Pardes Habib

All Publications

• In Vivo Polymer Mechanochemistry with Polynucleotides. Advanced materials (Deerfield Beach, Fla.) Ishaqat, A., Hahmann, J., Lin, C., Zhang, X., He, C., Rath, W. H., Habib, P., Sahnoun, S. E., Rahimi, K., Vinokur, R., Mottaghy, F. M., Göstl, R., Bartneck, M., Herrmann, A. 2024: e2403752

  Abstract

  Polymer mechanochemistry utilizes mechanical force to activate latent functionalities in macromolecules and widely relies on ultrasonication techniques. Fundamental constraints of frequency and power intensity have prohibited the application of the polymer mechanochemistry principles in a biomedical context up to now, although medical ultrasound is a clinically established modality. Here, a universal polynucleotide framework is presented that allows the binding and release of therapeutic oligonucleotides, both DNA- and RNA-based, as cargo by biocompatible medical imaging ultrasound. It is shown that the high molar mass, colloidal assembly, and a distinct mechanochemical mechanism enable the force-induced release of cargo and subsequent activation of biological function in vitro and in vivo. Thereby, we introduce a platform for the exploration of biological questions and therapeutics development steered by mechanical force. This article is protected by copyright. All rights reserved.

  View details for DOI 10.1002/adma.202403752

  View details for PubMedID 38804595

• The prostate-specific membrane antigen holds potential as a vascular target for endogenous radiotherapy with [177Lu]Lu-PSMA-I&T for triple-negative breast cancer. Breast cancer research : BCR Heesch, A., Florea, A., Maurer, J., Habib, P., Werth, L. S., Hansen, T., Stickeler, E., Sahnoun, S. E., Mottaghy, F. M., Morgenroth, A. 2024; 26 (1): 30

  Abstract

  Overexpression of prostate-specific membrane antigen (PSMA) on the vasculature of triple-negative breast cancer (TNBC) presents a promising avenue for targeted endogenous radiotherapy with [177Lu]Lu-PSMA-I&T. This study aimed to assess and compare the therapeutic efficacy of a single dose with a fractionated dose of [177Lu]Lu-PSMA-I&T in an orthotopic model of TNBC.Rj:NMRI-Foxn1nu/nu mice were used as recipients of MDA-MB-231 xenografts. The single dose group was treated with 1 × 60 ± 5 MBq dose of [177Lu]Lu-PSMA-I&T, while the fractionated dose group received 4 × a 15 ± 2 MBq dose of [177Lu]Lu-PSMA-I&T at 7 day intervals. The control group received 0.9% NaCl. Tumor progression was monitored using [18F]FDG-PET/CT. Ex vivo analysis encompassed immunostaining, TUNEL staining, H&E staining, microautoradiography, and autoradiography.Tumor volumes were significantly smaller in the single dose (p < 0.001) and fractionated dose (p < 0.001) groups. Tumor growth inhibition rates were 38% (single dose) and 30% (fractionated dose). Median survival was notably prolonged in the treated groups compared to the control groups (31d, 28d and 19d for single dose, fractionated dose and control, respectively). [177Lu]Lu-PSMA-I&T decreased the size of viable tumor areas. We further demonstrated, that [177Lu]Lu-PSMA-I&T binds specifically to the tumor-associated vasculature.This study highlights the potential of [177Lu]Lu-PSMA-I&T for endogenous radiotherapy of TNBC.

  View details for DOI 10.1186/s13058-024-01787-9

  View details for PubMedID 38378689

  View details for PubMedCentralID 9317013

• Point-of-Care Ultrasound to Detect Acute Large Vessel Occlusions in Stroke Patients: A Proof-of-Concept Study. The Canadian journal of neurological sciences. Le journal canadien des sciences neurologiques Habib, P., Dimitrov, I., Pinho, J., Schürmann, K., Bach, J. P., Wiesmann, M., Schulz, J. B., Reich, A., Nikoubashman, O. 2023; 50 (5): 656-661

  Abstract

  A primary admission of patients with suspected acute ischemic stroke and large vessel occlusion (LVO) to centers capable of providing endovascular stroke therapy (EVT) may induce shorter time to treatment and better functional outcomes. One of the limitations in this strategy is the need for accurately identifying LVO patients in the prehospital setting. We aimed to study the feasibility and diagnostic performance of point-of-care ultrasound (POCUS) for the detection of LVO in patients with acute stroke.We conducted a proof-of-concept study and selected 15 acute ischemic stroke patients with angiographically confirmed LVO and 15 patients without LVO. Duplex ultrasonography (DUS) of the common carotid arteries was performed, and flow profiles compatible with LVO were scored independently by one experienced and one junior neurologist.Among the 15 patients with LVO, 6 patients presented with an occlusion of the carotid-T and 9 patients presented with an M1 occlusion. Interobserver agreement between the junior and the experienced neurologist was excellent (kappa = 0.813, p < 0.001). Flow profiles of the CAA allowed the detection of LVO with a sensitivity of 73%, a positive predictive value of 92 and 100%, and a c-statistics of 0.83 (95%CI = 0.65-0.94) and 0.87 (95%CI = 0.69-0.94) (experienced neurologist and junior neurologist, respectively). In comparison with clinical stroke scales, DUS was associated with better trade-off between sensitivity and specificity.POCUS in acute stroke setting is feasible, it may serve as a complementary tool for the detection of LVO and is potentially applicable in the prehospital phase.

  View details for DOI 10.1017/cjn.2022.275

  View details for PubMedID 35872570

• Small-molecule modulators of TRMT2A decrease PolyQ aggregation and PolyQ-induced cell death. Computational and structural biotechnology journal Margreiter, M. A., Witzenberger, M., Wasser, Y., Davydova, E., Janowski, R., Metz, J., Habib, P., Sahnoun, S. E., Sobisch, C., Poma, B., Palomino-Hernandez, O., Wagner, M., Carell, T., Jon Shah, N., Schulz, J. B., Niessing, D., Voigt, A., Rossetti, G. 2022; 20: 443-458

  Abstract

  Polyglutamine (polyQ) diseases are characterized by an expansion of cytosine-adenine-guanine (CAG) trinucleotide repeats encoding for an uninterrupted prolonged polyQ tract. We previously identified TRMT2A as a strong modifier of polyQ-induced toxicity in an unbiased large-scale screen in Drosophila melanogaster. This work aimed at identifying and validating pharmacological TRMT2A inhibitors as treatment opportunities for polyQ diseases in humans. Computer-aided drug discovery was implemented to identify human TRMT2A inhibitors. Additionally, the crystal structure of one protein domain, the RNA recognition motif (RRM), was determined, and Biacore experiments with the RRM were performed. The identified molecules were validated for their potency to reduce polyQ aggregation and polyQ-induced cell death in human HEK293T cells and patient derived fibroblasts. Our work provides a first step towards pharmacological inhibition of this enzyme and indicates TRMT2A as a viable drug target for polyQ diseases.

