Tatsuya Mizoroki

In vivo evidence of CHIP up-regulation attenuating tau aggregation

The carboxyl terminus of heat‐shock cognate (Hsc)70‐interacting protein (CHIP) is a ubiquitin E3 ligase that can collaborate with molecular chaperones to facilitate protein folding and prevent protein aggregation. Previous studies showed that, together with heat‐shock protein (Hsp)70, CHIP can regulate tau ubiquitination and degradation in a cell culture system. Ubiquitinated tau is one component in neurofibrillary tangles (NFTs), which are a major histopathological feature of Alzheimers disease (AD). However, the precise sequence of events leading to NFT formation and the mechanisms involved remain unclear. To confirm CHIPs role in suppressing NFT formation in vivo, we performed a quantitative analysis of CHIP in human and mouse brains. We found increased levels of CHIP and Hsp70 in AD compared with normal controls. CHIP levels in both AD and controls corresponded directly to Hsp90 levels, but not to Hsp70 or Hsc70 levels. In AD samples, CHIP was inversely proportional to sarkosyl‐insoluble tau accumulation. In a JNPL3 mouse brain tauopathy model, CHIP was widely distributed but weakly expressed in spinal cord, which was the most prominent region for tau inclusions and neuronal loss. Protein levels of CHIP in cerebellar regions of JNPL3 mice were significantly higher than in non‐transgenic littermates. Human tau was more highly expressed in this region of mouse brains, but only moderate levels of sarkosyl‐insoluble tau were detected. This was confirmed when increased insoluble tau accumulation was found in mice lacking CHIP. These findings suggest that increases in CHIP may protect against NFT formation in the early stages of AD. If confirmed, this would indicate that the quality‐control machinery in a neuron might play an important role in retarding the pathogenesis of tauopathies.

GSK-3β Is Required for Memory Reconsolidation in Adult Brain

Activation of GSK-3β is presumed to be involved in various neurodegenerative diseases, including Alzheimers disease (AD), which is characterized by memory disturbances during early stages of the disease. The normal function of GSK-3β in adult brain is not well understood. Here, we analyzed the ability of heterozygote GSK-3β knockout (GSK+/−) mice to form memories. In the Morris water maze (MWM), learning and memory performance of GSK+/− mice was no different from that of wild-type (WT) mice for the first 3 days of training. With continued learning on subsequent days, however, retrograde amnesia was induced in GSK+/− mice, suggesting that GSK+/− mice might be impaired in their ability to form long-term memories. In contextual fear conditioning (CFC), context memory was normally consolidated in GSK+/− mice, but once the original memory was reactivated, they showed reduced freezing, suggesting that GSK+/− mice had impaired memory reconsolidation. Biochemical analysis showed that GSK-3β was activated after memory reactivation in WT mice. Intraperitoneal injection of a GSK-3 inhibitor before memory reactivation impaired memory reconsolidation in WT mice. These results suggest that memory reconsolidation requires activation of GSK-3β in the adult brain.

Hyperphosphorylated tau in parahippocampal cortex impairs place learning in aged mice expressing wild-type human tau

To investigate how tau affects neuronal function during neurofibrillary tangle (NFT) formation, we examined the behavior, neural activity, and neuropathology of mice expressing wild‐type human tau. Here, we demonstrate that aged (>20 months old) mice display impaired place learning and memory, even though they do not form NFTs or display neuronal loss. However, soluble hyperphosphorylated tau and synapse loss were found in the same regions. Mn‐enhanced MRI showed that the activity of the parahippocampal area is strongly correlated with the decline of memory as assessed by the Morris water maze. Taken together, the accumulation of hyperphosphorylated tau and synapse loss in aged mice, leading to inhibition of neural activity in parahippocampal areas, including the entorhinal cortex, may underlie place learning impairment. Thus, the accumulation of hyperphosphorylated tau that occurs before NFT formation in entorhinal cortex may contribute to the memory problems seen in Alzheimers disease (AD).

