REVIEW published: 18 January 2022 doi: 10.3389/fneur.2021.750543 Pathophysiological Underpinnings of Extra-Motor Neurodegeneration in Amyotrophic Lateral Sclerosis: New Insights From Biomarker Studies David Reyes-Leiva 1,2 , Oriol Dols-Icardo 3,4 , Sonia Sirisi 3,4 , Elena Cortés-Vicente 1,2 , Janina Turon-Sans 1,2 , Noemi de Luna 1,2 , Rafael Blesa 3,4 , Olivia Belbin 3,4 , Victor Montal 3,4 , Daniel Alcolea 3,4 , Juan Fortea 3,4 , Alberto Lleó 3,4 , Ricard Rojas-García 1,2 and Ignacio Illán-Gala 3,4* 1 Neuromuscular Diseases Unit, Department of Neurology, Hospital de la Santa Creu i Sant Pau, Universitat Autònoma de Barcelona, Barcelona, Spain, 2 Centro de Investigación Biomédica en Red de Enfermedades Raras, CIBERER, Valencia, Spain, 3 Sant Pau Memory Unit, Department of Neurology, Biomedical Research Institute Sant Pau, Hospital de la Santa Creu i Sant Pau, Universitat Autònoma de Barcelona, Barcelona, Spain, 4 Centro de Investigación Biomédica en Red de Enfermedades Neurodegenerativas, CIBERNED, Madrid, Spain Edited by: Amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) Umberto Manera, lie at opposing ends of a clinical, genetic, and neuropathological continuum. In University of Turin, Italy the last decade, it has become clear that cognitive and behavioral changes in Reviewed by: Maurizio Grassano, patients with ALS are more frequent than previously recognized. Significantly, these University of Turin, Italy non-motor features can impact the diagnosis, prognosis, and management of ALS. Fabiola De Marchi, Azienda Ospedaliero Universitaria Partially overlapping neuropathological staging systems have been proposed to Maggiore della Carità, Italy describe the distribution of TAR DNA-binding protein 43 (TDP-43) aggregates outside *Correspondence: the corticospinal tract. However, the relationship between TDP-43 inclusions and Ignacio Illán-Gala neurodegeneration is not absolute and other pathophysiological processes, such iillan@santpau.cat as neuroinflammation (with a prominent role of microglia), cortical hyperexcitability, Specialty section: and synaptic dysfunction also play a central role in ALS pathophysiology. In This article was submitted to the last decade, imaging and biofluid biomarker studies have revealed important Neuromuscular Disorders and Peripheral Neuropathies, insights into the pathophysiological underpinnings of extra-motor neurodegeneration a section of the journal in the ALS-FTLD continuum. In this review, we first summarize the clinical and Frontiers in Neurology pathophysiological correlates of extra-motor neurodegeneration in ALS. Next, we Received: 30 July 2021 discuss the diagnostic and prognostic value of biomarkers in ALS and their Accepted: 09 December 2021 Published: 18 January 2022 potential to characterize extra-motor neurodegeneration. Finally, we debate about Citation: how biomarkers could improve the diagnosis and classification of ALS. Emerging Reyes-Leiva D, Dols-Icardo O, Sirisi S, imaging biomarkers of extra-motor neurodegeneration that enable the monitoring of Cortés-Vicente E, Turon-Sans J, de Luna N, Blesa R, Belbin O, Montal V, disease progression are particularly promising. In addition, a growing arsenal of biofluid Alcolea D, Fortea J, Lleó A, biomarkers linked to neurodegeneration and neuroinflammation are improving the Rojas-García R and Illán-Gala I (2022) diagnostic accuracy and identification of patients with a faster progression rate. The Pathophysiological Underpinnings of Extra-Motor Neurodegeneration in development and validation of biomarkers that detect the pathological aggregates Amyotrophic Lateral Sclerosis: New of TDP-43 in vivo are notably expected to further elucidate the pathophysiological Insights From Biomarker Studies. Front. Neurol. 12:750543. underpinnings of extra-motor neurodegeneration in ALS. Novel biomarkers tracking the doi: 10.3389/fneur.2021.750543 different aspects of ALS pathophysiology are paving the way to precision medicine Frontiers in Neurology | www.frontiersin.org 1 January 2022 | Volume 12 | Article 750543 Reyes-Leiva et al. Extra-Motor Neurodegeneration in ALS approaches in the ALS-FTLD continuum. These are essential steps to improve the diagnosis and staging of ALS and the design of clinical trials testing novel disease-modifying treatments. Keywords: amyotrophic lateral sclerosis (ALS), neuroimage, cerebrospinal fluid (CSF), biomarker (BM), neuropathology, TDP-43 = TAR DNA-binding protein 43, frontotemporal lobar degeneration, frontotemporal dementia (FTD) INTRODUCTION: USING BIOMARKERS TO largely unexplored (10). This review aims to (i) summarize REVEAL THE EXTRA-MOTOR the clinical and pathophysiological correlates of extra-motor neurodegeneration in ALS; (ii) review the diagnostic and UNDERPINNINGS OF AMYOTROPHIC prognostic value of biomarkers and their potential to characterize LATERAL SCLEROSIS extra-motor neurodegeneration in ALS; and (iii) discuss how biomarkers could improve the diagnosis and classification Amyotrophic lateral sclerosis (ALS) is a progressive paralytic of ALS. disorder defined by the neurodegeneration of motor neurons, while frontotemporal dementia (FTD) is the most common presentation of frontotemporal lobar degeneration (FTLD) EXTRA-MOTOR INVOLVEMENT IN ALS and is characterized by progressive neurodegeneration of frontotemporal structures (1, 2). The diagnosis of ALS is Clinical Evidence for Extra-Motor based on the identification of motor signs and symptoms, Involvement in ALS while the diagnosis of FTD is based on cognitive, language, Although the first observation of the clinical overlap between and behavioral features (3–5). However, recent evidence ALS and FTD dates from the beginning of the twentieth century indicates that ALS can show varying degrees of cognitive and (1, 7–9, 23–26), the study of this clinical continuum has been behavioral changes at diagnosis (6–9). In ALS, the presence boosted in the last decade using the emerging neuropathological, of non-motor symptoms has been linked to frontotemporal molecular, and genetic evidence connecting the two processes neurodegeneration (10, 11), and current diagnostic criteria (10, 27–29). acknowledge the existence of a continuum of cognitive and behavioral changes between ALS without cognitive impairment Neuropsychological Evidence (ALSno-cbi) and full-blown FTD (12). However, the exact The development of specific tools to assess the cognitive status mechanisms driving extra-motor neurodegeneration in ALS of ALS patients with variable degrees of motor impairment has remain largely unknown. Filling this gap in our knowledge is led to a major breakthrough for the study of the ALS-FTD essential to improve the diagnosis and management of patients continuum (26, 30, 31). When studied with specifically designed with ALS and may also impact end-of-life legal decisions (13, instruments, such as the Edinburgh ALS cognitive and behavioral 14). screen (ECAS), the ALS cognitive behavioral screen, or the From a clinical perspective, ALS and FTLD are the Arrows and Colors Cognitive Test, up to 20% of patients with two heterogeneous diseases characterized by multiple clinical ALS can also be diagnosed with FTD, and up to 50% of presentations and highly variable rates of functional decline patients with ALS have a cognitive or behavioral impairment (15, 16). The observed phenotypic and prognostic heterogeneity (ALScbi) (6, 10, 32–36). The frequency and characteristics hampers the diagnosis and the prediction of disease progression of cognitive and behavioral impairment resemble those noted at the single-subject level and the design of disease-modifying in FTD, and recent neuropathological