l-Theanine attenuates cadmium-induced neurotoxicity through the inhibition of oxidative damage and tau hyperphosphorylation

Cadmium (Cd) has long been known to induce neurological degenerative disorders. We studied effects of l-theanine, one of the major amino acid components in green tea, on Cd-induced brain injury in mice. Male ICR mice were intraperitoneally injected with l-theanine (100 or 200 mg/kg/day) or saline and after one hour these mice were orally administrated with CdCl₂ (3.75–6 mg/kg). The treatment was conducted for 8 weeks. l-Theanine significantly reduced Cd level in the mouse brain and plasma. Cd-induced neuronal cell death in the mouse cortex and hippocampus were apparently inhibited by l-theanine treatment. l-Theanine also decreased the levels of malondialdehyde (MDA) and ROS, and obviously elevated the levels of glutathione (GSH) and activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) in the mouse brain. Hyperphosphorylation of tau protein is proposed to be an early event for the evolution of tau pathology, and may play an important role in Cd-induced neurodegeneration. Our results showed that l-theanine significantly suppressed Cd-induced tau protein hyperphosphorylation at Ser199, Ser202, and Ser396. Mechanism study showed that l-theanine inhibited the activation of glycogen synthase kinase-3β (GSK-3β) which contributed to the hyperphosphorylation of tau and Cd-induced cytotoxicity. Furthermore, l-theanine reduced Cd-induced cytotoxicity possibly by interfering with the Akt/mTOR signaling pathway. In conclusion, our study indicated that l-theanine protected mice against Cd-induced neurotoxicity through reducing brain Cd level and relieved oxidative damage and tau hyperphosphorylation. Our foundings provide a novel insight into the potential use of l-theanine as prophylactic and therapeutic agents for Cd-induced neurodegenerative diseases.     

Effect of amyloid-Β (25–35) in hyperglycemic and hyperinsulinemic rats,effects on phosphorylation and O-GlcNAcylation of tau protein

Aggregation of the amyloid beta (Aβ) peptide and hyperphosphorylation of tau protein, which are markers of Alzheimer's disease (AD), have been reported also in diabetes mellitus (DM). One regulator of tau phosphorylation is O-GlcNAcylation, whereas for hyperphosphorylation it could be GSK3beta, which is activated in hyperglycemic conditions. With this in mind, both O-GlcNAcylation and phosphorylation of tau protein were evaluated in the brain of rats with streptozotocin (STZ)-induced hyperglycemia and hyperinsulinemia and treated with the Aß25–35 peptide in the hippocampal region CA1. Weight, glycated hemoglobin, glucose, and insulin were determined. Male Wistar rats were divided in groups (N = 20): a) control, b) treated only with the Aβ25–35 peptide, c) treated with Aβ25–35 and STZ, and d) treated only with STZ. Results showed statistically significant differences in the mean weight, glucose levels, insulin concentration, and HbA1c percentage, between C- and D-treated groups and not STZ-treated A and B (P < 0.05). Interestingly, our results showed diminution of O-GlcNAcylation and increase in P-tau-Ser-396 in the hippocampal area of the Aβ25–35- and STZ-treated groups; moreover, enhanced expression of GSK3beta was observed in this last group. Our results suggest that hyperinsulinemia-Aβ25–35-hyperglycemia is relevant for the down regulation of O-GlcNAcylation and up-regulation of the glycogen synthase kinase-3 beta (GSK3beta), favoring Aβ25–35-induced neurotoxicity in the brain of rats.     

Cadmium-induced IL-6 and IL-8 expression and release from astrocytes are mediated by MAPK and NF-κB pathways