Promotion of CHIP-Mediated p53 Degradation Protects the Heart From Ischemic Injury

Rationale: The number of patients with coronary heart disease, including myocardial infarction, is increasing and novel therapeutic strategy is awaited. Tumor suppressor protein p53 accumulates in the myocardium after myocardial infarction, causes apoptosis of cardiomyocytes, and plays an important role in the progression into heart failure. Objectives: We investigated the molecular mechanisms of p53 accumulation in the heart after myocardial infarction and tested whether anti-p53 approach would be effective against myocardial infarction. Methods and Results: Through expression screening, we found that CHIP (carboxyl terminus of Hsp70-interacting protein) is an endogenous p53 antagonist in the heart. CHIP suppressed p53 level by ubiquitinating and inducing proteasomal degradation. CHIP transcription was downregulated after hypoxic stress and restoration of CHIP protein level prevented p53 accumulation after hypoxic stress. CHIP overexpression in vivo prevented p53 accumulation and cardiomyocyte apoptosis after myocardial infarction. Promotion of CHIP function by heat shock protein (Hsp)90 inhibitor, 17-allylamino-17-demethoxy geldanamycin (17-AAG), also prevented p53 accumulation and cardiomyocyte apoptosis both in vitro and in vivo. CHIP-mediated p53 degradation was at least one of the cardioprotective effects of 17-AAG. Conclusions: We found that downregulation of CHIP level by hypoxia was responsible for p53 accumulation in the heart after myocardial infarction. Decreasing the amount of p53 prevented myocardial apoptosis and ameliorated ventricular remodeling after myocardial infarction. We conclude that anti-p53 approach would be effective to treat myocardial infarction.

Molecular chaperone-mediated tau protein metabolism counteracts the formation of granular tau oligomers in human brain.

Intracellular accumulation of filamentous tau proteins is a defining feature of neurodegenerative diseases termed tauopathies. The pathogenesis of tauopathies remains largely unknown. Molecular chaperones such as heat shock proteins (HSPs), however, have been implicated in tauopathies as well as in other neurodegenerative diseases characterized by the accumulation of insoluble protein aggregates. To search for in vivo evidence of chaperone‐related tau protein metabolism, we analyzed human brains with varying degrees of neurofibrillary tangle (NFT) pathology, as defined by Braak NFT staging. Quantitative analysis of soluble protein levels revealed significant positive correlations between tau and Hsp90, Hsp40, Hsp27, α‐crystallin, and CHIP. An inverse correlation was observed between the levels of HSPs in each specimen and the levels of granular tau oligomers, the latter of which were isolated from brain as intermediates of tau filaments. We speculate that HSPs function as regulators of soluble tau protein levels, and, once the capacity of this chaperone system is saturated, granular tau oligomers form virtually unabated. This is expressed pathologically as an early sign of NFT formation. The molecular basis of chaperone‐mediated protection against neurodegeneration might lead to the development of therapeutics for tauopathies.

GABAA Receptor-Mediated Acceleration of Aging-Associated Memory Decline in APP/PS1 Mice and Its Pharmacological Treatment by Picrotoxin

Advanced age and mutations in the genes encoding amyloid precursor protein (APP) and presenilin (PS1) are two serious risk factors for Alzheimers disease (AD). Finding common pathogenic changes originating from these risks may lead to a new therapeutic strategy. We observed a decline in memory performance and reduction in hippocampal long-term potentiation (LTP) in both mature adult (9–15 months) transgenic APP/PS1 mice and old (19–25 months) non-transgenic (nonTg) mice. By contrast, in the presence of bicuculline, a GABAA receptor antagonist, LTP in adult APP/PS1 mice and old nonTg mice was larger than that in adult nonTg mice. The increased LTP levels in bicuculline-treated slices suggested that GABAA receptor-mediated inhibition in adult APP/PS1 and old nonTg mice was upregulated. Assuming that enhanced inhibition of LTP mediates memory decline in APP/PS1 mice, we rescued memory deficits in adult APP/PS1 mice by treating them with another GABAA receptor antagonist, picrotoxin (PTX), at a non-epileptic dose for 10 days. Among the saline vehicle-treated groups, substantially higher levels of synaptic proteins such as GABAA receptor α1 subunit, PSD95, and NR2B were observed in APP/PS1 mice than in nonTg control mice. This difference was insignificant among PTX-treated groups, suggesting that memory decline in APP/PS1 mice may result from changes in synaptic protein levels through homeostatic mechanisms. Several independent studies reported previously in aged rodents both an increased level of GABAA receptor α1 subunit and improvement of cognitive functions by long term GABAA receptor antagonist treatment. Therefore, reduced LTP linked to enhanced GABAA receptor-mediated inhibition may be triggered by aging and may be accelerated by familial AD-linked gene products like Aβ and mutant PS1, leading to cognitive decline that is pharmacologically treatable at least at this stage of disease progression in mice.

