In a relatively large cohort of ALS patients, we revealed that the pattern of amygdala abnormalities in ALS patients was substantially different among patients at different King’s clinical disease stages. In the present study, amygdala subnucleus volumes were unaltered at King’s stage 1. However, ALS patients at King’s stage 2 had significantly reduced left ABN and CAT volumes compared with HCs. Importantly, global amygdala atrophy and abnormal amygdala-based resting-state FC alterations were detected only in patients at King’s stage 3. Moreover, amygdala atrophy was associated with global cognition, and reduced volumes of specific subnuclei were independently correlated with anxiety, but not depression, in patients with ALS after controlling for age, sex, and TIV. Thus, amygdala abnormalities may play a more important role in emotional and cognitive impairments in ALS than was previously thought [20, 21].
Numerous neuroimaging studies have been conducted to examine the incidence of amygdala atrophy in ALS patients; however, these studies have generated inconsistent results [20–25]. Some previous studies, using either shape or volume analyses, reported that the amygdala did not differ significantly between ALS patients and HCs, which is consistent with our findings in patients with ALS at King’s Stage 1 [22, 25]. Consistent with the pattern observed in our patients with ALS at King’s Stage 2, Finegan and colleagues reported that, compared with HCs, ALS patients had reduced left amygdala volumes, whereas right amygdala volume remained unaffected in a group of advanced-stage ALS patients [with mean ALSFRS-R scores of 36.6] [21]. Moreover, using voxel-based morphometry analysis of grey matter structures, Menke et al. reported progressive reductions in bilateral amygdala volumes in patients with ALS during a longer period of follow-up, which is consistent with our findings in patients with ALS at King’s stage 3 [23]. Additionally, Pinkhardt et al. reported that a group of patients with definite ALS without dementia trended to have reduced amygdala volumes [24]. However, none of these studies analysed amygdala subnuclei volumes. Recently, Chipika et al. reported significantly reduced ABN and CoN volumes in a large cohort of patients with ALS compared with healthy participants, which is similar to our findings of the ALS patients at King’s stage 2 in the present study, and they suggested selective atrophy of amygdala subnuclei is a consistent feature of patients with ALS [20]. Our findings support their viewpoint that amygdala subnuclei may be nonuniformly affected by ALS pathology, and we further suggest that the inconsistencies among previous studies might largely result from averaged biophysical indices from affected and unaffected stages’ patients, because the pattern of amygdala atrophy in patients with ALS differs substantially at different disease stages. However, none of the previous studies used the well-validated King’s clinical staging system to divide ALS patients into homologous subgroups at different disease stages.
In the present study, right amygdala–cuneus connectivity was significantly increased in ALS patients at King’s stage 3. Similarly, in an 18fluorodeoxyglucose positron emission tomography study, Laere et al. reported that patients with ALS have clusters of relative hypermetabolism in the amygdala [36]. Using echo-planar spectroscopic imaging, Verma et al. demonstrated that the N-acetylaspartate/creatine ratio [a biomarker of neuronal integrity] is significantly lower in the right cuneus of patients with ALS [37]. The cuneus is a hub of the visual association cortex and may play an important role in visual information processing [37, 38]. Moreover, the cuneus also seems to participate in multisensory information integration and cognitive processes [37, 38]. Using 11C-flumazenil positron emission tomography, Wicks et al. reported that decreased 11C-flumazenil binding in the cuneus is related to confrontation naming impairment in patients with ALS [39]. Thus, the abnormal amygdala–cuneus resting-state FC in the present study may represent a compensatory change in response to structural damage in patients with ALS [23]. Consistent with our findings, Menke et al. recently also reported a mixed picture of widespread grey matter volume decreases and resting-state FC increases in patients with ALS over 2 years of follow-up, which is compatible with compensatory responses [23]. However, these findings need to be confirmed by further studies.
