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The amyloid-beta (Abeta) peptide, which likely plays a key role in Alzheimer disease, is derived from the amyloid-beta precursor protein (APP) through consecutive proteolytic cleavages by beta-site APP-cleaving enzyme and gamma-secretase. Unexpectedly gamma-secretase inhibitors can increase the secretion of Abeta peptides under some circumstances. This "Abeta rise" phenomenon, the same inhibitor causing an increase in Abeta at low concentrations but inhibition at higher concentrations, has been widely observed. Here we show that the Abeta rise depends on the beta-secretase-derived C-terminal fragment of APP (betaCTF) or C99 levels with low levels causing rises. In contrast, the N-terminally truncated form of Abeta, known as "p3," formed by alpha-secretase cleavage, did not exhibit a rise. In addition to the Abeta rise, low betaCTF or C99 expression decreased gamma-secretase inhibitor potency. This "potency shift" may be explained by the relatively high enzyme to substrate ratio under conditions of low substrate because increased concentrations of inhibitor would be necessary to affect substrate turnover. Consistent with this hypothesis, gamma-secretase inhibitor radioligand occupancy studies showed that a high level of occupancy was correlated with inhibition of Abeta under conditions of low substrate expression. The Abeta rise was also observed in rat brain after dosing with the gamma-secretase inhibitor BMS-299897. The Abeta rise and potency shift are therefore relevant factors in the development of gamma-secretase inhibitors and can be evaluated using appropriate choices of animal and cell culture models. Hypothetical mechanisms for the Abeta rise, including the "incomplete processing" and endocytic models, are discussed.

Herpes simplex virus (HSV) is neurotropic and when inoculated on the mouse footpad is retrogradely transported to the associated dorsal root ganglia (DRG), where infection is established. Previous observations suggest that, after HSV infection, sensory ganglion neurons may mount a sprouting response. In our HSV-infected DRG model, we investigate this issue by (1) examining expression of growth-associated protein (GAP-43), a molecule known to be induced by growing axons, and (2) determining ultrastructurally whether HSV-infected dorsal roots contain neurites. In a time course study, we show that GAP-43 is induced both in HSV-infected DRG and their central processes. The increase in GAP-43 is first seen 2 weeks following unilateral footpad inoculation in both cell bodies and dorsal roots, and is sustained at 1 month post inoculation in roots but not in perikarya. Large bundles of unmyelinated small caliber axons, lacking Schwann cell ensheathment, are observed by electron microscopy in dorsal roots 2 weeks and 1 month following inoculation. These profiles resemble developing or regenerating neurites and are rarely seen in roots of mock-infected or uninfected controls. The increased GAP-43 immunoreactivity and ultrastructural changes shown here, in conjunction with previously documented selective neuropeptide and enzyme alterations, confirm that a sprouting response is mounted in sensory ganglia following acute HSV infection.

Reduction of brain beta-amyloid peptide (Abeta) synthesis by gamma-secretase inhibitors is a promising approach for the treatment of Alzheimer's disease. However, measurement of central pharmacodynamic effects in the Alzheimer's disease patient will be a challenge. Determination of drug occupancy may facilitate the analysis of efficacy of gamma-secretase inhibitors in a clinical setting. In this study, the relationship of gamma-secretase site occupancy and brain Abeta40 reduction by gamma-secretase inhibitors was examined in Tg2576 mice. [3H](2R,3S)-2-Isobutyl-N1-((S)-1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diazepin-3-yl)-3-propylsuccinamide (IN973) was used as a gamma-secretase radioligand, since it has been shown to bind to gamma-secretase in rat, rhesus, and human brains with high affinity and specificity. We extended these findings by showing that [3H]IN973 bound to gamma-secretase in Tg2576 brains with an affinity, specificity, and regional localization very similar to the other species. To quantify gamma-secretase occupancy by gamma-secretase inhibitors, an ex vivo binding assay was developed using [3H]IN973 and frozen brain sections from drug-treated mice. Gamma-secretase occupancy and brain Abeta40 reduction were found to be highly correlated in animals dosed with either 2-[(1R)-1-[[4-chlorophenyl)-sulfonyl](2,5-difluorophenyl)amino] ethyl]-5-fluoro-benzenepropanoic acid (BMS-299897) or (S)-2-((S)-2-(3,5-difluorophenyl)-2-hydroxyacetamido)-N-((S,Z)-3-methyl-4-oxo-4,5-dihydro-3H-benzo[d][1,2]diazepin-5-yl)propanamide (BMS-433796) over a wide range of doses and times postdose, with the exception of the earliest times postdose. This lag in Abeta40 response to gamma-secretase inhibition is probably related to the delayed clearance of previously produced Abeta40. The excellent correlation between brain Abeta40 and gamma-secretase occupancy suggests that a positron emission tomography ligand for gamma-secretase could be a valuable biomarker to determine whether gamma-secretase inhibitors bind to their target in humans.

