Cytoplasmic aggregation of TDP43 and topographic correlation with tau and α-synuclein accumulation in the rTg4510 mouse model of tauopathy.

In patients with TDP43 proteinopathy, phosphorylated TDP43 (p-TDP43) accumulates in the cytoplasm of neurons. The accumulation of p-TDP43 has also been reported in patients with tauopathy and α-synucleinopathy. We investigated spatiotemporal changes in p-TDP43 accumulation in the brains of rTg4510 mice that overexpressed human mutant tau (P301L) and exhibited hyperphosphorylated tau (hp-tau) and phosphorylated αSyn (p-αSyn) accumulation. Immunohistochemically, p-TDP43 aggregates were observed in the cytoplasm of neurons, which increased with age. A significant positive correlation was observed between the number of cells with p-TDP43 aggregates and hp-tau and p-αSyn aggregates. Suppression of the human mutant tau (P301L) expression by doxycycline treatment reduces the accumulation of p-TDP43, hp-tau, and p-αSyn. Proteinase K-resistant p-TDP43 aggregates were found in regions with high hp-tau, and p-αSyn accumulation. Western blotting of the sarkosyl-insoluble fraction revealed bands of monomeric TDP43 and p-TDP43. These results indicate that the accumulation of mouse p-TDP43 is associated with the accumulation of human mutant tau (P301L) in rTg4510 mouse brains. The accumulation of hp-tau and p-αSyn may promote sarkosyl-insoluble p-TDP43 aggregates that are resistant to proteinase K. The synergistic effects of tau, TDP43, and αSyn may be involved in the pathology of proteinopathies, leading to the accumulation of multiple abnormal proteins.

Path integration deficits are associated with phosphorylated tau accumulation in the entorhinal cortex.

Alzheimer's disease is a devastating disease that is accompanied by dementia, and its incidence increases with age. However, no interventions have exhibited clear therapeutic effects. We aimed to develop and characterize behavioural tasks that allow the earlier identification of signs preceding dementia that would facilitate the development of preventative and therapeutic interventions for Alzheimer's disease. To this end, we developed a 3D virtual reality task sensitive to the activity of grid cells in the entorhinal cortex, which is the region that first exhibits neurofibrillary tangles in Alzheimer's disease. We investigated path integration (assessed by error distance) in a spatial navigation task sensitive to grid cells in the entorhinal cortex in 177 volunteers, aged 20-89 years, who did not have self-reported dementia. While place memory was intact even in old age, path integration deteriorated with increasing age. To investigate the relationship between neurofibrillary tangles in the entorhinal cortex and path integration deficit, we examined a mouse model of tauopathy (P301S mutant tau-overexpressing mice; PS19 mice). At 6 months of age, PS19 mice showed a significant accumulation of phosphorylated tau only in the entorhinal cortex, associated with impaired path integration without impairments in spatial cognition. These data are consistent with the idea that path integration deficit is caused by the accumulation of phosphorylated tau in the entorhinal cortex. This method may allow the early identification of individuals likely to develop Alzheimer's disease.

Tryptophan-tyrosine dipeptide improves tau-related symptoms in tauopathy mice.

Neurodegenerative diseases involving pathological tau protein aggregation are collectively known as tauopathies and include Alzheimer's disease and Pick's disease. Recent studies show that the intake of tryptophan-tyrosine (Trp-Tyr)-related ß-lactopeptides, including ß-lactolin, attenuates cognitive decline in the elderly and prevents the amyloid pathology in mouse models of Alzheimer's disease. However, the effects of Trp-Tyr-related ß-lactopeptides on tau-related pathology have not been investigated. In the present study, we examined the effects of Trp-Tyr dipeptide intake on tauopathy in PS19 transgenic mice, a well-established tauopathy model. Intake of Trp-Tyr dipeptide improved the behavioral deficits observed in the open field test, prevented tau phosphorylation, and increased the dopamine turnover and synaptophysin expression in the frontal cortex. Levels of short-chain fatty acids in the cecum were lower in PS19 mice than those in wild-type mice and were increased by treatment with Trp-Tyr dipeptide. In addition, intake of Trp-Tyr dipeptide extended the lifespan of PS19 mice. These findings suggest that the intake of Trp-Tyr-related peptides improves tauopathy symptoms, resulting in improvements in behavioral deficits and longevity. Hence, the intake of Trp-Tyr-related peptides, including ß-lactolin, may be beneficial for preventing dementia.