  View details for DOI 10.1016/j.csbj.2021.12.029

  View details for PubMedID 35070167

  View details for PubMedCentralID PMC8759985

• Standardized hypoxia-reoxygenation protocol to assess posthypoxic neurobehavioral impairments and molecular mechanisms in Drosophila melanogaster. STAR protocols Jung, J., Fehr, A. T., Voigt, A., Habib, P. 2022; 3 (3): 101634

  Abstract

  Hypoxia plays a pivotal role in the pathogenesis of major causes of mortality such as cerebral ischemia. Here, we present a standardized protocol for the induction of global hypoxia and reoxygenation in Drosophila melanogaster, with details on subsequent analysis of mortality, neurobehavioral impairments, and molecular mechanisms. This protocol emphasizes the importance of controlling and monitoring specific environmental parameters to ensure reproducible results. It also highlights profound differences that can arise from variations in the age and genotype of the flies. For complete details on the use and execution of this protocol, please refer to Habib et al. (2021).

  View details for DOI 10.1016/j.xpro.2022.101634

  View details for PubMedID 36035795

  View details for PubMedCentralID PMC9405534

• Erythropoietin Enhances Post-ischemic Migration and Phagocytosis and Alleviates the Activation of Inflammasomes in Human Microglial Cells. Frontiers in cellular neuroscience Arik, E., Heinisch, O., Bienert, M., Gubeljak, L., Slowik, A., Reich, A., Schulz, J. B., Wilhelm, T., Huber, M., Habib, P. 2022; 16: 915348

  Abstract

  Recombinant human erythropoietin (rhEPO) has been shown to exert anti-apoptotic and anti-inflammatory effects after cerebral ischemia. Inflammatory cytokines interleukin-1β and -18 (IL-1β and IL-18) are crucial mediators of apoptosis and are maturated by multiprotein complexes termed inflammasomes. Microglia are the first responders to post-ischemic brain damage and are a main source of inflammasomes. However, the impact of rhEPO on microglial activation and the subsequent induction of inflammasomes after ischemia remains elusive. To address this, we subjected human microglial clone 3 (HMC-3) cells to various durations of oxygen-glucose-deprivation/reperfusion (OGD/R) to assess the impact of rhEPO on cell viability, metabolic activity, oxidative stress, phagocytosis, migration, as well as on the regulation and activation of the NLRP1, NLRP3, NLRC4, and AIM2 inflammasomes. Administration of rhEPO mitigated OGD/R-induced oxidative stress and cell death. Additionally, it enhanced metabolic activity, migration and phagocytosis of HMC-3. Moreover, rhEPO attenuated post-ischemic activation and regulation of the NLRP1, NLRP3, NLRC4, and AIM2 inflammasomes as well as their downstream effectors CASPASE1 and IL-1β. Pharmacological inhibition of NLRP3 via MCC950 had no effect on the activation of CASPASE1 and maturation of IL-1β after OGD/R, but increased protein levels of NLRP1, NLRC4, and AIM2, suggesting compensatory activities among inflammasomes. We provide evidence that EPO-conveyed anti-inflammatory actions might be mediated via the regulation of the inflammasomes.

  View details for DOI 10.3389/fncel.2022.915348

  View details for PubMedID 35813499

  View details for PubMedCentralID PMC9263298

• Erythropoietin Abrogates Post-Ischemic Activation of the NLRP3, NLRC4, and AIM2 Inflammasomes in Microglia/Macrophages in a TAK1-Dependent Manner. Translational stroke research Heinisch, O., Zeyen, T., Goldmann, T., Prinz, M., Huber, M., Jung, J., Arik, E., Habib, S., Slowik, A., Reich, A., Schulz, J. B., Habib, P. 2022; 13 (3): 462-482

  Abstract

  Inflammasomes are known to contribute to brain damage after acute ischemic stroke (AIS). TAK1 is predominantly expressed in microglial cells and can regulate the NLRP3 inflammasome, but its impact on other inflammasomes including NLRC4 and AIM2 after AIS remains elusive. EPO has been shown to reduce NLRP3 protein levels in different disease models. Whether EPO-mediated neuroprotection after AIS is conveyed via an EPO/TAK1/inflammasome axis in microglia remains to be clarified. Subjecting mice deficient for TAK1 in microglia/macrophages (Mi/MΦ) to AIS revealed a significant reduction in infarct sizes and neurological impairments compared to the corresponding controls. Post-ischemic increased activation of TAK1, NLRP3, NLRC4, and AIM2 inflammasomes including their associated downstream cascades were markedly reduced upon deletion of Mi/MΦ TAK1. EPO administration improved clinical outcomes and dampened stroke-induced activation of TAK1 and inflammasome cascades, which was not evident after the deletion of Mi/MΦ TAK1. Pharmacological inhibition of NLRP3 in microglial BV-2 cells did not influence post-OGD IL-1β levels, but increased NLRC4 and AIM2 protein levels, suggesting compensatory activities among inflammasomes. Overall, we provide evidence that Mi/MΦ TAK1 regulates the expression and activation of the NLRP3, NLRC4, AIM2 inflammasomes. Furthermore, EPO mitigated stroke-induced activation of TAK1 and inflammasomes, indicating that EPO conveyed neuroprotection might be mediated via an EPO/TAK1/inflammasome axis.

