International Journal of Neuroscience, 2014; 124(12): 894–903 Copyright © 2014 Informa Healthcare USA, Inc. ISSN: 0020-7454 print / 1543-5245 online DOI: 10.3109/00207454.2014.890620
RESEARCH ARTICLE
Cortical degeneration in frontotemporal lobar degeneration with TDP-43 proteinopathy caused by progranulin gene mutation Richard A. Armstrong Int J Neurosci Downloaded from informahealthcare.com by Nyu Medical Center on 05/26/15 For personal use only.
Vision Sciences, Aston University, Birmingham, UK Familial frontotemporal lobar degeneration with transactive response (TAR) DNA-binding protein of 43 kDa (TDP43) proteinopathy (FTLD-TDP) is most commonly caused by progranulin (GRN) gene mutation. To characterize cortical degeneration in these cases, changes in density of the pathology across the cortical laminae of the frontal and temporal lobe were studied in seven cases of FTLD-TDP with GRN mutation using quantitative analysis and polynomial curve fitting. In 50% of gyri studied, neuronal cytoplasmic inclusions (NCI) exhibited a peak of density in the upper cortical laminae. Most frequently, neuronal intranuclear inclusions (NII) and dystrophic neurites (DN) exhibited a density peak in lower and upper laminae, respectively, glial inclusions (GI) being distributed in low densities across all laminae. Abnormally enlarged neurons (EN) were distributed either in the lower laminae or were more uniformly distributed across the cortex. The distribution of all neurons present varied between cases and regions, but most commonly exhibited a bimodal distribution, density peaks occurring in upper and lower laminae. Vacuolation primarily affected the superficial laminae and density of glial cell nuclei increased with distance across the cortex from pia mater to white matter. The densities of the NCI, GI, NII, and DN were not spatially correlated. The laminar distribution of the pathology in GRN mutation cases was similar to previously reported sporadic cases of FTLD-TDP. Hence, pathological changes initiated by GRN mutation, and by other causes in sporadic cases, appear to follow a parallel course resulting in very similar patterns of cortical degeneration in FTLD-TDP. KEYWORDS: Neuronal cytoplasmic inclusions (NCI), Neuronal intranuclear inclusions (NII), Dystrophic neurites (DN), Transactive response (TAR) DNA-binding protein of 43kDa (TDP-43), Vacuolation
Introduction A significant proportion of frontotemporal lobar degeneration (FTLD) with transactive response (TAR) DNA-binding protein of 43kDa (TDP-43) proteinopathy (FTLD-TDP) cases are familial [1–8]. Various genetic defects have been identified in these cases, the majority being caused by mutation of the progranulin (GRN) gene [2–9]. Familial FTLD-TDP can also be commonly caused by chromosome 9 open reading frame 72 (C9ORF72) gene [10,11] and more rarely by valosincontaining protein (VCP) gene mutation [12]. Several different frame-shift and premature termination mutations have been identified in GRN mutaReceived 7 November 2013; revised 7 January 2014; accepted 30 January 2014 Correspondence: R.A. Armstrong, Vision Sciences, Aston University, Birmingham B4 7ET, UK. Tel. 0121 359 3611. Fax: 0121 333 4220. E-mail: [email protected]
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tion cases [4,5,13]. Abnormal protein products may accumulate within the endoplasmic reticulum of the cell due to inefficient secretion or mutant RNA may have a lower expression within the cell at least in some mutants [4]. TDP-43 is a nuclear protein and exhibits a granular pattern of immunoreactivity in the nucleus of normal cells. The subsequent development of the pathology in FTLD-TDP is unclear, however, TDP43 may be redistributed from the nucleus to the cytoplasm, then ubiquinated, and hyperphosphorylated, and ultimately cleaved to generate C-terminal fragments [14]. These fragments could then accumulate to form inclusions that may ultimately lead to cell death and degeneration of anatomical pathways. The TDP-43-immunoreactive pathology in FTLD-TDP includes various types of inclusion including neuronal cytoplasmic inclusions (NCI) [1,7,15,17], neuronal intranuclear inclusions (NII) [16–18], dystrophic neurites (DN) [1,15,17], and oligodendroglial inclusions (GI)
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Table 1. Demographic features, gross brain weight, and genetics of the seven cases of familial frontotemporal lobar dementia with TDP-43 proteinopathy (FTLD-TDP) caused by progranulin (GRN) mutation. Braak score is based on density and distribution of neurofibrillary tangles. (M = Male, F = Female, DR = Duration, BW = Brain weight, IVS = intervening sequence.) Case
Sex
Age
Onset
DR
BW
Mutation
Location
Braak score
A B C D E F G
F F F M M F M
74 84 67 63 79 82 66
68 69 58 57 71 73 55
6 15 9 6 8 9 11
975 570 880 1080 1150 800 1050
g.4068C>A g.4068C>A g.4068C>A g.4068C>A g.4068C>A g.4068C>A g.5913A>G
Exon 1 Exon 1 Exon 1 Exon 1 Exon 1 Exon 1 IVS6
0 4 2 1 6 1
[16–18]. Vacuolation affecting the superficial regions of the cortex and the presence of abnormally enlarged neurons (EN) have also been observed [1,17,18]. In sporadic FTLD-TDP, NCI, DN, and vacuolation were abundant in upper cortical laminae of the frontal and temporal lobe and GI, NII, EN, and glial cell nuclei in the lower laminae [19]. In addition, variation in laminar distribution between cases was related to disease duration and varied with disease subtype [14,20,21]. Hence, to compare cortical degeneration in GRN cases with sporadic FTLD-TDP [19], changes in density of NCI, GI, NII, DN, all neurons present, EN, and vacuoles were studied across the cortical laminae in frontal and temporal lobe in seven cases of FTLD-TDP caused by GRN mutation using quantitative analysis and polynomial curve fitting.
laration of Helsinki (as modified Edinburgh, 2000). Blocks were taken from the frontal lobe at the level of the genu of the corpus callosum to study middle frontal gyrus (MFG) and the temporal lobe at the level of the lateral geniculate body to study inferior temporal gyrus (ITG) and parahippocampal gyrus (PHG). Tissue was fixed in 10% phosphate buffered formalsaline and subsequently embedded in paraffin wax. Sections were pretreated with formic acid (95%) for 5 min and then immunohistochemistry was carried out using a mouse monoclonal antibody that specifically recognizes phosphorylated TDP-43 (pTDP-43) (dilution 1:40,000; pS409/410–1, Clone 11–9, Cosmo Bio USA, Inc., Carlsbad, CA, USA). Sections were also stained with hematoxylin.
Morphometric methods
Materials and Methods Cases FTLD-TDP cases (N = 7) caused by GRN mutation (see Table 1) were obtained from the Departments of Neurology and Pathology and Immunology, Washington University, School of Medicine, St. Louis, MO, USA. Six of the cases carried the missense g.4068C>A mutation in exon 1 of the GRN gene [4] and one case the splice site g5913A>G (IVS6–2A>G) mutation located within intervening sequence (IVS) 6 [5]. All cases revealed the characteristic pathology of FTLD, viz., neuronal loss, microvacuolation of the superficial cortical laminae, and a reactive astrocytosis [1,4]. None of the cases had symptoms or pathology of motor neuron disease (FTLD-MND) [22,23] and none had coexisting hippocampal sclerosis (HS). Staging of disease was based on the Braak tangle score [24,25].
