SESSION 7A ROLE OF NON-NEURONAL CELLS

C40 GLIAL CELLS AND NEURONAL REPAIR: LESSONS FROM SPINAL CORD INJURY

RAISMAN G

Institute of Neurology, UCL, London, United Kingdom

E-mail address for correspondence: [email protected]

Keywords: glial cells, transplant, spinal cord

We have transplanted cultured adult olfactory ensheathing cells into lesions of intraspinal long tracts and spinal root avulsions in adult rats. The grafted cells encourage the growth of the cut nerve fibres, and suppress the excessive neuromatous branching found in untreated lesions. The grafted cells take up an elongated shape, and form a tightly aligned bridge between the ends of the cut fibre tract. The regenerating nerve fibres enter the graft and follow this new, aligned bridge pathway. Within the bridge the nerve fibres are intimately ensheathed by the Schwann-like cells, and enclosed in an outer, perineurial-like sheath of fibroblasts. In the case of the spinal tracts, once they reach the end of the graft they re-enter the host spinal cord, and continue along the distal part of the corticospinal tract to form terminal arborisations. The effect is to put a patch over the lesion, restoring the integrity of the original pathway, and results in the functional recovery of some specific functional tasks. In the case of the dorsal roots the fibres re-enter the spinal cord, arborise in the dorsal horn, and ascend in the dorsal columns. In the case of the ventral roots there is a 4–5 fold increase in the numbers of fibres entering the proximal part of the root.

C41 NON-NEURONAL NEUROPROTECTION IN ALS USING GLIAL RESTRICTED PRECURSOR TRANSPLANTATION: A NOVEL APPROACH FOR RESPIRATORY NEUROPROTECTION.

LEPORE A¹, DEJEA C¹, CAMPANELLI J², MARAGAKIS N¹

¹Johns Hopkins University, Baltimore, MD, United States, ²Q Therapeutics, Inc., Salt Lake City, UT, United States

E-mail address for correspondence: [email protected]

Keywords: non-neuronal cells, astrocytes, stem cells

Background: Studies in ALS models have suggested that cellular abnormalities are not limited to motor neurons and that non-neuronal cells play a role in disease progression. However, previous ALS stem cell transplantation studies have primarily focused mostly on motor neuron replacement. Since most ALS patients die from respiratory muscle paralysis, targeting the phrenic motor neuron pools for neuroprotection may be a strategy with clinical relevance in ALS trials. We have employed glial restricted precursors (GRPs) from both rodents and human tissues for transplantation into the cervical spinal cord. Given these observations and other findings of astrocyte dysfunction in ALS, this proposal aims to target the replacement of dysfunctional astrocytes using GRPs for possible therapeutic benefits.

Objectives: 1) Determine the fate and survival of rodent and human glial restricted precursors (GRPs) following transplantation into the SOD1^G93A rat model of ALS. 2) Determine the capacity for motor neuron protection and assessment of the important physiological properties of glial precursors following transplantation. 3) Determine the ability of GRPs to preserve forelimb strength, diaphragmatic function, and survival following transplantation into the SOD1^G93A rat.

Methods: GRPs from both rodent lines and human fetal tissue were transplanted into the ventral horn of the cervical spinal cords of presymptomatic SOD1^G93A rats at 3 levels (C4, C5, C6).

Results: The transplantation of GRPs results in differentiation into mature astrocytes which reside in the ventral gray matter adjacent to motor neuron soma and processes. SOD1^G93A rats transplanted with GRPs demonstrate a preservation of diaphragm function, a slowing of forelimb grip strength decline and a delay in the onset of forelimb weakness. The effect was focal with no change in hindlimb grip strength. Survival was prolonged in glial precursor-transplanted SOD1^G93A rats from the maintenance of respiratory function. The effects also appear related, at least in part, to the maintenance of glutamate transporter function—an astrocyte-specific property.

