Dichotomous scoring of TDP-43 proteinopathy from specific brain regions in 27 academic research centers: associations with Alzheimer’s disease and cerebrovascular disease pathologies

There is an evolving appreciation of a common brain disease with TAR-DNA binding protein 43 (TDP-43) proteinopathy that mimics Alzheimer’s disease (AD) clinically [5, 25, 26, 34, 39, 50] and affects 10–25% of persons aged 85 or older [5, 19, 21, 33, 50]. The defining characteristics of this disease are recognized by neuropathologic observations: TDP-43 pathology, often with comorbid neuronal loss and astrocytosis pathology in the hippocampus [1, 33], the latter two features collectively termed hippocampal sclerosis (HS). The literature that pertains to this disease was initially focused on HS pathology (TDP-43 pathology was discovered as a disease marker in 2006 [38]), but it is now recognized that TDP-43 pathology is the most sensitive and specific marker of the disease. For example, cases with HS pathology due to acute anoxia is immunonegative for TDP-43 and is considered a fundamentally different disease [2, 20, 33]. Importantly, the presence of TDP-43 proteinopathy, with or without comorbid HS pathology, is independently associated with cognitive impairment [5, 26, 29, 31].

“TDP-43 pathology” lacks a universally applied specific connotation, but refers to phosphorylated TDP-43 deposits in cytoplasmic (where it may appear like speckles, skeins, or tangles), intranuclear, perivascular, and/or neurite-like structures. TDP-43 pathology may also manifest as a decrease in the normal (non-phosphorylated) TDP-43 in the nucleus [4], or within twig-like deposits of phosphorylated TDP-43 detected immunohistochemically in the subpial or subependymal regions [11, 18, 30]. In prior published papers that have studied the spectrum of TDP-43 pathologies in aged brains (often with comorbid AD pathology), the severity of TDP-43 proteinopathy has been mostly graded according to stage-based classification systems where the presence of any TDP-43 pathology in a given region defines a particular stage [15, 17, 27, 29, 44]. For example, the amygdala seems to be affected very early so this is the first stage. By contrast, in cases with extensive pathology, the frontal neocortex may be affected and if this region has any detectable TDP-43 pathology, that is indicative of a late disease stage. Unfortunately, there currently is no consensus as to a specific antibody or combination of antibodies recommended for detecting TDP-43 proteinopathy. Further, the stage-based classification systems for common age-related disease differ from TDP-43 pathologic staging systems that were developed for amyotrophic lateral sclerosis (ALS) and/or frontotemporal lobar degeneration (FTLD)-TDP [6, 10, 45].

Prior published findings suggest that vascular factors may cause or exacerbate the disease process that manifests neuropathologically as TDP-43 (with or without HS pathology) in the aged brain [8, 41, 47, 49]. In prior work, arteriolosclerosis – dysmorphic changes in small arterioles – was preferentially associated with this disease [36]. Further, arteriolosclerosis was observed in regions outside of the hippocampal formation in cases with comorbid HS pathology, indicating a “whole-brain disease” rather than a disease process isolated to the medial temporal lobe [37]. However, the precise underlying mechanisms are not understood, and more work is required to determine how the clinical and pathologic endpoints are associated with each other.

The AD Centers (ADCs) program has constituted a critical resource for research on AD and related dementias in the U.S. This network derived from a National Institutes of Health (NIH)-funded initiative that started in 1984 and has included more than 30 different ADCs geographically dispersed across the U.S. Each ADC follows a longitudinal cohort of generally elderly individuals reflecting a broad spectrum of clinical diseases and pathologic manifestations. The National Alzheimer’s Coordinating Center (NACC) oversees data collection by the ADCs. For research subjects that died and came to autopsy, a standardized form was created by NACC to describe the neuropathology in a systematic manner, and for correlation with clinical, radiographic, genetic, and biochemical parameters in the same persons. The latest Neuropathology (NP) Form was updated in 2014, and is referred to as version 10 (v10). The NACC NP Form v10 incorporated detailed neuropathological data including Thal phase for Aβ plaques [43], relatively  newly categorized FTLD neuropathologic changes [23], ALS/motor neuron disease (MND), HS of the CA1 and/or subiculum, and distributions of TDP-43 immunoreactive inclusions in five brain regions. The summary data for the updated v10 form was recently described [3]. Here we focused on the clinical and pathologic correlates of TDP-43 pathology in the NACC NP v10 data set among individuals lacking unusual conditions such as FTLD.

Here we present data focusing on TDP-43 pathology in the aged human brain, using a large sample with autopsy confirmation, sourced from multiple high-quality research centers. Our data provide new information about comorbidities that are and are not apparently associated with TDP-43 pathologies in different brain regions. TDP-43 pathology is strongly associated with advanced AD and brain arteriolosclerosis pathologies.

