Rnd3 interacts with TAO kinases and contributes to mitotic cell rounding and spindle positioning.

Rnd3 is an atypical Rho family protein that is constitutively GTP bound, and acts on membranes to induce loss of actin stress fibers and cell rounding. Phosphorylation of Rnd3 promotes 14-3-3 binding and its relocation to the cytosol. Here, we show that Rnd3 binds to the thousand-and-one amino acid kinases TAOK1 and TAOK2 in vitro and in cells. TAOK1 and TAOK2 can phosphorylate serine residues 210, 218 and 240 near the C-terminus of Rnd3, and induce Rnd3 translocation from the plasma membrane to the cytosol. TAOKs are activated catalytically during mitosis and Rnd3 phosphorylation on serine 210 increases in dividing cells. Rnd3 depletion by RNAi inhibits mitotic cell rounding and spindle centralization, and delays breakdown of the intercellular bridge between two daughter cells. Our results show that TAOKs bind, phosphorylate and relocate Rnd3 to the cytosol and that Rnd3 contributes to mitotic cell rounding, spindle positioning and cytokinesis. Rnd3 can therefore participate in the regulation of early and late mitosis and may also act downstream of TAOKs to affect the cytoskeleton.

A new TAO kinase inhibitor reduces tau phosphorylation at sites associated with neurodegeneration in human tauopathies.

In Alzheimer's disease (AD) and related tauopathies, the microtubule-associated protein tau is highly phosphorylated and aggregates to form neurofibrillary tangles that are characteristic of these neurodegenerative diseases. Our previous work has demonstrated that the thousand-and-one amino acid kinases (TAOKs) 1 and 2 phosphorylate tau on more than 40 residues in vitro. Here we show that TAOKs are phosphorylated and active in AD brain sections displaying mild (Braak stage II), intermediate (Braak stage IV) and advanced (Braak stage VI) tau pathology and that active TAOKs co-localise with both pre-tangle and tangle structures. TAOK activity is also enriched in pathological tau containing sarkosyl-insoluble extracts prepared from AD brain. Two new phosphorylated tau residues (T123 and T427) were identified in AD brain, which appear to be targeted specifically by TAOKs. A new small molecule TAOK inhibitor (Compound 43) reduced tau phosphorylation on T123 and T427 and also on additional pathological sites (S262/S356 and S202/T205/S208) in vitro and in cell models. The TAOK inhibitor also decreased tau phosphorylation in differentiated primary cortical neurons without affecting markers of synapse and neuron health. Notably, TAOK activity also co-localised with tangles in post-mortem frontotemporal lobar degeneration (FTLD) brain tissue. Furthermore, the TAOK inhibitor decreased tau phosphorylation in induced pluripotent stem cell derived neurons from FTLD patients, as well as cortical neurons from a transgenic mouse model of tauopathy (Tau35 mice). Our results demonstrate that abnormal TAOK activity is present at pre-tangles and tangles in tauopathies and that TAOK inhibition effectively decreases tau phosphorylation on pathological sites. Thus, TAOKs may represent a novel target to reduce or prevent tau-associated neurodegeneration in tauopathies.

Targeting TAO Kinases Using a New Inhibitor Compound Delays Mitosis and Induces Mitotic Cell Death in Centrosome Amplified Breast Cancer Cells.

