Natural and designed proteins inspired by extremotolerant organisms can form condensates and attenuate apoptosis in human cells

ABSTRACTMany organisms can survive extreme conditions and successfully recover to normal life. This extremotolerant behavior has been attributed in part to repetitive, amphipathic, and intrinsically disordered proteins that are upregulated in the protected state. Here, we assemble a library of approximately 300 naturally-occurring and designed extremotolerance-associated proteins to assess their ability to protect human cells from chemically-induced apoptosis. We show that proteins from tardigrades, nematodes, and the Chinese giant salamander are apoptosis protective. Notably, we identify a region of the human ApoE protein with similarity to extremotolerance-associated proteins that also protects against apoptosis. This region mirrors the phase separation behavior seen with such proteins, like the tardigrade protein CAHS2. Moreover, we identify a synthetic protein, DHR81, that shares this combination of elevated phase separation propensity and apoptosis protection. Finally, we demonstrate that driving protective proteins into the condensate state increases apoptosis protection, and highlight the ability for DHR81 condensates to sequester caspase-7. Taken together, this work draws a link between extremotolerance-associated proteins, condensate formation, and human cellular protection.

Absence of Apolipoprotein E is associated with exacerbation of prion pathology and promotes microglial neurodegenerative phenotype

Prion diseases or prionoses are a group of rapidly progressing and invariably fatal neurodegenerative diseases. The pathogenesis of prionoses is associated with self-replication and connectomal spread of PrPSc, a disease specific conformer of the prion protein. Microglia undergo activation early in the course of prion pathogenesis and exert opposing roles in PrPSc mediated neurodegeneration. While clearance of PrPSc and apoptotic neurons have disease-limiting effect, microglia-driven neuroinflammation bears deleterious consequences to neuronal networks. Apolipoprotein (apo) E is a lipid transporting protein with pleiotropic functions, which include controlling of the phagocytic and inflammatory characteristics of activated microglia in neurodegenerative diseases. Despite the significance of microglia in prion pathogenesis, the role of apoE in prionoses has not been established. We showed here that infection of wild type mice with 22L mouse adapted scrapie strain is associated with significant increase in the total brain apoE protein and mRNA levels and also with a conspicuous cell-type shift in the apoE expression. There is reduced expression of apoE in activated astrocytes and marked upregulation of apoE expression by activated microglia. We also showed apoE ablation exaggerates PrPSc mediated neurodegeneration. Apoe−/− mice have shorter disease incubation period, increased load of spongiform lesion, pronounced neuronal loss, and exaggerated astro and microgliosis. Astrocytes of Apoe−/− mice display salient upregulation of transcriptomic markers defining A1 neurotoxic astrocytes while microglia show upregulation of transcriptomic markers characteristic for microglial neurodegenerative phenotype. There is impaired clearance of PrPSc and dying neurons by microglia in Apoe−/− mice along with increased level of proinflammatory cytokines. Our work indicates that apoE absence renders clearance of PrPSc and dying neurons by microglia inefficient, while the excess of neuronal debris promotes microglial neurodegenerative phenotype aggravating the vicious cycle of neuronal death and neuroinflammation.

Competition between distinct ApoE alleles and mCRP for the endothelial receptor CD31 differentially regulates neurovascular inflammation and Alzheimer's disease pathology

BACKGROUND: C-reactive protein (CRP) in peripheral inflammation is associated with increased Alzheimer disease (AD) risk in Apolipoprotein E4 (ApoE4), but not ApoE3 or E2, humans. It remains unknown whether peripheral monomeric CRP (mCRP) induces AD pathogenesis through some receptor of blood-facing endothelia in the brain in an ApoE genotype dependent fashion. METHODS: We used human samples, ApoE knock-in and deficient mouse models, and primary brain endothelia. Different ApoE mice were intraperitoneally (i.p.) injected with mCRP. The characterizations by immunostaining, proximity ligation assay (PLA) and siRNA were conducted to identify the receptor for mCRP. Brain microvessel and endothelia were isolated for RNA sequencing to explore the molecular pathway. RESULTS: We demonstrate that CD31 (PECAM-1), a blood-facing endothelial receptor in brain, is a competitive target of both mCRP and ApoE protein. ApoE2 competes more strongly with mCRP for CD31 than ApoE4 does, and expressing ApoE4 or knocking out ApoE gene results in higher levels of mCRP-CD31 binding, leading to a decrease of CD31 expression but an increase in CD31 phosphorylation, along with greater cerebrovascular damage and AD pathology. This competitive binding mediates differential endothelial molecular responses depending on ApoE genotype, increasing cerebrovascular inflammation and mitochondria impairment in ApoE4 mice, while inducing vasculogenesis and protective changes in the presence of ApoE2. CONCLUSIONS: Our study reveals a novel and dynamic endothelial ApoE-mCRP-CD31 pathway for AD pathogenesis during chronic inflammation and provides some insight into the opposing ApoE4-neurodegenerative and ApoE2-neuroprotective effects in AD.

