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Although the concepts of secondary injury and neuroprotection after neurotrauma are well-supported experimentally, clinical trials of neuroprotective agents in traumatic brain injury or spinal cord injury have been disappointing. Most strategies to date have used drugs directed toward a single pathophysiological mechanism that contributes to early necrotic cell death. Given these failures, recent research has increasingly focused on multifunctional (multipotential, pluripotential) agents that target multiple injury mechanisms, particularly those that occur later after the insult. Here we review two such approaches that show particular promise in experimental neurotrauma — cell cycle inhibitors and small cyclized peptides. Both show extended therapeutic windows for treatment and appear to share at least one important target.
Trauma to the central nervous system (CNS) causes both direct tissue damage and more delayed biochemical changes that lead to cell loss (secondary injury), demyelination and related functional deficits.1 Initiation of such biochemical cascades occurs from minutes to weeks after the insult. Numerous factors associated with delayed tissue loss have been identified from experimental studies of traumatic brain injury (TBI) and spinal cord injury (SCI); these include products of lipid degradation, disrupted ionic homeostasis, altered neurotransmitter release and receptor function, and inflammatory and immune changes.1–3 Together, these biochemical and associated metabolic effects result in loss of neuronal and oligodendroglial cells, reactive astrogliosis, and proliferation/activation of microglia.4,5
Most neuroprotective strategies have been directed at individual components of this delayed reactive cascade, such as reducing free radical-induced actions, excitotoxicity or inflammation. Whereas many such strategies have proven effective in experimental animal models of TBI or SCI, they have shown little or no neuroprotective actions in humans.2 However, the majority of clinical neuroprotective approaches to date have been directed at reducing neuronal necrosis – a relatively early event that is largely completed within 6–8 h.6 Yet only a relative minority of patients with neurotrauma can have treatment initiated within this time period. In addition, most therapies have aimed at modifying single components of the complex secondary injury cascade, even though it is recognized that many autodestructive biochemical changes occur in parallel. Use of multiple drug treatments, each directed to a different secondary injury component, has rarely been attempted, even experimentally, in neurotrauma,7 although multifactorial combination drug approaches have long been standard therapy for certain infectious diseases and cancers. However, even if such combination treatments showed promise in animal models, the methodological difficulties and costs associated with such multi-drug comparison studies in treating clinical neurotrauma would likely prove prohibitive.
An alternative approach would be to identify single agents that can modify diverse secondary injury cascades. A number of such multifunctional or multipotential treatments has been proposed and successfully tested in experimental neurotrauma models. These have included naturally occurring substances such as thyrotropin releasing hormone (TRH), progesterone, heat shock protein, neurotrophic factors and erythropoietin; drugs developed for other disorders such as statins or antibiotics; and agents developed through rational drug design.2
We have developed two multifunctional treatment approaches that have proved to be remarkably effective for the treatment of TBI and/or SCI. One has been developed through a rational drug design program and based upon the tripeptide hormone TRH. The other has adapted drugs used extensively in experimental oncology, with targets based upon data developed from extensive genomics profiling in experimental TBI and SCI.
In the early 1980s, we demonstrated that TRH – when used at higher than physiological concentrations – markedly improved outcome after experimental SCI, with a therapeutic window of at least 24 h.8,9 TRH inhibits multiple secondary injury factors or processes, including declines of blood flow and bioenergetics, lipid degradation products such as peptidyl leukotrienes and platelet activating factor, ionic dyshomeostasis (Na+, K+, Ca++, Mg++), endogenous opioids and excitotoxins.10–12 Subsequently, we found that TRH analogues that modified either the N-terminal or the middle amino acid of this tri-peptide hormone (pyroglamyl- histidyl- prolineamide) were even more effective than TRH, with longer biological half-lives and fewer undesirable physiological actions. Such analogues proved highly effective in improving functional recovery and reducing lesion volume after experimental SCI or TBI.13–17 The neuroprotective actions of TRH and TRH analogues in experimental neurotrauma have subsequently been confirmed by many laboratories.18–22 Moreover, a small clinical trial of TRH suggested protective effects after SCI.23
TRH is metabolized through two major pathways: endopeptidase cleavage of pyrogluamyl to produce cyclo-histidyly-proline diketopiperazine (CHP) or deamidation yielding the free acid form of TRH.24,25 Various TRH analogues have been developed that modify one of its amino acids (Fig. 1).26 Pyroglutamyl substitutions limit endopeptidase-mediated metabolism, resulting in compounds that have far longer biological half-lives than TRH (6–8 h versus 5 min); some of these are also more potent than TRH in terms of central nervous system (CNS) activity. For example, YM-14673 is longer acting than TRH (8–36 times) and much more potent (10–100 times).15 However, N-terminal substitutions retain the other physiological actions of TRH – endocrine, autonomic and analeptic. We have also evaluated modifications of the histidyl residue (imidazole substitution); certain substitutions reduced the cardiovascular and/or endocrine activity while maintaining the neuroprotective actions of TRH (Fig. 2).27 Critically, modification of the C-terminus results in compounds devoid of neuroprotective activity, although they retain endocrine, autonomic and analeptic activity similar to TRH.
