Josef Marksteiner

High Prevalence of Pathogenic Mutations in Patients with Early-Onset Dementia Detected by Sequence Analyses of Four Different Genes

Clinical differential diagnosis of early-onset dementia (EOD) includes familial Alzheimer disease (FAD) and hereditary prion disease. In both disease entities, postmortem brain histopathological examination is essential for unambiguous diagnosis. Mutations in the genes encoding the presenilins (PS1 and PS2) and amyloid precursor protein (APP) are associated with FAD, whereas mutations in the prion protein (PrP) gene are associated with prion disease. To investigate the proportion of EOD attributable to known genes, we prospectively (i.e., antemortem) screened these four genes for mutations by sequencing genomic PCR products from patients with EOD before age 60 years. Family history for dementia was positive (PFH) in 16 patients, negative (NFH) in 17 patients, and unknown (UFH) in 3 patients. In 12 patients, we found five novel mutations (in PS1, F105L; in PS2, T122P and M239I; and in PrP, Q160X and T188K) and five previously reported mutations (in APP, in three patients who were most likely unrelated, V717I; in PS1, A79V and M139V; and in PrP, P102L and T183A) that are all considered to be disease causing. Of these 12 patients, 9 had PFH. This indicates a detection rate of 56% (9/16) in patients with PFH. We found two mutations (APP V717I) in two of the three UFH patients, and only one mutation (PrP T188K) in 1 of the 17 patients with NFH. We conclude that because of the lack of specific antemortem diagnostic markers for FAD and hereditary prion disease, all four genes should be included in a molecular diagnostic program in patients with EOD who had PFH.

Neuropeptide Y biosynthesis is markedly induced in mossy fibers during temporal lobe epilepsy of the rat

Neuropeptide Y (NPY) immunoreactivity and gene expression was investigated in the hippocampus after kainic acid-induced seizures and pentylenetetrazol kindling in the rat. Pronounced increases of NPY immunoreactivity were found in the terminal field of mossy fibers in both animal models. In kainic acid-treated rats the peptide progressively accumulated in the hilus and the stratum lucidum of CA3, 5-60 days after injection of the toxin and, at the later intervals, extended to the supragranular molecular layer of the dentate gyrus indicating sprouting of these neurons. Unilateral injection of colchicine into the hilus abolished NPY staining of the mossy fibers. Using in situ hybridization, in both animal models markedly enhanced expression of prepro-NPY mRNA was observed in the granular layer, containing the perikarya of the mossy fibers. It is suggested that sustained expression of the neuromodulatory neuropeptide NPY, in addition to the observed plastic changes, may contribute to altered excitability of hippocampal mossy fibers in epilepsy. Neither somatostatin immunoreactivity nor gene expression were enhanced in granule cells/mossy fibers.

Distribution of secretoneurin, a peptide derived from secretogranin II, in rat brain: An immunocytochemical and radioimmunological study

The distribution of secretoneurin, a peptide derived from its precursor secretogranin II by proteolytic processing, was studied in the central nervous system of the rat by immunocytochemistry and radioimmunoassay and compared to the distribution of secretogranin II messenger RNA by using in situ hybridization. With a specific antiserum a distinct staining of fibers and to a lesser extent also of perikarya was observed throughout the central nervous system. A high density of immunoreactive fibers and terminals was found in several brain areas, i.e. the lateral septum, the medial parts of the amygdala, some medial thalamic nuclei, the hypothalamus, habenula, nucleus interpeduncularis, locus coeruleus, nucleus tractus solitarii, the substantiae gelatinosae of the caudal trigeminal nucleus and of the spinal cord. The quantitative distribution as measured by a radioimmunoassay agreed well with the varying densities of immunoreactivity found by immunocytochemistry. The highest concentrations of this peptide were present in the hypothalamus, in particular, in the median eminence and are comparable to those of the most highly concentrated neuropeptides. The distribution of immunopositive perikarya corresponded well with that of secretogranin II messenger RNA obtained by in situ hybridization. The pattern of secretoneurin expression in rat brain was widespread and unique, partially overlapping with established chemical transmitters and neuropeptides. The functional significance of this new brain peptide remains to be established.

Concomitant increase of somatostatin, neuropeptide y and glutamate decar☐ylase in the frontal cortex of rats with decreased seizure threshold

