Signaling through the Rho family of small GTPases has been increasingly investigated for their involvement in a wide variety of diseases such as cardiovascular, pulmonary, and neurological disorders as well as cancer. Rho GTPases are a subfamily of the Ras superfamily proteins which play essential roles in a number of biological processes, especially in the regulation of cell shape change, cytokinesis, cell adhesion, and cell migration. Many of these processes demonstrate a common theme: the rapid and dynamic reorganization of actin cytoskeleton of which Rho signaling has now emerged as a major switch control. The involvement of dynamic changes of Rho GTPases in disease states underscores the need to produce effective inhibitors for their therapeutic applications. Fasudil and Y-27632, with many newer additions, are two classes of widely used chemical compounds that inhibit Rho kinase (ROCK), an important downstream effector of RhoA subfamily GTPases. These inhibitors have been successful in many preclinical studies, indicating the potential benefit of clinical Rho pathway inhibition. On the other hand, except for Rac1 inhibitor NSC23766, there are few effective inhibitors directly targeting Rho GTPases, likely due to the lack of optimal structural information on individual Rho-RhoGEF, Rho-RhoGAP, or Rho-RhoGDI interaction to achieve specificity. Recently, LM11A-31 and other derivatives of peptide mimetic ligands for p75 neutrotrophoin receptor (p75NTR) show promising effects upstream of Rho GTPase signaling in neuronal regeneration. CCG-1423, a chemical compound showing profiles of inhibiting downstream of RhoA, is a further attempt for the development of novel pharmacological tools to disrupt Rho signaling pathway in cancer. Because of a rapidly growing number of studies deciphering the role of the Rho proteins in many diseases, specific and potent pharmaceutical modulators of various steps of Rho GTPase signaling pathway are critically needed to target for therapeutic intervention in cardiovascular disease, neurological disorders, and cancer progression.
Rho GTPase; ROCK; Y-27632; fasudil; LM11A-31; NSC23766; CCG-1423
Feline immunodeficiency virus (FIV) infection like human immunodeficiency virus (HIV), produces systemic and central nervous system disease in its natural host, the domestic cat, that parallels the pathogenesis seen in HIV-infected humans. The ability to culture feline nervous system tissue affords the unique opportunity to directly examine interactions of infectious virus with CNS cells for the development of models and treatments that can then be translated to a natural infectious model. To explore the therapeutic potential of a new p75 neurotrophin receptor ligand, LM11A-31, we evaluated neuronal survival, neuronal damage and calcium homeostasis in cultured feline neurons following inoculation with FIV. FIV resulted in the gradual appearance of dendritic beading, pruning of processes and shrinkage of neuronal perikarya in the neurons. Astrocytes developed a more activated appearance and there was an enhanced accumulation of microglia, particularly at longer times post-inoculation. Addition of 10 nM LM11A-31, to the cultures greatly reduced or eliminated the neuronal pathology as well as the FIV effects on astrocytes and microglia. LM11A-31 also, prevented the development of delayed calcium deregulation in feline neurons exposed to conditioned medium from FIV treated macrophages. The suppression of calcium accumulation prevented the development of foci of calcium accumulation and beading in the dendrites. FIV replication was unaffected by LM11A-31. The strong neuroprotection afforded by LM11A-31 in an infectious in vitro model indicates that LM11A-31 may have excellent potential for the treatment of HIV-associated neurodegeneration.