  View details for DOI 10.1007/s12975-021-00948-8

  View details for PubMedID 34628598

  View details for PubMedCentralID PMC9046144

• Increased Post-Hypoxic Oxidative Stress and Activation of the PERK Branch of the UPR in Trap1-Deficient Drosophila melanogaster Is Abrogated by Metformin. International journal of molecular sciences Kokott-Vuong, A., Jung, J., Fehr, A. T., Kirschfink, N., Noristani, R., Voigt, A., Reich, A., Schulz, J. B., Huber, M., Habib, P. 2021; 22 (21)

  Abstract

  Hypoxia is known to impair mitochondrial and endoplasmic reticulum (ER) homeostasis. Post-hypoxic perturbations of the ER proteostasis result in the accumulation of misfolded/unfolded proteins leading to the activation of the Unfolded Protein Response (UPR). Mitochondrial chaperone TNF receptor-associated protein 1 (TRAP1) is reported to preserve mitochondrial membrane potential and to impede reactive oxygen species (ROS) production thereby protecting cells from ER stress as well as oxidative stress. The first-line antidiabetic drug Metformin has been attributed a neuroprotective role after hypoxia. Interestingly, Metformin has been reported to rescue mitochondrial deficits in fibroblasts derived from a patient carrying a homozygous TRAP1 loss-of-function mutation. We sought to investigate a putative link between Metformin, TRAP1, and the UPR after hypoxia. We assessed post-hypoxic/reperfusion longevity, mortality, negative geotaxis, ROS production, metabolic activity, gene expression of antioxidant proteins, and activation of the UPR in Trap1-deficient flies. Following hypoxia, Trap1 deficiency caused higher mortality and greater impairments in negative geotaxis compared to controls. Similarly, post-hypoxic production of ROS and UPR activation was significantly higher in Trap1-deficient compared to control flies. Metformin counteracted the deleterious effects of hypoxia in Trap1-deficient flies but had no protective effect in wild-type flies. We provide evidence that TRAP1 is crucially involved in the post-hypoxic regulation of mitochondrial/ER stress and the activation of the UPR. Metformin appears to rescue Trap1-deficiency after hypoxia mitigating ROS production and downregulating the pro-apoptotic PERK (protein kinase R-like ER kinase) arm of the UPR.

  View details for DOI 10.3390/ijms222111586

  View details for PubMedID 34769067

  View details for PubMedCentralID PMC8583878

• CK1BP Reduces α-Synuclein Oligomerization and Aggregation Independent of Serine 129 Phosphorylation. Cells Elsholz, L., Wasser, Y., Ziegler, P., Habib, P., Voigt, A. 2021; 10 (11)

  Abstract

  The pathological accumulation of α-Synuclein (α-Syn) is the hallmark of neurodegenerative α-synucleinopathies, including Parkinsons's disease (PD). In contrast to the mostly non-phosphorylated soluble α-Syn, aggregated α-Syn is usually phosphorylated at serine 129 (S129). Therefore, S129-phosphorylation is suspected to interfere with α-Syn aggregation. Among other kinases, protein kinase CK1 (CK1) is known to phosphorylate α-Syn at S129. We overexpressed CK1 binding protein (CK1BP) to inhibit CK1 kinase activity. Using Bimolecular Fluorescence Complementation (BiFC) in combination with biochemical methods, we monitored the S129 phosphorylation and oligomerization of α-Syn in HEK293T cells. We found that CK1BP reduced the overall protein levels of α-Syn. Moreover, CK1BP concomitantly reduced S129 phosphorylation, oligomerization and the amount of insoluble α-Syn. Analyzing different α-Syn variants including S129 mutations, we show that the effects of CK1BP on α-Syn accumulation were independent of S129 phosphorylation. Further analysis of an aggregating polyglutamine (polyQ) protein confirmed a phosphorylation-independent decrease in aggregation. Our results imply that the inhibition of CK1 activity by CK1BP might exert beneficial effects on NDDs in general. Accordingly, CK1BP represents a promising target for the rational design of therapeutic approaches to cease or at least delay the progression of α-synucleinopathies.

  View details for DOI 10.3390/cells10112830

  View details for PubMedID 34831053

  View details for PubMedCentralID PMC8616157

• Dehydroepiandrosterone (DHEA) Serum Levels Indicate Cerebrospinal Fluid Levels of DHEA and Estradiol (E2) in Women at Term Pregnancy. Reproductive sciences (Thousand Oaks, Calif.) Habib, P., Neulen, J., Habib, S., Rösing, B. 2021; 28 (10): 2823-2829

  Abstract

  Neuroactive steroids such as dehydroepiandrosterone (DHEA), estradiol (E2), and progesterone (P4) are associated with structural and functional changes in the central nervous system (CNS). Measurement of steroid levels in the CNS compartments is restricted in accessibility. Consequently, there is only limited human data on the distributional equilibrium for steroid levels between peripheral and central compartments. While some neuroactive steroids including DHEA and E2 have been reported to convey excitatory and proconvulsant properties, the opposite was demonstrated for P4. We aimed to elucidate the correlation between peripheral and central DHEA, E2, and P4 levels in women at term pregnancy. CSF and serum samples of 27 healthy pregnant women (22-39 years) at term pregnancy were collected simultaneously under combined spinal and epidural anesthesia and used for DHEA ELISA and E2, and P4 ECLIA. All three neuroactive steroids were detected at markedly lower levels in CSF compared to their corresponding serum concentrations (decrease, mean ± SD, 97.66 ± 0.83%). We found a strong correlation for DHEA between its serum and the corresponding CSF levels (r = 0.65, p = 0.003). Serum and CSF levels of E2 (r = 0.31, p = 0.12) appeared not to correlate in the investigated cohort. DHEA serum concentration correlated significantly with E2 (r = 0.58, p = 0.0016) in CSF. In addition, a strong correlation was found between DHEA and E2, both measured in CSF (r = 0.65, p = 0.0002). Peripheral DHEA levels might serve as an indicator for central nervous levels of the neuroactive steroids DHEA and E2 in pregnant women.

