Paul Lingor - Academia.edu (original) (raw)

Papers by Paul Lingor

Journal of visualized experiments : JoVE, 2012

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder resulting in progressiv... more Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder resulting in progressive degeneration of motoneurons. Peak of onset is around 60 years for the sporadic disease and around 50 years for the familial disease. Due to its progressive course, 50% of the patients die within 30 months of symptom onset. In order to evaluate novel treatment options for this disease, genetic mouse models of ALS have been generated based on human familial mutations in the SOD gene, such as the SOD1 (G93A) mutation. Most important aspects that have to be evaluated in the model are overall survival, clinical course and motor function. Here, we demonstrate the clinical evaluation, show the conduction of two behavioural motor tests and provide quantitative scoring systems for all parameters. Because an in depth analysis of the ALS mouse model usually requires an immunohistochemical examination of the spinal cord, we demonstrate its preparation in detail applying the dorsal laminectomy meth...

Frontiers in cellular neuroscience, 2014

The Rho/ROCK pathway is a promising therapeutic target in neurodegenerative and neurotraumatic di... more The Rho/ROCK pathway is a promising therapeutic target in neurodegenerative and neurotraumatic diseases. Pharmacological inhibition of various pathway members has been shown to promote neuronal regeneration and survival. However, because pharmacological inhibitors are inherently limited in their specificity, shRNA-mediated approaches can add more information on the function of each single kinase involved. Thus, we generated adeno-associated viral vectors (AAV) to specifically downregulate Ras homologous member A (RhoA) via shRNA. We found that specific knockdown of RhoA promoted neurite outgrowth of retinal ganglion cells (RGC) grown on the inhibitory substrate chondroitin sulfate proteoglycan (CSPG) as well as neurite regeneration of primary midbrain neurons (PMN) after scratch lesion. In the rat optic nerve crush (ONC) model in vivo, downregulation of RhoA significantly enhanced axonal regeneration compared to control. Moreover, survival of RGC transduced with AAV expressing RhoA-...

Proceedings of the National Academy of Sciences, 2010

Neurobiology of Disease, 2010

Malfunction of the ubiquitin-proteasome system has been implicated as a causal factor in the path... more Malfunction of the ubiquitin-proteasome system has been implicated as a causal factor in the pathogenesis of aggregation-related disorders, e.g. Parkinson's disease. We show here that Transforming growth factorbeta 1 (TGF-β), a multifunctional cytokine and trophic factor for dopaminergic (DAergic) neurons modulates proteasome function in primary midbrain neurons. TGF-β differentially inhibited proteasomal subactivities with a most pronounced time-dependent inhibition of the peptidyl-glutamyl peptide hydrolyzing-like and chymotrypsin-like subactivity. Regulation of proteasomal activity could be specifically quantified in the DAergic subpopulation. Protein blot analysis revealed an accumulation of ubiquitinated proteins after TGF-β treatment. The identity of these enriched proteins was further analyzed by 2D-gel electrophoresis and mass spectrometry. We found epidermal fatty acid binding protein (EFABP) to be strongly increased and ubiquitinated after TGF-β treatment and confirmed this finding by co-immunoprecipitation. While application of TGF-β increased neurite regeneration in a scratch lesion model, downregulation of EFABP by siRNA significantly decreased this effect. We thus postulate that a differential regulation of proteasomal function, as demonstrated for TGF-β, can result in an enrichment of proteins, such as EFABP, that mediate physiological functions, such as neurite regeneration.

Nature Protocols, 2011

The user has requested enhancement of the downloaded file. All in-text references underlined in b... more The user has requested enhancement of the downloaded file. All in-text references underlined in blue are added to the original document and are linked to publications on ResearchGate, letting you access and read them immediately.

Molecular and Cellular Neuroscience, 2009

A b s t r a c t CNS regeneration is limited by lesion-induced neuronal apoptosis and an environme... more A b s t r a c t CNS regeneration is limited by lesion-induced neuronal apoptosis and an environment inhibiting axonal elongation. Inhibition of ROCK has been previously shown to promote regeneration in retinal ganglion cells (RGC) whereas Cdk5 inhibition mainly promoted survival. Therefore, we have evaluated the effects of combined treatment with inhibitors of ROCK and Cdk5. We show that in vitro, the co-application of the Cdk5 inhibitor, Indolinone A, and the ROCK inhibitor, Y-27632, potentiated the survival-promoting effect of either substance alone. However, neurite outgrowth in vitro was promoted only by the presence of Y-27632, not by Indolinone A alone. In the ex vivo explant and the in vivo optic nerve crush model the combination of both inhibitors significantly increased neurite outgrowth at small distances, but this effect leveled off for longer neurites. In summary, the combined treatment with the Cdk5 inhibitor Indolinone A and the ROCK inhibitor Y-27632 results in a strong additive effect on neuronal survival, but is not able to increase the regenerative response beyond the effect of the ROCK inhibitor.

