Josette Carnahan

BDNF Protein Measured by a Novel Enzyme Immunoassay in Normal Brain and after Seizure: Partial Disagreement with mRNA Levels

Messenger RNA for brain‐derived neurotrophic factor (BDNF) is distributed in many brain regions and regulated by excitatory neuronal activity. Despite numerous studies of BDNF mRNA, the distribution and regulation of BDNF protein are poorly understood because of the difficulty of its quantitative measurement. We have established a two‐site enzyme immunoassay that detects trace amounts of BDNF protein (>1 pg/assay) but not other neurotrophins or growth factors. The highest levels of BDNF in adult rat brain were found in the hippocampus, followed by the hypothalamus, neocortex, cerebellum, thalamus and striatum. This pattern is similar, but not identical, to the distribution of BDNF mRNA. A similar disparity between BDNF protein and mRNA levels was observed in their changes after hilus lesion‐induced limbic seizures. In limbic structures, BDNF concentrations remained elevated 4 days after seizure onset, whereas BDNF mRNA has been reported previously to return to basal levels within 46 h. The temporal and spatial differences between the dynamics of protein and mRNA levels suggest the importance of post‐translational and/or subcellular processes for BDNF production. The persistence of the increases in BDNF content was also reflected in its biological activity, e.g. peptidergic differentiation activity. After limbic seizures, neuropeptide Y content was most markedly and persistently elevated in the entorhinal/amygdaloid region, where the most sustained up‐regulation of BDNF protein was observed. These results suggest that the sustained increase of BDNF protein in these limbic structures is involved in prolonged post‐seizure phenomena, including peptidergic alterations.

Regulation of Neuropeptide Expression in Cultured Cerebral Cortical Neurons by Brain‐Derived Neurotrophic Factor

Abstract: The neuropeptide‐inducing activity of neurotrophic factors was tested in cultured cerebral cortical neurons. Brain‐derived neurotrophic factor (BDNF) specifically increased contents of the neuropeptides somatostatin (SOM) and neuropeptide Y (NPY), but its effect on contents of cholecystokinin octapeptide and GABA was much less significant. The maximal induction of NPY content (15‐fold increase) was achieved by 20 ng/ml of BDNF. These changes were also reproduced at the mRNA level. In contrast, neurotrophin‐3 was much less potent at increasing NPY and SOM contents, and nerve growth factor had no effect on them. The expression of mRNA for NPY and SOM was fully dependent on the presence of BDNF in culture but irrelevant to the survival‐promoting activity of BDNF, which has been reported previously. Most of the NPY immunoreactivity induced by BDNF was colocalized with glutamate decarboxylase immunoreactivity in cultured cortical neurons. These results suggest that BDNF regulates the peptidergic expression of GABAergic neurons in the cerebral cortex.

Glial cell line-derived neurotrophic factor (GDNF) is a neurotrophic factor for sensory neurons: Comparison with the effects of the neurotrophins

We compared the effects of glial cell line-derived neurotrophic factor (GDNF) on dorsal root ganglion (DRG) sensory neurons to that of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin 3 (NT-3). All of these factors were retrogradely transported to subpopulations of sensory neuron cell bodies in the L4/ L5 DRG of neonatal rats. The size distribution of 125I-GDNF-labeled neurons was variable and consisted of both small and large DRG neurons (mean of 506.60 microns2). 125I-NGF was preferentially taken up by small neurons with a mean cross-sectional area of 383.03 microns2. Iodinated BDNF and NT-3 were transported by medium to large neurons with mean sizes of 501.48 and 529.27 microns2, respectively. A neonatal, sciatic nerve axotomy-induced cell death model was used to determine whether any of these factors could influence DRG neuron survival in vivo. GDNF and NGF rescued nearly 100% of the sensory neurons. BDNF and NT-3 did not promote any detectable level of neuronal survival despite the fact that they underwent retrograde transport. We examined the in vitro survival-promoting ability of these factors on neonatal DRG neuronal cultures derived from neonatal rats. GDNF, NGF, and NT-3 were effective in vitro, while BDNF was not. The range of effects seen in the models described here underscores the importance of testing neuronal responsiveness in more than one model. The biological responsiveness of DRG neurons to GDNF in multiple models suggests that this factor may play a role in the development and maintenance of sensory neurons.

Brain-derived neurotrophic factor regulates the expression of AMPA receptor proteins in neocortical neurons.

