TY - JOUR
AU - Geula,, Changiz
AB - Abstract Diffusely stained phosphorylated 43-kDa TAR DNA-binding protein (TDP-43)-positive “pre-inclusions” have been described. This experiment investigated morphological subtypes of pre-inclusions and their relationship with TDP-43 inclusions in primary progressive aphasia (PPA), a dementia characterized by gradual dissolution of language. Brain sections from 5 PPA participants with postmortem diagnoses of frontotemporal lobar degeneration with TDP-43 pathology (FTLD-TDP) were immunohistochemically stained using an antibody to phosphorylated TDP-43 and quantitatively examined for regional and hemispheric distribution using unbiased stereology. Cortical TDP-43 pre-inclusions included smooth, granular/dot-like, or fibrillar staining with localization to the nucleus, cytoplasm, or both. Mature and pre-inclusions were quantified in a region with high and a region with low mature inclusion density, and contralateral homologs. Regions with lower mature inclusions were characterized by higher densities of pre-inclusions, while increasing burden of inclusions corresponded to lower densities of pre-inclusions (p < 0.05). Mature inclusions showed significant asymmetry that favored the language-dominant hemisphere (p < 0.01), while pre-inclusions displayed the opposite pattern (p < 0.01). Granular-type pre-inclusions were more abundant (p < 0.05) and drove the hemispheric and regional differences (p < 0.02). These results suggest that pre-inclusions are present in greater abundance prior to the formation of mature TDP-43 inclusions, and appear to develop through progressive stages into mature intracytoplasmic, or intranuclear aggregates. Frontotemporal dementia, Frontotemporal lobar degeneration, Neurodegeneration, Primary progressive aphasia, Proteinopathies, TDP-43 INTRODUCTION Primary progressive aphasia (PPA) is a clinical dementia syndrome in which abnormal language function is the primary salient clinical feature (1). The 2 major neuropathologic subtypes of PPA are Alzheimer disease (AD) and frontotemporal lobar degeneration (FTLD). A hallmark of FTLD is the abnormal aggregation of misfolded proteins into inclusions containing hyper-phosphorylated tau (FTLD-tau) or the 43-kDa transactive response element DNA-binding protein (TDP-43) (FTLD-TDP) (2). TDP-43 is a highly conserved, ubiquitously expressed DNA- and RNA-binding protein involved in transcriptional and translational regulation encoded by the TARDBP gene on chromosome 1 (3–5). In addition to hyper-phosphorylation, TDP-43 in its pathologic form is mislocalized from its mostly nuclear compartmentalization in healthy cells (6). Phosphorylated TDP-43 aggregates into fibrillar inclusions that conform in shape to the neuronal compartments within which they are found, namely neuronal intranuclear inclusions (NIIs), neuronal cytoplasmic inclusions (NCIs), and dystrophic neurites (DNs). The predominance of 1 or more of these inclusions defines 3 pathologic subtypes of FTLD-TDP. FTLD-TDP type A consists mostly of short DNs and NIIs, FTLD-TDP type B of a predominance of NCIs, and FTLD-TDP type C primarily of long DNs (7). In AD, formation of mature tangles appears to be preceded by concentration of phosphorylated tau in pre-tangles, consisting of accumulation of phosphorylated tau epitopes in otherwise normal appearing neurons (8). Similar to the progressive accumulation of phosphorylated tau in pre-tangles in AD, TDP-43-positive “pre-inclusions” have been described in FTLD-TDP—consisting of diffuse nuclear and/or cytoplasmic staining—suggesting that TDP-43 inclusions develop in distinct stages (8–11). However, the characterization of pre-inclusions has been limited to qualitative descriptions primarily in amyotrophic lateral sclerosis (ALS), with little attention to neocortical regions. Moreover, no study to date has quantitatively examined the density and distribution of these pre-inclusions, particularly in relation to the presence of mature (darkly stained, fibrillar) TDP-43 inclusions. In 5 clinically well-characterized cases of PPA with pathologic diagnoses of FTLD-TDP with no known genetic mutations, we examined the morphologic subtypes and densities of TDP-43 pre-inclusions bilaterally in a language region with high mature inclusion density and another region of low mature inclusion density. Mature inclusions were quantified as NIIs, NCIs, or DNs, while pre-inclusions