nNOS–CAPON interaction mediates amyloid‐β‐induced neurotoxicity,especially in the early stages

In neurons, increased protein–protein interactions between neuronal nitric oxide synthase (nNOS) and its carboxy‐terminal PDZ ligand (CAPON) contribute to excitotoxicity and abnormal dendritic spine development, both of which are involved in the development of Alzheimer's disease. In models of Alzheimer's disease, increased nNOS–CAPON interaction was detected after treatment with amyloid‐β in vitro, and a similar change was found in the hippocampus of APP/PS1 mice (a transgenic mouse model of Alzheimer's disease), compared with age‐matched background mice in vivo. After blocking the nNOS–CAPON interaction, memory was rescued in 4‐month‐old APP/PS1 mice, and dendritic impairments were ameliorated both in vivo and in vitro. Furthermore, we demonstrated that S‐nitrosylation of Dexras1 and inhibition of the ERK–CREB–BDNF pathway might be downstream of the nNOS–CAPON interaction.     

The nature of the cell cycle in neurons: Focus on a “non-canonical” pathway of DNA replication causally related to death

The mechanism whereby a reactivation of cell cycle in neurons causes cell death is beginning to be identified. In cellular models of Alzheimer's disease, activation of a non-canonical pathway of DNA replication contributes to neuronal death. This pathway involves the repair enzyme DNA polymerase-β, which is highly expressed in neurons of the Alzheimer's brain at early stages of the disease. Loading of DNA polymerase-β into the replication forks generates a death signal, which involves the tumor suppressor p53. The increasing knowledge of the main actors of the unscheduled DNA replication in neurons will pave the way for novel therapeutic interventions in Alzheimer's disease and other neurodegenerative disorders.     

Mitochondrial dysfunction is a trigger of Alzheimer's disease pathophysiology

Mitochondria are uniquely poised to play a pivotal role in neuronal cell survival or death because they are regulators of both energy metabolism and cell death pathways. Extensive literature exists supporting a role for mitochondrial dysfunction and oxidative damage in the pathogenesis of Alzheimer's disease. This review discusses evidence indicating that mitochondrial dysfunction has an early and preponderant role in Alzheimer's disease. Furthermore, the link between mitochondrial dysfunction and autophagy in Alzheimer's disease is also discussed. As a result of insufficient digestion of oxidatively damaged macromolecules and organelles by autophagy, neurons progressively accumulate lipofuscin that could exacerbate neuronal dysfunction. Since autophagy is the major pathway involved in the degradation of protein aggregates and defective organelles, an intense interest in developing autophagy-related therapies is growing among the scientific community. The final part of this review is devoted to discuss autophagy as a potential target of therapeutic interventions in Alzheimer's disease pathophysiology.     

The emerging roles of protein homeostasis‐governing pathways in Alzheimer's disease

Pathways governing protein homeostasis are involved in maintaining the structural, quantitative, and functional stability of intracellular proteins and involve the ubiquitin–proteasome system, autophagy, endoplasmic reticulum, and mTOR pathway. Due to the broad physiological implications of protein homeostasis pathways, dysregulation of proteostasis is often involved in the development of multiple pathological conditions, including Alzheimer's disease (AD). Similar to other neurodegenerative diseases that feature pathogenic accumulation of misfolded proteins, Alzheimer's disease is characterized by two pathological hallmarks, amyloid‐β (Aβ) plaques and tau aggregates. Knockout or transgenic overexpression of various proteostatic components in mice results in AD‐like phenotypes. While both Aβ plaques and tau aggregates could in turn enhance the dysfunction of these proteostatic pathways, eventually leading to apoptotic or necrotic neuronal death and pathogenesis of Alzheimer's disease. Therefore, targeting the components of proteostasis pathways may be a promising therapeutic strategy against Alzheimer's disease.     