The role of the neurotrophins; nerve growth factor, brain-derived neurotrophic factor, neurotrophin-3 and neurotrophin-4/5, in synaptic development and plasticity has been extensively investigated. The neurotrophins regulate synaptic transmission as well as neural development in the brain. However, the mechanisms underlying these processes are unknown. In this study we show that brain-derived neurotrophic factor triggers an increase in alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptor (GluR) proteins without significant changes in their messenger RNA levels. Brain-derived neurotrophic factor treatment specifically increased the protein levels of GluR1 (193+/-22%) and GluR2/3 (182+/-11%) in cultured rat neocortical neurons. In contrast, nerve growth factor and neurotrophin-3 failed to alter the protein levels of these neurons, and brain-derived neurotrophic factor effects on N-methyl-D-aspartate-type glutamate receptors were either modest or negligible. Immunocytochemical studies indicated that the increase in AMPA receptor proteins reflects the induction of their neuronal expression, but not selective neuronal survival. In agreement with these results, cortical neurons from brain-derived neurotrophic factor-knockout mice exhibited a reduction in AMPA receptor proteins in the cytoskeletal fraction containing postsynaptic proteins. Thus, the neurotrophin plays a crucial role in modulating the expression of AMPA receptors presumably at translational or post-translation levels and is implicated in synaptic development and plasticity.

Protective effects of brain-derived neurotrophic factor on the development of hippocampal kindling in the rat

Recent data have suggested the involvement of neurotrophins in the cascade of events occurring during seizure development. In particular, expression of both brain-derived neurotrophic factor (BDNF) and its receptor mRNAs increases in different brain structures after convulsive seizures. The physiological significance of this increase was investigated by chronic intrahippocampal perfusion of BDNF in the model of dorsal hippocampal kindling in the rat. A 7 day perfusion of BDNF, in the region of the stimulating electrode, blocked the development of kindling during the perfusion period and for the following 15 days. These results provide in vivo evidence for a protective role of BDNF in the regulation of plasticity involved in epileptogenesis in adult brain.

Dynamic changes of brain-derived neurotrophic factor protein levels in the rat forebrain after single and recurring kindling-induced seizures

Regional levels of brain-derived neurotrophic factor protein were measured in the rat brain using enzyme immunoassay following seizures evoked by hippocampal kindling stimulations. One stimulation, which induced a brief, single episode of epileptiform activity in hippocampus and piriform cortex but not in parietal cortex or striatum, gave rise to a transient increase of brain-derived neurotrophic factor levels in dentate gyrus and CA3 region and a decrease in piriform cortex. After 40 rapidly recurring seizures, with epileptiform activity also involving parietal cortex and striatum, increases were observed in dentate gyrus, CA3 and CA1 regions, piriform cortex and striatum. Maximum levels were reached at 2-24 h and brain-derived neurotrophic factor then returned to baseline except in dentate gyrus, where elevated protein content was sustained for four days. The differential regulation of brain-derived neurotrophic factor protein levels in various forebrain structures, which only partly correlates to messenger RNA changes, could indicate regional differences in protein release, antero- or retrograde transport, or brain-derived neurotrophic factor promotor activation. The dynamic changes of brain-derived neurotrophic factor levels in regions involved in the generation and spread of seizure activity may regulate excitability and trigger plastic responses in the post-seizure period.

STK33 Kinase Activity Is Nonessential in KRAS-Dependent Cancer Cells

Despite the prevalence of KRAS mutations in human cancers, there remain no targeted therapies for treatment. The serine-threonine kinase STK33 has been proposed to be required for the survival of mutant KRAS-dependent cell lines, suggesting that small molecule kinase inhibitors of STK33 may be useful to treat KRAS-dependent tumors. In this study, we investigated the role of STK33 in mutant KRAS human cancer cells using RNA interference, dominant mutant overexpression, and small molecule inhibitors. As expected, KRAS downregulation decreased the survival of KRAS-dependent cells. In contrast, STK33 downregulation or dominant mutant overexpression had no effect on KRAS signaling or survival of these cells. Similarly, a synthetic lethal siRNA screen conducted in a broad panel of KRAS wild-type or mutant cells identified KRAS but not STK33 as essential for survival. We also obtained similar negative results using small molecule inhibitors of the STK33 kinase identified by high-throughput screening. Taken together, our findings refute earlier proposals that STK33 inhibition may be a useful therapeutic approach to target human KRAS mutant tumors.

Regulation of neuropeptide expression in the brain by neurotrophins. Potential role in vivo.

