Treatment of motoneuron degeneration by intracerebroventricular delivery of VEGF in a rat model of ALS

Nature Neuroscience January 2005 - Vol 8 No 1

Neurotrophin treatment has so far failed to prolong the survival of individuals affected with amyotrophic lateral sclerosis (ALS), an incurable motoneuron degenerative disorder. Here we show that intracerebroventricular (i.c.v.) delivery of recombinant vascular endothelial growth factor (Vegf) in a SOD1 G93A rat model of ALS delays onset of paralysis by 17 d, improves motor performance and prolongs survival by 22 d, representing the largest effects in animal models of ALS achieved by protein delivery. By protecting cervical motoneurons, i.c.v. delivery of Vegf is particularly effective in rats with the most severe form of ALS with forelimb onset. Vegf has direct neuroprotective effects on motoneurons in vivo, because neuronal expression of a transgene expressing the Vegf receptor prolongs the survival of SOD1 G93A mice. On i.c.v. delivery, Vegf is anterogradely transported and preserves neuromuscular junctions in SOD1 G93A rats. Our findings in preclinical rodent models of ALS may have implications for treatment of neurodegenerative disease in general.

Related article:
VEGF: multitasking in ALS
Nature Neuroscience News and Views (01 Jan 2005)

CSX/NKX2.5 modulates differentiation of skeletal myoblasts and promotes differentiation into neuronal cells

J. Biol. Chem (Papers In Press, published online ahead of print January 13, 2005)

CSX/NKX2.5 modulates differentiation of skeletal myoblasts and promotes differentiation into neuronal cells in vitro

ABSTRACT:
CSX/Nkx2.5 transcription factor plays a pivotal role in cardiac development, however, its role in development and differentiation of other organs has not been investigated. In this study, we used C2C12 myoblasts and human fetal primary myoblasts to investigate the function of Nkx2.5 in skeletal myogenesis. The expression levels of Nkx2.5 decreased as C2C12 myoblasts elongated and fused to form myotubes. Expression of human NKX2.5 in C2C12 myoblasts inhibited myocyte differentiation and myotube formation, and up-regulated Gata4 and Tbx5 expression. Expression of NKX2.5 in terminally differentiated C2C12 myotubes resulted in a change in morphology and breakdown into smaller myotubes. Furthermore, over-expression of NKX2.5 in C2C12 cells and primary cultures of human fetal myoblasts led to differentiation of myoblasts into neuron-like cells and expression of neuronal markers. This study sheds light on previously unknown non-cardiac functions of Nkx2.5 transcription factor.

Quantum dots and other nanoparticles

Nanocrystals (quantum dots) and other nanoparticles (gold colloids, magnetic bars, nanobars, dendrimers and nanoshells) have been receiving a lot of attention recently with their unique properties for potential use in drug discovery, bioengineering and therapeutics.

Quantum dots (QDs) are semiconducting materials that are, in general, synthesized with II-VI or III-V column elements of the periodic table. They are neither atomic nor bulk semiconductors. Their properties originate from their physical size, which ranges from 10–100 Å in radius. Due to their bright fluorescence, narrow emission, broad UV excitation and high photostability [[4 and 5]], QDs have been adopted for in vitro bioimaging by many researchers as an alternative to organic based fluorophores [[4, 6, 7, 8, 9 and 10]]. Most recently, in vivo applications of these QDs have also been reported [[11, 12 and 13]].

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microRNAs may control more than one-third of human genes

More than one-third of human genes could be controlled by minuscule molecules called microRNAs. This claim by US scientists suggests that the molecules, which were first discovered in 2000, could play a role in almost every process from cell birth to cell death, and that they might even be useful in treating human disease.

MicroRNAs are rather like fragments of DNA, and are made up of around 22 chemical 'letters'. They act as controls by effectively blocking a gene from doing its normal job in a cell.

When a gene is switched on, its sequence is converted into messenger RNA, which carries the information to make a protein. MicroRNAs recognize and bind to particular messenger RNAs and stop them from making proteins.

Scientists know that human cells are swimming in microRNAs, but have been unsure how many of our 22,000 or so human genes they control. They might affect only a few hundred genes, say some, or as many as several thousand.

