Decreased mitochondrial priming determines chemoresistance of colon cancer stem cells

Tumor heterogeneity is in part determined by the existence of cancer stem cells (CSCs) and more differentiated tumor cells. CSCs are considered to be the tumorigenic root of cancers and suggested to be chemotherapy resistant. Here we exploited an assay that allowed us to measure chemotherapy-induced cell death in CSCs and differentiated tumor cells simultaneously. This confirmed that CSCs are selectively resistant to conventional chemotherapy, which we revealed is determined by decreased mitochondrial priming. In agreement, lowering the anti-apoptotic threshold using ABT-737 and WEHI-539 was sufficient to enhance chemotherapy efficacy, whereas ABT-199 failed to sensitize CSCs. Our data therefore point to a crucial role of BCLXL in protecting CSCs from chemotherapy and suggest that BH3 mimetics, in combination with chemotherapy, can be an efficient way to target chemotherapy-resistant CSCs.Colorectal cancer is the third most common cancer worldwide.^{1, 2} Patients with advanced stage colorectal cancer are routinely treated with 5-fluorouracil (5-FU), leucovorin and oxaliplatin (FOLFOX), or with 5-FU, leucovorin and irinotecan (FOLFIRI), often in combination with targeted agents such as anti-VEGF or anti-EGFR at metastatic disease.^{3, 4, 5, 6} Despite this intensive treatment, therapy is still insufficiently effective and chemotherapy resistance occurs frequently. Although still speculative, it has been suggested that unequal sensitivity to chemotherapy is due to an intratumoral heterogeneity that is orchestrated by cancer stem cells (CSCs) that can self-renew and give rise to more differentiated progeny.^{7, 8} When isolated from patients, CSCs efficiently form tumors upon xenotransplantation into mice which resemble the primary tumor from which they originated.^{9, 10, 11} In addition, many xenotransplantation studies have emphasized the importance of CSCs for tumor growth.^{9, 10, 11, 12} Colon CSCs were originally isolated from primary human colorectal tumor specimens using CD133 as cell surface marker and shown to be highly tumorigenic when compared with the non-CSCs population within a tumor.^{9, 10} Later, other cell surface markers as well as the activity of the Wnt pathway have been used to isolate colon CSCs from tumors.^{12, 13} Spheroid cultures have been established from human primary colorectal tumors that selectively enrich for the growth of colon CSCs,^{11, 12} although it is important to realize that these spheres also contain more differentiated tumor cells.¹² In agreement, we have shown that the Wnt activity reporter that directs the expression of enhanced green fluorescent protein (TOP-GFP) allows for the separation of CSCs from more differentiated progeny in the spheroid cultures.¹²CSCs are suggested to be responsible for tumor recurrence after initial therapy, as they are considered to be selectively resistant to therapy.^{11, 14} Conventional chemotherapy induces, among others, DNA damage and subsequent activation of the mitochondrial cell death pathway, which is regulated by a balance between pro- and anti-apoptotic BCL2 family members.¹⁵ Upon activation of apoptosis, pro-apoptotic BH3 molecules are activated and these may perturb the balance in favor of apoptosis initiated by mitochondrial outer membrane polarization (MOMP), release of cytochrome c and subsequent activation of a caspase cascade.The apoptotic balance of cancer cells can be measured with the use of a functional assay called BH3 profiling.¹⁶ BH3 profiling is a method to determine the apoptotic ‘priming'' level of a cell by exposing mitochondria to standardized amounts of roughly 20-mer peptides derived from the alpha-helical BH3 domains of BH3-only proteins and determining the rate of mitochondrial depolarization. Using this approach, priming was measured in various cancers and compared with normal tissues.^{17, 18} In all cancer types tested, the mitochondrial priming correlated well with the observed clinical response to chemotherapy. That is, cancers that are highly primed are more chemosensitive, whereas chemoresistant tumor cells and normal tissues are poorly primed.^{17, 18} This suggests that increasing mitochondrial priming can potentially increase chemosensitivity, which can be achieved by directly inhibiting the anti-apoptotic BCL2 family members.¹⁸ To this end, small-molecule inhibitors, so-called BH3 mimetics, have been developed. ABT-737 and the highly related ABT-263 both inhibit BCL2, BCLXL and BCLW^{19, 20, 21} and were shown to be effective in killing cancer cells in vitro and in vivo²¹ with a preference for BCL2.^{19, 22} As BCL2 protein expression is often upregulated in hematopoietic cancers, it represents a promising target, which was supported by high efficacy of these BH3 mimetics in animal experiments.²¹ However, in vivo efficacy is limited due to thrombocytopenia, which relates to a dependence of platelets on BCLXL for survival.^{23, 24} To overcome this toxicity, a BCL2-specific compound, ABT-199, was developed.²⁵ Souers et al.²⁵ showed that inhibition of BCL2 using ABT-199 blocks tumor growth of acute lymphoblastic leukemia cells in xenografts. In addition to the single compound effects of ABT-199, combination with rituximab inhibited growth of non-Hodgkin''s lymphoma, mantle cell lymphoma and acute lymphoblastic leukemia tumor cells growth in vivo.²⁵ Moreover, highly effective tumor lysis was observed in all three patients with chronic lymphocytic leukemia that were treated with ABT-199.²⁵ More recently, a BCLXL-specific compound, WEHI-539, was discovered using high-throughput chemical screening.²⁶As the apoptotic balance appears a useful target for the treatment of cancers and CSCs have been suggested to resist therapy selectively, we set out to analyze whether specifically colon CSCs are resistant to therapy and whether this is due to an enhanced anti-apoptotic threshold, specific to CSCs. To study chemosensitivity, we developed a robust single cell-based analysis in which we can measure apoptosis simultaneously in CSCs and their differentiated progeny. Utilizing this system we showed that colon CSCs and not their differentiated progeny are resistant to chemotherapeutic compounds and that this was due to the fact that these cells are less primed to mitochondrial death. Furthermore, inhibition of anti-apoptotic BCLXL molecule with either ABT-737 or WEHI-539, but not ABT-199, breaks this resistance and sensitizes the CSCs to chemotherapy.     

