Rivka Ravid

Axonal loss in multiple sclerosis lesions: Magnetic resonance imaging insights into substrates of disability

Magnetic resonance imaging (MRI) monitoring of disease progression in multiple sclerosis is limited by the lack of correlation of abnormalities seen on T2‐weighted imaging, and disability. We studied the histopathology of multiple sclerosis lesions, as depicted by MRI, in a large postmortem sample, focusing on axonal loss. Tissue samples from 17 patients were selected immediately postmortem for histopathological analysis on the basis of T2‐weighted imaging, including normal appearing white matter and T1 hypointense lesions. In each region, we measured magnetization transfer ratios (MTR), T1 contrast ratio, myelin, and axonal density. T2 lesions (109 samples) were heterogeneous with regard to MRI appearance on T1 and MTR, whereas axonal density ranged from 0% (no residual axons) to 100% (normal axonal density). Of 64 T2 lesions, 17 were reactive (mild perivascular inflammation only), 21 active, 15 chronically active, and 11 chronically inactive. MTR and T1 contrast ratio correlated strongly with axonal density. Also in normal appearing white matter (24 samples), MTR correlated with axonal density. In conclusion, postmortem tissue sampling by using MRI revealed a range of pathology, illustrating the high sensitivity and low specificity of T2‐weighted imaging. T1 hypointensity and MTR were strongly associated with axonal density, emphasizing their role in monitoring progression in multiple sclerosis.

GAB2 Alleles Modify Alzheimer's Risk in APOE ε4 Carriers

The apolipoprotein E (APOE) epsilon4 allele is the best established genetic risk factor for late-onset Alzheimers disease (LOAD). We conducted genome-wide surveys of 502,627 single-nucleotide polymorphisms (SNPs) to characterize and confirm other LOAD susceptibility genes. In epsilon4 carriers from neuropathologically verified discovery, neuropathologically verified replication, and clinically characterized replication cohorts of 1411 cases and controls, LOAD was associated with six SNPs from the GRB-associated binding protein 2 (GAB2) gene and a common haplotype encompassing the entire GAB2 gene. SNP rs2373115 (p = 9 x 10(-11)) was associated with an odds ratio of 4.06 (confidence interval 2.81-14.69), which interacts with APOE epsilon4 to further modify risk. GAB2 was overexpressed in pathologically vulnerable neurons; the Gab2 protein was detected in neurons, tangle-bearing neurons, and dystrophic neuritis; and interference with GAB2 gene expression increased tau phosphorylation. Our findings suggest that GAB2 modifies LOAD risk in APOE epsilon4 carriers and influences Alzheimers neuropathology.

Defects in IGF-1 receptor, insulin receptor and IRS-1/2 in Alzheimer's disease indicate possible resistance to IGF-1 and insulin signalling

Insulin like growth factor-1 receptor (IGF-1R) and insulin receptor (IR) signalling control vital growth, survival and metabolic functions in the brain. Here we describe specific and significant alterations in IGF-1R, IR, and their key substrate adaptor proteins IRS-1 and IRS-2 in Alzheimers disease (AD). Western immunoblot analysis detected increased IGF-1R levels, and decreased levels of IGF-1-binding protein-2 (IGFBP-2), a major IGF-1-binding protein, in AD temporal cortex. Increased IGF-1R was observed surrounding and within amyloid-beta (Abeta)-containing plaques, also evident in an animal model of AD, and in astrocytes in AD. However, despite the overall increase in IGF-1R levels, a significantly lower number of neurons expressed IGF-1R in AD, and IGF-1R was aberrantly distributed in AD neurons especially evident in those with neurofibrillary tangles (NFTs). IR protein levels were similar in AD and control cases, however, the IR was concentrated intracellularly in AD neurons, unlike its distribution throughout the neuronal cell soma and in dendrites in control brain. Significant decreases in IRS-1 and IRS-2 levels were identified in AD neurons, in association with increased levels of inactivated phospho(Ser312)IRS-1 and phospho(Ser616)IRS-1, where increased levels of these phosphoserine epitopes colocalised strongly with NFTs. Our results show that IGF-1R and IR signalling is compromised in AD neurons and suggest that neurons that degenerate in AD may be resistant to IGF-1R/IR signalling.

