SummaryDoublecortin (Dcx) defines a growing family of microtubule (MT)-associated proteins (MAPs) involved in neuronal migration and process outgrowth. We show that Dcx is essential for the function of Kif1a, a kinesin-3 motor protein that traffics synaptic vesicles. Neurons lacking Dcx and/or its structurally conserved paralogue, doublecortin-like kinase 1 (Dclk1), show impaired Kif1a-mediated transport of Vamp2, a cargo of Kif1a, with decreased run length. Human disease-associated mutations in Dcx's linker sequence (e.g., W146C, K174E) alter Kif1a/Vamp2 transport by disrupting Dcx/Kif1a interactions without affecting Dcx MT binding. Dcx specifically enhances binding of the ADP-bound Kif1a motor domain to MTs. Cryo-electron microscopy and subnanometer-resolution image reconstruction reveal the kinesin-dependent conformational variability of MT-bound Dcx and suggest a model for MAP-motor crosstalk on MTs. Alteration of kinesin run length by MAPs represents a previously undiscovered mode of control of kinesin transport and provides a mechanism for regulation of MT-based transport by local signals.

PCGEM1 is a prostate tissue-specific, and prostate cancer-associated noncoding RNA (ncRNA) gene. Previous results revealed a significant association of elevated PCGEM1 expression levels in prostate cancer cells of African-American patients, whose mortality rate is the highest among prostate cancer patients. Functional study of PCGEM1 demonstrated a marked increase in colony formation in LNCaP prostate cancer cells and NIH3T3 mouse fibroblast cells. This study demonstrates that PCGEM1 overexpression in LNCaP cell culture model results in the inhibition of apoptosis induced by doxorubicin (DOX). Induction of p53 and p21(Waf1/Cip1) by DOX were delayed in LNCaP cells stably overexpressing PCGEM1 (LNCaP-PCGEM1 cells) compared to control LNCaP cells. The protein levels of cleaved caspase 7, and cleaved PARP were attenuated in DOXtreated LNCaP-PCGEM1 cells compared to control LNCaP cells. Similar results were observed in LNCaP cells transiently overexpressing PCGEM1. The inhibition of PARP cleavage by PCGEM1 overexpression was also observed in LNCaP-PCGEM1 cells incubated with etoposide and sodium selenite. Fluorescence-Activated Cell Sorter Annexin-V analysis revealed significantly lower percentage of apoptotic cells in DOX-treated LNCaP-PCGEM1 cells compared to control LNCaP cells. The attenuation of apoptic response appears to be androgen dependent in this experimental model, as androgen-independent variants of LNCaP cells did not exhibit this response. In summary, this study provides new insights into cell biologic functions and novel features of an ncRNA. Further, these data unravel biological mechanisms of cell growth/cell survival-associated functions of this ncRNA in a widely used prostate cancer cell culture model.

SUMMARY Because molecular mechanisms underlying refractory focal epilepsy are poorly defined, we performed transcriptome analysis on human epileptogenic tissue. Compared with controls, expression of Circadian Locomotor Output Cycles Kaput (CLOCK) is decreased in epileptogenic tissue. To define the function of CLOCK, we generated and tested the Emx-Cre; Clockflox/flox and PV-Cre; Clockflox/flox mouse lines with targeted deletions of the Clock gene in excitatory and parvalbumin (PV)-expressing inhibitory neurons, respectively. The Emx-Cre; Clockflox/flox mouse line alone has decreased seizure thresholds, but no laminar or dendritic defects in the cortex. However, excitatory neurons from the Emx-Cre; Clockflox/flox mouse have spontaneous epileptiform discharges. Both neurons from Emx-Cre; Clockflox/flox mouse and human epileptogenic tissue exhibit decreased spontaneous inhibitory post-synaptic currents. Finally, video-EEG of Emx-Cre; Clockflox/flox mice reveals epileptiform discharges during sleep and also seizures arising from sleep. Altogether, these data show that disruption of CLOCK alters cortical circuits and may lead to generation of focal epilepsy.

