Parsons, Tammee M.

Aging and neurodegenerative disorders like Alzheimer’s Disease (AD) are associated with progressive dysfunction of the blood-brain barrier (BBB) and neurovascular unit (NVU), contributing to impaired vascular integrity and neuronal vulnerability. Apolipoprotein E (APOE) is a key regulator of neurovascular function, and the rare APOE3-R136S “Christchurch” variant ( APOE3Ch ) confers protection against AD. Mesenchymal stromal cells (MSCs) represent a promising cell-based therapy for the treatment of neurodegenerative diseases due to their paracrine effect exerted on vascular and neural cells. To investigate how APOE3Ch influences MSC-mediated neurovascular support, we used isogenic iPSC-derived MSCs (iMSCs) with homozygous APOE3Ch or APOE3 . We found that APOE3Ch iMSCs have stronger immunosuppressive effect on LPS-induced NFκB activation of THP1 cells. APOE3Ch iMSCs also enhanced endothelial barrier resistance and angiogenic capacity compared to APOE3 iMSCs when directly co-cultured with endothelial cells. In addition, conditioned medium from APOE3Ch iMSCs promoted neurite outgrowth more efficiently than that from APOE3 iMSCs. Metabolic profiling revealed differences between APOE3Ch and APOE3 iMSCs, suggesting altered metabolic resilience. Together, these findings demonstrate that iMSCs support vascular and neuronal function through paracrine mechanisms and suggest that APOE3Ch variant improves specific aspects of MSC-mediated neurovascular support. This work highlights the potential of combining MSC-based therapies with protective APOE variants to target BBB and NVU dysfunction in aging and neurodegeneration.

Abstract INTRODUCTION Loss‐of‐function variants of the ABCA7 gene are associated with an increased risk of Alzheimer's disease (AD). How neuronal ABCA7 contributes to AD pathogenesis is unknown. METHODS Using neuron‐specific Abca7 KO mice (n Abca7 −/− ) with or without 5×FAD amyloid model background and post mortem AD brains, we investigated AD‐related phenotypes through comprehensive approaches including transcriptomics and lipidomics. RESULTS Lipidomics analysis detected altered lipid profiles in the brains and synaptosomes of 5×FAD; n Abca7 −/− mice compared to controls. Transcriptomics profiling revealed that neuronal ABCA7 deficiency altered the expression of genes and pathways related to mitochondrial homeostasis and apoptosis, particularly in excitatory neurons. Consistently, synaptosomes isolated from 5×FAD; n Abca7 −/− mice showed diminished mitochondria respiration and reduced synaptic protein levels, which is further supported by results from human AD brains. DISCUSSION Our findings reveal that neuronal ABCA7 plays a critical role in mitochondrial homeostasis important for neuronal function and survival in the presence of AD pathology. Highlights Neuronal ABCA7 deficiency exacerbates Aβ pathology and neuronal damage in 5×FAD mice. Neuronal ABCA7 deficiency alters brain transcriptomes and lipidomes of 5×FAD mice. Neuronal ABCA7 deficiency disturbs mitochondria functions in synaptosomes from 5×FAD mice. Neuronal ABCA7 expression associates with genes and pathways related to mitochondrial homeostasis in AD brains.

AbstractBackgroundAlzheimer’s disease (AD) is a progressive neurodegenerative disease and the most prevalent form of late‐life dementia. The ε2 allele of the APOE gene encoding apolipoprotein E (APOE2) is associated with lower susceptibility to AD among the three genotypes (ε2, ε3, ε4), while APOE4 is the strongest genetic risk factor for late‐onset AD. APOE plays a critical role in maintaining synaptic plasticity and neuronal function by controlling lipid homeostasis, with APOE2 having a superior function. Gene therapy that increases APOE2 levels in the brain has, therefore, emerged as a potential therapeutic strategy to treat AD.MethodWe conjugated PEGylated liposomes with transferrin and Penetratin, a cell‐penetrating peptide, sufficiently deliver chitosan‐APOE2 cDNA plasmid complex into amyloid model AppNL‐G‐F knockin mice at 12‐month‐old. Biochemical studies and brain transcriptome analysis were employed to investigate how brain‐targeting liposome‐based APOE2 gene delivery influences amyloid‐β (Aβ)‐related pathologies in AppNL‐G‐F knockin mice.ResultOne month after APOE2 gene therapy, there was a trend of reduced insoluble Aβ levels in the mouse cortices. Furthermore, in the AppNL‐G‐F knockin mice that received the APOE2 gene therapy, brain transcriptome analysis through RNA‐sequencing identified the upregulation of genes/pathways related to neuronal development. This was supported by increases of Dlg4 and Syp mRNAs coding synaptic proteins in the experimental group. On the other hand, we found that APOE2 gene delivery increased soluble Aβ levels, including oligomers, as well as exacerbated neurite dystrophy and decreased synaptophysin.ConclusionOur results suggest that brain‐targeting liposome‐based APOE2 gene therapy is potentially beneficial for synaptic formation at the transcriptional level. Forced APOE2 expressions, however, may exacerbate Aβ toxicity by increasing the dissociation of Aβ oligomers from aggregates in the presence of considerable amyloid burden.

