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Aggregate-prone brain regions in Parkinson’s disease are rich in unique N-terminus α-synuclein conformers with high proteolysis susceptibility.

Wiseman JA, Murray HC, Faull RLMF, Dragunow M, Turner CP, Dieriks BV, Curtis, M. A. Aggregate-prone brain regions in Parkinson’s disease are rich in unique N-terminus α-synuclein conformers with high proteolysis susceptibility. Npj Park Dis. 2024;10:1–18 10.1038/s41531-023-00614-w. 

In Parkinson’s disease (PD), and other α-synucleinopathies, α-synuclein (α-Syn) aggregates form a myriad of conformational and truncational variants. Most antibodies used to detect and quantify α-Syn in the human brain target epitopes within the C-terminus (residues 96–140) of the 140 amino acid protein and may fail to capture the diversity of α-Syn variants present in PD. We sought to investigate the heterogeneity of α-Syn conformations and aggregation states in the PD human brain by labelling with multiple antibodies that detect epitopes along the entire length of α-Syn. We used multiplex immunohistochemistry to simultaneously immunolabel tissue sections with antibodies mapping the three structural domains of α-Syn. Discrete epitope-specific immunoreactivities were visualised and quantified in the olfactory bulb, medulla, substantia nigra, hippocampus, entorhinal cortex, middle temporal gyrus, and middle frontal gyrus of ten PD cases, and the middle temporal gyrus of 23 PD, and 24 neurologically normal cases. Distinct Lewy neurite and Lewy body aggregate morphologies were detected across all interrogated regions/cases. Lewy neurites were the most prominent in the olfactory bulb and hippocampus, while the substantia nigra, medulla and cortical regions showed a mixture of Lewy neurites and Lewy bodies. Importantly, unique N-terminus immunoreactivity revealed previously uncharacterised populations of (1) perinuclear, (2) glial (microglial and astrocytic), and (3) neuronal lysosomal α-Syn aggregates. These epitope-specific N-terminus immunoreactive aggregate populations were susceptible to proteolysis via time-dependent proteinase K digestion, suggesting a less stable oligomeric aggregation state. Our identification of unique N-terminus immunoreactive α-Syn aggregates adds to the emerging paradigm that α-Syn pathology is more abundant and complex in human brains with PD than previously realised. Our findings highlight that labelling multiple regions of the α-Syn protein is necessary to investigate the full spectrum of α-Syn pathology and prompt further investigation into the functional role of these N-terminus polymorphs.

Wrapping up the role of pericytes in Parkinson’s disease

Stevenson T. J. & Dieriks, B. V. (2023) Wrapping up the role of pericytes in Parkinson’s disease. Neural Regeneration Research 18(11): 2395-2396 doi:10.4103/1673‐5374.371362

Pericytes are classically defined as contractile cells within the central nervous system that regulate blood flow and permeability of the blood-brain barrier (BBB). This one-sided view is gradually changing, and pericytes are now considered versatile cells that can switch their function in response to different stimuli (Uemura et al., 2020). In addition to their role as gatekeepers of the BBB and maintaining homeostasis of the brain’s microenvironment through adjusting the vascular intraluminal diameter, pericytes are both sensors and initiators of inflammation, allowing communication between the cerebral parenchyma and the peripheral system (Dieriks et al., 2022). Pericytes can react quickly by releasing neurotrophins to promote neuroprotection or by secreting pro-inflammatory cytokines, which can exacerbate brain and BBB damage (Dieriks et al., 2022). BBB disruption, blood vessel alterations, and cerebral blood flow abnormalities are commonly seen in neurodegenerative disorders with loss of pericyte coverage present in Parkinson’s disease (PD) and Alzheimer’s disease (Uemura et al., 2020, Elabi et al., 2021).

