Potential antiviral effects of pantethine against SARS-CoV-2.

SARS-CoV-2 interacts with cellular cholesterol during many stages of its replication cycle. Pantethine was reported to reduce total cholesterol levels and fatty acid synthesis and potentially alter different processes that might be involved in the SARS-CoV-2 replication cycle. Here, we explored the potential antiviral effects of pantethine in two in vitro experimental models of SARS-CoV-2 infection, in Vero E6 cells and in Calu-3a cells. Pantethine reduced the infection of cells by SARS-CoV-2 in both preinfection and postinfection treatment regimens. Accordingly, cellular expression of the viral spike and nucleocapsid proteins was substantially reduced, and we observed a significant reduction in viral copy numbers in the supernatant of cells treated with pantethine. In addition, pantethine inhibited the infection-induced increase in TMPRSS2 and HECT E3 ligase expression in infected cells as well as the increase in antiviral interferon-beta response and inflammatory gene expression in Calu-3a cells. Our results demonstrate that pantethine, which is well tolerated in humans, was very effective in controlling SARS-CoV-2 infection and might represent a new therapeutic drug that can be repurposed for the prevention or treatment of COVID-19 and long COVID syndrome.

A simple novel approach for detecting blood-brain barrier permeability using GPCR internalization.

AIMS: Impairment of blood-brain barrier (BBB) is involved in numerous neurological diseases from developmental to aging stages. Reliable imaging of increased BBB permeability is therefore crucial for basic research and preclinical studies. Today, the analysis of extravasation of exogenous dyes is the principal method to study BBB leakage. However, these procedures are challenging to apply in pups and embryos and may appear difficult to interpret. Here we introduce a novel approach based on agonist-induced internalization of a neuronal G protein-coupled receptor widely distributed in the mammalian brain, the somatostatin receptor type 2 (SST2). METHODS: The clinically approved SST2 agonist octreotide (1 kDa), when injected intraperitoneally does not cross an intact BBB. At sites of BBB permeability, however, OCT extravasates and induces SST2 internalization from the neuronal membrane into perinuclear compartments. This allows an unambiguous localization of increased BBB permeability by classical immunohistochemical procedures using specific antibodies against the receptor. RESULTS: We first validated our approach in sensory circumventricular organs which display permissive vascular permeability. Through SST2 internalization, we next monitored BBB opening induced by magnetic resonance imaging-guided focused ultrasound in murine cerebral cortex. Finally, we proved that after intraperitoneal agonist injection in pregnant mice, SST2 receptor internalization permits analysis of BBB integrity in embryos during brain development. CONCLUSIONS: This approach provides an alternative and simple manner to assess BBB dysfunction and development in different physiological and pathological conditions.

Sstr2A: a relevant target for the delivery of genes into human glioblastoma cells using fiber-modified adenoviral vectors.

Glioblastomas are the most aggressive of the brain tumors occurring in adults and children. Currently available chemotherapy prolongs the median survival time of patients by only 4 months. The low efficiency of current treatments is partly owing to the blood-brain barrier, which restricts the penetration of most drugs into the central nervous system. Locoregional treatment strategies thus become mandatory. In this context, viral tools are of great interest for the selective delivery of genes into tumoral cells. Gliomas express high levels of type 2 somatostatin receptors (sstr2A), pinpointing them as suitable targets for the improvement of transduction efficiency in these tumors. We designed a new adenoviral vector based on the introduction of the full-length somatostatin (SRIF (somatotropin release-inhibiting factor)) sequence into the HI loop of the HAdV fiber protein. We demonstrate that (i) HAdV-5-SRIF uptake into cells is mediated by sstr2A, (ii) our vector drives high levels of gene expression in cells expressing endogenous sstr2A, with up to 65-fold enhancement and (iii) low doses of HAdV-5-SRIF are sufficient to infect high-grade human primary glioblastoma cells. Adenoviral vectors targeting SRIF receptors might thus represent a promising therapeutic approach to brain tumors.

IL-9/IL-9 receptor signaling selectively protects cortical neurons against developmental apoptosis.

