Kinin generation from exogenous kininogens at the surface of retinoic acid-differentiated human neuroblastoma IMR-32 cells after stimulation with interferon-γ

Bradykinin-related peptides, kinins, ubiquitously occur in the nervous system and together with other pro-inflammatory mediators contribute to pathological states of that tissue such as edema and chronic pain. In the current work we characterized the kinin-forming system of neuronal cells obtained by differentiation of human neuroblastoma cell line IMR-32 with retinoic acid. These cells were shown to concentrate exogenous kinin precursors, kininogens, on the surface, release kinins from kininogens and subsequently convert kinins to their des-Arg metabolites. Significantly higher amounts of kinins and des-Arg-kinins were produced after cell stimulation with interferon-γ, a potent pro-inflammatory mediator involved in many neurological disorders. The expression of the major tissue kininogenase (the human kallikrein 1) and the major cell membrane-bound kininase (the carboxypeptidase M) also increased after cell stimulation with interferon-γ, suggesting the involvement of these enzymes in the kinin production and degradation, respectively. Interferon-γ was also able to up-regulate the expression of two known subtypes of kinin receptors. On the protein level, the changes were only observed in the expression of the des-Arg-kinin-specific type 1 receptor which functions in the propagation of the inflammatory state. Taken together, these results suggest a novel way for local kinin and des-Arg-kinin generation in the nervous tissue during pathological states accompanied by interferon-γ release.     

What have we learned about the kallikrein-kinin and renin-angiotensin systems in neurological disorders?

The kallikrein-kinin system(KKS) is an intricate endogenous pathway involved in several physiological and pathological cascades in the brain. Due to the pathological effects of kinins in blood vessels and tissues, their formation and degradation are tightly controlled. Their components have been related to several central nervous system diseases such as stroke, Alzheimer's disease, Parkinson's disease, multiple sclerosis, epilepsy and others. Bradykinin and its receptors(B1R and B2R) may have a role in the pathophysiology of certain central nervous system diseases. It has been suggested that kinin B1R is up-regulated in pathological conditions and has a neurodegenerative pattern, while kinin B2R is constitutive and can act as a neuroprotective factor in many neurological conditions. The renin angiotensin system(RAS) is an important blood pressure regulator and controls both sodium and water intake. AngⅡ is a potent vasoconstrictor molecule and angiotensin converting enzyme is the major enzyme responsible for its release. AngⅡ acts mainly on the AT1 receptor, with involvement in several systemic and neurological disorders. Brain RAS has been associated with physiological pathways, but is also associated with brain disorders. This review describes topics relating to the involvement of both systems in several forms of brain dysfunction and indicates components of the KKS and RAS that have been used as targets in several pharmacological approaches.     

Altered neutrophil homeostasis in kinin B1 receptor-deficient mice

The kallikrein-kinin system is activated during inflammation and plays a major role in the inflammatory process. One of the main mechanisms of kinin action includes the modulation of neutrophil function employing both receptors for kinins, B1 and B2. In this report we show by the use of B1 receptor-deficient mice that neutrophil migration in inflamed tissues is dependent on kinin B1 receptors. However, there is no change in circulating leukocyte number and composition after genetic ablation of this receptor. Furthermore, apoptosis of neutrophils necessary for the resolution of persistent inflammatory processes is impaired in mice lacking the B1 receptor. We also show that this receptor is expressed on neutrophils, thus it may be directly involved in the induction of apoptosis in these cells after prolonged activation at inflamed sites. In conclusion, our data show that the kinin B1 receptor modulates migration and the life span of neutrophils at sites of inflammation and may be therefore an important drug target in the therapy of inflammatory diseases.     

