Introduction

Progressive supranuclear palsy (PSP) and Corticobasal degeneration (CBD) are rare progressive neurodegenerative movement disorders [1, 2]. PSP typically presents clinically with early falls, supranuclear vertical gaze palsy, parkinsonism, and dementia at about 65 years of age [3]. Individuals with CBD often present with progressive asymmetric rigidity and apraxia, loss of coordination, tremor, bradykinesia, akinesia, and occasionally alien limb syndrome [4, 5]. Both diseases have overlap** clinical symptoms with each other and other neurodegenerative diseases, such as Parkinson’s disease (PD) and Alzheimer’s disease (AD) [3, 6,7,8]. This can result in an inaccurate clinical diagnosis; definitive diagnosis is only achieved post-mortem using specific neuropathological diagnostic criteria [9, 10].

Neuropathologically, PSP and CBD are characterised as primary four-repeat (4R) tauopathies, with tau-positive aggregates in the form of neurofibrillary tangles (NFT), tufted astrocytes (TA), astrocytic plaques (AP), neuropil threads (NT), and oligodendroglia coiled bodies (CB), evident in the basal ganglia, diencephalon, and brainstem in PSP [11, 12], and in the substantia nigra and locus coeruleus in CBD [10]. Although generally considered sporadic disorders, MAPT, which encodes microtubule associated protein tau, is consistently documented as a strong genetic risk factor for both PSP and CBD [13, 14], and genetic variation in MAPT influences tau pathology severity in PSP [15]. Other genetic factors have also been identified however they do not explain complete disease aetiology [11, 13, 14, 16].

Age is the major risk factor for PSP and CBD, and mitochondrial health is well-established to contribute significantly to healthy ageing [17]. Mitochondrial dysfunction is also recognised in PSP pathogenesis as well as other clinically similar diseases such as PD and AD [18,19,20,21]. More specifically, defective mitochondria generate reactive oxygen species (ROS) which oxidise proteins, lipids, and nucleic acids, accelerating the ageing process [22]. ROS is suggested to contribute to the accumulation of insoluble proteinaceous deposits, such as Lewy bodies in PD, and senile plaques and NFT in AD [23,24,25], and dysfunction of complex I in the oxidative phosphorylation (OXPHOS) system has been shown to accelerate 4R tau isoform formation in PSP cell lines [26] and is defective in the substantia nigra of PD patients [27, 28].

Mitochondria contain their own double-stranded, circular 16.6 kilo-base pair genome (mtDNA), independent to nuclear DNA (nDNA). mtDNA encodes 37 polypeptides, of which 13 encode vital OXPHOS subunits. An individual cell can contain hundreds to thousands of mtDNA copies which significantly affects cellular metabolic background [29]. mtDNA also contains stable single nucleotide polymorphisms which define individuals to specific haplogroups. Individual mtDNA haplogroups have distinctive metabolic demands [30, 31] and haplogroup bioefficiency has also been shown to affect ageing and risk of develo** many neurodegenerative diseases, including PD and AD [31,32,33,34].

Despite evidence reporting mitochondrial dysfunction in PSP, no studies have examined if mtDNA background influences PSP or CBD risk or if mtDNA variation can contribute to overall tau pathology severity. Thus, herein we examine the association of mtDNA background with PSP and CBD risk and tau pathology severity in two autopsy-defined series.

Methods

Study design

1042 pathologically confirmed PSP cases, 171 pathologically confirmed CBD cases, and 910 neurologically healthy controls were included. All subjects were of self-reported European descent. PSP samples were donated between 1998 and 2016 and CBD samples were collected between 1994 and 2017. All samples were obtained from the CurePSP Brain Bank at Mayo Clinic Jacksonville and were rendered by a single neuropathologist (DWD) following published criteria [8, 10, 35]. Controls were recruited from 1998 to 2015 through the clinical Neurology department at Mayo Clinic Jacksonville, Florida. Demographic information is summarised in Table 1. Age of onset and disease duration was unavailable for 606 PSP and was not available for CBD cases. This study was approved by the Mayo Clinic Institutional Review Board and individual written consent was obtained from all subjects, or their next of kin, prior to commencement.

