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Biomedical subjects

F M Longo

Publications and source records attributed to F M Longo.

At least 19 recordsLinked to original sources
PubMed2001

The leukocyte common antigen-related protein tyrosine phosphatase receptor regulates regenerative neurite outgrowth in vivo.

Drosophila and leech models of nervous system development demonstrate that protein tyrosine phosphatase (PTP) receptors regulate developmental neurite outgrowth. Whether PTP receptors regulate neurite outgrowth in adult systems or in regenerative states remains unknown. The leukocyte common antigen-related (LAR) receptor is known to be present in rodent dorsal root ganglion (DRG) neurons; therefore, the well established model of postcrush sciatic nerve regeneration was used to test the hypothesis that LAR is required for neurite outgrowth in the adult mammalian nervous system. In uninjured sciatic nerves, no differences in nerve morphology and sensory function were detected between wild-type and LAR-deficient littermate transgenic mice. Sciatic nerve crush resulted in increased LAR protein expression in DRG neurons. In addition, nerve injury led to an increase in the proportion of LAR protein isoforms known to have increased binding affinity to neurite-promoting laminin-nidogen complexes. Two weeks after nerve crush, morphological analysis of distal nerve segments in LAR-deficient transgenic mice demonstrated significantly decreased densities of myelinated fibers, decreased axonal areas, and increased myelin/axon area ratios compared with littermate controls. Electron microscopy analysis revealed a significant twofold reduction in the density of regenerating unmyelinated fibers in LAR-/- nerves distal to the crush site. Sensory testing at the 2 week time point revealed a corresponding 3 mm lag in the proximal-to-distal progression of functioning sensory fibers along the distal nerve segment. These studies introduce PTP receptors as a major new gene family regulating regenerative neurite outgrowth in vivo in the adult mammalian system.

Animals↗
PubMed2000

Stress induces zinc finger immediate early genes in the rat adrenal gland.

The secretion of steroid hormones from the adrenal cortex as well as cathecolamines from the adrenal medulla is stimulated by stress. In this study, we studied the effect of capsaicin-induced stress on the expression of the immediate-early genes (IEGs) NGFI-A, -B, -C, egr-2, -3 and Nurr1 in the rat adrenal gland using in situ hybridization. All of these IEGs except egr-2 were rapidly induced in the adrenal cortex and medulla. The temporal patterns of the IEG induction in medulla varied significantly. NGFI-A was induced in medulla within 15 min after stress, NGFI-B, egr-3 and Nurr1 were induced by 30 min, whereas NGFI-C was induced by 2 h. Surprisingly, only NGFI-B and Nurr1 were induced in the glucocorticoid secreting regions of zonae reticularis and fasciculata of the cortex, starting 15 min after the stress. All of the inducible IEGs were induced in the aldosterone secreting zona glomerulosa 15-30 min after the capsaicin injection. NGFI-A, NGFI-B and Nurr1 expression persisted for 6 h. Since the IEGs studied had major differences in their temporospatial induction pattern, they are likely to be induced by distinct stress-elicited factors and have separate target genes and roles in stress-induced glucocorticoid and catecholamine secretion.

Adrenal Cortex↗
PubMed2000

Nerve growth factor (NGF) loop 4 dimeric mimetics activate ERK and AKT and promote NGF-like neurotrophic effects.

Previous work indicating that nerve growth factor (NGF) protein loops 2 and 4 interact with TrkA receptors raise the possibility that small molecule mimetics corresponding to TrkA-interacting domains that have NGF agonist activity can be developed. We applied our previously developed strategy of dimeric peptidomimetics to address the hypothesis that loop 4 small molecule dimeric mimetics would activate TrkA-related signal transduction and mimic NGF neurotrophic effects in a structure-specific manner. A loop 4 cyclized peptide dimer demonstrated NGF-like neurotrophic activity, whereas peptides with scrambled sequence, added or substituted residues, or cyclized in monomeric form were inactive. Activity was blocked by the TrkA inhibitors K252a and AG879 but not by NGF p75 receptor blocking antibody. Dimeric, but not monomeric, peptides partially blocked NGF activity. This profile was consistent with that of a NGF partial agonist. ERK and AKT phosphorylation was stimulated only by biologically active peptides and was blocked by K252a. The ERK inhibitor U0126 blocked the neurite- but not the survival-promoting activity of both NGF and active peptide. These studies support the proof of concept that small molecule NGF loop 4 mimetics can activate NGF signaling pathways and can mimic death-preventing and neurite-promoting effects of NGF. This finding will guide the rational design of NGF single-domain mimetics and contribute to elucidating NGF signal transduction mechanisms.

