Endogenous or exogenous insults can cause spinal cord injury (SCI), often resulting in the loss of motor, autonomic, sensory and reflex functions. The pathogenesis of SCI comprises two stages. The primary injury stage occurs at the moment of trauma and is characterized by hemorrhage and rapid cell death. The secondary injury stage occurs due to progression of primary damage and is characterized by tissue loss and functional disorder. One of the most important cellular mechanisms underlying secondary injury is glutamate excitotoxicity, which overactivates the calpain protease via excessive Ca2+ influx and induces neuronal apoptosis via p53 induction. Furthermore, Ca2+ influx elicits apoptosis by inducing p53, thus negatively affecting two pathways: the mitogenic extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) pathway and the survival phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) pathway. Speedy/rapid inducer of G2/M progression in oocytes (Speedy/RINGO) is a cell cycle regulatory protein that increases survival of p53-positive mitotic cells by inhibiting the apoptotic machinery. Moreover, this protein elicits p53-dependent anti-apoptotic effects on calpain-induced degeneration of primary hippocampal neurons, amyotrophic lateral sclerosis motor neurons, and astrocytes and microglia in spinal cord lesions. The pathophysiology of SCI has not been fully elucidated and this hinders the development of powerful therapeutic strategies. This review focuses on the cellular mechanisms underlying the anti-apoptotic effects of Speedy/RINGO and discusses how this protective function can possibly be exploited to facilitate recovery from SCI. Particular attention is paid to reversal of the negative effects on the ERK/MAPK and PI3K/AKT pathways via induction of p53.
Spinal cord injury (SCI) can be defined as an endogenous or exogenous trauma resulting in the loss of motor, autonomic, sensory and/or reflex functions. SCI is a major cause of permanent disability. Researchers estimate that 230,000 people in the United States are living with an SCI, and that 10,000 new patients are diagnosed each year[
The pathology of human spinal cord injury is the result of two main mechanisms known as “primary” and “secondary” injury. Primary injury begins at the moment of trauma and is characterized by hemorrhage and rapid cell death. Secondary injury is an extension of the original injury and occurs when vascular and biochemical effects cause tissue loss and functional disorders[
Among all, the most destructive cellular mechanism underlying secondary injury is glutamate excitotoxicity, which overactivates calpain protease via excessive Ca2+ influx and induces neuronal apoptosis via p53 induction[
Speedy/rapid inducer of G2/M progression in oocytes (Speedy/RINGO) is a cell cycle regulatory protein that increases survival of p53-positive mitotic cells by inhibiting the apoptotic machinery[
As yet, there is not any proven treatment regimen for SCI probably due to its lesser known pathophysiology. Revealing cellular mechanisms of SCI and correlating them with the clinical symptoms are of primary importance for developing effective SCI recovery treatments. In this regard, this review focuses on the underlying molecular mechanisms of Speedy/RINGO’s anti-apoptotic function by correlating these mechanisms with the complex pathophysiology of SCI. Furthermore, this review discusses how this protective function could possibly be exploited to facilitate recovery from SCI. Particular attention is paid to reversal of the negative effects on the ERK/MAPK and PI3K/AKT pathways via induction of p53. This new approach may assist in identifying the most promising molecular targets for effective treatment modalities and may also uncover the molecular basis of SCI.
