Gajanan Nilaver

Delivery of virus-sized iron oxide particles to rodent cns neurons

Delivery of viral particles to the brain is limited by the volume of distribution that can be obtained. Additionally, there is currently no way to non-invasively monitor the distribution of virus following delivery to the central nervous system (CNS). To examine the delivery of virus-sized particles across the blood-brain barrier (BBB), dextran coated, superparamagnetic monocrystalline iron oxide particles, with a hydrodynamic diameter of 20 +/- 4 nm, were delivered to rat brain by direct intracerebral inoculation or by osmotic BBB disruption with hypertonic mannitol. Delivery of these particles was documented by magnetic resonance (MR) imaging and, unexpectedly, neuronal uptake was demonstrated by histochemical staining. Electron microscopy (EM) confirmed iron particle delivery across the capillary basement membrane and localization within CNS parenchymal cells following administration with BBB disruption. This is the first histologic and ultrastructural documentation of the delivery of particles the size of virions across the blood-brain barrier. Additionally, these dextran-coated, iron oxide particles may be useful, in and of themselves, as vectors for diagnostic and/or therapeutic interventions directed at the CNS.

A-Kinase Anchoring Proteins

The cAMP-dependent protein kinase (PKA) regulates a variety of diverse biochemical events through the phosphorylation of target proteins. Because PKA is a multifunctional enzyme with a broad substrate specificity, its compartmentalization may be a key regulatory event in controlling which particular target substrates are phosphorylated. In recent years it has been demonstrated that differential localization of the type II holoenzyme is directed through interaction of the regulatory subunit (RII) with a family ofA-KinaseAnchoringProteins (AKAPs). In this report, we review evidence for PKA compartmentalization and discuss the structural and functional properties of AKAPs.

Genetically modified PC12 brain grafts: Survivability and inducible nerve growth factor expression

Neural transplantation of genetically modified cells has been successfully employed to reverse functional deficits in animal models of neurodegenerative disorders, including Parkinsons disease. While implanted PC12 cells secrete dopamine in vivo and can ameliorate dopamine deficiency in parkinsonian rat model systems, these cells either degenerate within 2-3 wk postimplantation (presumably due to the lack of neural trophic factor support at the site of implantation), or in some cases, form a tumor mass leading to the death of the host animal. To address these limitations, we have developed a genetically modified PC12 cell line that can synthesize nerve growth factor (NGF) under the control of a zinc-inducible metallothionein promoter. When implanted in the rat striatum and under in vivo zinc stimulation, these cells will neuro-differentiate, express tyrosine hydroxylase, and will undergo survival through potential autocrine trophic support. This regulatable cell line and general approach may provide additional insight on the potential utilization of cell transplants for treatment of Parkinsons disease and other neurodegenerative disorders.

Detection of low-molecular-weight polypeptides on nitrocellulose with monoclonal antibodies

An immunoblotting method to detect low-molecular-weight peptides with monoclonal antibodies that normally fail to demonstrate immunoreactivity using conventional blotting techniques is described. Detection of neurophysin, insulin, calcitonin, vasopressin, and beta-endorphin electroblotted on nitrocellulose membranes was optimized after introducing four modifications into the conventional procedure. These include renaturing the gels after sodium dodecyl sulfate electrophoresis, electroblotting the renatured gels in basic transfer buffer, fixing and/or heating the blots, and using avidin/alkaline phosphatase conjugates for antigen/antibody detection. This technique likely enables the denatured peptides to regain their native conformation and, therefore, restores antigenicity and recognition by highly structural specific monoclonal antibodies. Although the most dramatic improvement with this technique is with monoclonal antibodies, a modest improvement in sensitivity can be obtained when immunoblots are probed with polyclonal antibodies. The high resolution of this system will be useful in probing blots of partial proteolytic digests of proteins with both monoclonal and polyclonal antibodies.

Tyrosine hydroxylase expression in non-catecholaminergic cells in cerebellar cultures

Organotypic cerebellar cultures, some with incorporated portions of brainstem, were immunostained after 12-19 days in vitro with three different antibodies to tyrosine hydroxylase. Similar cultures were reacted with glyoxylic acid and examined for catecholamine histofluorescence. Locus coeruleus and other subcortical neurons were positive for tyrosine hydroxylase, as were Purkinje cells and outgrowth zone astrocytes. By contrast, only locus coeruleus neurons and their axons exhibited catecholamine histofluorescence after reaction with glyoxylic acid. These results confirm previously reported in vivo developmental studies indicating that tyrosine hydroxylase can be expressed in the absence of its normal biosynthetic products, and suggest that tyrosine hydroxylase cannot be considered to be a specific marker for catecholaminergic neurons in vitro, as well as during development in vivo.

Cochlear IgG in the C3H/lpr autoimmune strain mouse

The inner ear of the C3H/lpr autoimmune strain mouse was evaluated to identify potential mechanisms by which systemic autoimmune disease interferes with auditory function. The inner ears were immunohistochemically stained for IgG at ages before (2 months) and after (6-10 months) autoimmune disease onset and compared to age-matched nonautoimmune C3H/HeJ controls. Immunoreactivity for IgG was not seen in the 2 month C3H/lpr autoimmune mice or in either age group of the C3H/HeJ controls. On the other hand, all older C3H/lpr mice showed reaction product in the vessels of the cochlea, particularly the stria vascularis and bony capsule. Less frequent sites of staining were the geniculate ganglion, marrow cavities of the bony capsule, tensor tympani muscle, and on one occasion, a hair cell of the organ of Corti. These findings indicate that IgG is widespread within the cochlea and its vessels during systemic autoimmune disease and not directed against any specific sensorineural structure. This suggests a generalized or indirect mechanism whereby such systemic disease affects the inner ear.