High prevalence of mutations in the microtubule-associated protein tau in a population study of frontotemporal dementia in the Netherlands

Mutations in microtubule-associated protein tau recently have been identified in familial cases of frontotemporal dementia (FTD). We report the frequency of tau mutations in a large population-based study of FTD carried out in the Netherlands from January 1994 to June 1998. Thirty-seven patients had >/=1 first-degree relative with dementia. A mutation in the tau gene was found in 17.8% of the group of patients with FTD and in 43% of patients with FTD who also had a positive family history of FTD. Three distinct missense mutations (G272V, P301L, R406W) accounted for 15.6% of the mutations. These three missense mutations, and a single amino acid deletion (DeltaK280) that was detected in one patient, strongly reduce the ability of tau to promote microtubule assembly. We also found an intronic mutation at position +33 after exon 9, which is likely to affect the alternative splicing of tau. Tau mutations are responsible for a large proportion of familial FTD cases; however, there are also families with FTD in which no mutations in tau have been found, which indicates locus and/or allelic heterogeneity. The different tau mutations may result in disturbances in the interactions of the protein tau with microtubules, resulting in hyperphosphorylation of tau protein, assembly into filaments, and subsequent cell death.

Histopathologic correlates of white matter changes on MRI in Alzheimer's disease and normal aging

Article abstract—We investigated the histopathologic correlates of white matter changes in Alzheimers disease (AD) patients (n = 6) and controls (n = 9) using postmortem MRT. White matter changes were rated on a 0 to 3 scale in 51 regions. Histopathologically, we subjectively rated the loss of myelinated axons in the deep and periventricular white matter, denudation of the ventricular ependyma, gliosis, width of the perivascular spaces, and leptomeningeal con-gophilic angiopathy; we measured structural changes in the walls of the blood vessels in the white matter in micrometers. The AD brains displayed significantly more white matter hyperintensities on MRI than controls. Histopathologically, the denudation of the ventricular ependyma and the gliosis were significantly more severe in AD than in controls, and there was a trend toward more loss of myelinated axons in the deep white matter in the AD brains (p = 0.07). The MRI abnormalities correlated with the loss of myelinated axons in the deep white matter (r‘ = 0.37; p <0.01) and with the denudation of the ventricular lining (r’ = 0.54; p <0.01). We could not find any evidence for arteriolosclerosis, but the mean thickness of the adventitia of the arteries of the deep white matter in AD almost doubled the value in control brains (p = 0.0009). We conclude that white matter abnormalities in AD patients and controls consist of loss of myelinated axons, probably caused by arterial changes and breakdown of the ventricular lining. Since imaginghistopathologic correlation was similar in AD patients and controls, these changes probably represent some form of accelerated aging.

Activation of Akt/PKB, increased phosphorylation of Akt substrates and loss and altered distribution of Akt and PTEN are features of Alzheimer's disease pathology

Studies suggest that activation of phosphoinositide 3‐kinase‐Akt may protect against neuronal cell death in Alzheimers disease (AD). Here, however, we provide evidence of increased Akt activation, and hyperphosphorylation of critical Akt substrates in AD brain, which link to AD pathogenesis, suggesting that treatments aiming to activate the pathway in AD need to be considered carefully. A different distribution of Akt and phospho‐Akt was detected in AD temporal cortex neurons compared with control neurons, with increased levels of active phosphorylated‐Akt in particulate fractions, and significant decreases in Akt levels in AD cytosolic fractions, causing increased activation of Akt (phosphorylated‐Akt/total Akt ratio) in AD. In concordance, significant increases in the levels of phosphorylation of total Akt substrates, including: GSK3βSer9, tauSer214, mTORSer2448, and decreased levels of the Akt target, p27kip1, were found in AD temporal cortex compared with controls. A significant loss and altered distribution of the major negative regulator of Akt, PTEN (phosphatase and tensin homologue deleted on chromosome 10), was also detected in AD neurons. Loss of phosphorylated‐Akt and PTEN‐containing neurons were found in hippocampal CA1 at end stages of AD. Taken together, these results support a potential role for aberrant control of Akt and PTEN signalling in AD.

Gene expression profiling of parkinsonian substantia nigra pars compacta; alterations in ubiquitin-proteasome, heat shock protein, iron and oxidative stress regulated proteins, cell adhesion/cellular matrix and vesicle trafficking genes

Summary.Gene expression profiling of human substantia nigra pars compacta (SNpc) from Parkinson’s disease (PD) patients, was examined employing high density microarrays. We identified alterations in the expression of 137 genes, with 68 down regulated and 69 up regulated. The down regulated genes belong to signal transduction, protein degradation (e.g. ubiquitin-proteasome subunits), dopaminergic transmission/metabolism, ion transport, protein modification/phosphorylation and energy pathways/glycolysis functional classes. Up-regulated genes, clustered mainly in biological processes involving cell adhesion/cytoskeleton, extracellular matrix components, cell cycle, protein modification/phosphorylation, protein metabolism, transcription and inflammation/stress (e.g. key iron and oxygen sensor EGLN1). One major finding in the present study is the particular decreased expression of SKP1A, a member of the SCF (E3) ligase complex specifically in the substantia nigra (SN) of sporadic parkinsonian patients, which may lead to a wide impairment in the function of an entire repertoire of proteins subjected to regulatory ubiquitination. These findings reveal novel players in the neurodegenerative scenario and provide potential targets for the development of novel drug compounds.