Histological methods The consent of the next of kin was obtained after death for removal of the brain as determined by local Ethical Committee procedures and the 1995 Dec C
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The laminar distribution of the NCI, GI, NII, and DN together with the neurons, EN, vacuoles, and glial cell nuclei was studied across the cortex from pia matter to white matter using methods described by Armstrong et al. [19] and Duyckaerts et al. [26]. Five randomly positioned traverses from pia matter to the edge of the white matter were located along each gyrus. Histological features were counted in 50 × 250 μm sample fields arranged contiguously down the cortex, the larger dimension of the field being located parallel with the surface of the pia matter. An eye-piece micrometer was used as the sample field which was moved down each traverse one step at a time from pia matter to the edge of the white matter. The NCI were rounded, spicular, or skein-like [15,17,27], while the GI resembled similar inclusions reported in the tauopathies [28–32] The NII were lenticular, spindle-shaped, or circular [16,17] and the DN often long and contorted in shape [17,33]. Neurons had a larger shape, nonspherical outline and contained at least some cytoplasm [34], while glial cells comprised small spherical or asymmetrical nuclei without cytoplasm. EN had swollen perikarya, had a dystrophic nucleus usually displaced to the side of the cell,
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and maximum cell diameter was at least three times that of the nucleus [35]. Discrete vacuoles greater than 5 μm in diameter were also counted [36,37]. Vacuoles clearly associated with neuronal perikarya were not counted as they could be artifacts [36,37]. The mean of the counts from the five traverses was calculated to study variations in density of each histological feature across each gyrus. Data analysis It was difficult to locate precise boundaries between individual cortical laminae due to the degree of cortical degeneration present in many gyri. In addition, laminar identification is especially difficult in the frontal cortex because it exhibits a heterotypical structure, i.e., six laminae cannot always be clearly identified. Moreover, many pathological inclusions exhibited complex patterns of distribution across the cortex and were not confined to specific laminae [19]. Hence, to determine laminar distributions, variations in density across the cortex were analyzed using a polynomial curve-fitting procedure (STATISTICA software, Statsoft Inc., 2300 East 14th Street, Tulsa, OK 74104, USA) [16,36,37]. Hence, for each gyrus studied, polynomials up to the sixth order were fitted successively to the data, quadratic curves being parabolic, cubic curves ‘‘S” shaped, and quartic curves ‘‘double-peaked” or ‘‘bimodal” etc. Correlation coefficients (Pearson’s ‘‘r”), regression coefficients, standard errors (SE), values of t, and the residual mean squares were obtained at each stage of the analysis [38], the reduction in the sums
Figure 1. The pTDP-43-immunoreactive pathology in the upper
cortical laminae of a case of familial frontotemporal lobar dementia with TDP-43 proteinopathy (Case A) caused by progranulin (GRN) mutation showing the typical neuronal cytoplasmic inclusions (NCI, arrowhead) and a few dystrophic neurites (DN, arrow) in the upper cortical laminae corresponding to laminae II/III. Significant vacuolation is also evident. (pTDP-43 immunohistochemistry, bar = 20 μm).
Figure 2. Typical examples of (a) neuronal intranuclear inclusion (NII, arrow) in lower cortical laminae of Case B (pTDP43 immunohistochemistry, bar = 50 μm), (b) a glial inclusion (GI, arrow) in lower cortical laminae in a case (Case C) (pTDP-43 immunohistochemistry, bar = 50 μm), and (c) an abnormally enlarged neuron (EN, arrow) in lower cortical laminae of Case A (H/E bar = 20 μm).
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Pathology of GRN mutation cases of FTLD-TDP
of squares (SS) being tested for significance. A curve of best fit was established by observing when either a nonsignificant value of F was obtained or there was little gain in the explained variance [38,39]. Histological features were classified as having either a single peak of density (‘‘unimodal” distribution), peak density being located either in the upper cortex (approximating to laminae I, II, III), middle cortex (approximating to mainly lamina IV), or lower cortex (approximating to laminae V, VI) cortex or two peaks of density (“bimodal” distribution), with peaks of density occurring in upper and lower cortex. Bimodal distributions were classified further according to whether the density peaks were similar in upper and lower cortex. If no significant polynomial curves were obtained, then no significant trend in abundance across the cortex was assumed. To determine whether the frequencies of the different types of distribution exhibited by the pTDP-43immunoreactive inclusions varied with disease duration or Braak-tangle stage [25], data were compared using chi-square (χ 2 ) contingency table tests. In addition, correlations between the densities of all histological features were tested using Pearson’s correlation coefficient (r). To compare the frequency of different laminar distributions in GRN cases with those previously published
in sporadic cases of FTLD-TDP [19], the data for each histological feature were also analyzed using χ 2 contingency table tests. Four categories of distribution were used for this analysis, viz., a significant density peak in upper cortex, lower cortex, a bimodal distribution, or no significant change with distance from pia matter.
Results The pTDP-43-immunoreactive pathology in the upper laminae of a GRN mutation case of FTLD-TDP (Case A) is shown in Figure 1 and shows typical examples of NCI and DN in the upper cortical laminae. Some vacuolation of the upper laminae is also evident. Figure 2 illustrates typical examples of a pTDP-43immunoreactive NII in lower cortical laminae (Case B, Figure 2a), a GI in lower laminae (Case C, Figure 2b), and an EN in lower laminae (Case A, Figure 2c). Examples of the laminar distribution of the NCI and NII in a single gyrus (Case A, MFG) are shown in Figure 3. The distribution of the NCI was fitted by a thirdorder polynomial (r = 0.55, p < 0.01) with a single density peak in the upper laminae. The distribution of the NII was fitted with a fifth-order polynomial (r = 0.58,
Figure 3. Examples of the laminar distribution of the neuronal cytoplasmic inclusions (NCI) and neuronal
intranuclear inclusions (NII) in a single gyrus (Case A, MFG) of familial frontotemporal lobar dementia with TDP-43 proteinopathy caused by progranulin (GRN) mutation. Curve fitting; NCI, third-order polynomial (r = 0.55, p < 0.01), NII, fifth-order polynomial (r = 0.58, p < 0.01). C
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Figure 4. The laminar distribution of the neurons and vacuolation in a single gyrus (Case D, MFG) of a
case of familial frontotemporal lobar dementia with TDP-43 proteinopathy caused by progranulin (GRN) mutation. Curve fitting: neurons, fourth-order polynomial (r = 0.76, p < 0.001); vacuolation second-order polynomial (r = 0.90, p < 0.001).
p < 0.01), the distribution being bimodal, the peaks of density in the upper and lower laminae having approximately equal magnitude. Examples of the laminar distribution of the neurons and vacuolation in a single brain region (Case D, MFG) are shown in Figure 4. The distribution of the neurons was fitted by a fourth-order polynomial (r = 0.76, p < 0.001) suggesting a binomial distribution, with similar peaks of density in the upper and lower laminae. The distribution of the vacuoles (Case A, PHG) was fitted best by a second-order (quadratic) polynomial (r = 0.90, p < 0.001), vacuole density being greatest in the superficial laminae, density then declining rapidly with distance below the pia mater. The distributions of all histological features in each region and case are shown in Table 2 and summarized in Table 3. In approximately 50% of gyri studied, the density of the NCI was greatest in the upper laminae, four gyri having a unimodal and three a bimodal distribution. In five gyri, there were no significant differences in density with distance below the pia matter. The GI exhibited a peak of density in the upper laminae in 2/7 (29%) gyri, a peak in the lower laminae in 1/7 (14%), and there was no significant variation in density across the cortex
in 4/7 (57%) gyri. The NII exhibited a peak of density in the lower cortex in 5/17 (29%) of gyri and in 4/17 (24%) of gyri a bimodal distribution was present, density peaks being similar in the upper and lower cortex. In 8/17 (47%) of gyri, there was no significant change in density of NII across the cortex. The distribution of the DN varied between cases and gyri, a peak of density in the upper cortex present in approximately 50% of gyri. The EN were distributed in the lower laminae in 8/15 (53%) gyri or were uniformly distributed down the cortex in 5/15 (33%) gyri. The distribution of neurons was variable, the most common being a bimodal distribution with density peaks in the upper and lower cortex, the upper density peak being greater than the lower in 8/21 (38%) of gyri. The vacuoles were distributed largely in the upper laminae, 15/21 (71%) of gyri exhibiting this pattern, while the distribution of glial cell nuclei was primarily in the lower cortical laminae. In 19/21 (90%) of gyri, there was a linear increase in glial cell density across the cortex from pia matter to white matter. No consistent differences in laminar distribution were apparent between gyri in frontal and temporal lobes. The relationship between the distribution of the pTDP-43-immunoreactive inclusions, disease duration,
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Table 2. Results of the polynomial curve-fitting procedure for each histological feature (NCI = Neuronal cytoplasmic inclusions, GI = Glial inclusions, NII = Neuronal intranuclear inclusions, DN = Dystrophic neurites, EN = Abnormally enlarged neurons, V = Vacuolation, NR = Neurons, GL = Glial cell nuclei) in each cortical gyrus. The first figure of each entry is the order of polynomial fitted to the data (1 = linear, 2 = quadratic, 3 = cubic, 4 = quartic, etc and the second letter in parentheses, whether a density peak was located in the upper cortex (U) (approximating to laminae I,II, III), middle cortex (M) (approximating to lamina IV), or lower cortex (L) (approximating to laminae V,VI) cortex or whether two peaks of density (“bimodal” distribution) (B) were present with peaks of density occurring in upper and lower cortex (NS = no significant curve fitted the data, symbol “-” indicates insufficient density of a histological feature to quantify its distribution). Histological features Case
Region
NCI
GI
NII
DN
EN
V
NR
GL
A
MFG ITG PHG MFG ITG PHG MFG ITG PHG MFG ITG PHG MFG ITG PHG MFG ITG PHG MFG ITG PHG
4 (U) NS 1 (L) NS NS NS 4 (B) 1 (U) NS 4 (B) 4 (B) 1 (U) 4 (B) 3 (B) 2 (U)
NS NS NS 3 (U) 3 (U) 3 (L) NS -
4 (B) 2 (L) 1 (L) 4 (B) 1 (L) NS 4 (B) 3 (L) NS NS 3 (B) NS NS NS NS NS 3 (L)
4 (B) 4 (B) 1 (L) 4 (B) 4 (B) NS NS 3 (U) NS 2 (U) 2 (U) 3 (U) 1 (L) 2 (U) -
NS 1 (L) NS NS 1 (L) NS 1 (L) NS 3 (L) 3 (L) M 2 (L) M NS NS 1 (L) -
2 (U) 2 (U) 2 (U) 1 (L) 4 (B) 1 (U) 2 (U) 4 (B) 3 (B) 2 (U) 2 (U) 2 (U) 2 (U) 3 (B) 2 (U) 2 (U) 4 (B) 4 (B) 1 (U) 3 (U) 3 (U)
4 (B) 2 (U) 4 (B) 4 (B) 3 (B) 1 (M) NS 2 (L) 4 (B) 4 (B) 3 (U) 4 (B) 4 (B) 4 (B) 1 (U) 3 (B) 3 (B) 4 (B) 4 (B) 1 (L) 4 (B)
1 (L) 1 (L) 1 (L) 1 (L) 1 (L) 1 (L) 1 (L) NS 1 (L) 1 (L) 1 (L) 1 (L) 1 (L) 1 (L) 2 (M) 1 (L) 1 (L) 1 (L) 2 (L) 1 (L) 1 (L)
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B
C
D
E
F
G
and Braak tangle stage is shown in Table 4. There were no significant differences in the frequencies of unimodal or bimodal distributions when cases were classified according to disease duration (χ 2 = 1.32, 5DF, p > 0.05) or Braak tangle stage (χ 2 = 1.28, 5DF, p > 0.05). A summary of the spatial correlations in density of the various histological features across the gyri is shown in Table 5. There were few significant correlations between the NCI, GI, NII, and DN. However, there was a positive correlation between the densities of neurons and vacuoles in 7/21 (33%) gyri and a negative correlation between the densities of neurons and glial cell nuclei in 8/21 (38%) gyri. In addition, the densities of the vacuoles and glial cell nuclei were negatively correlated in 50% of gyri studied. A comparison of the laminar distributions in GRNmutation cases with those previously published for 10 sporadic cases of FTLD-TDP [16] is shown in Table 6. The laminar distributions were similar for GRN mutation and sporadic cases of the disease with the exception of the vacuolation, unimodal distributions with a density peak in the upper laminae being less frequent and bimodal distributions more frequent in sporadic cases. This difference between GRN and familial FTLD-TDP
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is also evident for the DN but does not reach statistical significance.