Animals↗
PubMed2000

Leukocyte common-antigen-related tyrosine phosphatase receptor: altered expression of mRNA and protein in the New England Deaconess Hospital rat line exhibiting spontaneous pheochromocytoma.

Regulation of cell proliferation by protein tyrosine phosphatases (PTPs) suggests that PTPs are important tumor suppressor genes. The gene encoding the leukocyte common-antigen-related (LAR) PTP receptor maps to chromosome 1p32-33, a region in which loss of heterozygosity is associated with human pheochromocytoma and other neuroectodermal tumors. The rat pheochromocytoma PC12 cell line was originally derived from the transplantable P259 tumor originating from the New England Deaconess Hospital (NEDH) line of Wistar inbred rats. Compared with their Wistar counterparts, 1-2-year-old NEDH rats exhibit a high incidence of spontaneous pheochromocytomas. This study investigates whether levels of LAR transcripts and protein are altered in NEDH adrenal tissue prior to tumor onset. In addition, alternative splicing of an LAR extracellular domain [LAR alternatively spliced element-c (LASE-c)], regulating LAR interaction with extracellular matrix components, was examined. These changes in LAR expression and alternative splicing were hypothesized to be more pronounced in tumor tissue and PC12 cells. Northern blot analysis demonstrated the presence of the approximately 5 kb LAR transcript in all cell lines examined, except PC12. In adrenal medulla tissue harvested from 2-3-month-old rats, LAR approximately 8 and approximately 5 kb transcript expression was decreased in NEDH compared with Wistar samples. RT-PCR demonstrated increased splicing of the LASE-c 27 bp alternatively spliced insert in the LAR extracellular domain in NEDH adrenal medulla tissue. Even greater LASE-c splicing was detected in adrenal medulla tumor tissue derived from 12-month-old NEDH rats and in PC12 cells. Western blot analysis demonstrated decreased levels of LAR protein and increased levels of LASE-c containing LAR protein isoforms in NEDH adrenal medulla tissue. These studies demonstrate that patterns of altered LAR expression present in PC12 cells and in pheochromocytoma tumor tissue are also present in adrenal tissue predisposed to a high incidence of spontaneous pheochromocytoma.

Adrenal Cortex↗
PubMed2000

Downregulation of LAR tyrosine phosphatase prevents apoptosis and augments NGF-induced neurite outgrowth.

The identity of the protein tyrosine phosphatases (PTPs) regulating cell death and responses to neurotrophins during neural development remain unknown. To determine if the leukocyte common antigen-related (LAR) PTP regulates these processes, PC12 cells were made LAR-deficient via stable transfection with an LAR antisense transgene. LAR-deficient cells demonstrated a stable novel phenotype, including a two-fold increase in nerve growth factor- but not fibroblast growth factor-induced neurite outgrowth. Upon serum-deprivation, LAR-deficient cells exhibited a two- to three-fold decrease in cell death. The findings that an endogenous PTP promotes cell death and counter-regulates neurotrophin actions introduce a major new receptor gene family to neurotrophic processes and suggest novel strategies for preventing cell death and augmenting neurotrophin function.

Animals↗
PubMed1999

Gene expression along the cerebral-spinal axis after regional gene delivery.