Thousand-and-one amino acid kinases (TAOK) 1 and 2 are activated catalytically during mitosis and can contribute to mitotic cell rounding and spindle positioning. Here, we characterize a compound that inhibits TAOK1 and TAOK2 activity with IC50 values of 11 to 15 nmol/L, is ATP-competitive, and targets these kinases selectively. TAOK inhibition or depletion in centrosome-amplified SKBR3 or BT549 breast cancer cell models increases the mitotic population, the percentages of mitotic cells displaying amplified centrosomes and multipolar spindles, induces cell death, and inhibits cell growth. In contrast, nontumorigenic and dividing bipolar MCF-10A breast cells appear less dependent on TAOK activity and can complete mitosis and proliferate in the presence of the TAOK inhibitor. We demonstrate that TAOK1 and TAOK2 localize to the cytoplasm and centrosomes respectively during mitosis. Live cell imaging shows that the TAOK inhibitor prolongs the duration of mitosis in SKBR3 cells, increases mitotic cell death, and reduces the percentages of cells exiting mitosis, whereas MCF-10A cells continue to divide and proliferate. Over 80% of breast cancer tissues display supernumerary centrosomes, and tumor cells frequently cluster extra centrosomes to avoid multipolar mitoses and associated cell death. Consequently, drugs that stimulate centrosome declustering and induce multipolarity are likely to target dividing centrosome-amplified cancer cells preferentially, while sparing normal bipolar cells. Our results demonstrate that TAOK inhibition can enhance centrosome declustering and mitotic catastrophe in cancer cells, and these proteins may therefore offer novel therapeutic targets suitable for drug inhibition and the potential treatment of breast cancers, where supernumerary centrosomes occur. Mol Cancer Ther; 16(11); 2410-21. ©2017 AACR.

Lamin B1 levels modulate differentiation into neurons during embryonic corticogenesis.

Lamin B1, a key component of the nuclear lamina, plays an important role in brain development. Ablation of endogenous Lamin B1 (Lmnb1) in the mouse strongly impairs embryonic brain development and corticogenesis. However, the mechanisms underlying these neurodevelopmental effects are unknown. Here, we report that Lamin B1 levels modulate the differentiation of murine neural stem cells (NSCs) into neurons and astroglial-like cells. In vitro, endogenous Lmnb1 depletion favors NSC differentiation into glial fibrillar acidic protein (GFAP)-immunoreactive cells over neurons, while overexpression of human Lamin B1 (LMNB1) increases the proportion of neurons. In Lmnb1-null embryos, neurogenesis is reduced, while in vivo Lmnb1 silencing in mouse embryonic brain by in utero electroporation of a specific Lmnb1 sh-RNA results in aberrant cortical positioning of neurons and increased expression of the astrocytic marker GFAP in the cortex of 7-day old pups. Together, these results indicate that finely tuned levels of Lamin B1 are required for NSC differentiation into neurons, proper expression of the astrocytic marker GFAP and corticogenesis.

Lamin B1 protein is required for dendrite development in primary mouse cortical neurons.

Lamin B1, a key component of the nuclear lamina, plays an important role in brain development and function. A duplication of the human lamin B1 (LMNB1) gene has been linked to adult-onset autosomal dominant leukodystrophy, and mouse and human loss-of-function mutations in lamin B1 are susceptibility factors for neural tube defects. In the mouse, experimental ablation of endogenous lamin B1 (Lmnb1) severely impairs embryonic corticogenesis. Here we report that in primary mouse cortical neurons, LMNB1 overexpression reduces axonal outgrowth, whereas deficiency of endogenous Lmnb1 results in aberrant dendritic development. In the absence of Lmnb1, both the length and complexity of dendrites are reduced, and their growth is unresponsive to KCl stimulation. This defective dendritic outgrowth stems from impaired ERK signaling. In Lmnb1-null neurons, ERK is correctly phosphorylated, but phospho-ERK fails to translocate to the nucleus, possibly due to delocalization of nuclear pore complexes (NPCs) at the nuclear envelope. Taken together, these data highlight a previously unrecognized role of lamin B1 in dendrite development of mouse cortical neurons through regulation of nuclear shuttling of specific signaling molecules and NPC distribution.

Messenger RNA processing is altered in autosomal dominant leukodystrophy.