Chronic exposure to cadmium has been linked to brain cancers, learning disabilities and memory deficits. Previous studies of cadmium toxicity in the central nervous system report cadmium induces oxidative stress in neurons and astrocytes. In the peripheral system, cadmium promotes interleukin-6 (IL-6) and IL-8 production and release. Elevation of IL-6 expression is linked to the pathogenesis of neurodegenerative diseases and astrogliosis. IL-8 plays a role in angiogenesis of gliomas and neurodegenerative diseases. Herein, the effects of non-toxic concentrations of cadmium on the production of IL-6 and IL-8 and the underlying mechanisms were investigated. U-87 MG human astrocytoma cells and primary human astrocytes were exposed to cadmium chloride. At 24 h post-exposure to 1 and 10 μM, levels of intracellular cadmium in U-87 MG cells were 11.89 ± 3.59 and 53.08 ± 7.59 μg/g wet weight, respectively. These concentrations had minimal effects on cell morphology and viability. IL-6 and IL-8 mRNA levels and secretion increased in dose- and time-dependent manners post cadmium exposure. Acute exposure to cadmium increased phosphorylation of ERK1/2, p38 MAPK, and p65 NF-κB. Pretreatment with U0126–an inhibitor of MEK1 and MEK2 kinases–SB203580–a p38 MAPK inhibitor–and SC-514–an IKKβ inhibitor–suppressed cadmium-induced IL-8 expression and release. Upregulation of cadmium-induced IL-6 was inhibited by U0126 and SC-514, but not SB203580. On the other hand, SP600125–a JNK inhibitor–and celecoxib–a selective COX-2 inhibitor–had no effect on production of both cytokines. In conclusion, non-toxic concentrations of cadmium can stimulate IL-6 and IL-8 release through MAPK phosphorylation and NF-κB activation. Suppressing IL-6 and IL-8 production could be novel approaches to prevent cadmium-induced angiogenesis in gliomas and inflammation in the brain.     

Matrix metalloproteinase-9 contributes to the increase of tau protein in serum during acute ischemic stroke

Previous studies indicate that tau protein, a marker of damage to neurons, is present in the serum of healthy patients at a concentration approximately 40% that of patients with ischemic stroke We assumed that increased serum activity of gelatinases (matrix metalloproteinase [MMP]-2 and MMP-9) can influence the level of tau protein in serum, probably due to disruption of the blood–brain barrier. We obtained blood sera from 31 patients admitted within the first 24 hours of ischemic stroke on days 1, 5 and 10, following the onset of stroke. Tau protein was detected in the serum of 12 patients (38.7%). The highest MMP-9 activity was recorded on day 5 (p < 0.05). Serum gelatinase activity did not differ between tau protein-positive or -negative individuals. However, a high degree of correlation between mean MMP-9 activity and the maximum tau protein level was observed for patients with detectable tau protein (r = 0.71, p = 0.009). Our study suggests that MMP-9 can increase the tau protein level in the sera of patients during acute ischemic stroke.     

Peripheral expression of MAPK pathways in Alzheimer’s and Parkinson’s diseases

Alteration of mitogen-activated protein kinase pathways may cause aberrant protein phosphorylation and enhanced apoptosis in Alzheimer’s disease (AD) and Parkinson’s disease (PD). Increased susceptibility of lymphocytes to apoptosis has been reported in AD. To our knowledge this is the first study to investigate the expression and phosphorylation status of p38 mitogen-activated protein kinase (p38MAPK) and c-Jun N-terminal kinase (JNK) in peripheral blood lymphocytes of 20 AD and 20 PD patients and 20 healthy controls using western blot analysis. Compared with controls, no significant difference of total p38MAPK or JNK levels were observed in AD and PD patients, whereas phosphorylated p38MAPK and phosphorylated JNK levels were significantly increased in the AD and PD groups (p < 0.001). However, the increased levels of the two phosphorylated kinases in AD versus PD patients presented no significant difference. Interestingly, phosphorylated p38MAPK and phosphorylated JNK levels were positively correlated with disease duration (r = 0.602, p = 0.005 and r = 0.561, p = 0.010, respectively) and negatively correlated with the Mini Mental State Examination score (r = −0.664, p = 0.001 and r = −0.578, p = 0.008, respectively) in AD patients. No correlations between protein levels and clinical variables were found in PD patients. Investigation of peripheral changes in the expression of p38MAPK and JNK may lead to the development of innovative biomarkers of neurodegenerative diseases, particularly for AD.     

