Prolonged Culturing of IPSC-derived Brain Endothelial-like Cells Is Associated With Quiescence, Downregulation of Glycolysis, and Resistance to Disruption by an Alzheimer’s Brain Milieu

Background: Human induced pluripotent stem cell (hiPSC)-derived brain endothelial-like cells (iBECs) are a robust, scalable, and translatable model of the human blood-brain barrier (BBB). Prior works have shown that high transendothelial electrical resistance (TEER) persists in iBECs for at least two weeks, emphasizing the utility of the model for longer term studies. However, most studies evaluate iBECs within the first few days of subculture, and little is known about their proliferative state, which could influence their functions. In this study, we characterized iBEC proliferative state in relation to key BBB properties at early (2 days) and late (9 days) post-subculture time points. Methods: hiPSCs were differentiated into iBECs using fully defined, serum-free medium. The proportion of proliferating cells was determined by BrdU assays. We evaluated TEER, expression of glycolysis enzymes and tight and adherens junction proteins (TJP and AJP), and glucose transporter-1 (GLUT1) function by immunoblotting, immunofluorescence, and quantifying radiolabeled tracer permeabilities. We also compared barrier disruption in response to TNF-α and conditioned medium (CM) from hiPSC-derived neurons harboring the Alzheimer’s disease (AD)-causing Swedish mutation (APPSwe/+).Results: A significant decline in iBEC proliferation over time in culture was accompanied by adoption of a more quiescent endothelial metabolic state, indicated by downregulation of glycolysis-related proteins and upregulation GLUT1. Interestingly, upregulation of GLUT1 was associated with reduced glucose transport rates in more quiescent iBECs. We also found significant decreases in claudin-5 (CLDN5) and vascular endothelial-cadherin (VE-Cad) and a trend toward a decrease in platelet endothelial cell adhesion molecule-1 (PECAM-1), whereas zona occludens-1 (ZO-1) increased and occludin (OCLN) remained unchanged. Despite differences in TJP and AJP expression, there was no difference in mean TEER on day 2 vs. day 9. TNF-α induced disruption irrespective of iBEC proliferative state. Conversely, APPSwe/+ CM disrupted only proliferating iBEC monolayers. Conclusion: iBECs can be used to study responses to disease-relevant stimuli in proliferating vs. more quiescent endothelial cell states, which may provide insight into BBB vulnerabilities in contexts of development, brain injury, and neurodegenerative disease.

Structural and Functional Alterations in Mitochondria-Associated Membranes (MAMs) and in Mitochondria Activate Stress Response Mechanisms in an In Vitro Model of Alzheimer’s Disease

Alzheimer’s disease (AD) is characterized by the accumulation of extracellular plaques composed by amyloid-β (Aβ) and intracellular neurofibrillary tangles of hyperphosphorylated tau. AD-related neurodegenerative mechanisms involve early changes of mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs) and impairment of cellular events modulated by these subcellular domains. In this study, we characterized the structural and functional alterations at MAM, mitochondria, and ER/microsomes in a mouse neuroblastoma cell line (N2A) overexpressing the human amyloid precursor protein (APP) with the familial Swedish mutation (APPswe). Proteins levels were determined by Western blot, ER-mitochondria contacts were quantified by transmission electron microscopy, and Ca2+ homeostasis and mitochondria function were analyzed using fluorescent probes and Seahorse assays. In this in vitro AD model, we found APP accumulated in MAM and mitochondria, and altered levels of proteins implicated in ER-mitochondria tethering, Ca2+ signaling, mitochondrial dynamics, biogenesis and protein import, as well as in the stress response. Moreover, we observed a decreased number of close ER-mitochondria contacts, activation of the ER unfolded protein response, reduced Ca2+ transfer from ER to mitochondria, and impaired mitochondrial function. Together, these results demonstrate that several subcellular alterations occur in AD-like neuronal cells, which supports that the defective ER-mitochondria crosstalk is an important player in AD physiopathology.

