An essential role for p38 MAPK in cerebellar granule neuron precursor proliferation.

Development of the cerebellum occurs postnatally and is marked by a rapid proliferation of cerebellar granule neuron precursors (CGNPs). CGNPs are the cells of origin for SHH-driven medulloblastoma, the most common malignant brain tumor in children. Here, we investigated the role of ERK, JNK, and p38 mitogen-activated protein kinases in CGNP proliferation. We found high levels of p38α in proliferating CGNPs. Concomitantly, members of the p38 pathway, such as ASK1, MKK3 and ATF-2, were also elevated. Inhibition of the Shh pathway or CGNP proliferation blunts p38α levels, irrespective of Shh treatment. Strikingly, p38α levels were high in vivo in the external granule layer of the postnatal cerebellum, Shh-dependent mouse medulloblastomas and human medulloblastomas of the SHH subtype. Finally, knocking down p38α by short hairpin RNA-carrying lentiviruses as well as the pharmacologically inhibiting of its kinase activity caused a marked decrease in CGNP proliferation, underscoring its requirement for Shh-dependent proliferation in CGNPs. The inhibition of p38α also caused a decrease in Gli1 and N-myc transcript levels, consistent with reduced proliferation. These findings suggest p38 inhibition as a potential way to increase the efficacy of treatments available for malignancies associated with deregulated SHH signaling, such as basal cell carcinoma and medulloblastoma.

ß-Arrestin-1 links mitogenic sonic hedgehog signaling to the cell cycle exit machinery in neural precursors.

Development of the cerebellum, a brain region regulating posture and coordination, occurs post-natally and is marked by rapid proliferation of granule neuron precursors (CGNPs), stimulated by mitogenic Sonic hedgehog (Shh) signaling. ß-Arrestin (ßArr) proteins play important roles downstream of Smoothened, the Shh signal transducer. However, whether Shh regulates ßArrs and what role they play in Shh-driven CGNP proliferation remains to be determined. Here, we report that Shh induces ßArr1 accumulation and localization to the nucleus, where it participates in enhancing expression of the cyclin dependent kinase (cdk) inhibitor p27, whose accumulation eventually drives CGNP cell cycle exit. ßArr1 knockdown enhances CGNP proliferation and reduces p27 expression. Thus, Shh-mediated ßArr1 induction represents a novel negative feedback loop within the Shh mitogenic pathway, such that ongoing Shh signaling, while required for CGNPs to proliferate, also sets up a cell-intrinsic clock programming their ultimate exit from the cell cycle.

Insulin receptor substrate 1 is an effector of sonic hedgehog mitogenic signaling in cerebellar neural precursors.

Sonic hedgehog (SHH) and insulin-like growth factor (IGF) signaling are essential for development of many tissues and are implicated in medulloblastoma, the most common solid pediatric malignancy. Cerebellar granule neuron precursors (CGNPs), proposed cells-of-origin for specific classes of medulloblastomas, require SHH and IGF signaling for proliferation and survival during development of the cerebellum. We asked whether SHH regulates IGF pathway components in proliferating CGNPs. We report that SHH-treated CGNPs showed increased levels of insulin receptor substrate 1 (IRS1) protein, which was also present in the germinal layer of the developing mouse cerebellum and in mouse SHH-induced medulloblastomas. Previous roles for IRS1, an oncogenic protein that is essential for IGF-mediated proliferation in other cell types, have not been described in SHH-mediated CGNP proliferation. We found that IRS1 overexpression can maintain CGNP proliferation in the absence of SHH. Furthermore, lentivirus-mediated knock down experiments have shown that IRS1 activity is required for CGNP proliferation in slice explants and dissociated cultures. Contrary to traditional models for SHH signaling that focus on gene transcription, SHH stimulation does not regulate Irs1 transcription but rather stabilizes IRS1 protein by interfering with mTOR-dependent IRS1 turnover and possibly affects Irs1 mRNA translation. Thus, we have identified IRS1 as a novel effector of SHH mitogenic signaling that may serve as a future target for medulloblastoma therapies. Our findings also indicate a previously unreported interaction between the SHH and mTOR pathways, and provide an example of a non-classical means for SHH-mediated protein regulation during development.

Nitric oxide synthase isoforms undertake unique roles during excitotoxicity.

BACKGROUND AND PURPOSE: Excitotoxicity is a component of many neurodegenerative diseases. The signaling events that lead from excitotoxic injury to neuronal death remain incompletely defined. Pharmacological approaches have shown that nitric oxide production is critical for the progression of neurodegeneration after the initiation of excitotoxicity by the glutamate analog kainate. Although nitric oxide additionally triggers blood-brain barrier (BBB) breakdown, the breakdown does not in itself inevitably lead to neuronal cell death, because neuroprotective pharmacological means can be used subsequently to prevent the neural death. METHODS: In this study, we use a genetic approach to analyze the contribution of 3 nitric oxide synthase (NOS) isoforms, neuronal NOS, endothelial NOS, and inducible NOS, to neurodegeneration and BBB breakdown in this setting. RESULTS: We find that neuronal NOS is critical for the progression of kainate-stimulated neurodegeneration, whereas endothelial NOS is required only for BBB breakdown. Inducible NOS is not required for either event. CONCLUSIONS: The observation that endothelial NOS-deficient mice undergo excitotoxic neurodegeneration in the absence of BBB breakdown unlinks the two processes. These findings suggest that it may be possible to achieve full amelioration of excitotoxic-triggered neurodegeneration through developing isoform-specific inhibitors solely for neuronal NOS.

Nitric oxide mediates neurodegeneration and breakdown of the blood-brain barrier in tPA-dependent excitotoxic injury in mice.

Stroke and many neurodegenerative diseases culminate in neuronal death through a mechanism known as excitotoxicity. Excitotoxicity proceeds through a complex signaling pathway that includes the participation of the serine protease tissue plasminogen activator (tPA). tPA mediates neurotoxic effects on resident central nervous system cells as well alters blood-brain barrier (BBB) permeability, which further promotes neurodegeneration. Another signaling molecule that promotes neurodegeneration and BBB dysfunction is nitric oxide (NO), although its precise role in pathological progression remains unclear. We examine here the potentially interrelated roles of tPA, NO and peroxynitrite (ONOO-), which is the toxic metabolite of NO, in BBB breakdown and neurodegeneration following intrahippocampal injection of the glutamate analog kainite (KA). We find that NO and ONOO- production are linked to tPA-mediated excitotoxic injury, and demonstrate that NO provision suffices to restore the toxic effects of KA in tPA-deficient mice that are normally resistant to excitotoxicity. NO also promotes BBB breakdown and excitotoxicity. Interestingly, BBB breakdown in itself does not suffice to elicit neurodegeneration; a subsequent ONOO(-)-mediated event is required. In conclusion, NO and ONOO- function as downstream effectors of tPA-mediated excitotoxicity.