Hypomyelinating leukodystrophies are heritable disorders defined by lack of development of brain myelin, but the cellular mechanisms of hypomyelination are often poorly understood. Mutations in TUBB4A, encoding the tubulin isoform tubulin beta class IVA (Tubb4a), result in the symptom complex of hypomyelination with atrophy of basal ganglia and cerebellum (H-ABC). Additionally, TUBB4A mutations are known to result in a broad phenotypic spectrum, ranging from primary dystonia (DYT4), isolated hypomyelination with spastic quadriplegia, and an infantile onset encephalopathy, suggesting multiple cell types may be involved. We present a study of the cellular effects of TUBB4A mutations responsible for H-ABC (p.Asp249Asn), DYT4 (p.Arg2Gly), a severe combined phenotype with hypomyelination and encephalopathy (p.Asn414Lys), as well as milder phenotypes causing isolated hypomyelination (p.Val255Ile and p.Arg282Pro). We used a combination of histopathological, biochemical and cellular approaches to determine how these different mutations may have variable cellular effects in neurons and/or oligodendrocytes. Our results demonstrate that specific mutations lead to either purely neuronal, combined neuronal and oligodendrocytic or purely oligodendrocytic defects that closely match their respective clinical phenotypes. Thus, the DYT4 mutation that leads to phenotypes attributable to neuronal dysfunction results in altered neuronal morphology, but with unchanged tubulin quantity and polymerization, with normal oligodendrocyte morphology and myelin gene expression. Conversely, mutations associated with isolated hypomyelination (p.Val255Ile and p.Arg282Pro) and the severe combined phenotype (p.Asn414Lys) resulted in normal neuronal morphology but were associated with altered oligodendrocyte morphology, myelin gene expression, and microtubule dysfunction. The H-ABC mutation (p.Asp249Asn) that exhibits a combined neuronal and myelin phenotype had overlapping cellular defects involving both neuronal and oligodendrocyte cell types in vitro. Only mutations causing hypomyelination phenotypes showed altered microtubule dynamics and acted through a dominant toxic gain of function mechanism. The DYT4 mutation had no impact on microtubule dynamics suggesting a distinct mechanism of action. In summary, the different clinical phenotypes associated with TUBB4A reflect the selective and specific cellular effects of the causative mutations. Cellular specificity of disease pathogenesis is relevant to developing targeted treatments for this disabling condition.

Doublecortin (Dcx) is the causative gene for X-linked lissencephaly, which encodes a microtubule (MT) binding protein. Axon tracts are abnormal in both affected individuals and in animal models. To determine the reason for the axon tract defect, we performed a semi-quantitative proteomic analysis of the corpus callosum in mice mutant for Dcx. In axons from mice mutant for Dcx, wide spread differences are found in actin-associated proteins as compared with wild type axons. Decreases in actin-binding proteins, α-actinin-1, α-actinin-4 and actin-related protein 2/3 complex subunit 3 (Arp3), are correlated with dysregulation in the distribution of filamentous actin (F-actin) in the mutant neurons with increased F-actin around the cell body and decreased F-actin in the neurites and growth cones. The actin distribution defect can be rescued, by full length Dcx, and further enhanced by Dcx S297A, the unphosphorylatable mutant, but not with the truncation mutant of Dcx missing the C terminal S/P rich domain. Thus, the C-terminal region of Dcx dynamically regulates formation of F-actin features in developing neurons, likely through interaction with spinophilin, but not through α-actinin-4 or Arp3. We show with that the phenotype of Dcx/Doublecortin Like Kinase 1 (Dclk1) deficiency is consistent with actin defect as these axons are selectively deficient in axon guidance, but not elongation.

After axotomy, neuronal survival and growth cone re-formation are required for axon regeneration. We discovered that doublecortin-like kinases (DCLKs), members of the doublecortin (DCX) family expressed in adult retinal ganglion cells (RGCs), play critical roles in both processes, through distinct mechanisms. Overexpression of DCLK2 accelerated growth cone re-formation in vitro and enhanced the initiation and elongation of axon re-growth after optic nerve injury. These effects depended on both the microtubule (MT)-binding domain and the serine-proline-rich (S/P-rich) region of DCXs in-cis in the same molecules. While the MT-binding domain is known to stabilize MT structures, we show that the S/P-rich region prevents F-actin destabilization in injured axon stumps. Additionally, while DCXs synergize with mTOR to stimulate axon regeneration, alone they can promote neuronal survival possibly by regulating the retrograde propagation of injury signals. Multifunctional DCXs thus represent potential targets for promoting both survival and regeneration of injured neurons.

The presence of petroleum acids in crude oils is a key problem in refineries due to corrosion to industrial units. To economically and effectively remove the petroleum acids from high acid crude oils is an urgent project. A theoretical and experimental study to develop a catalytic decarboxylation process to remove petroleum acids from high acid crude oils was conducted. By using the molecular simulation technology, a solid acid catalyst was constructed for removing petroleum acids. The experimental results indicated that the acid removal rate of a high acid crude oil with a total acid number (TAN) of 12.52 over the solid acid catalyst of MLC500 was up to 97% at a volume space velocity of 8 h−1, ratio of catalyst to oil of 7.5, and temperature of 460 °C in the fixed fluid bed reactor.

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