Discussion and Conclusions: The transplantation of GRPs results in the focal maintenance of respiratory physiology and function, a focal maintenance of forelimb strength and a slowing of the course of disease progression in the SOD1^G93A rat. This approach appears to be related to astrocyte-specific properties of transplanted cells and not a non-specific cellular effect. These data suggest that glutamatergic pathways may play at least a part in this neuroprotection. The use of human GRPs may offer a novel approach for stem cell replacement strategies in ALS patients with a focus on the preservation of respiratory function.

C42 FOCAL DEGENERATION OF GLUTAMATE-VULNERABLE ASTROCYTES IN AMYOTROPHIC LATERAL SCLEROSIS

ROSSI D¹, BRAMBILLA L¹, VALORI CF¹, RONCORONI C¹, CRUGNOLA A¹, YOKOTA T², BREDESEN DE², VOLTERRA A¹

¹Department of Pharmacological Sciences, University of Milan, Italy, ²Buck Institute for Age Research, California, United States

E-mail address for correspondence: [email protected]

Keywords: nonneuronal cells, degeneration, glutamate

Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disorder characterized by the loss of corticospinal and spinal motor neurons. The causes of the disease are mainly unknown, but about 2% of human cases have been associated with mutations in the gene encoding the antioxidant enzyme copper, zinc superoxide dismutase (SOD1). While the cascade of events ultimately responsible for motor neuron degeneration remains elusive, recent observations suggest that the death of motor cells implies a combination of cell-autonomous and non-cell-autonomous mechanisms that also involve glial cells, particularly microglia and astrocytes.

To study the morphological and structural changes astrocytes undergo during the progression of the disease, we performed immunohistochemical analysis of spinal cord sections from transgenic mice expressing the ALS-linked mutant SOD1 Gly93→Ala (hSOD1^G93A). In the neighbourhood of motor cells, we found that a subset of astrocytes harbouring protein inclusions underwent morphological and biochemical changes that were reminiscent of degenerating cells. These alterations show before the loss of motor neurons and the phenomenon becomes significant concomitant with the onset of neuronal degeneration and the appearance of ALS symptoms.

To investigate the impact of mutant SOD1s (mtSOD1s) on astroglial properties, we then expressed either the human wild-type (WT) or the two mtSOD1s, G93A and G85R, in primary cultures of rat spinal cord astrocytes. We found that mtSOD1s themselves induce no deleterious effects on cultured astrocytes but make them rather susceptible to the pro-apoptotic action of non-toxic concentrations of the excitatory neurotransmitter glutamate. Such effects are mediated by the metabotropic glutamate receptor 5 (mGluR5), as demonstrated by the fact that mGluR5 blockage is protective against the gliotoxic insult. Moreover, in vivo administration of an mGluR5 antagonist reduces astrocyte degeneration, delays the appearance of ALS symptoms and extends survival in hSOD1^G93A transgenic mice.

All of these data indicate that spinal cord astrocytes are endangered by the expression of ALS-linked mtSOD1s, become highly vulnerable to mildly toxic stimuli present in their microenvironment, and start to degenerate. This in turn may accelerate degeneration of the neighboring motor cells in an interactive process of reciprocal damage.

C43 MOTOR NEURON ROS MAY CONTRIBUTE TO ASTROCYTE PATHOLOGY IN A MUTANT SOD1 RAT MODEL OF ALS

WEISS J, RAO S, YIN H

University of California, Irvine, CA, United States

E-mail address for correspondence: [email protected]

Keywords: G93A, Ca2+ permeable AMPA channels, GLT-1

Background: Loss of astrocytic glutamate uptake in ALS may underlie excitotoxic motor neuron (MN) damage in the disease. However, the reason for the loss of astrocyte glutamate transporter function has been unknown. In past studies we have examined factors underlying a high susceptibility of MNs to excitotoxic injury, and found that MNs possess large numbers of Ca^2 + permeable AMPA-type glutamate (Ca-AMPA) channels (1), and that MNs buffer cytosolic Ca^2 + loads poorly such that much of the Ca^2 + entering through these channels is taken up by mitochondria, with consequent disruption of mitochondrial function and strong reactive oxygen species (ROS) generation (2).

Objectives: The aim of this study was to test the hypothesis that ROS produced in MNs contributes to the dysfunction of astrocytic glutamate transporters.