Kallikrein and kinin receptor expression in inflammation and cancer

The kallikrein family of serine proteases has been investigated in many inflammatory disorders as molecular mapping, gene characterisation and cloning of kinin receptor genes have unfolded experimentally. In the molecular events of the inflammatory response the kallikrein cascade plays a significant role, since it is considered to initiate and maintain systemic inflammatory responses and immune-modulated disorders. A primary event is the chemotactic attraction of neutrophils which deliver the kallikrein-kinin cascade to sites of cellular injury and carcinogenic transformation of cells. The present study establishes the casual involvement of the kallikrein cascade in infection, inflammatory joint disease, acute transplant rejection, renal glomerular diseases, angiogenesis and carcinoma. We provide strong evidence for new or enhanced expression of kinin B1 receptors in inflammation, and additionally the induction of kallikrein genes in angiogenesis and carcinoma. The results provide insights into possible roles of kallikrein inhibitors and kinin receptor antagonists.     

Lung cysteine cathepsins: intruders or unorthodox contributors to the kallikrein-kinin system?

The protease/antiprotease balance is tipped in favor of enhanced proteolysis in inflammatory lung disorders, promoting the spread and severity of inflammation. Cysteine cathepsins participate in the remodeling and/or degradation of the pulmonary extra cellular matrix and in lung homeostasis. There is now good evidence that cathepsins are involved in fibrosis, emphysema, asthma, and in bronchopulmonary dysplasia. Kinins are inflammatory mediators that induce edema, pain and vasodilatation, and participate in vascular homeostasis. Kinins may also contribute to the immune system by acting as danger signals, and activating bradykinin receptors. Kinins are believed to play a role in inflammatory obstructive airway diseases, asthma, and allergic rhinitis. Their release by plasma and tissue kallikreins is severely reduced at inflammatory sites, although local kinin production seems to remain intact. Such conflicting observations suggest that there are alternative mechanisms of kinin metabolism besides the classical pathways. This article reviews the biological and pathophysiological roles of lung cysteine cathepsins, kinins and their receptors, and summarizes the indications that cysteine cathepsins may contribute to kinin liberation and/or degradation.     

The role of mitochondria in inherited neurodegenerative diseases

In the past decade, the genetic causes underlying familial forms of many neurodegenerative disorders, such as Huntington's disease, Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis, Friedreich ataxia, hereditary spastic paraplegia, dominant optic atrophy, Charcot-Marie-Tooth type 2A, neuropathy ataxia and retinitis pigmentosa, and Leber's hereditary optic atrophy have been elucidated. However, the common pathogenic mechanisms of neuronal death are still largely unknown. Recently, mitochondrial dysfunction has emerged as a potential 'lowest common denominator' linking these disorders. In this review, we discuss the body of evidence supporting the role of mitochondria in the pathogenesis of hereditary neurodegenerative diseases. We summarize the principal features of genetic diseases caused by abnormalities of mitochondrial proteins encoded by the mitochondrial or the nuclear genomes. We then address genetic diseases where mutant proteins are localized in multiple cell compartments, including mitochondria and where mitochondrial defects are likely to be directly caused by the mutant proteins. Finally, we describe examples of neurodegenerative disorders where mitochondrial dysfunction may be 'secondary' and probably concomitant with degenerative events in other cell organelles, but may still play an important role in the neuronal decay. Understanding the contribution of mitochondrial dysfunction to neurodegeneration and its pathophysiological basis will significantly impact our ability to develop more effective therapies for neurodegenerative diseases.     

When amyloids become prions

The conformational diseases, linked to protein aggregation into amyloid conformations, range from non-infectious neurodegenerative disorders, such as Alzheimer''s disease (AD), to highly infectious ones, such as human transmissible spongiform encephalopathies (TSEs). They are commonly known as prion diseases. However, since all amyloids could be considered prions (from those involved in cell-to-cell transmission to those responsible for real neuronal invasion), it is necessary to find an underlying cause of the different capacity to infect that each of the proteins prone to form amyloids has. As proposed here, both the intrinsic cytotoxicity and the number of nuclei of aggregation per cell could be key factors in this transmission capacity of each amyloid.     