studies have reported treatments (15). These problems underscore the importance frontotemporal involvement consistent with the diagnosis of of developing precise, objective, and reproducible biomarkers FTLD in more than 30% of patients with ALS (37). Among to improve the diagnosis, prognosis, and disease monitoring the most common cognitive symptoms developed in patients (17–19). A biomarker can be defined as objective, quantifiable with ALS-FTD are executive dysfunction, language impairment, characteristics of a biological process (either physiological or and social cognition, all of which are linked to the frontal pathological) that can be objectively measured in vivo (20). and temporal lobes (6, 33). Some neuropsychological tools (i.e., Biomarkers have proven to be powerful tools to increase Sydney Language Battery, Test for Reception of Grammar) have diagnostic certainty and predict disease progression in other proven to be useful for characterizing the language performance neurodegenerative diseases (21). Biomarkers also have the in patients within the ALS-FTD clinical continuum. The presence potential to reveal the critical aspects of ALS pathophysiology of language impairment is almost universal in patients meeting and advance the field toward the implementation of precision the criteria for ALS-FTD. On the other hand, patients with medicine approaches (22). ALS who do not meet the criteria for ALS-ci or ALS-FTD may Despite the cumulative evidence suggesting that ALS and show only subtle language deficits (38–46). From a behavioral FTLD lie on a clinical, pathological, and genetic continuum, perspective, frontal behaviors like apathy, stereotypic behavior, the pathophysiological underpinnings of this continuum remain and disinhibition are the most common behavioral features in Frontiers in Neurology | www.frontiersin.org 2 January 2022 | Volume 12 | Article 750543 Reyes-Leiva et al. Extra-Motor Neurodegeneration in ALS patients with ALS (12, 47). However, behavioral changes in a loss of function through haploinsufficiency, RNA toxic gain- patients with ALS include subtle changes in social cognition. of-function through the sequestration and accumulation of These changes can be adequately measured with specific tests toxic dipeptide repeat proteins (DRP) (10, 58–63). As noted in such as the Reading the mind in the eyes test or the faux-pas Table 1, other genes causing both ALS and FTLD are related to test (41, 48). On the other hand, up to 15% of patients initially protein trafficking, cytoskeletal dynamics, protein degradation, diagnosed with FTD may also develop motor neuron signs during and mitophagy (12, 64). Similarly, genes such as TANK-binding follow-up (10, 49). It should be noted that the frequency of motor kinase-1 (TBK1) (65–69), Sequestosome-1 or p62 (SQTSM1) (70), neuron signs in patients with FTD is probably underestimated Optineurine protein (OPTN), and Valosin-containing protein because these signs are not always systematically investigated (VCP) that are associated with FTD, inclusion body myositis, during follow-up in FTD cohorts. motor neuron disease, and Paget’s disease encode proteins related Neuropsychological testing has proven to be helpful to protein degradation (71, 72). In addition, recent studies for the clinical characterization of patients with ALS, but have suggested that the apolipoprotein E (APOE) gene may neuropsychological evaluations are time-consuming and may play an important role in the development of cognitive and not be well-tolerated by some patients. These limitations behavioral impairment in ALS. The ε4 allele of the APOE gene reinforce the need for the biomarkers that help us to trace the is a major genetic factor in Alzheimer’s disease and has also development of extra-motor manifestations (30, 35). been shown to increase the risk of TAR DNA-binding protein 43 (TDP-43) proteinopathy and hippocampal sclerosis in a large Clinical Evidence community-based cohort. These results are in line with previous As we will discuss in Section 2.3, the neuropathological correlates evidence suggesting that the ε4 allele of the APOE gene may also of cognitive and behavioral changes in ALS are still a matter increase the risk of FTLD. Interestingly, recent evidence from of debate (6, 12, 33, 47, 49). Several studies have consistently well-characterized cohorts of ALS showed that the ε2 allele of noted that patients with ALS and having a bulbar onset of the APOE gene (together with the presence of C9orf72 repeat symptoms have an increased risk of cognitive and behavioral expansion) significantly increases the risk of FTD in participants impairment (6, 32, 50). Distal upper-limb weakness, sometimes with ALS (73–76). Recent studies on the evaluation of the with no upper motor neuron signs, is also frequently observed impact of Hungtingtin (HTT) pathologic expansions in the ALS- in patients meeting the criteria for ALS-FTD (51). Other FTD spectrum suggest an ethiopathological link (77). Similarly, reports link the development of bulbar symptoms during the rare variants in the GBA gene encoding glucocerebrosidase disease course with cognitive and behavioral symptoms (52, (previously described as a potential risk factor of cognitive 53). Interestingly, other studies have also reported a higher impairment in Parkinson’s disease) are also overrepresented in presence of abnormal oculomotor findings, extrapyramidal signs, patients with FTD-ALS and ALScbi compared to patients with and autonomic dysfunction in patients with ALS and having ALS and having no cognitive impairment (78). prominent bulbar symptoms at disease onset (54). A recent transcriptome-based study of postmortem frontal cortex tissue from patients with ALS reported a coordinated Genetic Determinants of Extra-Motor upregulation of transcripts from a sub-population of microglia Involvement (termed disease-associated microglia) and overexpressed Major Genetic Findings Associated With Extra-Motor neuroinflammatory molecules, such as YKL 40 (also known Impairment in ALS as CHI3L1) and CHI3L2. Thus, microglial and inflammatory Considerable progress has been made in unraveling the genetics pathways are involved in the pathogenesis of extra-motor of ALS and FTLD, and it is now clear that the genetics of impairment in ALS (79). these two neurodegenerative conditions overlap significantly In summary, genes associated with both ALS and FTD point (55). Table 1 summarizes the main characteristics of genes toward the involvement of pathophysiological processes outside associated with both ALS and FTD etiology. These genes shed the motor regions that are relevant for extra-motor involvement some light on several pathophysiological processes, which are in patients with ALS. relevant for extra-motor involvement in ALS. For instance, the genes involved in the lysosomal function and autophagy process Genetic Findings Associated With Extra-Motor may have a prominent role in both sides of the spectrum and Sparing in ALS other neurodegenerative diseases (56). It should be noted that the overlap in genes associated with ALS The C9orf72 hexanucleotide GGGGCC repeat intronic and FTLD is not complete. For example, superoxide dismutase expansion is the most common genetic cause of both ALS and 1 (SOD1), FUS, and TARDBP variants are most commonly FTLD. Some noteworthy studies reported that patients with ALS associated with ALS and only rarely cause FTLD. Similarly, GRN and carrying C9orf72 have a 4–5-fold higher risk of presenting is linked to the TDP-43 subtype of FTLD but not ALS (80). with cognitive or behavioral changes than non-carriers (57). Interestingly, the reduced