Takashi Saito

Somatostatin regulates brain amyloid beta peptide Abeta42 through modulation of proteolytic degradation.

Expression of somatostatin in the brain declines during aging in various mammals including apes and humans. A prominent decrease in this neuropeptide also represents a pathological characteristic of Alzheimer disease. Using in vitro and in vivo paradigms, we show that somatostatin regulates the metabolism of amyloid β peptide (Aβ), the primary pathogenic agent of Alzheimer disease, in the brain through modulating proteolytic degradation catalyzed by neprilysin. Among various effector candidates, only somatostatin upregulated neprilysin activity in primary cortical neurons. A genetic deficiency of somatostatin altered hippocampal neprilysin activity and localization, and increased the quantity of a hydrophobic 42-mer form of Aβ, Aβ42, in a manner similar to presenilin gene mutations that cause familial Alzheimer disease. These results indicate that the aging-induced downregulation of somatostatin expression may be a trigger for Aβ accumulation leading to late-onset sporadic Alzheimer disease, and suggest that somatostatin receptors may be pharmacological-target candidates for prevention and treatment of Alzheimer disease.

Domain-specific mutations in TGFB1 result in Camurati-Engelmann disease

Camurati-Engelmann disease (CED, MIM 131300) is an autosomal dominant, progressive diaphyseal dysplasia characterized by hyperosteosis and sclerosis of the diaphyses of long bones. We recently assigned the CED locus to an interval between D19S422 and D19S606 at chromosome 19q13.1–q13.3 (ref. 2), which two other groups confirmed. As the human transforming growth factor-β1 gene (TGFB1) is located within this interval, we considered it a candidate gene for CED.

Single App knock-in mouse models of Alzheimer's disease

Experimental studies of Alzheimers disease have largely depended on transgenic mice overexpressing amyloid precursor protein (APP). These mice, however, suffer from artificial phenotypes because, in addition to amyloid β peptide (Aβ), they overproduce other APP fragments. We generated knock-in mice that harbor Swedish and Beyreuther/Iberian mutations with and without the Arctic mutation in the APP gene. The mice showed typical Aβ pathology, neuroinflammation and memory impairment in an age-dependent manner.

ScaleS: an optical clearing palette for biological imaging

Optical clearing methods facilitate deep biological imaging by mitigating light scattering in situ. Multi-scale high-resolution imaging requires preservation of tissue integrity for accurate signal reconstruction. However, existing clearing reagents contain chemical components that could compromise tissue structure, preventing reproducible anatomical and fluorescence signal stability. We developed ScaleS, a sorbitol-based optical clearing method that provides stable tissue preservation for immunochemical labeling and three-dimensional (3D) signal rendering. ScaleS permitted optical reconstructions of aged and diseased brain in Alzheimers disease models, including mapping of 3D networks of amyloid plaques, neurons and microglia, and multi-scale tracking of single plaques by successive fluorescence and electron microscopy. Human clinical samples from Alzheimers disease patients analyzed via reversible optical re-sectioning illuminated plaque pathogenesis in the z axis. Comparative benchmarking of contemporary clearing agents showed superior signal and structure preservation by ScaleS. These findings suggest that ScaleS is a simple and reproducible method for accurate visualization of biological tissue.

In-frame Dystrophin Following Exon 51-Skipping Improves Muscle Pathology and Function in the Exon 52–Deficient mdx Mouse

