Chiho Hirata-Fukae

Females exhibit more extensive amyloid, but not tau, pathology in an Alzheimer transgenic model

Epidemiological studies indicate that women have a higher risk of Alzheimers disease (AD) even after adjustment for age. Though transgenic mouse models of AD develop AD-related amyloid beta (Abeta) and/or tau pathology, gender differences have not been well documented in these models. In this study, we found that female 3xTg-AD transgenic mice expressing mutant APP, presenilin-1 and tau have significantly more aggressive Abeta pathology. We also found an increase in beta-secretase activity and a reduction of neprilysin in female mice compared to males; this suggests that a combination of increased Abeta production and decreased Abeta degradation may contribute to higher risk of AD in females. In contrast to significantly more aggressive Abeta pathology in females, gender did not affect the levels of phosphorylated tau in 3xTg-AD mice. These results point to the involvement of Abeta pathways in the higher risk of AD in women. In addition to comparison of pathology between genders at 9, 16 and 23 months of age, we examined the progression of Abeta pathology at additional age points; i.e., brain Abeta load, intraneuronal oligomeric Abeta distribution and plaque load, in male 3xTg-AD mice at 3, 6, 9, 12, 16, 20 and 23 months of age. These findings confirm progressive Abeta pathology in 3xTg-AD transgenic mice, and provide guidance for their use in therapeutic research.

A neuronal microtubule-interacting agent, NAPVSIPQ, reduces tau pathology and enhances cognitive function in a mouse model of Alzheimer's disease.

Neurofibrillary tangles composed of aggregated, hyperphosphorylated tau in an abnormal conformation represent one of the major pathological hallmarks of Alzheimers disease (AD) and other tauopathies. However, recent data suggest that the pathogenic processes leading to cognitive impairment occur before the formation of classic tangles. In the earliest stages of tauopathy, tau detaches from microtubules and accumulates in the cytosol of the somatodendritic compartment of cells. Either as a cause or an effect, tau becomes hyperphosphorylated and aggregates into paired helical filaments that comprise the tangles. To assess whether an agent that modulates microtubule function can inhibit the pathogenic process and prevent cognitive deficits in a transgenic mouse model with AD-relevant tau pathology, we administered the neuronal tubulin-preferring agent, NAPVSIPQ (NAP). Three months of treatment with NAP at an early-to-moderate stage of tauopathy reduced the levels of hyperphosphorylated soluble and insoluble tau. A 6-month course of treatment improved cognitive function. Although nonspecific tubulin-interacting agents commonly used for cancer therapy are associated with adverse effects due to their anti-mitotic activity, no adverse effects were found after 6 months of exposure to NAP. Our results suggest that neuronal microtubule interacting agents such as NAP may be useful therapeutic agents for the treatment or prevention of tauopathies.

Intranasal NAP administration reduces accumulation of amyloid peptide and tau hyperphosphorylation in a transgenic mouse model of Alzheimer's disease at early pathological stage.

Accumulation of β-amyloid (Aβ) peptide and hyperphosphorylation of tau in the brain are pathological hallmarks of Alzheimers disease (AD). Agents altering these pathological events might modify clinical disease progression. NAP (Asn-Ala-Pro-Val-Ser-Ile-Pro-Gln) is an octapeptide that has shown neuroprotective effects in various in vitro and in vivo neurodegenerative models. Previous studies showed that NAP protected against Aβ-induced neurotoxicity, inhibited Aβ aggregation, and, by binding to tubulin, prevented disruption of microtubules. In this study, we investigated the effect of NAP on Aβ and tau pathology using a transgenic mouse model that recapitulates both aspects of AD. We administered NAP intranasally (0.5 μg/mouse per day, daily from Monday through Friday) for 3 mo, starting from 9 mo of age, which is a prepathological stage in these mice. NAP treatment significantly lowered levels of Aβ-1–40 and 1–42 in brain. In addition, NAP significantly reduced levels of hyperphosphorylated tau. Of particular interest, hyperphosphorylation at the threonine 231 site was reduced; phosphorylation at this site influences microtubule binding. Our results indicate that NAP treatment of transgenic mice initiated at an early stage reduced both Aβ and tau pathology, suggesting that NAP might be a potential therapeutic agent for AD.

BACE1 inhibition reduces endogenous Abeta and alters APP processing in wild-type mice.

