Sustained expression of PGC-1α in the rat nigrostriatal system selectively impairs dopaminergic function.

Mitochondrial dysfunction and oxidative stress have been implicated in the etiology of Parkinson's disease. Therefore, pathways controlling mitochondrial activity rapidly emerge as potential therapeutic targets. Here, we explore the neuronal response to prolonged overexpression of peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α), a transcriptional regulator of mitochondrial function, both in vitro and in vivo. In neuronal primary cultures from the ventral midbrain, PGC-1α induces mitochondrial biogenesis and increases basal respiration. Over time, we observe an increasing proportion of the oxygen consumed by neurons which are dedicated to adenosine triphosphate production. In parallel to enhanced oxidative phosphorylation, PGC-1α progressively leads to a decrease in mitochondrial polarization. In the adult rat nigrostriatal system, adeno-associated virus (AAV)-mediated overexpression of PGC-1α induces the selective loss of dopaminergic markers and increases dopamine (DA) catabolism, leading to a reduction in striatal DA content. In addition, PGC-1α prevents the labeling of nigral neurons following striatal injection of the fluorogold retrograde tracer. When PGC-1α is expressed at higher levels following intranigral AAV injection, it leads to overt degeneration of dopaminergic neurons. Finally, PGC-1α overexpression does not prevent nigrostriatal degeneration in pathologic conditions induced by α-synuclein overexpression. Overall, we find that lasting overexpression of PGC-1α leads to major alterations in the metabolic activity of neuronal cells which dramatically impair dopaminergic function in vivo. These results highlight the central role of PGC-1α in the function and survival of dopaminergic neurons and the critical need for maintaining physiological levels of PGC-1α activity.

Growth, efficiency, and yield of commercial broilers from 1957, 1978, and 2005.

The effect of commercial selection on the growth, efficiency, and yield of broilers was studied using 2 University of Alberta Meat Control strains unselected since 1957 and 1978, and a commercial Ross 308 strain (2005). Mixed-sex chicks (n = 180 per strain) were placed into 4 replicate pens per strain, and grown on a current nutritional program to 56 d of age. Weekly front and side profile photographs of 8 birds per strain were collected. Growth rate, feed intake, and measures of feed efficiency including feed conversion ratio, residual feed intake, and residual maintenance energy requirements were characterized. A nonlinear mixed Gompertz growth model was used to predict BW and BW variation, useful for subsequent stochastic growth simulation. Dissections were conducted on 8 birds per strain semiweekly from 21 to 56 d of age to characterize allometric growth of pectoralis muscles, leg meat, abdominal fat pad, liver, gut, and heart. A novel nonlinear analysis of covariance was used to test the hypothesis that allometric growth patterns have changed as a result of commercial selection pressure. From 1957 to 2005, broiler growth increased by over 400%, with a concurrent 50% reduction in feed conversion ratio, corresponding to a compound annual rate of increase in 42 d live BW of 3.30%. Forty-two-day FCR decreased by 2.55% each year over the same 48-yr period. Pectoralis major growth potential increased, whereas abdominal fat decreased due to genetic selection pressure over the same time period. From 1957 to 2005, pectoralis minor yield at 42 d of age was 30% higher in males and 37% higher in females; pectoralis major yield increased by 79% in males and 85% in females. Over almost 50 yr of commercial quantitative genetic selection pressure, intended beneficial changes have been achieved. Unintended changes such as enhanced sexual dimorphism are likely inconsequential, though musculoskeletal, immune function, and parent stock management challenges may require additional attention in future selection programs.

An in vivo ultrahigh field 14.1 T (1) H-MRS study on 6-OHDA and α-synuclein-based rat models of Parkinson's disease: GABA as an early disease marker.

