Neuropathic Pain: Central vs. Peripheral Mechanisms

Purpose of Review

Our goal is to examine the processes—both central and peripheral—that underlie the development of peripherally-induced neuropathic pain (pNP) and to highlight recent evidence for mechanisms contributing to its maintenance. While many pNP conditions are initiated by damage to the peripheral nervous system (PNS), their persistence appears to rely on maladaptive processes within the central nervous system (CNS). The potential existence of an autonomous pain-generating mechanism in the CNS creates significant implications for the development of new neuropathic pain treatments; thus, work towards its resolution is crucial. Here, we seek to identify evidence for PNS and CNS independently generating neuropathic pain signals.

Recent Findings

Recent preclinical studies in pNP support and provide key details concerning the role of multiple mechanisms leading to fiber hyperexcitability and sustained electrical discharge to the CNS. In studies regarding central mechanisms, new preclinical evidence includes the mapping of novel inhibitory circuitry and identification of the molecular basis of microglia-neuron crosstalk. Recent clinical evidence demonstrates the essential role of peripheral mechanisms, mostly via studies that block the initially damaged peripheral circuitry. Clinical central mechanism studies use imaging to identify potentially self-sustaining infra-slow CNS oscillatory activity that may be unique to pNP patients.

Summary

While new preclinical evidence supports and expands upon the key role of central mechanisms in neuropathic pain, clinical evidence for an autonomous central mechanism remains relatively limited. Recent findings from both preclinical and clinical studies recapitulate the critical contribution of peripheral input to maintenance of neuropathic pain. Further clinical investigations on the possibility of standalone central contributions to pNP may be assisted by a reconsideration of the agreed terms or criteria for diagnosing the presence of central sensitization in humans.

     

Instability of a dinucleotide repeat in the 3′‐untranslated region (UTR) of the microsomal prostaglandin E synthase‐1 (mPGES‐1) gene in microsatellite instability‐high (MSI‐H) colorectal carcinoma

DNA mismatch‐repair gene mutations, with consequent loss of functional protein expression, result in microsatellite instability (MSI). Microsatellite sequences are found in coding regions and in regulatory regions of genes (i.e., 5′‐UTRs and 3′‐UTRs). In addition to being a surrogate marker of defective mismatch repair, deletion or insertion microsatellite sequences can dysregulate gene expression in MSI‐H (microsatellite instability‐high) tumors. The microsomal prostaglandin E synthase‐1 (mPGES‐1) gene product, mPGES‐1, participates in prostaglandin E2 (PGE2) production. Moreover, mPGES‐1 is often overexpressed in human colorectal tumors, and is thought to contribute to progression of these tumors. Here we identified a dinucleotide repeat, (GT)24, in the mPGES‐1 gene 3′ untranslated region (3′‐UTR), and analyzed its mutation frequencies in MSI‐H and microsatellite stable (MSS) tumors. The (GT)24 repeat exhibited instability in all MSI‐H tumors examined (14), but not in any of the MSS tumors (13). In most cases, (GT)24 repeat instability resulted in insertion of additional GT units. We also determined mPGES‐1 mRNA levels in MSI‐H and MSS colorectal cancer cell lines. Three of four previously designated “MSI‐H” cell lines showed higher mPGES‐1 mRNA levels compared to MSS cell lines; correlations between elevated mPGES‐1 mRNA levels and microsatellite (GT)24 repeat characteristics are present for all six cell lines. Our results demonstrate that mPGES‐1 is a target gene of defective mismatch repair in human colorectal cancer, with functional consequence.     

Serological and virological studies of Japanese encephalitis in the Terai region of Nepal.

In 1987 and 1990, serum samples were collected from people living in the two districts (Itahari and Chitwan) of the Terai region of Nepal. Antibodies against Japanese encephalitis (JE) virus in these sera were detected by the hemagglutination inhibition (HI) and neutralization (N) tests. By the HI test, 26 out of 172 (15.1%) sera from Chitwan and 15 out of 137 (10.9%) sera from Itahari showed positive titers. Higher positive rates were shown by the N test, where 46 out of 172 (26.7%) sera from Chitwan and 22 out of 137 (16.1%) sera from Itahari had antibodies against JE virus. A JE strain was isolated from a blood specimen of a pig raised in Kathmandu. When the nucleotide sequence of the pre-M region of the strain was compared to the same region of the other JE virus strains reported, the highest similarity was observed to the strains isolated in Nepal in 1985. These results suggest that the Terai region has been an epidemic area of JE.     

