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Ageing is the greatest global healthcare challenge, as it underlies age-related functional decline and is the primary risk factor for a range of common diseases, including neurodegenerative conditions such as Alzheimer’s disease (AD). However, the molecular mechanisms defining chronological age versus biological age, and how these underlie AD pathogenesis, are not well understood. The objective of this study was to integrate common human genetic variation associated with human lifespan or AD from Genome-Wide Association Studies (GWAS) with co-expression networks altered with age in the central nervous system, to gain insights into the biological processes which connect ageing with AD and lifespan. Initially, we identified common genetic variation in the human population associated with lifespan and AD by performing a gene-based association study using GWAS data. We also identified preserved co-expression networks associated with age in the brains of C57BL/6J mice from bulk and single-cell RNA-sequencing (RNA-seq) data, and in the brains of humans from bulk RNA-seq data. We then intersected the human gene-level common variation with these co-expression networks, representing the different cell types and processes of the brain. We found that genetic variation associated with AD was enriched in both microglial and oligodendrocytic bulk RNA-seq gene networks, which show increased expression with ageing in the human hippocampus, in contrast to synaptic networks which decreased with age. Further, longevity-associated genetic variation was modestly enriched in a single-cell gene network expressed by homeostatic microglia. Finally, we performed a transcriptome-wide association study (TWAS), to identify and confirm new risk genes associated with ageing that show variant-dependent changes in gene expression. In addition to validating known ageing-related genes such as APOE and FOXO3, we found that Caspase 8 (CASP8) and APOC1 show genetic variation associated with longevity. We observed that variants contributing to ageing and AD balance different aspects of microglial function suggesting that ageing-related processes affect multiple cell types in the brain. Specifically, changes in homeostatic microglia are associated with lifespan, and allele-dependent expression changes in age-related genes control microglial activation and myelination influencing the risk of developing AD. We identified putative molecular drivers of these genetic networks, as well as module genes whose expression in relevant human tissues are significantly associated with AD-risk or longevity, and may drive “inflammageing.” Our study also shows allele-dependent expression changes with ageing for genes classically involved in neurodegeneration, including MAPT and HTT, and demonstrates that PSEN1 is a prominent member/hub of an age-dependent expression network. In conclusion, this work provides new insights into cellular processes associated with ageing in the brain, and how these may contribute to the resilience of the brain against ageing or AD-risk. Our findings have important implications for developing markers indicating the physiological age and pre-pathological state of the brain, and provide new targets for therapeutic intervention.

The APOE-ε4 allele is known to predispose to amyloid deposition and consequently is strongly associated with Alzheimer's disease (AD) risk. There is debate as to whether the APOE gene accounts for all genetic variation of the APOE locus. Another question which remains is whether APOE-ε4 carriers have other genetic factors influencing the progression of amyloid positive individuals to AD. We conducted a genome-wide association study in a sample of 5,390 APOE-ε4 homozygous (ε4ε4) individuals (288 cases and 5,102 controls) aged 65 or over in the UK Biobank. We found no significant associations of SNPs in the APOE locus with AD in the sample of ε4ε4 individuals. However, we identified a novel genome-wide significant locus associated to AD, mapping to DAB1 (rs112437613, OR=2.28, CI=1.73-3.01, p=5.4 × 10−9). This identification of DAB1 led us to investigate other components of the DAB1-RELN pathway for association. Analysis of the DAB1-RELN pathway indicated that the pathway itself was associated with AD, therefore suggesting an epistatic interaction between the APOE locus and the DAB1-RELN pathway.

Recently, we reported oligoadenylate synthetase 1 (OAS1) contributed to the risk of Alzheimer’s disease, by its enrichment in transcriptional networks expressed by microglia. However, the function of OAS1 within microglia was not known. Using genotyping from 1313 individuals with sporadic Alzheimer’s disease and 1234 control individuals, we confirm the OAS1 variant, rs1131454, is associated with increased risk for Alzheimer’s disease. The same OAS1 locus has been recently associated with severe coronavirus disease 2019 (COVID-19) outcomes, linking risk for both diseases. The single nucleotide polymorphisms rs1131454(A) and rs4766676(T) are associated with Alzheimer’s disease, and rs10735079(A) and rs6489867(T) are associated with severe COVID-19, where the risk alleles are linked with decreased OAS1 expression. Analysing single-cell RNA-sequencing data of myeloid cells from Alzheimer’s disease and COVID-19 patients, we identify co-expression networks containing interferon (IFN)-responsive genes, including OAS1, which are significantly upregulated with age and both diseases. In human induced pluripotent stem cell-derived microglia with lowered OAS1 expression, we show exaggerated production of TNF-α with IFN-γ stimulation, indicating OAS1 is required to limit the pro-inflammatory response of myeloid cells. Collectively, our data support a link between genetic risk for Alzheimer’s disease and susceptibility to critical illness with COVID-19 centred on OAS1, a finding with potential implications for future treatments of Alzheimer’s disease and COVID-19, and development of biomarkers to track disease progression.