Based upon these observations, we developed dual-substituted TRH analogues (modifications at both the N-terminal and histidyl moieties). Such compounds (53a, 57a) have limited endocrine, autonomic and analeptic effects while preserving or enhancing the neuroprotective actions (Fig. 2).16, 26 Compound 53a is at least two orders of magnitude more hydrophobic than either TRH or YM-14673, based upon their partition coefficients between n-octanol and water (logP); thus it should have enhanced cellular permeability to the CNS.16
TRH is metabolized to a cyclic dipeptide (CHP), which, like other diketopiperazines, retains considerable physiological activity.28 We have developed a series of diketopiperazines structurally related to CHP (Fig. 3).29 One of these (35b) has been extensively examined using in vitro and in vivo model systems.29–32 In neuronal cell culture models, 35b provides neuroprotection in necrotic cell death models (maitotoxin, glutamate, mechanical injury), as well as in apoptotic cell death models (staurosporine, beta amyloid) (Fig. 4).31 Given intravenously, 35b reduced lesion volume by nearly 70%. It also improved functional (cognitive and motor) outcomes after either fluid percussion-induced traumatic brain injury (FPI) in rats or controlled cortical impact (CCI) injury in mice.30, 31 Treatment also significantly reduced apoptotic cell death in rat hippocampus following FPI. The therapeutic window for the drug is at least 8 h, and it shows a relatively flat dose response for neuroprotection between 0.1 and 10 mg/kg. Optimal doses are between 1 and 3 mg/kg, with repeated dosing over time showing no added benefit as compared to single bolus dose treatment. 35b is currently being developed by RemeGenix, Inc., for clinical trials in head injury.
Using the NIMH Psychoactive Drug Screening Program, 35b does not have significant binding affinities for any of 50 classical receptors, channels and transporters tested.31 It also does not bind to either high or low affinity TRH receptors. To better address potential mechanisms, we performed temporal profiling using Affymetrix microarrays. Treatment with 35b after FPI up-regulated various endogenous neuroprotective factors (BDNF, HSP 70, HIF1, mGluR7) and down-regulated a number of recognized secondary injury factors (cyclins, calpain, cathepsin).29,32 These findings were confirmed by PCR and protein measurements. Particularly noteworthy were the effects of treatment on cell cycle proteins, whose up-regulation is associated with neuronal apoptosis, astrogliosis and microglial activation after TBI or SCI.33, 34 Administration of 35b suppressed expression of the major upstream cell cycle proteins including cyclin D1, the retinoblastoma protein Rb, and E2F5.29
Various other cyclic dipeptides reduce lesion volume and improve behavioral outcome after CCI in mice, with effects that are similar to those of 35b (Fig. 5).29 In compound 606 the histidine moiety was replaced by 3,5-di-tert-butyltyrosine (DBT), a phenolic amino acid that can trap reactive oxygen species. In contrast to 35b, 606 blocked free radical-mediated cell death in neuronal cultures induced by FeSO4. Compound 144 also showed substantial neuroprotective actions in vitro and was highly protective in both the FPI and CCI models (Fig. 5).29
Progression through the cell cycle is carefully regulated through the interplay of a number of cell cycle-related proteins, including cyclins, cyclin dependent kinases (CDKs) and CDK inhibitors. Early events include the synthesis of cyclin D, which binds to CDK4 and CDK6; in the nucleus, CDK4/6 phosphorylate the retinoblastoma protein (Rb), leading to release of E2F transcription factors and transition to G1.35, 36 Apoptosis and cell cycle pathways share several common regulatory elements, including the retinoblastoma protein (Rb), E2F, and p53.
From extensive temporal profiling studies of gene expression changes following rodent SCI,37,38 we identified a cluster of cell cycle genes that were coordinately regulated with the oncogene c-myc, which has been linked to neuronal cell death.37,39 These gene expression changes, detected using Affymetrix chips (Fig. 6), were confirmed using RT-PCR, Western blots and immunocytochemistry.34,37,38 Importantly, up-regulated cell cycle proteins include key upstream regulatory elements that lead to G1 transition – including cyclin D1, Rb and E2F5. Injury is also associated with down-regulation of endogenous cell cycle inhibitors such as p27.
