Associations of Mitochondrial Genomic Variation with Corticobasal Degeneration, Progressive Supranuclear Palsy, and Neuropathological Tau Measures

Mitochondrial health is important in ageing and dysfunctional oxidative phosphorylation (OXPHOS) accelerates ageing and influences neurodegeneration. Mitochondrial DNA (mtDNA) codes for vital OXPHOS subunits and mtDNA background has been associated with neurodegeneration; however, no study has characterised mtDNA variation in Progressive supranuclear palsy (PSP) or Corticobasal degeneration (CBD) risk or pathogenesis. In this case-control study, 916 (42.5% male) neurologically-healthy controls, 1051 (54.1% male) pathologically-confirmed PSP cases, and 173 (51.4% male) pathologically-confirmed CBD cases were assessed to determine how stable mtDNA polymorphisms, in the form of mtDNA haplogroups, were associated with risk of PSP, risk of CBD, age of PSP onset, PSP disease duration, and neuropathological tau pathology measures for neurofibrillary tangles (NFT), neuropil threads (NT), tufted astrocytes (TA), and oligodendroglial coiled bodies (CB). 767 PSP cases and 152 CBD cases had quantitative tau pathology scores. mtDNA was genotyped for 39 unique SNPs using Agena Bioscience iPlex technologies and mitochondrial haplogroups were defined to mitochondrial phylogeny. After adjustment for multiple testing, we observed a significant association with risk of CBD for mtDNA sub-haplogroup H4 (OR=4.49, P=0.001) and the HV/HV0a haplogroup was associated with a decreased severity of NT tau pathology in PSP cases (P=0.0023). Our study reports that mitochondrial genomic background may be associated with risk of CBD and may be influencing tau pathology measures in PSP. Replication of these findings will be important.

Familial globular glial tauopathy linked to MAPT mutations: molecular neuropathology and seeding capacity of a prototypical mixed neuronal and glial tauopathy

Globular glial tauopathy (GGT) is a progressive neurodegenerative disease involving the grey matter and white matter (WM) and characterized by neuronal deposition of hyper-phosphorylated, abnormally conformed, truncated, oligomeric 4Rtau in neurons and in glial cells forming typical globular astrocyte and oligodendrocyte inclusions (GAIs and GOIs, respectively) and coiled bodies. Present studies centre on four genetic GGT cases from two unrelated families bearing the P301T mutation in MAPT and one case of sporadic GGT (sGGT) and one case of GGT linked to MAPT K317M mutation, for comparative purposes. Clinical and neuropathological manifestations and biochemical profiles of phospho-tau are subjected to individual variations in patients carrying the same mutation, even in carriers of the same family, independently of the age of onset, gender, and duration of the disease. Immunohistochemistry, western blotting, transcriptomic, proteomics and phosphoproteomics, and intra-cerebral inoculation of brain homogenates to wild-type (WT) mice were the methods employed. In GGT cases linked to MAPT P301T mutation, astrocyte markers GFAP, ALDH1L1, YKL40 mRNA and protein, GJA1 mRNA, and AQ4 protein are significantly increased; glutamate transporter GLT1 (EAAT2) and glucose transporter (SLC2A1) decreased; mitochondrial pyruvate carrier 1 (MPC1) increased, and mitochondrial uncoupling protein 5 (UCP5) almost absent in GAIs in frontal cortex (FC). Expression of oligodendrocyte markers OLIG1 and OLIG2mRNA, and myelin-related genes MBP, PLP1, CNP, MAG, MAL, MOG, and MOBP are significantly decreased in WM; CNPase, PLP1, and MBP antibodies reveal reduction and disruption of myelinated fibres; and SMI31 antibodies mark axonal damage in the WM. Altered expression of AQ4, GLUC-t, and GLT-1 is also observed in sGGT and in GGT linked to MAPT K317M mutation. These alterations point to primary astrogliopathy and oligodendrogliopathy in GGT. In addition, GGT linked to MAPT P301T mutation proteotypes unveil a proteostatic imbalance due to widespread (phospho)proteomic dearrangement in the FC and WM, triggering a disruption of neuron projection morphogenesis and synaptic transmission. Identification of hyper-phosphorylation of variegated proteins calls into question the concept of phospho-tau-only alteration in the pathogenesis of GGT. Finally, unilateral inoculation of sarkosyl-insoluble fractions of GGT homogenates from GGT linked to MAPT P301T, sGGT, and GGT linked to MAPT K317M mutation in the hippocampus, corpus callosum, or caudate/putamen in wild-type mice produces seeding, and time- and region-dependent spreading of phosphorylated, non-oligomeric, and non-truncated 4Rtau and 3Rtau, without GAIs and GOIs but only of coiled bodies. These experiments prove that host tau strains are important in the modulation of cellular vulnerability and phenotypes of phospho-tau aggregates.