RTP801/REDD1 contributes to neuroinflammation severity and memory impairments in Alzheimer’s disease

RTP801/REDD1 is a stress-regulated protein whose upregulation is necessary and sufficient to trigger neuronal death. Its downregulation in Parkinson’s and Huntington’s disease models ameliorates the pathological phenotypes. In the context of Alzheimer’s disease (AD), the coding gene for RTP801, DDIT4, is responsive to Aβ and modulates its cytotoxicity in vitro. Also, RTP801 mRNA levels are increased in AD patients’ lymphocytes. However, the involvement of RTP801 in the pathophysiology of AD has not been yet tested. Here, we demonstrate that RTP801 levels are increased in postmortem hippocampal samples from AD patients. Interestingly, RTP801 protein levels correlated with both Braak and Thal stages of the disease and with GFAP expression. RTP801 levels are also upregulated in hippocampal synaptosomal fractions obtained from murine 5xFAD and rTg4510 mice models of the disease. A local RTP801 knockdown in the 5xFAD hippocampal neurons with shRNA-containing AAV particles ameliorates cognitive deficits in 7-month-old animals. Upon RTP801 silencing in the 5xFAD mice, no major changes were detected in hippocampal synaptic markers or spine density. Importantly, we found an unanticipated recovery of several gliosis hallmarks and inflammasome key proteins upon neuronal RTP801 downregulation in the 5xFAD mice. Altogether our results suggest that RTP801 could be a potential future target for theranostic studies since it could be a biomarker of neuroinflammation and neurotoxicity severity of the disease and, at the same time, a promising therapeutic target in the treatment of AD.Subject terms: Alzheimer''s disease, Alzheimer''s disease     

The role of STEP in Alzheimer's disease

Amyloid beta (Aβ), the putative causative agent in Alzheimer's disease, is known to affect glutamate receptor trafficking. Previous studies have shown that Aβ down-regulates the surface expression of N-methyl D-aspartate type glutamate receptors (NMDARs) by the activation of STriatal-Enriched protein tyrosine Phosphatase 61 (STEP₆₁). More recent findings confirm that STEP₆₁ plays an important role in Aβ-induced NMDAR endocytosis. STEP levels are elevated in human AD prefrontal cortex and in the cortex of several AD mouse models. The increase in STEP₆₁ levels and activity contribute to the removal of GluN1/GluN2B receptor complexes from the neuronal surface membranes. The elevation of STEP₆₁ is due to disruption in the normal degradation of STEP₆₁ by the ubiquitin proteasome system. Here, we briefly discuss additional studies in support of our hypothesis that STEP₆₁ contributes to aspects of the pathophysiology in Alzheimer's disease. Exogenous application of Aβ-enriched conditioned medium (7PA2-CM) to wild-type cortical cultures results in a loss of GluN1/GluN2B subunits from neuronal membranes. Aβ-mediated NMDAR internalization does not occur in STEP knock-out cultures, but is rescued by the addition of active TAT-STEP to the cultures prior to Aβ? treatment.     

Temporal relationship of autophagy and apoptosis in neurons challenged by low molecular weight β‐amyloid peptide

Alzheimer's disease (AD) is an aging‐related progressive neurodegenerative disorder. Previous studies suggested that various soluble Aβ species are neurotoxic and able to activate apoptosis and autophagy, the type I and type II programmed cell death, respectively. However, the sequential and functional relationships between these two cellular events remain elusive. Here we report that low molecular weight Aβ triggered cleavage of caspase 3 and poly (ADP‐ribose) polymerase to cause neuronal apoptosis in rat cortical neurons. On the other hand, Aβ activated autophagy by inducing autophagic vesicle formation and autophagy related gene 12 (ATG12), and up‐regulated the lysoso‐mal machinery for the degradation of autophagosomes. Moreover, we demonstrated that activation of autophagy by Aβ preceded that of apoptosis, with death associated protein kinase phosphorylation as the potential molecular link. More importantly, under Aβ toxicity, neurons exhibiting high level of autophagosome formation were absent of apoptotic features, and inhibition of autophagy by 3‐methylade‐nine advanced neuronal apoptosis, suggesting that autophagy can protect neurons from Aβ‐induced apoptosis.     

