Incidence and extent of TDP-43 accumulation in aging human brain

Tissue samples were collected at the Tokyo Metropolitan Geriatric Hospital, which provides community-based medical service to the elderly population. From February 2008 to July 2012, we obtained the 286 consecutive autopsy brains (165 men and 121 women) used for this study. Patient ages ranged from 43 to 104 years (mean ± SD, 82.0 ± 9.6 y). Clinical information was retrospectively obtained from the medical chart. The Mini-Mental State examination (MMSE) [15] was used for evaluation of cognitive function, and the clinical dementia scale (CDR) [16] was employed for the grading of cognitive decline as previously reported [17]. The brain samples used in this study were registered to the Brain Bank for Aging Research (BBAR) with the deceased’s relatives’ informed consent to carry out comprehensive research. The BBAR is approved by the ethics committee of the Tokyo Metropolitan Geriatric Hospital and Institute of Gerontology.

The brains and spinal cords were examined according to routine protocols used in our laboratory [18]. Briefly, the cerebral and cerebellar hemispheres as well as the brainstem were dissected in the midsagittal plane at the time of autopsy. In each case, part of the brain was stored at −80 °C for further biochemical and molecular analyses. The brains and spinal cords were fixed in 20 % buffered formalin (WAKO, Osaka, Japan) for 7–13 days. After fixation in formalin, the cerebral hemispheres, brainstem, and cerebellum were dissected along the coronal, axial, and sagittal planes, respectively. Representative anatomical areas were embedded in paraffin, and 6-μm-thick sections were obtained to diagnose each case neuropathologically. Sections were stained with hematoxylin and eosin (H&E) and the Klüver-Barrera methods. Selected sections were further examined with modified methenamine and Gallyas–Braak silver staining for senile changes, Congo red for amyloid deposition, and Elastica Masson trichrome stain for vascular changes. Selected sections were immunostained with a Ventana BenchMark GX autostainer (Ventana Medical Systems, Tucson, AZ, USA) and an I-View Universal DAB Detection Kit (Roche, Basel, Switzerland) in accordance with the manufacturer’s instructions. The antibodies employed are shown in Table 1. Histologic sections were observed under a research microscope (Eclipse 90i; Nikon, Tokyo, Japan).

The case selection procedure in this study is shown in Fig. 1. First, of the 286 consecutive autopsy cases, we selected cases that met all of the following conditions and defined them as control elderly cases that have minimal senile neuropathologic changes: Braak’s neurofibrillary tangle (NFT) stage II or less, senile plaque (SP) stage [19] A or less, AGD stage [17] II or less, and Lewy body stage [20] II or less. For comparison, we selected cases with AGD stage III as AGD and those with Lewy body stage above III as LBD. We also defined AD according to our brain bank definition (Braak NFT stage above IV and Braak’s SP stage C) [21].

As to the cases with comorbid pathologies and other neurological disorders, we also provided the results of TDP-43 pathology. However, they were not used for comparison with control elderly cases because they were limited in number: AD plus LBD (n = 6), AD plus AGD (n = 3), AD plus progressive supranuclear palsy (PSP) [22] (n = 1), AD plus hippocampal sclerosis (HS) (n = 1), LBD plus AGD (n = 1), LBD plus PSP (n = 1), AGD plus HS (n = 1), senile dementia with tangles (NFTD) (n = 6) [23], Creutzfeldt–Jakob disease (CJD) (n = 4), ALS/ALS with dementia (n = 4), multiple system atrophy (MSA) (n = 3), spinocerebellar ataxia type 6 (SCA6) (n = 2), spinocerebellar ataxia type 3 (SCA3) (n = 1), PSP (n = 2), HS (n = 2), dentatorubral–pallidoluysian atrophy (DRPLA) (n = 1), corticobasal degeneration (CBD) (n = 1), FTLD-TDP (n = 1), and mitochondrial encephalopathy with lactic acidosis and stroke-like episodes (MELAS) (n = 1). Of the remaining cases, we identified the followings as intermediate pathologies that did not fulfill diagnostic criteria: Braak NFT stage above III and Braak’s SP stage A as neurofibrillary tangle-predominant change (NFTC, n = 27), Braak NFT stage above III and Braak’s SP stage above B as AD type change (ADC, n = 18), and Braak NFT stage II or less and Braak’s SP stage above B as plaque dominant senile change (PSC, n = 12).

The presence and severity of TDP-43 immunoreactive structures were assessed in four brain sections including the anterior hippocampus, amygdala, medulla oblongata, and lumbar spinal cord. In fact, the anterior hippocampus is key anatomical structures of FTLD-TDP. In addition, TDP-43 immunoreactive deposits have been well analyzed in AD, DLB and AGD in the limbic regions [5, 6, 8‐11]. The medulla oblongata and spinal cord are important areas in association with ALS with TDP-43 [4]. Therefore, we considered that those four areas are appropriate for the study about TDP-43 pathology in control elderly brains. We assessed TDP-43 immunoreactive structures in each anatomical region shown in Table 2 and Fig. 2 under a 10× objective. Each anatomical region was identified using the neuroanatomy atlas [24]. The uncus was identified and analyzed at the level of amygdala and anterior hippocampus [25]. Neuronal cytoplasmic inclusions (NCIs), glial cytoplasmic inclusions (GCIs), and neuronal intranuclear inclusions (NIIs) immunoreactive for TDP-43 were quantitatively analyzed and scored as 0 to 3 depending on the total number of TDP-43 immunoreactive NCIs, GCIs, or NIIs: 0, none; 1, 1–3; 2, 4–9; and 3, ≥10. TDP-43 immunoreactive dystrophic neurites (DNs) were semi-quantitatively scored as 0 to 3: 0, absent; 1, sparse; 2, moderate; and 3, severe.

The Mann–Whitney U test was used to compare the age at death and brain weight between TDP-43–positive and TDP-43–negative groups. Fisher’s exact test was used to compare the male-to-female ratio and number of TDP-43 structures. A p value of less than 0.05 was considered statistically significant.