Therapeutic Potential For Neurological Disorders Research Articles

Making the case for prophylactic use of betaine to promote brain health in young (15-24 year old) athletes at risk for concussion.

Betaine supplementation in the context of human nutrition, athletic performance, and clinical therapy demonstrate that the osmolyte and methyl donor, betaine, is cytoprotective and beneficial to human health. These studies also demonstrate that betaine supplementation in healthy humans is straight-forward with no reported adverse effects. Here, we explore betaine uptake in the central nervous system (CNS) and contribute to evidence that betaine may be uniquely protective to the brain. We specifically describe the therapeutic potential of betaine and explore the potential implications of betaine on inhibition mediated by GABA and glycine neurotransmission. The influence of betaine on neurophysiology complement betaine's role as an osmolyte and metabolite and is consistent with clinical evidence of betaine-mediated improvements to cognitive function (reported in elderly populations) and its anti-convulsant properties. Betaine's therapeutic potential in neurological disorders including epilepsy and neurodegenerative diseases combined with benefits of betaine supplementation on athletic performance support the unique application of betaine as a prophylaxis to concussion. As an example, we identify young athletes (15-24 years old), especially females, for prophylactic betaine supplementation to promote brain health and resilience in a cohort at high risk for concussion and for developing Alzheimer's disease.

Calcium Triggered Compound Fusion at Ribbon Synapses During Exocytosis

AbstractBackgroundExocytosis in photoreceptors involves fusion of vesicles with the plasma membrane at the base of a specialized proteinaceous electron‐dense structure known as a synaptic ribbon. The molecular mechanisms underlying exocytosis at ribbon synapses are not well understood. Research suggests a compound fusion model where vesicles at ribbon synapses undergo homotypic fusion forming larger vesicles prior to fusion with the plasma membrane. But how this process is mediated remains unclear. Ca2+ triggers several forms of exocytosis in different types of eukaryotic cells through activation of the SNARE complex. SNARE‐mediated fusion of membranes is triggered by the calcium sensor Synaptotagmin, but not much is known about whether this applies to compound fusion. Here, we report that, like typical synaptic exocytosis, compound fusion is mediated by Ca2+ influx.MethodTo examine the role of calcium in compound fusion, we performed vesicle purification experiments from Mus musculus (C57BL/6 WT) retina. Brain tissue was also used as a control where we expected no compound fusion. Retina and brain tissue were dissected, homogenized, and ultracentrifuged. The supernatant was incubated in 2mM Ca2+ for 5 minutes then vesicles from the Ca2+samples and controls containing no Ca2 were isolated using magnetic beads coupled with rho1D4 monoclonal antibody. Vesicles were negatively stained and viewed under TEM. Vesicle sizes of the Ca2+ and control samples were determined via blind analysis using ImageJ.ResultsIn retina samples, exogenous Ca2+ caused a significant increase in vesicle size when viewed under TEM. In the retina Ca2+ samples, vesicle diameter ranged from 30nm to 250nm. This effect was not seen in retina controls where vesicle diameter ranged from 30nm to 60nm. No large vesicles were seen in any of the brain samples.ConclusionOur research supports the compound fusion model of vesicle exocytosis at ribbon synapses. We suggest that in retina, the larger vesicles formed during exocytosis are a result of smaller vesicles fusing with each other and that Ca2+ has a significant role in this process. Future studies investigating the role of calcium sensors such as synaptotagmin will help to further elucidate the mechanisms underlying compound fusion and its therapeutic potential in neurological disorders.

Mesenchymal stem-cell-derived microvesicles ameliorate MPTP-induced neurotoxicity in mice: a role of the gut-microbiota-brain axis.

