Ketamine may be a novel pharmacologic approach to enhance resilience and protect against stress-related disorders, but the molecular targets underlying this response remain to be fully characterized. The multifunctional protein p11 is crucial in the pathophysiology of depression and antidepressant responses. However, it is still unclear whether p11 plays a role in the pro-resilience effects induced by ketamine. Here, we demonstrated that prophylactic administration of ketamine buffers passive stress-induced maladaptive phenotypes induced by chronic stress exposure. Spatial neurotransmitter and metabolite analysis revealed that prophylactic ketamine was also effective in blunting stress-induced disturbances of tryptophan metabolism in dorsal raphe nuclei (DRN). Additionally, we demonstrated that ketamine prevented chronic restraint stress-induced p11 reduction in DRN, a highly p11-enriched region. Furthermore, we provide novel evidence indicating that p11 deficiency regulates susceptibility to stress-induced depression-related phenotypes, and these behavioral maladaptations are dependent, at least in part, on p11 function in serotonergic neurons. Spatial neurotransmitter and metabolite analysis also showed a reduction of tryptophan and dopamine metabolism in DRN of serotonergic p11-deficient mice. Viral-mediated downregulation of p11 within DRN induced a stress-susceptible phenotype. Finally, our results also unveiled that the ability of ketamine to elicit a pro-resilience response against stress-induced maladaptive phenotypes was occluded when p11 was selectively deleted in serotonergic neurons. Altogether, we showed a previously unexplored role of the DRN circuit in regulating stress susceptibility and resilience-enhancing actions of ketamine.

BACKGROUND:Recent studies identified increased cerebrospinal fluid (CSF) DOPA decarboxylase (DDC) as a promising biomarker for parkinsonian disorders, suggesting a compensation to dying dopaminergic neurons. A correlation with 123I-FP-CIT-SPECT (DaT-SPECT) imaging could shed light on this link. OBJECTIVE:The objective is to assess the relationship between CSF DDC levels and DaT-SPECT binding values. METHODS:A total of 51 and 72 Parkinson's disease (PD) subjects with available DaT-SPECT and CSF DDC levels were selected from the PPMI and Biopark cohorts, respectively. DDC levels were analyzed using proximity extension assay and correlated with DaT-SPECT striatal binding ratios (SBR). All analyses were corrected for age and sex. RESULTS:CSF DDC levels in PD patients correlated negatively with DaT-SPECT SBR in both putamen and caudate nucleus. Additionally, SBR decreased with increased DDC levels over time in PD patients. CONCLUSION:CSF DDC levels negatively correlate with DaT-SPECT SBR in levodopa-treated PD. © 2024 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Three-dimensional (3D) organoid models have been instrumental in understanding molecular mechanisms responsible for many cellular processes and diseases. However, established organic biomaterial scaffolds used for 3D hydrogel cultures, such as Matrigel, are biochemically complex and display significant batch variability, limiting reproducibility in experiments. Recently, there has been significant progress in the development of synthetic hydrogels for in vitro cell culture that are reproducible, mechanically tuneable, and biocompatible. Self-assembling peptide hydrogels (SAPHs) are synthetic biomaterials that can be engineered to be compatible with 3D cell culture. Here we investigate the ability of PeptiGel® SAPHs to model the mammary epithelial cell (MEC) microenvironment in vitro. The positively charged PeptiGel®Alpha4 supported MEC viability, but did not promote formation of polarised acini. Modifying the stiffness of PeptiGel® Alpha4 stimulated changes in MEC viability and changes in protein expression associated with altered MEC function, but did not fully recapitulate the morphologies of MECs grown in Matrigel. To supply the appropriate biochemical signals for MEC organoids, we supplemented PeptiGels® with laminin. Laminin was found to require negatively charged PeptiGel® Alpha7 for functionality, but was then able to provide appropriate signals for correct MEC polarisation and expression of characteristic proteins. Thus, optimisation of SAPH composition and mechanics allows tuning to support tissue-specific organoids.

