Inherited retinal degenerations, caused by mutations in over 100 individual genes, affect approximately 2 million people worldwide. Many of the underlying mutations cause protein misfolding or mistargeting in affected photoreceptors. This places an increased burden on the protein folding and degradation machinery, which may trigger cell death. We analyzed how these cellular functions are affected in degenerating rods of the transducin γ-subunit (Gγ ₁ ) knockout mouse. These rods produce large amounts of transducin β-subunit (Gβ ₁ ), which cannot fold without Gγ ₁ and undergoes intracellular proteolysis instead of forming a transducin βγ-subunit complex. Our data revealed that the most critical pathobiological factor leading to photoreceptor cell death in these animals is insufficient capacity of proteasomes to process abnormally large amounts of misfolded protein. A decrease in the Gβ ₁ production in Gγ ₁ knockout rods resulted in a significant reduction in proteasomal overload and caused a striking reversal of photoreceptor degeneration. We further demonstrated that a similar proteasomal overload takes place in photoreceptors of other mutant mice where retinal degeneration has been ascribed to protein mistargeting or misfolding, but not in mice whose photoreceptor degenerate as a result of abnormal phototransduction. These results establish the prominence of proteasomal insufficiency across multiple degenerative diseases of the retina, thereby positioning proteasomes as a promising therapeutic target for treating these debilitating conditions.

Transducin is a prototypic heterotrimeric G-protein mediating visual signaling in vertebrate photoreceptor cells. Despite its central role in phototransduction, little is known about the mechanisms that regulate its expression and maintain approximately stoichiometric levels of the α- and βγ-subunits. Here we demonstrate that the knock-out of transducin γ-subunit leads to a major downregulation of both α- and β-subunit proteins, despite nearly normal levels of the corresponding transcripts, and fairly rapid photoreceptor degeneration. Significant fractions of the remaining α- and β-subunits were mislocalized from the light-sensitive outer segment compartment of the rod. Yet, the tiny amount of the α-subunit present in the outer segments of knock-out rods was sufficient to support light signaling, although with a markedly reduced sensitivity. These data indicate that the γ-subunit controls the expression level of the entire transducin heterotrimer and that heterotrimer formation is essential for normal transducin localization. They further suggest that the production of transducin β-subunit without its constitutive γ-subunit partner sufficiently stresses the cellular biosynthetic and/or chaperone machinery to induce cell death.

Inherited retinal degenerations, affecting more than 2 million people worldwide, are caused by mutations in over 200 genes. This suggests that the most efficient therapeutic strategies would be mutation independent, i.e., targeting common pathological conditions arising from many disease-causing mutations. Previous studies revealed that one such condition is an insufficiency of the ubiquitin–proteasome system to process misfolded or mistargeted proteins in affected photoreceptor cells. We now report that retinal degeneration in mice can be significantly delayed by increasing photoreceptor proteasomal activity. The largest effect is observed upon overexpression of the 11S proteasome cap subunit, PA28α, which enhanced ubiquitin-independent protein degradation in photoreceptors. Applying this strategy to mice bearing one copy of the P23H rhodopsin mutant, a mutation frequently encountered in human patients, quadruples the number of surviving photoreceptors in the inferior retina of 6-month-old mice. This striking therapeutic effect demonstrates that proteasomes are an attractive target for fighting inherited blindness.

Background: Blood coagulation is a complex network of biochemical reactions, which is peculiar in that it is time- and space-dependent, and has to function in the presence of rapid flow. Recent experimental reports suggest that flow plays a significant role in its regulation. The objective of this study was to use systems biology techniques to investigate this regulation and to identify mechanisms creating a flow-dependent switch in the coagulation onset. Results: Using a detailed mechanism-driven model of tissue factor (TF)-initiated thrombus formation in a two-dimensional channel we demonstrate that blood flow can regulate clotting onset in the model in a threshold-like manner, in agreement with existing experimental evidence. Sensitivity analysis reveals that this is achieved due to a combination of the positive feedback of TF-bound factor VII activation by activated factor X (Xa) and effective removal of factor Xa by flow from the activating patch depriving the feedback of "ignition". The level of this trigger (i.e. coagulation sensitivity to flow) is controlled by the activity of tissue factor pathway inhibitor. Conclusions: This mechanism explains the difference between red and white thrombi observed in vivo at different shear rates. It can be speculated that this is a special switch protecting vascular system from uncontrolled formation and spreading of active coagulation factors in vessels with rapidly flowing blood.

