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Tauopathies, including Alzheimer's disease, corticobasal degeneration and progressive supranuclear palsy, are characterised by the aggregation of tau into insoluble neurofibrillary tangles in the brain. Tau is subject to a range of post-translational modifications, including proteolysis, that can promote its aggregation. Neuroinflammation is a hallmark of tauopathies and evidence is growing for a role of CD8+ T cells in disease pathogenesis. CD8+ T cells release granzyme proteases but what role these proteases play in neuronal dysfunction is currently lacking. Here, we identified that granzyme A (GzmA) is present in brain tissue and proteolytically cleaves tau. Mass spectrometric analysis of tau fragments produced on digestion of tau with GzmA identified three cleavage sites at R194-S195, R209-S210 and K240-S241. Mutation of the critical Arg or Lys residues at the cleavage sites in tau or chemical inhibition of GzmA blocked the proteolysis of tau by GzmA. Development of a semi-targeted mass spectrometry approach identified peptides in tauopathy brain tissue corresponding to proteolysis by GzmA at R209-S210 and K240-S241 in tau. When expressed in cells the GzmA-cleaved C-terminal fragments of tau were highly phosphorylated and aggregated upon incubation of the cells with tauopathy brain seed. The C-terminal fragment tau195–441 was able to transfer between cells and promote aggregation of tau in acceptor cells, indicating the propensity for such tau fragments to propagate between cells. Collectively, these results raise the possibility that GzmA, released from infiltrating cytotoxic CD8+ T cells, proteolytically cleaves tau into fragments that may contribute to its pathological properties in tauopathies. [ABSTRACT FROM AUTHOR]

Bone morphogenetic protein 1 (BMP1), a member of the astacin family of zinc‐metalloproteases, proteolytically cleaves the low‐density lipoprotein receptor (LDLR) within its ligand‐binding domain, reducing the binding and cellular uptake of LDL‐cholesterol. Here, we aimed to determine whether astacin proteases other than BMP1 may also cleave LDLR. Although human hepatocytes express all six astacin proteases, including the meprins and mammalian tolloid, we found through pharmacological inhibition and genetic knockdown that only BMP1 contributed to the cleavage of LDLR in its ligand‐binding domain. We also found that the minimum amino acid change required to render mouse LDLR susceptible to cleavage by BMP1 is mutation at the P1′ and P2 positions of the cleavage site. When expressed in cells, the resulting humanised‐mouse LDLR internalised LDL‐cholesterol. This work provides insight into the biological mechanisms regulating LDLR function. [ABSTRACT FROM AUTHOR]

Sporadic Alzheimer's disease (AD) is a neurodegenerative disorder that causes the most prevalent form of age-related dementia but its pathogenesis remains obscure. Altered regulation of metals, particularly pan-cerebral copper deficiency, and more regionally-localized perturbation of othermetals, are prominent in AD brain although data on how these CNS perturbations are reflected in the peripheral bloodstream are inconsistent to date. To assess the potential use of metal dysregulation to generate biomarkers in AD, we performed a case-control study of seven essential metals and selenium, measured by inductively coupled plasmamassspectrometry, in samples from AD and matched control cases. Metals were sodium, potassium, calcium, magnesium, iron, zinc, and copper. In the whole study-group and in female participants, plasma metal levels did not differ between cases and controls. In males by contrast, there was moderate evidence that zinc levels trended towards increase inAD[10.8 (10.2-11.5)] lmol/L, mean (± 95% CI; P = 0.021) compared with controls [10.2 (9.6-10.4)]. Thus alterations in plasma zinc levels differed between genders inAD. In correlational analysis, there was evidence for an increased number of 'strong' metal co-regulations in AD cases and differential comodulations of metal pairs: copper-sodium (Rcontrol- = - 0.03, RAD = 0.65; P = 0.009), and copper-calcium (Rcontrol = - 0.01, RAD = 0.65; P = 0.01) were significant in AD males, potentially consistent with reported evidence for dysregulation of copper in severely damaged brain regions in AD. In conclusion, our data suggest that the measurement of metals co-regulation in plasma may provide a useful representation of those metal perturbations taking place in the AD brain and therefore might be useful as plasma-based biomarkers. [ABSTRACT FROM AUTHOR]

Proteolysis of the amyloid precursor protein (APP) liberates various fragments including the proposed initiator of Alzheimer disease-associated dysfunctions, amyloid-β. However, recent evidence suggests that the accepted view of APP proteolysis by the canonical α-,β-, and γ-secretases is simplistic, with the discovery of a number of novel APP secretases (including ƍ- and η-secretases, alternative β-secretases) and additional metabolites, some of which may also cause synaptic dysfunction. Furthermore, various proteins have been identified that interact with APP and modulate its cleavage by the secretases. Here, we give an overview of the increasingly complex picture of APP proteolysis. [ABSTRACT FROM AUTHOR]

