Proteomics of the astrocyte secretome reveals changes in their response to soluble oligomeric Aß.

Astrocytes associate with amyloid plaques in Alzheimer's disease (AD). Astrocytes react to changes in the brain environment, including increasing concentrations of amyloid-ß (Aß). However, the precise response of astrocytes to soluble small Aß oligomers at concentrations similar to those present in the human brain has not been addressed. In this study, we exposed astrocytes to media from neurons that express the human amyloid precursor protein (APP) transgene with the double Swedish mutation (APPSwe), and which contains APP-derived fragments, including soluble human Aß oligomers. We then used proteomics to investigate changes in the astrocyte secretome. Our data show dysregulated secretion of astrocytic proteins involved in the extracellular matrix and cytoskeletal organization and increase secretion of proteins involved in oxidative stress responses and those with chaperone activity. Several of these proteins have been identified in previous transcriptomic and proteomic studies using brain tissue from human AD and cerebrospinal fluid (CSF). Our work highlights the relevance of studying astrocyte secretion to understand the brain response to AD pathology and the potential use of these proteins as biomarkers for the disease.

P2X7R influences tau aggregate burden in human tauopathies and shows distinct signalling in microglia and astrocytes.

The purinoceptor P2X7R is a promising therapeutic target for tauopathies, including Alzheimer's disease (AD). Pharmacological inhibition or genetic knockdown of P2X7R ameliorates cognitive deficits and reduces pathological tau burden in mice that model aspects of tauopathy, including mice expressing mutant human frontotemporal dementia (FTD)-causing forms of tau. However, disagreements remain over which glial cell types express P2X7R and therefore the mechanism of action is unresolved. Here, we show that P2X7R protein levels increase in human AD post-mortem brain, in agreement with an upregulation of P2RX7 mRNA observed in transcriptome profiles from the AMP-AD consortium. P2X7R protein increases mirror advancing Braak stage and coincide with synapse loss. Using RNAScope we detect P2RX7 mRNA in microglia and astrocytes in human AD brain, including in the vicinity of senile plaques. In cultured microglia, P2X7R activation modulates the NLRP3 inflammasome pathway by promoting the formation of active complexes and release of IL-1ß. In astrocytes, P2X7R activates NFκB signalling and increases production of the cytokines CCL2, CXCL1 and IL-6 together with the acute phase protein Lcn2. To further explore the role of P2X7R in a disease-relevant context, we expressed wild-type or FTD-causing mutant forms of tau in mouse organotypic brain slice cultures. Inhibition of P2X7R reduces insoluble tau levels without altering soluble tau phosphorylation or synaptic localisation, suggesting a non-cell autonomous role of glial P2X7R on pathological tau aggregation. These findings support further investigations into the cell-type specific effects of P2X7R-targeting therapies in tauopathies.

Artificial intelligence for neurodegenerative experimental models.

INTRODUCTION: Experimental models are essential tools in neurodegenerative disease research. However, the translation of insights and drugs discovered in model systems has proven immensely challenging, marred by high failure rates in human clinical trials. METHODS: Here we review the application of artificial intelligence (AI) and machine learning (ML) in experimental medicine for dementia research. RESULTS: Considering the specific challenges of reproducibility and translation between other species or model systems and human biology in preclinical dementia research, we highlight best practices and resources that can be leveraged to quantify and evaluate translatability. We then evaluate how AI and ML approaches could be applied to enhance both cross-model reproducibility and translation to human biology, while sustaining biological interpretability. DISCUSSION: AI and ML approaches in experimental medicine remain in their infancy. However, they have great potential to strengthen preclinical research and translation if based upon adequate, robust, and reproducible experimental data. HIGHLIGHTS: There are increasing applications of AI in experimental medicine. We identified issues in reproducibility, cross-species translation, and data curation in the field. Our review highlights data resources and AI approaches as solutions. Multi-omics analysis with AI offers exciting future possibilities in drug discovery.

Investigating key factors underlying neurodegeneration linked to alpha-synuclein spread.

