Proteostasis Disturbances and Inflammation in Neurodegenerative Diseases

Protein homeostasis (proteostasis) disturbances and inflammation are evident in normal aging and some age-related neurodegenerative diseases. While the proteostasis network maintains the integrity of intracellular and extracellular functional proteins, inflammation is a biological response to harmful stimuli. Cellular stress conditions can cause protein damage, thus exacerbating protein misfolding and leading to an eventual overload of the degradation system. The regulation of proteostasis network is particularly important in postmitotic neurons due to their limited regenerative capacity. Therefore, maintaining balanced protein synthesis, handling unfolding, refolding, and degrading misfolded proteins are essential to preserve all cellular functions in the central nervous sysytem. Failing proteostasis may trigger inflammatory responses in glial cells, and the consequent release of inflammatory mediators may lead to disturbances in proteostasis. Here, we review the mechanisms of proteostasis and inflammatory response, emphasizing their role in the pathological hallmarks of neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease, and amyotrophic lateral sclerosis. Furthermore, we discuss the interplay between proteostatic stress and excessive immune response that activates inflammation and leads to dysfunctional proteostasis

Proteostasis Disturbances and Inflammation in Neurodegenerative Diseases

Protein homeostasis (proteostasis) disturbances and inflammation are evident in normal aging and some age-related neurodegenerative diseases. While the proteostasis network maintains the integrity of intracellular and extracellular functional proteins, inflammation is a biological response to harmful stimuli. Cellular stress conditions can cause protein damage, thus exacerbating protein misfolding and leading to an eventual overload of the degradation system. The regulation of proteostasis network is particularly important in postmitotic neurons due to their limited regenerative capacity. Therefore, maintaining balanced protein synthesis, handling unfolding, refolding, and degrading misfolded proteins are essential to preserve all cellular functions in the central nervous sysytem. Failing proteostasis may trigger inflammatory responses in glial cells, and the consequent release of inflammatory mediators may lead to disturbances in proteostasis. Here, we review the mechanisms of proteostasis and inflammatory response, emphasizing their role in the pathological hallmarks of neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease, and amyotrophic lateral sclerosis. Furthermore, we discuss the interplay between proteostatic stress and excessive immune response that activates inflammation and leads to dysfunctional proteostasis

Synonymous site conservation in the HIV-1 genome

Background: Synonymous or silent mutations are usually thought to evolve neutrally. However, accumulating recent evidence has demonstrated that silent mutations may destabilize RNA structures or disrupt cis regulatory motifs superimposed on coding sequences. Such observations suggest the existence of stretches of codon sites that are evolutionary conserved at both DNA-RNA and protein levels. Such stretches may point to functionally important regions within protein coding sequences not necessarily reflecting functional constraints on the amino-acid sequence. The HIV-1 genome is highly compact, and often harbors overlapping functional elements at the protein, RNA, and DNA levels. This superimposition of functions leads to complex selective forces acting on all levels of the genome and proteome. Considering the constraints on HIV-1 to maintain such a highly compact genome, we hypothesized that stretches of synonymous conservation would be common within its genome. Results: We used a combined computational-experimental approach to detect and characterize regions exhibiting strong purifying selection against synonymous substitutions along the HIV-1 genome. Our methodology is based on advanced probabilistic evolutionary models that explicitly account for synonymous rate variation among sites and rate dependencies among adjacent sites. These models are combined with a randomization procedure to automatically identify the most statistically significant regions of conserved synonymous sites along the genome. Using this procedure we identified 21 conserved regions. Twelve of these are mapped to regions within overlapping genes, seven correlate with known functional elements, while the functions of the remaining four are yet unknown. Among these four regions, we chose the one that deviates most from synonymous rate homogeneity for in-depth computational and experimental characterization. In our assays aiming to quantify viral fitness in both early and late stages of the replication cycle, no differences were observed between the mutated and the wild type virus following the introduction of synonymous mutations. Conclusions: The contradiction between the inferred purifying selective forces and the lack of effect of these mutations on viral replication may be explained by the fact that the phenotype was measured in single-cycle infection assays in cell culture. Such a system does not account for the complexity of HIV-1 infections in vivo, which involves multiple infection cycles and interaction with the host immune system

