Manfred Schubert

A Cdk5 inhibitory peptide reduces tau hyperphosphorylation and apoptosis in neurons

The extracellular aggregation of amyloid β (Aβ) peptides and the intracellular hyperphosphorylation of tau at specific epitopes are pathological hallmarks of neurodegenerative diseases such as Alzheimers disease (AD). Cdk5 phosphorylates tau at AD‐specific phospho‐epitopes when it associates with p25. p25 is a truncated activator, which is produced from the physiological Cdk5 activator p35 upon exposure to Aβ peptides. We show that neuronal infections with Cdk5 inhibitory peptide (CIP) selectively inhibit p25/Cdk5 activity and suppress the aberrant tau phosphorylation in cortical neurons. Furthermore, Aβ1−42‐induced apoptosis of these cortical neurons was also reduced by coinfection with CIP. Of particular importance is our finding that CIP did not inhibit endogenous or transfected p35/Cdk5 activity, nor did it inhibit the other cyclin‐dependent kinases such as Cdc2, Cdk2, Cdk4 and Cdk6. These results, therefore, provide a strategy to address, and possibly ameliorate, the pathology of neurodegenerative diseases that may be a consequence of aberrant p25 activation of Cdk5, without affecting ‘normal’ Cdk5 activity.

Transduction of motor neurons and muscle fibers by intramuscular injection of HIV-1-based vectors pseudotyped with select rabies virus glycoproteins

For studies of motor neuron function or for therapeutic purposes, novel pseudotype HIV-1-based vectors were developed that are capable of expressing transgenes in motor neurons following injection into mouse hind limb muscles. To specifically target motor neurons, glycoproteins from two rabies virus (RV) isolates, the mouse-brain adapted challenge virus 24 (CVS-24) variants, CVS-N2c and CVS-B2c were evaluated for pseudotype formation with an HIV-1-based vector. Both RV glycoproteins incorporated into vector envelopes, and both pseudotypes yielded high titers with Hek293T and cortical plate neuron cultures. Increased neuronotropism by the CVS-N2c pseudotype was not observed, suggesting that vector tropism is not solely determined by the fusogenic viral glycoprotein. Vector injection into hind limb muscles resulted in EYFP reporter gene expression in the injected muscle fibers and in spinal cord motor neurons innervating the same muscle, indicating retrograde vector transport. Intramuscular vector injections into the soleus and tibialis anterior muscles transduced 26% and 16% of all motor neurons in each motor nucleus, respectively. These transduction efficiencies may allow novel approaches to functional studies of the motor system and the treatment of neuromuscular disease.

Gene delivery to the nervous system

The National Institute of Neurological Disorders and Stroke (NINDS) sponsored a workshop on gene delivery to the nervous system, which took place on 12–13 November 2007 in Washington, DC. The purpose of the workshop was to convene neuroscientists, molecular virologists/vectorologists, and surgical neurologists to assess the state of the art of gene therapy for neurologic diseases and brain tumors and to address the challenges for advancing promising preclinical studies to the clinic.

Inhibition of HIV‐1 Replication by Novel Multitarget Ribozymes

Ribozymes are small catalytic RNA molecules that can be targeted to cleave a substrate RNA at a specific nucleotide sequence. Expression of a single ribozyme can inhibit HIV-1 replication2 or revert the transformed phenotype of a cell by inhibiting expression of the fos ~ncogene .~ It is anticipated, however, that the effective use of ribozymes, particularly against RNA viruses, will be limited to only a few rounds of viral replication because of the high mutation rate of these viruses, allowing a rapid selection of resistant virus variants, probably even in the presence of the ribozyme. In addition, any therapeutic approach that employs ribozymes also has to be effective against preferably all virus isolates.

Trafficking of the NMDAR2B Receptor Subunit Distal Cytoplasmic Tail from Endoplasmic Reticulum to the Synapse

NMDA receptor NR2A/B subunits have PDZ-binding domains on their extreme C-termini that are known to interact with the PSD-95 family and other PDZ proteins. We explore the interactions between PSD-95 family proteins and the NR2A/B cytoplasmic tails, and the consequences of these interactions, from the endoplasmic reticulum (ER) through delivery to the synapse in primary rat hippocampal and cortical cultured neurons. We find that the NR2A/B cytoplasmic tails cluster very early in the secretory pathway and interact serially with SAP102 beginning at the intermediate compartment, and then PSD-95. We further establish that colocalization of the distal C-terminus of NR2B and PSD-95 begins at the trans-Golgi Network (TGN). Formation of NR2B/PSD-95/SAP102 complexes is dependent on the PDZ binding domain of NR2B subunits, but association with SAP102 and PSD-95 plays no distinguishable role in cluster pre-formation or initial targeting to the vicinity of the synapse. Instead the PDZ binding domain plays a role in restricting cell-surface clusters to postsynaptic targets.

