Locus coeruleus at asymptomatic early and middle Braak stages of neurofibrillary tangle pathology.

AIMS: The present study analyses molecular characteristics of the locus coeruleus (LC) and projections to the amygdala and hippocampus at asymptomatic early and middle Braak stages of neurofibrillary tangle (NFT) pathology. METHODS: Immunohistochemistry, whole-transcriptome arrays and RT-qPCR in LC and western blotting in hippocampus and amygdala in a cohort of asymptomatic individuals at stages I-IV of NFT pathology were used. RESULTS: NFTs in the LC increased in parallel with colocalized expression of tau kinases, increased neuroketal adducts and decreased superoxide dismutase 1 in neurons with hyperphosphorylated tau and decreased voltage-dependent anion channel in neurons containing truncated tau were found. These were accompanied by increased microglia and AIF1, CD68, PTGS2, IL1ß, IL6 and TNF-α gene expression. Whole-transcriptome arrays revealed upregulation of genes coding for proteins associated with heat shock protein binding and genes associated with ATP metabolism and downregulation of genes coding for DNA-binding proteins and members of the small nucleolar RNAs family, at stage IV when compared with stage I. Tyrosine hydroxylase (TH) immunoreactivity was preserved in neurons of the LC, but decreased TH and increased α2A adrenergic receptor protein levels were found in the hippocampus and the amygdala. CONCLUSIONS: Complex alteration of several metabolic pathways occurs in the LC accompanying NFT formation at early and middle asymptomatic stages of NFT pathology. Dopaminergic/noradrenergic denervation and increased expression of α2A adrenergic receptor in the hippocampus and amygdala occur at first stage of NFT pathology, suggesting compensatory activation in the face of decreased adrenergic input occurring before clinical evidence of cognitive impairment and depression.

Distribution of SNAP25, VAMP1 and VAMP2 in mature and developing deep cerebellar nuclei after estrogen administration.

Synaptosomal-associated protein of 25kDa (SNAP25), vesicle-associated membrane protein 1 (VAMP1) and 2 (VAMP2) are components of soluble N-ethylmaleimide-sensitive fusion attachment protein receptors (SNARE) complex which is involved in synaptic vesicle exocytosis, a fundamental step in neurotransmitter release. SNARE expression in cerebellum correlates with specific neurotransmitter pathways underlying synaptic diversification and defined synaptic properties. In this study we firstly characterized the distribution of SNAP25, VAMP1 and VAMP2 in the nerve terminals of a defined cerebellar region, the deep cerebellar nuclei (DCN), of adult and newborn rats. Then, given the pivotal role of estradiol (E2) in the synaptic organization of the cerebellar circuitry in early postnatal life, we examined whether administration of E2 in the newborn DCN affected synaptic density and changed the distribution of the presynaptic proteins SNAP25, VAMP1 and VAMP2, together with post synaptic density protein 95 (PSD95). Results showed that: (1) distribution of SNAP25, VAMP1 and VAMP2 in adult DCN differs significantly from that found in newborn DCN; (2) administration of E2 in the newborn DCN affected synaptic density and also changed the distribution of the pre- and postsynaptic proteins. The differential distribution of SNAP25, VAMP1 and VAMP2 in nerve terminals of adult and newborn rats may correlate with specific stages of neuronal phenotypic differentiation. The effects of E2 on SNAP25, VAMP1, VAMP2, PDS95 and synaptic density suggest that pre- and postsynaptic proteins are under estrogenic control during development and that synaptic maturation can also be related with the activity of this steroid.

Changes in expression and activity levels of ecto-5'-nucleotidase/CD73 along the mouse female estrous cycle.

AIM: Extracellular ATP and its hydrolysis product adenosine modulate various reproductive functions such as those requiring contraction, hormone synthesis and maintenance of fluid composition. Moreover, adenosine is a key molecule for sperm capacitation. Extracellular nucleotide and nucleoside levels are affected by cell surface ectonucleotidases, amongst which the ectonucleoside triphosphate diphosphohydrolase (E-NTPDase) family is the most abundant and effective to hydrolyse ATP and ADP to AMP. In the female reproductive tract three members of this family have been recently identified: NTPDase1, NTPDase2 and NTPDase3 (Histochem. Cell Biol.131, 2009, 615). The purpose of the present study was to characterize in this system the expression profile of ecto-5'-nucleotidase (CD73), the enzyme generating adenosine from AMP. METHODS: Immunological techniques and in situ enzymatic assays were used to characterize the ecto-5'-nucleotidase expression in the mouse female reproductive tract along the four stages of the estrous cycle, that were determined by vaginal smear examination. RESULTS: Ecto-5'-nucleotidase was abundantly detected in the corpora lutea of the ovaries, as well as in several epithelia, such as that of oviducts, uterus and endometrial glands. Marked changes in endometrial ecto-5'-nucleotidase expression and activity along the estrous cycle are described, these being maximum at estrus phase, coinciding with optimal female sexual receptivity. CONCLUSION: The adenosine generated thereby, besides other functions, might contribute to sperm capacitation, thus significantly influencing fertility.

