Yoshito Kinoshita

Alternating phases of FGF receptor and NGF receptor expression in the developing chicken nervous system

Patterns of expression of transcripts encoding receptors for fibroblast growth factor and nerve growth factor (FGF-R and NGF-R) in the developing chick nervous system are compared using in situ hybridization histochemistry. FGF-R transcripts are expressed abundantly in the germinal neuroepithelial layer. Expression ceases as cells migrate into the mantle layer and returns during late maturation of neuronal populations, including cholinergic nuclei of the basal forebrain, brainstem reticular and motor nuclei, and cerebellar Purkinje and granule neurons. The pattern of NGF-R expression is generally reciprocal to that of FGF-R in the CNS and in some phases of development of the PNS. These results suggest that FGF and NGF may act sequentially rather than in concert during neuronal development.

p53-Dependent Cell Death Signaling in Neurons

The p53 tumor suppressor gene is a sequence-specific transcription factor that activates the expression of genes engaged in promoting growth arrest or cell death in response to multiple forms of cellular stress. p53 expression is elevated in damaged neurons in acute models of injury such as ischemia and epilepsy and in brain tissue samples derived from animal models and patients with chronic neurodegenerative diseases. p53 deficiency or p53 inhibition protects neurons from a wide variety of acute toxic insults. Signal transduction pathways associated with p53-induced neuronal cell death are being characterized, suggesting that intervention may prove effective in maintaining neuronal viability and restoring function following neural injury and disease.

Positive and negative effects of neurotrophins on the isthmo-optic nucleus in chick embryos

The survival of neurons in the developing isthmo-optic nucleus (ION) is believed to depend on the retrograde transport of trophic molecules from the target, the contralateral retina. We now show that ION neurons transport nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) retrogradely and that BDNF and NT-3 support the survival of ION neurons in vivo and promote neurite outgrowth in vitro. Surprisingly, NGF enhanced normal developmental cell death in vivo in a dose-dependent way. These findings show that increased levels of NGF can have adverse effects on differentiated neurons. The negative effect of NGF could be mimicked by intraocular injection of antibodies that block binding of neurotrophins to the 75 kd neurotrophin receptor (p75). These data implicate a role for the p75 receptor in NGFs neurotoxicity and indicate that this receptor is involved in the mechanism by which ION neurons respond to BDNF and NT-3 in the target.

Evidence for involvement of Bax and p53, but not caspases, in radiation-induced cell death of cultured postnatal hippocampal neurons

Bax (a death‐promoting member of the bcl‐2 gene family), the tumor suppressor gene product p53, and the ICE/ced‐3‐related proteases (caspases) have all been implicated in programmed cell death in a wide variety of cell types. However, their roles in radiation‐induced neuronal cell death are poorly understood. In order to further elucidate the molecular mechanisms underlying radiation‐induced neuronal cell death, we have examined the ability of ionizing radiation to induce cell death in primary cultured hippocampal neurons obtained from wild‐type, p53‐deficient and Bax‐deficient newborn mice. Survival in neuronal cultures derived from wild‐type mice decreased in a dose‐dependent manner 24 hr after a single 10 Gy to 30 Gy dose of ionizing radiation. In contrast, neuronal survival in irradiated cultures derived from p53‐deficient or Bax‐deficient mice was equivalent to that observed in control, nonirradiated cultures. Western blot analyses indicated that neuronal p53 protein levels increased after irradiation in wild‐type cells. However, Bax protein levels did not change, indicating that other mechanisms exist for regulating Bax activity. Adenovirus‐mediated overexpression of p53 also caused neuronal cell death without increasing Bax protein levels. Irradiation resulted in a significant induction in caspase activity, as measured by increased cleavage of fluorogenic caspase substrates. However, specific inhibitors of caspase activity (zVAD‐fmk, zDEVD‐fmk and BAF) failed to protect postnatal hippocampal neurons from radiation‐induced cell death. Staurosporine (a potent inducer of apoptosis in many cell types) effectively induced neuronal cell death in wild‐type, p53‐deficient and Bax‐deficient hippocampal neurons, indicating that all were competent to undergo programmed cell death. These results demonstrate that both p53 and Bax are necessary for radiation‐induced cell death in postnatal cultured hippocampal neurons. The fact that cell death occurred despite caspase inhibition suggests that radiation‐induced neuronal cell death may occur in a caspase‐independent manner. J. Neurosci. Res. 54:721–733, 1998. 

