Production of High-Yield Adeno Associated Vector Batches Using HEK293 Suspension Cells.

Adeno-associated viral vectors (AAVs) are a remarkable tool for investigating the central nervous system (CNS). Innovative capsids, such as AAV.PHP.

CERT reduces C16 ceramide, amyloid-β levels, and inflammation in a model of Alzheimer's disease.

Background: Dysregulation of ceramide and sphingomyelin levels have been suggested to contribute to the pathogenesis of Alzheimer's disease (AD). Ceramide transfer proteins (CERTs) are ceramide carriers which are crucial for ceramide and sphingomyelin balance in cells. Extracellular forms of CERTs co-localize with amyloid-β (Aβ) plaques in AD brains.

Optimization of adeno-associated viral vector-mediated transduction of the corticospinal tract: comparison of four promoters.

Adeno-associated viral vectors are widely used as vehicles for gene transfer to the nervous system. The promoter and viral vector serotype are two key factors that determine the expression dynamics of the transgene. A previous comparative study has demonstrated that AAV1 displays efficient transduction of layer V corticospinal neurons, but the optimal promoter for transgene expression in corticospinal neurons has not been determined yet.

Small Scale Production of Recombinant Adeno-Associated Viral Vectors for Gene Delivery to the Nervous System.

Adeno-associated viral vectors have numerous applications in neuroscience, including the study of gene function in health and disease, targeting of light-sensitive proteins to anatomically distinct sets of neurons to manipulate neuronal activity (optogenetics), and the delivery of fluorescent protein to study anatomical connectivity in the brain. Moreover several phase I/II clinical trials for gene therapy of eye and brain diseases with adeno-associated viral vectors have shown that these vectors are well tolerated by human patients. In this chapter we describe a detailed protocol for the small scale production of recombinant adeno-associated viral vectors.

Repulsive Guidance Molecule a (RGMa) Induces Neuropathological and Behavioral Changes That Closely Resemble Parkinson's Disease.

Repulsive guidance molecule member a (RGMa) is a membrane-associated or released guidance molecule that is involved in axon guidance, cell patterning, and cell survival. In our previous work, we showed that RGMa is significantly upregulated in the substantia nigra of patients with Parkinson's disease. Here we demonstrate the expression of RGMa in midbrain human dopaminergic (DA) neurons.

Expression of a Mutant SEMA3A Protein with Diminished Signalling Capacity Does Not Alter ALS-Related Motor Decline, or Confer Changes in NMJ Plasticity after BotoxA-Induced Paralysis of Male Gastrocnemic Muscle.

Terminal Schwann cells (TSCs) are specialized cells that envelop the motor nerve terminal, and play a role in the maintenance and regeneration of neuromuscular junctions (NMJs). The chemorepulsive protein semaphorin 3A (SEMA3A) is selectively up-regulated in TSCs on fast-fatigable muscle fibers following experimental denervation of the muscle (BotoxA-induced paralysis or crush injury to the sciatic nerve) or in the motor neuron disease amyotrophic lateral sclerosis (ALS). Re-expression of SEMA3A in this subset of TSCs is thought to play a role in the selective plasticity of nerve terminals as observed in ALS and following BotoxA-induced paralysis.

Brain endothelial cells control fertility through ovarian-steroid-dependent release of semaphorin 3A.

Neuropilin-1 (Nrp1) guides the development of the nervous and vascular systems, but its role in the mature brain remains to be explored. Here we report that the expression of the 65 kDa isoform of Sema3A, the ligand of Nrp1, by adult vascular endothelial cells, is regulated during the ovarian cycle and promotes axonal sprouting in hypothalamic neurons secreting gonadotropin-releasing hormone (GnRH), the neuropeptide controlling reproduction. Both the inhibition of Sema3A/Nrp1 signaling and the conditional deletion of Nrp1 in GnRH neurons counteract Sema3A-induced axonal sprouting.

Mutant ubiquitin decreases amyloid β plaque formation in a transgenic mouse model of Alzheimer's disease.

The mutant ubiquitin UBB(+1) is a substrate as well as an inhibitor of the ubiquitin-proteasome system (UPS) and accumulates in the neuropathological hallmarks of Alzheimer's disease (AD). A role for the UPS has been suggested in the generation of amyloid β (Aβ) plaques in AD. To investigate the effect of UBB(+1) expression on amyloid pathology in vivo, we crossed UBB(+1) transgenic mice with a transgenic line expressing AD-associated mutant amyloid precursor protein (APPSwe) and mutant presenilin 1 (PS1dE9), resulting in APPPS1/UBB(+1) triple transgenic mice.

Alzheimer-associated mutant ubiquitin impairs spatial reference memory.