Parkinson's disease (PD) is a chronic and progressive neurodegenerative disorder. Increasing evidence suggests the role of the gut-microbiota-brain axis in the pathogenesis of PD. Mesenchymal stem-cell-derived microvesicles (MSC-MVs) have emerged as a therapeutic potential for neurological disorders over the last years. The objective of this study was to investigate whether MSC-MVs could improve PD-like neurotoxicity in mice after administration of MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine). MPTP-induced reductions in the dopamine transporter and tyrosine hydroxylase expressions in the striatum and substantia nigra (SNr) were attenuated after a subsequent single administration of MSC-MVs. Increases in the phosphorylated α-synuclein (p-α-Syn)/α-Syn ratio in the striatum, SNr, and colon after MPTP injection were also attenuated after MSC-MVs injection. Furthermore, MSC-MVs restored MPTP-induced abnormalities of the gut microbiota composition. Interestingly, positive correlations between the genus Dubosiella and the p-α-Syn/α-Syn ratio were observed in the brain and colon, suggesting their roles in the gut-microbiota-brain communication. Moreover, MSC-MVs attenuated MPTP-induced reduction of the metabolite, 3,6-dihydroxy-2-[3-methoxy-4-(sulfooxy)phenyl]-7-(sulfinooxy)-3,4-dihydro-2H-1-benzopyran-5-olate, in the blood. Interestingly, a negative correlation between this compound and the p-α-Syn/α-Syn ratio was observed in the brain and colon. These data suggest that MSC-MVs could ameliorate MPTP-induced neurotoxicity in the brain and colon via the gut-microbiota-brain axis. Therefore, MSC-MVs would have a new therapeutic potential for neurological disorders such as PD.

Safety and efficacy outcomes after intranasal administration of neural stem cells in cerebral palsy: a randomized phase 1/2 controlled trial

BackgroundNeural stem cells (NSCs) are believed to have the most therapeutic potential for neurological disorders because they can differentiate into various neurons and glial cells. This research evaluated the safety and efficacy of intranasal administration of NSCs in children with cerebral palsy (CP). The functional brain network (FBN) analysis based on electroencephalogram (EEG) and voxel-based morphometry (VBM) analysis based on T1-weighted images were performed to evaluate functional and structural changes in the brain.MethodsA total of 25 CP patients aged 3–12 years were randomly assigned to the treatment group (n = 15), which received an intranasal infusion of NSCs loaded with nasal patches and rehabilitation therapy, or the control group (n = 10) received rehabilitation therapy only. The primary endpoints were the safety (assessed by the incidence of adverse events (AEs), laboratory and imaging examinations) and the changes in the Gross Motor Function Measure-88 (GMFM-88), the Activities of Daily Living (ADL) scale, the Sleep Disturbance Scale for Children (SDSC), and some adapted scales. The secondary endpoints were the FBN and VBM analysis.ResultsThere were only four AEs happened during the 24-month follow-up period. There was no significant difference in the laboratory examinations before and after treatment, and the magnetic resonance imaging showed no abnormal nasal and intracranial masses. Compared to the control group, patients in the treatment group showed apparent improvements in GMFM-88 and ADL 24 months after treatment. Compared with the baseline, the scale scores of the Fine Motor Function, Sociability, Life Adaptability, Expressive Ability, GMFM-88, and ADL increased significantly in the treatment group 24 months after treatment, while the SDSC score decreased considerably. Compared with baseline, the FBN analysis showed a substantial decrease in brain network energy, and the VBM analysis showed a significant increase in gray matter volume in the treatment group after NSCs treatment.ConclusionsOur results showed that intranasal administration of NSCs was well-tolerated and potentially beneficial in children with CP.Trial registration: The study was registered in ClinicalTrials.gov (NCT03005249, registered 29 December 2016, https://www.clinicaltrials.gov/ct2/show/NCT03005249) and the Medical Research Registration Information System (CMR-20161129-1003).

