Proteomic analysis of peripheral nerve myelin during murine aging

Aging of the peripheral nervous system (PNS) is associated with structural and functional changes that lead to a reduction in regenerative capacity and the development of age-related peripheral neuropathy. Myelin is central to maintaining physiological peripheral nerve function and differences in myelin maintenance, degradation, formation and clearance have been suggested to contribute to age-related PNS changes. Recent proteomic studies have elucidated the complex composition of the total myelin proteome in health and its changes in neuropathy models. However, changes in the myelin proteome of peripheral nerves during aging have not been investigated. Here we show that the proteomes of myelin fractions isolated from young and old nerves show only subtle changes. In particular, we found that the three most abundant peripheral myelin proteins (MPZ, MBP, and PRX) do not change in old myelin fractions. We also show a tendency for high-abundance myelin proteins other than these three to be downregulated, with only a small number of ribosome-related proteins significantly downregulated and extracellular matrix proteins such as collagens upregulated. In addition, we illustrate that the peripheral nerve myelin proteome reported in this study is suitable for assessing myelin degradation and renewal during peripheral nerve degeneration and regeneration. Our results suggest that the peripheral nerve myelin proteome is relatively stable and undergoes only subtle changes in composition during mouse aging. We proffer the resultant dataset as a resource and starting point for future studies aimed at investigating peripheral nerve myelin during aging. Said datasets are available in the PRIDE archive under the identifier PXD040719 (aging myelin proteome) and PXD041026 (sciatic nerve injury proteome)

A Ponceau S Staining-Based Dot Blot Assay for Rapid Protein Quantification of Biological Samples

Despite the availability of a wide range of commercial kits, protein quantification is often unreliable, especially for tissue-derived samples, leading to uneven loading in subsequent experiments. Here we show that the widely used Bicinchoninic Acid (BCA) assay tends to underestimate protein concentrations of tissue samples. We present a Ponceau S staining-based dot-blot assay as an alternative for protein quantification. This method is simple, rapid, more reliable than the BCA assay, compatible with biological samples lysed in RIPA or 2x SDS gel-loading buffer, and also inexpensive

A Ponceau S Staining-Based Dot Blot Assay for Rapid Protein Quantification of Biological Samples

Despite the availability of a wide range of commercial kits, protein quantification is often unreliable, especially for tissue-derived samples, leading to uneven loading in subsequent experiments. Here we show that the widely used Bicinchoninic Acid (BCA) assay tends to underestimate protein concentrations of tissue samples. We present a Ponceau S staining-based dot-blot assay as an alternative for protein quantification. This method is simple, rapid, more reliable than the BCA assay, compatible with biological samples lysed in RIPA or 2x SDS gel-loading buffer, and also inexpensive

Developmental stage-dependent regulation of spine formation by calcium-calmodulin-dependent protein kinase IIα and Rap1

The roles of calcium-calmodulin-dependent protein kinase II-alpha (CaMKIIα) in the expression of long-term synaptic plasticity in the adult brain have been extensively studied. However, how increased CaMKIIα activity controls the maturation of neuronal circuits remains incompletely understood. Herein, we show that pyramidal neurons without CaMKIIα activity upregulate the rate of spine addition, resulting in elevated spine density. Genetic elimination of CaMKIIα activity specifically eliminated the observed maturation-dependent suppression of spine formation. Enhanced spine formation was associated with the stabilization of actin in the spine and could be reversed by increasing the activity of the small GTPase Rap1. CaMKIIα activity was critical in the phosphorylation of synaptic Ras GTPase-activating protein (synGAP), the dispersion of synGAP from postsynaptic sites, and the activation of postsynaptic Rap1. CaMKIIα is already known to be essential in learning and memory, but our findings suggest that CaMKIIα plays an important activity-dependent role in restricting spine density during postnatal development