Superior Frontal Gyrus TOMM40-APOE Locus DNA Methylation in Alzheimer’s Disease

Background: The APOE ɛ4 allele is the strongest known genetic risk factor for sporadic Alzheimer’s disease (AD). The neighboring TOMM40 gene has also been implicated in AD due to its close proximity to APOE. Objective: Here we tested whether methylation of the TOMM40-APOE locus may influence ApoE protein levels and AD pathology. Methods: DNA methylation levels across the TOMM40-APOE locus and ApoE levels were measured in superior frontal gyrus tissues of 62 human brains genotyped for APOE and scored for AD neuropathology. Results: Methylation levels within the TOMM40 CpG island in the promoter or APOE CpG island in Exon 4 did not differ between APOE ɛ4 carriers versus non-carriers. However, APOE ɛ4 carriers had significantly higher methylation the APOE promoter compared with non-carriers. Although DNA methylation at TOMM40, APOE promoter region, or APOE did not differ between AD pathological groups, there was a negative association between TOMM40 methylation and CERAD scores. ApoE protein concentrations did not significantly different between APOE ɛ4 carriers and non-carriers, or between AD pathological groups. Finally, there was no correlation between ApoE protein concentrations and DNA methylation levels. Conclusion: APOE gene methylation may not be affected by genotype, relate to AD pathology or ApoE protein levels in the superior frontal gyrus, though, DNA methylation at the ApoE promoter differed between genotype. DNA methylation at TOMM40 associated with amyloid-β plaques and longitudinal fluid intelligence. In sum, these results suggest a complicated regulation of the TOMM40-APOE locus in the brain in controlling ApoE protein levels and AD neuropathology.

New pharmacological strategies for the treatment of alzheimer’s disease

Introduction: Alzheimer’s disease (AD) is seen as the most important dementia, prevalent in the elderly over 60 years old. There is still no cure, and the pharmacological strategies are to delay the symptoms and development of the pathology. The pathophysiological mechanisms are: hyperphosphorylation of the tau protein and aggregation of amyloid-β. Update studies of the tested therapies target the main pathological mechanisms: accumulation of β amyloid (inhibitors and modulators of β-secretase and γ-secretase and active and passive anti-Aβ immunotherapies), tau protein (inhibition of abnormal hyperphosphorylation with GSK-3 inhibitors, passive and active immunotherapies and the use of intrathecal antisense oligonucleotides (ASOs) and correction of the ApoE protein (increase lipidation, correct structure, clearance of non-lipid ApoE and reduction of ApoE expression). Objectives and methodology: To develop a bibliographic review in order to address new drugs in the treatment of Alzheimer’s. Qualitative and descriptive study carried out by literary review with research on PubMed. Results: Several drugs have been tested in clinical trials, however, due to lack of effectiveness, none have been approved. Therefore, it’s important to understand the limitations of the tests developed as flaws in the methodology, insufficient understanding of the mechanisms involved and inclusion of patients in different stages of AD, so that future investigations can overcome these gaps. Conclusion: It’s important to investigate new pathophysiological mechanisms, as well as the factors that trigger AD. Diagnosis is essential, with further studies to identify new biomarkers of the disease that will also have an impact on the conduct of clinical trials.

Fragmentation of brain apolipoprotein E (ApoE) and its relevance in Alzheimer's disease

Alzheimer's disease (AD) is a very common cause of dementia in the elderly. It is characterized by progressive amnesia and accretions of neurofibrillary tangles (NFTs) of neurons and senile plaques in the neuropil. After aging, the inheritance of the apolipoprotein E (ApoE) epsilon 4 (ε4) allele is the greatest risk factor for late-onset AD. The ApoE protein is the translated product of the ApoE gene. This protein undergoes proteolysis, and the resulting fragments colocalize with neurofibrillary tangles and amyloid plaques, and for that matter may be involved in AD onset and/or progression. Previous studies have reported the pathogenic potential of various ApoE fragments in AD pathophysiology. However, the pathways activated by the fragments are not fully understood. In this review, ApoE fragments obtained from post-mortem brains and body fluids, cerebrospinal fluid (CSF) and plasma, are discussed. Additionally, current knowledge about the process of fragmentation is summarized. Finally, the mechanisms by which these fragments are involved in AD pathogenesis and pathophysiology are discussed.

