The γ-Protocadherins Regulate the Survival of GABAergic Interneurons during Developmental Cell Death.

Inhibitory interneurons integrate into developing circuits in specific ratios and distributions. In the neocortex, inhibitory network formation occurs concurrently with the apoptotic elimination of a third of GABAergic interneurons. The cell surface molecules that select interneurons to survive or die are unknown. Here, we report that members of the clustered Protocadherins (cPCDHs) control GABAergic interneuron survival during developmentally-regulated cell death. Conditional deletion of the gene cluster encoding the γ-Protocadherins (Pcdhgs) from developing GABAergic neurons in mice of either sex causes a severe loss of inhibitory populations in multiple brain regions and results in neurologic deficits such as seizures. By focusing on the neocortex and the cerebellar cortex, we demonstrate that reductions of inhibitory interneurons result from elevated apoptosis during the critical postnatal period of programmed cell death (PCD). By contrast, cortical interneuron (cIN) populations are not affected by removal of Pcdhgs from pyramidal neurons or glial cells. Interneuron loss correlates with reduced AKT signaling in Pcdhg mutant interneurons, and is rescued by genetic blockade of the pro-apoptotic factor BAX. Together, these findings identify the PCDHGs as pro-survival transmembrane proteins that select inhibitory interneurons for survival and modulate the extent of PCD. We propose that the PCDHGs contribute to the formation of balanced inhibitory networks by controlling the size of GABAergic interneuron populations in the developing brain.SIGNIFICANCE STATEMENT A pivotal step for establishing appropriate excitatory-inhibitory ratios is adjustment of neuronal populations by cell death. In the mouse neocortex, a third of GABAergic interneurons are eliminated by BAX-dependent apoptosis during the first postnatal week. Interneuron cell death is modulated by neural activity and pro-survival pathways but the cell-surface molecules that select interneurons for survival or death are unknown. We demonstrate that members of the cadherin superfamily, the clustered γ-Protocadherins (PCDHGs), regulate the survival of inhibitory interneurons and the balance of cell death. Deletion of the Pcdhgs in mice causes inhibitory interneuron loss in the cortex and cerebellum, and leads to motor deficits and seizures. Our findings provide a molecular basis for controlling inhibitory interneuron population size during circuit formation.

Activating transcription factor 4 regulates hypoxia inducible factor 1α in chronic hypoxia in pancreatic cancer cells.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive and difficult to treat cancers with tumors typically exhibiting high levels of chronic hypoxia. Hypoxia activates hypoxia-inducible factors (HIFs) that mediate cellular responses to adapt to low oxygen environments. Hypoxia also causes endoplasmic reticulum (ER) stress, increasing activating transcription factor 4 (ATF4), a master regulator of the unfolded protein response (UPR) pathway that mediates cellular response to ER stress. ATF4 is overexpressed in PDAC and is associated with poor prognoses. While ATF4 promotes cell proliferation and tumorigenesis, most studies have been conducted under normoxia or acute hypoxia. The functions of ATF4 in chronic hypoxia remain largely unexplored. Using siRNA knockdown experiments of healthy skin fibroblast cells WS1 and PDAC cell lines PANC-1 and Mia-PaCa2 to analyze mRNA and protein expression levels, a novel ATF4 function was identified, in which it decreases HIF2α mRNA and increases HIF1α mRNA in chronic hypoxia while having no effect in acute hypoxia. A scratch assay was used to show that ATF4 decreases cell migration in chronic hypoxia as opposed to the increase in cell migration ATF4 imparts in acute hypoxia. Colony formation assay and cell viability assay showed that ATF4 promotes colony formation and cell viability in both chronic and acute hypoxia. In addition to the differential response of ATF4 in chronic hypoxia compared with acute hypoxia, this is the first time ATF4 has been implicated in regulation of response to hypoxia via interaction with HIF proteins in PDAC.

Bilateral upregulation of α-synuclein expression in the mouse substantia nigra by intracranial rotenone treatment.