Journal of Neurochemistry, 2007

The anti-apoptotic Bcl-x L is a promising agent to prevent neurodegeneration in Parkinson's disea... more The anti-apoptotic Bcl-x L is a promising agent to prevent neurodegeneration in Parkinson's disease, which is characterized by a demise of dopaminergic neurons. We linked Bclx L to a peptide that allows its delivery across biological membranes and the blood-brain barrier. We tested the fusion protein in two models of Parkinson's Disease. Cell-permeable Bcl-x L protected neuroblastoma cells from the selective neurotoxin 1-methyl-4-phenylpyridinium. Furthermore, its systemic application in aged mice protected dopaminergic neurons following administration of MPTP as revealed by counting of tyrosine hydroxylase-immunoreactive neurons in the substantia nigra pars compacta. Hence, we present that a cell-permeable form of an anti-apoptotic protein can be delivered to CNS neurons through its systemic application, and we provide the proof that the delivery of this protein to the CNS neurons effectively prevents neuronal cell death in models of chronic neurodegenerative diseases.

Journal of Neurochemistry, 2006

We have recently shown that the hematopoietic Granulocyte-Colony Stimulating Factor (G-CSF) is ne... more We have recently shown that the hematopoietic Granulocyte-Colony Stimulating Factor (G-CSF) is neuroprotective in rodent stroke models, and that this action appears to be mediated via a neuronal G-CSF receptor. Here, we report that the G-CSF receptor is expressed in rodent dopaminergic substantia nigra neurons, suggesting that G-CSF might be neuroprotective for dopaminergic neurons and a candidate molecule for the treatment of Parkinson's disease. Thus, we investigated protective effects of G-CSF in 1-methyl-4-phenylpyridinium (MPP + )-challenged PC12 cells and primary neuronal midbrain cultures, as well as in the mouse 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of Parkinson's disease. Substantial protection was found against MPP + -induced dopaminergic cell death in vitro. Moreover, subcutaneous application of G-CSF at a dose of 40 lg/Kg body weight daily over 13 days rescued dopaminergic substantia nigra neurons from MPTP-induced death in aged mice, as shown by quantification of tyrosine hydroxylase-positive substantia nigra cells. Using HPLC, a corresponding reduction in striatal dopamine depletion after MPTP application was observed in G-CSF-treated mice. Thus our data suggest that G-CSF is a novel therapeutic opportunity for the treatment of Parkinson's disease, because it is well-tolerated and already approved for the treatment of neutropenic conditions in humans.

Journal of Molecular Neuroscience, 2011

Parkinson's disease is characterized by selective and progressive loss of midbrain DAergic neuron... more Parkinson's disease is characterized by selective and progressive loss of midbrain DAergic neurons (MDN) in the substantia nigra and degeneration of its nigrostriatal projections. Whereas the cellular pathophysiology has been closely linked to an activation of c-Jun N-terminal kinases (JNKs) and c-Jun, the involvement of JNKs in regenerative processes of the nigrostriatal pathway is controversially discussed. In our study, we utilized a mechanical scratch lesion paradigm of midbrain DAergic neurons in vitro and studied regenerative neuritic outgrowth. After a siRNAmediated knockdown of each of the three JNK isoforms, we found that JNKs differentially regulate neurite regeneration. Knockdown of JNK3 resulted in the most prominent neurite outgrowth impairment. This effect was attenuated again by plasmid overexpression of JNK3. We also evaluated cell survival of the affected neurons at the scratch border. JNK3 was found to be also relevant for survival of MDN which were lesioned by the scratch. Our data suggest that JNK isoforms are involved in differential regulation of cell death and regeneration in MDN depending on their neurite integrity. JNK3 appears to be required for regeneration and survival in the case of an environment permissive for regeneration. Future therapeutic approaches for the DAergic system may thus require isoform specific targeting of these kinases.

