Mass spectrometry identifies tau C-terminal phosphorylation cluster during neuronal hyperexcitation.

Tau is a microtubule-associated protein implicated in Alzheimer's disease (AD) and other neurodegenerative disorders termed tauopathies. Pathological, aggregated forms of tau form neurofibrillary tangles (NFTs), impairing its ability to stabilize microtubules and promoting neurotoxicity. Indeed, NFTs correlate with neuronal loss and cognitive impairment. Hyperphosphorylation of tau is seen in all tauopathies and mirrors disease progression, suggesting an essential role in pathogenesis. However, hyperphosphorylation remains a generic and ill-defined term, obscuring the functional importance of specific sites in different physiological or pathological settings. Here, we focused on global mapping of tau phosphorylation specifically during conditions of neuronal hyperexcitation. Hyperexcitation is a property of AD and other tauopathies linked to human cognitive deficits and increased risk of developing seizures and epilepsy. Moreover, hyperexcitation promotes extracellular secretion and trans-synaptic propagation of tau. Using unbiased mass spectrometry, we identified a novel phosphorylation signature in the C-terminal domain of tau detectable only during neuronal hyperactivity in primary cultured rat hippocampal neurons. These sites influenced tau localization to dendrites as well as the size of excitatory postsynaptic sites. These results demonstrate novel physiological tau functions at synapses and the utility of comprehensive analysis of tau phosphorylation during specific signaling contexts.

Resveratrol differentially affects MMP-9 release from neurons and glia; implications for therapeutic efficacy.

Resveratrol, a naturally occurring polyphenol that activates sirtuin 1 (SIRT1), has been shown to reduce overall levels of matrix metalloprotease-9 (MMP-9) in cerebrospinal fluid (CSF) samples from patients with Alzheimer's dementia (AD). Depending on the site of release, however, MMP-9 has the potential to improve or impair cognition. In particular, its release from microglia or pericytes proximal to the blood brain barrier can damage the basement membrane, while neuronal activity-dependent release of this protease from glutamatergic neurons can instead promote dendritic spine expansion and long-term potentiation of synaptic plasticity. In the present study, we test the hypothesis that resveratrol reduces overall MMP-9 levels in CSF samples from patients with APOE4, an allele associated with increased glial inflammation. We also examine the possibility that resveratrol reduces inflammation-associated MMP release from cultured glia but spares neuronal activity-dependent release from cultured cortical neurons. We observe that resveratrol decreases overall levels of MMP-2 and MMP-9 in CSF samples from AD patients. Resveratrol also reduces CSF levels of tissue inhibitor of metalloproteinases-1 (TIMP-1), glial-derived protein that restricts long-term potentiation of synaptic transmission, in individuals homozygous for APOE4. Consistent with these results, we observe that resveratrol reduces basal and lipopolysaccharide (LPS)-stimulated MMP and TIMP-1 release from cultured microglia and astrocytes. In contrast, however, resveratrol does not inhibit release of MMP-9 from cortical neurons. Overall, these results are consistent with the possibility that while resveratrol reduces potentially maladaptive MMP and TIMP-1 release from activated glia, neuroplasticity-promoting MMP release from neurons is spared. In contrast, resveratrol reduces release of neurocan and brevican, extracellular matrix components that restrict neuroplasticity, from both neurons and glia. These data underscore the diversity of resveratrol's actions with respect to affected cell types and molecular targets and also suggest that further studies may be warranted to determine if its effects on glial MMP release could make it a useful adjunct for AD- and/or anti-amyloid therapy-related damage to the blood brain barrier.

High-throughput Peptide epitope mapping using carbon nanotube field-effect transistors.

Label-free and real-time detection technologies can dramatically reduce the time and cost of pharmaceutical testing and development. However, to reach their full promise, these technologies need to be adaptable to high-throughput automation. To demonstrate the potential of single-walled carbon nanotube field-effect transistors (SWCNT-FETs) for high-throughput peptide-based assays, we have designed circuits arranged in an 8 × 12 (96-well) format that are accessible to standard multichannel pipettors. We performed epitope mapping of two HIV-1 gp160 antibodies using an overlapping gp160 15-mer peptide library coated onto nonfunctionalized SWCNTs. The 15-mer peptides did not require a linker to adhere to the non-functionalized SWCNTs, and binding data was obtained in real time for all 96 circuits. Despite some sequence differences in the HIV strains used to generate these antibodies and the overlapping peptide library, respectively, our results using these antibodies are in good agreement with known data, indicating that peptides immobilized onto SWCNT are accessible and that linear epitope mapping can be performed in minutes using SWCNT-FET.