were identified as smooth, granular, or fibrillar with localization to the nucleus, cytoplasm, or both. We present quantitative evidence for an inverse relationship between the densities of TDP-43 pre-inclusions and mature inclusions, suggesting that TDP-43 inclusions are converted through a series of stages from diffuse, dot-like staining to fully mature inclusions. MATERIALS AND METHODS Cohort Five Caucasian participants with a clinical diagnosis of PPA were enrolled through the PPA Program at the Mesulam Center for Cognitive Neurology and Alzheimer’s Disease of Northwestern University Feinberg School of Medicine. Participants were diagnosed with PPA based on gradual dissolution of language abilities spanning at least 2 years. Based on the specific symptoms, each participant was subtyped into 1 of 3 PPA variants (i.e. agrammatic, logopenic, semantic) using established classification guidelines (12–14). More information on each participant can be found in Table 1. Four PPA participants were right-handed, indicative of left hemisphere being language-dominant. A fifth participant was left-handed (Table 1, participant 5). Functional MRI and asymmetry of pathology indicated that language function was served by the right hemisphere in this participant (15, 16). Five additional cognitively characterized normal participants were used for comparison (81–90 years, 3 males, 2 females). TABLE 1. Demographic Features of PPA-TDP Participants (n = 5) Case # Clinical Subtype TDP Pathologic Subtype Gender Race Handedness Age at Death PMI (hours) Brain wt (grams) Disease Duration (years) Fixation Method 1 Gsp Type A Male C Right 70 4 999 4 Formalin 2 M Type A Female C Right 74 10 1035 7 Formalin 3 Gsp Type B Male C Right 55 29 1090 4 Formalin 4 L→G Type B Male C Right 59 12 1490 2 Formalin 5 L Type A Male C Left 65 6 1040 8 Formalin Case # Clinical Subtype TDP Pathologic Subtype Gender Race Handedness Age at Death PMI (hours) Brain wt (grams) Disease Duration (years) Fixation Method 1 Gsp Type A Male C Right 70 4 999 4 Formalin 2 M Type A Female C Right 74 10 1035 7 Formalin 3 Gsp Type B Male C Right 55 29 1090 4 Formalin 4 L→G Type B Male C Right 59 12 1490 2 Formalin 5 L Type A Male C Left 65 6 1040 8 Formalin PPA clinical subtypes: L, logopenic; G, agrammatic; Gsp, agrammatic with additional motor-speech deficits; M, mixed. Participant 4 progressed from logopenism to agrammatism. Abbreviations: C, Caucasian; PMI, postmortem interval. TABLE 1. Demographic Features of PPA-TDP Participants (n = 5) Case # Clinical Subtype TDP Pathologic Subtype Gender Race Handedness Age at Death PMI (hours) Brain wt (grams) Disease Duration (years) Fixation Method 1 Gsp Type A Male C Right 70 4 999 4 Formalin 2 M Type A Female C Right 74 10 1035 7 Formalin 3 Gsp Type B Male C Right 55 29 1090 4 Formalin 4 L→G Type B Male C Right 59 12 1490 2 Formalin 5 L Type A Male C Left 65 6 1040 8 Formalin Case # Clinical Subtype TDP Pathologic Subtype Gender Race Handedness Age at Death PMI (hours) Brain wt (grams) Disease Duration (years) Fixation Method 1 Gsp Type A Male C Right 70 4 999 4 Formalin 2 M Type A Female C Right 74 10 1035 7 Formalin 3 Gsp Type B Male C Right 55 29 1090 4 Formalin 4 L→G Type B Male C Right 59 12 1490 2 Formalin 5 L Type A Male C Left 65 6 1040 8 Formalin PPA clinical subtypes: L, logopenic; G, agrammatic; Gsp, agrammatic with additional motor-speech deficits; M, mixed. Participant 4 progressed from logopenism to agrammatism. Abbreviations: C, Caucasian; PMI, postmortem interval. Tissue Preparation and Immunohistochemistry Each brain was fixed in formalin for at least 2 weeks, cut into 2- to 3-cm-thick blocks and placed in increasing gradients of sucrose (10%–40%) for cryoprotection. Small blocks from various cortical and subcortical regions were embedded in paraffin, cut at a thickness of 5 µm, and used for pathological diagnosis and for qualitative comparison of TDP-43 inclusions and pre-inclusions between PPA and controls participants. For pathological diagnosis, standard staining protocols, including staining for phosphorylated tau, amyloid-β, α-synuclein, and TDP-43, were employed. All brains were examined for gross and microscopic pathology. Brains of cognitively normal participants were free of pathology except for age-appropriate changes, including low densities and restricted distribution of plaques and tangles. Brains of PPA cases were diagnosed