Immunofluorescence assay for demyelination, remyelination, and proliferation in an acute cuprizone mouse model.

Here, we present a protocol to assess demyelination in the corpus callosum of an acute cuprizone mouse model, which is routinely used to induce demyelination for studying myelin regeneration in the rodent brain. We describe the tracing of neural stem cells via intraperitoneal injection of tamoxifen into adult Gli1CreERT2;Ai9 mice and the induction of demyelination with cuprizone diet. We also detail EdU administration, cryosectioning of the mouse brain, EdU labeling, and immunofluorescence staining to examine proliferation and myelination. For complete details on the use and execution of this protocol, please refer to Radecki et al. (2020).1.

Wobbly hedgehog syndrome- a progressive neurodegenerative disease.

Wobbly hedgehog syndrome (WHS) has been long considered to be a myelin disease primarily affecting the four-toed hedgehog. In this study, we have shown for the first time that demyelination is accompanied by extensive remyelination in WHS. However, remyelination is not enough to compensate for the axonal degeneration and neuronal loss, resulting in a progressive neurodegenerative disease reminiscent of progressive forms of multiple sclerosis (MS) in humans. Thus, understanding the pathological features of WHS may shed light on the disease progression in progressive MS and ultimately help to develop therapeutic strategies for both diseases. Highlights: Wobbly hedgehog syndrome (WHS) is a progressive neurodegenerative disease.Spongy degeneration of the brain and spinal cord is the diagnostic feature of WHS.WHS affected brain and spinal cord show extensive demyelination and remyelination.Axonal degeneration is accompanied by loss of neurons in WHS.

Wobbly hedgehog syndrome- a progressive neurodegenerative disease.

Wobbly hedgehog syndrome (WHS) has been long considered to be a myelin disease primarily affecting the four-toed hedgehog. In this study, we have shown for the first time that demyelination is accompanied by extensive remyelination in WHS. However, remyelination is not enough to compensate for the axonal degeneration and neuronal loss, resulting in a progressive neurodegenerative disease reminiscent of progressive forms of multiple sclerosis (MS) in humans. Thus, understanding the pathological features of WHS may shed light on the disease progression in progressive MS and ultimately help to develop therapeutic strategies for both diseases.

Endogenous Neural Stem Cell Mediated Oligodendrogenesis in the Adult Mammalian Brain.

Oligodendrogenesis is essential for replacing worn-out oligodendrocytes, promoting myelin plasticity, and for myelin repair following a demyelinating injury in the adult mammalian brain. Neural stem cells are an important source of oligodendrocytes in the adult brain; however, there are considerable differences in oligodendrogenesis from neural stem cells residing in different areas of the adult brain. Amongst the distinct niches containing neural stem cells, the subventricular zone lining the lateral ventricles and the subgranular zone in the dentate gyrus of the hippocampus are considered the principle areas of adult neurogenesis. In addition to these areas, radial glia-like cells, which are the precursors of neural stem cells, are found in the lining of the third ventricle, where they are called tanycytes, and in the cerebellum, where they are called Bergmann glia. In this review, we will describe the contribution and regulation of each of these niches in adult oligodendrogenesis.

Isolation and culture of neural stem cells from adult mouse subventricular zone for genetic and pharmacological treatments with proliferation analysis.

Neural stem cells (NSCs) from the subventricular zone (SVZ) of the mouse brain can be expanded in vitro and grown as neurospheres, which can be stored long-term in liquid nitrogen. Here, we present a protocol for isolation and culture of NSCs from the adult mouse SVZ. We describe how to grow and expand primary NSCs to neurospheres, followed by differentiation and nucleofection/pharmacological treatments. Finally, we describe RNA extraction, EdU labeling of the cells, and immunofluorescent analysis to examine their proliferation. For complete details on the use and execution of this protocol, please refer to Radecki et al. (2020).

Relative Levels of Gli1 and Gli2 Determine the Response of Ventral Neural Stem Cells to Demyelination.

Enhancing repair of myelin is an important therapeutic goal in many neurological disorders characterized by demyelination. In the healthy adult brain, ventral neural stem cells (vNSCs) in the subventricular zone, marked by GLI1 expression, do not generate oligodendrocytes. However, in response to demyelination, their progeny are recruited to lesions where they differentiate into oligodendrocytes and ablation of GLI1 further enhances remyelination. GLI1 and GLI2 are closely related transcriptional activators but the role of GLI2 in remyelination by vNSCs is not clear. Here, we show that genetic ablation of Gli1 in vNSCs increases GLI2 expression and combined loss of both transcription factors decreases the recruitment and differentiation of their progeny in demyelinated lesions. These results indicate that GLI1 and GLI2 have distinct, non-redundant functions in vNSCs and their relative levels play an essential role in the response to demyelination.

Corticohippocampal Dysfunction In The OBiden Mouse Model Of Primary Oligodendrogliopathy.

Despite concerted efforts over decades, the etiology of multiple sclerosis (MS) remains unclear. Autoimmunity, environmental-challenges, molecular mimicry and viral hypotheses have proven equivocal because early-stage disease is typically presymptomatic. Indeed, most animal models of MS also lack defined etiologies. We have developed a novel adult-onset oligodendrogliopathy using a delineated metabolic stress etiology in myelinating cells, and our central question is, "how much of the pathobiology of MS can be recapitulated in this model?" The analyses described herein demonstrate that innate immune activation, glial scarring, cortical and hippocampal damage with accompanying electrophysiological, behavioral and memory deficits naturally emerge from disease progression. Molecular analyses reveal neurofilament changes in normal-appearing gray matter that parallel those in cortical samples from MS patients with progressive disease. Finally, axon initial segments of deep layer pyramidal neurons are perturbed in entorhinal/frontal cortex and hippocampus from OBiden mice, and computational modeling provides insight into vulnerabilities of action potential generation during demyelination and early remyelination. We integrate these findings into a working model of corticohippocampal circuit dysfunction to predict how myelin damage might eventually lead to cognitive decline.