Search

Familial dysautonomia (FD) is an autosomal recessive neurodegenerative disease caused by a splicing mutation in the gene encoding Elongator complex protein 1 (ELP1, also known as IKBKAP). This mutation results in tissue-specific skipping of exon 20 with a corresponding reduction of ELP1 protein, predominantly in the central and peripheral nervous system. Although FD patients have a complex neurological phenotype caused by continuous depletion of sensory and autonomic neurons, progressive visual decline leading to blindness is one of the most problematic aspects of the disease, as it severely affects their quality of life. To better understand the disease mechanism as well as to test the in vivo efficacy of targeted therapies for FD, we have recently generated a novel phenotypic mouse model, TgFD9; IkbkapΔ20/flox. This mouse exhibits most of the clinical features of the disease and accurately recapitulates the tissue-specific splicing defect observed in FD patients. Driven by the dire need to develop therapies targeting retinal degeneration in FD, herein, we comprehensively characterized the progression of the retinal phenotype in this mouse, and we demonstrated that it is possible to correct ELP1 splicing defect in the retina using the splicing modulator compound (SMC) BPN-15477.

Familial dysautonomia (FD) is a recessive neurodegenerative disease caused by a splice mutation in Elongator complex protein 1 (ELP1, also known as IKBKAP); this mutation leads to variable skipping of exon 20 and to a drastic reduction of ELP1 in the nervous system. Clinically, many of the debilitating aspects of the disease are related to a progressive loss of proprioception; this loss leads to severe gait ataxia, spinal deformities, and respiratory insufficiency due to neuromuscular incoordination. There is currently no effective treatment for FD, and the disease is ultimately fatal. The development of a drug that targets the underlying molecular defect provides hope that the drastic peripheral neurodegeneration characteristic of FD can be halted. We demonstrate herein that the FD mouse TgFD9;Ikbkap(Δ20/flox) recapitulates the proprioceptive impairment observed in individuals with FD, and we provide the in vivo evidence that postnatal correction, promoted by the small molecule kinetin, of the mutant ELP1 splicing can rescue neurological phenotypes in FD. Daily administration of kinetin starting at birth improves sensory-motor coordination and prevents the onset of spinal abnormalities by stopping the loss of proprioceptive neurons. These phenotypic improvements correlate with increased amounts of full-length ELP1 mRNA and protein in multiple tissues, including in the peripheral nervous system (PNS). Our results show that postnatal correction of the underlying ELP1 splicing defect can rescue devastating disease phenotypes and is therefore a viable therapeutic approach for persons with FD.

Familial dysautonomia (FD) is an autosomal recessive neurodegenerative disease caused by a splicing mutation in the gene encoding Elongator complex protein 1 (ELP1, also known asIKBKAP). This mutation results in tissue-specific skipping of exon 20 with a corresponding reduction of ELP1 protein, predominantly in the central and peripheral nervous system. Although FD patients have a complex neurological phenotype caused by continuous depletion of sensory and autonomic neurons, progressive visual decline leading to blindness is one of the most problematic aspect of the disease, as it severely affects their quality of life. To better understand the disease mechanism as well as to test thein vivoefficacy of targeted therapies for FD, we have recently generated a novel phenotypic mouse model,TgFD9; Elp1∆20/flox. This mouse exhibits most of the clinical features of the disease and accurately recapitulates the tissue-specific splicing defect observed in FD patients. Driven by the dire need to develop therapies targeting retinal degeneration in FD, herein, we comprehensively characterized the progression of the retinal phenotype in this mouse, and we demonstrated that it is possible to correctELP1splicing defect in the retina using the splicing modulator compound (SMC) BPN-15477.

Microtubule (MT)-based transport is an evolutionary conserved processed finely tuned by posttranslational modifications. Among them, α-tubulin acetylation, which is catalyzed by the α-tubulin N-acetyltransferase 1, Atat1, promotes the recruitment and processivity of molecular motors along MT tracks. However, the mechanisms that controls Atat1 activity remains poorly understood. Here, we show that a pool of vesicular ATP-citrate lyase Acly acts as a rate limiting enzyme to modulate Atat1 activity by controlling availability of Acetyl-Coenzyme-A (Acetyl-CoA). In addition, we showed that Acly expression is reduced upon loss of Elongator activity, further connecting Elongator to Atat1 in the pathway regulating α-tubulin acetylation and MT-dependent transport in projection neurons, across species. Remarkably, comparable defects occur in fibroblasts from Familial Dysautonomia (FD) patients bearing an autosomal recessive mutation in the gene coding for the Elongator subunit ELP1. Our data may thus shine new light on the pathophysiological mechanisms underlying FD.Competing Interest StatementThe authors have declared no competing interest.View Full Text