  View details for DOI 10.1007/s43032-021-00541-2

  View details for PubMedID 33772479

  View details for PubMedCentralID PMC8523509

• Alteration of miRNA Biogenesis Regulating Proteins in the Human Microglial Cell Line HMC-3 After Ischemic Stress. Molecular neurobiology Voelz, C., Habib, P., Köberlein, S., Beyer, C., Slowik, A. 2021; 58 (4): 1535-1549

  Abstract

  MicroRNAs (miRNA) are small noncoding sequences that control apoptosis, proliferation, and neuroinflammatory pathways in microglia cells. The expression of distinct miRNAs is altered after ischemia in the brain. Only minor information is available about the biogenesis and maturation of miRNAs after ischemia. We aimed at examining the impact of oxygen-glucose deprivation (OGD) and hydrogen peroxide (H2O2)-induced stress on the expression of miRNA regulating proteins such as DROSHA, DGCR8, XPO5, DICER, TARBP2, and AGO2 in the cultured human microglial cell line HMC-3 (human microglial cell line clone 3). OGD duration of 2.5 h or H2O2 stimulation at a concentration of 100 μM for 24 h resulted in a marked increase of the hypoxia sensor hypoxia-inducible factor1-α in HMC-3 cells. These treatments also led to an upregulation of DROSHA, DICER1, and AGO2 detected by semiquantitative real-time PCR (qrtPCR). XPO5 and TARBP2 were only upregulated after stimulation with H2O2, while DGCR8 responded only to OGD. We found elevated DICER1, DROSHA, and AGO2 protein levels by western blot and immunohistochemistry staining. Interestingly, the latter also exposed a colocalization of AGO2 with stress granules (G3BP1) after OGD. Our data indicate that DICER, DROSHA, and AGO2 are induced in microglial cells under hypoxia-like conditions. It might be speculated that their inductions might increase the miRNA synthesis rate. Future studies should investigate this correlation to determine which miRNAs are preferably expressed by microglia cells after ischemia and which functions they could exert.

  View details for DOI 10.1007/s12035-020-02210-y

  View details for PubMedID 33210205

  View details for PubMedCentralID 5363673

• Gaze Deviation and Paresis Score (GPS) Sufficiently Predicts Emergent Large Vessel Occluding Strokes. Journal of stroke and cerebrovascular diseases : the official journal of National Stroke Association Johannes, B., Habib, P., Schürmann, K., Nikoubashman, O., Wiesmann, M., Schulz, J. B., Reich, A. 2021; 30 (3): 105518

  Abstract

  The prognosis of patients with acute ischemic stroke (AIS) essentially depends on both prompt diagnosis and appropriate treatment. Endovascular stroke therapy (EST) proved to be highly efficient in the treatment of emergent large vessel occluding (ELVO) strokes in the anterior circulation. To achieve a timely diagnosis, a robust combination of few and simple signs to identify ELVOs in AIS patients applicable by paramedics in the prehospital triage is worthwhile.This retrospective single-center study included 904 AIS patients (324 ELVO, 580 non-ELVO) admitted between 2010 and 2015 in a tertiary stroke center. We re-evaluated two symptoms based on NIHSS items, gaze deviation and hemiparesis of the limbs ("Gaze deviation and Paresis Score, GPS") for the pre-hospital prediction of ELVO.A positive GPS AIS in patients predicted ELVO with a sensitivity of 0.89, specificity = 0.97, positive predictive value (PPV) = 0.95, negative predictive value (NPV) = 0.94 and diagnostic odds ratio (DOR) = 34.25 (CI: 20.75-56.53). The positive Likelihood-ratio (LR+) was 29.67, the negative Likelihood ratio (LR-) 0.11. NIHSS of patients with positive GPS (gaze palsy NIHSS ≥ 0, Motor arm NIHSS ≥2 and Motor leg NIHSS ≥2) was markedly higher compared to negative GPS patients (p < 0.001).The GPS proved to be similarly accurate in detecting ELVO in the anterior circulation of AIS patients and even more specific than other published clinical scores. Its simplicity and clarity might enable non-neurological medical staff to identify ELVO AIS patients with high certainty in a preclinical setting.

  View details for DOI 10.1016/j.jstrokecerebrovasdis.2020.105518

  View details for PubMedID 33388631

• Posthypoxic behavioral impairment and mortality of Drosophila melanogaster are associated with high temperatures, enhanced predeath activity and oxidative stress. Experimental & molecular medicine Habib, P., Jung, J., Wilms, G. M., Kokott-Vuong, A., Habib, S., Schulz, J. B., Voigt, A. 2021; 53 (2): 264-280

  Abstract

  Hypoxia is an underlying pathophysiological condition of a variety of devastating diseases, including acute ischemic stroke (AIS). We are faced with limited therapeutic options for AIS patients, and even after successful restoration of cerebral blood flow, the poststroke mortality is still high. More basic research is needed to explain mortality after reperfusion and to develop adjunct neuroprotective therapies. Drosophila melanogaster (D.m.) is a suitable model to analyze hypoxia; however, little is known about the impacts of hypoxia and especially of the subsequent reperfusion injury on the behavior and survival of D.m. To address this knowledge gap, we subjected two wild-type D.m. strains (Canton-S and Oregon-R) to severe hypoxia (<0.3% O2) under standardized environmental conditions in a well-constructed hypoxia chamber. During posthypoxic reperfusion (21% O2), we assessed fly activity (evoked and spontaneous) and analyzed molecular characteristics (oxidative stress marker abundance, reactive oxygen species (ROS) production, and metabolic activity) at various timepoints during reperfusion. First, we established standard conditions to induce hypoxia in D.m. to guarantee stable and reproducible experiments. Exposure to severe hypoxia under defined conditions impaired the climbing ability and reduced the overall activity of both D.m. strains. Furthermore, a majority of the flies died during the early reperfusion phase (up to 24 h). Interestingly, the flies that died early exhibited elevated activity before death compared to that of the flies that survived the entire reperfusion period. Additionally, we detected increases in ROS and stress marker (Catalase, Superoxide Dismutase and Heat Shock Protein 70) levels as well as reductions in metabolic activity in the reperfusion phase. Finally, we found that changes in environmental conditions impacted the mortality rate. In particular, decreasing the temperature during hypoxia or the reperfusion phase displayed a protective effect. In conclusion, our data suggest that reperfusion-dependent death might be associated with elevated temperatures, predeath activity, and oxidative stress.

