Clin Auton Res DOI 10.1007/s10286-015-0273-2
RESEARCH LETTER
Putative neuropathological interactions in MSA: focus in the rostral ventrolateral medulla E. E. Benarroch • A. M. Schmeichel J. E. Parisi • P. A. Low
•
Received: 22 December 2014 / Accepted: 9 February 2015 Ó Springer-Verlag Berlin Heidelberg 2015
Abstract We used double immunocytochemistry for a-synuclein and markers of sympathoexcitatory neurons, oligodendrocytes, iron metabolism, and autophagy to study putative neuropathological interactions in multiple system atrophy. We focused in the rostral ventrolateral medulla as a prototype vulnerable region. We found that loss of C1 neurons and oligodendrocytes related to glial cytoplasmic inclusion accumulation, downregulation of iron transport, and upregulation of autophagy and ferritin expression in these area. Keywords Multiple system atrophy Alpha-synuclein Ferroportin Beclin-1
Introduction The pathological hallmark of multiple system atrophy (MSA) is the accumulation of a-SYN containing glial cytoplasmic inclusions (GCIs), which are associated with E. E. Benarroch (&) A. M. Schmeichel J. E. Parisi P. A. Low Department of Neurology, Mayo Clinic, 200 First Street SW, Rochester, MN 55905, USA e-mail: [email protected] A. M. Schmeichel e-mail: [email protected] J. E. Parisi e-mail: [email protected] P. A. Low e-mail: [email protected] J. E. Parisi Division of Anatomical Pathology, Mayo Clinic, Rochester, MN, USA
neuronal loss in the striatonigral, olivopontocerebellar, and central autonomic systems. The relationship between oligodendroglial and neuronal pathology in central autonomic areas are poorly understood. Central sympathoexcitatory areas, such as the rostral ventrolateral medulla (RVLM) are highly vulnerable in MSA and provide the opportunity to address these issues. Whereas some studies show a correlation between GCI accumulation and neuronal loss in the striatonigral and pontocerebellar systems [1], GCI accumulation may not necessarily follow the pattern of neuronal changes [2], suggesting that neuronal and oligodendrocyte pathology may represent not only mutually interactive but also independent parallel processes. There is evidence of both dysregulation of iron metabolism and autophagy in MSA. Iron accumulates in the form of ferritin probably in activated microglia; there is also reduction of expression of the iron transport protein ferroportin 1 (Fp1) in the basis pontis and putamen [3]. One possible mechanism of a-SYN accumulation is failure in clearance mechanisms such as autophagy, which has been shown to be altered in MSA [4]. Neuronal loss and GCI accumulation are widespread in MSA, however, we focused specifically on the RVLM, studying: (1) the relationships between these two neuropathological processes with each other and (2) their relationship with markers of abnormal iron metabolism and autophagy by focusing on the RVLM. Although, the RVLM is only one of multiple brain regions affected in MSA, it contains highly vulnerable C1 sympathoexcitatory neurons that can be readily identified by their immunoreactivity for tyrosine hydroxylase (TH) using immunofluorescence. Whereas Simultaneous assessment of TH and a-SYN has been carried out in previous studies on other neurodegenerative disorders, but we decided to study the relationship between TH cell loss and abnormal a-SYN accumulation with the presence of markers of iron
123
Clin Auton Res
We studied the RVLM of one parkinsonism predominant MSA (MSA-P) patient (67-year-old man, disease duration 7 years), with severe autonomic failure documented on autonomic testing and one age-matched control (64/M). The MSA-P patient had neuropathological confirmation of MSA, with severe neuronal loss and GCIs affecting
