Early MRI observations of iron accumulation in the subcortical nuclei of MSA patients has well-established iron dysregulation in the pathophysiology of MSA.(31, 32) Elucidating its mechanism is crucial for the understanding of early disease pathogenesis. We used human post-mortem brain tissue of MSA patients to characterize pathologic iron deposition in the early-affected regions at the cellular level, revealing disease and subtype-specific alterations representing iron dyshomeostasis in MSA.
In the wide spectrum of neurodegenerative diseases that are associated with elevated brain iron levels, MSA is often simply thought of as a disease with a high burden of iron specifically in the putamen.(33) Our histological evaluation of the subcortical iron load, however, highlighted heterogeneous distribution of iron across the disease cohort that showed distinct patterns by the neuropathological disease subtypes (Fig. 1). Although examination in a larger cohort is required to confirm the stratification, a strong linear relationship between the relative inclusion and iron load in the putamen compared to the pons base and the GP (Fig. 1b), as well as consistent findings in iron-sensitive MRI of MSA-C and MSA-P patients support our results.(38, 39) Contrasting with the uniform deposition of subcortical α-syn pathology in early disease, distinct patterns of iron accumulation in the same regions suggest that iron-associated pathomechanisms may differ in the two disease types, further stratifying SND and OPCA subtypes as distinct pathogenic entities.
To further comprehensively explore these findings in more detail, we evaluated the distribution of subcortical iron deposition detected by DAB-enhanced Perl’s staining in different cell types: neurons, astroglia, microglia, and oligodendroglia. Consistent with observations in Alzheimer’s Disease,(40) PD,(41) and in normal aging,(42) iron accumulation in MSA was prominently observed in the microglia across all regions examined. As activated subcortical microglia is an early feature of MSA disease progression, phagocytic processing of cellular debris in the extracellular space most likely contributes to the observed microglial deposition of iron in the diseased brains. Such iron-infiltrated microglia may act as an important player in disease pathogenesis by ferroptosis-induced neurodegeneration. A recent study using human iPSC-derived tri-culture system of microglia, astrocytes, and neurons to examine the contribution of iron accumulation to neurodegeneration has demonstrated microglia to be highly sensitive for iron-related changes and susceptible to ferroptosis, consistent with findings in single cell culture vulnerability assays.(43, 44) Importantly, the removal of microglia from the same tri-culture system prevented neuronal cell death, further supporting an important role of microglial iron loading in the disease pathogenesis.(43) Our disease subtype-specific evaluation of cellular iron accumulation, however, reinforces different iron-related mechanisms by subtypes. In contrast to the SND subtype which consistently showed highest microglial iron load in the subcortical regions, the OPCA subtype shows a heterogeneous pattern across these regions in which astrocytes demonstrate either similar or higher burden of iron than that of the microglia. Astrocytes are found to be relatively resistant to iron-toxicity as well as serving a protective role against ferroptosis, further supporting distinct contributions of iron dyshomeostasis to disease progression in MSA-SND and MSA-OPCA.(43–45)
Examining iron accumulation in α-syn-affected cells distinctly, only 8.61% and 7.05% showed GCI-positivity for iron deposition in the GP and the putamen, and 28.93% in the SN. This is in sharp contrast with our previous observations in PSP,(34) where in the same subcortical regions, 74.50% and 65.25% of tau-affected astrocytes were positive for iron deposition in the very early-affected GP and the SN respectively.(34) Such distinction suggests that iron-associated pathomechanisms differ across neurodegenerative proteinopathies distinctively in relation to the build-up of protein pathologies. Furthermore, NCIs were negative for iron deposition in the examined nuclei of MSA brains. Although the loss of early-affected neuronal cells could account for the lack of association, evidence for neuronal mechanisms to minimize iron uptake in physiological and pathological conditions of elevated iron levels suggest otherwise.(43, 46, 47) Parallelly, the level of cellular iron deposition is also generally lower in MSA compared to that in PSP.