Sejt-autonóm és nem sejt-autonóm tényezők tanulmányozása a motoros idegsejtek degenerációja során = Study of the role of cell-autonomous and non cell-autonomous processes in the degeneration of motor neurons

Kísérleteink során a mozgató idegsejtek kalcium-kötő fehérje tartalmával jellemzett "sejt-autonóm" tulajdonsága és a környéki mikrogliális reakció mértékével jelzett "nem sejt-autonóm" sajátságok akut és krónikus stressz helyzetben kimutatható összefüggéseit vizsgáltuk. Akut sérülést előidéző modellen igazoltuk, hogy a természetesen különböző kalcium-kötő fehérje tartalmú mozgató idegsejtek kalcium-kötő fehérje tartalma és ellenálló képessége között korreláció áll fenn. Továbblépve, az idegsejtek egyfajta kalcium-kötő fehérje tartalmát (parvalbumin) génsebészeti módszerrel megemelve kimutattuk, hogy ezzel a beavatkozással a sejtek korlátozottan megnövelt ellenálló képességgel ruházhatók fel. Génsebészeti úton előidézett krónikus stressz alkalmazásával igazoltuk, hogy az ilyen jellegű ellenálló képesség növelés hatékonysága, kapacitása véges. Ezen kísérletek során derült fény arra, hogy az idegsejtek károsodásához, és pusztulásához a mozgató idegsejtek környéki mikroglia sejtjei aktívan hozzájárulnak, s igazoltuk, hogy a (mozgató) idegsejtek sérüléssel szembeni hatékony védelmének kidolgozásához a mikroglia sejteket is "kezelni" kell. Ezt, az állatkísérletekkel megfelelően alátámasztott stratégiát klinikai gyakorlatban is (csontvelő átültetéssel) kipróbáltuk. Bár az eljárásnak szignifikáns terápiás hatása nem volt, megmutattuk, hogy ezzel a beavatkozással a donor eredetű sejtek a sérülés helyére vándorolnak, melyek így felhasználhatók trofikus faktorok célzott bejuttatásához. | In our study, the interaction between the cell-autonomous properties (characterized by intracellular calcium buffering capacity) and non cell-autonomous properties (characterized by local microglial activation) of motor neurons has been examined experimentally in acute and chronic stress conditions. During acute lesion, evoked by nerve transection, the correlation between the naturally occurring elevated calcium binding protein capacity and increased resistance of motor neurons against injury has been demonstrated. Next, using transgenic technology, it has been proved that by this way though limited, but increased resistance could be transferred to motor neurons, by increasing their parvalbumin content. During these experiments it became also evident that, in certain stress conditions, the neighboring microglial cells could actively contribute to the destruction of motor neurons, thus, to develop an efficient protection against their injury, microglial cells has to be "treated", as well. This strategy, supported by firm experimental data from animal studies, has been tested clinically through bone marrow transplantation in (voluntary) patients with motor neuron disease. Although this intervention did not yield significant therapeutic benefit, it could be demonstrated that a certain population of cells of donor origin migrate to the site of injury, thus might be used as vehicles for targeted delivery of trophic factors to the lesion

Immun tényezők, parvalbumin és PARP aktiváció jelentősége neurodegeneratív kórképekben = The role of immune factors, parvalbumin and activation of PARP, neurodegenerative diseases

A neurodegeneratív betegségekben a károsodás közös útja az intraneuronális Ca emelkedése. Amyotrophiás laterálsclerosisban (ALS), /humán motoneuron (MN) betegség/, Parkinson kórban (PK) és állatkísérletes modelljeikben azok az idegsejtek, amelyek képesek kompenzatórikusan megemelni a Ca-kötő parvalbumin szintjüket, rezisztensek a károsodásra. A sérülékeny sejtek NMDA receptor antagonistákkal, Ca-csatorna gátlókkal és a parvalbumin gén beültetéssel megóvhatók a pusztulástól a kísérletes MN betegségben. DNS károsodás és az intraneuronális Ca emelkedés aktiválja a poly(ADP-ribose) polymerase (PARP) DNS javító enzymet ALS-ben a corticális MN-okban, PK-ban a dopaminerg sejtekben. A túlfokozott aktiváció elhasználva a sejt energia forrásait sejthalált okoz. A PARP aktiválódik a microgliában is, mely szabályozza a helyi immun-gyulladásos reakciót. A PARP gátlók adásának kedvező hatását tapasztaltuk a MN betegség immun-mediált kísérletes modelljében, s potenciálisan használhatónak tűnnek ALS-ben és PK-ban. Az autoimmun IgG intraneuronális felvételéből, a microglia aktiválódásából és antigén prezentáló dendritikus sejtekké alakulásából, T lymphocyták odavándorlásából álló immun-gyulladásos reakció figyelhető meg ALS-ből, PK-ból származó boncolási anyagban az érintett régiókban. A microglia aktiváció gátlók csökkentik a neuronokra káros anyagok elválasztását, akadályozzák a lokális antigén prezentációt és így a neuronokat károsító másodlagos autoimmun reakciót. | The rise in intraneuronal Ca is the common pathway of cell injury in neurodegenerative diseases. The neurons which are able to upregulate a calcium-buffering protein, parvalbumin are resistant to the damage in amyotrophic lateral sclerosis (ALS), in Parkinson disease (PD) and in their animal models. NMDA receptor antagonists, Ca-channel blockers and parvalbumin gene transfer were successfully used for ameliorating the MN damage in animal models. The rise in intraneuronal Ca upregulates the DNA repair enzyme poly(ADP-ribose) polymerase in the cortical MNs in ALS and in the dopaminergic neurons in PD. The overactivation of PARP exhausts the energy sources of the cells leading to death. The PARP is also upregulated in microglia, which dictates the local immune-inflammatory reaction. The beneficial effect of the use of PARP inhibitors is predicted in the treatment of the diseases and was experienced in an immune-mediated model of MN disease. An immune-inflammatory reaction consisting of autoimmune IgG uptake by the neurons, microglia activation and conversion to antigen presenting dendritic cells, and the recruitment of T lymphocytes was noted in autopsy materials from ALS, PD and from Alzheimer disease in the affected areas. The inhibitors of the activation of microglia may be useful to diminish the harmful effect of the materials secreted by it to damage neurons and can prevent the local antigen presentation and the secondary autoimmune attack targeting neurons

