Astrocytes from cortex and striatum show differential responses to mitochondrial toxin and BDNF: implications for protection of striatal neurons expressing mutant huntingtin.

BACKGROUND: Evidence shows significant heterogeneity in astrocyte gene expression and function. We previously demonstrated that brain-derived neurotrophic factor (BDNF) exerts protective effects on whole brain primary cultured rat astrocytes treated with 3-nitropropionic acid (3NP), a mitochondrial toxin widely used as an in vitro model of Huntington's disease (HD). Therefore, we now investigated 3NP and BDNF effects on astrocytes from two areas involved in HD: the striatum and the entire cortex, and their involvement in neuron survival. METHODS: We prepared primary cultured rat cortical or striatal astrocytes and treated them with BDNF and/or 3NP for 24 h. In these cells, we assessed expression of astrocyte markers, BDNF receptor, and glutamate transporters, and cytokine release. We prepared astrocyte-conditioned medium (ACM) from cortical and striatal astrocytes and tested its effect on a cellular model of HD. RESULTS: BDNF protected astrocytes from 3NP-induced death, increased expression of its own receptor, and activation of ERK in both cortical and striatal astrocytes. However, BDNF modulated glutamate transporter expression differently by increasing GLT1 and GLAST expression in cortical astrocytes but only GLT1 expression in striatal astrocytes. Striatal astrocytes released higher amounts of tumor necrosis factor-α than cortical astrocytes in response to 3NP but BDNF decreased this effect in both populations. 3NP decreased transforming growth factor-ß release only in cortical astrocytes, whereas BDNF treatment increased its release only in striatal astrocytes. Finally, we evaluated ACM effect on a cellular model of HD: the rat striatal neuron cell line ST14A expressing mutant human huntingtin (Q120) or in ST14A cells expressing normal human huntingtin (Q15). Neither striatal nor cortical ACM modified the viability of Q15 cells. Only ACM from striatal astrocytes treated with BDNF and ACM from 3NP + BDNF-treated striatal astrocytes protected Q120 cells, whereas ACM from cortical astrocytes did not. CONCLUSIONS: Data suggest that cortical and striatal astrocytes respond differently to mitochondrial toxin 3NP and BDNF. Moreover, striatal astrocytes secrete soluble neuroprotective factors in response to BDNF that selectively protect neurons expressing mutant huntingtin implicating that BDNF modulation of striatal astrocyte function has therapeutic potential against neurodegeneration.

Potentiation of Methylmercury-Induced Death in Rat Cerebellar Granular Neurons Occurs by Further Decrease of Total Intracellular GSH with BDNF via TrkB in Vitro.

Brain-derived neurotrophic factor (BDNF) is a principal factor for neurogenesis, neurodevelopment and neural survival through a BDNF receptor, tropomyosin-related kinase (Trk) B, while BDNF can also cause a decrease in the intracellular glutathione (GSH) level. We investigated the exacerbation of methylmercury-induced death of rat cerebellar granular neurons (CGNs) by BDNF in vitro. Since methylmercury can decrease intracellular GSH levels, we hypothesized that a further decrease of the intracellular GSH level is involved in the process of the exacerbation of neuronal cell death. In the present study, we established that in CGN culture, a decrease of the intracellular GSH level was further potentiated with BDNF in the process of the methylmercury-induced neuronal death and also in GSH reducer-induced neuronal death. BDNF treatment promoted the decrease in GSH levels induced by methylmercury and also by L-buthionine sulfoximine (BSO) and diethyl maleate (DEM). The promoting effect of BDNF was observed in a TrkB-vector transformant of the rat neuroblastoma B35 cell line but not in the mock-vector transformant. These results indicate that the exacerbating effect of BDNF on methylmercury-induced neuronal death in cultures of CGNs includes a further decrease of intracellular GSH levels, for which TrkB is essential.

proBDNF Attenuates Hippocampal Neurogenesis and Induces Learning and Memory Deficits in Aged Mice.

