Cellular

mechanosensitive pathway defects in ALS neurons

Riccardo Sirtori, Claudia Fallini

The maintenance of specific cell morphology and function within a tissue relies on the ability of cells to respond and adapt to dynamic changes in their micro-environment. This response consists in bidirectional feedback between microenvironmental signals, the contractile cytoskeleton that responds to these signals and determines cell shape, and the transcriptional circuitry that establishes and maintains cell identity. Extracellular forces exerted from the extracellular matrix (ECM) to the plasma membrane via adhesion molecules are transmitted to the cytoskeleton and then to the nucleus, ultimately resulting in changes to nuclear morphology, chromatin compaction/organization, and gene expression to adapt cell features to the environment mechanics. During aging occurs the accumulation of intra-intermolecular covalent bonds (crosslinks) between molecules with slow turnovers, such as collagen and elastin of the ECM. These crosslinks affect the physical properties of the ECM (i. e. stiffness) forcing cells to adapt to the changing environment. These physiologic changes of brain mechanical properties make the mechanical response of neurons crucial for the maintenance of their function and their survival. In the present study we show the effect of substrates (Matrigel and PDL/laminin) with different mechanical features on C9orf72 iNs (C9exp iNs) and isogenic controls (C9iso iNs). Our data demonstrate an alteration of the mechanical adaptation of C9exp iNs to high stiffness substrates affecting cell morphology and gene expression. Furthermore, we found the alteration of this complex pathway involves many different steps of the mechanical signaling cascade from the focal adhesions to the nucleus.

Development of nanoparticle-based targeted drug delivery system to mitigate alcoholrelated liver disease (ALD)

Janitha M Unagolla, Subarna Das, Jyothi U. Menon\

Alcohol-related liver disease (ALD) is the most prevalent form of liver disease commonly associated with hepatocellular carcinoma. The current anti-inflammatory, anti-viral, and immunosuppressive therapies for liver inflammation are not consistently effective and safe. Therefore, effective and timely interventions for targeted and sustained treatment of chronic ALD remain an urgent and unmet clinical need. The G-protein coupled bile acid receptor TGR5 (Gpbar1) expressed in Kupffer cells (KCs) in liver has a potent anti-inflammatory effect. Also, we incorporated INT 777, a highly selective and potent semisynthetic bile acid agonist of Gpbar1 attenuates pro-inflammatory cytokine production by KCs, into carboxymethyl chitosan (CMC) coated poly lactic co glycolic acid (PLGA) nanoparticle (NPs) to target KCs. In addition, CMC, a pH-responsive biodegradable polymer, was used to trigger the release of the anti-inflammatory drug dexamethasone (DEX) in response to the acidic pH in the inflamed liver microenvironment. Therefore, concurrent NP-mediated Gpbar1 stimulation and pH-responsive DEX release in the acidic microenvironment have an additive effect in mitigating chronic hepatic inflammation. The NPs were developed using acetone and dichloromethane (1:1 ratio) as a solvent using a single emulsion technique in a 5% (v/v) polyvinyl alcohol (PVA) solution. INT 777 was conjugated to NPs using a carbodiimide coupling reaction. The synthesized NPs showed good particle size (<300 nm), zeta potential, and stability. The INT conjugation was confirmed using x-ray photoelectron spectroscopy (XPS), and cytocompatibility studies confirmed that NPs were biocompatible. The DEX release study confirmed the pH-responsive release of DEX from NPs. The cell uptake study using THP1, a leukemia monocytic cell line representing KCs, showed that INT-conjugated particles targeted THP1 cells. The ALD was developed in a mouse model by giving a 5% (v/v) ethanol diet and injecting NPs with INT. The results confirmed the inflamed liver and accumulation of NPs in the liver.