Umbilical Cord Blood Cell Clearance Post-Infusion in Immune-Competent Children with Cerebral Palsy

Umbilical cord blood cells have therapeutic potential for neurological disorders, through a paracrine mechanism of action. A greater understanding of the safety and immunological effects of allogeneic donor cord blood cells in the context of a healthy recipient immune system, such as in cerebral palsy, is needed. This study aimed to determine how quickly donor cord blood cells were cleared from the circulation in children with cerebral palsy who received a single intravenous infusion of 12/12 human leucocyte antigen (HLA)-matched sibling cord blood cells. Twelve participants with cerebral palsy aged 2–12 years received cord blood cell infusions as part of a phase I trial of umbilical blood infusion for cerebral palsy. Digital droplet PCR analysis of DNA copy number variants specific to donor and recipient was used to assess donor DNA clearance at five timepoints post-infusion, a surrogate measure of cell clearance. Donor cells were cleared by 3 months post-infusion in 11/12 participants. When detected, donor DNA was at a fraction of 0.01–0.31% of total DNA with no signs of graft-versus-host disease in any participant. The donor DNA clearance times provided by this study have important implications for understanding the safety of allogeneic cord blood cell infusion for cerebral palsy and translational tissue engineering or regenerative medicine research in other disorders.

Astragalin attenuates AlCl3/D-galactose-induced aging-like disorders by inhibiting oxidative stress and neuroinflammation

Astragalin (AST) is a natural flavonoid with excellent antioxidant and anti-inflammatory activities. However, whether AST is an effective chemical for neuronal protection and its underlying mechanisms remain to be elucidated. In this study, we established a mouse model of cognitive impairment and aging-like phenotype induced by sequential administration of AlCl3 and D-galactose (Gal). We found that AST effectively ameliorated cognitive impairment in the model mice and improved their learning and memory performance in the Morris water maze (MWM) test. AlCl3/Gal-induced activation of astrocytes and microglia and inflammation were observed by immunohistochemistry and immunofluorescence, but could be attenuated by AST. In addition, alterations in oxidative stress-regulating enzymes or markers, including T-SOD, T-AOC, CAT, GSH-Px, and MDA, as well as the pro-inflammatory factors TNF-α, IL-1β, and IL-6, were restored. At the mechanistic level, AlCl3/Gal-intoxicated mice showed a significant elevation of Notch/HES-1 and NF-κB signaling axis corresponding to microglia activation and inflammation. AST attenuated the activation of Notch/HES-1 and NF-κB signaling axis, thus reducing the inflammation. In summary, AST is a promising natural product to protect neurons from toxin-induced injury, indicating its therapeutic potential for neurological disorders.

Safinamide protects against amyloid β (Aβ)-induced oxidative stress and cellular senescence in M17 neuronal cells

ABSTRACT Alzheimer’s disease (AD) is a neurodegenerative disorder that is pathologically related to oxidative stress and cellular senescence. Safinamide is one of the clinically prescribed monoamine oxidase B (MAOB) inhibitors. It has been reported to possess therapeutic potential in neurological disorders. However, the therapeutic potential of safinamide in AD is still under investigation. In this study, we explored the effect of safinamide in amyloid (Aβ)1–42 oligomers-stimulated M17 neuronal cells. We established the in vitro model with M17 cells by treating them with 1 μM Aβ1-42 oligomers with or without safinamide (100 or 200 nM). The results show that safinamide ameliorated Aβ1-42 oligomers-induced oxidative stress in M17 cells as revealed by the decreased reactive oxygen species (ROS) production and reduced glutathione (GSH) content. Safinamide treatment significantly ameliorated senescence-associated-β-galactosidase (SA-β-gal)-positive cells and telomerase activity. Further, we show that safinamide treatment resulted in decreased mRNA and protein expressions of p21 and plasminogen activator inhibitor-1 (PAI-1). Moreover, silencing of Sirtuin1 (SIRT1) abolished the effects of safinamide on the mRNA levels of p21 and PAI-1, as well as SA-β-gal-positive cells in Aβ1-42 oligomers-induced M17 cells. In conclusion, we reveal that safinamide exerted a protective function on M17 cells from Aβ1-42 oligomers induction-caused oxidative stress and cellular senescence through SIRT1 signaling. These present results provide meaningful evidence that safinamide may be medically developed for the prevention and therapy of AD.

