Clinical evaluation using the NMDAS rating scale showed that all 14 patients displayed symptoms consistent with cerebellar ataxia. In these patients, the severity of ataxia varied without any relation to the mtDNA defect type (), with the highest levels of impairment observed in patients with either m.3243A>G (Patient 6), m.8344A>G (Patient 8), single, large-scale mtDNA deletion (Patient 11), or recessive POLG mutations (Patients 12 - 14).
General Neuropathological Findings
The majority of patients had variable degrees of cerebellar abnormalities; there was variable and selective loss of one or more neuronal types among Purkinje cells, olivary and dentate neurons and/or granule cells. Evidence of myelin loss, dendritic abnormalities and axonal degeneration were apparent in patients with a high degree of neuronal cell loss (). In accordance with previous findings in Kearns-Sayre syndrome (KSS), we found myelin loss and spongiform degeneration of the white matter tracts of Patient 11 () (18
). We recently discussed the mechanisms underpinning loss of myelin in this patient and attribute these changes to oligodendrocyte dysfunction (27
). Other patients with different mtDNA defects also showed varying degrees of myelin loss but this was likely secondary to axonal/neuronal loss because, in general, it appeared to occur in regions affected by severe neuron loss. Patients with the m.3243A>G mutation typically exhibited intact myelin with only mild myelin pallor in 1 case (Patient 5; ) and selective myelin loss within regions of microinfarcts. Several patients had profound myelin loss of the dentate nucleus outflow tract, which correlated with marked neuron loss from the dentate nucleus (). These included patients with m.8344A>G, m.14709T>C and recessive POLG
Figure 1 Myelin loss, axonal and dendritic pathology in patients with mitochondrial DNA (mtDNA) defects are likely to be secondary to neuronal cell loss in all cases except Kearns-Sayre syndrome (KSS). (A) Relative to control (i; Loyez) there is mild myelin pallor (more ...)
Synaptic disorganization assessed using synaptophysin immunohistochemistry was prominent in many cases. The main changes were in the dentate nucleus. There was often an absence of presynaptic terminals on neurons in patients with extensive Purkinje cell loss compared to controls (), indicating loss of input to the dentate nucleus. In contrast, Patient 9 was the only case in which there were large, intensely labeled synaptic-like structures lacking morphologically distinguishable post-synaptic neurons, possibly surrounding “ghosts” of degenerating neurons (). This suggests relatively increased input to the dentate nucleus because the extent of the dentate neuron loss exceeded the Purkinje cell loss in this patient.
Axonal and dendritic abnormalities were observed in almost all patients and were particular prominent in Purkinje cells. Most notably, axonal ‘torpedoes,’ or swellings, were present in the proximal portion of Purkinje cell axons. These swellings largely consisted of neurofilaments and mitochondria and were localized in the Purkinje cell, granular cell and molecular cell layers (). Axonal spheroids in the deep white matter were often seen in conjunction (). Dendritic abnormalities consisted of an increase in dendritic arborization, thickening of the dendrites and evidence of ‘trapped’ mitochondria (). Often the dendritic trees became more expansive in those Purkinje cells neighboring regions where cell loss had occurred, suggesting a compensatory mechanism.
There was evidence of cerebellar atrophy with increased inter-folial spaces, mainly due to widespread atrophy of the molecular layer and loss of granule cells. This was typically observed in patients with the m.3243A>G mutations (Patient 5; ). The presence of small, shrunken and eosinophilic “dark” neurons was the most widespread morphological alteration (Patient 8; ).
Figure 2 Variation in neuron loss across the olivo-cerebellum in patients with mitochondrial DNA (mtDNA) mutations. (A) Inferior olivary neuronal densities vary remarkably according to the mtDNA defect vs. control tissues (n = 2) (i). Cell loss is most severe (more ...)