Discussion As in sporadic FTLD-TDP, the data suggest significant changes in density of the pathology across cortical gyri in GRN mutation cases. Changes in density varied between gyri and cases suggesting that the GRN cases do not conform to a single pathological subtype of FTLDTDP based on their laminar distribution [1,7,14,20]. The most consistent patterns of distribution were: (1) significant vacuolation in the superficial laminae, vacuoles declining in density with distance from the pia matter and (2) increasing densities of glial cell nuclei in the lower cortex. Of the pTDP-43-immunoreactive inclusions, the NCI and DN were most abundant in upper cortex and NII in lower cortex, while the GI exhibited no laminar preference. The distribution of the neurons was especially variable. In normal elderly brain, pyramidal neurons in frontal and temporal cortex often exhibit a bimodal distribution in which peak density in upper cortex
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Table 3. Summary of the laminar distribution of lesions (NCI = Neuronal cytoplasmic inclusions, GI = Glial inclusions, NII = Neuronal intranuclear inclusions, DN = Dystrophic neurites, EN = Abnormally enlarged neurons, V = Vacuolation, NR = Neurons, GN = Glial cell nuclei) in gyri of the frontal and temporal cortex in seven cases familial frontotemporal lobar dementia with TDP-43 proteinopathy caused by progranulin (GRN) mutation. (N = number of gyri analyzed. Data indicate the number of gyri in which a particular histological feature exhibited either a single peak of density in the upper cortex (U) (approximating to laminae I, II, III), middle cortex (M) (approximating to lamina IV), or lower cortex (L) (approximating to laminae V, VI) cortex or whether two peaks of density (‘bimodal’ distribution) (B) were present with peaks of density occurring in upper and lower cortex. NS = no significant change in density from pia matter to white matter). Unimodal distribution Lesion NCI GI NII DN EN NR V GL
Bimodal distribution
N
U
M
L
U≥L
U=L
L≥U
NS
15 7 17 14 15 21 21 21
4 2 0 5 0 5 15 0
0 0 0 1 2 1 0 1
1 1 5 2 8 1 0 19
3 0 0 2 0 8 6 0
2 0 4 1 0 3 0 0
0 0 0 0 0 2 0 0
5 4 8 3 5 1 0 1
(corresponding to laminae II/III) is usually greater than in lower cortex (corresponding to laminae V/VI) [40]. A similar bimodal distribution was observed in eight gyri in the GRN cases. In six further gyri, however, peak densities of neurons were similar in the upper and lower cortex, greater in lower than upper cortex, or were uniformly distributed across the cortex. All of these distributions suggest neuronal loss in upper cortical laminae. Neuronal loss in upper cortex could Table 4. Relationship between the distribution of TDP-43-immunoreactive inclusions, disease duration, and Braak stage in familial frontotemporal lobar dementia with TDP-43 proteinopathy caused by progranulin (GRN) mutation. Data show the frequency of gyri in which collectively, the TDP-43-immunoreactive inclusions exhibited a unimodal or a bimodal distribution, NS = No significant change in abundance across the cortex. Laminar distribution Variable Disease duration (years) Braak stage
Categories
Unimodal
Bimodal
NS
6–8 ≥9 0–2 4–6
8 13 11 6
7 5 10 2
8 11 12 4
Chi-square (χ 2 ) contingency table tests: Disease duration χ 2 = 1.32 (5DF, p > 0.05), Braak score χ 2 = 1.28 (5DF, p > 0.05).
Table 5. Summary of correlations (Pearson’s “r”) between the densities of histological features (NCI = Neuronal cytoplasmic inclusions, GI = Glial inclusions, NII = Neuronal intranuclear inclusions, DN = dystrophic neurites, EN = Abnormally enlarged neurons, V = vacuolation, NR = neurons, GN = glial cell nuclei). Frequency of correlation Variables
N
Positive ‘r’
Negative ‘r’
NS
NCI/GI NCI/NII NCI/DN NCI/EN NCI/NR NCI/V NCI/GL GI/NII GI/DN GI/EN GI/NR GI/V GI/GL NII/DN NII/EN NII/NR NII/V NII/GL DN/EN DN/NR DN/V DN/GL EN/NR EN/V EN/GL NR/V NR/GL V/GL
14 19 16 19 19 19 18 15 12 15 16 16 16 17 20 21 21 21 17 17 17 19 20 20 21 21 21 20
2 1 2 1 4 4 1 1 2 0 1 2 1 1 1 0 1 1 1 3 5 0 1 0 2 7 0 0
0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 1 1 0 0 1 0 2 0 1 0 0 8 10
12 18 14 18 15 15 17 15 10 15 16 14 15 16 19 20 19 20 16 13 12 157 19 19 19 14 13 10
be associated with degeneration of the feed-forward cortico-cortical projections, which have their cells of origin in laminae II/III [41]. By contrast, EN, which may reflect either a stress response or axonal degeneration [42,43], and glial cell nuclei, were frequently abundant in lower cortex. Pathological changes in lower cortex could be associated with degeneration of either the feedback cortico-cortical projections [41] or the afferent and efferent cortical-subcortical projections. Hence, as in sporadic FTLD-TDP [19], there is degeneration across all cortical laminae in GRN mutation cases potentially affecting multiple anatomical pathways. A notable feature of the data is the variability in laminar distributions across different GRN cases. pTDP-43immunoreactive pathology could spread between cortical regions via cell-to-cell transfer as has been suggested for tau and α-synuclein-immunoreactive pathologies [41,44]. Hence, variations in laminar distribution between cases and gyri could represent different stages of this process. However, no significant differences in the International Journal of Neuroscience
Pathology of GRN mutation cases of FTLD-TDP Table 6. Comparison of the frequencies of the various laminar distributions (NCI = Neuronal cytoplasmic inclusions, GI = Glial inclusions, NII = Neuronal intranuclear inclusions, DN = Dystrophic neurites, EN = Abnormally enlarged neurons, V = vacuolation, NR = Neurons, GN = glial cell nuclei) in gyri of the frontal and temporal cortex in seven cases of familial frontotemporal lobar degeneration with TDP-43 proteinopathy (FTLD-TDP) caused by progranulin (GRN) mutation with cases of sporadic FTLD-TDP. Histological feature NCI
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GI NII DN EN NR V GN
Laminar distribution
Patient group
Upper
Lower
Bimodal
NS
GRN Sporadic GRN Sporadic GRN Sporadic GRN Sporadic GRN Sporadic GRN Sporadic GRN Sporadic GRN Sporadic
4 7 2 1 0 1 5 2 0 2 5 4 15 8 1 0
1 1 1 1 5 6 2 2 8 6 1 0 0 0 19 28
5 9 0 3 4 6 3 8 0 2 13 18 6 21 0 2
5 6 4 1 8 8 3 1 5 10 1 8 0 1 1 0
Chi-square (χ 2 ) contingency table analysis comapring GRN and sporadic cases: NCI χ 2 = 0.38 (p > 0.05), GI χ 2 = 5.28 (p > 0.05), NII χ 2 = 1.08 (p > 0.05), DN χ 2 = 4.56 (p > 0.05), EN χ 2 = 4.67 (p > 0.05), SN χ 2 = 7.03 (p > 0.05), V χ 2 = 10.19, p < 0.01), GN χ 2 = 4.27 (p > 0.05).