Transfer of Central Nervous System Autoantigens and Presentation in Secondary Lymphoid Organs

Dendritic cells are thought to regulate tolerance induction vs immunization by transferring Ags and peripheral signals to draining lymph nodes (LN). However, whether myelin Ag transfer and presentation in LN occurs during demyelinating brain disease is unknown. In this study, we demonstrate redistribution of autoantigens from brain lesions to cervical LN in monkey experimental autoimmune encephalomyelitis (EAE) and in multiple sclerosis (MS). Immunohistochemical analysis revealed significantly more cells containing myelin Ags in cervical LN of monkeys with EAE compared with those of healthy control monkeys. Myelin Ags were observed in cells expressing dendritic cell/macrophage-specific markers, MHC class II, and costimulatory molecules. Moreover, these cells were directly juxtaposed to T cells, suggesting that cognate interactions between myelin-containing APC and T cells are taking place in brain-draining LN. Indeed, myelin Ag-reactive T cells were observed in cervical LN from marmosets and rhesus monkeys. Importantly, these findings were paralleled by our findings in human tissue. We observed significantly more myelin Ag-containing cells in LN of individuals with MS compared with those of control individuals. These cells expressed APC markers, as observed in marmosets and rhesus monkeys. These findings suggest that during MS and EAE, modulation of T cell reactivity against brain-derived Ags also takes place in cervical LN and not necessarily inside the brain. A major implication is that novel therapeutic strategies may be targeted to peripheral events, thereby circumventing the blood-brain barrier.

Expression of CCR7 in multiple sclerosis: Implications for CNS immunity

It is unclear how immune cells traffic between the lymphoid compartment and the central nervous system (CNS), which lacks lymphatic vessels and is shielded by the blood–brain barrier. We studied the expression of CCR7, a chemokine receptor required for migration of T cells and dendritic cells (DCs) to lymphoid organs, in the CNS of patients with multiple sclerosis (MS) to gain insight into pathways for CNS immune cell trafficking. Inflamed MS lesions contained numerous CCR7+ myeloid cells expressing major histocompatibility complex class II, CD68 and CD86, consistent with maturing DCs. CCR7+ DCs also were identified in cerebrospinal fluid (CSF). These observations suggested that the afferent limb of CNS immunity is comprised, in part, of DCs, which are generated within the CNS and migrate to deep cervical lymph nodes through the CSF after antigen capture. Ninety percent of CSF T cells expressed CCR7 and CSF from patients with MS was relatively depleted of CCR7‐negative effector‐memory T cells. In contrast, all T cells in parenchymal MS lesions lacked CCR7, indicating local retention and differentiation of central‐memory T cells upon restimulation by antigen within the CNS. These data suggested that the efferent limb of CNS immunity is executed by central‐memory T cells, which enter CSF directly from the circulation. Ann Neurol 2004

Toll‐like receptor 3 on adult human astrocytes triggers production of neuroprotective mediators

Toll‐like receptors (TLRs) are innate immunity receptors that are expressed on a wide range of cell types, including CNS glial cells. In general, TLR engagement by specific sets of microbial ligands triggers production of pro‐inflammatory factors and enhances antigen‐presenting cell functions. The functional roles of TLR in the CNS, however, are still poorly understood. While adult human astrocytes in culture dominantly express TLR4, they display a strikingly strong and selective induction of TLR3 when activated by pro‐inflammatory cytokines, TLR3 or TLR4 agonists, or oxidative stress. Gene profiling analysis of the astrocyte response to either TLR3 or TLR4 activation revealed that TLR3, but not TLR4, induces expression of a range of neuroprotective mediators and several other molecules that regulate cellular growth, differentiation, and migration. Also, TLR3 triggered enhanced production of anti‐inflammatory cytokines including interleukin‐9 (IL‐9), IL‐10, and IL‐11 and downregulation of the p40 subunit of IL‐12 and IL‐23. The collective TLR3‐induced products were found in functional assays to inhibit astrocyte growth, promote human endothelial cell growth, and importantly, to enhance neuronal survival in organotypic human brain slice cultures. Together, our data indicate that TLR3 is induced on human astrocytes upon inflammation and when activated, mediates a comprehensive neuroprotective response rather than a polarized pro‐inflammatory reaction.