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. 2014 Jul;76(1):66-81.
doi: 10.1002/ana.24185. Epub 2014 Jun 14.

Molecular Pathogenesis of Polymerase γ-Related Neurodegeneration

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Free PMC article

Molecular Pathogenesis of Polymerase γ-Related Neurodegeneration

Charalampos Tzoulis et al. Ann Neurol. .
Free PMC article

Abstract

Objective: Polymerase gamma (POLG) mutations are a common cause of mitochondrial disease and have also been linked to neurodegeneration and aging. We studied the molecular mechanisms underlying POLG-related neurodegeneration using postmortem tissue from a large number of patients.

Methods: Clinical information was available from all subjects. Formalin-fixed and frozen brain tissue from 15 patients and 23 controls was studied employing a combination of histopathology, immunohistochemistry, and molecular studies of microdissected neurons.

Results: The primary consequence of POLG mutation in neurons is mitochondrial DNA depletion. This was already present in infants with little evidence of neuronal loss or mitochondrial dysfunction. With longer disease duration, we found an additional, progressive accumulation of mitochondrial DNA deletions and point mutations accompanied by increasing numbers of complex I-deficient neurons. Progressive neurodegeneration primarily affected the cerebellar systems and dopaminergic cells of the substantia nigra. Superimposed on this chronic process were acute, focal cortical lesions that correlated with epileptogenic foci and that showed massive neuronal loss.

Interpretation: POLG mutations appear to compromise neuronal respiration via a combination of early and stable depletion and a progressive somatic mutagenesis of the mitochondrial genome. This leads to 2 distinct but overlapping biological processes: a chronic neurodegeneration reflected clinically by progressive ataxia and cognitive impairment, and an acute focal neuronal necrosis that appears to be related to the presence of epileptic seizures. Our findings offer an explanation of the acute-on-chronic clinical course of this common mitochondrial encephalopathy.