p53 and Mitochondrial Function in Neurons

The p53 tumor suppressor plays a central role in dictating cell survival and death as a cellular sensor for a myriad of stresses including DNA damage, oxidative and nutritional stress, ischemia and disruption of nucleolar function. Activation of p53-dependent apoptosis leads to mitochondrial apoptotic changes via the intrinsic and extrinsic pathways triggering cell death execution most notably by release of cytochrome c and activation of the caspase cascade. Although it was previously believed that p53 induces apoptotic mitochondrial changes exclusively through transcription-dependent mechanisms, recent studies suggest that p53 also regulates apoptosis via a transcription-independent action at the mitochondria. Recent evidence further suggests that p53 can regulate necrotic cell death and autophagic activity including mitophagy. An increasing number of cytosolic and mitochondrial proteins involved in mitochondrial metabolism and respiration are regulated by p53, which influences mitochondrial ROS production as well. Cellular redox homeostasis is also directly regulated by p53 through modified expression of pro- and anti-oxidant proteins. Proper regulation of mitochondrial size and shape through fission and fusion assures optimal mitochondrial bioenergetic function while enabling adequate mitochondrial transport to accommodate local energy demands unique to neuronal architecture. Abnormal regulation of mitochondrial dynamics has been increasingly implicated in neurodegeneration, where elevated levels of p53 may have a direct contribution as the expression of some fission/fusion proteins are directly regulated by p53. Thus, p53 may have a much wider influence on mitochondrial integrity and function than one would expect from its well-established ability to transcriptionally induce mitochondrial apoptosis. However, much of the evidence demonstrating that p53 can influence mitochondria through nuclear, cytosolic or intra-mitochondrial sites of action has yet to be confirmed in neurons. Nonetheless, as mitochondria are essential for supporting normal neuronal functions and in initiating/propagating cell death signaling, it appears certain that the mitochondria-related functions of p53 will have broader implications than previously thought in acute and progressive neurological conditions, providing new therapeutic targets for treatment.

Drp1 levels constitutively regulate mitochondrial dynamics and cell survival in cortical neurons.

Mitochondria exist as dynamic networks that are constantly remodeled through the opposing actions of fusion and fission proteins. Changes in the expression of these proteins alter mitochondrial shape and size, and may promote or inhibit the propagation of apoptotic signals. Using mitochondrially targeted EGFP or DsRed2 to identify mitochondria, we observed a short, distinctly tubular mitochondrial morphology in postnatal cortical neurons in culture and in retinal ganglion cells in vivo, whereas longer, highly interconnected mitochondrial networks were detected in cortical astrocytes in vitro and non-neuronal cells in the retina in vivo. Differential expression patterns of fusion and fission proteins, in part, appear to determine these morphological differences as neurons expressed markedly high levels of Drp1 and OPA1 proteins compared to non-neuronal cells. This finding was corroborated using optic tissue samples. Moreover, cortical neurons expressed several splice variants of Drp1 including a neuron-specific isoform which incorporates exon 3. Knockdown or dominant-negative interference of endogenous Drp1 significantly increased mitochondrial length in both neurons and non-neuronal cells, but caused cell death only in cortical neurons. Conversely, depletion of the fusion protein, Mfn2, but not Mfn1, caused extensive mitochondrial fission and cell death. Thus, Drp1 and Mfn2 in normal cortical neurons not only regulate mitochondrial morphology, but are also required for cell survival. The present findings point to unique patterns of Drp1 expression and selective vulnerability to reduced levels of Drp1 expression/activity in neurons, and demonstrate that the regulation of mitochondrial dynamics must be tightly regulated in neurons.

Global Analysis of the Cortical Neuron Proteome

In this study, a multidimensional fractionation approach was combined with MS/MS to increase the capability of characterizing complex protein profiles of mammalian neuronal cells. Proteins extracted from primary cultures of cortical neurons were digested with trypsin followed by fractionation using strong cation exchange chromatography. Each of these fractions was analyzed by microcapillary reversed-phase LC-MS/MS. The analysis of the MS/MS data resulted in the identification of over 15,000 unique peptides from which 3,590 unique proteins were identified based on protein-specific peptide tags that are unique to a single protein in the searched database. In addition, 952 protein clusters were identified using cluster analysis of the proteins identified by the peptides not unique to a single protein. This identification revealed that a minimum of 4,542 proteins could be identified from this experiment, representing ∼16% of all known mouse proteins. An evaluation of the number of false-positive identifications was undertaken by searching the entire MS/MS dataset against a database containing the sequences of over 12,000 proteins from archaea. This analysis allowed a systematic determination of the level of confidence in the identification of peptides as a function of SEQUEST cross correlation (Xcorr) and delta correlation (ΔCn) scores. Correlation charts were also constructed to show the number of unique peptides identified for proteins from specific classes. The results show that low-abundance proteins involved in signal transduction and transcription are generally identified by fewer peptides than high-abundance proteins that play a role in maintaining mammalian cellular structure and motility. The results presented here provide the broadest proteome coverage for a mammalian cell to date and show that MS-based proteomics has the potential to provide high coverage of the proteins expressed within a cell.