frequency of unimodal or bimodal-type distributions were observed when cases were classified according to either disease duration or Braak tangle score. Nevertheless, in sporadic cases, the pTDP-43-immunoreactive inclusions affected more of the cortical profile in the longer duration cases, which suggests a possible spread of TDP-43 pathology across the cortex [16]. pTDP-43-immunoreactive inclusions were not spatially correlated in most gyri suggesting that NCI, NII, DN, and GI are distributed relatively independently across the cortex and do not develop within the same laminae. NCI and neuron densities, however, were positively correlated in four gyri suggesting that a constant proportion of neurons develop NCI. In addition, vacuole and surviving neuron densities were positively correlated in approximately a third of gyri suggesting formation of vacuoles in laminae with high neuronal densities. Vacuolation also occurs in Creutzfeldt–Jakob disease (CJD), dementia with Lewy bodies (DLB), and advanced Alzheimer’s disease (AD) [17]. In sporadic CJD (sCJD), for example, vacuoles are clustered around neuronal perikarya [36] and in the cerebellar hemisphere of variant CJD (vCJD), clusters of vac C
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uoles in the molecular layer are negatively correlated with surviving Purkinje cells [37]. Hence, vacuolation within the superficial cortical laminae could be the result of neuronal degeneration within laminae II/III subsequently affecting the ascending projections [19]. With the exception of the vacuolation and possible the DN, the laminar distribution of the pTDP-43 pathology, EN, and glial cell nuclei in GRN mutation cases is similar to that previously reported in sporadic FTLD-TDP [19]. However, vacuolation may develop over more of the cortical profile in the GRN mutation compared with the sporadic cases, but there were no differences in age or disease duration which could account for these differences. Hence, GRN mutation does not uniquely determine laminar distribution of the pTDPimmunoreactive pathology in FTLD-TDP. Pathological changes initiated by GRN mutation and by other causes in sporadic cases appear to follow a parallel course resulting in very similar patterns of cortical degeneration in frontal and temporal lobes.
Conclusions No single pattern of laminar distribution is characteristic of the pathology of the seven GRN mutation cases studied. Most commonly, the pathology affected all laminae with significant vacuolation of the superficial cortical laminae and a gliosis largely affecting the lower laminae. Of the pTDP-43-immunoreactive inclusions, most frequently the NCI and DN were abundant in the upper cortex and NII and GI in the lower cortex. Laminar distributions of the pathology in GRN cases are similar to those reported in sporadic cases of the disease suggesting that GRN mutation does not result in a unique pattern of cortical degeneration of the frontal and temporal lobe in FTLD-TDP.
Acknowledgements The authors thanks clinical, genetic, pathology and technical staff of the Departments of Neurology and Pathology and Immunology, Washington University School of Medicine, St. Louis, MO, USA for making available cases for this study.
Declaration of Interest The author declares no conflicts of interest. The author alone is responsible for the content and writing of this paper.
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References 1. Cairns NJ, Bigio EH, Mackenzie IRA, et al. Neuropathologic diagnostic and nosological criteria for frontotemporal lobar degeneration: consensus of the Consortium for Frontotemporal Lobar Degeneration. Acta Neuropathol 2007;114:5–22. 2. Baker M, Mackenzie IR, Pickering-Brown SM, et al. Mutations in progranulin cause tau-negative frontotemporal dementia linked to chromosome 17. Nature 2006;442:916–9. 3. Cruts M, Gijselink I, van der ZJ, et al. Null mutations in progranulin cause ubiquitin-positive frontotemporal dementia linked to chromosome 17q21. Nature 2006;442: 920–4. 4. Mukherjee O, Pastor P, Cairns NJ, et al. HDDD2 is a familial frontotemporal lobar degeneration with ubiquitin-positive taunegative inclusions caused by a missense mutation in the signal peptide of progranulin. Ann Neurol 2006;60:314–22. 5. Behrens MI, Mukherjee O, Tu PH, et al. Neuropathologic heterogeneity in HDDD1: a familial frontotemporal lobar degeneration with ubiquitin-positive inclusions and progranulin mutation. Alz Dis Assoc Disord 2007;21:1–7. 6. Rademakers R, Hutton M. The genetics of frontotemporal lobar degeneration. Cur Neurol Neurosci Rep 2007;7:434–42. 7. Mackenzie IRA, Baker M, Pickering-Brown S, et al. The neuropathology of frontotemporal lobar degeneration caused by mutations in the progranulin gene. Brain 2006;129:3081–90. 8. Van der Zee J, Gyselinck I, Pirici D, et al. Frontotemporal lobar degeneration with ubiquitin-positive inclusions: a molecular genetic update. Neurodegen diseases 2007;4:227–35. 9. Van Deerlin VM, Wood EM, Moore P, et al. Clinical, genetic and pathologic characteristics of patients with frontotemporal dementia and progranulin mutation. Arch Neurol 2007;64:1148–53. 10. Luty AA, Kwok JBJ, Thompson EM, et al. Pedigree with frontotemporal lobar degeneration-motor neuron disease and Tar DNA binding protein-43 positive neuropathology: genetic linkage to chromosome 9. BMC Neurol 2008;8:32. 11. Renton AE, Majounie E, Waite A, et al. A hexanucleotide repeat expansion in C9ORF72 is the cause of chromosome 9p21linked ALS-FTD. Neuron 2011;72:257–68. 12. Forman MS, Mackenzie IR, Cairns NJ, et al. Novel ubiquitin neuropathology in frontotemporal dementia with valosincontaining protein gene mutations. J Neuropathol Exp Neurol 2006;65:571–81. 13. Beck J, Rohrer JD, Campbell T, et al. A distinct clinical, neuropsychological and radiological phenotype is associated with progranulin gene mutation in a large UK series. Brain 2008;131:706–20. 14. Neumann M, Igaz LM, Kwong LK, et al. Absence of heterogeneous nuclear riboproteins and survival neuron protein (TDP43) positive inclusions in frontotemporal lobar degeneration. Acta Neuropathol 2007;113:543–8. 15. Davidson Y, Kelley T, Mackenzie IRA, et al. Ubiquinated pathological lesions in frontotemporal lobar degeneration contain TAR DNA-binding protein, TDP-43. Acta Neuropathol 2007;113:521–33. 16. Pirici D, Vandenberghe R, Rademakers R, et al. Characterization of ubiquinated intraneuronal inclusions in a novel Belgian frontotemporal lobar degeneration family. J Neuropathol Exp Neurol 2006;65:289–301. 17. Armstrong RA, Ellis W, Hamilton RL, et al. Neuropathological heterogeneity in frontotemporal lobar degeneration with TDP-43 proteinopathy: a quantitative study of 94 cases using principal components analysis. J Neural Transm 2010;117:227– 239.