predominantly the striatonigral system, including the dopaminergic neurons of the substantia nigra pars compacta. We used double immunofluorescence techniques on 10 lm sections immunolabeled for markers of sympathoexcitatory C1 neurons (TH; rabbit monoclonal 1:1500 dilution; Millipore, Temecula, CA), oligodendrocytes (myelin basic protein, MBP; goat polyclonal 1:400, Santa Cruz Biotechnologies, Santa Cruz, CA); iron transport (FP1; rabbit polyclonal 1:5000; LSBio, Seattle, WA); iron accumulation (ferritin; rabbit polyclonal 1:1000, abcam, Cambridge, England); and autophagy (beclin-1; rabbit monoclonal 1:50; abcam, Cambridge, England) as well as a-SYN (mouse monoclonal 1:25; Invitrogen, Carisbad, CA). The sections were deparaffinized and antigen retrieval
Fig. 1 a Relationship between loss of tryosine hydroxylase (TH) immunoreactive (C1) neurons (green) and accumulation of asynuclein (a-SYN, red) in the form of glial cytoplasmic inclusions (GCIs) in the rostral ventrolateral medulla (RVLM) in a patient with MSA (right) compared to a control (left). Depletion of C1 neurons coincided with abundant GCI accumulation. b Comparison of expression of neuropathological markers in the RVLM the control (1,3,5,7) and the MSA (2,4,6,8) case. There was marked loss of myelin basic protein (MBP) immunoreactive oligodendrocytes
(green) associate with accumulation of a-SYN (red) in the MSA case (2) compared to the control (1). Loss of MBP expression (green) coincided with reduced expression of the iron transporter ferroporting1 (Fp1) red in the MSA case (4) compared to the control (3). Oligodendrocyte cell loss (green) corresponded to the area of upregulation of the autophagy marker beclin-1 (Bec1) in the MSA (6) compared to the control case (5). There was upregulation of ferritin expression the RVLM in the MSA (8) compared to the control (7) case, probably reflecting the presence of activated microglia
metabolism and autophagy in the RVLM. This approach could generate testable hypotheses for future studies on the spatial and temporal progression of MSA within central autonomic pathways.
Methods
123
Clin Auton Res
was performed with 1-mM EDTA buffer for 30 min in a steamer. Primary antibodies were incubated overnight at 4 °C, followed by appropriate secondaries for 1 h, and coverslipped with slow fade mounting media.
Results There were clear differences in the expression of the different markers in MSA and control case (Fig. 1). Not unexpectedly, there was severe loss of TH-immunoreactive neurons coincident with abundant accumulation of GCIs and marked loss of MBP-expressing oligodendrocytes. In our case we did not find double labeling for TH and abnormal a-SYN deposits in RVLM neurons. In the RVLM, TH neuronal loss and GCI accumulation were associated with downregulation of the iron transporter FP1 and upregulation of the autophagy marker beclin-1; as well as with accumulation of ferritin-containing cells, presumably activated microglia.
Discussion Our preliminary observations in the RVLM, one of the several selectively vulnerable regions affected in MSA, indicate that it is possible to apply double immunofluorescence techniques in autopsy tissue to explore the putative pathodynamic interactions among a-SYN accumulation, selective neuronal loss, oligodendrocyte pathology, and disturbances of iron metabolism and
Fig. 2 Putative Pathodynamic Interactions in MSA. Potential neuropathological interactions among neuronal and oligodendrocyte cell injury, alpha-synuclein (a-SYN) accumulation, iron metabolism