Excitotoxicity is defined as cell damage or cell death resulting from exposure to excitatory amino acids such as glutamate. Glutamate is a major neurotransmitter that plays an important role in the central nervous system[
A diagram depicting the mechanism of glutamate excitotoxicity. SCI: spinal cord injury
Ionic balance is essential to protecting the functional integrity of neural cells. Therefore, Ca2+ imbalance provides a mechanism for a severe secondary injury. The influx of Ca2+ is triggered by trauma through glutamate toxicity and continues for some time once it has been triggered. Ca2+ influx has serious negative effects in neural cells, including mitochondrial damage that further destabilizes Ca2+ balance, generation of free radicals, and activation of many enzymes, including calpain. This ultimately triggers degradation of cellular components, leading to p53 induction and subsequent caspase-dependent apoptosis[
Programmed cell signaling pathways play an important role in the pathobiology of neurological diseases such as SCI. After spinal cord trauma, a number of cells at the lesion site die via apoptosis or necrosis. Apoptosis is a programmed cell suicide mechanism which can be triggered by cytokines, post traumatic inflammation, free radicals and excitotoxicity. Recent studies confirm that cells of injured spinal cord tissue primarily die due to apoptosis[
Apoptosis is commonly observed in both neurons and oligodendrocytes, increasing the possibility of paralysis in patients with SCI. An experimental study in rats showed that apoptosis occurred 4 h after trauma, and that the effect of the injury could be decreased as late as 3 weeks after SCI[
In SCI, increased intracellular Ca2+ influx as a result of glutamate induction is one of the major apoptotic insults leading to overactivation of certain proteases which subsequently cause proteolytic degradation of myelin and cytoskeletal proteins and degeneration of axons. These are all hallmarks of secondary injury and contribute to the progression of SCI[
Based upon these findings, it is evident that glutamate-mediated p53 induction is the prominent reason for apoptosis in SCI. The p53-dependent anti-apoptotic function of Speedy/RINGO makes it an excellent therapeutic candidate for treatment of SCI.
Current research approaches for developing novel therapeutic regimens target both primary and secondary injuries, which are the hallmarks of SCI. Since more complex, multifaceted neurodegenerative progression occurs in secondary injury, the main aim of such investigations is to understand the underlying molecular mechanisms and find the potential key molecules to target for effective SCI treatment. Since the complex molecular mechanisms of SCI have only been partially elucidated, most efforts so far have had limited efficacy. These efforts mainly involve providing anti-neuro-inflammatory conditions[
Spinal cord injury results in loss of oligodendrocytes which, in turn, causes demyelination of axons. Since demyelination largely impedes functional recovery from SCI, an important treatment modality involves preventing oligodendrocytic death[
In addition to genetic and molecular-based studies, some researchers are studying the efficacy of transplanting stem cells, Schwann cells, peripheral blood stem cells and bone marrow to replace lost tissue[
Numerous studies on spinal cord injury in rodents, primates and humans have indicated that the level of inflammation increases as a result of glial cell activation and filtration of somatic immune cells through mechanically disrupted spinal cord tissue[
In addition to ERK/MAPK signaling, the other component responsible for this astrocytic proliferation was shown to be the mitotic regulator Speedy/RINGO[
Findings of these studies on astrocytic gliosis indicate that the interaction between Speedy/RINGO upregulation and ERK/MAPK hyper-phosphorylation leads to glial scar formation
A schematic diagram for proposed proliferative regulation of Speedy/rapid inducer of G2/M progression in oocytes (Speedy/RINGO) on extracellular signal-regulated kinase/mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase/protein kinase B (AKT) signaling cascades in astrocytic gliosis in spinal cord injury
Apart from the research studies on astrocytic gliosis, other experiments showed that, in SCI, the intraneuronal Ca2+ level increases as a result of glutamate induction. Consequent deregulation of Ca2+ homeostasis leads to abnormal activation of proteases, which subsequently cause proteolytic cleavage and degradation of myelin and cytoskeletal proteins, along with degeneration of axons. These are all hallmarks of secondary injury that contribute greatly to the progression of SCI[
As previously mentioned, one of these proteases, calpain, experimentally induces apoptosis in degenerating neurons through increasing p53 and caspase-3 activation[
Furthermore, there is strong evidence indicating that intracellular Ca2+ influx gives rise to apoptotic deregulation of mitogenic ERK/MAPK and survival PI3K/AKT pathways through p53 induction[
In another
Finally, a research group performed an
The aforementioned studies dealing with neurons and astrocytes demonstrate the controllable nature of ERK/MAPK and PI3K/AKT pathways through different effector molecules, including p53, NGF, CNTF and, most probably, Speedy/RINGO. This implies that properly balancing the activity of these pathways with respect to different neuropathological conditions and different cell types may help prevent neurodegeneration and apoptosis.
To recapitulate, deregulation of intraneuronal Ca2+ influx - which is one of the well-known triggering events of secondary injury in SCI - results in activation of calpain. This activation subsequently increases p53 levels, which abnormally regulate MAPK and PI3K/AKT pathways and lead to severe neurodegeneration and apoptotic death. A number of investigations have studied whether the reduction of apoptotic effects of Ca2+ influx can prevent or minimize secondary injury in SCI.