Mature brain-derived neurotrophic factor has shown promotive effect on neural cells in rodents, including neural proliferation, differentiation, survival, and synaptic formation. Conversely, the precursor of brain-derived neurotrophic factor (proBDNF) has been emerging as a differing protein against its mature form, for its critical role in aging process and neurodegenerative diseases. In the present study, we investigated the role of proBDNF in neurogenesis in the hippocampal dentate gyrus of aged mice and examined the changes in mice learning and memory functions. The results showed that the newborn cells in the hippocampus revealed a significant decline in proBDNF-treated group compared with bovine serum albumin group, but an elevated level in anti-proBDNF group. During the maturation period, no significant change was observed in the proportions of phenotype of the newborn cells among the three groups. In water maze, proBDNF-treated mice had poorer scores in place navigation test and probe test, compared with those from any other group. Thus, we conclude that proBDNF attenuates neurogenesis in the hippocampus and induces the deficits in learning and memory functions of aged mice.

Acceleration of methylmercury-induced cell death of rat cerebellar neurons by brain-derived neurotrophic factor in vitro.

Brain-derived neurotrophic factor (BDNF) is a member of the nerve growth factor (NGF) family and has been shown to promote neuronal survival and contribute to neural development. Although methylmercury, a neurotoxin, induces the cell death of neurons in vitro, there is little information regarding the effects of neurotrophins on the methylmercury-induced cell death of neurons. In the present study, we investigated the effect of BDNF on methylmercury-induced cell death in a primary culture of rat cerebellar granular cells. BDNF increased the viability of the cultured cells when treated alone, but unexpectedly accelerated the cell death induced by administration of methylmercury. Among other growth factors tested, only neurotrophin-4 (NT-4) demonstrated a similar acceleration of methylmercury-induced cell death. The cell death-accelerating effect of BDNF was inhibited by a BDNF-neutralizing antibody or a MAPK inhibitor. To determine whether the effect of BDNF occurs via TrkB, a receptor of BDNF and NT-4, we investigated the effects of BDNF and methylmercury in a TrkB transformant of rat neuroblastoma B35 cells. The methylmercury-induced cell death of the TrkB transformant was accelerated by BDNF, while that of the mock transformant was not. These results indicate that BDNF accelerates methylmercury-induced cell death via TrkB, at least in vitro, and suggest that BDNF and TrkB may also contribute to the sensitivity of neurons to methylmercury toxicity.

Synergistic neurochemical and behavioural effects of acute intrahippocampal injection of brain-derived neurotrophic factor and antidepressants in adult mice.

Preclinical data support the view that brain-derived neurotrophic factor (BDNF) and serotonergic systems regulate circuits involved in affective disorders. The present study examined neurochemical and behavioural consequences of an acute intrahippocampal injection of BDNF combined with an antidepressant by using in-vivo intracerebral microdialysis in the ventral hippocampus (vHi) in conscious mice and behavioural paradigms predictive of antidepressant and anxiolytic-like effects [the mouse forced swim test (FST), the open-field (OF) paradigm and the elevated plus maze (EPM)]. Neurochemical data revealed that BDNF (100 ng) potentiated the effects of the systemic administration of a serotonin selective reuptake inhibitor (SSRI; paroxetine 4 mg/kg i.p.) and that of a locally applied citalopram perfusion on dialysate 5-HT levels in the vHi. These neurochemical changes correlated with behavioural data since, in the FST, antidepressant-like activity of paroxetine as measured on swimming behaviour was potentiated by BDNF. These data suggest an interesting synergy between BDNF and SSRI on antidepressant-like activity. Furthermore, in both the OF and EPM paradigms BDNF induced an anxiogenic-like activity, whereas paroxetine prevented this effect. Finally, the neurochemical and behavioural effects of BDNF on the serotonergic system might occur at both pre- and post-synaptic levels since by using in-situ hybridization, we showed that TrkB-R mRNA was expressed in the hippocampus and the dorsal raphe nucleus in adult mice. Taken together the neurochemical and behavioural effects of BDNF suggest that these behavioural changes were mediated by increases in 5-HT neurotransmission in vHi. Thus a BDNF+SSRI combination may offer new alternatives to treat mood disorders.

