HDAC3 Regulates the Transition to the Homeostatic Myelinating Schwann Cell State.

The formation of myelinating Schwann cells (mSCs) involves the remarkable biogenic process, which rapidly generates the myelin sheath. Once formed, the mSC transitions to a stable homeostatic state, with loss of this stability associated with neuropathies. The histone deacetylases histone deacetylase 1 (HDAC1) and HDAC2 are required for the myelination transcriptional program. Here, we show a distinct role for HDAC3, in that, while dispensable for the formation of mSCs, it is essential for the stability of the myelin sheath once formed-with loss resulting in progressive severe neuropathy in adulthood. This is associated with the prior failure to downregulate the biogenic program upon entering the homeostatic state leading to hypertrophy and hypermyelination of the mSCs, progressing to the development of severe myelination defects. Our results highlight distinct roles of HDAC1/2 and HDAC3 in controlling the differentiation and homeostatic states of a cell with broad implications for the understanding of this important cell-state transition.

Inhibition of Signal Transducer and Activator of Transcription 3 (STAT3) reduces neonatal hypoxic-ischaemic brain damage.

Hypoxic-ischaemic encephalopathy is a leading cause of child death, with high mortality and morbidity, including cerebral palsy, epilepsy and cognitive disabilities. Hypoxia-ischaemia (HI) strongly up-regulates Signal Transducer and Activator of Transcription 3 (STAT3) in the immature brain. Our aim was to establish whether STAT3 up-regulation is associated with neonatal HI-brain damage and evaluate the phosphorylated STAT3-contribution from different cell types in eliciting damage. We subjected postnatal day seven mice to unilateral carotid artery ligation followed by 60 min hypoxia. Neuronal STAT3-deletion reduced cell death, tissue loss, microglial and astroglial activation in all brain regions. Astroglia-specific STAT3-deletion also reduced cell death, tissue loss and microglial activation, although not as strongly as the deletion in neurons. Systemic pre-insult STAT3-blockade at tyrosine 705 (Y705) with JAK2-inhibitor WP1066 reduced microglial and astroglial activation to a more moderate degree, but in a pattern similar to the one produced by the cell-specific deletions. Our results suggest that STAT3 is a crucial factor in neonatal HI-brain damage and its removal in neurons or astrocytes, and, to some extent, inhibition of its phosphorylation via JAK2-blockade reduces inflammation and tissue loss. Overall, the protective effects of STAT3 inactivation make it a possible target for a therapeutic strategy in neonatal HI. Current data show that neuronal and astroglial STAT3 molecules are involved in the pathways underlying cell death, tissue loss and gliosis following neonatal hypoxia-ischaemia, but differ with respect to the target of their effect. Y705-phosphorylation contributes to hypoxic-ischaemic histopathology. Protective effects of STAT3 inactivation make it a possible target for a therapeutic strategy in neonatal hypoxia-ischaemia.

SncRNA715 Inhibits Schwann Cell Myelin Basic Protein Synthesis.

Myelin basic proteins (MBP) are major constituents of the myelin sheath in the central nervous system (CNS) and the peripheral nervous system (PNS). In the CNS Mbp translation occurs locally at the axon-glial contact site in a neuronal activity-dependent manner. Recently we identified the small non-coding RNA 715 (sncRNA715) as a key inhibitor of Mbp translation during transport in oligodendrocytes. Mbp mRNA localization in Schwann cells has been observed, but has not been investigated in much detail. Here we could confirm translational repression of Mbp mRNA in Schwann cells. We show that sncRNA715 is expressed and its levels correlate inversely with MBP in cultured Schwann cells and in the sciatic nerve in vivo. Furthermore we could reduce MBP protein levels in cultured Schwann cells by increasing the levels of the inhibitory sncRNA715. Our findings suggest similarities in sncRNA715-mediated translational repression of Mbp mRNA in oligodendrocytes and Schwann cells.

c-Jun in Schwann cells promotes axonal regeneration and motoneuron survival via paracrine signaling.

The AP-1 transcription factor c-Jun is a master regulator of the axonal response in neurons. c-Jun also functions as a negative regulator of myelination in Schwann cells (SCs) and is strongly reactivated in SCs upon axonal injury. We demonstrate here that, after injury, the absence of c-Jun specifically in SCs caused impaired axonal regeneration and severely increased neuronal cell death. c-Jun deficiency resulted in decreased expression of several neurotrophic factors, and GDNF and Artemin, both of which encode ligands for the Ret receptor tyrosine kinase, were identified as novel direct c-Jun target genes. Genetic inactivation of Ret specifically in neurons resulted in regeneration defects without affecting motoneuron survival and, conversely, administration of recombinant GDNF and Artemin protein substantially ameliorated impaired regeneration caused by c-Jun deficiency. These results reveal an unexpected function for c-Jun in SCs in response to axonal injury, and identify paracrine Ret signaling as an important mediator of c-Jun function in SCs during regeneration.