Figures

FIGURE 1
FIGURE 1
Histopathological and immunohistochemical analysis of acute lesions in polymerase gamma encephalopathy. Brain histology from a representative patient (WS-1A) compares unaffected cortex (left side, A–D) and an acute cortical lesion (right side, E–I). Sections are either stained with hematoxylin and eosin (A, E, F), or react immunohistochemically to glial fibrillary acidic protein (B, G), the microglial marker HLA-DR (C, H), or complex I subunit NDUFB8 (D, I). Original magnification = ×100. (F) Magnified (original magnification = ×400) detail from E. The acute lesion is characterized by severe but incomplete neuronal loss (E and F) and vacuolation of the neuropil. Surviving neurons with normal morphological characteristics (arrows in F) are scattered throughout the acute lesion. There is pronounced astrocytosis (G) and diffuse microglial activation (H). In the neighboring, morphologically preserved cortex (D, arrows), there is a high proportion of complex I–negative neurons, but only complex I–positive cells survive within the acute lesion (I). Scale bars = 100μm (A–C, E, G, H), 50μm (D, F, I).
FIGURE 2
FIGURE 2
Cerebellar pathology in polymerase gamma encephalopathy: comparison of patients with the W748S and/or A467T mutations. Cerebellar sections from patients stained with hematoxylin and eosin (A–D) or porin immunohistochemistry (E, F) are shown. Patients are homozygous for the A467T (A, B), homozygous for the W748S (C, D), or compound heterozygous in transit for the A467T and W748S mutations (E, F). The cerebellum shows a combination of 2 types of pathology: diffuse neurodegeneration with Purkinje cell loss, Bergmann gliosis, and thinning of the molecular layer (A–F) and microscopic focal, sharply demarcated lesions (E, F). Diffuse degenerative changes are significantly more severe in the W748S homozygous (C, D) and compound heterozygous (E, F) compared to A467T homozygous (A, B) patients.
FIGURE 3
FIGURE 3
Respiratory chain dysfunction in polymerase gamma (POLG) encephalopathy. Serial sections of the hippocampal CA2 region (A–F) and inferior olivary nucleus (G–I) from a representative patient (AT-1B) are shown. There is severe and selective complex I (anti-NDUFB8) deficiency (A, G), with only a few complex IV–negative neurons in the hippocampus (D) and none in the olive (H), and normal staining for complexes II (B), III (C), and porin (E, I). Complex I stained all neurons in control hippocampus (F). Neuronal complex I deficiency is progressive in POLG encephalopathy (J). The proportion of complex I–negative neurons increased with age in all central nervous system areas studied. The diagrams in J show the percentage of complex I–negative neurons (y-axis) as a function of individual patient age in years (x-axis) in the frontal cortex, hippocampal CA2 region, substantia nigra, and anterior spinal horn. The dopaminergic neurons of the substantia nigra are the earliest among the studied areas to manifest complex I loss.
FIGURE 4
FIGURE 4
mtDNA relative quantification in microdissected neurons from patients with polymerase gamma encephalopathy. Each point is a pooled sample of 5 to 15 neurons. For the purposes of comparison, a control sample has been arbitrarily set to 1. Groups are compared by Mann–Whitney U test, and p values are shown above each graph; n = number of individuals in each group. Patient neurons from all central nervous system areas examined contain significantly less (∼50–60%) mtDNA than neurons of age-matched controls. The top left panel shows in addition that infants (≤1 year old) have lower neuronal mtDNA content compared to older individuals, in both the patients (infant 26% of postinfant values) and controls (infants 38% of postinfant values).
FIGURE 5
FIGURE 5
Neuronal mtDNA deletions in polymerase gamma encephalopathy. Long polymerase chain reaction analysis of mtDNA deletions in cortical homogenate (A) and single microdissected neurons (B) from the frontal cortex of patients and controls is shown. (A) Amplification of an 11kb mtDNA fragment in DNA from cortical homogenate. Samples from left to right: 2 controls and 4 patients aged 8, 44, 41, and 24 years, respectively. No deletions are seen in the child, whereas older patients show smears consistent with multiple deletions, which are more pronounced in the older individuals. (B) Results from single microdissected neurons. A nested amplification of an 8kb product was performed from an 11kb amplicon in patients and controls. All patient neurons contain ≥1 deleted species. Deletions are also detected in some of the control neurons, but quantification shows these to be at substantially lower levels than in patients (C, D). Arrows in A and B mark the normal band size. The ladders are 1kb (Gene Ruler). (C) The relative proportions of mtDNA depletion and deletion in microdissected neurons from various areas of the nervous system. Each bar represents the mean with standard deviations; blue bars show mean patient neuronal mtDNA copy number relative to controls, and red bars show levels of nondeleted neuronal mtDNA relative to controls. Depletion levels are similar throughout the nervous system, whereas excess deletion (compared to age-matched controls) is most pronounced in the substantia nigra. (D) The proportion of deleted mtDNA in patient neurons in excess of that seen in age-matched controls is plotted against patient age in years. The level of excess mtDNA deletions increases with patient age in frontal (r = 0.81), occipital (r = 0.78), and Purkinje (r = 0.97) neurons. A similar progressive increase in deletions was found in all brain areas examined.
FIGURE 6
FIGURE 6
Results of ultradeep resequencing-by-synthesis (UDS) in the brain of patients with polymerase gamma (POLG) encephalopathy. (A) Comparison of mtDNA point mutation burden (at >0.2% frequency) in the MT-HV2 region, in the brain of POLG encephalopathy patients and controls. Patients have a significantly higher burden of low-frequency mutations compared to age-matched controls (odds ratio [OR] = 3, p < 0.001). Patients homozygous for the A467T (AT) mutation have significantly more point mutations than patients homozygous for the W748S (WT) (OR = 1.86, p = 0.008). (B) The number of point mutations (x-axis) detected by UDS in the brain of individual patients is plotted against patient age in years (y-axis). A clear trend (r = 0.74, p = 0.09) for progressive age-dependent increase of mutations is seen in the patients, but not in the controls.

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