Deletion mapping analyses indicate that epitopes for monoclonal antibodies to the NS phosphoprotein of VSV are linear and clustered

Thirteen distinct monoclonal antibodies to the 30-kDa NS phosphoprotein of vesicular stomatitis virus were isolated and assayed by Western blot analysis and immune precipitation reactions. Epitopes recognized by the antibodies were mapped by immune precipitation of NS deletion proteins synthesized in vitro from cloned NS gene constructs. None of the epitopes recognized by the 13 antibodies could be mapped to the phosphorylated amino-terminal half of the NS protein. Twelve of the antibodies recognized epitopes located within the carboxy-terminal 142 amino acids of the protein. The great majority of epitopes appeared to consist of a linear array of amino acids.

Targeted Defective Interfering HIV-1 Particles as Renewable Antivirals?

Defective viruses are viral mutants which are incapable of replication because they lack essential regions of the viral genome. They are ubiquitous and can be detected within all DNA and RNA virus families.1 The replication of the defective virus requires functions which are carried out by gene products encoded in the missing regions. These functions must be provided in trans by a helper virus, which is usually the virus from which the defective virus originated. Many defective viral genomes contain all essential cis-acting nucleotide sequences, such as the origin of replication and the packaging signal necessary for viral assembly.2 With defective proviruses, some of these cis elements may also be deleted and replaced by host sequences. In such cases, the defective virus cannot be transmitted by virus particle formation. Initial observations with acute transforming retroviruses led to the discoveries of oncogenes and the roles of defective proviruses and their regulatory sequences in neoplastic transformation.3 Most defective proviruses, however, do not give rise to a recognizable cellular phenotype. Replication of these defective proviruses only occurs by cell division which the provirus itself can promote in some cases.

Targeted infection of HIV-1 Env expressing cells by HIV(CD4/CXCR4) vectors reveals a potential new rationale for HIV-1 mediated down-modulation of CD4

BackgroundEfficient targeted gene transfer and cell type specific transgene expression are important for the safe and effective expression of transgenes in vivo. Enveloped viral vectors allow insertion of exogenous membrane proteins into their envelopes, which could potentially aid in the targeted transduction of specific cell types. Our goal was to specifically target cells that express the T cell tropic HIV-1 envelope protein (Env) using the highly specific interaction of Env with its cellular receptor (CD4) inserted into the envelope of an HIV-1-based viral vector.ResultsTo generate HIV-1-based vectors carrying the CD4 molecule in their envelope, the CD4 ectodomain was fused to diverse membrane anchors and inserted together with the HIV-1 coreceptor CXCR4 into the envelopes of HIV-1 vector particles. Independent of the type of CD4 anchor, all chimeric CD4 proteins inserted into HIV-1 vector envelopes and the resultant HIV(CD4/CXCR4) particles were able to selectively confer neomycin resistance to cells expressing the fusogenic T cell tropic HIV-1 Env protein. Unexpectedly, in the absence of Env on the target cells, all vector particles carrying the CD4 ectodomain anchored in their envelope adhered to various cell types without infecting these cells. This cell adhesion was very avid. It was independent of the presence of Env on the target cell, the type of CD4 anchor or the presence of CXCR4 on the particle. In mixed cell populations with defined ratios of Env+/Env- cells, the targeted transduction of Env+ cells by HIV(CD4/CXCR4) particles was diminished in proportion to the number of Env- cells.ConclusionVector diversion caused by a strong, non-selective cell binding of CD4+-vector particles effectively prevents the targeted transduction of HIV-1 Env expressing cells in mixed cell populations. This Env-independent cell adhesion severely limits the effective use of targeted HIV(CD4/CXCR4) vectors designed to interfere with HIV-1 replication in vivo. Importantly, the existence of this newly described and remarkably strong CD4-dependent cell adhesion suggests that the multiple viral efforts to reduce CD4 cell surface expression may, in part, be to prevent cell adhesion to non-target cells and thereby to increase the infectivity of viral progeny. Preventing CD4 down-modulation by HIV-1 might be an effective component of a multi-faceted antiviral strategy.

Frequent generation of new 3'-defective interfering particles of vesicular stomatitis virus.

We have isolated and partially characterized a number of different genome types of defective interfering (DI) particles newly generated by a highly heat-resistant strain of vesicular stomatitis virus in either Rat(B77) or Vero cells. Northern blot analyses revealed that many of these DI genomes contain N gene sequences and/or sequences of the NS, M, and G genes. One type contains NS sequences without any indication for the presence of either N, M, or G sequences. Another type of DI particle genomes did not contain any detectable sequences of N, NS, M, or G, but contain panhandle-type sequences and, thus, most likely resembles the 5-panhandle-type DI particles. Unlike previously assumed, these data demonstrate that DI genomes which have the 3-terminal N, NS, M, and G genes or portions of these genes conserved do frequently arise together with 5-DI particle genomes after serial undiluted passages of the heat-resistant strain of vesicular stomatitis virus.

Gene Delivery to the Nervous System:NINDS Workshop on Gene Delivery to the Nervous System Washington, DC, 12–13 November 2007

The National Institute of Neurological Disorders and Stroke (NINDS) sponsored a workshop on gene delivery to the nervous system, which took place on 12–13 November 2007 in Washington, DC. The purpose of the workshop was to convene neuroscientists, molecular virologists/vectorologists, and surgical neurologists to assess the state of the art of gene therapy for neurologic diseases and brain tumors and to address the challenges for advancing promising preclinical studies to the clinic.