Related Fluoxetine and Methylprednisolone Changes of TNF-α and IL-6 Expression in The Hypothyroidism Rat Model of Spinal Cord Injury.

ObjectiveSpinal cord injury (SCI) is a serious clinical condition that leads to disability. Following primary injury, pro- inflammatory cytokines play an important role in the subsequent secondary events. The thyroid hormone (TH) is known as the modulator of inflammatory cytokines and acts as a neuroprotective agent. Methylprednisolone (MP) is used for the early treatment of SCI. Fluoxetine (FLX), also is known as a selective serotonin reuptake inhibitor (SSRI), has therapeutic potential in neurological disorders. The aim of the present study was to investigate the combined effects of MP and FLX on SCI in the rat hypothyroidism (hypo) model.Materials and MethodsIn this experimental study, 48 male Wistar rats with hypothyroidism were randomly divided into 6 groups (n=8/group): control (Hypo), Hypo+Surgical sham, Hypo+SCI, Hypo+SCI+MP, Hypo+SCI+FLX, and Hypo+SCI+MP+FLX. SCI was created using an aneurysm clip and Hypothyroidism was induced by 6-Propyl-2-thiouracil (PTU) at a dose of 10 mg/kg/day administered intraperitoneally. Following SCI induction, rats received MP and FLX treatments via separate intraperitoneal injections at a dose of 30 and 10 mg/kg/day respectively on the surgery day and FLX continued daily for 3 weeks. The expression levels of tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) were quantified by Real-time polymerase chain reaction (PCR) and Western blotting. Myelination and glutathione (GSH) levels were analyzed by Luxol Fast Blue (LFB) staining and ELISA respectively. Results Following combined MP and FLX treatments, the expression levels of TNF-α and IL-6 significantly decreased and GSH level considerably increased in the trial animals. ConclusionOur results show the neuroprotective effects of MP and FLX with better results in Hypo+SCI+MP+FLX group. Further study is required to identify the mechanisms involved.

Studies of involvement of G-protein coupled receptor-3 in cannabidiol effects on inflammatory responses of mouse primary astrocytes and microglia.

Cannabidiol (CBD) exhibits anti-inflammatory and neuroprotective properties and is suggested to be effective in the pre-clinical and clinical treatment of illnesses of the central nervous system (CNS). Two major types of CNS glial cells, astrocytes and microglia, play critical roles in the development and pathogenesis of CNS diseases. However, the mechanisms by which CBD plays an anti-inflammatory and neuroprotective role for these glial cells have not been fully elucidated. In this study, we examined the effects of CBD on the inflammatory response of mouse primary astrocytes and microglia. We also investigated whether the effect of CBD on cytokine release is mediated by the G protein coupled receptor 3 (GPR3), which was recently identified as a novel receptor for CBD. Our results showed that CBD inhibited inflammatory responses of astrocytes and microglia stimulated with lipopolysaccharide (LPS), a Toll-like receptor 4 (TLR4) ligand in vitro and in vivo. In addition, CBD reduced the phosphorylation of STAT3 and NF-κB signaling pathways in LPS-stimulated astrocytes. However, the inhibitory effect of CBD on pro-inflammatory cytokine production was independent of GPR3 expression in both types of glial cells. Thus, although CBD is effective in ameliorating the activation of astrocytes and microglia, its mechanism of action still requires further study. Our data support the concept that CBD may have therapeutic potential for neurological disorders that involve neuroinflammation.

Music as Add-On Therapy in the Rehabilitation Program of Parkinson's Disease Patients-A Romanian Pilot Study.