Methods: In vitro studies employed mixed spinal cultures (containing neurons on a monolayer of astrocytes). In vivo studies used G93A SOD1 mutant rat models in which drug was infused intrathecally during the late presymptomatic period (from ∼ 67 to 97 days).

Results: In cultures, we found glutamate to cause far more ROS generation in MNs than in other spinal neurons. Furthermore, this ROS appears able to exit the MNs and induce oxidation and rapid disruption of glutamate transport in adjacent astrocytes (3). Subsequently, to begin to assess the contribution of Ca-AMPA channel activation in vivo, we have carried out 30 day intrathecal infusion of the Ca-AMPA channel blocker, naphthyl acetylspermine (NAS), in G93A mutant SOD1 rats (4). In wild type animals, immunoreactivity for the astrocytic glutamate transporter, GLT-1, was particularly strong around ventral horn MNs. However, a marked loss of ventral horn GLT-1 was observed, along with substantial MN damage, prior to onset of symptoms (90–100 d) in the G93A rats. Conversely, labeling with the oxidative marker, nitrotyrosine, was increased in the neuropil surrounding MNs in the transgenic animals. Compared to sham treated G93A animals, 30 day NAS infusions (starting at 67±2 days of age) markedly diminished the loss of both MNs and of astrocytic GLT-1 labeling.

Discussion and Conclusions: Our culture studies provide precedent for the possibility that MN ROS can disrupt astrocytic glutamate transport. The finding that NAS, which can decrease glutamate triggered ROS generation in MNs, slows the loss of astrocytic glutamate transport in G93A rats, suggests that this mechanism may contribute to transporter loss in vivo. As astrocyte dysfunction can clearly damage MNs, we propose that reciprocal deleterious interactions between MNs and surrounding astrocytes underlie a final common pathway of disease progression in ALS (5).

C44 RILUZOLE AND DEXAMETHASONE BUT NOT CEFTRIAXONE UPREGULATE GLUTAMATE TRANSPORT ACTIVITY AND GLUTAMATE TRANSPORTER EXPRESSION IN STRIATAL ASTROCYTES

CARBONE M¹, DUTY S¹, RATTRAY M²

¹King's College London, United Kingdom, ²University of Reading, United Kingdom

E-mail address for correspondence: [email protected]

Keywords: astrocyte, transporter, neurodegeneration

Glutamate transporters expressed in astrocytes are critical for maintaining the extracellular concentration of glutamate below toxic levels in the central nervous system. In light of a number of prominent reports in this area, we selected a number of drugs which might potentiate the activity of glutamate transporters and therefore be beneficial as neuroprotective agents in Motor Neurone Disease.

The objective of the work was to determine the ability of a number of drugs to cause elevations of protein levels of the main glutamate transporter, GLT-1 and enhance ³H-glutamate uptake activity in primary cultures of striatal astrocytes. In particular we wished to directly compare ceftriaxone, which has been reported to be a positive modulator of GLT-1 levels with other agents that have been proposed to upregulate GLT-1.

Primary striatal astrocyte cultures from E16 mouse embryos after 7 days in vitro were grown for 4 days in medium supplemented with G5, a defined cocktail of growth factors then treated for three days with dexamethasone (0.1 and 1 µM), riluzole (1–100 µM), zonisamide (1–1000 µM), citicholine (10–1000 µM), ceftriaxone (100 and 1000 µM), or vehicle as a control. The vehicle-treated cultures showed low GLT-1 levels resulting from growth factor withdrawal, and Western blot analysis showed that only dexamethasone (1 µM) and riluzole (100 µ M) maintained or induced increases in GLT-1 protein, whereas zonisamide, citicoline and ceftriaxone treatment did not elevate the protein level. Dexamethasone and riluzole caused elevations in GLT-1 mediated ³H-glutamate uptake, determined by estimating the amount of uptake that is sensitive to WAY-213613.

Our results show that there are some compounds which show promise as a positive modulators of GLT-1 levels, although ceftriaxone does not regulate GLT-1 under these assay conditions.

The work was funded by the Parkinson's Disease Society (UK).