Another key finding of the present study was that amygdala atrophy was significantly related to anxiety [but not depression] and global cognitive deficits in ALS patients. The amygdala is an important hub of the limbic system and plays a pivotal role in cognitive and emotional processing. However, few studies have focused on the associations between amygdala abnormalities and neuropsychiatric symptoms that occur over the course of ALS [9, 22, 25]. The ABN has a cortical-like profile and forms a connection between LN and CeN, and its outputs project to several anxiety-related brain regions [11, 40, 41]. ABN activation can suppress high-anxiety states and fear-related freezing, whereas inhibition of the ABN increases anxiety and freezing [10, 11, 41]. The CoN can mediate aversive, defensive, and reproductive responses [40, 42]. Thus, it is not surprising that smaller ABN and CoN volumes were associated with anxiety in patients with ALS in the current study, although further studies are needed to investigate causality. Similar to our findings, Vriend et al. reported smaller left amygdala volumes in anxious PD patients, and suggested that amygdala volume was related to anxiety [19]. Furthermore, España et al. reported that intraneuronal β-amyloid accumulation in the amygdala may enhance anxiety and fear in an AD mouse model [43]. Previous studies have also demonstrated that an accumulation of AD pathology in the amygdala is detrimental to cognition in preclinical AD patients, and that amygdala atrophy may be an early marker of AD [44–46]. Moreover, Bouchard et al. reported that a smaller amygdala is associated with cognitive deficits in PD [47]. 48 Ahveninen et al. demonstrated that amygdala volumes are significantly reduced in patients with Huntington's disease, and are associated with global cognition [48]. Recent studies have suggested that cognitive impairments might worsen across King’s stages in patients with ALS, and may also correlate with pathological TDP-43 accumulation in corresponding cortical regions [5, 49]. Thus, our findings provide important evidence to support these studies, and further highlight that cognitive competency is not completely dependent on cortical integrity, but that subcortical abnormalities may also play a role in cognitive impairments in patients with ALS.
Overall, our findings suggest that amygdala abnormalities are an important feature of ALS, and that smaller amygdala volume is related to anxiety in these patients. Because medication currently available for ALS patients have limited efficacy, physicians should focus on improving prognosis and quality of life [2]. Importantly, if our findings are confirmed, they indicate that patient’s anxiety cannot be entirely attributed to receiving such a devastating diagnosis; the neurodegenerative process of ALS also seems to be involved [7, 8]. It may be possible to improve anxiety in ALS patients using both pharmacological and non-pharmacological approaches [50]. These approaches might alleviate disease progression and improve patient’s quality of life, and may become a key component of individualised therapy for ALS patients [50]. However, although ALS patients experience negative effects from anxiety, it remains under-recognised in clinical practice [8]. We therefore propose that the identification and management of anxiety in ALS should be given more attention.
Inevitably, the present study had several limitations. First, this study used a cross-sectional design, which prevented the establishment of causality between amygdala abnormalities, anxiety, and cognitive deficits. Thus, causality remains to be validated in further studies. Second, all included patients with ALS in the present study were newly diagnosed, and only three patients were classified as King’s stage 4. Thus, our study had a complete lack of patients with King’s stage 4 (nutritional or respiratory failure); however, ALS patients have less homogeneity in stage 4 compared with the other three stages, and it is commonly difficult for these patients to complete an MRI scan [27, 32]. Moreover, in this consecutive cohort, although there were no significant differences between the three patient subgroups in age and we used age, sex, and TIV as covariates, ALS patients at King stage 3 were (on average) 8.4 years older than patients at King stage 1, which is consistent with some previous studies [5, 51]. Similarly, in a population-based study, Manera et al. reported three regions were functionally involved in 196 patients with ALS (18.5%) at diagnosis, and 180 patients (91.8%) were older than 60 years [51]. The onset of ALS appears to involve a multistep process, and aging seem to be one of the processes and may accelerate the neurodegeneration of ALS. However, these findings need to be discussed by further studies. Third, we only used the MMSE, BNT, AVLT, and FAB to screen cognitive function in the present study, and we did not use ALS-specific tests, for example, Edinburgh cognitive and behavioural ALS screen [5]. However, these methods are included in our ongoing work, and only two patients with ALS were unable to complete the cognitive assessments in our study. Fourth, our results were also susceptible to selection bias, because the ALS patients who visit our centre commonly have a relatively short disease course (we are the largest ALS centre in the Shandong province). Thus, our findings need to be confirmed by population-based studies. Fifth, in the present study, we did not use any amygdala subnucleus as region of interest to explore resting-state FC alterations because of their relatively small size. Finally, we did not perform any genetic testing. However, the ALS patients included in this study were sporadic cases, and very few sporadic ALS patients in China carry known genetic mutations [52].
In conclusion, our study provides a comprehensive profile of amygdala abnormalities in ALS patients. The pattern of amygdala abnormalities in these patients differed greatly across King’s clinical disease stages; our findings suggest that amygdala abnormalities are an important feature in patients with ALS at relatively advanced stages. Moreover, specific amygdala subnucleus atrophy may play an important role in anxiety and cognitive impairment in these patients.