We demonstrate here that intracerebroventricular or spinal cord (intrathecal) injection of either plasmid DNA alone or cationic liposome: DNA complexes (CLDCs) produces significant levels of expression of both reporter genes and biologically relevant genes in nonparenchymal cells lining both the brain and the spinal cord. Gene expression was identified both within the spinal cord and the brain after intracerebroventricular or intrathecal injection of either CLDCs or plasmid DNA alone. Intracerebroventricular or intrathecal injection of CLDCs containing the beta-galactosidase (beta-Gal) gene produced patchy, widely scattered areas of beta-Gal expression. The chloramphenicol acetyltransferase (CAT) reporter gene product reached peak levels between 24 hr and 1 week postinjection, and was still present at significant levels 3 weeks after a single intracerebroventricular or intrathecal injection. Intrathecal injection of the human granulocyte colony-stimulating factor (G-CSF) gene produced high levels of hG-CSF activity in both the spinal cord and the brain. Intracerebroventricular injection of CLDCs containing the murine nerve growth factor (NGF) gene increased mNGF levels in the hippocampus, a target region for cholinergic neurons in the medial septum, and increased cholinergic neurotransmitter synthetic enzyme choline acetyltransferase (ChAT) activity within the brain, a well-characterized effect of both purified and recombinant NGF protein. These findings indicate that intracerebroventricular or intrathecal injection of CLDCs can produce significant levels of expression of biologically and therapeutically relevant genes within the CNS. Efficient gene transfer into the CNS will facilitate the evaluation of gene function and regulation within the brain and spinal cord. We attempted to transfer and express genes within the brain and spinal cord by direct CNS injection of either DNA alone or CLDCs into the intraventricular and subarachnoid compartments. We show that intracerebroventricular or spinal cord (intrathecal) injection of either plasmid DNA alone or CLDCs produces significant levels of expression of both reporter genes and biologically relevant genes in nonparenchymal cells lining both the brain and the spinal cord. Intrathecal injection of the hG-CSF gene produced high levels of hG-CSF activity in both the spinal cord and the brain. Intracerebroventricular injection of CLDCs containing the murine NGF gene increased mNGF levels in the hippocampus, and increased cholinergic neurotransmitter synthetic enzyme ChAT activity within the brain. Locoregional diffusion of gene products expressed by transfected meningeal lining cells into brain and spinal cord parenchyma could potentially target secreted proteins within brain and spinal cord regions relevant to neuropathological states while limiting peripheral side effects.

Animals↗
PubMed1999

Electromagnetic fields influence NGF activity and levels following sciatic nerve transection.

Pulsed electromagnetic fields (PEMF) have been shown to increase the rate of nerve regeneration. Transient post-transection loss of target-derived nerve growth factor (NGF) is one mechanism proposed to signal induction of early nerve regenerative events. We tested the hypothesis that PEMF alter levels of NGF activity and protein in injured nerve and/or dorsal root ganglia (DRG) during the first stages of regeneration (6-72 hr). Rats with a transection injury to the midthigh portion of the sciatic nerve on one side were exposed to PEMF or sham control PEMF for 4 hr/day for different time periods. NGF-like activity was determined in DRG, in 5-mm nerve segments proximal and distal to the transection site and in a corresponding 5-mm segment of the contralateral nonoperated nerve. NGF-like activity of coded tissue samples was measured in a blinded fashion using the chick DRG sensory neuron bioassay. Overall, PEMF caused a significant decrease in NGF-like activity in nerve tissue (P < 0.02, repeated measures analysis of variance, ANOVA) with decreases evident in proximal, distal, and contralateral nonoperated nerve. Unexpectedly, transection was also found to cause a significant (P=0.001) 2-fold increase in DRG NGF-like activity between 6 and 24 hr postinjury in contralateral but not ipsilateral DRG. PEMF also reduced NGF-like activity in DRG, although this decrease did not reach statistical significance. Assessment of the same nerve and DRG samples using ELISA and NGF-specific antibodies confirmed an overall significant (P < 0.001) decrease in NGF levels in PEMF-treated nerve tissue, while no decrease was detected in DRG or in nerve samples harvested from PEMF-treated uninjured rats. These findings demonstrate that PEMF can affect growth factor activity and levels, and raise the possibility that PEMF might promote nerve regeneration by amplifying the early postinjury decline in NGF activity.

Animals↗
PubMed1999

Leukocyte common antigen-related tyrosine phosphatase receptor: increased expression and neuronal-type splicing in breast cancer cells and tissue.

