Raffaella Mariotti

Natural killer cell-mediated lysis of dorsal root ganglia neurons via RAE1/NKG2D interactions

Natural killer cells have been reported to be able to kill various transformed and virus‐infected target cells. It was recently observed that NK cells also could kill syngeneic dorsal root ganglia (DRG) neurons by a perforin‐dependent mechanism. We demonstrate here that this phenomenon does not reflect a general ability of NK cells to kill neurons in culture. While DRG neurons of the peripheral nervous system were readily killed, ventral spinal cord neurons and hippocampal neurons of the central nervous system (CNS) were resistant to lysis. The resistance to NK cell‐mediated lysis of the latter neurons was not related to protection by MHC class I molecules, since similar β2‐microglobulin–/– neurons were equally resistant to lysis. While exploring other possible molecular mechanisms for the selective triggering of lysis of DRG neurons, we observed that the retinoic acid early inducible gene‐1 (RAE‐1), the product of which is a ligand for the NK cell‐activating receptor NKG2D, was expressed at high levels in the DRG neurons. In contrast, RAE‐1 was expressed only at very low levels in the resistant CNS‐derived neurons. Blocking NK cells withanti‐NKG2D antibodies inhibited NK cell‐mediated killing of the DRG neurons. Thus, we demonstrate that NK cell‐mediated lysis of DRG neurons correlates with the expression of RAE‐1 and that this lysis is dependent on activation of NK cells via NKG2D. This observation demonstrates that NK cells can kill non‐pathogen‐infected or non‐transformed syngeneic cells through activation of the NKG2D receptor.

Neuroinflammation and brain infections: historical context and current perspectives.

An overview of current concepts on neuroinflammation and on the dialogue between neurons and non-neuronal cells in three important infections of the central nervous systems (rabies, cerebral malaria, and human African trypanosomiasis or sleeping sickness) is here presented. Large numbers of cases affected by these diseases are currently reported. In the context of an issue dedicated to Camillo Golgi, historical notes on seminal discoveries on these diseases are also presented. Neuroinflammation is currently closely associated with pathogenetic mechanisms of chronic neurodegenerative diseases. Neuroinflammatory signaling in brain infections is instead relatively neglected in the neuroscience community, despite the fact that the above infections provide paradigmatic examples of alterations of the intercellular crosstalk between neurons and non-neuronal cells. In rabies, strategies of immune evasion of the host lead to silencing neuroinflammatory signaling. In the intravascular pathology which characterizes cerebral malaria, leukocytes and Plasmodium do not enter the brain parenchyma. In sleeping sickness, leukocytes and African trypanosomes invade the brain parenchyma at an advanced stage of infection. Both the latter pathologies leave open many questions on the targeting of neuronal functions and on the pathogenetic role of non-neuronal cells, and in particular astrocytes and microglia, in these diseases. All three infections are hallmarked by very severe clinical pictures and relative sparing of neuronal structure. Multidisciplinary approaches and a concerted action of the neuroscience community are needed to shed light on intercellular crosstalk in these dreadful brain diseases. Such effort could also lead to new knowledge on non-neuronal mechanisms which determine neuronal death or survival.

Exosome derived from murine adipose-derived stromal cells: Neuroprotective effect on in vitro model of amyotrophic lateral sclerosis

Therapeutic strategies for the fatal neurodegenerative disease amyotrophic lateral sclerosis (ALS) have not yet provided satisfactory results. Interest in stem cells for the treatment of neurodegenerative diseases is increasing and their beneficial action seems to be due to a paracrine effect via the release of exosomes, main mediators of cell-cell communication. Here we wished to assess, in vitro, the efficacy of a novel non-cell therapeutic approach based on the use of exosomes derived from murine adipose-derived stromal cells on motoneuron-like NSC-34 cells expressing ALS mutations, and used as in vitro models of disease. In particular, we set out to investigate the effect of exosomes on NSC-34 naïve cells and NSC-34 cells overexpressing human SOD1(G93A) or SOD1(G37R) or SOD1(A4V) mutants, exposed to oxidative stress. The data presented here indicate for the first time that exosomes (0.2 µg/ml) are able to protect NSC-34 cells from oxidative damage, which is one of the main mechanism of damage in ALS, increasing cell viability. These data highlight a promising role of exosomes derived from stem cells for potential therapeutic applications in motoneuron disease.