Estrogen is one inhibitor that has been used to protect cells in culture and in rat models against apoptosis. Researchers showed that estrogen and its analogs decreased the activity of calpain protease[
Melatonin, known for its antioxidant and anti-inflammatory properties, is another anti-apoptotic agent in SCI. It has been shown that melatonin promotes neuronal survival by preventing secondary injury through free oxygen radical scavenging[
Abnormal intracellular Ca2+ influx, as an integral part of SCI, has apoptotic effects such as aberrant regulation of MAPK and AKT signaling pathways via p53 induction. Because of this, it is vital to overcome, or at least minimize, this apoptotic effect of Ca2+ and provide neuroprotection to neurons at the injury site. In order to achieve this goal, the first challenge is to thoroughly understand the exact pro-apoptotic mechanisms driven by Ca2+ influx and the key factors involved in these mechanisms. From this point of view, superior neuronal protection in SCI involves a two-pronged approach: (1) reversal of the apoptotic effect on injured neurons caused by the apoptotic deregulation of the ERK/MAPK and PI3K/AKT pathways; and (2) inhibition of pathological calpain protease activation.
The goal of our laboratory is to understand the pro-apoptotic mechanism of Ca2+ deregulation and to prevent apoptosis by inhibiting the downstream effects of lethal Ca2+ influx in neurons. To this effect, we are studying the novel cell cycle regulatory protein Speedy/RINGO due to its p53-dependent anti-apoptotic function which has previously been observed in U2OS osteosarcoma cells[
The main function of Speedy/RINGO is the regulation of the cell cycle in mitotic cells. However, recent studies show that Speedy/RINGO also has an anti-apoptotic effect in DNA-damaged mitotic cells, allowing for their survival[
In eukaryotic cells, cell cycle progress is strictly controlled by cyclin-dependent kinases (CDKs) which are regulated by cyclins. Cyclins regulate CDK activity during different phases of the cell cycle by binding and phosphorylating them. Although cyclins are the key regulators of CDK activity, Speedy/RINGO, a novel cell cycle regulator, is shown to bind and regulate CDK activity in many eukaryotic cell types[
Speedy/RINGO was first identified in
Unlike cyclins, Speedy/RINGO binds and activates CDKs by a yet unelucidated phosphorylation-free mechanism[
There are at least three major branches in the Speedy/RINGO family (A, B and C), with a fourth branch (D) suspected. Speedy/RINGO A, the human homologue Spy1, is the most conserved and the most slowly evolving branch of Speedy/RINGO family. This is the branch used in our laboratory. Branch A is found in nearly all types of cells in fish, chickens, sea urchins and mammals. Expression levels are higher in testis tissue than in tissues such as brain, heart, lung, placenta, prostate, small intestine,
Although the main function of Speedy/RINGO is cell cycle regulation, studies have attributed a p53-dependent anti-apoptotic function to Speedy/RINGO in DNA-damaged mitotic cells, resulting in those cells evading apoptosis and, thus, surviving[
When apoptotic insult occurs during a cell cycle, the resulting DNA damage triggers cell cycle arrest. This arrest, in turn, activates checkpoint responses to allow cells to repair the DNA damage[
Speedy/RINGO has been shown to prevent p53-dependent apoptosis which is normally induced in response to DNA damage in a mitotic human osteosarcoma cell line, U2OS[
Although there are intrinsic regulatory mechanisms for calcium influx into neurons, a number of insults such as glutamate neurotoxicity cause deregulation of calcium homeostasis by increasing intraneuronal calcium influx, as in SCI. This increase in calcium influx induces cystein proteases, including calpain, and results in pathologic calpain activation. Pathological activation of calpain is known to be one of the most important neurodegenerative factors triggering apoptosis, which it does by inducing p53 and activating caspase-3.