Full news at Nature.com

Neurons Reach Out and Touch Everyone

3D synaptic contact_Brain
(Picture and full text from PNAS January 18, 2005 vol. 102 no. 3 880-885 )
Using multi-neuron whole-cell recordings and confocal microscopy images of rat somatosensory cortex, the authors found that the pyramidal neurons in a region called cortical layer V send out axons that touch all neighboring dendrites; they describe this process as "all-to-all" geometry. The functional synapses were characterized by the presence of synaptic boutons. These findings suggest that, within the neocortex, all possible connections already exist, though most are nonfunctional. These data provide the first direct experimental evidence for a tabula rasa-like structural matrix between neocortical pyramidal neurons and suggests that pre- and postsynaptic interactions shape the conversion between touches and synapses to form specific functional microcircuits. The authors suggest that this architecture provides the neocortex with its immense capacity for plasticity without requiring the wasteful process of axonal and dendritic remodeling.

Biotechnology, the brain and the future

Trends in Biotechnology Volume 23, Issue 1 , January 2005, Pages 34-41
Here is another interesting review.

Several new approaches to illness, inspired by recent advances in molecular biology, informatics and nanoscience, are readily applicable to diseases of the central nervous system. Novel classes of drugs will widen the scope of therapeutic action beyond merely modifying transmitter function and stem cell and gene therapies could offer an even more selective mode of targeting. Further into the future, nanotechnology has the potential to allow development of new medicines and novel access routes via miniaturized monitoring and screening devices: these systems, together with increasing use of carbon–silicon interfacing, will challenge traditional neuropharmacology. As the 21st century unfolds, the structure and function of the brain, which is incomparable with any other organ, will present unique technological and ethical questions.

Nanotechnology and Neuroscience

Self-Propagating, Molecular-Level Polymorphism in Alzheimer's ß-Amyloid Fibrils
Science, Vol 307, Issue 5707, 262-265 , 14 January 2005
ABSTRACT: Amyloid fibrils commonly exhibit multiple distinct morphologies in electron microscope and atomic force microscope images, often within a single image field. By using electron microscopy and solid-state nuclear magnetic resonance measurements on fibrils formed by the 40-residue ß-amyloid peptide of Alzheimer's disease (Aß1–40), we show that different fibril morphologies have different underlying molecular structures, that the predominant structure can be controlled by subtle variations in fibril growth conditions, and that both morphology and molecular structure are self-propagating when fibrils grow from preformed seeds. Different Aß1–40 fibril morphologies also have significantly different toxicities in neuronal cell cultures. These results have implications for the mechanism of amyloid formation, the phenomenon of strains in prion diseases, the role of amyloid fibrils in amyloid diseases, and the development of amyloid-based nano-materials.

Nanoscale velocity–drag force relationship in thin liquid layers measured by atomic force microscopy
Applied Physics Letters -- October 25, 2004 -- Volume 85, Issue 17, pp. 3881-3883
ABSTRACT: The relationship between velocity and drag force acting on a nanoprobe has been measured with an atomic force microscope (AFM). A special nanoprobe "whisker" was partially submerged in thin layers of glycerol–water mixtures and moved by using the AFM in scanning mode. The viscous drag force-caused torsion of the cantilever probe was recorded as a function of scanning speed and submersion depth. A linear drag force–velocity function was determined for cylindrical bodies with diameters of the order of 50 nm. The experimental results were supported by calculations for the torsional force exerted on an AFM probe dragged through a viscous medium. The viscosity was calculated for each experiment assuming no slip conditions and was in agreement with the macroscopically determined values. With some refinements, this offers a possible means of determining viscosity in thin liquid layers.

Integrated multiple patch-clamp array chip via lateral cell trapping junctions
Applied Physics Letters -- March 15, 2004 -- Volume 84, Issue 11, pp. 1973-1975
ABSTRACT: We present an integrated multiple patch-clamp array chip by utilizing lateral cell trapping junctions. The intersectional design of a microfluidic network provides multiple cell addressing and manipulation sites for efficient electrophysiological measurements at a number of patch sites. The patch pores consist of openings in the sidewall of a main fluidic channel, and a membrane patch is drawn into a smaller horizontal channel. This device geometry not only minimizes capacitive coupling between the cell reservoir and the patch channel, but also allows simultaneous optical and electrical measurements of ion channel proteins. Evidence of the hydrodynamic placement of mammalian cells at the patch sites as well as measurements of patch sealing resistance is presented. Device fabrication is based on micromolding of polydimethylsiloxane, thus allowing inexpensive mass production of disposable high-throughput biochips.

Nanotubular Highways for Intercellular Organelle Transport
Science -- February 13, 2004 -- Volume 303, Issue 5660, pp. 1007-1010
ABSTRACT: Cell-to-cell communication is a crucial prerequisite for the development and maintenance of multicellular organisms. To date, diverse mechanisms of intercellular exchange of information have been documented, including chemical synapses, gap junctions, and plasmodesmata. Here, we describe highly sensitive nanotubular structures formed de novo between cells that create complex networks. These structures facilitate the selective transfer of membrane vesicles and organelles but seem to impede the flow of small molecules. Accordingly, we propose a novel biological principle of cell-to-cell interaction based on membrane continuity and intercellular transfer of organelles.

