Ultrastructural Pathology, 2014; 38(1): 1–5 ! Informa Healthcare USA, Inc. ISSN: 0191-3123 print / 1521-0758 online DOI: 10.3109/01913123.2013.825689
ORIGINAL ARTICLE
Lewy Bodies under Atomic Force Microscope Agnieszka Tercjak, PhD1, Alberto Bergareche, MD2,3, Cristina Caballero, MD4, Teresa Tun˜on, MD5, and Gurutz Linazasoro, MD6 Department of Chemical and Environmental Engineering, University of the Basque Country, Spain, 2Neurology Department, University Hospital Donostia, Neurosciences Area, Biodonostia Institute, San Sebastia´n, Gipuzkoa, Spain, 3Center for Biomedical Research in Neurodegenerative Diseases Network (CIBERNED), Spain, 4 Department of Pathology, University Hospital Donostia, San Sebastia´n, Gipuzkoa, Spain, 5Department of Pathology, Brain Bank of Navarra, Navarra Hospital, Spain, and 6Centro de Investigacio´n Parkinson, Fundacio´n Inbiomed, San Sebastia´n, Spain
ABSTRACT Lewy bodies are the hallmark of Parkinson disease and their sophisticated analysis will undoubtedly elucidate the pathogenic process. They have been studied by using different microscopic tools. The authors have used atomic force microscopy (AFM) to study the ultramicrotom cut postmortem brain tissue of Parkinson disease patients. Under the same preparation conditions, they have found aggregated fibrillary nanostructures in Lewy bodies, as well as a loss of connections between neurons located in other parts of the substantia nigra. Although these results are preliminary and descriptive in nature, this paper reports the application of a novel and intriguing technique. Further studies including the study of cortical LB and Lewy neurites will be needed to determine the full potential of AFM in the study of the pathogenesis of cell death in Parkinson disease and other synucleinopathies. Keywords: Atomic force microscope, lewy bodies, neurodegeneration, Parkinson disease, a-synuclein
In addition, it seems unlikely that every dying neuron first goes through a stage of LB formation [2]. Furthermore, numerous in vitro and in vivo studies have shown posttranslational modifications and other changes of a-synuclein, leading to increased protein aggregation, which can be closely linked with neurotoxicity. Finally, LB pathology in sporadic PD disease progresses in a temporally and topographic ascending pattern. Thus, it has been suggested that the pathology is transmitted from neuron to neuron, presumably by spreading along axons by still unclear mechanisms [3]. Atomic force microscopy (AFM) is a powerful characterization technique with high-resolution imaging that makes it possible to distinguish between different objects on the surfaces of the investigated biomaterials at the nano and molecular scale due to the interactions between AFM-tip and investigated materials. This technique has been widely used in
Parkinson disease (PD) is one of the most common neurodegenerative disorders. It is characterized by a progressive degeneration of the nigrostriatal dopaminergic system, although other neuronal systems and organs are also affected. Degeneration is associated with the presence of intracytoplasmic aggregates of a-synuclein protein in the form of Lewy bodies (LB) and dystrophic Lewy neurites (LN) in surviving neurons. The formation and development of LBs, their relationship with the deposition of phosphorylated a-synuclein, and the interaction between a-synuclein and other pathogenic proteins are unclear. In particular, the biological significance of LBs, their impact on neurodegeneration, and their eventual role (detrimental or cytoprotective) are a hot topic of debate [1]. The loss of neurons in the substantia nigra pars compacta appears disproportionately high in comparison with the relatively small number of LBs.
20 14
Ultrastruct Pathol Downloaded from informahealthcare.com by Universitaet Zuerich on 06/30/14 For personal use only.