Table 1 Summary of cohort characteristics in N = 1042 PSP cases, 171 CBD cases, and N = 910 controls
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Neuropathological assessment

Semi-quantitative tau pathology scores in PSP and CBD cases were determined by a single neuropathologist (DWD) using standardized histopathologic methods and phospho-tau immunochemistry. Scores were generated in a subset of cases (PSP: N = 764, CBD: N = 150) on a four-point severity scale (0 = none, 1 = mild, 2 = moderate, and 3 = severe) [36]. All sections from all cases were processed in an identical manner with phospho-tau monoclonal antibody (CP13, from Dr. Peter Davies, Feinstein Institute, Long Island, NY) and immunohistochemistry using a DAKO Autostainer. NFT (PSP: N = 764, CBD: N = 150), CB (PSP: N = 763, CBD: N = 150), TA/AP (PSP: N = 734, CBD: N = 150), and NT (PSP: N = 763, CBD: N = 149) were assessed, and overall scores in 17–20 different, vulnerable neuroanatomical regions in PSP/CBD were generated for each separate tau pathology measure (Additional file 1: Table S1). Mean semi-quantitative measures were then calculated for each PSP/CBD patient across all anatomical regions, where a higher overall score indicated more severe tau pathology. PSP/CBD patients who did not have tau scores in a given region for a given tau pathology measure had their scores imputed by using the mean of the values of the patients in the given disease group (PSP or CBD) who did have scores. Any patients with missing data for > 50% of neuroanatomical regions for a given tau pathology measure were not included in any analysis involving tau pathology measures. PSP and CBD cases were additionally assessed for Alzheimer-type pathology with thioflavin-S fluorescent microscopy. Thioflavin-S predominantly binds to 3R and 4R tau molecules which form paired-helical filaments in AD pathology [37, 38]. As PSP/CBD are pure 4R tauopathies, with straight filament NFT [39], thioflavin S is specifically used to assign Braak NFT stage and should not detect PSP/CBD NFT. Braak NFT stage [40] and Thal amyloid phase [41] were generated for each case based on the density and distribution of plaques and tangles, as previously detailed [42, 43] (Table 1).

DNA preparation and genoty**

Genomic DNA and mtDNA was extracted from peripheral blood lymphocytes from control subjects and from frozen cerebellum brain tissue from PSP and CBD cases using Autogen Flex Star and Autogen 245T (Holliston, MA) methods respectively. mtDNA in all samples was genotyped on two high multiplex custom-designed iPlex assays (consisting of 39 mtDNA SNPs—Fig. 1) using Sequenom MassARRAY iPlex technology (MALDI-TOF MS) and iPlex® Gold chemistry technology [44]. PCR and sequence-specific extension primers were designed through Sequenom’s MassARRAY Typer 4.0 Designer software (version 0.2) (Additional file 1: Table S2), part of the Assay Design Suite (Agena Bioscience™, San Diego, USA), and were manufactured by Integrated DNA Technologies (IDT, San Diego, USA). Genotypes were determined by Sequenom MassARRAY® Bruker mass spectrometry (Agena Bioscience, San Diego, CA, USA) [44] and were accepted if intensities were > 5 from the base line intensity (< 5 was considered noise). Individuals with a genoty** call rate > 95% were accepted and had mtDNA haplogroups determined. Genoty** analysis was conducted using Sequenom’s Typer 4.0 Analyzer software (version 25.73). Additional details are provided in Additional file 1: Supplementary Methods.

Fig. 1
figure 1

Schematic overview of the mitochondrial phylotree and unique SNPs which define European mitochondrial haplogroups. Mitochondrial SNPs highlighted in red indicate SNPs genotyped using Sequenom iPlex technology (Agena Bioscience, San Diego, CA, USA) to determine mitochondrial DNA haplogroups. Adapted from Phylotree [45]

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Mitochondrial DNA haplogroup assignment

Mitochondrial DNA haplogroups were manually defined to mitochondrial phylogeny [45,

Conclusions

This is the first study to characterise the role of mtDNA background in susceptibility to PSP and CBD and in tau pathology severity in general. Our data suggests that mtDNA haplogroup background may influence CBD risk and may also modify tau aggregation formation in PSP. Though larger validation studies will be key (particularly for CBD due to the smaller sample size of this group), it will also be important for future studies to investigate how established nDNA risk factors, such as the MAPT H1 haplotype, interact with mtDNA genetic background with regard to susceptibility to disease and severity of tau pathology.