Galanin in Alzheimer disease

Galanin (GAL) and GAL receptors (GALR) are overexpressed in limbic brain regions associated with cognition in Alzheimer disease (AD). The functional consequences of this overexpression are unclear. Because GAL inhibits cholinergic transmission and restricts long-term potentiation in the hippocampus, GAL overexpression may exacerbate clinical features of AD. In contrast, GAL expression increases in response to neuronal injury, and galaninergic hyperinnervation prevents the decreased production of protein phosphatase 1 subtype mRNAs in cholinergic basal forebrain neurons in AD. Thus, GAL may also be neuroprotective for AD. Further elucidation of GAL activity in selectively vulnerable brain regions will help gauge the therapeutic potential of GALR ligands for the treatment of AD.     

Steroid imprinting and modulation of sexual dimorphism in the luteinizing hormone-releasing hormone neuronal system

Summary 1. Sex differences in the control of gonadotropin secretion and reproductive functions are a distinct characteristic in all mammalian species, including humans. Ovulation and cyclicity are among the most distinct neuroendocrine markers of female brain differentiation, along with sex behavioral traits that are also evident in different species.2. The luteinizing hormone-releasing hormone (LHRH) neuronal system is the prime regulator of neuroendocrine events leading to ovulation and hormonal changes during the menstrual cycle and, as such, is the potential site where many of these sex differences may be expressed or, at the very least, integrated. However, until recently, no significant differences were seen in LHRH neurons between male and female brains, including cell number, pattern of distribution, and expression of message or peptide (LHRH) levels.3. Recently, we reported that galanin (GAL), a brain-gut peptide, is coexpressed in LHRH neurons and that this coexpression is sexually dimorphic. When GAL is used as a marker for this neuronal system, it is clear that estradiol as well as progesterone profoundly affects the message and expression of the peptide and that this regulation, at least in rodents, is neonatally predetermined by gonadal steroid imprinting.4. Changes in GAL expression and message can also be seen at puberty, during pregnancy and lactation, and in aging, all situations that affect the function of the LHRH neuronal system. Using an immortalized LHRH neuronal cell line (GT1) we have recently observed that these neurons express estrogen receptor (ER) and GAL and that estradiol can increase the expression of GAL, indicating functional activation of the endogenous ER.     

Noradrenaline induces expression of peroxisome proliferator activated receptor gamma (PPARgamma) in murine primary astrocytes and neurons

Cerebral inflammatory events play an important part in the pathogenesis of Alzheimer's disease (AD). Agonists of the peroxisome proliferator-activated receptor gamma (PPARgamma), a nuclear hormone receptor that mediates anti-inflammatory actions of non-steroidal anti-inflammatory drugs (NSAIDs) and thiazolidinediones, have been therefore proposed as a potential treatment of AD. Experimental evidence suggests that cortical noradrenaline (NA) depletion due to degeneration of the locus ceruleus (LC) - a pathological hallmark of AD - plays a permissive role in the development of inflammation in AD. To study a possible relationship between NA depletion and PPARgamma-mediated suppression of inflammation we investigated the influence of NA on PPARgamma expression in murine primary cortical astrocytes and neurons. Incubation of astrocytes and neurons with 100 micro m NA resulted in an increase of PPARgamma mRNA as well as PPARgamma protein levels in both cell types. These effects were blocked by the beta-adrenergic antagonist propranolol but not by the alpha-adrenergic antagonist phentolamine, suggesting that they might be mediated by beta-adrenergic receptors. Our results indicate for the first time that PPARgamma expression can be modulated by the cAMP signalling pathway, and suggest that the anti-inflammatory effects of NA on brain cells may be partly mediated by increasing PPARgamma levels. Conversely, decreased NA due to LC cell death in AD may reduce endogenous PPARgamma expression and therefore potentiate neuroinflammatory processes.     