Funder: Cure Alzheimer's Fund; doi: http://dx.doi.org/10.13039/100007625, Funder: UK Dementia Research Institute (GB), Funder: Censejo Nacional de Ciencia Tecnilogia (MX), Funder: Alzheimerfonden; doi: http://dx.doi.org/10.13039/501100008599, Funder: Dolby Family Fund, Background: Microglia are active modulators of Alzheimer’s disease but their role in relation to amyloid plaques and synaptic changes due to rising amyloid beta is unclear. We add novel findings concerning these relationships and investigate which of our previously reported results from transgenic mice can be validated in knock-in mice, in which overexpression and other artefacts of transgenic technology are avoided. Methods: AppNL-F and AppNL-G-F knock-in mice expressing humanised amyloid beta with mutations in App that cause familial Alzheimer’s disease were compared to wild type mice throughout life. In vitro approaches were used to understand microglial alterations at the genetic and protein levels and synaptic function and plasticity in CA1 hippocampal neurones, each in relationship to both age and stage of amyloid beta pathology. The contribution of microglia to neuronal function was further investigated by ablating microglia with CSF1R inhibitor PLX5622. Results: Both App knock-in lines showed increased glutamate release probability prior to detection of plaques. Consistent with results in transgenic mice, this persisted throughout life in AppNL-F mice but was not evident in AppNL-G-F with sparse plaques. Unlike transgenic mice, loss of spontaneous excitatory activity only occurred at the latest stages, while no change could be detected in spontaneous inhibitory synaptic transmission or magnitude of long-term potentiation. Also, in contrast to transgenic mice, the microglial response in both App knock-in lines was delayed until a moderate plaque load developed. Surviving PLX5266-depleted microglia tended to be CD68-positive. Partial microglial ablation led to aged but not young wild type animals mimicking the increased glutamate release probability in App knock-ins and exacerbated the App knock-in phenotype. Complete ablation was less effective in altering synaptic function, while neither treatment altered plaque load. Conclusions: Increased glutamate release probability is similar across knock-in and transgenic mouse models of Alzheimer’s disease, likely reflecting acute physiological effects of soluble amyloid beta. Microglia respond later to increased amyloid beta levels by proliferating and upregulating Cd68 and Trem2. Partial depletion of microglia suggests that, in wild type mice, alteration of surviving phagocytic microglia, rather than microglial loss, drives age-dependent effects on glutamate release that become exacerbated in Alzheimer’s disease.

Genome-wide association studies of late-onset Alzheimer’s disease (AD) have highlighted the importance of variants associated with genes expressed by the innate immune system in determining risk for AD. Recently, we and others have shown that genes associated with variants that confer risk for AD are significantly enriched in transcriptional networks expressed by amyloid-responsive microglia. This allowed us to predict new risk genes for AD, including the interferon-responsive oligoadenylate synthetase 1 (OAS1). However, the function ofOAS1within microglia and its genetic pathway are not known. Using genotyping from 1,313 individuals with sporadic AD and 1,234 control individuals, we confirm that theOAS1variant, rs1131454, is associated with increased risk for AD and decreasedOAS1expression. Moreover, we note that the same locus was recently associated with critical illness in response to COVID-19, linking variants that are associated with AD and a severe response to COVID-19. By analysing single-cell RNA-sequencing (scRNA-seq) data of isolated microglia fromAPPNL-G-Fknock-in and wild-type C57BL/6J mice, we identify a transcriptional network that is significantly upregulated with age and amyloid deposition, and contains the mouse orthologueOas1a, providing evidence thatOas1aplays an age-dependent function in the innate immune system. We identify a similar interferon-related transcriptional network containingOAS1by analysing scRNA-seq data from human microglia isolated from individuals with AD. Finally, using human iPSC-derived microglial cells (h-iPSC-Mg), we see thatOAS1is required to limit the pro-inflammatory response of microglia. When stimulated with interferon-gamma (IFN-γ), we note that cells with lowerOAS1expression show an exaggerated pro-inflammatory response, with increased expression and secretion of TNF-α. Collectively, our data support a link between genetic risk for AD and susceptibility to critical illness with COVID-19 centred onOAS1and interferon signalling, a finding with potential implications for future treatments of both AD and COVID-19, and the development of biomarkers to track disease progression.

Mouse models of Alzheimer's disease (AD) are valuable but do not fully recapitulate human AD pathology, such as spontaneous Tau fibril accumulation and neuronal loss, necessitating the development of new AD models. The transgenic (TG) TgF344-AD rat has been reported to develop age-dependent AD features including neuronal loss and neurofibrillary tangles, despite only expressing APP and PSEN1 mutations, suggesting an improved modelling of AD hallmarks. Alterations in neuronal networks as well as learning performance and cognition tasks have been reported in this model, but none have combined a longitudinal, multimodal approach across multiple centres, which mimics the approaches commonly taken in clinical studies. We therefore aimed to further characterise the progression of AD-like pathology and cognition in the TgF344-AD rat from young-adults (6 months (m)) to mid- (12 m) and advanced-stage (18 m, 25 m) of the disease. Methods: TgF344-AD rats and wild-type (WT) littermates were imaged at 6 m, 12 m and 18 m with [18F]DPA-714 (TSPO, neuroinflammation), [18F]Florbetaben (Aβ) and [18F]ASEM (α7-nicotinic acetylcholine receptor) and with magnetic resonance spectroscopy (MRS) and with (S)-[18F]THK5117 (Tau) at 15 and 25 m. Behaviour tests were also performed at 6 m, 12 m and 18 m. Immunohistochemistry (CD11b, GFAP, Aβ, NeuN, NeuroChrom) and Tau (S)-[18F]THK5117 autoradiography, immunohistochemistry and Western blot were also performed. Results: [18F]DPA-714 positron emission tomography (PET) showed an increase in neuroinflammation in TG vs wildtype animals from 12 m in the hippocampus (+11%), and at the advanced-stage AD in the hippocampus (+12%), the thalamus (+11%) and frontal cortex (+14%). This finding coincided with strong increases in brain microgliosis (CD11b) and astrogliosis (GFAP) at these time-points as assessed by immunohistochemistry. In vivo [18F]ASEM PET revealed an age-dependent increase uptake in the striatum and pallidum/nucleus basalis of Meynert in WT only, similar to that observed with this tracer in humans, resulting in TG being significantly lower than WT by 18 m. In vivo [18F]Florbetaben PET scanning detected Aβ accumulation at 18 m, and (S)-[18F]THK5117 PET revealed subsequent Tau accumulation at 25m in hippocampal and cortical regions. Aβ plaques were low but detectable by immunohistochemistry from 6 m, increasing further at 12 and 18 m with Tau-positive neurons adjacent to Aβ plaques at 18 m. NeuroChrom (a pan neuronal marker) immunohistochemistry revealed a loss of neuronal staining at the Aβ plaques locations, while NeuN labelling revealed an age-dependent decrease in hippocampal neuron number in both genotypes. Behavioural assessment using the novel object recognition task revealed that both WT & TgF344-AD animals discriminated the novel from familiar object at 3 m and 6 m of age. However, low levels of exploration observed in both genotypes at later time-points resulted in neither genotype successfully completing the task. Deficits in social interaction were only observed at 3 m in the TgF344-AD animals. By in vivo MRS, we showed a decrease in neuronal marker N-acetyl-aspartate in the hippocampus at 18 m (-18% vs age-matched WT, and -31% vs 6 m TG) and increased Taurine in the cortex of TG (+35% vs age-matched WT, and +55% vs 6 m TG). Conclusions: This multi-centre multi-modal study demonstrates, for the first time, alterations in brain metabolites, cholinergic receptors and neuroinflammation in vivo in this model, validated by robust ex vivo approaches. Our data confirm that, unlike mouse models, the TgF344-AD express Tau pathology that can be detected via PET, albeit later than by ex vivo techniques, and is a useful model to assess and longitudinally monitor early neurotransmission dysfunction and neuroinflammation in AD.

