Biology subjects

Raleigh, D.

Publications and source records attributed to Raleigh, D..

5 recordsLinked to original sources

bioRxiv2026

Lag phase length of IAPP amyloid formation predicts the duration of β-cell toxicity

Aggregation of islet amyloid polypeptide (IAPP) contributes to pancreatic {beta}-cell dysfunction in diabetes, yet the identity and temporal persistence of the toxic species remain unresolved. Because amyloid formation proceeds through distinct kinetic phases, relating cytotoxicity to these phases provides a direct strategy to test competing mechanistic models. Here, we combine time-resolved {beta}-cell functional assays with concurrent biophysical measurements to quantitatively link IAPP aggregation kinetics to cellular dysfunction across multiple perturbations and sequence variants. We demonstrate that maximal toxicity occurs during the lag phase and establish aggregation kinetics as a predictive framework for the onset and duration of IAPP proteotoxicity. Across 22 independent experiments spanning more than a 450-fold range in lag times, the duration of {beta}-cell dysfunction scales linearly with lag phase length. Perturbations that alter aggregation kinetics, including changes in concentration, temperature, and sequence, predictably shift both the onset and duration of toxicity. Early lag-phase intermediates of the diabetes-associated S20G-IAPP mutant are more toxic than corresponding species formed by wild-type h-IAPP. Together, these results show that aggregation kinetics quantitatively define the temporal window of IAPP proteotoxicity and support a model in which transient pre-fibrillar intermediates, rather than mature fibrils, dominate {beta}-cell dysfunction.

biochemistry↗

bioRxiv2022

Increased mRNA expression of CDKN2A is a transcriptomic marker of clinically aggressive meningiomas

BackgroundHomozygous loss of CDKN2A/B is a genetic alteration found in many cancer types including meningiomas, where it is associated with poor clinical outcome. It is now also a diagnostic criterion for grade 3 meningiomas in the 2021 WHO classification for central nervous system tumors. However, as in other cancers, the relationship between copy number loss of CDKN2A/B and expression of its gene product is unclear and may be either commensurate or paradoxical in nature. Therefore, we aimed to investigate the association of CDKN2A mRNA expression with clinical prognosis, WHO grade, and other molecular biomarkers in meningiomas such as DNA methylation, molecular group, and proteomics. MethodsWe used multidimensional molecular data of 490 meningioma samples from 4 independent cohorts to examine the relationship between mRNA expression of CDKN2A and copy number status, its correlation to clinical outcome, the transcriptomic pathways altered in differential CDKN2A expression, and its relationship with DNA methylation, and proteomics using an integrated molecular approach. ResultsMeningiomas without any copy number loss were dichotomized into high (CDKN2Ahigh) and low (CDKN2Alow) CDKN2A mRNA expression groups. Patients with CDKN2Ahigh meningiomas had poorer progression free survival (PFS) compared to those with CDKN2Alow meningiomas. CDKN2A mRNA expression was increased in more aggressive molecular groups, and in higher WHO grade meningiomas across all cohorts. CDKN2Ahigh meningiomas and meningiomas with CDKN2A copy number loss shared common up-regulated cell cycling pathways. CDK4 mRNA expression was increased in CDKN2Ahigh meningiomas and both p16 and CDK4 protein were more abundant in CDKN2Ahigh meningiomas. CDKN2Ahigh meningiomas were frequently hypermethylated at the gene body and UTR compared to CDKN2Alow meningiomas and found be more commonly Rb-deficient. ConclusionsAn intermediate level of CDKN2A mRNA expression appears to be optimal as significantly low (CDKN2A deleted) or high expression (CDKN2Ahigh) are associated with poorer outcomes clinically. Though CDK4 is elevated in CDKN2Ahigh meningiomas, Rb-deficiency may be more common in this group, leading to lack of response to CDK inhibitors.

