Neuron Enriched Nuclear Proteome Isolated from Human Brain

The brain consists of diverse cell
types including neurons, astrocytes,
oligodendrocytes, and microglia. The isolation of nuclei from these
distinct cell populations provides an opportunity to identify cell-type-specific
nuclear proteins, histone modifications, and regulation networks that
are altered with normal brain aging or neurodegenerative disease.
In this study, we used a method by which intact neuronal and non-neuronal
nuclei were purified from human post-mortem brain employing a modification
of fluorescence activated cell sorting (FACS) termed fluorescence
activated nuclei sorting (FANS). An antibody against NeuN, a neuron
specific splicing factor, was used to isolate neuronal nuclei. Utilizing
mass spectrometry (MS) based label-free quantitative proteomics, we
identified 1755 proteins from sorted NeuN-positive and negative nuclear
extracts. Approximately 20% of these proteins were significantly enriched
or depleted in neuronal versus non-neuronal populations. Immunoblots
of primary cultured rat neuron, astrocyte, and oligodendrocyte extracts
confirmed that distinct members of the major nucleocytoplasmic structural
linkage complex (LINC), nesprin-1 and nesprin-3, were differentially
enriched in neurons and astrocytes, respectively. These comparative
proteomic data sets also reveal a number of transcription and splicing
factors that are selectively enriched in a cell-type-specific manner
in human brain.