Molecular forms of acetylcholinesterases in Alzheimer's disease

In this study, we examined 26 cases of Alzheimer's disease (AD) and 14 age-matched controls. In Brodmann area 21 cerebral cortex of the AD cases, there was no change in soluble G1 and G4 acetylcholinesterase (AChE) (EC 3.1.1.7), a significant 40% decrease in membrane-associated G4 AChE, significant 342 and 406% increases in A12 and A8 AChE, and a significant 71% decrease in choline acetyltransferase (ChAT) (EC 2.3.1.6). Our working hypothesis to account for these changes postulates that soluble globular forms are unchanged because they are primarily associated with intrinsic cortical neurons that are relatively unaffected by AD, that ChAT and membrane-associated G4 AChE decrease because they are primarily associated with incoming axons of cholinergic neurons that are abnormal in AD, and that asymmetric forms of AChE increase because of an acrylamide-type impairment of fast axonal transport in diseased incoming cholinergic axons. In the nucleus basalis of Meynert (nbM) of the 26 AD cases, there was a significant 61% decrease in the number of cholinergic neurons, an insignificant 23% decrease in nbM ChAT, a significant 298% increase in nbM ChAT per cholinergic neuron, and a significant 7% increase in the area of cholinergic perikarya. To account for the increased ChAT in cholinergic neurons and the enlargement of cholinergic perikarya, we propose that slow axonal transport may be impaired in nbM cholinergic neurons in AD.     

N epsilon-carboxymethyllysine in brain aging, diabetes mellitus, and Alzheimer's disease

Oxidative stress has been implicated in the pathophysiology of Alzheimer's disease (AD) and diabetes mellitus (DM). N epsilon-carboxymethyllysine (CML) is an advanced glycation end product (AGE) recently found to be associated with oxidative stress mechanisms. Using immunocytochemical methods we examined the distribution of CML in brain tissue from AD and DM subjects and aging controls. CML reactivity was present in the cytoplasm of neurons, but there were marked differences in the intensity of expression, number of cells, and topographical distribution. CML expression was higher in hippocampus than in frontal and temporal cortex. In the hippocampus, neuronal and, to an extent, glial expression was more marked in CA3 and CA4 than in CA1 and CA2. In AD, CML was found to be coexpressed with tau protein, showing the similar neurofibrillary tangle shape, as well as in neuritic plaques but not in the core of amyloid plaques. We noted an increasing degree of CML expression such that the highest reactivity was evident in those with both AD and DM, followed by AD, DM, and aging controls. There was an inverse relationship between Braak staging and topography of CML expression. Although DM cases did not show Abeta deposition or neurofibrillary tangles, these findings suggest increased CML expression is not limited to AD. Nonetheless, high CML expression in AD with coexistent DM suggests there are additive effects compared with AD alone. It is plausible that the microangiopathy also associated with DM could worsen AD pathogenesis.     

Processing of neuropeptide Y, galanin, and somatostatin in the cerebrospinal fluid of patients with Alzheimer’s disease and frontotemporal dementia

Alzheimer's disease (AD) and frontotemporal dementia (FTD) are two prevalent neurodegenerative disorders for which the causes are unknown, except in rare familial cases. Several changes in neuropeptide levels as measured by radioimmunoassay (RIA) have been observed in these illnesses. Somatostatin (SOM) levels in cerebrospinal fluid (CSF) are consistently decreased in AD and FTD. Neuropeptide Y (NPY) levels are decreased in AD, but normal in FTD. Galanin (GAL) levels increase with the duration of illness in AD patients. The majority of studies of neuropeptides in CSF have not been verified by HPLC. The observed decrease in a neuropeptide level as measured by RIA may therefore reflect an altered synthesis or extracellular processing, resulting in neuropeptide fragments that may or may not be detected by RIA. Matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-MS) has been shown to be a powerful technique in the analysis of biological materials without any pre-treatment, by detecting peptides and proteins at a specific mass-to-charge (m/z) ratio. We studied the processing of the neuropeptides NPY, NPY, SOM and GAL in the cerebrospinal fluid of patients with AD (n = 3), FTD (n = 3) and controls (n = 2) using MALDI-MS. We found that considerable inter-individual variability exists in the rate of neuropeptide metabolism in CSF, as well as the number of peptide fragments formed. Certain patients showed differences in the processing of specific neuropeptides, relative to other patients and controls. This analysis of the metabolic processing of neuropeptides in CSF yielded a large amount of data for each individual studied. Further studies are required to determine the changes in neuropeptide processing that can be associated with AD and FTD. With further investigations using MALDI-MS analysis, it may be possible to identify a neuropeptide fragment or processing enzyme that can be correlated to these disease states.     