(34) Accordingly, iron was notably observed in spaces outside the cellular bodies in the examined nuclei of MSA brains, which may function in pathological processes such as oligomer seeding, but importantly in neurodegeneration by reduction of ferric iron to ferrous iron by extracellular ferrireductases, and the production of toxic ROS by participation of ferrous iron in the Fenton reaction.(27, 48) Together with a general up-regulation of iron homeostatic genes in the same regions of MSA brains as revealed by Nanostring gene expression analysis, our findings suggest a mechanism of accelerated cellular iron cycling in MSA which builds up in the extracellular spaces to induce local toxicity (Fig. 5). The absence of significant changes in the mRNA expression levels of the cellular iron export protein, ferroportin, as well as its regulatory proteins, ceruloplasmin and hephaestin, supports the maintenance of an intact cellular iron cycling in the diseased brain.(48, 49) Moreover, our correlation findings between subcortical iron levels and shorter duration of disease further support this concept. In the SN, significant dysregulation in the gene expression of hemoglobin-alpha and neuroglobin provide novel insight to the possible underlying mechanisms of hypoxic pathways in MSA. Chronic hypoxia has been demonstrated to be involved in the pathophysiology of MSA by a significant increase in the protein levels of HIF2α marker in the SN compared to PD and age-matched controls.(50) Meanwhile, neuroglobin is thought to function in the scavenging of ROS and has been linked to neuroprotection in different neurodegenerative diseases by an overexpression in stroke, hypoxia, and ischemia.(51–55) The contradicting down-regulation of neuroglobin in MSA suggests a faulty compensation for oxidative stress which may exacerbate neurodegeneration and disease progression.
Evidence of the interchangeable relationship between iron and α-syn accumulation suggests a vicious feedback cycle contributing to early disease progression, although the primary event in the pathogenesis is yet undeciphered. We can propose four possible scenarios: 1) iron accumulation is consequent of α-syn pathology, 2) iron accumulation directly promotes α-syn pathology, 3) iron accumulation protects from, or hinders the accumulation of misfolded α-syn, and 4) iron dyshomeostasis and α-syn accumulation are parallel pathological phenomena which may converge over the course of the disease progression. Our first cellular mapping of iron accumulation in the vulnerable regions of human post-mortem MSA brains reveals selective cellular vulnerability patterns in pathologic iron accumulation that are distinct in MSA-OPCA and MSA-SND, demonstrating subtype-specific iron-associated disease mechanisms in MSA that has not been found for hypoxia-related events.(50) Neuropathological studies demonstrate that clear distinction of SND and OPCA type of MSA is difficult as the pathology shows significant overlap. A study on 100 MSA brains showed that 34% of the cases were SND- and 17% were OPCA-predominant, while the remainder (49%) had equivalent SND and OPCA pathology.(56) Accordingly, parkinsonian and cerebellar classification of MSA is made on the basis of early predominating clinical symptoms. However, our post-mortem study shows that even at end-stage disease, distinct patterns of regional iron burden correlate with, and characterize, the disease subtyping of MSA-P and MSA-C, suggesting that iron dysregulation may not be a consequence of α-syn pathology, but rather an early-effector that stratifies MSA subtypes. To further explore this phenomenon, we compared hierarchal clustering of our MSA cases using the deposition patterns of α-syn pathology and iron (Fig. 3). In line with the neuropathological studies discussed above, the subcortical α-syn load did not effectively cluster cases by disease subtypes. However, the cellular iron deposition in the four populations (regardless of α-syn pathology) perfectly clustered the MSA cases by subtypes, demonstrating closest proximity between cases of the same subtype. Therefore, we reveal cellular vulnerability pattern to pathological iron deposition as a novel neuropathological feature that effectively predicts MSA parkinsonian and cerebellar subtype, compared to α-syn pathology. Such relationship further reinforces the involvement of iron dysregulation in the disease pathogenesis. Accordingly, cellular iron burden in MSA associates better with neurodegeneration (as presented as clinical symptoms) than the α-syn load, further supported by MRI studies demonstrating correlation between iron deposition and local atrophy in MSA patients.(57–59)