Modeling of age-related neurological disease: utility of zebrafish.

Many age-related neurological diseases still lack effective treatments, making their understanding a critical and urgent issue in the globally aging society. To overcome this challenge, an animal model that accurately mimics these diseases is essential. To date, many mouse models have been developed to induce age-related neurological diseases through genetic manipulation or drug administration. These models help in understanding disease mechanisms and finding potential therapeutic targets. However, some age-related neurological diseases cannot be fully replicated in human pathology due to the different aspects between humans and mice. Although zebrafish has recently come into focus as a promising model for studying aging, there are few genetic zebrafish models of the age-related neurological disease. This review compares the aging phenotypes of humans, mice, and zebrafish, and provides an overview of age-related neurological diseases that can be mimicked in mouse models and those that cannot. We presented the possibility that reproducing human cerebral small vessel diseases during aging might be difficult in mice, and zebrafish has potential to be another animal model of such diseases due to their similarity of aging phenotype to humans.

Biology of cognitive aging across species.

Aging is associated with cognitive decline, which can critically affect quality of life. Examining the biology of cognitive aging across species will lead to a better understanding of the fundamental mechanisms involved in this process, and identify potential interventions that could help to improve cognitive function in aging individuals. This minireview aimed to explore the mechanisms and processes involved in cognitive aging across a range of species, from flies to rodents, and covers topics, such as the role of reactive oxygen species and autophagy/mitophagy in cognitive aging. Overall, this literature provides a comprehensive overview of the biology of cognitive aging across species, highlighting the latest research findings and identifying potential avenues for future research. Geriatr Gerontol Int 2024; 24: 15-24.

Small-molecule screening in aged Drosophila identifies mGluR as a regulator of age-related sleep impairment.

As a normal physiological phenomenon, aging has a significant impact on sleep. Aging leads to sleep impairment, including sleep loss, fragmented sleep, and a lower arousal threshold, leading to various diseases. Because sleep regulates memory consolidation, age-dependent sleep impairment also affects memory. However, the mechanisms underlying age-related sleep dysregulation and its impact on memory remain unclear. Using male and female Drosophila as a model, which possesses sleep characteristics similar to those of mammals and exhibits age-dependent sleep impairment, we performed small-molecule screening to identify novel regulators of age-dependent decline in sleep. The screening identified 3,3'-difluorobenzaldazine (DFB), a positive allosteric modulator of the metabotropic glutamate receptor (mGluR) 5, as a novel sleep-promoting compound in aged flies. We found that mutant flies of mGluR, a single mGluR gene in Drosophila, and decreased mGluR expression had significant impairment in sleep and memory due to olfactory conditioning. The decreased sleep phenotype in the mGluR mutants was not promoted by DFB, suggesting that the effects of DFB on age-dependent sleep impairment are dependent on mGluR. Although aging decreases the expression of mGluR and the binding scaffold proteins Homer and Shank, the transient overexpression of mGluR in neurons improves sleep in both young and aged flies. Overall, these findings indicate that age-dependent decreased expression or function of mGluR impairs sleep and memory in flies, which could lead to age-related sleep and memory impairment.

Neuropeptide diuretic hormone 31 mediates memory and sleep via distinct neural pathways in Drosophila.

Memory formation and sleep regulation are critical for brain functions in animals from invertebrates to humans. Neuropeptides play a pivotal role in regulating physiological behaviors, including memory formation and sleep. However, the detailed mechanisms by which neuropeptides regulate these physiological behaviors remains unclear. Herein, we report that neuropeptide diuretic hormone 31 (DH31) positively regulates memory formation and sleep in Drosophila melanogaster. The expression of DH31 in the dorsal and ventral fan-shaped body (dFB and vFB) neurons of the central complex and ventral lateral clock neurons (LNvs) in the brain was responsive to sleep regulation. In addition, the expression of membrane-tethered DH31 in dFB neurons rescued sleep defects in Dh31 mutants, suggesting that DH31 secreted from dFB, vFB, and LNvs acts on the DH31 receptor in the dFB to regulate sleep partly in an autoregulatory feedback loop. Moreover, the expression of DH31 in octopaminergic neurons, but not in the dFB neurons, is involved in forming intermediate-term memory. Our results suggest that DH31 regulates memory formation and sleep through distinct neural pathways.

Nitric oxide-soluble guanylyl cyclase pathway as a contributor to age-related memory impairment in Drosophila.