Acquisition of meiotic DNA repair regulators maintain genome stability in glioblastoma

Glioblastoma (GBM), the most prevalent type of primary intrinsic brain cancer in adults, remains universally fatal despite maximal therapy, including radiotherapy and chemotherapy. Cytotoxic therapy generates double-stranded DNA breaks (DSBs), most commonly repaired by homologous recombination (HR). We hypothesized that cancer cells coopt meiotic repair machinery as DSBs are generated during meiosis and repaired by molecular complexes distinct from genotoxic responses in somatic tissues. Indeed, we found that gliomas express meiotic repair genes and their expression informed poor prognosis. We interrogated the function of disrupted meiotic cDNA1 (DMC1), a homolog of RAD51, the primary recombinase used in mitotic cells to search and recombine with the homologous DNA template. DMC1, whose only known function is as an HR recombinase, was expressed by GBM cells and induced by radiation. Although targeting DMC1 in non-neoplastic cells minimally altered cell growth, DMC1 depletion in GBM cells decreased proliferation, induced activation of CHK1 and expression of p21^CIP1/WAF1, and increased RPA foci, suggesting increased replication stress. Combining loss of DMC1 with ionizing radiation inhibited activation of DNA damage responses and increased radiosensitivity. Furthermore, loss of DMC1 reduced tumor growth and prolonged survival in vivo. Our results suggest that cancers coopt meiotic genes to augment survival under genotoxic stress, offering molecular targets with high therapeutic indices.Glioblastomas (GBMs) rank among the deadliest of all human cancers, with only modest improvement in patient survival over recent decades. More than 12 000 GBM patients are diagnosed annually in the United States.^{1, 2} Despite aggressive treatment consisting of maximal safe surgical resection, concurrent radiotherapy and chemotherapy, and adjuvant chemotherapy, median survival remains dismal at 12–15 months.^{3, 4} Although numerous molecular targets have been identified in GBM, no molecularly targeted therapy has demonstrated a survival benefit. Radiotherapy remains the cornerstone of post-surgical GBM therapy with modest additional benefit offered by concurrent administration of the oral methylator, temozolomide. However, radioresistance and tumor recurrence is universal in GBM.^{4, 5, 6} Radiation also damages non-neoplastic brain tissue, resulting in cognitive impairment and decreased quality-of-life.⁷ Focal high-dose radiation reduces toxicity to non-neoplastic tissue, but tumor invasion into normal brain regions limits the survival benefit of highly focused radiotherapy techniques, like gamma knife and proton beam, establishing a need for improved combinatorial treatments, such as radiosensitizers.^{8, 9} To date, no radiosensitizer has successfully increased survival with acceptable toxicity in a clinical trial. Based on this background, we sought novel molecular targets that mediate responses to genotoxic stress and have limited function in normal cells.During mitosis, cells inspect the integrity of their DNA and repair replication errors through cell-state and error-specific mechanisms.¹⁰ Unrepaired or large regions of DNA damage overwhelm replication mechanisms to induce cell death.^{10, 11} DNA double-strand breaks (DSBs) are detrimental as they cause large-scale chromosomal rearrangements.¹⁰ The homologous recombination (HR) pathway is primarily used to repair DSBs during S- and G₂-phases, providing access to both sister and homologous chromosomes as repair templates.^{7, 12} RADiation sensitive 51 (RAD51) is a key recombinase important in HR and replication fork maintenance, functioning in both mitotic and meiotic cells.^{7, 12, 13, 14, 15} Phosphorylated RAD51 replaces replication protein A (RPA) upon DNA loading.¹⁶ Recombination mediated by RAD51 with the intact DNA template strand results in a relatively error-free repair.¹²In contrast to mitosis, germ cells undergoing meiosis actively generate genetic diversity through induction of programmed DSBs, which are repaired through HR.^{17, 18, 19} In meiotic HR, RAD51 functions in conjunction with the meiosis-specific recombinase, disrupted meiotic cDNA1 (DMC1). RAD51 and DMC1 are loaded onto DNA by a meiosis-specific accessory protein complex, homologous-pairing protein 2 (HOP2)–meiotic nuclear divisions 1 (MND1), to promote homologous strand invasion and dissociation-loop (D-loop) formation.^{20, 21} D-loops formed using the DMC1–RAD51 complex are more resistant to dissociation as opposed to D-loops formed by RAD51 alone, increasing the likelihood of DNA crossover events.²⁰ In addition, DMC1-directed crossovers preferentially utilize the homologous chromosome further increasing genetic variation.²²GBM cells commonly harbor genetic lesions that promote unrestrained proliferation but also stimulate genotoxic stress responses. Neoplastic cells do not require perfect fidelity of repair. In fact, dysfunctional repair accelerates genetic evolution of clones, but cancer cells must acquire mechanisms to bypass cell death or senescence in response to exogenous stressors.^{11, 23} Radiotherapy targets proliferating cancer cells by production of reactive oxygen species, leading to generation of DSBs and activation of the DNA damage response (DDR) pathway.^{11, 24} DSBs generated as a result of ionizing radiation (IR) are repaired through HR or non-homologous end joining (NHEJ).^{7, 12, 25, 26} Terminally differentiated neurons are post-mitotic and rely on NHEJ as a means to repair DNA DSBs. Therefore, inhibition of the NHEJ pathway may result in unfavorable normal neural cell toxicity.²⁶The HR pathway is an attractive target as it is linked to increased genetic variation and loss of heterozygosity (LOH).^{12, 27} Multiple HR checkpoints have been proposed as potential therapeutic targets for GBM.^{28, 29, 30, 31} Although the prognostic value of RAD51 expression in GBM is unresolved,^{29, 32, 33} RAD51 is consistently elevated in GBM compared with normal brain.³³ Reducing RAD51 expression radiosensitizes GBM cells,²⁹ but may have a limited therapeutic index because of the potentially toxic effects on non-neoplastic cells. In this study, we investigated the aberrant activity of meiotic HR regulators in glioma, focusing on the meiosis-specific DMC1. Activation of meiotic repair genes in neoplastic cells selectively provides tumor cells with a repair mechanism to evade cell death caused by DNA damage, yet increase genetic diversity to drive clonal evolution.     

A cellular screen identifies ponatinib and pazopanib as inhibitors of necroptosis

