The Down syndrome candidate dual-specificity tyrosine phosphorylation-regulated kinase 1A phosphorylates the neurodegeneration-related septin 4.

The dual-specific kinase DYRK1A (dual-specificity tyrosine phosphorylation-regulated kinase 1A) is the mammalian orthologue of the Drosophila minibrain (MNB) protein kinase and executes diverse roles in neuronal development and adult brain physiology. DYRK1A is overexpressed in Down syndrome (DS) and has recently been implicated in several neurodegenerative diseases. In an attempt to elucidate the molecular basis of its involvement in cognitive and neurodegeneration processes, we searched for novel proteins interacting with the kinase domain of DYRK1A in the adult mouse brain and identified septin 4 (SEPT4, also known as Pnutl2/CDCrel-2). SEPT4 is a member of the group III septin family of guanosine triphosphate hydrolases (GTPases), which has previously been found in neurofibrillary tangles of Alzheimer disease brains and in alpha-synuclein-positive cytoplasmic inclusions in Parkinson disease brains. In transfected mammalian cells, DYRK1A specifically interacts with and phosphorylates SEPT4. Phosphorylation of SEPT4 by DYRK1A was inhibited by harmine, which has recently been identified as the most specific inhibitor of DYRK1A. In support of a physiological relation in the brain, we found that Dyrk1A and Sept4 are co-expressed and co-localized in neocortical neurons. These findings suggest that SEPT4 is a substrate of DYRK1A kinase and thus provide a possible link for the involvement of DYRK1A in neurodegenerative processes and in DS neuropathologies.

Una nueva hipótesis para el origen del déficit neuronal y las alteraciones de la diferenciación neuronal asociadas al síndrome de Down: implicación del gen Minibrain / A new hypothesis for the neuronal deficit and the alterations in neuronal differentiation associated to Down syndrome: implication of the Minibrain gene

La disminución de neuronas, diversos defectos en la diferenciación neuronal y la aparición de síntomas neurodegenerativos están entre las alteraciones neuropatológicas que hacen del síndrome de Down (SD) la causa más frecuente de retraso mental. El SD se debe a la triplicación del cromosoma 21. En base a estudios genéticos y a la secuenciación de este cromosoma se han podido identificar los genes posiblemente más relevantes para la generación del SD, entre los cuales destaca Minibrain (Mnb). Dos han sido los objetivos de este trabajo: estudiar si Mnb podría estar implicado en la diferenciación neuronal y ver si la sobreexpresión de Mnb tiene efectos sobre muerte neuronal. Paralelamente se han intentado ver la relaciones de estas funciones del gen Mnb con las neuropatologías asociadas al SD. Exoerimentos llevados a cabo en modelos exaerimentales transgi!nicos demuestran que la sobreexpresión de Mnb genera muerte neuronal. Asimismo. los estudios de exaresión de Mnb durante el desarrollo tardío del cerebro sugiere; un papel de las Mnb-quinasas como elemento de señalización celular en el proceso de diferenciación neuronal. Todo ello contribuye a confeccionar una nueva hipótesis sobre las bases moleculares del déficit neuronal y las alteraciones de la diferenciación neuronal que se producen en el SD (AU)

The decrease of neuronal number, diverse defects in neuronal differentiation, and neurodegeneration are among the neuropathologic alterations which make DS the most frequent cause of mental retardation. DS is originated by triplication of chromosome 21. Based on genetic studies and the sequencing of chromosome 21, the possible most relevant genes for DS generation have been identified. Among them Minibrain (Mnb) appears the most likely candidate to explain some DS neuropathologies. Our work has approached two objectives: to study if Mnb could be involved in neuronal differentiation and find out if the overexpression of Mnb has an effect on cell death. In parallel, we have tried to establish the correlation of these functions of Mnb with the DS associated neuropathologies. By using transgenic experimental models, we have found that overexpression of Mnb induces neuronal death. Also, the expression of Mnb during late brain development suggests a role of Mnb-kinases as an important signaling element within the process of neuronal differentiation. Al1 to~ether.t hese results contribute to build a new hypothesis for the molecular basis of the neuronal deficit and alterations of neuronal differentiation associated to DS (AU)

Expression patterns and subcellular localization of the Down syndrome candidate protein MNB/DYRK1A suggest a role in late neuronal differentiation.

