Alphaviral vectors for gene transfer into neurons

Alphaviruses are small, enveloped positive-strand RNA viruses that have been successfully transformed into expression vectors in the case of Semliki Forest virus (SFV), Sindbis virus (SIN), and Venezuelan equine encephalitis virus. Compared to other viral vectors, their advantages are easy and fast generation of recombinant viral particles, rapid onset, and high-level transgene expression. When applied to neuronal tissue, SFV and SIN vectors possess the additional advantage of efficiently and preferentially transducing neurons rather than non-neuronal cells. This article gives an overview of the biology of SFV and SIN, their generation into expression vectors, and their application in neurobiology, with particular emphasis on the transduction of hippocampal neurons. In addition, it describes the more recent development of alphaviral vectors with decreased or absent cytotoxicity and lowered transgene expression, temperature-controllable gene expression, and altered host-cell specificity in the central nervous system (CNS). Finally, the review evaluates the use of SFV and SIN vectors in hippocampal tissue cultures vs recombinant lentivirus, adenovirus type 5, adeno-associated virus type 2, and measles viru

Shape oscillations of human neutrophil leukocytes: characterization and relationship to cell motility

When neutrophil leukocytes are stimulated by chemotactic factors or by substratum contact, they change their shape. Shape changes are a prerequisite for cellular migration and typically involve the extrusion of thin, veil-like lamellipods and the development of morphological polarity. Stimulation also leads to changes in the neutrophil content of filamentous actin (F-actin), which is the major cytoskeletal component. Suspensions of human neutrophils stimulated with chemoattractants exhibit sinusoidal light-scattering oscillations with a period of approximately 8 s at 37°C. These oscillations arise from periodic fluctuations in the cell body size caused by lamellipod extension and retraction cycles. The light-scattering oscillations are paralleled by corresponding oscillations in F-actin content. This raises the interesting possibility that cyclic actin polymerization constitutes the driving force for shape oscillations of suspended neutrophils. Similar periodic shape changes are present in neutrophils crawling on a surface, suggesting that shape oscillations are important for neutrophil motion. This review summarizes our present knowledge about shape oscillations in suspended and crawling neutrophils and discusses a possible role for these oscillations in neutrophil motility

The Transcriptional Corepressor NAB2 Inhibits NGF-induced Differentiation of PC12 Cells

The PC12 pheochromocytoma cell line responds to NGF by undergoing growth arrest and proceeding to differentiate toward a neuronal phenotype. Among the early genetic events triggered by NGF in PC12 cells are the rapid activation of the zinc finger transcription factor Egr1/NGFI-A, and a slightly delayed induction of NAB2, a corepressor that inhibits Egr1 transcriptional activity. We found that stably transfected PC12 cells expressing high levels of NAB2 do not differentiate, but rather continue to proliferate in response to NGF. Inhibition of PC12 differentiation by NAB2 overexpression was confirmed using two additional experimental approaches, transient transfection, and adenoviral infection. Early events in the NGF signaling cascade, such as activation of MAP kinase and induction of immediate-early genes, were unaltered in the NAB2-overexpressing PC12 cell lines. However, induction of delayed NGF response genes such as TGF-beta 1 and MMP-3 was inhibited. Furthermore, NAB2 overexpression led to downregulation of p21WAF1, a molecule previously shown to play a pivotal role in the ability of PC12 cells to undergo growth arrest and commit to differentiation in response to NGF. Cotransfection with p21WAF1 restored the ability of NAB2-overexpressing PC12 cells to differentiate in response to NGF

Homer/vesl proteins and their roles in CNS neurons

Since their initial discovery in 1997, Homer/Vesl proteins have become increasingly investigated as putative regulators of receptor and ion-channel function in the central nervous system. Within a relatively brief period, numerous research reports have described manifold effects of Homer proteins, including the modulation of the trafficking of type I metabotropic glutamate receptors (mGluRs), axonal pathfinding, mGluR coupling to calcium and potassium channels, agonist-independent mGluR activity, ryanodine receptor regulation, locomotor activity, and behavioral plasticity. This review summarizes our current knowledge on the induction, expression, and structure of the various forms of Homer proteins, as well as their roles in neuronal function. In addition, we provide an outlook on novel developments with regard to the involvement of Homer-1a in hippocampal synaptic functio

Novel Semliki Forest virus vectors with reduced cytotoxicity and temperature sensitivity for long-term enhancement of transgene expression.