The findings that protein tyrosine phosphatases (PTPs) regulate cell proliferation, response to growth factors, and cellular adhesion and the discovery that mutations in PTP genes are associated with breast cancer suggest that altered expression of PTPs contributes to the breast cancer cell phenotype. The leukocyte common antigen-related (LAR) PTP receptor is a prototype member of the class of PTP receptors containing cell adhesion domains. Full-length constitutively spliced LAR transcripts are expressed in breast and other tissues, whereas alternatively spliced isoforms are preferentially expressed in the nervous system. As a first step in evaluating the hypothesis that LAR-type PTPs influence breast cancer cell behavior, LAR expression and neuronal-type alternative splicing were examined in normal and breast cancer cell lines and tissues. Northern blot analysis demonstrated markedly increased LAR mRNA levels in breast cancer cell lines and tissues. Western blot analysis showed a greater than tenfold increase in LAR protein levels in breast cancer tissues. Reverse transcription-polymerase chain reaction was used to assess alternative splicing of extracellular and proximal membrane exons. Differential patterns of extracellular alternative splicing were found in normal versus carcinoma cell lines and tissues. Western blot analysis demonstrated increased levels of LAR protein isoforms encoded by alternatively spliced transcripts in breast cancer cell lines. This study is the first demonstration of increased LAR mRNA and LAR protein expression in breast cancer tissue and nontransformed cell lines and helps to elucidate the role of LAR in human breast cancer. The differential patterns of alternative splicing of LAR transcripts introduce LAR isoforms as candidate markers for future studies correlating differential gene expression and tumor behavior.

Alternative Splicing↗
PubMed1998

LAR tyrosine phosphatase receptor: proximal membrane alternative splicing is coordinated with regional expression and intraneuronal localization.

Examination of null-mutant Drosophila and Leukocyte Common Antigen-Related (LAR)-deficient transgenic mice has demonstrated that the LAR protein tyrosine phosphatase (PTP) receptor promotes neurite outgrowth. In the absence of known ligands, the mechanisms by which LAR-type PTP receptors are regulated are unknown. We hypothesized that an alternatively spliced eleven amino acid proximal membrane segment of LAR (LAR alternatively spliced element-a; LASE-a) contributes to regulation of LAR function. Human, rat and mouse LAR cDNA sequences demonstrated that the predicted eleven amino acid inserts in rat and mouse are identical and share nine of eleven residues with the human insert. LASE-a splicing led to the introduction of a Ser residue into LAR at a position analogous to Ser residues undergoing regulated phosphorylation in other PTPs. In-situ studies revealed increasingly region-specific expression of LASE-a containing LAR transcripts during postnatal development. RT-PCR analysis of cortical and hippocampal tissue confirmed that the proportion of LAR transcripts containing LASE-a decreases during development. Immunostaining of cultured PC12 cells, cerebellar granule neurons, dorsal root ganglia and sciatic nerve sections with antibody directed against the LASE-a insert demonstrated signal in cell bodies but little if any along neurites. In contrast, staining with antibody directed to a separate domain of LAR showed accumulation of LAR along neurites. The findings that LASE-a splicing is conserved across human, rat and mouse, that the LASE-a insert introduces a Ser at a site likely to be targeted for regulated phosphorylation and that developmentally regulated splicing is coordinated with specific regional and intraneuronal localization point to important novel potential mechanisms regulating LAR-type tyrosine phosphatase receptor function in the nervous system.

Alternative Splicing↗
PubMed1998

LAR tyrosine phosphatase receptor: a developmental isoform is present in neurites and growth cones and its expression is regional- and cell-specific.

Transgenic mice and Drosophila mutant studies demonstrate that the leukocyte common antigen-related (LAR) protein tyrosine phosphatase (PTPase) receptor is required for formation of neural networks. We assessed the hypothesis that alternative splicing of the LAR extracellular region contributes to this function by establishing temporospatial expression patterns of LAR isoforms containing an alternatively spliced extracellular nine amino acid segment (LAR alternatively spliced element-c; LASE-c). LASE-c was present in multiple alternatively spliced and truncated LAR transcripts. In contrast to LAR isoforms without LASE-c, levels of LAR transcripts and protein isoforms containing LASE-c were primarily present during development, suggesting a mechanism for developmental regulation of LAR function. In situ analysis demonstrated increasingly region- and cell-specific expression of LASE-c during maturation. Immunostaining revealed LASE-c-containing LAR protein along neurites and in growth cones. The discovery of highly regulated, temporospatial extracellular domain alternative splicing of LAR-type PTPase receptors points to a novel mechanism by which these receptors might influence network formation.