Proteomic Analysis in the Neurosciences

Proteomics is a field of study directed toward providing a comprehensive view of the characteristics and activity of every cellular protein. Rapid innovations in the core technologies required to characterize proteins on a global scale are poised to bring about a comprehensive understanding of how dynamic changes in protein expression, post-translational modification, and function affect complex signaling and regulatory networks. These advances have significant implications for understanding the multitude of pathways that govern behavior and cognition and the response of the nervous system to injury and disease.

Apoptotic actions of p53 require transcriptional activation of PUMA and do not involve a direct mitochondrial/cytoplasmic site of action in postnatal cortical neurons.

Recent studies in non-neuronal cells have shown that the tumor suppressor p53 can promote cell death through a transcription-independent mechanism involving its direct action with a subset of Bcl-2 family member proteins in the cytosol and at the mitochondria. In cultured cortical neurons, however, we could not find evidence supporting a significant contribution of the cytosolic/mitochondrial p53 pathway, and available evidence instead corroborated the requirement for the transcriptional activity of p53. When directly targeted to the cytosol/mitochondria, wild-type p53 lost its apoptosis-inducing activity in neurons but not in non-neuronal cells. The N-terminal p53 fragment (transactivation and proline-rich domains), which induces apoptosis in non-neuronal cells via the cytosolic/mitochondrial pathway, displayed no apoptogenic activity in neurons. In neuronal apoptosis induced by camptothecin or an MDM2 (murine double minute 2) inhibitor, nutlin-3, endogenous p53 protein did not accumulate in the cytosol/mitochondria, and transcriptional inhibition after p53 induction effectively blocked cell death. In addition, overexpression of a dominant-negative form of p53 (R273H) completely suppressed induction of proapoptotic p53 target genes and cell death. PUMA (p53-upregulated modulator of apoptosis) was one such gene induced by camptothecin, and its overexpression was sufficient to induce Bax (Bcl-2-associated X protein)-dependent neuronal death, whereas Noxa was not apoptogenic. These results collectively demonstrate that, in contrast to non-neuronal cells, the apoptotic activity of p53 in postnatal cortical neurons does not rely on its direct action at the cytosol/mitochondria but is exclusively mediated through its transcription-dependent functions. The uniqueness of p53-mediated apoptotic signaling in postnatal cortical neurons was further illustrated by the dispensable function of the proline-rich domain of p53.

Neurotrophic factor regulation of developing avian oculomotor neurons: differential effects of BDNF and GDNF.

Neurotrophic factors support the development of motoneurons by several possible mechanisms. Neurotrophins may act as target-derived factors or as afferent factors derived from the central nervous system (CNS) or sensory ganglia. We tested whether brain-derived neurotrophic factor (BDNF), neurotrophin 3 (NT-3), neurotrophin 4 (NT-4), and glial cell line-derived neurotrophic factor (GDNF) may be target-derived factors for neurons in the oculomotor (MIII) or trochlear (MIV) nucleus in chick embryos. Radio-iodinated BDNF, NT-3, NT-4, and GDNF accumulated in oculomotor neurons via retrograde axonal transport when the trophic factors were applied to the target. Systemic GDNF rescued oculomotor neurons from developmental cell death, while BDNF and NT-3 had no effect. BDNF enhanced neurite outgrowth from explants of MIII and MIV nuclei (identified by retrograde labeling in ovo with the fluorescent tracer DiI), while GDNF, NT-3, and NT-4 had no effect. The oculomotor neurons were immunoreactive for BDNF and the BDNF receptors p75(NTR) and trkB. To determine whether BDNF may be derived from its target or may act as an autocrine or paracrine factor, in situ hybridization and deprivation studies were performed. BDNF mRNA expression was detected in eye muscles, but not in CNS sources of afferent innervation to MIII, or the oculomotor complex itself. Injection of trkB fusion proteins in the eye muscle reduced BDNF immunoreactivity in the innervating motoneurons. These data indicate that BDNF trophic support for the oculomotor neurons was derived from their target.