Evaluation and Characterization of Modified K114 Method to Localize Plaques in Rodent and Plaques and Tangles in Human Brain Tissue.

BACKGROUND: A plethora of studies has shown the utility of several chemical dyes due to their affinity to bind Aß to enable visualization of plaques under light or fluorescence microscope, and some of them showed affinity to bind neurofibrillary tangles (NFT) as well. However, only a few of them have the propensity to bind both senile plaques (SP) and NFT simultaneously. OBJECTIVE: In our current study, we aimed to modify the K114 dye and the staining procedure to substantially improve the staining of amyloid plaques in both human and rodent brains and neurofibrillary tangles in the human brain. METHODS: We modified the K114 solution and the staining procedure using Sudan Black as a modifier. Additionally, to evaluate the target of the modified K114, we performed double labeling of K114 and increased Aß against three different epitopes. We used 5 different antibodies to detect phosphorylated tau to understand the specific targets that modified K114 binds. RESULTS: Dual labeling using hyperphosphorylated antibodies against AT8, pTau, and TNT1 revealed that more than 80% hyperphosphorylated tau colocalized with tangles that were positive for modified K114, whereas more than 70% of the hyperphosphorylated tau colocalized with modified K114. On the other hand, more than 80% of the plaques that were stained with Aß MOAB-2 were colocalized with modified K114. CONCLUSION: Our modified method can label amyloid plaques within 5 min in the rat brain and within 20 min in the human brain. Our results indicated that modified K114 could be used as a valuable tool for detecting amyloid plaques and tangles with high contrast and resolution relative to other conventional fluorescence markers.

Evaluation of Styrylbenzene analog- FSB and its affinity to bind parenchymal plaques and tangles in patients of Alzheimer's disease.

Although several histochemical markers for senile plaques (SP) and neurofibrillary tangles (NFTs) have been synthesized since the discovery of plaques in Alzheimer's disease (AD), only a handful of these markers stain both lesions in the human brain. Despite discovery of its ability to stain both SP and NFT over 13 years ago, the styrylbenzene derivative, (E,E)-1-fluoro-2,5-bis-(3-hydroxycarbonyl-4-hydroxy)styrylbenzene (FSB), has only recently gained attention, primarily due to its ability to function as a contrasting agent for MRI imaging of AD pathology in vivo. The structure of the compound is a nuclide with quantized angular momentum, which explains its value as a contrast agent. In the current study, modification of the established staining procedure produced meaningful improvement in the labeling of plaques and tangles in the human brain. We utilized two rodent models of AD to show FSB's value in labeling both Aß and tau lesions. Furthermore, our current modification allows us to detect SP in rodent brains in 15 min and both SP and NFT in human brains within 20 min. The study presents new evidence regarding potential binding targets for FSB as well as optimization protocols in which various parameters have been manipulated to show how section thickness, use of frozen versus paraffin-embedded sections, and selection of staining media can affect the intensity of the plaque and tangle staining in the brain. To determine the target FSB potentially binds, we performed double immunolabeling of FSB with mOC64 (a conformational antibody that label Aß1-42). Results indicated that all plaques in the brain colocalized with mOC64, suggesting that FSB has the potential to bind all Aß containing plaques, making it a very sensitive detector of multiple forms of SP... All antibodies were assessed for the degree of colocalization with FSB in order to better understand potential binding targets. We found more than 90% hyperphosphorylated Tau against AT8, AT180 and S214 colocalized with FSB labeled tangles. On the other hand, more than 90% of the mOC64 containing plaques colocalized with FSB stained plaques. Our results indicate that FSB is a valuable marker that can be used to detect AD pathologies in human and rodent brains with greater fluorescence intensity relative to other conventional fluorescence markers.

Tauroursodeoxycholic acid (TUDCA) is neuroprotective in a chronic mouse model of Parkinson's disease.

