Hiroko Yano

Neuregulin-1 Type III Determines the Ensheathment Fate of Axons

The signals that determine whether axons are ensheathed or myelinated by Schwann cells have long been elusive. We now report that threshold levels of neuregulin-1 (NRG1) type III on axons determine their ensheathment fate. Ensheathed axons express low levels whereas myelinated fibers express high levels of NRG1 type III. Sensory neurons from NRG1 type III deficient mice are poorly ensheathed and fail to myelinate; lentiviral-mediated expression of NRG1 type III rescues these defects. Expression also converts the normally unmyelinated axons of sympathetic neurons to myelination. Nerve fibers of mice haploinsufficient for NRG1 type III are disproportionately unmyelinated, aberrantly ensheathed, and hypomyelinated, with reduced conduction velocities. Type III is the sole NRG1 isoform retained at the axon surface and activates PI 3-kinase, which is required for Schwann cell myelination. These results indicate that levels of NRG1 type III, independent of axon diameter, provide a key instructive signal that determines the ensheathment fate of axons.

Akt1 Regulates a JNK Scaffold during Excitotoxic Apoptosis

Cell survival is determined by a balance among signaling cascades, including those that recruit the Akt and JNK pathways. Here we describe a novel interaction between Akt1 and JNK interacting protein 1 (JIP1), a JNK pathway scaffold. Direct association between Akt1 and JIP1 was observed in primary neurons. Neuronal exposure to an excitotoxic stimulus decreased the Akt1-JIP1 interaction and concomitantly increased association between JIP1 and JNK. Akt1 interaction with JIP1 inhibited JIP1-mediated potentiation of JNK activity by decreasing JIP1 binding to specific JNK pathway kinases. Consistent with this view, neurons from Akt1-deficient mice exhibited higher susceptibility to kainate than wild-type littermates. Overexpression of Akt1 mutants that bind JIP1 reduced excitotoxic apoptosis. These results suggest that Akt1 binding to JIP1 acts as a regulatory gate preventing JNK activation, which is released under conditions of excitotoxic injury.

A unique pathway for sustained neurotrophin signaling through an ankyrin‐rich membrane‐spanning protein

A major question in cell biology is how molecular specificity is achieved by different growth factor receptors that activate apparently identical signaling events. For the neurotrophin family, a distinguishing feature is the ability to maintain a prolonged duration of signal transduction. However, the mechanisms by which neurotrophin receptors assemble such a sustained signaling complex are not understood. Here we report that an unusual ankyrin‐rich transmembrane protein (ARMS+kidins220) is closely associated with Trk receptor tyrosine kinases, and not the EGF receptor. This association requires interactions between transmembrane domains of Trk and ARMS. ARMS is rapidly tyrosine phosphorylated after binding of neurotrophins to Trk receptors and provides a docking site for the CrkL–C3G complex, resulting in Rap1‐dependent sustained ERK activation. Accordingly, disruption of Trk–ARMS or the ARMS–CrkL interaction with dominant‐negative ARMS mutants, or treatment with small interference RNA against ARMS substantially reduce neurotrophin‐elicited signaling to ERK, but without any effect upon Ras or Akt activation. These findings suggest that ARMS acts as a major and neuronal‐specific platform for prolonged MAP kinase signaling by neurotrophins.

BDNF-mediated neurotransmission relies upon a myosin VI motor complex

Brain-derived neurotrophic factor (BDNF) has been implicated in higher-order cognitive functions and in psychiatric disorders such as depression and schizophrenia. BDNF modulates synaptic transmission and plasticity primarily through the TrkB receptor, but the molecules involved in BDNF-mediated synaptic modulation are largely unknown. Myosin VI (Myo6) is a minus end–directed actin-based motor found in neurons that express Trk receptors. Here we report that Myo6 and a Myo6-binding protein, GIPC1, form a complex that can engage TrkB. Myo6 and GIPC1 were necessary for BDNF-TrkB–mediated facilitation of long-term potentiation in postnatal day 12–13 (P12–13) hippocampus. Moreover, BDNF-mediated enhancement of glutamate release from presynaptic terminals depended not only upon TrkB but also upon Myo6 and GIPC1. Similar defects in basal synaptic transmission as well as presynaptic properties were observed in Myo6 and GIPC1 mutant mice. Together, these results define an important role for the Myo6-GIPC1 motor complex in presynaptic function and in BDNF-TrkB–mediated synaptic plasticity.