Cell Death and Differentiation, 2007

Under physiological conditions, mitochondrial morphology dynamically shifts between a punctuate a... more Under physiological conditions, mitochondrial morphology dynamically shifts between a punctuate appearance and tubular networks. However, little is known about upstream signal transduction pathways that regulate mitochondrial morphology. We show that mitochondrial fission is a very early and kinetically invariant event during neuronal cell death, which causally contributes to cytochrome c release and neuronal apoptosis. Using a small molecule CDK5 inhibitor, as well as a dominantnegative CDK5 mutant and RNAi knockdown experiments, we identified CDK5 as an upstream signalling kinase that regulates mitochondrial fission during apoptosis of neurons. Vice versa, our study shows that mitochondrial fission is a modulator contributing to CDK5-mediated neurotoxicity. Thereby, we provide a link that allows integration of CDK5 into established neuronal apoptosis pathways.

Cell and Tissue Research, 2012

Degeneration of the axon is an important step in the pathomechanism of traumatic, inflammatory an... more Degeneration of the axon is an important step in the pathomechanism of traumatic, inflammatory and degenerative neurological diseases. Increasing evidence suggests that axonal degeneration occurs early in the course of these diseases and therefore represents a promising target for future therapeutic strategies. We review the evidence for axonal destruction from pathological findings and animal models with particular emphasis on neurodegenerative and neurotraumatic disorders. We discuss the basic morphological and temporal modalities of axonal degeneration (acute, chronic and focal axonal degeneration and Wallerian degeneration). Based on the mechanistic concepts, we then delineate in detail the major molecular mechanisms that underlie the degenerative cascade, such as calcium influx, axonal transport, protein aggregation and autophagy. We finally concentrate on putative therapeutic targets based on the mechanistic prerequisites.

Annals of Neurology, 2009

Objective: The aim of this study was to investigate the role of voltage-dependent calcium channel... more Objective: The aim of this study was to investigate the role of voltage-dependent calcium channels (VDCCs) in axon degeneration during autoimmune optic neuritis. Methods: Calcium ion (Ca 2ϩ ) influx into the optic nerve (ON) through VDCCs was investigated in a rat model of optic neuritis using manganese-enhanced magnetic resonance imaging and in vivo calcium imaging. After having identified the most relevant channel subtype (N-type VDCCs), we correlated immunohistochemistry of channel expression with ON histopathology. In the confirmatory part of this work, we performed a treatment study using -conotoxin GVIA, an N-type specific blocker. Results: We observed that pathological Ca 2ϩ influx into ONs during optic neuritis is mediated via N-type VDCCs. By analyzing the expression of VDCCs in the inflamed ONs, we detected an upregulation of ␣ 1B , the pore-forming subunit of N-type VDCCs, in demyelinated axons. However, high expression levels were also found on macrophages/activated microglia, and lower levels were detected on astrocytes. The relevance of N-type VDCCs for inflammation-induced axonal degeneration and the severity of optic neuritis was corroborated by treatment with -conotoxin GVIA. This blocker led to decreased axon and myelin degeneration in the ONs together with a reduced number of macrophages/activated microglia. These protective effects were confirmed by analyzing the spinal cords of the same animals. Interpretation: We conclude that N-type VDCCs play an important role in inflammation-induced axon degeneration via two mechanisms: First, they directly mediate toxic Ca 2ϩ influx into the axons; and second, they contribute to macrophage/microglia function, thereby promoting secondary axonal damage.

Functional regeneration in the CNS is limited by lesion-induced neuronal apoptosis and an environ... more Functional regeneration in the CNS is limited by lesion-induced neuronal apoptosis and an environment inhibiting axonal elongation. A principal, yet unresolved question is the interaction between these two major factors. We thus evaluated the role of pharmacological inhibition of rho kinase (ROCK), a key mediator of myelinderived axonal growth inhibition and CNTF, a potent neurotrophic factor for retinal ganglion cells (RGC), in models of retinal ganglion cell apoptosis and neurite outgrowth/regeneration in vitro and in vivo. Here, we show for the first time that the ROCK inhibitor Y-27632 significantly enhanced survival of RGC in vitro and in vivo. In vitro, the co-application of CNTF and Y-27632 potentiated the effect of either substance alone. ROCK inhibition resulted in the activation of the intrinsic MAPK pathway, and the combination of CNTF and Y-27632 resulted in even more pronounced MAPK activation. While CNTF also induced STAT3 phosphorylation, the additional application of ROCK inhibitor surprisingly diminished the effects of CNTF on STAT3 phosphorylation. ROCK activity was also decreased in an additive manner by both substances. In vivo, both CNTF and Y-27632 enhanced regeneration of RGC into the non-permissive optic nerve crush model and additive effects were observed after combination treatment. Further evaluation using specific inhibitors delineate STAT3 as a negative regulator of neurite growth and positive regulator of cell survival, while MAPK and Akt support neurite growth. These results show that next to neurotrophic factors ROCK inhibition by Y-27632 potently supports survival of lesioned adult CNS neurons. Co-administration of CNTF and Y-27632 results in additive effects on neurite outgrowth and regeneration. The interaction of intracellular signalling pathways may, however, attenuate more pronounced synergy and has to be taken into account for future treatment strategies.