Aggregation of detergent-insoluble tau is involved in neuronal loss but not in synaptic loss

Neurofibrillary tangles (NFTs), which consist of highly phosphorylated tau, are hallmarks of neurodegenerative diseases including Alzheimer disease (AD). In neurodegenerative diseases, neuronal dysfunction due to neuronal loss and synaptic loss accompanies NFT formation, suggesting that a process associated with NFT formation may be involved in neuronal dysfunction. To clarify the relationship between the tau aggregation process and synapse and neuronal loss, we compared two lines of mice expressing human tau with or without an aggregation-prone P301L mutation. P301L tau transgenic (Tg) mice exhibited neuronal loss and produced sarcosyl-insoluble tau in old age but did not exhibit synaptic loss and memory impairment. By contrast, wild-type tau Tg mice neither exhibited neuronal loss nor produced sarcosyl-insoluble tau but did exhibit synaptic loss and memory impairment. Moreover, P301L tau was less phosphorylated than wild-type tau, suggesting that the tau phosphorylation state is involved in synaptic loss, whereas the tau aggregation state is involved in neuronal loss. Finally, increasing concentrations of insoluble tau aggregates leads to the formation of fibrillar tau, which causes NFTs to form.

Central nervous system-specific deletion of transcription factor Nrf1 causes progressive motor neuronal dysfunction.

Cap’n’Collar (CNC) proteins heterodimerize with small Maf proteins and regulate the transcription of various genes. Small Maf‐deficient mice develop severe neurodegeneration, and it remains unclear whether CNC proteins are involved in this process. In this study, we examined the contribution of Nrf1, one of the CNC proteins, to neuronal homeostasis in vivo. As Nrf1 gene knockout mice are embryonic lethal, we developed a central nervous system (CNS)‐specific Nrf1 knockout (CKO) mouse line using mice bearing an Nrf1flox allele and Nestin‐Cre allele. At birth, the CKO mice appeared indistinguishable from control mice, but thereafter they showed progressive motor ataxia and severe weight loss. All Nrf1 CKO mice died within 3 weeks. These phenotypes are similar to those reported in small Maf‐deficient mice, suggesting the presence of collaboration between Nrf1 and small Maf proteins. We also found aberrant accumulation of polyubiquitinated proteins in various CNS regions and apparent neuronal loss in the hippocampus of Nrf1 CKO mice. An oxidative stress marker was accumulated in the spinal cords of the mice, but the expression patterns of oxidative stress response genes regulated by Nrf2 did not change substantially. These results show that Nrf1 sustains the CNS homeostasis through regulating target genes distinct from those regulated by Nrf2.

Aluminum induces tau aggregation in vitro but not in vivo.

Etiological studies suggest that aluminum (Al) intake might increase an individuals risk of developing Alzheimers disease (AD). Biochemical analysis data on the effects of Al, however, are inconsistent. Hence, the pathological involvement of Al in AD remains unclear. If Al is involved in AD, then it is reasonable to hypothesize that Al might be involved in the formation of either amyloid plaques or neurofibrillary tangles (NFTs). Here, we investigated whether Al might be involved in NFT formation by using an in vitro tau aggregation paradigm, a tau-overexpressing neuronal cell line (N2a), and a tau-overexpressing mouse model. Although Al induced tau aggregation in a heparin-induced tau assembly assay, these aggregates were neither thioflavin T positive nor did they resemble tau fibrils seen in human AD brains. With cell lysates from stable cell lines overexpressing tau, the accumulation of SDS-insoluble tau increased when the lysates were treated with at least 100 muM Al-maltolate. Yet Al-maltolate caused illness or death in transgenic mice overexpressing human tau and in non-transgenic littermates well before the Al concentration in the brain reached 100 muM. These results indicate that Al has no direct link to AD pathology.

