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Abstract Blood-based gene expression signatures could potentially be used as biomarkers for PD. However, it is unclear whether genetically-regulated transcriptomic signatures can provide novel gene candidates for use as PD biomarkers. We leveraged on the Genotype-Tissue Expression (GTEx) database to impute whole-blood transcriptomic expression using summary statistics of three large-scale PD GWAS. A random forest classifier was used with the consensus whole-blood imputed gene signature (IGS) to discriminate between cases and controls. Outcome measures included Area under the Curve (AUC) of Receiver Operating Characteristic (ROC) Curve. We demonstrated that the IGS (n = 37 genes) is conserved across PD GWAS studies and brain tissues. IGS discriminated between cases and controls in an independent whole-blood RNA-sequencing study (1176 PD, 254 prodromal, and 860 healthy controls) with mean AUC and accuracy of 64.8% and 69.4% for PD cohort, and 78.8% and 74% for prodromal cohort. PATL2 was the top-performing imputed gene in both PD and prodromal PD cohorts, whose classifier performance varied with biological sex (higher performance for males and females in the PD and prodromal PD, respectively). Single-cell RNA-sequencing studies (scRNA-seq) of healthy humans and PD patients found PATL2 to be enriched in terminal effector CD8+ and cytotoxic CD4+ cells, whose proportions are both increased in PD patients. We demonstrated the utility of GWAS transcriptomic imputation in identifying novel whole-blood transcriptomic signatures which could be leveraged upon for PD biomarker derivation. We identified PATL2 as a potential biomarker in both clinical and prodromic PD.

Parkinson’s disease (PD) is a debilitating movement disorder characterised by the loss of dopaminergic neurons in the substantia nigra. As neuroprotective agents mitigating the rate of neurodegeneration are unavailable, the current therapies largely focus only on symptomatic relief. Here, we identified stress-inducible phosphoprotein 1 (STIP1) as a putative neuroprotective factor targeted by PD-specific autoantibodies. STIP1 is a co-chaperone with reported neuroprotective capacities in mouse Alzheimer’s disease and stroke models. With human dopaminergic neurons derived from induced pluripotent stem cells, STIP1 was found to alleviate staurosporine-induced neurotoxicity. A case-control study involving 50 PD patients (average age = 62.94 ± 8.48, Hoehn and Yahr >2 = 55%) and 50 age-matched healthy controls (HCs) (average age = 63.1 ± 8) further revealed high levels of STIP1 autoantibodies in 20% of PD patients compared to 10% of HCs. Using an overlapping peptide library covering the STIP1 protein, we identified four PD-specific B cell epitopes that were not recognised in HCs. All of these epitopes were located within regions crucial for STIP1’s chaperone function or prion protein association. Our clinical and neuro-immunological studies highlight the potential of the STIP1 co-chaperone as an endogenous neuroprotective agent in PD and suggest the possible involvement of autoimmune mechanisms via the production of autoantibodies in a subset of individuals.

Ion channels and ion fluxes control many aspects of tissue homeostasis. During oncogenic transformation, critical ion channel functions may be perturbed but conserved tumor specific ion fluxes remain to be defined. Here we used the tumoricidal protein-lipid complex HAMLET as a probe to identify ion fluxes involved in tumor cell death. We show that HAMLET activates a non-selective cation current, which reached a magnitude of 2.74±0.88 nA within 1.43±0.13 min from HAMLET application. Rapid ion fluxes were essential for HAMLET-induced carcinoma cell death as inhibitors (amiloride, BaCl2), preventing the changes in free cellular Na(+) and K(+) concentrations also prevented essential steps accompanying carcinoma cell death, including changes in morphology, uptake, global transcription, and MAP kinase activation. Through global transcriptional analysis and phosphorylation arrays, a strong ion flux dependent p38 MAPK response was detected and inhibition of p38 signaling delayed HAMLET-induced death. Healthy, differentiated cells were resistant to HAMLET challenge, which was accompanied by innate immunity rather than p38-activation. The results suggest, for the first time, a unifying mechanism for the initiation of HAMLET's broad and rapid lethal effect on tumor cells. These findings are particularly significant in view of HAMLET's documented therapeutic efficacy in human studies and animal models. The results also suggest that HAMLET offers a two-tiered therapeutic approach, killing cancer cells while stimulating an innate immune response in surrounding healthy tissues.

