Autophagic and lysosomal defects in human tauopathies: analysis of post-mortem brain from patients with familial Alzheimer disease, corticobasal degeneration and progressive supranuclear palsy
© Piras et al. 2016
Received: 17 February 2016
Accepted: 18 February 2016
Published: 2 March 2016
The accumulation of insoluble proteins within neurons and glia cells is a pathological hallmark of several neurodegenerative diseases. Abnormal aggregation of the microtubule-associated protein tau characterizes the neuropathology of tauopathies, such as Alzheimer disease (AD), corticobasal degeneration (CBD) and progressive supranuclear palsy (PSP). An impairment of the lysosomal degradation pathway called macroautophagy, hereafter referred to as autophagy, could contribute to the accumulation of aggregated proteins. The role of autophagy in neurodegeneration has been intensively studied in the context of AD but there are few studies in other tauopathies and it is not known if defects in autophagy is a general feature of tauopathies. In the present study, we analysed autophagic and lysosomal markers in human post-mortem brain samples from patients with early-onset familial AD (FAD) with the APP Swedish mutation (APPswe), CBD and PSP and control individuals.
FAD, CBD and PSP patients displayed an increase in LC3-positive vesicles in frontal cortex, indicating an accumulation of autophagic vesicles. Moreover, using double-immunohistochemistry and in situ proximity ligation assay, we observed colocalization of hyperphosphorylated tau with the autophagy marker LC3 in FAD, CBD and PSP patients but not in control individuals. Increased levels of the lysosomal marker LAMP1 was detected in FAD and CBD, and in addition Cathepsin D was diffusely spread in the cytoplasm in all tauopathies suggesting an impaired lysosomal integrity.
Taken together, our results indicate an accumulation of autophagic and lysosomal markers in human brain tissue from patients with primary tauopathies (CBD and PSP) as well as FAD, suggesting a defect of the autophagosome-lysosome pathway that may contribute to the development of tau pathology.
Abnormal intracellular aggregation and accumulation of the microtubule-associated protein tau is a common feature of many neurodegenerative disorders, including Alzheimer disease (AD), frontotemporal lobar degeneration, Pick´s disease, argyrophilic grain disease, corticobasal degeneration (CBD) and progressive supranuclear palsy (PSP) . Collectively, these neurodegenerative disorders are referred to as tauopathies. Under normal conditions, tau is predominantly expressed in neuronal axons where its main function is to promote microtubule assembly and stabilization, which is important for maintaining axonal transport and neuronal integrity [45, 60, 69]. The molecular mechanisms leading to intracellular tau aggregation in AD and other tauopathies are not fully understood, but abnormal post-translational modifications, such as hyperphosphorylation and acetylation [20, 47], and impaired degradation of tau  have been suggested.
Two major proteolytic systems contribute to tau degradation, the ubiquitin-proteasome system (UPS) and the autophagy-lysosome system [50, 52, 67]. The role of each of these pathways to the turnover of tau is an area of significant interest [16, 32, 38, 40, 66, 67]. The major lysosomal degradative pathway in eukaryotic cells, macroautophagy (hereafter referred to as autophagy), is responsible for degrading long-lived or aggregated proteins and is the principal mechanism for turning over cellular material too large to be degraded by the UPS [29, 75, 76]. Autophagy is a highly regulated process that involves the sequestration of cytoplasmic cargo, such as aggregated proteins and damaged organelles, within double-membrane vesicles called autophagosomes which are typically labelled by the microtubule-associated protein 1A/1B-light chain 3 (LC3) [23, 29, 30, 72, 76]. In order to achieve degradation of aggregated proteins in the autophagy-lysosome system, there must be a successful fusion of the autophagosomes with the lysosomes [17, 18, 29, 36, 73]. Functional autophagy is crucial for neuronal physiology and loss of autophagy in the central nervous system, for example by inactivation of key autophagy genes such as autophagy-related (Atg) proteins 5 (Atg5) or 7 (Atg7) leads to neurodegeneration [22, 31]. In Atg7 knockout mouse brains, there is a significant accumulation of hyperphosphorylated tau suggesting a role of autophagy in the clearance of pathological tau in adult neurons . Furthermore, increased accumulation of autophagic vesicles has been reported in human post-mortem AD brains and in mouse models of tauopathy [41, 54, 77]. Constitutive overexpression of mTor, (mammalian Target of rapamycin), a key negative regulator of the autophagic pathway, prevents activation of the autophagy pathway and increases the levels of hyperphosphorylated tau in a cell model of tauopathy . Conversely, autophagy enhancers like rapamycin (an mTor inhibitor) or trehalose (an mTor-independent autophagy activator) can promote the degradation of insoluble tau in mouse models of tauopathy [9, 56, 61]. Finally, post-translational modifications of tau can interfere with and impair the clearance mechanisms. For example, phosphorylation of tau at serine 422 (Tau/pS422) prevents tau cleavage by caspase-3 at aspartic acid 421 (D421), precluding tau degradation by the autophagy-lysosome system . Taken together these observations suggest that the autophagy-lysosome pathway plays an important role in the clearance of hyperphosphorylated tau. The majority of studies on human neurodegenerative disease and autophagy have included patients with Alzheimer disease where both Aβ and tau aggregations are key features (reviewed in [43, 55]), and only few studies have focused on other human tauopathies [19, 71]. Thus, in order to address the contribution of the autophagy-lysosomal system in different tauopathies, we studied human post-mortem brain tissue from patients with both tau and Aβ pathology [familial AD (FAD) cases with the Swedish double-mutation in the amyloid precursor protein (APPswe)] as well as brain tissue from patients with a primary tauopathies in the absence of significant amyloid pathology (CBD and PSP). In agreement with previous studies of sporadic AD cases [42, 51, 54], we found an accumulation of markers of the autophagy-lysosomal pathway in AD patients with the familial APPswe mutation. In addition, we showed that the autophagy-lysosomal system is impaired in patients with primary tauopathies suggesting that autophagic defects are a common feature of human tauopathies.