A promising therapeutic approach for Duchenne muscular dystrophy (DMD) is exon skipping using antisense oligonucleotides (AOs). In-frame deletions of the hinge 3 region of the dystrophin protein, which is encoded by exons 50 and 51, are predicted to cause a variety of phenotypes. Here, we performed functional analyses of muscle in the exon 52-deleted mdx (mdx52) mouse, to predict the function of in-frame dystrophin following exon 51-skipping, which leads to a protein lacking most of hinge 3. A series of AOs based on phosphorodiamidate morpholino oligomers was screened by intramuscular injection into mdx52 mice. The highest splicing efficiency was generated by a two-oligonucleotide cocktail targeting both the 5 and 3 splice sites of exon 51. After a dose-escalation study, we systemically delivered this cocktail into mdx52 mice seven times at weekly intervals. This induced 20-30% of wild-type (WT) dystrophin expression levels in all muscles, and was accompanied by amelioration of the dystrophic pathology and improvement of skeletal muscle function. Because the structure of the restored in-frame dystrophin resembles human dystrophin following exon 51-skipping, our results are encouraging for the ongoing clinical trials for DMD. Moreover, the therapeutic dose required can provide a suggestion of the theoretical equivalent dose for humans.

Neuronal Store-Operated Calcium Entry and Mushroom Spine Loss in Amyloid Precursor Protein Knock-In Mouse Model of Alzheimer's Disease

Alzheimers disease (AD) is the most common reason for elderly dementia in the world. We proposed that memory loss in AD is related to destabilization of mushroom postsynaptic spines involved in long-term memory storage. We demonstrated previously that stromal interaction molecule 2 (STIM2)-regulated neuronal store-operated calcium entry (nSOC) in postsynaptic spines play a key role in stability of mushroom spines by maintaining activity of synaptic Ca2+/calmodulin kinase II (CaMKII). Furthermore, we demonstrated previously that the STIM2–nSOC–CaMKII pathway is downregulated in presenilin 1 M146V knock-in (PS1–M146V KI) mouse model of AD, leading to loss of hippocampal mushroom spines in this model. In the present study, we demonstrate that hippocampal mushroom postsynaptic spines are also lost in amyloid precursor protein knock-in (APPKI) mouse model of AD. We demonstrated that loss of mushroom spines occurs as a result of accumulation of extracellular β-amyloid 42 in APPKI culture media. Our results indicate that extracellular Aβ42 acts by overactivating mGluR5 receptor in APPKI neurons, leading to elevated Ca2+ levels in endoplasmic reticulum, compensatory downregulation of STIM2 expression, impaired synaptic nSOC, and reduced CaMKII activity. Pharmacological inhibition of mGluR5 or overexpression of STIM2 rescued synaptic nSOC and prevented mushroom spine loss in APPKI hippocampal neurons. Our results indicate that downregulation of synaptic STIM2–nSOC–CaMKII pathway causes loss of mushroom synaptic spines in both presenilin and APPKI mouse models of AD. We propose that modulators/activators of this pathway may have a potential therapeutic value for treatment of memory loss in AD. SIGNIFICANCE STATEMENT A direct connection between amyloid-induced synaptic mushroom spine loss and neuronal store-operated calcium entry pathway is shown. These results provide strong support for the calcium hypothesis of neurodegeneration and further validate the synaptic store-operated calcium entry pathway as a potential therapeutic target for Alzheimers disease.

Interleukin-1β up-regulates TACE to enhance α-cleavage of APP in neurons: resulting decrease in Aβ production

The proinflammatory cytokine interleukin (IL)‐1β is up‐regulated in microglial cells surrounding amyloid plaques, leading to the hypothesis that IL‐1β is a risk factor for Alzheimer’s disease. However, we unexpectedly found that IL‐1β significantly enhanced α‐cleavage, indicated by increases in sAPPα and C83, but reduced β‐cleavage, indicated by decreases in sAPPβ and Aβ40/42, in human neuroblastoma SK‐N‐SH cells. IL‐1β did not significantly alter the mRNA levels of BACE1, ADAM‐9, and ADAM‐10, but up‐regulated that of TACE by threefold. The proform and mature form of TACE protein were also significantly up‐regulated. A TACE inhibitor (TAPI‐2) concomitantly reversed the IL‐1β‐dependent increase in sAPPα and decrease in sAPPβ, suggesting that APP consumption in the α‐cleavage pathway reduced its consumption in the β‐cleavage pathway. IL‐1Ra, a physiological antagonist for the IL‐1 receptor, reversed the effects of IL‐1β, suggesting that the IL‐1β‐dependent up‐regulation of α‐cleavage is mediated by the IL‐1 receptor. IL‐1β also induced this concomitant increase in α‐cleavage and decrease in β‐cleavage in mouse primary cultured neurons. Taken together we conclude that IL‐1β is an anti‐amyloidogenic factor, and that enhancement of its signaling or inhibition of IL‐1Ra activity could represent potential therapeutic strategies against Alzheimer’s disease.