Music has been proven to have therapeutic potential in neurological disorders, especially Parkinson’s disease (PD), since rhythmic auditory cueing can partially replace the progressive loss of rhythmicity and automaticity. Several reports have highlighted improvements in motor outcomes in PD patients undergoing music therapy, but only a few studies have evaluated non-motor outcomes, such as quality of life (QoL), which deteriorates with disease progression. The current pilot study aims to examine the effects of a multimodal rehabilitation program centered on physical therapy combined with listening to music on self-reported QoL in people with PD, compared to the same rehabilitation program alone. The study was conducted on patients with idiopathic PD who attended a specific rehabilitation program with a duration of 2.5 h daily for 14 days. The patients were divided into the study group (16 patients), who listened to background music during the rehabilitation program sessions, and the control group who did not listen to music during sessions. The patients were assessed using the self-report Parkinson’s Disease Questionnaire (PDQ-39) at the beginning of the program and 1 month after its initiation. The patients in the study group registered greater improvements in five of the eight areas of life assessed by PDQ-39 compared to the control group. In conclusion, listening to music combined with a multimodal rehabilitation program centered on physical therapy may be beneficial for the patients’ quality of life.

Neuromodulatory Effects of Transcranial Direct Current Stimulation on Motor Excitability in Rats.

Transcranial direct current stimulation (tDCS) is a noninvasive technique for modulating neural plasticity and is considered to have therapeutic potential in neurological disorders. For the purpose of translational neuroscience research, a suitable animal model can be ideal for providing a stable condition for identifying mechanisms that can help to explore therapeutic strategies. Here, we developed a tDCS protocol for modulating motor excitability in anesthetized rats. To examine the responses of tDCS-elicited plasticity, the motor evoked potential (MEP) and MEP input-output (IO) curve elicited by epidural motor cortical electrical stimulus were evaluated at baseline and after 30 min of anodal tDCS or cathodal tDCS. Furthermore, a paired-pulse cortical electrical stimulus was applied to assess changes in the inhibitory network by measuring long-interval intracortical inhibition (LICI) before and after tDCS. In the results, analogous to those observed in humans, the present study demonstrates long-term potentiation- (LTP-) and long-term depression- (LTD-) like plasticity can be induced by tDCS protocol in anesthetized rats. We found that the MEPs were significantly enhanced immediately after anodal tDCS at 0.1 mA and 0.8 mA and remained enhanced for 30 min. Similarly, MEPs were suppressed immediately after cathodal tDCS at 0.8 mA and lasted for 30 min. No effect was noted on the MEP magnitude under sham tDCS stimulation. Furthermore, the IO curve slope was elevated following anodal tDCS and presented a trend toward diminished slope after cathodal tDCS. No significant differences in the LICI ratio of pre- to post-tDCS were observed. These results indicated that developed tDCS schemes can produce consistent, rapid, and controllable electrophysiological changes in corticomotor excitability in rats. This newly developed tDCS animal model could be useful to further explore mechanical insights and may serve as a translational platform bridging human and animal studies, establishing new therapeutic strategies for neurological disorders.

In vitro and in vivo characterization of a synthetic scorpion toxin AmmTx3, a potent inhibitor of cardiac voltage-gated potassium channel Kv4.2

Voltage-gated potassium channel Kv4.2 (encoded by KCND2 gene) contributes to the cardiac transient outward potassium current (Ito1). This current is the main contributor to the repolarisation phase 1 of the cardiac action potential. The toxin AmmTx3, identified from the venom of the scorpion Androctonus mauretanicus , is a blocker of Kv4.x channels, and have interesting therapeutic potential for neurological disorders due to its effect in cerebellar granule neurons. Its effects on cardiac Kv4.2 channels remains unclear. The aim of this study was to: – produce a synthetic AmmTx3 (AmmTx3-NCL); – evaluate in vitro and in vivo, effects of this toxin on Kv4.2 channels compared to native toxin. We did chemical synthesis of AmmTx3 using the native chemical ligation strategy (NCL). We characterized its biological activity compared to native AmmTx3 using an automated patch-clamp system (Syncropatch 384 PE, Nanion) on HEK cells co-expressing Kv4.2 α subunit and two auxiliaries subunits (KChIP and DPP6). Effects of AmmTx3 and AmmTx3-NCL on cardiac cellular electrophysiology were studied by patch clamp on adult rat cardiomyocytes and hiPSC-CMs. Integrated cardiac effects of AmmTx3 were studied on ECG of wild-type mice. In this study, we report the total chemical synthesis of AmmTx3 and 3D structure was also determined. As expected, biological activity of folded AmmTx3-NCL is coherent to native toxin with a similar time course of Kv4.2 current inhibition without any change of activation kinetics. Characterisation of AmmTx3 effects on isolated cardiomyocytes, hiPSC-CMs and in vivo by ECG is ongoing. AmmTx3 toxin can be chemically synthesized and used as a Kv4.2 channel inhibitor to contributed to the better understanding of the exact role of Ito1 in cardiac electrophysiology. Those first results seem to be a promising evidence that AmmTx3 could a potential inhibitor of Ito current in early repolarisation syndrome.