Assessing Student, Pharmacist and Clinician Perspectives on Pharmacist Prescribed Hormonal Contraceptives

Audrey Whalen, Jeffrey Bratberg, Virginia Lemay

Background/Objectives: As of April 2023, 22 states and jurisdictions have a protocol in place to allow for autonomous prescribing of hormonal contraceptives by pharmacists. The American College of Obstetricians and Gynecologists (ACOG) has expressed their support for pharmacists prescribing hormonal contraceptives in addition to over-the-counter access to mitigate barriers to access. The objective of this study was to simultaneously assess student, pharmacist, and other clinician perspectives on pharmacist-prescribed hormonal contraceptives, to support expanded pharmacist authority to prescribe hormonal contraceptives in Rhode Island.

Methods: Researchers conducted an observational study via a cross-sectional survey distributed to students, licensed pharmacists and clinicians. This study was IRB approved through the University of Rhode Island as exempt and data were collected from October to December 2022. The survey required approximately 8 minutes to complete and assessed feelings and perspectives towards pharmacist prescribed hormonal contraception, likelihood of receiving pharmacist prescribed hormonal contraception and pharmacist comfort with prescribing hormonal contraceptives.

Results: Nearly 90% of students, 80% of pharmacists and 70% of clinicians either agree or strongly agree with pharmacist prescribed hormonal contraception. Sixty-one percent of pharmacists said that they are either very or somewhat comfortable prescribing hormonal contraception. The most frequently reported barriers in accessing contraception for all respondents, were time/delay in appointment, cost/copay, and lack of gynecologist. Approximately 76% of students said they were extremely or somewhat likely to obtain a hormonal contraception prescription from their pharmacist.

Implications/Conclusions: The majority of pharmacists, clinicians, students and in Rhode Island support pharmacist prescribed hormonal contraceptives and the most frequently reported barriers faced in accessing contraception can be overcome through pharmacist prescribing. The information and data gathered from this study has already had an impact to support pending legislation and implementation of prescriptive authority and expanded access to contraception in the State of Rhode Island.

Aβ40 and Aβ42 adopt distinct fibril structures that differentially stimulate astrocytes and microglia

Xiaoyue Zhu, Joseph M. Schrader, Brandon A. Irizarry, Steven O. Smith, William E. Van Nostrand

Fibrillar amyloid β-protein (Aβ) deposits in brain, which are primarily composed of Aβ40 or Aβ42 peptides, are key pathological features of Alzheimer’s disease and related disorders. Structural studies on mature A 40 fibrils formed in solution have suggested they adopt a two stranded (U-shaped) structure whereas studies on A 42 fibrils typically show a three-stranded structure. Fibrillar amyloid deposits in brain promote activation of astrocytes and microglia although the underlying mechanisms are still not clear. Here, we treated primary astrocyte and microglia cells with Aβ40 or Aβ42 fibrils, and bulk RNA sequencing was performed. According to the Pearson’s correlation result, astrocytes and microglia cells undergo a great number of Aβ peptides-induced changes in gene expression and Aβ42 and Aβ40 peptides showed similar effects on glia cells compared with the control group. To elucidate the gene changes after Aβ peptides treatment, we conducted the differential gene expression analysis to compare Aβ42 and Aβ40 glia cells. The expression data were set with limits of ≥5-fold increase in expression and p < 0.05. A greater number of genes were differentially expressed by Aβ42 treated glia cells compared with the Aβ40 treated glia cells. Immunohistochemical analysis validated our RNAseq data by performing in an AD rat model with parenchymal fibrillar Aβ42 deposits confirming the expression of MMP9, MMP12, PAI-1 and C1r in plaque-associated microglia and iNOS, GBP2 and C3D in plaque-associated astrocytes. Ingenuity Pathway Analysis further highlighted that Aβ42 treatment up-regulated cellular activation pathways and immune response pathways in glia cells. Interestingly, we found that astrocytes treated with Aβ fibrils up-regulated a larger number of A1 reactive genes compared with A2 reactive genes, and microglia treated with Aβ fibrils upregulated more M1 state genes than M2 state genes. In summary, ex vivo studies show that the distinct structures of Aβ40 and Aβ42 fibrils differentially stimulate the gene expression profile of glia cells and that Aβ42 fibrils are more potent stimulators, together with the intracerebral injection with Aβ fibrils, we are aiming to provide a detailed insight in the pathogenesis of AD and related amyloid-depositing disorders.