Abstract
Transmembrane Protein 230 (TMEM230) is a newly identified protein associated with Parkinson’s disease (PD) found in Lewy bodies and Lewy neurites of patients with PD or dementia with Lewy body disease. However, TMEM230 has not yet been investigated in the most common neurodegenerative disorder, Alzheimer’s disease (AD). Here, we demonstrate that the expression of TMEM230 is specifically increased in neurons in AD patients. Importantly, both granulovacuolar degeneration (GVD) and dystrophic neurites (DNs), two prominent characteristic pathological structures associated with AD, contain TMEM230 aggregates. TMEM230 immunoreactivity can be detected in neurofibrillary tangles-containing neurons and hyperphosphorylated tau positive DNs. TMEM230 accumulation is also noted around senile plaques. These findings identifying TMEM230 as a component of GVD and DNs suggest TMEM230 dysregulation as a likely mechanism playing an important role in the pathogenesis of AD.
Keywords
INTRODUCTION
Alzheimer’s disease (AD) is the leading cause of dementia in the elderly. It is characterized by two pathologic hallmarks: neurofibrillary tangles (NFTs) and senile plaques (SPs), and is usually accompanied by other prominent pathological changes such as neuronal loss, granulovacuolar degeneration (GVD), and dystrophic neurites (DNs) [1]. NFTs are intracellularaggregates composed of bundles of paired helical filaments, the major component of which is the hyperphosphorylated form of the microtubule-associated protein tau [1]. In contrast, SPs are spherical extracellular lesions made up of bundles of amyloid-β (Aβ) peptide fibrils, and can usually can be identified by silver, Congo red, or Thioflavin staining of AD brain [1]. Similar to NFTs, GVD bodies are also intracellular lesions, and defined as dense granules within large membrane-bound cytoplasmic vacuoles mainly seen in the CA1 and subiculum regions of the hippocampus [2, 3]. DNs are another prominent AD feature often found accumulating around SPs in the hippocampus and cortex, usually manifested as thickened and tortuous neuronal processes with either dendritic or axonal origin [4].
Despite their distinct neuropathological characteristics and largely uncharacterized heterogeneous molecular compositions, both GVD and DNs are closely associated with Aβ and especially tau or tau-related proteins [5]. For example, intraneuronal accumulation of hyperphosphorylated tau or some forms of Aβ aggregates have been found in GVD bodies [6, 7]. Previous studies have reported the presence of activated caspase 3, a protease involved in tau and amyloid-β protein precursor (AβPP) cleavage, or glycogen-synthase kinase 3 and casein kinase 1, kinases phosphorylating tau, in GVD bodies [8–11]. Further, a subset of DNs display AβPP immunoreactivity while most DNs surrounding dense-core SPs typically contain hyperphosphorylated tau [12, 13] Therefore, these AD pathological features are highly intermingled, indicating the likely crosstalk between different pathomechanistic events.
Genetic mutations in Transmembrane Protein 230, TMEM230 (also known as C20orf30), were recently discovered as a novel cause of autosomal dominant Parkinson’s disease (PD) [14]. The immunohistochemical survey in patients with PD and dementia with Lewy body (DLB) identified TMEM230 as a component of pathological hallmarks Lewy bodies and Lewy neurites [14], indicating the likely common role of TMEM230 in neurodegeneration. So far, there is no immunohistochemical study of TMEM230 in AD. Here, using postmortem normal and AD brains, we examined the pattern of localization and expression of TMEM230 as well as its relationship with the widely-studied AD-associated proteins, tau and Aβ, in cell bodies and neurites of degenerating neurons in AD.
METHODS
Tissue
Postmortem hippocampal tissues from histo-pathologically-confirmed AD and aged-matched control subjects were obtained from the University Hospitals Case Medical Center with the IRB approved protocol (Table 1). Brain tissues were stored frozen at –80°C or fixed in buffered formalin at 4°C. Fixed tissues were dehydrated through graded ethanol followed by xylene and embedded in paraffin. Microtome sections of 6 μm thickness were prepared.