18. Armstrong RA, Carter D, Cairns NJ. A quantitative study of the neuropathology of 32 sporadic and familial cases of frontotemporal lobar degeneration (FTLD) with TDP-43 proteinopathy (FTLD-TDP). Neuropathol Appl Neurobiol 2012;38: 25–38. 19. Armstrong RA, Hamilton RL, Mackenzie IRA, et al. Laminar distribution of the pathological changes in sporadic frontotemporal degeneration with TDP-43 proteinopathy: a quantitative study using polynomial curve fitting. Neuropathol Appl Neurobiol 2013;39:335–47. 20. Sampathu DM, Neumann M, Kwong LK, et al. Pathological heterogeneity of frontotemporal lobar degeneration with ubiquitin-positive inclusions delineated by ubiquitin immunohistochemistry and novel monoclonal antibodies. Am J Pathol 2006;169:1343–52. 21. Cairns NJ, Neumann M, Bigio EH, et al. TDP-43 familial and sporadic frontotemporal lobar degeneration with ubiquitin inclusions. Am J Pathol 2007;171:227–40. 22. Kersaitis C, Holliday GM, Xuereb JH, et al. Ubiquitin-positive inclusions and progression of pathology in FTD and MND identifies a group with mainly early pathology. Neuropathol Appl Neurobiol 2006;32:83–91. 23. Josephs KA, Whitwell JL, Jack CR, et al. Frontotemporal lobar degeneration without lobar atrophy. Arch Neurol 2006;63:1632–8. 24. Braak H, Braak E, Bohl J. Staging of Alzheimer-related cortical destruction. Eur Neurol 1993;33:403–8. 25. Braak H, Alafuzoff I, Arzberger T, et al. Staging of Alzheimer disease-associated neurofibrillary pathology using paraffin sections and immunocytochemistry. Acta Neuropathol 2006;112:389–404. 26. Duyckaerts C, Hauw JJ, Bastenaire F, et al. Laminar distribution of neocortical senile plaques in senile dementia of the Alzheimer type. Acta Neuropathol 1986;70:249–56. 27. Yaguchi M, Fujita Y, Amari M, et al. Morphological differences of intraneural ubiquitin positive inclusions in the dentate gyrus and parahippocampal gyrus of motor neuron disease with dementia. Neuropathology 2004;24:296–301. 28. Matsumoto S, Udaka F, Kameyama M, et al. Subcortical neurofibrillary tangles, neuropil threads and argentophilic glial inclusions in corticobasal degeneration. Clin Neuropath 1996;15:209–14. 29. Yamada T, McGeer PL, McGeer EG. Appearance of paired nucleated tau-positive glia in patients with progressive supranuclear palsy brain tissue. Neurosci Lett 1992;135:99–102. 30. Ikeda K, Akiyama H, Kondo H, et al. Thorn-shaped astrocytes: possibly secondarily induced tau-positive glial fibrillary tangles. Acta Neuropathol 1995;90:620–5. 31. Komori T. Tau positive glial inclusions in progressive supranuclear palsy, corticobasal degeneration and Pick’s disease. Brain Pathol 1999;9:663–79. 32. Probst A, Tolnay M. Argyrophilic grain disease, a frequent and largely underestimated cause of dementia in old patients. Rev Neurol 2002;158:155–65. 33. Hatanpaa KJ, Bigio EH, Cairns NJ, et al. TAR DNA-binding protein 43 immunohistochemistry reveals extensive neuritic pathology in FTLD-U: a Midwest-Southwest Consortium for FTLD-U study. J Neuropathol Exp Neurol 2008;67:271–9. 34. Armstrong RA. Correlations between the morphology of diffuse and primitive b-amyloid (Ab) deposits and the frequency of associated cells in Down’s syndrome. Neuropath Appl Neurobiol 1996;22:527–30. 35. Armstrong RA. A quantitative study of abnormally enlarged neurons in cognitively normal brain and neurodegenerative disease. Clin Neuropathol 2013;37:128–33.
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36. Armstrong RA, Lantos PL, Cairns NJ. Spatial correlations between the vacuolation, prion protein deposits, and surviving neurons in the cerebral cortex in sporadic Creutzfeldt-Jakob disease. Neuropathology 2001;21:266–71. 37. Armstrong RA, Ironside J, Lantos PL, Cairns NJ. A quantitative study of the pathological changes in the cerebellum of 15 cases of variant Creutzfeldt-Jakob disease. Neuropathol Appl Neurobiol 2009;35:36–45. 38. Armstrong RA, Hilton A. Statistical Analysis in Microbiology: Statnotes. Hoboken, NJ, USA: Wiley-Blackwell; 2011. 39. Snedecor GW, Cochran WG. Statistical Methods. Ames, IA USA: Iowa State University Press; 1980. 40. Armstrong RA, Slaven A. Does the neurodegeneration of Alzheimer’s disease spread between visual cortical regions B17 and B18 via the feedforward or feedback short cortico-cortical projections? Neurodegeneration 1994;3:191–6.
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41. De Lacoste M, White CL. The role of cortical connectivity in Alzheimer’s disease pathogenesis: a review and model system. Neurobiol Aging 1993;14:1–16. 42. Pierucci A, de Oliveira AL. Increased sensory neuron apoptotic death two weeks after peripheral axonotomy in C57BL/6J mice compared to A/J mice. Neurosci Lett 2006;396:127– 31. 43. Kato S, Hirano A, Umahara T, et al. Ultrastructural and immunohistochemical studies on ballooned cortical neurons in Creutzfeldt-Jakob disease: expression of alpha-B-crystallin, ubiquitin and stress response protein-27. Acta Neuropathol 1992;84:443–8. 44. Goedert M, Spillantini MG, Serpell LC, et al. From genetics to pathology: tau and alpha-synuclein assemblies in neurodegenerative diseases. Phil Trans Roy Soc Lond B. Biol Sci 2001;356:213–27.
RESEARCH ARTICLE
Cortical degeneration in frontotemporal lobar degeneration with TDP-43 proteinopathy caused by progranulin gene mutation Richard A. Armstrong Int J Neurosci Downloaded from informahealthcare.com by Nyu Medical Center on 05/26/15 For personal use only.
Vision Sciences, Aston University, Birmingham, UK Familial frontotemporal lobar degeneration with transactive response (TAR) DNA-binding protein of 43 kDa (TDP43) proteinopathy (FTLD-TDP) is most commonly caused by progranulin (GRN) gene mutation. To characterize cortical degeneration in these cases, changes in density of the pathology across the cortical laminae of the frontal and temporal lobe were studied in seven cases of FTLD-TDP with GRN mutation using quantitative analysis and polynomial curve fitting. In 50% of gyri studied, neuronal cytoplasmic inclusions (NCI) exhibited a peak of density in the upper cortical laminae. Most frequently, neuronal intranuclear inclusions (NII) and dystrophic neurites (DN) exhibited a density peak in lower and upper laminae, respectively, glial inclusions (GI) being distributed in low densities across all laminae. Abnormally enlarged neurons (EN) were distributed either in the lower laminae or were more uniformly distributed across the cortex. The distribution of all neurons present varied between cases and regions, but most commonly exhibited a bimodal distribution, density peaks occurring in upper and lower laminae. Vacuolation primarily affected the superficial laminae and density of glial cell nuclei increased with distance across the cortex from pia mater to white matter. The densities of the NCI, GI, NII, and DN were not spatially correlated. The laminar distribution of the pathology in GRN mutation cases was similar to previously reported sporadic cases of FTLD-TDP. Hence, pathological changes initiated by GRN mutation, and by other causes in sporadic cases, appear to follow a parallel course resulting in very similar patterns of cortical degeneration in FTLD-TDP. KEYWORDS: Neuronal cytoplasmic inclusions (NCI), Neuronal intranuclear inclusions (NII), Dystrophic neurites (DN), Transactive response (TAR) DNA-binding protein of 43kDa (TDP-43), Vacuolation
Introduction A significant proportion of frontotemporal lobar degeneration (FTLD) with transactive response (TAR) DNA-binding protein of 43kDa (TDP-43) proteinopathy (FTLD-TDP) cases are familial [1–8]. Various genetic defects have been identified in these cases, the majority being caused by mutation of the progranulin (GRN) gene [2–9]. Familial FTLD-TDP can also be commonly caused by chromosome 9 open reading frame 72 (C9ORF72) gene [10,11] and more rarely by valosincontaining protein (VCP) gene mutation [12]. Several different frame-shift and premature termination mutations have been identified in GRN mutaReceived 7 November 2013; revised 7 January 2014; accepted 30 January 2014 Correspondence: R.A. Armstrong, Vision Sciences, Aston University, Birmingham B4 7ET, UK. Tel. 0121 359 3611. Fax: 0121 333 4220. E-mail: [email protected]
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tion cases [4,5,13]. Abnormal protein products may accumulate within the endoplasmic reticulum of the cell due to inefficient secretion or mutant RNA may have a lower expression within the cell at least in some mutants [4]. TDP-43 is a nuclear protein and exhibits a granular pattern of immunoreactivity in the nucleus of normal cells. The subsequent development of the pathology in FTLD-TDP is unclear, however, TDP43 may be redistributed from the nucleus to the cytoplasm, then ubiquinated, and hyperphosphorylated, and ultimately cleaved to generate C-terminal fragments [14]. These fragments could then accumulate to form inclusions that may ultimately lead to cell death and degeneration of anatomical pathways. The TDP-43-immunoreactive pathology in FTLD-TDP includes various types of inclusion including neuronal cytoplasmic inclusions (NCI) [1,7,15,17], neuronal intranuclear inclusions (NII) [16–18], dystrophic neurites (DN) [1,15,17], and oligodendroglial inclusions (GI)
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Table 1. Demographic features, gross brain weight, and genetics of the seven cases of familial frontotemporal lobar dementia with TDP-43 proteinopathy (FTLD-TDP) caused by progranulin (GRN) mutation. Braak score is based on density and distribution of neurofibrillary tangles. (M = Male, F = Female, DR = Duration, BW = Brain weight, IVS = intervening sequence.) Case
Sex
Age
Onset
DR
BW
Mutation
Location
Braak score
A B C D E F G
F F F M M F M
74 84 67 63 79 82 66
68 69 58 57 71 73 55
6 15 9 6 8 9 11
975 570 880 1080 1150 800 1050
g.4068C>A g.4068C>A g.4068C>A g.4068C>A g.4068C>A g.4068C>A g.5913A>G
Exon 1 Exon 1 Exon 1 Exon 1 Exon 1 Exon 1 IVS6
0 4 2 1 6 1
[16–18]. Vacuolation affecting the superficial regions of the cortex and the presence of abnormally enlarged neurons (EN) have also been observed [1,17,18]. In sporadic FTLD-TDP, NCI, DN, and vacuolation were abundant in upper cortical laminae of the frontal and temporal lobe and GI, NII, EN, and glial cell nuclei in the lower laminae [19]. In addition, variation in laminar distribution between cases was related to disease duration and varied with disease subtype [14,20,21]. Hence, to compare cortical degeneration in GRN cases with sporadic FTLD-TDP [19], changes in density of NCI, GI, NII, DN, all neurons present, EN, and vacuoles were studied across the cortical laminae in frontal and temporal lobe in seven cases of FTLD-TDP caused by GRN mutation using quantitative analysis and polynomial curve fitting.
