Experimentally induced lysosomal dysfunction disrupts processing of hypothalamic releasing factors.

Previous studies have shown that experimentally induced lysosomal dysfunction elicits various features of aging in the cortical telencephalon. The present study used cultured slices to test if: (1) it causes similar changes in the hypothalamus, and/or (2) modifies the processing of two releasing factors important to aging. A 2-day exposure to N-CBZ-L-phenylalanyl-L-alanine-diazomethylketone (ZPAD), a selective inhibitor of cathepsins B and L, triggered a pronounced increase in the numbers of lysosomes in the ventromedial and dorsomedial nuclei, and in lateral hypothalamus. Continued incubation with the inhibitor for 3-12 days resulted in the spread of endosomes-lysosomes into dendrites and, in the lateral hypothalamus, the formation of massive, lysosome-filled expansions of neuronal processes (meganeurites). These effects did not occur in the arcuate nucleus, making it the first region so far examined in which lysosomal proliferation is not initiated by hydrolase inhibitors. Despite this, a dense plexus of axons and terminals in the median eminence was partially depleted of growth hormone releasing hormone (GHRH) within 48 hours after addition of ZPAD. Moreover, the inhibitor caused axonal GHRH to become collected into large puncta, an effect highly suggestive of a partial failure in axonal transport. GHRH mRNA levels were not greatly affected by 6 days of ZPAD exposure, indicating that reduced expression did not play a major role in the peptide changes seen at 48 hours. Similar but less pronounced immunocytochemical changes were recorded for the somatostatin system in the arcuate and periventricular nucleus. It is concluded that lysosome dysfunction: (1) has different consequences for the arcuate nucleus than other brain regions, and (2) disrupts transport of hypothalamic releasing factors. The potential significance of the results to endocrine senescence is discussed.

Integrins regulate neuronal neurotrophin gene expression through effects on voltage-sensitive calcium channels.

Integrin adhesion receptors regulate gene expression during growth and differentiation in various cell types. Recent work, implicating integrins in functional synaptic plasticity, suggest they may have similar activities in adult brain. The present study tested if integrins binding the arginine-glycine-aspartate (RGD) matrix sequence regulate neurotrophin and neurotrophin receptor gene expression in cultured hippocampal slices. The soluble RGD-containing peptide glycine-arginine-glycine-aspartate-serine-proline (GRGDSP) increased neurotrophin mRNA levels in transcript- and subfield-specific fashions. Integrin ligand effects were greatest for brain-derived neurotrophic factor transcripts I and II and barely detectable for transcript III. In accordance with increased nerve growth factor mRNA levels, GRGDSP increased c-fos expression as well. In contrast, growth-associated protein-43, amyloid precursor protein and fibroblast growth factor-1 mRNAs were not elevated. Ligand effects on brain-derived neurotrophic factor transcript II and c-fos mRNA did not depend on the integrity of the actin cytoskeleton, neuronal activity, or various signaling pathways but were blocked by L-type voltage-sensitive calcium-channel blockers. These results indicate that in mature hippocampal neurons integrin engagement regulates expression of a subset of growth-related genes at least in part through effects on calcium influx. Accordingly, these synaptic adhesion receptors may play the same role in maintaining an adult, differentiated state in brain as they do in other tissues and changes in integrin activation and/or engagement may contribute to dynamic changes in neurotrophin expression and to neuronal calcium signaling.

Localization and seizure-regulation of integrin beta 1 mRNA in adult rat brain.

Recent findings indicate that RGD-binding integrin receptors play a critical role in the maintenance of long-term potentiation but the identity and location of the integrin proteins involved are not known. The integrin beta1 is of particular interest in regard to synaptic plasticity because it is a component of many of the RGD-binding integrins and beta1-immunoreactivity has been localized within synaptic density fractions. The present study used in situ hybridization to evaluate the distribution of beta1 mRNA in adult rat brain and to determine if expression is altered by seizures. In untreated rats, beta1 mRNA is present at high levels in the ventricular epithelium and discrete neuronal groups including the magnocellular hypothalamic and efferent cranial nerve nuclei and the cerebellar Purkinje cells. Hybridization was less dense in the substantia nigra and hippocampal stratum pyramidale and low but present throughout the gray matter. Limbic seizures increased beta1 cRNA labeling of both neurons (e.g., hippocampal stratum pyramidale) and astroglial cells from 8 h through 48 h after seizure onset. These results indicate that in adult rat brain, beta1 mRNA is expressed by both neurons and glia; neuronal expression is highest in hypothalamic and peripherally projecting neurons capable of substantial morphological plasticity. Seizure effects demonstrate that beta1 is positively regulated by activity, and suggest that activity-dependent expression may play a role in synaptic plasticity in the adult brain.