Post-transplant erythrocytosis after kidney transplantation: A review

Post-transplant erythrocytosis (PTE) is defined as persistently elevated hemoglobin > 17 g/dL or hematocrit levels > 51% following kidney transplantation, independent of duration. It is a relatively common complication within 8 months to 24 months post-transplantation, occurring in 8%-15% of kidney transplant recipients. Established PTE risk factors include male gender, normal hemoglobin/hematocrit pre-transplant (suggestive of robust native kidney erythropoietin production), renal artery stenosis, patients with a well-functioning graft, and dialysis before transplantation. Many factors play a role in the development of PTE, however, underlying endogenous erythropoietin secretion pre-and post-transplant is significant. Other contributory factors include the renin-angiotensin- aldosterone system, insulin-like growth factors, endogenous androgens, and local renal hypoxia. Most patients with PTE experience mild symptoms like malaise, headache, fatigue, and dizziness. While prior investigations showed an increased risk of thromboembolic events, more recent evidence tells a different story-that PTE perhaps has lessened risk of thromboembolic events or negative graft outcomes than previously thought. In the evaluation of PTE, it is important to exclude other causes of erythrocytosis including malignancy before treatment. Angiotensin converting enzyme inhibitors (ACE-I) and angiotensin receptor blockers (ARBs) are the mainstays of treatment. Increased ACE-I/ARB use has likely contributed to the falling incidence of erythrocytosis. In this review article, we summarize the current literature in the field of post-transplant erythrocytosis after kidney transplantation.     

Nascent peptides that block protein synthesis in bacteria

Although the ribosome is a very general catalyst, it cannot synthesize all protein sequences equally well. For example, ribosomes stall on the secretion monitor (SecM) leader peptide to regulate expression of a downstream gene. Using a genetic selection in Escherichia coli, we identified additional nascent peptide motifs that stall ribosomes. Kinetic studies show that some nascent peptides dramatically inhibit rates of peptide release by release factors. We find that residues upstream of the minimal stalling motif can either enhance or suppress this effect. In other stalling motifs, peptidyl transfer to certain aminoacyl-tRNAs is inhibited. In particular, three consecutive Pro codons pose a challenge for elongating ribosomes. The translation factor elongation factor P, which alleviates pausing at polyproline sequences, has little or no effect on other stalling peptides. The motifs that we identified are underrepresented in bacterial proteomes and show evidence of stalling on endogenous E. coli proteins.We commonly think of the ribosome as capable of synthesizing any protein, regardless of its sequence. However, it turns out that some nascent peptides contain stalling motifs that inhibit core functions of the ribosome (1, 2). Why would a protein evolve to arrest its own synthesis? In one of the best-characterized examples, stalling in the secretion monitor (SecM) leader peptide up-regulates translation of SecA, a protein encoded downstream on the same mRNA (3). Known stalling motifs typically have been identified based on their function as genetic switches, regulating gene expression in response to levels of protein translocation factors (3, 4) or changes in the concentration of small-molecule metabolites (2, 5). Further understanding of the scope and mechanism of ribosome stalling may yield additional insight into programmed ribosome-stalling events that regulate gene expression in organisms from bacteria to humans (6, 7). In addition, by fine-tuning the rate of protein synthesis, stalling peptides may affect protein folding and function, as reported previously with rare codons (8, 9).Analyses of natural motifs have identified three sites of interaction within the ribosome that lead to stalling. First, conserved residues at a motif’s N terminus often interact with the ribosome near a constriction in the exit tunnel between proteins L4 and L22. Ribosomal mutations near this site were isolated in genetic screens for reduced levels of stalling (3, 10). Second, conserved residues near a motif’s C terminus interact with nucleotides surrounding the ribosomal active site, the peptidyl-transferase center or PTC (11). Third, some motifs encode a specific aminoacyl-tRNA that acts as a poor peptidyl acceptor when bound in the A site (12, 13). The SecM consensus motif (FxxxxWIxxxxGIRAGP) showcases all three of these interactions, each of which contributes to stalling. A Trp side chain 11 residues upstream of the stall site binds near the constriction in the tunnel, the Arg residue three residues upstream is positioned close to the PTC (3), and Pro-tRNA binds in the A site but does not react (14). A recent cryoelectron microscopy structure of the stalled SecM complex visualized these interactions directly and begins to provide some molecular rationale for how stalling is induced (15).A full understanding of the molecular mechanism underlying ribosome stalling has not been forthcoming because of the complexity of natural stalling motifs. In each case, stalling is reversible and is controlled by changes in the cellular environment. Stalling in TnaC, for example, is induced by high tryptophan concentrations through the binding of free tryptophan at an unknown site within the ribosome (5). Given that a single stalling motif interacts with the ribosome at multiple sites, deconvoluting the role of the conserved residues in the peptide is difficult enough without the added complexity of small-molecule binding. Moreover, even at well-validated sites of interaction, such as the L4/L22 constriction, different stalling motifs appear to work via different mechanisms. Ribosomal mutations that reduce stalling by one motif may have no effect or even increase stalling by another (13, 16). These complexities make it challenging to obtain general conclusions about the mechanism of ribosome stalling by natural stalling peptides.To characterize better the scope and mechanism of ribosome stalling, we set out to find a series of artificial motifs that inhibit translation during their own synthesis. We reasoned that, by selecting directly for stalling peptides, we might identify motifs that are simpler than natural ones because they are not required to stall reversibly or to regulate downstream genes. The fact that only a few residues are essential for stalling by SecM, TnaC, and ErmCL (1) and the fact that these motifs share little or no sequence similarity led us to believe that more stalling motifs exist but have not yet been identified. To this end, we developed a powerful genetic selection in Escherichia coli that ties stalling on a reporter protein to cellular survival.Here we report several stalling motifs, some that block peptide release during translational termination and others that block peptidyl transfer. Because these motifs are short, we are able to recapitulate the stalling phenomenon using pre–steady-state kinetic assays, an important step toward achieving mechanistic insights. Of particular interest, we show that polyproline sequences induce ribosome stalling. The translation factor elongation factor P (EF-P), which alleviates stalling at polyproline stretches, does not affect the other short motifs we identified, further defining its scope of action. Finally, our analysis of bacterial proteomes reveals that stalling motifs are underrepresented, implying that they have been selected against. Where they do occur in endogenous E. coli proteins, pausing is detectable by ribosome profiling. These findings argue that these short motifs have an impact on protein synthesis in bacteria.     