Different Aß43 deposition patterns in the brains of aged dogs, sea lions, and cats.

Cerebral amyloid ß (Aß) deposition is a pathological hallmark of Alzheimer's disease (AD). There are several molecular species of Aß, including Aß40, Aß42, and Aß43, and the pathological roles of Aß43 have attracted particular attention in recent years. Aß43 is mainly deposited as senile plaques (SPs) in AD brains, and is known to be more amyloidogenic and neurotoxic than Aß42 and Aß40. Aß40 and Aß42 deposition have been demonstrated in several animal species, while Aß43 deposition has not been studied in animals. The brains of sea lions, dogs, and cats exhibit unique age-related Aß pathologies. In the present study, the deposition patterns of Aß40, Aß42, and Aß43 were examined immunohistochemically in the brains of aged dogs (n=52), sea lions (n=5), and cats (n=17). In dogs, most cerebral amyloid angiopathy (CAA) lesions and primitive SPs were positive for Aß42, Aß43, and Aß40. However, diffuse SPs and capillary CAA lesions were negative for Aß40. In sea lions, all SPs and most CAA lesions were positive for Aß42, Aß43, and Aß40, while capillary CAA lesions were negative for Aß40. In cats, Aß42-immunopositive granular aggregates and arteriole and capillary CAA lesions were positive for Aß43, but negative for Aß40. Double-labelling immunohistochemistry revealed the co-localization of Aß42 and Aß43. These findings suggest that Aß43 and Aß42 are frequently deposited in the brains of Carnivora animals and may play an important role in Aß pathology.

Deposition of Phosphorylated α-Synuclein and Activation of GSK-3ß and PP2A in the PS19 Mouse Model of Tauopathy.

The simultaneous accumulation of multiple pathological proteins, such as hyperphosphorylated tau (hp-tau) and phosphorylated α-synuclein (p-αSyn), has been reported in the brains of patients with various neurodegenerative diseases. We previously demonstrated that hp-tau-dependent p-αSyn accumulation was associated with the activation of GSK-3ß in the brains of P301L tau transgenic mice. To confirm the effects of another mutant tau on p-αSyn accumulation in vivo, we herein examined the brains of PS19 mice that overexpress human P301S mutant tau. Immunohistochemically, hp-tau and p-αSyn aggregates were detected in the same neuronal cells in the cerebrum and brain stem of aged PS19 mice. A semiquantitative analysis showed a positive correlation between hp-tau and p-αSyn accumulation. Furthermore, an activated form of GSK-3ß was detected within cells containing both hp-tau and p-αSyn aggregates in PS19 mice. Western blotting showed a decrease in inactivated PP2A levels in PS19 mice. The present results suggest that the overexpression of human P301S mutant tau induces p-αSyn accumulation that is accompanied by not only GSK-3ß, but also PP2A activation in PS19 mice, and highlight the synergic effects between tau and αSyn in the pathophysiology of neurodegenerative diseases that show the codeposition of tau and αSyn.

Feline Spongy Encephalopathy With a Mutation in the ASPA Gene.

Canavan disease is an autosomal recessive leukodystrophy caused by mutations in the gene encoding aspartoacylase (ASPA), which hydrolyses N-acetylaspartate (NAA) to acetate and aspartate. A similar feline neurodegenerative disease associated with a mutation in the ASPA gene is reported herein. Comprehensive clinical, genetic, and pathological analyses were performed on 4 affected cats. Gait disturbance and head tremors initially appeared at 1 to 19 months of age. These cats eventually exhibited dysstasia and seizures and died at 7 to 53 months of age. Magnetic resonance imaging of the brain revealed diffuse symmetrical intensity change of the cerebral cortex, brainstem, and cerebellum. Gas chromatography-mass spectrometry analysis of urine showed significant excretion of NAA. Genetic analysis of the 4 affected cats identified a missense mutation (c.859G>C) in exon 6 of the ASPA gene, which was not detected in 4 neurologically intact cats examined as controls. Postmortem analysis revealed vacuolar changes predominantly distributed in the gray matter of the cerebrum and brain stem as well as in the cerebellar Purkinje cell layer. Immunohistochemically, these vacuoles were surrounded by neurofilaments and sometimes contained MBP- and Olig2-positive cells. Ultrastructurally, a large number of intracytoplasmic vacuoles containing mitochondria and electron-dense granules were detected in the cerebral cortex. All 4 cats were diagnosed as spongy encephalopathy with a mutation in the ASPA gene, a syndrome analogous to human Canavan disease. The histopathological findings suggest that feline ASPA deficiency induces intracytoplasmic edema in neurons and oligodendrocytes, resulting in spongy degeneration of the central nervous system.