Prodromal dysfunction of α5GABA-A receptor modulated hippocampal ripples in Alzheimer′s disease

Decades of research attempting to slow the onset of Alzheimer′s disease (AD) indicates that a better understanding of memory will be key to the discovery of effective therapeutic approaches. Here, we ask whether prodromal neural network dysfunction might occur in the hippocampal trisynaptic circuit by using α5IA as a selective negative modulator of extrasynaptic α5GABA-A receptors in TgF344-AD transgenic rats, a model for early onset AD. The results demonstrate that orally bioavailable α5IA, an established memory enhancer, increases CA1 pyramidal cell mean firing rates and peak CA1 ripple amplitude during wakeful immobility in wild type F344 rats resting in a familiar environment. We show that TgF344-AD rats, which express human amyloid-beta precursor protein (with the Swedish mutation) and human presenilin-1 (with a Δ exon 9 mutation), exhibit high serum Aβ42 and Aβ40 levels by 3 months of age. By 9 months of age, CA1 ripples in young adult TgF344-AD rats are nonresponsive to α5IA indicating network dysfunction prior to the onset of AD pathology and memory dysfunction. These results demonstrate, to the best of our knowledge, the first evidence for prodromal α5GABA-A receptor dysfunction in the AD hippocampal trisynaptic circuit. Moreover, as α5GABA-A receptors are located extrasynaptically and subserve the function of tonic inhibition we posit that an early stage of memory dysfunction involves the disruption of tonic inhibition in the hippocampus.

Cholesterol alters mitophagy by impairing optineurin recruitment and lysosomal clearance in Alzheimer’s disease

Background Emerging evidence indicates that impaired mitophagy-mediated clearance of defective mitochondria is a critical event in Alzheimer’s disease (AD) pathogenesis. Amyloid-beta (Aβ) metabolism and the microtubule-associated protein tau have been reported to regulate key components of the mitophagy machinery. However, the mechanisms that lead to mitophagy dysfunction in AD are not fully deciphered. We have previously shown that intraneuronal cholesterol accumulation can disrupt the autophagy flux, resulting in low Aβ clearance. In this study, we examine the impact of neuronal cholesterol changes on mitochondrial removal by autophagy. Methods Regulation of PINK1-parkin-mediated mitophagy was investigated in conditions of acute (in vitro) and chronic (in vivo) high cholesterol loading using cholesterol-enriched SH-SY5Y cells, cultured primary neurons from transgenic mice overexpressing active SREBF2 (sterol regulatory element binding factor 2), and mice of increasing age that express the amyloid precursor protein with the familial Alzheimer Swedish mutation (Mo/HuAPP695swe) and mutant presenilin 1 (PS1-dE9) together with active SREBF2. Results In cholesterol-enriched SH-SY5Y cells and cultured primary neurons, high intracellular cholesterol levels stimulated mitochondrial PINK1 accumulation and mitophagosomes formation triggered by Aβ while impairing lysosomal-mediated clearance. Antioxidant recovery of cholesterol-induced mitochondrial glutathione (GSH) depletion prevented mitophagosomes formation indicating mitochondrial ROS involvement. Interestingly, when brain cholesterol accumulated chronically in aged APP-PSEN1-SREBF2 mice the mitophagy flux was affected at the early steps of the pathway, with defective recruitment of the key autophagy receptor optineurin (OPTN). Sustained cholesterol-induced alterations in APP-PSEN1-SREBF2 mice promoted an age-dependent accumulation of OPTN into HDAC6-positive aggresomes, which disappeared after in vivo treatment with GSH ethyl ester (GSHee). The analyses in post-mortem brain tissues from individuals with AD confirmed these findings, showing OPTN in aggresome-like structures that correlated with high mitochondrial cholesterol levels in late AD stages. Conclusions Our data demonstrate that accumulation of intracellular cholesterol reduces the clearance of defective mitochondria and suggest recovery of the cholesterol homeostasis and the mitochondrial scavenging of ROS as potential therapeutic targets for AD.

Cholesterol Alters Mitophagy By Impairing Optineurin Recruitment And Lysosomal Clearance In Alzheimer’s Disease