Information of fixed and frozen brain tissues used in this study
Immunocytochemistry
Paraffin embedded tissue sections were deparaffinized in xylene followed by rehydration in graded ethanol. Endogenous peroxidase activity was blocked by the incubation with H2O2 for 30 min. Rehydrated tissue sections were then washed in Tris Buffered Saline (TBS, 50 mM Tris·HCl and 150 mM NaCl, pH = 7.6) for 10 min and placed in placed in 1X antigen decloaker (Biocare). Antigen retrieval was carried out under pressure using Biocare’s Decloaking Chamber by heating to 125°C for 10 s and cooling to 90°C for 30 s followed by heating to 22 psi at 128°C, and cooling to 0 psi at 94°C. The sections were quickly rinsed with distilled H2O. Nonspecific protein binding sites were blocked with 10% normal goat serum (NGS) in TBS for 30 min at room temperature, and sections were incubated with primary antibodies in TBS containing 1% NGS overnight at 4°C. After washes with 1% and 10% NGS in TBS, the sections were immunostained by the peroxidase-antiperoxidase method and developed using diaminobenzidine as chromogen as we described [15]. Primary antibodies used included rabbit polyclonal anti-TMEM230 (Proteintech, Cat No.: 21466-1-AP), mouse monoclonal anti-TMEM230 (Santa Cruz, Cat No.: sc-398561), mouse monoclonal anti-Aβ (6E10, BioLegend, Cat No.: SIG-39320-200), and mouse monoclonal anti-phosphorylated tau (AT8, Invitrogen, Cat No.: MN1020). The blocking peptide for mouse monoclonal anti-TMEM230 (Santa Cruz, Cat No.: sc-398561P) was used to confirm the specificity of immunostaining.
Immunofluorescence
Paraffin embedded tissue sections were deparaffinized and rehydrated followed by antigen retrieval similar to immunocytochemistry but with the omission of H2O2 incubation. After blocking with 10% NGS in phosphate buffered saline (PBS) and incubation with primary antibodies in PBS containing 1% NGS, sections were washed with PBS followed by 2-h incubation with Alexa Fluor 488 or 568 dye labeled second antibody (Invitrogen) at room temperature and subsequent PBS washes. All sections were mounted with Fluoromount-G mounting medium (SouthernBiotech).
Series of confocal images with optical thickness of 300 nm were captured with a Leica SP8 gSTED Super-Resolution Confocal equipped with confocal class objectives (100x), a motorized super Z galvo stage, two PMTs, 3 Hyd SP GaAsP detectors for gated imaging, and the AOBS system lasers including a 405 nm, Argon (458, 476, 488, 496, 514 nm), a tunable white light (470 to 670 nm), and a 592 nm STED depletion laser. 3D confocal images were reconstructed using Imaris.
Immunoblot
Frozen hippocampal tissues were quickly cut into small pieces and homogenized in 10 volume cell lysis buffer (Cell Signaling) plus 1 mM phenylmethylsulfonyl fluoride (Sigma-Aldrich) and Protease Inhibitor Cocktail (Roche). Equal amounts of total protein extract were resolved by SDS-PAGE and transferred to Immobilon-P (EMD Millipore). Following blocking with 10% nonfat dry milk, primary and secondary antibodies were applied as previously described [16]. The blots were developed with Immobilon Western Chemiluminescent HRP Substrate (EMD Millipore) and imaged by the Amersham imager 600 (GE Healthcare Life Sciences). Mouse monoclonal anti-actin was obtained from EMD Millipore.
