👤 Wanda L Pfeifer

CLN3 Batten disease is a lethal pediatric neurodegenerative disease caused by mutations in the CLN3 gene. Typically, the disease manifests as vision loss in early childhood and progresses to neurological dysfunction and death in young adulthood. Most therapeutic developments have focused on treating the brain and may not protect against vision loss, which greatly affects quality of life. We have previously shown that a splice-switching antisense oligonucleotide (ASO) delivered to the central nervous system can reduce neurological disease burden in mouse models of CLN3 disease. Here, we apply a similar ASO approach for treating retinal dysfunction in a pig model of CLN3 Batten disease, which is more representative of human vision. A single intravitreal injection of ASO induces robust exon skipping in the retina for up to 12 months. The ASO treatment resulted in higher amplitudes on electroretinograms, suggesting mitigation of retinal dysfunction at early timepoints of disease. One ASO that efficiently induces exon skipping in vivo was well-tolerated and targets a region conserved in humans, making it a promising candidate for clinical translation. Our findings demonstrate the utility of an ASO-based approach to treat retinal dysfunction in CLN3 Batten disease and support broader ASO applications for treating ocular diseases. Show less

Promoting brown adipose tissue (BAT) activity innovatively targets obesity and metabolic disease. While thermogenic activation of BAT is well understood, the rheostatic regulation of BAT to avoid excessive energy dissipation remains ill-defined. Here, we demonstrate that adenylyl cyclase 3 (AC3) is key for BAT function. We identified a cold-inducible promoter that generates a 5' truncated AC3 mRNA isoform (Adcy3-at), whose expression is driven by a cold-induced, truncated isoform of PPARGC1A (PPARGC1A-AT). Male mice lacking Adcy3-at display increased energy expenditure and are resistant to obesity and ensuing metabolic imbalances. Mouse and human AC3-AT are retained in the endoplasmic reticulum, unable to translocate to the plasma membrane and lack enzymatic activity. AC3-AT interacts with AC3 and sequesters it in the endoplasmic reticulum, reducing the pool of adenylyl cyclases available for G-protein-mediated cAMP synthesis. Thus, AC3-AT acts as a cold-induced rheostat in BAT, limiting adverse consequences of cAMP activity during chronic BAT activation. Show less

Mouse models of CLN3 Batten disease, a rare lysosomal storage disorder with no cure, have improved our understanding of CLN3 biology and therapeutics through their ease of use and a consistent display of cellular pathology. However, the translatability of murine models is limited by disparities in anatomy, body size, life span and inconsistent subtle behavior deficits that can be difficult to detect in CLN3 mutant mouse models, thereby limiting their use in preclinical studies. Here, we present a longitudinal characterization of a novel miniswine model of CLN3 disease that recapitulates the most common human pathogenic variant, an exon 7-8 deletion (CLN3Δex7/8). Progressive pathology and neuron loss is observed in various regions of the CLN3Δex7/8 miniswine brain and retina. Additionally, mutant miniswine present with retinal degeneration and motor abnormalities, similar to deficits seen in humans diagnosed with the disease. Taken together, the CLN3Δex7/8 miniswine model shows consistent and progressive Batten disease pathology, and behavioral impairment mirroring clinical presentation, demonstrating its value in studying the role of CLN3 and safety/efficacy of novel disease-modifying therapeutics. Show less

Heparan sulfate proteoglycans (HSPGs) possess various functions driving malignancy of tumors. However, their impact on tumor cell sensitivity to cytotoxic treatment is far less understood. Aiming to investigate this, we depleted HSPGs by downregulating Exostosin 1 (EXT1), a key enzyme in HS formation, or upregulating heparanase in human MV3 human melanoma cells, and investigated their response to cytotoxic drugs. Cytotoxicity of trametinib, doxorubicin, and mitoxantrone was detected by MTT assay. Insights into intracellular signaling was provided by kinome protein profiler array, and selected kinases were inhibited to investigate their impact on cell sensitization and migratory dynamics. EXT1 knockdown (EXT1kd) in MV3 cells affected the activity of doxorubicin and mitoxantrone, significantly increasing EC Show less

Transcriptome of papillary thyroid cancer (PTC) is well characterized and correlates with some prognostic and genotypic factors, but data addressing the interaction between PTC and tumor microenvironment (TME) are scarce. Therefore, in the present study, we aimed to assess the impact of TME on gene expression profile in PTC. We evaluated the gene expression profile in PTC and normal thyroid cells isolated by laser capture microdissection and in whole tissue slides corresponding to the entire tumor. We included 26 microdissected samples for gene expression analysis (HG-U133 PLUS 2.0, Affymetrix, currently Thermo Fisher Scientific USA): 15 PTC samples, 11 samples of normal thyrocytes, and 30 whole slides (15 PTC and 15 normal thyroid). Transcripts were divided into three groups: differentially expressed both in microdissected and whole slides, transcripts differently expressed in microdissected samples and not changed in whole slides, and transcripts differentially expressed in whole slides and not changed in microdissected samples. Eleven genes were selected for validation in an independent set of samples; among them, four genes differentiated only microdissected PTC and normal cells. Two genes (PTCSC and CTGF) were confirmed. One gene (FOS) was not confirmed by the validation, whereas EGR1 was also significant in whole slide analysis. The other seven genes (TFF3, FN1, MPPED2, MET, KCNJ2, TACSTD2, and GALE) showed differentiated expression in microdissected thyrocytes and in whole tumor slides. Most of identified genes were related to the tumor-microenvironment interaction and confirmed the crosstalk between TME and cancer cells. Show less