OBJECTIVE: Parkinson's disease (PD) is a progressive motor disease of unknown etiology. Although neuroprotective ability of endogenous bile acid, tauroursodeoxycholic acid (TUDCA), shown in various diseases, including an acute model of PD,the potential therapeutic role of TUDCA in progressive models of PD that exhibit all aspects of PD has not been elucidated. In the present study, mice were assigned to one of four treatment groups: (1) Probenecid (PROB); (2) TUDCA, (3) MPTP + PROB (MPTPp); and (3) TUDCA + MPTPp. Methods: Markers for dopaminergic function, neuroinflammation, oxidative stress and autophagy were assessed using high performance liquid chromatography (HPLC), immunohistochemistry (IHC) and western blot (WB) methods. Locomotion was measured before and after treatments. Results: MPTPp decreased the expression of dopamine transporters (DAT) and tyrosine hydroxylase (TH), indicating dopaminergic damage, and induced microglial and astroglial activation as demonstrated by IHC analysis. MPTPp also decreased DA and its metabolites as demonstrated by HPLC analysis. Further, MPTPp-induced protein oxidation; increased LAMP-1 expression indicated autophagy and the promotion of alpha-synuclein (α-SYN) aggregation. Discussion: Pretreatment with TUDCA protected against dopaminergic neuronal damage, prevented the microglial and astroglial activation, as well as the DA and DOPAC reductions caused by MPTPp. TUDCA by itself did not produce any significant change, with data similar to the negative control group. Pretreatment with TUDCA prevented protein oxidation and autophagy, in addition to inhibiting α-SYN aggregation. Although TUDCA pretreatment did not significantly affect locomotion, only acute treatment effects were measured, indicating more extensive assessments may be necessary to reveal potential therapeutic effects on behavior. Together, these results suggest that autophagy may be involved in the progression of PD and that TUDCA may attenuate these effects. The efficacy of TUDCA as a novel therapy in patients with PD clearly warrants further study.

Role of metals in Alzheimer's disease.

Metal homeostasis in the central nervous system (CNS) is a crucial component of healthy brain function, because metals serve as enzymatic cofactors and are key components of intra- and inter-neuronal signaling. Metal dysregulation wreaks havoc on neural networks via induction and proliferation of pathological pathways that cause oxidative stress, synaptic impairment, and ultimately, cognitive deficits. Thus, exploration of metal biology in relation to neurodegenerative pathology is essential in pursuing novel therapies for Alzheimer's Disease and other neurodegenerative disorders. This review covers mechanisms of action of aluminum, iron, copper, and zinc ions with respect to the progressive, toxic accumulation of extracellular ß-amyloid plaques and intracellular hyperphosphorylated neurofibrillary tau tangles that characterizes Alzheimer's Disease, with the goal of evaluating the therapeutic potential of metal ion interference in neurodegenerative disease prevention and treatment. As neuroscientific interest in the role of metals in neurodegeneration escalates-in large part due to emerging evidence substantiating the interplay between metal imbalances and neuropathology-it becomes clear that the use of metal chelating agents may be a viable method for ameliorating Alzheimer's Disease pathology, as its etiology remains obscure. We conclude that, although metal therapies can potentially deter neurodegenerative processes, the most promising treatments will remain elusive until further understanding of neurodegenerative etiology is achieved. New research directions may best be guided by animal models of neurodegeneration, which reveal specific insights into biological mechanisms underlying dementia.

In vivo demonstration of Congo Red labeled amyloid plaques via perfusion in the Alzheimer disease rat model.