RESULTS
The immunocytochemical analysis of hippocampal tissue sections from AD patients and age-matched control subjects revealed that the TMEM230 immunoreactivity was specifically high in AD neurons (Fig. 1A), compared with the relatively weak immunoreactivity in control neurons (Fig. 1B). Notably, a distinct pattern of TMEM230 immunostaining observed in AD patients was that TMEM230 immunostained GVD with typical vacuolar morphology (Fig. 1A, inset). In contrast, all control neurons had cytoplasmic TMEM230 immunostaining that was diffusive and not associated with vacuolar or large granules (Fig. 1B, inset). TMEM230-positive GVD bodies were specifically evident within pyramidal neurons of brain areas such as the hippocampus and the subiculum. TMEM230-positive GVD bodies in AD could be observed by two different TMEM230 antibodies. The TMEM230 immunoreactivity was abolished by the pre-absorption of the primaryantibody with antigen or the absence of the primary antibody (data not shown).
Immunocytochemical analysis of TMEM230 in a hippocampal section from an AD patient and an age-matched control subject. A) In AD, GVD lesions in the CA1 and subiculum region were readily detected with anti-TMEM230 antibodies. Asterisks denote enlargements shown in the right bottom. B) Diffusive TMEM230 immunostaining in pyramidal neurons of the hippocampus from age-matched control subject.
Double immunofluorescence staining of TMEM230 and hyperphosphorylated tau showed that TMEM230-positive punctate staining did not always occur in pyramidal neurons with AT8-positive NFTs (Fig. 2A, B). Yet other neurons with similar patterns of punctate TMEM230 staining displayed both TMEM230 punctate and hyperphosphorylated tau containing NFTs filling the cytoplasm and wrapping around the nucleus (Fig. 2B). Conversely, TMEM230 was always found highly co-localized with hyperphosphorylated tau at aggregating sites of DNs (Fig. 2C). The dense-cored or diffuse SPs, stained by 6E10, were abundantly present in all cases (data not shown). However, the TMEM230 accumulation only occurred peripherally around SPs, but did not colocalize with the amyloid component (Fig. 2D).
Double immunofluorescent staining of TMEM230 and phosphorylated tau (AT8) or Aβ (6E10) in a hippocampal section from an AD patient. A) Representative three-dimensional (3D) image showing the presence of TMEM230 accumulation in AD pyramidal neurons that do not contain phosphorylated tau (pointed by arrow). B) Representative 3D image showing TMEM230 aggregates in AD pyramidal neurons filled with NFTs (pointed by arrow). C) Representative 3D image showing TMEM230 aggregates in phosphorylated tau positive DNs (pointed by arrow). D) Representative 3D image showing the absence of TMEM230 within SPs. In all images, the nucleus is stained by DAPi as blue.
Immunoblot analysis of AD and control hippocampal grey matter tissues demonstrated a major band between 15–20 kDa, representing the isoform 1 of TMEM230 with the expected weight of 19 kDa (Fig. 3A). Consistent with immunocytochemical analysis showing a more intense immunoreactivity in AD neurons, further quantification demonstrated that there was a significant increase in the expression of TMEM230 in AD cases (Fig. 3B).
Increased expression of TMEM230 in AD. A) Representative western blot detection of TMEM230 in hippocampal gray matter tissues of AD and age-matched control cases. B) Quantification of the levels of TMEM230 relative to actin in AD and age-matched control cases. n = 6 per group. Data are means±s.e.m., representative of triplicate experiments. Student’s-t-test.