Pathogenesis of alcohol-related diseases such as alcoholic hepatitis involves gut barrier dysfunction, endotoxemia, and toxin-mediated cellular injury. Here we show that Lactobacillus plantarum not only blocks but also mitigates ethanol (EtOH)-induced gut and liver damage in mice. L. plantarum blocks EtOH-induced protein thiol oxidation, and down-regulation of antioxidant gene expression in colon L. plantarum also blocks EtOH-induced expression of TNF-α, IL-1β, IL-6, monocyte chemotactic protein 1 ( MCP1), C-X-C motif chemokine ligand ( CXCL)1, and CXCL2 genes in colon. Epidermal growth factor receptor (EGFR) signaling mediates the L. plantarum-mediated protection of tight junctions (TJs) and barrier function from acetaldehyde, the EtOH metabolite, in Caco-2 cell monolayers. In mice, doxycycline-mediated expression of dominant negative EGFR blocks L. plantarum-mediated prevention of EtOH-induced TJ disruption, mucosal barrier dysfunction, oxidative stress, and inflammatory response in colon. L. plantarum blocks EtOH-induced endotoxemia as well as EtOH-induced pathologic lesions, triglyceride deposition, oxidative stress, and inflammatory responses in the liver by an EGFR-dependent mechanism. L. plantarum treatment after injury accelerated recovery from EtOH-induced TJ, barrier dysfunction, oxidative stress, and inflammatory response in colon, endotoxemia, and liver damage. Results demonstrate that L. plantarum has both preventive and therapeutic values in treatment of alcohol-induced tissue injury, particularly in alcoholic hepatitis.-Shukla, P. K., Meena, A. S., Manda, B., Gomes-Solecki, M., Dietrich, P., Dragatsis, I., Rao, R. Lactobacillus plantarum prevents and mitigates alcohol-induced disruption of colonic epithelial tight junctions, endotoxemia, and liver damage by an EGF receptor-dependent mechanism.

Microtubules are polymerized dimers of α- and β-tubulin that underlie a broad range of cellular activities. Acetylation of α-tubulin by the acetyl-transferase ATAT1 modulates microtubule dynamics and functions in neurons. However, it remains unclear how and why this enzyme acetylates microtubules over long distances in axons. Here, we show that loss of ATAT1 impairs axonal transport in neurons and cell free motility assays confirm a requirement of tubulin acetylation for proper bidirectional vesicular transport. Moreover, we demonstrate that the main cellular pool of ATAT1 is transported at the cytosolic side of neuronal vesicles that are moving along axons. Altogether, our data suggest that axonal transport of ATAT1-enriched vesicles is the predominant driver of α-tubulin acetylation in axons.

Background Disruption of epithelial tight junctions (TJ), gut barrier dysfunction and endotoxemia play crucial role in the pathogenesis of alcoholic tissue injury. Occludin, a transmembrane protein of TJ, is depleted in colon by alcohol. However, it is unknown whether occludin depletion influences alcoholic gut and liver injury. Methods Wild type (WT) and occludin deficient (Ocln −/− ) mice were fed 1–6% ethanol in Lieber–DeCarli diet. Gut permeability was measured by vascular-to-luminal flux of FITC-inulin. Junctional integrity was analyzed by confocal microscopy. Liver injury was assessed by plasma transaminase, histopathology and triglyceride analyses. The effect of occludin depletion on acetaldehyde-induced TJ disruption was confirmed in Caco-2 cell monolayers. Results Ethanol feeding significantly reduced body weight gain in Ocln −/− mice. Ethanol increased inulin permeability in colon of both WT and Ocln −/− mice, but the effect was 4-fold higher in Ocln −/− mice. The gross morphology of colonic mucosa was unaltered, but ethanol disrupted the actin cytoskeleton, induced redistribution of occludin, ZO-1, E-cadherin and β-catenin from the junctions and elevated TLR4, which was more severe in Ocln −/− mice. Occludin knockdown significantly enhanced acetaldehyde-induced TJ disruption and barrier dysfunction in Caco-2 cell monolayers. Ethanol significantly increased liver weight and plasma transaminase activity in Ocln −/− mice, but not in WT mice. Histological analysis indicated more severe lesions and fat deposition in the liver of ethanol-fed Ocln −/− mice. Ethanol-induced elevation of liver triglyceride was also higher in Ocln −/− mice. Conclusion This study indicates that occludin deficiency increases susceptibility to ethanol-induced colonic mucosal barrier dysfunction and liver damage in mice.

Background Acetaldehyde, the toxic ethanol (EtOH) metabolite, disrupts intestinal epithelial barrier function. Aldehyde dehydrogenase (ALDH) detoxifies acetaldehyde into acetate. Subpopulations of Asians and Native Americans show polymorphism with loss-of-function mutations in ALDH2. We evaluated the effect of ALDH2 deficiency on EtOH-induced disruption of intestinal epithelial tight junctions and adherens junctions, gut barrier dysfunction, and liver injury. Methods Wild-type and ALDH2-deficient mice were fed EtOH (1 to 6%) in Lieber–DeCarli diet for 4 weeks. Gut permeability in vivo was measured by plasma-to-luminal flux of FITC-inulin, tight junction and adherens junction integrity was analyzed by confocal microscopy, and liver injury was assessed by the analysis of plasma transaminase activity, histopathology, and liver triglyceride. Results EtOH feeding elevated colonic mucosal acetaldehyde, which was significantly greater in ALDH2-deficient mice. ALDH2−/− mice showed a drastic reduction in the EtOH diet intake. Therefore, this study was continued only in wild-type and ALDH2+/− mice. EtOH feeding elevated mucosal inulin permeability in distal colon, but not in proximal colon, ileum, or jejunum of wild-type mice. In ALDH2+/− mice, EtOH-induced inulin permeability in distal colon was not only higher than that in wild-type mice, but inulin permeability was also elevated in the proximal colon, ileum, and jejunum. Greater inulin permeability in distal colon of ALDH2+/− mice was associated with a more severe redistribution of tight junction and adherens junction proteins from the intercellular junctions. In ALDH2+/− mice, but not in wild-type mice, EtOH feeding caused a loss of junctional distribution of tight junction and adherens junction proteins in the ileum. Histopathology, plasma transaminases, and liver triglyceride analyses showed that EtOH-induced liver damage was significantly greater in ALDH2+/− mice compared to wild-type mice. Conclusions These data demonstrate that ALDH2 deficiency enhances EtOH-induced disruption of intestinal epithelial tight junctions, barrier dysfunction, and liver damage.

