Ernest Arenas

Emerging roles of Wnts in the adult nervous system

The roles of the Wnt signalling pathway in several developmental processes, including synaptic differentiation, are well characterized. The expression of Wnt ligands and Wnt signalling components in the mature mammalian CNS suggests that this pathway might also play a part in synaptic maintenance and function. In fact, Wnts have a crucial role in synaptic physiology, as they modulate the synaptic vesicle cycle, the trafficking of neurotransmitter receptors and the interaction of these receptors with scaffold proteins in postsynaptic regions. In addition, Wnts participate in adult neurogenesis and protect excitatory synaptic terminals from amyloid-β oligomer toxicity. Here, the latest insights into the function of Wnt signalling in the adult nervous system and therapeutic opportunities for neurodegenerative diseases such as Alzheimers and Parkinsons disease are discussed.

Induction of a midbrain dopaminergic phenotype in Nurr1-overexpressing neural stem cells by type 1 astrocytes.

The implementation of neural stem cell lines as a source material for brain tissue transplants is currently limited by the ability to induce specific neurochemical phenotypes in these cells. Here, we show that coordinated induction of a ventral mesencephalic dopaminergic phenotype in an immortalized multipotent neural stem cell line can be achieved in vitro. This process requires both the overexpression of the nuclear receptor Nurr1 and factors derived from local type 1 astrocytes. Over 80% of cells obtained by this method demonstrate a phenotype indistinguishable from that of endogenous dopaminergic neurons. Moreover, this procedure yields an unlimited number of cells that can engraft in vivo and that may constitute a useful source material for neuronal replacement in Parkinsons disease.

GDNF prevents degeneration and promotes the phenotype of brain noradrenergic neurons in vivo

The locus coeruleus (LC), the main noradrenergic center in the brain, participates in many neural functions, as diverse as memory and motor output, and is severely affected in several neurodegenerative disorders of the CNS. GDNF, a neurotrophic factor initially identified as dopaminotrophic, was found to be expressed in several targets of central noradrenergic neurons in the adult rat brain. Grafting of genetically engineered fibroblasts expressing high levels of GDNF prevented > 80% of the 6-hydroxydopamine-induced degeneration of noradrenergic neurons in the LC in vivo. Moreover, GDNF induced a fasciculated sprouting and increased by 2.5-fold both tyrosine hydroxylase levels and the soma size of lesioned LC neurons. These findings reveal a novel and potent neurotrophic activity of GDNF that may have therapeutic applications in neurodegenerative disorders affecting central noradrenergic neurons, such as Alzheimers, Parkinsons, and Huntingtons diseases.

Differential regulation of midbrain dopaminergic neuron development by Wnt-1, Wnt-3a, and Wnt-5a

The Wnts are a family of glycoproteins that regulate cell proliferation, fate decisions, and differentiation. In our study, we examined the contribution of Wnts to the development of ventral midbrain (VM) dopaminergic (DA) neurons. Our results show that β-catenin is expressed in DA precursor cells and that β-catenin signaling takes place in these cells, as assessed in TOPGAL [Tcf optimal-promoter β-galactosidase] reporter mice. We also found that Wnt-1, -3a, and -5a expression is differentially regulated during development and that partially purified Wnts distinctively regulate VM development. Wnt-3a promoted the proliferation of precursor cells expressing the orphan nuclear receptor-related factor 1 (Nurr1) but did not increase the number of tyrosine hydroxylase-positive neurons. Instead, Wnt-1 and -5a increased the number of rat midbrain DA neurons in rat embryonic day 14.5 precursor cultures by two distinct mechanisms. Wnt-1 predominantly increased the proliferation of Nurr1+ precursors, up-regulated cyclins D1 and D3, and down-regulated p27 and p57 mRNAs. In contrast, Wnt-5a primarily increased the proportion of Nurr1+ precursors that acquired a neuronal DA phenotype and up-regulated the expression of Ptx3 and c-ret mRNA. Moreover, the soluble cysteine-rich domain of Frizzled-8 (a Wnt inhibitor) blocked endogenous Wnts and the effects of Wnt-1 and -5a on proliferation and the acquisition of a DA phenotype in precursor cultures. These findings indicate that Wnts are key regulators of proliferation and differentiation of DA precursors during VM neurogenesis and that different Wnts have specific and unique activity profiles.