laration of Helsinki (as modified Edinburgh, 2000). Blocks were taken from the frontal lobe at the level of the genu of the corpus callosum to study middle frontal gyrus (MFG) and the temporal lobe at the level of the lateral geniculate body to study inferior temporal gyrus (ITG) and parahippocampal gyrus (PHG). Tissue was fixed in 10% phosphate buffered formalsaline and subsequently embedded in paraffin wax. Sections were pretreated with formic acid (95%) for 5 min and then immunohistochemistry was carried out using a mouse monoclonal antibody that specifically recognizes phosphorylated TDP-43 (pTDP-43) (dilution 1:40,000; pS409/410–1, Clone 11–9, Cosmo Bio USA, Inc., Carlsbad, CA, USA). Sections were also stained with hematoxylin.
Morphometric methods
Materials and Methods Cases FTLD-TDP cases (N = 7) caused by GRN mutation (see Table 1) were obtained from the Departments of Neurology and Pathology and Immunology, Washington University, School of Medicine, St. Louis, MO, USA. Six of the cases carried the missense g.4068C>A mutation in exon 1 of the GRN gene [4] and one case the splice site g5913A>G (IVS6–2A>G) mutation located within intervening sequence (IVS) 6 [5]. All cases revealed the characteristic pathology of FTLD, viz., neuronal loss, microvacuolation of the superficial cortical laminae, and a reactive astrocytosis [1,4]. None of the cases had symptoms or pathology of motor neuron disease (FTLD-MND) [22,23] and none had coexisting hippocampal sclerosis (HS). Staging of disease was based on the Braak tangle score [24,25].
Histological methods The consent of the next of kin was obtained after death for removal of the brain as determined by local Ethical Committee procedures and the 1995 Dec C
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The laminar distribution of the NCI, GI, NII, and DN together with the neurons, EN, vacuoles, and glial cell nuclei was studied across the cortex from pia matter to white matter using methods described by Armstrong et al. [19] and Duyckaerts et al. [26]. Five randomly positioned traverses from pia matter to the edge of the white matter were located along each gyrus. Histological features were counted in 50 × 250 μm sample fields arranged contiguously down the cortex, the larger dimension of the field being located parallel with the surface of the pia matter. An eye-piece micrometer was used as the sample field which was moved down each traverse one step at a time from pia matter to the edge of the white matter. The NCI were rounded, spicular, or skein-like [15,17,27], while the GI resembled similar inclusions reported in the tauopathies [28–32] The NII were lenticular, spindle-shaped, or circular [16,17] and the DN often long and contorted in shape [17,33]. Neurons had a larger shape, nonspherical outline and contained at least some cytoplasm [34], while glial cells comprised small spherical or asymmetrical nuclei without cytoplasm. EN had swollen perikarya, had a dystrophic nucleus usually displaced to the side of the cell,
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and maximum cell diameter was at least three times that of the nucleus [35]. Discrete vacuoles greater than 5 μm in diameter were also counted [36,37]. Vacuoles clearly associated with neuronal perikarya were not counted as they could be artifacts [36,37]. The mean of the counts from the five traverses was calculated to study variations in density of each histological feature across each gyrus. Data analysis It was difficult to locate precise boundaries between individual cortical laminae due to the degree of cortical degeneration present in many gyri. In addition, laminar identification is especially difficult in the frontal cortex because it exhibits a heterotypical structure, i.e., six laminae cannot always be clearly identified. Moreover, many pathological inclusions exhibited complex patterns of distribution across the cortex and were not confined to specific laminae [19]. Hence, to determine laminar distributions, variations in density across the cortex were analyzed using a polynomial curve-fitting procedure (STATISTICA software, Statsoft Inc., 2300 East 14th Street, Tulsa, OK 74104, USA) [16,36,37]. Hence, for each gyrus studied, polynomials up to the sixth order were fitted successively to the data, quadratic curves being parabolic, cubic curves ‘‘S” shaped, and quartic curves ‘‘double-peaked” or ‘‘bimodal” etc. Correlation coefficients (Pearson’s ‘‘r”), regression coefficients, standard errors (SE), values of t, and the residual mean squares were obtained at each stage of the analysis [38], the reduction in the sums
Figure 1. The pTDP-43-immunoreactive pathology in the upper
cortical laminae of a case of familial frontotemporal lobar dementia with TDP-43 proteinopathy (Case A) caused by progranulin (GRN) mutation showing the typical neuronal cytoplasmic inclusions (NCI, arrowhead) and a few dystrophic neurites (DN, arrow) in the upper cortical laminae corresponding to laminae II/III. Significant vacuolation is also evident. (pTDP-43 immunohistochemistry, bar = 20 μm).
Figure 2. Typical examples of (a) neuronal intranuclear inclusion (NII, arrow) in lower cortical laminae of Case B (pTDP43 immunohistochemistry, bar = 50 μm), (b) a glial inclusion (GI, arrow) in lower cortical laminae in a case (Case C) (pTDP-43 immunohistochemistry, bar = 50 μm), and (c) an abnormally enlarged neuron (EN, arrow) in lower cortical laminae of Case A (H/E bar = 20 μm).
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Pathology of GRN mutation cases of FTLD-TDP
of squares (SS) being tested for significance. A curve of best fit was established by observing when either a nonsignificant value of F was obtained or there was little gain in the explained variance [38,39]. Histological features were classified as having either a single peak of density (‘‘unimodal” distribution), peak density being located either in the upper cortex (approximating to laminae I, II, III), middle cortex (approximating to mainly lamina IV), or lower cortex (approximating to laminae V, VI) cortex or two peaks of density (“bimodal” distribution), with peaks of density occurring in upper and lower cortex. Bimodal distributions were classified further according to whether the density peaks were similar in upper and lower cortex. If no significant polynomial curves were obtained, then no significant trend in abundance across the cortex was assumed. To determine whether the frequencies of the different types of distribution exhibited by the pTDP-43immunoreactive inclusions varied with disease duration or Braak-tangle stage [25], data were compared using chi-square (χ 2 ) contingency table tests. In addition, correlations between the densities of all histological features were tested using Pearson’s correlation coefficient (r). To compare the frequency of different laminar distributions in GRN cases with those previously published
in sporadic cases of FTLD-TDP [19], the data for each histological feature were also analyzed using χ 2 contingency table tests. Four categories of distribution were used for this analysis, viz., a significant density peak in upper cortex, lower cortex, a bimodal distribution, or no significant change with distance from pia matter.
