Role of PKN1 in Retinal Cell Type Formation.

We recently identified PKN1 as a developmentally active gatekeeper of the transcription factor neuronal differentiation-2 (NeuroD2) in several brain areas. Since NeuroD2 plays an important role in amacrine cell (AC) and retinal ganglion cell (RGC) type formation, we aimed to study the expression of NeuroD2 in the postnatal retina of WT and Pkn1-/- animals, with a particular focus on these two cell types. We show that PKN1 is broadly expressed in the retina and that the gross retinal structure is not different between both genotypes. Postnatal retinal NeuroD2 levels were elevated upon Pkn1 knockout, with Pkn1-/- retinae showing more NeuroD2+ cells in the lower portion of the inner nuclear layer. Accordingly, immunohistochemical analysis revealed an increased amount of AC in postnatal and adult Pkn1-/- retinae. There were no differences in horizontal cell, bipolar cell, glial cell and RGC numbers, nor defective axon guidance to the optic chiasm or tract upon Pkn1 knockout. Interestingly, we did, however, see a specific reduction in SMI-32+ α-RGC in Pkn1-/- retinae. These results suggest that PKN1 is important for retinal cell type formation and validate PKN1 for future studies focusing on AC and α-RGC specification and development.

PKN1 Exerts Neurodegenerative Effects in an In Vitro Model of Cerebellar Hypoxic-Ischemic Encephalopathy via Inhibition of AKT/GSK3ß Signaling.

We recently identified protein kinase N1 (PKN1) as a negative gatekeeper of neuronal AKT protein kinase activity during postnatal cerebellar development. The developing cerebellum is specifically vulnerable to hypoxia-ischemia (HI), as it occurs during hypoxic-ischemic encephalopathy, a condition typically caused by oxygen deprivation during or shortly after birth. In that context, activation of the AKT cell survival pathway has emerged as a promising new target for neuroprotective interventions. Here, we investigated the role of PKN1 in an in vitro model of HI, using postnatal cerebellar granule cells (Cgc) derived from Pkn1 wildtype and Pkn1-/- mice. Pkn1-/- Cgc showed significantly higher AKT phosphorylation, resulting in reduced caspase-3 activation and improved survival after HI. Pkn1-/- Cgc also showed enhanced axonal outgrowth on growth-inhibitory glial scar substrates, further pointing towards a protective phenotype of Pkn1 knockout after HI. The specific PKN1 phosphorylation site S374 was functionally relevant for the enhanced axonal outgrowth and AKT interaction. Additionally, PKN1pS374 shows a steep decrease during cerebellar development. In summary, we demonstrate the pathological relevance of the PKN1-AKT interaction in an in vitro HI model and establish the relevant PKN1 phosphorylation sites, contributing important information towards the development of specific PKN1 inhibitors.

Single nucleus RNA sequencing reveals glial cell type-specific responses to ischemic stroke.

Reactive neuroglia critically shape the brains response to ischemic stroke. However, their phenotypic heterogeneity impedes a holistic understanding of the cellular composition and microenvironment of the early ischemic lesion. Here we generated a single cell resolution transcriptomics dataset of the injured brain during the acute recovery from permanent middle cerebral artery occlusion. This approach unveiled infarction and subtype specific molecular signatures in oligodendrocyte lineage cells and astrocytes, which ranged among the most transcriptionally perturbed cell types in our dataset. Specifically, we characterized and compared infarction restricted proliferating oligodendrocyte precursor cells (OPCs), mature oligodendrocytes and heterogeneous reactive astrocyte populations. Our analyses unveiled unexpected commonalities in the transcriptional response of oligodendrocyte lineage cells and astrocytes to ischemic injury. Moreover, OPCs and reactive astrocytes were involved in a shared immuno-glial cross talk with stroke specific myeloid cells. In situ, osteopontin positive myeloid cells accumulated in close proximity to proliferating OPCs and reactive astrocytes, which expressed the osteopontin receptor CD44, within the perilesional zone specifically. In vitro, osteopontin increased the migratory capacity of OPCs. Collectively, our study highlights molecular cross talk events which might govern the cellular composition and microenvironment of infarcted brain tissue in the early stages of recovery.

PKN1 Is a Novel Regulator of Hippocampal GluA1 Levels.