In mammals, programmed cell death (PCD) is a central event during brain development. Trophic factors have been shown to prevent PCD in postmitotic neurons. Similarly, cytokines have neurotrophic effects involving regulation of neuronal survival. Nevertheless, neuronal PCD is only partially understood and host determinants are incompletely defined. The present study provides evidence that the cytokine interleukin-9 (IL-9) and its receptor specifically control PCD of neurons in the murine newborn neocortex. IL-9 antiapoptotic action appeared to be time-restricted to early postnatal stages as both ligand and receptor transcripts were mostly expressed in neocortex between postnatal days 0 and 10. This period corresponds to the physiological peak of apoptosis for postmitotic neurons in mouse neocortex. In vivo studies showed that IL-9/IL-9 receptor pathway inhibits apoptosis in the newborn neocortex. Furthermore, in vitro studies demonstrated that IL-9 and its receptor are mainly expressed in neurons. IL-9 effects were mediated by the activation of the JAK/STAT (janus kinase/signal transducer and activator of transcription) pathway, whereas nuclear factor-kappaB (NF-kappaB) or Erk pathways were not involved in mediating IL-9-induced inhibition of cell death. Finally, IL-9 reduced the expression of the mitochondrial pro-apoptotic factor Bax whereas Bcl-2 level was not significantly affected. Together, these data suggest that IL-9/IL-9 receptor signaling pathway represents a novel endogenous antiapoptotic mechanism for cortical neurons by controlling JAK/STAT and Bax levels.

The proprotein convertase PC2 is involved in the maturation of prosomatostatin to somatostatin-14 but not in the somatostatin deficit in Alzheimer's disease.

A somatostatin deficit occurs in the cerebral cortex of Alzheimer's disease patients without a major loss in somatostatin-containing neurons. This deficit could be related to a reduction in the rate of proteolytic processing of peptide precursors. Since the two proprotein convertases (PC)1 and PC2 are responsible for the processing of neuropeptide precursors directed to the regulated secretory pathway, we examined whether they are involved first in the proteolytic processing of prosomatostatin in mouse and human brain and secondly in somatostatin defect associated with Alzheimer's disease. By size exclusion chromatography, the cleavage of prosomatostatin to somatostatin-14 is almost totally abolished in the cortex of PC2 null mice, while the proportions of prosomatostatin and somatostatin-28 are increased. By immunohistochemistry, PC1 and PC2 were localized in many neuronal elements in human frontal and temporal cortex. The convertases levels were quantified by Western blot, as well as the protein 7B2 which is required for the production of active PC2. No significant change in PC1 levels was observed in Alzheimer's disease. In contrast, a marked decrease in the ratio of the PC2 precursor to the total enzymatic pool was observed in the frontal cortex of Alzheimer patients. This decrease coincides with an increase in the binding protein 7B2. However, the content and enzymatic activity of the PC2 mature form were similar in Alzheimer patients and controls. Therefore, the cortical somatostatin defect is not due to convertase alteration occuring during Alzheimer's disease. Further studies will be needed to assess the mechanisms involved in somatostatin deficiency in Alzheimer's disease.

Somatostatin receptor subtypes 2 and 4 affect seizure susceptibility and hippocampal excitatory neurotransmission in mice.