RESUMO Neste trabalho é avaliado o processo de corrosão das armaduras induzido pela ação de íons cloreto em concretos produzidos com adição do Resíduo do Beneficiamento de Rochas Ornamentais (RBRO). No estudo de caracterização, foram avaliadas propriedades físicas, químicas e mineralógicas do RBRO. Foram produzidos concretos com três níveis de relação água/cimento – 0,45; 0,55 e 0,65 – e quatro níveis de adição de RBRO – 0, 5, 10 e 15%- em relação à massa de cimento. No estado fresco foi avaliada a propriedade de consistência do concreto; no estado endurecido foi avaliada a resistência à compressão axial; as propriedades relacionadas com a durabilidade estudadas foram absorção de água por imersão e fervura, absorção por capilaridade, além de ciclos semanais de indução e aceleração da corrosão por ataque de cloretos e monitoramento do potencial de corrosão. Por último, foi feito o ensaio colorimétrico por aspersão do indicador AgNO3 a 0,1 mol/L. Os resultados mostraram que a adição de RBRO é vantajosa no teor de 5% de RBRO do ponto de vista da resistência mecânica e de durabilidade frente à ação de cloretos. ABSTRACT This research evaluates the reinforcement steel corrosion process induced by chloride ions in concrete containing ornamental rock waste (ORW) as mineral addition. Physical, chemical, mineralogical analysis and particle size distribution of ORW were performed. Concrete mixtures were produced with three levels of water / cement ratio – 0.45; 0.55 and 0.65 – and four levels of ORW addition – 0, 5, 10 and 15% - by weight of the binder. Consistency of fresh concrete and compressive strength of hardened concrete were evaluated; water absorption and capillary absorption of concrete were performed as durability features. The concrete specimens were exposed to wet and dry cycles and corrosion potential monitoring test. Finally, the colorimetric method (AgNO3 of 0,1 mol/L) was performed. Results show that the 5% ORW content ratio is beneficial to concrete at mechanical and durability perspectives against chloride ion attack.

We evaluated the impact of early embryonic deletion of huntingtin (htt) from pyramidal neurons on cortical development, cortical neuron survival and motor behavior, using a cre-loxP strategy to inactivate the mouse htt gene (Hdh) in emx1-expressing cell lineages. Western blot confirmed substantial htt reduction in cerebral cortex of these Emx-httKO mice, with residual cortical htt in all likelihood restricted to cortical interneurons of the subpallial lineage and/or vascular endothelial cells. Despite the loss of htt early in development, cortical lamination was normal, as revealed by layer-specific markers. Cortical volume and neuron abundance were, however, significantly less than normal, and cortical neurons showed reduced brain-derived neurotrophic factor (BDNF) expression and reduced activation of BDNF signaling pathways. Nonetheless, cortical volume and neuron abundance did not show progressive age-related decline in Emx-httKO mice out to 24 months. Although striatal neurochemistry was normal, reductions in striatal volume and neuron abundance were seen in Emx-httKO mice, which were again not progressive. Weight maintenance was normal in Emx-httKO mice, but a slight rotarod deficit and persistent hyperactivity were observed throughout the lifespan. Our results show that embryonic deletion of htt from developing pallium does not substantially alter migration of cortical neurons to their correct laminar destinations, but does yield reduced cortical and striatal size and neuron numbers. The Emx-httKO mice were persistently hyperactive, possibly due to defects in corticostriatal development. Importantly, deletion of htt from cortical pyramidal neurons did not yield age-related progressive cortical or striatal pathology.

Huntington’s Disease (HD) is an autosomal dominant progressive neurodegenerative disorder characterized by cognitive, behavioral and motor dysfunctions. HD is caused by a CAG repeat expansion in exon 1 of the HD gene that is translated into an expanded polyglutamine tract in the encoded protein, huntingtin (HTT). While the most significant neuropathology of HD occurs in the striatum, other brain regions are also affected and play an important role in HD pathology. To date there is no cure for HD, and recently strategies aiming at silencing HTT expression have been initiated as possible therapeutics for HD. However, the essential functions of HTT in the adult brain are currently unknown and hence the consequence of sustained suppression of HTT expression is unpredictable and can potentially be deleterious. Using the Cre-loxP system of recombination, we conditionally inactivated the mouse HD gene homologue at 3, 6 and 9 months of age. Here we show that elimination of Htt expression in the adult mouse results in behavioral deficits, progressive neuropathological changes including bilateral thalamic calcification, and altered brain iron homeostasis., Author summary Huntington’s Disease is a genetic disorder characterized by progressive cognitive, behavioral and motor dysfunctions. Usually the first symptoms appear around 40 years of age, and lead to death within 15–20 years after the onset of symptoms. To date there is no cure for Huntington’s Disease, and current therapeutic strategies are only palliative, and far from optimal. Gene silencing currently appears as the most attractive approach for the treatment of Huntington’s Disease. However, since normal and mutant huntingtin (the protein product of the Huntington’s disease gene) differ only on the polyglutamine length, unless allele-specific silencing is planned, normal huntingtin (that is neuroprotective) will also be inactivated with unknown implications. To address these questions, we investigated the consequences of elimination of normal huntingtin function in adulthood. In summary, our studies show that huntingtin plays a role in brain iron homeostasis, and that elimination of huntingtin in the adult mouse results in behavioral deficits and progressive neuropathological changes including bilateral thalamic calcification.

