Alzheimer disease (AD) is characterized by the progressive accumulation of extracellular amyloid plaques and intracellular neurofibrillary tangles. The protein subunit of the amyloid plaques, β-amyloid (Aβ), does not occur as a single molecular species; many different Aβ-containing peptides have been detected in human cerebrospinal fluid (CSF) and/or brain tissue.^1,2 The most common Aβ isoform in vivo is Aβ_1-40, ie, a peptide that begins at Asp1 and terminates at Val40 of the Aβ region of amyloid precursor protein (APP). Increased accumulation of Aβ_1-42, a peptide that differs from Aβ_1-40 by the inclusion of Ile41 and Ala42, is particularly associated with development of AD. The extra 2 hydrophobic amino acids of Aβ₄₂ greatly enhance its aggregation propensity,³ leading to accelerated formation of small (low-n) Aβ oligomers (oAβ), larger intermediate assemblies like protofibrils, and eventually the typical approximately 8-nm amyloid fibrils found abundantly in neuritic plaques and amyloid-bearing microvessels. Small, soluble oligomers of Aβ have been linked to neuronal toxic effects and synaptic failure (for review, see the article by Walsh and Selkoe⁴). How the oligomeric assemblies reach a balance with monomeric Aβ and large protofibrils and fibrils in the human brain is under investigation.

The testing of candidate drugs to slow progression of Alzheimer’s disease (AD) requires clinical trials that are lengthy and expensive. Efforts to model the biochemical milieu of the AD brain may be greatly facilitated by combining two cutting edge technologies to generate three-dimensional (3D) human neuro-spheroid from induced pluripotent stem cells (iPSC) derived from AD subjects. We created iPSC from blood cells of five AD patients and differentiated them into 3D human neuronal culture. We characterized neuronal markers of our 3D neurons by immunocytochemical staining to validate the differentiation status. To block the generation of pathologic amyloid β peptides (Aβ), the 3D-differentiated AD neurons were treated with inhibitors targeting β-secretase (BACE1) and γ-secretases. As predicted, both BACE1 and γ-secretase inhibitors dramatically decreased Aβ generation in iPSC-derived neural cells derived from all five AD patients, under standard two-dimensional (2D) differentiation conditions. However, BACE1 and γ-secretase inhibitors showed less potency in decreasing Aβ levels in neural cells differentiated under 3D culture conditions. Interestingly, in a single subject AD1, we found that BACE1 inhibitor treatment was not able to significantly reduce Aβ42 levels. To investigate underlying molecular mechanisms, we performed proteomic analysis of 3D AD human neuronal cultures including AD1. Proteomic analysis revealed specific reduction of several proteins that might contribute to a poor inhibition of BACE1 in subject AD1. To our knowledge, this is the first iPSC-differentiated 3D neuro-spheroid model derived from AD patients’ blood. Our results demonstrate that our 3D human neuro-spheroid model can be a physiologically relevant and valid model for testing efficacy of AD drug.

γ‐Secretase cleavage, mediated by a complex of presenilin, presenilin enhancer (Pen‐2), nicastrin, and Aph‐1, is the final proteolytic step in generating amyloid β protein found in brains of Alzheimer's disease patients and Notch intracellular domain critical for proper neuronal development. Here, we employ the zebrafish model to study the role of Pen‐2 in neuronal survival. We found that (i) knockdown of Pen‐2 using antisense morpholino led to a reduction of islet‐1 positive neurons, (ii) Notch signaling was reduced in embryos lacking Pen‐2 or other γ‐secretase components, (iii) neuronal loss in Pen‐2 knockdown embryos is not as a result of a lack of neuronal precursor cells or cell proliferation, (iv) absence of Pen‐2 caused massive apoptosis in the whole animal, which could be suppressed by simultaneous knockdown of the tumor suppressor p53, (v) loss of islet‐1 or acetylated tubulin positive neurons in Pen‐2 knockdown embryos could be partially rescued by knockdown of p53. Our results demonstrate that knockdown of Pen‐2 directly induces a p53‐dependent apoptotic pathway that contributes to neuronal loss and suggest that Pen‐2 plays an important role in promoting neuronal cell survival and protecting from apoptosis in vivo.

Mutations in the DJ-1 gene have been linked to autosomal recessive familial Parkinson's disease. To understand the function of DJ-1, we determined the DJ-1 expression in both zebrafish and post mortem human brains. We found that DJ-1 was expressed early during zebrafish development and throughout adulthood. Knock down (KD) of DJ-1 by injection of morpholino did not cause dramatic morphologic alterations during development, and no loss of dopaminergic neurons was observed in embryos lacking DJ-1. However, DJ-1 KD embryos were more susceptible to programmed cell death. While a slight reduction in staining for islet-1 positive neurons was observed in both DJ-1 KD and H₂O₂treated embryos, the number of apoptotic cells was significantly increased in both KD and H₂O₂treated embryos. Interestingly, DJ-1 expression was increased in brains of zebrafish under conditions of oxidative stress, indicating that DJ-1 is a part of stress-responsive machinery. Since oxidative stress is one of the major contributors to the development of Alzheimer's disease (AD), we also examined DJ-1 expression in AD brains. Using DJ-1 specific antibodies, we failed to detect a robust staining of DJ-1 in brain tissues from control subjects. However, DJ-1 immunoreactivity was detected in hippocampal pyramidal neurons and astrocytes of AD brains. Therefore, our results strongly suggest that DJ-1 expression is not necessary during zebrafish development but can be induced in zebrafish exposed to oxidative stress and is present in human AD brains.

Regulated intramembrane proteolysis is a novel mechanism involving proteases that hydrolyze their substrates in a hydrophobic environment. Presenilin (PS) 1 and PS 2 are required for intramembrane cleavage of an increasing number of type I membrane proteins, including the amyloid precursor protein of Alzheimer's disease and the Notch receptor, which signals during differentiation and development. Mutagenesis, affinity labeling, biochemical isolation, and reconstitution in cells reveal that PS, in complex with co-factors nicastrin, APH-1 and PEN-2, apparently contains the active site ofγ-secretase, a novel membrane aspartyl protease. In addition, other related aspartyl proteases have been identified. These include members of the type-4 prepilin peptidase family in bacteria, which are known proteases and carry a GD motif conserved in PS. A group of multi-pass membrane proteins found in eukaryotes also contain YD and LGXGD motifs in two transmembrane domains that are conserved in PS and postulated to constitute an aspartyl protease active site. Among these is signal peptide peptidase (SPP), which cleaves remnant signal peptides derived from signal-peptidase-mediated ectodomain shedding. SPP cuts type II membrane proteins, illustrating that PS-like proteases play a key role in intramembrane proteolysis of single-pass membrane proteins oriented in either direction.

Alzheimer’s disease (AD), the most prevalent form of dementia, is characterized by two pathological hallmarks: Tau-containing neurofibrillary tangles and amyloid-β protein (Aβ )-containing neuritic plaques. The goal of this study is to understand mil