Ha-Na Woo

Alzheimer’s disease and Notch signaling

Cleavage of the amyloid precursor protein (APP) by gamma-secretase generates a neurotoxic amyloid beta-peptide (Abeta) that is thought to be associated with the neurodegeneration observed in Alzheimers disease (AD) patients. Presenilin is the catalytic member of the gamma-secretase proteolytic complex and mutations in presenilins are the major cause of early-onset familial Alzheimers disease. In addition to APP, gamma-secretase substrates include Notch1 homologues, Notch ligands Delta and Jagged, and additional type I membrane proteins, raising concerns about mechanism-based toxicities that might arise as a consequence of inhibiting gamma-secretase. Notch signaling is involved in tumorigenesis as well as in determining the fates of neural and nonneural cells during development and in adults. Alterations in proteolysis of the Notch by gamma-secretase could be involved in the pathogenesis of AD. Inconsistently, several recent observations have indicated that enhanced Notch signaling and expression could be instrumental in neurodegeneration in AD. Therefore, detailed and precise study of Notch signaling in AD is important for elucidating diverse mechanisms of pathogenesis and potentially for treating and preventing Alzheimers disease.

Pro-apoptotic function of calsenilin/DREAM/KChIP3

Apoptotic cell death and increased production of amyloid β peptide (Aβ) are pathological features of Alzheimer’s disease (AD), although the exact contribution of apoptosis to the pathogenesis of the disease remains unclear. Here we describe a novel pro‐apoptotic function of calsenilin/DREAM/KChIP3. By antisense oligonucleotide‐induced inhibition of calsenilin/DREAM/KChIP3 synthesis, apoptosis induced by Fas, Ca2+‐ionophore, or thapsigargin is attenuated. Conversely, calsenilin/DREAM/KChIP3 expression induced the morphological and biochemical features of apoptosis, including cell shrinkage, DNA laddering, and caspase activation. Calsenilin/DREAM/KChIP3‐induced apoptosis was suppressed by caspase inhibitor z‐VAD and by Bcl‐xL, and was potentiated by increasing cytosolic Ca2+, expression of Swedish amyloid precursor protein mutant (APPsw) or presenilin 2 (PS2), but not by a PS2 deletion lacking its C‐terminus (PS2/411stop). In addition, calsenilin/DREAM/KChIP3 expression increased Aβ42 production in cells expressing APPsw, which was potentiated by PS2, but not by PS2/411stop, which suggests a role for apoptosis‐associated Aβ42 production of calsenilin/DREAM/KChIP3.

Evidence that γ-secretase mediates oxidative stress-induced β-secretase expression in Alzheimer's disease

Beta-secretase (BACE1), an enzyme responsible for the production of amyloid beta-peptide (Abeta), is increased by oxidative stress and is elevated in the brains of patients with sporadic Alzheimers disease (AD). Here, we show that oxidative stress fails to induce BACE1 expression in presenilin-1 (gamma-secretase)-deficient cells and in normal cells treated with gamma-secretase inhibitors. Oxidative stress-induced beta-secretase activity and sAPPbeta levels were suppressed by gamma-secretase inhibitors. Levels of gamma- and beta-secretase activities were greater in brain tissue samples from AD patients compared to non-demented control subjects, and the elevated BACE1 level in the brains of 3xTgAD mice was reduced by treatment with a gamma-secretase inhibitor. Our findings suggest that gamma-secretase mediates oxidative stress-induced expression of BACE1 resulting in excessive Abeta production in AD.

Inactivation of farnesyltransferase and geranylgeranyltransferase I by caspase-3: Cleavage of the common α subunit during apoptosis

Caspase plays an important role in apoptosis. We report here that farnesyltransferase/geranylgeranyltransferase (FTase/GGTase)-α, a common subunit of FTase (α/βFTase) and GGTase I (α/βGGTase), was cleaved by caspase-3 during apoptosis. FTase/GGTase-α (49 kDa) was cleaved to 35 kDa (p35) in the Rat-2/H-ras, W4 and Rat-1 cells treated with FTase inhibitor (LB42708), anti-Fas antibody and etoposide, respectively. This cleavage was inhibited by caspase-inhibitors (YVAD-cmk, DEVD-cho). Serial N-terminal deletions and site-directed mutagenesis showed that Asp59 of FTase/GGTase-α was cleaved by caspase-3. The common FTase/GGTase-α subunit, but not the β subunits, of the FTase or GGTase I protein complexes purified from baculovirus-infected SF-9 cells was cleaved to be inactivated by purified caspase-3. In contrast, FTase mutant protein complex [(D59A)α/βFTase] was resistant to caspase-3. Expression of either the cleavage product (60-379) or anti-sense of FTase/GGTase-α induced cell death in Rat-2/H-ras cells. Furthermore, expression of (D59A)FTase/GGTase-α mutant significantly desensitized cells to etoposide-induced death. Taken together, we suggest that cleavage of prenyltransferase by caspase contributes to the progression of apoptosis.