Hereditary sensory and autonomic neuropathies (HSANs) encompass a group of genetically inherited disorders characterized by sensory and autonomic dysfunctions. Familial dysautonomia (FD), also known as HSAN type III, is an autosomal recessive disorder that affects 1/3600 live births in the Ashkenazi Jewish population. The disease is caused by abnormal development and progressive degeneration of the sensory and autonomic nervous systems and is inevitably fatal, with only 50% of patients reaching the age of 40. FD is caused by a mutation in intron 20 of the Ikbkap gene that results in severe reduction in the expression of its encoded protein, inhibitor of kappaB kinase complex-associated protein (IKAP). Although the mutation that causes FD was identified in 2001, so far there is no appropriate animal model that recapitulates the disorder. Here, we report the generation and characterization of the first mouse models for FD that recapitulate the molecular and pathological features of the disease. Important for therapeutic interventions is also our finding that a slight increase in IKAP levels is enough to ameliorate the phenotype and increase the life span. Understanding the mechanisms underlying FD will provide insights for potential new therapeutic interventions not only for FD, but also for other peripheral neuropathies.

With spontaneous elongation of the CAG repeat in the R6/2 transgene to > or =335, resulting in a transgene protein too large for passive entry into nuclei via the nuclear pore, we observed an abrupt increase in lifespan to >20 weeks, compared to the 12 weeks common in R6/2 mice with 150 repeats. In the > or =335 CAG mice, large ubiquitinated aggregates of mutant protein were common in neuronal dendrites and perikaryal cytoplasm, but intranuclear aggregates were small and infrequent. Message and protein for the > or =335 CAG transgene were reduced to one-third that in 150 CAG R6/2 mice. Neurological and neurochemical abnormalities were delayed in onset and less severe than in 150 CAG R6/2 mice. These findings suggest that polyQ length and pathogenicity in Huntington's disease may not be linearly related, and pathogenicity may be less severe with extreme repeats. Both diminished mutant protein and reduced nuclear entry may contribute to phenotype attenuation.

Huntingtin (htt) is a 350 kDa protein of unknown function, with no homologies with other known proteins. Expansion of a polyglutamine stretch at the N-terminus of htt causes Huntington's disease (HD), a dominant neurodegenerative disorder. Although it is generally accepted that HD is caused primarily by a gain-of-function mechanism, recent studies suggest that loss-of-function may also be part of HD pathogenesis. Huntingtin is an essential protein in the mouse since inactivation of the mouse HD homolog (Hdh) gene results in early embryonic lethality. Huntingtin is widely expressed in embryogenesis, and associated with a number of interacting proteins suggesting that htt may be involved in several processes including morphogenesis, neurogenesis and neuronal survival. To further investigate the role of htt in these processes, we have inactivated the Hdh gene in Wnt1 cell lineages using the Cre-loxP system of recombination. Here we show that conditional inactivation of the Hdh gene in Wnt1 cell lineages results in congenital hydrocephalus, implicating huntingtin for the first time in the regulation of cerebral spinal fluid (CSF) homeostasis. Our results show that hydrocephalus in mice lacking htt in Wnt1 cell lineages is associated with increase in CSF production by the choroid plexus, and abnormal subcommissural organ.

α-Synuclein (α-syn), a protein implicated in Parkinson's disease pathogenesis, is a presynaptic protein suggested to regulate transmitter release. We explored how α-syn overexpression in PC12 and chromaffin cells, which exhibit low endogenous α-syn levels relative to neurons, affects catecholamine release. Overexpression of wild-type or A30P mutant α-syn in PC12 cell lines inhibited evoked catecholamine release without altering calcium threshold or cooperativity of release. Electron micrographs revealed that vesicular pools were not reduced but that, on the contrary, a marked accumulation of morphologically “docked” vesicles was apparent in the α-syn-overexpressing lines. We used amperometric recordings from chromaffin cells derived from mice that overexpress A30P or wild-type (WT) α-syn, as well as chromaffin cells from control and α-syn null mice, to determine whether the filling of vesicles with the transmitter was altered. The quantal size and shape characteristics of amperometric events were identical for all mouse lines, suggesting that overexpression of WT or mutant α-syn did not affect vesicular transmitter accumulation or the kinetics of vesicle fusion. The frequency and number of exocytotic events per stimulus, however, was lower for both WT and A30P α-syn-overexpressing cells. The α-syn-overexpressing cells exhibited reduced depression of evoked release in response to repeated stimuli, consistent with a smaller population of readily releasable vesicles. We conclude that α-syn overexpression inhibits a vesicle “priming” step, after secretory vesicle trafficking to “docking” sites but before calcium-dependent vesicle membrane fusion.

Leptin signaling in the brain regulates energy intake and expenditure. To test the degree of functional neuronal leptin signaling required for the maintenance of body composition, fertility, and cold tolerance, transgenic mice expressing Cre in neurons ( CaMKIIα-Cre) were crossed to mice carrying a floxed leptin receptor ( Lepr) allele to generate mice with neuron-specific deletion of Lepr in ∼50% ( C F/F mice) and ∼75% ( C Δ17/F mice) of hypothalamic neurons. Leptin receptor (LEPR)-deficient mice ( Δ17/Δ17) with heat-shock-Cre-mediated global Lepr deletion served as obese controls. At 16 wk, male C F/F, C Δ17/F, and Δ17/Δ17 mice were 13.2 ( P < 0.05), 45.0, and 55.9% ( P < 0.001) heavier, respectively, than lean controls, whereas females showed 31.6, 68.8, and 160.7% increases in body mass ( P < 0.001). Significant increases in total fat mass ( C F/F: P < 0.01; C Δ17/F and Δ17/Δ17: P < 0.001 vs. sex-matched, lean controls), and serum leptin concentrations ( P < 0.001 vs. controls) were present in proportion to Lepr deletion. Male C Δ17/F mice had significant elevations in basal serum insulin concentrations ( P < 0.001 vs. controls) and were glucose intolerant, as measured by glucose tolerance test (AUC P < 0.01 vs. controls). In contrast with previous observations in mice null for LEPR signaling, C F/F and C Δ17/F mice were fertile and cold tolerant. These findings support the hypothesis that body weight, adiposity, serum leptin concentrations, and glucose intolerance are proportional to hypothalamic LEPR deficiency. However, fertility and cold tolerance remain intact unless hypothalamic LEPR deficiency is complete.