Alterations in the processes that control α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) expression, assembly and trafficking are closely linked to psychiatric and neurodegenerative disorders. We have recently shown that the serine/threonine kinase Protein kinase N1 (PKN1) is a developmentally active regulator of cerebellar synaptic maturation by inhibiting AKT and the neurogenic transcription factor neurogenic differentiation factor-2 (NeuroD2). NeuroD2 is involved in glutamatergic synaptic maturation by regulating expression levels of various synaptic proteins. Here we aimed to study the effect of Pkn1 knockout on AKT phosphorylation and NeuroD2 levels in the hippocampus and the subsequent expression levels of the NeuroD2 targets and AMPAR subunits: glutamate receptor 1 (GluA1) and GluA2/3. We show that PKN1 is expressed throughout the hippocampus. Interestingly, not only postnatal but also adult hippocampal phospho-AKT and NeuroD2 levels were significantly elevated upon Pkn1 knockout. Postnatal and adult Pkn1 -/- hippocampi showed enhanced expression of the AMPAR subunit GluA1, particularly in area CA1. Surprisingly, GluA2/3 levels were not different between both genotypes. In addition to higher protein levels, we also found an enhanced GluA1 content in the membrane fraction of postnatal and adult Pkn1 -/- animals, while GluA2/3 levels remained unchanged. This points toward a very specific regulation of GluA1 expression and/or trafficking by the novel PKN1-AKT-NeuroD2 axis. Considering the important role of GluA1 in hippocampal development as well as the pathophysiology of several disorders, ranging from Alzheimer's, to depression and schizophrenia, our results validate PKN1 for future studies into neurological disorders related to altered AMPAR subunit expression in the hippocampus.

Protein kinase N1 critically regulates cerebellar development and long-term function.

Increasing evidence suggests that synapse dysfunctions are a major determinant of several neurodevelopmental and neurodegenerative diseases. Here we identify protein kinase N1 (PKN1) as a novel key player in fine-tuning the balance between axonal outgrowth and presynaptic differentiation in the parallel fiber-forming (PF-forming) cerebellar granule cells (Cgcs). Postnatal Pkn1-/- animals showed a defective PF-Purkinje cell (PF-PC) synapse formation. In vitro, Pkn1-/- Cgcs exhibited deregulated axonal outgrowth, elevated AKT phosphorylation, and higher levels of neuronal differentiation-2 (NeuroD2), a transcription factor preventing presynaptic maturation. Concomitantly, Pkn1-/- Cgcs had a reduced density of presynaptic sites. By inhibiting AKT with MK-2206 and siRNA-mediated knockdown, we found that AKT hyperactivation is responsible for the elongated axons, higher NeuroD2 levels, and reduced density of presynaptic specifications in Pkn1-/- Cgcs. In line with our in vitro data, Pkn1-/- mice showed AKT hyperactivation, elevated NeuroD2 levels, and reduced expression of PF-PC synaptic markers during stages of PF maturation in vivo. The long-term effect of Pkn1 knockout was further seen in cerebellar atrophy and mild ataxia. In summary, our results demonstrate that PKN1 functions as a developmentally active gatekeeper of AKT activity, thereby fine-tuning axonal outgrowth and presynaptic differentiation of Cgcs and subsequently the correct PF-PC synapse formation.

Protein Kinase C-Related Kinase (PKN/PRK). Potential Key-Role for PKN1 in Protection of Hypoxic Neurons.

Serine/threonine protein kinase C-related kinase (PKN/PRK) is a family of three isoenzymes (PKN1, PKN2, PKN3), which are widely distributed in eukaryotic organisms and share the same overall domain structure. The Nterminal region encompasses a conserved repeated domain, termed HR1a-c as well as a HR2/C2 domain. The serine/threonine kinase domain is found in the C-terminal region of the protein and shows high sequence homology to other members of the PKC superfamily. In neurons, PKN1 is the most abundant isoform and has been implicated in a variety of functions including cytoskeletal organization and neuronal differentiation and its deregulation may contribute to neuropathological processes such as amyotrophic lateral sclerosis and Alzheimer's disease. We have recently identified a candidate role of PKN1 in the regulation of neuroprotective processes during hypoxic stress. Our key findings were that: 1) the activity of PKN1 was significantly increased by hypoxia (1% O2) and neurotrophins (nerve growth factor and purine nucleosides); 2) Neuronal cells, deficient of PKN1 showed a decrease of cell viability and neurite formation along with a disturbance of the F-actinassociated cytoskeleton; 3) Purine nucleoside-mediated neuroprotection during hypoxia was severely hampered in PKN1 deficient neuronal cells, altogether suggesting a potentially critical role of PKN1 in neuroprotective processes. This review gives an up-to-date overview of the PKN family with a special focus on the neuroprotective role of PKN1 in hypoxia.

