Accumulation of APP C-terminal fragments causes endolysosomal dysfunction through the dysregulation of late endosome to lysosome-ER contact sites.

Neuronal endosomal and lysosomal abnormalities are among the early changes observed in Alzheimer's disease (AD) before plaques appear. However, it is unclear whether distinct endolysosomal defects are temporally organized and how altered γ-secretase function or amyloid precursor protein (APP) metabolism contribute to these changes. Inhibiting γ-secretase chronically, in mouse embryonic fibroblast and hippocampal neurons, led to a gradual endolysosomal collapse initiated by decreased lysosomal calcium and increased cholesterol, causing downstream defects in endosomal recycling and maturation. This endolysosomal demise is γ-secretase dependent, requires membrane-tethered APP cytoplasmic domains, and is rescued by APP depletion. APP C-terminal fragments (CTFs) localized to late endosome/lysosome-endoplasmic reticulum contacts; an excess of APP-CTFs herein reduced lysosomal Ca2+ refilling from the endoplasmic reticulum, promoting cholesterol accretion. Tonic regulation by APP-CTFs provides a mechanistic explanation for their cellular toxicity: failure to timely degrade APP-CTFs sustains downstream signaling, instigating lysosomal dyshomeostasis, as observed in prodromal AD. This is the opposite of substrates such as Notch, which require intramembrane proteolysis to initiate signaling.

Phospholipase D3 degrades mitochondrial DNA to regulate nucleotide signaling and APP metabolism.

Phospholipase D3 (PLD3) polymorphisms are linked to late-onset Alzheimer's disease (LOAD). Being a lysosomal 5'-3' exonuclease, its neuronal substrates remained unknown as well as how a defective lysosomal nucleotide catabolism connects to AD-proteinopathy. We identified mitochondrial DNA (mtDNA) as a major physiological substrate and show its manifest build-up in lysosomes of PLD3-defective cells. mtDNA accretion creates a degradative (proteolytic) bottleneck that presents at the ultrastructural level as a marked abundance of multilamellar bodies, often containing mitochondrial remnants, which correlates with increased PINK1-dependent mitophagy. Lysosomal leakage of mtDNA to the cytosol activates cGAS-STING signaling that upregulates autophagy and induces amyloid precursor C-terminal fragment (APP-CTF) and cholesterol accumulation. STING inhibition largely normalizes APP-CTF levels, whereas an APP knockout in PLD3-deficient backgrounds lowers STING activation and normalizes cholesterol biosynthesis. Collectively, we demonstrate molecular cross-talks through feedforward loops between lysosomal nucleotide turnover, cGAS-STING and APP metabolism that, when dysregulated, result in neuronal endolysosomal demise as observed in LOAD.

Characterisation of Gel-Forming Mucins Produced In Vivo and In Ex Vivo Conjunctival Explant Cultures.

One key element to the health of the ocular surface encompasses the presence of gel-forming mucins in the pre-ocular tear film. Conjunctival goblet cells are specialized epithelial cells that secrete mucins necessary for tear film stability and general homeostasis. Their dysfunction can be linked to a range of ocular surface inflammation disorders and chronic injuries. To obtain new perspectives and angles to tackle mucin deficiency, the need for an accurate evaluation of their presence and corresponding mucin secretion in ex vivo conjunctival cultures has become a requisite. In vitro, goblet cells show a significant decrease in the production and secretion of gel-forming mucins, accompanied by signs of dedifferentiation or transdifferentiation. Explant cultures on laminin-treated CLP-PEG hydrogels can, however, support the production of gel-forming mucins. Together, we challenge the current paradigm to evaluate the presence of cultured goblet cells solely based on their general mucin (MUC) content through imaging analyses, showing the need for additional techniques to assess the functionality of goblet cells. In addition, we broadened the gel-forming mucin profile of in vivo goblet cells with MUC5B and MUC6, while MUC2 and MUC6 is added to the profile of cultured goblet cells.

The microglial lysosomal system in Alzheimer's disease: Guardian against proteinopathy.

Microglia, the brain-resident immune cells, play an essential role in the upkeep of brain homeostasis. They actively adapt into specific activation states based on cues from the microenvironment. One of these encompasses the activated response microglia (ARMs) phenotype. It arises along a healthy aging process and in a range of neurodegenerative diseases, including Alzheimer's disease (AD). As the phenotype is characterized by an increased lipid metabolism, phagocytosis rate, lysosomal protease content and secretion of neuroprotective agents, it leaves to reason that the phenotype is adapted in an attempt to restore homeostasis. This is important to the conundrum of inflammatory processes. Inflammation per se may not be deleterious; it is only when microglial reactions become chronic or the microglial subtype is made dysfunctional by (multiple) risk proteins with single-nucleotide polymorphisms that microglial involvement becomes deleterious instead of beneficial. Interestingly, the ARMs up- and downregulate many late-onset AD-associated risk factor genes, the products of which are particularly active in the endolysosomal system. Hence, in this review, we focus on how the endolysosomal system is placed at the crossroad of inflammation and microglial capacity to keep pace with degradation.

