KO mice have not been performed to date. Here, we assessed the effect of the CB2-specific agonist JWH133 after a short-term (5 days in 3-month-old mice) or long-term (4 weeks in 6-month-old mice) treatment on structural, dynamic, and cellular bone morphometry obtained by μCT of the femur and histomorphometry of the vertebral bodies in p62 KO mice and their WT littermates in vivo . A genotype-independent stimulatory effect of CB2 on bone formation, trabecular number, and trabecular thickness after short-term treatment and on tissue mineral density after long-term treatment was detected, indicating a weak osteoanabolic function of this CB2 agonist. Moreover, after short-term systemic CB2 receptor activation, we found significant differences at the cellular level in the number of osteoblasts and osteoclasts only in p62 KO mice, together with a weak increase in trabecular number and a decrease in trabecular separation. Long-term treatment showed an opposite JWH133 effect on osteoclasts in WT versus p62 KO animals and decreased cortical thickness only in treated p62 KO mice. Our results provide new insights into CB2 receptor signaling in vivo and suggest that CB2 agonist activity may be regulated by the presence of its macromolecular binding partner p62., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Keller, Yorgan, Rading, Schinke and Karsak.)

Infantile nystagmus syndrome (INS) denominates early-onset, involuntary oscillatory eye movements with different etiologies. Nystagmus is also one of the symptoms in oculocutaneus albinism (OCA), a heterogeneous disease mainly caused by defects in melanin synthesis or melanosome biogenesis. Dopachrome tautomerase (DCT, also called TYRP2) together with tyrosinase (TYR) and tyrosin-related protein 1 (TYRP1) is one of the key enzymes in melanin synthesis. Although DCT´s role in pigmentation has been proven in different species, until now only mutations in TYR and TYRP1 have been found in patients with OCA. Detailed ophthalmological and orthoptic investigations identified a consanguineous family with two individuals with isolated infantile nystagmus and one family member with subtle signs of albinism. By whole-exome sequencing and segregation analysis, we identified the missense mutation c.176G > T (p.Gly59Val) in DCT in a homozygous state in all three affected family members. We show that this mutation results in incomplete protein maturation and targeting in vitro compatible with a partial or total loss of function. Subsequent screening of a cohort of patients with OCA (n = 85) and INS (n = 25) revealed two heterozygous truncating mutations, namely c.876C > A (p.Tyr292*) and c.1407G > A (p.Trp469*), in an independent patient with OCA. Taken together, our data suggest that mutations in DCT can cause a phenotypic spectrum ranging from isolated infantile nystagmus to oculocutaneous albinism.

The purpose of this study was to determine the role of Tctex1 (DYNLT1, dynein light chain-1) in the pathophysiology of glioblastoma (GBM). To this end, we performed immunohistochemical analyses on tissues from GBM patients ( n = 202). Tctex1 was additionally overexpressed in two different GBM cell lines, which were then evaluated in regard to their proliferative and invasive properties. We found that Tctex1 levels were significantly higher in GBM compared to healthy adjacent brain tissues. Furthermore, high Tctex1 expression was significantly associated with the short overall- ( p = 0.002, log-rank) and progression-free ( p = 0.028, log-rank) survival of GBM patients and was an independent predictor of poor overall survival in multivariate Cox-regression models. In vitro, Tctex1 promoted the metabolic activity, anchorage-independent growth and proliferation of GBM cells. This phenomenon was previously shown to occur via the phosphorylation of retinoblastoma protein (phospho-RB). Here, we found a direct and significant correlation between the levels of Tctex1 and phospho-RB (Ser807/801) in tissues from GBM patients ( p = 0.007, Rho = 0.284, Spearman's rank). Finally, Tctex1 enhanced the invasiveness of GBM cells and the release of pro-invasive matrix metalloprotease 2 (MMP2). These findings indicate that Tctex1 promotes GBM progression and therefore might be a useful therapeutic target in this type of cancer.

G-protein coupled cannabinoid CB2 receptor signaling and function is primarily mediated by its inhibitory effect on adenylate cyclase. The visualization and monitoring of agonist dependent dynamic 3',5'-cyclic adenosine monophosphate (cAMP) signaling at the single cell level is still missing for CB2 receptors. This paper presents an application of a live cell imaging while using a Förster resonance energy transfer (FRET)-based biosensor, Epac1-camps, for quantification of cAMP. We established HEK293 cells stably co-expressing human CB2 and Epac1-camps and quantified cAMP responses upon Forskolin pre-stimulation, followed by treatment with the CB2 ligands JWH-133, HU308, β-caryophyllene, or 2-arachidonoylglycerol. We could identify cells showing either an agonist dependent CB2-response as expected, cells displaying no response, and cells with constitutive receptor activity. In Epac1-CB2-HEK293 responder cells, the terpenoid β-caryophyllene significantly modified the cAMP response through CB2. For all of the tested ligands, a relatively high proportion of cells with constitutively active CB2 receptors was identified. Our method enabled the visualization of intracellular dynamic cAMP responses to the stimuli at single cell level, providing insights into the nature of heterologous CB2 expression systems that contributes to the understanding of Gαi-mediated G-Protein coupled receptor (GPCR) signaling in living cells and opens up possibilities for future investigations of endogenous CB2 responses.