Amyloidogenic processing of the amyloid precursor protein (APP) by β- and γ-secretases generates several biologically active products, including amyloid-β (Aβ) and the APP intracellular domain (AICD). AICD regulates transcription of several neuronal genes, especially the Aβ-degrading enzyme, neprilysin (NEP). APP exists in several alternatively spliced isoforms, APP695, APP751, and APP770. We have examined whether each isoform can contribute to AICD generation and hence up-regulation of NEP expression. Using SH-SY5Y neuronal cells stably expressing each of the APP isoforms, we observed that only APP695 up-regulated nuclear AICD levels (9-fold) and NEP expression (6-fold). Increased NEP expression was abolished by a β- or γ-secretase inhibitor but not an α-secretase inhibitor. This correlated with a marked increase in both Aβ1-40 and Aβ1-42 in APP695 cells as compared with APP751 or APP770 cells. Similar phenomena were observed in Neuro2a but not HEK293 cells. SH-SY5Y cells expressing the Swedish mutant of APP695 also showed an increase in Aβ levels and NEP expression as compared with wild-type APP695 cells. Chromatin immunoprecipitation revealed that AICD was associated with the NEP promoter in APP695, Neuro2a, and APPSwe cells but not APP751 nor APP770 cells where AICD was replaced by histone deacetylase 1 (HDAC1). AICD occupancy of the NEP promoter was replaced by HDAC1 after treatment of the APP695 cells with a β- but not an α-secretase inhibitor. The increased AICD and NEP levels were significantly reduced in cholesterol-depleted APP695 cells. In conclusion, Aβ and functional AICD appear to be preferentially synthesized through β-secretase action on APP695. [ABSTRACT FROM AUTHOR]

In Alzheimer disease amyloid-β (β) peptides derived from the amyloid precursor protein (APP) accumulate in the brain. Cleavage of APP by the β-secretase BACE1 is the rate-limiting step in the production of AlL We have reported previously that the cellular prion protein (PrPC) inhibited the action of BACE1 toward human wild type APP (APPWT) in cellular models and that the levels of endogenous murine AfJ were significantly increased in PrPC~null mouse brain. Here we investigated the molecular and cellular mechanisms underlying this observation. PrPC interacted directly with the prodomain of the immature Golgi-localized form of BACE1. This interaction decreased BACE1 at the cell surface and in endosomes where it preferentially cleaves APPWT but increased it in the Golgi where it preferentially cleaves APP with the Swedish mutation (APP5we). In transgenic mice expressing human APP with the Swedish and Indiana familial mutations (APPSweLnd), PrPC deletion had no influence on APP proteolytic processing, Aβ plaque deposition, or levels of soluble Aβ or Aβ oligomers. In cells, although PrPC inhibited the action of BACE1 on APPWT, it did not inhibit BACE1 activity toward ~ The differential subcellular location of the BACE1 cleavage of APPβ we relative to APPWT provides an explanation for the failure of PrPC deletion to affect Aβ accumulation in APPSwe,Ind mice. Thus, although PrPC exerts no control on cleavage of APPSwe by BACE1, it has a profound influence on the cleavage of APP\VT, suggesting that PrPC may be a key protective player against sporadic Alzheimer disease. [ABSTRACT FROM AUTHOR]

Alzheimer's disease (AD) is characterised by the aggregation and deposition of amyloid-β (Aβ) peptides in the human brain. In age-related late-onset AD, deficient degradation and clearance, rather than enhanced production, of Aβ contributes to disease pathology. In the present study, we assessed the contribution of the two key Aβ-degrading zinc metalloproteases, insulin-degrading enzyme (IDE) and neprilysin (NEP), to Aβ degradation in human induced pluripotent stem cell (iPSC)-derived cortical neurons. Using an Aβ fluorescence polarisation assay, inhibition of IDE but not of NEP, blocked the degradation of Aβ by human neurons. When the neurons were grown in a 3D extracellular matrix to visualise Aβ deposition, inhibition of IDE but not NEP, increased the number of Aβ deposits. The resulting Aβ deposits were stained with the conformation-dependent, anti-amyloid antibodies A11 and OC that recognise Aβ aggregates in the human AD brain. Inhibition of the Aβ-forming β-secretase prevented the formation of the IDE-inhibited Aβ deposits. These data indicate that inhibition of IDE in live human neurons grown in a 3D matrix increased the deposition of Aβ derived from the proteolytic cleavage of the amyloid precursor protein. This work has implications for strategies aimed at enhancing IDE activity to promote Aβ degradation in AD., Competing Interests: The authors declare that there are no competing interests associated with this manuscript., (© 2023 The Author(s).)

