Detecting Bulbar Involvement in Patients with Amyotrophic Lateral Sclerosis Based on Phonatory and Time-Frequency Features

The term bulbar involvement is employed in ALS to refer to deterioration of motor neurons within the corticobulbar area of the brainstem, which results in speech and swallowing dysfunctions. One of the primary symptoms is a deterioration of the voice. Early detection is crucial for improving the quality of life and lifespan of ALS patients suffering from bulbar involvement. The main objective, and the principal contribution, of this research, was to design a new methodology, based on the phonatory-subsystem and time-frequency characteristics for detecting bulbar involvement automatically. This study focused on providing a set of 50 phonatory-subsystem and time-frequency features to detect this deficiency in males and females through the utterance of the five Spanish vowels. Multivariant Analysis of Variance was then used to select the statistically significant features, and the most common supervised classifications models were analyzed. A set of statistically significant features was obtained for males and females to capture this dysfunction. To date, the accuracy obtained (98.01% for females and 96.10% for males employing a random forest) outperformed the models in the literature. Adding time-frequency features to more classical phonatory-subsystem features increases the prediction capabilities of the machine-learning models for detecting bulbar involvement. Studying men and women separately gives greater success. The proposed method can be deployed in any kind of recording device (i.e., smartphone)

Detection of Bulbar Involvement in Patients With Amyotrophic Lateral Sclerosis by Machine Learning Voice Analysis: Diagnostic Decision Support Development Study

Background: Bulbar involvement is a term used in amyotrophic lateral sclerosis (ALS) that refers to motor neuron impairment in the corticobulbar area of the brainstem, which produces a dysfunction of speech and swallowing. One of the earliest symptoms of bulbar involvement is voice deterioration characterized by grossly defective articulation; extremely slow, laborious speech; marked hypernasality; and severe harshness. Bulbar involvement requires well-timed and carefully coordinated interventions. Therefore, early detection is crucial to improving the quality of life and lengthening the life expectancy of patients with ALS who present with this dysfunction. Recent research efforts have focused on voice analysis to capture bulbar involvement. Objective: The main objective of this paper was (1) to design a methodology for diagnosing bulbar involvement efficiently through the acoustic parameters of uttered vowels in Spanish, and (2) to demonstrate that the performance of the automated diagnosis of bulbar involvement is superior to human diagnosis. Methods: The study focused on the extraction of features from the phonatory subsystem-jitter, shimmer, harmonics-to-noise ratio, and pitch-from the utterance of the five Spanish vowels. Then, we used various supervised classification algorithms, preceded by principal component analysis of the features obtained. Results: To date, support vector machines have performed better (accuracy 95.8%) than the models analyzed in the related work. We also show how the model can improve human diagnosis, which can often misdiagnose bulbar involvement. Conclusions: The results obtained are very encouraging and demonstrate the efficiency and applicability of the automated model presented in this paper. It may be an appropriate tool to help in the diagnosis of ALS by multidisciplinary clinical teams, in particular to improve the diagnosis of bulbar involvement

Voiceprint and machine learning models for early detection of bulbar dysfunction in ALS

