Decreased plasma nicotinamide and altered NAD+ metabolism in glial cells surrounding Aß plaques in a mouse model of Alzheimer's disease.

Alzheimer's disease (AD) is a progressive neurodegenerative disease and a leading cause of senile dementia. Amyloid-ß (Aß) accumulation triggers chronic neuroinflammation, initiating AD pathogenesis. Recent clinical trials for anti-Aß immunotherapy underscore that blood-based biomarkers have significant advantages and applicability over conventional diagnostics and are an unmet clinical need. To further advance ongoing clinical trials and identify novel therapeutic targets for AD, developing additional plasma biomarkers closely associated with pathogenic mechanisms downstream of Aß accumulation is critically important. To identify plasma metabolites reflective of neuroinflammation caused by Aß pathology, we performed untargeted metabolomic analyses of the plasma by capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS) and analyzed the potential roles of the identified metabolic changes in the brain neuroinflammatory response using the female App knock-in (AppNLGF) mouse model of Aß amyloidosis. The CE-TOFMS analysis of plasma samples from female wild-type (WT) and AppNLGF mice revealed that plasma levels of nicotinamide, a nicotinamide adenine dinucleotide (NAD+) precursor, were decreased in AppNLGF mice, and altered metabolite profiles were enriched for nicotinate/nicotinamide metabolism. In AppNLGF mouse brains, NAD+ levels were unaltered, but mRNA levels of NAD+-synthesizing nicotinate phosphoribosyltransferase (Naprt) and NAD+-degrading Cd38 genes were increased. These enzymes were induced in reactive astrocytes and microglia surrounding Aß plaques in the cortex and hippocampus of female AppNLGF mouse brains, suggesting neuroinflammation increases NAD+ metabolism. This study suggests plasma nicotinamide could be indicative of the neuroinflammatory response and that nicotinate and nicotinamide metabolism are potential therapeutic targets for AD, by targeting both neuroinflammation and neuroprotection.

Axonal distribution of mitochondria maintains neuronal autophagy during aging via eIF2ß.

Neuronal aging and neurodegenerative diseases are accompanied by proteostasis collapse, while cellular factors that trigger it are not identified. Impaired mitochondrial transport in the axon is another feature of aging and neurodegenerative diseases. Using Drosophila, we found that genetic depletion of axonal mitochondria causes dysregulation of translation and protein degradation. Axons with mitochondrial depletion showed abnormal protein accumulation, and autophagic defects. Lowering neuronal ATP levels by blocking glycolysis did not reduce autophagy, suggesting that autophagic defects are associated with mitochondrial distribution. We found eIF2ß was upregulated by depletion of axonal mitochondria via proteome analysis. Phosphorylation of eIF2α, another subunit of eIF2, was lowered, and global translation was suppressed. Neuronal overexpression of eIF2ß phenocopied the autophagic defects and neuronal dysfunctions, and lowering eIF2ß expression rescued those perturbations caused by depletion of axonal mitochondria. These results indicate the mitochondria-eIF2ß axis maintains proteostasis in the axon, of which disruption may underly the onset and progression of age-related neurodegenerative diseases.

Dissecting the daily feeding pattern: Peripheral CLOCK/CYCLE generate the feeding/fasting episodes and neuronal molecular clocks synchronize them.

A 24-h rhythm of feeding behavior, or synchronized feeding/fasting episodes during the day, is crucial for survival. Internal clocks and light input regulate rhythmic behaviors, but how they generate feeding rhythms is not fully understood. Here we aimed to dissect the molecular pathways that generate daily feeding patterns. By measuring the semidiurnal amount of food ingested by single flies, we demonstrate that the generation of feeding rhythms under light:dark conditions requires quasimodo (qsm) but not molecular clocks. Under constant darkness, rhythmic feeding patterns consist of two components: CLOCK (CLK) in digestive/metabolic tissues generating feeding/fasting episodes, and the molecular clock in neurons synchronizing them to subjective daytime. Although CLK is a part of the molecular clock, the generation of feeding/fasting episodes by CLK in metabolic tissues was independent of molecular clock machinery. Our results revealed novel functions of qsm and CLK in feeding rhythms in Drosophila.

Alzheimer's Disease-Related Phospho-Tau181 Signals Are Localized to Demyelinated Axons of Parvalbumin-Positive GABAergic Interneurons in an App Knock-In Mouse Model of Amyloid-ß Pathology.

