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Integrative computational analysis of the substantia nigra pars compacta and the locus coeruleus

ABSTRACT: The cells of the human brain show levels of functional specialization arguably unparalleled in biology. The recent advent of high-throughput, genome-wide assays to measure expression in individual cells offers an opportunity to characterize cellular variation in the human brain in a comprehensive, unbiased manner, providing a foundation for a brain-wide cell atlas. Crucial to the success of an atlas-scale project, however, is delivering a classification framework that is robust to variation across individuals, age and gender. Developing experimental and analytical tools that parse core, shared molecular signatures from inter-individual variation is therefore critically needed. The first step towards building these important tools is the acquisition and analysis of datasets from tissues known to possess biologically meaningful variation both within the tissue of one human being, and across the same tissue derived from different people. Here, we propose to molecularly profile two regions of the human brain known to display biologically meaningful cellular variation across individuals?the substantia nigra pars compacta (SNc) and the locus coeruleus (LC). These regions are highly susceptible to degeneration and are related to the two most common neurodegenerative diseases, Parkinson?s disease and Alzheimer?s disease. We will use high-throughput single-nucleus profiling of post-mortem human tissue to acquire 75,000 profiles each from SNc and LC, and analyze cellular diversity within and across individuals.

SUBMITTER: Evan Macosko

PROVIDER: S-SUBS14 | biostudies-other |

SECONDARY ACCESSION(S): SAMEA7283910

REPOSITORIES: biostudies-other

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Project description:This article gives a detailed description of a protocol using density gradient centrifugation for the enrichment of neuromelanin granules and synaptosomes from low amounts (?0.15g) of human substantia nigra pars compacta tissue. This has a great advantage compared to already existing methods as it allows for the first time (i) a combined enrichment of neuromelanin granules and synaptosomes and (ii) just minimal amounts of tissue necessary to enable donor specific analysis. Individual specimens were classified as control or diseased according to clinical evaluation and neuropathological examination. For the enrichment of synaptosomes and neuromelanin granules from the same tissue sample density gradient centrifugations using Percoll® and Iodixanol were performed. The purity of resulting fractions was checked by transmission electron microscopy. We were able to establish a reproducible and easy to handle protocol combining two different density gradient centrifugations: using an Iodixanol gradient neuromelanin granules were enriched and in parallel, from the same sample, a fraction of synaptosomes with high purity using a Percoll® gradient was obtained. Our subfractionation strategy will enable a subsequent in depth proteomic characterization of neurodegenerative processes in the substantia nigra pars compacta in patients with Parkinson's disease and dementia with Lewy bodies compared to appropriate controls.Key features of Parkinson's disease are the degeneration of dopaminergic neurons in the substantia nigra pars compacta, an associated loss of the brain pigment neuromelanin and a resulting impairment of the neuronal network. The accumulation of iron binding neuromelanin granules is age- and disease-dependent and disease specific alterations could affect the neuronal iron homeostasis leading to oxidative stress induced cell death. The focus of the described method is the analysis of neuromelanin granules as well as axonal cell-endings of nerve cells (synaptosomes) of individual donors (control and diseased). It is the basis for the identification of disease-relevant changes in the iron homeostasis and the generation of new insight into altered protein compositions or regulations which might lead to disturbed communications between nerve cells resulting in pathogenic processes.

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Integrative computational analysis of the substantia nigra pars compacta and the locus coeruleus

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