LAMTOR2-mediated modulation of NGF/MAPK activation kinetics during differentiation of PC12 cells.

LAMTOR2 (p14), a part of the larger LAMTOR/Ragulator complex, plays a crucial role in EGF-dependent activation of p42/44 mitogen-activated protein kinases (MAPK, ERK1/2). In this study, we investigated the role of LAMTOR2 in nerve growth factor (NGF)-mediated neuronal differentiation. Stimulation of PC12 (rat adrenal pheochromocytoma) cells with NGF is known to activate the MAPK. Pharmacological inhibition of MEK1 as well as siRNA-mediated knockdown of both p42 and p44 MAPK resulted in inhibition of neurite outgrowth. Contrary to expectations, siRNA-mediated knockdown of LAMTOR2 effectively augmented neurite formation and neurite length of PC12 cells. Ectopic expression of a siRNA-resistant LAMTOR2 ortholog reversed this phenotype back to wildtype levels, ruling out nonspecific off-target effects of this LAMTOR2 siRNA approach. Mechanistically, LAMTOR2 siRNA treatment significantly enhanced NGF-dependent MAPK activity, and this effect again was reversed upon expression of the siRNA-resistant LAMTOR2 ortholog. Studies of intracellular trafficking of the NGF receptor TrkA revealed a rapid colocalization with early endosomes, which was modulated by LAMTOR2 siRNA. Inhibition of LAMTOR2 and concomitant destabilization of the remaining members of the LAMTOR complex apparently leads to a faster release of the TrkA/MAPK signaling module and nuclear increase of activated MAPK. These results suggest a modulatory role of the MEK1 adapter protein LAMTOR2 in NGF-mediated MAPK activation required for induction of neurite outgrowth in PC12 cells.

Purine nucleosides: endogenous neuroprotectants in hypoxic brain.

Even a short blockade of oxygen flow in brain may lead to the inhibition of oxidative phosphorylation and depletion of cellular ATP, which results in profound deficiencies in cellular function. Following ischemia, dying, injured, and hypoxic cells release soluble purine-nucleotide and -nucleoside pools. Growing evidence suggests that purine nucleosides might act as trophic factors in the CNS and PNS. In addition to equilibrative nucleoside transporters (ENTs) regulating purine nucleoside concentrations intra- and extracellularly, specific extracellular receptor subtypes for these compounds are expressed on neurons, glia, and endothelial cells, mediating stunningly diverse effects. Such effects range from induction of cell differentiation, apoptosis, mitogenesis, and morphogenetic changes, to stimulation of synthesis and/or release of cytokines and neurotrophic factors under both physiological and pathological conditions. Multiple signaling pathways regulate the critical balance between cell death and survival in hypoxia-ischemia. A convergent pathway for the regulation of multiple modalities involved in O2 sensing is the mitogen activated protein kinase (p42/44 MAPK) or (ERK1/2 extracellular signal-regulated kinases) pathway terminating in a variety of transcription factors, for example, hypoxia-inducible factor 1α. In this review, the coherence of purine nucleoside-related pathways and MAPK activation in the endogenous neuroprotective regulation of the nervous system's development and neuroplasticity under hypoxic stress will be discussed.

Vital role of protein kinase C-related kinase in the formation and stability of neurites during hypoxia.

Exposure of pheochromocytoma cells to hypoxia (1% O(2)) favors differentiation at the expense of cell viability. Additional incubation with nerve growth factor (NGF) and guanosine, a purine nucleoside with neurotrophin characteristics, rescued cell viability and further enhanced the extension of neurites. In parallel, an increase in the activity of protein kinase C-related kinase (PRK1), which is known to be involved in regulation of the actin cytoskeleton, was observed in hypoxic cells. NGF and guanosine further enhanced PRK1 in normoxic and hypoxic cells. To study the role of PRK1 during cellular stress response and neurotrophin-mediated signaling, pheochromocytoma cells were transfected with small interfering RNA directed against PRK1. Loss of functional PRK1 initiated a significant loss of viability and inhibited neurite formation. SiRNA-mediated knockdown of PRK1 also completely stalled guanosine-mediated neuroprotective effects. Additionally, the F-actin-associated cytoskeleton and the expression of the plasticity protein growth associated protein-43 were disturbed upon PRK1 knockdown. A comparable dependency of neurite formation and growth associated protein-43 immunoreactivity on functional PRK1 expression was observed in cerebellar granule neurons. Based on these data, a putative role of PRK1 as a key-signaling element for the successive NGF- and purine nucleoside-mediated protection of hypoxic neuronal cells is hypothesized.

p42/44 MAPK is an essential effector for purine nucleoside-mediated neuroprotection of hypoxic PC12 cells and primary cerebellar granule neurons.

