Thomas Herdegen

Selective expression of Jun proteins following axotomy and axonal transport block in peripheral nerves in the rat : evidence for a role in the regeneration process

Expression of the protein products of the immediate-early genes (IEGs), members of the fos, jun and krox families (Jun, Fos, and Krox, resp.) was investigated in the spinal cord and sensory ganglia (DRG) of normal rats; and following transection of, block of axonal transport in, or electrical stimulation of their peripheral axons. The nuclei of many moto- and DRG neurons showed a faint basal immunoreactivity (IR) for Jun proteins, but not for Fos or Krox proteins. There was a strong and selective induction of Jun-IR in moto- and DRG neurons after peripheral nerve transection or crush, or colchicine- or vinblastine-induced block of axonal transport. The Jun-IR induced by nerve transection disappeared after nerve regeneration. In contrast, Jun, Fos and Krox proteins were all induced transynaptically in spinal dorsal horn neurons following electrical stimulation of the C-fibers in the afferent nerves. Thus in differentiated neurons in vivo these IEG proteins can be expressed either independently or concomitantly depending on the type of stimulus.

The transcription factors c-JUN, JUN D and CREB, but not FOS and KROX-24, are differentially regulated in axotomized neurons following transection of rat sciatic nerve

In adult rats, expression of c-JUN, JUN B, JUN D, c-FOS, FOS B, KROX-24 and CREB proteins was investigated by immunocytochemistry in L4 and L5 dorsal root ganglia and lumbar spinal cord for up to 300 days following transection of the left sciatic nerve. In dorsal root ganglia, expressions of c-JUN and JUN D were increased 10 h and 15 h after sciatic nerve transection, respectively. c-JUN was still at an elevated level after 300 days predominantly in small diameter neurons, whereas JUN D had declined to control levels after 100 days. In contrast to the JUN proteins, expression of CREB showed a delayed onset after 10 days and reached a maximum between 70 and 150 days. In motoneurons, expression of c-JUN and JUN D was increased 15 h and 25 h after sciatic nerve transection, respectively. Expression of c-JUN remained increased after 150 days, whereas JUN D had declined to control levels after 70 days. In contrast, expression of CREB declined within 30 h in axotomized motoneurons and remained on a reduced level for up to 150 days. JUN B, c-FOS, FOS B and KROX-24 were not induced either following axotomy or following a repeated nerve crush. Sciatic nerve transection including the surgical procedure transynaptically provoked a transient expression of all JUN, FOS and KROX-24 proteins in neurons of spinal dorsal horn which disappeared after 5 days except the expression of JUN D which lasted for up to 20 days. In contrast, CREB immunoreactivity was not at all altered in neurons of spinal dorsal horn. In untreated animals, CREB and to a lesser extent JUN D showed an ubiquitous expression in neurons and glia cells of spinal cord, whereas expression of c-JUN and a weak expression of FOS B were restricted to motoneurons. In neurons of the dorsal root ganglia, a basal expression was found for c-JUN, JUN D and CREB and, at a low level, for FOS B and KROX-24. c-JUN and JUN D were colocalized with CREB in many cells such as interneurons, motoneurons, dorsal root ganglion cells and glial cells indicating the possibility for both the control of c-jun and jun D expression by CREB and the competition of JUN and CREB proteins for CRE consensus sequences.

c-Jun N-terminal kinases (JNKs) mediate pro-inflammatory actions of microglia.