We have investigated the role of somatostatin receptor subtypes sst2 and sst4 in limbic seizures and glutamate-mediated neurotransmission in mouse hippocampus. As compared to wild-type littermates, homozygous mice lacking sst2 receptors showed a 52% reduction in EEG ictal activity induced by intrahippocampal injection of 30 ng kainic acid (P < 0.05). The number of behavioural tonic-clonic seizures was reduced by 50% (P < 0.01) and the time to onset of seizures was doubled on average (P < 0.05). Seizure-associated neurodegeneration was found in the injected hippocampus (CA1, CA3 and hilar interneurons) and sporadically in the ipsilateral latero-dorsal thalamus. This occurred to a similar extent in wild-type and sst2 knock-out mice. Intrahippocampal injection of three selective sst2 receptor agonists in wild-type mice (Octreotide, BIM 23120 and L-779976, 1.5-6.0 nmol) did not affect kainate seizures while the same compounds significantly reduced seizures in rats. L-803087 (5 nmol), a selective sst4 receptor agonist, doubled seizure activity in wild-type mice on average. Interestingly, this effect was blocked by 3 nmol octreotide. It was determined, in both radioligand binding and cAMP accumulation, that octreotide had no direct agonist or antagonist action at mouse sst4 receptors expressed in CCl39 cells, up to micromolar concentrations. In hippocampal slices from wild-type mice, octreotide (2 micro m) did not modify AMPA-mediated synaptic responses while facilitation occurred with L-803087 (2 micro m). Similarly to what was observed in seizures, the effect of L-803087 was reduced by octreotide. In hippocampal slices from sst2 knock-out mice, both octreotide and L-803087 were ineffective on synaptic responses. Our findings show that, unlike in rats, sst2 receptors in mice do not mediate anticonvulsant effects. Moreover, stimulation of sst4 receptors in the hippocampus of wild-type mice induced excitatory effects which appeared to depend on the presence of sst2 subtypes, suggesting these receptors are functionally coupled.

Cellular biology of somatostatin receptors.

Somatostatin, and the recently discovered neuropeptide cortistatin, exert their physiological actions via a family of six G protein-coupled receptors (sst1, sst2A, sst2B, sst3, sst4, sst5). Following the cloning of somatostatin receptors significant advances have been made in our understanding of their molecular, pharmacological and signaling properties although much progress remains to be done to define their physiological role in vivo. In this review, the present knowledge regarding neuroanatomical localization, signal transduction pathways, desensitization and internalization properties of somatostatin receptors is summarized. Evidence that somatostatin receptors can form homo- and heterodimers and can physically interact with members of the SSTRIP/Shank/ProSAP1/CortBP1 family is also discussed.

In vivo internalization of the somatostatin sst2A receptor in rat brain: evidence for translocation of cell-surface receptors into the endosomal recycling pathway.

To determine whether cellular compartmentalization of somatostatin receptors can be regulated in vivo, we examined the immunocytochemical distribution of the sst2A receptor (sst2AR) after stereotaxical injections of somatostatin analogs into the rat parietal cortex. Whereas CH-275, a sst1R agonist, failed to induce changes in the diffuse sst2AR immunostaining pattern characteristic of control animals, somatodendritic profiles displaying intracytoplasmic immunoreactive granules became apparent short-term after injection of either somatostatin or the sst2R agonist octreotide. Confocal microscopy revealed that 90% of sst2AR-immunoreactive endosome-like organelles displayed transferrin receptor immunoreactivity. At the electron microscopic level, the percentage of sst2AR immunoparticles dramatically decreased at the plasmalemma of perikarya and dendrites after octreotide injection. Conversely, it significantly increased in endosomes-like organelles. These results demonstrate that sst2ARs undergo, in vivo, rapid and massive internalization into the endocytic recycling compartment in response to acute agonist stimulation and provide important clues toward elucidating somatostatin receptor signaling in the mammalian brain.

Regional and cellular localization of the neuroendocrine prohormone convertases PC1 and PC2 in the rat central nervous system.

PC1 and PC2 are two major enzymes involved in the processing of protein precursors directed to the regulated secretory pathway. Whereas transcripts encoding both enzymes are widely distributed in the central nervous system, information regarding the localization of proteins themselves is still lacking. In an attempt to gain insight into the neurobiologic roles of PC1 and PC2, both enzymes were immunolocalized in the rat brain by using C-terminally directed antibodies, which respectively recognize the 87-kDa PC1 and the 75 and 68-kDa PC2 forms. Adjacent sections immunoreacted with PC1 or PC2 antibodies exhibited selective patterns of immunostaining in regions well characterized with respect to their biosynthesis of multiple neuropeptides such as the cerebral cortex, hippocampus, and hypothalamus. PC1 signal intensity was generally weaker than that of PC2, although both enzymes displayed extensive overlapping patterns of expression. As assessed by double-labeling experiments at the cellular level, PC1 and PC2 immunoreactive signals were localized within the trans-Golgi network and nerve terminals, in keeping with the biosynthetic pathways of neuropeptides. Immunoreactive fibers were detected in many areas throughout the brain but were particularly densely distributed in the hypothalamus and the brainstem. Both enzymes were also localized within dendrites of numerous neurons, supporting the hypothesis that dendritic neuropeptide maturation and release may occur in a large number of brain regions. Taken together, our results provide new evidence that both convertases are efficiently targeted to the neuronal regulated secretory pathway and are well poised to process protein precursors in biologically active end-products within the mammalian brain.