Galanin inhibits tyrosine hydroxylase expression in midbrain dopaminergic neurons

Galanin (GAL) inhibits midbrain dopamine (DA) activity in several experimental paradigms, yet the mechanism underlying this inhibition is unclear. We examined the effects of GAL on the expression of tyrosine hydroxylase (TH) in primary cultures of rat embryonic (E14) ventral mesencephalon (VM). One micromolar GAL had no effect on the number of TH-immunoreactive (ir) neurons in VM cultures. However, 1 micro m GAL reduced an approximately 100% increase in TH-ir neurons in 1 mm dibutyryl cAMP (dbcAMP)-treated cultures by approximately 50%. TH-ir neuron number in dbcAMP-treated VM cultures was dose-responsive to GAL and the GAL receptor antagonist M40 blocked GAL effects. Semi-quantitative RT-PCR and quantitative immunoblotting experiments revealed that GAL had no effect on TH mRNA levels in VM cultures but reduced TH protein. VM cultures expressed GALR1, GALR2, and GALR3 receptor mRNA. However, dbcAMP treatment resulted in a specific approximately 200% increase in GALR1 mRNA. GALR1 activity is linked to a pertussis toxin (PTX)-sensitive opening of G protein-gated K+ channels (GIRKs). GAL reduction of TH-ir neuron number in dbcAMP + GAL-treated cultures was sensitive to both PTX and tertiapin, a GIRK inhibitor. GAL inhibition of midbrain DA activity may involve a GALR1- mediated reduction of TH in midbrain dopaminergic neurons.     

Modulators of cytoskeletal reorganization in CA1 hippocampal neurons show increased expression in patients at mid-stage Alzheimer's disease

During the progression of Alzheimer's disease (AD), hippocampal neurons undergo cytoskeletal reorganization, resulting in degenerative as well as regenerative changes. As neurofibrillary tangles form and dystrophic neurites appear, sprouting neuronal processes with growth cones emerge. Actin and tubulin are indispensable for normal neurite development and regenerative responses to injury and neurodegenerative stimuli. We have previously shown that actin capping protein beta2 subunit, Capzb2, binds tubulin and, in the presence of tau, affects microtubule polymerization necessary for neurite outgrowth and normal growth cone morphology. Accordingly, Capzb2 silencing in hippocampal neurons resulted in short, dystrophic neurites, seen in neurodegenerative diseases including AD. Here we demonstrate the statistically significant increase in the Capzb2 expression in the postmortem hippocampi in persons at mid-stage, Braak and Braak stage (BB) III-IV, non-familial AD in comparison to controls. The dynamics of Capzb2 expression in progressive AD stages cannot be attributed to reactive astrocytosis. Moreover, the increased expression of Capzb2 mRNA in CA1 pyramidal neurons in AD BB III-IV is accompanied by an increased mRNA expression of brain derived neurotrophic factor (BDNF) receptor tyrosine kinase B (TrkB), mediator of synaptic plasticity in hippocampal neurons. Thus, the up-regulation of Capzb2 and TrkB may reflect cytoskeletal reorganization and/or regenerative response occurring in hippocampal CA1 neurons at a specific stage of AD progression.     

