Autonomic dysfunction is associated with disease progression and survival in amyotrophic lateral sclerosis: a prospective longitudinal cohort study

Background: Among non-motor symptoms, autonomic disturbances have been described in amyotrophic lateral sclerosis (ALS) and reported as mild to moderate in up to 75% of patients. However, no study has systematically investigated autonomic symptoms as prognostic factors. Objectives: The main aim of this longitudinal study was to examine the association of autonomic dysfunction with disease progression and survival in ALS. Methods: We enrolled newly diagnosed ALS patients and a healthy control group (HC). Time from disease onset to disease milestone (King’s stage 4) and death were calculated to assess disease progression and survival. Autonomic symptoms were assessed by a dedicated questionnaire. Longitudinal evaluation of parasympathetic cardiovascular activity was performed by the heart rate variability (HRV). Multivariable Cox proportional hazards regression models on the risk of the disease milestone and death were used. A mixed-effect linear regression model was used to compare autonomic dysfunction with a HC group as well as its impairment over time. Results: A total of 102 patients and 41 HC were studied. ALS patients, compared with HC, complained of more autonomic symptoms, especially in bulbar onset patients. Autonomic symptoms occurred in 69 (68%) patients at diagnosis and progressed over time (post-6: p = 0.015 and post-12: p < 0.001). A higher autonomic symptom burden was an independent marker of faster development of King’s stage 4 (HR 1.05; 95% CI 1.00–1.11; p = 0.022); whereas, urinary complaints were independent factors of a shorter survival (HR 3.12; 95% CI 1.22–7.97; p = 0.018). Moreover, HRV in ALS patients was lower than in HC (p = 0.018) and further decreased over time (p = 0.003), implying a parasympathetic hypofunction that progressed over time. Conclusion: Autonomic symptoms occur in most of the ALS patients at diagnosis and progress over time, implying that autonomic dysfunction represents an intrinsic non-motor feature of the disease. A higher autonomic burden is a poor prognostic factor, associated with a more rapid development of disease milestones and shorter survival

A novel SCN9A splicing mutation in a compound heterozygous girl with congenital insensitivity to pain, hyposmia and hypogeusia

Congenital insensitivity to pain (CIP) is a rare autosomal recessive disorder presenting with a spectrum of clinical features caused by mutations in different genes. A 10\u2010year\u2010old girl with CIP, hyposmia and hypogeusia, and her unaffected twin and parents underwent next generation sequencing of SCN9A exons and flanking splice sites. Transcript analysis from whole blood successfully assayed the effect of the mutation on the mRNA splicing by polymerase chain reaction amplification on cDNA and Sanger sequencing. We identified the novel splicing variant c.1108\u20102A>G compound with the p.Arg896Gln (c.2687G>A) missense mutation previously described in a homozygous patient. The new intronic variant was predicted to induce exon 10 skipping. Conversely, SCN9A mRNA assay demonstrated its partial deletion with a loss of 46 nucleotides causing a premature stop codon in position p.Gln369 (NP_002968). Genetic analysis showed that the two variants were biallelic, being the mother and brother heterozygous carriers of the missense mutation, and the father heterozygous for the splicing mutation. Skin biopsy showed lack of Meissner's corpuscles, loss of epidermal nociceptors and normal autonomic organ innervation. We report a novel splicing mutation and provide clues on its pathogenic effect, broadening the spectrum of genotypes and phenotypes associated to CIP

Cyclic nucleotide-dependent relaxation in human umbilical vessels

Umbilical vessels have a low sensitivity to dilate, and this property is speculated to have physiological implications. We aimed to investigate the different relaxing responses of human umbilical arteries (HUAs) and veins (HUVs) to agonists acting through the cAMP and cGMP pathways. Vascular rings were suspended in organ baths for isometric force measurement. Following precontraction with the thromboxane prostanoid (TP) receptor agonist U44069, concentration-response curves to the nitric oxide (NO) donor sodium nitroprusside (SNP), the soluble guanylate cyclase (sGC) stimulator BAY 41-2272, the adenylate cyclase (AC) activator forskolin, the \u3b2-adrenergic receptor agonists isoproterenol (ADRB1), salmeterol (ADRB2), and BRL37344 (ADRB3), and the phosphodiesterase (PDE) inhibitors milrinone (PDE3), rolipram (PDE4), and sildenafil (PDE5) were performed. None of the tested drugs induced a relaxation higher than 30% of the U44069-induced tone. Rings from HUAs and HUVs showed a similar relaxation to forskolin, SNP, PDE inhibitors, and ADRB agonists. BAY 41-2272 was significantly more efficient in relaxing veins than arteries. ADRB agonists evoked weak relaxations (&lt; 20%), which were impaired in endothelium-removed vessels or in the presence of the NO synthase inhibitor L-NAME, sGC inhibitor ODQ. PKA and PKG inhibitors impaired ADBR1-mediated relaxation but did not affect ADRB2-mediated relaxation. ADRB3-mediated relaxation was impaired by PKG inhibition in HUAs and by PKA inhibition in HUVs. Although HUA and HUV rings were relaxed by BRL37344, immunohistochemistry and RT-qPCR analysis showed that, compared to ADRB1 and ADRB2, ADRB3 receptors are weakly or not expressed in umbilical vessels. In conclusion, our study confirmed the low relaxing capacity of HUAs and HUVs from term infants. ADRB-induced relaxation is partially mediated by endothelium-derived NO pathway in human umbilical vessels