Animals↗
PubMed1997

Absence of p75NTR causes increased basal forebrain cholinergic neuron size, choline acetyltransferase activity, and target innervation.

Emerging evidence suggests that the p75 neurotrophin receptor (p75NTR) mediates cell death; however, it is not known whether p75NTR negatively regulates other neuronal phenotypes. We found that mice null for p75NTR displayed highly significant increases in the size of basal forebrain cholinergic neurons, including those that are TrkA-positive. Cholinergic hippocampal target innervation also was increased significantly. Activity of the cholinergic neurotransmitter synthetic enzyme choline acetyltransferase (ChAT) was increased in both the medial septum and hippocampus. Upregulation of these cholinergic features was not associated with increased basal forebrain or hippocampal target NGF levels. In contrast, striatal cholinergic neurons, which do not express p75NTR, showed no difference in neuronal number, size, or ChAT activity between wild-type and p75NTR null mutant mice. These findings indicate that p75NTR negatively regulates cholinergic neuronal phenotype of the basal forebrain cholinergic neurons, including cell size, target innervation, and neurotransmitter synthesis.

Animals↗
PubMed1997

Synthetic NGF peptide derivatives prevent neuronal death via a p75 receptor-dependent mechanism.

Cyclized peptides corresponding to beta-loop regions of NGF were purified by HPLC and assayed for neurotrophic activity using DRG neurons. Peptides with the highest activity corresponded to loop region 29-35, a domain likely to interact with the p75 receptor. Unexpectedly, activity was confined to late-eluting HPLC fractions containing peptide multimers and primarily promoted neuronal survival without neurite outgrowth. Directed synthesis of dimer and monomer cyclized peptides demonstrated that dimers acted as partial NGF agonists in that they had both survival-promoting and NGF-inhibiting activity while monomer and linear peptides were inactive. Dimer activity was not affected by the Trk inhibitor K252a but was blocked by p75 receptor antibody and absent using p75 null mutant neurons. These studies suggest that region 29-35 peptide derivatives inhibit neuronal death via a structure- and p75-dependent mechanism.

Animals↗
PubMed1997

Deficient LAR expression decreases basal forebrain cholinergic neuronal size and hippocampal cholinergic innervation.

A role in neural development for protein tyrosine phosphatase (PTPase) receptors has been suggested by the finding of aberrant neurite outgrowth in Drosophila mutants lacking functional leukocyte common antigen-related (LAR) PTPase receptors; however, PTPase functions in the mammalian nervous system remain to be established. In transgenic mice containing a gene trap in the LAR gene, only trace expression of full-length LAR transcripts was found. In these mice, the size of basal forebrain cholinergic neurons was significantly reduced and cholinergic innervation of the dentate gyrus was markedly decreased. These findings constitute the first demonstration of an aberrant neuronal phenotype in a mammalian PTPase mutant and support the hypothesis that LAR-type PTPase receptors function to establish and/or maintain neuronal networks.

Acetylcholine↗
PubMed1997

Aldose reductase inhibition increases CNTF-like bioactivity and protein in sciatic nerves from galactose-fed and normal rats.

The impact of exaggerated polyol pathway flux on ciliary neurotrophic factor (CNTF)-like bioactivity and expression of CNTF in rat sciatic nerve was examined after 2 months of galactose intoxication. Polyol content was elevated (P < 0.001) and motor nerve conduction velocity reduced (P < 0.05) in galactose-fed rats compared with control animals or control and galactose-fed rats treated with the aldose reductase inhibitor (ARI) Ponalrestat. CNTF-like bioactivity in the galactose-fed group was reduced to 30% of that assayed in the control group (P < 0.001). ARI treatment significantly increased CNTF-like bioactivity by 60% compared with the untreated galactose group (P < 0.05) but did not restore it to control levels. Unexpectedly, bioactivity in ARI-treated control animals was increased by nearly 250% compared with untreated controls (P < 0.005). In addition to the deficit in CNTF bioactivity in untreated galactose rats, the expression of protein, but not of mRNA, was reduced (P < 0.05). In ARI-treated control and galactose-fed rats, the expression of CNTF peptide was significantly enhanced above control levels (both P < 0.05). Concomitant with the reduction in CNTF levels, there was a shift in the axonal size-frequency distribution of myelinated fibers toward smaller axons in galactose-fed rats that was prevented by ARI treatment. Since galactose feeding has little impact on levels of CNTF mRNA, these observations suggest that deficits in CNTF-like bioactivity may result from a posttranscriptional modification of neurotrophic protein expression or turnover. Unlike other functional and structural disorders in galactose neuropathy, factors other than polyol accumulation may contribute to the deficit in CNTF-like bioactivity.

