Marc A. F. Sonnemans

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. These procedures are extremely time consuming and frequently result in a substantial loss of the infectious vector titer. As an alternative to CsCl we have investigated the use of Iodixanol, an X-ray contrast solution, as the density-gradient medium. Purification of rAAV vectors by Iodixanol shortened the centrifugation period to 3 hr and resulted in reproducible concentration and purification of rAAV-vector stocks. We show that injection of rAAV derived from an Iodixanol gradient can be used for in vivo gene transfer applications in the brain and spinal cord without detectable cytopathic effects and directing stable transgene expression for at least 2 months.

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. Using the ligase chain reaction to detect dinucleotide deletions, we report here that UBB+1 transcripts are present in each neurodegenerative disease studied (tauo‐ and synucleinopathies) and even in control brain samples. In contrast to UBB+1 transcripts, UBB+1 protein accumulation in the ubiquitin‐containing neuropathological hallmarks is restricted to the tauopathies such as Pick disease, frontotemporal dementia, progressive supranuclear palsy, and argyrophilic grain disease. Remarkably, UBB+1 protein is not detected in the major forms of synucleinopathies (Lewy body disease and multiple system atrophy). The neurologically intact brain can cope with UBB+1 as lentivirally delivered UBB+1 protein is rapidly degraded in rat hippocampus, whereas the K29,48R mutant of UBB+1, which is not ubiquitinated, is abundantly expressed. The finding that UBB+1 protein only accumulates in tauopathies thus implies that the ubiquitin‐proteasome system is impaired specifically in this group of neurodegenerative diseases and not in synucleinopathies and that the presence of UBB+1 protein reports proteasomal dysfunction in the brain.—Fischer, D. F., de Vos, R. A. I., van Dijk, R., de Vrij, F. M. S., Proper, E. A., Sonnemans, M. A. F., Verhage, M. C., Sluijs, J. A., Hobo, B., Zouambia, M., Jansen Steur, E. N. H., Kamphorst, W., Hol, E. M., van Leeuwen, F. W. Disease‐specific accumulation of mutant ubiquitin as a marker for proteasomal dysfunction in the brain. FASEB J. 17, 2014–2024 (2003)

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. We identified three novel GFAP splice forms: Δ 135 nt, Δ exon 6 and Δ 164 nt. Neuronal GFAP is mainly observed in the pyramidal neurons of the hippocampus of Alzheimer and Down syndrome patients and aged controls, but not in neurons of patients suffering from hippocampal sclerosis. Apparently, the hippocampal neurons in patients with Alzheimers disease pathology are capable of expressing glia-specific genes.

Endoplasmic reticulum derangement in hypothalamic neurons of rats expressing a familial neurohypophyseal diabetes insipidus mutant vasopressin transgene

Human familial neurohypophyseal diabetes insipidus (FNDI) is an autosomal dominant endocrine disorder that presents in early childhood as excessive drinking and urination as a consequence of a progressive loss of secretion of vasopressin (VP) from posterior pituitary nerve terminals. Mutations in the VP gene have been implicated as the cause of FNDI, but the mechanisms by which these mutants manifest their pathology, and prevent the secretion of the co‐expressed wild‐type protein, are unknown. One hypothesis suggests that mutant precursors are toxic, and stop the synthesis of wild‐type VP by killing expressing cells. Another hypothesis suggests that aberrant interactions between mutant and wild‐type precursors might directly inhibit the elaboration or secretion of the products of the normal allele. We have tested these hypotheses using new animal models‐‐transgenic rats that express an FNDI mutant VP gene that encodes a truncated precursor (Cys67stop). Cell‐specific and inducible expression of the Cys67stop mutation in rat VP hypothalamic neurons does not result in cell death or atrophy. Rather, expression of the FNDI mutant causes a neuronal pathology characterized by distorted structures in the cell body that are labeled by antisera that recognize endoplasmic reticulum (ER) markers, and that accumulate both mutant and wild‐type VP gene products. This is accompanied by an increase in the abundance of the mannose‐6‐phosphate receptor (MPR), a marker of endosome‐lysosome activity. We suggest that FNDI in humans may be initiated, as in our transgenic rat model, by the trapping of wild‐type VP gene products within an ER, which is targeted for lysosomal degradation by autophagy.

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. These findings suggest that the pituicyte may participate in auto- and/or cross-regulation of oxytocin and vasopressin release. Moreover, oxytocin and vasopressin precursor-derived peptides were found in the cytoplasm of some pituicytes, an unexpected finding that will be discussed.