BACKGROUND: The tau protein phosphorylated at Thr181 (p-tau181) in cerebrospinal fluid and blood is a sensitive biomarker for Alzheimer's disease (AD). Increased p-tau181 levels correlate well with amyloid-ß (Aß) pathology and precede neurofibrillary tangle formation in the early stage of AD; however, the relationship between p-tau181 and Aß-mediated pathology is less well understood. We recently reported that p-tau181 represents axonal abnormalities in mice with Aß pathology (AppNLGF). However, from which neuronal subtype(s) these p-tau181-positive axons originate remains elusive. OBJECTIVE: The main purpose of this study is to differentiate neuronal subtype(s) and elucidate damage associated with p-tau181-positive axons by immunohistochemical analysis of AppNLGF mice brains. METHODS: Colocalization between p-tau181 and (1) unmyelinated axons positive for vesicular acetylcholine transporter or norepinephrine transporter and (2) myelinated axons positive for vesicular glutamate transporter, vesicular GABA transporter, or parvalbumin in the brains of 24-month-old AppNLGF and control mice without Aß pathology were analyzed. The density of these axons was also compared. RESULTS: Unmyelinated axons of cholinergic or noradrenergic neurons did not overlap with p-tau181. By contrast, p-tau181 signals colocalized with myelinated axons of parvalbumin-positive GABAergic interneurons but not of glutamatergic neurons. Interestingly, the density of unmyelinated axons was significantly decreased in AppNLGF mice, whereas that of glutamatergic, GABAergic, or p-tau181-positive axons was less affected. Instead, myelin sheaths surrounding p-tau181-positive axons were significantly reduced in AppNLGF mice. CONCLUSION: This study demonstrates that p-tau181 signals colocalize with axons of parvalbumin-positive GABAergic interneurons with disrupted myelin sheaths in the brains of a mouse model of Aß pathology.

Drosophila Toll-9 is induced by aging and neurodegeneration to modulate stress signaling and its deficiency exacerbates tau-mediated neurodegeneration.

Drosophila Toll-9 is most closely related to mammalian Toll-like receptors; however, physiological functions of Toll-9 remain elusive. We examined the roles of Toll-9 in fly brains in aging and neurodegeneration. Toll-9 mRNA levels were increased in aged fly heads accompanied by activation of nuclear factor-kappa B (NF-kB) and stress-activated protein kinase (SAPK) signaling, and many of these changes were modulated by Toll-9 in glial cells. The loss of Toll-9 did not affect lifespan or brain integrity, whereas it exacerbated hydrogen peroxide-induced lethality. Toll-9 expression was also induced by nerve injury but did not affect acute stress response or glial engulfment activity, suggesting Toll-9 may modulate subsequent neurodegeneration. In a fly tauopathy model, Toll-9 deficiency enhanced neurodegeneration and disease-related tau phosphorylation with reduced SAPK activity, and blocking SAPK enhanced tau phosphorylation and neurodegeneration. In sum, Toll-9 is induced upon aging and nerve injury and affects neurodegeneration by modulating stress kinase signaling.

Distinct brain pathologies associated with Alzheimer's disease biomarker-related phospho-tau 181 and phospho-tau 217 in App knock-in mouse models of amyloid-ß amyloidosis.

Phospho-tau 217, phospho-tau 231 and phospho-tau 181 in cerebrospinal fluid and plasma are promising biomarkers for the diagnosis of Alzheimer's disease. All these p-tau proteins are detected in neurofibrillary tangles in brains obtained post-mortem from Alzheimer's disease patients. However, increases in p-tau levels in cerebrospinal fluid and plasma during the preclinical stage of Alzheimer's disease correlate with amyloid-ß burden and precede neurofibrillary tangles in brains, suggesting that these p-tau proteins are indicative of amyloid-ß-mediated brain pathology. In addition, phospho-tau 217 has greater sensitivity than phospho-tau 181, though it is unclear whether each of these p-tau variants contributes to the same or a different type of neuropathology prior to neurofibrillary tangle formation. In this study, we evaluated the intracerebral localization of p-tau in App knock-in mice with amyloid-ß plaques without neurofibrillary tangle pathology (AppNLGF ), in App knock-in mice with increased amyloid-ß levels without amyloid-ß plaques (AppNL ) and in wild-type mice. Immunohistochemical analysis showed that phospho-tau 217 and phospho-tau 231 were detected only in AppNLGF mice as punctate structures around amyloid-ß plaques, overlapping with the tau pathology marker, AT8 epitope phospho-tau 202/205/208. Moreover, phospho-tau 217 and phospho-tau 202/205/208 colocalized with the postsynaptic marker PSD95 and with a major tau kinase active, GSK3ß. In contrast and similar to total tau, phospho-tau 181 signals were readily detectable as fibre structures in wild-type and AppNL mice and colocalized with an axonal marker neurofilament light chain. In AppNLGF mice, these phospho-tau 181-positive structures were disrupted around amyloid-ß plaques and only partially overlapped with phospho-tau 217. These results indicate that phospho-tau 217, phospho-tau 231 and a part of phospho-tau 181 signals are markers of postsynaptic pathology around amyloid-ß plaques, with phospho-tau 181 also being a marker of axonal abnormality caused by amyloid-ß burden in brains.