The neuropeptides somatostatin and neuropeptide Y and the activity of glutamate decarboxylase were determined in the frontal cortex of rats subjected to experimental epilepsy. Two different animal models, (1) rats kindled for 4 weeks by daily injection of pentylenetetrazole, and (2) rats which had undergone strong limbic seizures induced by kainic acid, were used. Decreased seizure threshold, as shown by injection of a subconvulsive dose of pentylenetetrazole, was observed 10 days after the last kindling session and 1 month after injection of kainic acid, respectively. Significantly increased levels of somatostatin (by 60%), neuropeptide Y (135%) and increased activity of glutamate decarboxylase (22%) were found in the frontal cortex of rats previously treated with kainic acid. Separation of somatostatin-like immunoreactivity by size exclusion high-performance liquid chromatography showed a marked increase of immunoreactivity in fractions containing the somatostatin precursor (by 200%) and less prominently of somatostatin-14 and somatostatin-28 (by 60 and 80%, respectively). Michaelis-Menten kinetics of glutamate decarboxylase revealed an increased maximal velocity (Vmax) in the frontal cortex of kainic acid-treated rats, but no change in the Km value was found. Similar results were also obtained in pentylenetetrazole-kindled rats. Injection of cysteamine (100 mg/kg, i.p.) resulting in a 30% decrease of cortical somatostatin in kainic acid-pretreated rats markedly suppressed seizures induced by an otherwise subconvulsive dose of pentylenetetrazole.(ABSTRACT TRUNCATED AT 250 WORDS)

ENHANCED RATE OF EXPRESSION AND BIOSYNTHESIS OF NEUROPEPTIDE Y AFTER KAINIC ACID-INDUCED SEIZURES

Abstract: Recent studies have shown marked increases in brain content of neuropeptide Y (NPY) after seizures induced by intraperitoneal injection of kainic acid and after pentylenetetrazole kindling in the rat. We have now investigated possible changes in the rate of biosynthesis of NPY after kainic acid treatment, by using pulse‐labeling of the peptide and by determining prepro‐NPY mRNA concentrations. For pulse labeling experiments, [3H]tyrosine was injected into the frontal cortex, and the incorporation of the amino acid into NPY was determined after purifying the peptide by gel filtration chromatography, antibody affinity chromatography, and reversed‐phase HPLC. At 2 and 30 days after kainic acid treatment, the rate of tyrosine incorporation was enhanced by ∼380% in the cortex. In addition, concentrations of prepro‐NPY mRNA were determined in four different brain areas by hybridization of Northern blots with a complementary 32P‐labeled RNA probe 2, 10, 30, and 60 days after kainic acid treatment. Marked increases were observed in the frontal cortex (by up to 350% of controls), in the dorsal hippocampus (by 750%), and in the amygdala/pyriform cortex (by 280%) at all intervals investigated. In the striatum only a small, transient increase was observed. The data demonstrate increased expression of prepro‐NPY mRNA and an enhanced rate of in vivo synthesis of NPY as a result of seizures induced by the neurotoxin kainic acid.

Distribution of mRNAs for Chromogranins A and B and Secretogranin II in Rat Brain

The mRNA distribution of chromogranins A and B and secretogranin II was determined in rat brain. In Northern blots the oligonucleotide probes used hybridized with single mRNA species of the expected sizes. With tissue hybridization the mRNA signals for these three proteins were found throughout the brain. However, each of the three messages had a distinct distribution, which was exemplified by the fact that in the various regions either all three proteins, a combination of two or only one of them were apparently synthesized. Significant levels of all three mRNAs were found in several regions of the hippocampus and of the amygdala, in some thalamic nuclei and in the pyriform cortex. On the other hand the subiculum contained only the message for chromogranin A, the granule cell layer of the cerebellum only that for chromogranin B, and in posterior intralaminar thalamic and medial geniculate nuclei and in the nucleus of the solitary tract only secretogranin II mRNA was found. The distinct distributions of mRNAs for the chromogranins in various brain regions support the concept that these proteins are propeptides giving rise to functionally active components.

Differential increases in brain levels of neuropeptide Y and vasoactive intestinal polypeptide after kainic acid-induced seizures in the rat

SummaryChanges in immunoreactivities of neuropeptide Y (NPY) and vasoactive intestinal polypeptide (VIP) were investigated in the brain of rats after severe kainic acid (KA, 10 mg/kg, i.p.) induced limbic seizures. Decreased levels of both neuropeptides were observed in the frontal cortex, striatum, dorsal hippocampus and amygdala/pyriform cortex subsequently to the period of acute seizures (3 h after injection of the toxin). Then NPY increased consistently in the frontal cortex, hippocampus and amygdala/pyriform cortex. Highest levels (290% of controls) were found in the frontal cortex after two months. Anticonvulsant therapy with phenobarbital (20 mg/kg, i.p., twice daily for three weeks) partially suppressed the rise in NPY levels. Immunoreactivity of VIP increased (to 150%) in the frontal cortex only transiently 3 days after injection of kainic acid. At the subsequently examined time intervals (10–60 days after kainic acid) it declined to control values. Levels decreasing subsequently to acute seizures reflect increased release and degradation of the respective peptide. Increased NPY levels suggest “upregulation” of NPY/ somatostatin/GABA neurons due to the decreased seizure threshold of the animals. The early, reversible rise of VIP in the cortex points to a short-lasting activation of this peptide system contained in local cholinergic neurons. This may be a consequence either of the acute seizures or subsequent neuropathological changes.