Serum anti-myelin antibodies in chronic relapsing experimental allergic encephalomyelitis

To investigate the role of anti-myelin antibodies in chronic relapsing experimental allergic encephalomyelitis (CR-EAE), sera from SJL/J mice with CR-EAE actively induced by inoculation with spinal cord homogenate in complete Freunds adjuvant (CFA) were compared with sera from mice to whom CR-EAE was passively transferred by lymph node cells (LNC) stimulated with myelin basic protein (BP). Sera were obtained serially from mice during both remissions and relapses of disease and were evaluated for the presence of anti-myelin antibodies using an avidin-biotin-immunoperoxidase technique. Four of six mice with CR-EAE induced with cord-CFA were positive for anti-myelin antibodies 15-124 days after inoculation, with 16 of 18 sera positive in these four mice. Two mice inoculated with cord-CFA did not have detectable serum anti-myelin antibodies, despite a clinical and histopathological picture indistinguishable from the antibody-positive mice. None of seven mice with CR-EAE passively transferred by BP-stimulated LNC had detectable anti-myelin antibodies in 30 sera obtained 7-141 days after cell transfer. We conclude that serum anti-myelin antibodies probably do not play a significant role in the pathogenesis of CR-EAE in SJL/J mice.

Identification of neurophysin immunoreactivity in hypothalamus by a monoclonal antibody to a carcinoma cell surface antigen

Mouse monoclonal antibody (mAb) L6 identifies an antigen expressed on the cell surface of many different human carcinomas. While studying the binding activity of mAb L6 to intracerebral tumor xenografts of human lung carcinoma LX-1 cells in nude rats using immunohistological techniques, we observed that L6 can also bind to a cytoplasmic antigen expressed in the magnocellular component of the hypothalamo-neurohypophysial system. Double-labeling experiments with antisera to vasopressin and oxytocin confirmed the localization of L6 immunoreactivity within both peptide-containing cell groups. L6 immunoreactivity in Brattleboro rats (with genetic deletion in the vasopressin gene) was exclusively localized within oxytocin neurons. Oxytocin and vasopressin failed to block L6 staining which suggested that its target epitope resides within the neurophysin sequence, and this explanation was supported by the finding that adsorption of L6 with porcine neurophysin completely eliminated hypothalamic immunoreactivity. Western blot analysis of bovine neurophysin and human pituitary extracts identified L6-immunoreactive bands which corresponded to the position of neurophysin and pro-pressophysin, confirming that L6 immunoreactivity in hypothalamus is related to neurophysin. Thus, monoclonal antibody L6, which is highly reactive with a membrane antigen of human lung cancer cell line LX-1, recognizes a cytoplasmic epitope in hypothalamic neurons identified as neurophysin by immunohistochemistry and Western analysis.

Generation and Characterization of an Antiserum Directed Against Neurohypophyseal Prohormones

An antiserum to neurohypophyseal prohormones was generated by immunization of rabbits with a synthetic peptide fragment bridging the prohormone cleavage site between the vasopressin (AVP) and human AVP‐neurophysin sequences of pro‐pressophysin. Polyclonal antibodies directed against this peptide cross‐reacted with intact human pro‐pressophysin (ED50 of 260fmol), but not with either of the final products of enzymatic processing, AVP and human AVP‐neurophysin. Gel electrophoresis and Western immunoblotting of pituitary or hypothalamic extracts from multiple species including mouse, cow and man identified a protein band of molecular weight consistent with intact pro‐pressophysin; in hypothalamic extracts from normally‐hydrated rats no protein bands were stained, but in extracts from Brattleboro rats a faint band in the area of pro‐oxyphysin was identified. Immunohistochemical studies using the antiserum demonstrated the presence of only very small amounts of immunoreactive prohormone in a few widely scattered cells in the hypothalami of normally‐hydrated rats. However, after 5 days of solute loading with 2% NaCl as drinking solution, staining for intact prohormone was prominent in the supraoptic and paraventricular nuclei of the hypothalamus. Combined immunoperoxidase‐immunofluorescence labeling for prohormone and either AVP‐neurophysin or oxytocin‐neurophysin revealed prohormone staining in both types of magnocellular neurons in rat hypothalami. These studies suggest that during states of accelerated synthesis and secretion of neurohypophyseal hormones some accumulation of intact prohormone occurs in both AVP and oxytocin magnocellular neurons.

Immunological studies with a monoclonal antibody suggest a different conformation of neurophysin in parvicellular neurons of rat hypothalamus

A monoclonal antibody (mAb L6) to a carcinoma surface antigen has previously been shown to recognize neurophysins (NP), proteins associated with oxytocin and vasopressin. L6-reactivity in rat hypothalamus was confined to magnocellular neuronal systems. No staining was detected in parvicellular suprachiasmatic or paraventricular systems. mAb L6 immunoprecipitated vasopressin-neurophysin only under reducing conditions, and detected it in Western blots only after gel-renaturation and electroblotting in basic buffer. These findings suggest L6-reactivity to NP is conformation-sensitive, and imply NP expression in a unique configurational form in hypothalamic parvicellular systems.