Involvement of the Sst1 somatostatin receptor subtype in the intrahypothalamic neuronal network regulating growth hormone secretion: an in vitro and in vivo antisense study.

Five somatostatin (SRIH) receptors (sst1-5) have been cloned. Recent anatomical evidence suggests that sst1 and sst2 may be involved in the central regulation of GH secretion. Given the lack of specific receptor antagonists, we used selective antisense oligodeoxynucleotides (ODNs) to test the hypothesis that one or both of these subtypes are involved in the intrahypothalamic network regulating pulsatile GH secretion. In mouse neuronal hypothalamic cultures the proportion of GHRH neurons coexpressing sst1 or sst2 messenger RNAs (mRNAs) was identical. In contrast, sst1 mRNAs were more often present than sst2 in SRIH-expressing neurons. Firstly, sst1 antisense ODN in vitro treatment abolished sst1, but not sst2, receptor modulation of glutamate sensitivity and decreased sst1, but not sst2, mRNAs. The reverse was true after treatment with sst2 antisense. Sense ODNs did not alter the effects of SRIH agonists. In a second series of experiments, nonanaesthetized adult male rats were infused for 120 h intracerebroventricularly with ODNs. Only the sst1 antisense ODN diminished the amplitude of ultradian GH pulses without modifying their frequency. In parallel, sst1 antisense ODN strongly diminished sst1 immunoreactivity in the anterior periventricular nucleus and median eminence, as well as sstl periventricular nucleus mRNA levels. The effectiveness of the sst2 antisense ODN was attested by the inhibition of hypothalamic binding of [125I]Tyr0-D-Trp8-SRIH. Scrambled ODNs had no effect on GH secretion or on sst mRNAs or SRIH binding levels. These results favor a preferential involvement of sst1 receptors in the intrahypothalamic regulation of GH secretion by SRIH.

Cortistatin affects glutamate sensitivity in mouse hypothalamic neurons through activation of sst2 somatostatin receptor subtype.

Cortistatin is a 14-residue putative neuropeptide with strong structural similarity to somatostatin. Even if it shares several biological properties with somatostatin, the effects of cortistatin on cortical electrical activity and sleep are opposite to those elicited by somatostatin. We recently demonstrated that somatostatin could modulate glutamate sensitivity, either positively through activation of the sstl receptor subtype, or negatively through activation of the sst2 receptor subtype in hypothalamic neurons in culture which express almost exclusively these two sst subtypes. Thus, in the present study we compared the effects of cortistatin and somatostatin in hypothalamic neurons in culture, in order to define the former peptide activity on both subtypes. We first determined that the affinities of cortistatin and somatostatin were similar on cloned rat sstl and sst2 receptor subtypes in transfected cells and hypothalamic neurons membranes. We then found that cortistatin, like somatostatin, depresses the glutamate response but, unlike somatostatin, never potentiates glutamate sensitivity in hypothalamic neurons. The observed effect of cortistatin is strongly suggestive of an activation of the somatostatin sst2 receptor subtype in hypothalamic neurons in culture.

Loss of somatostatin-like immunoreactivity in the frontal cortex of Alzheimer patients carrying the apolipoprotein epsilon 4 allele.