Galanin is a ubiquitous neuropeptide that regulates a wide array of physiological processes via interaction with specific G protein-coupled receptors. A rat galanin receptor cDNA was cloned from the Rin14B insulinoma cell line. The isolated cDNA encodes a 346 amino acid G protein-coupled receptor that is 92% identical to the recently reported human GALR1 galanin receptor. [125I]Galanin binds with high affinity to two receptor states in COS1 cell membranes containing the rat GALR1 receptor, consistent with coupling of the receptor to a G protein in these membranes. N-terminal galanin fragments and the putative galanin receptor antagonists galantide, C7, M35 and M40 bind with high affinity to the rat GALR1 receptor. In contrast, C-terminal galanin fragments do not bind to this receptor. Galanin inhibits basal and forskolin-stimulated cAMP formation in CHO cells expressing the rat GALR1 receptor via a pertussis toxin-sensitive G protein. The GALR1 receptor is expressed in rat spinal cord, small intestine, Rin14B insulinoma cells and several brain regions, particularly ventral hippocampus, amygdala, supraoptic nucleus, hypothalamus, thalamus, lateral parabrachial nucleus and locus coeruleus. Cloning of the rat GALR1 galanin receptor cDNA will permit many new experimental strategies to be applied to studies of the structure and function of galanin receptors.

Visceral sensory neurons of the glossopharyngeal and vagus nerves are located in the petrosal and nodose ganglia, respectively. Our previous studies showed that peripheral axotomy which removes afferent input to visceral sensory perikarya decreased the number of calcitonin gene-related peptide (CGRP)-immunoreactive (ir) neurons in the petrosal but not the nodose ganglion [13]. To evaluate axotomy-induced changes in CGRP mRNA expression, we used in situ hybridization histochemistry with 35 S-labeled oligonucleotide probes. CGRP mRNA-containing neurons were studied 1, 3, 7 and 14 days after peripheral deafferentation of the left nodose and petrosal ganglia via transection of the left cervical vagus, superior laryngeal, glossopharyngeal and carotid sinus nerves. The numbers of CGRP mRNA-containing neurons in the deafferented petrosal ganglion were significantly reduced at 3, 7 and 14 days compared to either intact or sham-operated control ganglia. However, the density of hybridization product in the positively-labeled petrosal ganglion cells was not significantly changed. The numbers of CGRP mRNA-containing neurons in the deafferented nodose ganglion were significantly reduced at 3 and 7 days. These data suggest that axotomy-induced changes in CGRP-ir neurons of the petrosal ganglion correlate with changes in CGRP mRNA and probably result from altered CGRP gene expression. In addition, in situ hybridization histochemistry revealed changes in CGRP neurons of the nodose ganglion which were not apparent with immunocytochemistry.

We have examined the effects of herpes simplex virus type 2 (HSV-2) infection on neuron numbers in mouse dorsal root ganglia (DRG) following unilateral hind footpad inoculation. One month following HSV-2 strain MS inoculation, tissue sections of decalcified spine containing the paired 4th and 5th lumbar DRGs were stained with cresyl violet. Neuronal numbers, somal areas and ganglion volumes were determined for ganglia ipsilateral and contralateral to both HSV-2 and medium inoculations. One month following HSV-2 infection, 47–88% of neurons disappeared in the ipsilateral ganglia. The somal areas of the remaining neurons in these ganglia fell within the range of the uninfected population. Ganglionic shrinkage did not occur as a result of HSV-2 infection; neurons were replaced by large numbers of inflammatory cells. Neuron numbers in the contralateral control ganglia of the HSV-2 inoculated mice appeared slightly decreased. Mice inoculated with medium contained similar numbers of neurons in ipsilateral and contralateral ganglia. These results show that, in addition to other previously described host alterations, infection with HSV-2 strain MS results in neuronal death in the affected host ganglia. This is the first in vivo quantitative documentation of neuronal death induced by herpes simplex virus infection.