  View details for DOI 10.1038/s12276-021-00565-3

  View details for PubMedID 33564101

  View details for PubMedCentralID PMC8080651

• Gonadal Hormones E2 and P Mitigate Cerebral Ischemia-Induced Upregulation of the AIM2 and NLRC4 Inflammasomes in Rats. International journal of molecular sciences Habib, P., Harms, J., Zendedel, A., Beyer, C., Slowik, A. 2020; 21 (13)

  Abstract

  Acute ischemic stroke (AIS) is a devastating neurological condition with a lack of neuroprotective therapeutic options, despite the reperfusion modalities thrombolysis and thrombectomy. Post-ischemic brain damage is aggravated by an excessive inflammatory cascade involving the activation and regulation of the pro-inflammatory cytokines IL-1β and IL-18 by inflammasomes. However, the role of AIM2 and NLRC4 inflammasomes and the influence of the neuroprotective steroids 17β-estradiol (E2) and progesterone (P) on their regulation after ischemic stroke have not yet been conclusively elucidated. To address the latter, we subjected a total of 65 rats to 1 h of transient Middle Cerebral Artery occlusion (tMCAO) followed by a reperfusion period of 72 h. Moreover, we evaluated the expression and regulation of AIM2 and NLRC4 in glial single-cell cultures (astroglia and microglia) after oxygen-glucose deprivation (OGD). The administration of E2 and P decreased both infarct sizes and neurological impairments after cerebral ischemia in rats. We detected a time-dependent elevation of gene and protein levels (Western Blot/immunohistochemistry) of the AIM2 and NLRC4 inflammasomes in the post-ischemic brains. E2 or P selectively mitigated the stroke-induced increase of AIM2 and NLRC4. While both inflammasomes seemed to be exclusively abundant in neurons under physiological and ischemic conditions in vivo, single-cell cultures of cortical astrocytes and microglia equally expressed both inflammasomes. In line with the in vivo data, E and P selectively reduced AIM2 and NLRC4 in primary cortical astrocytes and microglial cells after OGD. In conclusion, the post-ischemic elevation of AIM2 and NLRC4 and their down-regulation by E2 and P may shed more light on the anti-inflammatory effects of both gonadal hormones after stroke.

  View details for DOI 10.3390/ijms21134795

  View details for PubMedID 32645874

  View details for PubMedCentralID PMC7370209

• Microglial-specific depletion of TAK1 is neuroprotective in the acute phase after ischemic stroke. Journal of molecular medicine (Berlin, Germany) Zeyen, T., Noristani, R., Habib, S., Heinisch, O., Slowik, A., Huber, M., Schulz, J. B., Reich, A., Habib, P. 2020; 98 (6): 833-847

  Abstract

  Transforming growth factor-β-activated kinase 1 (TAK1) is upregulated after cerebral ischemia and contributes to an aggravation of brain injury. TAK1 acts as a key regulator of NF-ΚB and the MAP kinases JNK and p38 and modulates post-ischemic neuroinflammation and apoptosis. Microglia are the main TAK1-expressing immunocompetent cells of the brain. However, little is known about the function and regulation of microglial TAK1 after cerebral ischemia. Tamoxifen-dependent conditional depletion of TAK1 in microglial cells was induced in Cx3cr1creER-Tak1fl/fl mice. The creER-negative Tak1fl/fl mice and vehicle-treated (corn oil) mice served as control groups. A transient intraluminal middle cerebral artery occlusion of 30 min followed by 6 h and 72 h of reperfusion was performed in male mice. Oxygen-glucose-deprivation (OGD) was performed with primary cortical glial cell cultures to examine the effect of microglial-specific and general (5Z-7-Oxozeaenol) TAK1 inhibition after different reperfusion times (1 h, 6 h, and 72 h). Cx3cr1creER-Tak1fl/fl mice showed reduced infarct sizes and improved neurological outcomes compared to the control group. The mRNA and protein levels of pro-inflammatory Il1b/IL-1β and Tnf/TNF-α in the peri-infarct zones of microglial-specific TAK1-depleted mice were significantly reduced. Furthermore, TAK1 depletion in vitro led to reduced cell death rates after OGD. Moreover, hypoxia-mediated activation of TAK1 and its downstream signalling proteins, JNK and p38, were dampened by microglial TAK1 depletion. In contrast, 5Z-7-Oxozeaenol-induced pharmacological inhibition of TAK1 completely diminished MAPK-signalling including the kinases JNK and p38 in all cells. Microglial TAK1 depletion abrogates post-ischemic neuroinflammation and apoptosis in the acute phase, hence might be considered as a potential target in the treatment of cerebral hypoxia. KEY MESSAGES: TAK1 is activated after cerebral ischemia and induces MAP kinases p38 and JNK. Activated TAK1 increases apoptosis rate and the level pro-inflammatory cytokines IL-1β and TNF-α. Microglial cells seem to be the main source of TAK1-mediated post-ischemic neuroinflammation. Microglial-specific TAK1-depletion mediates sustainable neuroprotective effects, which might be superior to global TAK1 inhibition.

  View details for DOI 10.1007/s00109-020-01916-9

  View details for PubMedID 32382778

  View details for PubMedCentralID PMC7297861

• Differential use of BTK and PLC in FcεRI- and KIT-mediated mast cell activation: A marginal role of BTK upon KIT activation. Biochimica et biophysica acta. Molecular cell research Simonowski, A., Wilhelm, T., Habib, P., Zorn, C. N., Huber, M. 2020; 1867 (4): 118622

  Abstract

  In mast cells (MCs), the TEC family kinase (TFK) BTK constitutes a central regulator of antigen (Ag)-triggered, FcεRI-mediated PLCγ phosphorylation, Ca2+ mobilization, degranulation, and pro-inflammatory cytokine production. Less is known about the function of BTK in the context of stem cell factor (SCF)-induced KIT signaling. In bone marrow-derived MCs (BMMCs), Ag stimulation caused intense phosphorylation of BTK at Y551 in its active center and at Y223 in its SH3-domain, whereas in response to SCF only Y223 was significantly phosphorylated. Further data using the TFK inhibitor Ibrutinib indicated that BTK Y223 is phosphorylated by a non-BTK TFK upon SCF stimulation. In line, SCF-induced PLCγ1 phosphorylation was stronger attenuated by Ibrutinib than by BTK deficiency. Subsequent pharmacological analysis of PLCγ function revealed a total block of SCF-induced Ca2+ mobilization by PLC inhibition, whereas only the sustained phase of Ca2+ flux was curtailed in Ag-stimulated BMMCs. Despite this severe stimulus-dependent difference in inducing Ca2+ mobilization, PLCγ inhibition suppressed Ag- and SCF-induced degranulation and pro-inflammatory cytokine production to comparable extents, suggesting involvement of additional TFK(s) or PLCγ-dependent signaling components. In addition to PLCγ, the MAPKs p38 and JNK were activated by Ag in a BTK-dependent manner; this was not observed upon SCF stimulation. Hence, FcεRI and KIT employ different mechanisms for activating PLCγ, p38, and JNK, which might strengthen their cooperation regarding pro-inflammatory MC effector functions. Importantly, our data clearly demonstrate that analyzing BTK Y223 phosphorylation is not sufficient to prove BTK activation.