The detection of Parkinson's disease (PD) in its preclinical stages prior to outright neurodegeneration is essential to the development of neuroprotective therapies and could reduce the number of misdiagnosed patients. However, early diagnosis is currently hampered by lack of reliable biomarkers. (1) H magnetic resonance spectroscopy (MRS) offers a noninvasive measure of brain metabolite levels that allows the identification of such potential biomarkers. This study aimed at using MRS on an ultrahigh field 14.1 T magnet to explore the striatal metabolic changes occurring in two different rat models of the disease. Rats lesioned by the injection of 6-hydroxydopamine (6-OHDA) in the medial-forebrain bundle were used to model a complete nigrostriatal lesion while a genetic model based on the nigral injection of an adeno-associated viral (AAV) vector coding for the human α-synuclein was used to model a progressive neurodegeneration and dopaminergic neuron dysfunction, thereby replicating conditions closer to early pathological stages of PD. MRS measurements in the striatum of the 6-OHDA rats revealed significant decreases in glutamate and N-acetyl-aspartate levels and a significant increase in GABA level in the ipsilateral hemisphere compared with the contralateral one, while the αSyn overexpressing rats showed a significant increase in the GABA striatal level only. Therefore, we conclude that MRS measurements of striatal GABA levels could allow for the detection of early nigrostriatal defects prior to outright neurodegeneration and, as such, offers great potential as a sensitive biomarker of presymptomatic PD.

Effect of holding temperature, shackling, sex, and age on broiler breast meat quality.

Antemortem holding temperature and flapping can affect broiler pectoralis major (referred to as p. major) meat quality. The influence of environmental temperature 12 to 14 h before processing and antemortem handling on p. major functional properties and color was tested at multiple processing ages in a 3 × 2 × 5 factorial design study. At 28, 35, 42, 49, and 56 d of age, mixed-sex broilers were crated and held in one of 3 target temperature treatments: heat (30°C), thermoneutral (21°C), or cool (7°C). Following feed withdrawal, birds were transported to a processing facility where either a long (120 s) or short (<10 s) shackling treatment was imposed. Sex was determined at processing. Lightness, redness, and yellowness values were measured at deboning [4.50 to 8.25 h postmortem (PM)] and at 24 h PM. Ultimate pH was measured at 24 h PM. Drip loss, cook loss, and Allo-Kramer shear force were determined at 72 h PM. Ultimate pH was highest in the cool treatment, with no difference between heat and thermoneutral treatments (5.97 vs. 5.87 and 5.90, respectively; P = 0.0004). Ultimate pH correlated negatively with drip loss (r = -0.47; P < 0.0001). Drip loss was lowest in the cool treatment, with no difference between the heat and thermoneutral treatments (2.06 vs. 2.24 and 2.19%, respectively; P = 0.007). The p. major from broilers in the heat treatment had higher Allo-Kramer shear force values than those in both the thermoneutral and cool treatments (4.64 kg/g vs. 4.21 and 4.21 kg/g, respectively; P = 0.023). With the exception of 49 d, broilers subjected to the long shackling treatment had higher redness values at deboning than broilers subjected to the short shackling treatment; by 24 h PM only the 28-d broilers subjected to the long shackling treatment had higher redness values. Higher hue angles at deboning and 24 h PM were observed in the short treatment compared with the long treatment (65.58 vs. 57.46 and 68.48 vs. 63.31, respectively; P < 0.0001), indicating that p. major from long-shackled broilers were redder in appearance. Ultimate pH was lower in female broilers than in male broilers (5.87 vs. 5.96, respectively; P < 0.0001) and subsequent drip loss was higher in females compared with males (2.34 vs. 1.99%, respectively; P < 0.0001). Although temperature and handling before slaughter affected p. major color and texture traits, the differences may not be large enough to have commercial significance. Differences in p. major quality attributable to sex of the bird may have greater commercial significance than previously reported.

Efficient transduction of non-human primate motor neurons after intramuscular delivery of recombinant AAV serotype 6.