Activation and response to axotomy of microglia in the facial motor nuclei of G93A superoxide dismutase transgenic mice.

Mice over-expressing a human mutation of Cu(2+)/Zn(2+) superoxide dismutase (SOD1) provide a model of amyotrophic lateral sclerosis. Using tomato lectin histochemistry, we analyzed microglia in the facial nuclei of SOD1(G93A) transgenic mice in the late stage of disease. In these animals, microglia was markedly activated, and ensheathed facial motoneurons as observed in wild-type mice 1 week after nerve transection. In the axotomized facial nucleus of transgenic mice at the same time point, microglia activation was enhanced and exhibited phagocytic features. The findings show that in the facial nucleus microglial cells react to motoneuron disease caused by the SOD1 mutation and to axotomy-induced damage of facial motoneurons.

Gene, Cell, and Axon Changes in the Familial Amyotrophic Lateral Sclerosis Mouse Sensorimotor Cortex

Lower motoneuron abnormalities have been extensively documented in the murine model of familial amyotrophic lateral sclerosis, whereas information on corticospinal neurons in these mice is very limited. We investigated 1) mRNA levels of inflammation-related molecules in the deep layers in which corticospinal neurons reside, 2) corticospinal neurons labeled from tracer injections in the corticospinal tract at the cervical level, 3) axonal damage revealed by &bgr;-amyloid precursor protein accumulation, and 4) glial cell activation in the sensorimotor cortex of presymptomatic and end-stage superoxide dismutase (SOD)-1 (G93A) mice. We demonstrated induction of inflammatory gene transcripts in the deep layers, early and progressive shrinkage of corticospinal cell bodies and activation of surrounding astrocytes and microglia with upregulation of major histocompatibility complex class I antigen. Accumulation of &bgr;-amyloid precursor protein in proximal axonal swellings indicating axonal injury was also evident at the terminal stage in the motor cortex and internal capsule. Glial and axon changes were not observed elsewhere in the cortex. These data reveal that the entire motor circuit is affected in this murine amyotrophic lateral sclerosis model as it is in human amyotrophic lateral sclerosis. Sensorimotor cortical inflammation and progressive corticospinal cell body and fiber damage may reflect transsynaptic signaling of damage from lower motoneurons.

Altered reaction of facial motoneurons to axonal damage in the presymptomatic phase of a murine model of familial amyotrophic lateral sclerosis

In transgenic mice carrying the G93A human mutation of Cu/Zn superoxide dismutase (SOD1), which provide a model of familial amyotrophic lateral sclerosis, we investigated, before the onset of symptoms, two parameters of the response of facial motoneurons to nerve transection, i.e. nitric oxide synthase induction and motoneuron loss. Axotomy elicited after 2 and 3 weeks high nitric oxide synthase expression in facial motoneurons of wild-type mice, whereas the induction was very weak or absent in transgenic mice. At 1 month post-axotomy, loss of facial motoneurons was significantly higher in mutant mice than in wild-type littermates. Thus, SOD1 mutation interferes with the oxidative cascade elicited by axonal injury in cranial motoneurons. The results also indicate that the adverse gain of function of the mutant SOD1 enhances the vulnerability of motoneurons to peripheral stressful conditions.