UBB(+1) is a mutant ubiquitin which accumulates in the hallmarks of tauopathies, including Alzheimer's disease. Transgenic mice expressing high levels of neuronal UBB(+1) exhibit moderately decreased proteasome activity and spatial reference memory deficits at 9months of age. In the present study, we characterized the behavioral phenotype of male UBB(+1) transgenic mice at different ages.

Low levels of mutant ubiquitin are degraded by the proteasome in vivo.

The ubiquitin-proteasome system fulfills a pivotal role in regulating intracellular protein turnover. Impairment of this system is implicated in the pathogenesis of neurodegenerative diseases characterized by ubiquitin- containing proteinaceous deposits. UBB(+1), a mutant ubiquitin, is one of the proteins accumulating in the neuropathological hallmarks of tauopathies, including Alzheimer's disease, and polyglutamine diseases.

Modest proteasomal inhibition by aberrant ubiquitin exacerbates aggregate formation in a Huntington disease mouse model.

UBB(+1), a mutant form of ubiquitin, is both a substrate and an inhibitor of the proteasome which accumulates in the neuropathological hallmarks of Huntington disease (HD). In vitro, expression of UBB(+1) and mutant huntingtin synergistically increase aggregate formation and polyglutamine induced cell death. We generated a UBB(+1) transgenic mouse line expressing UBB(+1) within the neurons of the striatum.

Long-term proteasome dysfunction in the mouse brain by expression of aberrant ubiquitin.

Many neurodegenerative diseases are characterized by deposits of ubiquitinated and aberrant proteins, suggesting a failure of the ubiquitin-proteasome system (UPS). The aberrant ubiquitin UBB(+1) is one of the ubiquitinated proteins accumulating in tauopathies such as Alzheimer's disease (AD) and polyglutamine diseases such as Huntington's disease. We have generated UBB(+1) transgenic mouse lines with post-natal neuronal expression of UBB(+1), resulting in increased levels of ubiquitinated proteins in the cortex.

Proteasome subunit proteins and neuropathology in tauopathies and synucleinopathies: Consequences for proteomic analyses.

Accumulation of proteins in inclusions in neurological disorders is partly due to dysfunction of the ubiquitin-proteasome system. Proteasomal dysfunction may be caused by misexpression of one or more of its subunits. A large number of antibodies reactive with proteasome subunits were screened on material from patients exhibiting tau- and synucleinopathies.

Alzheimer-associated APP+1 transgenic mice: frameshift beta-amyloid precursor protein is secreted in cerebrospinal fluid without inducing neuropathology.

Biomarkers present in the cerebrospinal fluid (CSF) of Alzheimer Disease patients could be instrumental in guiding diagnosis and monitoring of progression of the disease. We have previously reported on the secretion of a frameshifted form of amyloid-beta precursor protein, APP+1, into the CSF of Alzheimer patients and controls. APP+1 is secreted efficiently in controls, but during the progression of Alzheimer Disease, its secretion is reduced and APP+1 accumulates in tangle-bearing neurons.

Accumulation of aberrant ubiquitin induces aggregate formation and cell death in polyglutamine diseases.

Polyglutamine diseases are characterized by neuronal intranuclear inclusions (NIIs) of expanded polyglutamine proteins, indicating the failure of protein degradation. UBB(+1), an aberrant form of ubiquitin, is a substrate and inhibitor of the proteasome, and was previously reported to accumulate in Alzheimer disease and other tauopathies. Here, we show accumulation of UBB(+1) in the NIIs and the cytoplasm of neurons in Huntington disease and spinocerebellar ataxia type-3, indicating inhibition of the proteasome by polyglutamine proteins in human brain.

Disease-specific accumulation of mutant ubiquitin as a marker for proteasomal dysfunction in the brain.

Molecular misreading of the ubiquitin-B (UBB) gene results in a dinucleotide deletion in UBB mRNA. The resulting mutant protein, UBB+1, accumulates in the neuropathological hallmarks of Alzheimer disease. In vitro, UBB+1 inhibits proteasomal proteolysis, although it is also an ubiquitin fusion degradation substrate for the proteasome.

Frameshifted beta-amyloid precursor protein (APP+1) is a secretory protein, and the level of APP+1 in cerebrospinal fluid is linked to Alzheimer pathology.

Molecular misreading of the beta-amyloid precursor protein (APP) gene generates mRNA with dinucleotide deletions in GAGAG motifs. The resulting truncated and partly frameshifted APP protein (APP+1) accumulates in the dystrophic neurites and the neurofibrillary tangles in the cortex and hippocampus of Alzheimer patients. In contrast, we show here that neuronal cells transfected with APP+1 proficiently secreted APP+1.