5-Aminolevulinic acid and sodium ferrous citrate ameliorate muscle aging and extend healthspan in Drosophila.

Declines in mitochondrial functions are associated with aging. The combination of 5-aminolevulinic acid (5-ALA) and sodium ferrous citrate (SFC) improves mitochondrial functions in cultured cells. In this study, we investigated the effects of dietary supplementation with 5-ALA and SFC (5-ALA/SFC) on the healthspan and life span of Drosophila melanogaster. Adult Drosophila fruit flies were fed cornmeal food containing various concentrations of 5-ALA/SFC. Locomotor functions, life span, muscle architecture, and age-associated changes in mitochondrial function were analyzed. We found that feeding 5-ALA/SFC mitigated age-associated declines in locomotor functions and extended organismal life span. Moreover, 5-ALA/SFC preserved muscle architecture and maintained the mitochondrial membrane potential in aged animals. Since 5-ALA phosphate/SFC is used as a human dietary supplement, our results suggest that it could be used to slow the age-related declines in muscle functions, prevent age-associated clinical conditions such as frailty, and extend healthspan and life span.

Widespread Reduced Density of Noradrenergic Locus Coeruleus Axons in the App Knock-In Mouse Model of Amyloid-ß Amyloidosis.

BACKGROUND: The locus coeruleus (LC), a brainstem nucleus comprising noradrenergic neurons, is one of the earliest regions affected by Alzheimer's disease (AD). Amyloid-ß (Aß) pathology in the cortex in AD is thought to exacerbate the age-related loss of LC neurons, which may lead to cortical tau pathology. However, mechanisms underlying LC neurodegeneration remain elusive. OBJECTIVE: Here, we aimed to examine how noradrenergic neurons are affected by cortical Aß pathology in AppNL-G-F/NL-G-F knock-in mice. METHODS: The density of noradrenergic axons in LC-innervated regions and the LC neuron number were analyzed by an immunohistochemical method. To explore the potential mechanisms for LC degeneration, we also examined the occurrence of tau pathology in LC neurons, the association of reactive gliosis with LC neurons, and impaired trophic support in the brains of AppNL-G-F/NL-G-F mice. RESULTS: We observed a significant reduction in the density of noradrenergic axons from the LC in aged AppNL-G-F/NL-G-F mice without neuron loss or tau pathology, which was not limited to areas near Aß plaques. However, none of the factors known to be related to the maintenance of LC neurons (i.e., somatostatin/somatostatin receptor 2, brain-derived neurotrophic factor, nerve growth factor, and neurotrophin-3) were significantly reduced in AppNL-G-F/NL-G-F mice. CONCLUSION: This study demonstrates that cortical Aß pathology induces noradrenergic neurodegeneration, and further elucidation of the underlying mechanisms will reveal effective therapeutics to halt AD progression.

Phenotypic analysis of a transgenic Drosophila model of Alzheimer's amyloid-ß toxicity.

For decades, the fruit fly Drosophila melanogaster has been an efficient genetic model to investigate many aspects of human neurodegenerative diseases. Through genetic and pharmacologic approaches, these studies have revealed the molecular mechanisms underlying disease pathogenesis and provided therapeutic implications. Here, we describe a protocol for assessing Alzheimer's disease-related amyloid-ß toxicity in a transgenic fly model through biochemical, histological, and behavioral analyses. We also discuss the advantages and limitations of our protocols. For complete details on the use and execution of this protocol, please refer to Wang et al. (2021).