Survivin: A target from brain cancer to neurodegenerative disease

Apoptosis is an important contributing factor during neuronal death in a variety of neurodegenerative disorders, including multiple sclerosis, Parkinson’s disease and sciatic nerve injury. Whereas several clinical and preclinical studies have focused on the neuroprotective effects of caspase inhibitors, their clinical benefits are still unclear. Here, we discuss novel alternative strategies to protect neuronal cells from apoptotic death using members of the inhibitors of apoptosis (IAP) family. We specifically review the different roles of survivin, which is an important member of the IAP family that serves a dual role in the inhibition of apoptosis as well as a vital role in mitosis and cell division. Due to the various roles of survivin during cell division and apoptosis, targeting this protein illustrates a new therapeutic window for the treatment of neurodegenerative diseases.     

NF-κB in the regulation of epithelial homeostasis and inflammation

The IκB kinase/NF-κB signaling pathway has been implicated in the pathogenesis of several inflammatory diseases. Increased activation of NF-κB is often detected in both immune and non-immune cells in tissues affected by chronic inflammation, where it is believed to exert detrimental functions by inducing the expression of proinflammatory mediators that orchestrate and sustain the inflammatory response and cause tissue damage. Thus, increased NF-κB activation is considered an important pathogenic factor in many acute and chronic inflammatory disorders, raising hopes that NF-κB inhibitors could be effective for the treatment of inflammatory diseases. However, ample evidence has accumulated that NF-κB inhibition can also be harmful for the organism, and in some cases trigger the development of inflammation and disease. These findings suggested that NF-κB signaling has important functions for the maintenance of physiological immune homeostasis and for the prevention of inflammatory diseases in many tissues. This beneficial function of NF-κB has been predominantly observed in epithelial cells, indicating that NF-κB signaling has a particularly important role for the maintenance of immune homeostasis in epithelial tissues. It seems therefore that NF-κB displays two faces in chronic inflammation: on the one hand increased and sustained NF-κB activation induces inflammation and tissue damage, but on the other hand inhibition of NF-κB signaling can also disturb immune homeostasis, triggering inflammation and disease. Here, we discuss the mechanisms that control these apparently opposing functions of NF-κB signaling, focusing particularly on the role of NF-κB in the regulation of immune homeostasis and inflammation in the intestine and the skin.     

Neuroglobin and neuronal cell survival

The balance between neuronal apoptosis and survival sculpts the developing brain and has an important role in neurodegenerative diseases. Thus, the individuation of signals that could modulate the cell death machinery as well as enhance survival in neurons promises to provide multiple points of therapeutic intervention in neurodegenerative diseases. Neuroglobin (NGB), the first nerve globin identified in neuronal tissues of humans, seems to possess a protective role in the brain only after up-regulation. Here, the NGB physiological role in the control of neuronal survival is reviewed. In vitro studies suggested that cytosolic NGB could react very rapidly with cytochrome c released from mitochondria, thus interfering with the intrinsic pathway of apoptosis. Although very suggestive, these data do not explain either the role of NGB up-regulation in neuroprotection or the recently reported NGB localization into mitochondria. Recently, we identified the steroid hormone 17β-estradiol (E2) as an endogenous modulator of NGB levels in neuroblastoma SK-N-BE cell line. Upon E2 stimulation, NGB reallocates mainly into mitochondria where the association with the mitochondrial cytochrome c occurs. Remarkably, E2 treatment before an apoptotic stimulus strongly enhances the NGB:cytochrome c association reducing cytochrome c release into the cytosol. As a consequence, a decrease of caspase-3 activation and, in turn, of the apoptotic cascade activation take place. Besides E2, other compounds have been reported to up-regulate the NGB expression highlighting the possibility to develop NGB-mediated therapeutic strategies against stroke damage and neurodegenerative diseases. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins.     

