After traumatic brain lesion, microglial cells are rapidly activated, migrate toward the sites of injury, and cause secondary damage that accounts for most of the loss of brain function. In the present study, we have characterized a new macrophage/microglia activation factor (MA-F). Using the monocytic cell line U937, we were able to demonstrate that MAF is upregulated after TPA-induced differentiation into macrophages. We have generated a specific antibody against MAY In BV-2 microglial cells, MAF is partially co-localized with 1134, a classical microglial marker. In addition, we have analyzed the in vivo expression patterns of MAF after entorhinal cortex lesion. We were able to show a substantial upregulation of MAF on selected CD11b(+) and IB4(+) macrophages/microglial cells in the deafferented hippocampus and in the perilesional region, while no MAF expression was detectable on the contralateral side. Confocal microscopy revealed a lysosome-like expression pattern in BV-2 cells, as well as in ECL-associated macrophages/microglial cells in vivo. Furthermore, we were able to demonstrate that U937 cells with downregulated MAF converted slower and to a significantly reduced extent to the macrophageal phenotype after TPA treatment. In addition, MAF downregulation in BV-2 microglial cells substantially reduced the phagocytotic uptake of dextran beads. Our data indicate that MAF is expressed in selected macrophages/microglial cells around the lesion and in the degenerating hippocampus after ECL. Furthermore, MAF expression in monocytic cells seems to play a functional role in the differentiation to a phagocytosing phenotype and may be, at least partially, required for phagocytotic activity, specifically in lesioned tissue after brain trauma. (c) 2005 Wiley-Liss, Inc.

Multiple sclerosis (MS) is a chronic demyelinating disease in which it has only recently been suggested that damage to neuronal structures plays a key role. Here, we uncovered a link between the release of lipid breakdown products, found in the brain and cerebrospinal fluid (CSF) of MS patients as well as in experimental autoimmune encephalomyelitis, and neuronal damage mediated by microglial activation. The concentrations of the breakdown product 7-ketocholesterol detected in the CSF of MS patients were capable of inducing neuronal damage via the activation and migration of microglial cells in living brain tissue. 7-ketocholesterol rapidly entered the nucleus and activated poly(ADP-ribose)-polymerase (PARP)-1, followed by the expression of migration-regulating integrins CD11a and intercellular adhesion molecule 1. These findings reveal a novel mechanism linking demyelination and progressive neuronal damage, which might represent an underlying insidious process driving disease beyond a primary white matter phenomenon and rendering the microglial PARP-1 a possible antiinflammatory therapeutic target.

Excitotoxic brain lesions, including stroke and trauma, result initially in primary destruction of brain parenchyma and subsequently in secondary damage of neighboring neurons hours after the insult. This secondary damage of initially surviving neurons accounts for most of the volume of the infarcted area and the loss of brain function that a patient suffers after a stroke [1]. Therapeutic approaches must therefore focus on the protection of initially surviving neurons from this secondary damage. One major component of secondary neuronal damage is the migration of macrophages and microglial cells towards the sites of injury where they produce large amounts of toxic cytokines and oxygen radicals. To reduce excitotoxic neuronal cell death as well as ischemic damage, the use of inhibitors of poly(ADP-ribose)polymerase-1 (PARP-1) has been proposed as a protective therapy [2-4]. However, PARP is also involved in fast and efficient neuronal DNA repair [5], which is an essential mechanism in enabling neurons to cope with stress factors, including free radicals and cytotoxic cytokines as released by macrophages and microglial cells. Therefore, PARP inhibitors might well reduce the neuronal capability to carry out repair programs thereafter, resulting in further secondary damage. Since PARP is highly activated in phagocytosing microglia [6], its specific downregulation in these cells might therefore be a new strategy to protect neurons from this secondary damage. In order to test this idea, we used an approach which allows to study the effects of macrophage/microglial invasion [7, 8] into living organotypic brain tissue. We cloned an anti-sense-poly (ADP-ribose) polymerase-1 (antisense-PARP-1)-vector, established a stable transfected microglial cell line, and allowed these cells to invade organotypic hippocampal brain tissue cultures after the induction of primary neuronal damage by NMDA. We observed that control vector-transfected microglial cells invaded the tissue, migrated distinctly to the sites of neuronal injury, and caused severe secondary neuronal damage. In contrast, invading antisense-PARP-1 microglial cells were not capable of migrating to the injured neurons and were distributed diffusely in the hippocampal tissue. These cells did not cause any detectable secondary neuronal damage. Interestingly, the overall amount of neuronal damage in tissue invaded by antisense-PARP-1 microglial cells was significantly lower when compared to chemical inhibition of PARP in the entire tissue. These findings clearly demonstrate that selective inhibition of microglial PARP appears to be more effective to protect neurons after excitotoxic lesion than non-selective application of PARP inhibitors. FACS analysis of the adhesion molecule pattern which revealed a strongly reduced CD11 a-expression in antisense-PARP-1-microglial cells before and after stimulation by lipopolysaccharide or TNF-alpha. The expression of other adhesion molecules such as CD11 b, CD18 and ICAM-1 was not altered at all. Thus, we cloned an antisense-CD11 a vector (antisense-CD11 a) and established a stable transfected microglial cell line. Functional studies demonstrated that these antisense-CD11 a microglial cells were in fact not capable to migrate to sites of neuronal damage. In cell culture experiments, we were able to show that this microglial CD11 a-expression was dependent on both NF-kappaB-translocation into the nucleus and PARP activation. Furthermore, immunoprecipitation studies revealed a direct interaction of the activated and automodified PARP with the translocated NF-kappaB and the nuclear protein HMG-I(Y) in activated and CD11 a-expressing microglial cells. Here we demonstrate for the first time that the nuclear enzyme PARP regulates the expression of migration-relevant integrins in macrophages/microglial cells in dependence of their activation state. Thus, our results could further explain previously findings that inhibition of PARP attenuates neutrophil recruitment [9] and exhibits protective effects in tissues outside of the brain like after myocardial infarction [10, 11]. Therefore, our data render the specific inhibition of the microglial PARP a cellular target for preventing the primary injured brain tissue from severe secondary damage without altering the restorative function of neuronal PARP during post-injury DNA repair.