AIMS: It has long been considered that accumulation of pathological alpha-synuclein (aSyn) leads to synaptic/neuronal loss which then results in behavioural and cognitive dysfunction. To investigate this claim, we investigated effects downstream of aSyn preformed fibrils (PFFs) and 6-hydroxydopamine (6-OHDA), because aSyn PFFs induce spreading/accumulation of aSyn, and 6-OHDA rapidly causes local neuronal loss. METHODS: We injected mouse aSyn PFFs into the medial forebrain bundle (MFB) of Sprague-Dawley rats. We investigated spread of pathological aSyn, phosphorylation of aSyn and tau, oxidative stress, synaptic/neuronal loss and cognitive dysfunction 60, 90 and 120 days after injection. Similarly, we injected 6-OHDA into the MFB and examined the same parameters 1 and 3 weeks after injection. RESULTS: Following aSyn PFF injection, phosphorylated aSyn was found distant from the injection site in the hippocampus and frontal cortex. However, despite neuron loss being evident close to the site of injection in the substantia nigra at 120 days post injection, there were no other neurodegeneration-associated features associated with aSyn including synaptic loss. In contrast, 6-OHDA caused severe neuronal loss in the substantia nigra at 3 weeks post injection that was accompanied by phosphorylation of aSyn and tau, oxidative stress, loss of synaptic proteins, cognitive and motor dysfunction. CONCLUSIONS: Our results demonstrate that spread/replication and slow accumulation of pathological aSyn may not be sufficient to induce neurodegenerative changes. In contrast, oxidative stress responses in addition to aSyn accumulation were associated with other Parkinson's disease (PD)-associated abnormalities and cognitive dysfunction. Our results may be important when considering why only some PD patients develop dementia.

Tau in the gut, does it really matter?

The enteric nervous system plays a critical role in the regulation of gastrointestinal tract functions and is often referred to as the 'second brain' because it shares many features with the central nervous system. These similarities include among others a large panel of neurotransmitters, a large population of glial cells and a susceptibility to neurodegeneration. This close homology between the central and enteric nervous systems suggests that a disease process affecting the central nervous system could also involve its enteric counterpart. This was already documented in Parkinson's disease, the most common synucleinopathy, in which alpha-synuclein deposits are reported in the enteric nervous system in the vast majority of patients. Tau is another key protein involved in neurodegenerative disorders of the brain. Whether changes in tau also occur in the enteric nervous system during gut or brain disorders has just begun to be explored. The scope of the present article is therefore to review existing studies on the expression and phosphorylation pattern of tau in the enteric nervous system under physiological and pathological conditions and to discuss the possible occurrence of 'enteric tauopathies'.

Astrocytic C-X-C motif chemokine ligand-1 mediates ß-amyloid-induced synaptotoxicity.

BACKGROUND: Pathological interactions between ß-amyloid (Aß) and tau drive synapse loss and cognitive decline in Alzheimer's disease (AD). Reactive astrocytes, displaying altered functions, are also a prominent feature of AD brain. This large and heterogeneous population of cells are increasingly recognised as contributing to early phases of disease. However, the contribution of astrocytes to Aß-induced synaptotoxicity in AD is not well understood. METHODS: We stimulated mouse and human astrocytes with conditioned medium containing concentrations and species of human Aß that mimic those in human AD brain. Medium from stimulated astrocytes was collected and immunodepleted of Aß before being added to naïve rodent or human neuron cultures. A cytokine, identified in unbiased screens of stimulated astrocyte media and in postmortem human AD brain lysates was also applied to neurons, including those pre-treated with a chemokine receptor antagonist. Tau mislocalisation, synaptic markers and dendritic spine numbers were measured in cultured neurons and organotypic brain slice cultures. RESULTS: We found that conditioned medium from stimulated astrocytes induces exaggerated synaptotoxicity that is recapitulated following spiking of neuron culture medium with recombinant C-X-C motif chemokine ligand-1 (CXCL1), a chemokine upregulated in AD brain. Antagonism of neuronal C-X-C motif chemokine receptor 2 (CXCR2) prevented synaptotoxicity in response to CXCL1 and Aß-stimulated astrocyte secretions. CONCLUSIONS: Our data indicate that astrocytes exacerbate the synaptotoxic effects of Aß via interactions of astrocytic CXCL1 and neuronal CXCR2 receptors, highlighting this chemokine-receptor pair as a novel target for therapeutic intervention in AD.

Tau accumulates in Crohn's disease gut.

