Assessment of Preanalytical Cerebrospinal Fluid Handling and Storage Factors on Measurement of Aß1-42, Aß1-40, and pTau181 Using an Automated Chemiluminescent Platform.

BACKGROUND: Standardizing cerebrospinal fluid (CSF) laboratory protocols will improve the reliability and availability of clinical biomarker testing required for prescription of novel Alzheimer disease (AD) therapies. This study evaluated several preanalytical handling and storage factors common to ß-amyloid1-42 (Aß1-42), ß-amyloid1-40 (Aß1-40), and phosphorylated tau (pTau181) concentrations including storage at different temperatures, extended cap contact, various mixing methods, and multiple freeze-thaw cycles. METHODS: Aß1-42, Aß1-40, and pTau181 concentrations were measured using LUMIPULSE G1200 automated assays. Samples were collected in polypropylene tubes of various volumes. Sample cap-contact was evaluated by storing samples in upright and inverted positions at either 4°C for 1 week or -80°C for 1 month. To assess mixing methods, samples were freeze-thawed and mixed by inversion, vortex, horizontal roller, or unmixed prior to assay sampling. The impact of successive freeze-thaw cycles was assessed through freezing, thawing, and analyzing CSF samples. RESULTS: Short-term storage at 4°C did not affect Aß1-42, Aß1-40, or pTau181 measurements in any tube type. Tube cap contact affected Aß1-42 in 2.5 mL tubes and pTau181 levels in 10 mL tubes. No difference was observed between mixing methods. After 4 freeze-thaw cycles, Aß1-42 significantly decreased but Aß1-40 remained unchanged. Utilizing the Aß1-42/Aß1-40 ratio, Aß1-42 values normalized, maintaining ratio values within ±5% of baseline measurements. CONCLUSIONS: Storage of CSF at 4°C for 1 week or -80°C for 1 month did not significantly affect Aß1-42, Aß1-40, pTau181, or associated ratio measurements. Tube cap-contact impacted pTau181 and pTau181/Aß1-42 values in larger tubes. Mixing methods are equivalent. The Aß1-42/Aß1-40 ratio compensates for freeze-thaw variability up to 4 cycles.

Palmitoylation controls the stability of 190 kDa ankyrin-G in dendritic spines and is regulated by ZDHHC8 and lithium.

Introduction: AnkG, encoded by the ANK3 gene, is a multifunctional scaffold protein with complex isoform expression: the 480 and 270 kDa isoforms have roles at the axon initial segment and node of Ranvier, whereas the 190 kDa isoform (AnkG-190) has an emerging role in the dendritic shaft and spine heads. All isoforms of AnkG undergo palmitoylation, a post-translational modification regulating protein attachment to lipid membranes. However, palmitoylation of AnkG-190 has not been investigated in dendritic spines. The ANK3 gene and altered expression of AnkG proteins are associated with a variety of neuropsychiatric and neurodevelopmental disorders including bipolar disorder and are implicated in the lithium response, a commonly used mood stabilizer for bipolar disorder patients, although the precise mechanisms involved are unknown. Result: Here, we showed that Cys70 palmitoylation stabilizes the localization of AnkG-190 in spine heads and at dendritic plasma membrane nanodomains. Mutation of Cys70 impairs AnkG-190 function in dendritic spines and alters PSD-95 scaffolding. Interestingly, we find that lithium reduces AnkG-190 palmitoylation thereby increasing its mobility in dendritic spines. Finally, we demonstrate that the palmitoyl acyl transferase ZDHHC8, but not ZDHHC5, increases AnkG-190 stability in spine heads and is inhibited by lithium. Discussion: Together, our data reveal that palmitoylation is critical for AnkG-190 localization and function and a potential ZDHHC8/AnkG-190 mechanism linking AnkG-190 mobility to the neuronal effects of lithium.

Association between (GT)n Promoter Polymorphism and Recovery from Concussion: A Pilot Study.