The activation and function of c‐Jun N‐terminal kinases (JNKs) were investigated in primary microglia cultures from neonatal rat brain, which express all three JNK isoforms. Lipopolysaccharide (LPS), tumor necrosis factor‐α (TNF‐α), and thrombin preparations induced a rapid and lasting activation of JNKs in the cytoplasm. In the nucleus, the activation patterns were rather complex. In untreated microglia, the small pool of nuclear JNKs was strongly activated, while the high‐affinity JNK substrate c‐Jun was only weakly phosphorylated. Stimulation with LPS increased the total amount of nuclear JNKs and the phosphorylation of the transcription factor c‐Jun. Levels of activated JNKs in the nucleus, however, rapidly decreased. Analysis of the nuclear JNK isoforms revealed that the amount of JNK1 declined, while JNK2 increased, and the weakly expressed JNK3 did not vary. This observation suggests that JNK2 is mainly responsible for the activation of c‐Jun in this context. Upstream of JNKs, LPS induced a lasting activation of the constitutively present JNK kinase MKK4. The function of JNKs in LPS‐triggered cellular reactions was investigated using SP600125 (0.5–5 μM), a direct inhibitor of JNKs. Inhibition of JNKs reduced the LPS‐induced metabolic activity and induction of the AP‐1 target genes cyclooxygenase‐2 (Cox‐2), TNF‐α, monocyte chemoattractant protein‐1 (MCP‐1), and interleukin‐6 (IL‐6) in response to LPS, while ERK1/2 and p38α had a more pronounced effect on LPS‐induced cellular enlargement than JNKs. In summary, JNKs are essential mediators of relevant pro‐inflammatory functions in microglia with different contributions of the JNK isoforms.

Block of c-Fos and JunB Expression by Antisense Oligonucleotides Inhibits Light-induced-Phase Shifts of the Mammalian Circadian Clock

Light‐induced phase shifts of circadian rhythmic locomotor activity are associated with the expression of c‐Jun, JunB, c‐Fos and FosB transcription factors in the rat suprachiasmatic nucleus, as shown in the present study. In order to explore the importance of c‐Fos and JunB, the predominantly expressed AP‐1 proteins for the phase‐shifting effects of light, we blocked the expression of c‐Fos and JunB in the suprachiasmatic nucleus of male rats, housed under constant darkness, by intracerebroventricular application of 2 μ1 of 1 mM antisense phosphorothioate oligodeoxynucleotides (ASO) specifically directed against c‐fos and JunB mRNA. A light pulse (300 lux for 1 h) at circadian time 15 induced a significant phase shift (by 125 ± 15 min) of the circadian locomotor activity rhythm, whereas application of AS0 6 h before the light pulse completely prevented this phase shift. Application of control nonsense oligodeoxynucleotides had no effect. ASO strongly reduced the light‐induced expression of c‐Fos and JunB proteins. In contrast, light pulses with or without the control nonsense oligodeoxynucleotides evoked strong nuclear c‐Fos and JunB immunoreactivity in the rat suprachiasmatic nucleus. These results demonstrate for the first time that inducible transcription factors such as c‐Fos and JunB are an essential part of fundamental biological processes in the adult mammalian nervous system, e.g. of light‐induced phase shifts of the circadian pacemaker.

Specific pathophysiological functions of JNK isoforms in the brain

We have investigated the effect of JNK1 ko, JNK2 ko, JNK3 ko, JNK2+3 ko and c‐JunAA mutation on neuronal survival in adult transgenic mice following ischemia, 6‐hydroxydopamine induced neurotoxicity, axon transection and kainic acid induced excitotoxicity. Deletion of JNK isoforms indicated the compartment‐specific expression of JNK isoforms with 46‐kDa JNK1 as the main phosphorylated JNK isoform. Permanent occlusion of the MCA significantly enlarged the infarct area in JNK1 ko, which showed an increased expression of JNK3 in the penumbra. Survival of dopaminergic neurons in the substantia nigra compacta (SNC) following intrastriatal injection of 6‐hydroxydopamine was transiently improved in JNK3 ko and c‐JunAA mice after 7 days, but not 60 days. Following transection of the medial forebrain bundle, however, JNK3 ko conferred persisting neuroprotection of axotomised SNC neurons. None of the JNK ko and c‐JunAA mutation affected the survival of facial motoneurons following peripheral axotomy when investigated after 90 days. Finally, we determined the impact of JNK ko on the survival of animals and the degeneration of hippocampal neurons following kainic acid. JNK3 ko mice were substantially resistant against and survived kainic acid‐induced seizures. JNK3 ko and JNK1 ko showed a nonsignificant tendency for decreased or increased death of hippocampal neurons, respectively. Surprisingly, the deletion of a single JNK isoform did not attenuate the immunocytochemical signal of phosphorylated c‐Jun irrespective on the experimental set‐up. This comprehensive study provides novel insights into the context‐dependent physiological and pathological functions of JNK isoforms.