1
Received 7 June 2013; Revised 17 June 2013; Accepted 11 July 2013; Published online 15 October 2013 Correspondence: Dr. Alberto Bergareche, Neurology Department, University Hospital Donostia, San Sebastia´n, Gipuzkoa, Spain. E-mail: [email protected]
1
2
A. Tercjak et al.
biology as an imaging tool to determine morphological parameters, such as the height and length of amyloid fibrils formed from a-synuclein [4,5], insulin [6], and b-lactoglobulin [7]. Although the in situ self-assembly process of a-synuclein has been studied using AFM [8], to the best of our knowledge this is the first report of the use of AFM for the identification and analysis of LB on the postmortem brain tissue of PD patients using ultramicrotomy cut surfaces.
different regions of the specimens were scanned. Similar images were obtained, thus demonstrating the reproducibility of the results. For the analysis of the observed surface structures, nanoscope image processing software was used. In this study the AFM height images have been analyzed taking into account the very similar contrast of the AFM phase images. Optical microscopy (OM) measurements were performed in a Nikon Eclipse 600 using AFM samples.
RESULTS
Ultrastruct Pathol Downloaded from informahealthcare.com by Universitaet Zuerich on 06/30/14 For personal use only.
METHODS We selected two autopsied cases: a 79-year-old man with PD and a 71-year-old male without any neurological disorder. Both brains were acquired through explicit donation with appropriate ethical approval by the Brain Bank of Navarra (Spain), and meet the requirements of Spanish Real Decreto 1716/ 2011, which establishes the basic requirements for the authorization and operation of biobanks and the handling of biological samples of human origin. A hematoxilyn & eosin-stained slide from susbtantia nigra was checked in order to localize neurons and LB in the sample. We took a guided small sample from the paraffin block. The samples were placed in the stove at 60–70 C for 24 h, rehydrated in changes of xilol and descending alcohol (absolute, 96 C and 70 C), and washed in distilled water in the refrigerator. Next day, the samples were fixed in glutaraldehyde for at least 15 h, postfixed in osmium tetroxide for 2 h in the refrigerator, and washed with veronal buffer. After that, samples were dehydrated in ascending alcohol (30 C, 50 C, 70 C, 96 C, and absolute), washed in propylene oxide, and embedded in epoxy resin for 2 h. After the resin hardened, the material was cut into thick sections (semithin sections) in an ultramicrotome (LKB Bromma, Leika) and stained with toluidine blue in order to check for the presence of neurons with and without LB under light microscopy. After checking, the sections were cut into very small sections with a thickness of around 100– 200 nm. The cut sections were placed on the surface of the microscopic glass and analyzed directly without additional treatments. AFM was carried out using the Dimension 3100, Multimode from Digital Instruments, Veeco. AFM images were obtained by operating the AFM in tapping mode (TM-AFM) with a scanning probe equipped with an integrated silicon tip/cantilever having a resonance frequency of 300 kHz, from the same manufacturer. The height and phase images were obtained under ambient conditions with typical scan speeds of 0.7–1.2 line/s, using a hybrid scan head with a maximum range of 100 100 mm. Height and phase images were recorded simultaneously during scanning. In order to obtain repeatable results,
A representative AFM height image of the ultramicrotomy cut surface of these postmortem brain tissues is shown in Figure 1a. The optical microscopy (OM) image of the same region as the analyzed tissue is shown in Figure 1b. Both show the LB in the cytoplasm of the nerve cell. The AFM height image identifies two regions inside the LB forming a coreshell like structure. The size of the central core was around 8 nm in diameter and appeared as bright fibrillary nanostructured regions 400–800 nm in length and 60–80 nm in width. These regions could correspond to the amyloid fibrillary structure of a-synuclein. The shell-like region of LB was formed by the same fibrillary nanostructured proteins. However, in this case, intracellular proteins appearing as aggregates of 100–900 nm long could be clearly detected. Thus, the shell of LB contained denser fibrillary nanostructured regions than the central core-like region. Figure 1c shows that the shell of LB consisted of nanostructures that appeared white-colored, indicating that the aggregates were possibly constituted of similar material. As shown in the AFM height profile, the differences in width and height of these aggregates on the core and shell-like regions seem to be related to different extents of proteins assemblies. The left side of Figure 2 shows dark areas with the size of microseparated domains around 60–40 nm in diameter in the core of the LB. Likely, they correspond to spherical assemblies of a-synuclein molecules. AFM phase images can also detect variations in composition, adhesion, friction, viscoelasticity, and other properties. In our brain samples, AFM failed to show significant phase contrast between the small fibrillary and the smaller spherical nanostructures of the core-like region (Figure 2). This finding suggests that LB nanostructured aggregates are composed of similar materials, possibly a-synuclein molecules with different extents of assemblies: from spherical aggregates and small fibrillary nanostructures in the core to large fibrillary aggregates in the shell. Figure 3 shows a representative AFM height image of the prepared control brain. The optical microscopy (OM) image of the same region is shown in Figure 3b. A direct comparison of both postmortem brain tissue samples Ultrastructural Pathology
Ultrastruct Pathol Downloaded from informahealthcare.com by Universitaet Zuerich on 06/30/14 For personal use only.