CHROMOGRANINS IN RAT BRAIN : CHARACTERIZATION, TOPOGRAPHICAL DISTRIBUTION AND REGULATION OF SYNTHESIS

The properties and distribution of chromogranins A, B and secretogranin II in rat brain were analyzed by quantitative immunoblotting. In contrast to endocrine tissues brain contains a significant amount of the proteoglycan form of chromogranin A. For secretogranin II a significant degree of endogenous proteolytic processing is apparent. Chromogranin A and secretogranin II had a similar topographical distribution with the highest concentrations found in the hypothalamus, amygdala/piriform cortex and hippocampus, whereas for chromogranin B by far the highest concentration was found in the cerebellum. Compared with adrenal medulla the concentration of all three proteins is low, however, secretogranin II appears relatively enriched. The synthesis of chromogranin A in brain does not depend on glucocorticoids since neither adrenalectomy nor dexamethasone treatment changed its levels. This is in contrast to adrenal medulla and to the anterior pituitary. Three days after kainic acid-induced seizures the levels of chromogranin A in frontal cortex and hippocampus were significantly elevated. For frontal cortex there was also an increase of the respective mRNA. This result establishes that the synthesis of chromogranin A can be regulated like that of neuropeptides.

HUMAN AND RAT PRIMARY C-FIBRE AFFERENTS STORE AND RELEASE SECRETONEURIN, A NOVEL NEUROPEPTIDE

Secretoneurin is a recently discovered neuropeptide derived from secretogranin II (SgII). Since this peptide could be detected in the dorsal horn of the spinal cord we studied whether it is localized in and released from primary afferent neurons. Secretoneurin was investigated with immunocytochemistry and radioimmunoassay in spinal cord, dorsal root ganglia and peripheral organs. SgII mRNA was determined in dorsal root ganglia. Normal rats and rats pre‐treated neonatally with capsaicin to destroy selectively polymodal nociceptive (C‐) fibres were used. Slices of dorsal spinal cord were perfused in vitro for release experiments. Immunocytochemistry showed a distinct distribution of secretoneurin‐immunoreactivity (IR) in the spinal cord and lower brainstem. A particularly high density of fibres was found in lamina I and outer lamina II of the caudal trigeminal nucleus and of the spinal cord. This distribution was qualitatively identical in rat and human post‐mortem tissue. Numerous small diameter and some large dorsal root ganglia neurons were found to contain SgII mRNA. Capsaicin treatment led to a marked depletion of secretoneurin‐IR in the substantia gelatinosa, but not in other immunopositive areas of the spinal cord and to a substantial loss of small (<25 μm) SgII‐mRNA‐containing dorsal root ganglia neurons. Radioimmunoassay revealed a significant decrease of secretoneurin‐IR in the dorsal spinal cord, the trachea, heart and urinary bladder of capsaicin‐treated rats. Perfusion of spinal cord slices with capsaicin as well as with 60 mM potassium led to a release of secretoneurin‐IR. In conclusion, secretoneurin is a neuropeptide which is stored in and released from capsaicin‐sensitive, primary afferent (C‐fibre) neurons. It may, therefore, be a novel peptidergic modulator of pain transmission or of C‐fibre mediated non‐nociceptive information.

Temporal lobe epilepsy of the rat : differential expression of mRNAs of chromogranin B, secretogranin II, synaptin/synaptophysin and p65 in subfields of the hippocampus

We have investigated by in situ hybridization changes in the content of mRNAs encoding for chromogranin B, secretogranin II, synaptin/synaptophysin and p65 after kainic acid-induced seizures and pentylenetetrazol kindling. Kainic acid seizures resulted in marked but transient increases in secretogranin II mRNA concentrations in the granule cell layer and throughout the pyramidal cell layers of the hippocampus (by 100-500%) as well as in various areas of the cerebral cortex (by up to 900%) and the thalamus (up to 300%) 12 h after injection of the toxin. Chromogranin B mRNA concentrations were persistently increased in granule cells (but not in pyramidal cells) of the hippocampus (suprapyramidal blade, 450%) and in cortical areas (250%) at all time intervals after kainic acid injection (12 h to 60 days). Accordingly chromogranin B immunoreactivity was enhanced in the terminal field of mossy fibers and in the inner part of the molecular layer 30 days after kainic acid. Secretogranin II immunoreactivity was also markedly increased in CA1, the paraventricular thalamic nucleus and in the central amygdala. In rats kindled with pentylenetetrazol only chromogranin B (by 200%) but not secretogranin II mRNA was increased in dentate granule cells. In contrast to the mRNAs of these secretory proteins concentrations of mRNAs encoding synaptin/synaptophysin and p65, two membrane proteins of synaptic vesicles, were not altered in any of these brain structures. These data demonstrate that in brain the biosynthesis of chromogranin B and secretogranin II is regulated like that of neuropeptides which is consistent with a role of these secretory polypeptides as precursors of functional peptides. Activation of neurons induces an increased synthesis of neuropeptides but not a concomitant synthesis of membrane proteins of synaptic vesicle. This might lead to an increased quantal content available for transmission.