BACKGROUND: Congo Red (CR) has been used for its binding affinity to amyloid fibrils for the better part of a century. Recently, our laboratory has demonstrated its ability to bind to tau protein as well. NEW METHOD: Here we describe a novel methodology for fast, thorough, whole-brain labeling of amyloid plaques with CR via perfusion. We tested five different variants which altered the volume of CR, the speed of perfusion, and the solution CR was solubilized in to determine the best results. RESULTS AND CONCLUSION: We determined that intra-cardiac perfusion of animals with 0.5 % CR in 100 ml of 50 % ethanol or perfusion with 0.5 of CR in 100 ml of 10 % neutral buffer formalin both perfused at a rate of 30 ml/min for 3.3 min resulted in the clearest CR labeling, with little to no background noise. Both variants were compatible with subsequent immunolabeling procedures for NU-1, as well as Ferritin and GFAP. Compared to traditional CR plaque labeling methodology, this new method allows for quick whole brain CR-labeling. This reduces the amount of time from days to mere minutes. It also reduces potential for variability that would result from staining slides in batches. Thus, CR-perfusion is a rapid, thorough method that can be utilized to rapidly stain amyloid in the rodent brain.

Modification of methods to use Congo-red stain to simultaneously visualize amyloid plaques and tangles in human and rodent brain tissue sections.

Although there are multiple histochemical tracers available to label plaques and tangles in the brain to evaluate neuropathology in Alzheimer disease (AD), few of them are versatile in nature and compatible with immunohistochemical procedures. Congo Red (CR) is an anisotropic organic stain discovered to label amyloid beta (Aß) plaques in the brain. Unfortunately, its use is underappreciated due to its low resolution and brightness as stated in previous studies using bright field microscopy. Here, we modified a previous method to localize both plaques and tangles in brains from humans and a transgenic rodent model of AD for fluorescence microscopic visualization. The plaque staining affinities displayed by CR were compared with fibrillar pattern labeling seen with Thioflavin S. This study summarizes the optimization of protocols in which various parameters have been finetuned. To determine the target CR potentially binds, we have performed double labeling with different antibodies against Aß as well as phosphorylated Tau. The plaque staining affinities exhibited by CR are compared with those associated with the diffuse pattern of labeling seen with antibodies directed against different epitopes of Aß. Neither CP13, TNT2 or TOC1 binds all the neurofibrillary tangles as revealed by CR labeling in the human brain. Additionally, we also evaluated double labeling with AT8, AT180, and PHF1. Interestingly, PHF-1 shows 40% colocalization and AT8 shows 15% colocalization with NFT. Thus, CR is a much better marker to detect AD pathologies in human and rodent brains with higher fluorescence intensity relative to other conventional fluorescence markers.

Impaired Amyloid Beta Clearance and Brain Microvascular Dysfunction are Present in the Tg-SwDI Mouse Model of Alzheimer's Disease.

Alzheimer's disease (AD) pathology is characterized by amyloid plaques containing amyloid beta (Aß) peptides, neurofibrillary tangles containing hyperphosphorylated tau protein, and neuronal loss. In addition, Aß deposition in brain microvessels, known as cerebral amyloid angiopathy (CAA), increases blood-brain barrier (BBB) permeability and induces vascular dysfunction which aggravates AD pathology. The aim of the present study was to characterize neurovascular dysfunction in the Tg-SwDI mouse model of AD. Isolated brain capillaries from wild type (WT) and Tg-SwDI mice were used to evaluate the expression of monomeric and aggregated forms of Aß, P-glycoprotein (P-gp), the receptor for advance glycation end-products (RAGE) and the tight junction (TJs) proteins occludin and claudin-5. Cultured brain endothelial cells were used to analyze barrier function via fluorescein flux. Isolated capillaries from Tg-SwDI mice contained increased levels of aggregated and oligomeric Aß compared to WT animals. Isolated capillaries from Tg-SwDI had decreased levels of P-gp, which transports Aß from brain to blood, and increased levels of RAGE, which transports Aß from blood to brain. In addition, the TJ protein occludin was decreased in Tg-SwDI mice relative to WT mice, which correlated with an increase in BBB permeability in cultured brain endothelial cells. These findings demonstrated that Tg-SwDI mice exhibit Aß aggregation that is due, in part, to impaired Aß clearance driven by both a decrease in P-gp and increase in RAGE protein levels in brain capillaries. Aß aggregation promotes a decrease in the expression of the TJ protein occludin, and as consequence an increase in BBB permeability.