Injured adult motor and sensory axons regenerate into appropriate organotypic domains of neural progenitor grafts

Neural progenitor cell (NPC) transplantation has high therapeutic potential in neurological disorders. Functional restoration may depend on the formation of reciprocal connections between host and graft. While it has been reported that axons extending out of neural grafts in the brain form contacts onto phenotypically appropriate host target regions, it is not known whether adult, injured host axons regenerating into NPC grafts also form appropriate connections. We report that spinal cord NPCs grafted into the injured adult rat spinal cord self-assemble organotypic, dorsal horn-like domains. These clusters are extensively innervated by regenerating adult host sensory axons and are avoided by corticospinal axons. Moreover, host axon regeneration into grafts increases significantly after enrichment with appropriate neuronal targets. Together, these findings demonstrate that injured adult axons retain the ability to recognize appropriate targets and avoid inappropriate targets within neural progenitor grafts, suggesting that restoration of complex circuitry after SCI may be achievable.

Tetrahydroxystilbene Glucoside Improves Neurotrophic Factors Release in Cultured Astroglia.

Tetrahydroxystilbene glucoside (TSG), one of the main ingredients of Polygonum multiflorum, has a great number of beneficial effects for health including anti-oxidant, free radicalscavenging and anti-inflammatory properties. However, the potential effects of TSG on neurotrophic factors release remain unclear. In this study, rat primary astroglia cultures were applied to investigate TSG-mediated neurotrophic effects. The protein levels and production of glial cell-line derived neurotrophic factor (GDNF), brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) in astroglia and the culture medium were determined by western blotting assay and ELISA, respectively. Results indicated that TSG increased the production of neurotrophic factors in a concentration-dependent manner. At different time points of TSG treatment, the BDNF and NGF production in the culture medium was increased 48 h after treatment, while GDNF secretion was initially induced 24 h after TSG treatment. Consistent with the neurotrophic factors release, TSG significantly increased the BDNF, GDNF and NGF protein expressions in astroglia. Furthermore, TSG significantly induced the phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 and a specific ERK inhibitor-U0126 inhibited TSG-mediated secretion of BDNF, GDNF and NGF. Overall, this study demonstrated that TSG induces astroglia-derived neurotrophic factors release, suggesting TSG might hold a therapeutic potential for neurological disorders.

Comparative quantification of dietary supplemented neural creatine concentrations with 1H-MRS peak fitting and basis spectrum methods