Lewy Bodies under Atomic Force Microscope
3
FIGURE 1. (a) Tapping mode AFM height images of the postmortem brain tissue of PD patients (25 25 mm); (b) optical microscopy images of the postmortem brain tissue of PD patients (25 25 mm); (c) tapping mode AFM height images of the postmortem brain tissue of PD patients (10 10 mm); and (d) corresponding height profile.
FIGURE 2. Tapping mode AFM height/phase (left/right) images of the postmortem brain tissue of PD patients (3 3 mm).
(Figures 1a and 3a), shows a lack of continuity inside the neuron containig the LB. This phenomenon could be related to the formation of the LB, which could disrupt the interface between the affected nerve cell !
2014 Informa Healthcare USA, Inc.
and the substantia nigra (see Figures 1c and 2). In addition, Figure 3c shows that the the proteins that formed the unaffected neurons were well-organized in separated, spherical areas with a size around 0.8–1
Ultrastruct Pathol Downloaded from informahealthcare.com by Universitaet Zuerich on 06/30/14 For personal use only.
4
A. Tercjak et al.
FIGURE 3. (a) Tapping mode AFM height images of the postmortem brain tissue of patients without PD (25 25 mm); (b) optical microscopy images of the postmortem brain tissue of patients without PD (25 25 mm); (c) tapping mode AFM height images of the postmortem brain tissue of patients without PD (10 10 mm); and (d) corresponding height profile.
FIGURE 4. Tapping mode AFM height/phase (left/right) images of the postmortem brain tissue of patient without PD (3 3 mm).
micrometer in diameter. All of these areas consisted of a unique type of very regular spherical structure with a diameter of 20–60 nm (for a comparison see Figures 2 and 4). A representative profile of AFM height
image of this tissue (Figure 3d) confirmed a very regular structure since its roughness was very systematic and not higher than 50 nm. On the contrary, the roughness of the affected postmortem Ultrastructural Pathology
Lewy Bodies under Atomic Force Microscope brain tissue was higher than 80 nm owing to the presence of different types of aggregates in the nerve cell with LB.
Ultrastruct Pathol Downloaded from informahealthcare.com by Universitaet Zuerich on 06/30/14 For personal use only.
DISCUSSION The high sensibility of the AFM, as well as its ability to detect differences between components on the flat surface at nanoscale, makes this characterization technique a very useful tool for the rapid analysis of LB on the ultramicrotomy cut surface of postmortem brain tissue of PD patients. We have defined the coreand shell-like structure of LB. Likewise, we identified different extents of assemblies in these two regions of the LB. Likely, they occur due to the aggregation of a-synuclein molecules and oligomers, perhaps with other molecules. Two types of assemblies were detected in the core-like central region, fibrillary and spherical nanostructures. Aggregated fibrillary nanostructures were identified in the shell. Interestingly, all of these assemblies seem to have a constitution similar to the AFM height and phase images. Aggregated fibrillary nanostructures are not present in control brain tissue, thus showing the potential of AFM as a tool to gain insight into the pathophysiology of protein aggregation. Analysis of these images leads us to hypothesize about the process of formation and destruction of the LB in PD patients. Basically, our findings suggest that the formation of the LB seems to be the result of a low and progressive accumulation and organization of a-synuclein and other proteins in the environment of the aggresome [9]. This supports the idea that the unfolded protein response is a protective cellular mechanism triggered by rising levels of misfolded proteins. This might have important therapeutic implications not only in PD but also in many other neurodegenerative diseases characterized by protein misfolding [10]. In addition, the AFM might be a powerful tool in monitoring these strategies in animal models.
!
