Abstract
Keywords
INTRODUCTION
Neurodegenerative dementias are characterized by progressive neuronal loss, gliosis, and abnormal aggregation of proteins in the cortex, which are widely considered to underlie cognitive and behavioral symptoms [1, 2]. Apart from Alzheimer’s disease (AD), Lewy body dementias (LBD) are the most common forms of neurodegenerative dementias [3, 4]. LBD is a collective term for two major clinical diagnoses—dementia with Lewy bodies (DLB) and Parkinson’s disease dementia (PDD), distinguished on the basis of the temporal manifestation of cognitive impairments relative to parkinsonism. Therefore, patients developing dementia prior to, or within the first year of, parkinsonism are diagnosed with DLB. In PDD patients, dementia onset occurs more than one year after motor symptoms, and often manifest in the terminal stages of PD [5]. The typical pathological feature of AD, DLB, and PDD is the progressive accumulation of intra- or extra-neuronal protein aggregates. Neuropathological hallmarks of AD include the deposition of amyloid-β (Aβ) plaques composed of highly aggregated, fibrillar Aβ, neurofibrillary tangles (NFTs) and neuropil threads composed of hyperphosphorylated tau protein and neuritic plaques (NPs) which are Aβ plaques with dystrophic neurites that contain hyperphosphorylated tau protein. In contrast, DLB and PDD are characterized by Lewy bodies (LBs) and Lewy neurites containing aggregatedα-synuclein [6]. However, DLB frequently shows AD pathology, the severity of which may range from low to high AD neuropathologic burden [7], with the latter been referred to as mixed AD/DLB [8]. In PDD, AD pathology may also be present but both its prevalence and severity are lower than the ones observed in DLB [9–11].
In both AD and LBD, dysregulated neuroinflammation has been gaining recognition as a pathogenic factor and may be associated with neurodegeneration [12, 13]. Of the many inflammatory cytokines which have been implicated in neurodegenerative dementias, members of transforming growth factor β (TGFβ) family are multifunctional cytokines which have diverse roles in various organs, including regulation of cell growth, differentiation, and survival [14]. Effects of TGFβ are highly contextualized and, depending on cell type and environment, it may promote cell survival or induce apoptosis, stimulate cell proliferation or induce differentiation, and initiate or resolve inflammation [14, 15]. All three identified isoforms (TGFβ1, TGFβ2, and TGFβ3) are known to be expressed in mammalian brain, where they have been implicated in brain development, modulation of synaptic transmission, and neuroendocrine regulation [14]. While much of the research focus has been on the therapeutic potential of TGFβ1 due to in vitro as well as in vivo demonstration of its neuroprotective functions [14–17], later studies have suggested that TGFβ2 may also be relevant in the pathogenesis of neurodegenerative dementias. For example, immunohistochemical studies showed strong TGFβ2 immunoreactivity in NFT-bearing neurons, astrocytes, and neurites within NPs [18–20]. Moreover, recent in vitro and in vivo data suggest that the expression of TGFβ2 in both glial and neuronal cells can be induced by the 42-amino acid Aβ42, the major species of Aβ peptide in amyloid plaques [21], while other studies demonstrated a role for TGFβ2 in driving neuronal Aβ uptake and targeting, as well as impairing memory retrieval or consolidation [22]. However, while immunohistochemical studies have suggested increased TGFβ2 immunoreactivities in AD, it is unclear whether this represents a general upregulation of TGFβ2 levels, or specific enrichment of TGFβ2 immunoreactivity colocalizing to plaques, tangles, or activated glia. Furthermore, it remains unclear whether TGFβ2 levels are altered in DLB and PDD, which manifest varying degrees of amyloid plaque and NFT burden. In the present study, we measured TGFβ2 in the postmortem temporal cortex of AD, PDD, and DLB patients and correlated TGFβ2 levels with a range of disease severity markers, including neuropathological scores, cognitive scores, and Aβ42 concentrations.