Retrograde transport of viral vectors in the rodent spinal cord provides a powerful means to administer a therapeutic transgene from the innervated musculature. With the aim of scaling up this approach to non-human primates, we have injected recombinant adeno-associated vectors (rAAV) serotype 6 expressing enhanced green fluorescent protein (eGFP) into the gastrocnemius muscle of African green monkeys to determine whether this results in efficient transgene delivery to lumbar motor neurons. Cells expressing eGFP were observed across more than 1 cm of the spinal cord 4 weeks after intramuscular injection, reaching more than half of motor neurons in some cross-sections. Furthermore, quantitative PCR on the spinal cord tissue confirmed that eGFP expression within motor neurons was due to bona fide retrograde transport of the vector genome from the muscle. Although infiltrations of macrophages and lymphocytes were observed in the rAAV2/6-injected muscle, there was no detectable immune response within the transduced region of the spinal cord. These findings imply that retrograde delivery of rAAV serotype 6 in a primate species constitutes a non-invasive and robust approach to transduce motor neurons, a crucial target cell population in neurodegenerative disorders, such as amyotrophic lateral sclerosis and spinal muscular atrophy.

Linkage of replication to start by the Cdk inhibitor Sic1.

In Saccharomyces cerevisiae, three G1 cyclins (Clns) are important for Start, the event committing cells to division. Sic1, an inhibitor of C1b-Cdc28 kinases, became phosphorylated at Start, and this phosphorylation depended on the activity of Clns. Sic1 was subsequently lost, which depended on the activity of Clns and the ubiquitin-conjugating enzyme Cdc34. Inactivation of Sic1 was the only nonredundant essential function of Clns, because a sic1 deletion rescued the inviability of the cln1 cln2 cln3 triple mutant. In sic1 mutants, DNA replication became uncoupled from budding. Thus, Sic1 may be a substrate of Cln-Cdc28 complexes, and phosphorylation and proteolysis of Sic1 may regulate commitment to replication at Start.

Omega-3-enriched broiler meat: 2. Functional properties, oxidative stability, and consumer acceptance.

Consumers are becoming more aware of the impact on their health of the food they eat. One of the ways they hope to reduce their risk of cardiovascular disease is by consuming more foods enriched with polyunsaturated fatty acids (PUFA), particularly n-3 fatty acids. Flaxseed is a good source for increasing the n-3 PUFA in poultry meat because of the high content of alpha-linolenic acid. A study was conducted to identify an optimal process to enrich of broiler diets with n-3 PUFA by using 2 levels of flaxseed fed for various times before processing. The acceptability of broiler meat functional properties was tested to ensure that further processing efficiencies would not be compromised by the enrichment strategy. This experiment was conducted as a 2 x 8 factorial, with 2 dietary levels of ground flaxseed (10 and 17%) fed for 8 lengths of time before processing [0 (control), 4, 8, 12, 16, 20, 24, and 35 d]. Of 650 Ross x Ross 308 mixed-sex broilers reared in this study, 128 were used to evaluate breast and thigh meat functional properties, oxidative stability, and sensory analysis. No statistical interactions were found between treatments for chicken breast meat quality traits. The duration of feeding flaxseed strongly affected meat quality parameters. In particular, feeding flaxseed for 16 d resulted in a final pH of 5.65, compared with 5.93 in the control. The lower ultimate pH found in animals fed flaxseed affected meat cooking loss, drip loss, and shear value (P < 0.0001). Shear value significantly increased after 16 d of feeding flaxseed (P < 0.0001). Susceptibility to oxidation increased in both breast and thigh broiler meat with the duration of feeding flaxseed. Enriching the diet for less than 16 d did not result in perceivable sensory defects. Duration of flaxseed feeding significantly affected the color characteristics, functional properties, and oxidative stability of broiler meat.

Omega-3-enriched broiler meat: 1. Optimization of a production system.