The neuropeptide galanin mediates a diverse spectrum of biological activities by interacting with specific G-protein-coupled receptors. Through expression cloning, human and rat GALR1 receptor cDNA clones have previously been isolated and characterized. In this study, we have used homology screening to isolate a rat brain cDNA clone encoding a second galanin receptor subtype, the GALR2 receptor. The isolated cDNA encodes a 372-amino-acid G-protein-coupled receptor that shares 38% overall amino-acid identity with the rat GALR1 receptor. The pharmacological profile of the rat GALR2 receptor is similar to that of the rat GALR1 receptor. The rat GALR2 receptor binds galanin, N-terminal galanin fragments, and the putative galanin receptor antagonists galantide, C7, M35 and M40 with high affinity but it does not bind C-terminal galanin fragments. Galanin increases intracellular inositol phosphate levels in HEK 293 cells expressing the rat GALR2 receptor via a pertussis toxin-insensitive G-protein. The rat GALR2 receptor mRNA is highly expressed in several brain regions, including hypothalamus and hippocampus as well as the anterior pituitary, with lower levels of expression detected in amygdala, and regions of cortex. It is also highly expressed in the GH3 pituitary cell line and in gut tissues, and to a lower extent in spleen, lung, skeletal muscle, heart, kidney, liver and testis. These results suggest that GALR2 receptor mediates galanin's regulation of pituitary hormone secretion and possibly food intake.

Neurofilaments purified from adult rat brainstem by two methods were electrophoresed on NaDodSO4/polyacrylamide gels to separate the triplet proteins (approximate Mrs of 200,000, 155,000, and 68,000) which, in turn, were electroblotted onto nitrocellulose paper. On Coomassie blue-stained gels that were not electroblotted, the same banding pattern was seen with both methods of preparation. Immunocytochemical staining of the electroblots with each of five monoclonal antibodies revealed that three of the monoclonal antibodies were specific for the Mr 200,000 neurofilament protein and two, for both the Mrs 200,000 and 155,000 neurofilament proteins. None of the antibodies reacted with the Mr 68,000 band. The Mr 200,000 band could be resolved into doublet bands. Individual monoclonal antibodies reacted with either one or both of the Mr 200,000 doublets. The immunocytochemical staining of the neurofilament triplets on electroblots was compared to that of adult rat cerebellar paraffin sections. Each monoclonal antibody had a unique pattern of staining, reacting only with certain subpopulations of neurons or their processes. Correlation of the staining patterns in cerebellar tissue sections with those of neurofilament polypeptides on electroblots suggested that different neurofilament polypeptides can be localized to different structures and subpopulations of neurons and that molecular heterogeneity ("neurotypy") may be revealed within the Mrs 200,000 and 155,000 neurofilament polypeptides.

Two-dimensional immunoblots revealed a spectrum of 200 kDa neurofilament polypeptides (Nf-H) of apparent molecular weights ranging from 200 to 170 kDa. The entire spectrum was stained immunocytochemically by three monoclonal antibodies specific for nonphosphorylated neurofilaments, while more restricted staining was revealed by four monoclonal antibodies specific for phosphorylated neurofilament epitopes. Treatment with increasing amounts of phosphatase suggested the existence of various forms of partially phosphorylated neurofilaments that posses phosphoepitopes that differ in their ease of dephosphorylation. Immunoprecipitation in low detergent concentration confirmed the existence of microheterogeneous forms of Nf-H that differed in extent of phosphorylation or in distribution of phosphorylated sites.

The paradigm of IDPN neuropathy was produced in rats in order to examine the neurofilaments (NFs) that accumulate in the proximal motor and sensory axons of intoxicated animals, and to compare the aggregated NFs with control NFs and with the depleted populations of NFs in the distal portions of the same experimental nerves. NFs were probed biochemically and histochemically, using a large and well-characterized library of monoclonal antibodies that included antibodies that are monospecific for each of the rat NF protein subunits (NF-H, NF-M, and NF-L) as well as antibodies that recognized differential phosphorylated states of rat NF-H and NF-M. All antibodies tested showed enhanced immunostaining of enlarged axons and of large spheroids in the spinal cord and dorsal root ganglia of experimental animals. Biochemical analyses of IDPN-treated animals revealed enrichment of NF-H, NF-M, and NF-L in homogenates of dorsal root ganglia and of proximal motor and sensory nerve roots as well as depletion of the three subunits in distal nerve roots and in sciatic nerves. Immunoblot revealed a uniform enrichment of NF-H, NF-M, and NF-L in NF aggregates as well as the same admixture of phosphorylated and dephosphorylated epitopes of NF-H and NF-M in experimental and in control tissues. The global increase of immunoreactivity in axonal swellings to antibodies that react with phosphorylated, nonphosphorylated, and phosphorylation-independent NF epitopes suggests that IDPN induces an accumulation of NFs in proximal axons without necessarily altering the state of NF phosphorylation.