Adult-onset autosomal dominant leukodystrophy (ADLD) is a slowly progressive neurological disorder characterized by autonomic dysfunction, followed by cerebellar and pyramidal features. ADLD is caused by duplication of the lamin B1 gene (LMNB1), which leads to its increased expression. The molecular pathways involved in the disease are still poorly understood. Hence, we analyzed global gene expression in fibroblasts and whole blood of LMNB1 duplication carriers and used Gene Set Enrichment Analysis to explore their gene signatures. We found that LMNB1 duplication is associated with dysregulation of genes involved in the immune system, neuronal and skeletal development. Genes with an altered transcriptional profile clustered in specific genomic regions. Among the dysregulated genes, we further studied the role of RAVER2, which we found to be overexpressed at mRNA and protein level. RAVER2 encodes a putative trans regulator of the splicing repressor polypyrimidine tract binding protein (PTB) and is likely implicated in alternative splicing regulation. Functional studies demonstrated an abnormal splicing pattern of several PTB-target genes and of the myelin protein gene PLP1, previously demonstrated to be involved in ADLD. Mutant mice with different lamin B1 expression levels confirmed that Raver2 expression is dependent on lamin B1 in neural tissue and determines an altered splicing pattern of PTB-target genes and Plp1. Overall our results demonstrate that deregulation of lamin B1 expression induces modified splicing of several genes, likely driven by raver-2 overexpression, and suggest that an alteration of mRNA processing could be a pathogenic mechanism in ADLD.

Hyccin, the molecule mutated in the leukodystrophy hypomyelination and congenital cataract (HCC), is a neuronal protein.

"Hypomyelination and Congenital Cataract", HCC (MIM #610532), is an autosomal recessive disorder characterized by congenital cataract and diffuse cerebral and peripheral hypomyelination. HCC is caused by deficiency of Hyccin, a protein whose biological role has not been clarified yet. Since the identification of the cell types expressing a protein of unknown function can contribute to define the physiological context in which the molecule is explicating its function, we analyzed the pattern of Hyccin expression in the central and peripheral nervous system (CNS and PNS). Using heterozygous mice expressing the b-galactosidase (LacZ) gene under control of the Hyccin gene regulatory elements, we show that the gene is primarily expressed in neuronal cells. Indeed, Hyccin-LacZ signal was identified in CA1 hippocampal pyramidal neurons, olfactory bulb, and cortical pyramidal neurons, while it did not colocalize with oligodendroglial or astrocytic markers. In the PNS, Hyccin was detectable only in axons isolated from newborn mice. In the brain, Hyccin transcript levels were higher in early postnatal development (postnatal days 2 and 10) and then declined in adult mice. In a model of active myelinogenesis, organotypic cultures of rat Schwann cells (SC)/Dorsal Root Ganglion (DRG) sensory neurons, Hyccin was detected along the neurites, while it was absent from SC. Intriguingly, the abundance of the molecule was upregulated at postnatal days 10 and 15, in the initial steps of myelinogenesis and then declined at 30 days when the process is complete. As Hyccin is primarily expressed in neurons and its mutation leads to hypomyelination in human patients, we suggest that the protein is involved in neuron-to-glia signalling to initiate or maintain myelination.

Both Schwann cell and axonal defects cause motor peripheral neuropathy in Ebf2-/- mice.

Charcot-Marie-Tooth neuropathies are frequent hereditary disorders of the nervous system and most cases remain without a molecular definition. Mutations in transcription factors have been previously associated to various types of this disease. Mice carrying a null mutation in Ebf2 transcription factor present peripheral nerve abnormalities. To get insight into Ebf2 function in peripheral nervous system, here we characterize the peripheral neuropathy affecting these mice. We first show that Ebf2 is largely expressed in peripheral nerve throughout postnatal development, its expression being not only restricted to non-myelin forming Schwann cells, but also involving myelin forming Schwann cells and the perineurium. As a consequence, the onset of myelination is delayed and Schwann cell differentiation markers are downregulated in Ebf2-/- mice. Later in development, myelin pathology appears less severe and characterized by isolated clusters of hypomyelinated fibers. However, we find defects in the nerve architecture, such as abnormalities of the nodal region and shorter internodal length. Furthermore, we demonstrate a significant decrease in axonal calibre, with a lack of large calibre axons, and a severe impairment of motor nerve conduction velocity and amplitude, whereas the sensory nerve parameters are less affected. Interestingly, a clinical case with peripheral motor neuropathy and clinical features similar to Ebf2-/- mice phenotype was associated with a deletion encompassing EBF2 human genomic locus. These findings demonstrate that Ebf2 is a new molecule implicated in peripheral nerve development and a potential candidate gene for peripheral nerve disorders.