Neurotrophins, which are structurally related to nerve growth factor, have been shown to promote survival of various neurons. Recently, we found a novel activity of a neurotrophin in the brain: Brain-derived neurotrophic factor (BDNF) enhances expression of various neuropeptides. The neuropeptide differentiation activity was then compared among neurotrophins both in vivo and in vitro. In cultured neocortical neurons, BDNF and neurotrophin-5 (NT-5) remarkably increased levels of neuropeptide Y and somatostatin, and neurotrophin-3 (NT-3) also increased these peptides but required higher concentrations. At elevating substance P, however, NT-3 was as potent as BDNF. In contrast, NGF had negligible or no effect. Neurotrophins administered into neonatal brain exhibited slightly different potencies for increasing these neuropeptides: The most marked increase in neuropeptide Y levels was obtained in the neocortex by NT-5, whereas in the striatum and hippocampus by BDNF, although all three neurotrophins increased somatostatin similarly in all the brain regions examined. Overall spatial patterns of the neuropeptide induction were similar among the neurotrophins. Neurons in adult rat brain can also react with the neurotrophins and alter neuropeptide expression in a slightly different fashion. Excitatory neuronal activity and hormones are known to change expression of neurotrophins. Therefore, neurotrophins, neuronal activity, and hormones influence each other and all regulate neurotransmitter/peptide expression in developing and mature brain. Physiological implication of the neurotransmitter/peptide differentiation activities is also discussed.

Overexpression of neuropeptide Y induced by brain-derived neurotrophic factor in the rat hippocampus is long lasting

Brain‐derived neurotrophic factor (BDNF) plays an important role in hippocampal neuroplasticity. In particular, BDNF upregulation in the hippocampus by epileptic seizures suggests its involvement in the neuronal rearrangements accompanying epileptogenesis. We have shown previously that chronic infusion of BDNF in the hippocampus induces a long‐term delay in hippocampal kindling progression. Although BDNF has been shown to enhance the excitability of this structure upon acute application, long‐term transcriptional regulations leading to increased inhibition within the hippocampus may account for its suppressive effects on epileptogenesis. Therefore, the long‐term consequences of a 7‐day chronic intrahippocampal infusion of BDNF (12 μg/day) were investigated up to 2 weeks after the end of the infusion, on the expression of neurotransmitters contained in inhibitory hippocampal interneurons and which display anti‐epileptic properties. Our results show that BDNF does not modify levels of immunostaining for glutamic acid decarboxylase, the rate‐limiting enzyme for γ‐aminobutyric acid (GABA) synthesis, and somatostatin. Conversely, BDNF induces a long‐lasting increase of neuropeptide Y (NPY) in the hippocampus, measured by immunohistochemistry and radioimmunoassay, outlasting the end of the infusion by at least 7 days. The distribution of BDNF‐induced neuropeptide Y immunoreactivity is similar to the pattern observed in animals submitted to hippocampal kindling, with the exception of mossy fibres which only become immunoreactive following seizure activity. The enduring increase of neuropeptide Y expression induced by BDNF in the hippocampus suggests that this neurotrophin can trigger long‐term genomic effects, which may contribute to the neuroplasticity of this structure, in particular during epileptogenesis.

Mutual regulation between the intercellular messengers nitric oxide and brain‐derived neurotrophic factor in rodent neocortical neurons

The diffusible factors, nitric oxide (NO) and brain‐derived neurotrophic factor (BDNF) are both suggested to be intercellular messengers that have similar synaptic activities and developmental influences in the brain. In the present study, we have analysed their mutual regulation with respect to their production in rodent neocortical neurons. Some of the cultured rat neocortical neurons exhibited immunoreactivity for both neuronal NO synthase (NOS) and the BDNF receptor trkB. Neuronal NOS appeared to be activated autonomously and produced NO in culture as monitored by nitrite accumulation. Inhibition of the endogenous NO production in culture by a NOS inhibitor, NG‐monomethyl‐l‐arginine (NMMA), enhanced basal expression of BDNF mRNA and protein. Similarly, cerebroventricular administration of another NOS inhibitor, N‐ω‐nitro‐l‐arginine methylester (l‐NAME), but not d‐NAME or saline, increased BDNF content in the neocortex. In the opposite direction, however, BDNF appeared to function as a positive regulator for NO synthesis. Addition of BDNF upregulated the neuronal NOS expression as well as NO production in neocortical culture. In agreement, BDNF knock‐out mice exhibited significant impairment of neuronal NOS expression in the neocortex. Taken together, these observations suggest that the trans‐synaptic signalling molecules, NO and BDNF, influence the production of each other and mutually regulate the strength of their intercellular communications.