During incubation with phosphatase, the 200 kDa neurofilament protein in cytoskeletal preparations is degraded extensively. Degradation, which is divalent cation-independent, does not occur when inhibitors of phosphatase are added. The 160 kDa chymotryptic fragment of neurofilaments or affinity-purified 200 kDa protein are not degraded by phosphatase. The results suggest that (1) phosphorylated neurofilaments are protected against proteolysis, and (2) dephosphorylated neurofilaments are degraded by a calcium-independent, endogenous proteinase which is associated with assembled neurofilaments or with other cytoskeletal components, and not with the phosphatase used.

States of phosphorylation of neurofilament proteins were examined in the perikarya of rat sensory and motor neurons between 3 and 28 d following either a distal transection [6–7 cm from the L4-L5 dorsal root ganglia (DRG)] or a proximal transection (1–2 cm from the L4-L5 DRG) of the sciatic nerve. Paraffin sections of the right (experimental) and left (control) L4 and L5 DRG from animals with unilateral transection of the right distal sciatic nerve were stained immunocytochemically with monoclonal antibodies to phosphorylation- dependent (NF-P), dephosphorylation-dependent (NF-dP), or phosphorylation-independent (NF-ind) epitopes on the largest (NF200), mid-sized (NF150), or smallest (NF68) neurofilament protein subunits. Increased immunoreactivity to NF-P on NF200 and NF150 was detected in experimental DRC at 10 d, peaking by 20 d, and declining to near control levels by 28 d. Conversely, immunoreactivity to NF-dP declined in experimental DRG beginning at 6 d, reaching a maximum decline at 10– 16 d, and returning to near control levels by 28 d. Immunocytochemical changes were confirmed with biochemical studies on tissue homogenates that demonstrated an increase of immunoreactivity to NF-P and a decrease of reactivity to NF-dP in the experimental DRG. Changes in immunoreactivities to NF-P and NF-dP were observed only in the perikarya of large neurons and were closely associated with chromatolytic changes in these neurons. Marked enhancement of chromatolysis, as well as the immunoreactivities to NF-P and NF-dP, occurred following a proximal (left side) versus distal (right side) transection in the same animal.(ABSTRACT TRUNCATED AT 250 WORDS)

The control of neurofilament (NF) protein gene expression was studied by determining and comparing the levels of mRNA to the heavy (NF-H), mid-sized (NF-M) and light (NF-L) NF protein subunits in rat dorsal root ganglia (DRG) following sciatic nerve transection. mRNA to NF-H (4.5 kb), to NF-M (3.4 kb) and to NF-L (2.5 and 4.0 kb) were identified in Northern blots and quantitated in dot blot analyses, using specific cDNA probes for each NF protein. Following transection, there was a coordinated decline in mRNA levels to all 3 NF proteins, beginning as early as 3 days following transection and continuing for at least 28 days. The early and co-terminal fall in mRNAs suggests that the 3 NF genes are regulated by common factor(s) and that the function of these factor(s) is influenced by the state of axonal continuity with the target organ.

Adult and developing rat cerebella were stained immunocytochemically with six neuron-specific monoclonal antibodies obtained from spleen cells of BALB/c mice immunized with hypothalamus. Monoclonal peroxidase-antiperoxidase complex was used in the staining procedure. In the adult, three different staining patterns were seen with these antibodies. The general patterns have been designated as (1) neurofibrillar, (2) perikaryal-neurofribillar, and (3) synapse-associated, in addition, each antibody within a designated group showed a different immunocytochemical distribution. Some antibodies reacted widely, for example, with many neuronal perikarya and fibers, and their distribution increased with the development of the brain. Other antibodies had a more restricted distribution and stained only some structures within an area or pathway. To account for this type of restriction of distribution, we propose that there may be microheterogneity of some of the antigens visualized and have called this microheterogeneity ‘neurotypy’. A second type of restriction was also observed. With several antibodies a loss of staining occurred in the adult cerebellum in structures that had reacted during early development. These differences in staining probably reflect developmental regulation of the antigens (neurotypes).