The apolipoprotein E (APOE) gene is the most highly associated susceptibility locus for late onset Alzheimer's Disease (AD), and augmenting the beneficial physiological functions of apoE is a proposed therapeutic strategy. In a high throughput phenotypic screen for small molecules that enhance apoE secretion from human CCF-STTG1 astrocytoma cells, we show the chrysanthemic ester 82879 robustly increases expressed apoE up to 9.4-fold and secreted apoE up to 6-fold and is associated with increased total cholesterol in conditioned media. Compound 82879 is unique as structural analogues, including pyrethroid esters, show no effect on apoE expression or secretion. 82879 also stimulates liver x receptor (LXR) target genes including ATP binding cassette A1 (ABCA1), LXRα and inducible degrader of low density lipoprotein receptor (IDOL) at both mRNA and protein levels. In particular, the lipid transporter ABCA1 was increased by up to 10.6-fold upon 82879 treatment. The findings from CCF-STTG1 cells were confirmed in primary human astrocytes from three donors, where increased apoE and ABCA1 was observed along with elevated secretion of high-density lipoprotein (HDL)-like apoE particles. Nuclear receptor transactivation assays revealed modest direct LXR agonism by compound 82879, yet 10 μM of 82879 significantly upregulated apoE mRNA in mouse embryonic fibroblasts (MEFs) depleted of both LXRα and LXRβ, demonstrating that 82879 can also induce apoE expression independent of LXR transactivation. By contrast, deletion of LXRs in MEFs completely blocked mRNA changes in ABCA1 even at 10 μM of 82879, indicating the ability of 82879 to stimulate ABCA1 expression is entirely dependent on LXR transactivation. Taken together, compound 82879 is a novel chrysanthemic ester capable of modulating apoE secretion as well as apoE-associated lipid metabolic pathways in astrocytes, which is structurally and mechanistically distinct from known LXR agonists. Show less

Juvenile Neuronal Ceroid Lipofuscinosis (JNCL) presents with progressive vision loss at 4-7 years of age. Blindness results within 2 years, followed by inexorable neurologic decline and death. There is no treatment or cure. Neuroinflammation is postulated to play a role in the neurodegeneration. The JNCL mouse model demonstrated decreased neuroinflammation and improved motor skills with immunosuppression. Based on this work, a short-term human clinical trial of mycophenolate mofetil has begun, however longer term effects, and whether immunosuppression modulates vision loss, have not been studied. We report a JNCL patient treated with immunosuppressive therapy in whom visual function was comprehensively characterized over 2 years. Show less

The insulinotropic action of the incretin glucose-dependent insulinotropic polypeptide (GIP) is impaired in type 2 diabetes, while the effect of glucagon-like peptide-1 (GLP-1) is preserved. To evaluate the role of impaired GIP function in glucose homeostasis and development of the endocrine pancreas in a large animal model, we generated transgenic pigs expressing a dominant-negative GIP receptor (GIPR(dn)) in pancreatic islets. GIPR(dn) transgenic pigs were generated using lentiviral transgenesis. Metabolic tests and quantitative stereological analyses of the different endocrine islet cell populations were performed, and beta-cell proliferation and apoptosis were quantified to characterize this novel animal model. Eleven-week-old GIPR(dn) transgenic pigs exhibited significantly reduced oral glucose tolerance due to delayed insulin secretion, whereas intravenous glucose tolerance and pancreatic beta-cell mass were not different from controls. The insulinotropic effect of GIP was significantly reduced, whereas insulin secretion in response to the GLP-1 receptor agonist exendin-4 was enhanced in GIPR(dn) transgenic versus control pigs. With increasing age, glucose control deteriorated in GIPR(dn) transgenic pigs, as shown by reduced oral and intravenous glucose tolerance due to impaired insulin secretion. Importantly, beta-cell proliferation was reduced by 60% in 11-week-old GIPR(dn) transgenic pigs, leading to a reduction of beta-cell mass by 35% and 58% in 5-month-old and 1- to 1.4-year-old transgenic pigs compared with age-matched controls, respectively. The first large animal model with impaired incretin function demonstrates an essential role of GIP for insulin secretion, proliferation of beta-cells, and physiological expansion of beta-cell mass. Show less

The molecular mechanisms of cell cycle exit are poorly understood. Studies on lymphocytes at cell cycle exit after growth factor deprivation have predominantly focused on the initiation of apoptosis. We aimed to study gene expression profile of primary and immortalised IL-2-dependent human T cells forced to exit the cell cycle by growth factor withdrawal, before apoptosis could be evidenced. By the Affymetrix microarrays HG-U133 2.0 Plus, 53 genes were distinguished as differentially expressed before and soon after IL-2 deprivation. Among those, PIM1, BCL2, IL-8, HBEGF, DUSP6, OSM, CISH, SOCS2, SOCS3, LIF and IL13 were down-regulated and RPS24, SQSTM1, TMEM1, LRRC8D, ECOP, YY1AP1, C1orf63, ASAH1, SLC25A46 and MIA3 were up-regulated. Genes linked to transcription, cell cycle, cell growth, proliferation and differentiation, cell adhesion, and immune functions were found to be overrepresented within the set of the differentially expressed genes. Cell cycle exit of the growth factor-deprived T lymphocytes is characterised by a signature of differentially expressed genes. A coordinate repression of a set of genes known to be induced during T cell activation is observed. However, growth arrest following exit from the cell cycle is actively controlled by several up-regulated genes that enforce the non-dividing state. The identification of genes involved in cell cycle exit and quiescence provides new hints for further studies on the molecular mechanisms regulating the non-dividing state of a cell, the mechanisms closely related to cancer development and to many biological processes. Show less