Machine learning approaches for estimation of prediction interval for the model output.

A novel method for estimating prediction uncertainty using machine learning techniques is presented. Uncertainty is expressed in the form of the two quantiles (constituting the prediction interval) of the underlying distribution of prediction errors. The idea is to partition the input space into different zones or clusters having similar model errors using fuzzy c-means clustering. The prediction interval is constructed for each cluster on the basis of empirical distributions of the errors associated with all instances belonging to the cluster under consideration and propagated from each cluster to the examples according to their membership grades in each cluster. Then a regression model is built for in-sample data using computed prediction limits as targets, and finally, this model is applied to estimate the prediction intervals (limits) for out-of-sample data. The method was tested on artificial and real hydrologic data sets using various machine learning techniques. Preliminary results show that the method is superior to other methods estimating the prediction interval. A new method for evaluating performance for estimating prediction interval is proposed as well.     

In vitro maturation of oocytes

In vitro maturation of oocytes is a safe and effective treatment offered in some fertility centers for assisted reproduction, where immature oocytes are retrieved from unstimulated ovaries. Therefore, the procedure avoids ovarian stimulation with expensive gonadotropins, side effects of the medications, and risks such as ovarian hyperstimulation syndrome. Added advantages are reduced frequency of monitoring scans and shorter treatment regimen compared with in vitro fertilization. The candidates initially considered were women with polycystic ovaries having multiple antral follicles, but the indications are widening to include women with primarily poor quality embryos in repeated cycles and poor responders to stimulation. The two new applications for in vitro maturation we are now successfully implementing at McGill Reproductive Center are for oocyte donors and for fertility preservation, especially in women with cancer who are undergoing gonadotoxic therapy. In young women without partners needing this treatment for fertility preservation, it is combined with vitrification of the oocytes. We have achieved a 38% clinical pregnancy rate per cycle in women having IVM for infertility treatment up to the age of 35 years, and 50% clinical pregnancy rate per cycle in recipients of IVM egg donation.