with FTLD-TDP type A pathology or type B pathology (Table 1). FTLD-TDP type A is characterized by many NCIs and short DNs, predominantly in layer 2, while type B consists of moderate NCIs and few DNs throughout all cell layers (17). In addition to the primary TDP proteinopathy, some of the PPA brains contained secondary pathologies. These included ALS-type motor neuron loss (participants 3 and 4, Table 1), mild vascular changes (participants 1 and 3), cerebellar vermis and mammillary body atrophy (participant 1), hippocampal sclerosis, and brainstem Lewy bodies (participant 2). For determination of pre-inclusion types, distribution and quantitation, whole hemisphere blocks from brains of PPA participants containing regions of interest (ROIs) were cut in the coronal plane at 40-µm thickness on a freezing microtome (Leica SM2010R, Nussloch, Germany). Every 1 in 24 sections was immunohistochemically stained using the avidin-biotin peroxidase (ABC) method, with an antibody against TDP-43 phosphorylated at serine residues 409/410 (pS409/410-2, rabbit polyclonal, 1:3000, CosmoBio, Carlsbad, CA) to visualize inclusions and pre-inclusions. All sections were counterstained with 0.05% Cresyl violet to aid in subcellular localization of the TDP-43 inclusions in neurons. ROIs and Identification of Morphologic Subtypes of Pre-inclusions To gauge the relationship between mature TDP-43 inclusions and pre-inclusions, sections containing language and nonlanguage cortical regions in the language-dominant hemisphere were qualitatively assessed for pathologic severity of TDP-43 inclusions in all cases. Based on these evaluations, 1 region with high and another region with low mature TDP-43 inclusion density, respectively, were chosen for subsequent analyses (Table 2). Mature inclusions included NII, NCI, or DNs. The contralateral homologs of each region were also assessed. The pathologic severity of mature TDP-43 inclusions was confirmed quantitatively using unbiased stereological analysis. Regions with complete absence of mature inclusions were excluded from quantitative analysis. TABLE 2. Bilateral Regions Assessed in Each PPA Case Case # 1 2 3 4 5 Region with high mature TDP-inclusion density MFG aSTG IFG IFG STG Region with low mature TDP-inclusion density pSTG SPL IPL IPL SPL Case # 1 2 3 4 5 Region with high mature TDP-inclusion density MFG aSTG IFG IFG STG Region with low mature TDP-inclusion density pSTG SPL IPL IPL SPL Abbreviations: MFG, middle frontal gyrus; IFG, inferior frontal gyrus; aSTG, anterior superior temporal gyrus; pSTG, posterior superior temporal gyrus; IPL, inferior parietal lobule; SPL, superior parietal lobule. TABLE 2. Bilateral Regions Assessed in Each PPA Case Case # 1 2 3 4 5 Region with high mature TDP-inclusion density MFG aSTG IFG IFG STG Region with low mature TDP-inclusion density pSTG SPL IPL IPL SPL Case # 1 2 3 4 5 Region with high mature TDP-inclusion density MFG aSTG IFG IFG STG Region with low mature TDP-inclusion density pSTG SPL IPL IPL SPL Abbreviations: MFG, middle frontal gyrus; IFG, inferior frontal gyrus; aSTG, anterior superior temporal gyrus; pSTG, posterior superior temporal gyrus; IPL, inferior parietal lobule; SPL, superior parietal lobule. The same regions were subjected to a thorough microscopic analysis to devise a classification scheme of all pre-inclusion structures (see Results section), which displayed TDP pS409/410-2 reactivity. All pre-inclusion subtypes were quantified and compared with density of mature inclusions, as described below. Stereological Quantitation Densities of TDP-43 inclusions and pre-inclusions were quantified bilaterally using the StereoInvestigator software (MBF Biosciences, MicroBrightfield, Inc., Williston, VT) in the gray matter (all cell layers included) of a cortical region of high mature TDP-43 inclusion density and a region of low mature TDP-43 inclusion density in each case (Table 2). A 125 × 125 µm counting frame was used and the top and bottom 2 µm of each section were used as guard height. Every 1 in 24 sections (5–7 sections depending on ROI) was used for quantification. The section thickness was measured at every site. Cortical regions were traced at ×1 magnification and pathologic markers were quantified at ×60 magnification. Counts were expressed as pre- or mature inclusions per mm3. Stereologic parameters utilized resulted in a coefficient of error of less than 0.10. Statistical