Material and methods
Human brain samples
Age at death (years)
Age at onset (years)
Brain weight (g)
IHC, IF, PLA, WB
IHC, IF, PLA
IHC, IF, PLA
IHC, IF, PLA, WB
IHC, IF, PLA, WB
IHC, IF, PLA, WB
IHC, IF, PLA, WB
IHC, IF, PLA
IHC, IF, PLA
IHC, IF, PLA
IHC, IF, PLA
IHC, IF, PLA
List of primary antibodies
Cathepsin D (Cat D)
Cathepsin D (Cat D)
For immunofluorescence staining, all sections were processed under the same standardized conditions, following the method described above with minor modifications. After deparaffinization and antigen retrieval, the sections were blocked with Background Punisher (BP974, Biocare Medical) for 10 min at RT followed by washing and incubation with primary antibodies (Table 2) in TBS-T (overnight 4 °C, humid chamber). After washing in TBS-T, sections were incubated (1 h at RT) with appropriate secondary antibodies [anti-mouse and anti-rabbit IgG (H + L)] conjugated to Alexa Fluor 546 (A-11003, Invitrogen) or Alexa Fluor 488 (A-11008, Invitrogen) at a concentration of 1:500 in TBS-T. After washing in TBS-T (3 × 10 min) slides were incubated with (or without) Sudan Black B (199664, Sigma-Aldrich), 5 min at RT to reduce autofluorescence. Sections were then washed in TBS-T and incubated with DAPI for 5 min (D9564, Sigma-Aldrich). After washing in TBS-T, coverslips were mounted with Vectashield Hard Set (H-1200, Vector Laboratories) and the slides were stored at 4 °C. Sections from controls and patients were also incubated without primary antibody and used as negative control. Sections were examined using a laser scanning confocal microscope (LSM 510 META, ZEISS), and images were acquired using the same settings (laser intensity, detector gain and amplifier offset).
In situ Proximity Ligation Assay (PLA)
To specifically detect protein interaction (when the proteins are in close proximity <40 nm), PLA analyses were performed on section from FFPE frontal cortex of human brain from three patients of each group (Table 1). Deparaffinization and antigen retrieval were performed as described above. In situ PLA was performed according to the OLINK Bioscience instructions. To reduce non-specific signals, the sections were incubated with Blocking solution (82007, OLINK Bioscience) for 30 min at 37 °C. Sections were then incubated overnight at 4 °C with two primary antibodies (Table 2) in antibody diluent solution (82008, OLINK Bioscience) (80–100 μl/section). For each patient, two negative controls were performed by omitting one of the two primary antibodies (negative control 1 and negative control 2). After washing with TBS-T, sections were incubated with in situ PLA DNA-probes anti-rabbit PLUS and anti-mouse MINUS (82002 and 82004, OLINK, Bioscience) for 1 h at 37 °C. Ligation solution was added for 30 min at 37 °C, and washed twice with washing Buffer A (82047, OLINK, Bioscience). The amplification solution (92013, Detection reagents Far Red, OLINK Bioscience) was added for 100 min at 37 °C. Following incubation the sections were washed in Buffer B (82048, OLINK, Bioscience). The sections were allowed to dry and mounted with Vectashield Hard Set mounting medium with DAPI (H-1200, Vector Laboratories). Images from at least ten different fields of the frontal cortex for each sample were acquired using a laser scanning confocal microscope (LSM 510 Meta, ZEISS). Then, the images were analyzed with DUOLINK Image Tool software (OLINK Bioscience), which automatically counts the number of fluorescent signals (dots) per field. The signal was calculated as Dots = DotsPLA-(Dotsneg1 + Dotsneg2), where the DotsPLA is the number of dots of in situ PLA and Dotsneg1 and Dotsneg2 are respectively the dots obtained from negative control 1 [only primary antibody 1, anti-tau clone AT8 (pS202/T205)] and negative control 2 (only primary antibody 2, LC3) . Data were expressed as mean value ± Standard Deviation (S.D.) and ANOVA, followed by the Dunnett post-hoc test, was used for statistical analysis (IBM SPSS Software). Significance levels of *p < 0.05, **p < 0.01, *** p < 0.001 were used.