Seizure induced synthesis of fibronectin is rapid and age dependent: implications for long-term potentiation and sprouting.

Extracellular matrix proteins are induced by activity in adult brain but the time course of these responses, and hence the possibility of their involvement in use-dependent synaptic plasticity, is not known. To evaluate this issue, the influence of seizures on fibronectin expression was evaluated in the adult and developing hippocampus. In adult rats, kainic acid-induced seizures increased fibronectin mRNA and immunoreactivity (ir) by about 1 h after the first behavioral seizure. In situ hybridization analysis indicated that fibronectin mRNA was increased in broadly distributed glial cells as well as within discrete neuronal populations that normally express this transcript. Western blots demonstrated that increased fibronectin-ir was evident in both soluble and non-soluble fractions at the same time point. Immunocytochemical colocalization confirmed that fibronectin-ir was indeed elevated in broadly distributed glial fibrillary acidic protein-ir astroglia. Seizures had no detectable effect on fibronectin-ir in the hippocampus of nine day old rats. Together with previous results, the above findings suggest that intense physiological activity triggers a 'matrix response' (i.e., release proteases, activate integrins, secrete matrix proteins) that is sufficiently rapid to participate in the consolidation of long-term potentiation (LTP). The absence of such reactions in the immature hippocampus is in accord with the hypothesis that matrix proteins generated by mature astroglia impose temporal and spatial limitations on axonal remodeling.

Integrin subunit gene expression is regionally differentiated in adult brain.

Integrins are a diverse family of heterodimeric (alphabeta) adhesion receptors recently shown to be concentrated within synapses and involved in the consolidation of long-term potentiation. Whether neuronal types or anatomical systems in the adult rat brain are coded by integrin type was studied in the present experiments by mapping the relative densities of mRNAs for nine alpha and four beta subunits. Expression patterns were markedly different and in some regions complementary. General results and areas of notable labeling were as follows: alpha1-limited neuronal expression, neocortical layer V, hippocampal CA3; alpha3 and alpha5-diffuse neuronal and glial labeling, Purkinje cells, hippocampal stratum pyramidale, locus coeruleus (alpha3); alpha4- discrete limbic regions, olfactory cortical layer II, hippocampal CA2; alpha6-most prominently neuronal, neocortical subplate, endopiriform, subiculum; alpha7-discrete, all neocortical layers, hippocampal granule cells and CA3, cerebellar granule and Purkinje cells, all efferent cranial nerve nuclei; alpha8-discrete neuronal, deep cortex, hippocampal CA1, basolateral amygdala, striatum; alphaV-all cortical layers, striatum, Purkinje cells; beta4-dentate gyrus granule cells; beta5-broadly distributed, neocortex, medial amygdala, cerebellar granule and Purkinje cells, efferent cranial nerve nuclei; alpha2, beta2, and beta3-mRNAs not detected. These results establish that brain subfields express different balances of integrin subunits and thus different integrin receptors. Such variations will determine which matrix proteins are recognized by neurons and the types of intraneuronal signaling generated by matrix binding. They also could generate important differences in synaptic plasticity across brain systems.

Internal initiation of translation of five dendritically localized neuronal mRNAs.

In neurons, translation of dendritically localized mRNAs is thought to play a role in affecting synaptic efficacy. Inasmuch as components of the translation machinery may be limiting in dendrites, we investigated the mechanisms by which translation of five dendritically localized mRNAs is initiated. The 5' leader sequences of mRNAs encoding the activity-regulated cytoskeletal protein, the alpha subunit of calcium-calmodulin-dependent kinase II, dendrin, the microtubule-associated protein 2, and neurogranin (RC3) were evaluated for their ability to affect translation in the 5' untranslated region of a monocistronic reporter mRNA. In both neural and nonneural cell lines, the activity-regulated cytoskeletal protein, microtubule-associated protein 2, and alpha-CaM Kinase II leader sequences enhanced translation, whereas the dendrin and RC3 5' untranslated regions slightly inhibited translation as compared with controls. When cap-dependent translation of these constructs was suppressed by overexpression of a protein that binds the cap-binding protein eIF4E, it was revealed that translation of these mRNAs had both cap-dependent and cap-independent components. The cap-independent component was further analyzed by inserting the 5' leader sequences into the intercistronic region of dicistronic mRNAs. All five leader sequences mediated internal initiation via internal ribosome entry sites (IRESes). The RC3 IRES was most active and was further characterized after transfection in primary neurons. Although translation mediated by this IRES occurred throughout the cell, it was relatively more efficient in dendrites. These data suggest that IRESes may increase translation efficiency at postsynaptic sites after synaptic activation.