expression of GRN has been recently Physiologic C9orf72 protein is normally localized in the nuclei, identified as a possible contributing factor in the development and it regulates membrane trafficking. On the other hand, the of different neurodegenerative diseases including ALS (81). hexanucleotide repeat expansion is cytotoxic. While the precise In addition, experiments performed in animal models have molecular mechanisms underlying cytotoxicity are not fully identified the reduced expression of GRN as a sufficient factor understood, several mechanisms have been proposed, including to induce TDP-43 deposition (82). The gene encoding SOD1 is Frontiers in Neurology | www.frontiersin.org 3 January 2022 | Volume 12 | Article 750543 Reyes-Leiva et al. Extra-Motor Neurodegeneration in ALS TABLE 1 | Genes in close association with both amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) etiologies. Gene Location Pathway MI C9orf72 9p21.2 Nucleocytoplasmic transport/splicing AD TBK1 12q14.1 Autophagy/inflammation AD TARDBP 1p36.2 Nucleocytoplasmic transport/splicing AD SQSTM1 5q35.3 Autophagy AD/Risk VCP 9p13.3 Autophagy/mitochondrial function AD FUS 16p11.2 Nucleocytoplasmic transport/splicing AD/AR UBQLN2 Xp11 Autophagy/proteasome XL CHCHD10 22q11.2 Mitochondrial dysfunction/synaptic integrity AD MI, mode of inheritance; AAO, age at onset; DM, disease modifier; AD, autosomal dominant; AR, autosomal recessive; XL, X-linked. rarely related to extra-motor impairment in ALS albeit that some subtype in patients with motor neuron diseases and shows many mutations have been recently associated with an early cognitive granular TDP-43 inclusions with a few dystrophic neurites in all impairment (83). SOD1 is essential for antioxidant defense in the the cortical layers. Type C shows long dystrophic neurites and a cytosol and mitochondria (84). Non native formations of SOD1 few intracytoplasmic inclusions. Finally, type D histopathology have been detected in small granular SOD1-immunoreactive is associated with a mutation in VCP, which causes familial inclusions in the motor neurons of sporadic ALS patients inclusion body myositis, Paget’s disease of bone, FTD with or without pathogenic SOD1 variants and in patients carrying other without motor neuron disease involvement (98–100). Lee et al. ALS-associated genes (85, 86). These findings suggest that the reported a new histopathologic subtype for TDP-43 aggregates misfolding of SOD1 can be part of a joint downstream event in (type E) with specific TDP-43 aggregates, a uniform biochemical motor neuron neurodegeneration. profile, and a rapid clinical course (101). Finally, neuropathological findings in sporadic patients with Misfolded TDP-43 propagation is believed to follow a “prion- ALS and having FUS aggregates resemble those of patients with like” mechanism, seeding of the native protein misfolding in FTLD and having FUS deposition (87). However, mutations vitro and synaptic transmission to the next neuron (93, 94, 102– in the FUS gene are related to ALS and rarely cause FTLD 105). This protein deposition is associated with neuronal and (88). To date, the reasons behind the relative sparing of extra- synapse loss and affects the different brain regions in ALS and motor regions in patients with ALS and having FUS mutations FTD, at least initially. In patients with ALS, the phosphorylated remain unclear. TDP-43 inclusions spread initially from motor neurons located Interestingly, some genes that are strongly associated with in the motor cortex, spinal cord, and brainstem motor nuclei FTLD have scarcely been studied in the ALS-FTD spectrum, to other neocortical areas, cerebellum, and striatum (93). In where their role remains unclear. This is the case for MAPT contrast, in FTLD with TDP-43 inclusions, this spread begins in mutations, which cause the FTLD tau subtype, and GRN and the amygdala and orbitofrontal cortex, spreading to the temporal TREM2 mutations, which have been associated with the TDP-43 and frontal non-motor cortex first and motor cortex later, and subtype and Alzheimer’s disease (89–91). finally reaching the visual cortex (10, 93–95) (Figure 1). Most studies have shown moderate TDP-43 inclusions before synapse Neuropathological Underpinnings of and neuronal loss in animal models (94); however, an intriguing Extra-Motor Neurodegeneration minority of neurons may lack detectable nuclear TDP-43 despite Neuropathological Changes in ALS the apparent absence of a cytoplasmic TDP-43 inclusion. These From a pathological perspective, TDP-43 aggregation is a cells show neuronal atrophy comparable to inclusion-bearing frequent pathological feature of FTLD and the most typical neurons, suggesting that the loss of nuclear TDP-43 function neuropathological finding in patients with ALS (92–96). The promotes neurodegeneration even when TDP-43 aggregation is pathological hallmarks of TDP-43 proteinopathies include inconspicuous or absent (106). mislocalization from the nucleus to the cytoplasm, deposition of ubiquitinated and hyperphosphorylated TDP-43 into inclusion Neuropathological Correlates of Cognitive and bodies, protein truncation leading to the formation of toxic Behavioral Changes in ALS C-terminal TDP-43 fragments, and protein aggregation (97). Pathological aggregates of TDP-43 in extra-motor cortical and ALS and FTLD with TDP-43 inclusions can be subdivided into subcortical areas are believed to play an essential role in different subtypes based on the anatomical distribution and cognitive impairment in ALS. The burden of pathological TDP- morphology of abnormal TDP-43 aggregates. Type A is observed 43 pathology in extra-motor regions is higher in patients in patients with GRN mutations and is characterized by round with ALScbi and ALS-FTD than those with ALSno-cbi (39, “compact” intracytoplasmic TDP-43 inclusions, dystrophic short 107, 108). Also, synapse loss has been identified using high- neurites, and occasional lenticular intranuclear inclusions, resolution imaging of postmortem brain tissue samples in extra- mainly in the upper cortical layers. Type B is the most frequent motor cortical areas, such as the prefrontal cortex of sporadic Frontiers in Neurology | www.frontiersin.org 4 January 2022 | Volume 12 | Article 750543 Reyes-Leiva et al. Extra-Motor Neurodegeneration in ALS FIGURE 1 | Neuropathological staging schemes in: (A) FTLD-TDP (104) and (B) ALS-TDP (93). Adapted from Burrell et al. (10). FTLD-TDP, frontotemporal lobar degeneration with TAR DNA-binding protein inclusions; ALS-TDP, amyotrophic lateral sclerosis with TAR DNA-binding protein inclusions. BF, basal forebrain; A, Amygdala; H, Hippocampus; PC, precerebellar nuclei; C/P, Caudate nucleus and putamen; MD, Mediodorsal nucleus of the thalamus; T, Thalamus; LT, Lateral thalamus; R, Red nucleus; SN, Substantia nigra; RF, Reticular formation; IOC, Inferior olivary complex; XII, Hypoglossal nucleus. patients with ALS (109). Notably, the degree of synapse loss TDP-43 deposition in postmortem tissue is a common finding correlated with the severity of the patients’ cognitive impairment in multiple cortical and subcortical areas of patients with independent of cortical atrophy, supporting the idea that synapse ALS, but its significance remains uncertain (125, 126). Taken loss precedes neuronal loss (109, 110). Finally, Tau pathology together, these findings highlight the importance of concurring and hippocampal sclerosis may also play a secondary role in the neuroinflammatory processes in the pathophysiology of both presence of cognitive impairment in patients with ALS (37, 111, ALS and FTD. 112). Other Neuropathological Hallmarks of ALS: