Titer and product affect the distribution of gene expression after intraputaminal convection-enhanced delivery.

Background: The efficacy and safety of intracerebral gene therapy for brain disorders like Parkinson's disease depends on the appropriate distribution of gene expression.

Objectives: To assess whether the distribution of gene expression is affected by vector titer and protein type.

Methods: Four adult macaque monkeys seronegative for adeno-associated virus 5 (AAV5) received a 30-µl inoculation of a high- or a low-titer suspension of AAV5 encoding glial cell line-derived neurotrophic factor (GDNF) or green fluorescent protein (GFP) in the right and left ventral postcommissural putamen.

Frameshift proteins in autosomal dominant forms of Alzheimer disease and other tauopathies.

Frameshift (+1) proteins such as APP(+1) and UBB(+1) accumulate in sporadic cases of Alzheimer disease (AD) and in older subjects with Down syndrome (DS). We investigated whether these proteins also accumulate at an early stage of neuropathogenesis in young DS individuals without neuropathology and in early-onset familial forms of AD (FAD), as well as in other tauopathies, such as Pick disease (PiD) or progressive supranuclear palsy (PSP). APP(+1) is present in many neurons and beaded neurites in very young cases of DS, which suggests that it is axonally transported.

Frame-shifted amyloid precursor protein found in Alzheimer's disease and Down's syndrome increases levels of secreted amyloid beta40.

Frame-shifted amyloid precursor protein (APP(+1)), which has a truncated out-of-frame C-terminus, accumulates in the neuropathological hallmarks of patients with Alzheimer's disease pathology. To study a possible involvement of APP(+1) in the pathogenesis of Alzheimer's disease, we expressed APP695 and APP(+1) in the HEK293 cell-line and studied whether the processing of APP695 was affected. APP(+1) is a secretory protein, but high expression of APP695 and APP(+1) results in the formation of intracellular aggregate-like structures containing both proteins and Fe65, an adaptor protein that interacts with APP695.

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.

Neuronal expression of GFAP in patients with Alzheimer pathology and identification of novel GFAP splice forms.

Glial fibrillary acidic protein (GFAP) is considered to be a highly specific marker for glia. Here, we report on the expression of GFAP in neurons in the human hippocampus. Intriguingly, this neuronal GFAP is coded by out-of-frame splice variants and its expression is associated with Alzheimer pathology.

Molecular misreading of the ubiquitin B gene and hepatic mallory body formation.

Background & Aims: Molecular misreading of the ubiquitin B gene has been documented in the cerebral cortex of patients with Alzheimer's disease and Down syndrome. This novel process consists of the unfaithful conversion of genomic information into aberrant transcripts and its subsequent translation into +1 proteins.

Methods: Because Mallory bodies (MBs) also contain ubiquitinated proteins, we stained 11 autopsied and 6 biopsied MB-containing livers from patients with steatohepatitis with an antibody to ubiquitin(+1) to look for the presence of mutant (ubiquitin(+1)) protein.

Molecular misreading: a new type of transcript mutation expressed during aging.

Dinucleotide deletions (e.g. DeltaGA, DeltaGU) are created by molecular misreading in or adjacent to GAGAG motifs of neuronal mRNAs.

Molecular misreading in non-neuronal cells.

+1 Frame-shifted proteins such as amyloid precursor protein(+1) and ubiquitin-B(+1) have been identified in the neuropathological hallmarks of Alzheimer's disease. These frameshifts are caused by dinucleotide deletions in GAGAG motifs of messenger RNA encoded by genes that have maintained the unchanged wild-type DNA sequence. This process is termed 'molecular misreading'.

Purification of recombinant adeno-associated virus by iodixanol gradient ultracentrifugation allows rapid and reproducible preparation of vector stocks for gene transfer in the nervous system.

Recombinant adeno-associated virus (rAAV) vectors have become attractive tools for in vivo gene transfer. The production and purification of high-titer rAAV vector stocks for experimental and therapeutic gene transfer continue to undergo improvement. Standard rAAV vector purification protocols include the purification of the vector by cesium chloride (CsCl)-density gradient centrifugation followed by extensive desalination via dialysis against a physiological buffer for in vivo use.

Lack of translation of normal 7B2 mRNA levels in hypothalamic mutant vasopressin cells of the homozygous Brattleboro rat.

The homozygous Brattleboro rat (di/di) synthesizes a vasopressin (VP) precursor with an aberrant C-terminus, which causes a hypothalamic form of diabetes insipidus. The neuroendocrine polypeptide 7B2 is present in VP and oxytocin (OT) neurons of the supraoptic and paraventricular nucleus of the hypothalamus in wild type rats. However, in the di/di rat 7B2 immunoreactivity is absent in the VP cell population, whereas 7B2 levels within the OT cells are unaffected.

Frameshift mutants of beta amyloid precursor protein and ubiquitin-B in Alzheimer's and Down patients.

The cerebral cortex of Alzheimer's and Down syndrome patients is characterized by the presence of protein deposits in neurofibrillary tangles, neuritic plaques, and neuropil threads. These structures were shown to contain forms of beta amyloid precursor protein and ubiquitin-B that are aberrant (+1 proteins) in the carboxyl terminus. The +1 proteins were not found in young control patients, whereas the presence of ubiquitin-B+1 in elderly control patients may indicate early stages of neurodegeneration.

Adenoviral vector-mediated expression of B-50/GAP-43 induces alterations in the membrane organization of olfactory axon terminals in vivo.

B-50/GAP-43 is an intraneuronal membrane-associated growth cone protein with an important role in axonal growth and regeneration. By using adenoviral vector-directed expression of B-50/GAP-43 we studied the morphogenic action of B-50/GAP-43 in mature primary olfactory neurons that have established functional synaptic connections. B-50/GAP-43 induced gradual alterations in the morphology of olfactory synapses.