Neuroprotective roles of the P2Y2 receptor

Purinergic signaling plays a unique role in the brain by integrating neuronal and glial cellular circuits. The metabotropic P1 adenosine receptors and P2Y nucleotide receptors and ionotropic P2X receptors control numerous physiological functions of neuronal and glial cells and have been implicated in a wide variety of neuropathologies. Emerging research suggests that purinergic receptor interactions between cells of the central nervous system (CNS) have relevance in the prevention and attenuation of neurodegenerative diseases resulting from chronic inflammation. CNS responses to chronic inflammation are largely dependent on interactions between different cell types (i.e., neurons and glia) and activation of signaling molecules including P2X and P2Y receptors. Whereas numerous P2 receptors contribute to functions of the CNS, the P2Y(2) receptor is believed to play an important role in neuroprotection under inflammatory conditions. While acute inflammation is necessary for tissue repair due to injury, chronic inflammation contributes to neurodegeneration in Alzheimer's disease and occurs when glial cells undergo prolonged activation resulting in extended release of proinflammatory cytokines and nucleotides. This review describes cell-specific and tissue-integrated functions of P2 receptors in the CNS with an emphasis on P2Y(2) receptor signaling pathways in neurons, glia, and endothelium and their role in neuroprotection.     

Cannabinoids and Neuroprotection in Basal Ganglia Disorders

Cannabinoids have been proposed as clinically promising neuroprotective molecules, as they are capable to reduce excitotoxicity, calcium influx, and oxidative injury. They are also able to decrease inflammation by acting on glial processes that regulate neuronal survival and to restore blood supply to injured area by reducing the vasoconstriction produced by several endothelium-derived factors. Through one or more of these processes, cannabinoids may provide neuroprotection in different neurodegenerative disorders including Parkinson’s disease and Huntington’s chorea, two chronic diseases that are originated as a consequence of the degeneration of specific nuclei of basal ganglia, resulting in a deterioration of the control of movement. Both diseases have been still scarcely explored at the clinical level for a possible application of cannabinoids to delay the progressive degeneration of the basal ganglia. However, the preclinical evidence seems to be solid and promising. There are two key mechanisms involved in the neuroprotection by cannabinoids in experimental models of these two disorders: first, a cannabinoid receptor-independent mechanism aimed at producing a decrease in the oxidative injury and second, an induction/upregulation of cannabinoid CB2 receptors, mainly in reactive microglia, that is capable to regulate the influence of these glial cells on neuronal homeostasis. Considering the relevance of these preclinical data and the lack of efficient neuroprotective strategies in both disorders, we urge the development of further studies that allow that the promising expectatives generated for these molecules progress from the present preclinical evidence till a real clinical application.     

Unfolded proteins and endoplasmic reticulum stress in neurodegenerative disorders

The stimuli for neuronal cell death in neurodegenerative disorders are multi-factorial and may include genetic predisposition, environmental factors, cellular stressors such as oxidative stress and free radical production, bioenergy failure, glutamate-induced excitotoxicity, neuroinflammation, disruption of Ca(2+) -regulating systems, mitochondrial dysfunction and misfolded protein accumulation. Cellular stress disrupts functioning of the endoplasmic reticulum (ER), a critical organelle for protein quality control, leading to induction of the unfolded protein response (UPR). ER stress may contribute to neurodegeneration in a range of neurodegenerative disorders. This review summarizes the molecular events occurring during ER stress and the unfolded protein response and it specifically evaluates the evidence suggesting the ER stress response plays a role in neurodegenerative disorders.     

Visualisation of tissue kallikrein, kininogen and kinin receptors in human skin following trauma and in dermal diseases

During dermal injury and inflammation the serine proteases kallikreins cleave endogenous, multifunctional substrates (kininogens) to form bradykinin and kallidin. The actions of kinins are mediated by preferential binding to constitutively expressed kinin-B2 receptors or inducible kinin-B1 receptors. A feature of the kinin-B1 receptors is that they show low levels of expression, but are distinctly upregulated following tissue injury and inflammation. Because recent evidence suggested that kinin-B1 receptors may perform a protective role during inflammation, we investigated the specific occurrence of the kallikrein-kinin components in skin biopsies obtained from normal skin, patients undergoing surgery, basalioma, lichenificated atopic eczema, and psoriasis. The tissue was immunolabeled in order to determine the localisation of tissue pro-kallikrein, kallikrein, kininogen and kinin receptors. The kinin components were visualised in normal, diseased and traumatised skin, except that no labelling was observed for kininogen in normal skin. Of the five types of tissue examined, upregulation of kinin-B1 receptors was observed only in skin biopsies obtained following surgery. In essence, the expression of kinin-B1 receptors did not appear to be enhanced in the other biopsies. Within the multiple steps of the inflammatory cascade in wound healing, our results suggest an important regulatory role for kinin-B1 receptors during the first phase of inflammation following injury.     