The Minibrain (Mnb) gene belongs to a new protein kinase family, which is evolutionarily conserved, and probably plays several roles during brain development and in adulthood. In Drosophila, mnb is involved in postembryonic neurogenesis and in learning/memory. In humans, MNB has been mapped within the Down syndrome critical region of chromosome 21 and is overexpressed in the Down syndrome embryonic brain. It has been widely proposed that MNB is involved in the neurobiological alterations associated with Down syndrome. Nevertheless, little is known about the functional role that MNB plays in vertebrate brain development. We have recently shown [Hämmerle et al. (2002) Dev. Biol., 246, 259-273] that in early vertebrate embryos, Mnb is transiently expressed in neural progenitor cells during the transition from proliferating to neurogenic divisions. Here we have studied in detail a second wave of Mnb expression, which takes place in the brain of intermediate and late vertebrate embryos. In these stages, MNB seems to be restricted to certain populations of neurons, as no consistent expression was detected in astroglial or oligodendroglial cells. Interestingly, MNB expression takes place at the time of dendritic tree differentiation and is initiated by a transient translocation from the cytoplasm to the nucleus. Afterwards, MNB protein is transported to the growing dendritic tree, where it colocalizes with Dynamin 1, a putative substrate of MNB kinases. We propose that MNB kinase is involved in the signalling mechanisms that regulate dendrite differentiation. This functional role helps to build a new hypothesis for the implication of MNB/DYRK1A in the developmental aetiology of Down syndrome neuropathologies.

Bases moleculares de las neuropatologías del síndrome de Down: implicación del gen Minibrain / Molecular basis of Down syndrome's neuropathologies: implication of the Minibrain gene

El síndrome de Down (SD) genera un amplio número de anomalías, de las cuales las más graves son las neuropatologías que hacen del SD la causa más frecuente de retraso mental. El SD se debe a la triplicación del cromosoma 21. En base a estudios genéticos y a la secuenciación de este cromosoma se han podido identificar los genes posiblemente más relevantes para la generación del SD, entre los cuales destaca Minibrain (Mnb).El objetivo de este trabajo ha sido generar modelos experimentales in vivo para estudiar si las bases moleculares de las neuropatologías asociadas con el SD podían resistir en la alteración del gen Mnb. Con este fin, y para poder utilizar embriones de pollo como modelo experimental, se clonó el ortólogo de Mnb en pollo. Estudios de expresión de Mnb junto con experimentos de sobreexpresión llevados a cabo en transgénicos, sugieren que este gen está implicado en diversas funciones como proliferación y diferenciación a lo largo del desarrollo del cerebro (AU)

The MNB/DYRK1A protein kinase: neurobiological functions and Down syndrome implications.

Major attention is being paid in recent years to the genes harbored within the so called Down syndrome Critical Region of human chromosome 21. Among them, those genes with a possible brain function are becoming the focus of intense research due to the numerous neurobiological alterations and cognitive deficits that Down syndrome individuals have. MNB/DYRK1A is one of these genes. It encodes a protein kinase with unique genetic and biochemical properties, which have been evolutionarily conserved from insects to humans. MNB/DYRK1A is expressed in the developing brain where it seems to play a role in proliferation of neural progenitor cells, neurogenesis, and neuronal differentiation. Although at a lower level, MNB/DYRK1A is also expressed in the adult brain where, as judged by the phenotype of mutant and transgenic animals, it may be involved in learning and memory. Nevertheless, most of the molecular mechanisms underlying these functions remain to be unraveled. In this review we compile and discuss experimental evidences, which support the involvement of MNB/DYRK1A in several neuropathologies and cognitive deficits of Down syndrome.

Mnb/Dyrk1A is transiently expressed and asymmetrically segregated in neural progenitor cells at the transition to neurogenic divisions.