Background and Objective: Bulbar dysfunction is a term used in amyotrophic lateral sclerosis (ALS). It refers to motor neuron disability in the corticobulbar area of the brainstem which leads to a dysfunction of speech and swallowing. One of the earliest symptoms of bulbar dysfunction is voice deterioration characterized by grossly defective articulation, extremely slow laborious speech, marked hypernasality and severe harshness. Recently, research efforts have focused on voice analysis to capture this dysfunction. The main aim of this paper is to provide a new methodology to diagnose this dysfunction automatically at early stages of the disease, earlier than clinicians can do. Methods: The study focused on the creation of a voiceprint consisting of a pattern generated from the quasi-periodic components of a steady portion of the five Spanish vowels and the computation of the five principal and independent components of this pattern. Then, a set of statistically significant features was obtained using multivariate analysis of variance and the outcomes of the most common supervised classification models were obtained. Results: The best model (random forest) obtained an accuracy, sensitivity and specificity of 88.3%, 85.0% and 95.0% respectively when classifying bulbar vs. control participants but the results worsened when classifying bulbar vs. no-bulbar patients (accuracy, sensitivity and specificity of 78.7%, 80.0% and 77.5% respectively for support vector machines). Due to the great uncertainty found in the annotated corpus of the ALS patients without bulbar involvement, we used a safe semi-supervised support vector machine to relabel the ALS participants diagnosed without bulbar involvement as bulbar and no-bulbar. The performance of the results obtained increased, especially when classifying bulbar and no-bulbar patients obtaining an accuracy, sensitivity and specificity of 91.0%, 83.3% and 100.0% respectively for support vector machines. This demonstrates that our model can improve the diagnosis of bulbar dysfunction compared not only with clinicians, but also the methods published to date. Conclusions: The results obtained demonstrate the efficiency and applicability of the methodology presented in this paper. It may lead to the development of a cheap and easy-to-use tool to identify this dysfunction in early stages of the disease and monitor progress.This work was approved by the Research Ethics Committee for Biomedical Research Projects (CEIm) at the Bellvitge University Hospital in Barcelona and was supported by the Ministerio de Economía y Competitividad (TIN2017-84553-C2-2-R) and the Ministerio de Ciencia e Innovacion (PID2020-113614RBC22). AT is a member of CIMNE, a Severo Ochoa Centre of Excellence (2019-2023) under grant CEX2018-000797-S, funded by MCIN/AEI/10.13039/501100011033. The Neurology Department of the Bellvitge University Hospital in Barcelona permitted the recording of the voices of the participants in its facilities. The clinical records were provided by Carlos Augusto Salazar Talavera. Dr. Marta Fulla and Maria Carmen Majos Bellmunt contributed advice about the process of eliciting the sounds

Altered dynein axonemal assembly factor 1 expression in C-Boutons in bulbar and spinal cord motor-neurons in sporadic amyotrophic lateral sclerosis

Dyneins are major components of microtubules. Dynein assembly is modulated by a heterogeneous group of dynein axonemal assembly factors (DNAAFs). The present study analyzes dynein axonemal assembly factor 1 (DNAAF1) and leucine-rich repeat-containing protein 50 (LRRC50), the corresponding encoded protein, in lower motor neurons in spinal cord of sALS postmortem samples and hSOD1-G93A transgenic mice compared with controls. DNAAF1 mRNA is significantly reduced in the anterior horn in sALS, and LRRC50 immunoreactivity is significantly reduced in C-boutons of the remaining motor neurons of the anterior horn, dorsal nucleus of the vagus nerve, and hypoglossal nuclei at terminal stages of ALS. LRRC50 immunoreactivity has a perinuclear distribution in motor neurons in sALS thus suggesting a disorder of transport. The number of LRRC50-/S1R-immunoreactive structures is also significantly decreased in hSOD1-G93A transgenic mice at the age of 90days (preclinical stages), and the number of motor neurons with LRRC50immunoreactive structures is significantly reduced in animals aged 150days (clinical stages). These observations suggest cholinergic denervation of motor neurons as a pathogenic factor in motor neuron disease. LRRC50 protein levels were not detected in human CSF

TDP-43 Vasculopathy in the Spinal Cord in Sporadic Amyotrophic Lateral Sclerosis (sALS) and Frontal Cortex in sALS/FTLD-TDP

Sporadic amyotrophic lateral sclerosis (sALS) and FTLD-TDP are neurodegenerative diseases within the spectrum of TDP-43 proteinopathies. Since abnormal blood vessels and altered blood-brain barrier have been described in sALS, we wanted to know whether TDP-43 pathology also occurs in blood vessels in sALS/FTLD-TDP. TDP-43 deposits were identified in association with small blood vessels of the spinal cord in 7 of 14 cases of sALS and in small blood vessels of frontal cortex area 8 in 6 of 11 FTLD-TDP and sALS cases, one of them carrying a GRN mutation. This was achieved using single and double-labeling immunohistochemistry, and double-labeling immunofluorescence and confocal microscopy. In the sALS spinal cord, P-TDP43 Ser403-404 deposits were elongated and parallel to the lumen, whereas others were granular, seldom forming clusters. In the frontal cortex, the inclusions were granular, or elongated and parallel to the lumen, or forming small globules within or in the external surface of the blood vessel wall. Other deposits were localized in the perivascular space. The present findings are in line with previous observations of TDP-43 vasculopathy in a subset of FTLD-TDP cases and identify this pathology in the spinal cord and frontal cortex in a subset of cases within the sALS/FTLD-TDP spectrum