There are some potential pitfalls in our study sample, as we have discussed previously [3]. Contributory ADC cohorts tend to be enriched for rare, genetic, early-onset, and “pure” subtypes of diseases, including AD and many other degenerative conditions. In particular, this sample may be biased toward individuals with a clinical syndrome that mimics AD. The NACC-contributory ADCs also tend to recruit (and achieve autopsy consent for) Caucasian/white individuals of relatively high socioeconomic status; thus, there are relatively few non-Caucasian individuals or those lacking formal education. Further, ADCs apply exclusion criteria that can limit the number of autopsied participants with mental illness, substance abuse, physical disability, or other prevalent conditions. There also are challenges in data interpretation related to the lack of methodologic standardization between the ADCs in terms of TDP-43 IHC methods. This problem will probably plague multi-center studies for some time since our study confirms that different state-of-the-art research centers use different reagents to operationalize TDP-43 proteinopathy (~ 2/3rd of ADCs use phospho-specific TDP-43 antibodies, whereas the remaining ADCs use antibodies that recognize non-phosphorylated epitopes). We also recognize the current lack of knowledge about underlying mechanisms is a limitation, and our study does not describe how the brain arteriolosclerosis pathology spatially relates to the TDP-43 proteinopathy.

Despite the challenges and potential pitfalls, there also are considerable strengths related to this use of the NACC NP data set. We note that despite the abovementioned sources of data variability, our study found evidence of strong associations between TDP-43 proteinopathy and other factors (age, HS pathology, AD pathology, and arteriolosclerosis pathology). These data were collected from individuals who died and came to autopsy over the past 4 years (NACC NP Form v10), providing both fresh data and relatively up-to-date clinical and pathological testing modalities. The study of nearly 1000 brains with APOE genotype and TDP-43 pathology status in multiple brain regions is unusual, and the statistical power it provides is important. Further, the derivation of data from 27 different research centers with expertise in research in AD and related dementias is a strength because the autopsy data may be considered more generalizable than the results from a single neuropathologist or small group of pathologists. For the foreseeable future, it seems unlikely that all research centers will agree on a single protocol for TDP-43 IHC, and we consider it a strength that the current study incorporates results from multiple research centers using site-specific protocols.

There are three main findings that we describe: TDP-43 pathology is strongly associated with advanced AD pathology; TDP-43 pathology is associated with increasingly severe arteriolosclerosis pathology (particularly in non APOE ɛ4/ɛ4 carriers); and age-related TDP-43 pathology is predominantly seen in the medial temporal cortex, uncommon in frontal neocortex, and very rare in spinal cord.

There is a relatively large extant literature on the relatively common comorbidity of TDP-43 pathology with AD, providing a compelling evidence that the pathologies co-occur whether or not they directly interact mechanistically [14, 16, 27, 30, 48]. Staging schema have been proposed to describe how TDP-43 pathology is distributed in brains with comorbid AD pathology [15, 17, 27]. Notably, in multiple cohorts of aged persons, TDP-43 pathology is more strongly linked to HS than early AD pathology [5, 7, 26, 28, 33]. However, within the amygdala of subjects with advanced AD, protein misfolding (tau, Aβ, α-synuclein, and TDP-43 pathologies alike) tends to occur [14, 16].

Compared with AD, the literature on the association between TDP-43 pathology and cerebrovascular is smaller, and overlaps with the paradigm of hypoxia/ischemia. As stated by Zarow et al. [49], “HS has long been hypothesized to result from ischemic-hypoxic insult to the brain. The CA1 sector is fed by small end-arterioles from the anterior choroidal and posterior cerebral arteries and is known to be susceptible to hypoxic injury” (with citation to Ref [9]). Others have also published data compatible with a link between HS pathology and cerebrovascular disease [22, 41, 46, 47, 49]. However, we have found in various data sets previous evidence of a relatively specific association between the type of HS that frequently is comborbid with TDP-43 pathology, and brain arteriolosclerosis [12, 35–37]. In the present study, the specificity of that association was underscored since no other subtype of cerebrovascular pathology was linked to TDP-43 pathology. There currently is no proven mechanistic explanation for this association. We note that factors that are conventionally associated with arteriolosclerosis, such as diabetes or hypertension, do not appear to be specifically associated with TDP-43 pathology. Intriguingly, Montagne and colleagues recently showed that subtle blood-brain barrier dysfunction and “leaky vessels” in the human hippocampus precede cognitive impairment in advanced aging [24]. Winkler et al. [42] reported that pericyte damage could contribute to cognitive impairment through disruption of the neurovascular unit, which may relate to TDP-43 proteinopathy, rather than AD. There also have been described some genetic risk factors that may help explain the link between brain arteriolosclerosis and TDP-43 pathology [32], but more work is required in this area. We speculate that there may be some reason that the TDP-43 pathology is usually confined to the medial temporal lobe of aged individuals, perhaps analogous to how primary age-related tauopathy [13], in the absence of comorbid Aβ plaques, tends not to progress beyond Braak NFT stage IV. Considering this analogy, there may be, in some of the brains, a disease-accelerating factor, analogous to Aβ, which promotes TDP-43 pathology outside of the medial temporal lobe.