Selecting Appropriate Reference Genes for Quantitative Real-Time Polymerase Chain Reaction Studies in Isolated and Cultured Ocular Surface Epithelia.

The introduction of tissue engineering has allowed scientists to push the boundaries and treat seriously damaged ocular surface epithelia. They have managed to do this through the development of biological substitutes that restore, maintain or improve tissue function. To ensure the generation of a therapeutically safe and effective graft, knowledge on the transcriptional profile of native and cultured ocular surface epithelia is of undeniable value. Gene expression studies are, however, only as reliable as their proper selection of internal reaction controls or reference genes. In this study, we determined the expression stability of a number of reference genes: 18s rRNA, ACTB, ATP5B, CyC1, EIF4A2, GAPDH, RPL13A, SDHA, TOP1, UBC, and YWHAZ in primary isolates as well as in ex vivo cultured ocular surface epithelia explants (day 0 and/or day 14). Expression stability of the reference genes was assessed with both the geNorm and NormFinder software that use a pairwise comparison and a model-based approach, respectively. Our results extend the general recommendation of using multiple reference genes for normalization purposes to our model systems and provide an overview of several references genes that are likely to be stable in similar culture protocols.

Connecting the Dots in the Neuroglobin-Protein Interaction Network of an Unstressed and Ferroptotic Cell Death Neuroblastoma Model.

Neuroglobin is a heme protein of which increased levels provide neuroprotection against amyloid proteinopathy and hemorrhagic damage. These cellular stressors involve the promotion of ferroptosis-an iron-dependent, lipid peroxide-accreting form of cell death. Hence, we questioned whether neuroglobin could oppose ferroptosis initiation. We detected human neuroglobin (hNgb)-EGFP-expressing SH-SY5Y cells to be significantly more resistant to ferroptosis induction, identifying 0.68-fold less cell death. To elucidate the underlying pathways, this study investigated hNgb-protein interactions with a Co-IP-MS/MS approach both under a physiological and a ferroptotic condition. hNgb binds to proteins of the cellular iron metabolism (e.g., RPL15 and PCBP3) in an unstressed condition and shows an elevated binding ratio towards cell death-linked proteins, such as HNRNPA3, FAM120A, and ABRAXAS2, under ferroptotic stress. Our data also reveal a constitutive interaction between hNgb and the longevity-associated heterodimer XRCC5/XRCC6. Disentangling the involvement of hNgb and its binding partners in cellular processes, using Ingenuity Pathway Analysis, resulted in the integration of hNgb in the ubiquitination pathway, mTOR signaling, 14-3-3-mediated signaling, and the glycolysis cascade. We also detected a previously unknown strong link with motor neuropathies. Hence, this study contributes to the elucidation of neuroglobin's involvement in cellular mechanisms that provide neuroprotection and the upkeep of homeostasis.

CD56 Homodimerization and Participation in Anti-Tumor Immune Effector Cell Functioning: A Role for Interleukin-15.

A particularly interesting marker to identify anti-tumor immune cells is the neural cell adhesion molecule (NCAM), also known as cluster of differentiation (CD)56. Namely, hematopoietic expression of CD56 seems to be confined to powerful effector immune cells. Here, we sought to elucidate its role on various killer immune cells. First, we identified the high motility NCAM-120 molecule to be the main isoform expressed by immune cells. Next, through neutralization of surface CD56, we were able to (1) demonstrate the direct involvement of CD56 in tumor cell lysis exerted by CD56-expressing killer cells, such as natural killer cells, gamma delta (γδ) T cells, and interleukin (IL)-15-cultured dendritic cells (DCs), and (2) reveal a putative crosstalk mechanism between IL-15 DCs and CD8 T cells, suggesting CD56 as a co-stimulatory molecule in their cell-to-cell contact. Moreover, by means of a proximity ligation assay, we visualized the CD56 homophilic interaction among cancer cells and between immune cells and cancer cells. Finally, by blocking the mitogen-activated protein kinase (MAPK) pathway and the phosphoinositide 3-kinase (PI3K)-Akt pathway, we showed that IL-15 stimulation directly led to CD56 upregulation. In conclusion, these results underscore the previously neglected importance of CD56 expression on immune cells, benefiting current and future immune therapeutic options.

Neuroglobin Expression Models as a Tool to Study Its Function.

Neuroglobin (Ngb) is an evolutionary conserved member of the globin family with a primary expression in neurons of which the exact functions remain elusive. A plethora of in vivo and in vitro model systems has been generated to this day to determine the functional biological roles of Ngb. Here, we provide a comprehensive overview and discussion of the different Ngb models, covering animal and cellular models of both overexpression and knockout strategies. Intriguingly, an in-depth literature search of available Ngb expression models revealed crucial discrepancies in the outcomes observed in different models. Not only does the level of Ngb expression-either physiologically, overexpressed, or downregulated-alter its functional properties, the experimental setup, being in vitro or in vivo, does impact the functional outcome as well and, hence, whether or not a physiological and/or therapeutic role is ascribed to Ngb. These differences could highlight either technical or biological adaptations and should be considered until elucidation of the Ngb biology.