Increasing neuronal glucose uptake attenuates brain aging and promotes life span under dietary restriction in Drosophila.

Brain neurons play a central role in organismal aging, but there is conflicting evidence about the role of neuronal glucose availability because glucose uptake and metabolism are associated with both aging and extended life span. Here, we analyzed metabolic changes in the brain neurons of Drosophila during aging. Using a genetically encoded fluorescent adenosine triphosphate (ATP) biosensor, we found decreased ATP concentration in the neuronal somata of aged flies, correlated with decreased glucose content, expression of glucose transporter and glycolytic enzymes and mitochondrial quality. The age-associated reduction in ATP concentration did not occur in brain neurons with suppressed glycolysis or enhanced glucose uptake, suggesting these pathways contribute to ATP reductions. Despite age-associated mitochondrial damage, increasing glucose uptake maintained ATP levels, suppressed locomotor deficits, and extended the life span. Increasing neuronal glucose uptake during dietary restriction resulted in the longest life spans, suggesting an additive effect of enhancing glucose availability during a bioenergetic challenge on aging.

Transformative Network Modeling of Multi-omics Data Reveals Detailed Circuits, Key Regulators, and Potential Therapeutics for Alzheimer's Disease.

To identify the molecular mechanisms and novel therapeutic targets of late-onset Alzheimer's Disease (LOAD), we performed an integrative network analysis of multi-omics profiling of four cortical areas across 364 donors with varying cognitive and neuropathological phenotypes. Our analyses revealed thousands of molecular changes and uncovered neuronal gene subnetworks as the most dysregulated in LOAD. ATP6V1A was identified as a key regulator of a top-ranked neuronal subnetwork, and its role in disease-related processes was evaluated through CRISPR-based manipulation in human induced pluripotent stem cell-derived neurons and RNAi-based knockdown in Drosophila models. Neuronal impairment and neurodegeneration caused by ATP6V1A deficit were improved by a repositioned compound, NCH-51. This study provides not only a global landscape but also detailed signaling circuits of complex molecular interactions in key brain regions affected by LOAD, and the resulting network models will serve as a blueprint for developing next-generation therapeutic agents against LOAD.

Microtubule affinity-regulating kinase 4 with an Alzheimer's disease-related mutation promotes tau accumulation and exacerbates neurodegeneration.

Accumulation of the microtubule-associated protein tau is associated with Alzheimer's disease (AD). In AD brain, tau is abnormally phosphorylated at many sites, and phosphorylation at Ser-262 and Ser-356 plays critical roles in tau accumulation and toxicity. Microtubule affinity-regulating kinase 4 (MARK4) phosphorylates tau at those sites, and a double de novo mutation in the linker region of MARK4, ΔG316E317D, is associated with an elevated risk of AD. However, it remains unclear how this mutation affects phosphorylation, aggregation, and accumulation of tau and tau-induced neurodegeneration. Here, we report that MARK4ΔG316E317D increases the abundance of highly phosphorylated, insoluble tau species and exacerbates neurodegeneration via Ser-262/356-dependent and -independent mechanisms. Using transgenic Drosophila expressing human MARK4 (MARK4wt) or a mutant version of MARK4 (MARK4ΔG316E317D), we found that coexpression of MARK4wt and MARK4ΔG316E317D increased total tau levels and enhanced tau-induced neurodegeneration and that MARK4ΔG316E317D had more potent effects than MARK4wt Interestingly, the in vitro kinase activities of MARK4wt and MARK4ΔG316E317D were similar. When tau phosphorylation at Ser-262 and Ser-356 was blocked by alanine substitutions, MARK4wt did not promote tau accumulation or exacerbate neurodegeneration, whereas coexpression of MARK4ΔG316E317D did. Both MARK4wt and MARK4ΔG316E317D increased the levels of oligomeric forms of tau; however, only MARK4ΔG316E317D further increased the detergent insolubility of tau in vivo Together, these findings suggest that MARK4ΔG316E317D increases tau levels and exacerbates tau toxicity via a novel gain-of-function mechanism and that modification in this region of MARK4 may affect disease pathogenesis.

Disruption of a RAC1-centred network is associated with Alzheimer's disease pathology and causes age-dependent neurodegeneration.