Necroptosis is a form of regulated necrotic cell death mediated by receptor-interacting serine/threonine-protein kinase 1 (RIPK1) and RIPK3. Necroptotic cell death contributes to the pathophysiology of several disorders involving tissue damage, including myocardial infarction, stroke and ischemia-reperfusion injury. However, no inhibitors of necroptosis are currently in clinical use. Here we performed a phenotypic screen for small-molecule inhibitors of tumor necrosis factor-alpha (TNF-α)-induced necroptosis in Fas-associated protein with death domain (FADD)-deficient Jurkat cells using a representative panel of Food and Drug Administration (FDA)-approved drugs. We identified two anti-cancer agents, ponatinib and pazopanib, as submicromolar inhibitors of necroptosis. Both compounds inhibited necroptotic cell death induced by various cell death receptor ligands in human cells, while not protecting from apoptosis. Ponatinib and pazopanib abrogated phosphorylation of mixed lineage kinase domain-like protein (MLKL) upon TNF-α-induced necroptosis, indicating that both agents target a component upstream of MLKL. An unbiased chemical proteomic approach determined the cellular target spectrum of ponatinib, revealing key members of the necroptosis signaling pathway. We validated RIPK1, RIPK3 and transforming growth factor-β-activated kinase 1 (TAK1) as novel, direct targets of ponatinib by using competitive binding, cellular thermal shift and recombinant kinase assays. Ponatinib inhibited both RIPK1 and RIPK3, while pazopanib preferentially targeted RIPK1. The identification of the FDA-approved drugs ponatinib and pazopanib as cellular inhibitors of necroptosis highlights them as potentially interesting for the treatment of pathologies caused or aggravated by necroptotic cell death.Programmed cell death has a crucial role in a variety of biological processes ranging from normal tissue development to diverse pathological conditions.^{1, 2} Necroptosis is a form of regulated cell death that has been shown to occur during pathogen infection or sterile injury-induced inflammation in conditions where apoptosis signaling is compromised.^{3, 4, 5, 6} Given that many viruses have developed strategies to circumvent apoptotic cell death, necroptosis constitutes an important, pro-inflammatory back-up mechanism that limits viral spread in vivo.^{7, 8, 9} In contrast, in the context of sterile inflammation, necroptotic cell death contributes to disease pathology, outlining potential benefits of therapeutic intervention.¹⁰ Necroptosis can be initiated by death receptors of the tumor necrosis factor (TNF) superfamily,¹¹ Toll-like receptor 3 (TLR3),¹² TLR4,¹³ DNA-dependent activator of IFN-regulatory factors¹⁴ or interferon receptors.¹⁵ Downstream signaling is subsequently conveyed via RIPK1¹⁶ or TIR-domain-containing adapter-inducing interferon-β,^{8, 17} and converges on RIPK3-mediated^{13, 18, 19, 20} activation of MLKL.²¹ Phosphorylated MLKL triggers membrane rupture,^{22, 23, 24, 25, 26} releasing pro-inflammatory cellular contents to the extracellular space.²⁷ Studies using the RIPK1 inhibitor necrostatin-1 (Nec-1) ²⁸ or RIPK3-deficient mice have established a role for necroptosis in the pathophysiology of pancreatitis,¹⁹ artherosclerosis,²⁹ retinal cell death,³⁰ ischemic organ damage and ischemia-reperfusion injury in both the kidney³¹ and the heart.³² Moreover, allografts from RIPK3-deficient mice are better protected from rejection, suggesting necroptosis inhibition as a therapeutic option to improve transplant outcome.³³ Besides Nec-1, several tool compounds inhibiting different pathway members have been described,^{12, 16, 21, 34, 35} however, no inhibitors of necroptosis are available for clinical use so far.^{2, 10} In this study we screened a library of FDA approved drugs for the precise purpose of identifying already existing and generally safe chemical agents that could be used as necroptosis inhibitors. We identified the two structurally distinct kinase inhibitors pazopanib and ponatinib as potent blockers of necroptosis targeting the key enzymes RIPK1/3.     

Blood Supply to the Chicken Femoral Head

The purpose of this study was to conduct a comprehensive evaluation of the vascular supply to the femoral head, including the vessels that give rise to the terminal perfusing branches. Using a casting agent, we highlighted the anatomy of the external iliac and ischiatic arteries with their associated branches after anatomic dissection of 24 hips from 12 Leghorn chickens. We confirmed published findings regarding perfusion of the femoral head and identified 3 previously undescribed arterial branches to this structure. The first branch (the acetabular branch of the femoralis artery) was supplied by the femoralis artery and directly perfused the acetabulum and femoral head. The second branch (the lateral retinacular artery) was a tributary of the femoralis artery that directly supplied the femoral head. Finally, we found that the middle femoral nutrient artery supplies a previously undescribed ascending intraosseous branch (the ascending branch of the middle femoral nutrient artery) that perfuses the femoral head. Precise understanding of the major vascular branches to the femoral head would allow for complete or selective ligation of its blood supply and enable the creation of a reproducible bipedal model of femoral head osteonecrosis.Like humans, chickens are bipedal animals that rely on the hip joint to absorb the majority of the body''s weight. This anatomy, in concert with their high activity level, makes chickens an attractive model for the study of osteonecrosis of the femoral head in humans. The vast majority of animal research on osteonecrosis of the femoral head has been performed on quadrupedal animals,^{3,4,10,19,25,26,28,29,31,36,37,41,51,52} thus limiting its application to bipedal species because most quadruped models fail to progress to end-stage mechanical collapse similar to that in humans.⁶Avascular necrosis is the death of bone that occurs from ischemia due to disruption of the vascular supply to bone through direct or indirect mechanisms.³⁸ Avascular necrosis should be differentiated from the broader term of osteonecrosis, which refers to bone death in general.³² Causes of femoral head osteonecrosis include direct and indirect disruption of vascular supply (traumatic injury, intravascular coagulation, extrinsic compression) as well as changes in cellular differentiation and cellular apoptosis.^{4,7,12,15,17,18,24,30-32,38,49,50} Accordingly, causes of osteonecrosis are both traumatic and nontraumatic.^16,31,32The arterial anatomy in the chicken hindlimb has been outlined by several authors.^{20,22,27,35,42,44,45} Briefly, the external iliac and ischiatic artery arise from the abdominal aorta to provide blood supply to the chicken hind limb. The external iliac artery has 2 main branches—the femoralis and femoral circumflex arteries—that distribute blood to the chicken hindlimb. The ischiatic artery provides 3 main branches: the trochanteric artery, superior femoral nutrient artery, and middle femoral nutrient artery. Although the terminal vascular supply to the femoral head of Leghorn and Broiler chickens has been described,^46,47 the origin of these terminal arteries with reference to the ischiatic and femoralis arteries and their respective branches has not been addressed. The current study will describe the blood vessels that feed these terminal branches to the chicken femoral head.     

Induction of COX-2-PGE2 synthesis by activation of the MAPK/ERK pathway contributes to neuronal death triggered by TDP-43-depleted microglia

