We combined in vivo and ex vivo approaches to characterize and validate alterations in metal deposition that occur in the brain in HD; our findings suggest a complex alteration in metal homeostasis that may have profound clinical implications not only for HD but also for other neurodegenerative disorders. Using MR imaging, we found that concentrations of paramagnetic metals were elevated in pre-HD, long before the presence of clinical symptoms, but were distributed in a regionally selective pattern that recapitulates the neuropathology of HD. Indeed, the progressive regional increases in the MR imaging signal corresponded to those suggested previously, namely, the basal ganglia and cortex, including the sensorimotor, parietal, and occipital regions.21
The most significant cortical elevations were present in stage 3 HD, in which motor and cognitive symptoms are generally prominent but patients remain ambulatory and independent for many activities of daily living. The ex vivo work using inductively coupled plasma mass spectrometry confirms that these increases correspond to aberrant regional iron deposition in the brain in HD.
Iron, stored within ferritin, is thought to provide most of the paramagnetic metal signal in the brain; however, copper and manganese could also have contributed to our findings if they are sufficiently altered as well. Although ferritin is a storage form of iron, its increase suggests greater availability to do damage through some combination of more transport of iron into the brain, reduced efflux from cells, or redistribution from other compartments (ie, alteration of brain homeostasis). In addition to ferritin, iron is present in the brain, bound to various transporters, and is a cofactor for many metalloproteins.22
In its ferrous form, it can react with hydrogen peroxide (produced by mitochondria) and molecular oxygen to produce hydroxyl radicals (Fenton reaction). Hydroxyl radicals can further release iron (II) from iron-sulfur centers and other iron-containing compounds, promoting greater oxidative stress and perhaps further increasing the MR imaging signal if ferritin increases as a result. Any increase in the exposure of iron to cellular biomolecules could readily potentiate neurodegeneration, and indeed there is extensive evidence for early and profound oxidative damage to proteins, lipids, and nucleic acids in the HD brain.1
Even small metal elevations greatly increase cellular oxidative stress through redox reactions,23
have negative consequences for energy metabolism,24
and may affect signal transduction and gene transcription25
and result in DNA damage and eventually cell death.
At the cellular level, iron (ferritin) is predominantly stored in oligodendroglia in the brain but is also significantly present in microglia, astroglia, and neurons, each of which contain iron transport and storage systems. Increases that we have detected may or may not be pancellular depending on the underlying cause of the iron increase. Notably, the density of oligodendroglia is elevated in the HD brain,26
even from individuals with pre-HD,27
and it is possible that oligodendrogliosis contributes to the increase in FM values. The early aberrant accumulation of iron in the basal ganglia suggests that iron-induced oxidative stress, as well as altered regulation of iron-dependent enzymes, could have an important role in the early selective vulnerability of the basal ganglia during pre-HD.
Copper has been previously reported to be elevated in human HD postmortem putamen28
but not in other brain regions. It has also been reported to be increased in the R6/2 transgenic mouse model of HD but not in the CAG140 knockin model.11
In the present study, we did not detect increased copper in the human brain. Zinc was increased in the putamen and pallidum; however, it is a diamagnetic metal and is unlikely to contribute significantly to the FM signal. Notably, manganese was decreased in the cortex, perhaps because of depletion of the antioxidant manganese superoxide dismutase or displacement by iron of its transport by transferrin.
Modeling of the MR imaging data using a predictive formula for time to HD onset in individuals in the pre-HD group and for clinical progression in the HD group, as defined by the TFC, demonstrated the gradual accumulation of paramagnetic metals in the basoganglia as patients neared onset, followed by an apparent accelerated accumulation after clinical diagnosis. Notably, the DNA damage marker 8-hydroxy-2-deoxyguanosine, which is released from the brain in HD into the circulation and urine,29
is detected at low levels in pre-HD and at much higher levels once symptoms have manifested,30
a possible molecular correlate of oxidative damage that may be caused by accumulating metals. The upward inflection in both of these markers might indicate the onset of clinical disease.
In summary, our findings indicate that metal homeostasis is disturbed in HD, as measured by MR imaging and validated in postmortem brain. These results implicate metals as potential therapeutic targets and suggest that measurement of metals by MR imaging could provide useful markers of HD progression and response to treatment. Detailed examination of metal absorption, transport, and storage mechanisms, as well as the regulation of the metalloproteins that use metals, will be necessary to fully understand these findings.