autophagy. Since orthostatic hypotension is a major manifestation of MSA that reflects impairment in central sympathoexcitatory circuits, in this preliminary study we focussed on the C1 neurons of the RVLM. These neurons project to the preganglionic sympathetic neurons of the intermediolateral cell column and to the locus ceruleus, and receive inputs from areas of the hypothalamus and upper brainstem; all these areas are affected in this disorder. Sympathetic preganglionic neurons of the spinal cord are organized in a ladder-like fashion, which poses the risk of a sampling effect in paraffin sections used for this multiple immunofluorescence marker approach done in this study. In our present study we did not find double labeling for TH and abnormal a-SYN deposits in the RVLM, however, our sample was limited, and therefore these results do not necessarily indicate that a-SYN does not accumulate in C1 neurons. Given the increasing evidence that neuronal synuclein pathology occurs at sites of GCI accumulation, further studies are needed to definitively determine whether C1 neurons of the RVLM harbor abnormal a-SYN deposits. Our results suggest that neuronal and oligodendrocytes loss, together with GCI accumulation, may in part reflect impaired iron transport by Fp1 in these cells and upregulation of autophagy. There are multiple putative mechanisms by which oligodendrocyte neuropathology may lead to secondary neuronal loss [5]. Some of these interactions are shown in Fig. 2. Reduced iron export in neurons or oligodendrocytes due to downregulation of FP1 may result in intracellular iron accumulation and oxidative stress in both types of cells. Impaired iron metabolism may also have a negative
dysregulation, oxidative stress, autophagy, and neuroinflammation in multiple system atrophy (MSA)
123
Clin Auton Res
impact in the differentiation of oligodendrocyte precursors. Oxidative stress may trigger and/or potentiate a-SYN misfolding and accumulation in the oligodendrocytes, neurons, or both, and disrupt cell function by still undetermined mechanisms. Overload of the ubiquitin–proteasome system by misfolded a-SYN likely triggers upregulation of autophagy. Finally, products of neuronal and/or oligodendrocyte injury may activate microglia, which could upregulate iron uptake and accumulation in the form of ferritin and release a variety of products, including superoxide and cytokines, which could contribute to both neuronal and oligodendrocyte pathologies. Eventually, pathology in a specific region, such as the RVLM, may propagate in a prion-like fashion along central autonomic pathways. In comparison to mouse models, it is not possible to dissect a disease process solely on the basis of a postmortem evaluation. Simultaneous neuropathological assessment of multiple putative mechanisms in humans with a well-characterized phenotype may help in obtaining further insight into MSA pathogenesis in humans. Our preliminary results indicate that this approach can help explore testable hypotheses about the pathodynamics of MSA in central autonomic areas affected by the disease. Whereas there is nothing ‘‘unique’’ about the RVLM as a target in MSA, in this preliminary study we focused on this area as an initial step for further studies to characterize the evolution of neuronal loss, oligodendrocyte pathology, aSYN accumulation, iron metabolism, and autophagy both in space (by analyzing other components of the sympathetic pathway in individual cases) and time (by comparing the expression of the different markers in each region in cases with different disease duration). The complex interactions suggested by our findings emphasize the complexity of the pathodynamics of MSA. Our findings also reveal the necessity for a broader focus while we work to
123
generate testable hypotheses which can lead to the rational development of neuroprotective drugs and other clinically relevant approaches. Acknowledgments Selective cell vulnerability in MSA: insight from cases with associated lewy body disease, funded by the CurePSP foundation and Mayo Funds. Conflict of interest The authors declare that they have no conflict of interest. Study approved by the Mayo Clinic IRB#08-000783. Ethical standards All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Informed consent Informed consent was obtained from all individual participants included in the study.