2014 Informa Healthcare USA, Inc.
5
In summary, AFM could be of great value to better understand the mechanism of the formation of the LB. Further studies including the study of cortical LB and Lewy neurites will be needed to determine the full potential of AFM in the study of the pathogenesis of cell death in PD.
DECLARATION OF INTEREST The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
REFERENCES 1. Jellinger KA. Basic mechanisms of neurodegeneration: a critical update. J Cell Mol Med 2010;14: 457–87. 2. Terry RD. Do neuronal inclusions kill the cell? J Neural Transm Suppl 2000;59: 91–3. 3. Volpicelli-Daley LA, Luk KC, Patel TP, et al. Exogenous a-synuclein fibrils induce Lewy body pathology leading to synaptic dysfunction and neuron death. Neuron 2011;72: 57–71. 4. Van Raaij ME, Segers-Nolten IMJ, Subramaniam V. Quantitative morphological analysis reveals ultrastructural diversity of amyloid fibrils from a-synuclein mutants. Biophys J Biophys Lett 2006;91: L96–8. 5. Sweers K, van der Werf K, Bennink M, et al. Nanomechanical properties of a-synuclein amyloid fibrils: a comparative study by nanoindentation, harmonic force microscopy, and Peakforce QNM. Nanoscale Res Lett 2011;6: 270. 6. Jansen R, Dzwolak W, Winter R. Amyloidogenic selfassembly of insulin aggregates probed by high resolution atomic force microscopy. Biophys J 2005;88: 1344–53. 7. Adamcik J, Jung JM, Flakowski J, et al. Understanding amyloid aggregation by statistical analysis of atomic force microscopy images. Nat Nanotechnol 2010 Jun;5: 423–8. 8. Hoyer W, Cherny D, Subramaniam V, et al. Rapid selfassembly of a-synuclein observed by in situ atomic force microscopy. J Mol Biol 2004;340: 127–39. 9. Olanow CW, McNaught K. Parkinson’s disease, proteins, and prions: milestones. Mov Disord 2011; 26: 1056–71. 10. Moreno JA, Radford H, Peretti D, et al. Sustained translational repression by elF2a-P mediates prion neurodegeneration. Nature 2012;485: 507–11.
ORIGINAL ARTICLE
Lewy Bodies under Atomic Force Microscope Agnieszka Tercjak, PhD1, Alberto Bergareche, MD2,3, Cristina Caballero, MD4, Teresa Tun˜on, MD5, and Gurutz Linazasoro, MD6 Department of Chemical and Environmental Engineering, University of the Basque Country, Spain, 2Neurology Department, University Hospital Donostia, Neurosciences Area, Biodonostia Institute, San Sebastia´n, Gipuzkoa, Spain, 3Center for Biomedical Research in Neurodegenerative Diseases Network (CIBERNED), Spain, 4 Department of Pathology, University Hospital Donostia, San Sebastia´n, Gipuzkoa, Spain, 5Department of Pathology, Brain Bank of Navarra, Navarra Hospital, Spain, and 6Centro de Investigacio´n Parkinson, Fundacio´n Inbiomed, San Sebastia´n, Spain
ABSTRACT Lewy bodies are the hallmark of Parkinson disease and their sophisticated analysis will undoubtedly elucidate the pathogenic process. They have been studied by using different microscopic tools. The authors have used atomic force microscopy (AFM) to study the ultramicrotom cut postmortem brain tissue of Parkinson disease patients. Under the same preparation conditions, they have found aggregated fibrillary nanostructures in Lewy bodies, as well as a loss of connections between neurons located in other parts of the substantia nigra. Although these results are preliminary and descriptive in nature, this paper reports the application of a novel and intriguing technique. Further studies including the study of cortical LB and Lewy neurites will be needed to determine the full potential of AFM in the study of the pathogenesis of cell death in Parkinson disease and other synucleinopathies. Keywords: Atomic force microscope, lewy bodies, neurodegeneration, Parkinson disease, a-synuclein
In addition, it seems unlikely that every dying neuron first goes through a stage of LB formation [2]. Furthermore, numerous in vitro and in vivo studies have shown posttranslational modifications and other changes of a-synuclein, leading to increased protein aggregation, which can be closely linked with neurotoxicity. Finally, LB pathology in sporadic PD disease progresses in a temporally and topographic ascending pattern. Thus, it has been suggested that the pathology is transmitted from neuron to neuron, presumably by spreading along axons by still unclear mechanisms [3]. Atomic force microscopy (AFM) is a powerful characterization technique with high-resolution imaging that makes it possible to distinguish between different objects on the surfaces of the investigated biomaterials at the nano and molecular scale due to the interactions between AFM-tip and investigated materials. This technique has been widely used in
