In the current study, sNfL and cerebellar metabolite levels were determined in a cohort of 20 SCA3 patients to examine associations between the two measures. Compared with controls, we found high NfL levels and lower cerebellar metabolite levels, including NAA/Cr and NAA/Cho ratios, in 20 ATXN3 expansion carriers. Both measures—cerebellar metabolite and NfL levels—correlated with greater disease severity in our patients with SCA3. In addition, elevated NfL levels were associated with lower cerebellar metabolite levels; especially, low levels in NAA/Cr values are known to indicate cerebellar degeneration in ATXN3 repeat expansion carriers. As such, sNfL levels and cerebellar metabolites capture abnormalities in SCA3 patients, and thus, they may serve as complementary biomarkers for disease detection and future treatment monitoring.
In SCA3 disease, polyQ-expanded ATXN3 leads to protein neurotoxicity and causes selective neuronal cell death along with metabolic/cellular changes detected by MRS, including decreases in acetylcholine and NAA [5]. Emerging evidence has shown early neurochemical alterations in patients with SCA3 in the preclinical stage, even before brain atrophy [18]. In this regard, the measure of the NAA/Cr ratios has been found to be a good neuroimaging biomarker and can be used to monitor the clinical progression of SCA3 [4, 19]. In the current study, our findings demonstrated a decrease of the NAA/Cr and NAA/Cho ratios in the dentate nucleus, cerebellar vermis, and cerebellar hemisphere, suggesting that the degree of neuronal dysfunction in SCA3 would be consistent with previous findings [5, 20]. Associations between ataxia severity and metabolic/cellular changes have been described as reflected by NAA/Cr ratios. Our findings also showed that ICARS scores were negatively correlated with NAA/Cr ratios in the three ROIs of the dentate nucleus, cerebellar vermis, and cerebellar hemisphere in 20 SCA3 patients, which is in line with previous results [5, 21]. Furthermore, we found that SARA scores had an inverse relationship with NAA/Cr ratios in these ROIs. These results further confirmed that the metabolic alterations of the cerebellum were correlated to the predominant clinical features in SCA3 patients and suggested that NAA is a good marker for SCA3 [4, 5].
Neurofilament light protein can be released in significant quantities following neuro-axonal damage or neurodegeneration. The elevation of sNfL is considered a biomarker of clinical disease severity in SCA3 [12, 22, 23]. Higher NfL levels are not only correlated with ataxia symptoms and non-ataxia signs in ataxic SCA3 patients, but they show similar correlations in preclinical SCA3 individuals [13, 23]. Our findings were consistent with a previously published study on SCA3, which also found that NfL was elevated in patients with SCA3 and that NfL levels correlated with ataxia symptoms [13]. This correlation between elevated NfL levels and clinical severity could persist after accounting for the potential confounder of age.
In our patients with SCA3, both higher sNfL levels and decreased metabolites in the cerebellum significantly correlated with clinical severity, as reflected by the ICARS scores. Therefore, we hypothesized that the elevation of NfL values may be correlated with a lower cerebellar NAA/Cr ratio. Our study also explored the association between NfL and MRI features in a subgroup of ataxic SCA3 patients. First, our ROI analysis showed that certain cerebellar regions with a lower NAA/Cr ratio tended to be significantly associated with higher NfL levels, which included regions in the dentate nucleus, cerebellar vermis, and cerebellar hemisphere. Furthermore, higher NfL levels also showed an association with a reduced NAA/Cho ratio in the cerebellar vermis and cerebellar hemisphere, but a similar association was not found in the dentate nucleus in our analyses. Third, unexpectedly, there was no significant relationship between increased NfL and the Cho/Cr ratio in the cerebellum. One potential explanation for this result is that the Cho/Cr ratio was not observed to decrease in our patients. This finding suggests that the elevation of NfL was mainly associated with neuronal dysfunction rather than cell membrane or neurotransmitter metabolism impairment in our SCA3 patients, likely indicating the subtle impairment of neurofilaments in the cerebellum. Regarding these findings, further studies are needed to explore the association between the NAA/Cr ratio and sNfL in SCA3.