Genome-wide association studies have reported that, amongst other microglial genes, variants in TREM2 can profoundly increase the incidence of developing Alzheimer’s disease (AD). We have investigated the role of TREM2 in primary microglial cultures from wild type mice by using siRNA to decrease Trem2 expression, and in parallel from knock-in mice heterozygous or homozygous for the Trem2 R47H AD risk variant. The prevailing phenotype of Trem2 R47H knock-in mice was decreased expression levels of Trem2 in microglia, which resulted in decreased density of microglia in the hippocampus. Overall, primary microglia with reduced Trem2 expression, either by siRNA or from the R47H knock-in mice, displayed a similar phenotype. Comparison of the effects of decreased Trem2 expression under conditions of lipopolysaccharide (LPS) pro-inflammatory or IL-4 anti-inflammatory stimulation revealed the importance of Trem2 in driving a number of the genes up-regulated in the anti-inflammatory phenotype. RNA-seq analysis showed that IL-4 induced the expression of a program of genes including Arg1 and Ap1b1 in microglia, which showed an attenuated response to IL-4 when Trem2 expression was decreased. Genes showing a similar expression profile to Arg1 were enriched for STAT6 transcription factor recognition elements in their promoter, and Trem2 knockdown decreased levels of STAT6. LPS-induced pro-inflammatory stimulation suppressed Trem2 expression, thus preventing TREM2’s anti-inflammatory drive. Given that anti-inflammatory signaling is associated with tissue repair, understanding the signaling mechanisms downstream of Trem2 in coordinating the pro- and anti-inflammatory balance of microglia, particularly mediating effects of the IL-4-regulated anti-inflammatory pathway, has important implications for fighting neurodegenerative disease., Graphical Abstract Graphical Abstract

Genome-wide association studies of late-onset Alzheimer’s disease risk have previously identified genes primarily expressed in microglia that form a transcriptional network. Using transgenic mouse models of amyloid deposition, we previously showed that many of the mouse orthologues of these risk genes are co-expressed and associated with amyloid pathology. In this new study, we generate an improved RNA-seq-derived network that is expressed in amyloid-responsive mouse microglia and we statistically compare this with gene-level variation in previous human Alzheimer’s disease genome-wide association studies to predict at least four new risk genes for the disease (OAS1, LAPTM5, ITGAM/CD11b and LILRB4). Of the mouse orthologues of these genes Oas1a is likely to respond directly to amyloid at the transcriptional level, similarly to established risk gene Trem2, because the increase in Oas1a and Trem2 transcripts in response to amyloid deposition in transgenic mice is significantly higher than both the increase of the average microglial transcript and the increase in microglial number. In contrast, the mouse orthologues of LAPTM5, ITGAM/CD11b and LILRB4 (Laptm5, Itgam/CD11b and Lilra5) show increased transcripts in the presence of amyloid plaques similar in magnitude to the increase of the average microglial transcript and the increase in microglia number, except that Laptm5 and Lilra5 transcripts increase significantly quicker than the average microglial transcript as the plaque load becomes dense. This work suggests that genetic variability in the microglial response to amyloid deposition is a major determinant for Alzheimer’s disease risk, and identification of these genes may help to predict the risk of developing Alzheimer’s disease. These findings also provide further insights into the mechanisms underlying Alzheimer’s disease for potential drug discovery.