Secretases as therapeutic targets for Alzheimer's disease.

Accumulation of amyloid-β (Aβ) is widely accepted as the key instigator of Alzheimers disease (AD). The proposed mechanism is that accumulation of Aβ results in inflammatory responses, oxidative damages, neurofibrillary tangles and, subsequently, neuronal/synaptic dysfunction and neuronal loss. Given the critical role of Aβ in the disease process, the proteases that produce this peptide are obvious targets. The goal would be to develop drugs that can inhibit the activity of these targets. Protease inhibitors have proved very effective for treating other disorders such as AIDS and hypertension. Mutations in APP (amyloid-β precursor protein), which flanks the Aβ sequence, cause early-onset familial AD, and evidence has pointed to the APP-to-Aβ conversion as a possible therapeutic target. Therapies aimed at modifying Aβ-related processes aim higher up the cascade and are therefore more likely to be able to alter the progression of the disease. However, it is not yet fully known whether the increases in Aβ levels are merely a result of earlier events that were already causing the disease.

Characterization of subcellular localization and Ca2+ modulation of calsenilin/DREAM/KChIP3

Earlier reports found that calsenilin is a transcriptional repressor or a subunit of plasma membrane channel, and indicated that calsenilin was present in the nucleus or plasma membrane. Immunohistochemical and subcellular fractionation analysis, however, revealed that calsenilin/DREAM/KChIP3 was distributed throughout the cytoplasm of SK-N-BE2(C), Jurkat, and HeLa cells. In addition, the expression of calsenilin suppressed the ATP-induced increase in intracellular Ca2+ concentrations. By increase in intracellular calcium concentration, calsenilin was translocated into the nucleus.

Atypical role of proximal caspase-8 in truncated Tau-induced neurite regression and neuronal cell death.

Abnormal Tau protein is known to be closely associated with several neurodegenerative diseases. Previously, we showed that Tau was cleaved by caspase-3 to generate the cleavage product lacking the C-terminus (DeltaTau-1) during neuronal cell death. Here we characterized caspase-8-dependent neurotoxicity of the truncated Tau. Introduction of DeltaTau-1 into primary hippocampal neurons induced loss of neurites in a caspase-dependent manner. Caspase-8 and -6 were proteolytically activated during DeltaTau-1-triggered neuronal cell death, which was suppressed by IETD-fmk, caspase-8 inhibitor. Direct targeting of caspase-8 and its associated FADD with antisense approaches and transient expression of their dominant-negative mutants reduced DeltaTau-1-induced apopotosis. Cells deficient in caspase-8, but not caspase-3, became sensitized to DeltaTau-1-mediated toxicity upon reconstitution with caspase-8. In addition, ectopic expression of mitochondrial antiapoptotic Bcl-2, Bcl-X(L), or inactive caspase-9 short form suppressed DeltaTau-1 toxicity. These results suggest that the truncated Tau protein activates proximal caspase-8 through FADD as a necessary step leading to neuronal cell death and neurite regression, contributing to the progression of abnormal Tau-associated neurodegeneracy.

Calsenilin Contributes to Neuronal Cell Death in Ischemic Stroke

Calsenilin is a calcium sensor protein that interacts with presenilin and increases calcium‐triggered neuronal apoptosis, and γ‐secretase activity. Notch is a cell surface receptor that regulates cell‐fate decisions and synaptic plasticity in brain. The aim of the present study was to characterize the role of calsenilin as a regulator of the γ‐secretase cleavage of Notch in ischemic stroke. Here, we determined the modulation of expression level and cellular distribution of calsenilin in neurons subjected to ischemic‐like conditions. The levels of calsenilin and presenilin were increased in primary neurons after oxygen and glucose deprivation. Furthermore, calsenilin was found to enhance the γ‐secretase cleavage of Notch and to contribute to cell death under ischemia‐like conditions. The inhibition of γ‐secretase activity and a presenilin deficiency were both found to protect against calsenilin‐mediated ischemic neuronal death. The expression of calsenilin was found to be increased in brain following experimental ischemic stroke. These findings establish a specific molecular mechanism by which the induction of calsenilin enhances Notch activation in ischemic stroke, and identify calsenilin as an upstream of the γ‐secretase cleavage of Notch.