Absolute quantification of neuromelanin in formalin-fixed human brains using absorbance spectrophotometry

Chand DA, Scadeng M, Dieriks BV (2023) Absolute quantification of neuromelanin in formalin-fixed human brains using absorbance spectrophotometry. PLOS ONE 18 (7) 18:e0288327. doi: https://doi.org/10.1371/journal.pone.0288327

Parkinson’s disease is characterised by a visual, preferential degeneration of the pigmented neurons in the substantia nigra. These neurons are pigmented by neuromelanin which decreases in Parkinson’s disease. Not much is known about NM as it is difficult to study and quantify, primarily due to its insolubility in most solvents except alkali. Neuromelanin quantification could progress the development of biomarkers for prodromal Parkinson’s disease and provide insights into the presently unclear role of neuromelanin in Parkinson’s disease aetiology. Light microscopy with stereology can visualise pigmented neurons, but it cannot quantify neuromelanin concentrations. Absolute neuromelanin quantification using absorbance spectrophotometry is reported in the literature, but the methodology is dated and only works with fresh-frozen tissue. We have developed a quantification protocol to overcome these issues. The protocol involves breakdown of fixed tissue, dissolving the tissue neuromelanin in sodium hydroxide, and measuring the solution’s 350 nm absorbance. Up to 100 brain samples can be analysed in parallel, using as little as 2 mg of tissue per sample. We used synthetic neuromelanin to construct the calibration curve rather than substantia nigra neuromelanin. Our protocol enzymatically synthesises neuromelanin from dopamine and L-cysteine followed by high-heat ageing. This protocol enables successful lysis of the fixed substantia nigra tissue and quantification in three brains, with neuromelanin concentrations ranging from 0.23–0.55 μg/mg tissue. Quantification was highly reproducible with an interassay coefficient of variation of 6.75% (n = 5). The absorbance spectra and elemental composition of the aged synthetic neuromelanin and substantia nigra neuromelanin show excellent overlap. Our protocol can robustly and reliably measure the absolute concentration of neuromelanin in formalin-fixed substantia nigra tissue. This will enable us to study how different factors affect neuromelanin and provide the basis for further development of Parkinson’s disease biomarkers and further research into neuromelanin’s role in the brain.

Multiple system atrophy: α-Synuclein strains at the neuron-oligodendrocyte crossroad

Reddy K,. Dieriks, B. V. (2022) Multiple system atrophy: α-Synuclein strains at the neuron-oligodendrocyte crossroad Molecular Neurodegeneration doi: 10.1186/s13024-022-00579-z

Parkinson’s disease (PD) is a progressive, neurodegenerative disorder characterised by the abnormal accumulation of α-synuclein (α-syn) aggregates. Central to disease progression is the gradual spread of pathological α-syn. α-syn aggregation is closely linked to progressive neuron loss. As such, clearance of α-syn aggregates may slow the progression of PD and lead to less severe symptoms. Evidence that non-neuronal cells play a role in PD and other synucleinopathies such as Lewy body dementia and multiple system atrophy are increasing. Our previous work has shown that pericytes — vascular mural cells that regulate the blood-brain barrier — contain α-syn aggregates in human PD brains. Here, we demonstrate that pericytes efficiently internalise fibrillar α-syn irrespective of being in a monoculture or mixed neuronal cell culture. Pericytes efficiently break down α-syn aggregates in vitro, with clear differences in the number of α-syn aggregates/cell and average aggregate size when comparing five pure α-syn strains (Fibrils, Ribbons, fibrils65, fibrils91 and fibrils110). Furthermore, pericytes derived from PD brains have a less uniform response than those derived from control brains. Our results highlight the vital role brain vasculature may play in reducing α-syn burden in PD.

Human pericytes can degrade diverse alpha-synuclein aggregates.