Analysis Densities of TDP-43 mature inclusions were compared between the regions of high and low mature inclusion densities in the language-dominant hemisphere using a Wilcoxon matched-pairs signed rank test (did not pass normality). Similarly, a Wilcoxon matched-pairs signed rank test was conducted to assess differences in pre-inclusion densities in the regions of high and low mature inclusion densities within the language-dominant hemisphere. Hemispheric differences in TDP-43 mature and pre-inclusion densities were assessed, respectively, between the language- and nonlanguage-dominant hemispheres using 2-tailed paired t-tests. The non-parametric repeated measures Friedman test was conducted (data did not pass the test of normality, p > 0.05) in each region (language- and nonlanguage-dominant hemispheres of high and low mature inclusion density regions) to look for any significant differences between densities of pre-inclusion subtypes (i.e. smooth, granular, fibrillar). Dunn’s post-hoc tests were performed to adjust for multiple comparisons. Each region was also evaluated for hemispheric differences in densities for each pre-inclusion subtype using 2-tailed Wilcoxon matched-pairs signed rank tests. FTLD-TDP type A versus type B cases were compared for each pre-inclusion subtype using 2-tailed Mann-Whitney tests in each region. Lastly, a correlational analysis was conducted to determine the relationships between disease duration and mature or pre-inclusion densities. All data analyses were performed using GraphPad Prism software (version 5.03; GraphPad Software, Inc., La Jolla, CA). RESULTS Classification of TDP-43 Pre-inclusions Seven morphologic subtypes of neuronal TDP-43 pre-inclusions were identified as follows: (i) smooth staining in cytoplasm; (ii) smooth staining throughout (nucleus + cytoplasm); (iii) smooth + granular staining throughout; (iv) granular staining in cytoplasm; (v) granular staining throughout; (vi) fibrils in cytoplasm; and (vii) fibrils throughout (Fig. 1). FIGURE 1. View largeDownload slide Examples of morphologic subtypes of cortical TDP-43 pre-inclusions in PPA. (A) Smooth, nongranular, nonfibrillar immunoreactivity in cytoplasm and proximal dendrites (Case 4, FTLD-TDP type B); (B) smooth immunoreactivity in the entire neuron (Case 4, FTLD-TDP type B); (C) granular staining in cytoplasm (Case 4, FTLD-TDP type B); (D) granular staining throughout neuron (Case 3, FTLD-TDP type B); (E) granular staining confined to nucleus (Case 5, FTLD-TDP type A); (F) smooth and granular staining throughout neuron (Case 3, FTLD-TDP type B); (G) small fibrils in cytoplasm (Case 5, FTLD-TDP type A); (H) fibrils throughout neuron (Case 5, FTLD-TDP type A); and (I) fibrils throughout neuron and proximal dendrite (Case 5, FTLD-TDP type A). Scale bar: 10 µm. FIGURE 1. View largeDownload slide Examples of morphologic subtypes of cortical TDP-43 pre-inclusions in PPA. (A) Smooth, nongranular, nonfibrillar immunoreactivity in cytoplasm and proximal dendrites (Case 4, FTLD-TDP type B); (B) smooth immunoreactivity in the entire neuron (Case 4, FTLD-TDP type B); (C) granular staining in cytoplasm (Case 4, FTLD-TDP type B); (D) granular staining throughout neuron (Case 3, FTLD-TDP type B); (E) granular staining confined to nucleus (Case 5, FTLD-TDP type A); (F) smooth and granular staining throughout neuron (Case 3, FTLD-TDP type B); (G) small fibrils in cytoplasm (Case 5, FTLD-TDP type A); (H) fibrils throughout neuron (Case 5, FTLD-TDP type A); and (I) fibrils throughout neuron and proximal dendrite (Case 5, FTLD-TDP type A). Scale bar: 10 µm. “Smooth staining” was identified as faint cytoplasmic or whole-soma TDP-43 immunoreactivity. In “granular” or dot-like pre-inclusions, numerous immunoreactive granules were seen throughout the cytoplasm, nucleus, or the entirety of the soma. Due to the rarity of granules clearly confined to the nucleus only, we did not separately quantify this subtype of pre-inclusion, although an example can be seen in Figure 1E. The fibrils, referring to twisted, dark and smoothly stained thread-like structures with variable length, were present in the cytoplasm or filled the soma. The latter has been described as “skein-like” inclusions (9). However, due to their specific localization in the cell (clearly within the soma and not extended into an axon or dendrite), we classified these fibrils as pre-inclusions, perhaps