Age-related changes in the transcriptome lead to memory impairment. Several genes have been identified to cause age-dependent memory impairment (AMI) by changes in their expression, but genetic screens to identify genes critical for AMI have not been performed. The fruit fly is a useful model for studying AMI due to its short lifespan and the availability of consistent techniques and environments to assess its memory ability. We generated a list of candidate genes that act as AMI regulators by performing a comprehensive analysis of RNAsequencing data from young and aged fly heads and genome-wide RNAi screening data to identify memory-regulating genes. A candidate screen using temporal and panneuronal RNAi expression was performed to identify genes critical for AMI. We identified the guanylyl cyclase ß-subunit at 100B (gycß) gene, which encodes a subunit of soluble guanylyl cyclase (sGC), the only intracellular nitric oxide (NO) receptor in fruit flies, as a negative regulator of AMI. RNAi knockdown of gycß in neurons and NO synthase (NOS) in glia or neurons enhanced the performance of intermediate-term memory (ITM) without apparent effects on memory acquisition. We also showed that pharmacological inhibition of sGC and NOS enhanced ITM in aged individuals, suggesting the possibility that age-related enhancement of the NO-sGC pathway causes memory impairment.

The guppy (Poecilia reticulata) is a useful model for analyzing age-dependent changes in metabolism, motor function, and gene expression.

Aging is a major risk factor for many chronic diseases, causing a general decline in physiological function and loss of homeostasis. Recently, small teleost fish have been used as animal models of aging research because their genetic structures and organs closely resemble those of humans. Guppy (Poecilia reticulata), a small teleost fish, has a shorter lifespan than zebrafish. However, the age-dependent changes in physiology and genetics in guppies are not well understood. Here, we investigated the age-associated changes in metabolic rate, physical activity, and gene expression in guppies. Our results indicated that the resting metabolic rate and spontaneous motor activity in guppies decreased from an earlier age than those in mice. Moreover, the mRNA expression level of ppargc1a and the accumulation of lipofuscin were affected by age in the guppy livers; however, these changes were species-specific. On the other hand, in aged guppy brains, the mRNA expression changes of some genes were partly consistent with aged mammals. Although the process of senescence of the liver in guppies might vary from mammals, our findings suggest that guppy could be a useful animal model for age-related changes in physiological functions.

Appetitive Memory with Survival Benefit Is Robust Across Aging in Drosophila.

The formation of memory declines with advancing age. However, susceptibility to memory impairments depends on several factors, including the robustness of memory, the responsible neural circuits, and the internal state of aged individuals. How age-dependent changes in internal states and neural circuits affect memory formation remains unclear. Here, we show in Drosophila melanogaster that aged flies of both sexes form robust appetitive memory conditioned with nutritious sugar, which suppresses their high mortality rates during starvation. In contrast, aging impairs the formation of appetitive memory conditioned with non-nutritious sugar that lacks survival benefits for the flies. We found that aging enhanced the preference for nutritious sugar over non-nutritious sugar correlated with an age-dependent increase in the expression of Drosophila neuropeptide F, an ortholog of mammalian neuropeptide Y. Furthermore, a subset of dopaminergic neurons that signal the sweet taste of sugar decreases its function with aging, while a subset of dopaminergic neurons that signal the nutritional value of sugar maintains its function with age. Our results suggest that aging impairs the ability to form memories without survival benefits; however, the ability to form memories with survival benefits is maintained through age-dependent changes in the neural circuits and neuropeptides.SIGNIFICANCE STATEMENT The susceptibility to age-dependent memory impairments depends on the strength of the memory, changes in the responsible neurons, and internal states of aged individuals. How age-dependent changes in such internal states affect neural activity and memory formation remains unclear. We show in Drosophila melanogaster that aged flies of both sexes form robust appetitive memory conditioned with nutritious sugar, which has survival benefits for aged flies. In contrast, aging impairs the formation of appetitive memory conditioned with non-nutritious sugar that lacks survival benefits for the flies. Aging changes the neural circuits including dopamine neurons and neuropeptide F-expressing neurons, leading to the age-dependent impairment in memory with insufficient survival benefits and the preservation of the ability to form memory with survival benefits.

[New Research Development and Connection with Fellow Scholars through Study Abroad].

