PTEN negatively regulates the cell lineage progression from NG2+ glial progenitor to oligodendrocyte via mTOR-independent signaling.

Oligodendrocytes (OLs), the myelin-forming CNS glia, are highly vulnerable to cellular stresses, and a severe myelin loss underlies numerous CNS disorders. Expedited OL regeneration may prevent further axonal damage and facilitate functional CNS repair. Although adult OL progenitors (OPCs) are the primary players for OL regeneration, targetable OPC-specific intracellular signaling mechanisms for facilitated OL regeneration remain elusive. Here, we report that OPC-targeted PTEN inactivation in the mouse, in contrast to OL-specific manipulations, markedly promotes OL differentiation and regeneration in the mature CNS. Unexpectedly, an additional deletion of mTOR did not reverse the enhanced OL development from PTEN-deficient OPCs. Instead, ablation of GSK3ß, another downstream signaling molecule that is negatively regulated by PTEN-Akt, enhanced OL development. Our results suggest that PTEN persistently suppresses OL development in an mTOR-independent manner, and at least in part, via controlling GSK3ß activity. OPC-targeted PTEN-GSK3ß inactivation may benefit facilitated OL regeneration and myelin repair.

DJ-1 facilitates the interaction between STAT1 and its phosphatase, SHP-1, in brain microglia and astrocytes: A novel anti-inflammatory function of DJ-1.

Parkinson's disease (PD) is a progressive neurodegenerative movement disorder caused by the death of dopaminergic neurons in the substantia nigra. Importantly, altered astrocyte and microglial functions could contribute to neuronal death in PD. In this study, we demonstrate a novel mechanism by which DJ-1 (PARK7), an early onset autosomal-recessive PD gene, negatively regulates inflammatory responses of astrocytes and microglia by facilitating the interaction between STAT1 and its phosphatase, SHP-1 (Src-homology 2-domain containing protein tyrosine phosphatase-1). Astrocytes and microglia cultured from DJ-1-knockout (KO) mice exhibited increased expression of inflammatory mediators and phosphorylation levels of STAT1 (p-STAT1) in response to interferon-gamma (IFN-γ) compared to cells from wild-type (WT) mice. DJ-1 deficiency also attenuated IFN-γ-induced interactions of SHP-1 with p-STAT1 and STAT1, measured 1 and 12h after IFN-γ treatment, respectively. Subsequent experiments showed that DJ-1 directly interacts with SHP-1, p-STAT1, and STAT1. Notably, DJ-1 bound to SHP-1 independently of IFN-γ, whereas the interactions of DJ-1 with p-STAT1 and STAT1 were dependent on IFN-γ. Similar results were obtained in brain slice cultures, where IFN-γ induced much stronger STAT1 phosphorylation and inflammatory responses in KO slices than in WT slices. Moreover, IFN-γ treatment induced neuronal damage in KO slices. Collectively, these findings suggest that DJ-1 may function as a scaffold protein that facilitates SHP-1 interactions with p-STAT1 and STAT1, thereby preventing extensive and prolonged STAT1 activation. Thus, the loss of DJ-1 function may increase the risk of PD by enhancing brain inflammation.

PINK1 Deficiency Enhances Inflammatory Cytokine Release from Acutely Prepared Brain Slices.

Parkinson's disease (PD) is the second most common neurodegenerative motor disease caused by degeneration of dopaminergic neurons in the substantia nigra. Because brain inflammation has been considered a risk factor for PD, we analyzed whether PTEN induced putative kinase 1 (PINK1), an autosomal recessive familial PD gene, regulates brain inflammation during injury states. Using acutely prepared cortical slices to mimic injury, we analyzed expression of the pro-inflammatory cytokines tumor necrosis factor-α, interleukin (IL)-1ß, and IL-6 at the mRNA and protein levels. Both mRNA and protein expression of these cytokines was higher at 6-24 h after slicing in PINK1 knockout (KO) slices compared to that in wild-type (WT) slices. In serial experiments to understand the signaling pathways that increase inflammatory responses in KO slices, we found that IκB degradation was enhanced but Akt phosphorylation decreased in KO slices compared to those in WT slices. In further experiments, an inhibitor of PI3K (LY294002) upstream of Akt increased expression of pro-inflammatory cytokines. Taken together, these results suggest that PINK1 deficiency enhance brain inflammation through reduced Akt activation and enhanced IκB degradation in response to brain injury.

PINK1 deficiency attenuates astrocyte proliferation through mitochondrial dysfunction, reduced AKT and increased p38 MAPK activation, and downregulation of EGFR.

PINK1 (PTEN induced putative kinase 1), a familial Parkinson's disease (PD)-related gene, is expressed in astrocytes, but little is known about its role in this cell type. Here, we found that astrocytes cultured from PINK1-knockout (KO) mice exhibit defective proliferative responses to epidermal growth factor (EGF) and fetal bovine serum. In PINK1-KO astrocytes, basal and EGF-induced p38 activation (phosphorylation) were increased whereas EGF receptor (EGFR) expression and AKT activation were decreased. p38 inhibition (SB203580) or knockdown with small interfering RNA (siRNA) rescued EGFR expression and AKT activation in PINK1-KO astrocytes. Proliferation defects in PINK1-KO astrocytes appeared to be linked to mitochondrial defects, manifesting as decreased mitochondrial mass and membrane potential, increased intracellular reactive oxygen species level, decreased glucose-uptake capacity, and decreased ATP production. Mitochondrial toxin (oligomycin) and a glucose-uptake inhibitor (phloretin) mimicked the PINK1-deficiency phenotype, decreasing astrocyte proliferation, EGFR expression and AKT activation, and increasing p38 activation. In addition, the proliferation defect in PINK1-KO astrocytes resulted in a delay in the wound healing process. Taken together, these results suggest that PINK1 deficiency causes astrocytes dysfunction, which may contribute to the development of PD due to delayed astrocytes-mediated repair of microenvironment in the brain.