Role of fibrogenic markers in chronic hepatitis C and associated hepatocellular carcinoma

Detection and follow up of fibrogenesis in chronic hepatitis C (CHC) is mandatory for early treatment and risk stratification. The current study included 120 patients with CHC, of whom 30 had liver cirrhosis (LC) and 30 had hepatocellular carcinoma (HCC). 15 wedge liver biopsies, taken during laparoscopic cholecystectomy, were included as normal controls. Cases were subjected to laboratory investigations, serologic markers for viral hepatitis and assessment of circulating levels of hyaluronic acid (HA) and platelet-derived growth factor (PDGF). Immunohistochemical expression of connective tissue growth factor (CTGF), PDGF and transforming growth factor-β1 (TGF-β1) was also carried out. A significant increase (p < 0.01) in serum HA was noticed in CHC, LC and HCC compared to controls. Although, a significant decrease in serum PDGF was detected in CHC and LC compared to controls, HCC values were comparable. A significant up-regulation of CTGF was detected in CHC, LC and HCC (p < 0.01) in contrast to its limited mild expression in normal livers. Intense PDGF positive staining was noticed in CHC, LC and HCC compared to scattered faint expression in controls. The significant expression and marked intensity of PDGF staining matched the progress to tumorigenesis. A positive TGF-β1 immunostaining was also noticed in CHC, LC and HCC. An intense and extensive cytoplasmic expression of TGF-β1 was encountered in patients with LC revealing that CTGF, PDGF and TGF-β1 act synergistically in LC. Data revealed that HA and CTGF may be implicated as important diagnostic parameters for assessment of hepatic fibrosis and PDGF for monitoring malignant transformation in CHC.     

Down regulation of trk but not p75NTR gene expression in single cholinergic basal forebrain neurons mark the progression of Alzheimer's disease

Dysfunction of cholinergic basal forebrain (CBF) neurons of the nucleus basalis (NB) is a cardinal feature of Alzheimer's disease (AD) and correlates with cognitive decline. Survival of CBF neurons depends upon binding of nerve growth factor (NGF) with high-affinity (trkA) and low-affinity (p75(NTR)) neurotrophin receptors produced within CBF neurons. Since trkA and p75(NTR) protein levels are reduced within CBF neurons of people with mild cognitive impairment (MCI) and mild AD, trkA and/or p75(NTR) gene expression deficits may drive NB degeneration. Using single cell expression profiling methods coupled with custom-designed cDNA arrays and validation with real-time quantitative PCR (qPCR) and in situ hybridization, individual cholinergic NB neurons displayed a significant down regulation of trkA, trkB, and trkC expression during the progression of AD. An intermediate reduction was observed in MCI, with the greatest decrement in mild to moderate AD as compared to controls. Importantly, trk down regulation is associated with cognitive decline measured by the Global Cognitive Score (GCS) and the Mini-Mental State Examination (MMSE). In contrast, there is a lack of regulation of p75(NTR) expression. Thus, trk defects may be a molecular marker for the transition from no cognitive impairment (NCI) to MCI, and from MCI to frank AD.     

Neurofilament proteins NF-L, NF-M and NF-H in brain of patients with Down syndrome and Alzheimer's disease