After completing my doctoral training, I joined the Ronald Davis Laboratory at The Scripps Research Institute, Florida. At Scripps, I extended my research and worked to understand the mechanisms of age-related memory impairment. Three and a half years in Florida passed very quickly while I worked with a multi-national multi-cultural team of scientists. Since returning to Japan, I now lead my own research team. My research experience abroad helped me to expand my scope of research, and allowed me to combine the fields of aging and neurosciences to discover solutions for many newly emerging challenges to human health. Beyond the experience I gained from my research, working with researchers from different cultures and with different values broadened my horizons. One of my best memories while working in the USA was to enjoy going to the beach near my laboratory while waiting in between my experiments. Looking back, it was great to have so much time to myself to reflect on my challenging research topic, while also having a chance to make so many new friends. Having friends to share in and cherish each other's successes, or to discuss our research or a wide range of topics-even after returning to Japan-is indeed a great wealth I now possess as a result of having studied abroad.

Successive and discrete spaced conditioning in active avoidance learning in young and aged zebrafish.

We designed an automated device to study active avoidance learning abilities of zebrafish. Open source tools were used for the device control, statistical computing, and graphic outputs of data. Using the system, we developed active avoidance tests to examine the effects of trial spacing and aging on learning. Seven-month-old fish showed stronger avoidance behavior as measured by color preference index with discrete spaced training as compared to successive spaced training. Fifteen-month-old fish showed a similar trend, but with reduced cognitive abilities compared with 7-month-old fish. Further, in 7-month-old fish, an increase in learning ability during trials was observed with discrete, but not successive, spaced training. In contrast, 15-month-old fish did not show increase in learning ability during trials. Therefore, these data suggest that discrete spacing is more effective for learning than successive spacing, with the zebrafish active avoidance paradigm, and that the time course analysis of active avoidance using discrete spaced training is useful to detect age-related learning impairment.

Age-Related Changes in Insulin-like Signaling Lead to Intermediate-Term Memory Impairment in Drosophila.

Insulin and insulin-growth-factor-like signaling (IIS) plays important roles in the regulation of development, growth, metabolic homeostasis, and aging, as well as in brain functions such as learning and memory. The temporal-spatial role of IIS in learning and memory and its effect on age-dependent memory impairment remain unclear. Here, we report that intermediate-term memory (ITM), but not short-term memory (STM), in Drosophila aversive olfactory memory requires transient IIS during adulthood. The expression of Drosophila insulin-like peptide 3 (Dilp3) in insulin-producing cells and insulin receptor function in the fat body are essential for ITM. Although the expression of dilp3 decreases with aging, which is unique among dilp genes, the transient expression of dilp3 in aged flies enhances ITM. These findings indicate that ITM is systemically regulated by communication between insulin-producing cells and fat body and that age-dependent changes in IIS contribute to age-related memory impairment.

Mib1 modulates dynamin 2 recruitment via Snx18 to promote Dll1 endocytosis for efficient Notch signaling.

Notch signaling regulates normal development and tissue homeostasis. Ligand endocytosis plays critical roles in Notch signaling activation. Endocytic proteins such as epsin and dynamin participate in Notch ligand activity by mediating Notch ligand endocytosis. The ubiquitin ligase Mib1 also plays essential roles in Notch signaling via Notch ligand ubiquitination. However, the molecular links between Mib1 and endocytic proteins have not been fully defined. Here, we show that Mib1 is involved in dynamin 2 recruitment to Dll1 and that Snx18, which interacts with dynamin 2, modestly regulates Dll1 endocytosis. Furthermore, the ubiquitin ligase activity of Mib1 is induced by Notch ligand-receptor interactions. Mib1 promotes the interaction between dynamin 2 and Snx18 in an ubiquitin ligase activity-dependent manner. These results suggest that Mib1 modulates dynamin recruitment by regulating the interaction between Snx18 and dynamin 2, thereby helping to ensure the efficient signaling activity of Notch ligands.

Aging impairs protein-synthesis-dependent long-term memory in Drosophila.

Although aging is known to impair intermediate-term memory in Drosophila, its effect on protein-synthesis-dependent long-term memory (LTM) is unknown. We show here that LTM is impaired with age, not due to functional defects in synaptic output of mushroom body (MB) neurons, but due to connectivity defects of dorsal paired medial (DPM) neurons with their postsynaptic MB neurons. GFP reconstitution across synaptic partners (GRASP) experiments revealed structural connectivity defects in aged animals of DPM neurons with MB axons in the α lobe neuropil. As a consequence, a protein-synthesis-dependent LTM trace in the α/ß MB neurons fails to form. Aging thus impairs protein-synthesis-dependent LTM along with the α/ß MB neuron LTM trace by lessening the connectivity of DPM and α/ß MB neurons.