Downregulation of 14-3-3 Proteins in Alzheimer's Disease.

One of the most abundant proteins expressed in the brain, 14-3-3 comprises about 1% of the brain's total soluble proteins. The 14-3-3 isoforms bind to specific phosphoserine- and phosphothreonine-containing motifs found on a variety of signaling proteins (kinases and transcription factors, among others) to regulate a wide array of cellular processes including cell cycling, apoptosis, and autophagy. Previously, we described the expression of different 14-3-3 isoforms in the rat frontal cortex and reported their downregulation in a rodent model of neurodegeneration. To further investigate possible roles of 14-3-3 proteins in neurodegeneration, the present study examined different 14-3-3 isoforms in the frontal cortex of postmortem Alzheimer's disease (AD) patients and control subjects. Among the different 14-3-3 isoforms in the human frontal cortex, the relative abundance of expression is in the following order: 14-3-3-eta > tau > sigma > gamma > epsilon > zeta/delta > beta/alpha. These relative abundance levels of different 14-3-3 isoforms in human frontal cortex closely resemble those in rat frontal cortex, suggesting a conserved expression pattern of different 14-3-3 isoforms in mammalian species. In the AD samples, there was a significant decrease in total 14-3-3 levels and the 14-3-3-eta and 14-3-3-gamma isoforms, while no significant difference in the expression level of other 14-3-3 isoforms between AD and control brains was detected. Together, these results demonstrate an abundance of several 14-3-3 isoforms in the frontal cortex and that a downregulation of total 14-3-3 protein levels and specific 14-3-3 isoforms is associated with neurodegeneration. Given the known function of 14-3-3 proteins as inhibitors of apoptosis, the present results suggest that 14-3-3 proteins may play an important role in neurodegeneration and deserve further investigations into AD and other neurodegenerative disorders.

High Contrast and Resolution Labeling of Amyloid Plaques in Tissue Sections from APP-PS1 Mice and Humans with Alzheimer's Disease with the Zinc Chelator HQ-O: Practical and Theoretical Considerations.

BACKGROUND: Various methodologies have been employed for the localization of amyloid plaques in numerous studies on Alzheimer's disease. The majority of these stains are thought to label the plaques by virtue of their affinity for aggregated Aß. However, plaques are known to contain numerous other components, including multivalent metals such as zinc. OBJECTIVE: This investigates whether it is possible to localize the presence of zinc in parenchymal and vascular amyloid plaques in afflicted brains. To accomplish this, a novel fluorescent zinc chelator, HQO, was investigated to determine its mechanism of binding and to optimize a stain for the high contrast and resolution histological localization of amyloid plaques. METHODS: A novel zinc chelator, HQ-O, was developed for localizing zinc within amyloid plaques. The histology involves incubating tissue sections in a dilute aqueous solution of HQ-O. Its compatibility with a variety of other fluorescent methodologies is described. RESULTS: All amyloid plaques are stained in fine detail and appear bright green under blue light excitation. The staining of parenchymal plaques correlates closely with that seen following staining with antibodies to Aß, however, the HQ-O sometimes also label additional globular structures within blood vessels. In situ mechanistic studies revealed that fluorescent plaque-like structures are only observed with HQ-O when synthetic Aßx-42 is aggregated in the presence of zinc. CONCLUSION: Zinc is intimately bound to all amyloid plaques, which was demonstrated by its histological localization using a novel fluorescent zinc chelator, HQ-O. Additionally, the tracer is also capable of labeling intravascular leucocytes due to their high zinc content.

Neuroprotective effects of acetyl-l-carnitine (ALC) in a chronic MPTP-induced Parkinson's disease mouse model: Endothelial and microglial effects.