Negative regulator of ubiquitin-like protein 1 (NUB1) and its longer isoform NUB1L are ubiquitin-like (UBL)/ubiquitin-associated (UBA) proteins that facilitate the targeting of proteasomal substrates, including tau, synphilin-1 and huntingtin. Previous data revealed that NUB1 also mediated a reduction in tau phosphorylation and aggregation following proteasome inhibition, suggesting a switch in NUB1 function from targeted proteasomal degradation to a role in autophagy. Here, we delineate the mechanisms of this switch and show that NUB1 interacted specifically with p62 and induced an increase in p62 levels in a manner facilitated by inhibition of the proteasome. NUB1 moreover increased autophagosomes and the recruitment of lysosomes to aggresomes following proteasome inhibition. Autophagy flux assays revealed that NUB1 affected the autophagy–lysosomal pathway primarily via the UBA domain. NUB1 localized to cytosolic inclusions with pathological forms of tau, as well as LAMP1 and p62 in the hippocampal neurons of tauopathy mice. Finally, NUB1 facilitated the extracellular release of tau following proteasome inhibition. This study thus shows that NUB1 plays a role in regulating the autophagy–lysosomal pathway when the ubiquitin proteasome system is compromised, thus contributing to the mechanisms targeting the removal of aggregation-prone proteins upon proteasomal impairment.

Mouse models of Alzheimer's disease have commonly used transgenic overexpression of genes involved in production of amyloid β (APP and/or PSEN1/2) or Tau (MAPT) with mutations that result in familial forms of dementia. We discuss possible improvements that may create full models while avoiding the problems of overexpression and report synaptic results in APPKI models. We stress use of inappropriate controls without overexpression of the normal human protein and the mismatch between the learning deficits reported in mice with plaques but no tangles and the human condition. We focus on Tau overexpression, including new data that support previous reports of the grossly nonlinear relationship between Tau overexpression and neurofibrillary tangle load, with a twofold increase in Tau protein, resulting in a 100-fold increase in tangle density. These data also support the hypothesis that a high concentration of soluble Tau, in overexpression models, plays an important direct role in neurodegeneration, rather than only via aggregation. Finally, we hypothesize that there is an optimal concentration range over which Tau can bind to microtubules and a threshold beyond which much of the overexpressed protein is unable to bind. The excess thus causes toxicity in ways not necessarily related to the process in human dementias.

BackgroundProgression of Alzheimer’s disease is thought initially to depend on rising amyloidβ and its synaptic interactions. Transgenic mice (TASTPM; APPSwe/PSEN1M146V) show altered synaptic transmission, compatible with increased physiological function of amyloidβ, before plaques are detected. Recently, the importance of microglia has become apparent in the human disease. Similarly, TASTPM show a close association of plaque load with upregulated microglial genes.MethodsCA1 Synaptic transmission and plasticity were investigated using in vitro electrophysiology. Migroglial relationship to plaques was examined with immunohistochemistry. Behaviour was assessed with a forced-alternation T-maze, open field, light/dark box and elevated plus maze.FindingsThe most striking finding is the increase in microglial numbers in TASTPM, which, like synaptic changes, begins before plaques are detected. Further increases and a reactive phenotype occur later, concurrent with development of larger plaques. Long-term potentiation is initially enhanced at pre-plaque stages but decrements with the initial appearance of plaques. Finally, despite altered plasticity, TASTPM have little cognitive deficit, even with a heavy plaque load, although they show altered non-cognitive behaviours.InterpretationThe pre-plaque synaptic changes and microglial proliferation are presumably related to low, non-toxic amyloidβ levels in the general neuropil and not directly associated with plaques. However, as plaques grow, microglia proliferate further, clustering around plaques and becoming phagocytic. Like in humans, even when plaque load is heavy, without development of neurofibrillary tangles and neurodegeneration, these alterations do not result in cognitive deficits. Behaviours are seen that could be consistent with pre-diagnosis changes in the human condition.Research in contextEvidence before this studyThere is a large body of research examining many aspects of phenotypes associated with mouse models of Alzheimer’s disease – a PubMed search for the terms Alzheimer* AND mouse returns in excess of 21000 articles. However, there are few systematic articles pulling together pathological, functional (electrophysiological), and behavioural analyses across the life-span of such models. There is also a number of conflicting outcomes, for example reports of impaired versus enhanced synaptic plasticity; cognitive impairments or not.Recently, the importance of microglia in Alzheimer’s disease has come to the fore in human Genome Wide Association Studies (GWAS), with variants of a number of microglial genes identified as risk-factors for developing the disease. Interestingly, we have recently reported that Trem2 and other genes identified as risk-factors in humans are strongly up regulated in close association to plaque development in the mouse model used in this study. Moreover, this previous study predicted two of the most recently identified genes that were identified in GWAS since the publication of our paper.We have previously used this model to identify the earliest synaptic changes and shown changes in release of glutamate, the primary excitatory neurotransmitter in the brain, to occur even before plaques are detectable.Added value of this studyBy studying this transgenic mouse model of Alzheimer’s disease, throughout the development of plaques, from prior to detection through to heavy plaque loads, we have been able to identify a clear time course of key phenotypic changes associated with early disease. In particular, this study identifies the very early changes in microglia and can separate the time course of the microglial phenotype. In addition, we detail the changes in synaptic plasticity over time and importantly identify that, like in humans in the absence of Tau tangles or neurodegeneration, considerable synaptic changes can occur and a heavy plaque load without resulting in substantial cognitive loss.Implications of all the available evidenceOur data indicate that rising amyloid beta prior to detectable plaque deposition results in changes in synaptic function that likely reflects an enhanced physiological effect of amyloid beta. At this stage, microglia proliferate but do not activate. Once plaques begin to appear, microglia migrate to surround the plaque and become phagocytic, likely targeting dystrophic synapses and neurites caused by the cloud of highly-toxic amyloid beta around the plaque. Similarly to humans, who have plaques but no tangles and have yet to develop substantial neurodegeneration, cognitive deficits are not seen, even with a heavy plaque load; behavioural changes are limited to anxiety-like effects.This investigation of the parallel time-course of events highlights the probability that, if progression of disease can be reversed or slowed early enough, before Tau tangles and substantial neurodegeneration occur, the symptoms of cognitive decline could be very largely avoided. Moreover, it suggests that the substantial increases in microglia number and upregulation of their specific gene expression in association with plaques, is not associated with cognitive loss and may indeed be protective.