Role of the cellular prion protein in oligodendrocyte precursor cell proliferation and differentiation in the developing and adult mouse CNS.

There are numerous studies describing the signaling mechanisms that mediate oligodendrocyte precursor cell (OPC) proliferation and differentiation, although the contribution of the cellular prion protein (PrP(c)) to this process remains unclear. PrP(c) is a glycosyl-phosphatidylinositol (GPI)-anchored glycoprotein involved in diverse cellular processes during the development and maturation of the mammalian central nervous system (CNS). Here we describe how PrP(c) influences oligodendrocyte proliferation in the developing and adult CNS. OPCs that lack PrP(c) proliferate more vigorously at the expense of a delay in differentiation, which correlates with changes in the expression of oligodendrocyte lineage markers. In addition, numerous NG2-positive cells were observed in cortical regions of adult PrP(c) knockout mice, although no significant changes in myelination can be seen, probably due to the death of surplus cells.

Neuronal c-Jun is required for successful axonal regeneration, but the effects of phosphorylation of its N-terminus are moderate.

Although neural c-Jun is essential for successful peripheral nerve regeneration, the cellular basis of this effect and the impact of c-Jun activation are incompletely understood. In the current study, we explored the effects of neuron-selective c-Jun deletion, substitution of serine 63 and 73 phosphoacceptor sites with non-phosphorylatable alanine, and deletion of Jun N-terminal kinases 1, 2 and 3 in mouse facial nerve regeneration. Removal of the floxed c-jun gene in facial motoneurons using cre recombinase under control of a neuron-specific synapsin promoter (junΔS) abolished basal and injury-induced neuronal c-Jun immunoreactivity, as well as most of the molecular responses following facial axotomy. Absence of neuronal Jun reduced the speed of axonal regeneration following crush, and prevented most cut axons from reconnecting to their target, significantly reducing functional recovery. Despite blocking cell death, this was associated with a large number of shrunken neurons. Finally, junΔS mutants also had diminished astrocyte and microglial activation and T-cell influx, suggesting that these non-neuronal responses depend on the release of Jun-dependent signals from neighboring injured motoneurons. The effects of substituting serine 63 and 73 phosphoacceptor sites (junAA), or of global deletion of individual kinases responsible for N-terminal c-Jun phosphorylation were mild. junAA mutants showed decrease in neuronal cell size, a moderate reduction in post-axotomy CD44 levels and slightly increased astrogliosis. Deletion of Jun N-terminal kinase (JNK)1 or JNK3 showed delayed functional recovery; deletion of JNK3 also interfered with T-cell influx, and reduced CD44 levels. Deletion of JNK2 had no effect. Thus, neuronal c-Jun is needed in regeneration, but JNK phosphorylation of the N-terminus mostly appears to not be required for its function.

Bag1-L is a phosphorylation-dependent coactivator of c-Jun during neuronal apoptosis.

In the nervous system, cell death by apoptosis plays a critical role during normal development and pathological neurodegeneration. Jun N-terminal kinases (JNKs) are essential regulators of neuronal apoptosis. The AP-1 transcription factor c-Jun is phosphorylated at multiple sites within its transactivation domain by the JNKs, and c-Jun phosphorylation is required for JNK-induced neurotoxicity. While the importance of c-Jun as a mediator of apoptotic JNK signaling in neurons is firmly established, the molecular mechanism underlying the requirement for c-Jun N-terminal phosphorylation is enigmatic. Here we identify the multifunctional protein Bag1-L as a coactivator of phosphorylated c-Jun. Bag1-L preferentially interacts with N-terminally phosphorylated c-Jun, and Bag1-L greatly augments transcriptional activation by phosphorylated c-Jun. Chromatin immunoprecipitation experiments revealed binding of Bag1-L to the promoters of proapoptotic AP-1 target genes, and overexpression of Bag1-L augmented cell death in primary neurons. Therefore, Bag1-L functions as a coactivator regulating neurotoxicity mediated by phosphorylated c-Jun.

Radioimmunotherapy in relapsed follicular lymphoma previously treated by autologous bone marrow transplant: a report of eight new cases and literature review.