C9orf72, Neuroinflammation FUS-FET Neuroinflammation in ALS and FTD is primarily characterized As aforementioned (refer to Genetic determinants of extra- by the activation of innate immune sensing pathways in microglia motor features), patients carrying C9orf72 mutation have been and astrocytes resident to the central nervous system but also associated with more cognitive impairment and more extensive with the involvement of peripheral-derived inflammatory TDP-43 deposition following predominantly a Type B or a cells, such as T-lymphocytes, mast cells, monocyte-derived Type A + B pattern (93, 127). A p62 and ubiquitin negative macrophages, and dendritic cells (113–116). As part of the intracytoplasmic and intranuclear inclusions corresponding to innate immune system, some components of the innate dipeptide repeat are found in extra-motor areas, such as the complement system have been identified in postmortem studies frontal cortex, hippocampus, and cerebellum (12, 62, 110, 128). and their eventual role in disease progression (117, 118). Interestingly, some reports have also identified C9orf72 patients Also, microglial density and microglial activation measured with ALS and FTD presenting with cognitive impairment but using immunostaining for glial fibrillary acidic protein with minimally TDP-43 inclusions underscoring the influence of (GFAP) and CD6, show an increase in patients with ALS other physiopathological mechanisms such as dipeptide repeat and FTD and correlate well with synaptic, axonal, and accumulation and RNA foci in the appearance of cognitive neuronal loss and faster motor and extra-motor disease impairment (63, 108). progression (119–121). There is also a higher presence of Conversely, in the case of FUS protein deposition reactive astrocytes, which are believed to have neurotoxic neuropathology, it is believed that the pathological processes properties and have been described involving the gray matter underlying the aggregate formation and cell death between FTD of motor and non-motor cortex in patients with ALS and and ALS differ. While ALS with FUS mutations seems to be more also in affected cortical areas in FTD (122–124). In addition, restricted to the dysfunction of FUS protein, a more complex oligodendrocytes pathology mediated by phosphorylated dysregulation that includes the deposition of all FET proteins Frontiers in Neurology | www.frontiersin.org 5 January 2022 | Volume 12 | Article 750543 Reyes-Leiva et al. Extra-Motor Neurodegeneration in ALS (TAF15 and EWS) and Transportin-1 (Trn1) is thought to be ALS mimics, and healthy controls with similar accuracy to NfL involved in the subtypes of FTLD with FUS pathology (88). (155). Of note, a recent study showed that the difference in the abundance of NfH between ALS and FTD was markedly BIOMARKERS stronger for NfH than that of NfL (155). On the other hand, studies have reported a better correlation between the serum and Biomarkers can be used for four primary purposes: (i) to CSF for NfL compared to NfH, albeit that NfL has been more guide clinical diagnosis by identifying the key pathophysiological widely investigated (156). Overall, CSF NfL and NfH as well as changes of a particular disease (so-called diagnostic markers), serum NfL are equally suited for the differential diagnosis of (ii) to estimate the risk or speed of progression of a particular ALS, whereas serum NfH performs less well (155). Other serum disease (prognostic markers), (iii) to monitor the progression biomarkers, such as peptides linked with eating behavior and or response to therapy (theragnostic markers) (129), and (iv) metabolism, for instance, leptin and neuropeptide-Y, have been to characterize the relevant aspects of disease pathophysiology recently studied in the ALS-FTD continuum and show a promise (i.e., inflammation). Section Fluid Biomarkers will describe the as neurodegeneration biomarkers, but further investigations are currently available biomarkers for the study of extra-motor needed to clarify their role in the pathophysiology of ALS and neurodegeneration in the ALS-FTLD continuum and their FTD (157). diagnostic and prognostic value. Patients within the ALS-FTD clinical continuum also show increased levels of total tau (t-tau) in CSF but the diagnostic Fluid Biomarkers and prognostic value of this biomarker is lower than NfL The cerebrospinal fluid (CSF) represents an invaluable sample for (145, 158). Some studies have reported that low levels of both studying neurodegenerative diseases due to its close relationship phosphorylated tau at threonine 181 (p-tau) and the p-tau:t-tau with brain parenchyma (130). However, the study of CSF ratio in CSF may differentiate TDP-43 and tau subtypes of FTLD biomarkers may be limited because of their invasive nature. (159–162), but these findings seem to be driven by the higher Recently, blood biomarkers have shown promise as minimally levels of t-tau in patients with ALS (162–164) and not by lower invasive clinical tools in neurodegenerative diseases (129, 131). levels of p-tau in FTLD-TDP (165). Compared to CSF, the accessibility and cost-effectiveness of blood samples make them more suitable for first-line clinical use and Synapse Degeneration Markers facilitate clinical trial recruitment and monitoring. The amyloid precursor protein (APP) is a type I single-pass transmembrane protein with a large extracellular domain and a Neurodegeneration Biomarkers short cytoplasmic tail that undergoes very complex proteolytic Neurofilaments are the principal constituents of the neuro- processing, yielding biologically active fragments (166). These axonal cytoskeleton and play an essential part in axonal transport include the full-length Aβ1–42 and other N-terminal and C- and the synapse (132). Neurofilament light chain (NfL) is the terminal truncated forms of Aβ, sAPPα, sAPPβ, etc., that most abundant and soluble neurofilament subunit, and increased can be detected in human CSF (167–169). Numerous studies levels in CSF and blood reflect axonal damage in the CNS. The suggest that reduced neuronal-synaptic activity may also lead CSF and plasma levels of NfL are highly correlated and have been to less Aβ production (170, 171) and that Aβ may also found to increase in a wide variety of diseases (133, 134). Both have an important role in synaptic function (172). Recent CSF and plasma levels of NfL are increased in patients with ALS studies on autopsy-confirmed cases have reported reduced CSF and have proven helpful to differentiate between patients with concentrations of sAPPβ in FTLD in the absence of comorbid ALS and other ALS mimics (135–137). Importantly, multimodal Alzheimer’s pathology (173). Interestingly, CSF concentrations biomarker studies have shown that the CSF and plasma levels of sAPPβ are decreased in ALS and FTD compared to healthy of NfL correlate well with frontotemporal cortical thickness and controls and correlate with frontotemporal cortical thickness white matter microstructure in patients within the ALS-FTD and cognitive impairment suggesting that sAPPβ levels may continuum (138–140). The CSF and blood levels of NfL also reflect the neuronal loss in frontotemporal areas (140, 174). correlate with measures of disease severity, disease progression Unfortunately, plasma concentrations of sAPPβ poorly correlate rate, and the prediction of longitudinal clinical deterioration with CSF concentrations (175), potentially due to a confounding and survival (21, 137, 138, 141–148). The CSF NfL levels seem effect of abundant non-CNS-derived sAPPβ in the blood. On to increase over time but further longitudinal data are needed the other hand, CSF concentrations