Neuroinflammation is a striking hallmark of amyotrophic lateral sclerosis (ALS) and other neurodegenerative disorders. Previous studies have shown the contribution of glial cells such as astrocytes in TDP-43-linked ALS. However, the role of microglia in TDP-43-mediated motor neuron degeneration remains poorly understood. In this study, we show that depletion of TDP-43 in microglia, but not in astrocytes, strikingly upregulates cyclooxygenase-2 (COX-2) expression and prostaglandin E2 (PGE2) production through the activation of MAPK/ERK signaling and initiates neurotoxicity. Moreover, we find that administration of celecoxib, a specific COX-2 inhibitor, greatly diminishes the neurotoxicity triggered by TDP-43-depleted microglia. Taken together, our results reveal a previously unrecognized non-cell-autonomous mechanism in TDP-43-mediated neurodegeneration, identifying COX-2-PGE2 as the molecular events of microglia- but not astrocyte-initiated neurotoxicity and identifying celecoxib as a novel potential therapy for TDP-43-linked ALS and possibly other types of ALS.Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disease characterized by the degeneration of motor neurons in the brain and spinal cord.¹ Most cases of ALS are sporadic, but 10% are familial. Familial ALS cases are associated with mutations in genes such as Cu/Zn superoxide dismutase 1 (SOD1), TAR DNA-binding protein 43 (TARDBP) and, most recently discovered, C9orf72. Currently, most available information obtained from ALS research is based on the study of SOD1, but new studies focusing on TARDBP and C9orf72 have come to the forefront of ALS research.^{1, 2} The discovery of the central role of the protein TDP-43, encoded by TARDBP, in ALS was a breakthrough in ALS research.^{3, 4, 5} Although pathogenic mutations of TDP-43 are genetically rare, abnormal TDP-43 function is thought to be associated with the majority of ALS cases.¹ TDP-43 was identified as a key component of the ubiquitin-positive inclusions in most ALS patients and also in other neurodegenerative diseases such as frontotemporal lobar degeneration,^{6, 7} Alzheimer''s disease (AD)^{8, 9} and Parkinson''s disease (PD).^{10, 11} TDP-43 is a multifunctional RNA binding protein, and loss-of-function of TDP-43 has been increasingly recognized as a key contributor in TDP-43-mediated pathogenesis.^{5, 12, 13, 14}Neuroinflammation, a striking and common hallmark involved in many neurodegenerative diseases, including ALS, is characterized by extensive activation of glial cells including microglia, astrocytes and oligodendrocytes.^{15, 16} Although numerous studies have focused on the intrinsic properties of motor neurons in ALS, a large amount of evidence showed that glial cells, such as astrocytes and microglia, could have critical roles in SOD1-mediated motor neuron degeneration and ALS progression,^{17, 18, 19, 20, 21, 22} indicating the importance of non-cell-autonomous toxicity in SOD1-mediated ALS pathogenesis.Very interestingly, a vital insight of neuroinflammation research in ALS was generated by the evidence that both the mRNA and protein levels of the pro-inflammatory enzyme cyclooxygenase-2 (COX-2) are upregulated in both transgenic mouse models and in human postmortem brain and spinal cord.^{23, 24, 25, 26, 27, 28, 29} The role of COX-2 neurotoxicity in ALS and other neurodegenerative disorders has been well explored.^{30, 31, 32} One of the key downstream products of COX-2, prostaglandin E2 (PGE2), can directly mediate COX-2 neurotoxicity both in vitro and in vivo.^{33, 34, 35, 36, 37} The levels of COX-2 expression and PGE2 production are controlled by multiple cell signaling pathways, including the mitogen-activated protein kinase (MAPK)/ERK pathway,^{38, 39, 40} and they have been found to be increased in neurodegenerative diseases including AD, PD and ALS.^{25, 28, 32, 41, 42, 43, 44, 45, 46} Importantly, COX-2 inhibitors such as celecoxib exhibited significant neuroprotective effects and prolonged survival or delayed disease onset in a SOD1-ALS transgenic mouse model through the downregulation of PGE2 release.²⁸Most recent studies have tried to elucidate the role of glial cells in neurotoxicity using TDP-43-ALS models, which are considered to be helpful for better understanding the disease mechanisms.^{47, 48, 49, 50, 51} Although the contribution of glial cells to TDP-43-mediated motor neuron degeneration is now well supported, this model does not fully suggest an astrocyte-based non-cell autonomous mechanism. For example, recent studies have shown that TDP-43-mutant astrocytes do not affect the survival of motor neurons,^{50, 51} indicating a previously unrecognized non-cell autonomous TDP-43 proteinopathy that associates with cell types other than astrocytes.Given that the role of glial cell types other than astrocytes in TDP-43-mediated neuroinflammation is still not fully understood, we aim to compare the contribution of microglia and astrocytes to neurotoxicity in a TDP-43 loss-of-function model. Here, we show that TDP-43 has a dominant role in promoting COX-2-PGE2 production through the MAPK/ERK pathway in primary cultured microglia, but not in primary cultured astrocytes. Our study suggests that overproduction of PGE2 in microglia is a novel molecular mechanism underlying neurotoxicity in TDP-43-linked ALS. Moreover, our data identify celecoxib as a new potential effective treatment of TDP-43-linked ALS and possibly other types of ALS.     

New-Onset Diabetes Mellitus After Transplantation in a Cynomolgus Macaque (Macaca fasicularis)

A 5.5-y-old intact male cynomolgus macaque (Macaca fasicularis) presented with inappetence and weight loss 57 d after heterotopic heart and thymus transplantation while receiving an immunosuppressant regimen consisting of tacrolimus, mycophenolate mofetil, and methylprednisolone to prevent graft rejection. A serum chemistry panel, a glycated hemoglobin test, and urinalysis performed at presentation revealed elevated blood glucose and glycated hemoglobin (HbA1c) levels (727 mg/dL and 10.1%, respectively), glucosuria, and ketonuria. Diabetes mellitus was diagnosed, and insulin therapy was initiated immediately. The macaque was weaned off the immunosuppressive therapy as his clinical condition improved and stabilized. Approximately 74 d after discontinuation of the immunosuppressants, the blood glucose normalized, and the insulin therapy was stopped. The animal''s blood glucose and HbA1c values have remained within normal limits since this time. We suspect that our macaque experienced new-onset diabetes mellitus after transplantation, a condition that is commonly observed in human transplant patients but not well described in NHP. To our knowledge, this report represents the first documented case of new-onset diabetes mellitus after transplantation in a cynomolgus macaque.Abbreviations: NODAT, new-onset diabetes mellitus after transplantationNew-onset diabetes mellitus after transplantation (NODAT, formerly known as posttransplantation diabetes mellitus) is an important consequence of solid-organ transplantation in humans.^{7-10,15,17,19,21,25-28,31,33,34,37,38,42} A variety of risk factors have been identified including increased age, sex (male prevalence), elevated pretransplant fasting plasma glucose levels, and immunosuppressive therapy.^{7-10,15,17,19,21,25-28,31,33,34,37,38,42} The relationship between calcineurin inhibitors, such as tacrolimus and cyclosporin, and the development of NODAT is widely recognized in human medicine.^{7-10,15,17,19,21,25-28,31,33,34,37,38,42} Cynomolgus macaques (Macaca fasicularis) are a commonly used NHP model in organ transplantation research. Cases of natural and induced diabetes of cynomolgus monkeys have been described in the literature;^14,43,45 however, NODAT in a macaque model of solid-organ transplantation has not been reported previously to our knowledge.     