Acetylcholinesterase (AChE) expression in fast rat muscles is approximately fourfold higher than in slow muscles. We examined whether different muscle activation patterns are responsible for this difference and whether the calcineurin signaling pathway is involved in AChE regulation. The slow soleus and fast extensor digitorum longus (EDL) muscles were directly or indirectly stimulated by a tonic low-frequency or a phasic high-frequency pattern of electric impulses. The phasic, but not tonic, stimulation increased the AChE mRNA levels in denervated soleus muscles to those in the normal EDL and maintained high levels of AChE mRNA in denervated EDL muscles. Therefore, muscle activation pattern is the predominant regulator of extrajunctional AChE expression in rat muscles. Indirect phasic stimulation of innervated muscles, imposed on their natural pattern of neural activation, did not increase the AChE transcript levels in the soleus, whereas a 30% reduction was observed in the EDL muscles. A low number of impulses per day is therefore prerequisite for high AChE expression. Treatment by tacrolimus and cyclosporin A, two inhibitors of calcineurin (but not by a related substance rapamycin, which does not inhibit calcineurin), increased the levels of AChE transcripts in the control soleus muscles and in tonically electrically stimulated soleus and EDL muscles, even to reach those in the control EDL muscles. Therefore, tonic muscle activation reduces the extrajunctional levels of AChE transcripts by activating the calcineurin signaling pathway. In denervated soleus and EDL muscles, tacrolimus did not prevent the reduction of AChE mRNA levels, indicating that a calcineurin-independent suppressive mechanism was involved.

Retinal photoreceptor cells contain the highest concentration of docosahexaenoic acid (DHA) in our bodies, and it has been long assumed that this is critical for supporting normal vision. Indeed, early studies using DHA dietary restriction documented reduced light sensitivity by DHA-deprived retinas. Recently, it has been demonstrated that a major route of DHA entry in the retina is the delivery across the blood–retina barrier by the sodium-dependent lipid transporter, Mfsd2a. This discovery opened a unique opportunity to analyze photoreceptor health and function in DHA-deprived retinas using theMfsd2aknock-out mouse as animal model. Our lipidome analyses ofMfsd2a^−/−retinas and outer segment membranes corroborated the previously reported decrease in the fraction of DHA-containing phospholipids and a compensatory increase in phospholipids containing arachidonic acid. We also revealed an increase in the retinal content of monounsaturated fatty acids and a reduction in very long chain fatty acids. These changes could be explained by a combination of reduced DHA supply to the retina and a concomitant upregulation of several fatty acid desaturases controlled by sterol regulatory element-binding transcription factors, which are upregulated inMfsd2a^−/−retinas.Mfsd2a^−/−retinas undergo slow progressive degeneration, with ∼30% of photoreceptor cells lost by the age of 6 months. Despite this pathology, the ultrastructureMfsd2a^−/−photoreceptors and their ability to produce light responses were essentially normal. These data demonstrate that, whereas maintaining the lysophosphatidylcholine route of DHA supply to the retina is essential for long-term photoreceptor survival, it is not important for supporting normal phototransduction.SIGNIFICANCE STATEMENTPhospholipids containing docosahexaenoic acid (DHA) are greatly enriched in the nervous system, with the highest concentration found in the light-sensitive membranes of photoreceptor cells. In this study, we analyzed the consequences of impaired DHA transport across the blood–retina barrier. We have found that, in addition to a predictable reduction in the DHA level, the affected retinas undergo a complex, transcriptionally-driven rebuilding of their membrane lipidome in a pattern preserving the overall saturation/desaturation balance of retinal phospholipids. Remarkably, these changes do not affect the ability of photoreceptors to produce responses to light but are detrimental for the long-term survival of these cells.