of neurogranin, the most (149, 150). Moreover, the combination of plasma NfL with other studied synaptic marker to date, were not elevated in patients measurements of neuroinflammation in plasma may improve with ALS (176). In another study, label-free mass spectrometry- disease progression at the single-subject level (151–153). Because based proteomic analyses of CSF from patients with ALS biofluid levels of NfL are strongly correlated with age, recent revealed a concomitant decrease in proteins from biological multicenter studies have determined highly specific age-adjusted pathways related to synapse organization and inflammation- cut-offs (154). Overall, both CSF and plasma levels of NfL may be related proteins (177). Recently, other synaptic markers have helpful to support the diagnosis in clinically relevant scenarios, been identified by unbiased proteomics-based approaches. Still, disease staging, and the prediction of disease progression. In more studies are needed to clarify the role of these new addition to NfL, neurofilament heavy chain (NfH) has also been biomarkers and their potential use for the diagnosis, staging, and studied in patients with ALS and can discriminate between ALS, prognosis of patients with suspected ALS (178, 179). Frontiers in Neurology | www.frontiersin.org 6 January 2022 | Volume 12 | Article 750543 Reyes-Leiva et al. Extra-Motor Neurodegeneration in ALS Biomarkers Linked to Neuroinflammation and Glial and increased levels of C3 cleavage products in the serum of Activation patients with ALS (195, 196). A variety of systemic inflammatory responses (pro- and anti-inflammatory cytokine profiles, altered immune cell Biomarkers Linked to Abnormal TDP-43 Aggregation populations) have been consistently reported in patients with Cytoplasmic aggregation of ubiquitinated, phosphorylated, and ALS and FTD (113, 180). These responses do not seem to truncated TDP-43 is a unifying pathologic observation across be a mere passive phenomenon and may contribute to the the neuropathological continuum of ALS and FTLD. However, neurodegeneration/neuroprotection balance (181, 182). For standard immunoassays of TDP-43 have not proven to be useful instance, increased CSF and serum levels of some monocyte- to identify patients with pathological TDP-43 aggregation, and it derived cytokines linked with the inflammatory response such as is not yet clear whether TDP-43 would be expected to increase in TNF-α, IL-1β, IL-17, and TGF-β reveal a microglial and white the CSF due to the degeneration of nerve cells and the release blood cell participation in the ALS-FTD pathologic continuum of intracellular proteins or whether it would be expected to (113, 182, 183). In addition, increased CSF levels compared to be low in the CSF due to aggregate formation (197–199). This controls of Matrix Metalloproteinase-10 and decreased CSF is because most currently available techniques for quantifying levels of some chemokines and growth factors (such as IL-8, TDP-43 use commercially available antibodies, which only detect IL-12B, TGF-α among others) have been recently reported in a the full-length form of TDP-43 rather than the disease-specific multicentric study (184). Other surrogate markers of acute-phase forms of TDP-43 (200, 201). However, several recent advances systemic inflammation have been reported in ALS, including hold promise to the detection of abnormal TDP-43 aggregation lipopolysaccharide-binding protein, C reactive protein, and in vivo. A recent study used immuno-IR sensors to study the soluble CD14 (181, 185). secondary structure of all TDP-43 isoforms in the CSF (202). In In addition to a systemic inflammatory response, several addition, a recent study showed that real-time quaking-induced biomarkers in the blood and CSF have been found to be conversion reaction (RT-QuIC) antemortem prion detection was consistently altered in the ALS-FTD continuum (186). Some of a robust technique for prion amplification of TDP-43 (203). these biomarkers may be useful to predict disease progression Some promising biomarkers like truncated Stathmin-2 and survival (140, 187). Increased CSF levels of YKL-40 (also (STMN2) are linked to TDP-43 function and deposition. The known as Chitinase like-3 protein 1) in ALS were first observed expression of STMN2 is increased in the frontal cortex of patients in a small study (188), a finding that has been later replicated with the TDP subtype of FTLD, which makes it a good candidate in larger studies involving participants along the ALS-FTD biomarker related to TDP pathology (204). Finally, in C9orf72 continuum (140). Recent proteomic studies confirmed that three carriers, high CSF levels of DRPs can be detected even in pre- macrophage-derived chitinases (YKL-40, chitotriosidase, and symptomatic stages, suggesting a role of DRPs early in ALS-FTD chitinase like-3 protein 2) were increased in ALS and predicted pathogenesis in early stages (145, 205). Although not currently a faster disease course but only YKL-40 levels increased over relevant as clinical fluid biomarkers, DRPs may become more time in those with low initial levels (187). These results support widely used in detecting the C9orf72 expansion prior to genetic a crucial role for microglial activity in ALS (152), offering screening or in prodromal stages, which would make a valuable novel target engagement and pharmacodynamic biomarkers for addition to the biomarker arsenal in the future clinical trials. neuroinflammation-focused ALS therapy. However, YKL-40 is not specific to the central nervous system, and their levels Image-Based Biomarkers in the CSF and blood are poorly correlated, thus limiting Macrostructural and Microstructural Changes in MRI its use as a non-invasive biomarker. Conversely, GFAP is a Routine structural MRI has a limited utility in ALS as signal brain-specific protein and an established marker of astrogliosis. intensity, and gross volume changes in T1- and T2-weighted This protein can also be accurately measured in the blood images are not typically observed in most ALS cases. On the with new ultrasensitive technology (189). Interestingly, different contrary, quantitative analysis of MRI can improve the detection neuroinflammatory profiles between ALS and FTD have been of neurodegeneration-related abnormalities by measuring small recently reported, with increased CSF levels of the chitinase changes in cerebral structure. The in vivo quantification of chitotriosidase 1 in ALS and high levels of GFAP in FTD (190). motor and extra-motor cortical cerebral changes with MRI The microglial transmembrane receptor (TREM2) represents analyses is of great value in monitoring disease progression another important molecule linked to microglial activity. One in the ALS-FTD continuum (Figure 2) (206–208). In sporadic study reported that the TREM2 p.R47H genetic variant may ALS cases, recent meta-analyses of neuroimaging studies report represent a risk factor for sporadic ALS (191), but this finding consistent gray matter loss in the precentral gyrus, inferior frontal has not been replicated in subsequent studies (192, 193). In gyrus, cingulate/paracingulate gyrus, and rolandic operculum addition, the soluble part of the microglial transmembrane (209, 210). This cortical thinning in extra-motor cortical receptor (sTREM2) is measurable in the blood and CSF, but the areas correlates with cognitive and behavioral impairment in information regarding its role within the ALS-FTD continuum the neuropsychological test (211, 212). On the other hand, is scarce (194). Finally, immune innate complement factors C9orf72 cases and a subgroup of sporadic cases