Calpain overactivation directly or indirectly induces p53 expression and drives neurons into apoptosis. Indirectly, overactivated calpain cleaves p35 protein into p25 and p10 fractions. Under normal conditions, p35 is the partner for non-mitotic neuron-specific kinase cdk5, forming a cdk5/p35 complex. This complex functions in important cellular events such as neuronal development and maturation[
Calpain overactivation leads to increased p53 expression and activation which, in turn, triggers caspase-mediated apoptosis of neurons[
In addition to its cell cycle regulatory function, Speedy/RINGO has also been shown to function in preventing apoptosis by inhibiting caspase-3 activation in a p53-dependent manner in mitotic U2OS cells.
Since Speedy/RINGO is primarily a cell cycle regulatory protein, it is highly expressed in mitotic cells compared to post-mitotic cells such as neurons[
With this in mind, our laboratory designed an
In another recent study, Speedy/RINGO expression levels were shown to substantially decreased in ALS motor neurons compared with wild-type controls[
Even though the exact mechanism for the protective role of Speedy/RINGO in p53-mediated apoptosis requires further analysis, the effects are not due to the direct inhibition of calpain activity or p53 induction, as calpain-mediated p53 induction was maintained even in the presence of Speedy/RINGO[
In addition to the aforementioned studies on degenerating primary neurons and ALS motor neurons, results of carcinogenic[
p53 is a tumor suppressive transcription factor that inhibits tumorigenesis under genotoxic conditions by regulating gene expression. p53 induces anti-tumorigenesis mechanisms - including cell cycle arrest, senescence and apoptosis - according to cellular conditions, type and intensity of stress signals[
Under stress conditions, different signaling pathways can be triggered. For example, p38[
Another p53-related signaling pathway is PI3K/AKT. P13K/AKT has primarily been implicated in promoting cell survival in response to extracellular signals[
There is growing body of evidence indicating a negative regulatory function for p53 on cell survival in healthy cells. In this mechanism, p53 binds to the promoter site of PTEN (a phosphatase and tensin homolog deleted on chromosome ten)[
These two pathways have been shown to be equally important for neuronal survival and regeneration after nerve injury. Researchers found that 7 days after axotomy, ERK/MAPK and PI3K/AKT signaling activity was increased, causing reduced apoptosis[
Evidently, ERK/MAPK and PI3K/AKT signaling is very important for cell survival. Depending on the cellular context, cell type, and internal/external stimuli, however, p53 may act as a strong anti-apoptotic or pro-apoptotic regulator of both pathways.
Previous investigations by our lab indicate that Speedy/RINGO protects neurons against calpain-mediated p53-dependent apoptosis without decreasing p53 levels. This finding strongly implies that the anti-apoptotic effect of Speedy/RINGO is downstream of p53 activation, not directly on calpain or p53 itself. As explained above, the most remarkable downstream targets of p53, in terms of generating an apoptotic effect on neurons, are ERK/MAPK and PI3K/AKT pathways. Therefore, in degenerating neurons, Speedy/RINGO may use its ability to regulate ERK/MAPK and PI3K/AKT pathways to reverse the apoptosis-triggering effect of p53 induction on these pathways
A schematic diagram for the proposed mechanism of anti-apoptotic action of Speedy/rapid inducer of G2/M progression in oocytes (Speedy/RINGO) on mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase and phosphoinositide 3-kinase/protein kinase B (AKT) signaling cascades in degenerating neurons
Furthermore, cancer studies show promising evidence of direct or indirect interaction of Speedy/RINGO with ERK/MAP and PI3K/AKT pathways. Several studies on tumorigenesis in breast tissue show that ERK/MAPK pathway overactivation results in Speedy/RINGO overexpression. As a result of inhibition of enzyme MEK1, a member of the MAPK pathway, Speedy/RINGO expression is shown to decrease[
In addition, studies using testis tissue revealed that Speedy/RINGO overexpression causes an increase in Cyclin A2-cdk2 expression[
On the other hand, studies on glial scar formation through astrocytic gliosis, a hallmark of secondary injury that inhibits axonal regeneration[
Findings from these studies on astrocytic gliosis imply that there may be an interaction between upregulation of Speedy/RINGO and ERK/MAPK hyper-phosphorylation leading to glial scar formation. Taking these data into consideration, it is reasonable to think that Speedy/RINGO may have reversed the inhibitory effect of p53 on ERK/MAPK and PI3K/AKT pathways, and thus prevented apoptosis in degenerating hippocampal and ALS motor neurons. Hence, it is worthwhile to further explore p53-dependent anti-apoptotic regulatory function of Speedy/RINGO on these pathways.