Parkinson disease (PD) is one of the most common neurodegenerative disorders. It is characterized by a progressive degeneration of the nigrostriatal dopaminergic system, although other neuronal systems and organs are also affected. Degeneration is associated with the presence of intracytoplasmic aggregates of a-synuclein protein in the form of Lewy bodies (LB) and dystrophic Lewy neurites (LN) in surviving neurons. The formation and development of LBs, their relationship with the deposition of phosphorylated a-synuclein, and the interaction between a-synuclein and other pathogenic proteins are unclear. In particular, the biological significance of LBs, their impact on neurodegeneration, and their eventual role (detrimental or cytoprotective) are a hot topic of debate [1]. The loss of neurons in the substantia nigra pars compacta appears disproportionately high in comparison with the relatively small number of LBs.
20 14
Ultrastruct Pathol Downloaded from informahealthcare.com by Universitaet Zuerich on 06/30/14 For personal use only.
1
Received 7 June 2013; Revised 17 June 2013; Accepted 11 July 2013; Published online 15 October 2013 Correspondence: Dr. Alberto Bergareche, Neurology Department, University Hospital Donostia, San Sebastia´n, Gipuzkoa, Spain. E-mail: [email protected]
1
2
A. Tercjak et al.
biology as an imaging tool to determine morphological parameters, such as the height and length of amyloid fibrils formed from a-synuclein [4,5], insulin [6], and b-lactoglobulin [7]. Although the in situ self-assembly process of a-synuclein has been studied using AFM [8], to the best of our knowledge this is the first report of the use of AFM for the identification and analysis of LB on the postmortem brain tissue of PD patients using ultramicrotomy cut surfaces.
different regions of the specimens were scanned. Similar images were obtained, thus demonstrating the reproducibility of the results. For the analysis of the observed surface structures, nanoscope image processing software was used. In this study the AFM height images have been analyzed taking into account the very similar contrast of the AFM phase images. Optical microscopy (OM) measurements were performed in a Nikon Eclipse 600 using AFM samples.
RESULTS
Ultrastruct Pathol Downloaded from informahealthcare.com by Universitaet Zuerich on 06/30/14 For personal use only.
METHODS We selected two autopsied cases: a 79-year-old man with PD and a 71-year-old male without any neurological disorder. Both brains were acquired through explicit donation with appropriate ethical approval by the Brain Bank of Navarra (Spain), and meet the requirements of Spanish Real Decreto 1716/ 2011, which establishes the basic requirements for the authorization and operation of biobanks and the handling of biological samples of human origin. A hematoxilyn & eosin-stained slide from susbtantia nigra was checked in order to localize neurons and LB in the sample. We took a guided small sample from the paraffin block. The samples were placed in the stove at 60–70 C for 24 h, rehydrated in changes of xilol and descending alcohol (absolute, 96 C and 70 C), and washed in distilled water in the refrigerator. Next day, the samples were fixed in glutaraldehyde for at least 15 h, postfixed in osmium tetroxide for 2 h in the refrigerator, and washed with veronal buffer. After that, samples were dehydrated in ascending alcohol (30 C, 50 C, 70 C, 96 C, and absolute), washed in propylene oxide, and embedded in epoxy resin for 2 h. After the resin hardened, the material was cut into thick sections (semithin sections) in an ultramicrotome (LKB Bromma, Leika) and stained with toluidine blue in order to check for the presence of neurons with and without LB under light microscopy. After checking, the sections were cut into very small sections with a thickness of around 100– 200 nm. The cut sections were placed on the surface of the microscopic glass and analyzed directly without additional treatments. AFM was carried out using the Dimension 3100, Multimode from Digital Instruments, Veeco. AFM images were obtained by operating the AFM in tapping mode (TM-AFM) with a scanning probe equipped with an integrated silicon tip/cantilever having a resonance frequency of 300 kHz, from the same manufacturer. The height and phase images were obtained under ambient conditions with typical scan speeds of 0.7–1.2 line/s, using a hybrid scan head with a maximum range of 100 100 mm. Height and phase images were recorded simultaneously during scanning. In order to obtain repeatable results,