A sizeable body of evidence has recently emerged to suggest that gastrointestinal (GI) inflammation might be involved in the development of Parkinson's disease (PD). There is now strong epidemiological and genetical evidence linking PD to inflammatory bowel diseases and we recently demonstrated that the neuronal protein alpha-synuclein, which is critically involved in PD pathophysiology, is upregulated in inflamed segments of Crohn's colon. The microtubule associated protein tau is another neuronal protein critically involved in neurodegenerative disorders but, in contrast to alpha-synuclein, no data are available about its expression and phosphorylation patterns in inflammatory bowel diseases. Here, we examined the expression levels of tau isoforms, their phosphorylation profile and truncation in colon biopsy specimens from 16 Crohn's disease (CD) and 6 ulcerative colitis (UC) patients and compared them to samples from 16 controls. Additional experiments were performed in full thickness segments of colon of five CD and five control subjects, in primary cultures of rat enteric neurons and in nuclear factor erythroid 2-related factor (Nrf2) knockout mice. Our results show the upregulation of two main human tau isoforms in the enteric nervous system (ENS) in CD but not in UC. This upregulation was not transcriptionally regulated but instead likely resulted from a decrease in protein clearance via an Nrf2 pathway. Our findings, which provide the first detailed characterization of tau in CD, suggest that the key proteins involved in neurodegenerative disorders such as alpha-synuclein and tau, might also play a role in CD.

Disruption of endoplasmic reticulum-mitochondria tethering proteins in post-mortem Alzheimer's disease brain.

Signaling between the endoplasmic reticulum (ER) and mitochondria regulates a number of key neuronal functions, many of which are perturbed in Alzheimer's disease. Moreover, damage to ER-mitochondria signaling is seen in cell and transgenic models of Alzheimer's disease. However, as yet there is little evidence that ER-mitochondria signaling is altered in human Alzheimer's disease brains. ER-mitochondria signaling is mediated by interactions between the integral ER protein VAPB and the outer mitochondrial membrane protein PTPIP51 which act to recruit and "tether" regions of ER to the mitochondrial surface. The VAPB-PTPIP51 tethers are now known to regulate a number of ER-mitochondria signaling functions including delivery of Ca2+from ER stores to mitochondria, mitochondrial ATP production, autophagy and synaptic activity. Here we investigate the VAPB-PTPIP51 tethers in post-mortem control and Alzheimer's disease brains. Quantification of ER-mitochondria signaling proteins by immunoblotting revealed loss of VAPB and PTPIP51 in cortex but not cerebellum at end-stage Alzheimer's disease. Proximity ligation assays were used to quantify the VAPB-PTPIP51 interaction in temporal cortex pyramidal neurons and cerebellar Purkinje cell neurons in control, Braak stage III-IV (early/mid-dementia) and Braak stage VI (severe dementia) cases. Pyramidal neurons degenerate in Alzheimer's disease whereas Purkinje cells are less affected. These studies revealed that the VAPB-PTPIP51 tethers are disrupted in Braak stage III-IV pyramidal but not Purkinje cell neurons. Thus, we identify a new pathogenic event in post-mortem Alzheimer's disease brains. The implications of our findings for Alzheimer's disease mechanisms are discussed.

Direct Keap1-Nrf2 disruption as a potential therapeutic target for Alzheimer's disease.

Nrf2, a transcriptional activator of cell protection genes, is an attractive therapeutic target for the prevention of neurodegenerative diseases, including Alzheimer's disease (AD). Current Nrf2 activators, however, may exert toxicity and pathway over-activation can induce detrimental effects. An understanding of the mechanisms mediating Nrf2 inhibition in neurodegenerative conditions may therefore direct the design of drugs targeted for the prevention of these diseases with minimal side-effects. Our study provides the first in vivo evidence that specific inhibition of Keap1, a negative regulator of Nrf2, can prevent neuronal toxicity in response to the AD-initiating Aß42 peptide, in correlation with Nrf2 activation. Comparatively, lithium, an inhibitor of the Nrf2 suppressor GSK-3, prevented Aß42 toxicity by mechanisms independent of Nrf2. A new direct inhibitor of the Keap1-Nrf2 binding domain also prevented synaptotoxicity mediated by naturally-derived Aß oligomers in mouse cortical neurons. Overall, our findings highlight Keap1 specifically as an efficient target for the re-activation of Nrf2 in AD, and support the further investigation of direct Keap1 inhibitors for the prevention of neurodegeneration in vivo.