Genome-wide association studies have reported that, amongst other microglial genes, variants inTREM2can profoundly increase the incidence of developing Alzheimer’s disease (AD). We have investigated the role of TREM2 in primary microglial cultures from wild type mice by using siRNA to decreaseTrem2expression, and in parallel from knock-in mice heterozygous or homozygous for theTrem2R47H AD risk variant. The prevailing phenotype ofTrem2R47H knock-in mice was decreased expression levels ofTrem2in microglia, which resulted in decreased density of microglia in the hippocampus. Overall, primary microglia with reducedTrem2expression, either by siRNA or from the R47H knock-in mice, displayed a similar phenotype. Comparison of the effects of decreasedTrem2expression under conditions of LPS pro-inflammatory or IL-4 anti-inflammatory stimulation revealed the importance ofTrem2in driving a number of the genes up-regulated in the anti-inflammatory phenotype. RNA-seq analysis showed that IL-4 induced the expression of a programme of genes includingArg1andAp1b1in microglia, which showed an attenuated response to IL-4 whenTrem2expression was decreased. Genes showing a similar expression profile toArg1were enriched for STAT6 transcription factor recognition elements in their promoter, andTrem2knockdown decreased levels of the transcription factor STAT6. LPS-induced pro-inflammatory stimulation suppressedTrem2expression, thus preventing TREM2’s anti-inflammatory drive. Given that anti-inflammatory signaling is associated with tissue repair, understanding the signaling mechanisms downstream ofTrem2in coordinating the pro- and anti-inflammatory balance of microglia, particularly mediating effects of the IL-4-regulated anti-inflammatory pathway, has important implications for fighting neurodegenerative disease.Graphical abstract

Genetic analysis of late-onset Alzheimer’s disease risk has previously identified a network of largely microglial genes that form a transcriptional network. In transgenic mouse models of amyloid deposition we have previously shown that the expression of many of the mouse orthologs of these genes are co-ordinately up-regulated by amyloid deposition. Here we investigate whether systematic analysis of other members of this mouse amyloid-responsive network predicts other Alzheimer’s risk loci. This statistical comparison of the mouse amyloid-response network with Alzheimer’s disease genome-wide association studies identifies 5 other genetic risk loci for the disease (OAS1, CXCL10, LAPTM5, ITGAM and LILRB4). This work suggests that genetic variability in the microglial response to amyloid deposition is a major determinant for Alzheimer’s risk.One Sentence SummaryIdentification of 5 new risk loci for Alzheimer’s by statistical comparison of mouse Aβ microglial response with gene-based SNPs from human GWAS

BackgroundLate stage Alzheimer’s disease and other dementias are associated with neurofibrillary tangles and neurodegeneration. Here we describe a mouse (TauD35) carrying human Tau with the P301L mutation that results in Tau hyperphosphorylation and tangles. Previously we have compared gene expression in TauD35 mice to mice which develop plaques but no tangles. A similar comparison of other pathological features throughout disease progression is made here between amyloidβ and Tau mice described in Parts I and II of this study.MethodsIn vitro CA1 patch clamp and field recordings were used to investigate synaptic transmission and plasticity. Plaque load and microglia were investigated with immunohistochemistry. Cognition, locomotor activity and anxiety-related behaviours were assessed with a forced-alternation T-maze, open field and light/dark box.ResultsTransgene copy number in TauD35 mice fell into two groups (HighTAU and LowTAU), allowing assessment of dose-dependent effects of overexpression and resulting in tangle load increasing 100-fold for a 2-fold change in protein levels. Tangles were first detected at 8 (HighTAU) or 13 months (LowTAU) but the effects on synaptic transmission and plasticity and behaviour were subtle. However, severe neurodegeneration occurred in HighTAU mice at around 17 months, preceded by considerable proliferation and activation of microglia at 13 months of age; both increasing further at 17 months. LowTAU mice at 24 months of age showed a comparable tangle load and microglial proliferation to that occurring at 13 months in HighTAU mice. However, LowTAU mice showed no neurodegeneration at this stage and considerable microglial activation, stressing the dependence of these effects on overexpression and/or age.ConclusionsComparison of the effects of amyloidβ and plaques without tangles in a model of preclinical Alzheimer’s disease to the effects of tangles without amyloidβ plaques in the late stage model described here may clarify the progressive stages of Alzheimer’s disease. While Tau hyperphosphorylation and neurofibrillary tangles are eventually sufficient to cause severe neurodegeneration, initial effects on synaptic transmission and the immune response are subtle. In contrast while even with a heavy plaque load little if any neurodegeneration occurs, considerable effects on synaptic transmission and the immune system result, even before plaques are detectable.

Neuronal pentraxin 1 (NPTX1) has been implicated in Alzheimer's disease, being present in and around dystrophic neurons in plaques, affecting glutamatergic transmission postsynaptically and mediating effects of amyloidβ. Here, we confirm the presence of NPTX1 around plaques in postmortem Alzheimer's disease brain and report that acutely applied human NPTX1 increases paired-pulse ratio at mouse CA3-CA1 hippocampal synapses, indicating a decrease in glutamate release. In contrast, chronic exposure to NPTX1, NPTX2, or NPTX receptor decreases paired-pulse ratio, mimicking some of the earliest changes in mice expressing familial Alzheimer's disease genes. The peripheral pentraxin, serum amyloid P component (SAP), causes similar synaptic effects to NPTX1. The presence of SAP on amyloid plaques in Alzheimer's disease confirms that it can enter the brain. We show that SAP and neuronal pentraxins can interact and that SAP can enter the brain if the blood-brain barrier is compromised, suggesting that peripheral pentraxins could affect central synaptic transmission via this interaction, especially in the event of blood-brain barrier breakdown.