Both higher NfL levels and decreased cerebellar metabolites have been suggested to reflect neuronal dysfunction not only in SCA3 but also in other degenerative diseases [24–27]. In our samples, we found both measures—NAA/Cr ratios and sNfL levels—to be significant predictors of clinical severity, reflected as ICARS scores. However, it is difficult to estimate the relevance of sNfL levels because changes in these levels may possibly result from shifts of NAA/Cr, NAA/Cho, or both metabolites simultaneously. In this study, a mediation model based on a multiple regression approach helped us clarify how prominent the contribution of each component was. As the limited statistical power did not allow us to test a model with two mediators (NAA/Cr and NAA/Cho), we used two separate models with either the NAA/Cr ratio or NAA/Cho ratio as the mediator; the results indicated that in our patients with SCA3, the prediction of clinical severity by NfL was more likely to be driven by changes in the NAA/Cr ratio (Table 3; Fig. 3) but not changes in the NAA/Cho ratio (Supplemental Table 2; Supplemental Fig. 1). sNfL levels and cerebellar metabolites of NAA/Cr ratio are both the biomarkers of neuro-axonal damage in patients with SCA3. The serum NfL levels often reflect the neuron damage in the whole brain, while NAA/Cr ratio reflects the damage in part of the cerebellum in SCA3, which may explain why NAA/Cr ratio acts as a mediator. Considering the limited sample size and possible VOI localizations detected by MRS scanning, methods based on multiple regression analysis appear to improve our understanding of the cerebellar metabolic process in the SCA3 cohorts above conventional analysis of variance; they also provide more evidence of the putative mechanism by allowing testing of specific mechanistic models.
Table 3
Summary of Model Coefficients: Effect of serum NfL on clinical severity Mediated by NAA/Cr ratio Moderated
Model 1a | | NAA/Cr ratios |
| | F(2) = 4.9733, p = 0.0199*, R2 = 0.3691 |
| | coef | SE | t | p |
NfL | a | -0.0155 | 0.0053 | -2.9471 | 0.009 |
age | f | -0.0045 | 0.0042 | -1.0620 | 0.3031 |
Constant | | 1.6538 | 0.2501 | 6.6126 | < 0.001 |
Model 1b | | ICARS scores |
| | F(3) = 8.8199, p = 0.0011**, R2 = 0.632 |
| | coef | SE | t | p |
NfL | c' | 0.2232 | 0.2578 | 0.8656 | 0.3995 |
NAA/Cr | b | -25.7773 | 9.6889 | -2.6605 | 0.0171 |
age | g | 0.422 | 0.1738 | 2.4273 | 0.0274 |
Constant | | 33.9433 | 18.8833 | 1.7975 | 0.0911 |
Direct effect | | NfL-ICARS |
| | coef | SE | t | p |
| | 0.2232 | 0.2578 | 0.8656 | 0.3995 |
Indirect effect | | NfL-NAA/Cr-ICARS |
| | coef | BootSE | BootLLCI | BootULCI |
| | 0.3989 | 0.2538 | 0.0291 | 0.9927 |
Several potential limitations restrict the interpretability of our findings. First, the limited sample size was not sufficient to draw an affirmative conclusion in our cohort; thus, the findings need to be further validated in a larger cohort. Second, information on asymptomatic SCA3 patients was not available, which makes it difficult to analyze the correlation between cerebellar metabolites and sNfL levels. Third, not only should the neurochemical values of NAA, Cho, and Cr be included, but Gln, Glu, and Tau should also be determined; doing this could help to further explore the correction with sNfL in future research. Finally, while the mediated moderation analysis in our present study provides evidence for an associated effect, we need to acknowledge that effects were only significant for ICARS (but not SARA) scores and need to be replicated in future research.
In summary, we used a multimodal approach combining the serum biomarker with cerebellar metabolites to investigate the association between NfL and indicators of neurodegeneration in 20 SCA3 patients. To the best of our knowledge, this is the first study to examine correlated NfL levels with the cerebellar metabolites reflected by the NAA/Cr ratio in ATXN3 mutation carriers. Our results confirmed that sNfL levels are increased in SCA3 and that they are correlated with clinical disease severity, reflected as ICARS and SARA scores, as well as brain metabolic changes. We demonstrated that the NfL level and cerebellar metabolites were promising complementary biomarkers that capture the effects of the expansion ATXN3 mutation. In addition, our multiple regression models revealed that the decrease in the NAA/Cr ratio mediated this prediction effect. Additional longitudinal studies are strongly warranted to build on these results in the future.