Yukio Matsuba

19F and 1H MRI detection of amyloid |[beta]| plaques in vivo

Formation of senile plaques composed of amyloid β peptide, a pathological hallmark of Alzheimer disease, in human brains precedes disease onset by many years. Noninvasive detection of such plaques could be critical in presymptomatic diagnosis and could contribute to early preventive treatment strategies. Using amyloid precursor protein (APP) transgenic mice as a model of amyloid β amyloidosis, we demonstrate here that an intravenously administered 19F-containing amyloidophilic compound labels brain plaques and allows them to be visualized in living mice by magnetic resonance imaging (MRI) using 19F and 1H. Our findings provide a new direction for specific noninvasive amyloid imaging without the danger of exposure to radiation. This approach could be used in longitudinal studies in mouse models of Alzheimer disease to search for biomarkers associated with amyloid β pathology as well as to track disease course after treatment with candidate medications.

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.

Potent amyloidogenicity and pathogenicity of Aβ43

The amyloid-β peptide Aβ42 is known to be a primary amyloidogenic and pathogenic agent in Alzheimers disease. However, the role of Aβ43, which is found just as frequently in the brains of affected individuals, remains unresolved. We generated knock-in mice containing a pathogenic presenilin-1 R278I mutation that causes overproduction of Aβ43. Homozygosity was embryonic lethal, indicating that the mutation involves a loss of function. Crossing amyloid precursor protein transgenic mice with heterozygous mutant mice resulted in elevated Aβ43, impairment of short-term memory and acceleration of amyloid-β pathology, which accompanied pronounced accumulation of Aβ43 in plaque cores similar in biochemical composition to those observed in the brains of affected individuals. Consistently, Aβ43 showed a higher propensity to aggregate and was more neurotoxic than Aβ42. Other pathogenic presenilin mutations also caused overproduction of Aβ43 in a manner correlating with Aβ42 and with the age of disease onset. These findings indicate that Aβ43, an overlooked species, is potently amyloidogenic, neurotoxic and abundant in vivo.

Mechanistic involvement of the calpain-calpastatin system in Alzheimer neuropathology

The mechanism by which amyloid‐β peptide (Aβ) accumulation causes neurodegeneration in Alzheimers disease (AD) remains unresolved. Given that Aβ perturbs calcium homeostasis in neurons, we investigated the possible involvement of calpain, a calcium‐activated neutral protease. We first demonstrated close postsynaptic association of calpain activation with Aβ plaque formation in brains from both patients with AD and transgenic (Tg) mice overexpressing amyloid precursor protein (APP). Using a viral vector‐based tracer, we then showed that axonal termini were dynamically misdirected to calpain activation‐positive Aβ plaques. Consistently, cerebrospinal fluid from patients with AD contained a higher level of calpain‐cleaved spectrin than that of controls. Genetic deficiency of calpastatin (CS), a calpain‐specific inhibitor protein, augmented Aβ amyloidosis, tau phosphorylation, microgliosis, and somatodendritic dystrophy, and increased mortality in APP‐Tg mice. In contrast, brain‐specific CS overexpression had the opposite effect. These findings implicate that calpain activation plays a pivotal role in the Aβ‐triggered pathological cascade, highlighting a target for pharmacological intervention in the treatment of AD.—Higuchi, M., Iwata, N., Matsuba, Y., Takano, J., Suemoto, T., Maeda, J., Ji, B., Ono, M., Staufenbiel, M., Suhara, T., Saido, T. C. Mechanistic involvement of the calpain‐calpastatin system in Alzheimer neuropathology. FASEB J. 26, 1204‐1217 (2012). www.fasebj.org

Cerebrospinal fluid neprilysin is reduced in prodromal Alzheimer's disease

Amyloid β peptide (Aβ) has been implicated in Alzheimers disease (AD) as an initiator of the pathological cascades. Several lines of compelling evidence have supported major roles of Aβ‐degrading enzyme neprilysin in the pathogenesis of sporadic AD. Here, we have shown a substantial reduction of cerebrospinal fluid (CSF) neprilysin activity (CSF‐NEP) in patients with AD‐converted mild cognitive impairment and early AD as compared with age‐matched control subjects. The altered CSF‐NEP likely reflects changes in neuronal neprilysin, since transfer of neprilysin from brain tissue into CSF was demonstrated by injecting neprilysin‐carrying viral vector into the brains of neprilysin‐deficient mice. Interestingly, CSF‐NEP showed an elevation with the progression of AD. Along with a close association of CSF‐NEP with CSF tau proteins, this finding suggests that presynaptically located neprilysin can be released into CSF as a consequence of synaptic disruption. The impact of neuronal damages on CSF‐NEP was further demonstrated by a prominent increase of CSF‐NEP in rats exhibiting kainate‐induced neurodegeneration. Our results unequivocally indicate significance of CSF‐NEP as a biochemical indicator to pursue a pathological process that involves decreased neprilysin activity and Aβ‐induced synaptic toxicity, and the support the potential benefits of neprilysin up‐regulation in ameliorating neuropathology in prodromal and early AD. Ann Neurol 2005;57:832–842