Analysis of neuromuscular junctions and effects of anabolic steroid administration in the SOD1G93A mouse model of ALS

Several lines of evidence indicate that neuromuscular junction (NMJ) destruction and disassembly is an early phenomenon in amyotrophic lateral sclerosis (ALS). Here we analyzed by confocal and electron microscopy the NMJ structure in the diaphragm of SOD1G93A mice at symptom onset. In these mice, which provide a model for familial ALS, diaphragm denervation (~50%) as well as gastrocnemius denervation (~40%) was found. In addition, the size of the synaptic vesicle pool was reduced and alterations of mitochondria were observed in approximately 40% of the remaining presynaptic terminals. Chronic treatment of SOD1G93A mice with the anabolic steroid nandrolone during the presymptomatic stage preserved the diaphragm muscle mass and features indicative of synaptic activity. These features were represented by the number of vesicles docked within 200 nm from the presynaptic membrane and area of acetylcholine receptor clusters. Structural preservation of mitochondria was documented in presynaptic terminals. However, innervation of diaphragm muscle fibers was only slightly increased in nandrolone-treated SOD1-mutant mice. Altogether the results point out and define fine structural alterations of diaphragm NMJs in the murine model of familial ALS at symptom onset, and indicate that nandrolone may prevent or delay structural alterations in NMJ mitochondria and stimulate presynaptic activity but does not prevent muscle denervation during the disease.

The anabolic/androgenic steroid nandrolone exacerbates gene expression modifications induced by mutant SOD1 in muscles of mice models of amyotrophic lateral sclerosis

Graphical abstract

Voronoi-based spatial analysis reveals selective interneuron changes in the cortex of FALS mice.

The neurodegenerative disease amyotrophic lateral sclerosis affects lower motoneurons and corticospinal cells. Mice expressing human mutant superoxide dismutase (SOD)1 provide widely investigated models of the familial form of disease, but information on cortical changes in these mice is still limited. We here analyzed the spatial organization of interneurons characterized by parvalbumin immunoreactivity in the motor, somatosensory, and visual cortical areas of SOD1(G93A) mice. Cell number and sociological spatial behavior were assessed by digital charts of cell location in cortical samples, cell counts, and generation of two-dimensional Voronoi diagrams. In end-stage SOD1-mutant mice, an increase of parvalbumin-containing cortical interneurons was found in the motor and somatosensory areas (about 35% and 20%, respectively) with respect to wild-type littermates. Changes in cell spatial distribution, as documented by Voronoi-derived coefficients of variation, indicated increased tendency of parvalbumin cells to aggregate into clusters in the same areas of the SOD1-mutant cortex. Counts and coefficients of variation of parvalbumin cells in the visual cortex gave instead similar results in SOD1-mutant and wild-type mice. Analyses of motor and somatosensory areas in presymptomatic SOD1-mutant mice provided findings very similar to those obtained at end-stage, indicating early changes of interneurons in these cortical areas during the pathology. Altogether the data reveal in the SOD1-mutant mouse cortex an altered architectonic pattern of interneurons, which selectively affects areas involved in motor control. The findings, which can be interpreted as pathogenic factors or early disease-related adaptations, point to changes in the cortical regulation and modulation of the motor circuit during motoneuron disease.

Changes in the expression of P2X1 and P2X2 purinergic receptors in facial motoneurons after nerve lesions in rodents and correlation with motoneuron degeneration

Involvement of P2X1 and P2X2 purinergic receptors in motoneuron response to injury was investigated with Western blotting and immunohistochemistry and correlated with motoneuron loss, Bcl-2 expression, nitric oxide synthase induction and glial activation. P2X1 was highly induced in rat facial motoneurons after nerve resection, which causes slowly occurring neurodegeneration. P2X1 induction was lower and less persistent after nerve crush, permissive for fiber regeneration. P2X2 expression was found in nuclei of rat facial motoneurons, with nuclear export in the cytoplasm after nerve resection. P2X1 induction in axotomized facial motoneurons was impaired in superoxide dismutase (SOD)1-G93A-mutant mice, a model of motoneuron disease. The data in rats point to a correlation of P2X1 induction with motoneuron degeneration, which also involves P2X2 intracellular changes, rather than with axon regeneration effort. The data in mice show that the SOD1 mutation interferes with injury-elicited P2X1 induction, suggesting alterations of ATP release from mutant motoneurons after damage.