Background: Emerging evidence indicates that impaired mitophagy-mediated clearance of defective mitochondria is a critical event in Alzheimer’s disease (AD) pathogenesis. Amyloid-beta (Ab) metabolism and the microtubule-associated protein tau have been reported to regulate key components of the mitophagy machinery. However, the mechanisms that lead to mitophagy dysfunction in AD are not fully deciphered. We have previously shown that intraneuronal cholesterol accumulation can disrupt the autophagy flux, resulting in low Ab clearance. In this study, we examine the impact of neuronal cholesterol changes on mitochondrial removal by autophagy.Methods: Regulation of PINK1-parkin-mediated mitophagy was investigated in conditions of acute (in vitro) and chronic (in vivo) high cholesterol loading using cholesterol-enriched SH-SY5Y cells, cultured primary neurons from transgenic mice overexpressing active SREBF2 (sterol regulatory element binding factor 2), and mice of increasing age that express the amyloid precursor protein with the familial Alzheimer Swedish mutation (Mo/HuAPP695swe) and mutant presenilin 1 (PS1-dE9) together with active SREBF2.Results: In cholesterol-enriched SH-SY5Y cells and cultured primary neurons, high intracellular cholesterol levels stimulated mitochondrial PINK1 accumulation and mitophagosomes formation triggered by Ab while impairing lysosomal-mediated clearance. Antioxidant recovery of cholesterol-induced mitochondrial glutathione (GSH) depletion prevented mitophagosomes formation indicating mitochondrial ROS involvement. Interestingly, when brain cholesterol accumulated chronically in aged APP-PSEN1-SREBF2 mice the mitophagy flux was affected at the early steps of the pathway, with defective recruitment of the key autophagy receptor optineurin (OPTN). Sustained cholesterol-induced alterations in APP-PSEN1-SREBF2 mice promoted an age-dependent accumulation of OPTN into HDAC6-positive aggresomes, which disappeared after in vivo treatment with GSH ethyl ester (GSHee). The analyses in post-mortem brain tissues from individuals with AD confirmed these findings, showing OPTN in aggresome-like structures that correlated with high mitochondrial cholesterol levels in late AD stages. Conclusions: Our data demonstrate that accumulation of intracellular cholesterol reduces the clearance of defective mitochondria and suggest recovery of the cholesterol homeostasis and the mitochondrial scavenging of ROS as potential therapeutic targets for AD.

The Protective A673T mutation of Amyloid precursor protein (APP) in Alzheimer’s disease

Alzheimer's Disease is a progressive neurodegenerative disorder characterized by extracellular amyloid beta peptides, and neurofibrillary tangles consisted of intracellular hyperphosphorylated Tau in the hippocampus and cerebral cortex. Most of the mutations in key genes that code for amyloid precursor protein can lead to significant accumulation of these peptides in the brain and cause Alzheimer's Disease. Moreover, Some point mutations in amyloid precursor protein can cause familial Alzheimer's Disease, such as Swedish mutation and A673V mutation. However, recent studies have found that the A673T mutation in amyloid precursor protein gene can protect against Alzheimer's Disease, even it is located next to the Swedish mutation and at the same site as A673V mutation, which are pathogenic. It makes us curious about the protective A673T mutation. Here, we summarize the most recent insights of A673T mutation, focus on their roles in protective mechanisms against Alzheimer's Disease, and discuss their involvement in future treatment.

Cholesterol Alters Mitophagy by Impairing Optineurin Recruitment and Lysosomal Clearance in Alzheimer’s Disease

Background: Emerging evidence indicates that impaired mitophagy-mediated clearance of defective mitochondria is a critical event in Alzheimer’s disease (AD) pathogenesis. Amyloid-beta (Aβ) metabolism and the microtubule-associated protein tau have been reported to regulate key components of the mitophagy machinery. However, the mechanisms that lead to mitophagy dysfunction in AD are not fully deciphered. We have previously shown that intraneuronal cholesterol accumulation can disrupt the autophagy flux, resulting in low Aβ clearance. In this study, we examine the impact of neuronal cholesterol changes on mitochondrial removal by autophagy.Methods: Regulation of PINK1-parkin-mediated mitophagy was investigated in conditions of acute (in vitro) and chronic (in vivo) high cholesterol loading using cholesterol-enriched SH-SY5Y cells, cultured primary neurons from transgenic mice overexpressing active SREBF2 (sterol regulatory element binding factor 2), and mice of increasing age that express the amyloid precursor protein with the familial Alzheimer Swedish mutation (Mo/HuAPP695swe) and mutant presenilin 1 (PS1-dE9) together with active SREBF2.Results: In cholesterol-enriched SH-SY5Y cells and cultured primary neurons, high intracellular cholesterol levels stimulated mitochondrial PINK1 accumulation and mitophagosomes formation triggered by Aβ while impairing lysosomal-mediated clearance. Antioxidant recovery of cholesterol-induced mitochondrial glutathione (GSH) depletion prevented mitophagosomes formation indicating mitochondrial ROS involvement. Interestingly, when brain cholesterol accumulated chronically in aged APP-PSEN1-SREBF2 mice the mitophagy flux was affected at the early steps of the pathway, with defective recruitment of the key autophagy receptor optineurin (OPTN). Sustained cholesterol-induced alterations in APP-PSEN1-SREBF2 mice promoted an age-dependent accumulation of OPTN into HDAC6-positive aggresomes, which disappeared after in vivo treatment with GSH ethyl ester (GSHee). The analyses in post-mortem brain tissues from individuals with AD confirmed these findings, showing OPTN in aggresome-like structures that correlated with high mitochondrial cholesterol levels in late AD stages.Conclusions: Our data demonstrate that accumulation of intracellular cholesterol reduces the clearance of defective mitochondria and suggest recovery of the cholesterol homeostasis and the mitochondrial scavenging of ROS as potential therapeutic targets for AD.