  View details for DOI 10.1016/j.bbamcr.2019.118622

  View details for PubMedID 31837347

• EPO and TMBIM3/GRINA Promote the Activation of the Adaptive Arm and Counteract the Terminal Arm of the Unfolded Protein Response after Murine Transient Cerebral Ischemia. International journal of molecular sciences Habib, P., Stamm, A. S., Schulz, J. B., Reich, A., Slowik, A., Capellmann, S., Huber, M., Wilhelm, T. 2019; 20 (21)

  Abstract

  Ischemic stroke is known to cause the accumulation of misfolded proteins and loss of calcium homeostasis leading to impairment of endoplasmic reticulum (ER) function. The unfolded protein response (UPR) is an ER-located and cytoprotective pathway that aims to resolve ER stress. Transmembrane BAX inhibitor-1 motif-containing (TMBIM) protein family member TMBIM3/GRINA is highly expressed in the brain and mostly located at the ER membrane suppressing ER calcium release by inositol-1,4,5-trisphosphate receptors. GRINA confers neuroprotection and is regulated by erythropoietin (EPO) after murine cerebral ischemia. However, the role of GRINA and the impact of EPO treatment on the post-ischemic UPR have not been elucidated yet. We subjected GRINA-deficient (Grina-/-) and wildtype mice to transient (30 min) middle cerebral artery occlusion (tMCAo) followed by 6 h or 72 h of reperfusion. We administered EPO or saline 0, 24 and 48 h after tMCAo/sham surgery. Oxygen-glucose deprivation (OGD) and pharmacological stimulation of the UPR using Tunicamycin and Thapsigargin were carried out in primary murine cortical mixed cell cultures. Treatment with the PERK-inhibitor GSK-2606414, IRE1a-RNase-inhibitor STF-083010 and EPO was performed 1 h prior to either 1 h, 2 h or 3 h of OGD. We found earlier and larger infarct demarcations in Grina-/- mice compared to wildtype mice, which was accompanied by a worse neurological outcome and an abolishment of EPO-mediated neuroprotection after ischemic stroke. In addition, GRINA-deficiency increased apoptosis and the activation of the corresponding PERK arm of the UPR after stroke. EPO enhanced the post-ischemic activation of pro-survival IRE1a and counteracted the pro-apoptotic PERK branch of the UPR. Both EPO and the PERK-inhibitor GSK-2606414 reduced cell death and regulated Grina mRNA levels after OGD. In conclusion, GRINA plays a crucial role in post-ischemic UPR and the use of both GSK-2606414 and EPO might lead to neuroprotection.

  View details for DOI 10.3390/ijms20215421

  View details for PubMedID 31683519

  View details for PubMedCentralID PMC6862264

• EPO regulates neuroprotective Transmembrane BAX Inhibitor-1 Motif-containing (TMBIM) family members GRINA and FAIM2 after cerebral ischemia-reperfusion injury. Experimental neurology Habib, P., Stamm, A. S., Zeyen, T., Noristani, R., Slowik, A., Beyer, C., Wilhelm, T., Huber, M., Komnig, D., Schulz, J. B., Reich, A. 2019; 320: 112978

  Abstract

  Transmembrane BAX Inhibitor-1 Motif-containing (TMBIM) family members exert inhibitory activities in apoptosis and necroptosis. FAIM2 (TMBIM-2) is neuroprotective against murine focal ischemia and is regulated by erythropoietin (EPO). Similar to FAIM2, GRINA (TMBIM-3) is predominantly expressed in the brain. The role of GRINA in transient brain ischemia, its potential synergistic effects with FAIM2 and its regulation by EPO treatment were assessed.We performed transient (30 min) middle cerebral artery occlusion (tMCAo) followed by 72 h of reperfusion in GRINA-deficient (GRINA-/-), FAIM2-deficient (FAIM2-/-), double-deficient (GRINA-/-FAIM2-/-) and wildtype littermates (WT) mice. We administered EPO or saline 0, 24 and 48 h after tMCAo. We subjected primary murine cortical neurons (pMCN) of all mouse strains to oxygen-glucose deprivation (OGD) after GRINA and/or FAIM2 gene transfection.Compared to wildtype controls GRINA deficiency led to a similar increase in infarct volumes as FAIM2 deficiency (p < .01). We observed the highest neurological deficits and largest infarct sizes in double-deficient mice. EPO administration upregulated GRINA and FAIM2 mRNA levels in wildtype littermates. EPO decreased infarct sizes and abrogated neurological impairments in wildtype controls. GRINA and/or FAIM2 deficient mice showed increased expression levels of cleaved-caspase 3 and of pro-apoptotic BAX mRNA. Further, caspase 8 was upregulated in FAIM2-/- and caspase 9 in GRINA-/- mice. Overexpression of GRINA and FAIM2 in wildtype and in double deficient pMCN decreased cell death rate after OGD.GRINA and FAIM2 are highly expressed in the brain and convey EPO-mediated neuroprotection after ischemic stroke involving different caspases.