Summary. Neurofilaments (NFs) are integral constituents of the neuron playing a major role in brain development, maintenance, regeneration and the pattern of expression for NFs suggests their contribution to plasticity of the neuronal cytoskeleton and creating and maintaining neuronal architecture. Using immune-histochemical techniques the altered expression of NFs in Down syndrome (DS) and Alzheimer's disease (AD) has been already published but as no corresponding systematic immune-chemical study has been reported yet, we decided to determine proteins levels of three NFs in several brain regions of DS and AD brain. We evaluated immunoreactive NF-H, NF-M and NF-L levels using Western blotting in brain regions temporal, occipital cortex and thalamus of patients with DS (n = 9), AD (n = 9) and controls (n = 12). We found significantly increased NF-H in temporal cortex (controls: means 0.74 ± 0.39 SD; DS: means 3.01 ± 2.18 SD) of DS patients and a significant decrease of NF-L in occipital cortex of DS and AD patients (controls: means 1.19 ± 0.86 SD; DS: means 0.35 ± 0.20; AD: 0.20 ± 0.11 SD). We propose that the increase of NF-H in temporal cortex of DS brain is due to neuritic sprouting as observed in immune-histochemical studies. The increase may not be caused by the known accumulation of NFs in plaques, tangles or Lewy bodies due to our solubilization protocol. The decrease of NF-L in occipital cortex of DS and AD patients may well be reflecting neuronal loss. Altogether, however, we suggest that NFs are not reliable markers for neuronal death, a hallmark of both neurodegenerative diseases, in DS or AD. The increase of NF-H in DS or the decrease of NF-L in DS and AD leaves the other NFs unchanged, which points to dysregulation in DS and AD and raises the question of impaired structural assembly of neurofilaments. Received July 19, 2000 Accepted July 28, 2000     

Ischemia-related changes in galanin expression in the dentate hilar region after transient forebrain ischemia in gerbils.

Although galanin (GAL) protects hippocampal neurons from ischemic damage, no study has examined ischemia-related changes in endogenous GAL in the hippocampal dentate gyrus. We investigated the chronological changes of GAL, well-known as the potassium channel opener, expression in the dentate gyrus at various times after 5 min of transient forebrain ischemia in gerbils. A few GAL-immunoreactive (IR) neurons were found in the polymorphic layer of the sham-operated group. Three hours after ischemia-reperfusion, the pattern of GAL immunoreactivity was similar to that of the sham-operated group and the number of GAL-IR neurons and immunoreactivity were highest 12 h after ischemic insult. At this time, GAL-IR neurons in the polymorphic layer showed strong GAL immunoreactivity. Thereafter, GAL-IR neurons and immunoreactivity significantly decreased in the dentate hilar region. Four days after ischemic insult, GAL-IR neurons were not detectable. In addition, the results of a Western blot study showed a pattern of GAL expression similar to the immunohistochemical changes. GAL protein content also was highest 12 h after ischemia. In conclusion, the increased expression of endogenous GAL in the dentate gyrus after ischemia is related to response to the ischemic damage.     

Amyloid-beta-(1-42) increases ryanodine receptor-3 expression and function in neurons of TgCRND8 mice

Disruption of intracellular calcium homeostasis precedes the neurodegeneration that occurs in Alzheimer disease (AD). Of the many neuronal calcium-regulating proteins, we focused on endoplasmic reticulum (ER)-resident ryanodine receptors (RyRs) because they are increased in the hippocampus of mice expressing mutant presenilin-1 and are associated with neurotoxicity. Others have observed that ryanodine binding is elevated in human postmortem hippocampal regions suggesting that RyR(s) are involved in AD pathogenesis. Here we report that extracellular amyloid-beta(Abeta)-(1-42) specifically increased RyR-3, but not RyR-1 or RyR-2, gene expression in cortical neurons from C57Bl6 mice. Furthermore, endogenously produced Abeta-(1-42) increased RyR-3 mRNA and protein in cortical neurons from transgenic (Tg)CRND8 mice, a mouse model of AD. Increased RyR-3 mRNA and protein was also observed in brain tissue from 4- to 4.5-month-old Tg animals compared with non-Tg littermate controls. In experiments performed in nominal extracellular calcium, neurons from Tg mice had significant increases in intracellular calcium following ryanodine or glutamate treatment compared with littermate controls, which was abolished by treatment with small interfering RNA directed to RyR-3, indicating that the higher levels of calcium originated from RyR-3-regulated stores. Taken together, these observations suggest that Abeta-(1-42)-mediated changes in intracellular calcium homeostasis is regulated in part through a direct increase of RyR-3 expression and function.     