Inclusion body myositis (IBM) is characterised by infiltration of CD8+ T-cells and signs of protein aggregation such as rimmed vacuoles and inclusion bodies. Aggregated proteins include those present in neurodegenerative diseases, and also those involved in protein homeostasis. The aim of this review is to discuss the pathological effects of protein aggregates and the process of aggregation following immune attack in IBM. Immune attack is likely to cause protein aggregation by impairing endoplasmic reticulum (ER) and mitochondrial function. Apoptotic and necrotic pathways are activated, possibly leading to nucleo-cytoplasmic coagulation. Overexpression of nuclear and ribosomal proteins in rimmed vacuoles suggests that the vacuoles develop from the collapse of myonuclei and the surrounding ER. Aggregated proteins can activate the NLR family pyrin domain containing 3 (NLRP3) inflammasome or provoke a humoral immune response. Heat shock proteins, ribosomal proteins and protein fragments may provoke interferon-gamma and cytotoxic T-cell responses in a similar manner to Mycobacterium tuberculosis antigens. Persistent provocation can lead to T-cell large granular lymphocytic leukaemia which is resistant to immunosuppression, and would explain the progression from polymyositis to IBM. Protein aggregates may impair the cellular machinery, and proteins may propagate along a myocyte in a prion-like manner. These pathological mechanisms may prevent myocyte regeneration following damage from eccentric muscle contraction, causing weakness and atrophy in a characteristic pattern. Further understanding of the mechanisms of protein aggregation in IBM may lead to additional therapies as well as novel muscle and blood biomarkers. Earlier diagnosis and treatment may result in improved outcomes when effective therapies are available.

The neurovascular unit (NVU), consisting of neurons, glial cells, vascular cells (endothelial cells, pericytes and vascular smooth muscle cells (VSMCs)) together with the surrounding extracellular matrix (ECM), is an important interface between the peripheral blood and the brain parenchyma. Disruption of the NVU impacts on blood-brain barrier (BBB) regulation and underlies the development and pathology of multiple neurological disorders, including stroke and Alzheimer's disease (AD). The ability to differentiate induced pluripotent stem cells (iPSCs) into the different cell types of the NVU and incorporate them into physical models provides a reverse engineering approach to generate human NVU models to study BBB function. To recapitulate the in vivo situation such NVU models must also incorporate the ECM to provide a 3D environment with appropriate mechanical and biochemical cues for the cells of the NVU. In this review, we provide an overview of the cells of the NVU and the surrounding ECM, before discussing the characteristics (stiffness, functionality and porosity) required of hydrogels to mimic the ECM when incorporated into in vitro NVU models. We summarise the approaches available to measure BBB functionality and present the techniques in use to develop robust and translatable models of the NVU, including transwell models, hydrogel models, 3D-bioprinting, microfluidic models and organoids. The incorporation of iPSCs either without or with disease-specific genetic mutations into these NVU models provides a platform in which to study normal and disease mechanisms, test BBB permeability to drugs, screen for new therapeutic targets and drugs or to design cell-based therapies., Competing Interests: The authors declare that there are no competing interests associated with the manuscript., (© 2021 The Author(s).)

The cellular prion protein (PrP C ) is a key neuronal receptor for β-amyloid oligomers (AβO), mediating their neurotoxicity, which contributes to the neurodegeneration in Alzheimer's disease (AD). Similarly to the amyloid precursor protein (APP), PrP C is proteolytically cleaved from the cell surface by a disintegrin and metalloprotease, ADAM10. We hypothesized that ADAM10-modulated PrP C shedding would alter the cellular binding and cytotoxicity of AβO. Here, we found that in human neuroblastoma cells, activation of ADAM10 with the muscarinic agonist carbachol promotes PrP C shedding and reduces the binding of AβO to the cell surface, which could be blocked with an ADAM10 inhibitor. Conversely, siRNA-mediated ADAM10 knockdown reduced PrP C shedding and increased AβO binding, which was blocked by the PrP C -specific antibody 6D11. The retinoic acid receptor analog acitretin, which up-regulates ADAM10, also promoted PrP C shedding and decreased AβO binding in the neuroblastoma cells and in human induced pluripotent stem cell (iPSC)-derived cortical neurons. Pretreatment with acitretin abolished activation of Fyn kinase and prevented an increase in reactive oxygen species caused by AβO binding to PrP C Besides blocking AβO binding and toxicity, acitretin also increased the nonamyloidogenic processing of APP. However, in the iPSC-derived neurons, Aβ and other amyloidogenic processing products did not exhibit a reciprocal decrease upon acitretin treatment. These results indicate that by promoting the shedding of PrP C in human neurons, ADAM10 activation prevents the binding and cytotoxicity of AβO, revealing a potential therapeutic benefit of ADAM10 activation in AD., (© 2019 Jarosz-Griffiths et al.)

