Shunji Yamashita

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).

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.

Differential regional distribution of phosphorylated tau and synapse loss in the nucleus accumbens in tauopathy model mice

Tauopathies differ in terms of the brain regions that are affected. In Alzheimers disease, basal forebrain and hippocampus are mainly involved, while frontotemporal lobar degeneration affects the frontal and temporal lobes and subcortical nuclei including striatum. Over 90% of human cases of tauopathies are sporadic, although the majority of established tau-transgenic mice have had mutations. This prompted us to establish transgenic mice expressing wild-type human tau (Tg601). Old (>14 months old) Tg601 mice displayed decreased anxiety in the elevated plus maze test and impaired place learning in the Morris water maze test. Immunoblotting of brain tissue identified that soluble tau multimer was increased with aging even though insoluble tau was not observed. In the striatum of old Tg601, the level of AT8- or AT180-positive tau was decreased compared with that of other regions, while PHF-1-positive tau levels remained equal. Phosphorylated tau-positive axonal dilations were present mainly in layers V and VI of the prefrontal cortex. Loss of synaptic dendritic spine and decreased immunohistochemical level of synaptic markers were observed in the nucleus accumbens. In vivo 2-[(18)F]fluoro-2-deoxy-d-glucose positron emission tomography analysis also showed decreased activity exclusively in the nucleus accumbens of living Tg601 mice. In Tg601 mice, the axonal transport defect in the prefrontal cortex-nucleus accumbens pathway may lead to decreased anxiety behavior. Differential distribution of hyperphosphorylated tau may cause region-specific neurodegeneration.

Adaptive responses to alloxan-induced mild oxidative stress ameliorate certain tauopathy phenotypes

Oxidative stress is considered to promote aging and age‐related disorders such as tauopathy. Although recent reports suggest that oxidative stress under certain conditions possesses anti‐aging properties, no such conditions have been reported to ameliorate protein‐misfolding diseases. Here, we used neuronal and murine models that overexpress human tau to demonstrate that mild oxidative stress generated by alloxan suppresses several phenotypes of tauopathy. Alloxan treatment reduced HSP90 levels and promoted proteasomal degradation of tau, c‐Jun N‐amino terminal kinase, and histone deacetylase (HDAC) 6. Moreover, reduced soluble tau (phosphorylated tau) levels suppressed the formation of insoluble tau in tau transgenic mice, while reduced HDAC6 levels contributed to microtubule stability by increasing tubulin acetylation. Age‐dependent decreases in HDAC2 and phospho‐tau levels correlated with spatial memory enhancement in alloxan‐injected tau mice. These results suggest that mild oxidative stress, through adaptive stress responses, operates counteractively against some of the tauopathy phenotypes.

Examining the link between synaptosomal accumulation of tau protein and cognitive dysfunction

leading to restrictions of transport and promotion of amyloid plaques. Moreover, dendritic abnormalities like spine loss, shaft atrophy, bending, abrupt branch ending, varicosity formation, and sprouting are evoked. Methods: As a suitable transgenic model for Alzheimer’s disease, 3xTg-AD mice develop both beta amyloid (Aß) and tangle pathology, providing the opportunity to study degenerative processes resembling human AD-pathology. After crossing 3xTg-AD mice with a mouse line expressing yellow fluorescent protein in cortical and hippocampal neurons (YFP-H mice), we were able to perform 2-photon in vivo imaging of neurites down to the level of single dendritic spines. Investigating tau-dependent loss of spines in the somatosensory cortex, we found dendritic dystrophies which differed significantly from previously described degenerative neuritic changes: The dendritic swellings showed a distinct morphology resembling a pearl necklace and were positively stained for hyperphosphorylated tau as well as Aß. Huge varicosities surrounded a YFP-negative vacuole-like centre. In degenerative hippocampal neurites on the contrary, dystrophies near amyloid plaques contained neither hyperphosphorylated tau nor Aß, were YFP-positive throughout, and of rather spherical shape without visible interconnection. Results: By means of electron microscopy we analyzed the ultrastructural composition of the observed neuritic swellings, answering the following questions: What is inside the cortical dendritic swellings? What does their YFP-negative centre consist of? Is there an axodendritic shift of tau like in AD? Are the observed dystrophic changes specific for dendrites or also found in axons? How do cortical and hippocampal neuritic varicosities differ on the ultrastructural level? Conclusions: Our studies contribute to a better understanding of tau-dependent dendropathy in 3xTg-AD mice and may also help to elucidate the cause of neuritic dystrophies in AD.

P4-221: GABA-linked acceleration of aging-associated memory decline in APP/PS1 transgenic mouse and its pharmacological treatment by picrotoxin

gonadotropin-releasing hormone 1 (GnRH1) by producing luteinizing hormone (LH) that in turn induces neurosteroid synthesis. Methods: To test whether there may be an autocrine/paracrine feedback mechanism within the brain that modulates neurosteroid synthesis, as is the case for the regulation of gonadal sex steroid production by the HPG axis, 3-month old female BALB/c mice were either left intact, ovariectomized (OVX) or OVX and treated with a cholesterol (control), E2, P4 or E2 P4 pellet for 3 d. Results: Ovariectomy, which induces a dramatic increase in GnRH and gonadotropin synthesis, decreased the expression of GnRH receptor 1 (GnRHR1) and LH variants but did not alter estrogen receptor (ER ) expression. Ovariectomy had a differential affect on steroidogenic acute regulatory protein (StAR) expression, increasing the 30and 32-kDa StAR variants. Treatment of OVX animals with E2, P4 or E2 P4 all potently suppressed the expression of brain LH , GnRHR1 and ER , but increased the 37-kDa StAR variant. Conclusions: These results indicate that negative feedback loop(s) exist within the brain to regulate GnRH and LH signaling, and neurosteroid production via the regulation of StAR expression. Thus, circulating sex steroids can regulate the expression of components of the neurosteroid synthesis pathway in the brain, and may therefore play a major role in the regulation of neurosteroid synthesis. Given the importance of sex steroids to brain function, it is possible that autocrine/paracrine neurosteroid production may be a mechanism to fine-tune the level of sex steroids in the brain. Understanding the feedback loops in the brain that regulate neurosteroid production has important implications for developing therapeutic strategies to maintain brain sex steroid levels and cognition.

GABA-related acceleration of aging process in Alzheimer's model mouse

AD is characterized by accelerated memory loss in aging. GABA can suppress synaptic plasticity essential for memory. To test if that might explain memory impairment in PSAPP mouse model of AD, we estimated effect of GABA on LTP by using GABA receptor antagonist. Large GABA effect observed in ‘middle aged’ PSAPP resulted in low LTP and memory impairment by MWM. In contrast, GABA effect was reciprocally diminished at ‘old’ age of PSAPP. Consistent with its high LTP, ‘old’ PSAPP seemed to remember about repeated experience even better than ‘old’ nonTG, which showed large GABA effect. But, ‘old’ PSAPP mouse simultaneously showed less accurate learning and lack of memory flexibility. Large GABA effect in ‘old’ nonTG mouse is consistent with increasing GABA activity with aging in human brain. Large GABA effect in ‘middle aged’ PSAPP in contrast to such effect in ‘old’ nonTG indicates that acceleration of aging process in AD may be related to changing GABA activity.