Calpain Activation in Alzheimer's Model Mice Is an Artifact of APP and Presenilin Overexpression

Intraneuronal calcium stimulates the calpain-dependent conversion of p35 to p25, a CDK5 activator. It is widely believed that amyloid β peptide (Aβ) induces this conversion that, in turn, has an essential role in Alzheimers disease pathogenesis. However, in vivo studies on p25 generation used transgenic mice overexpressing mutant amyloid precursor protein (APP) and presenilin (PS). Here, using single App knock-in mice, we show that p25 generation is an artifact caused by membrane protein overexpression. We show that massive Aβ42 accumulation without overexpression of APP or presenilin does not produce p25, whereas p25 generation occurred with APP/PS overexpression and in postmortem mouse brain. We further support this finding using mice deficient for calpastatin, the sole calpain-specific inhibitor protein. Thus, the intracerebral environment of the APP/PS mouse brain and postmortem brain is an unphysiological state. SIGNIFICANCE STATEMENT We recently estimated using single App knock-in mice that accumulate amyloid β peptide without transgene overexpression that 60% of the phenotypes observed in Alzheimers model mice overexpressing mutant amyloid precursor protein (APP) or APP and presenilin are artifacts (Saito et al., 2014). The current study further supports this estimate by invalidating key results from papers that were published in Cell. These findings suggest that more than 3000 publications based on APP and APP/PS overexpression must be reevaluated.

Cell Surface Expression of the Major Amyloid-β Peptide (Aβ)-degrading Enzyme, Neprilysin, Depends on Phosphorylation by Mitogen-activated Protein Kinase/Extracellular Signal-regulated Kinase Kinase (MEK) and Dephosphorylation by Protein Phosphatase 1a

Background: Neprilysin is a major Aβ-degrading enzyme, the expression of which is reduced in the AD brain. Results: The phosphorylation status of the neprilysin intracellular domain regulates localization and cell surface activity. Conclusion: Regulation of neprilysin through phosphorylation influences Aβ levels. Significance: Our results indicate that neprilysin phosphorylation/dephosporylation could be one druggable target in the development of an AD-modifying treatment. Neprilysin is one of the major amyloid-β peptide (Aβ)-degrading enzymes, the expression of which declines in the brain during aging. The decrease in neprilysin leads to a metabolic Aβ imbalance, which can induce the amyloidosis underlying Alzheimer disease. Pharmacological activation of neprilysin during aging therefore represents a potential strategy to prevent the development of Alzheimer disease. However, the regulatory mechanisms mediating neprilysin activity in the brain remain unclear. To address this issue, we screened for pharmacological regulators of neprilysin activity and found that the neurotrophic factors brain-derived neurotrophic factor, nerve growth factor, and neurotrophins 3 and 4 reduce cell surface neprilysin activity. This decrease was mediated by MEK/ERK signaling, which enhanced phosphorylation at serine 6 in the neprilysin intracellular domain (S6-NEP-ICD). Increased phosphorylation of S6-NEP-ICD in primary neurons reduced the levels of cell surface neprilysin and led to a subsequent increase in extracellular Aβ levels. Furthermore, a specific inhibitor of protein phosphatase-1a, tautomycetin, induced extensive phosphorylation of the S6-NEP-ICD, resulting in reduced cell surface neprilysin activity. In contrast, activation of protein phosphatase-1a increased cell surface neprilysin activity and lowered Aβ levels. Taken together, these results indicate that the phosphorylation status of S6-NEP-ICD influences the localization of neprilysin and affects extracellular Aβ levels. Therefore, maintaining S6-NEP-ICD in a dephosphorylated state, either by inhibition of protein kinases involved in its phosphorylation or by activation of phosphatases catalyzing its dephosphorylation, may represent a new approach to prevent reduction of cell surface neprilysin activity during aging and to maintain physiological levels of Aβ in the brain.