representing the latest stage prior to becoming a neuritic mature inclusion (i.e. a DN). All morphologic subtypes of pre-inclusions were found in both FTLD-TDP type A and type B. Smooth pre-inclusions were seen mostly in the cytoplasm and rarely throughout the soma, whereas granular pre-inclusions were seen frequently in the cytoplasm and throughout the soma (Fig. 1A–D). Smooth and granular pre-inclusions never extended into dendrites. Fibrillar inclusions, in addition to being in the cytoplasm or throughout the soma, sometimes extended to the dendrites (Fig. 1I). TDP-43 inclusions (Fig. 2A–C), were absent from brains of cognitively normal participants. In addition, brains of 4 normal participants were free of TDP-43 pre-inclusions. One normal brain displayed scattered cortical pre-inclusions. In contrast, brains of all PPA participants contained significant densities of TDP-43-positive mature and pre-inclusions. FIGURE 2. View largeDownload slide Examples of mature (A) neuronal intracytoplasmic, (B) neuronal intranuclear, and (C) neurite TDP-43 inclusions (Case 4, FTLD-TDP type B). (D) Field of low mature inclusion density and high pre-inclusion (arrows) density (Case 4, FTLD-TDP type B). (E) Field of high mature inclusion density and low pre-inclusion (arrow) density (Case 5, FTLD-TDP type A). (F) CA3 of hippocampus exemplifies the presence of only pre-inclusions (arrows) and no mature inclusions (Case 3, FTLD-TDP type B). In contrast, we did not encounter any fields with only mature inclusions. Scale bars: A–C = 20 µm; D, E = 50 µm; F = 100 µm. FIGURE 2. View largeDownload slide Examples of mature (A) neuronal intracytoplasmic, (B) neuronal intranuclear, and (C) neurite TDP-43 inclusions (Case 4, FTLD-TDP type B). (D) Field of low mature inclusion density and high pre-inclusion (arrows) density (Case 4, FTLD-TDP type B). (E) Field of high mature inclusion density and low pre-inclusion (arrow) density (Case 5, FTLD-TDP type A). (F) CA3 of hippocampus exemplifies the presence of only pre-inclusions (arrows) and no mature inclusions (Case 3, FTLD-TDP type B). In contrast, we did not encounter any fields with only mature inclusions. Scale bars: A–C = 20 µm; D, E = 50 µm; F = 100 µm. Trial experiments using an antibody against normal human TDP-43 (mouse monoclonal, 1/5000; Abnova, Walnut, CA) in PPA cases resulted in identical staining of inclusions and pre-inclusions when compared with the phosphorylated TDP-43 antibody, as well as staining in neuronal nuclei. Different Morphologic Subtypes of Pre-inclusions Predominate Based on Density of Mature Inclusions Qualitative analysis PPA brains revealed that regions with lesser mature inclusions generally displayed pre-inclusions with smooth or granular staining confined to the cytoplasm (Fig. 1A, C), while regions with high densities of mature inclusions showed more granular or fibrillar pre-inclusions throughout the cell body (Fig. 1D, H). While we did not encounter regions that contained only mature inclusions with a complete absence of pre-inclusions, some regions, such as the CA3 of the hippocampus, contained only pre-inclusions (Fig. 2F). There was no significant correlation between disease duration and density of mature or pre-inclusion densities, regardless of morphologic subtype. Regions With Low Densities of TDP-43 Inclusions Display High Pre-inclusion Densities Stereological quantitation from the language-dominant hemisphere confirmed that the 2 regions chosen for analysis had significantly different densities of mature inclusions (p < 0.05), with greater density in the “high density” region (Fig. 3A), which was a language region as expected based on the PPA phenotype. When a similar analysis was conducted for pre-inclusions, an inverse pattern was observed, such that areas with higher density of mature inclusions displayed lower pre-inclusion counts (p < 0.05) (Figs. 2D, E and 3B). FIGURE 3. View largeDownload slide Densities of (A) TDP-43 mature and (B) pre-inclusions in regions of high versus low mature inclusion density from the language-dominant hemisphere, respectively. Bars represent mean stereological counts per cubic millimeter and standard errors. *p < 0.05. FIGURE 3. View largeDownload slide Densities of (A) TDP-43 mature and (B) pre-inclusions in regions of high versus low mature inclusion density from the language-dominant hemisphere, respectively. Bars represent mean stereological