We measured somatostatin-like immunoreactivity, using a radioimmunoassay which does not cross react with cortistatin-like immunoreactivity, in postmortem frontal cortex (Brodmann area 9) from 32 patients, of different apolipoprotein E genotypes, and presenting with different degrees of cognitive impairment. Eleven subjects and eight patients presented with no (controls) or limited memory impairments (Borderline), respectively. Six patients with clinical criteria for possible Alzheimer's disease also presented with clinical or brain imaging of cerebrovascular disease (mixed dementia) and seven patients were classified as Alzheimer's disease (AD). In the 6 months preceeding their deaths, all subjects had been evaluated by Folstein's Mini Mental State examination (MMS). Sixty nine percent of patients with MMS >20 did not carry the epsilon 4 allele while 66% of patients with MMS <10 did. Somatostatin concentrations (ng/mg wet weight) were significantly lower in the patients carrying the epsilon 4 allele (E2/3: 0.71 +/- 0.05, n = 19 vs. E4: 0.42 +/- 0.06, n = 13; mean +/- SEM, P < 0.001). These results, which are reminiscent of those obtained on cholinergic markers, suggest that apolipoprotein E4 is involved in the somatostatinergic dysfunction early after the onset in AD.

[Somatostatin: a ubiquitous peptide]. / La Somatostatine: un peptide ubiquitaire.

Somatostatin (SRIF) was discovered in 1973, in Roger Guillemin's laboratory as a Growth Hormone (GH) inhibiting neurohormone. It is widely distributed in mammals where it acts also as a peripheral hormone, an autocrine or paracrine factor and a neuropeptide. SRIF receptors are located on several human tumours and SRIF agonists are in clinical use to monitor GH secretion in acromegalic patients. This short review summarizes the properties of the central and peripheral somatostatinergic systems, the three peptides belonging to the somatostatin family: (SRIF14, SRIF28 and cortistatin), the pharmacology of the five SRIF receptor subtypes, some ontogenetic and phylogenetic aspects, as well as pathological findings.

Complementarity of radioautographic and immunohistochemical techniques for localizing neuroreceptors at the light and electron microscopy level.

To assess relationships between neuropeptide-binding sites and receptor proteins in rat brain, the distribution of radioautographically labeled somatostatin and neurotensin-binding sites was compared to that of immunolabeled sst2A and NTRH receptor subtypes, respectively. By light microscopy, immunoreactive sst2A receptors were either confined to neuronal perikarya and dendrites or diffusely distributed in tissue. By electron microscopy, areas expressing somatodendritic sst2A receptors displayed only low proportions of membrane-associated, as compared to intracellular, receptors. Conversely, regions displaying diffuse sst2A labeling exhibited higher proportions of membrane-associated than intracellular receptors. Furthermore, the former showed only low levels of radioautographically labeled somatostatin-binding sites whereas the latter contained high densities of somatostatin-binding suggesting that membrane-associated receptors are preferentially recognized by the radioligand. In the case of NTRH receptors, there was a close correspondence between the light microscopic distribution of NTRH immunoreactivity and that of labeled neurotensin-binding sites. Within the substantia nigra, the bulk of immuno- and autoradiographically labeled receptors were associated with the cell bodies and dendrites of presumptive DA neurons. By electron microscopy, both markers were detected inside as well as on the surface of labeled neurons. At the level of the plasma membrane, their distribution was highly correlated and characterized by a lack of enrichment at the level of synaptic junctions and by a homogeneous distribution along the remaining neuronal surface, in conformity with the hypothesis of an extra-synaptic action of this neuropeptide. Inside labeled dendrites, there was a proportionally higher content of immunoreactive than radiolabeled receptors. Some of the immunolabeled receptors not recognized by the radioligand were found in endosome-like organelles suggesting that, as in the case of sst2A receptors, they may have undergone endocytosis subsequent to binding to the endogenous peptide.

Interrelationships between somatostatin sst2A receptors and somatostatin-containing axons in rat brain: evidence for regulation of cell surface receptors by endogenous somatostatin.