Cholesterol Alters Mitophagy by Impairing Optineurin Recruitment and Lysosomal Clearance in Alzheimer’s Disease

Background: Emerging evidence indicates that impaired mitophagy-mediated clearance of defective mitochondria is a critical event in Alzheimer’s disease (AD) pathogenesis. Amyloid-beta (A) metabolism and the microtubule-associated protein tau have been reported to regulate key components of the mitophagy machinery. However, the mechanisms that lead to mitophagy dysfunction in AD are not fully deciphered. We have previously shown that intraneuronal cholesterol accumulation can disrupt the autophagy flux, resulting in low A clearance. In this study, we examine the impact of neuronal cholesterol changes on mitochondrial removal by autophagy.Methods: Regulation of PINK1-parkin-mediated mitophagy was investigated in conditions of acute (in vitro) and chronic (in vivo) high cholesterol loading using cholesterol-enriched SH-SY5Y cells, cultured primary neurons from transgenic mice overexpressing active SREBF2 (sterol regulatory element binding factor 2), and mice of increasing age that express the amyloid precursor protein with the familial Alzheimer Swedish mutation (Mo/HuAPP695swe) and mutant presenilin 1 (PS1-dE9) together with active SREBF2.Results: In cholesterol-enriched SH-SY5Y cells and cultured primary neurons, high intracellular cholesterol levels stimulated mitochondrial PINK1 accumulation and mitophagosomes formation triggered by A while impairing lysosomal-mediated clearance. Antioxidant recovery of cholesterol-induced mitochondrial glutathione (GSH) depletion prevented mitophagosomes formation indicating mitochondrial ROS involvement. Interestingly, when brain cholesterol accumulated chronically in aged APP-PSEN1-SREBF2 mice the mitophagy flux was affected at the early steps of the pathway, with defective recruitment of the key autophagy receptor optineurin (OPTN). Sustained cholesterol-induced alterations in APP-PSEN1-SREBF2 mice promoted an age-dependent accumulation of OPTN into HDAC6-positive aggresomes, which disappeared after in vivo treatment with GSH ethyl ester (GSHee). The analyses in post-mortem brain tissues from individuals with AD confirmed these findings, showing OPTN in aggresome-like structures that correlated with high mitochondrial cholesterol levels in late AD stages. Conclusions: Our data demonstrate that accumulation of intracellular cholesterol reduces the clearance of defective mitochondria and suggest recovery of the cholesterol homeostasis and the mitochondrial scavenging of ROS as potential therapeutic targets for AD.

Opposite changes in APP processing and human Aβ levels in rats carrying either a protective or a pathogenic APP mutation

Cleavage of APP by BACE1/β-secretase initiates the amyloidogenic cascade leading to Amyloid-β (Aβ) production. α-Secretase initiates the non-amyloidogenic pathway preventing Aβ production. Several APP mutations cause familial Alzheimer’s disease (AD), while the Icelandic APP mutation near the BACE1-cleavage site protects from sporadic dementia, emphasizing APP’s role in dementia pathogenesis. To study APP protective/pathogenic mechanisms, we generated knock-in rats carrying either the protective (Appp) or the pathogenic Swedish mutation (Apps), also located near the BACE1-cleavage site. α-Cleavage is favored over β-processing in Appp rats. Consequently, non-amyloidogenic and amyloidogenic APP metabolites are increased and decreased, respectively. The reverse APP processing shift occurs in Apps rats. These opposite effects on APP β/α-processing suggest that protection from and pathogenesis of dementia depend upon combinatorial and opposite alterations in APP metabolism rather than simply on Aβ levels. The Icelandic mutation also protects from aging-dependent cognitive decline, suggesting that similar mechanisms underlie physiological cognitive aging.