 

Dieriks, B. V., Highet B., Alik A., Bellande T., ..., Faull, R. L. Melki R., Curtis, M. A., Dragunow M. (2022) Human pericytes can degrade diverse alpha-synuclein aggregates. Plos One Vol.17(11), p.e0277658 doi:10.1371/journal.pone.0277658 

Parkinson’s disease (PD) is a progressive, neurodegenerative disorder characterised by the abnormal accumulation of α-synuclein (α-syn) aggregates. Central to disease progression is the gradual spread of pathological α-syn. α-syn aggregation is closely linked to progressive neuron loss. As such, clearance of α-syn aggregates may slow the progression of PD and lead to less severe symptoms. Evidence is increasing that non-neuronal cells play a role in PD and other synucleinopathies such as Lewy body dementia and multiple system atrophy. Our previous work has shown that pericytes—vascular mural cells that regulate the blood-brain barrier—contain α-syn aggregates in human PD brains. Here, we demonstrate that pericytes efficiently internalise fibrillar α-syn irrespective of being in a monoculture or mixed neuronal cell culture. Pericytes cleave fibrillar α-syn aggregates (Fibrils, Ribbons, fibrils65, fibrils91 and fibrils110), with cleaved α-syn remaining present for up to 21 days. The number of α-syn aggregates/cell and average aggregate size depends on the type of strain, but differences disappear within 5 five hours of treatment. Our results highlight the role brain vasculature may play in reducing α-syn aggregate burden in PD.

Huntingtin aggregates in the olfactory bulb in Huntington's disease.

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Highet B.#, Dieriks, B. V.# Murray, H. C., Faull, R. L. M., Curtis, M. A.  (2020) Huntingtin aggregates in the olfactory bulb in Huntington's disease. Frontiers in Aging Neuroscience 12(261) doi: 10.3389/fnagi.2020.00261. (# equally contributed first authors)

Olfactory deficits are an early and prevalent non-motor symptom of Huntington’s disease (HD). In other neurodegenerative diseases where olfactory deficits occur, such as Alzheimer’s disease and Parkinson’s disease, pathological protein aggregates (tau, β-amyloid, α-synuclein) accumulate in the anterior olfactory nucleus (AON) of the olfactory bulb (OFB). Therefore, in this study we determined whether aggregates are also present in HD OFBs; 13 HD and five normal human OFBs were stained for mutant huntingtin (mHtt), tau, β-amyloid, TDP-43, and α-synuclein. Our results show that mHtt aggregates detected with 1F8 antibody are present within all HD OFBs, and mHtt aggregate load in the OFB does not correlate with Vonsattel grading scores. The majority of the aggregates were located in the AON and in similar abundance in each anatomical segment of the AON. No mHtt aggregates were found in controls; 31% of HD cases also contained tau neurofibrillary tangles within the AON. This work demonstrates HD pathology in the OFB and indicates that disease-specific protein aggregation in the AON is a common feature of neurodegenerative diseases that show olfactory deficits.

α-synuclein inclusions are abundant in non-neuronal cells in the anterior olfactory nucleus of the Parkinson's disease olfactory bulb.

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Stevenson T. J., Murray, H. C., Turner, C., Faull, R. L. M., Dieriks, B. V.* & Curtis, M. A.* (2020). α-synuclein inclusions are abundant in non-neuronal cells in the anterior olfactory nucleus of the Parkinson's disease olfactory bulb. Scientific Reports, 10(1), 1-10. doi: 10.1038/s41598-020-63412-x. (* equally contributed co-last authors).

This paper is part of Scientific Reports' Editor's choice: neurodegenerative diseases' and part of Top 100 in Neuroscience. This collection highlights Scientific Reports most downloaded neuroscience papers published in 2020.

Reduced olfactory function (hyposmia) is one of the most common non-motor symptoms experienced by those living with Parkinson’s disease (PD), however, the underlying pathology of the dysfunction is unclear. Recent evidence indicates that α-synuclein (α-syn) pathology accumulates in the anterior olfactory nucleus of the olfactory bulb years before the motor symptoms are present. It is well established that neuronal cells in the olfactory bulb are affected by α-syn, but the involvement of other non-neuronal cell types is unknown. The occurrence of intracellular α-syn inclusions were quantified in four non-neuronal cell types – microglia, pericytes, astrocytes and oligodendrocytes as well as neurons in the anterior olfactory nucleus of post-mortem human PD olfactory bulbs (n = 11) and normal olfactory bulbs (n = 11). In the anterior olfactory nucleus, α-syn inclusions were confirmed to be intracellular in three of the four non-neuronal cell types, where 7.78% of microglia, 3.14% of pericytes and 1.97% of astrocytes were affected. Neurons containing α-syn inclusions comprised 8.60% of the total neuron population. Oligodendrocytes did not contain α-syn. The data provides evidence that non-neuronal cells in the PD olfactory bulb contain α-syn inclusions, suggesting that they may play an important role in the progression of PD.