Multi-modal Biomarkers Quantify Recovery in Autoimmune Autonomic Ganglionopathy

Objective: To evaluate patients with ganglionic acetylcholine receptor antibody (gAChR‐Ab) positive autoimmune autonomic ganglionopathy using a multi‐modal testing protocol to characterise their full clinical phenotype and explore biomarkers to quantify immunotherapy response. Methods: Cohort study of thirteen individuals (seven female; 21–69 years) with autonomic failure and gAChR‐Ab>100pM identified between 2005–2019. From 2018, all patients were longitudinally assessed with cardiovascular, pupillary, urinary, sudomotor, lacrimal and salivary testing, and COMPASS‐31 autonomic symptom questionnaires. The orthostatic intolerance ratio was calculated by dividing change in systolic blood pressure over time tolerated on head‐up tilt. Eleven patients received immunotherapy. Results: At first assessment, all 13 patients had cardiovascular and pupillary impairments, 7/8 had post‐ganglionic sudomotor dysfunction, 9/11 had urinary retention and xeropthalmia, and 6/8 had xerostomia. After immunotherapy, there were significant improvements in orthostatic intolerance ratio (33.3[17.8–61.3] to 5.2[1.4–8.2], P = .007), heart rate response to deep breathing (1.5[0.0–3.3] to 4.5[3.0–6.3], P = .02), pupillary constriction to light (12.0[5.5–18.0] to 19.0[10.6–23.8]%, P = .02), saliva production (0.01[0.01–0.05] to 0.08[0.02–0.20]g/min, P = .03) and COMPASS‐31 scores (52 to 17, P = .03). Orthostatic intolerance ratio correlated with autonomic symptoms at baseline (r = 0.841, P = .01) and following immunotherapy (r = 0.889, P = .02). Immunofluorescence analyses of skin samples from a patient 32 years after disease onset showed loss of nerve fibres supplying the dermal autonomic adnexa and epidermis, with clear improvements following immunotherapy. Interpretation: Patients with autoimmune autonomic ganglionopathy demonstrated objective evidence of widespread sympathetic and parasympathetic autonomic failure, with significant improvements after immunotherapy. Quantitative autonomic biomarkers should be used to define initial deficits, guide therapeutic decisions, and document treatment response

Comparison of Post-injection Site Pain Between Technetium Sulfur Colloid and Technetium Tilmanocept in Breast Cancer Patients Undergoing Sentinel Lymph Node Biopsy

BACKGROUND: No prior studies have examined injection pain associated with Technetium-99m Tilmanocept (TcTM). METHODS: This was a randomized, double-blinded study comparing postinjection site pain between filtered Technetium Sulfur Colloid (fTcSC) and TcTM in breast cancer lymphoscintigraphy. Pain was evaluated with a visual analogue scale (VAS) (0–100 mm) and the short-form McGill Pain Questionnaire (SF-MPQ). The primary endpoint was mean difference in VAS scores at 1-min postinjection between fTcSC and TcTM. Secondary endpoints included a comparison of SF-MPQ scores between the groups at 5 min postinjection and construction of a linear mixed effects model to evaluate the changes in pain during the 5-min postinjection period. RESULTS: Fifty-two patients underwent injection (27-fTcSC, 25-TcTM). At 1-min postinjection, patients who received fTcSC experienced a mean change in pain of 16.8 mm (standard deviation (SD) 19.5) compared with 0.2 mm (SD 7.3) in TcTM (p = 0.0002). At 5 min postinjection, the mean total score on the SF-MPQ was 2.8 (SD 3.0) for fTcSC versus 2.1 (SD 2.5) for TcTM (p = 0.36). In the mixed effects model, injection agent (p < 0.001), time (p < 0.001) and their interaction (p < 0.001) were associated with change in pain during the 5-min postinjection period. The model found fTcSC resulted in significantly more pain of 15.2 mm (p < 0.001), 11.3 mm (p = 0.001), and 7.5 mm (p = 0.013) at 1, 2, and 3 min postinjection, respectively. CONCLUSIONS: Injection with fTcSC causes significantly more pain during the first 3 min postinjection compared with TcTM in women undergoing lymphoscintigraphy for breast cancer

Light-evoked Somatosensory Perception of Transgenic Rats That Express Channelrhodopsin-2 in Dorsal Root Ganglion Cells

