Negative Regulator of Ubiquitin-Like Protein 1 modulates the autophagy-lysosomal pathway via p62 to facilitate the extracellular release of tau following proteasome impairment.

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.

Trem2 promotes anti-inflammatory responses in microglia and is suppressed under pro-inflammatory conditions.

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.

A genetic link between risk for Alzheimer's disease and severe COVID-19 outcomes via the OAS1 gene.

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.

FoxO6 regulates memory consolidation and synaptic function.

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.

Neuronal and Peripheral Pentraxins Modify Glutamate Release and may Interact in Blood-Brain Barrier Failure.

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 amyloidß. 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.

A FOXO-Pak1 transcriptional pathway controls neuronal polarity.

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.

First effects of rising amyloid-ß in transgenic mouse brain: synaptic transmission and gene expression.

Detecting and treating Alzheimer's disease, before cognitive deficits occur, has become the health challenge of our time. The earliest known event in Alzheimer's disease is rising amyloid-ß. Previous studies have suggested that effects on synaptic transmission may precede plaque deposition. Here we report how relative levels of different soluble amyloid-ß peptides in hippocampus, preceding plaque deposition, relate to synaptic and genomic changes. Immunoprecipitation-mass spectrometry was used to measure the early rise of different amyloid-ß peptides in a mouse model of increasing amyloid-ß ('TASTPM', transgenic for familial Alzheimer's disease genes APP/PSEN1). In the third postnatal week, several amyloid-ß peptides were above the limit of detection, including amyloid-ß40, amyloid-ß38 and amyloid-ß42 with an intensity ratio of 6:3:2, respectively. By 2 months amyloid-ß levels had only increased by 50% and although the ratio of the different peptides remained constant, the first changes in synaptic currents, compared to wild-type mice could be detected with patch-clamp recordings. Between 2 and 4 months old, levels of amyloid-ß40 rose by ∼7-fold, but amyloid-ß42 rose by 25-fold, increasing the amyloid-ß42:amyloid-ß40 ratio to 1:1. Only at 4 months did plaque deposition become detectable and only in some mice; however, synaptic changes were evident in all hippocampal fields. These changes included increased glutamate release probability (P < 0.001, n = 7-9; consistent with the proposed physiological effect of amyloid-ß) and loss of spontaneous action potential-mediated activity in the cornu ammonis 1 (CA1) and dentate gyrus regions of the hippocampus (P < 0.001, n = 7). Hence synaptic changes occur when the amyloid-ß levels and amyloid-ß42:amyloid-ß40 ratio are still low compared to those necessary for plaque deposition. Genome-wide microarray analysis revealed changes in gene expression at 2-4 months including synaptic genes being strongly affected but often showing significant changes only by 4 months. We thus demonstrate that, in a mouse model of rising amyloid-ß, the initial deposition of plaques does not occur until several months after the first amyloid-ß becomes detectable but coincides with a rapid acceleration in the rise of amyloid-ß levels and the amyloid-ß42:amyloid-ß40 ratio. Prior to acceleration, however, there is already a pronounced synaptic dysfunction, reflected as changes in synaptic transmission and altered gene expression, indicating that restoring synaptic function early in the disease progression may represent the earliest possible target for intervention in the onset of Alzheimer's disease.

FoxO transcription factors in the maintenance of cellular homeostasis during aging.

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.

Chromosome X-wide association study in case control studies of pathologically confirmed Alzheimer's disease in a European population.

Although there are several genome-wide association studies available which highlight genetic variants associated with Alzheimer's disease (AD), often the X chromosome is excluded from the analysis. We conducted an X-chromosome-wide association study (XWAS) in three independent studies with a pathologically confirmed phenotype (total 1970 cases and 1113 controls). The XWAS was performed in males and females separately, and these results were then meta-analysed. Four suggestively associated genes were identified which may be of potential interest for further study in AD, these are DDX53 (rs12006935, OR = 0.52, p = 6.9e-05), IL1RAPL1 (rs6628450, OR = 0.36, p = 4.2e-05; rs137983810, OR = 0.52, p = 0.0003), TBX22 (rs5913102, OR = 0.74, p = 0.0003) and SH3BGRL (rs186553004, OR = 0.35, p = 0.0005; rs113157993, OR = 0.52, p = 0.0003), which replicate across at least two studies. The SNP rs5913102 in TBX22 achieves chromosome-wide significance in meta-analysed data. DDX53 shows highest expression in astrocytes, IL1RAPL1 is most highly expressed in oligodendrocytes and neurons and SH3BGRL is most highly expressed in microglia. We have also identified SNPs in the NXF5 gene at chromosome-wide significance in females (rs5944989, OR = 0.62, p = 1.1e-05) but not in males (p = 0.83). The discovery of relevant AD associated genes on the X chromosome may identify AD risk differences and similarities based on sex and lead to the development of sex-stratified therapeutics.