Amyotrophic lateral sclerosis, gene deregulation in the anterior horn of the spinal cord and frontal cortex area 8: implications in frontotemporal lobar degeneration

Transcriptome arrays identifies 747 genes differentially expressed in the anterior horn of the spinal cord and 2,300 genes differentially expressed in frontal cortex area 8 in a single group of typical sALS cases without frontotemporal dementia compared with age-matched controls. Main up-regulated clusters in the anterior horn are related to inflammation and apoptosis; down-regulated clusters are linked to axoneme structures and protein synthesis. In contrast, up-regulated gene clusters in frontal cortex area 8 involve neurotransmission, synaptic proteins and vesicle trafficking, whereas main down-regulated genes cluster into oligodendrocyte function and myelin-related proteins. RT-qPCR validates the expression of 58 of 66 assessed genes from different clusters. The present results: a. reveal regional differences in de-regulated gene expression between the anterior horn of the spinal cord and frontal cortex area 8 in the same individuals suffering from sALS; b. validate and extend our knowledge about the complexity of the inflammatory response in the anterior horn of the spinal cord; and c. identify for the first time extensive gene up-regulation of neurotransmission and synaptic-related genes, together with significant down-regulation of oligodendrocyte-and myelin-related genes, as important contributors to the pathogenesis of frontal cortex alterations in the sALS/frontotemporal lobar degeneration spectrum complex at stages with no apparent cognitive impairment

Complex Inflammation mRNA-Related Response in ALS Is Region Dependent

Inflammatory changes are analyzed in the anterior spinal cord and frontal cortex area 8 in typical spinal-predominant ALS cases. Increased numbers of astrocytes and activated microglia are found in the anterior horn of the spinal cord and pyramidal tracts. Significant increased expression of TLR7, CTSS, and CTSC mRNA and a trend to increased expression of IL10RA, TGFB1, and TGFB2 are found in the anterior lumbar spinal cord in ALS cases compared to control cases, whereas C1QTNF7 and TNFRSF1A mRNA expression levels are significantly decreased. IL6 is significantly upregulated and IL1B shows a nonsignificant increased expression in frontal cortex area 8 in ALS cases. IL-6 immunoreactivity is found in scattered monocyte-derived macrophages/microglia and TNF-alpha in a few cells of unknown origin in ALS cases. Increased expression and abnormal distribution of IL-1 beta occurred in motor neurons of the lumbar spinal cord in ALS. Strong IL-10 immunoreactivity colocalizes with TDP-43-positive inclusions in motor neurons in ALS cases. The present observations show a complex participation of cytokines and mediators of the inflammatory response in ALS consistent with increased proinflammatory cytokines and sequestration of anti-inflammatory IL-10 in affected neurons

Inflammatory Gene Expression In Whole Peripheral Blood At Early Stage Of Sporadic Amyotrophic Lateral Sclerosis