Huntingtin-associated protein 1 (Hap1) is the first huntingtin interacting protein identified in a yeast two-hybrid screen. Although Hap1 expression has been demonstrated in neuronal and non-neuronal tissues, its molecular role is poorly understood. Recently, it has been shown that targeted disruption of Hap1 in mice results in early postnatal death as a result of depressed feeding behavior. Although this result clearly demonstrates an essential role of Hap1 in postnatal feeding, the mechanisms leading to this deficiency, as well as the role of Hap1 in adults, remain unclear. Here we show that Hap1 null mutants display suckling defects and die within the first days after birth due to starvation. Upon reduction of the litter size, some mutants survive into adulthood and display growth retardation with no apparent brain or behavioral abnormalities, suggesting that Hap1 function is essential only for early postnatal feeding behavior. Using a conditional gene repair strategy, we also show that the early lethality can be rescued if Hap1 expression is restored in neuronal cells before birth. Furthermore, no synergism was observed between Hap1 and huntingtin mutation during mouse development. Our results demonstrate that Hap1 has a fundamental role in regulating postnatal feeding in the first 2 weeks after birth and a non-essential role in the adult mouse.

Proteasomal dysfunction may underlie certain neuro-degenerative conditions such as Parkinson disease. We have shown that pharmacological inhibition of the proteasome in cultured neuronal cells leads to apoptotic death and formation of cytoplasmic ubiquitinated inclusions. These inclusions stain for alpha-synuclein and assume a fibrillar structure, as assessed by thioflavine S staining, and therefore resemble Lewy bodies. alpha-Synuclein is thought to be a central component of Lewy bodies. Whether alpha-synuclein is required for inclusion formation or apoptotic death has not been formally assessed. The present study examines whether alpha-synuclein deficiency in neurons alters their sensitivity to proteasomal inhibition-induced apoptosis or inclusion formation. Cortical neurons derived from alpha-synuclein-null mice showed a similar sensitivity to death induced by the proteasomal inhibitor lactacystin compared with neurons derived from wild-type mice. Furthermore, the absence of alpha-synuclein did not influence the percentage of lactacystin-treated neurons harboring cytoplasmic ubiquitinated inclusions or alter the solubility of such inclusions. In contrast, however, ubiquitinated inclusions in alpha-synuclein-deficient neurons lacked amyloid-like fibrillization, as determined by thioflavine S staining. This indicates that although alpha-synuclein deficiency does not affect the formation of ubiquitinated inclusions, it does significantly alter their structure. The lack of effect on survival in alpha-synuclein knock-out cultures further suggests that the fibrillar nature of the inclusions does not contribute to neuronal degeneration in this model.

α-Synuclein is a neuronal protein that is implicated in the control of synaptic vesicle function and in Parkinson's disease (PD). Consequently, alterations of α-synuclein levels may play a role in neurotransmission and in PD pathogenesis. However, the factors that regulate α-synuclein levels are unknown. Growth factors mediate neurotrophic and plasticity effects in CNS neurons, and may play a role in disease states. Here we examine the regulation of α-synuclein levels in primary CNS neurons, with particular emphasis on dopaminergic neurons. E18 rat cortical neurons and dopaminergic neurons of E14 rat ventral midbrain showed an induction of α-synuclein protein levels with maturation in culture. Application of basic Fibroblast growth factor (bFGF) promoted α-synuclein expression selectively within dopaminergic, and not GABAergic or cortical neurons. This induction was blocked by actinomycin D, but not by inhibition of bFGF-induced glial proliferation. α-Synuclein levels were not altered by glial-derived neurotrophic factor (GDNF), or by apoptotic stimuli. We conclude that bFGF promotes α-synuclein expression in cultured ventral midbrain dopaminergic neurons through a direct transcriptional effect. These results suggest that distinct growth factors may thus mediate plasticity responses or influence disease states in ventral midbrain dopaminergic neurons.

Huntington disease (HD) is an autosomal-dominant neurodegenerative disorder. HD is caused by a CAG repeat expansion, and the resulting extended polyglutamine stretch confers a deleterious gain-of-function to the protein (huntingtin), leading to extensive neurodegeneration, predominantly in the striatum. Recent experimental evidence derived from genetic models of HD suggests that loss of normal huntingtin function might also contribute to HD pathogenesis. Huntingtin is a predominantly cytoplasmic protein, widely expressed during development and enriched in the adult brain and testes. Analyses of genetically engineered mice indicate that huntingtin is an essential protein involved in multiple processes, such as iron transport in early development, ependymal cell differentiation, neuronal migration, neuroprotection, regulation of body weight, and spermatogenesis, among others. Understanding the normal function of hungtingtin not only provides insight into HD pathology but also offers guidance for the development of more efficient therapeutic strategies.

Huntingtin-associated protein 1 (HAP1) interacts with the product of the Huntington's disease gene. To investigate the function of Hap1 in development and in the adult mouse, we have examined the expression of Hap1 by northern analysis and in situ hybridization histochemistry. Hap1 expression is first detected in the embryonic day 8.5 (E8.5) neuroepithelium. Expression persists throughout development, predominantly in the brain and spinal cord, and to a lesser extent in enteric neurons and abdominal sympathetic ganglia. In the adult, Hap1 expression is detected not only in the brain but also in the ovary, testis, and the intermediate lobe of the pituitary.