Using an antipeptide antibody, we reported previously on the distribution of the somatostatin sst2A receptor subtype in rat brain. Depending on the region, immunolabeled receptors were either confined to neuronal perikarya and dendrites or distributed diffusely in tissue. To investigate the functional significance of these distribution patterns, we examined the regional and cellular relationships between somatostatin axons and sst2A receptors in the rat CNS, using double-labeling immunocytochemistry. Light and confocal microscopy revealed a significant correlation (p < 0.02) between the distribution of somatodendritic sst2A receptor immunoreactivity and that of somatostatin terminal fields, both quantitatively and qualitatively. Furthermore, in regions of somatodendritic labeling, a subpopulation of sst2A-immunoreactive cells was also immunopositive for somatostatin, suggesting that a subset of sst2A receptors consists of autoreceptors. By contrast, in regions displaying diffuse sst2A labeling only moderate to low densities of somatostatin terminals were observed, and no significant relationship was found between terminal density and receptor immunoreactivity. At the electron microscopic level, areas expressing somatodendritic sst2A labeling were found by immunogold cytochemistry to display low proportions of membrane-associated, as compared with intracellular, receptors. Conversely, in regions displaying diffuse sst2A receptor labeling, receptors were predominantly associated with neuronal plasma membranes, a finding consistent with the high density of sst2 binding sites previously visualized in these areas by autoradiography. Double-labeling studies demonstrated that in the former but not in the latter regions, sst2A-immunoreactive somata and dendrites were heavily contacted by somatostatin axon terminals. Taken together, these results suggest that the low incidence of membrane-associated receptors observed in regions of somatodendritic sst2A labeling may be caused by downregulation of cell surface receptors by endogenous somatostatin, possibly through ligand-induced receptor internalization.

Localization of the somatostatin receptor SST2A in rat brain using a specific anti-peptide antibody.

Biological actions of somatostatin are exerted via a family of receptors, for which five genes recently have been cloned. However, none of these receptor proteins has been visualized yet in the brain. In the present-study, the regional and cellular distribution of the somatostatin sst2A receptor was investigated via immunocytochemistry in the rat central nervous system by using an antibody generated against a unique sequence of the receptor protein. Specificity of the antiserum was demonstrated by immunoblot and immunocytochemistry on rat brain membranes and/or on cells transfected with cDNA encoding the different sst receptor subtypes. In rat brain sections, sst2A receptor immunoreactivity was concentrated either in perikarya and dendrites or in axon terminals distributed throughout the neuropil. Somatodendritic labeling was most prominent in the olfactory tubercle, layers II-III of the cerebral cortex, nucleus accumbens, pyramidal cells of CA1-CA2 subfields of the hippocampus, central and cortical amygdaloid nuclei, and locus coeruleus. Labeled terminals were detected mainly in the endopiriform nucleus, deep layers of the cortex, claustrum, substantia innominata, subiculum, basolateral amygdala, medial habenula, and periaqueductal gray. Electron microscopy confirmed the association of sst2A receptors with perikarya and dendrites in the former regions and with axon terminals in the latter. These results provide the first characterization of the cellular distribution of a somatostatin receptor in mammalian brain. The widespread distribution of the sst2A receptor in cerebral cortex and limbic structures suggests that it is involved in the transduction of both pre- and postsynaptic effects of somatostatin on cognition, learning, and memory.

Correlations between water maze performance and cortical somatostatin mRNA and high-affinity binding sites during ageing in rats.

Somatostatin levels and high-affinity (somatostatin-1) binding sites are decreased in post-mortem cortical samples of Alzheimer's disease patients but the relationships between such modifications and the cognitive deficits remain to be established. We investigated these relationships in the ageing rat. Three age groups (3-4, 14-15 and 26-27 months) were tested in a modified version of the Morris water maze. Somatostatin mRNA levels were quantified by in situ hybridization and somatostatin binding sites by radioautography using the selective agonist octreotide (SMS 201995) as a competing drug to evaluate high-affinity (somatostatin-1) and low-affinity (somatostatin-2) binding sites. The number of somatostatin mRNA-containing cells was not modified with age or memory performance in cortical, hippocampal and hypothalamic regions, but somatostatin mRNA densities were significantly decreased with age and with memory performance in the frontal and parietal cortex. In the frontal cortex somatostatin mRNA densities were already decreased in 14- to 15-month-old rats, whereas the decrease was observed only in 26- to 27-month-old rats in the parietal cortex. A decrease in somatostatin-1 binding was observed with memory performance, independently of age, in the basolateral amygdala only, while somatostatin-2 binding sites were not affected. In the frontal and parietal cortex, a significant correlation occurred between the latency to find the invisible platform in the water maze and somatostatin mRNA (r = -0.54 and 0.59 respectively, P < 0.02). These results indicate that ageing rats with memory impairments display some of the features of the somatostatinergic deficits observed in Alzheimer's disease.