without the involved in the inflammatory process have been studied in the C9orf72 expansion are characterized by widespread cortical CSF of patients with ALS finding an upregulation of C3 in CSF changes characterized by cortical thinning involving bilateral Frontiers in Neurology | www.frontiersin.org 7 January 2022 | Volume 12 | Article 750543 Reyes-Leiva et al. Extra-Motor Neurodegeneration in ALS FIGURE 2 | Group comparisons between those with amyotrophic lateral sclerosis (ALS) without cognitive or behavioral impairment (ALSno-cbi; left column), those with ALS with cognitive or behavioral impairment (ALScbi; middle column), and those with the behavioral variant of frontotemporal dementia (bvFTD; right column) compared to controls for (A) cortical thickness and (B) cortical mean diffusivity. Regions in blue represent thinner cortex; regions in green represent higher cortical mean diffusivity; and regions in purple represent lower cortical mean diffusivity. All analyses were adjusted for age, sex, education, and MRI equipment. Only clusters that survived family-wise error correction p < 0.05 are shown. ALSno-cbi, Amyotrophic Lateral Sclerosis without cognitive and behavioral impairment; ALScbi, Amyotrophic Lateral Sclerosis with cognitive and behavioral impairment; bvFTD, Behavioral variant of frontotemporal dementia. pars opercularis, fusiform, lingual and parietal cortex, and also changes in ALS, but more studies are needed to characterize smaller volumes in the right hippocampus and bilateral thalamus the longitudinal changes of cortical microstructure in ALS (158). (213, 214). On the other hand, longitudinal studies using DTI to study the Interestingly, Dadar et al. were able to replicate the white matter microstructure in ALS have shown the usefulness of neuropathological stages of TDP-43 in vivo with a novel this approach to detect extra-motor changes and monitor disease neuroimaging technique (215). However, some neuroimaging progression (222, 224–226). methods, which quantify brain atrophy (i.e., gray matter loss or cortical thickness), show limited sensitivity to the earliest cortical Functional Magnetic Resonance changes related to cortical dysfunction (158, 216). Diffusion fMRI based on the measurements of the fluctuation in tensor imaging (DTI) is a diffusion weighted imaging sequence blood flow and blood oxygen level as a consequence of that measures the diffusion of water molecules in the different neuronal activity helps us to trace the underlying networks directions of space (217). The main advantage of DTI is that it is between the areas that coactivate simultaneously. Assessing able to detect subtle changes at the microstructural level reflecting the resting-state brain connectivity in patients with ALS has the breakdown of biological barriers and neuroinflammation revealed either decreased or increased connectivity in premotor, (218, 219). Thus, DTI studies allow the study of microstructural sensorimotor, and basal ganglia (227, 228). Advanced network- changes of the cerebral cortex (i.e., cortical mean diffusivity) and based neuroimaging techniques based on connectomics and a the white matter (i.e., microstructural changes in different white whole-brain connectivity analysis have shown different patterns matter tracts). Several studies on neurodegenerative diseases, of motor and extra-motor involvement between different MND including FTD, have shown that DTI can reveal pathology phenotypes (229). that is not detected with cortical thickness measurements (216, 219–223). A recent cross-sectional study showed that Magnetic Resonance Spectroscopy ALScbi displayed cortical mean diffusivity changes in extra- MRS provides a means of measuring cerebral metabolites motor regions in the absence of cortical thinning and showed relevant to neurodegeneration in vivo [for a review, Kalra a better correlation with cognition than with cortical thickness. et al. (230), Ernst et al. (231)]. In ALS, neurochemical changes These results suggest that cortical mean diffusivity may be more reflecting neuronal loss or dysfunction (i.e., decreased N- sensitive than cortical thickness to detect extra-motor cortical acetylaspartate) are mostly significant in the motor cortex and Frontiers in Neurology | www.frontiersin.org 8 January 2022 | Volume 12 | Article 750543 Reyes-Leiva et al. Extra-Motor Neurodegeneration in ALS corticospinal tracts. Other neurochemical changes observed OTHER BIOMARKERS include increased myo-inositol, a putative marker of gliosis. Some studies also reported cerebral metabolite changes in extra- Neurophysiological Biomarkers motor regions. Previous studies performed in asymptomatic Neurophysiological studies have shown that hyperexcitability genetic carriers of mutations associated with FTLD using MRS- of the frontotemporal cortex is an early feature of ALS that derived markers exhibit an encouraging discriminatory ability to may precede motor neuron degeneration and can be assessed identify patients from healthy controls; however, more data are using transcranial magnetic stimulation with single or multiple needed to determine their ability to assist with the diagnosis in pulses (253). Transcranial magnetic stimulation with multiple early stages and in distinguishing from disease mimics (232–239). pulses evaluating the short-interval intracortical inhibition is one of the most robust tests developed for assessing cortical PET: 18F-Fluorodeoxyglucose Radiotracer hyperexcitability and has shown good sensitivity and specificity PET represents a powerful imaging biomarker for the study of for ALS diagnosis and a good correlation with other biomarkers neurodegenerative dementias (240, 241). An increasing number of peripheral neurodegeneration (254, 255). Recent studies of 18F-labeled tracers are not available for use at the clinical have demonstrated a more prominent cortical hyperexcitability site without the requirement of an on-site cyclotron, thus in patients with ALS presenting with cognitive symptoms turning brain PET scans into a widely applicable tool. Brain than in cognitively unimpaired patients with ALS (256). hypometabolism can be detected with PET and the 18F- Hyperexcitability in the sensory cortex may induce increased fluorodeoxyglucose radiotracer (18F-FDG-PET). Of note, 18F- amplitudes that can be detected early in ALS and correlate FDG-PET imaging has some advantages over MRI as it can be with a shorter disease survival (257). Some groups have tried performed in patients needing mechanical ventilation. Cerebral to assess cortical connectivity and the interaction between the metabolism reflects the local intensity of brain glutamatergic motor and non-motor network using other techniques, such as synaptic and astrocyte activity, and cerebral hypometabolism is electroencephalography and fMRI, but the clinical utility of these considered an indirect marker of neurodegeneration (240, 242). techniques is still limited (258, 259). In ALS, previous studies investigating 18F-FDG-PET revealed a marked frontal and prefrontal relative hypometabolism in Genetic Biomarkers MicroRNAs are short (about 22 nucleotides in length) non- ALScbi when compared to patients with ALSno-cbi (243, 244). coding RNA molecules that play a relevant role as endogenous Unexpectedly, previous studies also reported hypermetabolism regulators of gene expression (260). Emerging studies have shown in the midbrain, temporal poles, and hippocampus (245). It that circulating microRNAs can serve as potential biomarkers in has been hypothesized that hypermetabolism in patients with ALS. However, there are still many problems to be solved before ALS may be