Differential fatty acid-binding protein expression in persistent radial glia in the human and Sheep subventricular zone.

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Dieriks, B. V., Dean, J. M., Aronica, E., Waldvogel, H. J., Faull, R. L. M., & Curtis, M. A. (2018). Differential fatty acid-binding protein expression in persistent radial glia in the human and Sheep subventricular zone. Developmental neuroscience. 40(2):145-161. doi: 10.1159/000487633.

Fatty acid-binding proteins (FABPs) are a family of transport proteins that facilitate intracellular transport of fatty acids. Despite abundant expression in the brain, the role that FABPs play in the process of cell proliferation and migration in the subventricular zone (SVZ) remains unclear. Our results provide a detailed characterisation of FABP3, 5, and 7 expression in adult and fetal human and sheep SVZ. High FABP5 expression was specifically observed in the adult human SVZ and co-labelled with polysialylated neural cell adhesion molecule (PSA-NCAM), glial fibrillary acidic protein (GFAP), GFAPδ, and proliferating cell nuclear antigen (PCNA), indicating a role for FABP5 throughout the full maturation process of astrocytes and neuroblasts. Some FABP5+ cells had a radial glial-like appearance and co-labelled with the radial glia markers vimentin (40E-C) and GFAP. In the fetal human brain, FABP5 was expressed by radial glia cells throughout the ventricular zone. In contrast, radial glia-like cells in sheep highly expressed FABP3. Taken together, these differences highlight the species-specific expression profile of FABPs in the SVZ. In this study, we demonstrate the distribution of FABP in the adult human SVZ and fetal ventricular zone and reveal its expression on persistent radial glia that may be involved in adult neurogenesis.

α-synuclein transfer through tunneling nanotubes occurs in SH-SY5Y cells and primary brain pericytes from Parkinson's disease patients.

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Dieriks, B. V., Park, T. I. -H., Fourie, C., Faull, R. L., Dragunow, M., & Curtis, M. A. (2017). α-synuclein transfer through tunneling nanotubes occurs in SH-SY5Y cells and primary brain pericytes from Parkinson's disease patients. Scientific Reports, 7, 42984. doi: 10.1038/srep42984.

Parkinson’s disease (PD) is characterized by the presence of inclusions known as Lewy bodies, which mainly consist of α-synuclein (α-syn) aggregates. There is growing evidence that α-syn self-propagates in non-neuronal cells, thereby contributing to the progression and spread of PD pathology in the brain. Tunneling nanotubes (TNTs) are long, thin, F-actin-based membranous channels that connect cells and have been proposed to act as conduits for α-syn transfer between cells. SH-SY5Y cells and primary human brain pericytes, derived from postmortem PD brains, frequently form TNTs that allow α-syn transfer and long-distance electrical coupling between cells. Pericytes in situ contain α-syn precipitates like those seen in neurons. Exchange through TNTs was rapid, but dependent on the size of the protein. Proteins were able to spread throughout a network of cells connected by TNTs. Transfer through TNTs was not restricted to α-syn; fluorescent control proteins and labeled membrane were also exchanged through TNTs. Most importantly the formation of TNTs and transfer continued during mitosis. Together, our results provide a detailed description of TNTs in SH-SY5Y cells and human brain PD pericytes, demonstrating their role in α-syn transfer and further emphasize the importance that non-neuronal cells, such as pericytes play in disease progression.