In vertebrate somatosensory systems, each mode of touch-pressure, temperature or pain is sensed by sensory endings of different dorsal root ganglion (DRG) neurons, which conducted to the specific cortical loci as nerve impulses. Therefore, direct electrical stimulation of the peripheral nerve endings causes an erroneous sensation to be conducted by the nerve. We have recently generated several transgenic lines of rat in which channelrhodopsin-2 (ChR2) transgene is driven by the Thy-1.2 promoter. In one of them, W-TChR2V4, some neurons were endowed with photosensitivity by the introduction of the ChR2 gene, coding an algal photoreceptor molecule. The DRG neurons expressing ChR2 were immunohistochemically identified using specific antibodies to the markers of mechanoreceptive or nociceptive neurons. Their peripheral nerve endings in the plantar skin as well as the central endings in the spinal cord were also examined. We identified that ChR2 is expressed in a certain population of large neurons in the DRG of W-TChR2V4. On the basis of their morphology and molecular markers, these neurons were classified as mechanoreceptive but not nociceptive. ChR2 was also distributed in their peripheral sensory nerve endings, some of which were closely associated with CK20-positive cells to form Merkel cell-neurite complexes or with S-100-positive cells to form structures like Meissner's corpuscles. These nerve endings are thus suggested to be involved in the sensing of touch. Each W-TChR2V4 rat showed a sensory-evoked behavior in response to blue LED flashes on the plantar skin. It is thus suggested that each rat acquired an unusual sensory modality of sensing blue light through the skin as touch-pressure. This light-evoked somatosensory perception should facilitate study of how the complex tactile sense emerges in the brain

The +4G Site in Kozak Consensus Is Not Related to the Efficiency of Translation Initiation

The optimal context for translation initiation in mammalian species is GCCRCCaugG (where R = purine and “aug” is the initiation codon), with the -3R and +4G being particularly important. The presence of +4G has been interpreted as necessary for efficient translation initiation. Accumulated experimental and bioinformatic evidence has suggested an alternative explanation based on amino acid constraint on the second codon, i.e., amino acid Ala or Gly are needed as the second amino acid in the nascent peptide for the cleavage of the initiator Met, and the consequent overuse of Ala and Gly codons (GCN and GGN) leads to the +4G consensus. I performed a critical test of these alternative hypotheses on +4G based on 34169 human protein-coding genes and published gene expression data. The result shows that the prevalence of +4G is not related to translation initiation. Among the five G-starting codons, only alanine codons (GCN), and glycine codons (GGN) to a much smaller extent, are overrepresented at the second codon, whereas the other three codons are not overrepresented. While highly expressed genes have more +4G than lowly expressed genes, the difference is caused by GCN and GGN codons at the second codon. These results are inconsistent with +4G being needed for efficient translation initiation, but consistent with the proposal of amino acid constraint hypothesis

Biallelic mutations in neurofascin cause neurodevelopmental impairment and peripheral demyelination.

Axon pathfinding and synapse formation are essential processes for nervous system development and function. The assembly of myelinated fibres and nodes of Ranvier is mediated by a number of cell adhesion molecules of the immunoglobulin superfamily including neurofascin, encoded by the NFASC gene, and its alternative isoforms Nfasc186 and Nfasc140 (located in the axonal membrane at the node of Ranvier) and Nfasc155 (a glial component of the paranodal axoglial junction). We identified 10 individuals from six unrelated families, exhibiting a neurodevelopmental disorder characterized with a spectrum of central (intellectual disability, developmental delay, motor impairment, speech difficulties) and peripheral (early onset demyelinating neuropathy) neurological involvement, who were found by exome or genome sequencing to carry one frameshift and four different homozygous non-synonymous variants in NFASC. Expression studies using immunostaining-based techniques identified absent expression of the Nfasc155 isoform as a consequence of the frameshift variant and a significant reduction of expression was also observed in association with two non-synonymous variants affecting the fibronectin type III domain. Cell aggregation studies revealed a severely impaired Nfasc155-CNTN1/CASPR1 complex interaction as a result of the identified variants. Immunofluorescence staining of myelinated fibres from two affected individuals showed a severe loss of myelinated fibres and abnormalities in the paranodal junction morphology. Our results establish that recessive variants affecting the Nfasc155 isoform can affect the formation of paranodal axoglial junctions at the nodes of Ranvier. The genetic disease caused by biallelic NFASC variants includes neurodevelopmental impairment and a spectrum of central and peripheral demyelination as part of its core clinical phenotype. Our findings support possible overlapping molecular mechanisms of paranodal damage at peripheral nerves in both the immune-mediated and the genetic disease, but the observation of prominent central neurological involvement in NFASC biallelic variant carriers highlights the importance of this gene in human brain development and function

Retroviral matrix and lipids, the intimate interaction

Retroviruses are enveloped viruses that assemble on the inner leaflet of cellular membranes. Improving biophysical techniques has recently unveiled many molecular aspects of the interaction between the retroviral structural protein Gag and the cellular membrane lipids. This interaction is driven by the N-terminal matrix domain of the protein, which probably undergoes important structural modifications during this process, and could induce membrane lipid distribution changes as well. This review aims at describing the molecular events occurring during MA-membrane interaction, and pointing out their consequences in terms of viral assembly. The striking conservation of the matrix membrane binding mode among retroviruses indicates that this particular step is most probably a relevant target for antiviral research