Parkinson's disease (PD) is a progressive motor disease with clinical features emerging due to degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNc), which project to the caudate putamen (CPu) where they release dopamine (DA). The current study investigated whether acetyl-l-carnitine (ALC) could ameliorate the pathology seen in an in vivoin vivo chronic 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP)-induced mouse model of PD. Four treatment groups were included: 1) CONTROL receiving probenecid (PROB; 250 mg/kg) only, 2) MPTP (25 mg/kg) + PROB, 3) MPTP + ALC (100 mg/kg), and 4) ALC alone. MPTP-induced losses in tyrosine hydroxylase and DA transporter immunoreactivity in the SNc and CPu were significantly reduced by ALC. HPLC data further suggests that decreases in CPu DA levels produced by MPTP were also attenuated by ALC. Additionally, microglial activation and astrocytic reactivity induced by MPTP were greatly reduced by ALC, indicating protection against neuroinflammation. Glucose transporter-1 and the tight junction proteins occludin and zonula occludins-1 were also protected from MPTP-induced down-regulation by ALC. Together, data suggest that in this model, ALC protects against MPTP-induced damage to endothelial cells and loss of DA neurons in the SNc and CPu, suggesting that ALC therapy may have the potential to slow or ameliorate the progression of PD pathology in a clinical setting.

Characterization of Serum Exosomes from a Transgenic Mouse Model of Alzheimer's Disease.

BACKGROUND: Alzheimer's Disease (AD) is the most common type of dementia characterized by amyloid plaques containing Amyloid Beta (Aß) peptides and neurofibrillary tangles containing tau protein. In addition to neuronal loss, Cerebral Amyloid Angiopathy (CAA) commonly occurs in AD. CAA is characterized by Aß deposition in brain microvessels. Recent studies have suggested that exosomes (cell-derived vesicles containing a diverse cargo) may be involved in the pathogenesis of AD. OBJECTIVE: Isolate and characterize brain-derived exosomes from a transgenic mouse model of AD that presents CAA. METHODS: Exosomes were isolated from serum obtained from 13-month-old wild type and AD transgenic female mice using an exosome precipitation solution. Characterization of exosomal proteins was performed by western blots and dot blots. RESULTS: Serum exosomes were increased in transgenic mice compared to wild types as determined by increased levels of the exosome markers flotillin and alix. High levels of neuronal markers were found in exosomes, without any difference any between the 2 groups. Markers for endothelial-derived exosomes were decreased in the transgenic model, while astrocytic-derived exosomes were increased. Exosome characterization showed increased levels of oligomeric Aß and oligomeric and monomeric forms tau on the transgenic animals. Levels of amyloid precursor protein were also increased. In addition, pathological and phosphorylated forms of tau were detected, but no difference was observed between the groups. CONCLUSION: These data suggest that monomeric and oligomeric forms of Aß and tau are secreted into serum via brain exosomes, most likely derived from astrocytes in the transgenic mouse model of AD with CAA. Studies on the implication of this event in the propagation of AD are underway.

ERK/MAP Kinase Activation is Evident in Activated Microglia of the Striatum and Substantia Nigra in an Acute and Chronically-Induced Mouse Model of Parkinson's Disease.

INTRODUCTION: Parkinson's Disease (PD) is a debilitating, age-related disorder characterized by selective degeneration of dopaminergic neurons in the midbrain substantia nigra (SNc). Dopaminergic neurons originating in the midbrain project to the striatum (Caudate-putamen-CPU). Although studies have suggested that the extracellular signal-regulated kinase ½ (ERK ½) in the brain is activated after 1-Methyl-4-phenyl-1, 2,3,6-tetrahydropyridine (MPTP) exposure, to our knowledge no study has yet been done to demonstrate whether such activation occurs in neurons or in glia. MATERIAL AND METHODS: In the current study, we utilized both an acute and a repeat dose mouse model of PD using the neurotoxicant MPTP as the causative agent. Immunohistochemical studies using phospho ERK ½ antibody suggested that ERK ½ activation takes place in the striatum (CPU) and SNc of both animal models. Moreover, double immunolabeling studies using phospho ERK ½ and the microglial marker, CD11b or the astrocyte marker, Glial Fibrillary Acidic Protein (GFAP) suggested that the phospho ERK ½ was present exclusively in the microglia and not in the astrocytes. RESULTS: Western Blot results suggested that there were no alterations in ERK in either MPTPtreated animals or in control animals; however, phospho ERK ½ was found to be significantly increased in the striatum and SNc in both acute chronic mouse PD models. Tyrosine Hydroxylase (TH) immunolabeling revealed significant decreases in dopaminergic neurons in the SNc in both animal models' concomitant with activation of microglia and ERK activation. CONCLUSION: These observations suggest that ERK activation takes place following MPTP treatment and that activation of ERK occurs primarily in the microglia. The data provided also suggest that ERK activation may be involved in transcriptional activation of microglia following neurotoxicant insults.