Lactadherin/MFG‐E8 is essential for microglia‐mediated neuronal loss and phagoptosis induced by amyloid β

Nanomolar β‐amyloid peptide (Aβ) can induce neuronal loss in culture by activating microglia to phagocytose neurons. We report here that this neuronal loss is mediated by the bridging protein lactadherin/milk‐fat globule epidermal growth factor‐like factor 8 (MFG‐E8), which is released by Aβ‐activated microglia, binds to co‐cultured neurons and opsonizes neurons for phagocytosis by microglia. Aβ stimulated microglial phagocytosis, but did not opsonize neurons for phagocytosis. Aβ (250 nM) induced delayed neuronal loss in mixed glial‐neuronal mouse cultures that required microglia and occurred without increasing neuronal apoptosis or necrosis. This neuronal death/loss was prevented by antibodies to MFG‐E8 and was absent in cultures from Mfge8 knockout mice (leaving viable neurons), but was reconstituted by addition of recombinant MFG‐E8. Thus, nanomolar Aβ caused neuronal death by inducing microglia to phagocytose otherwise viable neurons via MFG‐E8. The direct neurotoxicity of micromolar Aβ was not affected by MFG‐E8. The essential role of MFG‐E8 in Aβ‐induced phagoptosis, suggests this bridging protein as a potential therapeutic target to prevent neuronal loss in Alzheimer's disease.     

Alzheimer – Mechanismen und therapeutische Ansätze. Warum wir im Alter dement werden

Alzheimer's disease is an age‐dependent neurodegenerative disorder, which is characterized by the accumulation of Amyloid plaques and neurofibrillary tangles. Amyloid β‐peptide is responsible for the massive neuronal cell death and is generated from the Amyloid precursor protein by proteolysis. Secretases generate Amyloid β‐peptide in a physiologically normal pathway. Secretases are targets for therapeutic intervention, since their inhibition leads to a dramatic reduction of Amyloid β‐peptide and at least in animal models to memory stabilization. Similarly vaccination against Aβ leads to a significant reduction of the amyloid plaque burdon. The pros and cons of current therapeutic efforts are discusses based on first results from clinical trials.     

Neurochemical characteristics of early and late onset types of Alzheimer's disease.

Brains of 49 patients who had died with Alzheimer''s disease and 54 controls were examined. The Alzheimer group exhibited noticeably reduced activity of the cholinergic marker enzyme choline acetyltransferase in the cerebral cortex, but cortical concentrations of noradrenaline, gamma-aminobutyric acid, and somatostatin were also significantly reduced. Analysis of the results according to age at death showed that the older patients, dying in their 9th and 10th decades, had a relatively pure cholinergic deficit confined to temporal lobe and hippocampus, together with a reduced concentration of somatostatin confined to temporal cortex. By contrast, the younger patients, dying in their 7th and 8th decades, had a widespread and severe cholinergic deficit together with the abnormalities of noradrenaline, gamma-aminobutyric acid, and somatostatin, and the younger patients accounted for most of the abnormalities in these systems observed in the overall group. Comparison of the young subjects with Alzheimer''s disease with the older controls did not support the concept of Alzheimer''s disease representing an acceleration of the aging process. These results suggest that Alzheimer''s disease in people aged under 80 may represent a distinct form of presenile dementia which differs in important respects from the dementia of old age.     

How neurons die in Alzheimer's disease: Implications for neuroinflammation

Despite the long-standing observation of vast neuronal loss in Alzheimer's disease (AD) our understanding of how and when neurons are eliminated is incomplete. While previous investigation has focused on apoptosis, several novel forms of cell death (i.e. necroptosis, parthanatos, ferroptosis, cuproptosis) have emerged that require further investigation. This review aims to collect evidence for different modes of neuronal cell death in AD and to also discuss how these different forms of cell death may impact the neuroinflammatory environment that prevails in the AD brain. Improved understanding of how neurons die may help to delineate disease pathogenesis, provide insights toward treatment, and aid in the development of improved animal models of AD.     