Accumulation of amyloid beta peptide (Abeta) in brain is a hallmark of Alzheimers disease (AD). Inhibition of beta‐site amyloid precursor protein (APP)‐cleaving enzyme‐1 (BACE1), the enzyme that initiates Abeta production, and other Abeta‐lowering strategies are commonly tested in transgenic mice overexpressing mutant APP. However, sporadic AD cases, which represent the majority of AD patients, are free from the mutation and do not necessarily have overproduction of APP. In addition, the commonly used Swedish mutant APP alters APP cleavage. Therefore, testing Abeta‐lowering strategies in transgenic mice may not be optimal. In this study, we investigated the impact of BACE1 inhibition in non‐transgenic mice with physiologically relevant APP expression. Existing Abeta ELISAs are either relatively insensitive to mouse Abeta or not specific to full‐length Abeta. A newly developed ELISA detected a significant reduction of full‐length soluble Abeta 1–40 in mice with the BACE1 homozygous gene deletion or BACE1 inhibitor treatment, while the level of x‐40 Abeta was moderately reduced due to detection of non‐full‐length Abeta and compensatory activation of alpha‐secretase. These results confirmed the feasibility of Abeta reduction through BACE1 inhibition under physiological conditions. Studies using our new ELISA in non‐transgenic mice provide more accurate evaluation of Abeta‐reducing strategies than was previously feasible.

A humanin derivative reduces amyloid beta accumulation and ameliorates memory deficit in triple transgenic mice.

Humanin (HN), a 24-residue peptide, was identified as a novel neuroprotective factor and shows anti-cell death activity against a wide spectrum of Alzheimers disease (AD)-related cytotoxicities, including exposure to amyloid beta (Abeta), in vitro. We previously demonstrated that the injection of S14G-HN, a highly potent HN derivative, into brain ameliorated memory loss in an Abeta-injection mouse model. To fully understand HNs functions under AD-associated pathological conditions, we examined the effect of S14G-HN on triple transgenic mice harboring APPswe, tauP310L, and PS-1M146V that show the age-dependent development of multiple pathologies relating to AD. After 3 months of intranasal treatment, behavioral analyses showed that S14G-HN ameliorated cognitive impairment in male mice. Moreover, ELISA and immunohistochemical analyses showed that Abeta levels in brains were markedly lower in S14G-HN-treated male and female mice than in vehicle control mice. We also found the expression level of neprilysin, an Abeta degrading enzyme, in the outer molecular layer of hippocampal formation was increased in S14G-HN-treated mouse brains. NEP activity was also elevated by S14G-HN treatment in vitro. These findings suggest that decreased Abeta level in these mice is at least partly attributed to S14G-HN-induced increase of neprilysin level. Although HN was identified as an anti-neuronal death factor, these results indicate that HN may also have a therapeutic effect on amyloid accumulation in AD.

Triazole-linked reduced amide isosteres: an approach for the fragment-based drug discovery of anti-Alzheimer's BACE1 inhibitors.

In the course of a β-site APP-cleaving enzyme 1 (BACE1) inhibitor discovery project an in situ synthesis/screening protocol was employed to prepare 120 triazole-linked reduced amide isostere inhibitors. Among these compounds, four showed modest (single digit micromolar) BACE1 inhibition. Our ligand design was based on a potent reduced amide isostere 1, wherein the P(2) amide moiety was replaced with an anti-1,2,3-triazole unit. Unfortunately, this replacement resulted in a 1000-fold decrease in potency. Docking studies of triazole-linked reduced amide isostere A3Z10 and potent oxadiazole-linked tertiary carbinamine 2a with BACE1 suggests that the docking poses of A3Z10 and 2a in the active sites are quite similar, with one exception. In the docked structures the placement of the protonated amine that engages D228 differs considerably between 2a and A3Z10. This difference could account for the lower BACE1 inhibition potency of A3Z10 and related compounds relative to 2a.

Deglycosylated anti‐amyloid beta antibodies reduce microglial phagocytosis and cytokine production while retaining the capacity to induce amyloid beta sequestration