Identification of altered microRNAs in serum of a mouse model of Parkinson's disease.

Parkinson's disease (PD) is the second most prevalent neurodegenerative disease, whose hallmark is the loss of dopamine terminals in the substantia nigra pars compacta (SNpc). PD is usually diagnosed after the appearance of motor symptoms, when about 70% of neurons in the SNpc have already been lost. Because of that, it is important to search for new methods that aid in the early diagnosis of PD. In recent years, microRNAs (miRs) have emerged as potential biomarkers for a variety of diseases and hold the potential to be used to aid in the diagnosis of PD. Therefore, the aim of this study was to characterize if specific miRs are differentially expressed in serum in a mouse model of PD. To induce PD-like damage, mice were subcutaneously injected with 25 mg/kg of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine hydrochloride (MPTP) by administering 10 doses over a period of 5 weeks, with 3.5 days between doses. Expression of 71 different microRNAs was quantified in serum separated from blood collected at day 35, using next-generation sequencing. Histological analysis and quantification of neurotransmitters were performed to confirm dopaminergic neurodegeneration. Chronic MPTP treatment induced loss of dopaminergic terminals in the SNpc and caudate putamen, confirmed by a decrease in the number of tyrosine hydroxylase and dopamine transporter positive cells. In addition, MPTP decreased the concentration of dopamine and its metabolites in the SNpc, simulating the damage observed in PD. From the 71 miRs analyzed, only 4 were differentially expressed after MPTP treatment. Serum levels of miR19b, miR124, miR126a and miR133b were significantly decreased in MPTP-treated mice compared to control. These data suggest that specific miRs are downregulated in a pre-clinical model of PD and hold the potential to be used as biomarkers to aid in the diagnosis of this disease. Further experiments need to be conducted to validate the use of these miRs as biomarkers of PD in additional pre-clinical models as well as in samples from patients diagnosed with PD.

Downregulation of 14-3-3 Proteins in a Kainic Acid-Induced Neurotoxicity Model.

The 14-3-3 proteins are among the most abundant proteins expressed in the brain, comprising about 1% of the total amount of soluble brain proteins. Through phosphoserine- and phosphothreonine-binding motifs, 14-3-3 proteins regulate many signaling proteins and cellular processes including cell death. In the present study, we utilized a well-known kainic acid (KA)-induced excitotoxicity rat model and examined the expression of 14-3-3 and its isoforms in the frontal cortex of KA-treated and control animals. Among the different 14-3-3 isoforms, abundant levels of eta and tau were detected in the frontal cortex, followed by sigma, epsilon, and gamma, while the expression levels of alpha/beta and zeta/delta isoforms were low. Compared to the control animals, KA treatment induced a significant downregulation of the overall 14-3-3 protein level as well as the levels of the abundant isoforms eta, tau, epsilon, and gamma. We also investigated two 14-3-3-interacting proteins that are involved in the cell death process: Bcl-2-associated X (BAX) and extracellular signal-regulated kinase (ERK). Both BAX and phosphorylated ERK showed increased levels following KA treatment. Together, these findings demonstrate an abundance of several 14-3-3 isoforms in the frontal cortex and that KA treatment can cause a downregulation of 14-3-3 expression and an upregulation of 14-3-3-interacting proteins BAX and phospho-ERK. Thus, downregulation of 14-3-3 proteins could be one of the early molecular events associated with excitotoxicity. This could lead to subsequent upregulation of 14-3-3-binding proteins such as BAX and phospho-ERK that contribute to further downstream apoptosis processes, eventually leading to cell death. Maintaining sufficient levels of 14-3-3 expression and function may become a target of therapeutic intervention for excitotoxicity-induced neurodegeneration.