  View details for DOI 10.1016/j.expneurol.2019.112978

  View details for PubMedID 31211943

• α1-antitrypsin mitigates NLRP3-inflammasome activation in amyloid β1-42-stimulated murine astrocytes. Journal of neuroinflammation Ebrahimi, T., Rust, M., Kaiser, S. N., Slowik, A., Beyer, C., Koczulla, A. R., Schulz, J. B., Habib, P., Bach, J. P. 2018; 15 (1): 282

  Abstract

  Neuroinflammation has an essential impact on the pathogenesis and progression of Alzheimer's disease (AD). Mostly mediated by microglia and astrocytes, inflammatory processes lead to degeneration of neuronal cells. The NLRP3-inflammasome (NOD-like receptor family, pyrin domain containing 3) is a key component of the innate immune system and its activation results in secretion of the proinflammatory effectors interleukin-1β (IL-1β) and interleukin-18 (IL-18). Under physiological conditions, cytosolic NLRP3-inflammsome is maintained in an inactive form, not able to oligomerize. Amyloid β1-42 (Aβ1-42) triggers activation of NLRP3-inflammasome in microglia and astrocytes, inducing oligomerization and thus recruitment of proinflammatory proteases. NLRP3-inflammasome was found highly expressed in human brains diagnosed with AD. Moreover, NLRP3-deficient mice carrying mutations associated with familial AD were partially protected from deficits associated with AD. The endogenous protease inhibitor α1-antitrypsin (A1AT) is known for its anti-inflammatory and anti-apoptotic properties and thus could serve as therapeutic agent for NLRP3-inhibition. A1AT protects neurons from glutamate-induced toxicity and reduces Aβ1-42-induced inflammation in microglial cells. In this study, we investigated the effect of Aβ1-42-induced NLRP3-inflammasome upregulation in primary murine astrocytes and its regulation by A1AT.Primary cortical astrocytes from BALB/c mice were stimulated with Aβ1-42 and treated with A1AT. Regulation of NLRP3-inflammasome was examined by immunocytochemistry, PCR, western blot and ELISA. Our studies included an inhibitor of NLRP3 to elucidate direct interactions between A1AT and NLRP3-inflammasome components.Our study revealed that A1AT reduces Aβ1-42-dependent upregulation of NLRP3 at the mRNA and protein levels. Furthermore, A1AT time-dependently mitigated the expression of caspase 1 and its cleavage product IL-1β in Aβ1-42-stimulated astrocytes.We conclude that Aβ1-42-stimulation results in an upregulation of NLRP3, caspase 1, and its cleavage products in astrocytes. A1AT time-dependently hampers neuroinflammation by downregulation of Aβ1-42-mediated NLRP3-inflammasome expression and thus may serve as a pharmaceutical opportunity for the treatment of Alzheimer's disease.

  View details for DOI 10.1186/s12974-018-1319-x

  View details for PubMedID 30261895

  View details for PubMedCentralID PMC6158809

• Estrogen serum concentration affects blood immune cell composition and polarization in human females under controlled ovarian stimulation. The Journal of steroid biochemistry and molecular biology Habib, P., Dreymueller, D., Rösing, B., Botung, H., Slowik, A., Zendedel, A., Habib, S., Hoffmann, S., Beyer, C. 2018; 178: 340-347

  Abstract

  Estrogens modulate the immune system and possess anti-inflammatory properties. In line, immune cells express a variety of estrogen receptors (ER) including ER-alpha and -beta. In the present study, we examined the influence of 17beta-estradiol (E2) serum concentrations on blood leukocyte composition and their ex vivo polarization/activation status by FACS analysis in sub-fertile human females under controlled ovarian stimulation (COS). Using a set of cell-type and polarization-specific markers, we demonstrate that increased 17ß-estradiol (E2) serum concentrations yield an overall increase in leukocytes, neutrophils and monocytes but decreased lymphocytes. There was a clear ratio shift towards an increase in M2 monocytes with a protective quality and an increase in T-helper cells compared to a decrease in cytotoxic T-cells. These data support experimental findings and clinical trials, i.e. related to multiple sclerosis and other autoimmune-related diseases, that have shown a down-regulation of CD8(+) T cells and up-regulation of T-regulatory cells. Further studies have to pinpoint to which extent the immune system/-responsiveness of otherwise healthy female patients is affected by medium-term systemic E2 variations.

  View details for DOI 10.1016/j.jsbmb.2018.02.005

  View details for PubMedID 29448043

• Regulation of brain microglia by female gonadal steroids. The Journal of steroid biochemistry and molecular biology Habib, P., Beyer, C. 2015; 146: 3-14

  Abstract

  Microglial cells are the primary mediators of the CNS immune defense system and crucial for shaping inflammatory responses. They represent a highly dynamic cell population which is constantly moving and surveying their environment. Acute brain damage causes a local attraction and activation of this immune cell type which involves neuron-to-glia and glia-to-glia interactions. The prevailing view attributes microglia a "negative" role such as defense and debris elimination. More topical studies also suggest a protective and "positive" regulatory function. Estrogens and progestins exert anti-inflammatory and neuroprotective effects in the CNS in acute and chronic brain diseases. Recent work revealed that microglial cells express subsets of classical and non-classical estrogen and progesterone receptors in a highly dynamic way. In this review article, we would like to stress the importance of microglia for the spreading of neural damage during hypoxia, their susceptibility to functional modulation by sex steroids, the potency of sex hormones to switch microglia from a pro-inflammatory M1 to neuroprotective M2 phenotype, and the regulation of pro- and anti-inflammatory properties including the inflammasome. We will further discuss the possibility that the neuroprotective action of sex steroids in the brain involves an early and direct modulation of local microglia cell function. This article is part of a Special Issue entitled 'Sex steroids and brain disorders'.

  View details for DOI 10.1016/j.jsbmb.2014.02.018

  View details for PubMedID 24607811

• Omega-3 polyunsaturated fatty acids ameliorate neuroinflammation and mitigate ischemic stroke damage through interactions with astrocytes and microglia. Journal of neuroimmunology Zendedel, A., Habib, P., Dang, J., Lammerding, L., Hoffmann, S., Beyer, C., Slowik, A. 2015; 278: 200-11

  Abstract

  Omega-3 polyunsaturated fatty acids (PUFA n3) provide neuroprotection due to their anti-inflammatory and anti-apoptotic properties as well as their regulatory function on growth factors and neuronal plasticity. These qualities enable PUFA n3 to ameliorate stroke outcome and limit neuronal damage. Young adult male rats received transient middle cerebral artery occlusion (tMCAO). PUFA n3 were intravenously administered into the jugular vein immediately after stroke and 12h later. We analyzed stroke volume and behavioral performance as well as the regulation of functionally-relevant genes in the penumbra. The extent of ischemic damage was reduced and behavioral performance improved subject to applied PUFA n3. Expression of Tau and growth-associated protein-43 genes were likewise restored. Ischemia-induced increase of cytokine mRNA levels was abated by PUFA n3. Using an in vitro approach, we demonstrate that cultured astroglial and microglia directly respond to PUFA n3 administration by preventing ischemia-induced increase of cyclooxygenase 2, hypoxia-inducible factor 1alpha, inducible nitric oxide synthase, and interleukin 1beta. Cultured cortical neurons also appeared as direct targets, since PUFA n3 shifted the Bcl-2-like protein 4 (Bax)/B-cell lymphoma 2 (Bcl 2) ratio towards an anti-apoptotic constellation. Thus, PUFA n3 reveal a high neuroprotective and anti-inflammatory potential in an acute ischemic stroke model by targeting astroglial and microglial function as well as improving neuronal survival strategies. Our findings signify the potential clinical feasibility of PUFA n3 therapeutic treatment in stroke and other acute neurological diseases.

