# Tracking a Fast-Moving Disease: Longitudinal Markers, Monitoring, and Clinical Trial Endpoints in ALS

Rangariroyashe Hannah Chipika, Eoin Finegan, Stacey Li Hi Shing, Orla Hardiman and Peter Bede\*

Computational Neuroimaging Group, Biomedical Sciences Institute, Trinity College Dublin, Dublin, Ireland

Amyotrophic lateral sclerosis (ALS) encompasses a heterogeneous group of phenotypes with different progression rates, varying degree of extra-motor involvement and divergent progression patterns. The natural history of ALS is increasingly evaluated by large, multi-time point longitudinal studies, many of which now incorporate presymptomatic and post-mortem assessments. These studies not only have the potential to characterize patterns of anatomical propagation, molecular mechanisms of disease spread, but also to identify pragmatic monitoring markers. Sensitive markers of progressive neurodegenerative change are indispensable for clinical trials and individualized patient care. Biofluid markers, neuroimaging indices, electrophysiological markers, rating scales, questionnaires, and other disease-specific instruments have divergent sensitivity profiles. The discussion of candidate monitoring markers in ALS has a dual academic and clinical relevance, and is particularly timely given the increasing number of pharmacological trials. The objective of this paper is to provide a comprehensive and critical review of longitudinal studies in ALS, focusing on the sensitivity profile of established and emerging monitoring markers.

Edited by: Francesca Trojsi, Università degli Studi della Campania

Luigi Vanvitelli Caserta, Italy

### Reviewed by:

Judith Machts, Universitätsklinikum Magdeburg, Germany Monica Consonni, Istituto Neurologico Carlo Besta (IRCCS), Italy

\*Correspondence:

Peter Bede<EMAIL_ADDRESS>
### Specialty section:

This article was submitted to Neurodegeneration, a section of the journal Frontiers in Neurology

Received: 28 October 2018 Accepted: 22 February 2019 Published: 19 March 2019

### Citation:

Chipika RH, Finegan E, Li Hi Shing S, Hardiman O and Bede P (2019) Tracking a Fast-Moving Disease: Longitudinal Markers, Monitoring, and Clinical Trial Endpoints in ALS. Front. Neurol. 10:229. doi: 10.3389/fneur.2019.00229 Keywords: motor neuron disease, amyotrophic lateral sclerosis, biomarkers, magnetic resonance imaging, neuroimaging

# INTRODUCTION

Amyotrophic lateral sclerosis (ALS) is a clinically, genetically, and pathologically heterogeneous neurodegenerative condition (1–3). Clinical heterogeneity in ALS is multidimensional owing to variations in upper motor neuron (UMN) and lower motor neuron (LMN) involvement, extra-motor symptoms, age of onset, survival, and progression-rates. Disease heterogeneity hinders biomarker development (3, 4) which in turn impedes the reliable assessment of candidate drugs in clinical trials (1). Current clinical trials recruit relatively heterogeneous cohorts of symptomatic patients, despite the notion that considerable pathological changes can already be detected at the time of diagnosis (5, 6). The considerable variability in progression rates in ALS is another confounding factor in clinical trial designs (1, 7–10). Imaging and electrophysiological markers have been repeatedly proposed as candidate monitoring markers (11, 12), but it is increasingly clear that a panel of several "wet" and "dry" biomarkers may be required to capture subtle changes over short periods of time (13, 14). The objective of this paper is the comprehensive and critical review of longitudinal studies in ALS, focusing on study designs, statistical power, clinical correlations, the sensitivity profile of proposed monitoring markers and their applicability to clinical trials.

## METHODS

A formal literature search was performed on PubMed using the core search terms "amyotrophic lateral sclerosis" and "longitudinal" combined with each of the following keywords

**Abbreviations:** 2D-DIGE, two-dimensional fluorescence difference gel electrophoresis; ABG, arterial blood gas; ACE-3, Addenbrookes Cognitive Examination - Third Edition; ACE-R, Addenbrooke's Cognitive Examinationrevised; AD, axial diffusivity; ADM, abductor digiti minimi; ADQ, abductor digiti quinti; AGA, arterial gas analyses; AHB, abductor halluces brevis; ALS, amyotrophic lateral sclerosis; ALSAQ-40, ALS assessment questionnaire; ALS-CBS, ALS Cognitive Behavior Screen; ALS-CFB, ALS computerized frontal battery; ALS-FBI, ALS-Frontal Behavioral Inventory; ALSFRS-r, revised ALS functional rating scale; ALSS, ALS severity scale; ALSSQoL-R, revised ALSspecific Quality of Life questionnaire; APB, abductor pollicis brevis; ARSLA, Association pour la recherche sur la SLA; ATLIS, accurate test of limb isometric strength; BAI, body adiposity index; BMI, body mass index; CALR, Calreticulin; CALSNIC, Canadian ALS Neuroimaging Consortium; CHI3L1, chitinase-3-like protein 1; CHI3L2, chitinase-3-like protein 2; CHIT1, chitotriosidase-1; Cho, Choline; CIDP, chronic inflammatory demyelinating polyneuropathy; CIS20-R, checklist individual strength; CK, creatinine kinase; CLIC1, Chloride intracellular channel protein 1; CMAP, compound muscle action potential; CMCT, central motor conduction time; CMT, Charcot-Marie-Tooth disease; CNS-BFS, Center for Neurologic Study-Bulbar Function Scale; CNS-LS, Center for Neurologic Study-Lability Scale; COWAT, controlled oral word association test; Cr, creatinine; CSF, cerebrospinal fluid; CSP, cortical silence period; CypA, peptidyl-prolyl cistrans isomerase A; DCMAP, distal compound muscle action potential; D-KEFS, Delis–Kaplan Executive Function System; DTI, diffusion tensor imaging; DWI, diffusion-weighted imaging; ECAS, Edinburgh Cognitive and Behavioral ALS Screen; ECL, electrochemiluminescence; EDB, extensor digitorum brevis; EMG, electromyography; ERp57, protein disulfide-isomerase A3; EURALS, European Registry of ALS Consortium; EUROMOTOR, European multidisciplinary ALS network identification to cure motor neurone degeneration; FA, fractional anisotropy; FAB, frontal assessment battery; FBI, frontal behavioral inventory; FD, fiber density; FEV1, forced expiratory volume; fibs-sw, fibrillation/sharpwaves; FM-ADP, fat mass air displacement plethysmography; FPs, fasciculation potentials; FSS, fatigue severity scale; FUBP1, far upstream element-binding protein 1; FVC, forced vital capacity; GM, gray matter; GSTO1, glutathione S-transferase omega-1; HADS, hospital anxiety and depression scale; HDAC4, histone deacetylase 4; HHD, handheld dynamometry; HSC70, heat shock cognate 71 kDa protein; IL, interleukin; IFN, interferon; IRAK4, Interleukin-1 receptorassociated kinase 4; JPND, EU Joint Programme for Neurodegenerative Disease Research; LGVF, letter guided verbal fluency; LMN, lower motor neuron; MAS, modified Ashworth scale; McDESPOT, multi-component driven equilibrium single pulse observation of T1/T2; MDRS-2, Mattis Dementia Rating scale-Second Edition; MEP, maximal static expiratory mouth pressure; MiND-B, motor neuron disease behavior scale; MIP, maximal inspiratory pressure; MiRNAs, micro-RNAs; MITOS, Milano-Torino staging system; MMSE, mini mental state examination; MMT, manual muscle testing; MND, Motor neuron disease; MoCA, Montreal Cognitive Assessment; MR, magnetic resonance; MRC, Medical Research Council Scale for muscle strength; MRCSS-LL, Medical Research Council sum score; MRI, magnetic resonance imaging; MRS, magnetic resonance spectroscopy; MS, multiple sclerosis; MU, motor unit; MUNE, motor unit number estimation; MUNIX, motor unit number index; MUPs, motor unit potentials; MUSIX, motor unit size index; NAA, N-acetylaspartate; NEALS, Northeast ALS Consortium; NF-L, neurofilament light chain; NI, neurophysiology index; NISALS, Neuroimaging Society in Amyotrophic Lateral Sclerosis; NMR, nuclear magnetic resonance; nUHPLC LC-MS, nano ultra-high performance liquid chromatography tandem mass spectrometry; p75ECD, neurotrophin receptor p75 extracellular domain; PA28a, proteasome activator complex subunit 1; PBA, pseudobulbar affect; PCR, polymerase chain reaction; PDI, protein disulfide-isomerase; PEFT, peak expiratory flow time; PET, positron emission tomography; PGGM, precentral gyruses gray matter; PGRN, progranulin; PLS, primary lateral sclerosis; PMA, progressive muscular atrophy; pNFH, Phosphorylated neurofilament heavy chain; PRDX2, peroxiredoxin-2; PRO-ACT, Pooled Resource Open-Access ALS Clinical Trials; QoL, quality of life; RD, radial diffusivity; RMN, Research Motor separately: "staging," "monitoring," "outcomes," "clinical," "clinical trials," "electrophysiology," "neurophysiology," "electromyography," "transcranial magnetic stimulation," "motor unit number estimation," "motor unit number index," "positon emission tomography," "single photon emission computed tomography," "magnetic resonance imaging," "neuroimaging," "imaging," "blood," "urine," "cerebrospinal fluid," "saliva," and "muscle." A supplementary search combined the core search terms with the following keywords: "presymptomatic," "asymptomatic," and "post-mortem." Inclusion criteria included longitudinal studies investigating imaging, neurophysiological, clinical, or biofluid biomarkers in ALS. Animal studies, review papers, opinion pieces, editorials, case reports, and case series were excluded. Only articles written in English and published between January 1980 and August 2018 were reviewed. Based on the above criteria a total of 118 original research papers were selected and reviewed in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) recommendations.

# RESULTS

### Neuroimaging

The sample size characteristics, study design features, followup intervals of longitudinal neuroimaging, neurophysiology, and clinical studies are summarized in **Table 1**. Whilst most longitudinal imaging studies in ALS evaluate cerebral alterations (10), a number of promising spinal studies have now also been published. Spinal imaging has gradually overcome the technical challenges of physiological motion, small crosssectional dimensions and susceptibility gradients (19, 110–118). The majority of longitudinal studies in ALS are single-center studies eliminating the need for cross-platform MR sequence harmonization and inter-rater reliability tests. Given the low incidence of certain phenotypes such as primary lateral sclerosis (PLS), progressive muscular atrophy (PMA), and spinal and bulbar muscular atrophy (SBMA) however, multisite collaboration is often necessary (119). The infrastructure, funding and governance of such multicenter collaborations are now established via international consortia like the Neuroimaging Society in Amyotrophic Lateral Sclerosis (NISALS) or the Northeast ALS Consortium (NEALS) (16, 23, 120, 121). The need to include disease-controls in addition to healthy controls to describe ALS-specific changes

Neuron; ROA2, Heterogeneous nuclear ribonucleoproteins A2/B1; RSA, relative surface area; rsfMRI, resting state functional magnetic resonance imaging; SCA, spinocerebellar ataxia; SBMA, spinal and bulbar muscular atrophy; SEIQOL-DW, Schedule for the Evaluation of the Individual Quality of Life-Direct Weighting; SF-36, 36-Item short form health survey; SMA, spinal muscular atrophy; SMUAP, single motor unit action potential; SNIP, sniff nasal inspiratory pressure; SOD1, superoxide dismutase 1; SOP, Standard operating procedure; SPECT, single photon emission computed tomography; SPO2, peripheral capillary oxygen saturation; SVC, slow vital capacity; TA, tibialis anterior; TDP-43, TAR DNAbinding protein 43; TiM, Telehealth in Motor Neuron disease; TMS, transcranial magnetic stimulation; TNF, tumor necrosis factor; TUG, timed up and go test; Tw Pdi, twitch trans-diaphragmatic pressure; TWBC, total white blood cell count; UMN, upper motor neuron; VC, vital capacity; WALS, Western ALS Consortium; WVFI, Written Verbal Fluency Index.

### TABLE 1 | Longitudinal "dry biomarker" studies in ALS: Neuroimaging, Neurophysiology and Clinical Studies.

Studies detecting progressive changes are listed first followed by studies not capturing longitudinal changes. \*indicates clinical trial.

is increasingly recognized (30, 43, 44). With few exceptions (122–124), most ALS imaging studies use 3 Tesla platforms and 7 Tesla systems are more commonly used in post-mortem studies (125, 126). Disease progression has been detected across a range of MR imaging metrics including structural (22, 26), diffusion (16, 18), functional (28, 40), and spectroscopy (41, 42) measures. As the majority of studies have a two-timepoint design, it is often unclear if specific imaging metrics show linear or exponential changes. The few existing multi-timepoint studies suggest that pathological change is not linear (10). The revised ALS functional rating scale (ALSFRS-r) is the most commonly reported clinical measure (16, 18–20), with only few imaging studies reporting associations with staging (15) or neuropsychological performance (15, 24).