Synaptic Localisation of Tau.

The microtubule-associated protein tau has been identified in several intraneuronal compartments, including in association with synapses. In Alzheimer's disease, frontotemporal dementia and related tauopathies, highly phosphorylated tau accumulates as intraneuronal protein aggregates that are likely responsible for the demise of neurons and the subsequent progressive cognitive decline. However, the molecular mechanisms underlying such tau-mediated damage in the tauopathies is not fully understood. Tauopathy induces loss of synapses, which is one of the earliest structural correlates of cognitive dysfunction and disease progression. Notably, altered post-translational modifications of tau, including increased phosphorylation and acetylation, augment the mislocalisation of tau to synapses, impair synaptic vesicle release and might influence the activity-dependent release of tau from neurons. Thus, disease-associated accumulation of modified tau at the synapse adversely affects critical neuronal processes that are linked to neuronal activity and synaptic function. These findings emphasise the importance of gaining a comprehensive understanding of the diverse roles of tau at distinct intraneuronal locations. An improved knowledge of the impact of synaptic tau under physiological and pathological conditions and how tau localisation impacts on neuronal function will provide valuable insights that may lead to the development of new therapies for the tauopathies.

ALS/FTD-associated FUS activates GSK-3ß to disrupt the VAPB-PTPIP51 interaction and ER-mitochondria associations.

Defective FUS metabolism is strongly associated with amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD), but the mechanisms linking FUS to disease are not properly understood. However, many of the functions disrupted in ALS/FTD are regulated by signalling between the endoplasmic reticulum (ER) and mitochondria. This signalling is facilitated by close physical associations between the two organelles that are mediated by binding of the integral ER protein VAPB to the outer mitochondrial membrane protein PTPIP51, which act as molecular scaffolds to tether the two organelles. Here, we show that FUS disrupts the VAPB-PTPIP51 interaction and ER-mitochondria associations. These disruptions are accompanied by perturbation of Ca(2+) uptake by mitochondria following its release from ER stores, which is a physiological read-out of ER-mitochondria contacts. We also demonstrate that mitochondrial ATP production is impaired in FUS-expressing cells; mitochondrial ATP production is linked to Ca(2+) levels. Finally, we demonstrate that the FUS-induced reductions to ER-mitochondria associations and are linked to activation of glycogen synthase kinase-3ß (GSK-3ß), a kinase already strongly associated with ALS/FTD.

Roles of tau protein in health and disease.

Tau is well established as a microtubule-associated protein in neurons. However, under pathological conditions, aberrant assembly of tau into insoluble aggregates is accompanied by synaptic dysfunction and neural cell death in a range of neurodegenerative disorders, collectively referred to as tauopathies. Recent advances in our understanding of the multiple functions and different locations of tau inside and outside neurons have revealed novel insights into its importance in a diverse range of molecular pathways including cell signalling, synaptic plasticity, and regulation of genomic stability. The present review describes the physiological and pathophysiological properties of tau and how these relate to its distribution and functions in neurons. We highlight the post-translational modifications of tau, which are pivotal in defining and modulating tau localisation and its roles in health and disease. We include discussion of other pathologically relevant changes in tau, including mutation and aggregation, and how these aspects impinge on the propensity of tau to propagate, and potentially drive neuronal loss, in diseased brain. Finally, we describe the cascade of pathological events that may be driven by tau dysfunction, including impaired axonal transport, alterations in synapse and mitochondrial function, activation of the unfolded protein response and defective protein degradation. It is important to fully understand the range of neuronal functions attributed to tau, since this will provide vital information on its involvement in the development and pathogenesis of disease. Such knowledge will enable determination of which critical molecular pathways should be targeted by potential therapeutic agents developed for the treatment of tauopathies.

α-Synuclein binds to the ER-mitochondria tethering protein VAPB to disrupt Ca2+ homeostasis and mitochondrial ATP production.