Rett syndrome (RTT) results from loss-of-function mutations in the gene encoding the methyl-CpG-binding protein 2 (MeCP2) and is characterized by abnormal motor, respiratory and autonomic control, cognitive impairment, autistic-like behaviors and increased risk of seizures. RTT patients and Mecp2 null mice exhibit reduced expression of Brain-Derived Neurotrophic Factor (BDNF), which has been linked in mice to increased respiratory frequency, a hallmark of RTT. The present study was undertaken to test the hypotheses that BDNF deficits in Mecp2 mutants are associated with reduced activation of the BDNF receptor, TrkB, and that pharmacologic activation of TrkB would improve respiratory function. We characterized BDNF protein expression, TrkB activation and respiration in heterozygous female Mecp2 mutant mice (Het), a model that recapitulates the somatic mosaicism for mutant Mecp2 found in typical RTT patients, and evaluated the ability of a small molecule TrkB agonist, LM22A-4, to ameliorate biochemical and functional abnormalities in these animals. We found that Het mice exhibit 1) reduced BDNF expression and TrkB activation in the medulla and pons and 2) breathing dysfunction, characterized by increased frequency due to periods of tachypnea, and increased apneas, as in RTT patients. Treatment of Het mice with LM22A-4 for 4 weeks rescued wildtype levels of TrkB phosphorylation in the medulla and pons and restored wildtype breathing frequency. These data provide new insight into the role of BDNF signaling deficits in the pathophysiology of RTT and highlight TrkB as a possible therapeutic target in this disease.
The lack of effective therapies for spinal cord injury points to the need for identifying novel targets for therapeutic intervention. Here we report that a small molecule, LM11A-31, developed to block proNGF-p75 interaction and p75-mediated cell death crosses the blood–brain barrier efficiently when delivered orally. Administered starting 4 h postinjury, LM11A-31 promotes functional recovery without causing any toxicity or increased pain in a mouse model of spinal contusion injury. In both weight-bearing open-field tests and nonweight-bearing swim tests, LM11A-31 was effective in improving motor function and coordination. Such functional improvement correlated with a >50% increase in the number of surviving oligodendrocytes and myelinated axons. We also demonstrate that LM11A-31 indeed inhibits proNGF-p75 interaction in vivo, thereby curtailing the JNK3-mediated apoptotic cascade. These results thus highlight p75 as a novel therapeutic target for an orally delivered treatment for spinal cord injury.
CSPGs (chondroitin sulfate proteoglycans) are a family of extracellular matrix molecules with various functions in regulating tissue morphogenesis, cell division and axon guidance. A number of CSPGs are highly upregulated by reactive glial scar tissues after injuries and form a strong barrier for axonal regeneration in the adult vertebrate CNS. Although CSPGs may negatively regulate axonal growth via binding and altering activity of other growth-regulating factors, the molecular mechanisms by which CSPGs restrict axonal elongation are not well understood. Here, we identified a novel receptor mechanism whereby CSPGs inhibit axonal growth via interactions with neuronal transmembrane LAR (the leukocyte common antigen-related phosphatase). CSPGs bind LAR with high affinity in transfected COS-7 cells and co-immunoprecipitate with LAR expressed in various tissues including the brain and spinal cord. CSPG stimulation enhances activity of LAR phosphatase in vitro. Deletion of LAR in knockout mice or blockade of LAR with sequence-selective peptides significantly overcomes neurite growth restrictions of CSPGs in neuronal cultures. Intracellularly, CSPG-LAR interaction mediates axonal growth inhibition of neurons partially via inactivating Akt and activating RhoA signals. Systemic treatments with LAR-targeting peptides in mice with thoracic spinal cord transection injuries induce significant axon growth of descending serotonergic fibers in the vicinity of the lesion and beyond in the caudal spinal cord and promote locomotor functional recovery. Identification of LAR as a novel CSPG functional receptor provides a therapeutic basis for enhancing axonal regeneration and functional recovery after CNS injuries in adult mammals.