Purine nucleosides protect neurons against hypoxic insult, but the signaling mechanisms have not yet been fully elucidated. We studied the role of the p42/44 MAPK pathway in purine nucleoside-mediated protection of cultured PC12 cells and primary cerebellar granule neurons from hypoxia-induced cell death. Incubation with adenosine reduced hypoxia-evoked cell death morphology, and increased the activity of the MAPK pathway. Inosine, a metabolic derivative of adenosine was generally less potent in PC12 cells. Pharmacological inhibition of the MAPK pathways severely hampered adenosine-mediated induction of cell viability and neurite outgrowth. Consistently, siRNA-mediated knockdown of p42 and p44 MAPK completely blocked adenosine-mediated rescue of hypoxic PC12 cells. The role of MAPK activation was further studied in primary neurons. Cells were significantly rescued by adenosine and inosine and siRNA-mediated knockdown severely affected purine-mediated rescue of neuronal viability after hypoxic insult. Results point to the important role of p42/44 MAPK for adenosine receptor-mediated neuroprotection.

HIF-1 alpha is an essential effector for purine nucleoside-mediated neuroprotection against hypoxia in PC12 cells and primary cerebellar granule neurons.

Hypoxia-inducible factor-1 alpha (HIF-1alpha) and purine nucleosides adenosine and inosine are critical mediators of physiological responses to acute and chronic hypoxia. The specific aim of this paper was to evaluate the potential role of HIF-1alpha in purine-mediated neuroprotection. We show that adenosine and inosine efficiently rescued clonal rat pheochromocytoma (PC12) cells (up to 43.6%) as well as primary cerebellar granule neurons (up to 25.1%) from hypoxic insult, and furthermore, that HIF-1alpha is critical for purine-mediated neuroprotection. Next, we studied hypoxia or purine nucleoside increased nuclear accumulation of HIF-1alpha in PC12 cells. As a possible result of increased protein stabilization or synthesis an up to 2.5-fold induction of HIF-1alpha accumulation was detected. In cerebellar granule neurons, purine nucleosides induced an up to 3.1-fold HIF-1alpha accumulation in cell lysates. Concomitant with these results, small interfering RNA-mediated reduction of HIF-1alpha completely abolished adenosine- and inosine-mediated protection in PC12 cells and severely hampered purine nucleoside-mediated protection in primary neurons (up to 94.2%). Data presented in this paper thus clearly demonstrate that HIF-1alpha is a key regulator of purine nucleoside-mediated rescue of hypoxic neuronal cells.

Purine nucleoside-mediated protection of chemical hypoxia-induced neuronal injuries involves p42/44 MAPK activation.

Hypoxia in brain may lead to cell death by apoptosis and necrosis. Concomitant is the formation of purine nucleosides, e.g. adenosine, a powerful endogenous neuroprotectant. Despite vigorous studies, many aspects of the mechanisms involved in purine-based protection are still unclear. In this study, we wanted to investigate the effect of purine nucleosides on cellular responses to chemical hypoxia. O(2)-sensitive neuronal pheochromocytoma (PC12)-cells, which are widely used as a model system for sympathetic ganglion-like neurons, were subjected to chemical hypoxia induced with rotenone, an inhibitor of mitochondrial complex I. Adenosine and its relatives guanosine and inosine were tested for their neuroprotective capability to improve neurite outgrowth and viability. In addition, cell lysates were analyzed for mitogen-activated-protein-kinases (MAPK) activation by anti-active and anti-total MAPKinase immunoblotting. Adenosine, guanosine and inosine significantly inhibited the loss of viability after hypoxic insult. In combination with NGF, purine nucleosides also partially rescued neurite outgrowth. The MEK-1/-2 inhibitor PD098059 inhibited purine nucleoside-mediated protection up to 85.23% and also markedly decreased neurite formation induced by NGF and purine nucleosides in hypoxic cells. Immunoblot analysis revealed a strong activation of MAPKinase upon incubation of cells with adenosine, guanosine or inosine. In combination with NGF an additive effect was observed. Results suggested that activation of the MAPKinase pathway plays a vital role in purine nucleoside-mediated protection of neuronal cells following hypoxic insult.

Purine nucleosides support the neurite outgrowth of primary rat cerebellar granule cells after hypoxia.