Microglia produce the amyloidogenic ABri peptide in familial British dementia.

Mutations in ITM2B cause familial British, Danish, Chinese and Korean dementias. In familial British dementia (FBD) a mutation in the stop codon of the ITM2B gene (also known as BRI2 ) causes a C-terminal cleavage fragment of the ITM2B/BRI2 protein to be extended by 11 amino acids. This fragment, termed amyloid-Bri (ABri), is highly insoluble and forms extracellular plaques in the brain. ABri plaques are accompanied by tau pathology, neuronal cell death and progressive dementia, with striking parallels to the aetiology and pathogenesis of Alzheimer's disease. The molecular mechanisms underpinning FBD are ill-defined. Using patient-derived induced pluripotent stem cells, we show that expression of ITM2B/BRI2 is 34-fold higher in microglia than neurons, and 15-fold higher in microglia compared with astrocytes. This cell-specific enrichment is supported by expression data from both mouse and human brain tissue. ITM2B/BRI2 protein levels are higher in iPSC-microglia compared with neurons and astrocytes. Consequently, the ABri peptide was detected in patient iPSC-derived microglial lysates and conditioned media but was undetectable in patient-derived neurons and control microglia. Pathological examination of post-mortem tissue support ABri expression in microglia that are in proximity to pre-amyloid deposits. Finally, gene co-expression analysis supports a role for ITM2B/BRI2 in disease-associated microglial responses. These data demonstrate that microglia are the major contributors to the production of amyloid forming peptides in FBD, potentially acting as instigators of neurodegeneration. Additionally, these data also suggest ITM2B/BRI2 may be part of a microglial response to disease, motivating further investigations of its role in microglial activation. This has implications for our understanding of the role of microglia and the innate immune response in the pathogenesis of FBD and other neurodegenerative dementias including Alzheimer's disease.

Whole genome analysis in APOE4 homozygotes identifies the DAB1-RELN pathway in Alzheimer's disease pathogenesis.

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 5102 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.

Knock-in models related to Alzheimer's disease: synaptic transmission, plaques and the role of microglia.

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.

Prodromal neuroinflammatory, cholinergic and metabolite dysfunction detected by PET and MRS in the TgF344-AD transgenic rat model of AD: a collaborative multi-modal study.

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 (Aß) 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, Aß, 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 Aß accumulation at 18 m, and (S)-[18F]THK5117 PET revealed subsequent Tau accumulation at 25m in hippocampal and cortical regions. Aß plaques were low but detectable by immunohistochemistry from 6 m, increasing further at 12 and 18 m with Tau-positive neurons adjacent to Aß plaques at 18 m. NeuroChrom (a pan neuronal marker) immunohistochemistry revealed a loss of neuronal staining at the Aß 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.

IGF-independent effects of insulin-like growth factor binding protein-5 (Igfbp5) in vivo.

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.

Genetic variability in response to amyloid beta deposition influences Alzheimer's disease risk.

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.

Effects of rising amyloidß levels on hippocampal synaptic transmission, microglial response and cognition in APPSwe/PSEN1M146V transgenic mice.

BACKGROUND: Progression of Alzheimer's disease is thought initially to depend on rising amyloidß and its synaptic interactions. Transgenic mice (TASTPM; APPSwe/PSEN1M146V) show altered synaptic transmission, compatible with increased physiological function of amyloidß, 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. METHODS: CA1 synaptic transmission and plasticity were investigated using in vitro electrophysiology. Microglial 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. FINDINGS: The 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. INTERPRETATION: The pre-plaque synaptic changes and microglial proliferation are presumably related to low, non-toxic amyloidß 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. FUNDING: GlaxoSmithKline; BBSRC; UCL; ARUK; MRC.

Improving Mouse Models for Dementia. Are All the Effects in Tau Mouse Models Due to Overexpression?

Mouse models of Alzheimer's disease have commonly used transgenic overexpression of genes involved in production of amyloid ß (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.

Akt2, a novel functional link between p38 mitogen-activated protein kinase and phosphatidylinositol 3-kinase pathways in myogenesis.

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.

Insulin-like growth factor-binding protein-5 induces a gender-related decrease in bone mineral density in transgenic mice.

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.