Paula Garcia-Esparcia

Neuroinflammatory signals in alzheimer disease and APP/PS1 transgenic mice: Correlations with plaques, tangles, and oligomeric species

Abstract To understand neuroinflammation-related gene regulation during normal aging and in sporadic Alzheimer disease (sAD), we performed functional genomics analysis and analyzed messenger RNA (mRNA) expression by quantitative reverse transcription–polymerase chain reaction of 22 genes involved in neuroinflammation-like responses in the cerebral cortex of wild-type and APP/PS1 transgenic mice. For direct comparisons, mRNA expression of 18 of the same genes was then analyzed in the entorhinal cortex, orbitofrontal cortex, and frontal cortex area 8 of middle-aged human subjects lacking Alzheimer disease–related pathology and in older subjects with sAD pathology covering Stages I–II/0(A), III–IV/A–B, and V–VI/C of Braak and Braak classification. Modifications of cytokine and immune mediator mRNA expression were found with normal aging in wild-type mice and in middle-aged individuals and patients with early stages of sAD-related pathology; these were accompanied by increased protein expression of certain mediators in ramified microglia. In APP/PS1 mice, inflammatory changes coincided with &bgr;-amyloid (A&bgr;) deposition; increased levels of soluble oligomers paralleled the modified mRNA expression of cytokines and mediators in wild-type mice. In patients with sAD, regulation was stage- and region-dependent and not merely acceleration and exacerbation of mRNA regulation with aging. Gene regulation at first stages of AD was not related to hyperphosphorylated tau deposition in neurofibrillary tangles, A&bgr; plaque burden, concentration of A&bgr;1–40 (A&bgr;40) and A&bgr;1–42 (A&bgr;42), or fibrillar A&bgr; linked to membranes but rather to increased levels of soluble oligomers. Thus, species differences and region- and stage-dependent inflammatory responses in sAD, particularly at the initial stages, indicate the need to identify new anti-inflammatory compounds with specific molecular therapeutic targets.

Human DNA methylomes of neurodegenerative diseases show common epigenomic patterns.

Different neurodegenerative disorders often show similar lesions, such as the presence of amyloid plaques, TAU-neurotangles and synuclein inclusions. The genetically inherited forms are rare, so we wondered whether shared epigenetic aberrations, such as those affecting DNA methylation, might also exist. The studied samples were gray matter samples from the prefrontal cortex of control and neurodegenerative disease-associated cases. We performed the DNA methylation analyses of Alzheimers disease, dementia with Lewy bodies, Parkinsons disease and Alzheimer-like neurodegenerative profile associated with Downs syndrome samples. The DNA methylation landscapes obtained show that neurodegenerative diseases share similar aberrant CpG methylation shifts targeting a defined gene set. Our findings suggest that neurodegenerative disorders might have similar pathogenetic mechanisms that subsequently evolve into different clinical entities. The identified aberrant DNA methylation changes can be used as biomarkers of the disorders and as potential new targets for the development of new therapies.

Functional genomics reveals dysregulation of cortical olfactory receptors in Parkinson disease: novel putative chemoreceptors in the human brain.

Parkinson disease (PD) is no longer considered a complex motor disorder but rather a systemic disease with variable nonmotor deficits that may include impaired olfaction, depression, mood and sleep disorders, and altered cortical function. Increasing evidence indicates that multiple metabolic defects occur in regions outside the substantia nigra, including the cerebral cortex, even at premotor stages of the disease. We investigated changes in gene expression in the frontal cortex in PD patient brains using a transcriptomics approach. Functional genomics analysis indicated that cortical olfactory receptors (ORs) and taste receptors (TASRs) are altered in PD patients. Olfactory receptors OR2L13, OR1E1, OR2J3, OR52L1, and OR11H1 and taste receptors TAS2R5 and TAS2R50 were downregulated, but TAS2R10 and TAS2R13 were upregulated at premotor and parkinsonian stages in the frontal cortex area 8 in PD patient brains. Furthermore, we present novel evidence that, in addition to the ORs, obligate downstream components of OR function adenylyl cyclase 3 and olfactory G protein (Gαolf), OR transporters, receptor transporter proteins 1 and 2 and receptor expression enhancing protein 1, and OR xenobiotic removing UDP-glucuronosyltransferase 1 family polypeptide A6 are widely expressed in neurons of the cerebral cortex and other regions of the adult human brain. Together, these findings support the concept that ORs and TASRs in the cerebral cortex may have novel physiologic functions that are affected in PD patients.

Complex deregulation and expression of cytokines and mediators of the immune response in Parkinson's disease brain is region dependent.