Importance Vaginal fibroblast function is altered in people with pelvic organ prolapse. Thus, it is important to study vaginal fibroblasts to better understand the pathophysiology of prolapse. Objective This study aimed to compare 3 culturing methods of primary vaginal fibroblasts. Study Design This was an in vitro study. Patients who were undergoing surgery for vaginal prolapse were recruited. Excess vaginal epithelial tissue that would have otherwise been discarded was collected. The vaginal fibroblasts from each participant were cultured via (1) 3-hour digest, (2) coverslip, and (3) gelatin-coat methods. Differences in the efficiency of cell isolation, expression of known fibroblast-associated genes, and cellular function were compared between the 3 methods using one-way analysis of variance and Tukey test for post hoc pairwise comparisons ( P < 0.05). Results Five patients with pelvic organ prolapse were recruited. Fibroblasts cultured via the 3-hour digest method became confluent within 3–5 days in a 100-mm dish compared to 2–3 weeks in a 6-well dish for the coverslip and gelatin-coat methods. Cells from all culture methods expressed similar amounts of vimentin and α smooth muscle actin. There were no significant differences in morphology; gene expression levels of MMP1 , MMP2 , ACTA2 , COL1A1 , COL3A1 , and LOXL1 on qPCR; cell viability; proliferation; and migration between the 3 culturing methods. Conclusion Culturing primary vaginal fibroblasts via the 3-hour digest, coverslip, and gelatin-coat methods similarly resulted in reliable primary vaginal fibroblast growth and function.

Abstract Introduction: Radiation therapy (RT) is the standard of care for central nervous system (CNS) cancers. Despite the success of RT in reducing tumor mass, RT-induced side effects such as ECM remodeling, vasculopathies and neuroinflammation are well documented that cause reduced neurogenecity and premature aging like complications during post-cancer survivorship. Since mesenchymal stromal cells (MSCs) are multipotent adult stem cells that support tissue integrity, homeostasis and repair through factors they release, we here investigated the potential of iPSC-derived MSC (iMSC)-secretome in immunomodulation and vasculature repair in response to radiation-induced alterations utilizing models of human monocytic and microglial cells (THP1, HMC-3) and human brain endothelial cells (HCMEC/D3). Approaches: (a) Investigate the impact of iMSC secretome on vasculature damage caused by radiation-induced cell injury and RT-induced inflammation, by evaluating its effects on (i) endothelial cell survival and morphogenesis (ii) activation of peripheral monocytes stimulated by X-ray radiation therapy (RT), RT-induced microglial injury, and vasculature injury mediated inflammation (b) Identify factors and pathways that may contribute to the observed phenotype. Results: Decline in intact angiogenic tube formation and less compact spheroid formation was observed for HCMEC/D3 cells after irradiation at doses (Gy): 2.5, 5, 10, 15, and 30, with maximal effect observed at 30Gy. Evaluating the effects of iMSC secretome on radiation induced endothelial cell damage and inflammation revealed increased cell viability with reduced apoptosis, higher cell adherance and better spheroid compactness in 5Gy irradiated HCMEC/D3 cells after iMSC-conditioned media (iMSC-CM) treatment. Reduction in NFκB promoter activity was observed in irradiated THP1- NFκB-Luc2 reporter line, with reduced levels of TNF-α release per unit cell viability in THP1 after iMSC-CM treatment. NFκB promoter activity was also reduced in effect of iMSC treatment in THP1- NFκB-Luc2 reporter cells subjected to irradiated vasculature cells (D3), with or without iMSC coculture and, irradiated microglial cells (HMC3), mimicking vasculature injury, microglial injury, and immune cell crosstalk, respectively. iMSC secretome facilitated a more networked tube formation in HCMEC/D3 cells in coculture of D3+iMSC cells that was longer retained. Cytokine arrays revealed iMSC-CM to have proangiogenic, immunomodulatory (immune suppressive) and chemotactic factors: MCP1, ANG (angiogenin), IL6, IL8, GRO, GROα, RANTES, and SDF1. Our initial pathway analysis using human phospho-proteome profiling showed iMSC secretome elevates levels of beta-catenin, and PRAS40 (AKT/mTOR modulator) and lowers level of pRSK1/2/3 in HCMEC/D3. Conclusions: Taken together, we demonstrated that iMSC-secretome complements the proangiogenic and immunosuppressive effects of iMSC during radiation induced injury. Further studies are required to validate the in vivo potential of iMSC-secretome as safe biological therapy in long-term cancer survivors. Citation Format: Kshama Gupta, Ralph B. Perkerson III, Tammee M Parsons, Prasanna Vibhute, Abba C. Zubair, Alfredo Quinones-Hinojosa, Takahisa Kanekiyo. Impact of human iPSC-derived MSC secretome in modulating radiation induced inflammation and brain vascular endothelial cell damage [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C116.