Cells respond to stress by synthesizing chaperone proteins that seek to correct protein misfolding and maintain function. However, abrogation of protein homeostasis is a hallmark of aging, leading to loss of function and the formation of proteotoxic aggregates characteristic of pathology. Consequently, discovering the underlying molecular causes of this deterioration in proteostasis is key to designing effective interventions to disease or to maintaining cell health in regenerative medicine strategies. Here, we examined primary human mesenchymal stem cells, cultured to a point of replicative senescence and subjected to heat shock, as an in vitro model of the aging stress response. Multi -omics analysis showed how homeostasis components were reduced in senescent cells, caused by dysregulation of a functional network of chaperones, thereby limiting proteostatic competence. Time-resolved analysis of the primary response factors, including those regulating heat shock protein 70 kDa (HSPA1A), revealed that regulatory control is essentially translational. Senescent cells have a reduced capacity for chaperone protein translation and misfolded protein (MFP) turnover, driven by downregulation of ribosomal proteins and loss of the E3 ubiquitin ligase CHIP (C-terminus of HSP70 interacting protein) which marks MFPs for degradation. This limits the cell’s stress response and subsequent recovery. A kinetic model recapitulated these reduced capacities and predicted an accumulation of MFP, a hypothesis supported by evidence of systematic changes to the proteomic fold state. These results thus establish a specific loss of regulatory capacity at the protein, rather than transcript, level and uncover underlying systematic links between aging and loss of protein homeostasis.

Background Glioblastoma is the most common and aggressive primary brain tumour in adults. Hallmarks of glioblastoma include its intra-tumoural heterogeneity and extensive invasion of the surrounding brain. Glioblastoma is known to remodel the extracellular matrix (ECM) of the brain, resulting in altered mechanical properties and the establishment of a tumour-promoting microenvironment. How changes in the expression and spatial distribution of ECM constituents within glioblastoma contribute to invasion and disease progression is still unclear. Methods Here we report on a protocol for laser-capture microdissection coupled with mass spectrometry (LCM-proteomics) that allowed a spatially resolved and unbiased analysis of the regional ECM proteome (matrisome) in formalin-fixed and paraffin-embedded (FFPE) samples of human glioblastoma. We investigated five molecularly characterised hemispheric adult glioblastomas where the brain/tumour interface and tumour epicentre were represented in the surgical specimens and snap-frozen tissue was available. LCM-proteomic analysis was validated with immunohistochemistry. Results LCM-proteomics identified 53 matrisome proteins in FFPE tissue, demonstrating comparable performance with conventional analysis of snap-frozen tissue. The analysis revealed distinct matrisome components in the brain/tumour interface versus the tumour epicentre. Guided by data from LCM-proteomic analysis, immunostaining for tenascin-R confirmed greater staining in the brain/tumour interface, whilst expression of fibronectin was higher in the tumour epicentre. Conclusion The protocol described in this work allowed for accurate, spatially resolved analysis of ECM in FFPE tissues, with performance comparable to analysis of snap-frozen tissue. While the focus for this work was on the regional ECM composition of glioblastoma, we found that the LCM-proteomics protocol is also applicable to the study of the wider proteome, and represents a valuable tool for investigating tumour/tissue heterogeneity. This protocol opens the possibility to apply LCM-proteomics to retrospective studies with the advantage of accessing clinical history and follow-up information, providing a valuable resource for translational research in glioblastoma.

ABSTRACT Cells respond to stress by synthesising chaperone proteins that correct protein misfolding to maintain function. However, protein homeostasis is lost in ageing, leading to aggregates characteristic of protein-folding diseases. Whilst much is known about how these diseases progress, discovering what causes protein-folding to deteriorate could be key to their prevention. Here, we examined primary human mesenchymal stem cells (hMSCs), cultured to a point of replicative senescence and subjected to heat shock, as an in vitro model of the ageing stress response. We found through proteomic analysis that the maintenance of homeostasis deteriorated in senescent cells. Time-resolved analysis of factors regulating heat shock protein 70 kDa (HSPA1A) revealed a lack of capacities for protein turnover and translation to be key factors in limiting the stress response during senescence. A kinetic model predicted a consequence of these reduced capacities to be the accumulation of misfolded protein, a hypothesis supported by evidence of systematic changes to protein fold state. These results thus further our understanding of the underlying mechanistic links between ageing and loss of protein homeostasis.