### Neurophysiology

Most longitudinal neurophysiology studies are single center studies, reducing the risk of inter-rater and inter-center variability (127). As presented in **Table 1**, follow-up interval ranges between 7 days (65) and 3 years (66), and up to 7 follow-up time-points have been included in some studies (57, 60). Surprisingly few studies include disease controls such as peripheral neuropathy (60) or benign fasciculation syndrome (50). Clinical assessments performed in conjunction with neurophysiology typically include ALSFRS-r (51), forced vital capacity (FVC) (55), slow vital capacity (SVC) (56), grip strength (64), pinch strength (58), and manual muscle testing (MMT) (58), however, correlations between neurophysiological measures and clinical assessments are seldom reported. The majority of longitudinal neurophysiological studies focus on upper limb muscles, e.g., abductor pollicis brevis, deltoid, first dorsal interrosseus, extensor digitorum brevis, abductor digiti minimi (51, 52, 55, 60, 61) with relatively few studies evaluating lower limb muscles such as abductor hallicus brevis and tibialis anterior (50, 56, 57, 66). The most commonly reported longitudinal neurophysiological indices include compound muscle action potential (CMAP) (51, 52), single motor unit action potential (SMUAP) (60), MUNE (55, 59), MUNIX (49, 56), neurophysiology index (NI) (54, 55), TMS measures (57, 58), and axonal excitability (52). Progressive neurophysiological changes have been detected by MUNIX (49, 51, 56), MUNE (51, 58, 60), CMAP (52, 61), NI (54), and TMS measures (57) and allowing for study-design limitations, the consensus is that degenerative changes are not linear.

### Clinical Biomarkers and Instruments

Robust clinical longitudinal studies in ALS have up to 6 followup time points (88, 89, 91), the interval between the assessments can be as short as 3 months (95) and the sample size can be as big as several thousands (70, 93) (**Table 1**). Few multi-timepoint studies include disease controls such as motor neuropathies (91), alternative neuromuscular diseases (78), or neurodegenerative conditions (83). Large, multi-timepoint longitudinal studies invariably suffer from considerable attrition rates, but these are rarely explicitly reported in the manuscript abstracts (10). Detailed genotyping is only available in a minority of longitudinal studies (15, 77, 79, 94). The most widely utilized rating scale in longitudinal studies is the ALSFRS-r (70, 71, 128) which provides a composite score of bulbar, limb and respiratory dysfunction, and is invariably evaluated in clinical trials (72, 105). Quality of life (QoL) in ALS is increasingly evaluated by disease-specific instruments such as the 40-item ALS assessment questionnaire (ALSAQ-40) or the revised ALS-specific Quality of Life questionnaire (ALSSQoL-R) (129–131). A number of symptom-specific instruments are also commonly used such as the Center for Neurologic Study-Bulbar Function Scale (CNS-BFS), a 21-item self-report scale of bulbar function, and the Center for Neurologic Study-Lability Scale (CNS-LS), a 7-item self-report scale of pseudobulbar affect (PBA) (132). Tapping rates, composite reflex scores, The Penn UMN Score (133), the Modified Ashworth scale (MAS) are often used as proxies of UMN degeneration (132).

In clinical trials, muscle strength is often estimated by handheld dynamometry (HHD) (134), manual muscle testing (MMT) (105), scoring systems such as the Medical Research Council (MRC) Scale for muscle strength (135) and some studies also report limb circumference (136). Respiratory function in ALS is typically monitored by sniff nasal inspiratory pressure (SNIP), SVC, or FVC in addition to measures such as early morning arterial blood gas (ABG) and overnight pulse-oximetry (137, 138). Measures of typing ability (91), tongue movements (90), vital capacity (VC) (96), FVC (99), SNIP (97), and diaphragm amplitude (97) all show progressive longitudinal changes. Nutritional markers such as body mass index (BMI) and lipid profile are now established prognostic indicators (139, 140). Cognitive and behavioral domains are routinely assessed thanks to the availability of validated screening instruments such as the Edinburgh Cognitive and Behavioral ALS Screen (ECAS) (141), the Beaumont Behavioral Inventory (BBI) (142) and the ALS Cognitive Behavioral Screen (ALS-CBS) (143). In contrast to the relentlessly progressive motor deficits of ALS, the trajectory of cognitive and behavioral deficits is less clear due to considerable individual variations, genotype-associated profiles (144, 145), differences in assessment strategies and practiceeffects (146). Several longitudinal neuropsychology studies do not detect progression (77, 147, 148), progressive behavioral impairment has been noted in the absence of cognitive change (149), and some studies report improved performance as a result of practice effects (77).

# Wet Biomarkers

The findings, study design characteristics, and follow-up intervals of longitudinal biofluid studies are summarized in **Table 2**. Phosphorylated neurofilament heavy chain (pNFH), neurofilament light chain (NF-L), progranulin (PGRN), cytokines, TAR DNA-binding protein 43 (TDP-43), cystatin C, creatinine, micro-RNAs (miRNAs), chitotriosidase-1 (CHIT1), chitinase-3-like protein 1 (CHI3L1), chitinase-3-like protein 2 (CHI3L2) have been evaluated in both research studies (152, 153, 157, 158, 162, 164, 168, 171) and clinical trials (150, 156, 157, 160, 161). Markers of iron metabolism and ferroptosis are relatively recent domains of ALS biomarker research (172, 173). Most biofluid studies are either serum (150, 157) or CSF studies (152, 167), but urine (155) and skeletal

Chipika et al. Longitudinal and Monitoring Markers of MND

muscle-based (153) studies have now also been published. Quantitative enzyme-linked immunosorbent assay (ELISA) is the most commonly used antibody-based technique (13, 174) which can be performed with one antibody (indirect ELISA), or with two antibodies (sandwich ELISA). Increased CSF (13) and serum (175) pNFH detected by ELISA is thought to be a sensitive marker of axonal degeneration in ALS (152, 171, 176, 177). The specificity of this marker however may be inadequate to reliably differentiate ALS from other neurodegenerative conditions (13, 176). Other antibody-based techniques such as Western blot (171) and electrochemiluminescence (ECL) (153, 168) may improve detection sensitivity and reliability (13). Panels of multiple proteins can be evaluated by multiplex immunoassays such as planar or microbead assays (13). Mass spectrometry based methods using chromatin-immunoprecipitation-based surfaces, two-dimensional gel electrophoresis or high-resolution mass spectrometry have identified cystatin-C and transthyretin as candidate biomarkers (178–180). The longest wet biomarker study followed patients for 4 years (164). The majority of studies have at least 2 follow-up timepoints (155, 162, 170) and one study included 13 follow-up timepoints (156, 159). Large multi-center trials include as much as 1,000 participants (156). One of the most striking shortcomings of existing longitudinal studies is that very few included disease controls such as Parkinson's disease cohorts, patients with multifocal motor neuropathy with conduction block, Kennedy's disease, chronic inflammatory demyelinating polyneuropathy (CIDP), cervical or lumbar radiculopathy, Charcot-Marie-Tooth disease (CMT), benign fasciculation, and cramp syndrome etc. (152, 159, 162). Another limitation of many longitudinal studies is the lack of comprehensive genotyping (12) as very few studies report comprehensive screening for ALS-associated mutations (153, 159, 169, 171). Exhaustive clinical profiling, such as medications (152, 164), neuropsychological assessments (171), quality of life indices are rarely reported in longitudinal studies. The majority of studies limit their clinical descriptions to ALSFRS-r, FVC, MRC, and Ashworth scores (153, 161, 162). Serum and plasma biomarkers such as creatinine (150, 156), pNfH (158, 159), and micro-RNAs (157), CSF biomarkers such as CHI3L1 (152), tau (160, 161), and cystatin-C (162), and urinary (155) and skeletal muscle (153) biomarkers are some of the promising tools for detecting disease progression. While no progressive changes have been detected in NFL levels, it is likely to be a useful as a diagnostic biomarker (168, 171).

### Studies of Asymptomatic Mutation Carriers

Current clinical trials only recruit symptomatic cases despite accruing evidence that ALS has a long presymptomatic phase (5). Imaging studies of asymptomatic mutation carriers have consistently confirmed disease-specific cerebral and spinal cord changes prior to symptom onset (181–184) indicating that this disease-phase may represent a crucial window for therapeutic or neuroprotective intervention. The majority of presymptomatic studies assess a single time-point, as opposed to the longitudinal tracking of asymptomatic carriers of ALS-causing mutations (15). While the overwhelming majority of presymptomatic studies focus on C9orf72 hexanucleotide carriers (183, 185– 187), no prognostic markers have been validated to predict whether single patients will develop ALS or FTD. Compared to imaging studies, strikingly few presymptomatic neurophysiology studies have been undertaken (66). Studies of asymptomatic ALScausing mutation carriers have enormous potential for academic research and may pave the way for asymptomatic pharmaceutical trials (5, 181).

# DISCUSSION

Clinical trials currently evaluate the efficacy of candidate drugs using the revised ALS functional rating scale (ALSFRS-r), muscle strength assessment tools such as manual muscle testing (MMT), respiratory function indices such as forced vital capacity (FVC), slow vital capacity (SVC) and sniff nasal inspiratory pressure (SNIP), neurophysiological measures and survival (102, 116, 120, 188, 189). These measures however primarily reflect latestage functional impairment and are not indicative of early stage pathology. Brain and spinal cord imaging has been evaluated as early-stage biomarkers with both diagnostic and monitoring potential (116, 120, 190). The core neuroimaging signature of ALS, irrespective of the disease-stage, includes corticospinal tract (191, 192), corpus callosum (193) and motor cortex degeneration (194). Atrophy in frontotemporal regions has been primarily associated with neuropsychological deficits (195–197) and linked to hexanucleotide repeats in C9orf72 (145, 198). Longitudinal imaging studies are superior to crosssectional studies as they readily detect dynamic structural and functional changes and may elucidate compensatory processes (10, 14, 23, 28, 40, 120, 199). The emergence of multi-timepoint study designs (14, 20) enable the characterization of anatomical propagation patterns (200) and provide invaluable temporal insights into the disease trajectory of late-stage ALS. Interscan intervals as short as 3 months can detect longitudinal changes (14, 18, 120). Many longitudinal studies make use of multiple magnetic resonance (MR) metrics which is particularly useful in establishing an optimal panel of monitoring markers (120). Several longitudinal studies have indicated that white matter degeneration can be detected relatively early in the course of ALS with restricted further progression over time, whereas gray matter pathology shows relentless progression in the symptomatic phase of the disease (4, 14, 120). In addition to structural imaging studies, connectivity-based, metabolic, peripheral nerve, and, whole body muscle imaging have contributed to our understanding of longitudinal changes (20, 201–203).

Needle electromyography and nerve conduction studies play an important clinical role in ruling out alternative conditions and confirming a suspected diagnosis of ALS. Despite variations in local protocols, neurophysiological tests are recognized as objective, reliable and cost-effective tests of neuromuscular dysfunction, and have also been repeatedly proposed as longitudinal markers (55, 204). CMAP is generated by depolarization of muscle fibers through the stimulation of a single nerve, where amplitude reductions are interpreted as loss of motor axons (205, 206). While CMAP measurements capture longitudinal decline, it is confounded by variations in temperature, limb positioning and electrode placement (56, 207). CMAP-derived measures such as MUNE and MUNIX are now extensively utilized to characterize progressive changes in ALS. MUNE estimates motor neuron numbers, and may detect the rate of motor neuron loss, making it a more reliable method of appraising disease progression than CMAP (208, 209). However, its early-phase sensitivity has been questioned, as its use is limited to distal muscles, and the technique requires considerable training, especially for inter-rater and multi-site comparisons (205, 210). TMS allows the characterization of upper motor neuron dysfunction, and may be particularly useful in detecting progressive changes (57, 205).

Functional rating-scales are often the monitoring instruments of choice in clinical trials (55), as they are easy to administer, cost-effective to utilize and have acceptable inter- and intrarater reliability profiles (7). The most widely used rating scale in clinical longitudinal studies is the ALSFRS-r. Despite its ease of administration, it has considerable limitations, as it may be disproportionately influenced by LMN dysfunction, does not account for laterality or asymmetry of symptoms, omits cognitive impairment, and may be affected by medications (14, 128, 188, 211).

Proteomics, metabolomics and lipidomics have seen significant advances in ALS research and CSF and serum markers are now used in longitudinal academic and pharmacological studies (172). Potential biomarkers for the detection of disease progression include serum and plasma biomarkers such as creatinine (150, 156), pNfH (158, 159), and micro-RNAs (157), CSF biomarkers such as CHI3L1 (152), tau (160, 161), and cystatin-C (162), and urinary (155) and skeletal muscle (153) biomarkers.

### Prediction Analyses

Age at symptom onset (212), BMI (139), bulbar involvement (213), cognitive impairment (214), C9orf72 genotype status (144), respiratory insufficiency (215), "definite ALS" by the El Escorial criteria (216), and functional disability (217) are the most commonly cited determinants of poor prognosis in ALS. SNIP (218) and less commonly used measures such as twitch trans-diaphragmatic pressure (Tw Pdi) (219) and maximal static expiratory mouth pressure (MEP) were shown to be good predictors of ventilator-free survival (219). A combined panel of several clinical, wet, and dry biomarkers is likely to offer the most accurate prognostic information (115, 120, 216, 217, 220). While cerebral (217, 221, 222) and spinal (115) imaging measures have been repeatedly linked to survival outcomes, these have not been utilized in a clinical setting. Neurophysiological variables, such as phrenic nerve stimulation outcomes (223) and biofluid markers, such as pNFH and NFL (165, 168, 224–226) are also thought to be accurate predictors.