Amyloid ß and tau pathology in brains of aged pinniped species (sea lion, seal, and walrus).

Alzheimer's disease (AD) is characterized by the accumulation of amyloid-ß (Aß) as senile plaques and cerebral amyloid angiopathy, and hyperphosphorylated tau (hp-tau) as neurofibrillary tangles in the brain. The AD-related pathology has been reported in several non-human animals, and most animals develop only the Aß or tau pathology. We herein describe the Aß and hp-tau pathology in the brains of aged pinniped species (seal, sea lion, and walrus). Molecular analyses revealed that the sequence of pinniped Aß was identical to that of human Aß. Histopathological examinations detected argyrophilic plaques composed of Aß associated with dystrophic neurites in the cerebral cortex of aged pinnipeds. Astrogliosis and microglial infiltration were identified around Aß plaques. Aß deposits were observed in the blood vessel walls of the meninges and cerebrum. Pinniped tau protein was physiologically subjected to alternative splicing at exons 2, 3, and 10, and presented as five isoforms: two 3-repeat tau isoforms (1N3R, 2N3R) and three 4-repeat tau isoforms (0N4R, 1N4R, 2N4R); 0N3R tau isoform was absent. Histopathological examinations revealed argyrophilic fibrillar aggregates composed of hp-tau in the neuronal somata and neurites of aged pinniped brains. Few hp-tau aggregates were found in oligodendrocytes and microglia. Biochemically, hp-tau of the 3-repeat and 4-repeat isoforms was detected in brain sarkosyl-insoluble fractions. Aß and hp-tau both predominantly accumulated in the neocortex, particularly the frontal cortex. Furthermore, the activation of GSK-3ß was detected within cells containing hp-tau aggregates, and activated GSK-3ß was strongly expressed in cases with severe hp-tau pathologies. The present results suggest that, in association with Aß deposition, the activation of GSK-3ß contributes to hp-tau accumulation in pinniped brains. Here, we report that pinniped species naturally accumulate Aß and tau with aging, similar to the human AD pathology.

Memory formation in old age requires GSK-3ß.

Glycogen synthase kinase 3ß (GSK-3ß) is a therapeutic target for various age-related neurodegenerative diseases. It is linked to the two main pathological features of Alzheimer's disease (AD), tau and amyloid ß (Aß); GSK-3ß is a major candidate to pathologically hyperphosphorylate tau and modulate Aß production. However, inhibition of GSK-3ß in clinical studies in humans has been found to not significantly improve cognitive function of AD patients, prompting us to study the physiological role of GSK-3ß in old mice. Using a contextual fear-conditioning paradigm, we now report that old gsk-3ß+/- mice are deficient in both short-term and long-term memory formation, suggesting that GSK-3ß is required for memory formation at old age. Biochemical and immunohistochemical analyses showed that the number of synapses does not differ between gsk-3ß+/- and age-matched wild-type (wt) littermate mice. Based on these observations, we propose that, GSK-3ß may contribute to help maintain brain function during aging. Our results may explain the poor efficacy of GSK-3ß inhibitors in preserving memory capacity in AD patients.

Phosphorylation and oligomerization of α-synuclein associated with GSK-3ß activation in the rTg4510 mouse model of tauopathy.

Neurodegenerative diseases are characterized by the accumulation of specific phosphorylated protein aggregates in the brain, such as hyperphosphorylated tau (hp-tau) in tauopathies and phosphorylated α-synuclein (p-αSyn) in α-synucleinopathies. The simultaneous accumulation of different proteins is a common event in many neurodegenerative diseases. We herein describe the detection of the phosphorylation and dimerization of αSyn and activation of GSK-3ß, a major kinase known to phosphorylate tau and αSyn, in the brains of rTg4510 mice that overexpress human P301L mutant tau. Immunohistochemistry showed p-αSyn aggregates in rTg4510 mice, which were suppressed by doxycycline-mediated decreases in mutant tau expression levels. A semi-quantitative analysis revealed a regional correlation between hp-tau and p-αSyn accumulation in rTg4510 mice. Furthermore, proteinase K-resistant αSyn aggregates were found in the region with excessive hp-tau accumulation in rTg4510 mice, and these aggregates were morphologically different from proteinase K-susceptible p-αSyn aggregates. Western blotting revealed decreases in p-αSyn monomers in TBS- and sarkosyl-soluble fractions and increases in ubiquitinated p-αSyn dimers in sarkosyl-soluble and insoluble fractions in rTg4510 mice. Furthermore, an activated form of GSK-3ß was immunohistochemically detected within cells containing both hp-tau and p-αSyn aggregates. A semi-quantitative analysis revealed that increased GSK-3ß activity strongly correlated with hp-tau and p-αSyn accumulation in rTg4510 mice. Collectively, the present results suggest that the overexpression of human P301L mutant tau promoted the phosphorylation and dimerization of endogenous αSyn by activating GSK-3ß in rTg4510 mice. This synergic effect between tau, αSyn, and GSK-3ß may be involved in the pathophysiology of several neurodegenerative diseases that show the accumulation of both tau and αSyn.