Cholinergic deficit in Alzheimer's disease: A study based on CSF and autopsy data

Cholinesterase (ChE) activity was measured as a possible marker of cholinergic neurotransmission of the brain in CSF of 93 patients with probable Alzheimer's disease/senile dementia of the Alzheimer type (AD/SDAT) and of 29 control patients. ChE activity in CSF was decreased significantly in the AD/SDAT patients as compared to the controls. This reduction correlated significantly with the various measures of the severity of dementia. However, the reduction of ChE activity was only moderate (25–30%) even in patients with the most severe dementia and nonsignificant in patients with early symptoms of AD/SDAT. The significance of various confounding factors, which may interfere with CSF ChE measurements is discussed. Our findings seem to indicate that the deficiency of cholinergic neurons is not directly reflected in CSF and that the measurements of ChE activities in CSF are not helpful in diagnosing AD/SDAT. In the autopsy study the activities of cholineacetyltransferase (ChAT) and ChE were determined for ten brain areas of 20 AD/SDAT patients and of 14 controls. In AD/SDAT patients ChAT activity was profoundly decreased (50–85% decrease) in the cortical areas and hippocampus, but was unchanged or only mildly reduced in other subcortical brain areas. This study further confirms that the affection of cholinergic neurons is limited to projections from nucleus basalis to cortex and hippocampus, whereas other cholinergic neurons, like in striatum, seem to be relatively spared. In general, the activities of ChAT and ChE were lower in Alzheimer patients dying at younger age suggesting more severe disease process with these patients.     

Ceruloplasmin immunoreactivity in neurodegenerative disorders

Ceruloplasmin (CP) is a 132kd cuproprotein which, together with transferrin, provides the majority of anti-oxidant capacity in serum. Increased iron deposition and lipid peroxidation in the basal ganglia of subjects with hereditary CP deficiency suggest that CP may serve as an anti-oxidant in the brain as well. The present study compared CP immunoreactivity in brain specimens from normal controls and subjects with neurodegenerative disorders (Alzheimer's disease [AD], Parkinson's disease [PD], progressive supranuclear palsy [PSP], and Huntington's disease [HD]) (n = 5 per group). The relative intensity of neuronal CP staining and the numbers of CP-stained neurons per 25x microscope field were determined in hippocampus (CA1, subiculum, and parahippocampal gyrus), parietal cortex, frontal cortex, substantia nigra, and caudate. CP was detected in both neurons and astrocytes in all specimens, and in senile plaques and occasional neurofibrillary tangles in AD brain. Neuronal CP staining intensity tended to increase in most AD brain regions, but was statistically significant vs controls only in the CA1 region of hippocampus (p = .016). Neuronal CP staining in brain specimens from other neurodegenerative disorders showed a slight but nonsignificant increase vs controls. The numbers of CP-stained neurons per field did not differ between the various neurodegenerative disorders and controls. These results suggest that a modest increase in neuronal CP content is present in the AD brain, and lesser elevations in neuronal CP occur in the other neurodegenerative disorders in this study. Though CP functions as both an acute phase protein and an anti-oxidant in peripheral tissues, whether it does so in the brain remains to be determined.     

Targeted expression of tetanus neurotoxin interferes with behavioral responses to sensory input in Drosophila.

Targeted inactivation of neurons by expression of toxic gene products is a useful tool to assign behavioral functions to specific neurons or brain structures. Of a variety of toxic gene products tested, tetanus neurotoxin light chain (TNT) has the least severe side effects and can completely block chemical synapses. By using the GAL4 system to drive TNT expression in a subset of chemo- and mechanosensory neurons, we detected walking and flight defects consistent with blocking of relevant sensory information. We also found, for the first time, an olfactory behavioral phenotype associated with blocking of a specific subset of antennal chemoreceptors. Similar behavioral experiments with GAL4 lines expressing in different subsets of antennal chemoreceptors should contribute to an understanding of olfactory coding in Drosophila. To increase the utility of the GAL4 system for such purposes, we have designed an inducible system that allows us to circumvent lethality caused by TNT expression during early development.