APP mouse models for Alzheimer's disease preclinical studies

Animal models of human diseases that accurately recapitulate clinical pathology are indispensable for understanding molecular mechanisms and advancing preclinical studies. The Alzheimers disease (AD) research community has historically used first‐generation transgenic (Tg) mouse models that overexpress proteins linked to familial AD (FAD), mutant amyloid precursor protein (APP), or APP and presenilin (PS). These mice exhibit AD pathology, but the overexpression paradigm may cause additional phenotypes unrelated to AD. Second‐generation mouse models contain humanized sequences and clinical mutations in the endogenous mouse App gene. These mice show Aβ accumulation without phenotypes related to overexpression but are not yet a clinical recapitulation of human AD. In this review, we evaluate different APP mouse models of AD, and review recent studies using the second‐generation mice. We advise AD researchers to consider the comparative strengths and limitations of each model against the scientific and therapeutic goal of a prospective preclinical study.

An aberrant sugar modification of BACE1 blocks its lysosomal targeting in Alzheimer's disease

The β‐site amyloid precursor protein cleaving enzyme‐1 (BACE1), an essential protease for the generation of amyloid‐β (Aβ) peptide, is a major drug target for Alzheimers disease (AD). However, there is a concern that inhibiting BACE1 could also affect several physiological functions. Here, we show that BACE1 is modified with bisecting N‐acetylglucosamine (GlcNAc), a sugar modification highly expressed in brain, and demonstrate that AD patients have higher levels of bisecting GlcNAc on BACE1. Analysis of knockout mice lacking the biosynthetic enzyme for bisecting GlcNAc, GnT‐III (Mgat3), revealed that cleavage of Aβ‐precursor protein (APP) by BACE1 is reduced in these mice, resulting in a decrease in Aβ plaques and improved cognitive function. The lack of this modification directs BACE1 to late endosomes/lysosomes where it is less colocalized with APP, leading to accelerated lysosomal degradation. Notably, other BACE1 substrates, CHL1 and contactin‐2, are normally cleaved in GnT‐III‐deficient mice, suggesting that the effect of bisecting GlcNAc on BACE1 is selective to APP. Considering that GnT‐III‐deficient mice remain healthy, GnT‐III may be a novel and promising drug target for AD therapeutics.

Brain endothelial cells produce amyloid {beta} from amyloid precursor protein 770 and preferentially secrete the O-glycosylated form.

Deposition of amyloid β (Aβ) in the brain is closely associated with Alzheimer disease (AD). Aβ is generated from amyloid precursor protein (APP) by the actions of β- and γ-secretases. In addition to Aβ deposition in the brain parenchyma, deposition of Aβ in cerebral vessel walls, termed cerebral amyloid angiopathy, is observed in more than 80% of AD individuals. The mechanism for how Aβ accumulates in blood vessels remains largely unknown. In the present study, we show that brain endothelial cells expressed APP770, a differently spliced APP mRNA isoform from neuronal APP695, and produced Aβ40 and Aβ42. Furthermore, we found that the endothelial APP770 had sialylated core 1 type O-glycans. Interestingly, Ο-glycosylated APP770 was preferentially processed by both α- and β-cleavage and secreted into the media, suggesting that O-glycosylation and APP processing involved related pathways. By immunostaining human brain sections with an anti-APP770 antibody, we found that APP770 was expressed in vascular endothelial cells. Because we were able to detect O-glycosylated sAPP770β in human cerebrospinal fluid, this unique soluble APP770β has the potential to serve as a marker for cortical dementias such as AD and vascular dementia.