Neuroprotective effects of edaravone on cognitive deficit,oxidative stress and tau hyperphosphorylation induced by intracerebroventricular streptozotocin in rats

Oxidative stress is implicated as an important factor in the development of Alzheimer's disease (AD). In the present study, we have investigated the effects of edaravone (9 mg/kg, 3-methyl-1-phenyl-2-pyrazolin-5-one), a free radical scavenger, in a streptozotocin (STZ-3 mg/kg) induced rat model of sporadic AD (sAD). Treatment with edaravone significantly improved STZ-induced cognitive damage as evaluated in Morris water maze and step-down tests and markedly restored changes in malondialdehyde (MDA), 4-hydroxy-2-nonenal (4-HNE) adducts, hydroxyl radical (OH), hydrogen peroxide (H₂O₂), total superoxide dismutase (T-SOD), reduced glutathione (GSH), glutathione peroxidase (GPx) and protein carbonyl (PC) levels. In addition, histomorphological observations confirmed the protective effect of edaravone on neuronal degeneration. Moreover, hyperphosphorylation of tau resulting from intracerebroventricular streptozotocin (ICV-STZ) injection was decreased by the administration of edaravone. These results provide experimental evidence demonstrating preventive effects of edaravone on cognitive dysfunction, oxidative stress and hyperphosphorylation of tau in ICV-STZ rats. Since edaravone has been used for treatment of patients with stroke, it represents a safe and established therapeutic intervention that has the potential for a novel application in the treatment of age-related neurodegenerative disorders associated with cognitive decline, such as AD.     

Inhibition of cyclin-dependent kinase 5 but not of glycogen synthase kinase 3-β prevents neurite retraction and tau hyperphosphorylation caused by secretable products of human T-cell leukemia virus type I-infected lymphocytes

Human T-cell leukemia virus type I (HTLV-I)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a neurodegenerative disease characterized by selective loss of axons and myelin in the corticospinal tracts. This central axonopathy may originate from the impairment of anterograde axoplasmic transport. Previous work showed tau hyperphosphorylation at T(181) in cerebrospinal fluid of HAM/TSP patients. Similar hyperphosphorylation occurs in SH-SY5Y cells incubated with supernatant from MT-2 cells (HTLV-I-infected lymphocytes secreting viral proteins, including Tax) that produce neurite shortening. Tau phosphorylation at T(181) is attributable to glycogen synthase kinase 3-β (GSK3-β) and cyclin-dependent kinase 5 (CDK5) activation. Here we investigate whether neurite retraction in the SH-SY5Y model associates with concurrent changes in other tau hyperphosphorylable residues. Threonine 181 turned out to be the only tau hyperphosphorylated residue. We also evaluate the role of GSK3-β and CDK5 in this process by using specific kinase inhibitors (LiCl, TDZD-8, and roscovitine). Changes in both GSK3-β active and inactive forms were followed by measuring the regulatory phosphorylable sites (S(9) and Y(216) , inactivating and activating phosphorylation, respectively) together with changes in β-catenin protein levels. Our results showed that LiCl and TDZD-8 were unable to prevent MT-2 supernatant-mediated neurite retraction and also that neither Y(216) nor S(9) phosphorylations were changed in GSK3-β. Thus, GSK3-β seems not to play a role in T(181) hyperphosphorylation. On the other hand, the CDK5 involvement in tau phosphorylation was confirmed by both the increase in its enzymatic activity and the absence of MT-2 neurite retraction in the presence of roscovitine or CDK5 siRNA transfection.     

Dynamic of neurochemical alterations in striatum,hippocampus and cortex after the 6-OHDA mesostriatal lesion

Immediate neurochemical alterations produced by 6-OHDA could explain the general toxic pattern in the central nervous system. However, no evidences describe the effects of 6-OHDA on early changes of neurotransmitters in rats’ striatum, cortex and hippocampus. In our study, unilateral 6-OHDA injection into medial forebrain bundle (MFB) was used in rats, then five neurotransmitters were analyzed at 3, 6, 12, 24, 48 and 72 h, respectively. Results showed that 6-OHDA injection caused a sharp decline of striatal dopamine (DA) levels in the first 12 h followed by a further reduction between 12 and 48 h. However, striatal levels of homovanillic acid (HVA) were stable in the first 12 h and showed a marked reduction between 12 and 24 h. Striatal levels of 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) decreased linearly for 72 h, whereas levels of norepinephrine (NE) showed a slight reduction in the first 48 h, and returned back to normal afterwards. Striatal HVA/DA ratio increased significantly in the first 12 h, but 5-HIAA/5-HT ratio showed a sharp increase between 12 and 72 h. Besides, neurochemical alterations were also found in hippocampus and cortex, and the correlations of neurotransmitters were analyzed. Our study indicated that NE system had little influence in the early phase of 6-OHDA injection, moreover, early neurochemical alterations were involved with striatum, hippocampus and cortex.     