Results The pTDP-43-immunoreactive pathology in the upper laminae of a GRN mutation case of FTLD-TDP (Case A) is shown in Figure 1 and shows typical examples of NCI and DN in the upper cortical laminae. Some vacuolation of the upper laminae is also evident. Figure 2 illustrates typical examples of a pTDP-43immunoreactive NII in lower cortical laminae (Case B, Figure 2a), a GI in lower laminae (Case C, Figure 2b), and an EN in lower laminae (Case A, Figure 2c). Examples of the laminar distribution of the NCI and NII in a single gyrus (Case A, MFG) are shown in Figure 3. The distribution of the NCI was fitted by a thirdorder polynomial (r = 0.55, p < 0.01) with a single density peak in the upper laminae. The distribution of the NII was fitted with a fifth-order polynomial (r = 0.58,
Figure 3. Examples of the laminar distribution of the neuronal cytoplasmic inclusions (NCI) and neuronal
intranuclear inclusions (NII) in a single gyrus (Case A, MFG) of familial frontotemporal lobar dementia with TDP-43 proteinopathy caused by progranulin (GRN) mutation. Curve fitting; NCI, third-order polynomial (r = 0.55, p < 0.01), NII, fifth-order polynomial (r = 0.58, p < 0.01). C
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Figure 4. The laminar distribution of the neurons and vacuolation in a single gyrus (Case D, MFG) of a
case of familial frontotemporal lobar dementia with TDP-43 proteinopathy caused by progranulin (GRN) mutation. Curve fitting: neurons, fourth-order polynomial (r = 0.76, p < 0.001); vacuolation second-order polynomial (r = 0.90, p < 0.001).
p < 0.01), the distribution being bimodal, the peaks of density in the upper and lower laminae having approximately equal magnitude. Examples of the laminar distribution of the neurons and vacuolation in a single brain region (Case D, MFG) are shown in Figure 4. The distribution of the neurons was fitted by a fourth-order polynomial (r = 0.76, p < 0.001) suggesting a binomial distribution, with similar peaks of density in the upper and lower laminae. The distribution of the vacuoles (Case A, PHG) was fitted best by a second-order (quadratic) polynomial (r = 0.90, p < 0.001), vacuole density being greatest in the superficial laminae, density then declining rapidly with distance below the pia mater. The distributions of all histological features in each region and case are shown in Table 2 and summarized in Table 3. In approximately 50% of gyri studied, the density of the NCI was greatest in the upper laminae, four gyri having a unimodal and three a bimodal distribution. In five gyri, there were no significant differences in density with distance below the pia matter. The GI exhibited a peak of density in the upper laminae in 2/7 (29%) gyri, a peak in the lower laminae in 1/7 (14%), and there was no significant variation in density across the cortex
in 4/7 (57%) gyri. The NII exhibited a peak of density in the lower cortex in 5/17 (29%) of gyri and in 4/17 (24%) of gyri a bimodal distribution was present, density peaks being similar in the upper and lower cortex. In 8/17 (47%) of gyri, there was no significant change in density of NII across the cortex. The distribution of the DN varied between cases and gyri, a peak of density in the upper cortex present in approximately 50% of gyri. The EN were distributed in the lower laminae in 8/15 (53%) gyri or were uniformly distributed down the cortex in 5/15 (33%) gyri. The distribution of neurons was variable, the most common being a bimodal distribution with density peaks in the upper and lower cortex, the upper density peak being greater than the lower in 8/21 (38%) of gyri. The vacuoles were distributed largely in the upper laminae, 15/21 (71%) of gyri exhibiting this pattern, while the distribution of glial cell nuclei was primarily in the lower cortical laminae. In 19/21 (90%) of gyri, there was a linear increase in glial cell density across the cortex from pia matter to white matter. No consistent differences in laminar distribution were apparent between gyri in frontal and temporal lobes. The relationship between the distribution of the pTDP-43-immunoreactive inclusions, disease duration,
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Table 2. Results of the polynomial curve-fitting procedure for each histological feature (NCI = Neuronal cytoplasmic inclusions, GI = Glial inclusions, NII = Neuronal intranuclear inclusions, DN = Dystrophic neurites, EN = Abnormally enlarged neurons, V = Vacuolation, NR = Neurons, GL = Glial cell nuclei) in each cortical gyrus. The first figure of each entry is the order of polynomial fitted to the data (1 = linear, 2 = quadratic, 3 = cubic, 4 = quartic, etc and the second letter in parentheses, whether a density peak was located in the upper cortex (U) (approximating to laminae I,II, III), middle cortex (M) (approximating to lamina IV), or lower cortex (L) (approximating to laminae V,VI) cortex or whether two peaks of density (“bimodal” distribution) (B) were present with peaks of density occurring in upper and lower cortex (NS = no significant curve fitted the data, symbol “-” indicates insufficient density of a histological feature to quantify its distribution). Histological features Case
Region
NCI
GI
NII
DN
EN
V
NR
GL
A
MFG ITG PHG MFG ITG PHG MFG ITG PHG MFG ITG PHG MFG ITG PHG MFG ITG PHG MFG ITG PHG
4 (U) NS 1 (L) NS NS NS 4 (B) 1 (U) NS 4 (B) 4 (B) 1 (U) 4 (B) 3 (B) 2 (U)
NS NS NS 3 (U) 3 (U) 3 (L) NS -
4 (B) 2 (L) 1 (L) 4 (B) 1 (L) NS 4 (B) 3 (L) NS NS 3 (B) NS NS NS NS NS 3 (L)
4 (B) 4 (B) 1 (L) 4 (B) 4 (B) NS NS 3 (U) NS 2 (U) 2 (U) 3 (U) 1 (L) 2 (U) -
NS 1 (L) NS NS 1 (L) NS 1 (L) NS 3 (L) 3 (L) M 2 (L) M NS NS 1 (L) -
2 (U) 2 (U) 2 (U) 1 (L) 4 (B) 1 (U) 2 (U) 4 (B) 3 (B) 2 (U) 2 (U) 2 (U) 2 (U) 3 (B) 2 (U) 2 (U) 4 (B) 4 (B) 1 (U) 3 (U) 3 (U)
4 (B) 2 (U) 4 (B) 4 (B) 3 (B) 1 (M) NS 2 (L) 4 (B) 4 (B) 3 (U) 4 (B) 4 (B) 4 (B) 1 (U) 3 (B) 3 (B) 4 (B) 4 (B) 1 (L) 4 (B)
1 (L) 1 (L) 1 (L) 1 (L) 1 (L) 1 (L) 1 (L) NS 1 (L) 1 (L) 1 (L) 1 (L) 1 (L) 1 (L) 2 (M) 1 (L) 1 (L) 1 (L) 2 (L) 1 (L) 1 (L)
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B
C
D
E
F
G
and Braak tangle stage is shown in Table 4. There were no significant differences in the frequencies of unimodal or bimodal distributions when cases were classified according to disease duration (χ 2 = 1.32, 5DF, p > 0.05) or Braak tangle stage (χ 2 = 1.28, 5DF, p > 0.05). A summary of the spatial correlations in density of the various histological features across the gyri is shown in Table 5. There were few significant correlations between the NCI, GI, NII, and DN. However, there was a positive correlation between the densities of neurons and vacuoles in 7/21 (33%) gyri and a negative correlation between the densities of neurons and glial cell nuclei in 8/21 (38%) gyri. In addition, the densities of the vacuoles and glial cell nuclei were negatively correlated in 50% of gyri studied. A comparison of the laminar distributions in GRNmutation cases with those previously published for 10 sporadic cases of FTLD-TDP [16] is shown in Table 6. The laminar distributions were similar for GRN mutation and sporadic cases of the disease with the exception of the vacuolation, unimodal distributions with a density peak in the upper laminae being less frequent and bimodal distributions more frequent in sporadic cases. This difference between GRN and familial FTLD-TDP
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is also evident for the DN but does not reach statistical significance.