Rare coding variants in the triggering receptor expressed on myeloid cells-2 (TREM2) gene have been associated with Alzheimer disease (AD) and homozygous TREM2 loss-of-function variants have been reported in families with monogenic frontotemporal-like dementia with/without bone abnormalities. In a whole-exome sequencing study of a family with probable AD-type dementia without pathogenic variants in known autosomal dominant dementia disease genes and negative for the apolipoprotein E (APOE) ε4 allele, we identified an extremely rare TREM2 coding variant, that is, a glycine-to-tryptophan substitution at amino acid position 145 (NM_018965.3:c.433G>T/p.[Gly145Trp]). This alteration is found in only 1 of 251,150 control alleles in gnomAD. It was present in both severely affected as well as in another putatively affected and one 61 years old as yet unaffected family member suggesting incomplete penetrance and/or a variable age of onset. Gly145 maps to an intrinsically disordered region (IDR) of TREM2 between the immunoglobulin-like and transmembrane domain. Subsequent cellular studies showed that the variant led to IDR shortening and structural changes of the mutant protein resulting in an impairment of cellular responses upon receptor activation. Our results, suggest that a p.(Gly145Trp)-induced structural disturbance and functional impairment of TREM2 may contribute to the pathogenesis of an AD-like form of dementia., (© 2019 The Authors. Human Mutation published by Wiley Periodicals, Inc.)

Myeloid cells are essential for the initiation and termination of innate and adaptive immunity that create homeostasis in the liver. Smad7 is an inhibitor of the transforming growth factor β (TGF-β) signaling pathway, which regulates inflammatory cellular processes. Knockdown of Smad7 in hepatocytes has been shown to promote liver fibrosis, but little is known about the effects of Smad7 in myeloid cells during inflammatory responses in the liver. Using mice with a myeloid-specific knockdown of Smad7 (LysM-Cre Smad7 fl/fl ), we investigated the impact of Smad7 deficiency in myeloid cells on liver inflammation and regeneration using the well-established model of CCl 4 -mediated liver injury. Early (24/48 h) and late (7 d) time points were analyzed. We found that CCl 4 induces severe liver injury, with elevated serum ALT levels, centrilobular and periportal necrosis, infiltrating myeloid cells and an increase of inflammatory cytokines in the liver. Furthermore, as expected, inflammation peaked at 24 h and subsided after 7 d. However, the knockdown of Smad7 in myeloid cells did not affect any of the investigated parameters in the CCl 4 -treated animals. In summary, our results suggest that the inhibition of TGF-β signaling via Smad7 expression in myeloid cells is dispensable for the induction and control of acute CCl 4 -induced liver injury.

The endocannabinoid system (ECS) consists particularly of cannabinoid receptors 1 and 2 (CB1 and CB2), their endogenous ligands, and enzymes that synthesize and degrade their ligands. It acts in a variety of organs and disease states ranging from cancer progression over neuropathic pain to neurodegeneration. Protein components engaged in the signaling, trafficking, and homeostasis machinery of the G-protein coupled CB2, are however largely unknown. It is therefore important to identify further interaction partners to better understand CB2 receptor functions in physiology and pathophysiology. For this purpose, we used an affinity purification and mass spectrometry-based proteomics approach of Strep-HA-CB2 receptor in HEK293 cells. After subtraction of background interactions and protein frequency library assessment we could identify 83 proteins that were classified by the identification of minimally 2 unique peptides as highly probable interactors. A functional protein association network analysis obtained an interaction network with a significant enrichment of proteins functionally involved in protein metabolic process, in endoplasmic reticulum, response to stress but also in lipid metabolism and membrane organization. The network especially contains proteins involved in biosynthesis and trafficking like calnexin, Sec61A, tubulin chains TUBA1C and TUBB2B, TMED2, and TMED10. Six proteins that were only expressed in stable CB2 expressing cells were DHC24, DHRS7, GGT7, HECD3, KIAA2013, and PLS1. To exemplify the validity of our approach, we chose a candidate having a relatively low number of edges in the network to increase the likelihood of a direct protein interaction with CB2 and focused on the scaffold/phagosomal protein p62/SQSTM1. Indeed, we independently confirmed the interaction by co-immunoprecipitation and immunocytochemical colocalization studies. 3D reconstruction of confocal images furthermore showed CB2 localization in close proximity to p62 positive vesicles at the cell membrane. In summary, we provide a comprehensive repository of the CB2 interactome in HEK293 cells identified by a systematic unbiased approach, which can be used in future experiments to decipher the signaling and trafficking complex of this cannabinoid receptor. Future studies will have to analyze the exact mechanism of the p62-CB2 interaction as well as its putative role in disease pathophysiology., (Copyright © 2019 Sharaf, Mensching, Keller, Rading, Scheffold, Palkowitsch, Djogo, Rezgaoui, Kestler, Moepps, Failla and Karsak.)