Natural HLA Class I Polymorphism Controls the Pathway of Antigen Presentation and Susceptibility to Viral Evasion

HLA class I polymorphism creates diversity in epitope specificity and T cell repertoire. We show that HLA polymorphism also controls the choice of Ag presentation pathway. A single amino acid polymorphism that distinguishes HLA-B*4402 (Asp116) from B*4405 (Tyr116) permits B*4405 to constitutively acquire peptides without any detectable incorporation into the transporter associated with Ag presentation (TAP)-associated peptide loading complex even under conditions of extreme peptide starvation. This mode of peptide capture is less susceptible to viral interference than the conventional loading pathway used by HLA-B*4402 that involves assembly of class I molecules within the peptide loading complex. Thus, B*4402 and B*4405 are at opposite extremes of a natural spectrum in HLA class I dependence on the PLC for Ag presentation. These findings unveil a new layer of MHC polymorphism that affects the generic pathway of Ag loading, revealing an unsuspected evolutionary trade-off in selection for optimal HLA class I loading versus effective pathogen evasion

Intranasal administration of adenoviral vaccines expressing SARS-CoV-2 spike protein improves vaccine immunity in mouse models

The ongoing SARS-CoV-2 pandemic is controlled but not halted by public health measures and mass vaccination strategies which have exclusively relied on intramuscular vaccines. Intranasal vaccines can prime or recruit to the respiratory epithelium mucosal immune cells capable of preventing infection. Here we report a comprehensive series of studies on this concept using various mouse models, including HLA class II-humanized transgenic strains. We found that a single intranasal (i.n.) dose of serotype-5 adenoviral vectors expressing either the receptor binding domain (Ad5-RBD) or the complete ectodomain (Ad5-S) of the SARS-CoV-2 spike protein was effective in inducing i) serum and bronchoalveolar lavage (BAL) anti-spike IgA and IgG, ii) robust SARS-CoV-2-neutralizing activity in the serum and BAL, iii) rigorous spike-directed T helper 1 cell/cytotoxic T cell immunity, and iv) protection of mice from a challenge with the SARS-CoV-2 beta variant. Intramuscular (i.m.) Ad5-RBD or Ad5-S administration did not induce serum or BAL IgA, and resulted in lower neutralizing titers in the serum. Moreover, prior immunity induced by an intramuscular mRNA vaccine could be potently enhanced and modulated towards a mucosal IgA response by an i.n. Ad5-S booster. Notably, Ad5 DNA was found in the liver or spleen after i.m. but not i.n. administration, indicating a lack of systemic spread of the vaccine vector, which has been associated with a risk of thrombotic thrombocytopenia. Unlike in otherwise genetically identical HLA-DQ6 mice, in HLA-DQ8 mice Ad5-RBD vaccine was inferior to Ad5-S, suggesting that the RBD fragment does not contain a sufficient collection of helper-T cell epitopes to constitute an optimal vaccine antigen. Our data add to previous promising preclinical results on intranasal SARS-CoV-2 vaccination and support the potential of this approach to elicit mucosal immunity for preventing transmission of SARS-CoV-2

The Gag Cleavage Product, p12, is a Functional Constituent of the Murine Leukemia Virus Pre-Integration Complex