Mammalian neurons require a constant supply of oxygen to maintain adequate cellular functions and survival. Following sustained hypoxia during ischemic events in brain, the energy status of neurons and glia is compromised, which may subsequently lead to cell death by apoptosis and necrosis. Concomitant with energy depletion is the formation of the purine nucleoside adenosine, a powerful endogenous neuroprotectant. In this paper the effect of chemical hypoxia on cell survival and neurite outgrowth of primary cerebellar granule cells was investigated. Rotenone, a mitochondrial complex I inhibitor, induced a 30.4 +/- 3.6% loss of viable cells and a 35.0 +/- 4.4% loss of neurite formation of cerebellar granule cells, which was partially restored by the addition of purine nucleosides adenosine, inosine and guanosine. Inosine had the most striking effect of 37.7 +/- 2.9% rescue of viability and 71.2 +/- 18.4% rescue of neurite outgrowth. Data confirm the suggested role of purine nucleosides for the neuronal regeneration of primary brain cells following hypoxic insult.

Evidence of functional modulation of the MEKK/JNK/cJun signaling cascade by the low density lipoprotein receptor-related protein (LRP).

Lipoprotein receptors, such as LRP, have been shown to assemble multiprotein complexes containing intracellular signaling molecules; however, in vivo, their signaling function is poorly understood. Using a novel LRP receptor fusion construct, a type I transmembrane protein chimera, termed sIgG-LRP (bearing the intracellular COOH-terminal tail of human LRP as recombinant fusion to a transmembrane region plus the extracellular IgG-F(c) domain), we here investigated LRP signal transduction specificity in intact cells. First and similar to activated alpha2-macroglobulin as agonist of endogenous LRP, expression of sIgG-LRP demonstrated significant apoptosis protection. Second and similar to alpha2-macroglobulin-induced endogenous LRP, sIgG-LRP is sufficient to negatively modulate mitogen-induced Elk-1 and cJun (but not NF-kappaB) transcriptional activity. Third, expression of sIgG-LRP also impaired cJun transactivation mediated by constitutive active mutants of Rac-1 and MEKK-1. Fourth and unexpectedly, sIgG-LRP expression was found to be associated with a marked enhancement of mitogen-induced cJun amino-terminal kinase (JNK) activation. Fifth, confocal microscopic examination and subcellular fractionation demonstrated that sIgG-LRP and JNK co-localize in transfected cells. Therefore, sIgG-LRP expression was found to significantly impair activation-induced translocation of JNK into the nucleus. Taken together, we here demonstrate that sIgG-LRP protein sequesters activated JNK into the plasma membrane compartment in intact cells, inhibiting nuclear activation of the JNK-dependent transcription factors Elk-1 and cJun.

7,8-Dihydroneopterin induces apoptosis of Jurkat T-lymphocytes via a Bcl-2-sensitive pathway.

Activated cell-mediated immunity is known to be accompanied by elevated concentrations of 7,8-dihydroneopterin which in high concentrations was found to interfere with the oxidant-antioxidant balance. In this study we investigated whether 7,8-dihydroneopterin mediates apoptosis of Jurkat T-lymphocytes via a CrmA- or Bcl-2-sensitive pathway. Transient transfection assays with CrmA and Bcl-2 expression constructs showed that apoptosis was not affected by CrmA whereas it was significantly decreased upon cotransfection with Bcl-2 constructs. Results suggest that 7,8-dihydroneopterin-induced apoptosis of T-lymphocytes is mediated by a Bcl-2-sensitive pathway.

Reduced pteridine derivatives induce apoptosis in PC12 cells.

In cerebrospinal fluid of patients with cerebral infections, elevated concentrations of the pteridine compounds neopterin and 7,8-dihydroneopterin were detected. Here, the potential of pteridines to induce apoptosis of the rat pheochromocytoma cells (PC12) was investigated. In contrast to aromatic pteridines like neopterin, the reduced forms 7,8-dihydroneopterin, 5,6,7,8-tetrahydrobiopterin and 7,8-dihydrobiopterin led to a significant increase of apoptotic cells. After terminal differentiation, cells were less sensitive to incubation with pteridines. A noticeable augmentation of apoptosis was observed upon incubation with 7,8-dihydroneopterin and 7,8-dihydrofolic acid. Antioxidants partly protected PC12 cells from pteridine-induced apoptosis, suggesting the involvement of reactive oxygen intermediates. Exposure of cells to 7,8-dihydroneopterin led to activation of the mitogen-activated protein (MAP) kinase and to a lesser degree also of JUN/SAP kinase. Results implicate that high concentrations of reduced pteridines, might contribute to the pathogenesis involved in neurodegeneration.