DISCUSSION
This study provides immunocytochemical evidence that TMEM230 accumulates in two prominent AD pathological features, GVD and DNs. GVD has long been suggested to represent autophagic abnormalities [17]. And autophagic markers such as LC3 and p62/sequestosome 1 (SQSTM 1) have been reported in GVD [18]. Like GVD, DNs also contain LC3 and p62 and the predominant organelles in DNs are largely autophagosomes [19]. Interestingly, a very recent study of TMEM230 has revealed that it is directly involved in autophagy by regulating LC3 and p62 expression [20]. Given expanding evidence revealing the important role of autophagy in the pathogenesis of AD [21], it is conceivable that through the autophagic pathways, the accumulation of TMEM230 may contribute to the localized aggregation of tau and Aβ in GVD and DNs. In addition to autophagy, GVD bodies also exhibit properties of endocytic and lysosomal vesicles [18]. Some DNs also are enriched in mitochondria, synaptic vesicles and lysosomal proteins [4], indicating their heterogeneous composition. Along this line, it has been reported that TMEM230 mediates retromer trafficking, Golgi-derived vesicle secretion, and synaptic vesicle transport [14, 20]. Therefore, the widespread existence of different types of GVD or DNs could be attributed to the multifaceted functions of TMEM230.
The intraneuronal accumulation of TMEM230 showed the typical morphological pattern of GVD. TMEM230-positive GVD were mainly found in neurons of subicular and CA1 neurons, consistent with previous description. Either GVD or DNs could be found in many other neurodegenerative diseases such as amyotrophic lateral sclerosis, multiple system atrophy with Parkinsonism, progressive supranuclear palsy, and Down syndrome [22]. In PD and DLB, TMEM230 labels large vacuolar-like structures or dense granules in cell bodies and neurites of midbrain neurons positive for alpha-synuclein, encoded by the firstly identified gene associated with PD [14]. Thus, TMEM230-positive GVD or DNs are unlikely to be a clear-cut pathology of AD. Nevertheless, future studies will be needed to investigate whether TMEM230-positive GVD or DNs are also present in other neurodegenerative diseases, and explore the potential association of TMEM230 with other commonly studied proteinopathies such as those with altered TDP-43.
TMEM230 accumulation was observed in many hyperphosphorylated tau positive neurons and nearly all tau-positive DNs, implying a likely close relationship between TMEM230 and tau. TMEM230 aggregates in the cytoplasm or the neurites may interfere with vesicle secretion or trafficking, impairing the sorting or axonal transport of tau and contributing to the initiation or acceleration of tau aggregation. Or, as phosphorylated tau can directly regulate axonal transport by binding to kinesin-1 [23], hyperphosphorylated tau aggregation may disrupt TMEM230 transport and cause TMEM230 accumulation. Thus, the concurrent accumulation of TMEM230 and hyperphosphorylated tau in subsets of neurons or neurites may represent neurons at different stages of the disease. Although TMEM230 accumulation was not noted within amyloid plaques or neurons with intense Aβ or AβPP immunoreactivity (data not shown), TMEM230-positive punctae could be observed in close proximity with SPs. However, the relationship between TMEM230 and SPs or Aβ remains unknown and worthy of furtherinvestigation.
Another interesting aspect of our findings is that levels of TMEM230 were increased in AD brain and specifically in neurons. TMEM230 is a putative transmembrane protein, suggesting that autophagy may play a role in altered TMEM230 turnover in AD. TMEM230 and hyperphosphorylated tau are closely associated. Also, hyperphosphorylated tau has been reported to inhibit proteasome activity in AD [24]. Thus, the potential involvement of the ubiquitin–proteasome pathway in elevated levels of TMEM230 could not been ruled out. It is worth mentioning that TMEM230 is a multi-functional protein and the loss of TMEM230 disrupts vesicle trafficking and autophagic cargo degradation [20]. TMEM230 upregulation in AD neurons may be a compensatory response to offset the decline in autophagy or proteasome activities.
In sum, this study shows that PD-associated protein TMEM230 accumulates in GVD and DNs in AD. The similar accumulation of TMEM230 in cell bodies and neurites has been noted in PD and DLB,suggesting that TMEM230 accumulation or TMEM230 proteinopathy could be present in a wide range of neurodegenerative diseases. Further studies will be needed to investigate the role of TMEM230 in the pathogenesis of these devastating diseases.
DISCLOSURE STATEMENT
Authors’ disclosures available online (http://j-alz.com/manuscript-disclosures/17-0190r1).