Frozen tissue from frontal cortex (~145 mg/sample) of human brains from FAD (n = 3), CBD (n = 2) and Control (n = 3) (Table 1) were homogenized in RIPA buffer (50 mM Tris pH 7.5; 150 mM NaCl; 0.5 % sodium deoxycholate; 0.1 % SDS; 1 % NP40) containing Benzonase Nuclease (70664–3, Millipore, 1:1000), Phosphatase and Protease inhibitor cocktails (P8340 and P0044, Sigma-Aldrich, 1:100) on ice. Samples were centrifuged at 7000xg for 10 min at 4 °C. Supernatants were stored at −20 °C.
After determination of the protein content by BCA protein assay (23227, Thermo Fischer Scientific), homogenates (30–50 μg total protein) were separated on NuPAGE® 12 % Bis-Tris Gels, 1.0 mm, 10 wells (NP0341, Life Technologies) by electrophoreses in running buffer MOPS [3-(N-morpholino) propanelsulfonic acid] (NP0001, Life Technologies) and blotted onto iBlot Gel Transfer Stacks Nitrocellulose (IB301001, Life Technologies). The membranes were blocked in 5 % non-fat milk (70166, Sigma-Aldrich) in TBS-T. Membranes were incubated overnight at 4 °C with primary antibodies (see Table 2), washed in TBS-T and then incubated with secondary species-specific antibodies (NA934 anti-rabbit, NA931 anti-mouse, GE Healthcare) in TBS-T + 5 % non-fat milk for 1 h at RT. Blots were visualized with enhanced chemiluminescence reagent (34076, Pierce). Images were obtained with a FujiFilm LAS-3000 camera and semiquantitative analysis was performed using ImageJ software. Data were expressed as mean intensity of an arbitrary unit (AU) ± S.D. and were obtained from the average of at least 3 replications of the experiment. For quantification of immunoblots, protein levels were normalized to GAPDH. ANOVA, followed by the Dunnett post-hoc test, was used to compute statistical significance and significance levels of *p < 0.05, **p < 0.01, *** p < 0.001 were used.
Characterization of tau pathology in frontal cortex from patients diagnosed with different tauopathies
Accumulation of p62/SQSTM1 and colocalization with hyperphosphorylated tau in frontal cortex from cases with tauopathies
Accumulation of LC3-positive structures in frontal cortex from patients with taoupathies and colocalization with hyperphosphorylated tau
Next we investigated if hyperphosphorylated tau colocalized with LC3 in patients with tauopathies. In FAD patients, colocalization between hyperphosphorylated tau (Tau/pS422) and LC3 was found in tangle-like structures (arrows in Fig. 3j, n; see also Additional file 2: Figure S2) and in neuropil threads (asterisks in Fig. 3j and high magnification Fig. 3n, q). CBD and PSP patients showed colocalization of hyperphosphorylated tau and LC3 mainly in threads (asterisks in Fig. 3k, l and high magnifications in Fig. 3o, r, s), although we cannot exclude that this is a result of the low levels of hyperphosphorylated tau in neuronal soma in CBD and PSP patients.
Accumulation of lysosomal markers and impairment of lysosomal integrity in tauopathies
Next, we analysed the expression of Cathepsin D (Cat D), a major lysosomal hydrolase involved in the proteolytic degradation of proteins in lysosomes, by immunofluorescence. In frontal cortex from controls, we observed small perinuclear Cat D-positive punctate structures, corresponding to lysosomes (Fig. 5j, n, t, asterisks and Additional file 3: Figure S3). In contrast, the Cat D immunoreactivity was stronger in FAD, CBD and PSP patients compared to control individuals (Fig. 5g-i and Additional file 3: Figure S3). Two different patterns of Cat D immunoreactivity were observed: (1) a staining pattern of small vesicular structures, corresponding to lysosomes (Fig. 5q-s, asterisks) and (2) a diffuse Cat D staining pattern showing a fluorescent signal throughout the cytoplasm (Fig. 5q-s, arrows, and Additional file 3: Figure S3), suggesting that Cat D could have leaked out of the lysosomes (reviewed in [8, 74, 78]). Taken together the Cat D results suggest that the lysosomal integrity may be impaired in all three patient groups.