Nanotechnology for neuronal ion channels
Journal of Neurology Neurosurgery and Psychiatry 2003;74:1466-1475
ABSTRACT: Ion channels provide the basis for the regulation of electrical excitability in the central and peripheral nervous systems. This review deals with the techniques that make the study of structure and function of single channel molecules in living cells possible. These are the patch clamp technique, which was derived from the conventional voltage clamp method and is currently being developed for automated and high throughput measurements; and fluorescence and nano-techniques, which were originally applied to non-biological surfaces and are only recently being used to study cell membranes and their proteins, especially in combination with the patch clamp technique. The characterisation of the membrane channels by techniques that resolve their morphological and physical properties and dynamics in space and time in the nano range is termed nanoscopy.

Botulinum toxin type B micromechanosensor
PNAS November 11, 2003 vol. 100 no. 23 13621-13625
ABSTRACT: Botulinum neurotoxin (BoNT) types A, B, E, and F are toxic to humans; early and rapid detection is essential for adequate medical treatment. Presently available tests for detection of BoNTs, although sensitive, require hours to days. We report a BoNT-B sensor whose properties allow detection of BoNT-B within minutes. The technique relies on the detection of an agarose bead detachment from the tip of a micromachined cantilever resulting from BoNT-B action on its substratum, the synaptic protein synaptobrevin 2, attached to the beads. The mechanical resonance frequency of the cantilever is monitored for the detection. To suspend the bead off the cantilever we use synaptobrevin's molecular interaction with another synaptic protein, syntaxin 1A, that was deposited onto the cantilever tip. Additionally, this bead detachment technique is general and can be used in any displacement reaction, such as in receptor-ligand pairs, where the introduction of one chemical leads to the displacement of another. The technique is of broad interest and will find uses outside toxicology.

Simultaneous imaging of ionic conductivity and morphology of a microfluidic system
Journal of Applied Physics -- June 15, 2003 -- Volume 93, Issue 12, pp. 10134-10136
ABSTRACT: We present a method for nanoscale simultaneous measurements of the conductivity and morphology of microfluidic systems. While device morphology is imaged by atomic force microscopy (AFM), the AFM tip is used as an electrode probe to measure the conductivity through a buffer in the fluidic channels to a reference electrode. Connectivity to a reference electrode can be probed simultaneously at a large number of test points along micro- and nanofluidic channels without the requirement of external fluidic connections. Since the placement of microelectrodes is essential to a number of microfluidic applications, this technique allows for the AFM tip to be used as a rapid prototyping tool.

Nanomechanics of Microtubules
Phys. Rev. Lett. 89, 248101 (2002)
ABSTRACT: We have determined the mechanical anisotropy of a single microtubule by simultaneously measuring the Young's and the shear moduli in vitro. This was achieved by elastically deforming the microtubule deposited on a substrate tailored by electron-beam lithography with a tip of an atomic force microscope. The shear modulus is 2 orders of magnitude lower than the Young's, giving rise to a length-dependent flexural rigidity of microtubules. The temperature dependence of the microtubule's bending stiffness in the (5–40) °C range shows a strong variation upon cooling coming from the increasing interaction between the protofilaments.

RGM is a repulsive guidance molecule for retinal axons
Nature -- September 26, 2002 -- Volume 419, Issue 6905, pp. 392-395
ABSTRACT: Axons rely on guidance cues to reach remote targets during nervous system development. A well-studied model system for axon guidance is the retinotectal projection. The retina can be divided into halves; the nasal half, next to the nose, and the temporal half. A subset of retinal axons, those from the temporal half, is guided by repulsive cues expressed in a graded fashion in the optic tectum, part of the midbrain. Here we report the cloning and functional characterization of a membrane-associated glycoprotein, which we call RGM (repulsive guidance molecule). This molecule shares no sequence homology with known guidance cues, and its messenger RNA is distributed in a gradient with increasing concentration from the anterior to posterior pole of the embryonic tectum. Recombinant RGM at low nanomolar concentration induces collapse of temporal but not of nasal growth cones and guides temporal retinal axons in vitro, demonstrating its repulsive and axon-specific guiding activity.

beta-Lactam antibiotics offer neuroprotection by increasing glutamate transporter expression

Nature 433, 73 - 77 (06 January 2005); doi:10.1038/nature03180

Correspondence and requests for materials should be addressed to J.D.R. (jrothste@jhmi.edu).