Normal feeding behavior, body weight and leptin response require the neuropeptide Y Y2 receptor

Neuropeptide Y (NPY), a 36-amino-acid peptide widely expressed in the brain is involved in many physiological responses, including hypothalamic control of food intake and cardiovascular homeostasis. NPY mediates its effects through binding to the Y1, Y2 and Y5 G-protein-coupled receptors. Little is known of the role of the Y2 receptor in mediating the different NPY effects. We inactivated the Y2 receptor subtype in mice and found that these mice developed increased body weight, food intake and fat deposition. The null mutant mice showed an attenuated response to leptin administration but a normal response to NPY-induced food intake and intact regulation of re-feeding and body weight after starvation. An absence of the Y2 receptor subtype also affected the basal control of heart rate, but did not influence blood pressure. These findings indicate an inhibitory role for the Y2 receptor subtype in the central regulation of body weight and control of food intake.

A Wnt1-regulated genetic network controls the identity and fate of midbrain-dopaminergic progenitors in vivo

Midbrain neurons synthesizing the neurotransmitter dopamine play a central role in the modulation of different brain functions and are associated with major neurological and psychiatric disorders. Despite the importance of these cells, the molecular mechanisms controlling their development are still poorly understood. The secreted glycoprotein Wnt1 is expressed in close vicinity to developing midbrain dopaminergic neurons. Here, we show that Wnt1 regulates the genetic network, including Otx2 and Nkx2-2, that is required for the establishment of the midbrain dopaminergic progenitor domain during embryonic development. In addition, Wnt1 is required for the terminal differentiation of midbrain dopaminergic neurons at later stages of embryogenesis. These results identify Wnt1 as a key molecule in the development of midbrain dopaminergic neurons in vivo. They also suggest the Wnt1-controlled signaling pathway as a promising target for new therapeutic strategies in the treatment of Parkinsons disease.

Neurogenin 2 is required for the development of ventral midbrain dopaminergic neurons

Proneural genes are crucial regulators of neurogenesis and subtype specification in many areas of the nervous system; however, their function in dopaminergic neuron development is unknown. We report that proneural genes have an intricate pattern of expression in the ventricular zone of the ventral midbrain, where mesencephalic dopaminergic neurons are generated. Neurogenin 2 (Ngn2) and Mash1 are expressed in the ventral midline, while Ngn1, Ngn2 and Mash1 are co-localized more laterally in the ventricular zone. Ngn2 is also expressed in an intermediate zone immediately adjacent to the ventricular zone at the ventral midline. To examine the function of these genes, we analyzed mutant mice in which one or two of these genes were deleted (Ngn1, Ngn2 and Mash1) or substituted (Mash1 in the Ngn2 locus). Our results demonstrate that Ngn2 is required for the differentiation of Sox2+ ventricular zone progenitors into Nurr1+ postmitotic dopaminergic neuron precursors in the intermediate zone, and that it is also likely to be required for their subsequent differentiation into tyrosine hydroxylase-positive dopaminergic neurons in the marginal zone. Although Mash1 normally has no detectable function in dopaminergic neuron development, it could partially rescue the generation of dopaminergic neuron precursors in the absence of Ngn2. These results demonstrate that Ngn2 is uniquely required for the development of midbrain dopaminergic neurons.