Alphaviral vectors inhibit host cell protein synthesis and are cytotoxic. To overcome these limitations, we modified the nonstructural protein-2 (nsP2) gene in the Semliki Forest virus (SFV) vector, pSFV1. Packaging of SFV replicons with two point mutations in nsP2 resulted in high-titer recombinant SFV(PD) particles. SFV(PD) led to more efficient host cell protein synthesis, exhibited reduced cytotoxicity and improved cell survival, and allowed greater and prolonged transgene expression than the original vector, SFV. In dissociated hippocampal neurons and organotypic rat hippocampal slices, SFV(PD) infection preserved neuronal morphology and synaptic function more efficiently than SFV. Combination of the two point mutations with a replication-persistent mutation in nsP2 resulted in a highly temperature-sensitive vector, SFV(PD713P), which efficiently transduced neurons in hippocampal slice cultures. At 31 °C, SFV(PD713P) allowed continuous transgene expression in BHK cells, at amounts comparable to SFV(PD). These new SFV mutants are expected to substantially broaden the application of alphaviral vectors in neurons and other mammalian cells

Alphaviral Vectors for Gene Transfer into Neurons

Alphaviruses are small, enveloped positive-strand RNA viruses that have been successfully transformed into expression vectors in the case of Semliki Forest virus (SFV), Sindbis virus (SIN), and Venezuelan equine encephalitis virus. Compared to other viral vectors, their advantages are easy and fast generation of recombinant viral particles, rapid onset, and high-level transgene expression. When applied to neuronal tissue, SFV and SIN vectors possess the additional advantage of efficiently and preferentially transducing neurons rather than non-neuronal cells. This article gives an overview of the biology of SFV and SIN, their generation into expression vectors, and their application in neurobiology, with particular emphasis on the transduction of hippocampal neurons. In addition, it describes the more recent development of alphaviral vectors with decreased or absent cytotoxicity and lowered transgene expression, temperature-controllable gene expression, and altered host-cell specificity in the central nervous system (CNS). Finally, the review evaluates the use of SFV and SIN vectors in hippocampal tissue cultures vs recombinant lentivirus, adenovirus type 5, adeno-associated virus type 2, and measles viru

Advanced modular self‐inactivating lentiviral expression vectors for multigene interventions in mammalian cells and in vivo transduction

In recent years, lentiviral expression systems have gained an unmatched reputation among the gene therapy community for their ability to deliver therapeutic transgenes into a wide variety of difficult‐to‐transfect/transduce target tissues (brain, hematopoietic system, liver, lung, retina) without eliciting significant humoral immune responses. We have cloned a construction kit‐like self‐inactivating lentiviral expression vector family which is compatible to state‐of‐the‐art packaging and pseudotyping technologies and contains, besides essential cis‐acting lentiviral sequences, (i) unparalleled polylinkers with up to 29 unique sites for restriction endonucleases, many of which recognize 8 bp motifs, (ii) strong promoters derived from the human cytomegalovirus immediate‐early promoter (PhCMV) or the human elongation factor 1α (PhEF1α), (iii) PhCMV- or PPGK- (phosphoglycerate kinase promoter) driven G418 resistance markers or fluorescent protein‐based expression tracers and (iv) tricistronic expression cassettes for coordinated expression of up to three transgenes. In addition, we have designed a size‐optimized series of highly modular lentiviral expression vectors (pLenti Module) which contain, besides the extensive central polylinker, unique restriction sites flanking any of the 5′U3, R‐U5‐ψ+‐SD, cPPT‐RRE‐SA and 3′LTRΔU3 modules or placed within the 5′U3 (-78 bp) and 3′LTRΔU3 (8666 bp). pLentiModule enables straightforward cassette‐type module swapping between lentiviral expression vector family members and facilitates the design of Tat‐independent (replacement of 5′LTR by heterologous promoter elements), regulated and self‐excisable proviruses (insertion of responsive operators or LoxP in the 3′LTRΔU3 element). We have validated our lentiviral expression vectors by transduction of a variety of insect, chicken, murine and human cell lines as well as adult rat cardiomyocytes, rat hippocampal slices and chicken embryos. The novel multi‐purpose construction kit‐like vector series described here is compatible with itself as well as many other (non‐viral) mammalian expression vectors for straightforward exchange of key components (e.g. promoters, LTRs, resistance genes) and will assist the gene therapy and tissue engineering communities in developing lentiviral expression vectors tailored for optimal treatment of prominent human disease