Stable mutated tau441 transfected SH-SY5Y cells as screening tool for Alzheimer's disease drug candidates

The role of hyperphosphorylation of the microtubule-associated protein tau in the pathological processes of several neurodegenerative diseases is becoming better understood. Consequently, development of new compounds capable of preventing tau hyperphosphorylation is an increasingly hot topic. For this reason, dependable in vitro and in vivo models that reflect tau hyperphosphorylation in human diseases are needed. In this study, we generated and validated an in vitro model appropriate to test potential curative and preventive compound effects on tau phosphorylation. For this purpose, a stably transfected SH-SY5Y cell line was constructed over-expressing mutant human tau441 (SH-SY5Y-TMHT441). Analyses of expression levels and tau phosphorylation status in untreated cells confirmed relevance to human diseases. Subsequently, the effect of different established kinase inhibitors on tau phosphorylation (e.g., residues Thr231, Thr181, and Ser396) was examined. It was shown with several methods including immunosorbent assays and mass spectrometry that the phosphorylation pattern of tau in SH-SY5Y-TMHT441 cells can be reliably modulated by these compounds, specifically targeting JNK, GSK-3, CDK1/5, and CK1. These four protein kinases are known to be involved in in vivo tau phosphorylation and are therefore authentic indicators for the suitability of this new cell culture model for tauopathies.     

Early-postnatal iron deficiency impacts plasticity in the dorsal and ventral hippocampus in piglets

In this study, we investigated whether alterations in plasticity markers such as brain-derived neurotrophic factor (BDNF), p75 neurotrophin receptor (p75^NTR) and tyrosine receptor kinase B (TrkB) are underlying iron deficiency (ID)-induced cognitive impairments in iron depleted piglets. Newborn piglets were either fed an iron-depleted diet (21 mg Fe/kg) or an iron-sufficient diet (88 mg Fe/kg) for four weeks. Subsequently, eight weeks after iron repletion (190–240 mg Fe/kg) we found a significant decrease in mature BDNF (14 kDa) and proBDNF (18 kDa and 24 kDa) protein levels in the ventral hippocampus, whereas we found increases in the dorsal hippocampus. The phosphorylation of cAMP response element binding protein (CREB) follows the mature BDNF protein level pattern. No effects were found on BDNF and CREB protein levels in the prefrontal cortex. The protein levels of the high affinity BDNF receptor, TrkB, was significantly decreased in both dorsal and ventral hippocampus of ID piglets, whereas it was increased in the prefrontal cortex. Together, our data suggest a disrupted hippocampal plasticity upon postnatal ID.     

Methylmercury-induced developmental toxicity is associated with oxidative stress and cofilin phosphorylation. Cellular and human studies

Environmental exposure to methylmercury (MeHg) during development is of concern because it is easily incorporated in children’s body both pre- and post-natal, it acts at several levels of neural pathways (mitochondria, cytoskeleton, neurotransmission) and it causes behavioral impairment in child. We evaluated the effects of prolonged exposure to 10–600 nM MeHg on primary cultures of mouse cortical (CCN) and of cerebellar granule cells (CGC) during their differentiation period. In addition, it was studied if prenatal MeHg exposure correlated with altered antioxidant defenses and cofilin phosphorylation in human placentas (n = 12) from the INMA cohort (Spain).Exposure to MeHg for 9 days in vitro (DIV) resulted in protein carbonylation and in cell death at concentrations ≥200 nM and ≥300 nM, respectively. Exposure of CCN and CGC to non-cytotoxic MeHg concentrations for 5 DIV induced an early concentration-dependent decrease in cofilin phosphorylation. Furthermore, in both cell types actin was translocated from the cytosol to the mitochondria whereas cofilin translocation was found only in CGC. Translocation of cofilin and actin to mitochondria in CGC occurred from 30 nM MeHg onwards. We also found an increased expression of cortactin and LIMK1 mRNA in CGC but not in CCN. All these effects were prevented by the antioxidant probucol.Cofilin phosphorylation was significantly decreased and a trend for decreased activity of glutathione reductase and glutathione peroxidase was found in the fetal side of human placental samples from the highest (20–40 μg/L) MeHg-exposed group when compared with the low (<7 μg/L) MeHg-exposed group.In summary, cofilin dephosphorylation and oxidative stress are hallmarks of MeHg exposure in both experimental and human systems.     