To explore the potential of a simple and rapid approach for ubiquitous conditional gene disruption, we have generated Cre-producer mouse transgenic lines (Hs-cre1, 6 and 7) expressing a recombinase transgene (cre) from a heat shock gene promoter and tested their performance in Cre-mediated excision of target DNA in crosses with Cre-responder strains carrying loxP-modified alleles of the genes encoding the Huntington's disease gene homolog (Hdh), the epidermal growth factor receptor (Egfr), and the type 1 insulin-like growth factor receptor (Igf1r). Analyses of progeny possessing various transgene/reporter combinations showed that cre expression can occur without heat shock in early embryos, but this constitutive transcription is stochastic and transgene dependent. Thus, Hs-cre1 behaves predominantly as a "deleter" strain, since the majority of progeny (approximately 70-85%) exhibit complete recombination, regardless of reporter locus. Lines Hs-cre6 and Hs-cre7, however, function successfully as "mosaicking" strains because, in addition to two extreme classes of progeny with 0% or 100% recombination, they generate an intermediate class of mosaics exhibiting various degrees of partial DNA excision. Notably, the frequency of offspring in each class varies between reporters, but mosaic embryos are consistently obtained in adequate numbers (approximately 30-60%). The Hs-cre6 transgene is also inducible and can be used to introduce mosaicism into adult tissues at preselected developmental times by heat shock treatment of mice with 0% recombination in tail DNA. By bypassing the lethality resulting from some gene knockouts, mosaic embryos and mice make particular mutational analyses possible and are also very useful for the identification of cell lineage-specific gene functions.

During neural development in vertebrates, a spatially ordered array of neurons is generated in response to inductive signals derived from localized organizing centres. One organizing centre that has been proposed to have a role in the control of neural patterning is the roof plate. To define the contribution of signals derived from the roof plate to the specification of neuronal cell types in the dorsal neural tube, we devised a genetic strategy to ablate the roof plate selectively in mouse embryos. Embryos without a roof plate lack all the interneuron subtypes that are normally generated in the dorsal third of the neural tube. Using a genetically based lineage analysis and in vitro assays, we show that the loss of these neurons results from the elimination of non-autonomous signals provided by the roof plate. These results reveal that the roof plate is essential for specifying multiple classes of neurons in the mammalian central nervous system.

Neuregulin-1 (NRG-1) signaling has been implicated in inductive interactions between pre- and postsynaptic partners during synaptogenesis. We used gene targeting to selectively disrupt cysteine-rich domain– (CRD-) containing NRG-1 isoforms. In CRD-NRG-1 −/− mice, peripheral projections defasciculated and displayed aberrant branching patterns within their targets. Motor nerve terminals were transiently associated with broad bands of postsynaptic ACh receptor (AChR) clusters. Initially, Schwann cell precursors accompanied peripheral projections, but later, Schwann cells were absent from axons in the periphery. Following initial stages of synapse formation, sensory and motor nerves withdrew and degenerated. Our data demonstrate the essential role of CRD-NRG-1-mediated signaling for coordinating nerve, target, and Schwann cell interactions in the normal maintenance of peripheral synapses, and ultimately in the survival of CRD-NRG-1-expressing neurons.

The splice site mutation in the IKBKAP gene coding for IKAP protein leads to the tissue-specific skipping of exon 20, with concomitant reduction in IKAP protein production. This causes the neurodevelopmental, autosomal-recessive genetic disorder - Familial Dysautonomia (FD). The molecular hallmark of FD is the severe reduction of IKAP protein in the nervous system that is believed to be the main reason for the devastating symptoms of this disease. Our recent studies showed that in the brain of two FD patients, genes linked to oligodendrocyte differentiation and/or myelin formation are significantly downregulated, implicating IKAP in the process of myelination. However, due to the scarcity of FD patient tissues, these results awaited further validation in other models. Recently, two FD mouse models that faithfully recapitulate FD were generated, with two types of mutations resulting in severely low levels of IKAP expression. Here we demonstrate that IKAP deficiency in these FD mouse models affects a similar set of genes as in FD patients' brains. In addition, we identified two new IKAP target genes involved in oligodendrocyte cells differentiation and myelination, further underscoring the essential role of IKAP in this process. We also provide proof that IKAP expression is needed cell-autonomously for the regulation of expression of genes involved in myelin formation since knockdown of IKAP in the Oli-neu oligodendrocyte precursor cell line results in similar deficiencies. Further analyses of these two experimental models will compensate for the lack of human postmortem tissues and will advance our understanding of the role of IKAP in myelination and the disease pathology.

Huntington disease (HD) is a devastating autosomal-dominant neurodegenerative disorder. It is caused by expansion of a CAG repeat in the first exon of the huntingtin (HTT) gene that encodes a mutant HTT protein with a polyglutamine (polyQ) expansion at the amino terminus. Here, we demonstrate that WT HTT regulates ciliogenesis by interacting through huntingtin-associated protein 1 (HAP1) with pericentriolar material 1 protein (PCM1). Loss of Htt in mouse cells impaired the retrograde trafficking of PCM1 and thereby reduced primary cilia formation. In mice, deletion of Htt in ependymal cells led to PCM1 mislocalization, alteration of the cilia layer, and hydrocephalus. Pathogenic polyQ expansion led to centrosomal accumulation of PCM1 and abnormally long primary cilia in mouse striatal cells. PCM1 accumulation in ependymal cells was associated with longer cilia and disorganized cilia layers in a mouse model of HD and in HD patients. Longer cilia resulted in alteration of the cerebrospinal fluid flow. Thus, our data indicate that WT HTT is essential for protein trafficking to the centrosome and normal ciliogenesis. In HD, hypermorphic ciliogenesis may affect signaling and neuroblast migration so as to dysregulate brain homeostasis and exacerbate disease progression.