Kinin receptors in cultured rat microglia

Kinins are produced and act at the site of injury and inflammation in various tissues. They are likely to initiate a particular cascade of inflammatory events, which evokes physiological and pathophysiological responses including an increase in blood flow and plasma leakage. In the central nervous system (CNS), kinins are potent stimulators of the production and release of pro-inflammatory mediators represented by prostanoids and cytotoxins. They are known to induce neural tissue damage. Many of the cytotoxins such as cytokines and free radicals and prostanoids are released from glial cells. Among glial cells, astrocytes and oligodendrocytes are known to possess bradykinin (BK) B(2) receptors that phosphoinositide (PI) turnover and raise intracellular Ca(2+) concentration. The presence of bradykinin receptors in microglia has been of great significance. We recently showed that rat primary microglia express kinin receptors. In resting microglia, B(2) receptors but not B(1) receptors are expressed. When the microglia are activated by bradykinin, B(1) receptors are up-regulated, while B(2) receptors are down-regulated. As observed in other glial cells, electrophysiological measurements suggest that B(2) receptors in phosphoinositide turnover and intracellular Ca(2+) concentration in microglia. Release of cytotoxins is likely consequent upon the activation of BK receptors. Our study provides the first evidence that microglia express functional kinin receptors and suggests that microglia play an important role in CNS inflammatory responses.     

Angiotensin-converting enzyme limits inflammation elicited by Trypanosoma cruzi cysteine proteases: a peripheral mechanism regulating adaptive immunity via the innate kinin pathway

Tissue injury by pathogens induces a stereotyped inflammatory response that alerts the innate immune system of the potential threat to host integrity. Here, we review knowledge emerging from investigations of the role of the kinin system in the mechanisms that link innate to the adaptive phase of immunity. Progress in this field started with results demonstrating that bradykinin is an endogenous danger signal that induces dendritic cell (DC) maturation via G protein-coupled bradykinin B2 receptors (B2R). The immunostimulatory role of kinins was recently confirmed in two different mouse models of Trypanosoma cruzi infection, a parasitic protozoan equipped with kinin-releasing cysteine proteases (cruzipain). Infection by the intraperitoneal route showed that DCs from B2R-/- mice (susceptible phenotype) failed to sense kinin 'danger' signals proteolytically released by parasites, explaining why these mutant mice display lower frequencies of interferon-gamma-producing effector T-cells. Studies of the dynamics of inflammation in the subcutaneous model of infection revealed that the balance between cruzipain and angiotensin-converting enzyme, respectively acting as kinin-generating and degrading enzymes, governs extent of DC maturation and TH1 development via the B2R-dependent innate pathway. Studies of the kinin role in immunity may shed light on the relationship between proteolytic networks and the cytokine circuits that guide T-cell development.     

Neuroprotective effects of genistein in VSC4.1 motoneurons exposed to activated microglial cytokines

Pro-inflammatory cytokines released from activated microglia may be responsible for neuronal damage and resulting motor deficits associated with CNS disorders such as spinal cord injury, Parkinson’s disease, and multiple sclerosis. Estrogen (17β-estradiol) is capable of ameliorating motoneuron death following spinal cord injury, but has a number of deleterious side effects. Genistein (GEN), an estrogen receptor beta agonist and potent antioxidant, may represent an alternative to estrogen in treating neurodegenerative disorders. However, little is known about the neuroprotective effects of GEN. We therefore tested whether GEN would prevent apoptosis in cultured motoneurons following exposure to pro-inflammatory cytokines released from IFN-γ activated microglia. Exposure of ventral spinal cord 4.1 motoneurons to microglial cytokine supernatant in vitro caused significant apoptosis and reduced mitochondrial membrane potential. An increase in reactive oxygen species, intracellular Ca²⁺, calpain, caspases, cytochrome c, and the bax:bcl-2 ratio were also noted. GEN treatment reversed apoptotic death and cellular changes following cytokine exposure and was associated with increased expression of estrogen receptor β suggesting that GEN may promote neuroprotection via receptor-mediated pathways. The addition of ICI 182, 780, an estrogen receptor antagonist following GEN treatment attenuated neuroprotection, suggesting that GEN may act mainly via estrogen receptor β to protect VSC4.1 motoneurons. We conclude that GEN protects cultured ventral spinal cord 4.1 cells from inflammatory insult and thus may represent a potential beneficial therapy in the treatment of neurodegenerative disorders.     