Synergistic effect of ALOX5AP polymorphisms and cigarette smoking on the risk of atherosclerotic cerebral infarction in a Northern Han Chinese population

The effect of activating 5-lipoxygenase (ALOX5AP) gene polymorphisms on stroke risk may be influenced by the coexistence of modifiable predisposing conditions. We explored the interactions of ALOX5AP polymorphisms and cigarette smoking in a case-control study of patients with atherosclerotic cerebral infarction (ACI). Three polymorphisms of the ALOX5AP gene (rs10507391, rs4769874, and rs9551963) were analyzed in 420 ACI patients and 488 unrelated healthy controls matched for age and sex from a Northern Han Chinese population. Among the three single nucleotide polymorphisms, only rs10507391 genotype TT/TA was observed to be associated with an increased risk of ACI on multivariate analysis (odds ratio [OR] = 1.82, 95% confidence interval [CI] = 1.14–2.92, p = 0.012) compared with the AA genotype. However, after stratifying by smoking status, multivariate logistic regression analysis revealed that rs10507391 genotype TT/TA and rs9551963 genotype CC/CA had a 5.63-fold (OR = 5.63, 95%CI = 2.00–15.84, p = 0.001) and a 2.71-fold (OR = 2.71, 95%CI = 1.28–5.73, p = 0.009) increased risk for ACI patients who smoked compared with the AA genotype, respectively. Additionally, according to the haplotype analysis, the risk of haplotype TGC (OR = 3.12, 95%CI = 2.00–4.88, p < 0.001, corrected p [pc] < 0.001) increased for ACI patients who smoked compared to the data (OR = 1.60, 95%CI = 1.28–1.98, p < 0.001, pc < 0.001) in total samples. These results suggest that ALOX5AP polymorphisms are associated with ACI, and cigarette smoking along with ALOX5AP could increase the risk of ACI.     

Effect of the lipid peroxidation product acrolein on tau phosphorylation in neural cells

A hallmark of several neurodegenerative disorders, including Alzheimer's disease and tauopathies, is the hyperphosphorylation of the microtubule-associated protein tau. Tau phosphorylation by proline-directed and non-proline-directed protein kinases has been tested using antibodies PHF1 and 12E8, respectively. The effect of the lipid peroxidation product acrolein on these modes of phosphorylation has been assayed. We have found that acrolein, a peroxidation product from arachidonic acid, increases the phosphorylation of tau at the site recognized by PHF-1 both in human neuroblastoma cells and in primary cultures of mouse embryo cortical neurons. Whereas the basal phosphorylation of tau protein at the PHF1 site seems to be largely mediated by glycogen synthase kinase-3 (which is also activated in response to Abeta peptide), the acrolein-induced tau hyperphosphorylation at the same site is also due to p38 stress-activated kinase. These results support the view that oxidative stress and subsequent formation of lipid peroxidation products may contribute to tau protein phosphorylation in Alzheimer's disease and tauopathies.     

Effects of cyclin-dependent kinase-5 activity on apoptosis and tau phosphorylation in immortalized mouse brain cortical cells

Cyclin-dependent kinase-5 (CDK5), a unique CDK family member, is active primarily in the central nervous system (CNS). Previous studies suggest that CDK5 is proapoptotic and contributes to tau hyperphosphorylation and neurodegeneration in Alzheimer's disease. The objective of this study was to examine CDK5 effects on apoptotic progression and tau phosphorylation. Immortalized embryonic mouse brain cortical cells were used to establish a stable cell line that overexpressed wild-type human tau. In these studies, thapsigargin, which induces endoplasmic reticulum stress and can cause accumulation of misfolded proteins, was used to induce apoptosis. Caspase-3 activity and poly-(ADP-ribose)-polymerase (PARP) cleavage, as measures of apoptosis, were significantly increased 24 and 48 hr after thapsigargin treatment, and these events were unaffected by tau expression. Although transient coexpression of CDK5 and its activator, p25, increased CDK5 activity greater than tenfold, increases in caspase-3 activity in response to thapsigargin treatment were unaffected by the presence of CDK5/p25. Tau phosphorylation at the PHF-1 epitope, but not the Tau-1 epitope, was increased significantly in CDK5/p25-transfected cells compared to cells transfected with dominant negative CDK5 (DNCDK5). The PHF-1 epitope remained phosphorylated until 48 hr after thapsigargin treatment in the CDK5/p25-transfected cells. Over the course of apoptosis in this model, phosphorylation of the Tau-1 epitope was unaffected in cells transfected with DNCDK5, vector, or CDK5/p25. In summary, these results demonstrate that CDK5 does not have a significant impact on tau phosphorylation and thapsigargin-induced apoptosis in this neuronal cell model.     