Consumer awareness of the health benefits of n-3 fatty acids is growing and is driving consumer demand for enriched food products. Enrichment of meat with n-3 fatty acids is an opportunity for the broiler production sector to add value to their product, but enrichment can increase the cost of production. A study was conducted to determine an optimal production strategy for n-3 enrichment of broiler meat using ground full-fat flaxseed. Low and high levels of dietary flaxseed (10 and 17%, respectively) were fed to broilers for 8 lengths of time (0, 4, 8, 12, 16, 20, 24, or 35 d) before processing at 35 d. Increasing the level or duration decreased feed intake, BW, and the percentage yield of carcass and breast. Flaxseed level and duration of feeding increased feed conversion ratios and the cost of production. Feeding flaxseed at 10 and 17% increased breast n-3 fatty acid levels by 7.65 and 13.70 mg/100 g of meat per day, respectively. In breast meat, the threshold level of 300 mg/g, required in Canada for labeling foods as a source of n-3 fatty acids, was reached at 12.1 and 24.1 d in the high and low flaxseed treatments, respectively. This was due primarily to a tripling of alpha-linolenic acid (18:3n-3) levels in the breast meat. Levels of the long-chain n-3 fatty acid eicosapentaenoic acid increased significantly in the breast meat with increased level and duration of flax feeding, indicating that birds were able to desaturate and elongate alpha-linolenic acid to eicosapentaenoic acid. To minimize cost, while achieving adequate breast meat n-3 enrichment, carcass weight, and meat yield, feeding 10% flaxseed for 24.1 d before processing is recommended as an optimal breast meat n-3 enrichment strategy. The optimal thigh meat n-3 enrichment strategy was to feed 10% flaxseed for 4.54 d before processing.

Metabolic context and possible physiological themes of sigma(54)-dependent genes in Escherichia coli.

Sigma(54) has several features that distinguish it from other sigma factors in Escherichia coli: it is not homologous to other sigma subunits, sigma(54)-dependent expression absolutely requires an activator, and the activator binding sites can be far from the transcription start site. A rationale for these properties has not been readily apparent, in part because of an inability to assign a common physiological function for sigma(54)-dependent genes. Surveys of sigma(54)-dependent genes from a variety of organisms suggest that the products of these genes are often involved in nitrogen assimilation; however, many are not. Such broad surveys inevitably remove the sigma(54)-dependent genes from a potentially coherent metabolic context. To address this concern, we consider the function and metabolic context of sigma(54)-dependent genes primarily from a single organism, Escherichia coli, in which a reasonably complete list of sigma(54)-dependent genes has been identified by computer analysis combined with a DNA microarray analysis of nitrogen limitation-induced genes. E. coli appears to have approximately 30 sigma(54)-dependent operons, and about half are involved in nitrogen assimilation and metabolism. A possible physiological relationship between sigma(54)-dependent genes may be based on the fact that nitrogen assimilation consumes energy and intermediates of central metabolism. The products of the sigma(54)-dependent genes that are not involved in nitrogen metabolism may prevent depletion of metabolites and energy resources in certain environments or partially neutralize adverse conditions. Such a relationship may limit the number of physiological themes of sigma(54)-dependent genes within a single organism and may partially account for the unique features of sigma(54) and sigma(54)-dependent gene expression.

A novel multiple affinity purification tag and its use in identification of proteins associated with a cyclin-CDK complex.

A novel multiple affinity purification (MAFT) or tandem affinity purification (TAP) tag has been constructed. It consists of the calmodulin binding peptide, six histidine residues, and three copies of the hemagglutinin epitope. This 'CHH' MAFT tag allows two or three consecutive purification steps, giving high purity. Active Clb2-Cdc28 kinase complex was purified from yeast cells after inserting the CHH tag into Clb2. Associated proteins were identified using mass spectrometry. These included the known associated proteins Cdc28, Sic1 and Cks1. Several other proteins were found including the 70 kDa chaperone, Ssa1.

Survival of encapsulated human primary fibroblasts and erythropoietin expression under xenogeneic conditions.