The G-protein coupled cannabinoid receptor 2 (CB2) has been implicated in the regulation of adult neurogenesis in the hippocampus. The contribution of CB2 towards basal levels of proliferation and the number of neural progenitors in the subgranular zone (SGZ) of the dentate gyrus, however, remain unclear. We stained hippocampal brain sections of 16- to 17-week-old wildtype and CB2-deficient mice, for neural progenitor and immature neuron markers doublecortin (DCX) and calretinin (CR) and for the proliferation marker Ki67 and quantified the number of positive cells in the SGZ. The quantification revealed that CB2 deficiency neither altered overall cell proliferation nor the size of the DCX+ or DCX and CR double-positive populations in the SGZ compared to control animals. The results indicate that CB2 might not contribute to basal levels of adult neurogenesis in four-month-old healthy mice. CB2 signaling might be more relevant in conditions where adult neurogenesis is dynamically regulated, such as neuroinflammation.

The activator of G protein signalling AGS2 (Tctex-1) forms protein complexes with Gβγ, and controls cell proliferation by regulating cell cycle progression. A direct interaction of Tctex-1 with various G protein-coupled receptors has been reported. Since the carboxyl terminal portion of CB2 carries a putative Tctex-1 binding motif, we investigated the potential interplay of CB2 and Tctex-1 in the absence and presence of Gβγ. The supposed interaction of cannabinoid receptor CB2 with Tctex-1 and the influence of CB2 on the formation of Tctex-1-Gβγ-complexes were studied by co- and/or immunoprecipitation experiments in transiently transfected HEK293 cells. The analysis on Tctex-1 protein was performed in the absence and presence of the ligands JWH 133, 2-AG, and AM 630, the protein biosynthesis inhibitor cycloheximide or the protein degradation blockers MG132, NH4Cl/leupeptin or bafilomycin. Our results show that CB2 neither directly nor indirectly via Gβγ interacts with Tctex-1, but competes with Tctex-1 in binding to Gβγ. The Tctex-1-Gβγ protein interaction was disrupted by CB2 receptor expression resulting in a release of Tctex-1 from the complex, and its degradation by the proteasome and partly by lysosomes. The decrease in Tctex-1 protein levels is induced by CB2 expression "dose-dependently" and is independent of stimulation by agonist or blocking by an inverse agonist treatment. The results suggest that CB2 receptor expression independent of its activation by agonists is sufficient to competitively disrupt Gβγ-Tctex-1 complexes, and to initiate Tctex-1 degradation. These findings implicate that CB2 receptor expression modifies the stability of intracellular protein complexes by a non-canonical pathway., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Introduction: Failure of acetabular components often leads to bone loss with extensive elongated defects in the surrounding bone. In these cases, reconstruction is challenging and stable fixation of the revision implant difficult. The use of an oblong cup has been described as an option for acetabular reconstruction in such revisions. We report the first long-term results obtained with this implant to date., Materials and Methods: Fifty-six longitudinal oblong revision cups (LOR) were evaluated clinically and radiologically after a follow-up of 8-12 years (average 9 years). The defects treated with the LOR cup ranged from Paprosky type 1-3. Allogenic cancellous bone chips were additionally used in 31 reconstructions to fill cavitary defects., Results: Based on radiological criteria, 50 acetabular implants underwent osseointegration without any definitive signs of loosening; 2 consistently exhibited zonal radiolucent lines that were always smaller than 2 mm, 1 migrated by around 3 mm. None of these cases exhibited any clinical symptoms. In 11 cases where acetabular defects manifested postoperatively, 8 were remodeled completely and 3 partially at final follow-up. Three revision implants migrated farther than 5 mm and had to be revised before 32-month follow-up. In addition, 1 septic implant failure occurred. After an average follow-up of 9 years, 93% of the investigated implants remained in situ without further revision and 95% without aseptic implant failure., Conclusion: This 12-year clinical study demonstrates that the LOR cup offers a successful concept for the revision of failed acetabular components that also promotes the biological reconstruction of bony defects. Compared with other methods with similarly long follow-ups, our long-term results prove this procedure has a very low rate of revision and aseptic implant failure.