SUMO1‐conjugation is altered during normal aging but not by increased amyloid burden

A proper equilibrium of post‐translational protein modifications is essential for normal cell physiology, and alteration in these processes is key in neurodegenerative disorders such as Alzheimer's disease. Recently, for instance, alteration in protein SUMOylation has been linked to amyloid pathology. In this work, we aimed to elucidate the role of protein SUMOylation during aging and increased amyloid burden in vivo using a His₆‐HA‐SUMO1 knock‐in mouse in the 5XFAD model of Alzheimer's disease. Interestingly, we did not observe any alteration in the levels of SUMO1‐conjugation related to Alzheimer's disease. SUMO1 conjugates remained localized to neuronal nuclei upon increased amyloid burden and during aging and were not detected in amyloid plaques. Surprisingly however, we observed age‐related alterations in global levels of SUMO1 conjugation and at the level of individual substrates using quantitative proteomic analysis. The identified SUMO1 candidate substrates are dominantly nuclear proteins, mainly involved in RNA processing. Our findings open novel directions of research for studying a functional link between SUMOylation and its role in guarding nuclear functions during aging.     

Taxol protects against calcium-mediated death of differentiated rat pheochromocytoma cells

Elevated levels of intraneuronal calcium may contribute to neuronal death in both Alzheimer's disease and stroke. In part, this neuronal death may be due to calcium-induced disruption of microtubules and inhibition of axonal transport. Taxol stabilizes microtubules to disaggregation. To determine whether taxol could protect against calcium-mediated neuron cell death, a test system was established using a nerve growth factor-differentiated rat pheochromocytoma cell line (PC12 cells). PC12 cells were cultured with nerve growth factor to induce a neuronal phenotype. After 15 days, the cells were exposed to taxol, the calcium ionophore, A23187, or taxol plus ionophore for up to 24 h. Taxol alone reduced cell survival in a concentration dependent manner. At a concentration of 50 nM survival was reduced to between 63% and 84% of control after 4 h of exposure. The ionophore (1 μM) variably reduced cell survival to between 10 and 55% at 4h. However, when tacol was added to the ionophore the cell survival was significantly increased by 1.5 to 4-fold. The protective effect of taxol lasted up to 24h. We conclude that taxol has a protective effect on calcium-mediated neurotoxicity. Drugs targeting underlying cellular mechanisms involved in calcium-mediated neuronal death may lead to successful therapy for Alzheimer's disease and stroke.     

Guanosine Triphosphate Binding to β-Subunit of Tubulin in Alzheimer's Disease Brain: Role of Microtubule-Associated Protein τ

Abstract: In Alzheimer's disease, paired helical filaments composed mainly of abnormally phosphorylated τ accumulate in certain selected neurons of the brain, and microtubules are rarely seen in the affected cells. In the present study, the binding of ³²P-labeled 8-azidoguanosine triphosphate ([γ-³²P]8N₃GTP), the photoaffinity analogue of GTP, to the β-subunit of tubulin in brain homogenates was found to be markedly lower in patients with Alzheimer's disease than in aged control human cases. No significant differences were observed in the levels of the α- and β-subunits of tubulin between Alzheimer's disease and control brains obtained 2–7 h postmortem. In nine of 19 Alzheimer's disease and 11 of 12 control autopsied brains (2–7 h postmortem and stored at ?75°C) tubulin was isolated successfully from brain cytosol by in vitro polymerization induced with DEAE-dextran. The GTP binding was observed in the two cycled assembled microtubule preparations from all the normal control, and in eight of nine Alzheimer's disease cases. Alzheimer's disease microtubule preparations contained varying amounts of abnormally phosphorylated τ, whereas no abnormal τ was detected in the control brain preparations. Addition of bovine τ to bovine, normal human, and Alzheimer's disease brain tubulin preparations markedly increased GTP binding to the β-subunit. An alkaline phosphatase-treated paired helical filament-enriched preparation increased by approximately twofold the GTP binding to bovine brain tubulin. GTP binding to tubulin prepared by phosphocellulose chromatography of two cycled microtubules from three Alzheimer's disease and three normal control brains, revealed insignificant differences between the two groups. These findings have suggested that (1) τ protein promotes the GTP binding to the β-subunit of tubulin, and (2) the breakdown of the microtubule system in brains of patients with Alzheimer's disease might in part be due to the abnormal phosphorylation of τ which depresses the GTP binding.     