Variability in recovery among concussed athletes can be attributed to several risk factors. One risk factor not definitively explored is genetic variation. Genetic variations such as variable number tandem repeats (VNTR) in the promotor region are normal in the population, and can lead to disparities in the amount of protein produced, which could be associated with neuronal recovery. Little research has been conducted to investigate promoter VNTRs within genes responsible for recovery following a concussion. The authors implemented a prospective cohort design using a standardized concussion protocol to diagnose and follow 93 athletes to full recovery at three different sites to determine the association between promotor GT(n) VNTR polymorphisms and recovery time within concussed athletes. The GT(n) VNTR within the promoter region of glutamate ionotropic receptor N-methyl-d-aspartate (NMDA) type subunit 2A (GRIN2A), potassium voltage-gated channel subfamily H member 2 (KCNH2), glutamate ionotropic receptor kainate type subunit 1 (GRIK1), and neurofilament light (NEFL) were genotyped using capillary electrophoresis. GT(n) VNTR promotor polymorphisms were dichotomized into long (L) and short (s) alleles. Using adjusted negative binomial regression models we found that athletes carrying the LL GRIN2A GT(n) VNTR within the promoter region were more likely to experience a prolonged concussion recovery, which resulted in their not being able to return to play for ∼60 days. Additionally, there was a trend toward significance, in which the ss NEFL GT(n) Caucasian athletes had prolonged concussion recovery. This could presumably be attributed to altered proteins or protein levels that disrupt neuronal recovery. This pilot study suggests that these VNTRs are associated with prolonged concussion recovery. In future studies, we plan to measure the extent to which the L or s alleles alter the level and the activity of the GluNR2a and NEFL proteins that GRIN2A and NEFL produce, respectively.

mTORC1 Signaling Is Palmitoylation-Dependent in Hippocampal Neurons and Non-neuronal Cells and Involves Dynamic Palmitoylation of LAMTOR1 and mTOR.

The mechanistic target of rapamycin (mTOR) Complex 1 (mTORC1) controls growth and proliferation of non-neuronal cells, while during neuronal development mTORC1 responds to glutamate and neurotrophins to promote neuronal migration and dendritic arborization. Recent studies reveal that mTORC1 signaling complexes are assembled on lysosomal membranes, but how mTORC1 membrane targeting is regulated is not fully clear. Our examination of palmitoyl-proteomic databases and additional bioinformatic analyses revealed that several mTORC1 proteins are predicted to undergo covalent modification with the lipid palmitate. This process, palmitoylation, can dynamically target proteins to specific membranes but its roles in mTORC1 signaling are not well described. Strikingly, we found that acute pharmacological inhibition of palmitoylation prevents amino acid-dependent mTORC1 activation in HEK293T cells and brain-derived neurotrophic factor (BDNF)-dependent mTORC1 activation in hippocampal neurons. We sought to define the molecular basis for this finding and found that the mTORC1 proteins LAMTOR1 and mTOR itself are directly palmitoylated, while several other mTORC1 proteins are not palmitoylated, despite strong bioinformatic prediction. Interestingly, palmitoylation of LAMTOR1, whose anchoring on lysosomal membranes is important for mTORC1 signaling, was rapidly increased prior to mTORC1 activation. In contrast, mTOR palmitoylation was decreased by stimuli that activate mTORC1. These findings reveal that specific key components of the mTOR pathway are dynamically palmitoylated, suggesting that palmitoylation is not merely permissive for mTOR activation but is instead actively involved in mTORC1-dependent signaling.

Alcohol-Mediated Missplicing of Mcl-1 Pre-mRNA is Involved in Neurotoxicity.