Repressor element-1 silencing transcription factor (REST) is a transcriptional repressor of neuro... more Repressor element-1 silencing transcription factor (REST) is a transcriptional repressor of neuron-specific genes that binds to a conserved DNA element, the neuron restrictive silencer element (NRSE ⁄ RE1). Interestingly, increased REST activity is found in several neurological diseases like Huntington's disease and cerebral ischemia. Recently, it was shown that NRSE dsRNA, a double-stranded non-coding RNA can bind to REST during a defined period of neuronal differentiation, and thereby changes REST from a transcriptional repressor to an activator of neuron-specific genes. Here, we analyzed the effects of NRSE dsRNA expression in primary retinal ganglion cells. We found that NRSE dsRNA expression vectors significantly enhance neurite outgrowth even when axonal degeneration is induced by neurotrophin deprivation. Transfection of HEK cells with NRSE dsRNAexpressing vectors altered their morphology leading to the formation of thin processes and induced the expression of neurofilament-68. Surprisingly, control vectors containing REST-binding sites, but not expressing NRSE dsRNA, resulted in the same effects, also in the retinal ganglion cell model. Reporter assays and retention of REST in the cytoplasm with a labeled NRSE ⁄ RE1-containing plasmid incapable of entering the nucleus suggest that sequestration of REST in the cytoplasm is the reason for the observed effects. No evidence for a biological function of NRSE dsRNA could be found in these models. We conclude that sequestration of REST leads to enhanced neurite outgrowth in retinal ganglion cells and that an increased activity of REST, as it is found in several neurodegenerative diseases, can be effectively modulated by sequestration of REST with plasmids containing NRSE ⁄ RE1 sites.

Cell death & disease, 2014

The Rho/ROCK/LIMK pathway is central for the mediation of repulsive environmental signals in the ... more The Rho/ROCK/LIMK pathway is central for the mediation of repulsive environmental signals in the central nervous system. Several studies using pharmacological Rho-associated protein kinase (ROCK) inhibitors have shown positive effects on neurite regeneration and suggest additional pro-survival effects in neurons. However, as none of these drugs is completely target specific, it remains unclear how these effects are mediated and whether ROCK is really the most relevant target of the pathway. To answer these questions, we generated adeno-associated viral vectors to specifically downregulate ROCK2 and LIM domain kinase (LIMK)-1 in rat retinal ganglion cells (RGCs) in vitro and in vivo. We show here that specific knockdown of ROCK2 and LIMK1 equally enhanced neurite outgrowth of RGCs on inhibitory substrates and both induced substantial neuronal regeneration over distances of more than 5 mm after rat optic nerve crush (ONC) in vivo. However, only knockdown of ROCK2 but not LIMK1 increas...

Frontiers in neuroscience, 2014

Disease progression in amyotrophic lateral sclerosis (ALS) is characterized by degeneration of mo... more Disease progression in amyotrophic lateral sclerosis (ALS) is characterized by degeneration of motoneurons and their axons which results in a progressive muscle weakness and ultimately death from respiratory failure. The only approved drug, riluzole, lacks clinical efficacy so that more potent treatment options are needed. We have identified rho kinase (ROCK) as a target, which can be manipulated to beneficially influence disease progression in models of ALS. Here, we examined the therapeutic potential of the ROCK inhibitor Y-27632 in both an in vitro and in an in vivo paradigm of motoneuron disease. Application of Y-27632 to primary motoneurons in vitro increased survival and promoted neurite outgrowth. In vivo, SOD1(G93A) mice were orally treated with 2 or 30 mg/kg body weight of Y-27632. The 2 mg/kg group did not benefit from Y-27632 treatment, whereas treatment with 30 mg/kg resulted in improved motor function in male mice. Female mice showed only limited improvement and overall...

Frontiers in Aging Neuroscience, 2014

FEBS Journal, 2011