### Patient Stratification

Attempts to enroll patients in the early stages of the disease are hampered by the universally long diagnostic delay in ALS (227). Patient stratification in trials is typically based on site of onset (228), instead of other variables which have an established prognostic impact (138, 229). Admixed patient cohorts within a trial may hamper the ability to detect how different phenotypes and genotypes may exhibit a different response to a candidate drug (230–232). The stratification of heterogeneous cohorts is now aided by the development of validated staging systems, such as the King's (233), Milano-Torino (MITOS) (234) or the Fine'til 9 (FT9) (235) staging systems. The King's Staging system is based on the number of body regions affected, and the presence of nutritional or respiratory failure (233). The MITOS staging system is based on the ALSFRS-r, and is particularly sensitive to changes in later stages of the disease (236, 237). However, none of these staging systems account for cognitive or behavioral changes (236). Pathological staging systems suggest a four-stage model of ALS based on anatomical patterns of pTDP-43 load (238, 239). This system has now been validated by in vivo neuroimaging studies (240) and signals that accurate pathological staging and patient stratification may be possible based on neuroimaging (199, 240).

# International Consortia

Only few ALS centers maintain dedicated biobanking facilities to store and process molecular markers in human biofluid locally. Similarly, relatively few centers are in a position to generate sufficient number of MRI and neurophysiology data sets of rare phenotypes to make meaningful inferences in a single center setting. Brain and tissue banks are also challenging to establish, maintain and fund, despite their invaluable contribution to ALS research (241–243).

Biospecimen samples are also often collected during clinical trials, and discarded after negative outcomes, despite their enormous potential for biomarker discovery (172). One of the most important achievements of biomarker development efforts is the establishment of national and international research consortia such as Association pour la recherche sur la SLA (ARSLA), Neuroimaging Society in ALS (NISALS), Research Motor Neuron (RMN), Canadian ALS Neuroimaging Consortium (CALSNIC), EU Joint Programme for Neurodegenerative Disease Research (JPND), European multidisciplinary ALS network identification to cure motor neurone degeneration (EUROMOTOR) which maintain vital biobanking facilities, registries, data repositories for multicenter data interpretation (121, 244). Clinical trial networks are also increasingly recognized as valuable platforms for multisite data collection and interpretation as they operate with carefully standardized protocols. Consortia such as the European Registry of ALS (EURALS) Consortium, the Western ALS (WALS) Consortium and the Northeast ALS (NEALS) Consortium are other examples (245). NEALS is one of the largest consortia with over 100 member sites from the US, Canada, Mexico, Italy, Lebanon and Australia (246). EURALS coordinates research studies and clinical trials relying on population-based European registries and include centers from Scotland, England, Netherlands, Spain, Ireland, Serbia, Italy, France, and Germany (241, 247, 248). ALS research consortia promote patient-oriented research, maintain biofluid, imaging and DNA banks, and have the potential to translate scientific advances into pragmatic clinical interventions.

### Telehealth

Novel trends in longitudinal data collection include telemedicine-based technologies, wearable sensors and mobile phone applications (230). The continuous collection of data via telephone or telemedicine applications such as the Telehealth in Motor Neuron disease (TiM) system circumvent the inconvenience of patients and caregivers traveling long distances for research appointments (249). Once local data-protection and governance guidelines are complied with, information uploaded from these systems can be made available to healthcare professionals of multidisciplinary teams in real time (249). The feasibility of telehealth for ALS patients via live videoconferencing has also been evaluated (250) and is considered a particularly promising clinical and research platform (249, 250). A number of cognitive-behavioral screening tools have also been adapted for phone administration (251) including modified versions of the ALS Cognitive Behavior Screen (ALS-CBS), the Controlled Oral Word Association Test (COWAT), the Center for Neurologic Study-Lability Scale (CNS-LS) and found to be statistically equivalent to face-to-face assessments (251). Performance on other tests however, such as the telephone versions of the ALS-Frontal Behavioral Inventory (ALS-FBI) caregiver interview and the Written Verbal Fluency Index (WVFI) was not equivalent to clinic-based assessments (251). The continued development of telephone and internet-enabled

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# CONCLUSIONS

While clinical indicators of disease progression remain indispensable, neuroimaging, neurophysiology, and biofluid measures are particularly promising, objective, quantitative biomarker candidates. The validation of combined "wet" and "dry" biomarker panels will not only enable the detection of subtle progressive changes in ALS, but allow precision stratification of heterogeneous patient cohorts in clinical trials and improve existing prediction algorithms.

# AUTHOR CONTRIBUTIONS

The manuscript was drafted by RC. EF, SL, OH and PB contributed to the conceptualization, editing, and revision of this paper.

### FUNDING

This work was supported by the Andrew Lydon Scholarship, the Health Research Board (HRB–Ireland; HRB EIA-2017-019), the Irish Institute of Clinical Neuroscience (IICN)–Novartis Ireland Research Grant, the Iris O'Brien Foundation, the Perrigo Clinician-Scientist Research Fellowship, and the Research Motor Neuron (RMN–Ireland) Foundation.

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**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Chipika, Finegan, Li Hi Shing, Hardiman and Bede. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Pathological Crying and Laughing in Motor Neuron Disease: Pathobiology, Screening, Intervention

Eoin Finegan, Rangariroyashe H. Chipika, Stacey Li Hi Shing, Orla Hardiman and Peter Bede\*

Computational Neuroimaging Group, Biomedical Sciences Institute, Trinity College Dublin, Dublin, Ireland

Pathological crying and laughing (PCL) has significant quality-of-life implications in amyotrophic lateral sclerosis (ALS); it can provoke restrictive life-style modifications and lead to social isolation. Despite its high prevalence and quality of life implications, it remains surprisingly understudied. Divergent pathophysiological models have been proposed, centered on corticobulbar tract degeneration, prefrontal cortex pathology, sensory deafferentation, and impaired cerebellar gate-control mechanisms. Quantitative MRI techniques and symptom-specific clinical instruments offer unprecedented opportunities to elucidate the anatomical underpinnings of PCL pathogenesis. Emerging neuroimaging studies of ALS support the role of cortico–pontine–cerebellar network dysfunction in context-inappropriate emotional responses. The characterization of PCL-associated pathophysiological processes is indispensable for the development of effective pharmacological therapies.

### Edited by:

Francesca Trojsi, Università Degli Studi Della Campania Luigi Vanvitelli Caserta, Italy

### Reviewed by:

Susanne Petri, Hannover Medical School, Germany Judith Machts, Universitätsklinikum Magdeburg, Germany

> \*Correspondence: Peter Bede<EMAIL_ADDRESS>
### Specialty section:

This article was submitted to Neurodegeneration, a section of the journal Frontiers in Neurology

Received: 20 December 2018 Accepted: 26 February 2019 Published: 21 March 2019

### Citation:

Finegan E, Chipika RH, Li Hi Shing S, Hardiman O and Bede P (2019) Pathological Crying and Laughing in Motor Neuron Disease: Pathobiology, Screening, Intervention. Front. Neurol. 10:260. doi: 10.3389/fneur.2019.00260 Keywords: pathological crying and laughing, pseudobulbar affect, emotional lability, involuntary emotional expression disorder, motor neuron disease, amyotrophic lateral sclerosis, biomarkers, magnetic resonance imaging

# INTRODUCTION

The terms "pathological crying and laughing," "pseudobulbar affect," "emotional lability," and "involuntary emotional expression disorder" are often used interchangeably depending on geographical location and year of publication. Despite the differences in terminology, recurrent episodes of involuntary or exaggerated emotional expression, particularly in the form of crying and laughing, are common in several neurological conditions including, in order of prevalence: motor neuron disease (MND), traumatic brain injury (TBI), multiple sclerosis (MS), stroke, multiple system atrophy-cerebellar type (MSA-C), Alzheimer's disease (AD), and Parkinson's disease (PD) (1–6).

The disorder is particularly common in motor neuron diseases (MND): amyotrophic lateral sclerosis (ALS) and primary lateral sclerosis (PLS). Prevalence estimates vary, but between one quarter and one half of MND patients are thought to be affected (5, 7–11). While there is a paucity of studies comparing PCL prevalence across the spectrum of MND phenotypes, a recent study confirmed the relationship between clinical upper motor neuron dysfunction and PCL prevalence; PCL was most commonly identified in PLS and UMN-predominant patients (39%), followed by typical ALS (29%) and lower motor neuron (LMN) predominant groups (10%). Consistent with this pattern, there was a single case of PCL in a group of 12 patients with progressive muscular atrophy (PMA) (12). While the manifestations of the episodes may be congruent with the person's contemporary emotional state (10, 13, 14), the magnitude of emotional responses is disproportionate to the emotive stimulus and the social context. Such episodes may cause significant distress, embarrassment, and ultimately may lead to social withdrawal (15).

The primary objective of this review is to systematically appraise PCL-related studies in MND from a dual academic and clinical perspective. We outline established and emerging disease-models based on neuroimaging, neurosurgical and neurophysiological studies. The spectrum of clinical presentations, diagnostic challenges, functional impact, and treatment options are also discussed. We preferentially use the term "pathological crying and laughing" (PCL) in this review, in its broadest sense: encompassing the entire spectrum of presentations. Furthermore, while PCL in ALS is the primary focus of this review, we draw further information from PCL studies in other neurological conditions to discuss unifying, symptom-specific, pathophysiological concepts.

## METHODS

A formal literature search was conducted using PubMed/Medline and Embase using the terms "pathological crying and laughing," "pseudobulbar affect," "emotional lability," involuntary emotional expression disorder" separately, and in combination with terms "amyotrophic lateral sclerosis," "motor neuron disease," "pseudobulbar palsy," "clinical trials," "treatment," and "pathology." Only articles in English, published between 1988 and October 2018 were included. A total of 220 articles met these criteria; these were systematically reviewed for information relating to diagnosis, disease-mechanisms, anatomical localization, and treatment options.

### Historical Context and Terminology

The abundance of terms used to describe this syndrome epitomizes the tireless efforts to characterize the underpinnings of both physiological and pathological emotional expression. Charles Darwin observed in 1872 that "certain brain-diseases, as hemiplegia, brain-wasting, and senile decay, have a special tendency to induce weeping" (16). Oppenheim (17) used the term "pseudobulbar affect" (PBA), based on the important observation that the disorder commonly occurs in association with motor features of pseudobulbar palsy, a condition resulting from bilateral corticobulbar tract pathology. Although this association is still well-recognized (13, 18), the term PBA may be misleading; new evidence suggests that corticobulbar tract dysfunction alone is neither necessary, nor sufficient to cause PCL (19– 23). "Emotional Lability" (EL) was described by Pierre-Marie as early as 1892, a term still commonly used in the literature (24). The term pathological crying and laughing (PCL) was used by Wilson in his influential essay of 1924, in which he introduced his unifying theory of the disorder (25). More recently, the term "involuntary emotional expression disorder" (IEED) has been proposed to encompass the wider range of emotional symptoms which may accompany the disorder (26).

# Clinical Presentations, Diagnosis and Monitoring

A useful conceptualization of PCL is that symptoms may lie on a spectrum, (27) with infrequent, mood congruent but disproportionate episodes at one end, and frequent, mood incongruent episodes at the other. While there is considerable variability in episode type and character across patients, within individuals episodes tend to be consistent over time in terms of symptom type (i.e., uncontrolled laughing or crying), the context in which the episodes recur, the severity, duration, and the degree of voluntary control that the individual retains over the episodes (26, 28). The wide range of presentations coupled with the lack of unifying terminology, has hampered efforts to establish widely adopted diagnostic criteria. Several criteria have been proposed and later revised, reflecting evolving concepts of the PCL (29). Poeck's 1969 criteria (30), focus on episodes that are entirely situation inappropriate or unrelated to the patient's internal emotional state.  Revised criteria proposed by Cummings et al. for "involuntary emotional expression disorder" (IEED) (26) are inclusive of episodes that are either disproportionate to the emotive stimulus or to the individual's internal emotional state. The episodes must represent a distinct change from the patient's emotional reactivity prior to the onset of a neurological disease. These criteria specifically require the exclusion of alternative causes for the symptoms; mood, facial tics, dystonia, or substance effects. More recently, Miller et al. proposed to include supportive features such as the presence of pseudobulbar signs and a proneness to anger (27). The latter reflects accruing observations from several studies (14, 23, 27, 31).

PCL needs to be carefully distinguished from mood disorders through careful clinical evaluation (1, 9, 32). A key difference is that crying in depression or excessive laughter in mania occur in the context of pervasive low or elevated mood, respectively (33). In the case of clinical depression, associated symptoms can also be identified such as anhedonia and insomnia (27, 34). Although some studies report an association between depression and PCL (12), more commonly, no significant association is found (8, 13, 35). The clinical distinction may be particularly challenging in cases where pathological crying co-exists with depressive symptoms (2, 36). Emerging evidence suggests that PCL may respond to selective serotonin reuptake

**Abbreviations:** AD, Alzheimer's disease; ALS, Amyotrophic lateral sclerosis; B.I.D., Bis in die/twice daily; CNS-LS, Center for Neurologic Study-lability scale; DxQ, Dextromethorphan-quinidine; EL, Emotional lability; ELQ, Emotional lability questionnaire; EMA, European Medicines Agency; FDA, US Food and Drug Administration; LMN, Lower motor neuron; MND, Motor neuron disease; MRI, Magnetic resonance imaging; MS, Multiple sclerosis; MSA-C, Multiple system atrophy-cerebellar type; NMDA, N-methyl-D-aspartate; PBA, Pseudobulbar affect; PC, Pathological crying; PCL, Pathological crying and laughing; PD, Parkinson's disease; PLACS, The pathological laughing and crying scale; PLS, Primary lateral sclerosis; PMA, Progressive muscular atrophy; SPECT, Single-photon emission computed tomography; SSRI, Selective serotonin reuptake inhibitor; TBI, Traumatic brain injury; TCA, Tricyclic anti-depressant; UMN, Upper motor neuron.

inhibitors (SSRIs) within days, whereas depression typically only responds to pharmacological intervention after several weeks (37).