Inflammatory responses are not sufficient to cause delayed neuronal death in ATP-induced acute brain injury.

BACKGROUND: Brain inflammation is accompanied by brain injury. However, it is controversial whether inflammatory responses are harmful or beneficial to neurons. Because many studies have been performed using cultured microglia and neurons, it has not been possible to assess the influence of multiple cell types and diverse factors that dynamically and continuously change in vivo. Furthermore, behavior of microglia and other inflammatory cells could have been overlooked since most studies have focused on neuronal death. Therefore, it is essential to analyze the precise roles of microglia and brain inflammation in the injured brain, and determine their contribution to neuronal damage in vivo from the onset of injury. METHODS AND FINDINGS: Acute neuronal damage was induced by stereotaxic injection of ATP into the substantia nigra pars compacta (SNpc) and the cortex of the rat brain. Inflammatory responses and their effects on neuronal damage were investigated by immunohistochemistry, electron microscopy, quantitative RT-PCR, and stereological counting, etc. ATP acutely caused death of microglia as well as neurons in a similar area within 3 h. We defined as the core region the area where both TH(+) and Iba-1(+) cells acutely died, and as the penumbra the area surrounding the core where Iba-1(+) cells showed activated morphology. In the penumbra region, morphologically activated microglia arranged around the injury sites. Monocytes filled the damaged core after neurons and microglia died. Interestingly, neither activated microglia nor monocytes expressed iNOS, a major neurotoxic inflammatory mediator. Monocytes rather expressed CD68, a marker of phagocytic activity. Importantly, the total number of dopaminergic neurons in the SNpc at 3 h (∼80% of that in the contralateral side) did not decrease further at 7 d. Similarly, in the cortex, ATP-induced neuron-damage area detected at 3 h did not increase for up to 7 d. CONCLUSIONS: Different cellular components (microglia, astrocytes, monocytes, and neutrophils) and different factors (proinflammatory and neurotrophic) could be produced in inflammatory processes depending on the nature of the injury. The results in this study suggest that the inflammatory responses of microglia and monocytes in response to ATP-induced acute injury could not be neurotoxic.

Enhanced phosphatidylinositol 4-phosphate 5-kinase alpha expression and PI(4,5)P2 production in LPS-stimulated microglia.

Microglia are the major glial cells responsible for immune responses against harmful substances in the central nervous system. Type I phosphatidylinositol 4-phosphate 5-kinase alpha (PIP5Kalpha) and its lipid product, phosphatidylinositol 4,5-bisphosphate (PI[4,5]P(2)), regulate important cell surface functions. Here, we report that lipopolysaccharide (LPS) significantly enhanced PIP5Kalpha mRNA and protein expression levels in a time- and concentration-dependent manner in microglia. Furthermore, LPS stimulation led to a robust increase in PI(4,5)P(2) in the plasma membrane, demonstrated by PI(4,5)P(2) immunostaining or PI(4,5)P(2) imaging using a PI(4,5)P(2)-specific probe, tubby (R332H), fused to yellow fluorescent protein. Phosphatidylinositol 3-kinase, p38 mitogen-activated protein kinase (MAPK), p42/44 MAPK, and c-Jun N-terminal kinase signaling pathway inhibitors clearly reduced PIP5Kalpha expression, indicating that these pathways are necessary for LPS-induced PIP5Kalpha expression. In addition, inhibition of nuclear factor-kappaB and Sp1 transcription factors interfered with the LPS-induced upregulation of PIP5Kalpha. Delivery of PI(4,5)P(2) into microglia increased the expression of interleukin-1beta and tumor necrosis factor alpha. These findings indicate that PIP5Kalpha upregulation and the subsequent rise in PI(4,5)P(2) in LPS-stimulated microglia may positively regulate microglial inflammatory responses.

Dual-specificity tyrosine(Y)-phosphorylation regulated kinase 1A-mediated phosphorylation of amyloid precursor protein: evidence for a functional link between Down syndrome and Alzheimer's disease.

Most individuals with Down Syndrome (DS) show an early-onset of Alzheimer's disease (AD), which potentially results from the presence of an extra copy of a segment of chromosome 21. Located on chromosome 21 are the genes that encode beta-amyloid (Abeta) precursor protein (APP ), a key protein involved in the pathogenesis of AD, and dual-specificity tyrosine(Y)-phosphorylation regulated kinase 1A (DYRK1A ), a proline-directed protein kinase that plays a critical role in neurodevelopment. Here, we describe a potential mechanism for the regulation of AD pathology in DS brains by DYRK1A-mediated phosphorylation of APP. We show that APP is phosphorylated at Thr668 by DYRK1A in vitro and in mammalian cells. The amounts of phospho-APP and Abeta are increased in the brains of transgenic mice that over-express the human DYRK1A protein. Furthermore, we show that the amounts of phospho-APP as well as those of APP and DYRK1A are elevated in human DS brains. Taken together, these results reveal a potential regulatory link between APP and DYRK1A in DS brains, and suggest that the over-expression of DYRK1A in DS may play a role in accelerating AD pathogenesis through phosphorylation of APP.