Caspase inhibition in select olfactory neurons restores innate attraction behavior in aged Drosophila.

Sensory and cognitive performance decline with age. Neural dysfunction caused by nerve death in senile dementia and neurodegenerative disease has been intensively studied; however, functional changes in neural circuits during the normal aging process are not well understood. Caspases are key regulators of cell death, a hallmark of age-related neurodegeneration. Using a genetic probe for caspase-3-like activity (DEVDase activity), we have mapped age-dependent neuronal changes in the adult brain throughout the lifespan of Drosophila. Spatio-temporally restricted caspase activation was observed in the antennal lobe and ellipsoid body, brain structures required for olfaction and visual place memory, respectively. We also found that caspase was activated in an age-dependent manner in specific subsets of Drosophila olfactory receptor neurons (ORNs), Or42b and Or92a neurons. These neurons are essential for mediating innate attraction to food-related odors. Furthermore, age-induced impairments of neural transmission and attraction behavior could be reversed by specific inhibition of caspase in these ORNs, indicating that caspase activation in Or42b and Or92a neurons is responsible for altering animal behavior during normal aging.

Homeostatic epithelial renewal in the gut is required for dampening a fatal systemic wound response in Drosophila.

Effective defense responses involve the entire organism. To maintain body homeostasis after tissue damage, a systemic wound response is induced in which the response of each tissue is tightly orchestrated to avoid incomplete recovery or an excessive, damaging response. Here, we provide evidence that in the systemic response to wounding, an apoptotic caspase pathway is activated downstream of reactive oxygen species in the midgut enterocytes (ECs), cells distant from the wound site, in Drosophila. We show that a caspase-pathway mutant has defects in homeostatic gut cell renewal and that inhibiting caspase activity in fly ECs results in the production of systemic lethal factors after wounding. Our results indicate that wounding remotely controls caspase activity in ECs, which activates the tissue stem cell regeneration pathway in the gut to dampen the dangerous systemic wound reaction.

Aging impairs intermediate-term behavioral memory by disrupting the dorsal paired medial neuron memory trace.

How the functional activity of the brain is altered during aging to cause age-related memory impairments is unknown. We used functional cellular imaging to monitor two different calcium-based memory traces that underlie olfactory classical conditioning in young and aged Drosophila. Functional imaging of neural activity in the processes of the dorsal paired medial (DPM) and mushroom body neurons revealed that the capacity to form an intermediate-term memory (ITM) trace in the DPM neurons after learning is lost with age, whereas the capacity to form a short-term memory trace in the α'/ß' mushroom body neurons remains unaffected by age. Stimulation of the DPM neurons by activation of a temperature-sensitive cation channel between acquisition and retrieval enhanced ITM in aged but not young flies. These data indicate that the functional state of the DPM neurons is selectively altered with age to cause an age-related impairment of ITM, and demonstrate that altering the excitability of DPM neurons can restore age-related memory impairments.

Aging causes distinct characteristics of polyglutamine amyloids in vivo.

Polyglutamine diseases, including Machado-Joseph disease and Huntington's disease, typically appear in midlife and are characterized by amyloid accumulations of abnormally expanded polyglutamine proteins. Although there is growing evidence that aging has an important role in the occurrence of such diseases, the role of aging in the late onset of these diseases is not well understood. Recent studies showed that differences in amyloid conformation from different brain regions lead to differing toxicity. We hypothesized that higher amyloid toxicity at later ages might cause the late onset of polyglutamine diseases. Using a method for temporal and regional gene expression targeting (TARGET) in Drosophila, we showed that transient polyglutamine expression caused more severe neurodegeneration in older flies than in younger flies. Moreover, the polyglutamine amyloids themselves showed distinct characteristics in relation to age; those from older flies were less resistant to SDS and more effective at seeding polymerization than those from younger flies, suggesting that the polyglutamine amyloids in aged individuals may have higher toxicity. These findings show that age-related changes in amyloid characteristics may be a trigger for late-onset polyglutamine diseases.

[Genetic approach for understanding of the late-onset mechanisms of polyglutamine disease].