Enzymatic Characteristics of I213T Mutant Presenilin-1/γ-Secretase in Cell Models and Knock-in Mouse Brains FAMILIAL ALZHEIMER DISEASE-LINKED MUTATION IMPAIRS γ-SITE CLEAVAGE OF AMYLOID PRECURSOR PROTEIN C-TERMINAL FRAGMENT β

Presenilin (PS)/γ-secretase-mediated intramembranous proteolysis of amyloid precursor protein produces amyloid β (Aβ) peptides in which Aβ species of different lengths are generated through multiple cleavages at the γ-, ζ-, and ϵ-sites. An increased Aβ42/Aβ40 ratio is a common characteristic of most cases of familial Alzheimer disease (FAD)-linked PS mutations. However, the molecular mechanisms underlying amyloid precursor protein proteolysis leading to increased Aβ42/Aβ40 ratios still remain unclear. Here, we report our findings on the enzymatic analysis of γ-secretase derived from I213T mutant PS1-expressing PS1/PS2-deficient (PS–/–) cells and from the brains of I213T mutant PS1 knock-in mice. Kinetics analyses revealed that the FAD mutation reduced de novo Aβ generation, suggesting that mutation impairs the total catalytic rate of γ-secretase. Analysis of each Aβ species revealed that the FAD mutation specifically reduced Aβ40 levels more drastically than Aβ42 levels, leading to an increased Aβ42/Aβ40 ratio. By contrast, the FAD mutation increased the generation of longer Aβ species such as Aβ43, Aβ45, and >Aβ46. These results were confirmed by analyses of γ-secretase derived from I213T knock-in mouse brains, in which the reduction of de novo Aβ generation was mutant allele dose-dependent. Our findings clearly indicate that the mechanism underlying the increased Aβ42/Aβ40 ratio observed in cases of FAD mutations is related to the differential inhibition of γ-site cleavage reactions, in which the reaction producing Aβ40 is subject to more inhibition than that producing Aβ42. Our results also provide novel insight into how enhancing the generation of longer Aβs may contribute to Alzheimer disease onset.Presenilin (PS)/gamma-secretase-mediated intramembranous proteolysis of amyloid precursor protein produces amyloid beta (Abeta) peptides in which Abeta species of different lengths are generated through multiple cleavages at the gamma-, zeta-, and epsilon-sites. An increased Abeta42/Abeta40 ratio is a common characteristic of most cases of familial Alzheimer disease (FAD)-linked PS mutations. However, the molecular mechanisms underlying amyloid precursor protein proteolysis leading to increased Abeta42/Abeta40 ratios still remain unclear. Here, we report our findings on the enzymatic analysis of gamma-secretase derived from I213T mutant PS1-expressing PS1/PS2-deficient (PS(-/-)) cells and from the brains of I213T mutant PS1 knock-in mice. Kinetics analyses revealed that the FAD mutation reduced de novo Abeta generation, suggesting that mutation impairs the total catalytic rate of gamma-secretase. Analysis of each Abeta species revealed that the FAD mutation specifically reduced Abeta40 levels more drastically than Abeta42 levels, leading to an increased Abeta42/Abeta40 ratio. By contrast, the FAD mutation increased the generation of longer Abeta species such as Abeta43, Abeta45, and >Abeta46. These results were confirmed by analyses of gamma-secretase derived from I213T knock-in mouse brains, in which the reduction of de novo Abeta generation was mutant allele dose-dependent. Our findings clearly indicate that the mechanism underlying the increased Abeta42/Abeta40 ratio observed in cases of FAD mutations is related to the differential inhibition of gamma-site cleavage reactions, in which the reaction producing Abeta40 is subject to more inhibition than that producing Abeta42. Our results also provide novel insight into how enhancing the generation of longer Abetas may contribute to Alzheimer disease onset.