Variations in Neuronal Cell Loss in Different Areas of the Olivary-Cerebellum
Quantification of neuronal cell density revealed significant variations in cell loss across the olivary-cerebellum in relation to mtDNA defects (). Percentage levels of cell loss are shown in . Patient 5 (m.3243A>G) was the only patient that demonstrated an increase in cell density for the inferior olivary and dentate nucleus neurons vs. controls. This was likely due to severe cerebellar atrophy; the percentage values for cell loss are listed as 0% in . Cell loss was typically most profound in all regions in association with the m.8344A>G and POLG mutations. For example, Patient 12 (p.Ala47Thr and p.Trp748Ser POLG mutations) exhibited 81%, 91% and 96% loss of neurons from Purkinje cells (), inferior olivary and dentate nucleus, respectively. Although patients with the m.3243A>G demonstrated inter-patient variability in cell density, the main finding was of severe Purkinje cell loss (likely due to the presence of ischemic-like lesions), with an average ~50% of cells lost (; ). Cell density was generally reduced in all other patients with different mtDNA defects. It appeared that for Patient 9 (m.14709T>C), neuronal cell loss was most severe in the dentate nucleus with 74% loss whereas the inferior olivary nucleus and Purkinje cells were only moderately affected. Approximately 50% of neurons were lost from the olivo-cerebellum in Patient 10 with an m.13094T>C mutation. Purkinje cells and inferior olivary neurons were predominantly affected in Patient 11 due to the single, large-scale mtDNA deletion where cell loss reached 52% and 60%, respectively. In that case cell loss in the dentate nucleus was minimal ().
Quantification of neuronal cell density revealed that patients with the common m.3243A>G mutation associated with the mitochondrial encephalopathy with lactic acidosis and stroke-like episodes syndrome (MELAS) displayed microinfarcts or ischemic-like lesions in the cerebellar cortex (). Typically, these lesions affect not only the Purkinje cells but also the molecular layer, granular cell layer and adjacent white matter. In m.3243A>G patients, neuronal cell loss was predominately due to the microinfarcts. In cases with the myoclonic epilepsy with ragged red fibers-associated mutation, m.8344A>G and recessive p.Ala467Thr and p.Trp748Ser POLG mutations, microinfarcts were also found but there was also more widespread Purkinje cell loss not associated with microinfarcts (). Furthermore, assessment of the cerebellar lobules suggested that in all cases evaluated microinfarcts typically occurred in the posterior inferior lobules of the cerebellum.
Immunohistochemistry with for glial fibrillary acidic protein, a marker of astrocytes, revealed proliferation of the astroglial cell populations in regions where cell loss was greatest. This was particularly obvious microinfarcts in which proliferation of the Bergmann glia was prominent. In addition, deficiency of components of the mitochondrial respiratory chain in the astroglia was evident in serial sections (). This suggests that astroglia may harbor respiratory-compromised mitochondria.
Figure 3 Astrogliosis in regions of microinfarcts with evidence of severe respiratory chain deficiency in a patient with m.3243A>G. There are numerous microinfarcts in the cerebellar cortex (cresyl fast violet, [CFV)]); typically, these are accompanied (more ...)
Correlation between Neuronal Cell Loss and a High NMDAS Score for Cerebellar Ataxia
We observed a strong correlation between a high NMDAS score for ataxia and evidence of a low neuronal cell density in all 3 areas of the olivary-cerebellum, suggesting that the greater the impairment the less chance that cellular density is maintained. Our data indicates that a high functional impairment is accompanied by a physical loss of neurons (because low cell density indicates cell loss). This correlation was found to be significant in the inferior olivary nucleus (Pearson’s correlation = −0.902, p = 0.000), the Purkinje cell (Pearson’s correlation = −0.628, p = 0.029) and dentate nucleus (Pearson’s correlation = −0.577, p = 0.049) neuronal populations.