Huntington's disease (HD) is an autosomal dominant progressive neurodegenerative disorder that prominently affects the basal ganglia, leading to affective, cognitive, behavioral and motor decline. The basis of HD is a CAG repeat expansion to >35 CAG in a gene that codes for a ubiquitous protein known as huntingtin, resulting in an expanded N-terminal polyglutamine tract. The size of the expansion is correlated with disease severity, with increasing CAG accelerating the age of onset. A variety of possibilities have been proposed as to the mechanism by which the mutation causes preferential injury to the basal ganglia. The present chapter provides a basic overview of the genetics and pathology of HD.

SummaryHuntingtin is the protein mutated in Huntington's disease, a devastating neurodegenerative disorder. We demonstrate here that huntingtin is essential to control mitosis. Huntingtin is localized at spindle poles during mitosis. RNAi-mediated silencing of huntingtin in cells disrupts spindle orientation by mislocalizing the p150Glued subunit of dynactin, dynein, and the large nuclear mitotic apparatus NuMA protein. This leads to increased apoptosis following mitosis of adherent cells in vitro. In vivo inactivation of huntingtin by RNAi or by ablation of the Hdh gene affects spindle orientation and cell fate of cortical progenitors of the ventricular zone in mouse embryos. This function is conserved in Drosophila, the specific disruption of Drosophila huntingtin in neuroblast precursors leading to spindle misorientation. Moreover, Drosophila huntingtin restores spindle misorientation in mammalian cells. These findings reveal an unexpected role for huntingtin in dividing cells, with potential important implications in health and disease.

The ribosomal RNA genes of two species of Trypanosoma, Trypanosoma cruzi, the etiological agent of Chagas' disease, and Trypanosoma conorhini, a non-pathogenic rodent trypanosome, were cloned and partially characterized. The physical maps derived for their rRNA genes were similar throughout the region that encompasses the SSU-and LSU-rRNA coding sequences. However, the non-transcribed spacer DNA of both T. cruzi and T. conorhini was found to be polymorphic for several restriction enzyme sites. We show that strains of T. cruzi can be typed according to the characteristic restriction fragment length polymorphism of their NTS DNAs.

alpha-Synuclein is an abundant presynaptic protein implicated in neuronal plasticity and neurodegenerative diseases. Although the function of alpha-synuclein is not thoroughly elucidated, we found that alpha-synuclein regulates dopamine synthesis by binding to and inhibiting tyrosine hydroxylase, the rate limiting enzyme in dopamine synthesis. Understanding alpha-synuclein function in dopaminergic cells should add to our knowledge of this key protein, which is implicated in Parkinson's disease and other disorders. Herein, we report a mechanism by which alpha-synuclein diminishes tyrosine hydroxylase phosphorylation and activity in stably transfected dopaminergic cells. Short-term regulation of tyrosine hydroxylase depends on the phosphorylation of key seryl residues in the amino-terminal regulatory domain of the protein. Of these, Ser40 contributes significantly to tyrosine hydroxylase activation and dopamine synthesis. We observed that alpha-synuclein overexpression caused reduced Ser40 phosphorylation in MN9D cells and inducible PC12 cells. Ser40 is phosphorylated chiefly by the cyclic AMP-dependent protein kinase PKA and dephosphorylated almost exclusively by the protein phosphatase, PP2A. Therefore, we measured the impact of alpha-synuclein overexpression on levels and activity of PKA and PP2A in our cells. PKA was unaffected by alpha-synuclein. PP2A protein levels also were unchanged, however, the activity of PP2A increased in parallel with alpha-synuclein expression. Inhibition of PP2A dramatically increased Ser40 phosphorylation only in alpha-synuclein overexpressors in which alpha-synuclein was also found to co-immunoprecipitate with PP2A. Together the data reveal a functional interaction between alpha-synuclein and PP2A that leads to PP2A activation and underscores a key role for alpha-synuclein in protein phosphorylation.

Information regarding the mechanism underlying Huntington's disease (HD) has come primarily from the analysis of transgenic mice overexpressing additional copies of full-length or truncated mutant huntingtin. However, unraveling the molecular causes of neuronal dysfunction in transgenic mouse models requires caution since over expression of the transgene (either truncated or full-length) may cause abnormalities by mechanisms other than those in HD. From a genetic perspective, the most accurate strategy for replicating HD in the mouse is to insert pathogenic expanded CAG repeats into the mouse homolog of the HD gene. These mouse models have provided new tools to investigate the early events of the disease as well as its progression. Another important issue regarding HD pathogenesis is whether HD results from a gain-of-function mechanism or loss of the wild-type function contributes to the disease. Recent evidence stemming from hypomorphic and knock-out mouse models suggests that loss of wild-type protein function may also play a role in HD. Knock-in mouse models have provided valuable insights into the early events and progressive alterations that are associated with HD pathogenesis. Although motor deficits are mild compared to transgenic mice and neuronal loss is limited, the cellular and molecular changes and the neuropathology of knock-in mice are similar to those observed in HD patients. This finding not only is important for understanding the mechanism that leads to HD pathology but also impacts the development of therapeutic strategies.