The Fcp1-Wee1-Cdk1 axis affects spindle assembly checkpoint robustness and sensitivity to antimicrotubule cancer drugs

To grant faithful chromosome segregation, the spindle assembly checkpoint (SAC) delays mitosis exit until mitotic spindle assembly. An exceedingly prolonged mitosis, however, promotes cell death and by this means antimicrotubule cancer drugs (AMCDs), that impair spindle assembly, are believed to kill cancer cells. Despite malformed spindles, cancer cells can, however, slip through SAC, exit mitosis prematurely and resist killing. We show here that the Fcp1 phosphatase and Wee1, the cyclin B-dependent kinase (cdk) 1 inhibitory kinase, play a role for this slippage/resistance mechanism. During AMCD-induced prolonged mitosis, Fcp1-dependent Wee1 reactivation lowered cdk1 activity, weakening SAC-dependent mitotic arrest and leading to mitosis exit and survival. Conversely, genetic or chemical Wee1 inhibition strengthened the SAC, further extended mitosis, reduced antiapoptotic protein Mcl-1 to a minimum and potentiated killing in several, AMCD-treated cancer cell lines and primary human adult lymphoblastic leukemia cells. Thus, the Fcp1-Wee1-Cdk1 (FWC) axis affects SAC robustness and AMCDs sensitivity.The spindle assembly checkpoint (SAC) delays mitosis exit to coordinate anaphase onset with spindle assembly. To this end, SAC inhibits the ubiquitin ligase Anaphase-Promoting Complex/Cyclosome (APC/C) to prevent degradation of the anaphase inhibitor securin and cyclin B, the major mitotic cyclin B-dependent kinase 1 (cdk1) activator, until spindle assembly.¹ However, by yet poorly understood mechanisms, exceedingly prolonging mitosis translates into cell death induction.^{2, 3, 4, 5, 6, 7} Although mechanistic details are still missing on how activation of cell death pathways is linked to mitosis duration, prolongation of mitosis appears crucial for the ability of antimicrotubule cancer drugs (AMCDs) to kill cancer cells.^{2, 3, 4, 5, 6, 7} These drugs, targeting microtubules, impede mitotic spindle assembly and delay mitosis exit by chronically activating the SAC. Use of these drugs is limited, however, by toxicity and resistance. A major mechanism for resistance is believed to reside in the ability of cancer cells to slip through the SAC and exit mitosis prematurely despite malformed spindles, thus resisting killing by limiting mitosis duration.^{2, 3, 4, 5, 6, 7} Under the AMCD treatment, cells either die in mitosis or exit mitosis, slipping through the SAC, without or abnormally dividing.^{2, 3, 4} Cells that exit mitosis either die at later stages or survive and stop dividing or proliferate, giving rise to resistance.^{2, 3, 4} Apart from a role for p53, what dictates cell fate is still unknown; however, it appears that the longer mitosis is protracted, the higher the chances for cell death pathway activation are.^{2, 3, 4, 5, 6, 7}Although SAC is not required per se for killing,⁶ preventing SAC adaptation should improve the efficacy of AMCD by increasing mitosis duration.^{2, 3, 4, 5, 6, 7} Therefore, further understanding of the mechanisms by which cells override SAC may help to improve the current AMCD therapy. Several kinases are known to activate and sustain SAC, and cdk1 itself appears to be of primary relevance.^{1, 8, 9} By studying mitosis exit and SAC resolution, we recently reported a role for the Fcp1 phosphatase to bring about cdk1 inactivation.^{10, 11} Among Fcp1 targets, we identified cyclin degradation pathway components, such as Cdc20, an APC/C co-activator, USP44, a deubiquitinating enzyme, and Wee1.^{10, 11} Wee1 is a crucial kinase that controls the G2 phase by performing inhibitory phosphorylation of cdk1 at tyr-15 (Y15-cdk1). Wee1 is also in a feedback relationship with cdk1 itself that, in turn, can phosphorylate and inhibit Wee1 in an autoamplification loop to promote the G2-to-M phase transition.¹² At mitosis exit, Fcp1 dephosphorylated Wee1 at threonine 239, a cdk1-dependent inhibitory phosphorylation, to dampen down the cdk1 autoamplification loop, and Cdc20 and USP44, to promote APC/C-dependent cyclin B degradation.^{10, 11, 12} In this study we analysed the Fcp1 relevance in SAC adaptation and AMCD sensitivity.     

Plasma membrane poration by opioid neuropeptides: a possible mechanism of pathological signal transduction

Neuropeptides induce signal transduction across the plasma membrane by acting through cell-surface receptors. The dynorphins, endogenous ligands for opioid receptors, are an exception; they also produce non-receptor-mediated effects causing pain and neurodegeneration. To understand non-receptor mechanism(s), we examined interactions of dynorphins with plasma membrane. Using fluorescence correlation spectroscopy and patch-clamp electrophysiology, we demonstrate that dynorphins accumulate in the membrane and induce a continuum of transient increases in ionic conductance. This phenomenon is consistent with stochastic formation of giant (~2.7 nm estimated diameter) unstructured non-ion-selective membrane pores. The potency of dynorphins to porate the plasma membrane correlates with their pathogenic effects in cellular and animal models. Membrane poration by dynorphins may represent a mechanism of pathological signal transduction. Persistent neuronal excitation by this mechanism may lead to profound neuropathological alterations, including neurodegeneration and cell death.Neuropeptides are the largest and most diverse family of neurotransmitters. They are released from axon terminals and dendrites, diffuse to pre- or postsynaptic neuronal structures and activate membrane G-protein-coupled receptors. Prodynorphin (PDYN)-derived opioid peptides including dynorphin A (Dyn A), dynorphin B (Dyn B) and big dynorphin (Big Dyn) consisting of Dyn A and Dyn B are endogenous ligands for the κ-opioid receptor. Acting through this receptor, dynorphins regulate processing of pain and emotions, memory acquisition and modulate reward induced by addictive substances.^{1, 2, 3, 4} Furthermore, dynorphins may produce robust cellular and behavioral effects that are not mediated through opioid receptors.^{5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29} As evident from pharmacological, morphological, genetic and human neuropathological studies, these effects are generally pathological, including cell death, neurodegeneration, neurological dysfunctions and chronic pain. Big Dyn is the most active pathogenic peptide, which is about 10- to 100-fold more potent than Dyn A, whereas Dyn B does not produce non-opioid effects.^{16, 17, 22, 25} Big Dyn enhances activity of acid-sensing ion channel-1a (ASIC1a) and potentiates ASIC1a-mediated cell death in nanomolar concentrations^{30, 31} and, when administered intrathecally, induces characteristic nociceptive behavior at femtomolar doses.^{17, 22} Inhibition of endogenous Big Dyn degradation results in pathological pain, whereas prodynorphin (Pdyn) knockout mice do not maintain neuropathic pain.^{22, 32} Big Dyn differs from its constituents Dyn A and Dyn B in its unique pattern of non-opioid memory-enhancing, locomotor- and anxiolytic-like effects.²⁵Pathological role of dynorphins is emphasized by the identification of PDYN missense mutations that cause profound neurodegeneration in the human brain underlying the SCA23 (spinocerebellar ataxia type 23), a very rare dominantly inherited neurodegenerative disorder.^{27, 33} Most PDYN mutations are located in the Big Dyn domain, demonstrating its critical role in neurodegeneration. PDYN mutations result in marked elevation in dynorphin levels and increase in its pathogenic non-opioid activity.^{27, 34} Dominant-negative pathogenic effects of dynorphins are not produced through opioid receptors.ASIC1a, glutamate NMDA (N-methyl-d-aspartate) and AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid)/kainate ion channels, and melanocortin and bradykinin B2 receptors have all been implicated as non-opioid dynorphin targets.^{5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 30, 31, 35, 36} Multiplicity of these targets and their association with the cellular membrane suggest that their activation is a secondary event triggered by a primary interaction of dynorphins with the membrane. Dynorphins are among the most basic neuropeptides.^{37, 38} The basic nature is also a general property of anti-microbial peptides (AMPs) and amyloid peptides that act by inducing membrane perturbations, altering membrane curvature and causing pore formation that disrupts membrane-associated processes including ion fluxes across the membrane.³⁹ The similarity between dynorphins and these two peptide groups in overall charge and size suggests a similar mode of their interactions with membranes.In this study, we dissect the interactions of dynorphins with the cell membrane, the primary event in their non-receptor actions. Using fluorescence imaging, correlation spectroscopy and patch-clamp techniques, we demonstrate that dynorphin peptides accumulate in the plasma membrane in live cells and cause a profound transient increase in cell membrane conductance. Membrane poration by endogenous neuropeptides may represent a novel mechanism of signal transduction in the brain. This mechanism may underlie effects of dynorphins under pathological conditions including chronic pain and tissue injury.     