α-Synuclein is strongly linked to Parkinson's disease but the molecular targets for its toxicity are not fully clear. However, many neuronal functions damaged in Parkinson's disease are regulated by signalling between the endoplasmic reticulum (ER) and mitochondria. This signalling involves close physical associations between the two organelles that are mediated by binding of the integral ER protein vesicle-associated membrane protein-associated protein B (VAPB) to the outer mitochondrial membrane protein, protein tyrosine phosphatase-interacting protein 51 (PTPIP51). VAPB and PTPIP51 thus act as a scaffold to tether the two organelles. Here we show that α-synuclein binds to VAPB and that overexpression of wild-type and familial Parkinson's disease mutant α-synuclein disrupt the VAPB-PTPIP51 tethers to loosen ER-mitochondria associations. This disruption to the VAPB-PTPIP51 tethers is also seen in neurons derived from induced pluripotent stem cells from familial Parkinson's disease patients harbouring pathogenic triplication of the α-synuclein gene. We also show that the α-synuclein induced loosening of ER-mitochondria contacts is accompanied by disruption to Ca2+ exchange between the two organelles and mitochondrial ATP production. Such disruptions are likely to be particularly damaging to neurons that are heavily dependent on correct Ca2+ signaling and ATP.

Alzheimer-related decrease in CYFIP2 links amyloid production to tau hyperphosphorylation and memory loss.

Characteristic features of Alzheimer's disease are memory loss, plaques resulting from abnormal processing of amyloid precursor protein (APP), and presence of neurofibrillary tangles and dystrophic neurites containing hyperphosphorylated tau. Currently, it is not known what links these abnormalities together. Cytoplasmic FMR1 interacting protein 2 (CYFIP2) has been suggested to regulate mRNA translation at synapses and this may include local synthesis of APP and alpha-calcium/calmodulin-dependent kinase II, a kinase that can phosphorylate tau. Further, CYFIP2 is part of the Wiskott-Aldrich syndrome protein-family verprolin-homologous protein complex, which has been implicated in actin polymerization at synapses, a process thought to be required for memory formation. Our previous studies on p25 dysregulation put forward the hypothesis that CYFIP2 expression is reduced in Alzheimer's disease and that this contributes to memory impairment, abnormal APP processing and tau hyperphosphorylation. Here, we tested this hypothesis. First, in post-mortem tissue CYFIP2 expression was reduced by ∼50% in severe Alzheimer's hippocampus and superior temporal gyrus when normalized to expression of a neuronal or synaptic marker protein. Interestingly, there was also a trend for decreased expression in mild Alzheimer's disease hippocampus. Second, CYFIP2 expression was reduced in old but not in young Tg2576 mice, a model of familial Alzheimer's disease. Finally, we tested the direct impact of reduced CYFIP2 expression in heterozygous null mutant mice. We found that in hippocampus this reduced expression causes an increase in APP and ß-site amyloid precursor protein cleaving enzyme 1 (BACE1) protein, but not mRNA expression, and elevates production of amyloid-ß42 Reduced CYFIP2 expression also increases alpha-calcium/calmodulin-dependent kinase II protein expression, and this is associated with hyperphosphorylation of tau at serine-214. The reduced expression also impairs spine maturity without affecting spine density in apical dendrites of CA1 pyramidal neurons. Furthermore, the reduced expression prevents retention of spatial memory in the water maze. Taken together, our findings indicate that reduced CYFIP2 expression triggers a cascade of change towards Alzheimer's disease, including amyloid production, tau hyperphosphorylation and memory loss. We therefore suggest that CYFIP2 could be a potential hub for targeting treatment of the disease.

Tauopathy induced by low level expression of a human brain-derived tau fragment in mice is rescued by phenylbutyrate.