Aldehyde Reductase↗
PubMed1995

Cloning of rat grp75, an hsp70-family member, and its expression in normal and ischemic brain.

Following metabolic stress a variety of gene products are induced in cells in the brain, some of which may protect the tissue from subsequent stresses. The heat shock proteins (hsps), in particular hsp70, have been widely studied in this context, but evidence for the involvement of known hsps in protection of the CNS is inconclusive. We have therefore undertaken the search for other stress-induced proteins which may mitigate ischemic injury. Beginning with degenerate RT-PCR, we have isolated a rat-brain cDNA encoding a protein highly similar to human grp75, a mitochondrial member of the hsp70-family of stress proteins. It is also highly similar to two non-mitochondrial proteins; mortalin, a senescence-related gene product, and pbp74, a protein implicated in B-cell peptide processing. Sequence structure and phylogenetic analyses predict mitochondrial localization and induction by a calcium ionophore and glucose deprivation in PC12 cells support its identification as rat grp75. In situ analysis of normal brain reveals an unusual distribution, with very high expression in neurons of the basal forebrain, reticular and subthalamic nuclei, globus pallidus, amygdala and elsewhere. grp75-mRNA is upregulated following focal brain ischemia in a distinctive fashion. When the degree of injury is small, induction occurs in the area of injury, similar to the pattern observed for hsp70. However, when the injury is extensive, hsr is upregulated in neurons outside the ischemic area. The induction of grp75 may represent a sensitive marker of metabolically compromised tissue.

Amino Acid Sequence↗
PubMed1995

LAR tyrosine phosphatase receptor: alternative splicing is preferential to the nervous system, coordinated with cell growth and generates novel isoforms containing extensive CAG repeats.

Receptor-linked tyrosine phosphatases regulate cell growth by dephosphorylating proteins involved in tyrosine kinase signal transduction. The leukocyte common antigen-related (LAR) tyrosine phosphatase receptor has sequence similarity to the neural cell adhesion molecule N-CAM and is located in a chromosomal region (1p32-33) frequently altered in neuroectodermal tumors. To understand the function of receptor-linked tyrosine phosphatases in neural development, we sought to identify LAR isoforms preferentially expressed in the nervous system and cellular processes regulating LAR alternative splicing. We report here the isolation of a series of rat LAR cDNA clones arising from complex combinatorial alternative splicing, not previously demonstrated for the tyrosine phosphatase-receptor gene family in general. Isoforms included: (a) deletions of the fourth, sixth and seventh fibronectin type III-like domains; (b) an alternatively spliced novel cassette exon in the fifth fibronectin type III-like domain; (c) two alternatively spliced novel cassette exons in the juxtamembrane region; (d) a retained intron in the extracellular region with in-frame stop codons predicting a secreted LAR isoform; and (e) an LAR transcript including an alternative 3' untranslated region containing multiple stretches of tandem CAG repeats up to 21 repeats in length. This number of repeats was in the range found in normal alleles of genes in which expansions of repeats are associated with neurodegenerative disease and the genetic phenomenon of anticipation. RT-PCR and Northern analysis demonstrated that LAR alternative splicing occurred preferentially in neuromuscular tissue in vivo and in neurons compared to astrocytes in vitro and was developmentally regulated. Alternative splicing was also regulated in PC12 cells by NGF, in 3T3 fibroblasts by cell confluence and in sciatic nerve and muscle subsequent to nerve transection. Western blot analysis demonstrated that alternatively spliced cassette exons result in the presence of corresponding amino acid segments of LAR protein in vivo. These studies suggest specialized functions of LAR isoforms in the nervous system and support our hypothesis that LAR-like tyrosine phosphatase receptors play a role in neural development and regeneration.

3T3 Cells↗