related to microglial activity in these areas but microRNAs could be used in clinical practice [for a detailed more multimodal biomarker studies are needed to understand review please refer to Wang and Zhang (261)]. the pathophysiological underpinnings of cortical and subcortical metabolism in ALS (206, 245, 246). Overall, previous studies support the view that 18F-FDG-PET could be a useful tool to DISCUSSION: ADVANCING ALS assess the spread of brain pathology in vivo. CLASSIFICATION WITH BIOMARKERS PET to Detect Neuroinflammation Phenotypic classification of ALS relies on clinical observations Multiple imaging studies have reported in vivo microglial such as site and region of onset and predominance of upper or activation in motor and extra-motor regions in both ALS and lower motor neuron signs, while the diagnosis of FTD is based FTD using different radiotracers with affinity to the translocator on clinical criteria (2, 24). Although ALS and FTD share partially protein or TSPO (which is expressed in the mitochondria overlapping patterns of neurodegeneration, the generation of of activated microglia) (247–250). Nevertheless, some caveats clinically defined subtypes is not straightforward due to the of those radiotracers might influence their usefulness; the observed clinical and prognostic heterogeneity. The definition of most used radiotracer 11C-PK11195, which labels peripheral precise and reproducible classification systems for both ALS and benzodiazepine binding sites, has a low signal-to-background FTD is urgently needed to guide present and future treatments, ratio and binding of the subsequent generation of radiotracers provide an indication of prognosis and enable the analysis in (11C-PBR28, 18F-DPA714, and 18F-FEPPA) is influenced by the clinical trials of homogenous groups for a more personalized TSPO polymorphism (247, 250–252). Thus, more work is needed approach to therapy (24, 262). These refined classification for the validation of radiotracers that can detect and monitor systems would ideally consider the etiology (i.e., proteinopathy microglial activation in vivo. responsible for the patient’s symptoms), other clinical, or biological features for robust disease staging at the single-subject PET to Detect TDP-43 Pathology level (i.e., scales measuring a motor or cognitive function, or The development of a radiotracer able to bind phosphorylated frontotemporal atrophy burden on neuroimage) and finally the TDP-43 and allow the in vivo visualization of pathological quantification of other pathophysiological processes that are TDP inclusions would be a major breakthrough in the field. relevant for prognosis (i.e., microglial activation at diagnosis). Unfortunately, such a biomarker is not available at this In the following section, we will discuss how biomarkers (alone moment (251). or in combination with multimodal biomarkers studies) could Frontiers in Neurology | www.frontiersin.org 9 January 2022 | Volume 12 | Article 750543 Reyes-Leiva et al. Extra-Motor Neurodegeneration in ALS be integrated into multidimensional classification systems to parallels the neuropathological stages of TDP-43 (215). further improve the diagnosis and prognosis of these complex However, neuroimaging studies investigating the structural neurodegenerative diseases. correlates of cognitive and behavioral impairment have failed to define a consistent topography of extra-motor Etiology: Early Identification of TDP-43 involvement (210, 212, 273). One limitation of previous studies is the application of neuroimaging techniques that may not Proteinopathies capture the earliest cortical changes driving cognitive and There is no definitive diagnostic test for ALS or due to behavioral impairment in ALS such as gray matter density abnormal TDP-43 aggregation (1). Because clinical-pathological or cortical thickness (274). On the contrary, other DTI- correlations are imperfect, biomarkers, which can detect based MRI biomarkers have been shown to be sensitive to abnormal TDP-43 aggregates in vivo, are needed to provide an the earliest neurodegeneration-related changes and may be early etiological diagnosis (i.e., ALS caused due to abnormal of great value to monitor disease progression (158, 207). TDP-43 vs. FUS or tau aggregates in FTD). Recently developed More studies are needed to determine the potential utility RT-QuIC assays for TDP-43 are promising (203). Concomitant of DTI-based imaging techniques for quantifying disease TDP-43 pathology can also be seen in a significant number of burden within first and second motor neurons and extra- patients with Alzheimer’s disease neuropathological changes, motor neurodegeneration. This information could allow the as well as in a smaller proportion of other neurodegenerative refinement of current classification systems and advance the field diseases (263–268). Thus, an understanding of how abnormal toward a multidimensional approach for the classification of TDP-43 aggregates impact brain function may also be neurodegenerative diseases (275). valuable to understanding its role in other neurodegenerative dementias. Of note, pathological TDP-43 aggregates are a key neuropathological finding in the recently described The Relationship Between Astroglial neuropathological entity named “Limbic predominant Age- Activity and Disease Progression in the Related TDP-43 pathology encephalopathy” or simply “LATE” ALS-FTD Continuum (269). This new neuropathological construct may account for As discussed previously, several biofluid biomarkers have up to 22% of the attributable risk of developing dementia of proven to be useful to predict disease progression in the Alzheimer’s type and has shown to impact the clinical patients with ALS. Due to the role of microglial reactivity presentation and prognosis in patients with Alzheimer’s disease in the neurodegenerative process, YKL-40 might be neuropathological changes. However, it is unclear whether useful for the prognostic stratification in the ALS-FTD “LATE” is the underlying pathology in cases with the FTLD spectrum (187). The combination of multiple biomarkers, TDP-43 subtype (263, 270). such as CSF cytokines and innate complement system A recent study on neuropathologically confirmed FTLD fragments in a single study may provide important clues suggests the existence of disparate patterns of gray and white to understand the role of neuroinflammation in ALS and matter involvement in FTLD subtypes, with more prominent FTD pathophysiology. However, one important limitation of involvement of white matter in tauopathies than in TDP- biofluid biomarkers is that they do not provide information 43 proteinopathies (271). We envision that the combination regarding the topography of neurodegeneration (190). A of cortical mean diffusivity with additional measures of recent study combined structural MRI with the use of a subcortical white matter microstructure and the use of fluid- PET ligand to measure microglial activation and showed based biomarkers of neurodegeneration and synaptic loss (i.e., that the areas of ligand binding were correlated with motor NfL, sAPPb) could reveal important insights into the etiology signs, cortical thinning, reduced fractional anisotropy and of neurodegeneration by identifying specific neurodegenerative increased diffusivity (248). Interestingly, this study did not signatures in vivo. This approach may also improve clinical- identify a change in ligand binding in subjects undergoing pathological correlations in some challenging clinical scenarios. repeat imaging over 6 months despite clinical progression, For example, it may help identify patients presenting with a