Reference 1. Ozawa T, Paviour D, Quinn NP et al (2004) The spectrum of pathological involvement of the striatonigral and olivopontocerebellar systems in multiple system atrophy: clinicopathological correlations. Brain 127:2657–2671 2. Armstrong RA, Lantos PL, Cairns NJ (2004) Spatial patterns of alpha-synuclein positive glial cytoplasmic inclusions in multiple system atrophy. Mov Disord 19:109–112 3. Visanji NP, Collingwood JF, Finnegan ME, Tandon A, House E, Hazrati LN (2013) Iron deficiency in parkinsonism: region-specific iron dysregulation in Parkinson’s disease and multiple system atrophy. J Parkinsons Dis 3:523–537 4. Schwarz L, Goldbaum O, Bergmann M, Probst-Cousin S, RichterLandsberg C (2012) Involvement of macroautophagy in multiple system atrophy and protein aggregate formation in oligodendrocytes. J Mol Neurosci 47:256–266 5. Wenning GK, Stefanova N, Jellinger KA, Poewe W, Schlossmacher MG (2008) Multiple system atrophy: a primary oligodendrogliopathy. Ann Neurol 64:239–246
RESEARCH LETTER
Putative neuropathological interactions in MSA: focus in the rostral ventrolateral medulla E. E. Benarroch • A. M. Schmeichel J. E. Parisi • P. A. Low
•
Received: 22 December 2014 / Accepted: 9 February 2015 Ó Springer-Verlag Berlin Heidelberg 2015
Abstract We used double immunocytochemistry for a-synuclein and markers of sympathoexcitatory neurons, oligodendrocytes, iron metabolism, and autophagy to study putative neuropathological interactions in multiple system atrophy. We focused in the rostral ventrolateral medulla as a prototype vulnerable region. We found that loss of C1 neurons and oligodendrocytes related to glial cytoplasmic inclusion accumulation, downregulation of iron transport, and upregulation of autophagy and ferritin expression in these area. Keywords Multiple system atrophy Alpha-synuclein Ferroportin Beclin-1
Introduction The pathological hallmark of multiple system atrophy (MSA) is the accumulation of a-SYN containing glial cytoplasmic inclusions (GCIs), which are associated with E. E. Benarroch (&) A. M. Schmeichel J. E. Parisi P. A. Low Department of Neurology, Mayo Clinic, 200 First Street SW, Rochester, MN 55905, USA e-mail: [email protected] A. M. Schmeichel e-mail: [email protected] J. E. Parisi e-mail: [email protected] P. A. Low e-mail: [email protected] J. E. Parisi Division of Anatomical Pathology, Mayo Clinic, Rochester, MN, USA
neuronal loss in the striatonigral, olivopontocerebellar, and central autonomic systems. The relationship between oligodendroglial and neuronal pathology in central autonomic areas are poorly understood. Central sympathoexcitatory areas, such as the rostral ventrolateral medulla (RVLM) are highly vulnerable in MSA and provide the opportunity to address these issues. Whereas some studies show a correlation between GCI accumulation and neuronal loss in the striatonigral and pontocerebellar systems [1], GCI accumulation may not necessarily follow the pattern of neuronal changes [2], suggesting that neuronal and oligodendrocyte pathology may represent not only mutually interactive but also independent parallel processes. There is evidence of both dysregulation of iron metabolism and autophagy in MSA. Iron accumulates in the form of ferritin probably in activated microglia; there is also reduction of expression of the iron transport protein ferroportin 1 (Fp1) in the basis pontis and putamen [3]. One possible mechanism of a-SYN accumulation is failure in clearance mechanisms such as autophagy, which has been shown to be altered in MSA [4]. Neuronal loss and GCI accumulation are widespread in MSA, however, we focused specifically on the RVLM, studying: (1) the relationships between these two neuropathological processes with each other and (2) their relationship with markers of abnormal iron metabolism and autophagy by focusing on the RVLM. Although, the RVLM is only one of multiple brain regions affected in MSA, it contains highly vulnerable C1 sympathoexcitatory neurons that can be readily identified by their immunoreactivity for tyrosine hydroxylase (TH) using immunofluorescence. Whereas Simultaneous assessment of TH and a-SYN has been carried out in previous studies on other neurodegenerative disorders, but we decided to study the relationship between TH cell loss and abnormal a-SYN accumulation with the presence of markers of iron
123
Clin Auton Res
We studied the RVLM of one parkinsonism predominant MSA (MSA-P) patient (67-year-old man, disease duration 7 years), with severe autonomic failure documented on autonomic testing and one age-matched control (64/M). The MSA-P patient had neuropathological confirmation of MSA, with severe neuronal loss and GCIs affecting
