Summary Anamniotes, rodents, and young humans maintain neural stem cells in the ependymal zone (EZ) around the central canal of the spinal cord, representing a possible endogenous source for repair in mammalian lesions. Cell diversity and genes specific for this region are ill defined. A cellular and molecular resource is provided here for the mouse and human EZ based on RNA profiling, immunostaining, and fluorescent transgenic mice. This uncovered the conserved expression of 1,200 genes including 120 transcription factors. Unexpectedly the EZ maintains an embryonic-like dorsal-ventral pattern of expression of spinal cord developmental transcription factors (ARX, FOXA2, MSX1, and PAX6). In mice, dorsal and ventral EZ cells express Vegfr3 and are derived from the embryonic roof and floor plates. The dorsal EZ expresses a high level of Bmp6 and Gdf10 genes and harbors a subpopulation of radial quiescent cells expressing MSX1 and ID4 transcription factors.

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Traumatic brain injury (TBI) is an injury to the brain caused by an external mechanical force, affecting millions of people worldwide. The disease course and prognosis are often unpredictable, and it can be challenging to determine an early diagnosis in case of mild injury as well as to accurately phenotype the injury. There is currently no cure for TBI-drugs having failed repeatedly in clinical trials-but an intense effort has been put to identify effective neuroprotective treatment. The detection of novel biomarkers, to understand more of the disease mechanism, facilitates early diagnosis, predicts disease progression, and develops molecularly targeted therapies that would be of high clinical interest. Over the last decade, there has been an increasing effort and initiative toward finding TBI-specific biomarker candidates. One promising strategy has been to use state-of-the-art neuroproteomics approaches to assess clinical biofluids and compare the cerebrospinal fluid (CSF) and blood proteome between TBI and control patients or between different subgroups of TBI. In this chapter, we summarize and discuss the status of biofluid proteomics in TBI, with a particular focus on the latest findings.

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The Human Genome Project in 2003 has resulted in the complete sequence of ~99% of the human genome paving the road for the Human Proteome Project (HPP) assessing the full characterization of the translated protein map of the 20,300 protein-coding genes. Consequently, the emerging of the proteomics field has successfully been adopted as the method of choice for the proteome characterization. Proteomics is a term that is used to encompass multidisciplinary approaches combining different technologies that aim to study the entire spectrum of protein changes at a specific physiological condition. Proteomics research has shown excellent outcomes in different fields, among which is neuroscience; however, the complexity of the nervous systems necessitated the genesis of a new subdiscipline of proteomics termed as "neuroproteomics." Neuroproteomics studies involve assessing the quantitative and qualitative aspects of nervous system components encompassing global dynamic events underlying various brain-related disorders ranging from neuropsychiatric disorders, degenerative disorders, mental illness, and most importantly brain-specific neurotrauma-related injuries. In this introductory chapter, we will provide a brief historical perspective on the field of neuroproteomics. In doing so, we will highlight on the recent applications of neuroproteomics in the areas of neurotrauma, an area that has benefitted from neuroproteomics in terms of biomarker research, spatiotemporal injury mechanism, and its use to translate its findings from experimental settings to human translational applications. Importantly, this chapter will include some recommendation to the general studies in the area of neuroproteomics and the need to move from this field from being a descriptive, hypothesis-free approach to being an independent mature scientific discipline.

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Ependymal cells reside in the adult spinal cord and display stem cell properties in vitro. They proliferate after spinal cord injury and produce neurons in lower vertebrates but predominantly astrocytes in mammals. The mechanisms underlying this glial-biased differentiation remain ill-defined. We addressed this issue by generating a molecular resource through RNA profiling of ependymal cells before and after injury. We found that these cells activate STAT3 and ERK/MAPK signaling post injury and downregulate cilia-associated genes and FOXJ1, a central transcription factor in ciliogenesis. Conversely, they upregulate 510 genes, seven of them more than 20-fold, namely Crym, Ecm1, Ifi202b, Nupr1, Rbp1, Thbs2 and Osmr—the receptor for oncostatin, a microglia-specific cytokine which too is strongly upregulated after injury. We studied the regulation and role of Osmr using neurospheres derived from the adult spinal cord. We found that oncostatin induced strong Osmr and p-STAT3 expression in these cells which is associated with reduction of proliferation and promotion of astrocytic versus oligodendrocytic differentiation. Microglial cells are apposed to ependymal cells in vivo and co-culture experiments showed that these cells upregulate Osmr in neurosphere cultures. Collectively, these results support the notion that microglial cells and Osmr/Oncostatin pathway may regulate the astrocytic fate of ependymal cells in spinal cord injury.

Direct neuronal reprogramming, the process whereby a terminally differentiated cell is converted into an induced neuron without traversing a pluripotent state, has tremendous therapeutic potential for a host of neurodegenerative diseases. While there is strong evidence for astrocyte-to-neuron conversion in vitro, in vivo studies in the adult brain are less supportive or controversial. Here, we set out to enhance the efficacy of neuronal conversion of adult astrocytes in vivo by optimizing the neurogenic capacity of a driver transcription factor encoded by the proneural gene Ascl1. Specifically, we mutated six serine phospho-acceptor sites in Ascl1 to alanines (Ascl1SA6) to prevent phosphorylation by proline-directed serine/threonine kinases. Native Ascl1 or Ascl1SA6 were expressed in adult, murine cortical astrocytes under the control of a glial fibrillary acidic protein (GFAP) promoter using adeno-associated viruses (AAVs). When targeted to the cerebral cortex in vivo, mCherry+ cells transduced with AAV8-GFAP-Ascl1SA6-mCherry or AAV8-GFAP-Ascl1-mCherry expressed neuronal markers within 14 days post-transduction, with Ascl1SA6 promoting the formation of more mature dendritic arbors compared to Ascl1. However, mCherry expression disappeared by 2-months post-transduction of the AAV8-GFAP-mCherry control-vector. To circumvent reporter issues, AAV-GFAP-iCre (control) and AAV-GFAP-Ascl1 (or Ascl1SA6)-iCre constructs were generated and injected into the cerebral cortex of Rosa reporter mice. In all comparisons of AAV capsids (AAV5 and AAV8), GFAP promoters (long and short), and reporter mice (Rosa-zsGreen and Rosa-tdtomato), Ascl1SA6 transduced cells more frequently expressed early- (Dcx) and late- (NeuN) neuronal markers. Furthermore, Ascl1SA6 repressed the expression of astrocytic markers Sox9 and GFAP more efficiently than Ascl1. Finally, we co-transduced an AAV expressing ChR2-(H134R)-YFP, an optogenetic actuator. After channelrhodopsin photostimulation, we found that Ascl1SA6 co-transduced astrocytes exhibited a significantly faster decay of evoked potentials to baseline, a neuronal feature, when compared to iCre control cells. Taken together, our findings support an enhanced neuronal conversion efficiency of Ascl1SA6 vs. Ascl1, and position Ascl1SA6 as a critical transcription factor for future studies aimed at converting adult brain astrocytes to mature neurons to treat disease.