Molecular Genetic Investigation of Levels of Mutated mtDNA
Molecular genetic analysis was performed on individual isolated Purkinje cells and neurons of the inferior olivary and dentate nuclei to determine mutation load. Pyrosequencing of mtDNA point mutations revealed high percentage levels in remaining cells throughout the olivo-cerebellum (). High levels of point mutation did not correlate with the degree of cell loss. This was particularly evident in Patient 9 where the m.14709T>C mutation levels were near homoplasmy yet only dentate nucleus neurons reveal severe cell loss. Patients with the m.3243A>G mutation generally showed higher percentage levels of mutated mtDNA that are typically greater than 73%. A lack of correlation between high mutation load and cell loss is clearly demonstrated by Patient 4 (m.3243A>G) in which the level of m.3243A>G reached 88% in inferior olivary neurons despite there being only 42% cell loss in this region (, ).
Percentage Level Heteroplasmy for Mutated or Deleted mtDNA
In contrast, patients with recessive POLG mutations and accumulated multiple mtDNA deletions showed relatively low levels of mtDNA deletion following RT-PCR. The highest level (50.7%) of mtDNA deletion was seen in the Purkinje cells from Patient 13 and the lowest (19.9%) in Purkinje cells from Patient 12. This was despite the extreme cell loss seen in these patients. Neuron cell loss did not correlate with the level of mutated mtDNA in remaining neurons. Therefore, the level of mutation alone does not appear to determine where neuron cell loss will occur. However, we did not evaluate mtDNA depletion in these patients.
Mitochondrial Protein Changes
Various mitochondrial markers were employed to investigate the selective vulnerability of neurons. Porin immunohistochemistry revealed punctate, uniformly high staining within neuronal cytoplasm in both control and patients, indicating that mitochondrial mass was abundant. However, neurons within the inferior olivary nucleus were the exception showing unusual mitochondrial localization in both patients and controls. Generally, mitochondria within these neurons were distributed around the periphery of the cell body or were perinuclear, whereas the cytoplasm remained devoid of mitochondria.
Quantification of the respiratory chain proteins revealed marked respiratory chain deficiency; there was complex I deficiency within remaining neuronal populations but only low levels of complex IV-deficiency (). The percentage level neuronal respiratory chain deficiency correlated with the degree of cell loss, particularly in the Purkinje cell and dentate nucleus neuronal populations. Typically patients with extreme cell loss demonstrate high percentage levels of complex I-deficient neurons and moderate levels of complex IV-deficient neurons. The data obtained from Patient 11 (m.14709T>C) support these findings with severe neuron loss from the dentate nucleus at 74% with a complex I and complex IV severity index of 60% and 30%, respectively (). Conversely, patients with the m.3243A>G mutation where cell loss was moderate, a lower percentage of neurons were deficient for complex I and complex IV was unaffected. For example, Patient 3 (m.3243A>G) demonstrated a 26% reduction in neuron density in the dentate nucleus with remaining cells revealing a complex I and complex IV severity index of 30% and 20%. This was also seen in Patient 10 (m.13094T>C) and Patient 13 (POLG mutations and multiple mtDNA deletions) where Purkinje cell and dentate nucleus cell loss reached 40% to 45% and the complex I and complex IV severity index revealed moderate and mild scores, respectively (; ). Intriguingly, there was a stronger relationship between complex IV deficiency and cell loss, suggesting that development of complex IV deficiency is particularly detrimental to neuronal viability (Purkinje cells; Pearson’s correlation coefficient = 0.736, p = 0.006. Dentate nucleus; Pearson’s correlation coefficient = 0.606, p = 0.048).
Figure 4 High levels of complex I-deficient neurons can be detected in remaining neuronal populations. The inferior olivary nucleus shows an unusual distribution of mitochondria (m.3243A>G, first column) (porin), whereas mitochondria appear abundant throughout (more ...)
The neurons within the inferior olivary nucleus showed widespread deficiency for complex I in the absence of neuronal cell loss (; Patient 4). Therefore, all patients had a high complex I severity index, often reaching 100% ().