Feline Niemann-Pick Disease With a Novel Mutation of SMPD1 Gene.

A 4-month-old female mixed-breed cat showed gait disturbance and eventual dysstasia with intention tremor and died at 14 months of age. Postmortem histological analysis revealed degeneration of neuronal cells, alveolar epithelial cells, hepatocytes, and renal tubular epithelial cells. Infiltration of macrophages was observed in the nervous system and visceral organs. The cytoplasm of neuronal cells was filled with Luxol fast blue (LFB)-negative and periodic acid-Schiff (PAS)-negative granules, and the cytoplasm of macrophages was LFB-positive and PAS-negative. Ultrastructurally, concentric deposits were observed in the brain and visceral organs. Genetic and biochemical analysis revealed a nonsense mutation (c.1017G>A) in the SMPD1 gene, a decrease of SMPD1 mRNA expression, and reduced acid sphingomyelinase immunoreactivity. Therefore, this cat was diagnosed as having Niemann-Pick disease with a mutation in the SMPD1 gene, a syndrome analogous to human Niemann-Pick disease type A.

ß-Lactolin, a Whey-Derived Lacto-Tetrapeptide, Prevents Alzheimer's Disease Pathologies and Cognitive Decline.

The prevention of age-related memory decline and dementia has been becoming a high priority because of the rapid growth in aging populations. Accumulating epidemiological and clinical studies indicate that intake of fermented dairy products rich in ß-lactolin improves memory retrieval and executive function and attenuates cognitive decline in the elderly. However, the effects of long-term consumption of ß-lactolin on Alzheimer's disease (AD) pathologies have not been investigated. In the present study, we examined the effects of ß-lactolin and whey digestion rich in ß-lactolin on AD pathology in 5×FAD transgenic mice and PS19 tauopathy mice. Intake of ß-lactolin and whey digestion rich in ß-lactolin reduced the levels of inflammatory cytokines, suppressed the infiltration of activated microglia, decreased the levels of amyloid-ß, ameliorated impaired long-term object memory, and attenuated decreased synaptophysin, dopamine, brain-derived neurotrophic factor, and insulin-like growth factor 1 levels in the cortex in 5×FAD transgenic mice. In addition, intake of ß-lactolin and whey digestion rich in ß-lactolin improved behavioral abnormality and reduced the ratio of phosphorylated tau to total tau in the cortex in PS19 tauopathy mice. These findings indicate that consumption with ß-lactolin and whey digestion rich in ß-lactolin suppresses inflammation and attenuates AD pathology and cognitive impairment.

Iso-α-Acids, Bitter Components in Beer, Suppress Inflammatory Responses and Attenuate Neural Hyperactivation in the Hippocampus.

Due to the growth in aging populations worldwide, prevention and therapy for age-related cognitive decline and dementia are in great demand. We previously demonstrated that long-term intake of iso-α-acids, which are hop-derived bitter compounds found in beer, prevent Alzheimer's pathology in a rodent model. On the other hand, the effects of iso-α-acids on neural activity in Alzheimer's disease model mice have not been investigated. Here, we demonstrated that short-term intake of iso-α-acids suppresses inflammation in the hippocampus and improves memory impairment even after disease onset. Importantly, we demonstrated that short-term administration of iso-α-acids attenuated the neural hyperactivation in hippocampus. In 6-month-old 5 × FAD mice exhibiting hippocampus inflammation and memory impairment, oral administration of iso-α-acids for 7 days reduced inflammatory cytokines, including MIP-1α and soluble Aß and improved object memory in the novel object recognition test. In 12-month-old J20 mice, intake of iso-α-acids for 7 days also suppressed inflammatory cytokines and soluble Aß in the brain. Manganese-enhanced magnetic resonance imaging (MEMRI) of hippocampi of J20 mice showed increased manganese compared with wild type mice, but iso-α-acids canceled this increased MEMRI signal in J20 mice, particularly in the hippocampus CA1 and CA3 region. Taken together, these findings suggest that short-term intake of iso-α-acids can suppress hippocampus inflammation even after disease onset and improve hyper neural activity in Alzheimer's disease model mice.