Similarly to the multitude of diagnostic criteria proposed for PCL, several screening and symptom severity scoring instruments have been developed. While not all of these have been extensively validated, these tools have been used in both academic research and clinical trials. The pathological laughing and crying scale (PLACS) was validated for use in stroke patients with "PBA" in 1993. The authors used it as an efficacy measure in a small placebo-controlled trial of nortriptyline; treatment was associated with symptom reduction (38). The Center for Neurologic Study-Lability Scale (CNS-LS) was introduced as a self-reported measure of "affective lability" in ALS (39). This short, self-administered questionnaire consists of 7 items; 4 relating to labile laughing and 3 relating to crying. The scale evaluates subjective burden of symptoms over the preceding week, in terms of episode burden and severity. While this scale relies on retrospective patient account, it has been shown to be an accurate indicator of episode frequency (40). The CNS-LS has been adopted as an efficacy measure in several recent clinical trials (41–44). Another assessment tool, the "emotional lability questionnaire" (ELQ) has also been validated in ALS. It extends the period over which symptoms are assessed from one up to four weeks prior to screening, which helps to capture patients who experience less frequent episodes (45, 46). In addition to PCL, it also includes a specific section on abnormal smiling. One of the strengths of the ELQ is that it includes the caregiver's perspective, which helps to identify lack of insight if significantly discordant scores are given by the patient and the carer. Interestingly, in ALS, there is significant agreement between patient and caregiver scores, indicating that patients are keenly aware of PCL symptoms (45). This concordance contrasts with behavioral deficits in ALS-FTD, where patient reports may differ substantially from caregivers reports (47).

Although screening instruments are valuable tools for identifying and monitoring PCL, they don't evaluate the impact of PCL on individual patients. There is evidence, that PCL impacts on the quality-of-life and social functioning of affected individuals (48) and may contribute to carer distress (49). There is a growing effort to understand the individualized experiences of patients with PCL (14, 35, 50, 51).

### DISEASE MODELS

### Traditional and Revised Hypotheses

The traditional PCL model, proposed by Wilson in 1924 has, until recently been the most widely accepted one (25). Under physiological circumstances, it contends, emotional expression is influenced by both voluntary motor and involuntary emotional centers. These pathways descend onto the medullary "faciorespiratory" centers, which mediate the facial movements and breathing patterns necessary to convey emotion. This model predicts that disruption of the descending voluntary inputs to the brainstem, such as may occur in pseudobulbar palsy, results in disinhibition of involuntary emotional influence on expression. In support of this model, Wilson cited cases of dissociated emotional and voluntary facial expression, such as the observation that some patients with pseudobulbar palsy are unable to make voluntary facial movements but can be observed to smile, laugh or cry in response to emotional stimuli. This phenomenon is usually termed "voluntary facial palsy" (52). The contrary, "mimic palsy" or "emotional facial palsy" is sometimes observed, in which a patient with entirely normal voluntary facial movements, exhibits a complete lack of movement on one side of the face when reacting to emotional stimuli (53).

Parvizi et al. highlighted several limitations of the traditional model (54). Patients with bilateral voluntary facial paralysis, as in Wilson's example do not seem to be excessively prone to PBA, as would be predicted by the "disinhibition" model. Furthermore, patients with severe PBA symptoms are usually able to voluntarily mimic laughing or crying, indicating that involuntary expression can occur alongside intact voluntary control.

The revised model of PCL draws on the increasing appreciation of the role of the cerebellum in cognitive processes (55, 56). This model suggests that the cerebellum plays a key role in gating and modulating emotional output in response to contextual cues from cortical and limbic areas (54). Modulation is believed to be mediated through corticoponto-cerebellar pathways. In normal motor control, the cerebellum is known to modulate motor output in response to multiple sensory inputs; disruption of these inputs produces motor modulation deficits, including dysmetria. The involuntary emotional expression resulting from disruption of corticoponto-cerebellar emotional circuits has, analogously been termed "affective dysmetria" (57).

### Insights From Imaging and Neurophysiological Studies Across Neurological Diseases

Research over the past 30 years has led to a revision of the traditional model of PCL; studies have provided strong evidence to refute a simple causal relationship between PCL and corticobulbar tract dysfunction (58). Instead, a disruption within a widely dispersed network of emotional control appears to underlie the disorder. **Tables 1**, **2** provide an overview of studies highlighting key anatomical regions implicated in the pathogenesis of PCL. An MRI-based lesion study in poststroke pathological crying classified patients based on symptom severity (70). It found a positive pathoanatomical correlation between lesion size and location and pathological crying severity. Bilateral pontine infarcts were associated with greatest severity, while bilateral anterior hemispheric infarcts were associated with moderate PCL severity.

Among the likely neurotransmitters involved in the physiological expression of emotion, serotonin and glutamate have received particular attention (27, 71, 74). Evidence for dysfunction within these neurotransmitter pathways in PCL comes from the success of serotonergic (31, 38, 75), and anti-glutamatergic drugs (42) in PCL treatment. The selective serotonin re-uptake inhibitors (SSRIs) and tricyclic antidepressants (TCAs), are thought to work by increasing TABLE 1 | Neuroanatomical regions implicated in PCL circuitry based on clinical observations.

CPC, cortico-ponto-cerebellar pathways; CPM, Central Pontine Myelinolysis; Dx, Diagnosis; HC, Healthy control; MSA-C, Multiple system atrophy, cerebellar type; n, sample size; PBP,Pseudobulbar Palsy; PC, Pathological crying; PL, pathological laughing; PM, Post-mortem/Autopsy; SERT, Serotonin transporter; STN-DBS, Subthalamic nucleus, deep brain stimulation; UMN, Upper motor neuron; WM, White matter.

availability of serotonin at synapses in corticolimbic and cerebellar circuits (32).

A SPECT study found reduced brainstem serotonin receptor (SERT) densities in post-stroke PCL (71), providing further evidence of the role of serotonergic transmission in PCL. Serotonergic neurons from the raphe nuclei have widespread projections from the paramedian brainstem to cortical, subcortical, and cerebellar targets (76, 77). Also consistent with the serotonin hypothesis, lesions involving these nuclei and their projections are frequently associated with PCL (70). An MRI study of PBA in MS patients showed an association between symptoms and lesions in key regions: brainstem, bilateral inferior parietal, and medial frontal regions (23).

There is increasing recognition of the role of sensory deafferentation of the cerebellum in PCL (78). Evidence from neurophysiological studies in PCL suggests that the cerebellum may filter emotional output through a "gate-control" mechanism (79). At a cellular level, cerebellar Golgi cells may play a crucial role in gate-control. It has been demonstrated that Golgi cells, when activated from various peripheral inputs, show decreased firing rate, thereby reducing inhibition of granule cells (80). This finding, suggests, that rather than providing "gain-control," Golgi cells may act as a "context-specific gate" on transmission through the mossy fiber–granule cell pathway. A study of PCL in MSA-cerebellar type found a prevalence of 36% in this condition, in which clinically-significant cerebellar dysfunction is apparent (59).This prevalence estimate exceeds those of studies in idiopathic Parkinson's disease (5), suggesting that cerebellar pathology is linked to PCL in Parkinsonian disorders. Several neurological and neurosurgical case-reports have linked PCL to cerebellar pathology, especially in association with vermis pathology (3, 62, 67, 81). These studies support the cerebellar TABLE 2 | Anatomical conclusions of neuroimaging studies in PCL.

CPC, cortico-ponto-cerebellar pathways; CPM, Central Pontine Myelinolysis; Dx, Diagnosis; HC, Healthy control; MSA-C, Multiple system atrophy, cerebellar type; n, sample size; PBP, Pseudobulbar Palsy; PC, Pathological crying; PL, pathological laughing; PM, Post-mortem/Autopsy; SERT, Serotonin transporter; IC, Internal Capsule; STN-DBS, Subthalamic nucleus, deep brain stimulation; UMN, Upper motor neuron; WM, White matter.

gate-control theory of emotional expression, indicating that the disruption to cortico-ponto-cerebellar emotional circuitry may underlie this disorder.

## Insights From PCL Studies in Motor Neuron Disease

Corticobulbar tract dysfunction in ALS has been linked to cognitive impairment, and in particular to executive dysfunction (82). A study of PCL in ALS found an association between PCL and poor performance in executive tasks, implicating pre-frontal cortical areas in the disorder (20). Other studies, in contrast have found no such associations (8, 45). Systematic studies of social cognition in patients with PCL are lacking. Despite the conflicting findings, evidence from imaging and neurophysiology studies support the involvement of frontal cortical dysfunction in PCL (73, 79).

Advanced neuroimaging techniques enable the characterization of symptom-specific structural (83, 84), and functional (85, 86) alterations, providing insights into disease mechanisms (87, 88). Given the high prevalence of PCL in MND, it provides unique opportunities to explore PCL-specific network alterations (58). Floeter et al. used MRI diffusion methods to explore the white matter signature of PCL in ALS and PLS (10). Both ALS and PLS patients exhibited considerable white matter pathology in the corticospinal tracts and the corpus callosum. PCL-associated white matter changes were identified in frontotemporal regions, transverse pontine fibers and the middle cerebellar peduncles. A recent multimodal MRI study by Christidi et al. used the CNS-LS to divide a large group of ALS patients into PCL-positive and PCL-negative groups (73). The PCL-positive group showed significant gray and white matter changes compared with the PCL-negative group. The gray matter assessment found reduced volume of left orbitofrontal cortex, operculum, putamen, and of bilateral frontal poles. White matter analyses revealed diffusion abnormalities in the left cingulum, the posterior corona radiata and in the left middle and bilateral inferior cerebellar peduncles. The finding of cerebellar involvement in PCL in ALS again implicates cerebellar dysfunction in the pathophysiology of PCL, across a range of neurological diseases. While it is challenging to detect cerebellar signs clinically in the presence of pyramidal and lower motor neuron degeneration in ALS, imaging studies suggest that cerebellar degeneration is an important feature of ALS pathology, which is likely to contribute to PCL (89–91).

### THERAPEUTIC OPTIONS

### Antidepressant Medication in the Management of PCL

Divergent pharmacological strategies have been explored in the management of PCL. SSRIs and TCAs are the most frequently used off-label medications (75). Surprisingly, we did not identify any placebo-controlled trial of any antidepressants for PCL in ALS. In the absence of robust clinical trial data in ALS, evidence from other neurological conditions, mostcommonly stroke, is used to guide treatment. There have been positive results in small placebo-controlled trials of SSRIs including citalopram (92), fluoxetine, (31) sertraline (93) and of the TCA nortriptyline (38), in post-stroke PCL. Case-reports and uncontrolled trials reported symptom improvement with amitriptyline (94) and duloxetine (95) in ALS; memantine in AD (96); and mirtazapine in post-stoke PCL (97). A 2010 Cochrane review of treatments for "emotionalism" after stroke concluded that there is "suggestive but not definitive" evidence that antidepressants reduce frequency of symptoms, although it highlighted "several methodological deficiencies" in available studies (98).

### Management of PCL in Motor Neuron Disease

In 2010, dextromethorphan/quinidine (Dx/Q) became the first FDA-approved treatment for PCL, following more than a decade of research into the potential benefits of the commonly-used anti-tussive for this indication (99). Dextromethorphan acts as a non-competitive glutamate antagonist on NMDA-receptors and as an agonist on sigma receptors (100). When administered alone, it is rapidly metabolized by first-pass metabolism through the cytochrome P450-2D6 system. The addition of the CYP-2D6 inhibitor, quinidine, dramatically increases the bioavailability of dextromethorphan (101). In 2004, a randomized, doubleblinded study compared treatment with Dx/Q (30/30 mg twice daily) with dextromethorphan alone in ALS patients with PBA, defined by a CNS-LS score ≥13 (43). The combination not only reduced CNS-LS scores and episode frequency but also led to improvements in quality-of-life measures. Treatmentrelated side effects including nausea, dizziness, somnolence, and loose stools were relatively common however; about one quarter of patients withdrew from treatment, the majority within 1 week (102). A follow-up study in 2010 assessed whether a lower quinidine dose would reduce adverse effects relative to the earlier trial, while maintaining efficacy (42, 103) The study randomized ALS (n = 197) and MS (n = 129) patients with PBA to Dx/Q 30/10 mg BID, Dx/Q 20/10 mg BID or placebo BID. Both Dx/Q doses were found to reduce episode frequency, CNS-LS scores and to improve the likelihood of symptom remission compared to placebo. There was a lower discontinuation rate than in the earlier trial. It is interesting to note the considerable placebo response rate across efficacy endpoints in both trials. It must also be pointed-out that CYP450 2D6-poor metabolizers (104) were excluded from the efficacy and safety analyses raising questions about the requirement to screen for this phenotype prior to prescribing (105). Efficacy outcomes were also maintained in a 12-week open label extension study (106). A 52-week open-label study in 553 patients, including 199 patients with ALS, reported no serious drug-related adverse effects (75). However, clinicians must be cognizant of underlying cardiac conditions as quinidine can cause serious QT-interval prolongation (107).

