Shiv S. Prasad

Role of NMDA receptor–dependent activation of SREBP1 in excitotoxic and ischemic neuronal injuries

Excitotoxic neuronal damage caused by overactivation of N-methyl-D-aspartate glutamate receptors (NMDARs) is thought to be a principal cause of neuronal loss after stroke and brain trauma. Here we report that activation of sterol regulatory element binding protein-1 (SREBP-1) transcription factor in affected neurons is an essential step in NMDAR-mediated excitotoxic neuronal death in both in vitro and in vivo models of stroke. The NMDAR-mediated activation of SREBP-1 is a result of increased insulin-induced gene-1 (Insig-1) degradation, which can be inhibited with an Insig-1–derived interference peptide (Indip) that we have developed. Using a focal ischemia model of stroke, we show that systemic administration of Indip not only prevents SREBP-1 activation but also substantially reduces neuronal damage and improves behavioral outcome. Our study suggests that agents that reduce SREBP-1 activation such as Indip may represent a new class of neuroprotective therapeutics against stroke.

Localization of M3 Muscarinic Receptor Subtype and mRNA in the Human Eye

Cholinergic agents used to lower intraocular pressure in the medical management of glaucoma act on muscarinic receptors in the eye. Recently, subtypes of muscarinic receptors have been recognized in many tissues including the eye. To localize the M3 muscarinic receptor subtype and m3 messenger RNA (mRNA) in the human eye, we used in vitro ligand binding and in situ hybridization techniques on post-mortem sections. We used an M3 antagonist, [3H]-4-DAMP ([3H]-4-diphenylacetoxy-N-methylpiperidine methiodide) to identify M3-binding sites and a [35S]-labelled oligonucleotide probe to detect and localize m3 mRNA. M3 and m3 mRNA were both localized in the ciliary muscle, ciliary epithelium, iris, corneal epithelium and anterior lens epithelium. The m3 transcript was also detected in the trabecular meshwork and corneal endothelium.

Anisomycin activates p38 MAP kinase to induce LTD in mouse primary visual cortex

Anisomycin is both a well-established protein synthesis inhibitor and a potent activator of the p38/JNK MAPK pathway. It has been used to block the late phase of long-term potentiation (LTP) and long-term depression (LTD) in hippocampus. In this study, we have found that anisomycin produces a time-dependent decline in the magnitude of the field EPSP (fEPSP) in acute brain slices of mouse primary visual cortex. This anisomycin-mediated fEPSP depression occludes NMDA receptor-dependent LTD induced by low-frequency stimulation (LFS). In contrast, two other protein synthesis inhibitors, emetine and cycloheximide, have no effect either on baseline synaptic transmission or on LTD. Moreover, the decline of the fEPSP caused by anisomycin can be rescued by the application of the p38 inhibitor SB203580 but not by the JNK inhibitor SP600125. These results indicate that activation of p38 MAPK by anisomycin induces LTD and subsequently occludes electrically induced LTD. Also, the occlusion of LFS-LTD by anisomycin suggests that common mechanisms may be shared between the two forms of synaptic depression. Consistent with this view, bath application of a membrane permeant peptide derived from the carboxyl tail of GluR2 subunit of AMPA receptor, which specifically blocks regulated AMPA receptor endocytosis, thereby preventing the expression of LFS-induced LTD, significantly reduced the anisomycin-induced decline of the fEPSP. In conclusion, our results indicate that anisomycin produces long-lasting depression of AMPA receptor-mediated synaptic transmission by activating p38 MAPK-mediated endocytosis of APMA receptors in mouse primary visual cortex.

Identification of cDNA clones expressed selectively during the critical period for visual cortex development by subtractive hybridization.

We have used the method of subtractive hybridization to isolate cDNA clones of mRNAs expressed in abundance in the visual cortex of 30-day-old kittens but absent or in lower abundance in the adult cat visual cortex. Of 12,000 colonies screened, 200 clones which hybridized to the subtracted probe were isolated and characterized. Northern blots confirmed the specificity of the vast majority of the isolated clones. 120 of the 200 clones were sequenced and the EMBL and GenBank (release 76) database were searched for known identities using FASTA and BLAST programs. Twenty-seven of these sequenced clones were identifiable. The identities showed that these sequences code for proteins involved in a variety of cellular processes. These include cell-cell interaction (TAPA-1, contactin, tachykinin receptor, phospholipase A2), cellular remodeling (C1q beta isoform, heat shock protein), neurofilament assembly (alpha tubulin and alpha internexin), neurotransmitter release (VAMP-2, amphiphysin, carboxypeptidase E, scg 10 and proton channel), energy metabolism (mitochondrial hinge protein, ADP/ATP transporter, cytochrome oxidase subunits), RNA processing (helix destabilizing protein, ribonucleoprotein) and protein synthesis (eIF-4A initiation factor, ribosomal protein S27). The results show that gene expression in the kitten visual cortex differs rather little from that of the adult visual cortex since over 98% of the sequences appear common. The relatively rare kitten-specific sequences are likely to form the basis for the critical period plasticity in this system.