Chondroitin sulfate proteoglycans; LAR; receptor; axon regeneration; spinal cord injury
Alzheimer's disease (AD), the most common form of dementia, is an age-dependent progressive neurodegenerative disorder. β-amyloid, a metabolic product of the amyloid precursor protein (APP), plays an important role in the pathogenesis of AD. The Thy1-hAPPLond/Swe+ (line 41) transgenic mouse overexpresses human APP751 and contains the London (V717I) and Swedish (K670M/N671L) mutations. Here, we used a battery of behavioral tests to evaluate general activity, cognition, and social behavior in six-month-old male Thy1-hAPPLond/Swe+ mice. We found hyperactivity in a novel environment as well as significant deficits in spontaneous alternation behavior. In fear conditioning (FC), Thy1-hAPPLond/Swe+ mice did not display deficits in acquisition or in memory retrieval in novel context of tone-cued FC, but they showed significant memory retrieval impairment during contextual testing in an identical environment. Surprisingly, in a standard hidden platform water maze, no significant deficit was detected in mutant mice. However, a delayed-matching-to-place paradigm revealed a significant deficit in Thy1-hAPPLond/Swe+ mice. Lastly, in the social novelty session of a three-chamber test, Thy1-hAPPLond/Swe+ mice exhibited a significantly decreased interest in a novel versus a familiar stranger compared to control mice. This could possibly be explained by decreased social memory or discrimination and may parallel disturbances in social functioning in human AD patients. In conclusion, the Thy1-hAPPLond/Swe+ mouse model of AD displayed a behavioral phenotype that resembles, in part, the cognitive and psychiatric symptoms experienced in AD patients.
Alzheimer's disease; amyloid precursor protein; behavior; learning and memory; neurodegenerative disorder; social interaction
The ability to regulate neurogenesis in the adult dentate gyrus will require further identification and characterization of the receptors regulating this process. In vitro and in vivo studies have demonstrated that neurotrophins and the p75 neurotrophin receptor (p75NTR) can promote neurogenesis; therefore we tested the hypothesis that p75NTR is expressed by adult dentate gyrus progenitor cells and is required for their proliferation and differentiation.
In a first series of studies focusing on proliferation, mice received a single BrdU injection and were sacrificed 2, 10 and 48 hours later. Proliferating, BrdU-positive cells were found to express p75NTR. In a second series of studies, BrdU was administered by six daily injections and mice were sacrificed 1 day later. Dentate gyrus sections demonstrated a large proportion of BrdU/p75NTR co-expressing cells expressing either the NeuN neuronal or GFAP glial marker, indicating that p75NTR expression persists at least until early stages of maturation. In p75NTR (-/-) mice, there was a 59% decrease in the number of BrdU-positive cells, with decreases in the number of BrdU cells co-labeled with NeuN, GFAP or neither marker of 35%, 60% and 64%, respectively.
These findings demonstrate that p75NTR is expressed by adult dentate progenitor cells and point to p75NTR as an important receptor promoting the proliferation and/or early maturation of not only neural, but also glial and other cell types.
Brain-derived neurotrophic factor (BDNF) activates the receptor tropomyosin-related kinase B (TrkB) with high potency and specificity, promoting neuronal survival, differentiation, and synaptic function. Correlations between altered BDNF expression and/or function and mechanism(s) underlying numerous neurodegenerative conditions, including Alzheimer disease and traumatic brain injury, suggest that TrkB agonists might have therapeutic potential. Using in silico screening with a BDNF loop–domain pharmacophore, followed by low-throughput in vitro screening in mouse fetal hippocampal neurons, we have efficiently identified small molecules with nanomolar neurotrophic activity specific to TrkB versus other Trk family members. Neurotrophic activity was dependent on TrkB and its downstream targets, although compound-induced signaling activation kinetics differed from those triggered by BDNF. A selected prototype compound demonstrated binding specificity to the extracellular domain of TrkB. In in vitro models of neurodegenerative disease, it prevented neuronal degeneration with efficacy equal to that of BDNF, and when administered in vivo, it caused hippocampal and striatal TrkB activation in mice and improved motor learning after traumatic brain injury in rats. These studies demonstrate the utility of loop modeling in drug discovery and reveal what we believe to be the first reported small molecules derived from a targeted BDNF domain that specifically activate TrkB.We propose that these compounds constitute a novel group of tools for the study of TrkB signaling and may provide leads for developing new therapeutic agents for neurodegenerative diseases.