Co-Authored Papers (since 2017)

  1. Stevenson T. J., Johnson R. H., …, Melki R., Dieriks, B. V., Curtis, M. A, Dragunow M. (2022) Pericytes take up and degrade α-synuclein but succumb to apoptosis under cellular stress. Sci Rep. 2022;12(17314). Doi:10.1038/s41598-022-20261-0 

  2. Thumbadoo, K. M., Dieriks, B. V., Murray, H. C., and Swanson, M. E. V,…, Scotter E. L. (2022). Hippocampal protein aggregation signatures fully distinguish pathogenic and wildtype UBQLN2 in amyotrophic lateral sclerosis. Preprint. doi: 10.1101/2022.01.12.475792.

  3. Murray, H., Johnson K., Sedlock A., Highet, B., Dieriks, B.V., Vikas Anekal, P., Faull, R. L. M., Curtis, M. A., Koretsky A., Maric D. (2022). Lamina-specific immunohistochemical signatures in the olfactory bulb of healthy, Alzheimer's and Parkinson's disease patients. Commun Biol. 5 (88), 1–12. doi: 10.1038/s42003-022-03032-5.

  4. Waters S, Swanson ME V., Dieriks B V., Zhang YB, Grimsey NL, Murray HC,..., Scotter E. L. (2021). Blood-spinal cord barrier leakage is independent of motor neuron pathology in ALS. Acta Neuropathol Commun. 9(1): 1–17. doi.org/10.1186/s40478-021-01244-0.

  5. Jansson, D., Dieriks, B. V., Rustenhoven, J., Leon, C. D., Scotter, E., Aalderink, M., …, Dragunow, M. (2021). Recycling old drugs : cardiac glycosides as therapeutics to target barrier inflammation of the vasculature , meninges and choroid plexus. Commun Biol. 4(260), 1–17. doi: 10.1038/s42003-021-01787-x

  6. Highet, B., Vikas Anekal, P., Ryan, B., Murray, H., Coppieters, N., Dieriks, B.V., …, Curtis, M. A. (2021). fISHing with immunohistochemistry for housekeeping gene changes in Alzheimer's disease using an automated quantitative analysis workflow. J. Neurochem., 157 (4), 1270-1283. doi: 10.1111/jnc.15283.

  7. Park TI-H.#, Schweder P.#, Lee K.#, Dieriks B. V., Jung Y., Smyth L., Rustenhoven J., …, Montgomery J. M., Dragunow M. (2020). Isolation and culture of functional adult human neurons from neurosurgical brain specimens. Brain Communication 2(2). doi: 10.1093/braincomms/fcaa171. (# equally contributed first authors)

  8. H. C. Murray, B. V. Dieriks, M. E. V. Swanson, P. V. Anekal, C. Turner, R. L. M. Faull, L. Belluscio, A. Koretsky, M. A. Curtis (2020) The unfolded protein response is activated in the olfactory system in Alzheimer's disease. Acta Neuropathologica Communications 8:109 doi: 10.1186/s40478-020-00986-7.

  9. Wu J. J., Cai A., Greenslade J. E., Higgins N. R., Fan C., Le N. T.T., Tatman M., Whiteley A. A., Prado M. A., Dieriks B. V., …, Monteiro M. J. (2020). ALS/FTD mutations in UBQLN2 impede autophagy by reducing autophagosome acidification through loss-of-function. PNAS 201917371. doi: 10.1073/pnas.1917371117.

  10. Zapiec, B., Dieriks, B., Tan, S., Faull, R., Mombaerts, P., & Curtis, M. (2017). A ventral glomerular deficit in Parkinson's disease revealed by whole olfactory bulb reconstruction. Brain, 140(10), 2722-2736. doi:10.1093/brain/awx208.

  11. Gardner, B., Dieriks, B. V., Cameron, S., Mendis, L. H., ...., Faull, R. L., & Curtis, M. A. (2017). Metal concentrations and distributions in the human olfactory bulb in Parkinson's disease. Scientific Reports, 7 (1), 1-14. doi:10.1038/s41598-017-10659-6.

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