Magnetic resonance spectroscopy (MRS) is an analytical procedure that can be used to non-invasively measure the concentration of a range of neural metabolites. Creatine is an important neurometabolite with dietary supplementation offering therapeutic potential for neurological disorders with dysfunctional energetic processes. Neural creatine concentrations can be probed using proton MRS and quantified using a range of software packages based on different analytical methods.This experiment examines the differences in quantification performance of two commonly used analysis packages following a creatine supplementation strategy with potential therapeutic application. Human participants followed a seven day dietary supplementation regime in a placebo-controlled, cross-over design interspersed with a five week wash-out period. Spectroscopy data were acquired the day immediately following supplementation and analyzed with two commonly-used software packages which employ vastly different quantification methods.Results demonstrate that neural creatine concentration was augmented following creatine supplementation when analyzed using the peak fitting method of quantification (105.9%±10.1). In contrast, no change in neural creatine levels were detected with supplementation when analysis was conducted using the basis spectrum method of quantification (102.6%±8.6). Results suggest that software packages that employ the peak fitting procedure for spectral quantification are possibly more sensitive to subtle changes in neural creatine concentrations.The relative simplicity of the spectroscopy sequence and the data analysis procedure suggest that peak fitting procedures may be the most effective means of metabolite quantification when detection of subtle alterations in neural metabolites is necessary. The straightforward technique can be used on a clinical magnetic resonance imaging system.

Molecular mechanism of ERK dephosphorylation by striatal‐enriched protein tyrosine phosphatase

Striatal-enriched tyrosine phosphatase (STEP) is an important regulator of neuronal synaptic plasticity, and its abnormal level or activity contributes to cognitive disorders. One crucial downstream effector and direct substrate of STEP is extracellular signal-regulated protein kinase (ERK), which has important functions in spine stabilisation and action potential transmission. The inhibition of STEP activity toward phospho-ERK has the potential to treat neuronal diseases, but the detailed mechanism underlying the dephosphorylation of phospho-ERK by STEP is not known. Therefore, we examined STEP activity toward para-nitrophenyl phosphate, phospho-tyrosine-containing peptides, and the full-length phospho-ERK protein using STEP mutants with different structural features. STEP was found to be a highly efficient ERK tyrosine phosphatase that required both its N-terminal regulatory region and key residues in its active site. Specifically, both kinase interaction motif (KIM) and kinase-specific sequence of STEP were required for ERK interaction. In addition to the N-terminal kinase-specific sequence region, S245, hydrophobic residues L249/L251, and basic residues R242/R243 located in the KIM region were important in controlling STEP activity toward phospho-ERK. Further kinetic experiments revealed subtle structural differences between STEP and HePTP that affected the interactions of their KIMs with ERK. Moreover, STEP recognised specific positions of a phospho-ERK peptide sequence through its active site, and the contact of STEP F311 with phospho-ERK V205 and T207 were crucial interactions. Taken together, our results not only provide the information for interactions between ERK and STEP, but will also help in the development of specific strategies to target STEP-ERK recognition, which could serve as a potential therapy for neurological disorders. Regulation of phospho-ERK by STEP underlies important neuronal activities. A detailed enzymologic characterisation and cellular studies of STEP revealed that specific residues in KIM and active site mediated ERK recognition. Structural differences between the KIM-ERK interfaces and the active site among different ERK phosphatases could be targeted to develop specific STEP inhibitor, which has therapeutic potential for neurological disorders. PKA, protein kinase A & NGF, nerve growth factor.

AMPA receptors as drug targets in neurological disease – advantages, caveats, and future outlook

Most excitatory transmission in the brain is mediated by the AMPA receptor subtype of the ionotropic glutamate receptors. In many neurological diseases, synapse structure and AMPA receptor function are altered, thus making AMPA receptors potential therapeutic targets for clinical intervention. The work summarized in this review suggests a link between AMPA receptor function and debilitating neuropathologies, and discusses the current state of therapies targeting AMPA receptors in four diseases. In amyotrophic lateral sclerosis, AMPA receptors allow cytotoxic levels of calcium into neurons, leading to motor neuron death. Likewise, in some epilepsies, overactivation of AMPA receptors leads to neuron damage. The same is true for ischemia, where oxygen deprivation leads to excitotoxicity. Conversely, Alzheimer's disease is characterized by decreased AMPA activation and synapse loss. Unfortunately, many clinical studies have had limited success by directly targeting AMPA receptors in these diseases. We also discuss how the use of AMPA receptor modulators, commonly known as ampakines, in neurological diseases initially seemed promising in animal studies, but mostly ineffective in clinical trials. We propose that indirectly affecting AMPA receptors, such as by modulating transmembrane AMPA receptor regulatory proteins or, more generally, by regulating glutamatergic transmission, may provide new therapeutic potential for neurological disorders.

