Molecular Genetics of DNA Polymerase Gamma‐associated Neurodegeneration

Abstract

Polymerase gamma (POLG) mutations are a common cause of mitochondrial disease and have also been linked to neurodegeneration and ageing. POLG mutations impair the maintenance of mitochondrial deoxyribonucleic acid (mtDNA) in cells causing a combination of quantitative mtDNA depletion, which can present in infancy, and the accumulation of deletions and point mutations, which gradually build up over time. In neurones, this increased somatic mtDNA mutagenesis compromises neuronal respiration, primarily in the form of complex I deficiency, which in turn primes neurones for injury. These two distinct but overlapping biological processes are best described as acute on chronic neurodegeneration. The chronic process affects the cerebellum, deep nuclei such as the dopaminergic cells of the substantia nigra and spinal cord and is reflected clinically by progressive spinocerebellar ataxia. In contract, the acute cortical lesions associated with focal neuronal necrosis occur widely and appear to be triggered and or worsened by epileptic seizures.

Key Concepts

  • Polymerase gamma is the only enzyme that replicates and repairs mitochondrial DNA and comprises one catalytic subunit (pol γ A) encoded by the POLG gene and two accessory subunits (pol γ B) encoded by the POLG1 gene.
  • POLG dysfunction is a common cause of mitochondrial disease and has been associated with ageing and neurodegeneration.
  • POLG mutations impair mtDNA homeostasis.
  • MtDNA loss, depletion, is an early event often present from infancy, whereas deletions and point mutations accumulate gradually during the course of the disease.
  • Accumulating mtDNA damage leads to respiratory dysfunction and in particular complex I deficiency, compromising ATP production in neurones.
  • Neuronal populations that are most vulnerable to ATP deficiency, including those in the cerebellum, thalamus and substantia nigra, exhibit a chronic progressive neurodegeneration and a progressive spinocerebellar ataxia.
  • In spite of severe substantia nigra degeneration and nigrostriatal depletion, patients do not develop clinical parkinsonism, suggesting this is compensated by dysfunction elsewhere in the brain.
  • Acute cortical lesions appear to be triggered by epileptic seizures and underlie episodes with severe, rapidly progressive encephalopathy, focal neurological dysfunction and high morbidity and mortality.
  • The most important predictors of prognosis are the presence of a compound heterozygous genotype, an age of onset before 2 years and the presence of epilepsy; these are associated with severe disease, rapid progression and highest mortality rate.
  • No disease modulating therapy exists for POLG encephalopathy. Efficient control of the epilepsy is the cornerstone of treatment.

Keywords: mitochondria; mitochondrial disease; polymerase gamma; POLG; encephalopathy; neurodegeneration