Optogenetic manipulation of medullary neurons in the locust optic lobe

The locust is a widely used animal model for studying sensory processing and its relation to behavior. Due to the lack of genomic information, genetic tools to manipulate neural circuits in locusts are not yet available. We examined whether Semliki Forest virus is suitable to mediate exogenous gene expression in neurons of the locust optic lobe. We subcloned a channelrhodopsin variant and the yellow fluorescent protein Venus into a Semliki Forest virus vector and injected the virus into the optic lobe of locusts (Schistocerca americana). Fluorescence was observed in all injected optic lobes. Most neurons that expressed the recombinant proteins were located in the first two neuropils of the optic lobe, the lamina and medulla. Extracellular recordings demonstrated that laser illumination increased the firing rate of medullary neurons expressing channelrhodopsin. The optogenetic activation of the medullary neurons also triggered excitatory postsynaptic potentials and firing of a postsynaptic, looming-sensitive neuron, the lobula giant movement detector. These results indicate that Semliki Forest virus is efficient at mediating transient exogenous gene expression and provides a tool to manipulate neural circuits in the locust nervous system and likely other insects

THE PREVALENCE OF PALPABLE FINGER JOINT NODULES IN DIFFUSE IDIOPATHIC SKELETAL HYPEROSTOSIS (DISH). A CONTROLLED STUDY

The presence of clinically palpable finger joint nodules a(Heberden's and Bouchard's nodes) was documented in 123 consecutive cases with diffuse idiopathic skeletal hyperostosis (DISH) of the thoracic spine and 191 matched DISH negative controls. The prevalence of palpable finger joint nodules was almost twice as frequent in cases with spinal DISH compared to controls (46% versus 31%, X2 = 7.67, P<0.01; multivariate adjusted odds ratio OR = 1.84; 95% CI: 1.14-2.98). This increase was most marked at the proximal interphalangeal joint, in males and in patients up to the age of 65 years. In addition and independent of other variables such as hyperostotic features, age and sex, the prevalence of palpable finger joint nodules was about twice as high in probands with a history of physically heavy work compared to those without (43% ver sus 26%, X = 9.18, P<0.005; multivariate adjusted odds ratio OR = 2.10; 95% CI: 1.26-3.52). From these results we con clude that DISH should be considered as an independent risk factor in the development of finger joint nodule

DIFFUSE IDIOPATHIC SKELETAL HYPEROSTOSIS (DISH) OF THE SPINE: A CAUSE OF BACK PAIN? A CONTROLLED STUDY

This is the first controlled study of the frequency of back pain in a European caucasian population with diffuse idiopathic skeletal hyperostosis (DISH). Elderly patients admitted to hospital for reasons other than back pain were assessed for the presence of spinal DISH using the routine lateral chest radiograph films. A total of 106 probands (82 males, 24 females) with a mean age of 70 years fulfilled the criteria for DISH as defined previously. One hundred and seventyeight patients (117 males, 61 females) not meeting these criteria were used as controls. The prevalence of back pain was assessed by a blinded interviewer using a structured questionnaire. Our primary hymthesis was that spinal DISH positive probands had not had back pain more often than controls. The controlled study showed no statistically significant difference in pain frequency between spinal DISH positive probands and controls at any spinal level. We conclude that back pain does not occur more often in radiographically defined DISH positive probands than in controls. The radiological finding of spinal DISH, as far as it does not lead to stenosis of the spinal canal or dysphagia, thus seems to be a finding without clinical relevanc