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.e. those brain areas in which sex differences have been reported, with female rats showing a greater estrogen binding capacity than male rats. The highest densities of androgen receptor immunoreactivity were found in the septohypothalamic nucleus, the medial preoptic area, the posterior division of the bed nucleus of the stria terminalis and the posterodorsal division of the medial amygdaloid nucleus. No significant differences in distribution or total numbers of androgen receptors were found between neonatally estrogen-deprived males and control males. These findings suggest that neonatal estrogens, derived from the neural aromatization of testosterone, are involved in the sexual differentiation of the estrogen receptor system in the periventricular preoptic area and the ventromedial hypothalamus. The role of neonatal estrogens in the development of the forebrain androgen receptor system is less clear.

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. In older DS patients (>37 years), a mixed pattern of APP+1 immunoreactivity was observed in healthy looking neurons and neurites, dystrophic neurites, in association with neuritic plaques, as well as neurofibrillary tangles. UBB+1 immunoreactivity was exclusively present in AD type of neuropathology. A similar pattern of APP+1 and UBB+1 immunoreactivity was also observed for FAD and much less explicit in nondemented controls after the age of 51 years. Furthermore, we observed accumulation of +1 proteins in other types of tauopathies, such as PiD, frontotemporal dementia, PSP and argyrophylic grain disease. These data suggest that accumulation of +1 proteins contributes to the early stages of dementia and plays a pathogenic role in a number of diseases that involve the accumulation of tau.

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 Alzheimers disease. These frameshifts are caused by dinucleotide deletions in GAGAG motifs of mes· senger RNA encoded by genes that have maintained the unchanged wild‐type DNA sequence. This process is termed ‘molecular misreading’. A key question is whether this process is confined to neurons or whether it could also occur in non‐neuronal cells. A transgenic mouse line (MV‐B) carrying multiple copies of a rat vasopressin minigene as a reporter driven by the MMTV‐LTR promotor was used to screen non‐neuronal tissues for molecular misreading by means of detection of the rat vasopressin+1 protein and mutated mRNA. Molecular misreading was demonstrated to occur in several organs (e.g., epididymis and the parotid gland) where transgenic vasopressin expression is abundant, but its penetrance is variable both between and within tissues. This implies that non‐neural tissues too, could be affected by cellular derangements caused by molecular misreading.–van Leeuwen, F. W., Hol, E. M., Hermanussen, R. W. H., Sonnemans, M. A. F., Moraal, E., Fischer, D. F., Evans, D. A. P., Chooi, K.‐F., Burbach, J. P. H., Murphy, D. Molecular misreading in non‐neuronal cells. FASEB J. 14, 1595–1602 (2000)

Chapter 27 Dinucleotide deletions in neuronal transcripts: A novel type of mutation in non-familial Alzheimer's disease and Down syndrome patients

Publisher Summary Familial Alzheimers disease (FAD) represents about 40% of the total Alzheimers disease (AD) cases. Most of these FAD cases ( 35% of all AD patients) do not inherit AD as an autosomaldominant trait. Although these patients have at least one other relative in the first degree suffering from the disease, the genetic factor causing AD in these cases is not known. Families with an autosomal-dominant inheritance pattern of AD, account for only 5% of the total number of AD patients. In a subset of these families, missense mutations in the genes for β-amyloid precursor protein (β-APP), presenilin-1, and -2 underlie the AD pathogenesis. The non-familial or sporadic form of AD comprises approximately 60% of the total AD cases. Aging is probably an important factor in the AD etiology. The central nervous system (CNS), however, displays a high degree of plasticity, such that initial or minor damage to the CNS will not directly lead to neuropathology and can be compensated for. On the other side, the aging CNS is very vulnerable, because it is not capable to compensate for lost neurons that are especially prominent in a number of areas. Thus, during life-time irreversible cellular damage caused by somatic mutations, oxidative stress, or synapse loss accumulate in the post-mitotic neurons. Especially in AD most of these neurons do not die, but appear to become less active and show shrinkage. This chapter proposes that transcript mutations occurring in neuronal genes might be one of these unknown aging factors and might be a part of a general mechanism that could contribute to the neuropathogenesis in the majority of the AD cases, apart from the autosomal dominant forms. Recently this process was designated as molecular misreading.

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. This results in two + 1 di-vasopressin precursors that are still partially mutated within the neurophysin region. The present study provides immunocytochemical evidence which demonstrates that, within magnocellular solitary neurons of the supraoptic and paraventricular nuclei of the di/di rat, the + 1 di-vasopressin precursors can enter the secretory pathway followed by their enzymatic processing into vasopressin during axonal transport to the neural lobe. However, the cellular characteristics of biosynthesis are different from those of wild-type rats. Immunoelectron microscopical localization of vasopressin gene products in the neural lobe of did/di rats revealed their presence in neurosecretory granules, the diameter of which is intermediate (116 nm) between those of the neurosecretory granules in the di/di (80-100 nm) and wild-type (160 nm) rats.