Sex-dependent effects of lead and prenatal stress on post-translational histone modifications in frontal cortex and hippocampus in the early postnatal brain

Environmental lead (Pb) exposure and prenatal stress (PS) are co-occurring risk factors for impaired cognition and other disorders/diseases in adulthood and target common biological substrates in the brain. Sex-dependent differences characterize the neurochemical and behavioral responses of the brain to Pb and PS and sexually dimorphic histone modifications have been reported to occur in at-risk brain regions (cortex and hippocampus) during development. The present study sought to examine levels and developmental timing of sexually dimorphic histone modifications (i.e., H3K9/14Ac and H3K9Me3) and the extent to which they may be altered by Pb ± PS. Female C57/Bl6 mice were randomly assigned to receive distilled deionized drinking water containing 0 or 100 ppm Pb acetate for 2 months prior to breeding and throughout lactation. Half of the dams in each group were exposed to restraint stress (PS, three restraint sessions in plastic cylindrical devices 3×/day at for 30 min/day (1000, 1300, and 1600 h)) from gestational day 11–19 or no stress (NS). At delivery (PND0) and postnatal day 6 (PND6), pups were euthanized and frontal cortex and hippocampus were removed, homogenized, and assayed for levels of H3K9/14Ac and H3K9Me3. Sex-dependent differences in both levels of histone modifications as well as the developmental trajectory of changes in these levels were observed in both structures and these parameters were differentially affected by Pb ± PS in a sex and brain-region-dependent manner. Disruptions of these epigenetic processes by developmental Pb ± PS may underlie some of the sex-dependent neurobehavioral differences previously observed in these animals.     

Epistasis between tau phosphorylation regulating genes (CDK5R1 and GSK‐3β) and Alzheimer’s disease risk

Objective – Glycogen synthase kinase‐3β (GSK‐3β) and cyclin‐dependent kinase 5 (CDK5) have been implicated as two major protein kinases involved in the abnormal hyperphosphorylation of tau in Alzheimer’s disease (AD) brain, and the development of neurofibrillary tangles. CDK5 regulatory subunit 1 (CDK5R1) encodes for p35, a protein required for activation of CDK5. As both CDK5R1 and GSK‐3β genes are related to phosphorylation of tau, we examined the combined contribution of these genes to the susceptibility for AD. Methods – In a case–control study in 283 AD patients and 263 healthy controls, we examined the combined effects between CDK5R1 (3′‐UTR, rs735555) and GSK‐3β (?50, rs334558) polymorphisms on susceptibility to AD. Results – Subjects carrying both the CDK5R1 (3′‐UTR, rs735555) AA genotype and the GSK‐3β (?50, rs334558) CC genotype had a 12.5‐fold decrease in AD risk (adjusted by age, sex and APOE status OR = 0.08, 95% CI = 0.01–0.76, P = 0.03), suggesting synergistic effects (epistasis) between both genes. Conclusion – These data support a role for tau phosphorylation regulating genes in risk for AD.     