The molecular biological mechanisms of Alzheimer's disease (AD) involve disease-associated crosstalk through many genes and include a loss of normal as well as a gain of abnormal interactions among genes. A protein domain network (PDN) is a collection of physical bindings that occur between protein domains, and the states of the PDNs in patients with AD are likely to be perturbed compared to those in normal healthy individuals. To identify PDN changes that cause neurodegeneration, we analysed the PDNs that occur among genes co-expressed in each of three brain regions at each stage of AD. Our analysis revealed that the PDNs collapsed with the progression of AD stage and identified five hub genes, including Rac1, as key players in PDN collapse. Using publicly available as well as our own gene expression data, we confirmed that the mRNA expression level of the RAC1 gene was downregulated in the entorhinal cortex (EC) of AD brains. To test the causality of these changes in neurodegeneration, we utilized Drosophila as a genetic model and found that modest knockdown of Rac1 in neurons was sufficient to cause age-dependent behavioural deficits and neurodegeneration. Finally, we identified a microRNA, hsa-miR-101-3p, as a potential regulator of RAC1 in AD brains. As the Braak neurofibrillary tangle (NFT) stage progressed, the expression levels of hsa-miR-101-3p were increased specifically in the EC. Furthermore, overexpression of hsa-miR-101-3p in the human neuronal cell line SH-SY5Y caused RAC1 downregulation. These results highlight the utility of our integrated network approach for identifying causal changes leading to neurodegeneration in AD.

Cdk5 increases MARK4 activity and augments pathological tau accumulation and toxicity through tau phosphorylation at Ser262.

Hyperphosphorylation of the microtubule-associated protein tau is associated with many neurodegenerative diseases, including Alzheimer's disease. Microtubule affinity-regulating kinases (MARK) 1-4 and cyclin-dependent kinase 5 (Cdk5) are tau kinases under physiological and pathological conditions. However, their functional relationship remains elusive. Here, we report a novel mechanism by which Cdk5 activates MARK4 and augments tau phosphorylation, accumulation and toxicity. MARK4 is highly phosphorylated at multiple sites in the brain and in cultured neurons, and inhibition of Cdk5 activity reduces phosphorylation levels of MARK4. MARK4 is known to be activated by phosphorylation at its activation loop by liver kinase B1 (LKB1). In contrast, Cdk5 increased phosphorylation of MARK4 in the spacer domain, but not in the activation loop, and enhanced its kinase activity, suggesting a novel mechanism by which Cdk5 regulates MARK4 activity. We also demonstrated that co-expression of Cdk5 and MARK4 in mammalian cultured cells significantly increased the levels of tau phosphorylation at both Cdk5 target sites (SP/TP sites) and MARK target sites (Ser262), as well as the levels of total tau. Furthermore, using a Drosophila model of tau toxicity, we demonstrated that Cdk5 promoted tau accumulation and tau-induced neurodegeneration via increasing tau phosphorylation levels at Ser262 by a fly ortholog of MARK, Par-1. This study suggests a novel mechanism by which Cdk5 and MARK4 synergistically increase tau phosphorylation and accumulation, consequently promoting neurodegeneration in disease pathogenesis.

Amyloid-ß plaque formation and reactive gliosis are required for induction of cognitive deficits in App knock-in mouse models of Alzheimer's disease.

BACKGROUND: Knock-in (KI) mouse models of Alzheimer's disease (AD) that endogenously overproduce Aß without non-physiological overexpression of amyloid precursor protein (APP) provide important insights into the pathogenic mechanisms of AD. Previously, we reported that AppNL-G-F mice, which harbor three familial AD mutations (Swedish, Beyreuther/Iberian, and Arctic) exhibited emotional alterations before the onset of definitive cognitive deficits. To determine whether these mice exhibit deficits in learning and memory at more advanced ages, we compared the Morris water maze performance of AppNL-G-F and AppNL mice, which harbor only the Swedish mutation, with that of wild-type (WT) C57BL/6J mice at the age of 24 months. To correlate cognitive deficits and neuroinflammation, we also examined Aß plaque formation and reactive gliosis in these mice. RESULTS: In the Morris water maze, a spatial task, 24-month-old AppNL-G-F/NL-G-F mice exhibited significantly poorer spatial learning than WT mice during the hidden training sessions, but similarly to WT mice during the visible training sessions. Not surprisingly, AppNL-G-F/NL-G-F mice also exhibited spatial memory deficits both 1 and 7 days after the last training session. By contrast, 24-month-old AppNL/NL mice had intact spatial learning and memory relative to WT mice. Immunohistochemical analyses revealed that 24-month-old AppNL-G-F/NL-G-F mice developed massive Aß plaques and reactive gliosis (microgliosis and astrocytosis) throughout the brain, including the cortex and hippocampus. By contrast, we observed no detectable brain pathology in AppNL/NL mice despite overproduction of human Aß40 and Aß42 in their brains. CONCLUSIONS: Aß plaque formation, followed by sustained neuroinflammation, is necessary for the induction of definitive cognitive deficits in App-KI mouse models of AD. Our data also indicate that introduction of the Swedish mutation alone in endogenous APP is not sufficient to produce either AD-related brain pathology or cognitive deficits in mice.

