Using Laser Tweezers For Manipulating Isolated Neurons In Vitro

In this paper and video, we describe the protocols used in our laboratory to study the targeting preferences of regenerating cell processes of adult retinal neurons in vitro. Procedures for preparing retinal cell cultures start with the dissection, digestion and trituration of the retina, and end with the plating of isolated retinal cells on dishes made especially for use with laser tweezers. These dishes are divided into a cell adhesive half and a cell repellant half. The cell adhesive side is coated with a layer of Sal-1 antibodies, which provide a substrate upon which our cells grow. Other adhesive substrates could be used for other cell types. The cell repellant side is coated with a thin layer of poly-HEMA. The cells plated on the poly-HEMA side of the dish are trapped with the laser tweezers, transported and then placed adjacent to a cell on the Sal-1 side to create a pair. Formation of cell groups of any size should be possible with this technique. "Laser-tweezers-controlled micromanipulation" means that the investigator can choose which cells to move, and the desired distance between the cells can be standardized. Because the laser beam goes through transparent surfaces of the culture dish, cell selection and placement are done in an enclosed, sterile environment. Cells can be monitored by video time-lapse and used with any cell biological technique required. This technique may help investigations of cell-cell interactions

ROCK inhibitors in ocular disease

Rho kinases (ROCKs) have a crucial role in actin-cytoskeletal reorganization and thus are involved in broad aspects of cell motility, from smooth muscle contraction to neurite outgrowth. The first marketed ROCK inhibitor, called fasudil, has been used safely for treatment of cerebral vasospasm since 1995 in Japan. During the succeeding decades ROCK inhibitors have been applied in many pathological conditions from central nervous system disorders to cardiovascular disease as potential therapeutic agents or experimental tools to help understand the underlying (patho)mechanisms. In 2014, a fasudil derivate named ripasudil was accepted for clinical use in glaucoma and ocular hypertension. Since ROCK kinases are widely expressed in ocular tissues, they have been implicated in the pathology of many ocular conditions such as corneal dysfunction, glaucoma, cataract, diabetic retinopathy, age-related macular degeneration, and retinal detachment. This paper aims to provide an overview of the most recent status/application of ROCK inhibitors in the field of eye disease

Lim kinase, a bi-functional effector in injury-induced structural plasticity of synapses

The structural plasticity of synaptic terminals contributes to normal nervous system function but also to neural degeneration, in the form of terminal retraction, and regeneration, due to process growth. Synaptic morphological change is mediated through the actin cytoskeleton, which is enriched in axonal and dendritic terminals. Whereas the three RhoGTPases, RhoA, Cdc42 and Rac, function as upstream signaling nodes sensitive to extracellular stimuli, LIMK-cofilin activity serves as a common downstream effector to up-regulate actin turnover, which is necessary for both polymerization and depolymerization. The dual effects of LIMK activity make LIMK a potential target of therapeutic intervention for injury-induced synaptic plasticity, as LIMK inhibition can stabilize actin cytoskeleton and preserve existing structure. This therapeutic benefit of LIMK inhibition has been demonstrated in animal models of injury-induced axon retraction and neuritic sprouting by rod photoreceptors. A better understanding of the regulation of LIMK-cofilin activity and the interaction with the microtubular cytoskeleton may open new ways to promote synaptic regeneration that can benefit neuronal degenerative disease

ROCK inhibitors in ocular disease

Rho kinases (ROCKs) have a crucial role in actin-cytoskeletal reorganization and thus are involved in broad aspects of cell motility, from smooth muscle contraction to neurite outgrowth. The first marketed ROCK inhibitor, called fasudil, has been used safely for treatment of cerebral vasospasm since 1995 in Japan. During the succeeding decades ROCK inhibitors have been applied in many pathological conditions from central nervous system disorders to cardiovascular disease as potential therapeutic agents or experimental tools to help understand the underlying (patho)mechanisms. In 2014, a fasudil derivate named ripasudil was accepted for clinical use in glaucoma and ocular hypertension. Since ROCK kinases are widely expressed in ocular tissues, they have been implicated in the pathology of many ocular conditions such as corneal dysfunction, glaucoma, cataract, diabetic retinopathy, age-related macular degeneration, and retinal detachment. This paper aims to provide an overview of the most recent status/application of ROCK inhibitors in the field of eye disease

RhoA Inactivation Prevents Photoreceptor Axon Retraction in an In Vitro Model of Acute Retinal Detachment

Axon retraction by rod cells is an early response to detachment that likely contributes to poor outcomes after reattachment. In mammalian retina, blocking RhoA signaling prevents axon retraction. Effective treatment can occur as much as 6 hours after the detachment

Cell Specific Post-Translational Processing of Pikachurin, A Protein Involved in Retinal Synaptogenesis