Iso-α-Acids, the Bitter Components of Beer, Suppress Microglial Inflammation in rTg4510 Tauopathy.

Due to the growth in aging populations, prevention for cognitive decline and dementia are in great demand. We previously demonstrated that the consumption of iso-α-acids (IAA), the hop-derived bitter compounds in beer, prevents inflammation and Alzheimer's disease pathology in model mice. However, the effects of iso-α-acids on inflammation induced by other agents aside from amyloid ß have not been investigated. In this study, we demonstrated that the consumption of iso-α-acids suppressed microglial inflammation in the frontal cortex of rTg4510 tauopathy mice. In addition, the levels of inflammatory cytokines and chemokines, including IL-1ß and MIP-1ß, in the frontal cortex of rTg4510 mice were greater than those of wild-type mice, and were reduced in rTg4510 mice fed with iso-α-acids. Flow cytometry analysis demonstrated that the expression of cells producing CD86, CD68, TSPO, MIP-1α, TNF-α, and IL-1ß in microglia was increased in rTg4510 mice compared with wild-type mice. Furthermore, the expression of CD86- and MIP-1α-producing cells was reduced in rTg4510 mice administered with iso-α-acids. Moreover, the consumption of iso-α-acids reduced the levels of phosphorylated tau in the frontal cortex. Collectively, these results suggest that the consumption of iso-α-acids prevents the inflammation induced in tauopathy mice. Thus, iso-α-acids may help in preventing inflammation-related brain disorders.

Novel lactopeptides in fermented dairy products improve memory function and cognitive decline.

Alongside the rapid growth in aging populations, prevention of age-related memory decline and dementia has become a high priority. Several epidemiological and clinical studies have concluded that fermented dairy products can help to prevent cognitive decline; furthermore, intake of Camembert cheese prevents Alzheimer's pathology in model mice. To elucidate molecular mechanisms underlying the preventive effects of fermented dairy products, here we screened peptides from digested fermented dairy products for ability to improve memory function in a scopolamine-induced amnesia mouse model. We found that Trp-Tyr (WY)-containing peptides from whey protein improved memory function in the mice, and the effects were confirmed in aged mice. The WY-containing peptides directly inhibited monoamine oxidase-B activity and increased dopamine levels in brain tissue. Pretreatment with dopamine receptor antagonist abolished the improvement in memory function due to WY-containing peptides. These results suggest that WY-containing peptides in fermented dairy products increase monoamine levels by inhibiting monoamine oxidase-B activity, helping to prevent age-related cognitive decline.

Deposition of Phosphorylated α-Synuclein in the rTg4510 Mouse Model of Tauopathy.

The accumulation of specific phosphorylated protein aggregates in the brain is a hallmark of severe neurodegenerative disorders. Specifically, hyperphosphorylated tau (hp-tau) accumulates in Alzheimer disease, frontotemporal dementia with Parkinsonism linked to chromosome 17, and progressive supranuclear palsy; furthermore, phosphorylated α-synuclein (p-αSyn) accumulates in Parkinson disease, dementia with Lewy bodies, and multiple system atrophy. Moreover, codeposition of different pathological protein aggregates is common in the brains of individuals with neurodegenerative diseases. In the present report, we describe the detection of p-αSyn aggregates in the brain of rTg4510 mice that overexpress human P301L mutant tau. Immunohistochemistry showed that hp-tau and p-αSyn aggregates were found within the same neuronal cells in rTg4510 mice and increased with age. Moreover, semiquantitative analysis revealed a significant regional correlation between hp-tau and p-αSyn accumulation. These results indicate that endogenous mouse αSyn protein is phosphorylated and accumulates with hp-tau aggregation in neurons and suggest that the overexpression of human P301L mutant tau may enhance endogenous αSyn phosphorylation and aggregation via a similar hyperphosphorylation mechanism in vivo. This synergic effect between tau and αSyn accumulation may exacerbate the pathology of several neurodegenerative disorders that show a cooccurrence of hp-tau and p-αSyn aggregation.