The FoxO family of transcription factors is known to slow aging downstream from the insulin/IGF (insulin-like growth factor) signaling pathway. The most recently discovered FoxO isoform in mammals, FoxO6, is highly enriched in the adult hippocampus. However, the importance of FoxO factors in cognition is largely unknown. Here we generated mice lacking FoxO6 and found that these mice display normal learning but impaired memory consolidation in contextual fear conditioning and novel object recognition. Using stereotactic injection of viruses into the hippocampus of adult wild-type mice, we found that FoxO6 activity in the adult hippocampus is required for memory consolidation. Genome-wide approaches revealed that FoxO6 regulates a program of genes involved in synaptic function upon learning in the hippocampus. Consistently, FoxO6 deficiency results in decreased dendritic spine density in hippocampal neurons in vitro and in vivo. Thus, FoxO6 may promote memory consolidation by regulating a program coordinating neuronal connectivity in the hippocampus, which could have important implications for physiological and pathological age-dependent decline in memory.

SummaryIn the nervous system, neural stem cells (NSCs) are necessary for the generation of new neurons and for cognitive function. Here we show that FoxO3, a member of a transcription factor family known to extend lifespan in invertebrates, regulates the NSC pool. We find that adult FoxO3−/− mice have fewer NSCs in vivo than wild-type counterparts. NSCs isolated from adult FoxO3−/− mice have decreased self-renewal and an impaired ability to generate different neural lineages. Identification of the FoxO3-dependent gene expression profile in NSCs suggests that FoxO3 regulates the NSC pool by inducing a program of genes that preserves quiescence, prevents premature differentiation, and controls oxygen metabolism. The ability of FoxO3 to prevent the premature depletion of NSCs might have important implications for counteracting brain aging in long-lived species.

IGF activity is regulated tightly by a family of IGF binding proteins (IGFBPs). IGFBP-5 is the most conserved of these and is up-regulated significantly during differentiation of several key lineages and in some cancers. The function of IGFBP-5 in these physiological and pathological situations is unclear, however, several IGFBP-5 sequence motifs and studies in vitro suggest IGF-independent actions. Therefore, we aimed to compare the phenotypes of mice overexpressing wild-type Igfbp5 or an N-terminal mutant Igfbp5 with negligible IGF binding affinity. Both significantly inhibited growth, even at low expression levels. Even though wild-type IGFBP-5 severely disrupted the IGF axis, we found no evidence for interaction of mutant IGFBP-5 with the IGF system. Further, overexpression of wild-type IGFBP-5 rescued the lethal phenotype induced by "excess" IGF-II in type 2 receptor-null mice; mutant IGFBP-5 overexpression could not. Therefore, wild-type IGFBP-5 provides a very effective mechanism for the inhibition of IGF activity and a powerful in vivo mechanism to inhibit IGF activity in pathologies such as cancer. This study is also the first to suggest significant IGF-independent actions for IGFBP-5 during development.

The FoxO family of Forkhead transcription factors functions at the interface of tumor suppression, energy metabolism, and organismal longevity. FoxO factors are key downstream targets of insulin, growth factor, nutrient, and oxidative stress stimuli that coordinate a wide-range of cellular outputs. FoxO-dependent cellular responses include gluconeogenesis, neuropeptide secretion, atrophy, autophagy, apoptosis, cell cycle arrest, and stress resistance. This review will discuss the roles of the mammalian FoxO family in a variety of cell-types, from stem cells to mature cells, in the context of the whole organism. Given the overwhelming evidence that the FoxO factors promote longevity in invertebrates, this review will also discuss the potential role of the FoxO factors in the aging of mammalian organisms.

Activation of either the phosphatidylinositol 3-kinase (PI 3-kinase)/Akt or the p38 mitogen-activated protein kinase (MAPK) signaling pathways accelerates myogenesis but only when the reciprocal pathway is functional. We therefore examined the hypothesis that cross-activation between these signaling cascades occurs to orchestrate myogenesis. We reveal a novel and reciprocal cross-talk and activation between the PI 3-kinase/Akt and p38 MAPK pathways that is essential for efficient myoblast differentiation. During myoblast differentiation, Akt kinase activity correlated with S473 but not T308 phosphorylation and occurred 24 h after p38 activation. Inhibition or activation of p38 with SB203580, dominant-negative p38, or MKK6EE regulated Akt kinase activity. Analysis of Akt isoforms revealed a specific increase in Akt2 protein levels that coincided with AktS473 phosphorylation during myogenesis and an enrichment of S473-phosphorylated Akt2. Akt2 promoter activity and protein levels were regulated by p38 activation, thus providing a mechanism for communication. Subsequent Akt activation by S473 phosphorylation was PI 3-kinase dependent and specific for Akt2 rather than Akt1. Complementary to p38-mediated transactivation of Akt, activation or inhibition of PI 3-kinase regulated p38 activity upstream of MKK6, demonstrating reciprocal communication and positive feedback characteristic of myogenic regulation. Our findings have identified novel communication between p38 MAPK and PI 3-kinase/Akt via Akt2.