MATERIALS AND METHODS
Participants, cognitive assessments, and brain tissues
All dementia subjects for this study were selected on the basis of clinicopathological consensus diagnoses. Clinical AD diagnosis was based on Consortium to Establish a Registry for Alzheimer’s disease (CERAD) criteria [23], while the DLB Consortium’s “one-year rule” [5] and the Movement Disorders Society criteria [24] were used to distinguish between DLB and PDD. Annual assessments of cognitive function using the Mini-Mental State Examination (MMSE) [25] were also available in a subset of patients, with the average decline per year (MMSE decline) from the time of dementia diagnosis to death used as a measure of dementia severity. At death, informed consent was sought from next-of-kin before removal of brains, which were collected via University Hospital Stavanger, Norway, and the Brains for Dementia Research network sites, United Kingdom, which include the Thomas Willis Oxford Brain Collections, the London Neurodegenerative Diseases Brain Bank, and Newcastle University. The collection and study of brain tissues have received Institutional Review Board approval in both the UK (08/H1010/4) and Singapore institutions (NUS 12-062E). Brains of 20 DLB patients, 20 PDD patients, and 14 AD patients were divided into hemispheres, with one formalin fixed for neuropathological assessments (see below), while the other hemisphere was dissected to obtain 1 cm3 blocks from temporal cortex (Brodmann Area, BA21) followed by fresh freezing and storage at –80°C. Brains from 16 aged-matched controls were also included in this study. Control cases were neurologically and cognitively normal, had only age-associated neuropathological changes and no history of psychiatric diseases. Due to limited tissue availability, not all neurochemical variables were performed for all samples, and respective N values are listed in the table and figure legends.
Neuropathological assessments
All cases underwent standardized neuropathological assessments, including Braak stages [26], the Newcastle/McKeith criteria for LBD [5], and the National Institute on Aging–Alzheimer’s Association guidelines which combine phases of Aβ deposition, Braak stages, and CERAD scores [7, 27]. In addition, semi-quantitative pathology scoring for NPs (immunostaining with the 4G8 antibody), NFTs/neuropil threads (phosphotau immunohistochemistry), and LBs/Lewy neurites (α-synuclein immunohistochemistry) in BA21 sections were performed as previously described [11], and scored by experienced neuropathologists blinded to clinical diagnosis on a four point scale: 0 (none), 1 (sparse), 2 (moderate), and 3 (severe).
Brain TGFβ2 measurements
All chemicals were of reagent grade and purchased from Sigma-Aldrich Co. (St Louis, MO, USA) unless otherwise stated. Processing of brain tissues for TGFβ2 measurements by Luminex assays (Merck Millipore, Billerica, MA, USA) was as previously described [28]. Briefly, grey matterdissected free of meninges and blood vessels were homogenized in 50 mM Tris buffer (pH 7.4) with added Complete ULTRAtrademark protease inhibitor tablets and PhosSTOPtrademark phosphatase inhibitor (Roche Life Science, Penzberg, Germany), with the resultant homogenate subjected to agitation on a plate shaker (800 rpm, 40 min) followed by centrifugation at 6000 g (4°C, 20 min) to obtain supernatants for TGFβ2 measurements in duplicates according to manufacturer’s instructions. Although similar to ELISAs in principle, Luminex assays use an xMAP®-based technology for efficient and accurate analyte measurements with multiplexing capabilities, and have been widely used for cytokine/chemokine profiling both in the brain as well as in the periphery [28, 29]. In this study, Luminex measurements of TGFβ2 levels were interpolated from standard curves (detection range = 6.6–10 000 pg/mL), and protein in supernatants determined by Piercetrademark Coomassie assay (ThermoFisher Scientific, Waltham, MA, USA), for the conversion of TGFβ2 concentrations into pg/mg total brain protein.
Brain Aβ42 and tau measurements
Processing of brain tissues for Aβ42 measurements by enzyme-linked immunosorbent assay (ELISA) kit (Invitrogen, Carlsbad, CA, USA) was as previously reported [30, 31]. Briefly, tissues from BA21 were homogenized in Tris HCl buffer (pH 8.0) with or without 5M guanidine to obtain total and soluble fractions, respectively. Brain homogenates were then assayed in duplicates (minimum detectable range around 10 pg/mL) according to manufacturer’s instructions, and expressed in pg/mg total brain protein after protein determination as described above. Similarly, ELISA measurements of tau phosphorylated at serine-396 (pS396 tau) and total tau (Invitrogen, Carlsbad, CA, USA) were performed in the same samples according to manufacturer’s instructions and expressed in ng/mL.