Neuroinflammation is common in neurodegenerative diseases including Parkinson disease (PD). Expression of 25 mRNAs was assessed with TaqMan‐PCR including members of the complement system, colony stimulating factors, Toll family, cytokines IL‐8, IL‐6, IL‐6ST, IL‐1B, TNF‐α family, IL‐10, TGFβ family, cathepsins and integrin family, in the substantia nigra pars compacta, putamen, frontal cortex area 8 and angular gyrus area 39, in a total of 43 controls and 56 cases with PD‐related pathology covering stages 1–6 of Braak. Up‐regulation of IL‐6ST was the only change in the substantia nigra at stages 1–2. Down‐regulation of the majority of members examined occurred in the substantia nigra from stage 4 onwards. However, region‐dependent down‐ and up‐regulation of selected mRNAs occurred in the putamen and frontal cortex, whereas only mRNA up‐regulated mRNAs were identified in the angular cortex from stage 3 onwards in PD cases. Protein studies in frontal cortex revealed increased IL6 expression and reduced IL‐10 with ELISA, and increased IL‐6 with western blotting in PD. Immunohistochemistry revealed localization of IL‐5, IL‐6 and IL‐17 receptors in glial cells, mainly microglia; IL‐5, IL‐10 and M‐CSF in neurons; TNF‐α in neurons and microglia; and active NF‐κB in the nucleus of subpopulations of neurons and glial cells in PD. Distinct inflammatory responses, involving pro‐ and anti‐inflammatory cytokines, and variegated mediators of the immune response occur in different brain regions at the same time in particular individuals. Available information shows that altered α‐synuclein solubility and aggregation, Lewy body formation, oxidative damage and neuroinflammation converge in the pathogenesis of PD.

Dysregulation of brain olfactory and taste receptors in AD, PSP and CJD, and AD-related model

Recently, we have shown the expression of novel chemoreceptors corresponding to the olfactory receptor (OR) and taste receptor (TASR) families in the human brain. We have also shown dysregulation of ORs and TASRs in the cerebral cortex in Parkinsons disease. The present study demonstrates the presence of OR mRNA and mRNA of obligated downstream components of OR signaling adenylyl cyclase 3 (ADYLC3) and olfactory G protein (Gnal) in the cerebral cortex of the mouse. Dysregulation of selected ORs and TASRs has been found in the entorhinal cortex and frontal cortex in Alzheimers disease (AD) in a gradient compatible with Braak and Braak staging; frontal cortex in terminal stages of Progressive Supranuclear Palsy; and frontal cortex and cerebellum in Creutzfeldt-Jakob disease subtypes methionine/methionine at codón 129 of PRNP (MM1) and valine/valine at codón 129 of PRNP (VV2). Altered OR, ADYLC3 and Gnal mRNA expression with disease progression has also been found in APP/PS1 transgenic mice, used as a model of AD. The function of these orphan receptors is not known, but probably related to cell signaling pathways responding to unidentified ligands. Variability in the drift, either down- or up-regulation, of dysregulated genes, suggests that central ORs and TASRs are vulnerable to variegated neurodegenerative diseases with cortical involvement, and that altered expression of ORs and TASRs is not a mere reflection of neuronal loss but rather a modulated pathological response.

Genetic and Transcriptomic Profiles of Inflammation in Neurodegenerative Diseases: Alzheimer, Parkinson, Creutzfeldt-Jakob and Tauopathies

Polymorphisms in certain inflammatory-related genes have been identified as putative differential risk factors of neurodegenerative diseases with abnormal protein aggregates, such as sporadic Alzheimer’s disease (AD) and sporadic Parkinson’s disease (sPD). Gene expression studies of cytokines and mediators of the immune response have been made in post-mortem human brain samples in AD, sPD, sporadic Creutzfeldt-Jakob disease (sCJD) subtypes MM1 and VV2, Pick’s disease (PiD), progressive supranuclear palsy (PSP) and frontotemporal lobar degeneration linked to mutation P301L in MAPT Frontotemporal lobar degeneration-tau (FTLD-tau). The studies have disclosed variable gene regulation which is: (1) disease-dependent in the frontal cortex area 8 in AD, sPD, sCJD MM1 and VV2, PiD, PSP and FTLD-tau; (2) region-dependent as seen when comparing the entorhinal cortex, orbitofrontal cortex, and frontal cortex area 8 (FC) in AD; the substantia nigra, putamen, FC, and angular gyrus in PD, as well as the FC and cerebellum in sCJD; (3) genotype-dependent as seen considering sCJD MM1 and VV2; and (4) stage-dependent as seen in AD at different stages of disease progression. These observations show that regulation of inflammation is much more complicated and diverse than currently understood, and that new therapeutic approaches must be designed in order to selectively act on specific targets in particular diseases and at different time points of disease progression.

Deregulation of purine metabolism in Alzheimer's disease

The neuroprotective role of adenosine and the deregulation of adenosine receptors in Alzheimers disease (AD) have been extensively studied in recent years. However, little is known about the involvement of purine metabolism in AD. We started by analyzing gene expression in the entorhinal cortex of human controls and AD cases with whole-transcript expression arrays. Once we identified deregulation of the cluster purine metabolism, messenger RNA expression levels of 23 purine metabolism genes were analyzed with qRT-PCR in the entorhinal cortex, frontal cortex area 8, and precuneus at stages I-II, III-IV, and V-VI of Braak and Braak and controls. APRT, DGUOK, POLR3B, ENTPD3, AK5, NME1, NME3, NME5, NME7, and ENTPD2 messenger RNAs were deregulated, with regional variations, in AD cases when compared with controls. In addition, liquid chromatography mass spectrometry based metabolomics in the entorhinal cortex identified altered levels of dGMP, glycine, xanthosine, inosine diphosphate, guanine, and deoxyguanosine, all implicated in this pathway. Our results indicate stage- and region-dependent deregulation of purine metabolism in AD.

