A novel blood test for the early detection of Alzheimer's disease.

Despite a variety of testing approaches, it is often difficult to make an accurate diagnosis of Alzheimer's disease (AD), especially at an early stage of the disease. Diagnosis is based on clinical criteria as well as exclusion of other causes of dementia but a definitive diagnosis can only be made at autopsy. We have investigated the diagnostic value of a 96-gene expression array for detection of early AD. Gene expression analysis was performed on blood RNA from a cohort of 203 probable AD and 209 cognitively healthy age matched controls. A disease classification algorithm was developed on samples from 208 individuals (AD = 103; controls = 105) and was validated in two steps using an independent initial test set (n = 74; AD = 32; controls = 42) and another second test set (n = 130; AD = 68; controls = 62). In the initial analysis, diagnostic accuracy was 71.6 ± 10.3%, with sensitivity 71.9 ± 15.6% and specificity 71.4 ± 13.7%. Essentially the same level of agreement was achieved in the two independent test sets. High agreement (24/30; 80%) between algorithm prediction and subjects with available cerebrospinal fluid biomarker was found. Assuming a clinical accuracy of 80%, calculations indicate that the agreement with underlying true pathology is in the range 85%-90%. These findings suggest that the gene expression blood test can aid in the diagnosis of mild to moderate AD, but further studies are needed to confirm these findings.

Modulation of DRAK2 autophosphorylation by antigen receptor signaling in primary lymphocytes.

Death-associated protein-related apoptotic kinase-2 (DRAK2), a member of the death-associated protein-like family of serine/threonine kinases, is highly expressed in lymphoid organs and is a negative regulator of T cell activation. To investigate the regulation of DRAK2 activity in primary lymphocytes, we employed mass spectrometry to identify sites of autophosphorylation on DRAK2. These studies have revealed a key site of autophosphorylation on serine 12. Using a phospho-specific antibody to detect Ser(12) phosphorylation, we found that autophosphorylation is induced by antigen receptor stimulation in T and B cells. In Jurkat T cells, resting B cells and thymocytes, DRAK2 was hypophosphorylated on Ser(12) but rapidly phosphorylated with antigen receptor ligation. This increase in phosphorylation was dependent on intracellular calcium mobilization, because BAPTA-AM blocked DRAK2 kinase activity, whereas the SERCA inhibitor thapsigargin promoted Ser(12) phosphorylation. Our results show that DRAK2 kinase activity is regulated in a calcium-dependent manner and that Ser(12) phosphorylation is necessary for optimal suppression of T cell activation by this kinase, suggesting a potential feedback loop may act to modulate the activity of this kinase following antigen receptor signaling.

FOXO transcription factors cooperate with delta EF1 to activate growth suppressive genes in B lymphocytes.

Forkhead transcription factors regulate many aspects of lymphocyte development and function. The FOXO subgroup of Forkhead factors opposes proliferation and survival, and FOXO inactivation is an important outcome of Ag receptor signaling. FOXO activity at target promoters is modulated by other transcription factors in a manner dependent on cell type and external stimulus. We have investigated the mechanisms by which FOXO proteins activate the promoters of two target genes in murine B lymphocytes, Ccng2 (encoding cyclin G2) and Rbl2 (p130), each of which has been implicated in cell cycle arrest. FOXO proteins bound directly to both promoters in vitro and in vivo, augmented transcriptional activity in reporter assays, and increased expression of the endogenous genes. Each of the promoter sequences has consensus binding sites for the deltaEF1 transcription factor, previously shown to either repress or activate different promoters. deltaEF1 bound to the Ccng2 and Rbl2 promoters in vitro and in vivo and increased reporter activity as well as endogenous mRNA levels for these genes. Strikingly, deltaEF1 synergized with FOXO proteins to strongly activate transcription from both promoters. Coexpression of deltaEF1 enhanced FOXO-induced cell cycle arrest in B lymphoma cells. These findings establish a novel mechanism of FOXO function at target promoters: cooperation with deltaEF1.