Finally we analysed the colocalization of hyperphosphorylated tau with Cat D using double immunofluorescence. In all three patient groups, we found that Cat D-positive staining rarely colocalized with hyperphosphorylated tau and only a few cells were immunoreactive for both Tau/pS422 (red) and Cat D (green) (arrowheads and inset in FAD and PSP, Fig. 5k, m, o, p). This suggests that hyperphosphorylated tau is not localized to lysosomes which is in line with previous studies in sporadic AD patients [5, 15].
Here we used human post-mortem brain tissue to demonstrate that abnormal accumulation of markers of the autophagy-lysosomal pathway is a common feature of different tauopathies. We found that patients with primary (CBD and PSP) and secondary (early onset FAD) tauopathies show an abnormal accumulation of the autophagy markers p62/SQSTM1 and LC3, and that both these markers colocalized with hyperphosphorylated tau. In FAD patients, hyperphosphorylated tau colocalized with LC3-positive structures and p62/SQSTM1 both in the soma and in neuritic threads, while in CBD and PSP patients, colocalization was found mainly in threads. Previous studies have demonstrated that hyperphosphorylated tau impairs microtubule-based axonal retrograde transport [27, 44, 46, 49], and that disruption of microtubule-based vesicle transport, either by the microtubule-depolymerizing drug vinblastine or by deleting the motor-protein dynein, results in a massive accumulation of autophagosomes in neurites [7, 28]. Thus, it is tempting to speculate that the accumulation of hyperphosphorylated tau, p62/SQSTM1 and LC3 in threads in the tauopathy patients are, at least partly, due to impaired axonal transport. It should be noted that in CBD and PSP patients, hyperphosphorylated tau is found both in neurons and in glial cells, and although the autophagy-lysosomal pathway has been demonstrated to be the main degradative pathway of tau in neurons, other mechanisms (including the ubiquitin-proteasome system) may be involved in degradation of tau in other cell types such as glia [32, 67, 68].
Our analysis of lysosomal markers showed increased protein levels of LAMP1 and Cat D in FAD patients as well as in patients with primary tauopathies, suggesting a defective lysosomal clearance which is in line with previous reports in AD brain [10, 12, 48, 53, 58]. In contrast to the control cases where Cat D staining was typically punctate, indicating localization within lysosomes, we observed diffuse cytoplasmic Cat D immunoreactivity in the tauopathy patients. Diffuse Cat D staining in the soma is indicative of lysosomal membrane rupture , and can be induced by oxidative stress [26, 59]. It has also been reported that tau can interact with lysosomal membranes and trigger lysosomal permeability in vitro  and that small tau fibrils can bind to lysosomal membranes resulting in lysosomal damage in a transgenic mouse model of AD . Further studies are needed to determine if there is a leakage of Cat D into the cytoplasm in human tauopathies, but our data indicate that defective lysosomal integrity is prominent in FAD, CBD and PSP patients. It should be noted that the increased LAMP1 levels could be attributable to gliosis in the tauopathy patients [5, 11, 13].
In recent years, several studies have indicated that (pharmacological) induction of autophagy can be beneficial for treatment of neurodegenerative diseases, and increased autophagy has been shown to ameliorate pathology in various disease models by enhancing the clearance of intracytoplasmic protein aggregates, including hyperphosphorylated tau [22, 66]. It is possible that an impaired retrograde transport could explain the observed accumulation of LC3-positive structures containing hyperphosphorylated tau in threads in tauopathy patients. On the other hand, our findings from the FAD cases suggest that hyperphosphorylated tau can be transported to the soma where we observed colocalization with LC3 and p62/SQSTM1.
In conclusion, our findings give support for an impairment of the autophagy-lysosomal system in patients with primary tauopathies as well as in familial AD caused by the Swedish APP mutation. Although the autophagosomal-lysosomal clearance pathway is compromised in all three tauopathies, our observations also indicate that the precise location of the impairment along this pathway is not necessarily the same. Thus, our study highlights the fact that therapeutic strategies targeting the autophagosomal-lysosomal pathway may need to be specifically tailored for different tauopathies.
All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee (the Regional Ethical Review Board in Stockholm, Sweden) and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.
AP was supported by the Roche Postdoctoral Fellowship Program. We thank Anna-Karin Lindström for excellent technical support, neuropathologist Inger Nennesmo for the clinical neuropathological characterization of the patients included in the study. We also thank Helena Karlström for guidance on the Western blot analyses and Sophia Schedin-Weiss for her technical guidance of the in situ PLA analyses.
This study was funded by the Roche Postdoc Fellowship program.
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