<div><p>Pikachurin is a recently identified, highly conserved, extracellular matrix-like protein. Murine pikachurin has 1,017 amino acids (∼110 kDa), can bind to α-dystroglycan, and has been found to localize mainly in the synaptic cleft of photoreceptor ribbon synapses. Its knockout selectively disrupts synaptogenesis between photoreceptor and bipolar cells. To further characterize this synaptic protein, we used an antibody raised against the N-terminal of murine pikachurin on Western blots of mammalian and amphibian retinas and found, unexpectedly, that a low weight ∼60-kDa band was the predominant signal for endogenous pikachurin. This band was predicted to be an N-terminal product of post-translational cleavage of pikachurin. A similar sized protein was also detected in human Y79 retinoblastoma cells, a cell line with characteristics of photoreceptor cells. In Y79 cells, endogenous pikachurin immunofluorescence was found on the cell surface of living cells. The expression of the N-fragment was not significantly affected by dystroglycan overexpression in spite of the biochemical evidence for pikachurin-α-dystroglycan binding. The presence of a corresponding endogenous C-fragment was not determined because of the lack of a suitable antibody. However, a protein of ∼65 kDa was detected in Y79 cells expressing recombinant pikachurin with a C-terminal tag. In contrast, in QBI-HEK 293A cells, whose endogenous pikachurin protein level is negligible, recombinant pikachurin did not appear to be cleaved. Instead pikachurin was found either intact or as dimers. Finally, whole and N- and C-fragments of recombinant pikachurin were present in the conditioned media of Y79 cells indicating the secretion of pikachurin. The site of cleavage, however, was not conclusively determined. Our data suggest the existence of post-translational cleavage of pikachurin protein as well as the extracellular localization of cleaved protein specifically by retinal cells. The functions of the pikachurin N- and C-fragments in the photoreceptor ribbon synapse are unknown.</p> </div

C-terminal fragment detected in Y79 cells expressing exogenous pikachurin.

<p>(A) Schematic of c-Myc/His tagged murine pikachurin (PK). SS, signal sequence; FN3, fibronectin type 3 domain; LamG, laminin G domain. The numbers indicate the end positions of three N-terminal mutants. (B) Western blots of Y79 cell lysates after transfection of double-tagged pikachurin (dPK). pCIG, pCIG-transfected controls. Note the presence of pikachurin monomer (open arrow), dimer (filled arrow), and fragments (arrowheads). PK antibody- and His antibody-labeled fragments are from the N and C-terminal respectively; PK antibody labels both endogenous and exogenous protein. (C) Western blots of QBI-HEK 293A cell lysates after transfection of dPK. There are no fragments of pikachurin but monomers (open arrow) and dimers (filled arrow) are present. A small band (*), found in both control and dPK-transfected samples, is believed to be non-specific. (D) N-terminal mutants (P39, P55 and P66) were used to transfect QBI-HEK 293A cells and their expression was confirmed on Western blot. Similar to dPK, P55 and P66 not only existed in monomers but also formed dimers (arrows). However, P39 did not dimerize indicating the loss of the site of dimerization. (E) Pikachurin (PK) immunofluorescence of dPK-transfected Y79 cells. Nuclear GFP marks transfected cells. Labeling is primarily due to overexpressed exogenous PK. Non-permeabilized immunolabeling highlights cell surface label. Bar = 10 µm. (F) Co-immunolabeling of PK and His tag in dPK-transfected Y79 cells. Immunofluorescence of PK and His tag is generally overlapping. However, some cells have relatively stronger His signals than their pikachurin counterparts (arrows). Bar = 10 µm. (G) Co-immunolabeling of FLAG and His tags in dPK-transfected QBI-HEK 293A cells. Large cell size allows the separation of signals into cytoplasmic and perinuclear regions respectively. Bar = 20 µm.</p

Pikachurin fragments present in the conditioned medium of Y79 cells.

<p>(A) Western blots of the conditioned medium from pCIG and dPK transfected cells. PK antibody labeled N fragments and whole protein and His antibody labeled C fragments and whole protein. (B) Inclusion of protease inhibitors in culture medium (dPK/PIs) didn’t affect the expression of dPK in the cell lysates (CL) but decreased its levels in the conditioned medium (CM). Note: c-Myc labels only recombinant protein. Short exposure reveals the high levels of recombinant pikachurin monomers and oligomers on the CL blot. Small fragments on this blot were visible with prolonged exposure but on a much darker background (data not shown). (C) Morphological changes in Y79 cells induced by incubation with protease inhibitors for 48 hrs. The solvent DMSO was diluted 1∶400 in the culture medium for control cells. Bar = 10 µm. (D) Density ratios of Western blot bands (N-fragments/monomers, Fig. 4B). N = 3 experiments. * = significant difference between protease inhibition treatment group (dPK/PIs) and control group (dPK) (p<0.05, one-way ANOVA test).</p