  View details for DOI 10.1016/j.jneuroim.2014.11.007

  View details for PubMedID 25468770

• Hypoxia-induced gene expression of aquaporin-4, cyclooxygenase-2 and hypoxia-inducible factor 1α in rat cortical astroglia is inhibited by 17β-estradiol and progesterone. Neuroendocrinology Habib, P., Dang, J., Slowik, A., Victor, M., Beyer, C. 2014; 99 (3-4): 156-67

  Abstract

  17β-Estradiol (E2) and progesterone (P) are neuroprotective in acute brain injury by attenuating neuropathophysiological processes and regulating local glial function. Besides controlling brain-intrinsic immune responses, astrocytes are cellular targets for sex steroids in health and disease and typically resist to hypoxic damage. In this in vitro study, we aimed at uncovering astroglia-specific reactions to sublethal hypoxic conditions and astroglia-specific effects of both sex steroid hormones on these parameters. Short-term hypoxia for 3 h increased reactive oxygen species production, but had no influence on cell viability of cerebral cortical rat astroglia. Astrocytes expressed classical estrogen receptors (ER), progesterone receptor (PR), and a set of nonclassical steroid hormone receptors. Hypoxia specifically induced ERα and PR isoform A gene expression. Oxygen deprivation increased gene expression of aquaporin-4 (AQP4), hypoxia-inducible factor 1α (Hif1α), and cyclooxygenase-2 (COX2). The application of E2 and P selectively prevented this induction. Effects on protein levels of these genes appeared to be delayed. These data show that astrocytes change their receptivity for sex steroid hormones by switching steroid hormone receptor expression and that E2 and P modify or antagonize proinflammatory COX2 synthesis, edema-promoting AQP4 expression, and the Hif1α increase. In vivo studies have to address whether these cell responses contribute to steroid-mediated neuroprotection in stroke.

  View details for DOI 10.1159/000362279

  View details for PubMedID 24685982

• Regulation of hypoxia-induced inflammatory responses and M1-M2 phenotype switch of primary rat microglia by sex steroids. Journal of molecular neuroscience : MN Habib, P., Slowik, A., Zendedel, A., Johann, S., Dang, J., Beyer, C. 2014; 52 (2): 277-85

  Abstract

  Microglia cells are the primary mediators of the CNS immune defense system and crucial for the outcome of shaping inflammatory responses. They are highly dynamic, moving constantly, and become activated by neuronal signaling under pathological conditions. They fulfill a dual role by not only regulating local neuroinflammation but also conferring neuronal protection. Gonadal steroids are known to exert anti-inflammatory effects in the CNS. Recently, we have shown that the microglial-like cell line BV-2 is hypoxia-sensitive and regulated by gonadal steroids. The present study used primary rat cerebral cortex-derived microglia to analyze whether this cell type directly perceive and respond to acute hypoxia. Second, we investigated whether 17β-estradiol (E2) and progesterone (P) interfere with hypoxia-induced changes. Short-term hypoxia increased the expression of a subset of pro-inflammatory (TNFa, IL1b) and oxidative stress-related (Hif1a) genes. The induction of TNFa and IL1b was counteracted by P. Hypoxia shifted the primary microglia to the pro-inflammatory M1 phenotype. The administration of E2 and P favored the neuroprotective M2 phenotype. Our findings extend previous data obtained with BV-2 cells and show that the primary microglia directly perceive hypoxia which increase their inflammatory activity. Both steroid hormones directly and indirectly interact with the microglia cells by reducing the inflammatory scenario and stimulating neuroprotection.

  View details for DOI 10.1007/s12031-013-0137-y

  View details for PubMedID 24163150

• Sex steroid hormone-mediated functional regulation of microglia-like BV-2 cells during hypoxia. The Journal of steroid biochemistry and molecular biology Habib, P., Dreymueller, D., Ludwig, A., Beyer, C., Dang, J. 2013; 138: 195-205

  Abstract

  17β-estradiol (E2) and progesterone (P) are neuroprotective hormones in different neurological disorders and in particular under hypoxic conditions in the brain. Both hormones dampen brain-intrinsic immune responses and regulate local glial cell function. Besides astrocytes which are functionally regulated in a manifold and complex manner, especially microglial cells are in the focus of steroid-mediated neuroprotection. In previous studies using a transient brain artery occlusion model, we demonstrated that microglial characteristics are critically modified after the administration of either E2 or P. We here studied the influence of sex steroids on the murine BV-2 microglia cell line under hypoxic conditions. Hypoxia changed the cell morphology from an amoeboid-like phenotype with processes to a rounded shape of secreting cell type. BV-2 cells expressed both estrogen receptor-β and progesterone receptors under each condition. Oxygen deprivation increased the expression of inducible nitric oxide synthetase (iNOS) and up-regulated selected cytokines and chemokines. Both hormones selectively prevented the induction of pro-inflammatory iNOS, interleukin IL-1ß, and chemokine ligand CCL5, whereas anti-inflammatory IL-10 and protective TREM 2 were up-regulated by sex steroids. Sex hormones abrogated hypoxia-dependent reduction of BV-2 phagocytic activity. We demonstrate that BV-2 microglia cells respond to hypoxia by enhanced pro-inflammatory cytokine secretion and reduced phagocytic activity. This effect is prevented by sex steroids resulting in a switch of BV-2 cells from a pro-inflammatory to a more anti-inflammatory phenotype. Anti-inflammatory effects of gonadal steroids might directly be mediated through hormone-microglia interactions in addition to known effects via astroglial regulation.

  View details for DOI 10.1016/j.jsbmb.2013.06.003

  View details for PubMedID 23792783