Specific expression of secretogranin II in magnocellular vasopressin neurons of the rat supraoptic and paraventricular nucleus in response to osmotic stimulation.

As secretogranin II is considered to be a marker for the regulated secretory pathway, its distribution in the hypothalamo-neurohypophyseal system of salt-loaded Wistar rats was studied in detail by immunocytochemistry. Although after an osmotic challenge both vasopressin and oxytocin neurons are stimulated, secretogranin II was exclusively expressed in a subpopulation of vasopressinergic magnocellular neurons in the supraoptic and paraventricular nucleus of Wistar rats. Secretogranin II was only surely visualized after a combination of osmotic challenge and blockade of axonal transport by colchicine treatment.

Quantitative estimation of estrogen and androgen receptor-immunoreactive cells in the forebrain of neonatally estrogen-deprived male rats.

Using quantitative immunocytochemical procedures, the total number of estrogen and androgen receptors was estimated in a large number of hypothalamic and limbic nuclei of male rats, in which brain estrogen formation was inhibited neonatally by treatment with the aromatase inhibitor 1,4,6-androstatriene-3,17-dione. The highest densities of estrogen receptor immunoreactivity were observed in the periventricular preoptic area and the medial preoptic area. Neonatally estrogen-deprived males showed a higher estrogen receptor immunoreactivity than control males in the periventricular preoptic area and the ventrolateral portion of the ventromedial nucleus of the hypothalamus, i.

Immunocytochemical evidence for the presence of vasopressin in intermediate sized neurosecretory granules of solitary neurohypophyseal terminals in the homozygous Brattleboro rat.

A single base deletion (delta G) in the vasopressin gene is the cause of diabetes insipidus in the homozygous Brattleboro rat (di/di). The resulting frameshift leads to the expression of an aberrant vasopressin precursor which is unable to enter the secretory pathway, thereby preventing vasopressin biosynthesis. In a small number of solitary magnocellular hypothalamic neurons within the supraoptic and paraventricular nuclei, the reading frame is restored by a dinucleotide (delta GA) frameshift mutation, at two separate GAGAG motifs downstream of the original G-deletion.

Localization of vasopressin mRNA and immunoreactivity in pituicytes of pituitary stalk-transected rats after osmotic stimulation.

The presence of [arginine] vasopressin (AVP) mRNA and AVP immunoreactivity in pituicytes of the neural lobe (NL) of intact and pituitary stalk-transected rats, with and without osmotic stimulation, was examined. AVP mRNA was analyzed by Northern blotting, as well as by in situ hybridization in combination with immunocytochemistry using anti-glial fibrillary acidic protein (GFAP) as a marker for pituicytes. In intact rats, a poly(A) tail-truncated 0.

Rat oxytocin receptor in brain, pituitary, mammary gland, and uterus: partial sequence and immunocytochemical localization.

Partial complementary DNAs of an oxytocin (OT) receptor were cloned from rat brain and uterus. The complementary DNAs encoded for the same amino acid sequence, which showed a high degree of homology with the human and porcine uterine OT receptors, except for a region in the third intracellular loop. Antibodies were raised against nonoverlapping sequences of the third intracellular loop of this rat OT receptor.

Frameshift mutations at two hotspots in vasopressin transcripts in post-mitotic neurons.

Mutations in DNA underlie carcinogenesis, inherited pathology, and aging and are generally thought to be introduced during meiosis and mitosis. Here we report that in post-mitotic neurons specific frameshift mutations occur at high frequency. These mutations were identified in vasopressin transcripts in magnocellular neurons of the homozygous Brattleboro rat and predominantly consist of a GA deletion in GAGAG motifs.

Differential neurophysin immunoreactivities in solitary magnocellular neurons of the homozygous Brattleboro rat indicate an altered neurophysin moiety.

In the homozygous Brattleboro rat (di/di) a single base deletion in the vasopressin (VP) gene causes diabetes insipidus, resulting in the synthesis of a VP precursor with a different C-terminus. We reported previously that a small number of post-mitotic VP neurons in di/di rats undergo a switch to a heterozygous phenotype, suggesting the existence of VP mRNAs with a restored reading frame coding for a normal VP precursor. In the present study we report that the increase in the number of these revertant cells declines after 79 weeks of age.

Immunocytochemical localization of neuropeptide FF (FMRF amide-like peptide) in the hypothalamo-neurohypophyseal system of Wistar and Brattleboro rats by light and electron microscopy.

Neuropeptide FF (F8Famide, FMRFamide-like, or morphine modulating peptide) immunoreactivity was localized by light and electron microscopy in the hypothalamo-neurohypophyseal system of Wistar and Brattleboro rats. In Wistar rats neuropeptide FF was present in part of the magnocellular neurones of the paraventricular and supraoptic nuclei in which it was coexpressed with vasopressin. Neuropeptide FF containing fibres were present in the paraventricular and the supraoptic nuclei, and in the central part of the neural lobe.

Immunoelectron microscopic demonstration of oxytocin and vasopressin in pituicytes and in nerve terminals forming synaptoid contacts with pituicytes in the rat neural lobe.

A pre-embedding immunoelectron microscopic technique was used to obtain morphological evidence for a role of oxytocin and vasopressin in the regulation of their own or each others release from the neural lobe. No synaptoid contacts of oxytocin- or vasopressin-containing axons with other neuronal structures were observed. However, synaptoid contacts of oxytocin- and vasopressin-containing nerve terminals and Herring bodies with pituicytes were frequently observed.