Allogeneic cells are the most attractive source for cell transplantation, as the use of xenogeneic cells is hampered by safety concerns and the use of autologous cells involves practical difficulties. The immune rejection of allogeneic cells can be overcome by physical immunoprotection provided by polymer encapsulation. To study the variability of cell and donor sources, we compared different primary human cells as candidates for gene therapy-mediated delivery of human erythropoietin (hEpo). DARC-3.1 fibroblasts, MDX-01 fibroblasts, and ARPE-19 retinal pigment epithelial cells were encapsulated into polyethersulfone hollow fibers and implanted for 1 month in nude mice as well as in immunocompetent and FK506-immunosuppressed mice to test their in vivo resistance, with the assumption that xenogeneic conditions constitute a stringent model for human application. DARC-3.1 fibroblasts showed the best survival, prompting us to evaluate cell lineages from the same donor (DARC-3.2) or another donor (DARC-4.3 and DARC-4.4). With the exception of DARC-4.3, the remaining three lineages showed comparable survival in immunocompetent C3H and DBA/2J mice. DARC-3.1 fibroblasts were retrovirally engineered with hEpo cDNA, reaching a secretion level of 170 IU of hEpo per 10(6) cells per day. Encapsulated DARC-3.1-hEpo cells led to significantly increased hematocrits in the various hosts and under various transplantation conditions. The present study shows that encapsulated primary human DARC-3.1 fibroblasts are able to survive under xenogeneic conditions and, once engineered with hEpo cDNA, to increase the hematocrit of transplanted mice.

7,12-Dimethylbenz[a]anthracene-induced mouse keratinocyte malignant transformation independent of Harvey ras activation.

Independent clones of mouse keratinocytes initiated in vitro gave rise to tumor phenotypes typical of mouse skin multistage carcinogenesis and histologically similar to human tumors of the skin, and head and neck. High-molecular-weight genomic DNAs isolated from two 7,12-dimethylbenz[a]anthracene (DMBA)-initiated murine epithelial carcinoma cell lines and one papilloma cell line were examined for transforming activity by transfection into NIH3T3 cells. DNAs from each of these cell lines resulted in the formation of foci morphologically unlike foci containing an activated c-Ha-ras oncogene. Following polymerase chain reaction amplification of the c-Ha-ras gene, Xba I restriction analysis and oligonucleotide differential hybridization did not detect 61st, 12th, or 13th codon mutations. Southern and Northern analysis confirmed that the normal c-Ha-ras gene was not activated by amplification or overexpression. These results provide evidence that 7,12-dimethylbenz[a]anthracene-induced malignant transformation of murine keratinocytes occurred independent of point mutations associated with c-Ha-ras activation. The absence of an activated c-Ha-ras oncogene in these cell lines distinguishes our model from other mouse models of carcinogenesis and may provide a model for functional genetic changes during initiation and progression of human epithelial cancers.

Long-term host unresponsiveness to encapsulated xenogeneic myoblasts after transient immunosuppression.

BACKGROUND: Encapsulating cells prevents the immune destruction of allogeneic cells in the subcutaneous site as well as allogeneic and xenogeneic cells in the central nervous system. However, when encapsulated xenogeneic cells are implanted s.c., they may be subject to rejection by the host. METHODS: Murine C2C12 myoblasts engineered to secrete mouse erythropoietin (mEpo) were used to evaluate the response of control versus FK506-treated xenogeneic recipients (Fischer rats) to encapsulated myoblasts implanted in the s.c. site. RESULTS: Encapsulated C2C12 mEpo cells were rapidly eliminated in immunocompetent Fischer rats. Devices transplanted into nude rats induced a sustained increase in the hematocrit, associated with an extended viability of the encapsulated cells. Short-term immunosuppression with FK506, for periods lasting either 1, 2, or 4 weeks after implantation, permitted the long-term survival of encapsulated C2C12 mEpo cells in Fischer rats. Animals increased their hematocrits to more than 70% and maintained these levels for 13 weeks, independent of the duration of FK506 treatment. Unencapsulated C2C12 mEpo cells injected i.m. in immunosuppressed animals were rejected over this same period. CONCLUSIONS: Encapsulation alone cannot protect xenogeneic myoblasts from immune destruction in the s.c. site. These results highlight the importance of combining the technique of cell encapsulation with transient immunosuppression to achieve long-term survival of xenografted myoblasts in a peripheral immunoreactive site.