Tolerability of recombinant-methionyl human neurotrophin-3 (r-metHuNT3) in healthy subjects.

This phase I, double-blind, randomized, placebo-controlled study evaluated the safety of single and multiple (daily for 7 days) subcutaneous administrations of recombinant-methionyl human neurotrophin-3 (r-metHuNT3) in healthy human volunteers at seven doses, ranging from 3 to 500 microg/kg/day. No serious or life-threatening adverse events occurred. The most frequently recorded adverse effects were mild injection-site pain, diarrhea, and elevation of liver function tests. No change in neurologic function was noted with these dosing regimens. We conclude that r-metHuNT3 is safe and well tolerated in the dosages used in this study.

Granule neuron regulation of Purkinje cell development: striking a balance between neurotrophin and glutamate signaling.

Granule neurons, presynaptic afferents of Purkinje cells, are potent regulators of Purkinje cell development. Purified Purkinje cells survive and differentiate poorly, whereas coculture with granule neurons enhances their survival and dendritic development. Here we investigate the role of neurotrophins in granule-Purkinje cell interactions. BDNF or NT-4 improves, but NT-3 or CNTF reduces, survival of isolated Purkinje cells. When granule neurons are present, however, BDNF or NT-4 treatment leads to Purkinje cell loss. This decrease is overcome by anti-BDNF or TrkB-IgG-blocking reagents or by CNQX, a non-NMDA glutamate receptor antagonist. Furthermore, BDNF increases the spine density on the surviving Purkinje cells. These results suggest that Purkinje cell survival and differentiation are context-dependent and require a balance between neurotrophin- and activity-dependent signaling.

Kinetic and safety studies on intrathecally infused recombinant-methionyl human brain-derived neurotrophic factor in dogs.

To define the kinetics and safety of spinally infused recombinant-methionyl human brain-derived neurotrophic factor (r-metHuBDNF), beagle dogs were prepared with lumbar intrathecal catheters passed through the cisternal membrane to the L1-L4 lumbar level. For kinetic studies, r-metHuBDNF was delivered by bolus or infusion through one catheter and lumbar CSF was sampled periodically through a second. As a lumbar bolus, r-metHuBDNF displayed a biphasic clearance with t(1/2)a = 0.7 hr and t(1/2)b = 7. 9 hr. Lumbar to cisternal concentrations after bolus delivery were approximately 60:1. For safety studies, dogs received continuous intrathecal infusion (2.4 ml/day) for 28 days of saline (n = 6), r-metHuBDNF at 200 (n = 6), 800 (n = 6), or 2000 (n = 7) microg/day. Control dogs showed no changes. Intrathecally infused r-metHuBDNF produced a dose-dependent increase in muscle tone and decreased coordination. Low-dose r-metHuBDNF was associated with moderate increases in muscle tone after 22-28 days of infusion. No clinically important changes were noted in rectal temperature, arterial pressure, respiration and heart rate, body weight, food consumption, stool or urine output, or change in blood chemistries measured throughout the study. Cisternal CSF protein and glucose sampled at 28 days were not different between dose groups and all cultures were negative. Histopathological examination of the spinal cord typically revealed some degree of chronic inflammation around the catheter, including fibrotic adhesions and focal accumulations of lymphoid and plasma cells, but these effects were not dose dependent. In other dogs receiving r-metHuBDNF (2000 or 4000 microg/day), termination of infusion resulted in significant recovery.

Neuronal heterotopias in the developing cerebral cortex produced by neurotrophin-4.

The marginal zone (MZ) of embryonic neocortex is crucial to its normal development. We report that neurotrophin-4 (but not NT3 or NGF), applied to embryonic rodent cortex in vitro or in vivo, produces heterotopic accumulations of neurons in the MZ. Although heterotopia production is TrkB mediated, BDNF is >10-fold less effective than NT4. Heterotopic neurons have the same birth date and phenotype as normal MZ neurons; they are not the result of NT4-induced proliferation or rescue from apoptosis. We suggest that NT4 causes excess neurons to migrate into the MZ and thus may play a role in normal MZ formation as well as in the pathogenesis of certain human cortical dysplasias.