Our laboratory is currently investigating the function of Speedy/RINGO on the ERK/MAPK and PI3K/AKT pathways using undifferentiated p53- and Speedy/RINGO-expressing neuronal-like neuroblastoma cells. Preliminary data give remarkable clues indicating that Speedy/RINGO plays an essential role on the regulation of ERK/MAPK and PI3K/AKT signaling pathways that directly affect the apoptotic state and survival rate of neuroblastoma cells. More precisely, silencing of the Speedy/RINGO gene significantly alters expression levels and phosphorylation states of certain members of the ERK/MAPK and PI3K/AKT pathways. This, in turn, leads to apoptotic death of neuroblastoma cells, likely due to the absence of Speedy/RINGO’s regulatory function on these two pathways.
SCI is a critical clinical issue whose ongoing destructive path affects patients for life. It is one of the most important causes of disability and mortality around the world[
It has long been known that glutamate-induced Ca2+ influx through glutamate receptors, known as glutamate excitotoxicity, is indispensable for SCI. This influx ultimately causes the p53-mediated apoptotic death of neurons. It is most likely that p53 exerts its apoptotic function on the members of ERK/MAPK and PI3K/AKT signaling cascades[
The goal of our laboratory is to elucidate and prevent the pro-apoptotic intracellular Ca2+ deregulation in neurons. We are therefore optimistic about Speedy/RINGO, a novel cell cycle regulatory protein proven to have a p53-dependent anti-apoptotic function in different cell types, including U2OS osteosarcoma cells[
Although the mechanism of Speedy/RINGO’s anti-apoptotic function in degenerating neurons is not yet known, Speedy/RINGO most probably exhibits its protective function on downstream targets of p53, rather than on p53 levels directly[
Overexpressing Speedy/RINGO in
As described elsewhere in this paper, we believe that Speedy/RINGO is likely to exhibit anti-apoptotic activity in the neuron and glia cells of areas affected by SCI, making this protein a strong potential candidate for therapeutic treatment of SCI patients. In order to confirm the presumed anti-apoptotic function of Speedy/RINGO in SCI, further studies should be performed with both
It is important to remember that Speedy/RINGO’s anti-apoptotic function in neurons and astrocytes may be advantageous or disadvantageous depending on the cell type and function in SCI. Speedy/RINGO’s anti-apoptotic function is desirable in neurons, but undesirable for astrocytes, since it causes glial scar formation and thereby prevents axonal regeneration. In developing an effective SCI recovery regimen, Speedy/RINGO will need to be regulated differentially depending on the therapeutic target.
The pathophysiology of SCI has not yet been fully elucidated, making it difficult to develop effective treatment methods. Overcoming this problem will require collaboration between basic and clinical researchers. Basic research must take place to gain a clear understanding of the basic neuronal and glial mechanisms seen in SCI before these mechanisms can be linked to clinical SCI symptoms and recovery. Versatile molecules like Speedy/RINGO are an excellent tool for increasing our understanding of the molecular mechanisms of SCI with the goal of developing effective treatment strategies.
We sincerely thank Prof. Arzu Karabay for her invaluable contributions as an advisor to our studies on primary hippocampal neurons. We are grateful to Prof. Daniel J. Donoghue for his generous gift of myc-tagged Speedy A-pCS3 construct for neuronal transfection studies. We also warmly thank Sharon Page for editing this paper.
Made substantial contributions to the conception and design of the study, performed data analysis and interpretation, and wrote the paper: Yildiz A
Performed data acquisition and provided technical support: Kaya Y
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Studies mentioned here that were performed in our laboratory were supported by grants to Ayşegül Yıldız from Mugla Sitki Kocman University Scientific Research Project Office, Research and Development Projects (17/023), to Arzu Karabay from The Turkish Academy of Sciences Distinguished Young Scientist Award (TÜBA-GEBIP), and The Scientific and Technological Research Council of Turkey (TÜBİTAK), The Basic Sciences Research Group (TBAG) (108T811).
Both authors declared that there are no conflicts of interest.
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© The Author(s) 2019.