Cell adhesion is tightly regulated by specific molecular interactions and detachment from the extracellular matrix modifies proliferation and survival. HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells) is a protein-lipid complex with tumoricidal activity that also triggers tumor cell detachment in vitro and in vivo, suggesting that molecular interactions defining detachment are perturbed in cancer cells. To identify such interactions, cell membrane extracts were used in Far-western blots and HAMLET was shown to bind α-actinins; major F-actin cross-linking proteins and focal adhesion constituents. Synthetic peptide mapping revealed that HAMLET binds to the N-terminal actin-binding domain as well as the integrin-binding domain of α-actinin-4. By co-immunoprecipitation of extracts from HAMLET-treated cancer cells, an interaction with α-actinin-1 and -4 was observed. Inhibition of α-actinin-1 and α-actinin-4 expression by siRNA transfection increased detachment, while α-actinin-4-GFP over-expression significantly delayed rounding up and detachment of tumor cells in response to HAMLET. In response to HAMLET, adherent tumor cells rounded up and detached, suggesting a loss of the actin cytoskeletal organization. These changes were accompanied by a reduction in β1 integrin staining and a decrease in FAK and ERK1/2 phosphorylation, consistent with a disruption of integrin-dependent cell adhesion signaling. Detachment per se did not increase cell death during the 22 hour experimental period, regardless of α-actinin-4 and α-actinin-1 expression levels but adherent cells with low α-actinin levels showed increased death in response to HAMLET. The results suggest that the interaction between HAMLET and α-actinins promotes tumor cell detachment. As α-actinins also associate with signaling molecules, cytoplasmic domains of transmembrane receptors and ion channels, additional α-actinin-dependent mechanisms are discussed.

BackgroundCell membrane interactions rely on lipid bilayer constituents and molecules inserted within the membrane, including specific receptors. HAMLET (human alpha-lactalbumin made lethal to tumor cells) is a tumoricidal complex of partially unfolded alpha-lactalbumin (HLA) and oleic acid that is internalized by tumor cells, suggesting that interactions with the phospholipid bilayer and/or specific receptors may be essential for the tumoricidal effect. This study examined whether HAMLET interacts with artificial membranes and alters membrane structure.Methodology/principal findingsWe show by surface plasmon resonance that HAMLET binds with high affinity to surface adherent, unilamellar vesicles of lipids with varying acyl chain composition and net charge. Fluorescence imaging revealed that HAMLET accumulates in membranes of vesicles and perturbs their structure, resulting in increased membrane fluidity. Furthermore, HAMLET disrupted membrane integrity at neutral pH and physiological conditions, as shown by fluorophore leakage experiments. These effects did not occur with either native HLA or a constitutively unfolded Cys-Ala HLA mutant (rHLA(all-Ala)). HAMLET also bound to plasma membrane vesicles formed from intact tumor cells, with accumulation in certain membrane areas, but the complex was not internalized by these vesicles or by the synthetic membrane vesicles.Conclusions/significanceThe results illustrate the difference in membrane affinity between the fatty acid bound and fatty acid free forms of partially unfolded HLA and suggest that HAMLET engages membranes by a mechanism requiring both the protein and the fatty acid. Furthermore, HAMLET binding alters the morphology of the membrane and compromises its integrity, suggesting that membrane perturbation could be an initial step in inducing cell death.

Parkinson's disease (PD) is a chronic neurodegenerative disease underpinned by both genetic and environmental etiologic factors. Recent findings suggest that inflammation may be a pathogenic factor in the onset and progression of both familial and sporadic PD. Understanding the precise role of inflammatory factors in PD will likely lead to understanding of how the disease arises. In vivo evidence for inflammation in PD includes dysregulated molecular mediators such as cytokines, complement system and its receptors, resident microglial activation, peripheral immune cells invasion, and altered composition and phenotype of peripheral immune cells. The growing awareness of these factors has prompted novel approaches to modulate the immune system, although it remains whether these approaches can be used in humans. Influences of ageing and differential exposure to environmental agents suggest potential host-pathogen specific pathophysiologic factors. There is a clear need for research to further unravel the pathophysiologic role of immunity in PD, with the potential of developing new therapeutic targets for this debilitating condition. [ABSTRACT FROM AUTHOR]

Background: Cell membrane interactions rely on lipid bilayer constituents and molecules inserted within the membrane, including specific receptors. HAMLET (human α-lactalbumin made lethal to tumor cells) is a tumoricidal complex of partially unfolded α-lactalbumin (HLA) and oleic acid that is internalized by tumor cells, suggesting that interactions with the phospholipid bilayer and/or specific receptors may be essential for the tumoricidal effect. This study examined whether HAMLET interacts with artificial membranes and alters membrane structure. Methodology/Principal Findings: We show by surface plasmon resonance that HAMLET binds with high affinity to surface adherent, unilamellar vesicles of lipids with varying acyl chain composition and net charge. Fluorescence imaging revealed that HAMLET accumulates in membranes of vesicles and perturbs their structure, resulting in increased membrane fluidity. Furthermore, HAMLET disrupted membrane integrity at neutral pH and physiological conditions, as shown by fluorophore leakage experiments. These effects did not occur with either native HLA or a constitutively unfolded Cys-Ala HLA mutant (rHLAall-Ala). HAMLET also bound to plasma membrane vesicles formed from intact tumor cells, with accumulation in certain membrane areas, but the complex was not internalized by these vesicles or by the synthetic membrane vesicles. Conclusions/Significance: The results illustrate the difference in membrane affinity between the fatty acid bound and fatty acid free forms of partially unfolded HLA and suggest that HAMLET engages membranes by a mechanism requiring both the protein and the fatty acid. Furthermore, HAMLET binding alters the morphology of the membrane and compromises its integrity, suggesting that membrane perturbation could be an initial step in inducing cell death. [ABSTRACT FROM AUTHOR]