Passive transfer of blood sera from ALS patients with identified mutations results in elevated motoneuronal calcium level and loss of motor neurons in the spinal cord of mice

Introduction: Previously, we demonstrated the degeneration of axon terminals in mice after repeated injections of blood sera from amyotrophic lateral sclerosis (ALS) patients with identified mutations. However, whether a similar treatment affects the cell body of motor neurons (MNs) remained unresolved. Methods: Sera from healthy individuals or ALS patients with a mutation in different ALS-related genes were intraperitoneally injected into ten-week-old male Balb/c mice (n = 3/serum) for two days. Afterward, the perikaryal calcium level was measured using electron microscopy. Furthermore, the optical disector method was used to evaluate the number of lumbar MNs. Results: The cytoplasmic calcium level of the lumbar MNs of the ALS-serum-treated mice, compared to untreated and healthy-serum-treated controls, was significantly elevated. While injections of the healthy serum did not reduce the number of MNs compared to the untreated control group, ALS sera induced a remarkable loss of MNs. Discussion: Similarly to the distant motor axon terminals, the injection of blood sera of ALS patients has a rapid degenerative effect on MNs. Analogously, the magnitude of the evoked changes was specific to the type of mutation; furthermore, the degeneration was most pronounced in the group treated with sera from ALS patients with a mutation in the chromosome 9 open reading frame 72 gene

Passive transfer of sera from als patients with identified mutations evokes an increased synaptic vesicle number and elevation of calcium levels in motor axon terminals, similar to sera from sporadic patients

Previously, we demonstrated increased calcium levels and synaptic vesicle densities in the motor axon terminals (MATs) of sporadic amyotrophic lateral sclerosis (ALS) patients. Such alterations could be conferred to mice with an intraperitoneal injection of sera from these patients or with purified immunoglobulin G. Later, we confirmed the presence of similar alterations in the superoxide dismutase 1 G93A transgenic mouse strain model of familial ALS. These consistent observations suggested that calcium plays a central role in the pathomechanism of ALS. This may be further reinforced by completing a similar analytical study of the MATs of ALS patients with identified mutations. However, due to the low yield of muscle biopsy samples containing MATs, and the low incidence of ALS patients with the identified mutations, these examinations are not technically feasible. Alternatively, a passive transfer of sera from ALS patients with known mutations was used, and the MATs of the inoculated mice were tested for alterations in their calcium homeostasis and synaptic activity. Patients with 11 different ALS-related mutations participated in the study. Intraperitoneal injection of sera from these patients on two consecutive days resulted in elevated intracellular calcium levels and increased vesicle densities in the MATs of mice, which is comparable to the effect of the passive transfer from sporadic patients. Our results support the idea that the pathomechanism underlying the identical manifestation of the disease with or without identified mutations is based on a common final pathway, in which increasing calcium levels play a central role

Talampanel reduces the level of motoneuronal calcium in transgenic mutant SOD1 mice only if applied presymptomatically

We tested the efficacy of treatment with talampanel in a mutant SOD1 mouse model of ALS by measuring intracellular calcium levels and loss of spinal motor neurons. We intended to mimic the clinical study; hence, treatment was started when the clinical symptoms were already present. The data were compared with the results of similar treatment started at a presymptomatic stage. Transgenic and wild-type mice were treated either with talampanel or with vehicle, starting in pre-symptomatic or symptomatic stages. The density of motor neurons was determined by the physical disector, and their intracellular calcium level was assayed electron microscopically. Results showed that motor neurons in the SOD1 mice exhibited an elevated calcium level, which could be reduced, but not restored, with talampanel only when the treatment was started presymptomatically. Treatment in either presymptomatic or symptomatic stages failed to rescue the motor neurons. We conclude that talampanel reduces motoneuronal calcium in a mouse model of ALS, but its efficacy declines as the disease progresses, suggesting that medication initiation in the earlier stages of the disease might be more effective

Experimental Motor Neuron Disease Induced in Mice with Long-Term Repeated Intraperitoneal Injections of Serum from ALS Patients

In an earlier study, signs of commencing degeneration of spinal motor neurons were induced in mice with short-term intraperitoneal injections of immunoglobulin G (IgG) taken from patients with amyotrophic lateral sclerosis (ALS). Since in that study, neither weakness nor loss of motor neurons was noted, to test whether the ALS IgG in this paradigm has the potential to evoke relentless degeneration of motor neurons, treatment with repeated injections over a longer period was carried out. Mice were systematically injected intraperitoneally with serum taken from ALS patients over a 75-day period. At selected time points, the isometric force of the limbs, number of spinal motor neurons and their intracellular calcium levels were determined. Furthermore, markers of glial activation and the motoneuronal uptake of human IgG were monitored. During this period, gliosis and progressive motoneuronal degeneration developed, which led to gradual loss of spinal motor neurons, more than 40% at day 21, along with decreasing muscle strength in the limbs. The inclusion-like accumulation of IgG appeared in the perikarya with the increase of intracellular calcium in the cell bodies and motor nerve terminals. Our results demonstrate that ALS serum can transfer motor neuron disease to mice