Trophoblast Deportation to the Lungs of Cotton Rats (Sigmodon hispidus)

Cotton rats (Sigmodon hispidus) have been used to study a variety of infectious agents, particularly human respiratory viral pathogens. During the course of comprehensive pathologic evaluations of aging breeders from our breeding colony, 6 of 22 (27%) female cotton rats had histologic evidence, limited to the lungs, of embolized cells that were confirmed to be trophoblastic in origin by HSD3B1 immunoreactivity. When pulmonary trophoblast emboli were numerous, they usually were associated with additional histologic findings in the lungs, including pulmonary edema and hemorrhage, endothelial hypertrophy, fibrinoid vascular necrosis, and abundant alveolar macrophages containing fresh fibrin and hemolyzing erythrocytes. Of the 6 cotton rats with pulmonary trophoblast emboli, 5 (83%) were at 8 to 18 d of the 27-d gestation period, with the greatest number of emboli per lung present between days 10 through 14. The remaining cotton rat had a focal pulmonary trophoblast embolus and was not pregnant but had delivered a litter 3 mo previously. Three other cotton rats in either the early or late stages of gestation showed no histologic evidence of pulmonary trophoblast deportation. This report is the first to document pulmonary trophoblast emboli in cotton rats. This finding suggests that cotton rats may be an alternative animal model for the study of normal and aberrant trophoblast deportation in routine pregnancies and gestational pathologic conditions in women.Abbreviations: HSD3B1, hydroxyl-C-5-steroid dehydrogenaseCotton rats (Sigmodon hispidus) are a relevant animal model for the study of human respiratory²³ viral pathogens, with increasing usage by academic and industrial institutions. The hemochorial placentation in Sigmodontinae²² is similar to that of humans and several laboratory animal species including mice, rats, hamsters, rabbits, guinea pigs, chinchillas, and nonhuman primates.^{10,20,40,42,44,49} In these species, one or more layers of analogous trophoblast types comprise the interhemal barrier between maternal and fetal blood supplies. Placental trophoblasts perform a number of critical functions during gestation, including mediation of uterine implantation and invasion, nutrient exchange, regulation of maternal blood flow, and hormone production.^{1,19,26-28,35,38,46,47}As a consequence of their inherent invasiveness, placental trophoblasts migrate into maternal uterine blood vessels, after which syncytiotrophoblasts (syncytial knots) are normally deported daily to the lungs in humans.^2,3,17 Deportation increases with frequency as gestation progresses,^3,4 with gestational pathologic conditions such as preeclampsia and eclampsia,^2,3,18,36 and after cesarean sections⁵⁰ and abortions.⁴⁸ The current thinking is that these syncytial knots undergo programmed cell death and apoptotic shedding during routine pregnancy, in contrast to conditions like preeclampsia and eclampsia, during which aberrant intervillous hemodynamics resulting in hypoxia favor necrosis and associated inflammation.^18,25,29,30 In addition, spontaneous trophoblast emboli have been documented in the lungs and a few other tissues, including uterus, adrenal gland, spleen, and liver of chinchillas,^6,11,52 hamsters,^7,41 and porcupines.²⁴ Experimentally, trophoblast invasion has been further studied in mice^8,9 and hamsters.⁵ To our knowledge, pulmonary trophoblast emboli in cotton rats have not previously been reported.Pairs of cotton rat breeders were maintained for the production of animals to be used in various studies investigating human respiratory viruses, including measles, respiratory syncytial, and parainfluenza viruses. During the course of comprehensive pathologic evaluations of aging breeders, 6 female cotton rats were incidentally found to have pulmonary trophoblast emboli. The purposes of the present case series were to characterize the embolized trophoblasts and associated pulmonary histopathology in these cotton rats and to correlate the incidence with gestational stage.     

β-Catenin and NF-κB co-activation triggered by TLR3 stimulation facilitates stem cell-like phenotypes in breast cancer