Accumulation of amyloid beta (Abeta) is a pathological hallmark of Alzheimers disease, and lowering Abeta is a promising therapeutic approach. Intact anti‐Abeta antibodies reduce brain Abeta through two pathways: enhanced microglial phagocytosis and Abeta transfer from the brain to the periphery (Abeta sequestration). While activation of microglia, which is essential for microglial phagocytosis, is necessarily accompanied by undesired neuroinflammatory events, the capacity for sequestration does not seem to be linked to such effects. We and other groups have found that simple Abeta binding agents are sufficient to reduce brain Abeta through the sequestration pathway. In this study, we aimed to eliminate potentially deleterious immune activation from antibodies without affecting the ability to induce sequestration. The glycan portion of immunoglobulin is critically involved in interactions with immune effectors including the Fc receptor and complement c1q; deglycosylation eliminates these interactions, while antigen (Abeta)‐binding affinity is maintained. In this study, we investigated whether deglycosylated anti‐Abeta antibodies reduce microglial phagocytosis and neuroinflammation without altering the capacity to induce Abeta sequestration. Deglycosylated antibodies maintained Abeta binding affinity. Deglycosylated antibodies did not enhance Abeta phagocytosis or cytokine release in primary cultured microglia, whereas intact antibodies did so significantly. Intravenous injection of deglycosylated antibodies elevated plasma Abeta levels and induced Abeta sequestration to a similar or greater degree compared with intact antibodies in an Alzheimers transgenic mouse model without or with Abeta plaque pathology. We conclude that deglycosylated antibodies effectively induced Abeta sequestration without provoking neuroinflammation; thus, these deglycosylated antibodies may be optimal for sequestration therapy for Alzheimers disease.

Levels of soluble and insoluble tau reflect overall status of tau phosphorylation in vivo

The clinical progression of Alzheimers disease is closely related to tau pathology. Hyperphosphorylation of tau precedes histopathological evidence of tangle formation, and modulation of tau phosphorylation is a promising therapeutic target. Although some phosphorylation sites are more critical in pathological processes, the importance of each phosphorylation site is unclear. In this study, we found that levels of phosphorylated tau drastically increased in crude and insoluble tau fractions with aging in a transgenic mouse model of Alzheimer-type tauopathy. However, changes in the soluble tau fraction were minor and phosphorylation at some sites was even reduced with aging. Total soluble (presumably functional) tau was reduced, while insoluble tau increased with aging. Synaptic proteins were reduced as insoluble tau increased. Taken together, these findings suggest that levels of soluble and insoluble tau are indicative of overall levels of tau phosphorylation, and may be useful markers to evaluate the effects of anti-tau therapeutic strategies in vivo.

In vivo protein transduction to the CNS.

Proteins and peptides are useful research and therapeutic tools, however applications are limited because delivery to the desired location is not easily achievable. There are two hurdles in protein/peptide delivery to the brain: the blood-brain barrier and intracellular penetration. Penetration to both brain and the intracellular space can be achieved by adjusting hydrophilicity, and small molecule pharmacological agents have been successfully developed using this approach. But with proteins and peptides, it is difficult to modify the hydrophilicity without influencing biological functions. Trans-acting factor protein from the human immunodeficiency virus contains a highly conserved cationic peptide sequence necessary for transduction across the cell membrane. While trans-acting factor peptide has been used for in vitro protein transduction, its in vivo application is very limited because it is rapidly degraded by proteolysis. Polyethylenimine is a chemically synthesized small molecule cationization agent; the charge density is greater than a peptide-based cationic cluster such as trans-acting factor, and it is resistant to proteolysis in vivo. We first tested intracellular protein transduction following direct brain injection in mice using polyethylenimine-conjugated green fluorescence protein and beta-galactosidase (molecular weights 29 and 540 kDa, respectively). Polyethylenimine-conjugates penetrated to the intracellular space immediately surrounding the injection site within one hour. We further tested polyethylenimine-mediated protein transduction following intranasal administration, which bypasses the blood-brain barrier. Polyethylenimine-conjugates in pH 7.5 solution did not reach the brain, probably because the polyethylenimine-conjugates penetrated into the intracellular space where first exposed to the tissue, i.e. at the nasal mucosae. We temporarily reduced the electrostatic interaction between cationized polyethylenimine-conjugates and cellular surfaces by adjusting the pH to 4.5; solution rapidly reached the brain and penetrated to the intracellular space. This study suggests that polyethylenimine is a useful protein transduction agent in the brain in vivo, and adjusting cationic charge interaction can determine the extent of brain penetration.

Beta-site amyloid precursor protein-cleaving enzyme-1 (BACE1)-mediated changes of endogenous amyloid beta in wild-type and transgenic mice in vivo

Beta-site amyloid precursor protein-cleaving enzyme-1 (BACE1) initiates generation of amyloid beta (Abeta), a pathological hallmark of Alzheimers disease. We investigated the impact of BACE1 protein level on endogenous Abeta. Endogenous Abeta and BACE1 protein levels were concurrently and significantly reduced during early life. However, Abeta levels were similar between BACE1 transgenic and wildtype mice. This suggests that BACE1 protein level has a minimal effect on the level of endogenous Abeta. Consequently, other factors must be involved in modulation of Abeta production in adult and ageing brain and investigation of such factors may yield therapeutic targets. Further, these results suggest that substantial inhibition of BACE1 in brain may be required for clinical benefit in Alzheimers disease.