Multicenter, retrospective study of standard-dose RIT in eight heavily pre-treated patients with CD20-positive follicular lymphoma who had relapsed after previous autologous bone marrow transplantation (ABMT). Patients underwent nine courses of (90)Y-ibritumomab tiuxetan (0.3 or 0.4 mCi/kg body weight). Responses included five CR, two PR, one SD and one PD. Median DFS was 12 months with median follow-up of 17 months and 1-year OS was 83% (7/8 patients). Grade 4 thrombocytopenia occurred in 7/9 treatments, with no episodes of bleeding, and only two patients received a platelet transfusion. One patient, who had 20% bone marrow involvement at the time of relapse diagnosis, presented with Grade 4 thrombocytopenia and Grade 4 neutropenia and died of septic shock 6 months after RIT. One other case of Grade 4 neutropenia, without a serious infectious syndrome, was observed. Standard-dose RIT seems feasible and potentially effective after ABMT in correctly selected patients with follicular lymphoma.

Bcl-2 overexpression delays caspase-3 activation and rescues cerebellar degeneration in prion-deficient mice that overexpress amino-terminally truncated prion.

Prnp knockout mice that overexpress an amino-truncated form of PrPc (deltaPrP) are ataxic and display cerebellar cell loss and premature death. Studies on the molecular and intracellular events that trigger cell death in these mutants may contribute to elucidate the functions of PrPc and to the design of treatments for prion disease. Here we examined the effects of Bcl-2 overexpression in neurons on the development of the neurological syndrome and cerebellar pathology of deltaPrP. We show that deltaPrP overexpression activates the stress-associated kinases ERK1-2 in reactive astroglia, p38 and the phosphorylation of p53, which leads to the death of cerebellar neurons in mutant mice. We found that the expression of deltaPrP in cell lines expressing very low levels of PrPc strongly induces the activation of apoptotic pathways, thereby leading to caspase-3 activation and cell death, which can be prevented by coexpressing Bcl-2. Finally, we corroborate in vivo that neuronal-directed Bcl-2 overexpression in deltaPrP mice (deltaPrP Bcl-2) markedly reduces caspase-3 activation, glial activation, and neuronal cell death in cerebellum by improving locomotor deficits and life expectancy.

Semaphorin 6C leads to GSK-3-dependent growth cone collapse and redistributes after entorhino-hippocampal axotomy.

We studied the changes in the distribution of a specific variant of Semaphorin Y/6C (Sema6C) in mouse forebrain after axotomy of the entorhino-hippocampal perforant pathway. We found this isoform to be widely expressed during development, remaining in the adult and showing variations in distribution when the perforant pathway was axotomized. These changes were detected in both the hippocampal and entorhinal cortices. Sema6C1 immunoreactivity (IR) was high in the stratum radiatum of the hippocampus proper and the inner molecular layer of the dentate gyrus; the entorhinal cortex showed Sema6C1 IR in both cell bodies and in fibers of the II/III and V/VI layers. In axotomized animals, the IR of the ipsilateral, but not the contralateral, hemisphere showed that IR had moved into the stratum lacunosum-moleculare, the medial molecular layer of the dentate gyrus and the fibers, but not the cell bodies, of the entorhinal cortex. These results were not reproduced after lateral axotomy of the fimbria fornix, indicating a specific role for Sema6C variants in the generation and/or stability of entorhino-hippocampal synapses. Growth cone collapse of entorhinal and pyramidal neurons, as well as activation of glycogen synthase kinase-3 (GSK-3) through depletion of the inactive pool, induced by diffusible Sema6C1 further supports this view.

Cell proliferation in the adult hippocampal formation of rodents and its modulation by entorhinal and fimbria-fornix afferents.

New granule neurons are produced in the dentate gyrus (DG) of rodents throughout adult life. Recent studies have also reported adult neurogenesis in the cerebral cortex in normal animals or after brain injury. However, few of these studies focused on the hippocampal formation (HF), a cortical area involved in learning and memory in which extensive cell death occurs in neurodegenerative diseases. Thus, we studied cell proliferation in the HF of rodents and the intrinsic putative neurogenic potential of entorhinal cortex (EC) progenitors. We show that only the DG generates new neurons in the HF. In addition, neurospheres from the EC differentiate into neurons and glia in vitro and after transplantation in the adult DG. We also analyzed whether the absence of the synaptic input from the main hippocampal afferents induces neuronal generation in the HF outside the DG and/or regulates the proliferation of DG neuroprogenitors. We show that the denervation of the hippocampus does not induce neurogenesis in HF regions other than the DG. However, neuroprogenitor proliferation in the DG is reduced after fimbria-fornix lesions but not after entorhinal deafferentation, which supports the view that neuroprogenitor proliferation and/or differentiation in the DG are controlled from basal forebrain/septal neurons.

Expression, synaptic localization, and developmental regulation of Ack1/Pyk1, a cytoplasmic tyrosine kinase highly expressed in the developing and adult brain.