Cancer stem cells (CSCs) are responsible for tumor initiation and progression. Toll-like receptors (TLRs) are highly expressed in cancer cells and associated with poor prognosis. However, a linkage between CSCs and TLRs is unclear, and potential intervention strategies to prevent TLR stimulation-induced CSC formation and underlying mechanisms are lacking. Here, we demonstrate that stimulation of toll-like receptor 3 (TLR3) promotes breast cancer cells toward a CSC phenotype in vitro and in vivo. Importantly, conventional NF-κB signaling pathway is not exclusively responsible for TLR3 activation-enriched CSCs. Intriguingly, simultaneous activation of both β-catenin and NF-κB signaling pathways, but neither alone, is required for the enhanced CSC phenotypes. We have further identified a small molecule cardamonin that can concurrently inhibit β-catenin and NF-κB signals. Cardamonin is capable of effectively abolishing TLR3 activation-enhanced CSC phenotypes in vitro and successfully controlling TLR3 stimulation-induced tumor growth in human breast cancer xenografts. These findings may provide a foundation for developing new strategies to prevent the induction of CSCs during cancer therapies.Despite incessant efforts to combat cancer over decades, breast cancer is still the second leading cause of death in women, remaining high with over 39 000 deaths in 2012 in the United States alone.¹ Conventional interventions, such as radiation or chemotherapy, may eliminate the bulk of the tumor but spare rare aggressive cancer cells that have an exceptional capacity to survive, self-renew, and advance the malignancy. These residual tumor cells have recently been found to possess key stem-like properties and have thus been termed ‘cancer stem cells (CSCs)''.^{2, 3, 4, 5} Breast CSCs, characterized by expression of CD44^high/CD24^−/low surface markers, are proposed to be largely responsible for cancer progression and metastasis.^3,6,7 These CD44^high/CD24^−/low cells possess stem cell-like properties and tumor-initiating capacity. Furthermore, these cells resist standard therapies^3,6,8,9 and can be converted from non-CSC cells under certain conditions.^10,11 Therefore, specific targeting of CSCs within a tumor will be imperative to prevent disease progression and recurrence.⁵ However, the conditions and mechanisms underlying CSC formation remain poorly understood. Although the majority of cancers arise from de novo oncogenic and epigenetic alterations, most tumors display signals of unremitting inflammatory activity,¹² which occurs even in the absence of infection or autoimmunity.¹³Toll-like receptors (TLRs) are a key family of microbial sensors in the host innate and adaptive immunity as well as in tissue repair and regeneration. They are also involved in the inflammatory signaling triggered by endogenous macromolecules released by injured tissue.^14,15 Ten TLRs are encoded by the human genome. TLRs detecting nucleic acids (TLR3, TLR7, TLR8, and TLR9) are localized in the endosomal compartment in nearly all cell types, while TLRs mainly detecting proteolipidic structures (TLR1, TLR2, TLR4, TLR5, TLR6, and TLR10) are exposed on the cell surface.^14,16 In cancer, TLRs have emerged as important participants in tumorigenesis. TLR3, 4, 7, and 9 were overexpressed in 70, 72, 67, and 78% of patients with esophageal cancer.¹⁷ The -196 to -174del/del genotype of TLR2 may increase the risk of gastric cancer,¹⁸ and TLR4+896A>G polymorphism is a risk factor for non-cardia gastric carcinoma.¹⁹ Functions of epithelial-expressed TLR2 and 5 in promoting epithelial cell survival, proliferation, migration,²⁰ and angiogenesis (TLR2 only)²¹ may be usurped by tumor cells to facilitate progression and metastasis. Although TLR3, 5, 7, 8, and 9 may achieve antitumor effects by converting immune tolerance into antitumor immunity,¹⁴ considerable discrepancies have been reported. For instance, high TLR3 expression in esophageal cancer cells was significantly associated with a higher probability of lymph-node metastasis and increased depth of invasion.¹⁷ Elevated TLR3 expression in breast cancer was also associated with poor prognosis.^22,23Several clinical trials using TLR agonists for cancer treatment are currently in progress. Among all anticancer immunotherapy agents, TLR agonists are classified as the ones with highest potential. However, clinical outcomes are inconsistent and repeatedly disappointing.²⁴ Specifically, high expectations were placed on TLR3 agonists for their ability to boost host immune systems to fight diseases. TLR3 is located in intracellular endosomes for the recognition of double-stranded RNA (dsRNA) and polyinosinic-polycytidylic acid (poly(I:C), a synthetic analog of dsRNA).²⁵ In addition to upregulating immune response, a broader range of functions of TLR3 have been revealed recently, especially in stem cells. For instances, activation of TLR3 was found to amplify mesenchymal stem cell trophic factors and enhance therapeutic potency.²⁶ Recently, Lee et al.²⁷ also showed that TLR3 stimulation caused rapid and global changes in the expression of epigenetic modifiers to enhance chromatin remodeling and nuclear reprogramming when converting adult cells to induced pluripotent stem cells. Nevertheless, the role of TLR3 in cancer remains inconsistent, and its function in breast CSCs is unclear.Here, we demonstrate that TLR3 activation in breast cancer cells leads to a preferential enrichment of a subset of cells with CSC phenotypes in vitro and in vivo. Conventional NF-κB signaling is not fully responsible for the enhanced CSC properties. Unexpectedly, β-catenin pathway is required for the promotion of CSC phenotypes in breast cancer cells following TLR3 activation. Our results provide new tantalizing strategies to effective target breast and other CSCs with elevated TLR3 expression to prevent progression and relapse.     

Tyr1068-phosphorylated epidermal growth factor receptor (EGFR) predicts cancer stem cell targeting by erlotinib in preclinical models of wild-type EGFR lung cancer

