patch-clamp-recording-starburst-amacrine-cells-flat-mount-preparation

Patch Clamp Recording of Starburst Amacrine Cells in a Flat-mount Preparation of Deafferentated Mouse Retina

The mammalian retina is a layered tissue composed of multiple neuronal types. To understand how visual signals are processed within its intricate synaptic ...

Baylor College of Medicine

Protein aggregation is one of the characteristic steps in a number of neurodegenerative diseases eventually leading to neuronal death and thorough study of aggregation is required for the development of effective therapy. We apply fluorescence lifetime imaging for the characterization of the fluorescence dynamics of the enhanced green fluorescent protein (eGFP) in fusion with the polyQ-expanded polyglutamine stretch. At the expansion of polyQ above 39 residues, it has an inherent propensity to form amyloid-like fibrils and aggregates, and is responsible for Huntington's disease. The results of the experiments show that expression of the eGFP in fusion with the 97Q protein leads to the decrease of the eGFP fluorescence lifetime by approximately 300 ps. This phenomenon does not appear in Hsp104-deficient cells, where the aggregation in polyQ is prevented. We demonstrate that the lifetime decrease observed is related to the aggregation per se and discuss the possible role of refractive index and homo-FRET in these dynamics.

Fluorescence lifetime dynamics of enhanced green fluorescent protein in protein aggregates with expanded polyglutamine.

Tauopathies are neurodegenerative diseases based on pathological tau-aggregation including Alzheimer's disease, frontotemporal dementia (FTD) and Pick's disease. In general, cargo (e.g., β-amyloid precursor protein, tau, neurofilaments) accumulation is a commonly observed phenomenon in degenerated neurons. Therefore, it is crucial to investigate the interaction between cargo, microtubule-binding proteins and molecular motors. We report the effect of tau/PTL-1 (protein with tau-like repeats) on the transport characteristics of the major axonal transporter kinesin-3 KIF1A/UNC-104 in the nervous system of Caenorhabditis elegans. Using confocal spinning disk time-lapse imaging we analyzed the motility of UNC-104::mRFP in ptl-1 knockout worms and found that predominantly retrograde moving characteristics are affected (rather than the motor's anterograde displacements). A similar motility pattern was observed for synaptobrevin-1-containing vesicles, a major cargo of UNC-104. Moreover, UNC-104 and PTL-1 colocalize and occasionally co-migrate. We further confirmed physical interactions between PTL-1 and UNC-104 in living animals using the bimolecular fluorescence complementation assay (BiFC) as well as in co-immunoprecipitation experiments. Though this study focuses on PTL-1/UNC-104 interactions, we extended our research on monitoring conventional kinesin-1 (UNC-116) as well as dynein motility pattern and found that in ptl-1 mutants retrograde displacements were also affected for UNC-116, while for dynein, interestingly, its anterograde movements were affected.

Tau/PTL-1 associates with kinesin-3 KIF1A/UNC-104 and affects the motor's motility characteristics in C. elegans neurons.

At hippocampal synapses, activation of group I metabotropic glutamate receptors (mGluRs) induces long-term depression (LTD), which requires new protein synthesis. However, the underlying mechanism remains elusive. Here we describe the translational program that underlies mGluR-LTD and identify the translation factor eIF2α as its master effector. Genetically reducing eIF2α phosphorylation, or specifically blocking the translation controlled by eIF2α phosphorylation, prevented mGluR-LTD and the internalization of surface AMPA receptors (AMPARs). Conversely, direct phosphorylation of eIF2α, bypassing mGluR activation, triggered a sustained LTD and removal of surface AMPARs. Combining polysome profiling and RNA sequencing, we identified the mRNAs translationally upregulated during mGluR-LTD. Translation of one of these mRNAs, oligophrenin-1, mediates the LTD induced by eIF2α phosphorylation. Mice deficient in phospho-eIF2α-mediated translation are impaired in object-place learning, a behavioral task that induces hippocampal mGluR-LTD in vivo. Our findings identify a new model of mGluR-LTD, which promises to be of value in the treatment of mGluR-LTD-linked cognitive disorders.

Chih-Chun Hsu

Department of Ophthalmology,
Department of Neuroscience

Baylor College of Medicine

Patch Clamp Recording of Starburst Amacrine Cells in a Flat-mount Preparation of Deafferentated Mouse Retina

Chih-Chun Hsu

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Ophthalmology,Department of Neuroscience

Baylor College of Medicine

Patch Clamp Recording of Starburst Amacrine Cells in a Flat-mount Preparation of Deafferentated Mouse Retina

Department of Ophthalmology,
Department of Neuroscience