The p12 protein is a cleavage product of the Gag precursor of the murine leukemia virus (MLV). Specific mutations in p12 have been described that affect early stages of infection, rendering the virus replication-defective. Such mutants showed normal generation of genomic DNA but no formation of circular forms, which are markers of nuclear entry by the viral DNA. This suggested that p12 may function in early stages of infection but the precise mechanism of p12 action is not known. To address the function and follow the intracellular localization of the wt p12 protein, we generated tagged p12 proteins in the context of a replication-competent virus, which allowed for the detection of p12 at early stages of infection by immunofluorescence. p12 was found to be distributed to discrete puncta, indicative of macromolecular complexes. These complexes were localized to the cytoplasm early after infection, and thereafter accumulated adjacent to mitotic chromosomes. This chromosomal accumulation was impaired for p12 proteins with a mutation that rendered the virus integration-defective. Immunofluorescence demonstrated that intracellular p12 complexes co-localized with capsid, a known constituent of the MLV pre-integration complex (PIC), and immunofluorescence combined with fluorescent in situ hybridization (FISH) revealed co-localization of the p12 proteins with the incoming reverse transcribed viral DNA. Interactions of p12 with the capsid and with the viral DNA were also demonstrated by co-immunoprecipitation. These results imply that p12 proteins are components of the MLV PIC. Furthermore, a large excess of wt PICs did not rescue the defect in integration of PICs derived from mutant p12 particles, demonstrating that p12 exerts its function as part of this complex. Altogether, these results imply that p12 proteins are constituent of the MLV PIC and function in directing the PIC from the cytoplasm towards integration

Transduction of Human Immunodeficiency Virus Type 1 Vectors Lacking Encapsidation and Dimerization Signals▿

The encapsidation signal (Ψ) and the nested dimerization initiation site are important for efficient packaging of human immunodeficiency virus type 1 (HIV-1) genomic RNA dimers. Consequently, these signals are included in all HIV-1 vectors. Here, we provide evidence demonstrating that these elements in such vectors are not absolutely required for vector transduction. In single-cycle infection assays, vectors with Ψ deleted (ΔΨ) were transduced with only a two- to fivefold reduction compared to the wild type. The transduction of ΔΨ showed typical products of reverse transcription and vector integration; however, in vitro and in vivo dimerization assays demonstrated the lack of normal dimerization of the ΔΨ vector. The reduction in transduction reflected a similar reduction in packaging. Nevertheless, a relatively high specificity of packaging was retained, as the ΔΨ vector was encapsidated at a level 4 orders of magnitude higher than that for overexpressed, nonretroviral cellular mRNA and 15 orders of magnitude higher than that for a murine leukemia virus (MLV)-based vector, all containing the same reporter gene, suggesting a Ψ-independent mechanism of packaging. The fact that HIV-1 and MLV vectors were encapsidated with a much higher level of efficiency than the cellular RNA suggests that the genomic RNAs of different retroviruses share common features and/or pathways that target them to encapsidation. Overall, these results formally demonstrate that packaging and dimerization signals are not required for the early stages of infection and can be deleted without risking a total loss of vector transduction. Deletion of these signals should enhance the safety of these vectors

Differential Regulation of Vascular Endothelial Growth Factors by Promoter-targeted shRNAs

Vascular endothelial growth factors (VEGFs) and their receptors (VEGF-R) are central regulators of vasculogenesis, angiogenesis, and lymphangiogenesis. They contribute to many vascular-related pathologies, and hence VEGF-targeted therapies have been widely sought after. In this study, the authors investigated the ability of promoter-targeted small hairpin RNAs (shRNAs) to regulate VEGF-A, VEGF-C and VEGF-R1 in different cell lines. The authors identified shRNAs that can upregulate hVEGF-C at both the mRNA and protein levels, and differentially regulate hVEGF-A depending on the cell type. Likewise, the authors identified shRNA that downregulated VEGF-R1 gene expression. Hence, promoter-targeted shRNAs can affect endogenous gene expression not only bimodally, but also differentially in a cell-type specific manner. Importantly, all three genes tested were regulated by at least one shRNA, supporting the idea that nuclear RNA interference is a widespread phenomenon. The level of regulation across the panel of shRNAs varied maximally from a 2.2-fold increase to a 4-fold decrease. This level of change should be useful in fine-tuning and modulating target gene expression, which for potent molecules, such as VEGF-A and VEGF-C, can be very beneficial. These promoter-targeted shRNAs may facilitate the design and development of targeted, context-dependent strategies for both pro- and antiangiogenic therapies for the treatment of vascular-related pathologies