Inducing host acceptance to encapsulated xenogeneic myoblasts.

BACKGROUND: Cell encapsulation holds promise for the chronic delivery of recombinant proteins such as erythropoietin. Encapsulated xenogeneic mouse C2C12 myoblasts display long-term survival in the central nervous system whereas they do not in the subcutaneous tissue, suggesting that encapsulation only partially prevents affector and effector mechanisms of the host immune response. Transient immunosuppression with FK506 at the time of subcutaneous implantation leads, however, to their long-term survival. The nature of this acceptance was further investigated in this report. METHODS: Fischer rats were rendered unresponsive to encapsulated murine C2C12 myoblasts secreting mouse erythropoietin by either a 1- or 4-week initial treatment of FK506. To examine the extent of xenograft acceptance, animal were challenged with a second implant 9 weeks after the initial implantation. RESULTS: Challenging animals treated only 1 week with FK506 led to rejection of both primary and secondary implants. Animals administered FK506 for 4 weeks accepted both implants over the period investigated. However, these animals rejected unencapsulated xenogeneic cells injected at a later time, highlighting the requirement of the polymer membrane for immune protection. Developed unresponsiveness to encapsulated xenogeneic myoblasts lasted over extended periods (at least 7 months), in the absence of both immunosuppression and stimulating xenoantigens. CONCLUSIONS: These findings reveal that host acceptance of encapsulated but not unencapsulated xenogeneic myoblasts can be developed in the subcutaneous tissue after transient FK506 immunosuppression. This may have direct clinical relevance as it enables capsules to be replaced without additional immunosuppression, facilitating long-term cell-based therapies.

Selective degeneration of a putative cholinergic pathway in the chinchilla cochlea following infusion with ethylcholine aziridinium ion.

Ethylcholine aziridinium ion (AF64A) diluted in artificial perilymph, or artificial perilymph alone was infused into the cochlea of chinchillas. After a survival time of 7 days, the cochleas were fixed with aldehydes, post-fixed in osmium and embedded in epoxy resin for light and electron microscopy. The ultrastructure of the cochleas infused with artificial perilymph was normal. Infusion of 1 microM AF64A resulted in massive degeneration of the axons of the lateral efferent system, a putative cholinergic pathway that originates in the brainstem and terminates on dendrites of the spiral ganglion innervating cochlear inner hair cells. The axons and terminals of a second putative cholinergic pathway, the medial efferent system which terminates on the outer hair cells, were normal. Infusion of AF64A in a concentration of 10 microM resulted in significant pathology of cochlear and supporting cells as well as the loss of efferent terminals at both inner and outer hair cell regions. The results suggest that AF64A is a selective neurotoxin when used under low-dosage conditions, and that certain pathways may be more susceptible to the effects of AF64A than others. One interpretation of these findings is that lateral efferent axons may have a higher rate of high-affinity choline uptake than terminals of the medial efferent axons.

Location, substructure, and composition of basal laminar drusen compared with drusen associated with aging and age-related macular degeneration.