A representative AFM height image of the ultramicrotomy cut surface of these postmortem brain tissues is shown in Figure 1a. The optical microscopy (OM) image of the same region as the analyzed tissue is shown in Figure 1b. Both show the LB in the cytoplasm of the nerve cell. The AFM height image identifies two regions inside the LB forming a coreshell like structure. The size of the central core was around 8 nm in diameter and appeared as bright fibrillary nanostructured regions 400–800 nm in length and 60–80 nm in width. These regions could correspond to the amyloid fibrillary structure of a-synuclein. The shell-like region of LB was formed by the same fibrillary nanostructured proteins. However, in this case, intracellular proteins appearing as aggregates of 100–900 nm long could be clearly detected. Thus, the shell of LB contained denser fibrillary nanostructured regions than the central core-like region. Figure 1c shows that the shell of LB consisted of nanostructures that appeared white-colored, indicating that the aggregates were possibly constituted of similar material. As shown in the AFM height profile, the differences in width and height of these aggregates on the core and shell-like regions seem to be related to different extents of proteins assemblies. The left side of Figure 2 shows dark areas with the size of microseparated domains around 60–40 nm in diameter in the core of the LB. Likely, they correspond to spherical assemblies of a-synuclein molecules. AFM phase images can also detect variations in composition, adhesion, friction, viscoelasticity, and other properties. In our brain samples, AFM failed to show significant phase contrast between the small fibrillary and the smaller spherical nanostructures of the core-like region (Figure 2). This finding suggests that LB nanostructured aggregates are composed of similar materials, possibly a-synuclein molecules with different extents of assemblies: from spherical aggregates and small fibrillary nanostructures in the core to large fibrillary aggregates in the shell. Figure 3 shows a representative AFM height image of the prepared control brain. The optical microscopy (OM) image of the same region is shown in Figure 3b. A direct comparison of both postmortem brain tissue samples Ultrastructural Pathology
Ultrastruct Pathol Downloaded from informahealthcare.com by Universitaet Zuerich on 06/30/14 For personal use only.
Lewy Bodies under Atomic Force Microscope
3
FIGURE 1. (a) Tapping mode AFM height images of the postmortem brain tissue of PD patients (25 25 mm); (b) optical microscopy images of the postmortem brain tissue of PD patients (25 25 mm); (c) tapping mode AFM height images of the postmortem brain tissue of PD patients (10 10 mm); and (d) corresponding height profile.
FIGURE 2. Tapping mode AFM height/phase (left/right) images of the postmortem brain tissue of PD patients (3 3 mm).
(Figures 1a and 3a), shows a lack of continuity inside the neuron containig the LB. This phenomenon could be related to the formation of the LB, which could disrupt the interface between the affected nerve cell !
2014 Informa Healthcare USA, Inc.
and the substantia nigra (see Figures 1c and 2). In addition, Figure 3c shows that the the proteins that formed the unaffected neurons were well-organized in separated, spherical areas with a size around 0.8–1
Ultrastruct Pathol Downloaded from informahealthcare.com by Universitaet Zuerich on 06/30/14 For personal use only.
4
A. Tercjak et al.
FIGURE 3. (a) Tapping mode AFM height images of the postmortem brain tissue of patients without PD (25 25 mm); (b) optical microscopy images of the postmortem brain tissue of patients without PD (25 25 mm); (c) tapping mode AFM height images of the postmortem brain tissue of patients without PD (10 10 mm); and (d) corresponding height profile.