counts per cubic millimeter and standard errors. *p < 0.05. TDP-43 Mature and Pre-inclusions Show Reversed Hemispheric Asymmetry When all selected regions (Table 2) in each hemisphere were combined to assess hemispheric asymmetry for mature inclusions and pre-inclusions, mature TDP-43 inclusions displayed significant asymmetry, such that the language-dominant hemisphere contained significantly higher TDP-43 inclusions than did the nonlanguage-dominant hemisphere (p < 0.05) (Fig. 4A). In contrast, the nonlanguage-dominant hemisphere displayed significantly higher densities of pre-inclusions than did the language-dominant hemisphere (p < 0.05) (Fig. 4B). FIGURE 4. View largeDownload slide Bilateral densities of TDP-43 (A) mature and (B) pre-inclusions when the regions assessed were combined. Bars represent mean stereological counts per cubic millimeter and standard errors. *p < 0.05; **p < 0.005. LD, language-dominant hemisphere; NLD, nonlanguage-dominant hemisphere. FIGURE 4. View largeDownload slide Bilateral densities of TDP-43 (A) mature and (B) pre-inclusions when the regions assessed were combined. Bars represent mean stereological counts per cubic millimeter and standard errors. *p < 0.05; **p < 0.005. LD, language-dominant hemisphere; NLD, nonlanguage-dominant hemisphere. Granular-type Pre-inclusions Drive Distribution Patterns In all selected regions, there was a significant pre-inclusion subtype effect (p < 0.05). Post-hoc analyses revealed that granular-type pre-inclusions were the most abundant morphologic subtype when compared to smooth and fibrillar pre-inclusions (Fig. 5). This pattern was statistically significant when comparing granular versus smooth pre-inclusions in the language-dominant hemisphere of the low mature inclusion density region (p < 0.05), and granular versus fibrillar pre-inclusions in the language- and nonlanguage-dominant hemispheres of the high mature inclusion density region (p < 0.05 for both). FIGURE 5. View largeDownload slide Densities of each pre-inclusion subtype (smooth, granular, fibrillar) are shown for each case in (A) language-dominant (LD) hemisphere of high mature inclusion density region, (B) LD hemisphere of low mature inclusion density region, (C) nonlanguage-dominant (NLD) hemisphere of high mature inclusion density region, and (D) NLD hemisphere of low mature inclusion density region. Granular-type pre-inclusions were more abundant than smooth or fibrillar-type pre-inclusions (p < 0.05), and there were no significant differences in pre-inclusion density between FTLD-TDP type A and type B for any of the pre-inclusion subtypes (p > 0.05). Bars represent mean stereological counts per cubic millimeter. LD, language-dominant hemisphere; NLD, nonlanguage-dominant hemisphere. FIGURE 5. View largeDownload slide Densities of each pre-inclusion subtype (smooth, granular, fibrillar) are shown for each case in (A) language-dominant (LD) hemisphere of high mature inclusion density region, (B) LD hemisphere of low mature inclusion density region, (C) nonlanguage-dominant (NLD) hemisphere of high mature inclusion density region, and (D) NLD hemisphere of low mature inclusion density region. Granular-type pre-inclusions were more abundant than smooth or fibrillar-type pre-inclusions (p < 0.05), and there were no significant differences in pre-inclusion density between FTLD-TDP type A and type B for any of the pre-inclusion subtypes (p > 0.05). Bars represent mean stereological counts per cubic millimeter. LD, language-dominant hemisphere; NLD, nonlanguage-dominant hemisphere. When the high and low mature inclusion density regions were evaluated for hemispheric differences in densities for each pre-inclusion subtype, there were no significant differences in smooth and fibrillar pre-inclusion densities across hemispheres (p > 0.05). However, granular-type pre-inclusions were significantly more abundant in the nonlanguage-dominant hemisphere, in both the high (p = 0.0059) and low (p = 0.0137) mature inclusion density regions. Pre-inclusion Morphologic Subtypes Do Not Differ Between FTLD-TDP Type A and Type B In each of the 4 regions assessed (language- and nonlanguage-dominant hemispheres of high and low mature inclusion density regions), there were no significant differences in smooth, granular, or fibrillar-type pre-inclusion densities between FTLD-TDP type A and type B cases (p > 0.05). DISCUSSION The