Human neurodegenerative tauopathies exhibit pathological tau aggregates in the brain along with diverse clinical features including cognitive and motor dysfunction. Post-translational modifications including phosphorylation, ubiquitination and truncation, are characteristic features of tau present in the brain in human tauopathy. We have previously reported an N-terminally truncated form of tau in human brain that is associated with the development of tauopathy and is highly phosphorylated. We have generated a new mouse model of tauopathy in which this human brain-derived, 35 kDa tau fragment (Tau35) is expressed in the absence of any mutation and under the control of the human tau promoter. Most existing mouse models of tauopathy overexpress mutant tau at levels that do not occur in human neurodegenerative disease, whereas Tau35 transgene expression is equivalent to less than 10% of that of endogenous mouse tau. Tau35 mice recapitulate key features of human tauopathies, including aggregated and abnormally phosphorylated tau, progressive cognitive and motor deficits, autophagic/lysosomal dysfunction, loss of synaptic protein, and reduced life-span. Importantly, we found that sodium 4-phenylbutyrate (Buphenyl®), a drug used to treat urea cycle disorders and currently in clinical trials for a range of neurodegenerative diseases, reverses the observed abnormalities in tau and autophagy, behavioural deficits, and loss of synapsin 1 in Tau35 mice. Our results show for the first time that, unlike other tau transgenic mouse models, minimal expression of a human disease-associated tau fragment in Tau35 mice causes a profound and progressive tauopathy and cognitive changes, which are rescued by pharmacological intervention using a clinically approved drug. These novel Tau35 mice therefore represent a highly disease-relevant animal model in which to investigate molecular mechanisms and to develop novel treatments for human tauopathies.

Loss of c-Jun N-terminal kinase-interacting protein-1 does not affect axonal transport of the amyloid precursor protein or Aß production.

Disruption to axonal transport is an early pathological feature in Alzheimer's disease. The amyloid precursor protein (APP) is a key axonal transport cargo in Alzheimer's disease since perturbation of its transport increases APP processing and production of amyloid-ß peptide (Aß) that is deposited in the brains of Alzheimer's disease patients. APP is transported anterogradely through axons on kinesin-1 motors. One favoured route for attachment of APP to kinesin-1 involves the scaffolding protein c-Jun N-terminal kinase-interacting protein-1 (JIP1), which has been shown to bind both APP and kinesin-1 light chain (KLC). However, direct experimental evidence to support a role of JIP1 in APP transport is lacking. Notably, the effect of loss of JIP1 on movement of APP through axons of living neurons, and the impact of such loss on APP processing and Aß production has not been reported. To address these issues, we monitored how siRNA mediated loss of JIP1 influenced transport of enhanced green fluorescent protein (EGFP)-tagged APP through axons and production of endogenous Aß in living neurons. Surprisingly, we found that knockdown of JIP1 did not affect either APP transport or Aß production. These results have important implications for our understanding of APP trafficking in Alzheimer's disease.

Physiological release of endogenous tau is stimulated by neuronal activity.

Propagation of tau pathology is linked with progressive neurodegeneration, but the mechanism underlying trans-synaptic spread of tau is unknown. We show that stimulation of neuronal activity, or AMPA receptor activation, induces tau release from healthy, mature cortical neurons. Notably, phosphorylation of extracellular tau appears reduced in comparison with intracellular tau. We also find that AMPA-induced release of tau is calcium-dependent. Blocking pre-synaptic vesicle release by tetanus toxin and inhibiting neuronal activity with tetrodotoxin both significantly impair AMPA-mediated tau release. Tau secretion is therefore a regulatable process, dysregulation of which could lead to the spread of tau pathology in disease.

Prostate-derived sterile 20-like kinases (PSKs/TAOKs) phosphorylate tau protein and are activated in tangle-bearing neurons in Alzheimer disease.

In Alzheimer disease (AD), the microtubule-associated protein tau is highly phosphorylated and aggregates into characteristic neurofibrillary tangles. Prostate-derived sterile 20-like kinases (PSKs/TAOKs) 1 and 2, members of the sterile 20 family of kinases, have been shown to regulate microtubule stability and organization. Here we show that tau is a good substrate for PSK1 and PSK2 phosphorylation with mass spectrometric analysis of phosphorylated tau revealing more than 40 tau residues as targets of these kinases. Notably, phosphorylated residues include motifs located within the microtubule-binding repeat domain on tau (Ser-262, Ser-324, and Ser-356), sites that are known to regulate tau-microtubule interactions. PSK catalytic activity is enhanced in the entorhinal cortex and hippocampus, areas of the brain that are most susceptible to Alzheimer pathology, in comparison with the cerebellum, which is relatively spared. Activated PSK is associated with neurofibrillary tangles, dystrophic neurites surrounding neuritic plaques, neuropil threads, and granulovacuolar degeneration bodies in AD brain. By contrast, activated PSKs and phosphorylated tau are rarely detectible in immunostained control human brain. Our results demonstrate that tau is a substrate for PSK and suggest that this family of kinases could contribute to the development of AD pathology and dementia.