Abstract: Glutamate is the principal excitatory neurotransmitter in the nervous system. Inactivation of synaptic glutamate is handled by the glutamate transporter GLT1 (also known as EAAT2; refs 1, 2), the physiologically dominant astroglial protein. In spite of its critical importance in normal and abnormal synaptic activity, no practical pharmaceutical can positively modulate this protein. Animal studies show that the protein is important for normal excitatory synaptic transmission, while its dysfunction is implicated in acute and chronic neurological disorders, including amyotrophic lateral sclerosis (ALS)3, stroke4, brain tumours5 and epilepsy6. Using a blinded screen of 1,040 FDA-approved drugs and nutritionals, we discovered that many -lactam antibiotics are potent stimulators of GLT1 expression. Furthermore, this action appears to be mediated through increased transcription of the GLT1 gene7. -Lactams and various semi-synthetic derivatives are potent antibiotics that act to inhibit bacterial synthetic pathways8. When delivered to animals, the -lactam ceftriaxone increased both brain expression of GLT1 and its biochemical and functional activity. Glutamate transporters are important in preventing glutamate neurotoxicity1, 9-11. Ceftriaxone was neuroprotective in vitro when used in models of ischaemic injury and motor neuron degeneration, both based in part on glutamate toxicity11. When used in an animal model of the fatal disease ALS, the drug delayed loss of neurons and muscle strength, and increased mouse survival. Thus these studies provide a class of potential neurotherapeutics that act to modulate the expression of glutamate neurotransmitter transporters via gene activation.

Dopaminergic neurons generated from monkey embryonic stem cells function in a Parkinson primate model

Abstract

Parkinson disease (PD) is a neurodegenerative disorder characterized by loss of midbrain dopaminergic (DA) neurons. ES cells are currently the most promising donor cell source for cell-replacement therapy in PD. We previously described a strong neuralizing activity present on the surface of stromal cells, named stromal cell–derived inducing activity (SDIA). In this study, we generated neurospheres composed of neural progenitors from monkey ES cells, which are capable of producing large numbers of DA neurons. We demonstrated that FGF20, preferentially expressed in the substantia nigra, acts synergistically with FGF2 to increase the number of DA neurons in ES cell–derived neurospheres. We also analyzed the effect of transplantation of DA neurons generated from monkey ES cells into 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine–treated (MPTP-treated) monkeys, a primate model for PD. Behavioral studies and functional imaging revealed that the transplanted cells functioned as DA neurons and attenuated MPTP-induced neurological symptoms.

J. Clin. Invest. 115:102-109 (2005). doi:10.1172/JCI200521137.

Antibiotic Shown to Protect Nerves in Animal Study

January 5, 2005 10:38:39 AM PST , Reuters

Antibiotics could one day be used for more than killing bacteria and may help patients suffering from neurological diseases, scientists said on Wednesday.

If a family of antibiotics, which include penicillin, produces the same effect in humans as they did in mice, researchers from Johns Hopkins University in Maryland believe the drugs could help to prevent nerve damage and death in illnesses such dementia, stroke and epilepsy.
"We're very excited by these drugs' abilities," Jeffrey Rothstein, a professor of neurology and neuroscience at the university, said in a statement.

In studies of mice genetically engineered to develop amyotrophic lateral sclerosis (ALS), or Lou Gehrig's disease, Rothstein and his team discovered that daily injections of the drug ceftriaxone improved survival and reduced symptoms of the progressive disease that attacks nerve cells in the brain and spinal cord causing paralysis and death.

Ceftriaxone is produced by Roche Holding AG under the brand name Rocephin.

They found that the drug turned on a gene that increased the number of transporters that remove the brain chemical glutamate from nerves. Glutamate usually helps electrical signals travel from one nerve to another but too much of the chemical can kill nerves.

"Because we study ALS, we tested the drugs in a mouse model of that disease, but this is much bigger than ALS. This approach has potential applications in numerous neurological and psychiatric conditions that arise from abnormal control of glutamate," Rothstein, who reported the findings in the science journal Nature, explained.

Although it is early research and clinical trials in humans are needed to prove if the drugs can help patients, the scientists said the results are encouraging despite the fact that the mice treated with the antibiotic were eventually paralyzed.

"If we can find drugs that protect against other causes of nerve death in ALS, the combination might offer a real therapy, much like using drug combinations to treat cancer," said Rothstein.

Clones may aid work on motor neuron disease
 

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