PURPOSE: To determine whether basal laminar drusen differ in their location, ultrastructure, or composition from drusen associated with aging and age-related macular degeneration. METHODS: A paraffin-embedded block from an eye of a patient with basal laminar drusen was obtained. Sections were examined immunohistochemically using a battery of antibodies and lectins directed against drusen-associated proteins and glycoconjugates, respectively. Thin sections were examined by electron microscopy and compared with eyes with age-related macular degeneration. RESULTS: Drusen in the eye with basal laminar drusen are located between the basal lamina of the retinal pigment epithelium and the inner collagenous layer of Bruch membrane, just as they are in age-related macular degeneration. Two distinct ultrastructural phenotypes are observed in the eye with basal laminar drusen; their substructure is indistinguishable from drusen phenotypes in age-related macular degeneration. Both basal laminar drusen and drusen associated with age-related macular degeneration are bound by the lectins Ricinis communis agglutinin and Arachis hypogea agglutinin (after neuraminidase digestion) and by antivitronectin, anti-HLA-DR, anti-serum amyloid P, and anti-C5 antibodies, but not by antibodies directed against basement membrane-associated heparan sulfate proteoglycan, laminin, fibrinogen, or collagen type IV. CONCLUSIONS: These data support the notion that cuticular or basal laminar drusen are similar to, and perhaps indistinguishable from, drusen associated with age-related macular degeneration and are not nodular or diffuse thickenings of Bruch membrane, as previously suggested. Thus, we suggest basal laminar drusen is a misnomer. This clinical phenotype should be identified as "early adult onset, grouped drusen" or by the eponym "Gass syndrome." Features of basal laminar drusen, such as uniform drusen size, clustered distribution, and angiographic features, do not appear to be related to differences in drusen location, composition, or substructure.

Measurement of autophagy flux in the nervous system in vivo.

Accurate methods to measure autophagic activity in vivo in neurons are not available, and most of the studies are based on correlative and static measurements of autophagy markers, leading to conflicting interpretations. Autophagy is an essential homeostatic process involved in the degradation of diverse cellular components including organelles and protein aggregates. Autophagy impairment is emerging as a relevant factor driving neurodegeneration in many diseases. Moreover, strategies to modulate autophagy have been shown to provide protection against neurodegeneration. Here we describe a novel and simple strategy to express an autophagy flux reporter in the nervous system of adult animals by the intraventricular delivery of adeno-associated viruses (AAV) into newborn mice. Using this approach we efficiently expressed a monomeric tandem mCherry-GFP-LC3 construct in neurons of the peripheral and central nervous system, allowing the measurement of autophagy activity in pharmacological and disease settings.

Rab1A over-expression prevents Golgi apparatus fragmentation and partially corrects motor deficits in an alpha-synuclein based rat model of Parkinson's disease.

Although the overabundance of human alpha-synuclein in nigral dopaminergic neurons is considered to play a pathogenic role in Parkinson's disease (PD), it remains unclear how alpha-synuclein leads to neuronal degeneration and motor symptoms. Here, we explored the effect of human alpha-synuclein in the rat substantia nigra following AAV-mediated gene delivery inducing a moderate loss of dopaminergic neurons together with motor impairments. A significant fraction of the surviving nigral neurons were found to express human αSyn and displayed a pathological fragmentation of the Golgi apparatus. This observation prompted further investigation on the role of the secretory pathway, in particular at the ER/Golgi level, in alpha-synuclein toxicity. To address this question, we co-expressed human alpha-synuclein with Rab1A, a regulator of ER-to-Golgi vesicular trafficking, and found a significant reduction of Golgi fragmentation. Rab1A did not protect the dopaminergic neurons from the alpha-synuclein-induced degeneration that occurred within several months following vector injection. However, we observed in animals co-expressing Rab1A an improvement of motor behavior that correlates with the rescue of normal Golgi morphology in alpha-synuclein-expressing dopaminergic neurons. The non-prenylable mutant Rab1A-DeltaCC did not produce any of the effects observed with the wild-type form of Rab1A, linking the protective role of Rab1A with its activity in ER-to-Golgi vesicular trafficking. In conclusion, Rab1A can rescue the Golgi fragmentation caused by the overabundance of alpha-synuclein in nigral dopaminergic neurons, improving the ability of the surviving neurons to control motor function in hemiparkinsonian animals.

Salmonella typhimurium nit is nadE: defective nitrogen utilization and ammonia-dependent NAD synthetase.

S. typhimurium nit mutants are defective in nitrogen assimilation, despite having normal levels of assimilatory enzymes. Complementation, enzyme assays, and genetic mapping show that nit is nadE. We present evidence that ammonia, not glutamine, is the physiological substrate for eubacterial NAD synthetases and that low activity completely accounts for the mutant phenotype.