Spatial learning impairment, enhanced CDK5/p35 activity, and downregulation of NMDA receptor expression in transgenic mice expressing tau-tubulin kinase 1.

Tau-tubulin kinase-1 (TTBK1) is involved in phosphorylation of tau protein at specific Serine/Threonine residues found in paired helical filaments, suggesting its role in tauopathy pathogenesis. We found that TTBK1 levels were upregulated in brains of human Alzheimer' disease (AD) patients compared with age-matched non-AD controls. To understand the effects of TTBK1 activation in vivo, we developed transgenic mice harboring human full-length TTBK1 genomic DNA (TTBK1-Tg). Transgenic TTBK1 is highly expressed in subiculum and cortical pyramidal layers, and induces phosphorylated neurofilament aggregation. TTBK1-Tg mice show significant age-dependent memory impairment as determined by radial arm water maze test, which is associated with enhancement of tau and neurofilament phosphorylation, increased levels of p25 and p35, both activators of cyclin-dependent protein kinase 5 (CDK5), enhanced calpain I activity, and reduced levels of hippocampal NMDA receptor types 2B (NR2B) and D. Enhanced CDK5/p35 complex formation is strongly correlated with dissociation of F-actin from p35, suggesting the inhibitory mechanism of CDK5/p35 complex formation by F-actin. Expression of recombinant TTBK1 in primary mouse cortical neurons significantly downregulated NR2B in a CDK5- and calpain-dependent manner. These data suggest that TTBK1 in AD brain may be one of the underlying mechanisms inducing CDK5 and calpain activation, NR2B downregulation, and subsequent memory dysfunction.

HIV-1 transforms the monocyte plasma membrane proteome.

How HIV-1 affects the monocyte proteome is incompletely understood. We posit that one functional consequence of virus-exposure to the monocyte is the facilitation of protein transformation from the cytosol to the plasma membrane (PM). To test this, cell surface labeling with CyDye fluorophores followed by 2 dimensional differential in-gel electrophoresis (2D DIGE) and liquid chromatography tandem mass spectrometry (LC-MS/MS) was performed. Fifty three percent of HIV-1 induced proteins were PM associated. These were linked, in large measure, to cellular activation and oxidative stress. They included, but not limited to, biliverdin reductase, leukotriene hydrolase A(4), heat shock protein 70, and cystatin B. HIV-1 induced PM protein translocation was associated with cathepsin B- and caspase 9, 3-dependent apoptosis. In contrast, PMA-treated monocytes bypassed caspase 3, 9 pathways and lead to cathepsin B-dependent necrosis. These results demonstrate that HIV-1 uniquely affects monocyte activation and oxidative stress. These do not affect viral infection dynamics but are linked to stress-induced cell death.

Multidimensional protein fractionation using ProteomeLab PF 2D for profiling amyotrophic lateral sclerosis immunity: A preliminary report.

BACKGROUND: The ProteomeLab PF 2D platform is a relatively new approach to global protein profiling. Herein, it was used for investigation of plasma proteome changes in amyotrophic lateral sclerosis (ALS) patients before and during immunization with glatiramer acetate (GA) in a clinical trial. RESULTS: The experimental design included immunoaffinity depletion of 12 most abundant proteins from plasma samples with the ProteomeLab IgY-12 LC10 column kit as first dimension separation, also referred to as immuno-partitioning. Second and third dimension separations of the enriched proteome were performed on the PF 2D platform utilizing 2D isoelectric focusing and RP-HPLC with the resulting fractions collected for analysis. 1D gel electrophoresis was added as a fourth dimension when sufficient protein was available. Protein identification from collected fractions was performed using nano-LC-MS/MS approach. Analysis of differences in the resulting two-dimensional maps of fractions obtained from the PF 2D and the ability to identify proteins from these fractions allowed sensitivity threshold measurements. Masked proteins in the PF 2D fractions are discussed. CONCLUSION: We offer some insight into the strengths and limitations of this emerging proteomic platform.

HIV-1-infected astrocytes and the microglial proteome.

The human immunodeficiency virus (HIV) invades the central nervous system early after viral exposure but causes progressive cognitive, behavior, and motor impairments years later with the onset of immune deficiency. Although in the brain, HIV preferentially replicates productively in cells of mononuclear phagocyte (MP; blood borne macrophage and microglia), astrocytes also can be infected, at low and variable frequency, particularly in patients with encephalitis. Among their many functions, astrocytes network with microglia to provide the first line of defense against microbial infection; however, very little is known about astrocytes' consequences on MP. Here, we addressed this question using co-culture systems of HIV-infected mouse astrocytes and microglia. Pseudotyped vesicular stomatis virus/HIV was used to circumvent the absence of viral receptors and ensure cell genotypic uniformity for studies of intercellular communication. The study demonstrated that infected astrocytes show modest changes in protein elements compared to uninfected cells. In contrast, infected astrocytes induce robust changes in the proteome of HIV-1-infected microglia. Accelerated cell death and redox proteins, among others, were produced in abundance. The observations confirmed the potential of astrocytes to influence the neuropathogenesis of HIV-1 infection by specifically altering the neurotoxic potential of infected microglia and regulating viral maturation.

Genomic and proteomic microglial profiling: pathways for neuroprotective inflammatory responses following nerve fragment clearance and activation.

Microglia, a primary immune effector cell of the central nervous system (CNS) affects homeostatic, neuroprotective, regenerative and degenerative outcomes in health and disease. Despite these broad neuroimmune activities linked to specific environmental cues, a precise cellular genetic profile for microglia in the context of disease and repair has not been elucidated. To this end we used nucleic acid microarrays, proteomics, immunochemical and histochemical tests to profile microglia in neuroprotective immune responses. Optic and sciatic nerve (ON and SN) fragments were used to stimulate microglia in order to reflect immune consequences of nervous system injury. Lipopolysaccharide and latex beads-induced microglial activation served as positive controls. Cytosolic and secreted proteins were profiled by surface enhanced laser desorption ionization-time of flight (SELDI-TOF) ProteinChip, 1D and 2D difference gel electrophoresis. Proteins were identified by peptide sequencing with tandem mass spectrometry, ELISA and western blot tests. Temporal expression of pro-inflammatory cytokines, antioxidants, neurotrophins, and lysosomal enzyme expression provided, for the first time, a unique profile of secreted microglia proteins with neuroregulatory functions. Most importantly, this molecular and biochemical signature supports a broad range of microglial functions for debris clearance and promotion of neural repair after injury.