predominantly the striatonigral system, including the dopaminergic neurons of the substantia nigra pars compacta. We used double immunofluorescence techniques on 10 lm sections immunolabeled for markers of sympathoexcitatory C1 neurons (TH; rabbit monoclonal 1:1500 dilution; Millipore, Temecula, CA), oligodendrocytes (myelin basic protein, MBP; goat polyclonal 1:400, Santa Cruz Biotechnologies, Santa Cruz, CA); iron transport (FP1; rabbit polyclonal 1:5000; LSBio, Seattle, WA); iron accumulation (ferritin; rabbit polyclonal 1:1000, abcam, Cambridge, England); and autophagy (beclin-1; rabbit monoclonal 1:50; abcam, Cambridge, England) as well as a-SYN (mouse monoclonal 1:25; Invitrogen, Carisbad, CA). The sections were deparaffinized and antigen retrieval
Fig. 1 a Relationship between loss of tryosine hydroxylase (TH) immunoreactive (C1) neurons (green) and accumulation of asynuclein (a-SYN, red) in the form of glial cytoplasmic inclusions (GCIs) in the rostral ventrolateral medulla (RVLM) in a patient with MSA (right) compared to a control (left). Depletion of C1 neurons coincided with abundant GCI accumulation. b Comparison of expression of neuropathological markers in the RVLM the control (1,3,5,7) and the MSA (2,4,6,8) case. There was marked loss of myelin basic protein (MBP) immunoreactive oligodendrocytes
(green) associate with accumulation of a-SYN (red) in the MSA case (2) compared to the control (1). Loss of MBP expression (green) coincided with reduced expression of the iron transporter ferroporting1 (Fp1) red in the MSA case (4) compared to the control (3). Oligodendrocyte cell loss (green) corresponded to the area of upregulation of the autophagy marker beclin-1 (Bec1) in the MSA (6) compared to the control case (5). There was upregulation of ferritin expression the RVLM in the MSA (8) compared to the control (7) case, probably reflecting the presence of activated microglia
metabolism and autophagy in the RVLM. This approach could generate testable hypotheses for future studies on the spatial and temporal progression of MSA within central autonomic pathways.
Methods
123
Clin Auton Res
was performed with 1-mM EDTA buffer for 30 min in a steamer. Primary antibodies were incubated overnight at 4 °C, followed by appropriate secondaries for 1 h, and coverslipped with slow fade mounting media.
Results There were clear differences in the expression of the different markers in MSA and control case (Fig. 1). Not unexpectedly, there was severe loss of TH-immunoreactive neurons coincident with abundant accumulation of GCIs and marked loss of MBP-expressing oligodendrocytes. In our case we did not find double labeling for TH and abnormal a-SYN deposits in RVLM neurons. In the RVLM, TH neuronal loss and GCI accumulation were associated with downregulation of the iron transporter FP1 and upregulation of the autophagy marker beclin-1; as well as with accumulation of ferritin-containing cells, presumably activated microglia.
Discussion Our preliminary observations in the RVLM, one of the several selectively vulnerable regions affected in MSA, indicate that it is possible to apply double immunofluorescence techniques in autopsy tissue to explore the putative pathodynamic interactions among a-SYN accumulation, selective neuronal loss, oligodendrocyte pathology, and disturbances of iron metabolism and
Fig. 2 Putative Pathodynamic Interactions in MSA. Potential neuropathological interactions among neuronal and oligodendrocyte cell injury, alpha-synuclein (a-SYN) accumulation, iron metabolism
autophagy. Since orthostatic hypotension is a major manifestation of MSA that reflects impairment in central sympathoexcitatory circuits, in this preliminary study we focussed on the C1 neurons of the RVLM. These neurons project to the preganglionic sympathetic neurons of the intermediolateral cell column and to the locus ceruleus, and receive inputs from areas of the hypothalamus and upper brainstem; all these areas are affected in this disorder. Sympathetic preganglionic neurons of the spinal cord are organized in a ladder-like fashion, which poses the risk of a sampling effect in paraffin sections used for this multiple immunofluorescence marker approach done in this study. In our present study we did not find double labeling for TH and abnormal a-SYN deposits in the RVLM, however, our sample was limited, and therefore these results do