Roles of tau pathology in the locus coeruleus (LC) in age-associated pathophysiology and Alzheimer's disease pathogenesis: Potential strategies to protect the LC against aging.

The locus coeruleus (LC) is the noradrenaline (norepinephrine, NE)-containing nucleus in the brainstem and innervates into widespread brain regions. This LC-NE system plays a critical role in a variety of brain functions, including attention, arousal, emotion, cognition, and the sleep-wake cycle. The LC is one of the brain regions vulnerable to the occurrence of neurofibrillary tangles (NFTs), which is associated with "primary age-related tauopathy (PART)" that describes the pathology commonly observed in the brains of aged individuals. In Alzheimer's disease (AD), the LC is one of the first places to develop NFTs, which may act as a seed for subsequent spreading of the pathology throughout the brain upon amyloid-ß (Aß) accumulation. As AD progresses, significant neuron loss occurs in the LC. Moreover, LC neurodegeneration is not only a consequence of AD, but also drives clinical and pathological manifestations of AD, such as microglial dysregulation, sleep disturbance, cognitive decline, and neurovascular dysfunction. Therefore, prevention of NFT pathology and neuron loss in the LC-NE system is critical for suppressing the progression of AD. We propose that targeting aging itself may be a proactive intervention against age-associated changes in the LC. Such an approach could open the way for novel interventions against age-associated neurodegenerative disorders, in particular, AD.

S6K/p70S6K1 protects against tau-mediated neurodegeneration by decreasing the level of tau phosphorylated at Ser262 in a Drosophila model of tauopathy.

Abnormal accumulation of the microtubule-associated protein tau is thought to cause neuronal cell death in a group of age-associated neurodegenerative disorders. Tau is phosphorylated at multiple sites in diseased brains, and phosphorylation of tau at Ser262 initiates tau accumulation and toxicity. In this study, we sought to identify novel factors that affect the metabolism and toxicity of tau phosphorylated at Ser262 (pSer262-tau). A biased screen using a Drosophila model of tau toxicity revealed that knockdown of S6K, the Drosophila homolog of p70S6K1, increased the level of pSer262-tau and enhanced tau toxicity. S6K can be activated by the insulin signaling, however, unlike knockdown of S6K, knockdown of insulin receptor or insulin receptor substrate nonselectively decreased total tau levels via autophagy. Importantly, activation of S6K significantly suppressed tau-mediated axon degeneration, whereas manipulation of either the insulin signaling pathway or autophagy did not. Our results suggest that activation of S6K may be an effective therapeutic strategy for selectively decreasing the levels of toxic tau species and suppressing neurodegeneration.

Cognitive and emotional alterations in App knock-in mouse models of Aß amyloidosis.