# Limitations of Currently Available Treatments

While the emergence of the first FDA-approved drug for PCL is an important advancement, the effective treatment of PCL remains challenging. Unfortunately, there have been no head-to-head trials of Dx/Q and any commonly used antidepressant. This knowledge-gap is particularly problematic given the current cost of Dx/Q, which may be prohibitive. Not only is the price of the approved combination product higher than alternative options, it is dramatically more expensive than the combined cost of its individual components (108). Finally, although Dx/Q was granted approval by the European Medicines Agency (EMA) in 2013, it was subsequently withdrawn by the manufacturer in 2016, on commercial grounds. (109, 110) A 2017 Cochrane review of "symptomatic treatments" in ALS highlighted emotional lability, as a symptom for which there is a "significant gap" in studies regarding the effectiveness of available treatments (111). There is a pressing and unmet need for robust clinical trials of antidepressants in the management of PCL. Finally, there is evidence that ALS patients and carers, lack awareness of the association between PCL and their underlying neurological condition (112), highlighting the importance of enquiring about PCL symptoms in patients with high-risk conditions, such as ALS.

# CONCLUSIONS

Pathological crying and laughing is a shared symptom of many neurological conditions across infective, vascular, inflammatory, and neurodegenerative etiologies. Various terminologies have been used to encompass the heterogeneity of symptoms, which vary in severity, emotional congruity, frequency, and degree of control. After centuries of insightful observations, lesion studies and case reports, neuroimaging methods now provide long-awaited in-vivo insights into the specific pathophysiological mechanisms underlying the disorder. Patho-anatomical correlations indicate, that irrespective of the pathology (i.e., neurodegeneration, stroke, demyelination, TBI), the disorder occurs due to disruption in circuits involved in the initiation and modulation of emotional output. Key components of the network include sensori-motor cortical regions and their pontine and cerebellar connections. Further research is needed to elucidate the specific role of individual components within the network and their interactions. Effective symptomatic treatments are available; however, further studies are needed to establish individualized treatment strategies for patients experiencing impaired social or occupational functioning.

# AUTHOR CONTRIBUTIONS

The manuscript was drafted by EF. RC, SLHS, OH and PB contributed to the conceptualization, editing, and revision of this paper.

# ACKNOWLEDGMENTS

Peter Bede and the Computational Neuroimaging Group is supported by the Health Research Board (HRB–Ireland; HRB

### REFERENCES

EIA-2017-019), the Andrew Lydon scholarship, the Irish Institute of Clinical Neuroscience IICN–Novartis Ireland Research Grant, the Iris O'Brien Foundation, and the Research Motor Neuron (RMN-Ireland) Foundation.

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Finegan, Chipika, Li Hi Shing, Hardiman and Bede. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Ethical Principles in Patient-Centered Medical Care to Support Quality of Life in Amyotrophic Lateral Sclerosis

Dorothée Lulé<sup>1</sup> \*, Andrea Kübler <sup>2</sup> and Albert C. Ludolph<sup>1</sup>

<sup>1</sup> Department of Neurology, University of Ulm, Ulm, Germany, <sup>2</sup> Interventional Psychology, Psychology III, University of Würzburg, Würzburg, Germany

It is one of the primary goals of medical care to secure good quality of life (QoL) while prolonging survival. This is a major challenge in severe medical conditions with a prognosis such as amyotrophic lateral sclerosis (ALS). Further, the definition of QoL and the question whether survival in this severe condition is compatible with a good QoL is a matter of subjective and culture-specific debate. Some people without neurodegenerative conditions believe that physical decline is incompatible with satisfactory QoL. Current data provide extensive evidence that psychosocial adaptation in ALS is possible, indicated by a satisfactory QoL. Thus, there is no fatalistic link of loss of QoL when physical health declines. There are intrinsic and extrinsic factors that have been shown to successfully facilitate and secure QoL in ALS which will be reviewed in the following article following the four ethical principles (1) Beneficence, (2) Non-maleficence, (3) Autonomy and (4) Justice, which are regarded as key elements of patient centered medical care according to Beauchamp and Childress. This is a JPND-funded work to summarize findings of the project NEEDSinALS (www.NEEDSinALS.com) which highlights subjective perspectives and preferences in medical decision making in ALS.

Keywords: ethics, quality of life (QoL), care, amyotrophic lateral sclerosis (ALS), well-being, depression, coping, psychosocial adaptation

# QUALITY OF LIFE IN ALS

Amyotrophic lateral sclerosis (ALS) is among the most devastating neurological conditions: patients lose the ability to speak, to walk and eventually to breathe. On average, patients die within 3 years after symptom onset. If life-sustaining measures such as invasive ventilation are taken, patients may terminate in a locked-in state with a clear mind in a paralyzed body. There is no cure for ALS and care focuses on maintaining functional ability and providing palliative and symptomatic interventions to relieve the burden of symptoms (1). The communication of the diagnosis is a major stressful event for patients, families and caretakers and thus most challenging with regard to medical counseling (2).

There are different ways of how patients cope with these major changes. Quality of life (QoL) is one possible measure of good psychosocial adaptation to disability such as ALS, similarly to depression (3). There have been contradictory reports whether QoL is lost in the course of physical decline (4–6). This discrepancy is partly attributed to selection of patient subgroups (e.g., shortly after diagnosis vs. long-term survivors) and the use of different QoL definitions. QoL is the general well-being of a person and includes physical (individuals' perception of their physical state), psychological (individuals' perception of their cognitive and affective state) and social

Edited by:

Peter Bede, Trinity College Dublin, Ireland

### Reviewed by:

Anne Hogden, University of Tasmania, Australia Foteini Christidi, National and Kapodistrian University of Athens Medical School, Greece

### \*Correspondence:

Dorothée Lulé<EMAIL_ADDRESS>
### Specialty section:

This article was submitted to Neurodegeneration, a section of the journal Frontiers in Neurology

Received: 11 January 2019 Accepted: 26 February 2019 Published: 22 March 2019

### Citation:

Lulé D, Kübler A and Ludolph AC (2019) Ethical Principles in Patient-Centered Medical Care to Support Quality of Life in Amyotrophic Lateral Sclerosis. Front. Neurol. 10:259. doi: 10.3389/fneur.2019.00259

**249**

dimensions (individuals' perception of the interpersonal relationships and social roles in their life). It is therefore not simply a state of physical integrity (7). QoL is increasingly used to supplement objective clinical or biological measures to evaluate health care provision and interventions in research and clinical trials (8).

There is a debate which QoL measure is truly patient centered. Measures are either based on hedonic concepts focusing on subjective factors and emotional evaluation or eudaimonic concepts with more objective factors of QoL such as physical health or economic status (9). As physical health declines in ALS and mobility becomes heavily restricted, these QoL measures provide evidence for low QoL in ALS simply by the nature of the underlying concept. These clinimetric endpoints are increasingly considered overly reductionistic (10) as they include aspects, which are no longer relevant or are out of range of an immobile patient, e.g., physical activity (11); thus, patients often prefer more subjective scales of QoL as these better capture their emotional state of well-being (**Table 1**). They might as well be regarded as possible outcome measures in clinical trials to determine the subjective benefit of a treatment for a patient. Observations concerning hedonic QoL are often counterintuitive: simultaneous deterioration of physical integrity and well-being does not necessarily occur (19). Accordingly, ALS patients may experience a surprisingly high subjective QoL and an only moderately increased affective state as compared to healthy subjects (6, 20–27) which can be maintained throughout the course of ALS (27–29). This may even be true in the final state of complete immobility, the locked-in state [LIS; (30, 31)].

The lack of association of severity of illness and subjective QoL has been shown for several diseases and is referred to as the "well-being-paradox" (32). Prerequisite for this paradox is a process of psychosocial adaptation to the altered circumstances of severe physical function loss. According to the theory of homeostasis in quality of life, everybody has his/her individual level of well-being which he/she aims to reach which is usually in the range of 70–80% of the maximum QoL (33). Provided that sufficient time (29) and intrinsic (e.g., successful coping) and extrinsic resources (e.g., strong family support) are given, patients may show a process of ongoing change and adaptation of their expectations to the actual circumstances [TOTE model; (34)]. The capability of adaptation is not simply a matter of disease state or general personality traits (21). It can be successfully supported by medical teams through patient centered medical care. The different intrinsic and extrinsic factors in medical care to facilitate QoL in ALS and the individualistic perspective in medical decision making have been evaluated within the JPND-funded project NEEDSinALS (www.NEEDSinALS.com). These factors may be subsumed under the four ethical principles of good medical care according to Beauchamp and Childress (35), namely beneficence, non-maleficence, autonomy and justice (**Figure 1**).

### Beneficence

This principle requires that everything should be done in the best interest of the patient. Therapeutic interventions are usually introduced by the physician and their interdisciplinary teams to facilitate QoL in ALS. No cure is available yet, but different therapeutic interventions e.g., non-invasive ventilation (NIV) may be means also to prolong survival (36). Previous studies have provided extensive evidence that ALS patients with NIV have an increased QoL (20, 37, 38). Permanent respiratory insufficiency may lead to disturbed sleep, fatigue and reduced physical fitness, all these symptoms may be relieved by ventilation (39). Thus, ventilation may positively impact QoL and patients with ventilation may show even higher QoL than those without (20). Further, nutritional support is a major element of beneficence in ALS as a loss in BMI is a negative prognostic factor (40). Unfortunately, fear of choking during meals is widely prevalent in patients with bulbar symptoms (41), so many patients fear to eat at all. Thus, introduction of a PEG may be a highly useful approach to improve QoL as it allows for weight control while relieving the patient from the pressure to eat. As patients may nevertheless be able to have oral food intake, the pleasure and sensuality of eating can be maintained which additionally supports QoL. However, in some patients the positive effect of PEG insertion might be outbalanced by "particularly strong feelings of loss of control" (41), highlighting the subjective perspective on patient centered care (8). Other therapeutic interventions may as well-facilitate QoL such as application of botox to stop the debilitating syndrome of "drooling" (sialorrhoea). Also, therapies such as physiotherapy, occupational therapy (ergotherapy) and speech therapy (logopedics) may relieve physical symptoms of pain, muscle tension and stiffness which all finally may help to improve QoL (42).

Apart from therapeutics, there is one major extrinsic factor which may substantially improve QoL which is social support (23). Family is the most frequently named aspect of individual QoL in ALS (26, 29, 43). And, as satisfaction with family was often good among patients, the patient's QoL is also often good (44). For healthy subjects, there are other factors which are important for well-being (finances, career) whereas most ALS show a response shift toward social support in the course (20). Thus, inclusion of family members in clinical counseling and supporting the patient in refocusing on social resources may facilitate QoL.

Apart from these extrinsic factors, QoL in serious illness is highly depending on intrinsic factors, such as resilience which is a general characteristics addressing the capacity to recover quickly from difficulties (45). In this context, there has been evidence in ALS that appraisal of coping potential and mental attitudes may be crucial to adapt (23). A re-set of preferences referred to as response shift (46) may support these inner processes with the ability to see what is still there and what is untouched by the disease [e.g., spiritual well-being; (20)]. Further, reframing, the ability to see the same situation from a different perspective [e.g., instead of looking at what you lose, you pay attention to what is spared such as your emotions, feelings and desires; (20)] can be highly supportive for psychosocial adaptation. Finally, many years of research about adapting to and living with chronic diseases suggest that mindfulness in the sense of accepting the circumstances which cannot be changed without judgement and focus on the present (47) may reduce the negative psychological impact of the illness (48, 49).

TABLE 1 | Examples of most widely used measures of subjective, patient-centered QoL.

Patients can be encouraged to use these inner resources mentioned above. Psychotherapeutic interventions may help to improve the QoL of patients and may even prolong survival as the psychoemotional state of the patient has impact on QoL (50) and survival time (49). The beneficence of the above mentioned intrinsic factors may as well be addressed in clinical routine by physicians and medical staff to encourage patients to give more room in life for any beneficial intrinsic process.

Beneficence requires the knowledge of the patient's wishes as peer evaluations might not meet the patient's actual needs. There is evidence for discrepancy between patient's well-being and the perspective on patient's well-being of people without neurodegenerative conditions (27, 29). Peers judgement of patient's well-being is primarily based on personal opinion when they anticipate a low QoL in severely disabled patients. This is true for people without experience in ALS and is even true for caregivers and physicians if they lack experience with ALS (51). Healthy subjects may be blind toward the patient's process of ongoing change and adaptation and instead they may conclude from their personal perspective. The more experienced healthcare professionals are, the more they know about the capacity to adjust and are thus abled to correctly anticipate patient's quality of life, affective state, and wish for hastened death (51).

Thus, the knowledge of and believe in beneficence in ALS is a matter of the physician's experience. As many patients gradually adjust to their situation and also possibly change their therapeutic preferences in the course of the disease (52), beneficence from the patient's perspective is a dynamic construct which needs to be recognized and may be supported by caretakers and medical care teams.

### Non-maleficence

Primum non-nocere, refrain from harm is the other side of the coin of beneficence and thus, similar aspects concern maleficence than beneficence. Non-maleficence needs to be considered the moment the diagnosis is communicated. "Breaking the news" is a highly delicate balance between patient's need to be informed which requires veracity and fidelity on the one hand and the right for denial on the other hand which can be a helpful strategy at least shortly after diagnosis (23). Maleficence in the sense of the emotional burden of diagnosis can be reduced by using a thorough approach for breaking the news as it may attenuate negative impact on QoL (53). But also providing sufficient information can prevent maleficence: patients with sudden respiratory insufficiency in an emergency situation who have been informed on all aspects of respiratory support may feel more competent to take the right decision (41).