Apolipoprotein E promoter genotypes are not associated with white matter hyperintensity development in high-altitude careers

Objective This study sought to determine if there is an association between variants in the apolipoprotein E (ApoE) promoter regions and development of white matter hyperintensities (WMH) in military subjects who have been exposed to high altitude. In an earlier study, we found that ApoE status did not correlate with WMH development, and here we hypothesized that regulation of APOE protein expression may be protective. Results Our cohort of 92 subjects encountered altitude exposures above 25,000 feet mean sea level through their occupations as pilots or altitude chamber technicians. Using Taqman-style polymerase chain reaction genotyping and t-tests and two-way analyses of variance we found no significant association between ApoE promoter genotypes and the presence, volume, or quantity of WMHs after high altitude exposure. Taken together, the observations that neither ApoE genotype status nor promoter status are associated with WMH properties, we believe that the mechanism of action for developing WMH does not derive from ApoE, nor would therapies for ApoE-mediated neurodegeneration likely benefit high altitude operators.

Germline genetic determination of cancer progression and survival

We report the surprising finding that common germline polymorphisms of APOE, present in approximately 39% of Caucasians, predict survival outcomes in human melanoma. Analysis of The Cancer Genome Atlas revealed that carriers of the APOE2 variant experienced shorter survival relative to APOE3 homozygotes, while APOE4 variant carriers exhibited increased survival. Consistent with this, melanoma growth in human APOE knock-in mice followed the order of APOE2 > APOE3 > APOE4, revealing causal regulation of progression by APOE variants. Mechanistically, recombinant ApoE protein variants differentially suppressed melanoma cell invasion and endothelial recruitment phenotypes. Moreover, tumors in APOE4 mice exhibited greater immune cell infiltration and activation relative to tumors of APOE2 mice. These findings support the notion that human germline genetic makeup can impact the trajectory of a future malignancy.

Development of a new biochip array for APOE4 classification from plasma samples using immunoassay-based methods

Background:Apolipoprotein E (APOE) is a key player in lipid transport and metabolism and exists in three common isoforms: APOE2, APOE3 and APOE4. The presence of theE4allelic variant is recognized as a major genetic risk factor for dementia and other chronic (neuro)degenerative diseases. The availability of a validated assay for rapid and reliable APOE4 classification is therefore advantageous.Methods:Biochip array technology (BAT) was successfully applied to identify directly the APOE4 status from plasma within 3 h, through simultaneous immunoassay-based detection of both specific APOE4 and total APOE levels.Results:Samples (n=432) were first genotyped by polymerase chain reaction (PCR), and thereafter, using BAT, the corresponding plasma was identified as null, heterozygous or homozygous for theE4allele by calculating the ratio of APOE4 to total APOE protein. Two centers based in Austria and Ireland correctly classified 170 and 262 samples, respectively, and achieved 100% sensitivity and specificity.Conclusions:This chemiluminescent biochip-based sandwich immunoarray provides a novel platform to detect rapidly and accurately an individual’sAPOE4status directly from plasma. TheE4genotype of individuals has been shown previously to affect presymptomatic risk, prognosis and treatment response for a variety of diseases, including Alzheimer’s disease. The biochip’s potential for being incorporated in quantitative protein biomarker arrays capable of analyzing disease stages makes it a superior alternative to PCR-basedAPOEgenotyping and may deliver additional protein-specific information on a variety of diseases in the future.

The Effect of the APOE4 Gene on Accumulation of Aβ 40 After Brain Injury Cannot Be Reversed by Increasing apoE4 Protein

The apolipoprotein E (apoE) protein is involved in clearance of β-amyloid (Aβ) from the brain; and the APOE4 gene is associated with Aβ plaque formation in humans following traumatic brain injury (TBI). Here, we examined the association between apoE and Aβ 40 after experimental TBI and the effects of APOE alleles on this relationship. We report a biphasic response of soluble apoE protein after TBI with an acute reduction at 1 day postinjury followed by an increase at 7 days postinjury. TBI-induced Aβ 40 levels decreased as soluble apoE levels increased. In APOE4 mice there was a diminished apoE response to TBI that corresponded to prolonged accumulation of TBI-induced Aβ 40 versus that in APOE3 mice. Amyloid precursor protein processing was similar in APOE3 and APOE4 mice suggesting that impaired clearance was responsible for the abnormal accumulation of Aβ 40 in the latter. Treatment of APOE4 mice with bexarotene for 7 days increased apoE4 protein levels but was not sufficient to reduce TBI-induced Aβ 40 . Thus, rapid clearance of TBI-induced Aβ 40 occurs in mice but these pathways are impaired in APOE4 carriers. These data may help explain the deposition of Aβ in APOE4 carriers and the increased incidence of brain Aβ plaques following TBI.