Differential correlation between neurochemical deficits, neuropathology, and cognitive status in Alzheimer's disease.

The relationships between neurofibrillary tangles (NFT), senile plaques (SP), and the deficits in somatostatin (SRIH) and choline acetyltransferase (ChAT) levels were determined in Brodmann area 9, 40, 22, and 17/18 in 12 women whose Blessed test score (BTS) ranged from 27 to 1. NFT density correlated with the cognitive decline in areas 9, 40, and 22 and with SP number in area 22 and 17/18. ChAT levels were linked to the BTS in area 9, 40, and 22 and SRIH levels in area 9 only. ChAT, but not SRIH, did correlate with SP (area 22) and NFT (area 40 and 22). Decreases in ChAT and SRIH were correlated in areas 9 and 22. These results indicate that the somatostatinergic deficit in Alzheimer's disease is more regionally restricted than the cholinergic one. The correlation between SRIH and ChAT as observed in area 9 and 22 may indicate that somatostatin- and acetylcholine-containing elements in the frontal and temporal lobes are particularly relevant to the cognitive decline as observed in Alzheimer's disease.

Choline acetyltransferase and somatostatin levels in aged Microcebus murinus brain.

beta-Amyloid protein (beta-AP) deposits, analoguous to those found in Alzheimer's disease (AD) are observed in the brain of aging Microcebus murinus. Because choline acetyltransferase (ChAT) activity and somatostatin (SRIH) content are consistently decreased in AD, we tested whether such changes could be observed in middle aged to aged Microcebus cerebral cortex and whether they were accompanied by beta-AP deposits. A positive correlation was observed between age and ChAT activity. By HPLC, SRIH immunoreactivity eluted as four peaks, two of which being identical with SRIH-28 and SRIH-14 while the other two likely represented precursor forms. Cortical SRIH content was not significantly affected by age. ChAT activity and SRIH content were not significantly correlated. Amyloid angiopathy was observed in every brain examined and the presence of cortical lesions analoguous to senile plaques observed in the oldest case only which did not demonstrate important alterations in ChAT and somatostatin levels.

Somatostatin messenger RNA-containing neurons in Alzheimer's disease: an in situ hybridization study in hippocampus, parahippocampal cortex and frontal cortex.

The level of expression of somatostatin messenger RNA-containing neurons in human brain was visualized and quantified by in situ hybridization with a 35S-labelled oligonucleotide complementary to amino acids 96-111 of the preprosomatostatin complementary DNA sequence. The analysis was carried out in the frontal and parahippocampal cortices and hippocampus of six age- and post mortem delay-matched Alzheimer's disease and control brains. By northern blot analysis, in frontal cortex samples, 18S rRNA degradation was identical in control and Alzheimer brains and somatostatin messenger RNAs migrated as a single band of 1 kb. By in situ hybridization, specificity was demonstrated by abolition of the signal using either an excess of unlabelled antisense probe or using a labelled sense probe. Somatostatin messenger RNA-containing neurons displayed a similar regional and subregional distribution in control subjects and patients with Alzheimer's disease, being more abundant in the frontal cortex, followed by the hippocampus and the parahippocampal cortex. An overall reduction of labelled cell density was observed in patients with Alzheimer's disease (frontal cortex gray matter:--41%; white matter:--66%; hippocampus:--44%; parahippocampal cortex white matter:--40%). Due to a great variation between brains, this decrease only reached significance in the parahippocampal cortex (-59%, P < 0.05). A significantly lower level of expression of somatostatin messenger RNA per somatostatinergic cell was observed in the hippocampus of Alzheimer's disease patients (-47%, P < 0.05), but not in frontal cortex gray (-17%) and white (-36%) matter and parahippocampal cortex gray (-42%) and white (-29%) matter. These data are in accordance with the distribution of somatostatin cells as visualized by immunohistochemistry in human brain. They indicate that the ability of cortical cells to express somatostatin messenger RNA is partially preserved in Alzheimer disease brains and that the decrease in the amount of somatostatin messenger RNA per cell is restricted to the hippocampal formation.