The intracellular accumulation of unfolded or misfolded proteins is believed to contribute to aging and age-related neurodegenerative diseases. However, the links between age-dependent proteotoxicity and cellular protein degradation systems remain poorly understood. Here, we show that 26S proteasome activity and abundance attenuate with age, which is associated with the impaired assembly of the 26S proteasome with the 19S regulatory particle (RP) and the 20S proteasome. In a genetic gain-of-function screen using Drosophila, we characterized Rpn11, which encodes a subunit of the 19S RP, as a suppressor of expanded polyglutamine-induced progressive neurodegeneration. Rpn11 overexpression suppressed the age-related reduction of the 26S proteasome activity, resulting in the extension of flies' life spans with suppression of the age-dependent accumulation of ubiquitinated proteins. On the other hand, the loss of function of Rpn11 caused an early onset of reduced 26S proteasome activity and a premature age-dependent accumulation of ubiquitinated proteins. It also caused a shorter life span and an enhanced neurodegenerative phenotype. Our results suggest that maintaining the 26S proteasome with age could extend the life span and suppress the age-related progression of polyglutamine diseases.

Genetic evidence linking age-dependent attenuation of the 26S proteasome with the aging process.

The intracellular accumulation of unfolded or misfolded proteins is believed to contribute to aging and age-related neurodegenerative diseases. However, the links between age-dependent proteotoxicity and cellular protein degradation systems remain poorly understood. Here, we show that 26S proteasome activity and abundance attenuate with age, which is associated with the impaired assembly of the 26S proteasome with the 19S regulatory particle (RP) and the 20S proteasome. In a genetic gain-of-function screen, we characterized Rpn11, which encodes a subunit of the 19S RP, as a suppressor of expanded polyglutamine-induced progressive neurodegeneration. Rpn11 overexpression suppressed the age-related reduction of the 26S proteasome activity, resulting in the extension of flies' life spans with suppression of the age-dependent accumulation of ubiquitinated proteins. On the other hand, the loss of function of Rpn11 caused an early onset of reduced 26S proteasome activity and a premature age-dependent accumulation of ubiquitinated proteins. It also caused a shorter life span and an enhanced neurodegenerative phenotype. Our results suggest that maintaining the 26S proteasome with age could extend the life span and suppress the age-related progression of neurodegenerative diseases.

Local initiation of caspase activation in Drosophila salivary gland programmed cell death in vivo.

Programmed cell death, or apoptosis, is an essential event in animal development. Spatiotemporal analysis of caspase activation in vivo could provide new insights into programmed cell death occurring during development. Here, using the FRET-based caspase-3 indicator, SCAT3, we report the results of live-imaging analysis of caspase activation in developing Drosophila in vivo. In Drosophila, the salivary gland is sculpted by caspase-mediated programmed cell death initiated by the steroid hormone 20-hydroxyecdysone (ecdysone). Using a SCAT3 probe, we observed that caspase activation in the salivary glands begins in the anterior cells and is then propagated to the posterior cells in vivo. In vitro salivary gland culture experiments indicated that local exposure of ecdysone to the anterior salivary gland reproduces the caspase activation gradient as observed in vivo. In betaFTZ-F1 mutants, caspase activation was delayed and occurred in a random pattern in vivo. In contrast to the in vivo response, the salivary glands from betaFTZ-F1 mutants showed a normal in vitro response to ecdysone, suggesting that betaFTZ-F1 may be involved in ecdysteroid biosynthesis and secretion of ecdysone from the ring gland for local initiation of programmed cell death. These results imply a role of betaFTZ-F1 in coordinating the initiation of salivary gland apoptosis in development.

Drosophila IKK-related kinase regulates nonapoptotic function of caspases via degradation of IAPs.

Caspase activation has been extensively studied in the context of apoptosis. However, caspases also control other cellular functions, although the mechanisms regulating caspases in nonapoptotic contexts remain obscure. Drosophila IAP1 (DIAP1) is an endogenous caspase inhibitor that is crucial for regulating cell death during development. Here we describe Drosophila IKK-related kinase (DmIKKvarepsilon) as a regulator of caspase activation in a nonapoptotic context. We show that DmIKKvarepsilon promotes degradation of DIAP1 through direct phosphorylation. Knockdown of DmIKKvarepsilon in the proneural clusters of the wing imaginal disc, in which nonapoptotic caspase activity is required for proper sensory organ precursor (SOP) development, stabilizes endogenous DIAP1 and affects Drosophila SOP development. Our results demonstrate that DmIKKvarepsilon is a determinant of DIAP1 protein levels and that it establishes the threshold of activity required for the execution of nonapoptotic caspase functions.