Decreased Mcl-1 protein level in the striatum of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mice.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is a well-known neurotoxicant that can selectively destroy dopaminergic neurons and MPTP-treated animals are often used as models for studying aspects of Parkinson's disease (PD). While apoptosis has been suggested as a possible mechanism underlying MPTP-induced cell death and several apoptosis-associated proteins have been implicated in MPTP-animal models, relevant information regarding the possible involvement of Mcl-1 (myeloid cell leukemia 1) protein is missing. Mcl-l is an important member of the Bcl-2 family that is thought to be a highly regulated controller of cell death and survival. However, the expression level of Mcl-1 in response to MPTP-treatment has not been examined in any area of the brain previously. In the present study, an acute MPTP treatment regimen was utilized with male C57BL/6 mice (10 mg/kg i.p. injections, 4 times with 2 h intervals) and several protein markers were examined 24-h after the initial injection. Dramatic decreases in the immunoreactivities of tyrosine hydroxylase and dopamine transporters were observed. Western-blot analysis and immunocytochemical labeling demonstrated an MPTP-induced decrease in Mcl-1 protein levels in the striatum. In addition, the two proteins BAX and ERK, both of which are also involved in apoptosis signaling, were examined. While the total BAX levels showed no significant difference between the control and MPTP-treated groups, levels of phosphorylated ERK were significantly increased following MPTP-treatment. Since Mcl-1 is an anti-apoptotic protein, down-regulation of Mcl-1 following MPTP-treatment would be expected to lead to increased apoptotic activities processes, leading to increased neurodegeneration.

Changes in the metabolome and microRNA levels in biological fluids might represent biomarkers of neurotoxicity: A trimethyltin study.

Neurotoxicity has been linked with exposure to a number of common drugs and chemicals, yet efficient, accurate, and minimally invasive methods to detect it are lacking. Fluid-based biomarkers such as those found in serum, plasma, urine, and cerebrospinal fluid have great potential due to the relative ease of sampling but at present, data on their expression and translation are lacking or inconsistent. In this pilot study using a trimethyl tin rat model of central nervous system toxicity, we have applied state-of-the-art assessment techniques to identify potential individual biomarkers and patterns of biomarkers in serum, plasma, urine or cerebral spinal fluid that may be indicative of nerve cell damage and degeneration. Overall changes in metabolites and microRNAs were observed in biological fluids that were associated with neurotoxic damage induced by trimethyl tin. Behavioral changes and magnetic resonance imaging T2 relaxation and ventricle volume changes served to identify animals that responded to the adverse effects of trimethyl tin. Impact statement These data will help design follow-on studies with other known neurotoxicants to be used to assess the broad applicability of the present findings. Together this approach represents an effort to begin to develop and qualify a set of translational biochemical markers of neurotoxicity that will be readily accessible in humans. Such biomarkers could prove invaluable for drug development research ranging from preclinical studies to clinical trials and may prove to assist with monitoring of the severity and life cycle of brain lesions.

Introducing Euro-Glo, a rare earth metal chelate with numerous applications for the fluorescent localization of myelin and amyloid plaques in brain tissue sections.