The insulin-like growth factors (IGFs) are essential for development; bioavailable IGF is tightly regulated by six related IGF-binding proteins (IGFBPs). Igfbp5 is the most conserved and is developmentally up-regulated in key lineages and pathologies; in vitro studies suggest that IGFBP-5 functions independently of IGF interaction. Genetic ablation of individual Igfbps has yielded limited phenotypes because of substantial compensation by remaining family members. Therefore, to reveal Igfbp5 actions in vivo , we generated lines of transgenic mice that ubiquitously overexpressed Igfbp5 from early development. Significantly increased neonatal mortality, reduced female fertility, whole-body growth inhibition, and retarded muscle development were observed in Igfbp5 -overexpressing mice. The magnitude of the response in individual transgenic lines was positively correlated with Igfbp5 expression. Circulating IGFBP-5 concentrations increased a maximum of only 4-fold, total and free IGF-I concentrations increased up to 2-fold, and IGFBP-5 was detected in high M r complexes; however, no detectable decrease in the proportion of free IGF-I was observed. Thus, despite only modest changes in IGF and IGFBP concentrations, the Igfbp5 -overexpressing mice displayed a phenotype more extreme than that observed for other Igfbp genetic models. Although growth retardation was obvious prenatally, maximal inhibition occurred postnatally before the onset of growth hormone-dependent growth, regardless of Igfbp5 expression level, revealing a period of sensitivity to IGFBP-5 during this important stage of tissue programming.

We provide microarray data comparing genome-wide differential expression and pathology throughout life in four lines of "amyloid" transgenic mice (mutant human APP, PSEN1, or APP/PSEN1) and "TAU" transgenic mice (mutant human MAPT gene). Microarray data were validated by qPCR and by comparison to human studies, including genome-wide association study (GWAS) hits. Immune gene expression correlated tightly with plaques whereas synaptic genes correlated negatively with neurofibrillary tangles. Network analysis of immune gene modules revealed six hub genes in hippocampus of amyloid mice, four in common with cortex. The hippocampal network in TAU mice was similar except that Trem2 had hub status only in amyloid mice. The cortical network of TAU mice was entirely different with more hub genes and few in common with the other networks, suggesting reasons for specificity of cortical dysfunction in FTDP17. This Resource opens up many areas for investigation. All data are available and searchable at http://www.mouseac.org.

Neuronal polarity is essential for normal brain development and function. However, cell-intrinsic mechanisms that govern the establishment of neuronal polarity remain to be identified. Here, we report that knockdown of endogenous FOXO proteins in hippocampal and cerebellar granule neurons, including in the rat cerebellar cortex in vivo, reveals a requirement for the FOXO transcription factors in the establishment of neuronal polarity. The FOXO transcription factors, including the brain-enriched protein FOXO6, play a critical role in axo–dendritic polarization of undifferentiated neurites, and hence in a switch from unpolarized to polarized neuronal morphology. We also identify the gene encoding the protein kinase Pak1, which acts locally in neuronal processes to induce polarity, as a critical direct target gene of the FOXO transcription factors. Knockdown of endogenous Pak1 phenocopies the effect of FOXO knockdown on neuronal polarity. Importantly, exogenous expression of Pak1 in the background of FOXO knockdown in both primary neurons and postnatal rat pups in vivo restores the polarized morphology of neurons. These findings define the FOXO proteins and Pak1 as components of a cell-intrinsic transcriptional pathway that orchestrates neuronal polarity, thus identifying a novel function for the FOXO transcription factors in a unique aspect of neural development.

Cell-cell contact is essential for appropriate co-ordination of development and it initiates significant signalling events. During myogenesis, committed myoblasts migrate to sites of muscle formation, align and form adhesive contacts that instigate cell-cycle exit and terminal differentiation into multinucleated myotubes; thus myogenesis is an excellent paradigm for the investigation of signals derived from cell-cell contact. PI3-K and p38 MAPK are both essential for successful myogenesis. Pro-myogenic growth factors such as IGF-II activate PI3-K via receptor tyrosine kinases but the extracellular cues and upstream intermediates required for activation of the p38 MAPK pathway in myoblast differentiation are not known. Initial observations suggested a correlation between p38 MAPK phosphorylation and cell density, which was also related to N-cadherin levels and Igf2 expression. Subsequent studies using N-cadherin ligand, dominant-negative N-cadherin, constitutively active and dominant-negative forms of RhoA, and MKK6 and p38 constructs, reveal a novel pathway in differentiating myoblasts that links cell-cell adhesion via N-cadherin to Igf2 expression (assessed using northern and promoter-reporter analyses) via RhoA and p38α and/or β but not γ. We thus define a regulatory mechanism for p38 activation that relates cell-cell-derived adhesion signalling to the synthesis of the major fetal growth factor, IGF-II.

IGF-binding protein-5 (IGFBP-5) is abundant in serum and bone during normal skeletal development, but levels decrease in osteoporosis. Studies have shown that IGFBP-5 stimulates markers of bone formation by potentiating IGF actions and by IGF-independent actions. To test the hypothesis that IGFBP-5 promotes the acquisition of bone mineral density (BMD), we generated transgenic (Tg) mice overexpressing Igfbp5 using a cytomegalovirus enhancer and beta-actin promoter (CMV/betaA). Tg animals showed an increase in serum IGFBP-5 concentrations by 7.7- to 3.5-fold at 3-8 wk of age, respectively. Concentrations were 6-49% higher for males compared with females in both wild-type and Tg mice. Surprisingly, BMD decreased in a gender-dependent manner, with Tg male adults affected more severely than Tg females (31.3% vs. 19.2% reduction, respectively, compared with wild-type mice, assessed by dual energy x-ray absorptiometry). Significant gender differences in BMD were confirmed by peripheral quantitative computed tomography. Histomorphometry revealed that although the bone formation rate and mineralizing surface at the periosteum decreased in Tg mice, they increased at the endosteum, suggesting opposing effects of IGFBP-5 on periosteal and endosteal osteoblasts (by altering proliferation or survival). These findings differ from previous observations in Igf1- and Igf2-null animals. In conclusion, IGFBP-5 has a significant influence on BMD acquisition and maintenance that is dependent on gender and age. The phenotype of Igfbp5 mice cannot be explained solely by IGF inhibition; thus, this study provides the first in vivo evidence, by genetic manipulation, for IGF-independent actions of IGFBP-5 in bone function. These findings have implications for the gender-biased progression of osteoporosis.