cancer biology↗

bioRxiv2022

The cilia enriched oxysterol 7β,27-DHC is required for polycystin activation

PC-1 and PC-2 form a heteromeric ion channel complex (hereafter called the Polycystin complex) that is abundantly expressed on primary cilia of renal epithelial cells. Mutations within the polycystin complex cause Autosomal Dominant Polycystic Kidney Disease (ADPKD). The Polycystin complex forms a non-selective cation channel, yet the spatial and temporal regulation of the polycystin complex within the ciliary membrane remains poorly understood, partially due to technical limitations posed by the tiny ciliary compartment. Here, we employ our novel assays to functionally reconstitute the polycystin complex in the plasma membrane. Using whole-cell and ciliary patch-clamp recordings we identified a ciliary enriched oxysterol, 7{beta},27-DHC, as a critical component required for activation of the polycystin complex. We identified a novel oxysterol binding pocket in PC-2 using molecular docking simulation. We also identified two amino acids within the PC-2 oxysterol binding pocket, E208 and R581, to be critical for 7{beta},27-DHC dependent polycystin activation in both the plasma membrane and ciliary compartment. Further, we can show that the pharmacological and genetic inhibition of oxysterol synthesis by carbenoxolone (CNX) reduces channel activity in primary cilia. Our findings identified a unique second messenger that regulates the polycystin complex. We hypothesize that cilia-enriched lipids license the polycystin complex to be functional only in the ciliary organelle, thus providing novel insights into the spatial regulation of the polycystin complex. Our results also establish a framework to target the same allosteric regulatory site in the polycystin complex to identify activators of the polycystin channels as novel therapeutic strategies for ADPKD.

physiology↗

bioRxiv2021

The Fluorescent Dye 1,6-Diphenyl-1,3,5-Hexatriene Binds to Amyloid Fibrils Formed by Human Amylin and Provides a New Probe of Amylin Amyloid Kinetics

The fluorescent dye 1,6-diphenyl-1,3,5-hexatriene (DPH) is widely used as a probe of membrane order. We show that DPH also interacts with amyloid fibrils formed by human amylin (also known as islet amyloid polypeptide) in solution and this results in a 100-fold increase in DPH fluorescence for a sample of 20M human amylin and 0.25 M DPH. No increase in DPH fluorescence is observed with the non-amyloidogenic rat amylin or with freshly dissolved, non-fibrillar human amylin. The time course of amyloid formation by amylin was followed by monitoring the fluorescence of added DPH as a function of time and was similar to that monitored by the standard fluorescent probe thioflavin-T. The inclusion of DPH in the buffer did not perturb the time course of amyloid formation under the conditions examined and the time course was independent of the range of DPH concentrations tested (0.25 to 5 M). Maximum final fluorescence intensity is observed at substoichiometric ratios of DPH to amylin. No significant increase in fluorescence was observed during the lag phase of amyloid formation, and the implications for the structure of amylin pre-fibril oligomers are discussed. Human amylin contains three aromatic residues. A triple aromatic to leucine mutant forms amyloid and DPH binds to the resulting fibrils, indicating that interactions with aromatic side chains are not required for DPH amylin amyloid interactions. DPH may be especially useful for studies on mutant amylins and other polypeptides in which changes in charged residues might complicate interpretation of thioflavin-T fluorescence.

biophysics↗

bioRxiv2021

Glioblastoma remodeling of neural circuits in the human brain decreases survival

Gliomas synaptically integrate into neural circuits. Prior work has demonstrated bidirectional interactions between neurons and glioma cells, with neuronal activity driving glioma growth and gliomas increasing neuronal excitability. In this study we wanted to know how glioma induced neuronal changes influence neural circuits underlying cognition and whether these interactions influence patient survival. We use intracranial brain recordings during lexical retrieval language tasks in awake humans in addition to site specific tumor tissue biopsies and cell biology experiments. We find that gliomas remodel functional neural circuitry such that task-relevant neural responses activate tumor-infiltrated cortex, beyond cortical excitation normally recruited in the healthy brain. Site-directed biopsies from functionally connected regions within the tumor are enriched for a glioblastoma subpopulation that exhibits a distinct synaptogenic and neuronotrophic phenotype. Tumor cells from functionally connected regions secrete the synaptogenic factor thrombospondin-1, which contributes to the differential neuron-glioma interactions observed in functionally connected tumor regions compared to tumor regions with less functional connectivity. The degree of functional connectivity between glioblastoma and the normal brain negatively impacts both patient survival and language task performance. These data demonstrate that high-grade gliomas functionally remodel neural circuits in the human brain, which both promotes tumor proliferation and impairs cognition.

cancer biology↗