Discussion As in sporadic FTLD-TDP, the data suggest significant changes in density of the pathology across cortical gyri in GRN mutation cases. Changes in density varied between gyri and cases suggesting that the GRN cases do not conform to a single pathological subtype of FTLDTDP based on their laminar distribution [1,7,14,20]. The most consistent patterns of distribution were: (1) significant vacuolation in the superficial laminae, vacuoles declining in density with distance from the pia matter and (2) increasing densities of glial cell nuclei in the lower cortex. Of the pTDP-43-immunoreactive inclusions, the NCI and DN were most abundant in upper cortex and NII in lower cortex, while the GI exhibited no laminar preference. The distribution of the neurons was especially variable. In normal elderly brain, pyramidal neurons in frontal and temporal cortex often exhibit a bimodal distribution in which peak density in upper cortex
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Table 3. Summary of the laminar distribution of lesions (NCI = Neuronal cytoplasmic inclusions, GI = Glial inclusions, NII = Neuronal intranuclear inclusions, DN = Dystrophic neurites, EN = Abnormally enlarged neurons, V = Vacuolation, NR = Neurons, GN = Glial cell nuclei) in gyri of the frontal and temporal cortex in seven cases familial frontotemporal lobar dementia with TDP-43 proteinopathy caused by progranulin (GRN) mutation. (N = number of gyri analyzed. Data indicate the number of gyri in which a particular histological feature exhibited either a single peak of density in the upper cortex (U) (approximating to laminae I, II, III), middle cortex (M) (approximating to lamina IV), or lower cortex (L) (approximating to laminae V, VI) cortex or whether two peaks of density (‘bimodal’ distribution) (B) were present with peaks of density occurring in upper and lower cortex. NS = no significant change in density from pia matter to white matter). Unimodal distribution Lesion NCI GI NII DN EN NR V GL
Bimodal distribution
N
U
M
L
U≥L
U=L
L≥U
NS
15 7 17 14 15 21 21 21
4 2 0 5 0 5 15 0
0 0 0 1 2 1 0 1
1 1 5 2 8 1 0 19
3 0 0 2 0 8 6 0
2 0 4 1 0 3 0 0
0 0 0 0 0 2 0 0
5 4 8 3 5 1 0 1
(corresponding to laminae II/III) is usually greater than in lower cortex (corresponding to laminae V/VI) [40]. A similar bimodal distribution was observed in eight gyri in the GRN cases. In six further gyri, however, peak densities of neurons were similar in the upper and lower cortex, greater in lower than upper cortex, or were uniformly distributed across the cortex. All of these distributions suggest neuronal loss in upper cortical laminae. Neuronal loss in upper cortex could Table 4. Relationship between the distribution of TDP-43-immunoreactive inclusions, disease duration, and Braak stage in familial frontotemporal lobar dementia with TDP-43 proteinopathy caused by progranulin (GRN) mutation. Data show the frequency of gyri in which collectively, the TDP-43-immunoreactive inclusions exhibited a unimodal or a bimodal distribution, NS = No significant change in abundance across the cortex. Laminar distribution Variable Disease duration (years) Braak stage
Categories
Unimodal
Bimodal
NS
6–8 ≥9 0–2 4–6
8 13 11 6
7 5 10 2
8 11 12 4
Chi-square (χ 2 ) contingency table tests: Disease duration χ 2 = 1.32 (5DF, p > 0.05), Braak score χ 2 = 1.28 (5DF, p > 0.05).
Table 5. Summary of correlations (Pearson’s “r”) between the densities of histological features (NCI = Neuronal cytoplasmic inclusions, GI = Glial inclusions, NII = Neuronal intranuclear inclusions, DN = dystrophic neurites, EN = Abnormally enlarged neurons, V = vacuolation, NR = neurons, GN = glial cell nuclei). Frequency of correlation Variables
N
Positive ‘r’
Negative ‘r’
NS
NCI/GI NCI/NII NCI/DN NCI/EN NCI/NR NCI/V NCI/GL GI/NII GI/DN GI/EN GI/NR GI/V GI/GL NII/DN NII/EN NII/NR NII/V NII/GL DN/EN DN/NR DN/V DN/GL EN/NR EN/V EN/GL NR/V NR/GL V/GL
14 19 16 19 19 19 18 15 12 15 16 16 16 17 20 21 21 21 17 17 17 19 20 20 21 21 21 20
2 1 2 1 4 4 1 1 2 0 1 2 1 1 1 0 1 1 1 3 5 0 1 0 2 7 0 0
0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 1 1 0 0 1 0 2 0 1 0 0 8 10
12 18 14 18 15 15 17 15 10 15 16 14 15 16 19 20 19 20 16 13 12 157 19 19 19 14 13 10
be associated with degeneration of the feed-forward cortico-cortical projections, which have their cells of origin in laminae II/III [41]. By contrast, EN, which may reflect either a stress response or axonal degeneration [42,43], and glial cell nuclei, were frequently abundant in lower cortex. Pathological changes in lower cortex could be associated with degeneration of either the feedback cortico-cortical projections [41] or the afferent and efferent cortical-subcortical projections. Hence, as in sporadic FTLD-TDP [19], there is degeneration across all cortical laminae in GRN mutation cases potentially affecting multiple anatomical pathways. A notable feature of the data is the variability in laminar distributions across different GRN cases. pTDP-43immunoreactive pathology could spread between cortical regions via cell-to-cell transfer as has been suggested for tau and α-synuclein-immunoreactive pathologies [41,44]. Hence, variations in laminar distribution between cases and gyri could represent different stages of this process. However, no significant differences in the International Journal of Neuroscience
Pathology of GRN mutation cases of FTLD-TDP Table 6. Comparison of the frequencies of the various laminar distributions (NCI = Neuronal cytoplasmic inclusions, GI = Glial inclusions, NII = Neuronal intranuclear inclusions, DN = Dystrophic neurites, EN = Abnormally enlarged neurons, V = vacuolation, NR = Neurons, GN = glial cell nuclei) in gyri of the frontal and temporal cortex in seven cases of familial frontotemporal lobar degeneration with TDP-43 proteinopathy (FTLD-TDP) caused by progranulin (GRN) mutation with cases of sporadic FTLD-TDP. Histological feature NCI
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GI NII DN EN NR V GN
Laminar distribution
Patient group
Upper
Lower
Bimodal
NS
GRN Sporadic GRN Sporadic GRN Sporadic GRN Sporadic GRN Sporadic GRN Sporadic GRN Sporadic GRN Sporadic
4 7 2 1 0 1 5 2 0 2 5 4 15 8 1 0
1 1 1 1 5 6 2 2 8 6 1 0 0 0 19 28
5 9 0 3 4 6 3 8 0 2 13 18 6 21 0 2
5 6 4 1 8 8 3 1 5 10 1 8 0 1 1 0
Chi-square (χ 2 ) contingency table analysis comapring GRN and sporadic cases: NCI χ 2 = 0.38 (p > 0.05), GI χ 2 = 5.28 (p > 0.05), NII χ 2 = 1.08 (p > 0.05), DN χ 2 = 4.56 (p > 0.05), EN χ 2 = 4.67 (p > 0.05), SN χ 2 = 7.03 (p > 0.05), V χ 2 = 10.19, p < 0.01), GN χ 2 = 4.27 (p > 0.05).
frequency of unimodal or bimodal-type distributions were observed when cases were classified according to either disease duration or Braak tangle score. Nevertheless, in sporadic cases, the pTDP-43-immunoreactive inclusions affected more of the cortical profile in the longer duration cases, which suggests a possible spread of TDP-43 pathology across the cortex [16]. pTDP-43-immunoreactive inclusions were not spatially correlated in most gyri suggesting that NCI, NII, DN, and GI are distributed relatively independently across the cortex and do not develop within the same laminae. NCI and neuron densities, however, were positively correlated in four gyri suggesting that a constant proportion of neurons develop NCI. In addition, vacuole and surviving neuron densities were positively correlated in approximately a third of gyri suggesting formation of vacuoles in laminae with high neuronal densities. Vacuolation also occurs in Creutzfeldt–Jakob disease (CJD), dementia with Lewy bodies (DLB), and advanced Alzheimer’s disease (AD) [17]. In sporadic CJD (sCJD), for example, vacuoles are clustered around neuronal perikarya [36] and in the cerebellar hemisphere of variant CJD (vCJD), clusters of vac C
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uoles in the molecular layer are negatively correlated with surviving Purkinje cells [37]. Hence, vacuolation within the superficial cortical laminae could be the result of neuronal degeneration within laminae II/III subsequently affecting the ascending projections [19]. With the exception of the vacuolation and possible the DN, the laminar distribution of the pTDP-43 pathology, EN, and glial cell nuclei in GRN mutation cases is similar to that previously reported in sporadic FTLD-TDP [19]. However, vacuolation may develop over more of the cortical profile in the GRN mutation compared with the sporadic cases, but there were no differences in age or disease duration which could account for these differences. Hence, GRN mutation does not uniquely determine laminar distribution of the pTDPimmunoreactive pathology in FTLD-TDP. Pathological changes initiated by GRN mutation and by other causes in sporadic cases appear to follow a parallel course resulting in very similar patterns of cortical degeneration in frontal and temporal lobes.
Conclusions No single pattern of laminar distribution is characteristic of the pathology of the seven GRN mutation cases studied. Most commonly, the pathology affected all laminae with significant vacuolation of the superficial cortical laminae and a gliosis largely affecting the lower laminae. Of the pTDP-43-immunoreactive inclusions, most frequently the NCI and DN were abundant in the upper cortex and NII and GI in the lower cortex. Laminar distributions of the pathology in GRN cases are similar to those reported in sporadic cases of the disease suggesting that GRN mutation does not result in a unique pattern of cortical degeneration of the frontal and temporal lobe in FTLD-TDP.
Acknowledgements The authors thanks clinical, genetic, pathology and technical staff of the Departments of Neurology and Pathology and Immunology, Washington University School of Medicine, St. Louis, MO, USA for making available cases for this study.
Declaration of Interest The author declares no conflicts of interest. The author alone is responsible for the content and writing of this paper.
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