Ubiquilin-1 and protein quality control in Alzheimer disease

Single nucleotide polymorphisms in the ubiquilin-1 gene may confer risk for late-onset Alzheimer disease (AD). We have shown previously that ubiquilin-1 functions as a molecular chaperone for the amyloid precursor protein (APP) and that protein levels of ubiquilin-1 are decreased in the brains of AD patients. We have recently found that ubiquilin-1 regulates APP trafficking and subsequent secretase processing by stimulating non-degradative ubiquitination of a single lysine residue in the cytosolic domain of APP. Thus, ubiquilin-1 plays a central role in regulating APP biosynthesis, trafficking and ultimately toxicity. As ubiquilin-1 and other ubiquilin family members have now been implicated in the pathogenesis of numerous neurodegenerative diseases, these findings provide mechanistic insights into the central role of ubiquilin proteins in maintaining neuronal proteostasis.     

Is lipid signaling through cannabinoid 2 receptors part of a protective system?

The mammalian body has a highly developed immune system which guards against continuous invading protein attacks and aims at preventing, attenuating or repairing the inflicted damage. It is conceivable that through evolution analogous biological protective systems have been evolved against non-protein attacks. There is emerging evidence that lipid endocannabinoid signaling through cannabinoid 2 (CB₂) receptors may represent an example/part of such a protective system/armamentarium. Inflammation/tissue injury triggers rapid elevations in local endocannabinoid levels, which in turn regulate signaling responses in immune and other cells modulating their critical functions. Changes in endocannabinoid levels and/or CB₂ receptor expressions have been reported in almost all diseases affecting humans, ranging from cardiovascular, gastrointestinal, liver, kidney, neurodegenerative, psychiatric, bone, skin, autoimmune, lung disorders to pain and cancer, and modulating CB₂ receptor activity holds tremendous therapeutic potential in these pathologies. While CB₂ receptor activation in general mediates immunosuppressive effects, which limit inflammation and associated tissue injury in large number of pathological conditions, in some disease states activation of the CB₂ receptor may enhance or even trigger tissue damage, which will also be discussed alongside the protective actions of the CB₂ receptor stimulation with endocannabinoids or synthetic agonists, and the possible biological mechanisms involved in these effects.     

Fluc‐EGFP reporter mice reveal differential alterations of neuronal proteostasis in aging and disease

The cellular protein quality control machinery is important for preventing protein misfolding and aggregation. Declining protein homeostasis (proteostasis) is believed to play a crucial role in age‐related neurodegenerative disorders. However, how neuronal proteostasis capacity changes in different diseases is not yet sufficiently understood, and progress in this area has been hampered by the lack of tools to monitor proteostasis in mammalian models. Here, we have developed reporter mice for in vivo analysis of neuronal proteostasis. The mice express EGFP‐fused firefly luciferase (Fluc‐EGFP), a conformationally unstable protein that requires chaperones for proper folding, and that reacts to proteotoxic stress by formation of intracellular Fluc‐EGFP foci and by reduced luciferase activity. Using these mice, we provide evidence for proteostasis decline in the aging brain. Moreover, we find a marked reaction of the Fluc‐EGFP sensor in a mouse model of tauopathy, but not in mouse models of Huntington’s disease. Mechanistic investigations in primary neuronal cultures demonstrate that different types of protein aggregates have distinct effects on the cellular protein quality control. Thus, Fluc‐EGFP reporter mice enable new insights into proteostasis alterations in different diseases.