BACKGROUND: Heavy and chronic ethanol (EtOH) exposure can cause significant structural and functional damage to the adult brain. The most devastating consequence of EtOH exposure is the neurotoxicity associated with the depletion of neurons. Regulation of splice variants in the brain can modulate protein functions, which may ultimately affect behaviors associated with alcohol dependence and EtOH-mediated neurotoxicity. As alcohol consumption is associated with neurotoxicity, it is possible that altered splicing of survival and pro-survival factors during the development of alcoholism may contribute to the neurotoxicity. METHODS: Primary human neurons and a neuroblastoma cell line were exposed to different concentrations of EtOH for various time periods. Cell viability and neuronal marker expression were analyzed by MTT assay and immunoblotting, respectively. Effect of EtOH exposure on splicing regulatory protein expression and alternative splicing of candidate genes was analyzed by a biochemical approach. Transcriptional activity of serine/arginine-rich splicing factor 1 (SRSF1) gene was determined by reporter gene analysis. RESULTS: Our results suggest that EtOH exposure to neuronal cells at 25 mM and higher concentrations are detrimental. In addition, EtOH exposure caused a dramatic reduction in SRSF1 expression levels. Furthermore, EtOH exposure led to pre-mRNA missplicing of Mcl-1, a pro-survival member of the Bcl-2 family, by down-regulating the expression levels of SRSF1. Moreover, ectopic expression of both SRSF1 and Mcl-1L isoform was able to recover EtOH-mediated neurotoxicity. CONCLUSIONS: Our results suggest that EtOH exposure can lead to pre-mRNA missplicing of Mcl-1 in neuronal cells. Our results indicate that EtOH exposure of neurons leads to a decrease in the ratio of Mcl-1L/Mcl-1S by favoring pro-apoptotic Mcl-1S splicing over anti-apoptotic Mcl-1L isoform suggesting that Mcl-1S may play a crucial role in neurotoxicity associated with alcohol consumption.

HIV-1 Tat and Cocaine Impair Survival of Cultured Primary Neuronal Cells via a Mitochondrial Pathway.

Addictive stimulant drugs, such as cocaine, are known to increase the risk of exposure to HIV-1 infection and hence predispose towards the development of AIDS. Previous findings suggested that the combined effect of chronic cocaine administration and HIV-1 infection enhances cell death. Neuronal survival is highly dependent on the health of mitochondria providing a rationale for assessing mitochondrial integrity and functionality following cocaine treatment, either alone or in combination with the HIV-1 viral protein Tat, by monitoring ATP release and mitochondrial membrane potential (ΔΨm). Our results indicate that exposing human and rat primary hippocampal neurons to cocaine and HIV-1 Tat synergistically decreased both mitochondrial membrane potential and ATP production. Additionally, since previous studies suggested HIV-1 infection alters autophagy in the CNS, we investigated how HIV-1 Tat and cocaine affect autophagy in neurons. The results indicated that Tat induces an increase in LC3-II levels and the formation of Parkin-ring-like structures surrounding damaged mitochondria, indicating the possible involvement of the Parkin/PINK1/DJ-1 (PPD) complex in neuronal degeneration. The importance of mitochondrial damage is also indicated by reductions in mitochondrial membrane potential and ATP content induced by HIV-1 Tat and cocaine.

Immune suppression of JC virus gene expression is mediated by SRSF1.

Progressive multifocal leukoemcephalopathy (PML) is a fatal demyelinating disease caused by the human neurotropic JC virus (JCV). JCV infects the majority of the human population during childhood and establishes a latent/persistent life-long infection. The virus reactivates under immunosuppressive conditions by unknown mechanisms, resulting in productive infection of oligodendrocytes in the central nervous system (CNS). Given the fact that the natural occurrence of PML is strongly associated with immunosuppression, the functional and molecular interaction between glial cells and neuroimmune signaling mediated by soluble immune mediators is likely to play a major role in reactivation of JCV and the progression of the lytic viral life cycle leading to the development of PML. In order to explore the effect of soluble immune mediators secreted by peripheral blood mononuclear cells (PBMCs) on JCV transcription, primary human fetal glial (PHFG) cells were treated with conditioned media from PBMCs. We observed a strong suppression of JCV early as well as late gene transcription in cells treated with conditioned media from induced PBMCs. Using a variety of virological and molecular biological approaches, we demonstrate that immune mediators secreted by PBMCs induce the expression of SRSF1, a strong inhibitor of JCV gene expression, and inhibit the replication of JCV. Our results show that downregulation of SRSF1 in glial cells overcomes the suppression of JCV gene expression and its replication mediated by soluble immune mediators. These findings suggest the presence of a novel immune signaling pathway between glial cells and PBMCs that may control JCV gene expression during the course of viral reactivation.