The vast majority of fluorochromes are organic in nature and none of the few existing chelates have been applied as histological tracers for localizing brain anatomy and pathology. NEW METHOD: In this study we have developed and characterized a Europium chelate with the ability to fluorescently label normal and pathological myelin in control and toxicant-exposed rats, as well as the amyloid plaques in aged AD/Tg mice. RESULTS: This study demonstrates how Euro-Glo can be used for the detailed labeling of both normal myelination in the control rat as well as myelin pathology in the kainic acid exposed rat. In addition, this study demonstrates how E-G will label the shell of amyloid plaques in an AD/Tg mouse model of Alzheimer's disease a red color, while the plaque core appears blue in color. The observed E-G staining pattern is compared with that of well characterized tracers specific for the localization of myelin (Black-Gold II), degenerating neurons (Fluoro-Jade C), A-beta aggregates (Amylo-Glo) and glycolipids (PAS). COMPARISONS WITH EXISTING METHODS: This study represents the first time a rare earth metal (REM) chelate has been used as a histochemical tracer in the brain. This novel tracer, Euro-Glo (E-G), exhibits numerous advantages over conventional organic fluorophores including high intensity emission, high resistance to fading, compatibility with multiple labeling protocols, high Stoke's shift value and an absence of bleed-through of the signal through other filters. CONCLUSIONS: Euro-Glo represents the first fluorescent metal chelate to be used as a histochemical tracer, specifically to localize normal and pathological myelin as well as amyloid plaques.

Neuroprotective and Therapeutic Strategies against Parkinson's Disease: Recent Perspectives.

Parkinsonism is a progressive motor disease that affects 1.5 million Americans and is the second most common neurodegenerative disease after Alzheimer's. Typical neuropathological features of Parkinson's disease (PD) include degeneration of dopaminergic neurons located in the pars compacta of the substantia nigra that project to the striatum (nigro-striatal pathway) and depositions of cytoplasmic fibrillary inclusions (Lewy bodies) which contain ubiquitin and α-synuclein. The cardinal motor signs of PD are tremors, rigidity, slow movement (bradykinesia), poor balance, and difficulty in walking (Parkinsonian gait). In addition to motor symptoms, non-motor symptoms that include autonomic and psychiatric as well as cognitive impairments are pressing issues that need to be addressed. Several different mechanisms play an important role in generation of Lewy bodies; endoplasmic reticulum (ER) stress induced unfolded proteins, neuroinflammation and eventual loss of dopaminergic neurons in the substantia nigra of mid brain in PD. Moreover, these diverse processes that result in PD make modeling of the disease and evaluation of therapeutics against this devastating disease difficult. Here, we will discuss diverse mechanisms that are involved in PD, neuroprotective and therapeutic strategies currently in clinical trial or in preclinical stages, and impart views about strategies that are promising to mitigate PD pathology.

Oral Administration of Thioflavin T Prevents Beta Amyloid Plaque Formation in Double Transgenic AD Mice.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder and the fourth leading cause of death in the United States and most common cause of adult-onset dementia. The major hallmarks of AD are the formation of senile amyloid plaques made of beta amyloid and neurofibrillary tangles (NFT) which are primarily composed of phosphorylated tau protein. Although numerous agents have been considered as providing protection against AD, identification of potential agents with neuroprotective ability is limited. Thioflavin T has been used in the past to stain amyloid beta plaques in brain. In this study, Thioflavin T (ThT) and vehicle (infant formula) were administered orally by gavage to transgenic (B6C3 APP PS1; AD-Tg) mice beginning at 4 months age and continuing until sacrifice at 9 months of age at 40 mg/kg dose. The number of amyloid plaques was reduced dramatically by ThT treatment in both male and female transgenic mice compared to those in control mice. Additionally, GFAP and Amylo-Glo labeling suggest that astrocytic hypertrophy is minimized in ThT-treated animals. Similarly, CD68 labeling, which detects activated microglia, along with Amylo-Glo labeling, suggests that microglial activation is significantly less in ThT-treated mice. Both Aß-40 and Aß-42 concentrations in blood rose significantly in the ThT-treated animals suggesting that ThT may inhibit the deposition, degradation, and/or clearance of Aß plaques in brain.