deposition of phosphorylated TDP-43 aggregates is at the core of pathologies for ALS/FTD-spectrum disorders and represent a hallmark of the majority of brains with FTLD. Yet, the lifetime of a TDP-43 inclusion, from formation to supposed disappearance, remains unclear. To understand the progression of pathologic TDP-43 deposition, it is essential that the process of its formation and spread is better understood both within the cell and throughout distinct anatomic pathways, similar to descriptions of phosphorylated tau aggregation and anatomic propagation in AD (8, 18). We addressed the former issue in this study by characterizing and quantifying the various types of TDP-43 “pre-inclusions” and evaluating their relationship to the burden of “mature” inclusions in PPA. Progressive Formation of a TDP-43 Inclusion Our observations suggest several stages in the progressive formation of a mature perikaryal TDP-43 inclusion. In such a hypothetical model, the initial stages of inclusion formation are characterized by smooth/diffuse staining in either the cytoplasm or nucleus. In the next stage, the diffuse immunoreactivity becomes concentrated in granular, dot-like structures. This appears to be followed by formation of fibrils in the cytoplasm, eventually including the nucleus. The final stage would then consist of fully mature inclusions in the cytoplasm or nucleus. The description of immunopositive “pre-inclusions” we provide here are similar to what has been described in ALS and behavioral variant frontotemporal dementia, implying that the subtypes of TDP pre-inclusions we describe are common to multiple, if not all, TDP-43 proteinopathies (9, 19). We occasionally observed fibrillar pre-inclusions that extended into the dendrites, suggesting that TDP-43 precipitates may migrate from the cytoplasm to neurites. However, due to their small size, we were not always able to morphologically distinguish potential pre-inclusions from mature inclusions in neurites. Nevertheless, it is reasonable to assume that TDP-43 pre-inclusions extend into neurites prior to formation of mature neuritic TDP-43 inclusions. Our stereological quantifications support the proposed scheme of TDP-43 inclusion formation described above. We observed an inverse relationship between the densities of mature and pre-inclusions, such that regions with higher densities of mature inclusions showed lower densities of pre-inclusions, and vice versa. Consistent with this and previous findings (20, 21), mature TDP-43 inclusions displayed a clinically concordant distribution that favored the language-dominant hemisphere, while pre-inclusions displayed the reverse pattern favoring the non-dominant hemisphere. This suggests that with increasing pathological severity, a greater number of pre-inclusions may have converted into mature inclusions, leading to a decreased presence of pre-inclusions. Moreover, the observations that some regions contained only pre-inclusions and a complete absence of mature inclusions—while the reverse was not observed—further supports our hypothesis that the mature inclusions are preceded by “pre-inclusions.” Previous studies of post-translational modifications, including ubiquitination and p62 positivity, have also been supportive of this idea of progression of various TDP-43 inclusions. One particular study of similar pre-inclusions in ALS reported colocalized ubiquitination in more mature inclusions, but not in what we have here called “pre-inclusions,” which directly supports our quantitative findings and stages of progression of TDP-43 inclusion formation (9). While our findings suggest sequential steps in the formation of mature TDP-43 inclusions, the nature of histopathologic analysis precludes knowledge of the length of time associated with the stages of inclusion formation and subsequent neuron loss. Thus, we cannot rule-out the possibility that some of the smooth or dot-like pre-inclusions, which are supposedly soluble, may be cleared instead of progressing to mature inclusions. The fibrillar type pre-inclusions identified in this report have been described by others as “skein-like” inclusions, found in higher densities in ALS with or without FTLD (9). However, because they lack the dense and compact characteristics of mature inclusions, we propose that they form an intermediate entity in our scheme of progression of TDP-43 inclusion formation. Furthermore, the