IFN-Gamma Inhibits JC Virus Replication in Glial Cells by Suppressing T-Antigen Expression.

OBJECTIVE: Patients undergoing immune modulatory therapies for the treatment of autoimmune diseases such as multiple sclerosis, and individuals with an impaired-immune system, most notably AIDS patients, are in the high risk group of developing progressive multifocal leukoencephalopathy (PML), an often lethal disease of the brain characterized by lytic infection of oligodendrocytes in the central nervous system (CNS) with JC virus (JCV). The immune system plays an important regulatory role in controlling JCV reactivation from latent sites by limiting viral gene expression and replication. However, little is known regarding the molecular mechanisms responsible for this regulation. METHODS AND RESULTS: Here, we investigated the impact of soluble immune mediators secreted by activated PBMCs on viral replication and gene expression by cell culture models and molecular virology techniques. Our data revealed that viral gene expression and viral replication were suppressed by soluble immune mediators. Further studies demonstrated that soluble immune mediators secreted by activated PBMCs inhibit viral replication induced by T-antigen, the major viral regulatory protein, by suppressing its expression in glial cells. This unexpected suppression of T-antigen was mainly associated with the suppression of translational initiation. Cytokine/chemokine array studies using conditioned media from activated PBMCs revealed several candidate cytokines with possible roles in this regulation. Among them, only IFN-γ showed a robust inhibition of T-antigen expression. While potential roles for IFN-ß, and to a lesser extent IFN-α have been described for JCV, IFN-γ has not been previously implicated. Further analysis of IFN-γ signaling pathway revealed a novel role of Jak1 signaling in control of viral T-antigen expression. Furthermore, IFN-γ suppressed JCV replication and viral propagation in primary human fetal glial cells, and showed a strong anti-JCV activity. CONCLUSIONS: Our results suggest a novel role for IFN-γ in the regulation of JCV gene expression via downregulation of the major viral regulatory protein, T-antigen, and provide a new avenue of research to understand molecular mechanisms for downregulation of viral reactivation that may lead to development of novel strategies for the treatment of PML.

HIV-1 Tat protein induces glial cell autophagy through enhancement of BAG3 protein levels.

BAG3 protein has been described as an anti-apoptotic and pro-autophagic factor in several neoplastic and normal cells. We previously demonstrated that BAG3 expression is elevated upon HIV-1 infection of glial and T lymphocyte cells. Among HIV-1 proteins, Tat is highly involved in regulating host cell response to viral infection. Therefore, we investigated the possible role of Tat protein in modulating BAG3 protein levels and the autophagic process itself. In this report, we show that transfection with Tat raises BAG3 levels in glioblastoma cells. Moreover, BAG3 silencing results in highly reducing Tat- induced levels of LC3-II and increasing the appearance of sub G0/G1 apoptotic cells, in keeping with the reported role of BAG3 in modulating the autophagy/apoptosis balance. These results demonstrate for the first time that Tat protein is able to stimulate autophagy through increasing BAG3 levels in human glial cells.

The agnoprotein of polyomavirus JC is released by infected cells: evidence for its cellular uptake by uninfected neighboring cells.

Poliomavirus JC replicates in glial cells in the brain, and causes the fatal demyelinating disease, progressive multifocal leukoencephalopathy (PML). PML is usually seen in patients with underlying immunocompromised conditions, notably among AIDS patients and those on chronic immunosuppressive regimens. The late leader sequence of JC virus contains an open reading frame encoding a small regulatory protein called agnoprotein. Agnoprotein contributes to progressive viral infection by playing significant roles in viral replication cycle. Here, we demonstrate that agnoprotein can be detected in cell-free fractions of glial cultures infected with JCV, transfected with expression plasmids or transduced with an adenovirus expression system. We also provide evidence that extracellular agnoprotein can be taken up by uninfected neighboring cells. These studies have revealed a novel phenomenon of agnoprotein during the viral life cycle with a potential of developing diagnostic and therapeutic interventions.