Prevention of neuropathic pain in an animal model of spare nerve injury following oral immunization with recombinant adenovirus serotype 5-mediated NR2B gene transfer

Spinal N-methyl-D-aspartate receptor 2B subunit (NR2B)-increased expression plays an important role in the facilitation and maintenance of the persistent pain state due to peripheral nerve injury. A vaccination strategy to reduce the expression of brain protein is feasible and may have therapeutic potential for neurological disorders. Thus, we investigated the effect of oral immunization with recombinant adenovirus serotype 5-mediated NR2B gene transfer (rAd5/NR2B) for the modulation of neuropathic pain. After peroral administration of the rAd5/NR2B vaccine, transgene NR2B expression persisted for at least a week and was associated with the induction of high serum titers of NR2B-specific antibodies. Following the occurrence of mechanical allodynia due to peripheral nerve injury, NR2B-specific antibodies could pass the blood-brain barrier, transport and subsequently bind to the spinal NR2B protein. The humoral immunoresponse results in the strong antiallodynia in the spared nerve injury animal model. These data proved the feasibility of oral immunization with rAd5/NR2B for the prevention of neuropathic pain.

Brain transplantation of human neural stem cells transduced with tyrosine hydroxylase and GTP cyclohydrolase 1 provides functional improvement in animal models of Parkinson disease

Parkinson disease is a neurodegenerative disease characterized by loss of midbrain dopaminergic neurons resulting in movement disorder. Neural stem cells (NSC) of the CNS have recently aroused a great deal of interest, not only because of their importance in basic research of neural development, but also for their therapeutic potential in neurological disorders. We have recently generated an immortalized human NSC cell line, HB1.F3, via retrovirus-mediated v-myc transfer. This line is capable of self-renewal, is multipotent, and expresses cell specific markers for NSC, ATP-binding cassettes transporter (ABCG2) and nestin. Next, we co-transduced the F3 NSC line with genes encoding tyrosine hydroxylase (TH) and GTP cyclohydrolase 1 (GTPCH1) in order to generate dopamine-producing NSC. The F3.TH.GTPCH human NSC line expresses TH and GTPCH phenotypes as determined by RT-PCR, western blotting and immunocytochemistry, and shows a 800 to 2000-fold increase in production of L-dihydroxyphenyl alanine in HPLC analysis. A marked improvement in amphetamine-induced turning behavior was observed in parkinsonian rats implanted with F3.TH.GTPCH cells, but not in control rats receiving F3 NSC. In the animals showing functional improvement, a large number of TH-positive F3.TH.GTPCH NSC were found at injection sites. These results indicate that human NSC, genetically transduced with TH and GTPCH1 genes, have great potential in clinical utility for cell replacement therapy in patients suffering from Parkinson disease.

An oral vaccine against NMDAR1 with efficacy in experimental stroke and epilepsy.

The brain is generally considered immunoprivileged, although increasing examples of immunological responses to brain antigens, neuronal expression of major histocompatibility class I genes, and neurological autoimmunity have been recognized. An adeno-associated virus (AAV) vaccine generated autoantibodies that targeted a specific brain protein, the NR1 subunit of the N-methyl-D-aspartate (NMDA) receptor. After peroral administration of the AAV vaccine, transgene expression persisted for at least 5 months and was associated with a robust humoral response in the absence of a significant cell-mediated response. This single-dose vaccine was associated with strong anti-epileptic and neuroprotective activity in rats for both a kainate-induced seizure model and also a middle cerebral artery occlusion stroke model at 1 to 5 months following vaccination. Thus, a vaccination strategy targeting brain proteins is feasible and may have therapeutic potential for neurological disorders.