Advance directives and living wills are crucial to prevent maleficence, e.g., insertion of a tracheostomy in an emergency if the patient does not want to [possibly because he/she is afraid of the burden for others; (52)]. Many therapeutic options secure QoL (e.g., ventilation) but most patients are unable to anticipate this shortly after diagnosis. During the course of ALS, some might dismiss the idea of maleficence of invasive ventilation and might realize the beneficial effect of this therapeutic treatment (29). Therefore, dynamic adjustment to living wills is a key aspect to prevent maleficence.

Preferences regarding therapeutic measures are highly determined by patient's personal values, religious beliefs and cultural background (54).

Cultural differences exist: in Japan, invasive ventilation is more regarded as routine therapeutic treatment than in other countries [33% in Japan; (55)]. In some countries, life prolongation might be regarded as maleficence and more life-shortening treatments are suggested (56). Thus, social context may define what is beneficence and maleficence in the context of cultural norms (54, 57).

### Patient's Autonomy

Patient's sense of autonomy is a key issue of quality of life and goes beyond being physically autonomous to perform an action. Autonomy also encompasses the sense of capability to take decisions and the feeling of being an author of one's own action which is a key feature of self-efficacy and thus for QoL (58). Taking decisions also sustains the feeling of social embeddedness disregarding physical disability, e.g., the patient can be included in family decisions and may participate in daily routine if possible (59). This allows the patient to be an active part of daily routine: to participate in decision making, to be asked questions, to express concerns, address fears and anxieties, express wishes, values, desires, and hopes. It is noteworthy that possible minor cognitive deficits in some patients do not interfere with the competency to decide and participate (60).

It is especially challenging to secure patient's autonomy in LIS as there is lack of direct means to communicate in this state. Assisted communication (20, 29) becomes important for individual QoL in the course of ALS, but is not mentioned by patients in early stages of the disease (20). Many patients use letter board for communication which requires considerable effort from a second person to record which item the patient selected from the board. Technical devices may allow for communication but these are time consuming and strenuous to use and also additional assistance is required (59). Thus, knowledge on patient's wishes, desires and thoughts in advanced stages is sparse and there is substantial lack of understanding which factors may impact the dynamics of QoL and affective state in the course of ALS (30). Communication via eye-gaze control is possible, including standardized interviews (61, 62) but the latter are rarely performed. Brain Computer Interfaces (BCI) are promising technologies for communication and interaction (63, 64) but in a subgroup of patients only (65). Other means of BCI-use such as unrestricted access to web browsers of which some are adaptable to home based BCI systems (66) secure new degrees of freedom in severe paralysis (67–70). Some patients already use these techniques in their home environment for communication and painting (71–76) and first evidence support the notion that these techniques positively impact QoL (71, 77). In the future, with major advances in communication technology well-being in ALS might possibly be facilitated. BCIs might also be indirectly used in evaluation and recognition of well-being and emotional state in highly advanced patients (78) such as the amplitude of the N400, a negative deflection of the EEG curve following a meaningful event (79), which was higher in patients with high QoL compared to those with a poor. Thus, the N400 may serve as an objective physiological indicator of individual QoL in non-responsive ALS patients (80). Overall, there is still a long way to go until BCI will be a standard tool for home care for a majority of ALS patients (63, 81). But for patient-centered care, compensation for progressive loss of

verbal speech is mandatory to secure patient's autonomy and QoL (52).

### Justice

This ethical principle of care requires that all patients are treated in an equal way without prejudice or social discrimination.. In the sense of justice, patients in similar situations should have access to the same care options. Palliative care intervention improves quality of life in patients and caregivers (82) and medical care may facilitate this positive dynamics by offering this care to every patient. ALS patients expect dignified care (82) but instead, patients are often dissatisfied with health care services (83). Every patient needs to be treated differently according to the actual preferences and needs (54). There is no justice in defining every person by the diagnosis with a nihilistic view of the disease which has to be prevented under all circumstances (48). Instead, to grant justice every patient has to be regarded as an individual with specific needs and the right to be treated the same according to his/her preferences, disregarding mental, societal or financial status.

Further, providing sufficient information according to the patient's needs as outlined above is also a matter of justice. Thus, granting the patient the right for information is similarly a matter of justice as granting the right for not-knowing. In this sense, it is a matter of justice to grant patient's will even if it interferes with the physician's personal and professional opinion.

Finally, justice in medical care is secured in many countries as most healthcare systems secure this kind of justice by providing coverage of (most) costs. Despite that most medical systems are based on a solidary idea allowing for justice, the impact of the disease may vary between patients thus justice in clinical care is not easy to accomplish. There are basic settings which significantly hamper justice in care provision which cannot be changed by the medical team, e.g., there is variance of the paid costs by the insurance companies. Further, in some instances, only basic technical equipment is provided which possibly don't meet the patient's actual needs. Thus, patients have to cover the extra charges for the devices which fully meet their needs. And finally, there are personal settings (e.g., living and working conditions) which may heavily impact patient's life with ALS and which interfere with the principle of justice (**Figure 2**).

# CONSEQUENCES AND FUTURE DIRECTIONS

There is evidence that considerate medical care within multidisciplinary teams (84) helps patients to find their own way of coping with the disease to gain or maintain a satisfactory QoL (48). Living with a fatal disease creates a crisis loaded environment and adapting to the disease is a psychological process rendering mandatory a strong support from these specialists' teams (41). Following the ethical principles of medical care as outlined in this text allows for a holistic support of the patient to secure QoL.

### DATA AVAILABILITY

The raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher.

## AUTHOR CONTRIBUTIONS

DL, AK, and AL have collected data underlying this review and discussion of data. Text writing was done by DL, thorough revision of the manuscript was performed by all authors.

### REFERENCES

### ACKNOWLEDGMENTS

We thank all the patients and families for supporting our research. This is an EU Joint Programme–Neurodegenerative Disease Research (JPND) project. The project is supported through the following organizations under the aegis of JPND www.jpnd.eu, e.g., Germany, Bundesministerium für Bildung und Forschung (BMBF, FKZ), Sweden, Vetenskapr å det Sverige, and Poland, Narodowe Centrum Badan i Rozwoju (NCBR). This ´ work was supported by the Deutsche Forschungsgemeinschaft and the Bundesministerium für Bildung und Forschung (BMBF #01GM1103A, MND-Net).

quality of life in people with ALS. Amyotroph Lateral Scler. (2008) 9:99–107. doi: 10.1080/17482960701875805

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Lulé, Kübler and Ludolph. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Lipid Biomarkers for Amyotrophic Lateral Sclerosis

Jose-Luis González De Aguilar 1,2 \*

<sup>1</sup> Université de Strasbourg, UMR\_S1118, Strasbourg, France, <sup>2</sup> INSERM, U1118, Mécanismes Centraux et Périphériques de la Neurodégénerescence, Strasbourg, France

Amyotrophic lateral sclerosis (ALS) is a fatal degenerative disease primarily characterized by the selective loss of upper and lower motor neurons. To date, there is still an unmet need for robust and practical biomarkers that could estimate the risk of the disease and its progression. Based on metabolic modifications observed at the level of the whole body, different classes of lipids have been proposed as potential biomarkers. This review summarizes investigations carried out over the last decade that focused on changes in three major lipid species, namely cholesterol, triglycerides and fatty acids. Despite some contradictory findings, it is becoming increasingly accepted that dyslipidemia, and related aberrant energy homeostasis, must be considered as essential components of the pathological process. Therefore, it is tempting to envisage dietary interventions as a means to counterbalance the metabolic disturbances and ameliorate the patient's quality of life.

### Edited by:

Pierre-francois Pradat, Hôpitaux Universitaires Pitié Salpêtrière, France

### Reviewed by:

Débora Lanznaster, INSERM U1253Imagerie et Cerveau (iBrain), France Philippe Couratier, Centre Hospitalier Universitaire de Limoges, France

> \*Correspondence: Jose-Luis González De Aguilar

#<EMAIL_ADDRESS>
### Specialty section:

This article was submitted to Neurodegeneration, a section of the journal Frontiers in Neurology

Received: 03 November 2018 Accepted: 05 March 2019 Published: 04 April 2019

### Citation:

González De Aguilar J-L (2019) Lipid Biomarkers for Amyotrophic Lateral Sclerosis. Front. Neurol. 10:284. doi: 10.3389/fneur.2019.00284 Keywords: amyotrophic lateral sclerosis, biomarker, cholesterol, fatty acid, triglyceride

# ALS AND THE NEED FOR BIOMARKERS

Amyotrophic lateral sclerosis (ALS) is a degenerative disease of upper and lower motor neurons mainly characterized by progressive muscle wasting, fasciculations, dysarthria, dysphagia, altered reflexes, and spasticity. It affects about 2 per 100,000 people per year, and usually appears at 40–70 years of age. A significant proportion of cases also presents cognitive or behavioral abnormalities typical of frontotemporal dementia (FTD). The etiology of ALS still remains elusive. About 90% of cases are considered as sporadic. The remaining 10% are inherited mostly in an autosomal dominant manner. Most familial cases can be explained by mutations in four major genes, including C9ORF72, SOD1, FUS, and TARDBP. Based on this genetic diversity, multiple pathogenic mechanisms have been implicated in triggering motor neuron degeneration, adding considerable complexity to the understanding of the disease (1).

From a clinical point of view, ALS is easily recognized in its full-blown presentation. However, the diagnostic process may be challenging at very early stages. The diagnosis is based on clinical examination, electrophysiological findings, medical history, and exclusion of confounding disorders. In practice, a correct diagnosis may take as long as 1 year (2). Moreover, disease progression is very heterogeneous. Death may occur between 1 and 5 years after diagnosis, but 20% of patients live longer than 5 years, and 10% survive for more than 10 years (3). Promising biomarkers of diagnosis and prognosis have been proposed based on advanced neurophysiological and neuroimaging techniques. However, many of these practices still lack validation and standardization between clinical centers, and they have been applied only to small cohorts of patients [(4–6), and references therein].

As far as molecular biomarkers are concerned, a great number of molecules have been isolated from patient material, including cerebrospinal fluid, blood and tissues, that recognized and/or monitored ALS with more or less accuracy [(7, 8), and references therein]. To date, changes in the amounts of neurofilament proteins found in cerebrospinal fluid and blood have been postulated as the most promising candidates [(9), and references therein]. However, these and other proposed molecular markers have not reached routine clinical application. Therefore, there is still an incontestable lack of robust and practical biomarkers that could facilitate an earlier diagnosis and improve the prognosis of ALS.

### ALTERED ENERGY HOMEOSTASIS AND LIPID METABOLISM IN ALS

Amyotrophic lateral sclerosis was classically attributed to an intrinsic defect of upper and lower motor neurons. Now it is generally accepted that non-neuronal cells surrounding motor neurons, additional neuronal cell types, as well as other cells outside the nervous system participate actively in the pathological process [(10–12), and references therein]. In particular, compelling evidence has emerged over the last decade showing a characteristic imbalance between energy intake and consumption, which is associated with metabolic alterations at the level of the whole body of yet unexplained etiology [(13), and references therein]. Seminal studies revealed that many ALS patients show an increase in energy expenditure, or hypermetabolism, which could account, at least in part, for the decline of their nutritional status (14, 15). It was also recently reported that hypermetabolic patients have a worse prognosis than normometabolic ones (16), which could be related to a detrimental weight loss. In fact, patients that lost more than 5% of body mass at the time of diagnosis had an increased risk of death (17). In addition, a lower body mass index appeared to precede the symptomatic stage of the disease (18). Overall, these studies strongly support that the energy imbalance in ALS could contribute to the rapid deterioration of the patients.

The origin of the hypermetabolism in ALS is currently unknown, although recent studies have pointed to the altered function of hypothalamic neurons involved in the regulation of food intake and energy homeostasis (19, 20). From a therapeutic point of view, pioneering preclinical studies conducted on an ALS mouse model, which carries a mutation in the Sod1 gene, provided part of the answer to this question. An increase in energy consumption occurs in these mice well-before the onset of the first motor symptoms. This is accompanied by a reduction of adiposity and lower levels of circulating leptin. Most importantly, these studies revealed that sustaining the hypermetabolic rate of ALS mice with a highly-energetic highfat diet partially protected motor neurons and extended lifespan (21). Likewise, a higher premorbid intake of high-fat food was observed in ALS patients (18). Moreover, a moderate increase in fat mass over the course of the disease was associated with a decreased risk of death, and increasing circulating levels of leptin were positively associated with longer survival (17, 22). Although there is no conclusive evidence of a mechanistic link between the hypermetabolism present in ALS and altered levels of lipids, altogether, these studies suggest that the utilization of lipids as energy substrates could offer benefit, by counteracting an increased metabolic rate and compensating the associated weight loss. In this respect, several pilot studies reported positive effects of highly caloric fat supplements on ALS patients (23, 24). It was also shown that the administration of acetyl-Lcarnitine, which supports the transport of fatty acids into mitochondria for being used as energy substrates, retarded the worsening of the patients (25). Recent research has made efforts to identify specific changes in lipid metabolism that could provide clues for future nutritional interventions, as well as serve as robust biomarkers for the disease. This review covers some of the most significant findings published during the last decade.