Expression of mutant ubiquitin (UBB+1) and p62 in myotilinopathies and desminopathies.

Protein aggregates in muscle cells are the morphological hallmark of myofibrillar myopathies, including myotilinopathies and desminopathies. The aim of the present study is to analyse the expression of mutant ubiquitin (UBB+1), an aberrant form of ubiquitin which accumulates in certain disorders characterized by intracellular aggregates of proteins, and p62, a multimeric signal protein which plays an active role in aggregate formation, in muscle biopsies from patients suffering from myotilinopathy and desminopathy in order to gain understanding of the mechanisms leading to protein aggregation in these disorders. Single immunohistochemistry, and single- and double-labelling immunofluorescence and confocal microscopy for UBB+1 and p62, has been performed in muscle biopsies from patients suffering from myotilinopathy and desminopathy. Strong UBB+1 immunoreactivity, colocalizing with myotilin aggregates, was found in muscle fibres in myotilinopathies. UBB+1 accumulation, colocalizing with desmin aggregates, also occurs in desminopathies. In addition, strong p62 immunoreactivity colocalizing with myotilin aggregates was observed in myotilinopathies. Similarly, p62 immunoreactivity colocalizing with desmin aggregates was found in desminopathies. The present findings suggest that accumulation of protein aggregates in myotilinopathies and in desminopathies may be related with UBB+1/abnormal protein complexes which are resistant to proteasome degradation. Furthermore, these observations suggest a relationship between the presence of p62 and the formation of inclusions in different subtypes of myofibrillar myopathies.

Involvement of clusterin and the aggresome in abnormal protein deposits in myofibrillar myopathies and inclusion body myositis.

Myofibrillar myopathies (MM) are characterized morphologically by the presence of non-hyaline structures corresponding to foci of dissolution of myofibrils, and hyaline lesions composed of aggregates of compacted and degraded myofibrillar elements. Inclusion body myositis (IBM) is characterized by the presence of rimmed vacuoles, eosinophilic inclusions in the cytoplasm, rare intranuclear inclusions, and by the accumulation of several abnormal proteins. Recent studies have demonstrated impaired proteasomal expression and activity in MM and IBM, thus accounting, in part, for the abnormal protein accumulation in these diseases. The present study examines other factors involved in protein aggregation in MM and IBM. Clusterin is a multiple-function protein which participates in Abeta-amyloid, PrP(res) and a-synuclein aggregation in Alzheimer disease, prionopathies and a-synucleinopathies, respectively. gamma-Tubulin is present in the centrosome and is an intracellular marker of the aggresome. Moderate or strong clusterin immunoreactivity has been found in association with abnormal protein deposits, as revealed by immunohistochemistry, single and double-labeling immunofluorescence and confocal microscopy, in MM and IBM, and in target structures in denervation atrophy. Gamma-Tubulin has also been observed in association with abnormal protein deposits in MM, IBM, and in target fibers in denervation atrophy. These morphological findings are accompanied by increased expression of clusterin and gamma-tubulin in muscle homogenates of MM and IBM cases, as revealed by gel electrophoresis and Western blots. Together, these observations demonstrate involvement of clusterin in protein aggregates, and increased expression of aggresome markers in association with abnormal protein inclusions in MM and IBM and in targets, as crucial events related to the pathogenesis of abnormal protein accumulation and degradation in these muscular diseases.

Expression of stress-activated kinases c-Jun N-terminal kinase (SAPK/JNK-P) and p38 kinase (p38-P), and tau hyperphosphorylation in neurites surrounding betaA plaques in APP Tg2576 mice.