Histone H2AX-dependent GABA A receptor regulation of stem cell proliferation

Stem cell self-renewal implies proliferation under continued maintenance of multipotency. Small changes in numbers of stem cells may lead to large differences in differentiated cell numbers, resulting in significant physiological consequences. Proliferation is typically regulated in the G1 phase, which is associated with differentiation and cell cycle arrest. However, embryonic stem (ES) cells may lack a G1 checkpoint. Regulation of proliferation in the ‘DNA damage’ S/G2 cell cycle checkpoint pathway is known for its role in the maintenance of chromatin structural integrity. Here we show that autocrine/paracrine γ-aminobutyric acid (GABA) signalling by means of GABAA receptors negatively controls ES cell and peripheral neural crest stem (NCS) cell proliferation, preimplantation embryonic growth and proliferation in the boundary-cap stem cell niche, resulting in an attenuation of neuronal progenies from this stem cell niche. Activation of GABAA receptors leads to hyperpolarization, increased cell volume and accumulation of stem cells in S phase, thereby causing a rapid decrease in cell proliferation. GABAA receptors signal through S-phase checkpoint kinases of the phosphatidylinositol-3-OH kinase-related kinase family and the histone variant H2AX. This signalling pathway critically regulates proliferation independently of differentiation, apoptosis and overt damage to DNA. These results indicate the presence of a fundamentally different mechanism of proliferation control in these stem cells, in comparison with most somatic cells, involving proteins in the DNA damage checkpoint pathway.

Differential effects of glial cell line-derived neurotrophic factor and neurturin on developing and adult substantia nigra dopaminergic neurons.

Abstract: Neurturin (NTN) and glial cell line‐derived neurotrophic factor (GDNF), two members of the GDNF family of growth factors, exert very similar biological activities in different systems, including the substantia nigra. Our goal in the present work was to compare their function and define whether nonoverlapping biological activities on midbrain dopaminergic neurons exist. We first found that NTN and GDNF are differentially regulated during postnatal development. NTN mRNA progressively decreased in the ventral mesencephalon and progressively increased in the striatum, coincident with a decrease in GDNF mRNA expression. This finding suggested distinct physiological roles for each factor in the nigrostriatal system. We therefore examined their function in ventral mesencephalon cultures and found that NTN promoted survival comparable with GDNF, but only GDNF induced sprouting and hypertrophy of developing dopaminergic neurons. We subsequently examined the ability of NTN to prevent the 6‐hydroxydopamine‐induced degeneration of adult dopaminergic neurons in vivo. Fibroblasts genetically engineered to deliver high levels of GDNF or NTN were grafted supranigrally. NTN was found to be as potent as GDNF at preventing the death of nigral dopaminergic neurons, but only GDNF induced tyrosine hydroxylase staining, sprouting, or hypertrophy of dopaminergic neurons. In conclusion, our results show selective survival‐promoting effects of NTN over wider survival, neuritogenic, and hypertrophic effects of GDNF on dopaminergic neurons in vitro and in vivo. Such differences are likely to underlie unique roles for each factor in postnatal development and may ultimately be exploited in the treatment of Parkinson’s disease.

Brain-derived neurotrophic factor, neurotrophin-3, and neurotrophin-4/5 prevent the death of striatal projection neurons in a rodent model of Huntington's disease

Abstract: Intrastriatal injection of quinolinate has been proven to be a very useful animal model to study the pathogenesis and treatment of Huntingtons disease. To determine whether growth factors of the neurotrophin family are able to prevent the degeneration of striatal projection neurons, cell lines expressing brain‐derived neurotrophic factor (BDNF), neurotrophin‐3 (NT‐3), or neurotrophin‐4/5 (NT‐4/5) were grafted in the adult rat striatum before quinolinate injection. Three days after lesioning, ongoing cell death was assessed by in situ detection of DNA fragmentation. In animals grafted with the control cell line, quinolinate injection induced a gradual cell loss that was differentially prevented by intrastriatal grafting of BDNF‐, NT‐3‐, or NT‐4/5‐secreting cells. Seven days after lesioning, we characterized striatal projection neurons that were protected by neurotrophins. Quinolinate injection, alone or in combination with the control cell line, induced a selective loss of striatal projection neurons. Grafting of a BDNF‐secreting cell line prevented the loss of all types of striatal projection neurons analyzed. Glutamic acid decarboxylase 67‐, preproenkephalin‐, and preprotachykinin A‐ but not prodynorphin‐expressing neurons were protected by grafting of NT‐3‐ or NT‐4/5‐secreting cells but with less efficiency than the BDNF‐secreting cells. Our findings show that neurotrophins are able to promote the survival of striatal projection neurons in vivo and suggest that BDNF might be beneficial for the treatment of striatonigral degenerative disorders, including Huntingtons disease.