Identification of differentially expressed genes in the visual structures of brain using high-density cDNA grids.

The hybridization patterns of 18,371 high-density-grid-arrayed non-redundant complementary DNA (cDNA) clones were examined using three different sources of cDNA probes. The first set of probes was synthesized from mRNA isolated from visual brain areas MT and V4 of Vervet monkey. The second set of probes was derived from cDNA libraries constructed from two micro dissected sets of layers of the monkey Lateral Geniculate Nucleus layers within the visual pathway, namely the magnocellular and parvocellular layers. The third set of cDNA probes was synthesized from the subtracted fractions of the cDNAs enriched for either the magnocellular or the parvocellular layers of the Lateral Geniculate Nucleus. Software, linked directly to the Genbank database, was developed to aid in the rapid identification of both expressed and differentially expressed genes. Our results indicate that both the cDNA probes synthesized from mRNA and cDNA libraries can identify similar fractions of expressed genes. However, the subtracted cDNA probes improve the efficiency of detection for those genes that are expressed at much lower abundance. Analyses of these results for the differential expression patterns of these genes were validated by semi-quantitative PCR on the DNA derived from the whole tissue cDNA libraries. A list of some known genes that are statistically differentially expressed within the magnocellular layers of the LGN and area MT in the primate visual areas is derived.

Identification of a Novel Truncated Isoform of trkB in the Kitten Primary Visual Cortex

Neurotrophins have been shown to play important roles in development and plasticity of the visual cortex (VC). Since signal transduction of neurotrophins is mediated through neurotrophin receptors, we attempted to analyze neurotrophin receptors in the VC. In this study, we isolated cDNAs encoding the intracellular regions of truncated isoforms of the trkB receptor from 30-d-old kitten primary VC. Two distinct truncated isoforms of trkB were isolated and characterized by sequence analyses. One of the isoforms corresponds to the previously described truncated trkB in several mammalian species. The second isoform represents a novel truncated trkB variant form in the kitten VC. Sequence analysis revealed that this contains a sequence that has not yet been reported in any species. This novel isoform, designated trkB.T4, results from alternative splicing 189-bp (63 amino acids) downstream from the splice site giving rise to the first known truncated isoforms of trkB. In the context of recent hypotheses regarding the function of truncated trkB receptors, sequence analysis indicates that trkB.T4 may bear putative signaling/internalization sequences.

Immunohistochemical investigations of neurofilament M′ and αβ-crystallin in the magnocellular layers of the primate lateral geniculate nucleus

Abstract The magnocellular and parvocellular pathways are two major processing streams in the primate visual system. Using high-density grid arrayed cDNA clones to hybridize to cDNA probes from cortical regions of each pathway, a list of candidate differentially expressed genes was produced [Mol. Brain Res. 82 (2000) 11–24]. Magnocellular pathway candidates include neurofilament M′ and αβ-crystallin. Using antibodies generated against these proteins, immunohistochemical analysis revealed preferential staining of the magnocellular layers in the primate lateral geniculate nucleus, providing verification of two candidate magnocellular-enriched genes.

Molecular analysis of trkC in the cat visual cortex

AbstracttrkC belongs to the trk family of neurotrophin receptors. Several isoforms of trkC have been cloned to date; a full-length catalytic form containing a tyrosine kinase (TK) domain, three full-length isoforms with amino-acid insertions (14, 25, and 39 amino acids) in the TK domain, and five noncatalytic truncated forms that completely lack the TK domain. These isoforms have been studied in several mammalian species, including the pig, rat, mouse, monkey, and human.In this article we report the cloning and sequencing of five trkC isoforms isolated from 30-d postnatal cat visual cortex. The first isoform corresponded to the previously reported full-length trkC transcript containing the 14 amino-acid insert. To search for the presence of other inserts, reverse transcription polymerase chain reaction (RT-PCR) was performed on 30-d postnatal cat visual cortex mRNA using primers that flank the insertion site in the TK domain. Both the isoform containing the 14 amino-acid insert and the isoform lacking any insertion were present in abundant amounts, whereas the other two insert containing isoforms (TK25 and TK39) were much less abundant. The fifth isoform discovered corresponds to the previously reported truncated transcript. Overall, there is a high degree of identity (89–98%) and homology (97–99%) between the cat trkC nucleotide and amino-acid sequences among all mammals. The extracellular juxtamembrane domain was found to be highly divergent among all mammals that have been studied to date. This divergent region also included a proline deletion in the cat trkC sequence. This is the first report of the cloning, sequencing, and RT-PCR analysis of trkC in cat visual cortex, a system extensively studied using anatomical and physiological approaches.