MAPK/Erk-dependent phosphorylation of synapsin mediates formation of functional synapses and short-term homosynaptic plasticity.

MAPK/Erk is a protein kinase activated by neurotrophic factors involved in synapse formation and plasticity, which acts at both the nuclear and cytoplasmic level. Synapsin proteins are synaptic-vesicle-associated proteins that are well known to be MAPK/Erk substrates at phylogenetically conserved sites. However, the physiological role of MAPK/Erk-dependent synapsin phosphorylation in regulating synaptic formation and function is poorly understood. Here, we examined whether synapsin acts as a physiological effector of MAPK/Erk in synaptogenesis and plasticity. To this aim, we developed an in vitro model of soma-to-soma paired Helix B2 neurons, that establish bidirectional excitatory synapses. We found that the formation and activity-dependent short-term plasticity of these synapses is dependent on the MAPK/Erk pathway. To address the role of synapsin in this pathway, we generated non-phosphorylatable and pseudo-phosphorylated Helix synapsin mutants at the MAPK/Erk sites. Overexpression experiments revealed that both mutants interfere with presynaptic differentiation, synapsin clustering, and severely impair post-tetanic potentiation, a form of short-term homosynaptic plasticity. Our findings show that MAPK/Erk-dependent synapsin phosphorylation has a dual role both in the establishment of functional synaptic connections and their short-term plasticity, indicating that some of the multiple extranuclear functions of MAPK/Erk in neurons can be mediated by the same multifunctional presynaptic target.

Synapsin-I- and synapsin-II-null mice display an increased age-dependent cognitive impairment.

Synapsin I (SynI) and synapsin II (SynII) are major synaptic vesicle (SV) proteins that function in the regulation of the availability of SVs for release in mature neurons. SynI and SynII show a high level of sequence similarity and share many functions in vivo, although distinct physiological roles for the two proteins have been proposed. Both SynI(-/-) and SynII(-/-) mice have a normal lifespan, but exhibit a decreased number of SVs and synaptic depression upon high-frequency stimulation. Because of the role of the synapsin proteins in synaptic organization and plasticity, we studied the long-lasting effects of synapsin deletion on the phenotype of SynI(-/-) and SynII(-/-) mice during aging. Both SynI(-/-) and SynII(-/-) mice displayed behavioural defects that emerged during aging and involved emotional memory in both mutants, and spatial memory in SynII(-/-) mice. These abnormalities, which were more pronounced in SynII(-/-) mice, were associated with neuronal loss and gliosis in the cerebral cortex and hippocampus. The data indicate that SynI and SynII have specific and non-redundant functions, and that synaptic dysfunctions associated with synapsin mutations negatively modulate cognitive performances and neuronal survival during senescence.

Nitric oxide-evoked cGMP production in Purkinje cells in rat cerebellum: an immunocytochemical and pharmacological study.

The cerebellar cells that account for glutamate-dependent cyclic GMP (cGMP) production, involving activation of the ionotropic glutamate receptors/nitric oxide synthase/soluble guanylyl cyclase pathway, are not fully established. In the present paper we have searched for the localisation of the cGMP response to the nitric oxide (NO) donor S-nitroso-penicillamine (SNAP 1muM), expected to generate local NO concentrations in the low nanomolar physiological range and evoking a cGMP response dependent on glutamate release and on the consequent activation of ionotropic glutamate NMDA/non-NMDA receptors, in cerebellar slices from adult rat. We have found that low concentration of exogenous NO evoked cGMP accumulation in Purkinje cells in an ionotropic glutamate receptor-dependent and tetrodotoxin-sensitive manner. Such immunocytochemical localisation appears consistent with functional evidence for physiologically relevant glutamate-dependent cGMP production in Purkinje cells in rat cerebellar cortex.