non- suggesting that glial activation is present early and does fluent agrammatic variant of primary progressive aphasia with not change at least in terms of TSPO imaging. This study underlying TDP-43 pathology and possibly benefit from future illustrated the potential of multimodal biomarker studies protein-specific treatments (272). to understand the role of neuroinflammation in ALS. More studies and better tools are needed to clearly define how Disease Staging: Looking Beyond neuronal-glial signaling occurs at the different stages of Corticospinal Tract Neurodegeneration the disease, and where potential therapeutic interventions Imaging studies have consistently reported that extra- can be made to modulate inflammation to slow disease motor neurodegeneration in ALS is related to cognitive progression (113). impairment and that the frequency of cognitive and behavioral impairment seems to increase with disease progression Beyond TDP-43: Identifying Fast and Slow (33). Prefrontal changes detected with MRI in vivo predict Progressors the progression rate, and recent studies suggest that the Although TDP-43 is the major neuropathological hallmark topography of extra-motor atrophy in patients with ALS of the disease in most ALS cases, other pathophysiological Frontiers in Neurology | www.frontiersin.org 10 January 2022 | Volume 12 | Article 750543 Reyes-Leiva et al. Extra-Motor Neurodegeneration in ALS other hand, the development of novel imaging biomarkers more sensitive than conventional imaging biomarkers may increase our sensitivity for the detection of FTLD- related neurodegenerative changes by lowering the detection threshold. This, in turn, may allow at diagnosis the earliest clinical stages. Finally, pathophysiological biomarkers measuring the key aspects of the neurodegenerative process (i.e., neuroinflammation or FTLD subtype) could play an important role in future multimodal classification schemes, similar to the recently proposed biomarker- based classification systems for other neurodegenerative diseases (278). CONCLUSIONS FIGURE 3 | Representation of the increasing neurodegenerative burden in patients with ALS over time depending on their pathophysiological profile. The use of biomarkers may help to improve the current detection threshold The fact that some patients diagnosed with ALS develop (detection threshold and diagnostic window 1) from 1 to earlier clinical stages cognitive and behavioral changes while others remain and differentiating between profiles (detection threshold and diagnostic cognitively intact is intriguing. This observation may window 2). provide important clues to the specific vulnerability of the frontotemporal cortex in neurodegenerative dementias. As we have shown in this review, biomarkers have the potential to improve our understanding of the pathophysiological processes play an important role. Clinical-pathological studies underpinnings of extra-motor neurodegeneration in ALS. investigating the relationship between pathological aggregates Emerging imaging biomarkers have shown a significant of TDP-43 and cognitive impairment have revealed conflicting potential to improve the diagnosis and staging of patients results (33, 108). However, a recent study reported that within the ALS-FTD clinical continuum. In addition, pre-mortem cognitive performance was better correlated several biofluid biomarkers have the potential to increase with postmortem synaptic density than with postmortem the diagnostic certainty of underlying ALS or FTLD, cortical thickness or neuronal loss (109). In addition, cognitive predict disease progression at the single-subject level, and behavioral changes were better correlated with cortical and characterize the pathophysiological underpinnings of microstructure than with cortical thickness in a recent extra-motor neurodegeneration in ALS. Future studies for neuroimaging study involving patients from the ALS-FTD determining the benefit of current and future biomarkers to clinical continuum (158). advance ALS classification and monitoring are the essential Of note, early neurodegenerative changes and synapse loss steps for improving the design of clinical trials testing novel can be captured with novel radiotracers capturing synaptic disease-modifying treatments. density but to date, these novel imaging methods have not been assessed in ALS (276, 277). Taken together, these findings suggest that synaptic loss and other neurodegeneration-related changes AUTHOR CONTRIBUTIONS may antedate cortical atrophy and pathological aggregates of TDP-43 but additional studies are needed to clarify the II-G and DR-L designed and drafted the manuscript. OD- relationship between non-motor symptoms, synaptic density, I, EC-V, JT-S, NL, RB, OB, VM, DA, JF, AL, and RR-G cortical microstructure, and cerebral metabolism. In addition, the provided critical feedback and helped review and edit the exact significance of cortical mean diffusivity changes in ALS is manuscript. All authors contributed to the article and approved unknown. Future multimodal biomarker studies combining 18F- the submitted version. FDG-PET, cortical mean diffusivity, and measures of microglial activity and neuroinflammation will be helpful to understand FUNDING the pathophysiology of the earliest microstructural changes in ALS. This study was supported by the Fondo de Investigaciones Figure 3 illustrates the potential use of biomarkers to Sanitario (FIS), Instituto de Salud Carlos III (PI21/00791 advance the diagnosis of ALS in earlier clinical stages to II-G, PI13/01532 and PI16/01825 to RB, PI14/1561 and when future targeted therapies may be more effective. PI17/01896 to AL, and PI15/01618 and PI19/01543 to RR- The information provided by biomarkers at diagnosis G), the CIBERNED program (Program 1, Alzheimer’s Disease could also be considered for the prediction of disease to AL and SIGNAL study, www.signalstudy.es), and partly progression and the selection of candidates for clinical jointly funded by Fondo Europeo de Desarrollo Regional, trials. This information may be crucial for the candidate Unión Europea, Una manera de hacer Europa. This work selection for future trials targeting a specific aspect of ALS was supported by Departament de Salut de la Generalitat pathophysiology (i.e., anti-inflammatory drugs). On the de Catalunya, Pla Estratègic de Recerca i Innovació en Salut Frontiers in Neurology | www.frontiersin.org 11 January 2022 | Volume 12 | Article 750543 Reyes-Leiva et al. Extra-Motor Neurodegeneration in ALS (SLT002/16/00408 to AL), Fundació La Marató de TV3 (044412 ACKNOWLEDGMENTS to RB and 201437.10 to RR-G). II-G was supported by the Juan Rodes grant (JR20/0018) and the Pilot Awards for DR-L, EC-V, JT-S, and RR-G are members of the European Global Brain Health Leaders (GBHI ALZ UK-21-720973). This Reference Network for Neuromuscular Diseases and work was also supported by an Alzheimer’s Association Grant members of Xarxes d’Unitat d’Expertesa Clínica en Malalties (AACSF-21-850193). Minoritàries (XUECs). REFERENCES frontotemporal dementia and association with clinical deterioration. JAMA Netw Open. (2021) 4:e211290. doi: 10.1001/jamanetworkopen.2021.1290 1. Brown RH, Al-Chalabi A. Amyotrophic lateral sclerosis. N Engl J Med. (2017) 17. Woolley JD, Khan BK, Murthy NK, Miller BL, Rankin KP. The 377:162–72. doi: 10.1056/NEJMra1603471 diagnostic challenge of psychiatric symptoms in neurodegenerative disease: 2. Elahi FM, Miller BL. A clinicopathological approach to the diagnosis of rates of and risk factors for prior psychiatric diagnosis in patients dementia. Nat Rev Neurol. 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