Tyrosine kinase inhibitors (TKIs) have shown strong activity against non-small-cell lung cancer (NSCLC) patients harboring activating epidermal growth factor receptor (EGFR) mutations. However, a fraction of EGFR wild-type (WT) patients may have an improvement in terms of response rate and progression-free survival when treated with erlotinib, suggesting that factors other than EGFR mutation may lead to TKI sensitivity. However, at present, no sufficiently robust clinical or biological parameters have been defined to identify WT-EGFR patients with greater chances of response. Therapeutics validation has necessarily to focus on lung cancer stem cells (LCSCs) as they are more difficult to eradicate and represent the tumor-maintaining cell population. Here, we investigated erlotinib response of lung CSCs with WT-EGFR and identified EGFR phosphorylation at tyrosine1068 (EGFR^tyr1068) as a powerful biomarker associated with erlotinib sensitivity both in vitro and in preclinical CSC-generated xenografts. In contrast to the preferential cytotoxicity of chemotherapy against the more differentiated cells, in EGFR^tyr1068 cells, erlotinib was even more active against the LCSCs compared with their differentiated counterpart, acquiring potential value as CSC-directed therapeutics in the context of WT-EGFR lung cancer. Although tumor growth was inhibited to a similar extent during erlotinib or chemotherapy administration to responsive tumors, erlotinib proved superior to chemotherapy in terms of higher tolerability and reduced tumor aggressiveness after treatment suspension, substantiating the possibility of preferential LCSC targeting, both in adenocarcinoma (ADC) and squamous cell carcinoma (SCC) tumors. We conclude that EGFR^tyr1068 may represent a potential candidate biomarker predicting erlotinib response at CSC-level in EGFR-WT lung cancer patients. Finally, besides its invariable association with erlotinib sensitivity in EGFR-WT lung CSCs, EGFR^tyr1068 was associated with EGFR-sensitizing mutations in cell lines and patient tumors, with relevant diagnostic, clinical and therapeutic implications.Non-small-cell lung cancer (NSCLC) accounts for ∼80% of lung cancer subtypes and is the leading cause of cancer-related death worldwide.¹ In recent years, molecular characterization of NSCLC has reached an unprecedented detail and has allowed segregating NSCLC into discrete molecular subgroups, characterized by specific oncogenic drivers, such as epidermal growth factor receptor (EGFR), BRAF, KRAS, epidermal growth factor receptor 2 (HER2) mutations, MET amplification and anaplastic lymphoma kinase gene rearrangements (ALK).^{2, 3} Consequently, the understanding of NSCLC biology has brought two new classes of targeted agents into the clinical setting: EGFR tyrosine kinase inhibitors (TKIs) and ALK inhibitors.^{4, 5} In particular, clinical trials have shown that NSCLC patients whose tumors harbor sensitizing EGFR mutations significantly benefit from the upfront use of an EGFR TKI, rather than conventional chemotherapy.^{6, 7, 8, 9, 10, 11} Although licensed for clinical use in chemotherapy-pretreated patients, regardless of EGFR mutational status, the EGFR TKI erlotinib has limited efficacy when compared with standard chemotherapy in patients with WT-EGFR NSCLC.^{12, 13, 14}However, a fraction of patients on erlotinib treatment may achieve clinically significant objective responses and prolonged disease control, despite the lack of detectable EGFR mutations.¹⁵ Nevertheless, no biomarker investigated so far was felt sufficiently robust to select for the use of erlotinib in the maintenance or refractory setting.¹⁶ Thus, it would be crucial to identify molecular predictors of TKI sensitivity in EGFR wild-type (WT) tumors in order to prospectively select the subgroup of patients who may benefit from erlotinib therapy. Moreover, EGFR TKIs have also shown a modest therapeutic effect in lung squamous cell carcinoma (SCC), where EGFR mutations are very rare and patients have limited therapeutic options in the maintenance and relapsed settings.^{16, 17, 18, 19, 20}Even more importantly, in order to obtain meaningful clinical responses it is crucial to effectively target the population of cells that are able to escape treatment and maintain the growth of a resistant tumor.²¹ Cancer stem cells (CSCs) have been in fact identified within most solid tumors, including lung tumors, and are associated with increased resistance to therapies.^{22, 23, 24, 25, 26, 27, 28, 29, 30} Thus, the efficacy of innovative therapeutic strategies should be validated against these more aggressive, tumor-maintaining cells.^{23, 27, 31} Importantly, TKI response has never been determined at the level of the tumor-maintaining CSCs. Thus, we investigated erlotinib response of EGFR mutation-negative lung cancer stem cells (LCSCs) and LCSC-based xenografts with the attempt to evaluate their sensitivity to the drug and correlate it with their molecular pattern in order to identify potential biomarkers predictive of erlotinib response in a WT-EGFR context at the CSC level.     

Paravascular pathways contribute to vasculitis and neuroinflammation after subarachnoid hemorrhage independently of glymphatic control

Subarachnoid hemorrhage (SAH) is a devastating disease with high mortality. The mechanisms underlying its pathological complications have not been fully identified. Here, we investigate the potential involvement of the glymphatic system in the neuropathology of SAH. We demonstrate that blood components rapidly enter the paravascular space following SAH and penetrate into the perivascular parenchyma throughout the brain, causing disastrous events such as cerebral vasospasm, delayed cerebral ischemia, microcirculation dysfunction and widespread perivascular neuroinflammation. Clearance of the paravascular pathway with tissue-type plasminogen activator ameliorates the behavioral deficits and alleviates histological injury of SAH. Interestingly, AQP4^−/− mice showed no improvements in neurological deficits and neuroinflammation at day 7 after SAH compared with WT control mice. In conclusion, our study proves that the paravascular pathway dynamically mediates the pathological complications following acute SAH independently of glymphatic control.Cerebral aneurysm rupture causes subarachnoid hemorrhage (SAH), which is associated with a high mortality due to its secondary complications, including hemorrhage, hydrocephalus and delayed cerebral ischemia (DCI).^{1, 2, 3} Therapeutic interventions against the secondary complications, especially DCI, are yet limited, as the pathological mechanism underlying that is not fully understood.^{2, 3, 4, 5, 6, 7} Current hypotheses of the development of the secondary complications mainly include cerebral vasospasm (CVS) and the microcirculation disturbance, as well as parenchymal arterial lesions, microthrombosis and neuroinflammation.^{1, 2, 4, 7, 8, 9}Previous studies have shown that the blockade of cerebral lymphatic drainage deteriorated the secondary cerebral ischemia after SAH, suggesting that the cerebral lymphatic drainage pathway could be involved in the pathological mechanism of SAH.^{10, 11} However, the central nervous system (CNS) was considered lack of a conventional lymphatic drainage system in the past. Recently, several studies have shown that the brain has in fact the proper lymphatic system, including sinus-associated lymphatic vessels and the glymphatic system (GS).^{12, 13, 14, 15} Sinus-associated lymphatic vessels express all of the molecular hallmarks of lymphatic endothelial cells, contain cerebrospinal fluid (CSF) and immune cells, and drain into the deep cervical lymph nodes.^{12, 13}There is a histologically defined space in the brain, the Virchow–Robin space, where the subarachnoid space meets the paravascular space (or perivascular space in somewhere, PVS).¹⁶ The GS is a specialized brain-wide anatomic structure locating at the PVS surrounding the brain vasculature, which is ensheathed with the astroglial endfeet and astroglial water channel aquaporin-4 (AQP4).^{14, 15} The GS facilitates the efficient lymphatic clearance of extracellular solutes and fluid in the brain through astroglial-mediated interstitial fluid bulk flow.¹⁴Impairment of GS involves neurological conditions including traumatic brain injuries,¹⁷ ischemic stroke¹⁸ and aged brain.¹⁹ Interestingly, brain imaging study with magnetic resonance imaging reported weakened GS perfusion following acute stroke or SAH.^{18, 20} However, little is known about whether the GS is involved in the secondary complications of SAH. Here, we examined the potential involvement of GS in SAH-associated pathology progression with in vivo two-photon microscopy and CLARITY technique.^{21, 22} Our data showed that subarachnoid blood flowed into the brain parenchyma rapidly through the PVS, causing CVS, vasculitis, widespread microinfraction and neuroinflammation in the animal model of SAH and SAH patients. Prevention of CVS with Fasudil²³ did not improve the neurological impairment nor alleviated the pathology, while the PVS clearance with tissue-type plasminogen activator (tPA) infusion improved the behavioral recovery and reduced neuroinflammation in the brain. Interestingly, AQP4^−/− mice showed no improvements in neurological deficits and neuroinflammation at day 7 after SAH compared with WT control mice. Our study therefore suggested that the paravacular pathway dynamically mediates the pathological complications following acute SAH independently of glymphatic control.