Oligomeric forms of amyloid-β(1–42) (Aβ) are thought to play a causal role in Alzheimer’s disease (AD) and the p75 neurotrophin receptor (p75NTR) has been implicated in Aβ-induced neurodegeneration. To further define the functions of p75NTR in AD, we examined the interaction of oligomeric Aβ with p75NTR, and the effects of that interaction on neurite integrity in neuron cultures and in a chronic AD mouse model. Atomic force microscopy was used to ascertain the aggregated state of Aβ, and fluorescence resonance energy transfer (FRET) analysis revealed that Aβ oligomers interact with the extracellular domain of p75NTR. In vitro studies of Aβ-induced death in neuron cultures isolated from wildtype and p75NTR −/− mice, in which the p75NTR extracellular domain is deleted, showed reduced sensitivity of mutant cells to Aβ-induced cell death. Interestingly, Aβ-induced neuritic dystrophy and activation of c-Jun, a known mediator of Aβ-induced deleterious signaling, were completely prevented in p75NTR −/− neuron cultures. Thy1-hAPPLond/Swe X p75NTR−/− mice exhibited significantly diminished hippocampal neuritic dystrophy and complete reversal of basal forebrain cholinergic neurite degeneration relative to those expressing wild type p75NTR. Aβ levels were not affected, suggesting that removal of p75NTR extracellular domain reduced the ability of excess Aβ to promote neuritic degeneration. These findings indicate that while p75NTR likely does not mediate all Aβ effects, it does play a significant role in enabling Aβ-induced neurodegeneration in vitro and in vivo, establishing p75NTR as an important therapeutic target for AD.
p75NTR; amyloid-β; Alzheimer’s disease; neuritic dystrophy; neurodegeneration; basal forebrain cholinergic neurons
The neurotrophin receptor p75 is induced by various injuries to the nervous system, but its role after injury has remained unclear. Here, we report that p75 is required for the death of oligodendrocytes following spinal cord injury, and its action is mediated mainly by proNGF. Oligodendrocytes undergoing apoptosis expressed p75, and the absence of p75 resulted in a decrease in the number of apoptotic oligodendrocytes and increased survival of oligodendrocytes. ProNGF is likely responsible for activating p75 in vivo, since the proNGF from the injured spinal cord induced apoptosis among p75+/+, but not among p75-/-, oligodendrocytes in culture, and its action was blocked by proNGF-specific antibody. Together, these data suggest that the role of proNGF is to eliminate damaged cells by activating the apoptotic machinery of p75 after injury.
Many chemotherapy drugs are known to cause significant clinical neurotoxicity, which can result in the early cessation of treatment. To identify and develop more effective means of neuroprotection it is important to understand the toxicity of these drugs at the molecular and cellular levels. In the present study, we examine the effects of paclitaxel (taxol), cisplatin, and methotrexate on primary rat neurons including hippocampal, cortical, and dorsal horn/dorsal root ganglion neuronal cultures. We found that all of these anti-cancer drugs induce substantial neurotoxicity evidenced by neurite degeneration. The neurons are capable of recovering after treatment withdrawal, but taxol exerts a biphasic effect that results in the collapse of processes days after treatment is withdrawn. After cisplatin and methotrexate treatment, we observed the degeneration of neuronal processes including the reduction of dendritic branching, length, and altered growth cone formation, indicating an abnormal arrangement of the actin cytoskeleton consistent with the involvement of Rho family small GTPases. Inhibiting RhoA downstream effector p160ROCK/Rho kinase using Y-27632, or activating p75 neurotrophin receptor (p75NTR) using non-peptide mimetic LM11A-31, were able to reverse the degeneration caused by cisplatin and methotrexate. Therefore, the neurotoxicity resulting from exposure to the anti-cancer drugs cisplatin and methotrexate can be alleviated by inhibiting Rho signaling pathway.