Proteasomal dysfunction may contribute to neurodegenerative diseases; however, its effects on primary neurons are largely unknown. We have previously reported that pharmacological proteasomal inhibition leads to apoptosis and cytoplasmic ubiquitinated inclusions in primary rat cortical neurons. In cell lines the transcription factor p53 is regulated by the proteasome and in some cases it mediates death following proteasomal inhibition. It is unclear, however, if this is the case in primary neurons. Here we show in proteasome inhibitor-treated cortical neurons an early increase of p53 levels, accompanied by nuclear translocation. At later time points p53 is found sequestered within ubiquitinated inclusions. Compared to controls, p53-deficient mouse neurons show delayed apoptosis, but increased numbers of inclusions, likely secondary to enhanced survival. We conclude that p53 plays a role in cortical neuron apoptosis induced by proteasomal inhibition and, despite the fact that it localizes to inclusions, it is not necessary for their formation.

Body weight regulation is mediated through several major signaling pathways, some of which have been delineated by positional cloning of spontaneous genetic mutations in mice. Lepr(db/db) mice are obese due to a defect in the signaling portion of the leptin receptor, which has led to extensive study of this highly conserved system over the past several years. We have created an allelic series at Lepr for the further examination of LEPR signaling phenotypes using both the FLP /frt and CRE /loxP systems. By inserting a frt-PGK-neo-frt sequence in Lepr intron 16, we have generated a conditional gene repair Lepr allele ( Lepr-neo) that elicits morbid obesity, diabetes, and infertility in homozygous mice, recapitulating the obesity syndrome of Lepr(db/db) mice. Thus, in vivo excision of the PGK-neo cassette with a FLP recombinase transgene restores the lean and fertile phenotype to Lepr(flox/flox) mice. In the same construct, we have also inserted loxP sites that flank Lepr coding exon 17, a region that encodes a JAK docking site required for STAT3 signaling. CRE-mediated excision of Lepr coding exon 17 from Lepr with a frameshift in subsequent exons results in a syndrome of obesity, diabetes, and infertility in LeprDelta17/Delta17 mice, which is indistinguishable from Lepr(neo/neo) and Lepr(db/db) mice. We conclude that suppression of Lepr gene expression by PGK-neo is phenotypically equivalent to deletion of the Lepr signaling motifs, and therefore the Lepr(neo/neo) mouse may be used to investigate conditional gene repair of Lepr signaling deficiency.

Alpha-synuclein is a neuronal protein that is implicated in the control of synaptic vesicle function and in Parkinson's disease (PD). Consequently, alterations of alpha-synuclein levels may play a role in neurotransmission and in PD pathogenesis. However, the factors that regulate alpha-synuclein levels are unknown. Growth factors mediate neurotrophic and plasticity effects in CNS neurons, and may play a role in disease states. Here we examine the regulation of alpha-synuclein levels in primary CNS neurons, with particular emphasis on dopaminergic neurons. E18 rat cortical neurons and dopaminergic neurons of E14 rat ventral midbrain showed an induction of alpha-synuclein protein levels with maturation in culture. Application of basic Fibroblast growth factor (bFGF) promoted alpha-synuclein expression selectively within dopaminergic, and not GABAergic or cortical neurons. This induction was blocked by actinomycin D, but not by inhibition of bFGF-induced glial proliferation. alpha-Synuclein levels were not altered by glial-derived neurotrophic factor (GDNF), or by apoptotic stimuli. We conclude that bFGF promotes alpha-synuclein expression in cultured ventral midbrain dopaminergic neurons through a direct transcriptional effect. These results suggest that distinct growth factors may thus mediate plasticity responses or influence disease states in ventral midbrain dopaminergic neurons.

Familial Dysautonomia (FD) is an autosomal recessive disorder that affects 1/3,600 live births in the Ashkenazi Jewish population, and leads to death before the age of 40. The disease is characterized by abnormal development and progressive degeneration of the sensory and autonomic nervous system. A single base pair substitution in intron 20 of the Ikbkap gene accounts for 98% of FD cases, and results in the expression of low levels of the full-length mRNA with simultaneous expression of an aberrantly spliced mRNA in which exon 20 is missing. To date, there is no animal model for the disease, and the essential cellular functions of IKAP - the protein encoded by Ikbkap - remain unknown. To better understand the normal function of IKAP and in an effort to generate a mouse model for FD, we have targeted the mouse Ikbkap gene by homologous recombination. We created two distinct alleles that result in either loss of Ikbkap expression, or expression of an mRNA lacking only exon 20. Homozygosity for either mutation leads to developmental delay, cardiovascular and brain malformations, accompanied with early embryonic lethality. Our analyses indicate that IKAP is essential for expression of specific genes involved in cardiac morphogenesis, and that cardiac failure is the likely cause of abnormal vascular development and embryonic lethality. Our results also indicate that deletion of exon 20 abolishes gene function. This implies that the truncated IKAP protein expressed in FD patients does not retain any significant biological function.

The transcription start point (tsp) of the ribosomal RNA(rRNA)-encoding gene of Trypanosoma cruzi was mapped at 1550 bp upstream from the 18S rRNA coding sequence. The + 1 nucleotide (tsp) was determined to be a guanosine. As described for other eukaryotes, no consensus sequence was found when the putative promoter sequence (-200 to + 50) was compared with that described for Trypanosoma brucei and Crithidia fasciculata. However, a repeated element was found in the upstream intergenic spacer sequence (IGS) of T. cruzi. Motifs, present in this element, exhibit significant homology to the T. cruzi promoter sequence. Furthermore, the same motifs could be found, in a similar sequence organization, within the T. brucei promoter region. Therefore, the data described in this paper strongly indicate that the IGS rDNA (DNA coding for rRNA) organization in trypanosomatids appears similar to that found in higher eukaryotes.