The Melatonin MT1 Receptor Axis Modulates Mutant Huntingtin-Mediated Toxicity

Melatonin mediates neuroprotection in several experimental models of neurodegeneration. It is not yet known, however, whether melatonin provides neuroprotection in genetic models of Huntingtons disease (HD). We report that melatonin delays disease onset and mortality in a transgenic mouse model of HD. Moreover, mutant huntingtin (htt)-mediated toxicity in cells, mice, and humans is associated with loss of the type 1 melatonin receptor (MT1). We observe high levels of MT1 receptor in mitochondria from the brains of wild-type mice but much less in brains from HD mice. Moreover, we demonstrate that melatonin inhibits mutant htt-induced caspase activation and preserves MT1 receptor expression. This observation is critical, because melatonin-mediated protection is dependent on the presence and activation of the MT1 receptor. In summary, we delineate a pathologic process whereby mutant htt-induced loss of the mitochondrial MT1 receptor enhances neuronal vulnerability and potentially accelerates the neurodegenerative process.

Neurotrophin receptor structure and interactions.

Although ligand-induced dimerization or oligomerization of receptors is a well established mechanism of growth factor signaling, increasing evidence indicates that biological responses are often mediated by receptor trans-signaling mechanisms involving two or more receptor systems. These include G protein-coupled receptors, cytokine, growth factor and trophic factor receptors. Greater flexibility is provided when different signaling pathways are merged through multiple receptor signaling systems. Trophic factors exemplified by NGF and its family members, ciliary neurotrophic factor (CNTF) and glial derived neurotrophic factor (GDNF) all utilize increased tyrosine phosphorylation of cellular substrates to mediate neuronal cell survival. Actions of the NGF family of neurotrophins are not only dictated by ras activation through the Trk family of receptor tyrosine kinases, but also a survival pathway defined by phosphatidylinositol-3-kinase activity (Yao and Cooper, 1995), which gives rise to phosphoinositide intermediates that activate the serine/threonine kinase Akt/PKB (Dudek et al., 1997). Induction of the serine-threonine kinase activity is critical for cell survival, as well as cell proliferation. Hence, for many trophic factors, multiple proteins constitute a functional multisubunit receptor complex that activates ras-dependent and ras-independent intracellular signaling. The NGF receptors provide an example of bidirectional crosstalk. In the presence of TrkA receptors, p75 can participate in the formation of high affinity binding sites and enhanced neurotrophin responsiveness leading to a survival or differentiation signal. In the absence of TrkA receptors, p75 can generate, in only specific cell populations, a death signal. These activities include the induction of NF kappa B (Carter et al., 1996); the hydrolysis of sphingomyelin to ceramide (Dobrowsky et al., 1995); and the pro-apoptotic functions attributed to p75. Receptors are generally drawn and viewed as isolated integral membrane proteins which span the lipid bilayer, with signal transduction proceeding in a linear step-wise fashion. There are now numerous examples which indicate that each receptor acts not only in a linear, independent manner, but can also influence the activity of other cell surface receptors, either directly or through signaling intermediates. Which step and which intermediates are utilized for crosstalk between the receptors is a critical question. For neurotrophins, their primary function in sustaining the viability of neurons is counterbalanced by a receptor mechanism to eliminate cells by an apoptotic mechanism. It is conceivable that this bidirectional system may be utilized selectively during development and in neurodegenerative diseases.

Inhibition of mitochondrial protein import by mutant huntingtin

Mitochondrial dysfunction is associated with neuronal loss in Huntingtons disease (HD), a neurodegenerative disease caused by an abnormal polyglutamine expansion in huntingtin (Htt). However, the mechanisms linking mutant Htt and mitochondrial dysfunction in HD remain unknown. We identify an interaction between mutant Htt and the TIM23 mitochondrial protein import complex. Remarkably, recombinant mutant Htt directly inhibited mitochondrial protein import in vitro. Furthermore, mitochondria from brain synaptosomes of presymptomatic HD model mice and from mutant Htt-expressing primary neurons exhibited a protein import defect, suggesting that deficient protein import is an early event in HD. The mutant Htt–induced mitochondrial import defect and subsequent neuronal death were attenuated by overexpression of TIM23 complex subunits, demonstrating that deficient mitochondrial protein import causes mutant Htt-induced neuronal death. Collectively, these findings provide evidence for a direct link between mutant Htt, mitochondrial dysfunction and neuronal pathology, with implications for mitochondrial protein import–based therapies in HD.