Cerebral ischemia is known to be a major cause of death and the later development of Alzheimer's disease and vascular dementia. However, ischemia induced cellular damage that initiates these diseases remain poorly understood. This is primarily due to lack of clinically relevant models that are highly reproducible. Here, we have optimised a murine model of global cerebral ischaemia with multiple markers to determine brain pathology, neurochemistry and correlated memory deficits in these animals. Cerebral ischaemia in mice was induced by bilateral common carotid artery occlusion. Following reperfusion, the mice were either fixed with 4% paraformaldehyde or decapitated under anaesthesia. Brains were processed for Western blotting or immunohistochemistry for glial (GLT1) and vesicular (VGLUT1, VGLUT2) glutamate transporters and paired helical filament (PHF1) tau. The PHF1 tau is the main component of neurofibrillary tangle, which is the pathological hallmark of Alzheimer's disease and vascular dementia. The novel object recognition behavioural assay was used to investigate the functional cognitive consequences in these mice. The results show consistent and selective neuronal and glial cell changes in the hippocampus and the cortex together with significant reductions in GLT1 (***P < 0.001), VGLUT1 (**P < 0.01) and VGLUT2 (***P < 0.001) expressions in the hippocampus in occluded mice as compared to sham-operated animals. These changes are associated with increased PHF1 (***P < 0.0001) protein and a significant impairment of performance (*p < 0.0006, N = 6/group) in the novel object recognition test. This model represents a useful tool for investigating cellular, biochemical and molecular mechanisms of global cerebral ischaemia and may be an ideal preclinical model for vascular dementia., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Background: Tissue non-specific alkaline phosphatase (TNAP) has been shown to promote the neurotoxicity of extracellular tau which contributes to the spread of pathology in Alzheimer's disease (AD)., Objective: To investigate changes in TNAP activity in the hippocampus in both sporadic and familial AD, and to examine whether changes in neuronal TNAP are reflected systemically by looking at changes in plasma TNAP activity in AD., Methods: We measured the activity of TNAP in the hippocampus in sporadic AD, familial AD and appropriate age-matched controls, and in an ageing series (age: 25-88 years) of brains. In addition, we measured TNAP activity in plasma from 110 AD and 110 non-demented control participants., Results: TNAP activity was significantly increased in the hippocampus in sporadic (by 56%; p = 0.038) and familial AD (by 121%; p = 0.042) compared with the age-matched controls. However, there was no correlation of TNAP activity with age. Furthermore, plasma TNAP activity was increased in AD (by 13%; p = 0.018) and inversely correlated with cognitive function (r(s) = -0.211; p = 0.027)., Conclusion: Together, these data indicate that TNAP is increased in both sporadic and familial AD but not in the aged brain, indicating that the increase is likely a consequence of AD-associated changes in the brain. The neuronal change in TNAP is reflected in an increase in plasma TNAP in AD and is inversely correlated with cognitive function., (Copyright © 2011 S. Karger AG, Basel.)

A novel series of isatin-based inhibitors of beta-secretase (BACE-1) have been identified using a virtual high-throughput screening approach. Structure-activity relationship studies revealed structural features important for inhibition. Docking studies suggest these inhibitors may bind within the BACE-1 active site through H-bonding interactions involving the catalytic aspartate residues.

Alzheimer and prion diseases are neurodegenerative disorders characterised by the abnormal processing of amyloid-beta (Abeta) peptide and prion protein (PrP(C)), respectively. Recent evidence indicates that PrP(C) may play a critical role in the pathogenesis of Alzheimer disease. PrP(C) interacts with and inhibits the beta-secretase BACE1, the rate-limiting enzyme in the production of Abeta. More recently PrP(C) was identified as a receptor for Abeta oligomers and the expression of PrP(C) appears to be controlled by the amyloid intracellular domain (AICD). Here we review these observations and propose a feedback loop in the normal brain where PrP(C) exerts an inhibitory effect on BACE1 to decrease both Abeta and AICD production. In turn, the AICD upregulates PrP(C) expression, thus maintaining the inhibitory effect of PrP(C) on BACE1. In Alzheimer disease, this feedback loop is disrupted, and the increased level of Abeta oligomers bind to PrP(C) and prevent it from regulating BACE1 activity.