Treatment of different human leukemia cell variants with the anthracycline adriamycin was associated with a rapid activation of the proteasome. Thus, proliferating U937, TUR, and retrodifferentiated U937 cells exhibited a 4.3-fold, 5.8-fold, and 4.3-fold proteasome activation within 15 minutes after adriamycin treatment, respectively. In contrast, little if any proteasome activation was detectable in a growth-arrested differentiated U937 population following adriamycin treatment. Further analysis of this mechanism revealed a significant reduction of adriamycin-induced proteasome activity after inhibition of poly(ADP-ribose) polymerase (PARP) by 3-aminobenzamide (3-ABA) in the proliferating leukemic cell types. These findings suggested that PARP is involved in the regulation of drug-induced proteasome activation. Indeed, anti-PARP immunoprecipitation experiments of adriamycin-treated cells revealed increasing levels of coprecipitated, enzymatically active proteasome particularly in the proliferating cell variants in contrast to the differentiated U937 cells, with a maximum after 15 minutes, and sensitivity to PARP inhibition by 3-ABA. The specific role of the PARP was investigated in U937 and TUR cell clones stably transfected with a constitutively active antisense PARP (asPARP) vector. Thus, asPARP-TUR cells developed a 25-fold increased sensitivity to adriamycin treatment. Furthermore, we investigated leukemic blasts isolated from acute myelogenous leukemia patients and obtained a similarly enhanced proteasome activity after adriamycin treatment, which was dependent on the PARP and thus could be coprecipitated with anti-PARP antibodies. Transient transfection of leukemic blasts with the asPARP vector significantly reduced the adriamycin-induced proteasome activation. These data suggest that the PARP-associated nuclear proteasome activation represents a potential target within chemotherapeutic defense mechanisms developed by leukemia cells.

During neuroinflammation, activated microglial cells migrate to the sites of neuronal injury, phagocytose neighboring cells, and produce large amounts of oxygen free radicals, which might contribute to severe cell damage and death. It is interesting that microglial cells have withstood this cytotoxic action of free radicals, which indicates that there is an intracellular mechanism that apparently enables microglial cells to cope with such oxidative challenges. In this study, we investigated the capability of BV-2 murine microglial cells to cope with oxidatively damaged proteins by the proteasomal proteolytic system. To induce a highly activated state, we used the proinflammatory cytokine tumor necrosis factor-alpha, which acts as a priming signal for microglial superoxide radical production. We showed that activation of the nuclear enzyme poly(ADPribose)polymerase (PARP) enabled activated microglial cells to resist oxidative damage by an up-regulation of the nuclear proteasome. Activated microglial cells revealed an efficient recognition and degradation of oxidatively damaged proteins during an enhanced endogenous protein turnover. The impairment of PARP function by inhibitor or antisense experiments resulted in an accumulation of damaged proteins and subsequently cell death. In contrast, this was not the case in resting microglial cells. These findings demonstrate the crucial role of the PARP in microglial cell survival during activation and renders it a potential anti-inflammatory target.