Long-lasting increase of nitric oxide synthase immunoreactivity, NADPH-diaphorase reaction and c-JUN co-expression in rat dorsal root ganglion neurons following sciatic nerve transection

Changes of NADPH-diaphorase reaction (NDP) and nitric oxide synthase immunoreactivity (NOS-IR) in neurons of dorsal root ganglia (DRG) were investigated following transection and ligation of rat sciatic nerve. In untreated rats, 2.7% of L4/L5 DRG neurons were labelled by NDP. After 3 days, intensity of NDP and number of labelled neurons increased and reached a maximal level between 10 and 20 days in 26.8% neurons which persisted up to 50 days. After 150 days, 8.7% of DRG neurons were still labelled. In contralateral L4/L5 DRG, but not L1 and T10 DRG, the number but not the intensity of NDP labelled neurons slightly increased between 10 and 50 days. The patterns of NOS-IR and NDP were congruent. Ipsilaterally, 76% to 92% of NDP neurons showed co-expression with the c-JUN transcription factor which is supposed to play a crucial role in the regeneration process. NDP accumulated in the peripheral nerve stump and was increased in the superficial dorsal horn between 10 and 30 days, whereas motoneurons were not labelled by NOS and NDP.

AP-1 proteins in the adult brain: facts and fiction about effectors of neuroprotection and neurodegeneration

Jun and Fos proteins are induced and activated following most physiological and pathophysiological stimuli in the brain. Only few data allow conclusions about distinct functions of AP-1 proteins in neurodegeneration and neuroregeneration, and these functions mainly refer to c-Jun and its activation by JNKs. Apoptotic functions of activated c-Jun affect hippocampal, nigral and primary cultured neurons following excitotoxic stimulation and destruction of the neuron-target-axis including withdrawal of trophic molecules. The inhibition of JNKs might exert neuroprotection by subsequent omission of c-Jun activation. Besides endogenous neuronal functions, the c-Jun/AP-1 proteins can damage the nervous system by upregulation of harmful programs in non-neuronal cells (e.g. microglia) with release of neurodegenerative molecules. In contrast, the differentiation with neurite extension and maturation of neural cells in vitro indicate physiological and potentially neuroprotective functions of c-Jun and JNKs including sensoring for alterations in the cytoskeleton. This review summarizes the multiple molecular interfunctions which are involved in the shift from the physiological role to degenerative effects of the Jun/JNK-axis such as cell type-specific expression and intracellular localization of scaffold proteins and upstream activators, antagonistic phosphatases, interaction with other kinase systems, or the activation of transcription factors competing for binding to JNK proteins and AP-1 DNA elements.

Activation of cerebral peroxisome proliferator-activated receptors gamma promotes neuroprotection by attenuation of neuronal cyclooxygenase-2 overexpression after focal cerebral ischemia in rats

Up‐regulation of cyclooxygenase (COX)‐2 exacerbates neuronal injury after cerebral ischemia and contributes to neuronal cell death. The present study clarifies the function of cerebral peroxisome‐proliferator‐activated receptor(s) gamma (PPARγ) in the expression of COX‐2 in neurons of the rat brain after middle cerebral artery occlusion (MCAO) with reperfusion by immunohistochemistry, Western blot, and immunofluorescence staining. In peri‐infarct cortical areas the PPARγ was located in both microglia and neurons, whereas COX‐2 was almost exclusively expressed in neurons. PPARγ immunolabeling reached the peak 12 h after MCAO, whereas the number of COX‐2 immunostained cells gradually rose and reached its peak at 48 h. Intracerebroventricular infusion of pioglitazone, an agonist of the PPARγ, over a 5‐day period before and 2 days after MCAO, reduced the infarct size, the expression of tumor necrosis factor α (TNF‐α), COX‐2, and the number of cells positively stained for COX‐1 and COX‐2 in the peri‐infarct cortical regions. COX‐2 induction was also attenuated in the ipsilateral but not in the contralateral hippocampus. In primary cortical neurons expressing the PPARγ, pioglitazone suppressed COX‐2 expression in response to oxidative stress. This protective effect was reversed after cotreatment with GW 9662, a selective antagonist of the PPARγ, clearly demonstrating a PPARγ‐dependent mechanism. Our data provide evidence that activation of neuronal PPARγ considerably contributes to neuroprotection by prevention of COX‐2 up‐regulation in vitro and in peri‐infarct brain areas.—Zhao, Y., Patzer, A., Herdegen, T., Gohlke, P., Culma, J. Activation of cerebral peroxisome proliferator‐activated receptors gamma (PPARγ) promotes neuroprotection by attenuation of neuronal cyclooxygenase‐2 overexpression after focal cerebral ischemia in rats. FASEB J. 20, 1162–1175 (2006)