Immunoblotting for α-synuclein
Brain homogenates were added 1:1 vol/vol to Laemmli buffer (Bio-Rad, Hercules, CA, USA) and boiled at 95°C for 5 min, then loaded on a 12% SDS-polyacrylamide gel and transferred to nitrocellulose membrane using the iBlot® dry-transfer system (Invitrogen, Carlsbad, CA, USA). Membranes were blocked with 5% bovine serum albumin (BSA) in phosphate-buffered saline with 0.1% Tween® 20 (PBST) at 25°C for 1 h, then incubated with polyclonal anti-α-synuclein (1:1000 dilution, Santa Cruz Biotechnology, Dallas, TX, USA) overnight at 4°C degree in PBST. At the end of primary antibody incubation, membranes were washed with PBST for 3×10 min and incubated with the appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies (1:5,000 dilution, Jackson ImmunoResearch, West Grove, PA, USA) for 1 h at room temperature. Immunoblots were stripped and re-probed with monoclonal anti-β-actin (1:5000 dilution, Sigma Aldrich, St Louis, MO, USA) as a loading control. Immunoreactivities were visualized with Luminatatrademark Crescendo Western HRP substrate (Merck Millipore, Billerica, MA, USA) and quantified with the Alliance 4.7 image analyser (UVItec, Cambridge, UK).
Statistical analysis
Data were first assessed for normality using Kolmogorov-Smirnov tests, and differences in TGFβ2 and Aβ42 levels between groups were compared by one way-analyses of variance (ANOVA) followed by post-hoc Bonferroni corrections for multiple comparisons, or Kruskal-Wallis ANOVA followed by post-hoc Dunn’s tests, as appropriate. Correlations with semi-quantitative pathological scores were performed using Spearman’s rank correlation. All analyses were performed using SPSS Statistics (version 21, IBM Inc., Armonk, NY, USA), with p < 0.05 considered statistically significant.
RESULTS
Demographics of study participants
Table 1 shows that the neurologically normal controls were well matched with the dementia groups in age at death, brain pH (as a measure of agonal state and postmortem tissue quality [32]) and postmortem delay not significantly different. There were more females (64%) in the AD group, while the other groups were evenly matched for gender distribution. For distribution of Braak stages [26], controls showed low scores of between Braak stage 0-II as expected, while all but two AD patients were Braak stage V-VI. For LBD, Braak scores were higher in DLB than in PDD, corroborating previous studies showing higher AD pathological burden in DLB compared to PDD [9–11]. Similarly, semi-quantitative scores of NPs and NFTs were highest in AD, while scores for DLB were higher than those for PDD (Fig. 1). LB scores were highest in DLB, lowest in AD and controls, whilst PDD showed intermediate levels (Fig. 1). For the dementia groups, clinical measurements of dementia severity (predeath MMSE and MMSE decline) were not significantly different among PDD, DLB, and AD (Table 1).
Increased TGFβ2 is correlated with clinical and neuropathological markers of disease severity
TGFβ2 concentrations in BA21 were significantly elevated in DLB and AD compared to aged controls,while PDD showed a nonsignificant trend toward increase (Fig. 2). To assess whether TGFβ2 alterations may be related to disease severity, we correlated, in the combined dementia cohort (AD, DLB, and PDD), TGFβ2 levels with several neuropathological and clinical measures, namely Braak staging (stratified into stages “0-II”, “III-IV”, and “V-VI”), MMSE decline, as well as NP and NFT scores for BA21. Furthermore, because α-synuclein-containing LBs were usually undetectable in AD (see Fig. 1), LB scores for BA21 were correlated within the LBD (DLB + PDD) subgroups only. Table 2 shows that TGFβ2 levels correlated with MMSE decline as well as with NFT and LB, but not NP, scores.
Increased TGFβ2 is correlated with soluble Aβ42, but not total Aβ42, tau or α-synuclein levels
Next, we correlated TGFβ2 concentrations with soluble and total fractions of Aβ42 in brain homogenates using a previously described guanidine treatment protocol [30, 31], and found that TGFβ2 correlated with soluble, but not total, Aβ42 in BA21 (Fig. 3). TGFβ2 also did not correlate with pS396 tau (Fig. 4), or with monomeric α-synuclein, which showed a non-significant trend toward decreases in AD (Fig. 5). The lack of significant alterations in α-synuclein immunoreactivities corroborates previous reports for both PDD and DLB [33–35]. As the soluble fractions are known to consist of oligomeric or other low molecular weight Aβ species, while guanidine treated (total) fractions contained predominantly insoluble, highly aggregated fibrils of Aβ42 found in NPs [31], our data suggest that increased TGFβ2 correlated specifically with non-plaque-associated, soluble forms of Aβ.