BACKGROUND: Alzheimer's disease (AD), the most common cause of dementia, is characterized by the progressive deposition of amyloid-ß (Aß) peptides and neurofibrillary tangles. Mouse models of Aß amyloidosis generated by knock-in (KI) of a humanized Aß sequence provide distinct advantages over traditional transgenic models that rely on overexpression of amyloid precursor protein (APP). In App-KI mice, three familial AD-associated mutations were introduced into the endogenous mouse App locus to recapitulate Aß pathology observed in AD: the Swedish (NL) mutation, which elevates total Aß production; the Beyreuther/Iberian (F) mutation, which increases the Aß42/Aß40 ratio; and the Arctic (G) mutation, which promotes Aß aggregation. AppNL-G-F mice harbor all three mutations and develop progressive Aß amyloidosis and neuroinflammatory response in broader brain areas, whereas AppNL mice carrying only the Swedish mutation exhibit no overt AD-related pathological changes. To identify behavioral alterations associated with Aß pathology, we assessed emotional and cognitive domains of AppNL-G-F and AppNL mice at different time points, using the elevated plus maze, contextual fear conditioning, and Barnes maze tasks. RESULTS: Assessments of emotional domains revealed that, in comparison with wild-type (WT) C57BL/6J mice, AppNL-G-F/NL-G-F mice exhibited anxiolytic-like behavior that was detectable from 6 months of age. By contrast, AppNL/NL mice exhibited anxiogenic-like behavior from 15 months of age. In the contextual fear conditioning task, both AppNL/NL and AppNL-G-F/NL-G-F mice exhibited intact learning and memory up to 15-18 months of age, whereas AppNL-G-F/NL-G-F mice exhibited hyper-reactivity to painful stimuli. In the Barnes maze task, AppNL-G-F/NL-G-F mice exhibited a subtle decline in spatial learning ability at 8 months of age, but retained normal memory functions. CONCLUSION: AppNL/NL and AppNL-G-F/NL-G-F mice exhibit behavioral changes associated with non-cognitive, emotional domains before the onset of definitive cognitive deficits. Our observations consistently indicate that AppNL-G-F/NL-G-F mice represent a model for preclinical AD. These mice are useful for the study of AD prevention rather than treatment after neurodegeneration.

Integrated biology approach reveals molecular and pathological interactions among Alzheimer's Aß42, Tau, TREM2, and TYROBP in Drosophila models.

BACKGROUND: Cerebral amyloidosis, neuroinflammation, and tauopathy are key features of Alzheimer's disease (AD), but interactions among these features remain poorly understood. Our previous multiscale molecular network models of AD revealed TYROBP as a key driver of an immune- and microglia-specific network that was robustly associated with AD pathophysiology. Recent genetic studies of AD further identified pathogenic mutations in both TREM2 and TYROBP. METHODS: In this study, we systematically examined molecular and pathological interactions among Aß, tau, TREM2, and TYROBP by integrating signatures from transgenic Drosophila models of AD and transcriptome-wide gene co-expression networks from two human AD cohorts. RESULTS: Glial expression of TREM2/TYROBP exacerbated tau-mediated neurodegeneration and synergistically affected pathways underlying late-onset AD pathology, while neuronal Aß42 and glial TREM2/TYROBP synergistically altered expression of the genes in synaptic function and immune modules in AD. CONCLUSIONS: The comprehensive pathological and molecular data generated through this study strongly validate the causal role of TREM2/TYROBP in driving molecular networks in AD and AD-related phenotypes in flies.

Knockdown of wfs1, a fly homolog of Wolfram syndrome 1, in the nervous system increases susceptibility to age- and stress-induced neuronal dysfunction and degeneration in Drosophila.

Wolfram syndrome (WS), caused by loss-of-function mutations in the Wolfram syndrome 1 gene (WFS1), is characterized by juvenile-onset diabetes mellitus, bilateral optic atrophy, and a wide spectrum of neurological and psychiatric manifestations. WFS1 encodes an endoplasmic reticulum (ER)-resident transmembrane protein, and mutations in this gene lead to pancreatic ß-cell death induced by high levels of ER stress. However, the mechanisms underlying neurodegeneration caused by WFS1 deficiency remain elusive. Here, we investigated the role of WFS1 in the maintenance of neuronal integrity in vivo by knocking down the expression of wfs1, the Drosophila homolog of WFS1, in the central nervous system. Neuronal knockdown of wfs1 caused age-dependent behavioral deficits and neurodegeneration in the fly brain. Knockdown of wfs1 in neurons and glial cells resulted in premature death and significantly exacerbated behavioral deficits in flies, suggesting that wfs1 has important functions in both cell types. Although wfs1 knockdown alone did not promote ER stress, it increased the susceptibility to oxidative stress-, excitotoxicity- or tauopathy-induced behavioral deficits, and neurodegeneration. The glutamate release inhibitor riluzole significantly suppressed premature death phenotypes induced by neuronal and glial knockdown of wfs1. This study highlights the protective role of wfs1 against age-associated neurodegeneration and furthers our understanding of potential disease-modifying factors that determine susceptibility and resilience to age-associated neurodegenerative diseases.