Colocalization of BRCA1 with Tau Aggregates in Human Tauopathies

The mechanism of neuronal dysfunction via tau aggregation in tauopathy patients is controversial. In Alzheimer’s disease (AD), we previously reported mislocalization of the DNA repair nuclear protein BRCA1, its coaggregation with tau, and the possible importance of the subsequent DNA repair dysfunction. However, whether this dysfunction in BRCA1 also occurs in other tauopathies is unknown. The aim of this study was to evaluate whether BRCA1 colocalizes with tau aggregates in the cytoplasm in the brains of the patients with tauopathy. We evaluated four AD, two Pick’s disease (PiD), three progressive supranuclear palsy (PSP), three corticobasal degeneration (CBD), four normal control, and four disease control autopsy brains. Immunohistochemistry was performed using antibodies against BRCA1 and phosphorylated tau (AT8). Colocalization was confirmed by immunofluorescence double staining. Colocalization of BRCA1 with tau aggregates was observed in neurofibrillary tangles and neuropil threads in AD, pick bodies in PiD, and globose neurofibrillary tangles and glial coiled bodies in PSP. However, only partial colocalization was observed in tuft-shaped astrocytes in PSP, and no colocalization was observed in CBD. Mislocalization of BRCA1 was not observed in disease controls. BRCA1 was mislocalized to the cytoplasm and colocalized with tau aggregates in not only AD but also in PiD and PSP. Mislocalization of BRCA1 by tau aggregates may be involved in the pathogenesis of PiD and PSP.

Argyrophilic grain disease: An underestimated tauopathy

Argyrophilic grain disease (AGD) is an under-recognized, distinct, highly frequent sporadic tauopathy, with a prevalence reaching 31.3% in centenarians. The most common AGD manifestation is slowly progressive amnestic mild cognitive impairment, accompanied by a high prevalence of neuropsychiatric symptoms. AGD diagnosis can only be achieved postmortem based on the finding of its three main pathologic features: argyrophilic grains, oligodendrocytic coiled bodies and neuronal pretangles. AGD is frequently seen together with Alzheimer's disease-type pathology or in association with other neurodegenerative diseases. Recent studies suggest that AGD may be a defense mechanism against the spread of other neuropathological entities, particularly Alzheimer's disease. This review aims to provide an in-depth overview of the current understanding on AGD.

Localization of ASFV DNA replication and morphological study of subnuclear compartments during viral infection

African Swine Fever Virus (ASFV) is one of the most threatening agents of domestic pig diseases, without a vaccine or treatment, being its control exclusively based on compulsive sanitary measures. Until now, ASFV was thought to perform its viral life cycle within the cytoplasm although recent evidences indicate the presence of viral DNA material inside the host cell nucleus promoting ASFV reclassification into the Nucleocytoplasmic Large DNA Viruses group (NCLDV). So far, no studies have been performed regarding ASFV genome replication phenomena or the host nuclear compartments morphology during cellular infection. Therefore, we aim to unveil the spatiotemporal localization of ASFV DNA replication and the distribution patterns of three subnuclear domains (PML, speckles and coiled bodies), in order to improve knowledge on host nucleus-viral interactions.For the viral DNA replication foci staining, Vero cells were synchronized at a G2/M stage with nocodazole (250 ng/ml, 24h). Following synchronization, cells were grown on glass coverslips (5,0x104 cells/cm2) and infected with ASFV-Ba71V isolate (1h adsorption period with a multiplicity of infection of 5). Afterwards, at specific time-points of viral infection, BrdU (150 µM/ml) was added for a short pulse (30 min) and immediate fixation was performed. For BrdU and ASFV immunodetection, the following primary and secondary antibodies were used: sheep polyclonal anti-BrdU (GTX21893, Genetex, USA; 1:100), an in-house clarified swine anti-ASFV whole-serum (1:100); Alexa Fluor 594 donkey anti-sheep IgG (A-11016, Life Technologies, USA; 1:500) and a FITC rabbit anti-swine IgG (ab6773, Abcam, UK; 1:400). For PML, speckles and coiled nuclear bodies immunolabeling a rabbit polyclonal anti-PML (ab53773, Abcam; 1:100), a goat polyclonal anti-SC35 (sc-10252, Santa Cruz Biotech, USA; 1:50) and a rabbit popyclonal anti-coilin (sc-32860, Santa Cruz Biotech; 1:50) were used, while DyLight 594 donkey anti-rabbit IgG (ab98490, Abcam; 1:500) and Alexa Fluor 594 chicken anti-goat IgG (A-21468, Molecular Probes, 1:400) were used as the secondary antibodies. Microscopic analysis of cells was performed with a Leica epifluorescence microscope (model DM R HC, Germany). De novo synthesized viral DNA was identified in a scattered nuclear localization (discreet dots) during the initial period of infection (Fig. 1, row 1), whereas in a later phase of infection (8h pi), most of the BrdU signal accumulated in the cytoplasmic viral factory (Fig. 1, row 2). PML bodies and nuclear speckles presented a morphological enlargement and a decreasing number in ASFV infected Vero cells (Fig. 2, rows 1 and 2). In an opposite manner, the coiled bodies increased their number during infection (Fig.2, row 3).Our results provide the first evidence that ASFV DNA replication also occurs inside the host nucleus. Nuclear discrete replication foci, at the initial onset of the viral infection, contrast to an accumulation of synthesized viral DNA inside the bigger cytoplasmic viral factory, suggesting that these two distinct patterns are related to the viral life cycle demands. The early PML reorganization can probably be related with cellular antiviral defense mechanisms, given that DNA damage response and p53 are activated during ASFV infection. The low number of nuclear speckles observed in ASFV-infected cells, associated with their enlargement, is most possibly related to the relocation of host splicing factors, since ASFV genome lacks intronic regions.This study was supported by Fundação para a Ciência e Tecnologia through the Project (PTDC/CVT/105630/2008) and the PhD fellowship (SFRH/BD/65532/2009).