Cytosolic tyrosine kinases play a critical role both in neural development and in adult brain function and plasticity. Here we isolated a cDNA with high homology to human Ack1 and mouse Tnk2. This cDNA directs the expression of a 125-kD protein that can be autophosphorylated in tyrosines. Initially, this clone was named Pyk1 for proline-rich tyrosine kinase (Lev et al., 1995); however, since it corresponds to the mouse homolog of Ack1, here we called it Ack1/Pyk1. In this study we show that Ack1/Pyk1 mRNA and protein is highly expressed in the developing and adult brain. The highest levels of Ack1/Pyk1 expression were detected in the hippocampus, neocortex, and cerebellum. Electron microscopy studies showed that Ack1/Pyk1 protein is expressed in these regions both at dendritic spines and presynaptic axon terminals, indicating a role in synaptic function. Furthermore, we demonstrate that Ack1/Pyk1 mRNA levels are strongly upregulated by increased neural activity, produced by intraperitoneal kainate injections. During development, Ack1/Pyk1 was also expressed in the proliferative ventricular zones and in postmitotic maturing neurons. In neuronal cultures, Ack1/Pyk1 was detected in developing dendrites and axons, including dendritic tips and growth cones. Moreover, Ack1/Pyk1 colocalized with Cdc42 GTPase in neuronal cultures and coimmunoprecipitated with Cdc42 in HEK 293T cells. Altogether, our findings indicate that Ack1/Pyk1 tyrosine kinase may be involved both in adult synaptic function and plasticity and in brain development.

Overexpression of myelin-associated glycoprotein after axotomy of the perforant pathway.

Myelin-associated glycoprotein (MAG) contributes to the prevention of axonal regeneration in the adult central nervous system (CNS). However, changes in MAG expression following lesions and the involvement of MAG in the failure of cortical connections to regenerate are still poorly understood. Here, we show that MAG expression is differently regulated in the entorhinal cortex (EC) and the hippocampus in response to axotomy of the perforant pathway. In the EC, MAG mRNA is transiently overexpressed by mature oligodendrocytes after lesion. In the hippocampus, MAG overexpression is accompanied by an increase in the number of MAG-expressing cells. Lastly, the participation of MAG in preventing axonal regeneration was tested in vitro, where neuraminidase treatment of axotomized entorhino-hippocampal cultures potentiates axonal regeneration. These results demonstrate that MAG expression is regulated in response to cortical axotomy, and indicate that it may limit axonal regeneration after CNS injury.

Bcl-2 overexpression does not promote axonal regeneration of the entorhino-hippocampal connections in vitro after axotomy.

CNS lesions trigger cell death in injured neurons and glia. Genes of the bcl-2 family play crucial roles in the control of apoptosis and cell survival in the CNS. Recently, it has been suggested that overexpression of bcl-2 induces axonal elongation and regeneration in vitro and in vivo. Here, we analyze the regenerative potential of bcl-2 overexpression in the axotomized entorhino-hippocampal connection in organotypic slice cocultures. Our results show that in slice cocultures from bcl-2-overexpressing mice, there is a decrease in the number of dead neurons in the entorhinal cortex. In addition, axonal regeneration is not enhanced after axotomy. Thus, in the entorhino-hippocampal formation in vitro, bcl-2 overexpression rescues neurons from axotomy-induced cell death but fails to enhance the regeneration of the entorhino-hippocampal connection.

Regulation of Nogo and Nogo receptor during the development of the entorhino-hippocampal pathway and after adult hippocampal lesions.

Axonal regeneration in the adult CNS is limited by the presence of several inhibitory proteins associated with myelin. Nogo-A, a myelin-associated inhibitor, is responsible for axonal outgrowth inhibition in vivo and in vitro. Here we study the onset and maturation of Nogo-A and Nogo receptor in the entorhino-hippocampal formation of developing and adult mice. We also provide evidence that Nogo-A does not inhibit embryonic hippocampal neurons, in contrast to other cell types such as cerebellar granule cells. Our results also show that Nogo and Nogo receptor mRNA are expressed in the adult by both principal and local-circuit hippocampal neurons, and that after lesion, Nogo-A is also transiently expressed by a subset of reactive astrocytes. Furthermore, we analyzed their regulation after kainic acid (KA) treatment and in response to the transection of the entorhino-hippocampal connection. We found that Nogo-A and Nogo receptor are differentially regulated after kainic acid or perforant pathway lesions. Lastly, we show that the regenerative potential of lesioned entorhino-hippocampal organotypic slice co-cultures is increased after blockage of Nogo-A with two IN-1 blocking antibodies. In conclusion, our results show that Nogo and its receptor might play key roles during development of hippocampal connections and that they are implicated in neuronal plasticity in the adult.