Objective: Characterization of altered expression of selected transcripts linked to inflammation in the peripheral blood of sporadic amyotrophic lateral sclerosis (sALS) patients at early stage of disease to increase knowledge about peripheral inflammatory response in sALS. Methods: RNA expression levels of 45 genes were assessed by RT-qPCR in 22 sALS cases in parallel with 13 age-matched controls. Clinical and serum parameters were assessed at the same time. Results: Upregulation of genes coding for factors involved in leukocyte extravasation (ITGB2, INPP5D, SELL, and ICAM1) and extracellular matrix remodeling (MMP9 and TIMP2), as well as downregulation of certain chemokines (CCL5 and CXC5R), antiinflammatory cytokines (IL10, TGFB2, and IL10RA), pro-inflammatory cytokines (IL-6), and T-cell regulators (CD2 and TRBC1) was found in sALS cases independently of gender, clinical symptoms at onset (spinal, respiratory, or bulbar), progression, peripheral leukocyte number, and integrity of RNA. MMP9 levels positively correlated with age, whereas CCR5, CCL5, and TRBC1 negatively correlated with age in sALS but not in controls. Relatively higher TNFA expression levels correlate with higher creatinine kinase protein levels in plasma. Conclusion: Present findings show early inflammatory responses characterized by upregulation of factors enabling extravasation of leukocytes and extracellular matrix remodeling in blood in sALS cases, in addition to increased TNFA levels paralleling skeletal muscle damage

HPLC-MS/MS Oxylipin Analysis of Plasma from Amyotrophic Lateral Sclerosis Patients

Oxylipins play a critical role in regulating the onset and resolution phase of inflammation. Despite inflammation is a pathological hallmark in amyotrophic lateral sclerosis (ALS), the plasma oxylipin profile of ALS patients has not been assessed yet. Herein, we develop an oxylipin profile-targeted analysis of plasma from 74 ALS patients and controls. We found a significant decrease in linoleic acid-derived oxylipins in ALS patients, including 9-hydroxy-octadecadienoic acid (9-HODE) and 13-HODE. These derivatives have been reported as important regulators of inflammation on different cell systems. In addition, some 5-lipoxygenase metabolites, such as 5-hydroxy- eicosatetraenoic acid also showed a significant decrease in ALS plasma samples. Isoprostanes of the F2 alpha family were detected only in ALS patients but not in control samples, while the hydroxylated metabolite 11-HETE significantly decreased. Despite our effort to analyze specialized pro-resolving mediators, they were not detected in plasma samples. However, we found the levels of 14-hydroxy-docosahexaenoic acid, a marker pathway of the Maresin biosynthesis, were also reduced in ALS patients, suggesting a defective activation in the resolution programs of inflammation in ALS. We further analyze oxylipin concentration levels in plasma from ALS patients to detect correlations between these metabolites and some clinical parameters. Interestingly, we found that plasmatic levels of 13-HODE and 9-HODE positively correlate with disease duration, expressed as days since onset. In summary, we developed a method to analyze (oxy)lipidomics in ALS human plasma and found new profiles of metabolites and novel lipid derivatives with unknown biological activities as potential footprints of disease onset

Estimation of the prevalence and incidence of motor neuron diseases in two Spanish regions: Catalonia and Valencia

According to the degree of upper and lower motor neuron degeneration, motor neuron diseases (MND) can be categorized into amyotrophic lateral sclerosis (ALS), primary lateral sclerosis (PLS) or progressive muscular atrophy (PMA). Although several studies have addressed the prevalence and incidence of ALS, there is a high heterogeneity in their results. Besides this, neither concept has been previously studied in PLS or PMA. Thus, the objective of this study was to estimate the prevalence and incidence of MND, (distinguishing ALS, PLS and PMA), in the Spanish regions of Catalonia and Valencia in the period 2011-2019. Two population-based Spanish cohorts were used, one from Catalonia and the other from Valencia. Given that the samples that comprised both cohorts were not random, i.e., leading to a selection bias, we used a two-part model in which both the individual and contextual observed and unobserved confounding variables are controlled for, along with the spatial and temporal dependence. The prevalence of MND was estimated to be between 3.990 and 6.334 per 100,000 inhabitants (ALS between 3.248 and 5.120; PMA between 0.065 and 0.634; and PLS between 0.046 and 1.896), and the incidence between 1.682 and 2.165 per 100,000 person-years for MND (ALS between 1.351 and 1.754; PMA between 0.225 and 0.628; and PLS between 0.409-0.544). Results were similar in the two regions and did not differ from those previously reported for ALS, suggesting that the proposed method is robust and that neither region presents differential risk or protective factors