Animal models for Alzheimer's disease and frontotemporal dementia: a perspective

In dementia research, animal models have become indispensable tools. They not only model aspects of the human condition, but also simulate processes that occur in humans and hence provide insight into how disease is initiated and propagated. The present review discusses two prominent human neurodegenerative disorders, Alzheimer''s disease and frontotemporal dementia. It discusses what we would like to model in animals and highlights some of the more recent achievements using species as diverse as mice, fish, flies and worms. Advances in imaging and therapy are explored. We also discuss some anticipated new models and developments. These will reveal how key players in the pathogenesis of Alzheimer''s disease and frontotemporal dementia, such as the peptide Aβ (amyloid β) and the protein tau, cause neuronal dysfunction and eventually, neuronal demise. Understanding these processes fully will lead to early diagnosis and therapy.     

A mutation protective against Alzheimer's disease renders amyloid β precursor protein incapable of mediating neurotoxicity

Expression of a familial Alzheimer's disease (AD)‐linked mutant of amyloid β precursor protein (APP) or the binding of transforming growth factor β2 to wild‐type (wt)‐APP causes neuronal death by activating an intracellular death signal (a APP‐mediated intracellular death signal) in the absence of the involvement of amyloid β (Aβ) toxicity in vitro. These neuronal death models may therefore be regarded as Aβ‐independent neuronal death models related to AD. A recent study has shown that the A673T mutation in the APP isoform APP₇₇₀, corresponding to the A598T mutation in the most prevalent neuronal APP isoform APP₆₉₅ (an AD‐protective mutant of APP), is linked to a reduction in the incidence rate of AD. Consistent with this, cells expressing the AD‐protective mutant of APP produce less Aβ than cells expressing wt‐APP. In this study, transforming growth factor β2 caused death in cultured neuronal cells expressing wt‐APP, but not in those expressing the AD‐protective mutant of APP. This result suggests that the AD‐protective mutation of APP reduces the incidence rate of AD by attenuating the APP‐mediated intracellular death signal. In addition, a mutation that causes hereditary cerebral hemorrhage with amyloidosis‐Dutch type also attenuated the APP‐mediated intracellular death signal.

     

HT22 hippocampal neuronal cell line possesses functional cholinergic properties

AimsHippocampal cholinergic hypofunction is known to be involved in the cognitive deficits of Alzheimer's disease, but the detailed mechanisms remain to be elucidated. In order to establish an in vitro hippocampal cholinergic neuronal model for the relevant mechanistic studies, we have characterized a widely used hippocampal neuronal cell line, HT22, a sub-line derived from parent HT4 cells that were originally immortalized from primary mouse hippocampal neuronal culture.Main methodsWestern blot and immunocytochemistry were used to examine expression of cholinergic markers in HT22 cells. High potassium-evoked [³H]ACh release was used to evaluate the cholinergic functional properties of the cells.Key findingsWe found that HT22 cells express essential cholinergic markers, such as the high affinity choline transporter, choline acetyltransferase, vesicular acetylcholine transporter, and muscarinic acetylcholine receptors. Exposure of HT22 cells to high potassium evoked [³H]ACh release in a dose-dependent manner. In addition, the [³H]ACh release was significantly potentiated when presynaptic autoreceptors were blocked.SignificanceOur results suggest that HT22 cells possess functional cholinergic properties, and can be used for an in vitro model for defining the mechanisms in cognitive deficits of Alzheimer's disease.     

Aging impact on amyloid precursor protein neuronal trafficking

Neurons live a lifetime. Neuronal aging may increase the risk of Alzheimer's disease. How does neuronal membrane trafficking maintain synapse function during aging? In the normal aged brain, intraneuronal beta-amyloid (Aβ) accumulates without Alzheimer's disease mutations or risk variants. However, do changes with neuronal aging potentiate Aβ accumulation? We reviewed the membrane trafficking of the amyloid precursor protein in neurons and highlighted its importance in Aβ production. Importantly, we reviewed the evidence supporting the impact of aging on neuronal membrane trafficking, APP processing, and consequently Aβ production. Dissecting the molecular regulators of APP trafficking during neuronal aging is required to identify strategies to delay synaptic decline and protect from Alzheimer's disease.