# APOLIPOPROTEIN E AND THE RISK OF ALS

Apolipoprotein E (APOE) is a constituent of lipoprotein particles primarily involved in the transport of triglycerides and their clearance from the bloodstream. It is mainly synthesized in the liver but it is also produced by astrocytes in the brain, where APOE is the most important cholesterol carrier. The human APOE gene exists as three major alleles called ε2, ε3, and ε4. The identification of APOE ε4 as a risk factor for Alzheimer's disease represented a major breakthrough in the field [(26), and references therein]. On the contrary, most studies on ALS did not observe any association of APOE ε4 with an increased risk (27–30), excepted some recent findings (31). Additional reports showed complex interactions between particular APOE alleles and other genetic or physiopathological variables. Penco and collaborators identified a combination of seven genetic variants, inluding one affecting APOE, that distinguished between ALS patients and control subjects (32). It was also found that individuals who had suffered from head trauma in the adulthood were more prone to have ALS, and this association was stronger in the presence of APOE ε4 (33). In contrast, the frequency of APOE ε2, which is a priori neuroprotective, was higher in ALS patients that had practiced sport regularly (30). APOE ε2 also increased the risk of developing FTD in a cohort of patients with ALS (34). It must be noted, however, that the implication of APOE in the incidence of this form of dementia is rather controversial. The increase in the probability of having FTD was associated with APOE ε2 in some cases, and with APOE ε4 in other cases [(35), and references therein].

The influence of APOE on the course of ALS has also been contradictory. Initial reports revealed that APOE ε4 was associated with earlier age at onset but not with disease duration (27). However, follow-up studies failed to show any relationship between APOE ε4 and age at onset or rate of progression, although this allele was more frequent in men with bulbar-onset ALS (28). Parallel investigations did not find any association between the APOE genotype and age of onset, site of onset, rate of progression, cognitive impairment or survival (36). Overall, the implication of APOE in the incidence and progression of ALS is therefore not clearly established.

### THE INTRIGUING CASE OF CHOLESTEROL

Cholesterol is an essential lipid molecule, which is transported through the bloodstream by several types of lipoprotein particles. In clinical practice, increased levels of total cholesterol or lowdensity lipoprotein cholesterol (LDL-c), in combination with decreased levels of high-density lipoprotein cholesterol (HDL-c), are indicative of a higher risk of atherosclerotic cardiovascular disease. In the case of ALS, hypercholesterolemia, as detected prior to the onset of motor symptoms, was initially associated with a lower risk (37). However, follow-up studies contradicted these findings. An increase in the premorbid intake of cholesterol was associated with a higher incidence of the disease, as shown after examination of dietary habits obtained from food frequency questionnaires (18). In addition, individuals with increased levels of LDL-c and a higher LDL-c/HDL-c ratio were more prone to develop ALS later (38). Finally, the analysis of GWAS databases revealed that particular alleles predisposing to elevated levels of LDL-c and total cholesterol appeared associated with an increased risk (39).

In many studies, the proportion of hypercholesterolemia individuals or the average contents of total cholesterol and LDL-c were shown to be higher in the ALS population and, in some cases, this increase was noticeable at the time of diagnosis (40– 44). In agreement with these findings, a detailed analysis of circulating lipoprotein particles also showed increased levels of LDL-1, which is a LDL subfraction very enriched in cholesterol (44). Other reports, however, did not find clear-cut differences (45–48), or even revealed opposite results (49).

From a prognostic point of view, decreased levels of total cholesterol or LDL-c and a lower LDL-c/HDL-c ratio were associated with a severe respiratory impairment (42, 45). Contrasting with these findings, Delaye and collaborators did not observe any association between several cholesterol parameters and disease progression (44). Yet, most authors agree that hypercholesterolemia, present as elevated levels of total cholesterol and LDL-c or a higher LDL-c/HDL-c ratio, associates with longer survival. This association, however, did not reach significance after adjusting for potential confounding demographic and clinical factors (37, 40, 43, 49–51).

In addition to the biomarker potential of cholesterol per se, a few studies have focused on the implication of oxysterols, which are oxidized derivatives mainly involved in maintaining cholesterol homeostasis. Levels of several oxysterol metabolites, including 27-hydroxycholesterol, 24-hydroxycholesterol esters, and 3β,7α-dihydroxycholest-5-en-26-oic acid and other related compounds, were shown to be lower in ALS patients. These changes were detected in blood or cerebrospinal fluid, or both, and they were attributed to a deficit in the metabolism of excess cholesterol, which would result in subsequent toxicity in the brain (46, 52, 53). On the other hand, additional studies reported increased levels of 25-hydroxycholesterol in cerebrospinal fluid and serum of ALS patients. The accumulation of this toxic oxysterol derivative was associated, at least in serum, with a higher rate of disease progression (54).

### THE ENERGIZING TRIGLYCERIDES IN ALS

Triglycerides are a primary source of energy for the body but, when accumulated in an excessive manner, they represent an important risk factor for cardiovascular disease. Triglyceride contents should be expected to change in ALS patients according to their characteristic high rate of energy expenditure. In this respect, the proportion of hypertriglyceridemia individuals was more important among ALS patients than in the normal population (55). Hypertriglyceridemia was also found in ALS women (42), and higher triglyceride levels were associated with a better functional status (48). Other reports, however, failed to reproduce these findings (40, 47, 48). Moreover, Blasco and collaborators identified a lipidomic signature in the cerebrospinal fluid of ALS patients, in which certain triglyceride species were found reduced at levels associated with a better prognosis (56). Finally, as in the case of cholesterol, hypertriglyceridemia was associated with longer survival, but this association appeared to have no effect after adjusting for confounding factors (47, 55).

### THE ENTRANCE OF FATTY ACIDS ON STAGE

Fatty acids are lipid molecules key for sustaining the structural integrity of cell membranes, providing energy and serving in signaling pathways. They can be mainly transported through the bloodstream attached to a glycerol molecule (that is, in the form of triglycerides) or as non-esterified free fatty acids. The studies relating to the implication of fatty acids as biomarkers for ALS are scarce. Based on food frequency questionnaires, Fitzgerald and collaborators showed that a higher intake of ω3 polyunsaturated fatty acids, which are considered as neuroprotective factors, were associated with a reduced risk of ALS (57). Similar studies did not find the same association but rather reported a higher premorbid intake of trans- and saturated fatty acids associated with an increased risk (18). On average, the proportion of polyunsaturated fatty acids in the lipid fraction of clotted blood was decreased in ALS patients while that of monounsaturated fatty acids was concomitantly increased (58). Polyunsaturated fatty acids were also lower in the free fatty acid fraction of plasma (59). Finally, a higher palmitoleic/palmitic fatty acid ratio, indicative of increased adiposity, correlated with a better functional status, and was associated with longer survival (58).

### CONCLUSION

Over the last decade, many lipid molecules have been proposed as promising biomarkers for ALS, but none of them has been translated into effective tools in clinical practice. There are several issues of concern that still need to be addressed. On the one hand, the etiology of ALS is multifactorial, and it is likely that the pathological process in subpopulations of patients, with different genetic and environmental backgrounds, is not the same. In the future, the use of cohorts of well-defined patients should improve statistical robustness. It would also be interesting to compare between patients with ALS and other patients suffering from mimic conditions. On the other hand, lipid changes at the level of the whole body can be affected by a myriad of factors, including genetic, nutritional, physical and pathological factors, which can introduce bias on the results. It is also noteworthy to mention that for those studies that used food frequency questionnaires to estimate food preferences and evaluate eating behavior, they depend, at least in part, on their interpretation probing the patient's perception of food intake, hence lacking sensitivity and objectiveness. Therefore, protocols and measurements need to be standardized between study centers.

Despite some conflicting findings, most studies presented in this review show important alterations of the circulating contents of cholesterol (and related lipoprotein particles), triglycerides and fatty acids, which occur prior to and over the course of ALS. These changes seem to reflect a metabolic environment, which would be appropriate to meet the high energy demands imposed by the increased metabolic rate present in the disease. The understanding of the mechanisms underlying this "lowgrade dyslipidemia" is still insufficient but, from a clinical point of view, it leaves open the possibility for therapeutic nutritional intervention. In this respect, recent studies that analyzed the eating behavior of ALS patients revealed marked modifications in their food preferences. In particular, an increase in the

### REFERENCES

intake of saturated fat and meat protein was associated with longer survival (60–62). Moreover, two clinical trials have been initiated, which aim at retarding disease progression by using high-caloric food supplements. The first trial (NCT02306590) is a randomized, parallel-group, double-blind study that compares between placebo and a treatment consisting of a high caloric fatty diet, which is equivalent to an additional intake of 45 g fat per day. The primary objective of this study is to evaluate the impact on survival. The second trial is a randomized, parallel-group, open label study that will determine the effects of a high-protein, high-energy supplement on the functional status of newly diagnosed ALS patients (NCT02152449). The results of these trials as well as the ongoing research on lipid biomarkers and on the understanding of their implication in ALS will certainly pave the way for developing new therapeutic tools.

### AUTHOR CONTRIBUTIONS

The author confirms being the sole contributor of this work and has approved it for publication.

# FUNDING

J-LG received funds from Association pour la Recherche sur la Sclérose Latérale Amyotrophique et autres Maladies du Motoneurone (ARSLA).

onset in amyotrophic lateral sclerosis. J Neurol Neurosurg Psychiatr. (2017) 88:1033–41. doi: 10.1136/jnnp-2017-315795

of amyotrophic lateral sclerosis: a more than 20-year follow-up of the Swedish AMORIS cohort. Ann Neurol. (2017) 81:718–28. doi: 10.1002/ana.24936

**Conflict of Interest Statement:** The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 González De Aguilar. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Spinal Cord Imaging in Amyotrophic Lateral Sclerosis: Historical Concepts—Novel Techniques

Mohamed Mounir El Mendili 1,2, Giorgia Querin2,3, Peter Bede2,3,4† and Pierre-François Pradat 2,3 \* †

<sup>1</sup> Department of Neurology, Icahn School of Medicine at Mount Sinai, New York, NY, United States, <sup>2</sup> Biomedical Imaging Laboratory (LIB), Sorbonne University, CNRS, INSERM, Paris, France, <sup>3</sup> Department of Neurology, Pitié-Salpêtrière University Hospital (APHP), Paris, France, <sup>4</sup> Computational Neuroimaging Group, Trinity College Dublin, Dublin, Ireland

### Edited by:

Justin John Yerbury, University of Wollongong, Australia

### Reviewed by:

Virginie Callot, Centre National de la Recherche Scientifique (CNRS), France David Devos, Université de Lille, France

### \*Correspondence:

Pierre-François Pradat<EMAIL_ADDRESS>
†These authors have contributed equally as co-senior authors

### Specialty section:

This article was submitted to Neurodegeneration, a section of the journal Frontiers in Neurology

Received: 30 December 2018 Accepted: 21 March 2019 Published: 12 April 2019

### Citation:

El Mendili MM, Querin G, Bede P and Pradat P-F (2019) Spinal Cord Imaging in Amyotrophic Lateral Sclerosis: Historical Concepts—Novel Techniques. Front. Neurol. 10:350. doi: 10.3389/fneur.2019.00350 Amyotrophic lateral sclerosis (ALS) is the most common adult onset motor neuron disease with no effective disease modifying therapies at present. Spinal cord degeneration is a hallmark feature of ALS, highlighted in the earliest descriptions of the disease by Lockhart Clarke and Jean-Martin Charcot. The anterior horns and corticospinal tracts are invariably affected in ALS, but up to recently it has been notoriously challenging to detect and characterize spinal pathology in vivo. With recent technological advances, spinal imaging now offers unique opportunities to appraise lower motor neuron degeneration, sensory involvement, metabolic alterations, and interneuron pathology in ALS. Quantitative spinal imaging in ALS has now been used in cross-sectional and longitudinal study designs, applied to presymptomatic mutation carriers, and utilized in machine learning applications. Despite its enormous clinical and academic potential, a number of physiological, technological, and methodological challenges limit the routine use of computational spinal imaging in ALS. In this review, we provide a comprehensive overview of emerging spinal cord imaging methods and discuss their advantages, drawbacks, and biomarker potential in clinical applications, clinical trial settings, monitoring, and prognostic roles.

Keywords: ALS (Amyotrophic lateral sclerosis), MRI—magnetic resonance imaging, MND, spinal cord, neuroimaging

# INTRODUCTION

Amyotrophic lateral sclerosis (ALS) is a relentlessly progressive neurodegenerative disorder. Anterior horn pathology and corticospinal tract degeneration has been identified as a core feature of ALS since the earliest descriptions of the condition (1, 2). Despite repeated attempts to detect and characterize spinal cord pathology in vivo (3), technological constraints have traditionally precluded reliable quantitative spinal imaging in ALS. Due to the plethora of methodological challenges, such as the small cross-sectional area of the human spinal cord, respiratory, and cardiac movement effects, the overwhelming majority of imaging studies have focused on cerebral alterations in ALS (4).

**263**

The diagnosis of ALS is primarily clinical and requires the careful exclusion of ALS-mimics (5). Given the heterogeneity of clinical presentations and the prevalence of atypical phenotypes, diagnostic delay in ALS is not uncommon, and the average period between symptom onset and definite diagnosis is ∼12 months worldwide (6). The median survival from symptom onset ranges from 20 to 48 months (7–9). Progression rates in ALS show considerable variation, and prognosis depends on age at onset, region of onset, co-morbid cognitive impairment, nutritional status, and certain genotypes are associated with faster progression (10–16). Given the considerable clinical, cognitive, and genetic heterogeneity of ALS, there is an unmet need for early diagnostic biomarkers to aid patient stratification into specific phenotypes (17). Clinical trials of ALS continue to rely on survival, functional scores and respiratory measures as outcome measures despite the potential of candidate imaging markers (18).