Association of the Abl tyrosine kinase with the Trk nerve growth factor receptor.

Nerve growth factor (NGF) initiates the majority of its biological effects by promoting the dimerization and activation of the tyrosine kinase receptor TrkA. In addition to rapid increases in the phosphorylation of phosphatidylinositol 3′‐kinase (PI 3‐kinase) and phospholipase C‐γ and increased ras activity, phosphorylation of c‐Crk and paxillin proteins has been observed upon TrkA activation. The c‐Abl tyrosine kinase is involved in the control of the axonal cytoskeleton and is known to interact with c‐Crk proteins. Here we have tested the possibility that TrkA receptors might form an association with the c‐Abl protein. After transfection in 293T cells, TrkA and c‐Abl kinases could be coimmunoprecipitated. This interaction did not require TrkA receptors to be autophosphorylated. Mapping analysis indicated that the region of c‐Abl association was confined to the juxtamembrane region of TrkA. The interaction of c‐Abl with TrkA was also observed in differentiated pheochromocytoma PC12 cells. These results suggest that c‐Abl may be recruited to the NGF receptor complex and be involved in regulating specific phosphorylation events that occur during neuronal differentiation. J. Neurosci. Res. 59:356–364, 2000

Identification of a Switch in Neurotrophin Signaling by Selective Tyrosine Phosphorylation

Neurotrophins, such as nerve growth factor and brain-derived neurotrophic factor, activate Trk receptor tyrosine kinases through receptor dimerization at the cell surface followed by autophosphorylation and recruitment of intracellular signaling molecules. The intracellular pathways used by neurotrophins share many common protein substrates that are used by other receptor tyrosine kinases (RTK), such as Shc, Grb2, FRS2, and phospholipase C-γ. Here we describe a novel RTK mechanism that involves a 220-kilodalton membrane tetraspanning protein, ARMS/Kidins220, which is rapidly tyrosine phosphorylated in primary neurons after neurotrophin treatment. ARMS/Kidins220 undergoes multiple tyrosine phosphorylation events and also serine phosphorylation by protein kinase D. We have identified a single tyrosine (Tyr1096) phosphorylation event in ARMS/Kidins220 that plays a critical role in neurotrophin signaling. A reassembled complex of ARMS/Kidins220 and CrkL, an upstream component of the C3G-Rap1-MAP kinase cascade, is SH3-dependent. However, Tyr1096 phosphorylation enables ARMS/Kidins220 to recruit CrkL through its SH2 domain, thereby freeing the CrkL SH3 domain to engage C3G for MAP kinase activation in a neurotrophin dependent manner. Accordingly, mutation of Tyr1096 abolished CrkL interaction and sustained MAPK kinase activity, a response that is not normally observed in other RTKs. Therefore, Trk receptor signaling involves an inducible switch mechanism through an unconventional substrate that distinguishes neurotrophin action from other growth factor receptors.

Apoptotic Functions of PDCD10/CCM3, the Gene Mutated in Cerebral Cavernous Malformation 3

Background and Purpose— Mutations in the Programmed Cell Death 10 (PDCD10) gene cause autosomal dominant familial cerebral cavernous malformations (CCM3). To date, little is known about the function of this gene and its role in disease pathogenesis. Methods— We examined the effects of overexpression of wild-type and 2 human disease-causing variants of PDCD10 on cell death using 3 different methods (TUNEL and MTT assays and caspase-3 activation). We analyzed expression of CCM3, activated caspase-3, and p38 in endothelial cell lines using the serum deprivation model of apoptosis induction. Finally, we assayed the effects of siRNA-mediated inhibition of endogenous PDCD10 expression on cell death in endothelial cell cultures. Results— Overexpression of wild-type CCM3, but not disease-linked mutant forms, induced apoptosis as confirmed by TUNEL and increased levels of activated caspase-3. Serum starvation of endothelial cells, an inducer of apoptosis, led to increased expression of CCM3 and activation of p38 and ultimately activated caspase-3. siRNA-mediated inhibition of CCM3 expression resulted in decreased levels of p38 and activated caspase-3, and decreased cell death. Conclusions— CCM3 is both necessary and sufficient to induce apoptosis in vitro in well-defined cell culture systems. Even though it is currently unclear whether this effect on apoptosis is direct or indirect through modulation of cell cycle, these results led to the novel hypothesis that CCM lesions may form as a consequence of aberrant apoptosis, potentially altering the balance between the endothelium and neural cells within the neurovascular unit.