Patil, S. V. | Search bioRxiv

bioRxiv2025

A human pluripotent stem cell tri-culture platform to elucidate microglial regulation of retinal ganglion cells in neuroinflammation

Optic neuropathies, including glaucoma, are characterized by the progressive degeneration of retinal ganglion cells (RGCs), ultimately leading to irreversible vision loss. Increasing evidence implicates microglia, the resident immune cells of the central nervous system, as key modulators of RGC health and disease progression. However, the precise mechanisms by which microglia influence RGCs remain poorly understood, particularly in the human context. In this study, we established human pluripotent stem cell (hPSC)-derived co-culture systems incorporating microglia, astrocytes, and RGCs to explore how microglia shape RGC growth and maturation under physiological conditions. We first examined the impact of homeostatic microglia on RGCs in both co-culture and tri-culture systems, revealing distinct influences of cell types in co-culture compared to when they were grown individually. We then modeled inflammatory states by activating microglia with lipopolysaccharide (LPS) and evaluated their effects on RGCs both directly and in the context of astrocyte co-culture. This stepwise, reductionist approach enabled us to dissect the cellular interactions driving RGC vulnerability in inflammatory conditions relevant to optic neuropathies. Our findings provide new insight into the complex neuroimmune landscape that underlies RGC degeneration and identify key pathways that may serve as therapeutic targets across a range of optic nerve diseases.

neuroscience↗

bioRxiv2018

Direct Measurement of Dissipation in a Single Protein using Small Amplitude Atomic Force Microscopy

In Krammers theory, stiffness and dissipation coefficient of a protein determine the rate of their conformational change. Using atomic force microscope, it is possible to measure viscoelasticity of a single protein, wherein its dissipative and elastic nature is directly and independently measured. Such measurements are performed, either by measuring the thermal fluctuations of the protein held under a constant force, or by providing small modulations to the protein by dithering the cantilever and measuring its response. In small amplitude approximation, where dither amplitude is comparable to persistence length of polymers, it is possible to measure the proteins viscoelastic response accurately. We measured dissipation in I27 at extremely low pulling speeds ([~] 50 nm/s) and low dither frequencies ([~]100 Hz). At these experimental parameters the dissipation is found to be [~]10-5 kg/s, well above the detection limit of conventional AFM and upper limit predicted by Benedetti et al. Our stiffness data clearly reveals unfolding intermediate of titins individual immunoglobulin units. The intermediate is elongation of folded domains by [~]8 [A], wherein two hydrogen bonds are broken between beta sheets. It was possible to measure this elongation in our experiments. The directly measured internal friction of unfolded polymer chain shows a scaling with tension on the chain. The measurements show that it is possible to measure internal friction in single molecules unambiguously using small amplitude AFM. It suggests that systematic experiments to unravel the relation between directly measured internal friction and folding rates of proteins are possible.

biophysics↗