cancer drug; neurotoxicity; neuroprotectant; cisplatin; methotrexate; Rho GTPases
During development of the visual system, retinal ganglion cells (RGCs) require cell-cell adhesion molecules and extracellular matrix proteins for axon growth. In this study, we demonstrate that the classical cadherin, E-cadherin, is expressed in RGCs from E6 to E12 and promotes neurite outgrowth from all regions of the chick retina at E6, E8 and E10. E-cadherin is also expressed in the optic tectum. E-cadherin adhesion blocking antibodies specifically inhibit neurite outgrowth on an E-cadherin substrate. The receptor-type protein tyrosine phosphatase, PTPμ, associates with E-cadherin. In this manuscript, we demonstrate that antisense-mediated down regulation of PTPμ, overexpression of catalytically inactive PTPμ, and perturbation of endogenous PTPμ using a specific PTPμ inhibitor peptide results in a substantial reduction in neurite outgrowth on E-cadherin. Taken together these findings demonstrate that E-cadherin is an important adhesion molecule for chick RGC neurite outgrowth and suggest that PTPμ expression and catalytic activity are required for outgrowth on an E-cadherin substrate.
neurite outgrowth; receptor protein tyrosine phosphatase (PTPμ); cadherin; retina; tectum; axon guidance
Background and Purpose
Stroke is the leading cause of long-term disability in the United States, yet no drugs are available that are proven to improve recovery. Brain-derived neurotrophic factor (BDNF) stimulates neurogenesis and plasticity, processes that are implicated in stroke recovery. It binds to both the tropomyosin-related kinase B (TrkB) and p75 neurotrophin (p75NTR) receptors. However, BDNF is not a feasible therapeutic agent, and no small molecule exists that can reproduce its binding to both receptors. We tested the hypothesis that a small molecule (LM22A-4) that selectively targets TrkB would promote neurogenesis and functional recovery after stroke.
Four-month-old mice were trained on motor tasks prior to stroke. After stroke, functional test results were used to randomize mice into two equally, and severely, impaired groups. Beginning 3 days after stroke, mice received LM22A-4 or saline vehicle daily for ten weeks.
LM22A-4 treatment significantly improved limb swing speed and accelerated the return to normal gait accuracy after stroke. LM22A-4 treatment also doubled both the number of new mature neurons and immature neurons adjacent to the stroke. Drug-induced differences were not observed in angiogenesis, dendritic arborization, axonal sprouting, glial scar formation, or neuroinflammation.
A small molecule agonist of TrkB improves functional recovery from stroke and increases neurogenesis when administered beginning three days after stroke. These findings provide proof-of-concept that targeting of TrkB alone is capable of promoting one or more mechanisms relevant to stroke recovery. LM22A-4 or its derivatives might therefore serve as “pro-recovery” therapeutic agents for stroke.
Stroke recovery; neurotrophin; small molecule
The p75 neurotrophin receptor (p75NTR) is expressed by neurons particularly vulnerable in Alzheimer's disease (AD). We tested the hypothesis that non-peptide, small molecule p75NTR ligands found to promote survival signaling might prevent Aβ-induced degeneration and synaptic dysfunction. These ligands inhibited Aβ-induced neuritic dystrophy, death of cultured neurons and Aβ-induced death of pyramidal neurons in hippocampal slice cultures. Moreover, ligands inhibited Aβ-induced activation of molecules involved in AD pathology including calpain/cdk5, GSK3β and c-Jun, and tau phosphorylation, and prevented Aβ-induced inactivation of AKT and CREB. Finally, a p75NTR ligand blocked Aβ-induced hippocampal LTP impairment. These studies support an extensive intersection between p75NTR signaling and Aβ pathogenic mechanisms, and introduce a class of specific small molecule ligands with the unique ability to block multiple fundamental AD-related signaling pathways, reverse synaptic impairment and inhibit Aβ-induced neuronal dystrophy and death.