DISCUSSION
The major forms of neurodegenerative dementias, AD and LBD (together with its two subtypes, DLB and PDD), have been broadly defined neuropathologically by the differential burden of amyloid plaques, NFTs, and α-synuclein containing LBs. Although AD-associated plaque and tangle pathologies are present in LBD, they are widely considered to be higher in DLB than PDD [9–11], while LBs, the characteristic pathological finding in LBD, are low or absent in AD-only diagnoses [36]. These observations have generally been corroborated in the present study (see Fig. 1), indicating the representative nature of our cohort. As part of ongoing efforts to uncover neurochemical substrates which may be pathogenically relevant in LBD, we report here an increase in temporal cortical TGFβ2 in DLB (similar to AD), but not PDD (Fig. 2), suggesting that some of the neurochemical differences between the clinical subtypes may be partly related to differences in neuropathological burden. Indeed, TGFβ2 positively correlated with NFT scores in the combined dementia cohort, and with LB scores in the LBD cohort (Table 2). Interestingly, TGFβ2 did not correlate with NP scores (Table 2), and when we followed up with measurements of Aβ42, only the soluble fraction thought to consist mainly of smaller molecular weight, non-fibrillar Aβ42 [31], correlated with TGFβ2 (Fig. 3). Furthermore, TGFβ2 also correlated with MMSE decline in the combined dementia cohort (Table 2). On the other hand, while phosphorylated pS396 tau, a marker for NFTs [37] showed similar increases in DLB and AD as Aβ42, TGFβ2 levels did not correlate with phosphorylated tau (Fig. 4), suggesting that the associations with NFT may be indirect. Taking into consideration previous studies which showed the pathogenicity and neurotoxicity of soluble, non-fibrillar Aβ42 [38, 39], together with the observation that Aβ-induced neuronal death was mediated via TGFβ2 upregulation [21], our study thus suggests the potential involvement of TGFβ2 in Aβ-related neurodegeneration as well as the utility of targeting TGFβ2 as a therapeutic strategy for neurodegenerative dementias with relatively high Aβ loads, such as AD and DLB.
While the current data are in line with previous immunohistochemical studies on increased TGFβ2 in AD neocortex [18–20] and further extends these observations to DLB, several limitations are apparent. First, due to issues with tissue availability we chose a region (BA21 of the temporal cortex) widely known to be affected by AD [40] for assessment. However, it is unclear whether this region is affected to a similar extent in DLB and PDD, and follow-up studies of other brain regions should be carried out. Furthermore, while previous studies have shown mechanistic links between Aβ (as well as its precursor protein, AβPP), TGFβ2, neuronal death, and associated memory impairments [21, 41], it is unclear whether specific mechanisms also underlie the currently observed associations of TGFβ2 with NFT and LB pathology, or rather, reflect indirect associations and general disease states. Furthermore, the unchanged levels of monomeric soluble α-synuclein and lack of correlation with TGFβ2 (see Fig. 5) suggest that only specific α-synuclein forms may be differentially affected in LBD (for example, insoluble α-synuclein phosphorylated at serine-129 [42]), and follow-up studies are needed to elucidate the associations between the various α-synuclein species and TGFβ2. Finally, while the preponderance of evidence point to a neurotoxic, pathogenic role for increased TGFβ2 [21, 41], some studies have also demonstrated potential pro-survival functions [14, 43], and further studies are needed to elucidate the effects of increased TGFβ2 in ADand DLB.
Conclusions
Using a sensitive Luminex-based platform on postmortem cortical tissues from a well-characterized patient cohort, we confirmed previous immunohistochemical finding of increased TGFβ2 in AD, and found a similar change in DLB (which has high NP burden) but not PDD (with low plaque burden). We further showed that the increased TGFβ2 correlated with NFT and LB pathology scores, soluble Aβ42, and dementia severity. Our data support a potential pathogenic role for increased TGFβ2 in AD and DLB, and point to the need for further assessment of TGFβ2 as a therapeutic target.
Footnotes
ACKNOWLEDGMENTS
This study was funded by the National Medical Research Council of Singapore
(NMRC/CSA/032/2011) and the Yong Loo Lin School of Medicine, National University of
Singapore (R-184-000-223-133). CGB would like to thank the National Institute for Health
Research (NIHR) Mental Health Biomedical Research Centre and Dementia Unit at South London
and Maudsley NHS Foundation Trust; and the Institute of Psychiatry, King’s College London.
Tissues for this study were collected through the Newcastle Brain Tissue Resource (NBTR);
the London Neurodegenerative Brain Bank; the Thomas Willis Oxford Brain Collection; and
Stavanger University Hospital, Norway. The UK tissue repositories are supported the UK
Medical Research Council and by Brains for Dementia Research, (
), a joint venture between
Alzheimer’s Society and Alzheimer’s Research UK. The NBTR is further supported by the NIHR
Newcastle Biomedical Research Unit based at Newcastle upon Tyne Hospitals NHS Foundation
Trust and Newcastle University.