It is known that up-regulation of cell cycle proteins in post-mitotic cells such as neurons or oligodendroglia results in caspase-mediated cell death.40 Consistent with this view, we found that increased cell cycle expression in neurons was associated with active caspase 3 expression and/or TUNEL positive staining after TBI 33,40 or SCI37 (Fig. 7). Up-regulation of cell cycle proteins is also readily observed in primary neuronal culture models following stimulation of caspase-dependent apoptosis with ceramide,41 β-amyloid,42 KCl withdrawal,43 or DNA damage.44–46 In addition, kainic acid-induced excitotoxicity of cerebellar granule cells is associated with increased expression of cyclins D and E, PCNA and E2F1, as well as with increased expression of caspases 3 and 9.47 Similarly, trophic withdrawal-induced cell death is associated with increased expression of both cyclins and cyclin dependent kinases.48
SCI and TBI cause active astrogliosis that causes glial scar formation and proliferation/activation of microglia. After injury, cell cycle proteins are highly expressed in GFAP positive cells, as well as in activated microglia. For example, cyclin D1 expression is found to be increased in microglia after transient forebrain ischemia in the rat49 and global ischemia in the gerbil.50 Astrocyte proliferation was also associated with increased cell cycle proteins after ischemia.50 Following SCI, microglia and astrocytes demonstrate a marked reduction in p27, an endogenous cell cycle inhibitor.51 Up-regulation of cell cycle proteins is observed in primary cell culture models, including astroglial proliferation after exposure to serum52 or microglial proliferation/activation following exposure to lipopolysaccharide (LPS).53 Following CNS injury, astrocytes undergo rapid proliferation and contribute to the formation of the glial scar.54 This scar may provide a physical barrier to axonal growth55 as well as a ‘wall’ to prevent migration of inflammatory cells into undamaged tissue.56,57 Microglia, the primary immunological cell in the CNS, undergo rapid proliferation and transition from a resting, ramified phenotype to an ameboid phagocytic phenotype that is nearly indistinguishable from infiltrating macrophages.58 Activated microglia produce pro-inflammatory molecules such as interleukin (IL)-1β, IL6, inducible nitric oxide synthase,59 complement components60 and reactive oxygen species,61 which serve to modify both secondary injury and endogenous neuroprotective responses.
Increases in cell cycle protein expression have also been reported in chronic neurodegenerative disorders. For example, both neurons and glia show increased PCNA and cyclin D expression in human Alzheimer’s patients.62,63 Furthermore, DNA replication has been identified in apoptotic neurons in human Alzheimer’s patients.64 In an animal model of Alzheimer’s disease, genetic APP23 mice demonstrate an increase of cell cycle-related proteins in astrocytes.65
Cell cycle inhibitors have been developed and extensively evaluated in experimental cancer models, and several have been tested in humans. The best characterized and studied among these are flavopiridol, a semi-synthetic flavonoid derived from rohitukin bark 66, and the purine analogues roscovitine and olomoucine.67 Flavopiridol blocks all the CDKs and also inhibits the transcription of cyclin D1.68, 69 In contrast, the purine analogues preferentially inhibit CDK 2 and CDK5, although at higher concentrations they may inhibit other kinases.70
Each of these agents shows neuroprotection in vitro, such as against etoposide-induced neuronal apoptosis 33 or apoptosis of cerebellar granule cells following KCl withdrawal.43 Moreover, olomoucine inhibits hypoxia-induced neuronal cell death in culture,71,whereas flavopiridol inhibits kainite-mediated or colchicines-mediated apoptotic cell death.72, 73 A recent study in our laboratory determined that inhibition of multiple cyclin-dependent kinases reduces etoposide-induced neuronal apoptosis, including CDK1 and CDK4.74
Cell cycle inhibitors show inhibitory effects on the proliferation and activation of mitotic cells, such as microglia and astrocytes in vitro. For example, stimulation of microglia with LPS induces proliferation. Pre-treatment of microglia with cell cycle inhibitors, such as flavopiridol or roscovitine, for 1 h prior to the addition of LPS results in a significant suppression of microglial proliferation 33 and nitric oxide production.74 Importantly, roscovitine treatment of microglial cells stimulated with LPS reduced microglial-induced neurotoxicity.74 Similarly, proliferation of astrocytes induced by the addition of 10% serum was completely inhibited by flavopiridol.33
In vivo, cell cycle inhibition using pharmacological approaches has shown neuroprotective effects. For example, early treatment with flavopiridol, administered centrally, showed remarkable neuroprotection after FPI in rats.40 Lesion volume was reduced by approximately 70% and chronic behavioral recovery (motor and cognitive) was indistinguishable from sham-injured controls. Caspase-mediated neuronal cell death after TBI was nearly completely attenuated. In addition to neuroprotection, significant effects on mitotic cells were also observed. GFAP expression and markers of microglial activation were markedly reduced. These changes were associated with near complete suppression of cell cycle proteins in neurons, astroglia and microglia, respectively.40 Delayed administration of flavopiridol was similarly found to have neuroprotective effects. In a follow up study, flavopiridol was administered centrally at 30 min or 4 h after FPI, or systemically (intraperitoneally) at 24 h after FPI;33 each of these treatments resulted in markedly reduced lesion volumes – approximately 90%, 50% and 60%, respectively (Fig. 8).