Soft-diet feeding after weaning affects behavior in mice: Potential increase in vulnerability to mental disorders.

Mastication is one of the most important oral functions, and the period during which mastication is acquired overlaps with the term of rapid development and maturation of the neural systems. In particular, the acquisition period after weaning is related to the potential onset of mental disorders. However, the roles of mastication during this period for brain development remain largely unknown. Therefore, we used a series of standard behavioral analyses, assessment of hippocampal cell proliferation, and the expression of brain-derived neurotrophic factor (BDNF), TrkB, and Akt1 in the hippocampus and frontal cortex of mice to investigate the effects of post-weaning mastication on brain function. We fed 21-day-old C57BL6/J male mice either a hard or a soft diet for 4weeks and conducted a series of standard behavioral tests from 7weeks of age. Further, histological analysis with bromodeoxyuridine was performed to compare hippocampal cell proliferation at 7 and 14weeks of age. Real-time polymerase chain reaction was performed to compare BDNF, TrkB, and Akt1 expression in the hippocampus and frontal cortex of 14-week-old mice. Compared to mice fed a hard diet (HDM), soft-diet mice (SDM) showed behavioral impairments, including decreased home cage activity, increased open field test activity, and deficits in prepulse inhibition. These results were similar to those observed in mouse models of schizophrenia. However, no effects were observed on anxiety-like behaviors or memory/learning tests. Compared to HDM, SDM showed significantly decreased hippocampal cell proliferation and hippocampal BDNF and Akt1 gene expression at 14weeks of age. A soft diet after weaning may have resulted in histological and molecular changes in the hippocampus and influenced outcomes of behavioral tests related to mental disorders. Our findings suggest that soft-diet feeding after weaning may affect both physical and mental development of mice, and may increase vulnerability to mental disorders.

Introduction of pathogenic mutations into the mouse Psen1 gene by Base Editor and Target-AID

Base Editor (BE) and Target-AID (activation-induced cytidine deaminase) are engineered genome-editing proteins composed of Cas9 and cytidine deaminases. These base-editing tools convert C:G base pairs to T:A at target sites. Here, we inject either BE or Target-AID mRNA together with identical single-guide RNAs (sgRNAs) into mouse zygotes, and compare the base-editing efficiencies of the two distinct tools in vivo. BE consistently show higher base-editing efficiency (10.0–62.8%) compared to that of Target-AID (3.4–29.8%). However, unexpected base substitutions and insertion/deletion formations are also more frequently observed in BE-injected mice or zygotes. We are able to generate multiple mouse lines harboring point mutations in the mouse presenilin 1 (Psen1) gene by injection of BE or Target-AID. These results demonstrate that BE and Target-AID are highly useful tools to generate mice harboring pathogenic point mutations and to analyze the functional consequences of the mutations in vivo.CRISPR-guided cytidine deaminases, including BE3 (Base Editor 3) and Target-AID (activation-induced cytidine deaminase), can covert C:G base pairs to T:A at target site. Here, the authors generate missense mutations of mouse Psen1 gene and find BE3 has higher editing efficiency than Target-AID.

Generation of App knock-in mice reveals deletion mutations protective against Alzheimer’s disease-like pathology

Although, a number of pathogenic mutations have been found for Alzheimer’s disease (AD), only one protective mutation has been identified so far in humans. Here we identify possible protective deletion mutations in the 3′-UTR of the amyloid precursor protein (App) gene in mice. We use an App knock-in mouse model carrying a humanized Aβ sequence and three AD mutations in the endogenous App gene. Genome editing of the model zygotes using multiple combinations of CRISPR/Cas9 tools produces genetically mosaic animals with various App 3′-UTR deletions. Depending on the editing efficiency, the 3′-UTR disruption mitigates the Aβ pathology development through transcriptional and translational regulation of APP expression. Notably, an App knock-in mouse with a 34-bp deletion in a 52-bp regulatory element adjacent to the stop codon shows a substantial reduction in Aβ pathology. Further functional characterization of the identified element should provide deeper understanding of the pathogenic mechanisms of AD.To date, only one mutation in the gene for amyloid-beta precursor protein APP has been suggested to be protective against Alzheimer’s disease. Here, authors found using gene editing of a mutant App knock-in mouse line that deletion of the 3’UTR region is protective against amyloid-β accumulation in vivo, and subsequently identify a 52-bp element in the 3’UTR region that is responsible for this effect.