Induction of immediate early gene encoded proteins in the rat hippocampus after bicuculline-induced seizures: differential expression of KROX-24, FOS and JUN proteins.

Immunocytochemistry with specific antisera was used to assess regional levels of six immediate early gene encoded proteins (KROX-24, c-FOS, FOS B, c-JUN, JUN B and JUN D) in the rat hippocampus after 15 min of bicuculline-induced seizures. Serial sections of the dorsal hippocampus were examined at various postictal recovery periods up to 24 h. The results demonstrate a complex temporal and spatial pattern of immediate early gene synthesis and accumulation. Three major categories of immediate early gene products could best be distinguished in the dentate gyrus: KROX-24 and c-FOS showed a concurrent rapid rise with peak levels at 2 h and a return to baseline levels within 8 h after seizure termination. FOS B, c-JUN and JUN B levels increased more gradually with peak intensities in the dentate gyrus reached at 4 h. These immediate early gene products showed above normal levels in various hippocampal subpopulations up to 24 h. JUN D exhibited the most delayed onset combined with a prolonged increase of seizure-induced immunoreactivity. Irrespective of this differential temporal expression profile of individual transcription factors, the sequence of induction in the hippocampal subpopulations was identical for all immediate early gene-encoded proteins examined: first in the dentate gyrus granule cells followed by CA1 and CA3 neurons, respectively. Our data indicate an asynchronous synthesis of several immediate early gene-encoded proteins in the brain after status epilepticus. FOS and JUN proteins act via homo- or heterodimer complexes at the AP-1 and other DNA binding sites. The different time-courses for individual immediate early gene products strongly suggest, that at different time-points after status epilepticus, different AP-1 complexes are effective. In vitro studies have shown that different AP-1 complexes possess different DNA binding affinities as well as different transcriptional regulatory effects. Our results suggest that these molecular mechanisms are also effective in vivo.

Differential Transcription and Translation of Immediate Early Genes in the Gerbil Hippocampus after Transient Global Ischemia

Excitotoxic activation of glutamate receptors is thought to be a key event for the molecular pathogenesis of postischemic delayed neuronal death of CA-1 neurons in the gerbil hippocampus. Glutamate receptor stimulation also causes induction of transcription factors that belong to the class of immediate early genes. We examined the expression of six different immediate early genes in the gerbil hippocampus after transient global ischemia. Comparative analysis of c-fos and Krox-24 expression was carried out in the same animals at the transcriptional and translational level by in situ hybridization and immunocytochemistry. Postischemic synthesis of four additional immediate early gene (IEG)–encoded proteins (FOS-B, c-JUN, JUN-B, and JUN-D) was investigated by immunocytochemistry at recirculation intervals between 1 and 48 h. After 5 min of ischemia, transcription of c-fos and Krox-24 mRNA was induced in all hippocampal subpopulations with peak expression at 1 h after recirculation. In vulnerable CA-1 neurons, increased transcription of c-fos and Krox-24 was not followed by translation into protein. Induction of immediate early gene-encoded proteins was restricted to neuronal populations less vulnerable to brief ischemia and identified neurons that are targets of glutamate receptor-mediated neurotoxicity but that are destined to survive. Our data indicate an asynchronous synthesis and persistence of individual IEG-encoded proteins in these neurons. The staggered induction implies that combinatorial changes of transcription factors allow a differential postischemic regulation of target gene expression both spatially and over time.