The more specific cell cycle inhibitor roscovitine, which does not have potentially confusing effects on gene transcription, has similar actions after FPI. Administration of roscovitine 30 min after FPI resulted in highly significant reductions in lesion volume and improved behavioral outcome (motor and cognitive). This cell cycle inhibitor also reduced astrogliosis and produced a marked inhibition of microglial activation-related inflammation74 (Fig. 9).
Research in SCI confirms the strong beneficial effects of treatment with cell cycle inhibitors. We have shown that flavopiridol treatment – administered centrally by mini-osmotic pump beginning 30 min post-trauma and continuing over 7 days – significantly improved motor recovery and reduced lesion volume at 28 days.34 Treatment reduced cell cycle protein induction in neurons and astrocytes; this reduction was associated with decreased cleaved caspase-3 labeling in neurons and oligodendrocytes, as well as reduction in glial scar. Neuronal loss, measured by MAP-2 staining, was alleviated by flavopiridol treatment, and tissue loss overall was significantly reduced. Further, treatment with the cell cycle inhibitor markedly limited microglial activation and associated inflammatory factors (Figs. 10, ,11).11). The cell cycle inhibitor olomoucine has also been shown to decrease lesion volume and improve function after SCI.75 Reductions in microglial-related inflammation76 and astrocytic scar75 were found with olomoucine treatment, supporting the beneficial effect of cell cycle inhibition after SCI. Preliminary work using cyclin D1 knockout mice subjected to SCI has shown decreased lesion volumes in knockouts as compared to wild type controls, consistent with the pharmacological inhibition studies.34
Research in experimental cerebral ischemia is highly consistent with the TBI and SCI studies. Dominant negative CDK4/5 animals show reduced neuronal cell death after focal or global brain ischemia,77 as does treatment with CDK inhibitors.78, 79 Moreover, after focal cerebral ischemia, cyclin D1 knockouts or animals treated with olomoucine show reduced astrocyte proliferation. Excitotoxic cell death following kainic acid administration is attenuated by treatment with anti-sense oligonucleotides directed against CDK4 or cyclin D1.
Activation of cell cycle proteins in the CNS causes proliferation of mitotic cells such as astroglia or microglia, but induces apoptosis in post-mitotic cells such as neurons or oligodendroglia. Acute injuries to the CNS, including TBI and SCI, cause up-regulation of many cell cycle proteins in both mitotic and post-mitotic cells. These changes cause neuronal and oligodendroglial cell death, astroglial scar formation and proliferation/activation of microglia with the release of associated inflammatory factors. Treatment with cell cycle inhibitors results in striking neuroprotection, likely related to its multifunctional actions on these diverse cell types. Because cell cycle proteins have such diverse effects, even selective inhibitors of these pathways may serve as multifunctional neuroprotective agents.
Another approach to multi-potential drug treatment of CNS injury is to use compounds that modulate different signal transduction pathways that are involved in secondary injury. TRH is a naturally occurring brain hormone which, when used at higher than physiological levels as a drug, can inhibit many factors and mechanisms implicated in delayed cell death. Thus, TRH and TRH analogues can improve blood flow and bioenergetic state; limit loss of ionic homeostasis; reduce lipid degradation; and inhibit the actions of endogenous opioids, leukotrienes, platelet activating factor and possibly glutamate.29,32 The neuroprotective effects do not appear to be mediated by TRH receptors, as they occur at supraphysiological doses and can be dissociated from the other physiological effects of TRH (endocrine, analeptic, autonomic).
Diketopiperazines that are structurally related to a metabolic product of TRH have marked neuroprotective activity but do not act on either high or low affinity TRH receptors. They also have diverse multifunctional neuroprotective actions. Like cell cycle inhibitors, the prototype compound 35b inhibits the activation of many cell cycle proteins after injury. But they also reduce other known secondary injury factors, including calpains and cathepsins, while up-regulating several well-established endogenous neuroprotective factors including BDNF, HSP 70 and HIF 1. Each of the latter factors has considerable protective activity in animal models. These findings underscore the attractiveness of multifunctional drug approaches for the treatment of neurotrauma and other neurodegenerative disorders.
This work has been supported by NIH grants 5R01NS054221 to AIF.
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