Figure 1. The structure of the catalytic pol γ subunit (pol γA). (a) Linearised schematic depiction. (b) Three‐dimensional figure showing secondary and tertiary protein structure when the polymerase is not bound to DNA (deoxyribonucleic acid). Alpha‐helices are depicted as springs and beta‐sheets as flat arrows. The catalytic subunit comprises five subdomains which, starting from the N‐terminus, are mitochondrial leading sequence (light blue), exonuclease (dark blue), palm (red), fingers (orange), thumb (green) and spacer (yellow). Protein databank ID: 3ikm (Lee et al., ).
Figure 2. Survival (Kaplan–Meier) curves in POLG (polymerase gamma) encephalopathy. (a) Survival according to genotype in a total of 161 patients with POLG encephalopathy. Patients with infantile onset (≤2 years) have a significantly worse prognosis with a rapid course and early mortality (n = 76, median survival 1 year) compared to patients with later (juvenile/adult) onset (n = 85, median survival 30 years) (p < 0.0001). (b) Survival according to genotype in a total of 79 patients with POLG encephalopathy caused by the common A467T and/or W748S mutations. The data show that A467T homozygous patients (n = 23, median survival 29 years) and W748S homozygous patients (n = 43, median survival 30 years) live longest, whereas compound heterozygous (n = 13, median survival 9 years) have the worse prognosis with significantly shorter survival than the other two groups (p = 0.03). (c) Survival according to epilepsy in a total of 161 patients with POLG encephalopathy. Patients with epilepsy (n = 115, median survival 4.7 years) have a significantly shorter lifespan compared to patients without epilepsy (n = 46) who can survive for at least 60 years from disease onset (p = 0.01). Curve comparison is done by Gehan–Breslow–Wilcoxon test.
Figure 3. MRI (magnetic resonance imaging) and MRS (magnetic resonance spectroscopy) findings in POLG encephalopathy. (a) Sagittal T1 image showing cerebellar atrophy. (b–d) Axial T2 images showing cerebellar changes. (b) Atrophy of the dentate nucleus (arrow), (c) cerebellar white matter hyperintensity and (d) bilateral hypertrophic olivary degeneration. (e) Axial T2 FLAIR image showing bilateral thalamic and cortical occipital lesions. (f) Axial DWI (b = 1000) showing an acute SLL in the right cerebellar cortex of patient AL‐1A. (g) Axial T1 image showing linear, gyriform hyperintensity in the right medial occipital cortex of patient CP‐3A (laminar necrosis). (h) Axial T2‐weighted image showing the natural evolution of an occipital stroke‐like lesion. Times on the images refer to intervals between episode onset and MRI. The lesion progressed inexorably, while the patient's condition gradually worsened. He eventually became comatose and died a little over 3 months after episode onset. (i) MRS measurement in the right occipital lesion of the same patient as in (h). Spectra show a decrease in N‐acetyl‐aspartate (NAA) and a prominent lactate peak at 1.3 ppm, inverting at 144 ms echo time. Spectra from the contralateral, unaffected occipital area were normal (not shown). Cho: choline, Cr: creatine.
Figure 4. Diffusion evolution of cortical stroke‐like lesions during exacerbation episodes. Sequential ADC (analogue to digital converter) measurements are performed in evolving frontal and parietal stroke‐like lesions of a patient with POLG encephalopathy. Cortex with normal MRI appearance is used as control. At 8 days after episode onset, both lesions have low ADCs consistent with restricted diffusion and cytotoxic oedema. Subsequently, ADC values increase, suggesting development of extracellular oedema, exceed those of control cortex and remain elevated at day 70. A new right occipital lesion appears on the scan on day 70. Representative DWI sequences (b = 1000) are shown for each scan.
Figure 5. Histopathological and immunohistochemical analysis of acute lesions in POLG encephalopathy. Brain histology from a representative patient with POLG encephalopathy, comparing unaffected cortex (a–d) and an acute cortical lesion (e–i). Sections are either stained with HE (a, e, f) or react immunohistochemically for GFAP (glial fibrillary acidic protein) (b, g), the microglial marker HLA‐DR (c, h) or complex I subunit NDUFB8 (d, i). Magnification is 100×. (f) A magnified (400×) detail from (e). The acute lesion is characterised 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 neighbouring, 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). Reproduced with permission from Tzoulis et al. 2014 © John Wiley & Sons Ltd.
Figure 6. Cerebellar pathology in POLG encephalopathy: comparison of patients with the W748S and/or A467T mutations. Cerebellar sections from patients stained with HE (a–d) or porin immunohistochemistry (e, f). Patients are homozygous for the A467T (a, b), homozygous for the W748S (c, d) or compound heterozygous in trans for the A467T and W748S mutations (e, f). The cerebellum shows a combination of two 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.Reproduced with permission from Tzoulis et al. 2014 © John Wiley & Sons Ltd.
Figure 7. MtDNA relative quantification in microdissected neurons from patients with POLG encephalopathy. Each point is a pooled sample of 5–15 neurons. For the purposes of comparison, one control sample has been arbitrarily set to one. 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 CNS (central nervous system) areas examined contain significantly less (∼50–60%) mtDNA (mitochondrial deoxyribonucleic acid) than neurons of age‐matched controls. The top left panel shows in addition that infants (≤1 year) 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). Reproduced with permission from Tzoulis et al. 2014 © John Wiley & Sons Ltd.
Figure 8. Neuronal mtDNA deletions in POLG encephalopathy. Long PCR (polymerase chain reaction) analysis of mtDNA deletions in cortical homogenate (a) and single microdissected neurons (b) from the frontal cortex of POLG patients and controls. (a) Amplification of an 11 kb mtDNA fragment in DNA from cortical homogenate. Samples from left to right: two controls and four 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. All patient neurons contain one or more 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 mark the normal band size. The ladders are 1 kb (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; 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. (c) 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. Reproduced with permission from Tzoulis et al. 2014 © John Wiley & Sons Ltd.
Figure 9. Ultradeep resequencing‐by‐synthesis (UDS) in the brain of patients with 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 (OR 3, p < 0.001). Patients homozygous for the A467T mutation have significantly more point mutations than patients homozygous for the W748S (OR 1.86, p = 0.008). (b) The number of point mutations (X) detected by UDS in the brain of individual patients is plotted against patient age in years (Y). 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. Reproduced with permission from Tzoulis et al. 2014 © John Wiley & Sons Ltd.
Figure 10. Respiratory chain dysfunction in POLG encephalopathy. Serial sections of the hippocampal CA2 region (a–f) and inferior olivary nucleus (g–i) from a representative patient (AT‐1B). 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 CNS 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.Reproduced with permission from Tzoulis et al. 2014 © John Wiley & Sons Ltd.
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Further Reading

Bindoff LA and Engelsen BA (2012) Mitochondrial diseases and epilepsy. Epilepsia 53 (Suppl 4): 92–97.

Cohen BH and Chinnery PF (2014) POLG‐related disorders. GeneReviews. PMID: 20301791.

DiMauro S, Hirano M and Schon EA (eds) (2006) Mitochondrial Medicine. Abingdon: Informa.

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Tzoulis, Charalampos, and Bindoff, Laurence A(Nov 2016) Molecular Genetics of DNA Polymerase Gamma‐associated Neurodegeneration. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0026904]