The Minibrain (Mnb) gene encodes a new family of protein kinases that is evolutionarily conserved from insects to humans. In Drosophila, Mnb is involved in postembryonic neurogenesis. In humans, MNB has been mapped within the Down's Syndrome (DS) critical region of chromosome 21 and is overexpressed in DS embryonic brain. In order to study a possible role of Mnb on the neurogenesis of vertebrate brain, we have cloned the chick Mnb orthologue and studied the spatiotemporal expression of Mnb in proliferative regions of the nervous system. In early embryos, Mnb is expressed before the onset of neurogenesis in the three general locations where neuronal precursors are originated: neuroepithelia of the neural tube, neural crest, and cranial placodes. Mnb is transiently expressed during a single cell cycle of neuroepithelial progenitor (NEP) cells. Mnb expression precedes and widely overlaps with the expression of Tis21, an antiproliferative gene that has been reported to be expressed in the onset of neurogenic divisions of NEP cells. Mnb transcription begins in mitosis, continues during G(1), and stops before S-phase. Very interestingly, we have found that Mnb mRNA is asymmetrically localized during the mitosis of these cells and inherited by one of the sibling cells after division. We propose that Mnb defines a transition step between proliferating and neurogenic divisions of NEP cells.

A method for pulse and chase BrdU-labeling of early chick embryos.

Pulse and chase BrdU labeling of early chick embryos represents a serious technical problem due to the hindrance of removing unincorporated BrdU after the pulse. We have developed a simple method that allows BrdU washout and control of pulse/chase duration. In this method, BrdU pulses are carried out in ovo. Afterwards, embryos are removed from the yolk, BrdU is washed out, and the embryos are maintained in a wholemount culture. Under these conditions, HH8-12 embryos continue with their normal development for at least 30 h. Morphological development of the nervous system and cell cycle kinetics of precursor cells seem to be normally maintained in cultured embryos. To prove the feasibility of the method, it has been applied to determine the onset of TUJ1 expression. TUJ1 is frequently considered an early neuronal marker, yet some reports have shown its expression in dividing progenitor cells and differentiating neurons. The application of this new method demonstrates that TUJ1 is expressed in newborn neurons as early as 1 h after cell cycle exit.

scully, an essential gene of Drosophila, is homologous to mammalian mitochondrial type II L-3-hydroxyacyl-CoA dehydrogenase/amyloid-beta peptide-binding protein.

The characterization of scully, an essential gene of Drosophila with phenocritical phases at embryonic and pupal stages, shows its extensive homology with vertebrate type II L-3-hydroxyacyl-CoA dehydrogenase/ERAB. Genomic rescue demonstrates that four different lethal mutations are scu alleles, the molecular nature of which has been established. One of them, scu3127, generates a nonfunctional truncated product. scu4058 also produces a truncated protein, but it contains most of the known functional domains of the enzyme. The other two mutations, scu174 and scuS152, correspond to single amino acid changes. The expression of scully mRNA is general to many tissues including the CNS; however, it is highest in both embryonic gonadal primordia and mature ovaries and testes. Consistent with this pattern, the phenotypic analysis suggests a role for scully in germ line formation: mutant testis are reduced in size and devoid of maturing sperm, and mutant ovarioles are not able to produce viable eggs. Ultrastructural analysis of mutant spermatocytes reveals the presence of cytoplasmic lipid inclusions and scarce mitochondria. In addition, mutant photoreceptors contain morphologically aberrant mitochondria and large multilayered accumulations of membranous material. Some of these phenotypes are very similar to those present in human pathologies caused by beta-oxidation disorders.

Diverse expression and distribution of Shaker potassium channels during the development of the Drosophila nervous system.

The spatio-temporal expression of Shaker (Sh) potassium channels (Kch) in the developing and adult nervous system of Drosophila has been studied at the molecular and histological level using specific antisera. Sh Kch are distributed in most regions of the nervous system, but their expression is restricted to only certain populations of cells. Sh Kch have been found in the following three locations: in synaptic areas of neuropile, in axonal fiber tracks, and in a small number of neuronal cell bodies. This wide subcellular localization, together with a diverse distribution, implicates Sh Kch in multiple neuronal functions. Experiments performed with Sh mutants that specifically eliminate a few of the Sh Kch splice variants clearly demonstrate an abundant differential expression and usage of the wide repertoire of Sh isoforms, but they do not support the idea of extensive segregation of these isoforms among different populations of neurons. Sh Kch are predominantly expressed at late stages of postembryonic development and adulthood. Strikingly, wide changes in the repertoire of Sh splice isoforms occur some time after the architecture of the nervous system is complete, indicating that the expression of Sh Kch contributes to the final refinements of neuronal differentiation. These late changes in the expression and distribution of Sh Kch seem to correlate with activity patterns suggesting that Sh Kch may be involved in adaptative mechanisms of excitability.