Altered Machinery of Protein Synthesis in Alzheimer's: From the Nucleolus to the Ribosome

Ribosomes and protein synthesis have been reported to be altered in the cerebral cortex at advanced stages of Alzheimers disease (AD). Modifications in the hippocampus with disease progression have not been assessed. Sixty‐seven cases including middle‐aged (MA) and AD stages I–VI were analyzed. Nucleolar chaperones nucleolin, nucleophosmin and nucleoplasmin 3, and upstream binding transcription factor RNA polymerase I gene (UBTF) mRNAs are abnormally regulated and their protein levels reduced in AD. Histone modifications dimethylated histone H3K9 (H3K9me2) and acetylated histone H3K12 (H3K12ac) are decreased in CA1. Nuclear tau declines in CA1 and dentate gyrus (DG), and practically disappears in neurons with neurofibrillary tangles. Subunit 28 ribosomal RNA (28S rRNA) expression is altered in CA1 and DG in AD. Several genes encoding ribosomal proteins are abnormally regulated and protein levels of translation initiation factors eIF2α, eIF3η and eIF5, and elongation factor eEF2, are altered in the CA1 region in AD. These findings show alterations in the protein synthesis machinery in AD involving the nucleolus, nucleus and ribosomes in the hippocampus in AD some of them starting at first stages (I–II) preceding neuron loss. These changes may lie behind reduced numbers of dendritic branches and reduced synapses of CA1 and DG neurons which cause hippocampal atrophy.

PrP mRNA and protein expression in brain and PrPc in CSF in Creutzfeldt-Jakob disease MM1 and VV2

Creutzfeldt-Jakob disease (CJD) is a heterogenic neurodegenerative disorder associated with abnormal post-translational processing of cellular prion protein (PrPc). CJD displays distinctive clinical and pathological features which correlate with the genotype at the codon 129 (methionine or valine: M or V respectively) in the prion protein gene and with size of the protease-resistant core of the abnormal prion protein PrPsc (type 1: 20/21 kDa and type 2: 19 kDa). MM1 and VV2 are the most common sporadic CJD (sCJD) subtypes. PrP mRNA expression levels in the frontal cortex and cerebellum are reduced in sCJD in a form subtype-dependent. Total PrP protein levels and PrPsc levels in the frontal cortex and cerebellum accumulate differentially in sCJD MM1 and sCJD VV2 with no relation between PrPsc deposition and spongiform degeneration and neuron loss, but with microgliosis, and IL6 and TNF-α response. In the CSF, reduced PrPc, the only form present in this compartment, occurs in sCJD MM1 and VV2. PrP mRNA expression is also reduced in the frontal cortex in advanced stages of Alzheimer disease, Lewy body disease, progressive supranuclear palsy, and frontotemporal lobe degeneration, but PrPc levels in brain varies from one disease to another. Reduced PrPc levels in CSF correlate with PrP mRNA expression in brain, which in turn reflects severity of degeneration in sCJD.

Olfactory receptors in non-chemosensory organs: the nervous system in health and disease

Olfactory receptors (ORs) and down-stream functional signaling molecules adenylyl cyclase 3 (AC3), olfactory G protein α subunit (Gαolf), OR transporters receptor transporter proteins 1 and 2 (RTP1 and RTP2), receptor expression enhancing protein 1 (REEP1), and UDP-glucuronosyltransferases (UGTs) are expressed in neurons of the human and murine central nervous system (CNS). In vitro studies have shown that these receptors react to external stimuli and therefore are equipped to be functional. However, ORs are not directly related to the detection of odors. Several molecules delivered from the blood, cerebrospinal fluid, neighboring local neurons and glial cells, distant cells through the extracellular space, and the cells’ own self-regulating internal homeostasis can be postulated as possible ligands. Moreover, a single neuron outside the olfactory epithelium expresses more than one receptor, and the mechanism of transcriptional regulation may be different in olfactory epithelia and brain neurons. OR gene expression is altered in several neurodegenerative diseases including Parkinson’s disease (PD), Alzheimer’s disease (AD), progressive supranuclear palsy (PSP) and sporadic Creutzfeldt-Jakob disease (sCJD) subtypes MM1 and VV2 with disease-, region- and subtype-specific patterns. Altered gene expression is also observed in the prefrontal cortex in schizophrenia with a major but not total influence of chlorpromazine treatment. Preliminary parallel observations have also shown the presence of taste receptors (TASRs), mainly of the bitter taste family, in the mammalian brain, whose function is not related to taste. TASRs in brain are also abnormally regulated in neurodegenerative diseases. These seminal observations point to the need for further studies on ORs and TASRs chemoreceptors in the mammalian brain.