Endo-lysosomal dysregulations and late-onset Alzheimer's disease: impact of genetic risk factors.

Increasing evidence supports that cellular dysregulations in the degradative routes contribute to the initiation and progression of neurodegenerative diseases, including Alzheimer's disease. Autophagy and endolysosomal homeostasis need to be maintained throughout life as they are major cellular mechanisms involved in both the production of toxic amyloid peptides and the clearance of misfolded or aggregated proteins. As such, alterations in endolysosomal and autophagic flux, as a measure of degradation activity in these routes or compartments, may directly impact as well on disease-related mechanisms such as amyloid-ß clearance through the blood-brain-barrier and the interneuronal spreading of amyloid-ß and/or Tau seeds, affecting synaptic function, plasticity and metabolism. The emerging of several genetic risk factors for late-onset Alzheimer's disease that are functionally related to endocytic transport regulation, including cholesterol metabolism and clearance, supports the notion that in particular the autophagy/lysosomal flux might become more vulnerable during ageing thereby contributing to disease onset. In this review we discuss our current knowledge of the risk genes APOE4, BIN1, CD2AP, PICALM, PLD3 and TREM2 and their impact on endolysosomal (dys)regulations in the light of late-onset Alzheimer's disease pathology.

Non-Methylation-Linked Mechanism of REST-Induced Neuroglobin Expression Impacts Mitochondrial Phenotypes in a Mouse Model of Amyotrophic Lateral Sclerosis.

Neuroglobin (Ngb) is a REST/NRSF-regulated protein, active in reactive oxygen species detoxification and cytochrome c inhibition, which provides a beneficial outcome in pathologies as Alzheimer's disease and strokes. Considering that oxidative stress and cell death are typical hallmarks of amyotrophic lateral sclerosis (ALS), we sought to explore Ngb's involvement along this disease progression. Ngb transcription was detected to be two-fold down-regulated in late-stage SODG93A mice, similarly as previously described for Alzheimer disease. Interestingly, in accordance with REST/NRSF transcription, Ngb expression is higher in spinal cords than in cortices. Hence, downstream REST/NRSF mechanisms were studied. A methylation cluster in Ngb's exon 1 (Chr12:87101763-87102586) was selected to assess methylation alterations, based on significantly altered positions in GEO DataSets of human c9orf72 and sporadic ALS cases. However, only the methylation percentage on position Chr12.87102586 was significantly increased in SODG93A mice. A larger impact can therefore be expected from the detected altered REST splicing; with levels of alternatively spliced, gene-activating REST4 to be lower than those of the gene-inhibitory full variant. To look further into the link between Ngb and ALS, we generated a double mutant Ngb-/-SODG93A mouse model, which shows an earlier onset and severity of hind limb deficits. Mitochondria derived thereof showed an altered mean volume, granularity and Ca2+-induced swelling as compared to NgbWt/WtSODG93A mice. These results indicate Ngb to be involved in and affected by the SOD1G93A pathology, which could in part be attributed to its role in halting destabilizing events of mitochondrial swelling and phenotypes.

Neuroglobin Expression in the Brain: a Story of Tissue Homeostasis Preservation.

After its discovery in 2000, the notion grew that neuroglobin, a neuronal specific heme protein, is involved in cytoprotection. To date, neuroglobin levels have been positively correlated with a beneficial outcome in a plethora of neurotoxic insults, e.g., ischemic and traumatic brain injuries and Alzheimer's disease. The first part of this review goes further into these changes of neuroglobin expression upon different neuronal insults as well as the underlying regulation. In the second part, we shed light on the mechanisms by which neuroglobin contributes to neuroprotection, being (i) the scavenging and detoxification of reactive oxygen/nitrogen species, (ii) the augmentation of the threshold for apoptosis initiation, (iii) its contribution to an anti-inflammatory milieu, and (iv) tissue regeneration. We also consider different neuroglobin models to address as yet unanswered questions. Based on the recent findings and progress in the field, we invigorate the avenues of neuroglobin in neurological ailments to increase in the coming years.

Impaired hypoxic tolerance in APP23 mice: a dysregulation of neuroprotective globin levels.

Although neuroglobin confers neuroprotection against Alzheimer's disease (AD) pathology, its expression becomes downregulated in late-stage AD. Here, we provide evidence that indicates that this decrease is associated with the AD-linked angiopathy. While wild-type mice of different ages show upregulated cerebral neuroglobin expression upon whole-body hypoxia, APP23 mice exhibit decreased cerebral transcription of neuroglobin. Interestingly, transcription of cytoglobin, whose involvement in amyloid pathology still needs to be elucidated, follows a similar pattern. To further unravel the underlying mechanism, we examined the expression levels of the RE-1-silencing transcription factor (REST/NRSF) after identifying a recognition site for it in the regulatory region of both globins. Neuroglobin-cytoglobin-REST/NRSF expression correlations are detected mainly in the cortex. This raises the possibility of REST/NRSF being an upstream regulator of these globins.