Hyperphosphorylated tau in neurites surrounding beta-amyloid (betaA) deposits, as revealed with phospho-specific anti-tau antibodies, are found in amyloid precursor protein (APP) Tg2576 mice. Because betaA is a source of oxidative stress and may be toxic for cultured cells, the present study examines the expression of phosphorylated (active) stress-activated kinase c-Jun N-terminal kinase (SAPK/JNK-P) and p38 kinase (p38-P), which have the capacity to phosphorylate tau at specific sites, and their specific substrates c-Jun and ATF-2, which are involved in cell death and survival in several paradigms, in Tg2576 mice. The study was planned to shed light about the involvement of these kinases in tau phosphorylation in cell processes surrounding amyloid plaques, as well as in the possible phosphorylation (activation) of c-Jun and activating transcription factor-2 (ATF-2) in relation to betaA deposition. Moderate increase in the expression of phosphorylated mitogen-activated protein kinase and extracelullar signal-regulated kinase (MAPK/ERK-P) occurs in a few amyloid plaques. However, strong expression of SAPK/JNK-P and p38-P is found in the majority of, if not all, amyloid plaques, as seen in serial consecutive sections stained for betaA and stress kinases. Moreover, confocal microscopy reveals colocalization of phospho-tau and SAPK/JNK-P, and phospho-tau and p38-P in many dystrophic neurites surrounding amyloid plaques. Increased expression levels of nonbound tau, SAPK/JNK-P and p38-P are corroborated by Western blots of total cortical homogenate supernatants in Tg2576 mice when compared with age-matched controls. No increase in phosphorylated c-JunSer63 (c-Jun-P) and ATF-2Thr71 (ATF-2-P) is found in association with betaA deposits. In addition, no expression of active (cleaved) caspase-3 (17 kDa) has been found in transgenic mice. Taken together, these observations provide a link between betaA-induced oxidative stress, activation of stress kinases SAPK/JNK and p38, and tau hyperphosphorylation in neurites surrounding amyloid plaques, but activation of these kinases is not associated with accumulation of c-Jun-P and ATF-2-P, nor with activation of active caspase-3 in the vicinity of betaA deposits.

Clusterin solubility and aggregation in Creutzfeldt-Jakob disease.

Prion protein (PrPC) is a glycolipid-anchored cell membrane syaloglycoprotein that localizes in presynaptic membranes. PrP has the property of aggregating into amyloid fibrils and being deposited in the brains in cases with transmissible encephalopathies (TSEs), when PrPC is converted into abnormal protease-resistant PrP (PrPRES). Clusterin is a heterodimeric glycoprotein, the expression of which is enhanced in astrocytes in association with punctate-type PrPRES deposits during TSE progression. In addition, clusterin co-localizes in PrPRES plaques in several human TSEs, including Creutzfeldt-Jakob disease (CJD). Clusterin is up-regulated in the cerebral cortex and cerebellum in CJD as revealed by DNA micro-array technology. Clusterin expression was examined in seven sporadic cases of CJD (codon 129 genotype, PrP type: 4 MM1, 1 MV1, 1 MV2, 1 VV2) and three age-matched controls by immunohistochemistry, Western blotting and solubility. In addition to small punctate clusterin deposition in the neuropil, single- and double-labeling immunohistochemistry disclosed clusterin localization in PrPRES plaques, which predominated in the cerebellum of cases MV1, MV2 and VV2. Moreover, clusterin in plaques, but not punctate clusterin deposits, was resistant to protease digestion, as revealed in tissue sections pre-incubated with proteinase K. Clusterin in CJD, but not clusterin in control brains, was partially resistant to protease digestion in Western blots of total brain homogenates immunostained with anti-clusterin antibodies, which were processed in parallel with Western blots to PrP, without and with pre-incubation with proteinase K. Protein aggregation was analyzed in brain homogenates subjected to several solvents. PrP was recovered in the deoxycholate fraction in control and CJD cases, but in the SDS fraction only in CJD, thus indicating differences in PrP solubility between CJD and controls. Clusterin was recovered in the cytosolic, deoxycholate and SDS fraction in both CJD and control cases, but only clusterin from CJD was recovered in the urea-soluble fraction and, especially, in the remaining pellet. These findings demonstrate the capacity of clusterin to form aggregates and interact with PrPRES aggregates. The implications of this property are not known, but it can be suggested that clusterin participates in PrP clustering and sequestration, thus modifying PrP toxicity in CJD.