FIGURE 4. Tapping mode AFM height/phase (left/right) images of the postmortem brain tissue of patient without PD (3 3 mm).
micrometer in diameter. All of these areas consisted of a unique type of very regular spherical structure with a diameter of 20–60 nm (for a comparison see Figures 2 and 4). A representative profile of AFM height
image of this tissue (Figure 3d) confirmed a very regular structure since its roughness was very systematic and not higher than 50 nm. On the contrary, the roughness of the affected postmortem Ultrastructural Pathology
Lewy Bodies under Atomic Force Microscope brain tissue was higher than 80 nm owing to the presence of different types of aggregates in the nerve cell with LB.
Ultrastruct Pathol Downloaded from informahealthcare.com by Universitaet Zuerich on 06/30/14 For personal use only.
DISCUSSION The high sensibility of the AFM, as well as its ability to detect differences between components on the flat surface at nanoscale, makes this characterization technique a very useful tool for the rapid analysis of LB on the ultramicrotomy cut surface of postmortem brain tissue of PD patients. We have defined the coreand shell-like structure of LB. Likewise, we identified different extents of assemblies in these two regions of the LB. Likely, they occur due to the aggregation of a-synuclein molecules and oligomers, perhaps with other molecules. Two types of assemblies were detected in the core-like central region, fibrillary and spherical nanostructures. Aggregated fibrillary nanostructures were identified in the shell. Interestingly, all of these assemblies seem to have a constitution similar to the AFM height and phase images. Aggregated fibrillary nanostructures are not present in control brain tissue, thus showing the potential of AFM as a tool to gain insight into the pathophysiology of protein aggregation. Analysis of these images leads us to hypothesize about the process of formation and destruction of the LB in PD patients. Basically, our findings suggest that the formation of the LB seems to be the result of a low and progressive accumulation and organization of a-synuclein and other proteins in the environment of the aggresome [9]. This supports the idea that the unfolded protein response is a protective cellular mechanism triggered by rising levels of misfolded proteins. This might have important therapeutic implications not only in PD but also in many other neurodegenerative diseases characterized by protein misfolding [10]. In addition, the AFM might be a powerful tool in monitoring these strategies in animal models.
!
2014 Informa Healthcare USA, Inc.
5
In summary, AFM could be of great value to better understand the mechanism of the formation of the LB. Further studies including the study of cortical LB and Lewy neurites will be needed to determine the full potential of AFM in the study of the pathogenesis of cell death in PD.
DECLARATION OF INTEREST The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
REFERENCES 1. Jellinger KA. Basic mechanisms of neurodegeneration: a critical update. J Cell Mol Med 2010;14: 457–87. 2. Terry RD. Do neuronal inclusions kill the cell? J Neural Transm Suppl 2000;59: 91–3. 3. Volpicelli-Daley LA, Luk KC, Patel TP, et al. Exogenous a-synuclein fibrils induce Lewy body pathology leading to synaptic dysfunction and neuron death. Neuron 2011;72: 57–71. 4. Van Raaij ME, Segers-Nolten IMJ, Subramaniam V. Quantitative morphological analysis reveals ultrastructural diversity of amyloid fibrils from a-synuclein mutants. Biophys J Biophys Lett 2006;91: L96–8. 5. Sweers K, van der Werf K, Bennink M, et al. Nanomechanical properties of a-synuclein amyloid fibrils: a comparative study by nanoindentation, harmonic force microscopy, and Peakforce QNM. Nanoscale Res Lett 2011;6: 270. 6. Jansen R, Dzwolak W, Winter R. Amyloidogenic selfassembly of insulin aggregates probed by high resolution atomic force microscopy. Biophys J 2005;88: 1344–53. 7. Adamcik J, Jung JM, Flakowski J, et al. Understanding amyloid aggregation by statistical analysis of atomic force microscopy images. Nat Nanotechnol 2010 Jun;5: 423–8. 8. Hoyer W, Cherny D, Subramaniam V, et al. Rapid selfassembly of a-synuclein observed by in situ atomic force microscopy. J Mol Biol 2004;340: 127–39. 9. Olanow CW, McNaught K. Parkinson’s disease, proteins, and prions: milestones. Mov Disord 2011; 26: 1056–71. 10. Moreno JA, Radford H, Peretti D, et al. Sustained translational repression by elF2a-P mediates prion neurodegeneration. Nature 2012;485: 507–11.