Abstract Background Mesenchymal stromal cells (MSCs) have a dynamic secretome that plays a critical role in tissue repair and regeneration. However, studying the MSC secretome in mixed-culture disease models remains challenging. This study aimed to develop a mutant methionyl-tRNA synthetase-based toolkit (MetRSL274G) to selectively profile secreted proteins from MSCs in mixed-culture systems and demonstrate its potential for investigating MSC responses to pathological stimulation.Methods We used CRISPR/Cas9 homology-directed repair to stably integrate MetRSL274G into cells, enabling the incorporation of the non-canonical amino acid, azidonorleucine (ANL), and facilitating selective protein isolation using click chemistry. MetRSL274G was integrated into both in H4 cells and induced pluripotent stem cells (iPSCs) for a series of proof-of-concept studies. Following iPSC differentiation into induced-MSCs, we validated their identity and co-cultured MetRSL274G-expressing iMSCs with naïve or lipopolysaccharide- (LPS) treated THP-1 cells. We then profiled the iMSC secretome using antibody arrays.Results Our results showed successful integration of MetRSL274G into targeted cells, allowing specific isolation of proteins from mixed-culture environments. We also demonstrated that the secretome of MetRSL274G-expressing iMSCs can be differentiated from that of THP-1 cells in co-culture, and is altered when co-cultured with LPS-treated THP-1 cells compared to naïve THP-1 cells.Conclusions The MetRSL274G-based toolkit we have generated enables selective profiling of the MSC secretome in mixed-culture disease models. This approach has broad applications for examining not only MSC responses to models of pathological conditions, but any other cell type that can be differentiated from iPSCs. This can potentially reveal novel MSC-mediated repair mechanisms and advancing our understanding of tissue regeneration processes.

Cerebrovasculature is critical in maintaining brain homeostasis; its dysregulation often leads to vascular cognitive impairment and dementia (VCID) during aging. VCID is the second most prevalent cause of dementia in the elderly, after Alzheimer’s disease (AD), with frequent cooccurrence of VCID and AD. While multiple factors are involved in the pathogenesis of AD and VCID, APOE4 increases the risk for both diseases. A major apolipoprotein E (apoE) receptor, the low-density lipoprotein receptor-related protein 1 (LRP1), is abundantly expressed in vascular mural cells (pericytes and smooth muscle cells). Here, we investigated how deficiency of vascular mural cell LRP1 affects the cerebrovascular system and cognitive performance using vascular mural cell–specific Lrp1 -KO mice ( smLrp1 –/– ) in a human APOE3 or APOE4 background. We found that spatial memory was impaired in the 13- to 16-month-old APOE4 smLrp1 –/– mice but not in the APOE3 smLrp1 –/– mice, compared with their respective littermate control mice. These disruptions in the APOE4 smLrp1 –/– mice were accompanied with excess paravascular glial activation and reduced cerebrovascular collagen IV. In addition, blood-brain barrier (BBB) integrity was disrupted in the APOE4 smLrp1 –/– mice. Together, our results suggest that vascular mural cell LRP1 modulates cerebrovasculature integrity and function in an APOE genotype–dependent manner.

Abstract Genetic variants that define two distinct haplotypes at the TMEM106B locus have been implicated in multiple neurodegenerative diseases and in healthy brain ageing. In frontotemporal dementia (FTD), the high expressing TMEM106B risk haplotype was shown to increase susceptibility for FTD with TDP-43 inclusions (FTD-TDP) and to modify disease penetrance in progranulin mutation carriers (FTD-GRN). To elucidate the biological function of TMEM106B and determine whether lowering TMEM106B may be a viable therapeutic strategy, we performed brain transcriptomic analyses in 8-month-old animals from our recently developed Tmem106b−/− mouse model. We included 10 Tmem106b+/+ (wild-type), 10 Tmem106b+/− and 10 Tmem106−/− mice. The most differentially expressed genes (153 downregulated and 60 upregulated) were identified between Tmem106b−/− and wild-type animals, with an enrichment for genes implicated in myelination-related cellular processes including axon ensheathment and oligodendrocyte differentiation. Co-expression analysis also revealed that the most downregulated group of correlated genes was enriched for myelination-related processes. We further detected a significant loss of OLIG2-positive cells in the corpus callosum of Tmem106b−/− mice, which was present already in young animals (21 days) and persisted until old age (23 months), without worsening. Quantitative polymerase chain reaction revealed a reduction of differentiated but not undifferentiated oligodendrocytes cellular markers. While no obvious changes in myelin were observed at the ultrastructure levels in unchallenged animals, treatment with cuprizone revealed that Tmem106b−/− mice are more susceptible to cuprizone-induced demyelination and have a reduced capacity to remyelinate, a finding which we were able to replicate in a newly generated Tmem106b CRISPR/cas9 knock-out mouse model. Finally, using a TMEM106B HeLa knock-out cell line and primary cultured oligodendrocytes, we determined that loss of TMEM106B leads to abnormalities in the distribution of lysosomes and PLP1. Together these findings reveal an important function for TMEM106B in myelination with possible consequences for therapeutic strategies aimed at lowering TMEM106B levels.