not necessarily indicate that a-SYN does not accumulate in C1 neurons. Given the increasing evidence that neuronal synuclein pathology occurs at sites of GCI accumulation, further studies are needed to definitively determine whether C1 neurons of the RVLM harbor abnormal a-SYN deposits. Our results suggest that neuronal and oligodendrocytes loss, together with GCI accumulation, may in part reflect impaired iron transport by Fp1 in these cells and upregulation of autophagy. There are multiple putative mechanisms by which oligodendrocyte neuropathology may lead to secondary neuronal loss [5]. Some of these interactions are shown in Fig. 2. Reduced iron export in neurons or oligodendrocytes due to downregulation of FP1 may result in intracellular iron accumulation and oxidative stress in both types of cells. Impaired iron metabolism may also have a negative
dysregulation, oxidative stress, autophagy, and neuroinflammation in multiple system atrophy (MSA)
123
Clin Auton Res
impact in the differentiation of oligodendrocyte precursors. Oxidative stress may trigger and/or potentiate a-SYN misfolding and accumulation in the oligodendrocytes, neurons, or both, and disrupt cell function by still undetermined mechanisms. Overload of the ubiquitin–proteasome system by misfolded a-SYN likely triggers upregulation of autophagy. Finally, products of neuronal and/or oligodendrocyte injury may activate microglia, which could upregulate iron uptake and accumulation in the form of ferritin and release a variety of products, including superoxide and cytokines, which could contribute to both neuronal and oligodendrocyte pathologies. Eventually, pathology in a specific region, such as the RVLM, may propagate in a prion-like fashion along central autonomic pathways. In comparison to mouse models, it is not possible to dissect a disease process solely on the basis of a postmortem evaluation. Simultaneous neuropathological assessment of multiple putative mechanisms in humans with a well-characterized phenotype may help in obtaining further insight into MSA pathogenesis in humans. Our preliminary results indicate that this approach can help explore testable hypotheses about the pathodynamics of MSA in central autonomic areas affected by the disease. Whereas there is nothing ‘‘unique’’ about the RVLM as a target in MSA, in this preliminary study we focused on this area as an initial step for further studies to characterize the evolution of neuronal loss, oligodendrocyte pathology, aSYN accumulation, iron metabolism, and autophagy both in space (by analyzing other components of the sympathetic pathway in individual cases) and time (by comparing the expression of the different markers in each region in cases with different disease duration). The complex interactions suggested by our findings emphasize the complexity of the pathodynamics of MSA. Our findings also reveal the necessity for a broader focus while we work to
123
generate testable hypotheses which can lead to the rational development of neuroprotective drugs and other clinically relevant approaches. Acknowledgments Selective cell vulnerability in MSA: insight from cases with associated lewy body disease, funded by the CurePSP foundation and Mayo Funds. Conflict of interest The authors declare that they have no conflict of interest. Study approved by the Mayo Clinic IRB#08-000783. Ethical standards All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Informed consent Informed consent was obtained from all individual participants included in the study.
Reference 1. Ozawa T, Paviour D, Quinn NP et al (2004) The spectrum of pathological involvement of the striatonigral and olivopontocerebellar systems in multiple system atrophy: clinicopathological correlations. Brain 127:2657–2671 2. Armstrong RA, Lantos PL, Cairns NJ (2004) Spatial patterns of alpha-synuclein positive glial cytoplasmic inclusions in multiple system atrophy. Mov Disord 19:109–112 3. Visanji NP, Collingwood JF, Finnegan ME, Tandon A, House E, Hazrati LN (2013) Iron deficiency in parkinsonism: region-specific iron dysregulation in Parkinson’s disease and multiple system atrophy. J Parkinsons Dis 3:523–537 4. Schwarz L, Goldbaum O, Bergmann M, Probst-Cousin S, RichterLandsberg C (2012) Involvement of macroautophagy in multiple system atrophy and protein aggregate formation in oligodendrocytes. J Mol Neurosci 47:256–266 5. Wenning GK, Stefanova N, Jellinger KA, Poewe W, Schlossmacher MG (2008) Multiple system atrophy: a primary oligodendrogliopathy. Ann Neurol 64:239–246