Igfbp5 is upregulated during the differentiation of several key cell lineages and in some tumours; the function of IGFBP-5 in these physiological and pathological situations is unknown. Since IGFBP-5 contains sequence motifs consistent with IGF-independent actions, the aim of these studies was to distinguish between IGF-dependent and -independent actions of IGFBP-5. Myc-tagged wild-type (termed wtIGFBP-5) and non-IGF binding mouse Igfbp5 (termed mutIGFBP-5) cDNAs were generated and used to transfect C2 myoblasts, a cell line that undergoes differentiation to myotubes in an IGF- and IGFBP-5-regulated manner. WtIGFBP-5, but not mutIGFBP-5, inhibited myogenesis, as assessed by cell morphology, MHC immunocytochemistry and caveolin 3 expression. However, both wt- and mutIGFBP-5 increased cell survival and decreased apoptosis, as indicated by decreased caspase-3 activity and cell surface annexin V binding. Further examination of apoptotic pathways revealed that wt- and mutIGFBP-5 ameliorated the increase in caspase-9 but not the modest increase in caspase-8 during myogenesis, suggesting that IGFBP-5 increased cell survival via inhibition of intrinsic cell death pathways in an IGF-independent manner. The relationship between IGF-II and IGFBP-5 was examined further by cotransfecting C2 myoblasts with antisense Igf2 (previously established to induce increased cell death) and Igfbp5; both wt- and mutIGFBP-5 conferred equivalent protection against the decreased cell survival and increased apoptosis. In conclusion, we have partitioned IGFBP-5 action in myogenesis into IGF-dependent inhibition of differentiation and IGF-independent cell survival. Our findings suggest that, by regulation of cell survival, IGFBP-5 has an autonomous role in the regulation of cell fate in development and in tumourigenesis.

We provide microarray data comparing genome-wide differential expression and pathology throughout life in four lines of "amyloid" transgenic mice (mutant human APP, PSEN1, or APP/PSEN1) and "TAU" transgenic mice (mutant human MAPT gene). Microarray data were validated by qPCR and by comparison to human studies, including genome-wide association study (GWAS) hits. Immune gene expression correlated tightly with plaques whereas synaptic genes correlated negatively with neurofibrillary tangles. Network analysis of immune gene modules revealed six hub genes in hippocampus of amyloid mice, four in common with cortex. The hippocampal network in TAU mice was similar except that Trem2 had hub status only in amyloid mice. The cortical network of TAU mice was entirely different with more hub genes and few in common with the other networks, suggesting reasons for specificity of cortical dysfunction in FTDP17. This Resource opens up many areas for investigation. All data are available and searchable at http://www.mouseac.org. Fil: Matarin, Mar. Queen Square. Institute of Neurology. Department of Clinical and Experimental Epilepsy; Reino Unido. Physiology and Pharmacology. Department of Neuroscience; Reino Unido Fil: Salih, Dervis A.. Physiology and Pharmacology. Department of Neuroscience; Reino Unido Fil: Yasvoina, Marina. Physiology and Pharmacology. Department of Neuroscience; Reino Unido Fil: Cummings, Damian M.. Physiology and Pharmacology. Department of Neuroscience; Reino Unido Fil: Guelfi, Sebastian. Institute of Neurology. Reta Lila Research Laboratories and Department of Molecular Neuroscience; Reino Unido Fil: Liu, Wenfei. Physiology and Pharmacology. Department of Neuroscience; Reino Unido Fil: NahabooSolim, Muzammil A.. Physiology and Pharmacology. Department of Neuroscience; Reino Unido. Queen Square. UCL Institute of Neurology. Department of Neuroinflammation; Reino Unido Fil: Moens, Thomas G.. Physiology and Pharmacology. Department of Neuroscience; Reino Unido Fil: Paublete, Rocio Moreno. Physiology and Pharmacology. Department of Neuroscience; Reino Unido Fil: Ali, Shabinah S.. Physiology and Pharmacology. Department of Neuroscience; Reino Unido Fil: Perona, Marina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Queen Square. Institute of Neurology. Department of Clinical and Experimental Epilepsy; Reino Unido Fil: Desai, Roshni. Queen Square. UCL Institute of Neurology. Department of Neuroinflammation; Reino Unido Fil: Smith, Kenneth J.. Queen Square. UCL Institute of Neurology. Department of Neuroinflammation; Reino Unido Fil: Latcham, Judy. GlaxoSmithKline R&D. Department of Laboratory Animal Science; Reino Unido Fil: Fulleylove, Michael. GlaxoSmithKline R&D. Department of Laboratory Animal Science; Reino Unido Fil: Richardson, Jill C.. GlaxoSmithKline R&D. Neurosciences Therapeutic Area; Reino Unido Fil: Hardy, John. Queen Square. Institute of Neurology. Department of Clinical and Experimental Epilepsy; Reino Unido Fil: Edwards, Frances A.. Queen Square. Institute of Neurology. Department of Clinical and Experimental Epilepsy; Reino Unido