The pesticide rotenone has been shown to cause systemic inhibition of mitochondrial complex I activity, with consequent degeneration of dopamine neurons along the nigrostriatal pathway, as observed in Parkinson's disease (PD). Recently, intracranial infusion of rotenone was found to increase the protein levels of the Lewy body constituents, α-synuclein and small ubiquitin-related modifier-1(SUMO-1), in the lesioned hemisphere of the mouse brain. These findings are supportive of a mouse model of PD, but information about the dopamine-synthesizing enzyme, tyrosine hydroxylase (TH), an essential marker of dopaminergic status, was not reported. Clarification of this issue is important because an intracranial rotenone mouse model of Parkinson's disease has not been established. Towards this end, the present study examined the effects of intracranial rotenone treatment on TH and α-synuclein immunohistochemistry in addition to forelimb motor function. Mice were unilaterally infused with either vehicle or rotenone (2µg/site) in both the medial forebrain bundle and the substantia nigra. The forelimb asymmetry (cylinder) test indicated a significant decrease in use of the contralateral forelimb in lesioned animals as compared to the sham group. Densitometric analysis revealed a significant depletion of TH immunofluorescence within the ipsilateral striatum and substantia nigra of lesioned animals. Moreover, a significant bilateral increase in α-synuclein immunofluorescence was found in the substantia nigra of lesioned mice, as compared to control animals. These findings indicate that this intracranial rotenone mouse model will be useful for studies of neurodegenerative disorders such as PD.

Chronic low-dose melatonin treatment maintains nigrostriatal integrity in an intrastriatal rotenone model of Parkinson's disease.

Parkinson's disease is a major neurodegenerative disorder which primarily involves the loss of dopaminergic neurons in the substantia nigra and related projections in the striatum. The pesticide/neurotoxin, rotenone, has been shown to cause systemic inhibition of mitochondrial complex I activity in nigral dopaminergic neurons, with consequent degeneration of the nigrostriatal pathway, as observed in Parkinson's disease. A novel intrastriatal rotenone model of Parkinson's disease was used to examine the neuroprotective effects of chronic low-dose treatment with the antioxidant indoleamine, melatonin, which can upregulate neurotrophic factors and other protective proteins in the brain. Sham or lesioned rats were treated with either vehicle (0.04% ethanol in drinking water) or melatonin at a dose of 4 µg/mL in drinking water. The right striatum was lesioned by stereotactic injection of rotenone at three sites (4 µg/site) along its rostrocaudal axis. Apomorphine administration to lesioned animals resulted in a significant (p<0.001) increase in ipsilateral rotations, which was suppressed by melatonin. Nine weeks post-surgery, animals were sacrificed by transcardial perfusion. Subsequent immunohistochemical examination revealed a decrease in tyrosine hydroxylase immunoreactivity within the striatum and substantia nigra of rotenone-lesioned animals. Melatonin treatment attenuated the decrease in tyrosine hydroxylase in the striatum and abolished it in the substantia nigra. Stereological cell counts indicated a significant (p<0.05) decrease in dopamine neurons in the substantia nigra of rotenone-lesioned animals, which was confirmed by Nissl staining. Importantly, chronic melatonin treatment blocked the loss of dopamine neurons in rotenone-lesioned animals. These findings strongly support the therapeutic potential of long-term and low-dose melatonin treatment in Parkinson's disease.

Altered melatonin MT1 receptor expression in the ventral midbrain following 6-hydroxydopamine lesions in the rat medial forebrain bundle.

The indoleamine hormone melatonin protects dopamine neurons in the rat nigrostriatal pathway following 6-hydroxydopamine lesioning, and an increase in striatal melatonin levels has been detected in this model of Parkinson's disease. Melatonin induces the expression of tyrosine hydroxylase, the rate-limiting enzyme for dopamine synthesis, in the ventral midbrain, where G protein-coupled melatonin receptors are present. Based on the interaction between the melatonergic and dopaminergic systems, we hypothesized that 6-hydroxydopamine-induced degeneration of dopamine neurons would affect the expression of melatonin receptors in the nigrostriatal pathway. Following unilateral injection of 6-hydroxydopamine into the rat striatum or medial forebrain bundle, there was a significant increase in apomorphine-induced contralateral rotations in lesioned animals as compared to sham controls. A loss of tyrosine hydroxylase immunoreactivity and/or immunofluorescence in the striatum and substantia nigra was seen in animals lesioned in either the striatum or medial forebrain bundle, indicating degeneration of dopamine neurons. There were no significant differences in melatonin MT1 receptor protein expression in the striatum or substantia nigra, between intrastriatally lesioned animals and sham controls. In contrast, lesions in the medial forebrain bundle caused a significant increase in MT1 receptor mRNA expression (p<0.03) on the lesioned side of the ventral midbrain, as compared with the contralateral side. Given the presence of MT1 receptors on neurons in the ventral midbrain, these results suggest that a compensatory increase in MT1 transcription occurs to maintain expression of this receptor and neuroprotective melatonergic signaling in the injured brain.