Probenecid treatment enhances retinal and brain delivery of N-4-benzoylaminophenylsulfonylglycine: An anionic aldose reductase inhibitor

Anion efflux transporters are expected to minimize target tissue delivery of N-[4-(benzoylaminophenyl)sulfonyl]glycine (BAPSG), a novel carboxylic acid aldose reductase inhibitor, which exists as a monocarboxylate anion at physiological conditions. Therefore, the objective of this study was to determine whether BAPSG delivery to various eye tissues including the retina and the brain can be enhanced by probenecid, a competitive inhibitor of anion transporters. To determine the influence of probenecid on eye and brain distribution of BAPSG, probenecid was administered intraperitoneally (120 mg/kg body weight; i.p.) 20 min prior to BAPSG (50 mg/kg; i.p.) administration. Drug disposition in various eye tissues including the retina and the brain was determined at 15 min, 1, 2 and 4 h after BAPSG dose in male Sprauge–Dawley rats. To determine whether probenecid alters plasma clearance of BAPSG, influence of probenecid (120 mg/kg; i.p.) on the plasma pharmacokinetics of intravenously administered BAPSG (15 mg/kg) was studied as well. Finally, the effect of probenecid co-administration on the ocular tissue distribution of BAPSG was assessed in rabbits following topical (eye drop) administration. Following pretreatment with probenecid in the rat study, retinal delivery at 1 h was increased by about 11-fold (2580 ng/g vs. 244 ng/g; p < 0.05). Further, following probenecid pretreatment, significant BAPSG levels were detectable in the brain (45 ± 20 ng/g) at 1 h, unlike controls where the drug was not detectable. Plasma concentrations, plasma elimination half-life, and total body clearance of intravenously administered BAPSG were not altered by i.p. probenecid pretreatment. In the topical dosing study, a significant decline in BAPSG delivery was observed in the iris–ciliary body but no significant changes were observed in other tissues of the anterior segment of the eye including tears. Thus, inhibition of anion transporters is a useful approach to elevate retinal and brain delivery of BAPSG.     

An unbiased,staged, multicentre,validation strategy for Alzheimer's disease CSF tau levels

Newly proposed diagnostic criteria for Alzheimer's disease include cerebrospinal fluid (CSF) tau levels as one core supportive criterion. The published high sensitivity and specificity figures for CSF tau levels in Alzheimer's disease are offset by the large range of proposed cutoff values (9.6 pg/mL to 1140 pg/mL). This study aimed to provide guidance on how to establish, validate and audit CSF tau cutoff values using an unbiased, two-stage multicentre strategy. Both receiver operator characteristics (ROC) optimised and population-based cutoff values were calculated on a pilot dataset (n = 99), validated in a large dataset (n = 560) and then compared to the literature. The data suggest using an ROC optimised cutoff level of 323 ± 51.7 pg/mL allowing for the published inter-laboratory coefficient of variation of 16%. This cutoff level was confirmed in a prospective audit (n = 100). As demand for CSF tau levels will increase globally, the accuracy of local CSF hTau cutoff levels can be compared against this benchmark.     

Evidence in support of using a neurochemistry approach to identify therapy for both epilepsy and associated depression

The present study aimed to develop a neurochemistry-based single or adjuvant therapy approach for comprehensive management of epilepsy and associated depression employing pentylenetetrazole-kindled animals. Kindling was induced in two-month-old male Swiss albino mice by administering a subconvulsant pentylenetetrazole dose (35 mg/kg, i.p.) at an interval of 48 ± 2 h. These kindled animals were treated with saline and sodium valproate (300 mg/kg/day, i.p.) for 15 days. Except for the naïve group, all other groups were challenged with pentylenetetrazole (35 mg/kg, i.p.) on days 5, 10, and 15 to evaluate the seizure severity. Depression was evaluated in all experimental groups after normalization of locomotor activity, using tail suspension and forced swim test on days 1, 5, 10, and 15. Four hours after behavioral evaluations on day 15, all animals were euthanized to collect their serum and discrete brain parts. Corticosterone levels were estimated in all the experimental groups as a marker of a dysregulated hypothalamus pituitary adrenal axis. Neurochemical alterations (norepinephrine, dopamine, tryptophan, kynurenine, serotonin, glutamate, GABA, and total nitrate levels) were also estimated in the cortical and hippocampal areas of the mouse brain. Results revealed that saline-treated kindled animals were associated with significant depression and altered neurochemical milieu in comparison with naïve animals. Chronic valproate treatment in kindled animals significantly reduced seizure severity score bud did not ameliorate associated depression or completely restore altered biochemical and neurochemical milieu. Based on the observation of neurochemical changes in all the groups, we propose that restoration of altered neurochemical milieu, elevated indoleamine 2,3-dioxygenase enzyme activity, and corticosterone levels using pharmacological tools with/out valproic acid may be explored for management of both epilepsy and comorbid depression.