granular type pre-inclusions have recently been reported to be more abundant in TDP-43 type B cases (22). However, we did not observe significant differences in the density of granular pre-inclusions in our type A when compared with type B cases. It should be noted, however, that separation into subtypes resulted in small sample sizes, and therefore, future studies with larger samples will be required to provide definitive conclusions in this regard. Potential Toxicity of TDP-43 Pre-inclusions In many neurodegenerative disorders, the soluble oligomeric forms of abnormal proteins that aggregate in inclusions, such as amyloid-β and phosphorylated tau in AD, have been suggested to exert toxic effects (23, 24). Evidence suggests that TDP-43, similarly, forms soluble oligomers and fibrils in vitro (25, 26). However, it remains unknown which form of TDP-43—the soluble form, intermediate forms, or fibrillary mature inclusions—is the primary toxic component. The results of our study, in which mature inclusions showed higher densities in the language-dominant hemisphere and thus concordance with disease phenotype, suggest that the mature form is likely to exert greater toxic effects. However, an independent toxic effect of soluble TDP-43 oligomers cannot be excluded. Several reports indicate that TDP-43 and its fragments are capable of forming amyloid fibrils (27, 28), which likely exerts toxic effects similar to fibrillary forms of other pathologic proteins, such as the amyloid-β peptide (29). We have found that Thioflavin S binds to mature TDP-43 inclusions, indicative of β-pleated sheet conformation of fibrillary proteins (30). Interestingly, we did not observe Thioflavin S positivity in pre-inclusions, except the type containing fibrils. Thioflavin S positivity in skein-like inclusions of ALS has also been reported by others (31). Ultrastructurally, mature TDP-43 inclusions and fibrillary pre-inclusions have been shown to consist of fibrils (9). However, granular pre-inclusions appear to accumulate in organelles and lack fibrils. Whether the toxicity of TDP-43 is caused by a loss of normal function or a toxic gain of function remains unclear. The observations that modifications of cytoplasmic TDP-43, such as truncation, are sufficient to cause toxicity (32, 33) have led to speculation that TDP-43 pathology leads to FTLD through a toxic gain of function (34). The finding that expression of wild-type or mutated human TDP-43 gene in transgenic animals also results in inclusion formation and neuronal loss (35–37) has reinforced this speculation. However, the fact that TDP-43 regulates a large number of RNAs, and that deletion of the TARDBP gene in embryonic development or conditional deletion in mature organisms results in lethality, suggests that the mislocalization of TDP-43 from the nucleus to cytoplasm results in neuronal abnormalities and degeneration in FTLD through a loss of normal function (34). Based on the available evidence at present, the only viable conclusion is that TDP-43 pathology exerts detrimental effects through both a loss of normal function and a toxic gain of function (34, 38). The extent to which this toxicity is due to aggregated TDP-43 fibrils or soluble oligomers remains to be elucidated. This work was supported by grants from the National Institute of Neurological Disorders and Stroke (NS085770), National Institute on Deafness and Other Communication Disorders (DC008552), the Louis Family Foundation, Alzheimer’s Disease Center Grant from the National Institute on Aging (AG013854), National Institute on Aging (T32 AG20506), and the Florane and Jerome Rosenstone Fellowship. Footnotes The authors have no duality or conflicts of interest to declare. REFERENCES 1 Mesulam MM , Weintraub S , Rogalski EJ , et al. . Asymmetry and heterogeneity of Alzheimer's and frontotemporal pathology in primary progressive aphasia . Brain 2014 ; 137 : 1176 – 92 Google Scholar Crossref Search ADS PubMed 2 Cairns NJ , Bigio EH , Mackenzie IR , et al. . 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TI - Morphology and Distribution of TDP-43 Pre-inclusions in Primary Progressive Aphasia
JF - Journal of Neuropathology & Experimental Neurology
DO - 10.1093/jnen/nlz005
DA - 2019-03-01
UR - https://www.deepdyve.com/lp/oxford-university-press/morphology-and-distribution-of-tdp-43-pre-inclusions-in-primary-IhpHHOUGYc
SP - 229
VL - 78
IS - 3
DP - DeepDyve
ER -