Magnetic resonance imaging (MRI) not only contributed to the characterization of ALS-associated cerebral changes, it has also contributed important pathophysiological insights, such as the role of inflammation (19), patterns of spread (20, 21), inhibitory dysfunction (22, 23), and network-wise propagation (24, 25). In addition to describing unifying disease-associated signatures, imaging studies of ALS have gradually characterized the features of specific genotypes (26, 27), phenotypes (28, 29), the substrate of cognitive and extra-pyramidal impairments (30), as well as presymptomatic (31) and longitudinal changes (32). Despite the momentous advances however, the overwhelming majority of imaging studies in ALS remain cerebral, overlooking a disease-defining site of ALS pathology; the spinal cord (3).

### SPINAL CORD IMAGING

One of the key challenges of spinal cord imaging stems from its elongated dimensions, small cross-sectional area in the axial plane coupled with long sagittal and coronal expansion (33). Furthermore, the cord is surrounded by tissues that have very different magnetic susceptibility profiles and is it subject to both direct (cardiac and respiratory) and fluidmediated [cerebrospinal fluid (CSF)] movement effects. The main challenges of quantitative spinal cord imaging include (i) partial volume effects, (ii) an inhomogeneous magnetic field environment, and (iii) physiological and patient motion (34).

# METHODOLOGICAL CHALLENGES

### Partial Volume Effects

Partial volume refers to scenarios where different tissues contribute to the same voxel. In spinal cord imaging this occurs when a voxel is at the CSF/white matter, white matter/gray matter, CSF/vascular, white matter/vascular interfaces. Signals from different tissue densities with different amounts of spins contribute to the total MR signal in these voxels, which results in indistinct tissue-boundaries. Partial volume effects can be reduced by increasing the spatial resolution, but this in turn results in lower signal-to-noise (SNR) and contrast-to-noise ratios (CNR). Magnetic fields strengths of three or seven Tesla compared to conventional 1.5 Tesla platforms (35–38), higher number of phased-array coils with parallel imaging (35, 38, 39), and corrections for physiological motion improves spatial resolution, SNR, and CNR (35, 38, 39).

# Physiological and Patient Motion

Due to its proximity to the lungs and the heart, almost the entire spinal cord undergoes repetitive displacement due to respiration, CSF, and cardiac pulsation (40–43). The movement of the human spinal cord linearly increases caudally with distance from the head. The available literature suggest that physiological anteriorposterior (A-P) cord movement (0.60 ± 0.34 mm) exceeds those observed in superior-inferior (SI) (0.4 ± 0.1 mm) and rightleft (RL) direction (0.17 ± 0.09 mm) (44, 45). Spinal imaging is also susceptible to movement artifacts from swallowing and patient movements during long MR acquisitions which can create ghosting artifacts (42, 46). By "gating" the acquisition, i.e., synchronizing with the respiratory or cardiac cycles, the effect of periodical movements can be significantly reduced (38, 39, 47). Motion artifacts can also be reduced using "saturation bands" that cover the esophagus, chest, and abdomen, by attenuating signals from moving structures so that it does not corrupt the signal from the spinal cord itself. Velocity compensating gradient sequences and signal averaging across multiple phases of motion can also be applied to minimize motion artifacts. Reducing acquisition time by using fast sequences, i.e., fastspin-echo, parallel imaging that increases acquisition speed by factors from 1.5 to 3, i.e., SENSitivity Encoding/GeneRalized Autocalibration Partial Parallel Acquisition-type reconstructions, partial Fourier imaging, reducing the size of the phase-encoded direction, and decreasing the k-space matrix size effectively reduce both physiological and subject motion effects (48–53). MRI compatible cervical collars, which minimize involuntary neck movements, may also reduce movement artifacts (46). Co-registration of all data when dealing with multiple series acquisition, e.g., diffusion tensor imaging (DTI) and functional MRI (fMRI), can also be performed to limit the inconsistency in derived maps (54, 55).

### Inhomogeneous Magnetic Field Environment

The spinal canal is surrounded by bones, ligaments, disks, arteries, and venous plexi.  Adipose tissue, bone, and air have different magnetic susceptibility profiles, and respiration-induced B0 field fluctuations (43) also contribute to the inhomogeneity of the magnetic field around the spinal cord, resulting in geometric distortions and signal intensity loss (56). To some extent, these artifacts can be counteracted with "shimming." Shimming aims at compensating for field inhomogeneities by creating an auxiliary magnetic field via shim coils (57). While shimming improves overall field homogeneity, it is limited to smooth variations across larger regions and cannot fully compensate for small, and localized field variations, such as those observed at cartilaginous discs between the vertebral bodies. Echo planar imaging sequences, such as DTI, are particularly sensitive to geometric distortions around vertebral disks. In addition to shimming, parallel imaging, and careful slices positioning may reduce magnetic field inhomogeneity, i.e., slices centered in the middle of each vertebral body and perpendicular to the spinal cord (38, 47, 58). The specific geometry of the magnetic field inhomogeneities should be considered in order to correct for its effect (59–61).

# SPINAL CORD IMAGING IN ALS

The role of conventional spinal MRI in ALS is to rule of alternative structural, inflammatory or neoplastic pathologies which may result in a combination of upper and lower motor neuron involvement mimicking ALS (62). Compressive myelopathies and radiculopathies are relatively common and early, predominantly lower limb presentations of ALS are sometimes attributed to these radiological findings resulting in laminectomies and other invasive procedures (63, 64). Conventional, clinical spinal sequences are typically only qualitatively interpreted without specific measurements. The majority of clinical spinal scans in ALS are reported as normal, but non-specific signs such as high signal along the corticospinal tracts are occasionally observed on T2-weighted imaging (65–67).

In sharp contrast with clinical sequences, advanced quantitative spinal protocols allow for the detailed and quantitative characterization of spinal gray and white matter integrity (38, 47, 58, 68). These protocols provide high resolution, high SNR, and high CNR images compared to standard clinical sequences. Furthermore, purpose-designed spinal protocols are based on mathematical MR signal modeling (e.g., diffusionbased methods, quantitative magnetization transfer, and MR spectroscopy) and the derived outputs can be quantitatively interpreted to provide accurate, motion-corrected white, and gray matter metrics.

### Cord Morphometry

Gross axonal and gray matter loss have traditionally been estimated by measuring spinal cord cross-sectional areas at specific levels and interpreted as a proxy of atrophy in the context of reference normative values (69–72). The "cross-sectional approach" consists of estimating a mean cord cross-sectional area over a representative number of slices at a given vertebral level (70, 71, 73, 74), which can be relatively easily calculated from conventional MR sequences such as T1- or T2-weighted images. A variety of indexes, such as A-P dimension, L-R width, and radial distance can be derived from the cross-sectional area (CSA) approach. These measures reflect on different aspects of pathology, such as global vs. regional, lateral vs. anterior tissue loss, and are often interpreted as predominantly motor or sensory involvement (70, 75). More specific gray and white matter measures can be derived from higher resolution images followed by tissue-type segmentation methods (72, 76, 77). Novel quantitative approaches, such as tensor based morphometry and surface based-morphometry permit a more fine-grained characterization of cord topography and the definition of diseaseassociated signatures (74, 78). Recent studies demonstrated that spinal cord atrophy, especially gray matter atrophy, correlates with disability and disease progression and may be predictive of respiratory failure and of survival in ALS (58, 70, 72, 73, 79). The main findings of structural spinal cord studies are summarized in **Table 1**.

# Diffusion Weighted Imaging

Diffusion weighted imaging (DWI) relies on the evaluation of water diffusion in CNS tissues and is primarily used to characterize white matter integrity (90, 91). DWI-derived metrics, such as axial diffusivity (AD), mean diffusivity (MD), fractional anisotropy (FA), radial diffusivity (RD) enable the quantitative characterization of white matter integrity. Novel high-directional approaches, such as high-angular resolution diffusion imaging (92), q-ball imaging (93), diffusion kurtosis imaging (94), diffusion basis spectrum imaging (DBSI) (95) are particularly well-suited to assess the integrity of crossingfibers (96, 97). Emerging diffusion techniques such as neurite orientation dispersion and density imaging (NODDI) (98) help to estimate the microstructural attributes of dendrites and axons (99). While in ALS NODDI has been primarily used in cerebral studies in ALS (100, 101), it also has been also piloted in spinal applications (90, 102). Specific DTI indices (AD, RD) have been associated with specific pathological processes, such as axonal (103, 104) vs. myelin-related (105, 106) degeneration, but this interpretation is likely to be simplistic, as DTI measures are affected by axonal density, axonal diameter, myelin thickness and fiber orientation, fiber coherence, and acquisition parameters. DTI has been extensively used to study cerebral changes in ALS and describe phenotype-associated (107), genotype-specific (27), presymptomatic (32), and longitudinal white matter changes in the brain (81). In contrast to the plethora of cerebral DTI studies, relatively few spinal DTI studies have been published in ALS to date (58, 69, 73, 80–82, 85). These have consistently highlighted both motor and sensory tract alterations (**Table 1**).

# Magnetization Transfer Imaging

Hydrogen nuclei linked to macromolecules such as the proteins and lipids of the myelin sheet have an extremely short T2 signal. While these macromolecules are not directly detectable by standard MRI sequences, magnetization transfer (MT) imaging enables the characterization of these structures. Macromolecular spins can be saturated using an off-resonance RF pulse, then the magnetization transfer between bound and free pools TABLE 1 | Quantitative spinal imaging studies in ALS, ALS, amyotrophic lateral sclerosis; ALSFRS-r, the revised ALS functional scale; FA, fractional anisotropy; CSA, cross-sectional area; CST, corticospinal tract; FVC, force vital capacity; ihMT, inhomogeneous magnetization transfer; ihMTR, inhomogeneous magnetization transfer ratio; MD, mean diffusivity; MT, magnetization transfer; MTR, magnetization transfer ratio; MMT, manual muscle testing; SC, spinal cord; SOD1, superoxide dismutase 1 gene.

can be measured (108). Magnetization transfer occurs by means of cross relaxation processes, such as dipole-dipole interactions and chemical exchange. Magnetization transfer ratio (MTR) is calculated as the percentage difference of MT images with macromolecules signal saturation and one without. MTR enables inferences on myelin content, axonal count, and density as shown by three MS histological studies, and has been used extensively to assess demyelination, remyelination, and degeneration in MS (109–111). Conversely, relatively few studies have used cerebral MT imaging in ALS, and the majority of these focused on corticospinal tract alterations (112–115). Relatively few studies evaluated spinal MT changes in ALS, but they have shown progressive reduction overt time and correlation with muscle weakness (58, 69, 73). The key findings of spinal MT imaging studies in ALS and associated technical challenges are summarized in **Tables 1**, **2**.

# Inhomogeneous Magnetization Transfer Imaging

Inhomogeneous magnetization transfer (ihMT) imaging is a novel method (116, 117), which allows the unprecedented characterization of myelin integrity, by isolating key myelin components from the broader macromolecular pool. ihMT shows unparalleled potential to detect and quantify demyelination (118) and may be adapted to spinal applications. ihMT imaging has already been applied to ALS cohorts and demonstrated significant correlation with muscle strength and disability profiles (58).

### MR Spectroscopy

Magnetic resonance spectroscopy (MRS) is well-established, non-invasive imaging tool which provides neurochemical insights based on the concentration and relaxation profile of specific metabolites in cerebral and spinal tissues. MRS has been extensively used in cerebral studies of ALS (119), used to assess the therapeutic effect of Riluzole (120, 121), and also used to study brainstem metabolic alterations (122). Crosssectional and longitudinal (123), single voxel and whole brain multi-voxel studies have both contributed to our understanding of ALS pathophysiology (124). The main targets of proton spectroscopy (1H-MRS) include the following metabolites; N-Acetyl Aspartate (NAA), creatine (Cr), choline (Cho), and myo-Inositol (Myo). These metabolites are typically associated with neuronal integrity/viability (NAA), tissue energy metabolism (Cr), membrane integrity (Cho), and glial function (Myo). (125). Relatively few studies have used 1H-MRS to characterize metabolic changes at the spinal level, and the majority of these studies focused on multiple sclerosis (126, 127) MRS however seems particularly applicable to ALS cohorts, where it promises the characterization of presymptomatic changes and by including both the upper and lower motor components of the motor system, it has led to particularly significant clinico-radiological correlations (31, 83, 84). For the contribution of MRS studies to

ALS, amyotrophic lateral sclerosis; ALSFRS-r, revised ALS functional scale; FA, fractional anisotropy; CST, corticospinal tract; FVC, force vital capacity; MD, mean diffusivity; MMT, manual muscle testing.

ALS research and specific methodological considerations please see **Tables 1**, **2**.

### Functional MRI

Functional MRI (fMRI) detects local variations in blood oxygenation level-dependent MR signal at rest and during activation paradigms (128). FMRI has been extensively applied to ALS cohorts to describe network changes and assess altered activation patterns when performing motor or cognitive tasks (129–131). Following decades of successful cerebral studies, the first spinal fMRI studies have now been published (55, 132). Emerging spinal cord fMRI studies in healthy controls provide proof of feasibility and the first studies using spinal fMRI in neurological conditions are underway (133).

### THE CONTRIBUTION OF SPINAL IMAGING TO ALS RESEARCH

### Evidence for Motor Involvement in ALS