Lafora disease in the Indian population: EPM2A and NHLRC1 gene mutations and their impact on subcellular localization of laforin and malin

Lafora disease (LD) is a fatal form of teenage-onset autosomal recessive progressive myoclonus epilepsy. LD is more common among geographic isolates and in populations with a higher rate of consanguinity. Mutations in two genes, EPM2A encoding laforin phosphatase, and NHLRC1 encoding malin ubiquitin ligase, have been shown to cause the LD. We describe here a systematic analysis of the EPM2A and the NHLRC1 gene sequences in 20 LD families from the Indian population. We identified 12 distinct mutations in 15 LD families. The identified novel mutations include 4 missense mutations (K140N, L310W, N148Y, and E210 K) and a deletion of exon 3 for EPM2A, and 4 missense mutations (S22R, L279P, L279P, and L126P) and a single base-pair insertional mutation (612insT) for NHLRC1. The EPM2A gene is known to encode two laforin isoforms having distinct carboxyl termini; a major isoform localized in the cytoplasm, and a minor isoform that targeted the nucleus. We show here that the effect of the EPM2A gene mutation L310W was limited to the cytoplasmic isoform of laforin, and altered its subcellular localization. We have also analyzed the impact of NHLRC1 mutations on the subcellular localization of malin. Of the 6 distinct mutants tested, three targeted the nucleus, one formed perinuclear aggregates, and two did not show any significant difference in the subcellular localization as compared to the wild-type malin. Our results suggest that the altered subcellular localization of mutant proteins of the EPM2A and NHLRC1 genes could be one of the molecular bases of the LD phenotype.     

Resident alveolar macrophages are replaced by recruited monocytes in response to endotoxin-induced lung inflammation

In the acute respiratory distress syndrome, recruitment of peripheral blood monocytes results in expansion of the total pool of resident alveolar macrophages. The fate of resident macrophages, or whether recruited monocytes are selectively eliminated from the alveolar airspace or differentiate into resident alveolar macrophages during the resolving phase of inflammation, has not been determined. Here, we analyzed the kinetics of resident and recruited macrophage turnover within the alveolar airspace of untreated and LPS-challenged mice. Using bone marrow chimeric CD45.2 mice that were generated by lethal irradiation of CD45.2 alloantigen-expressing recipient mice and bone marrow transplantation from CD45.1 alloantigen-expressing donor mice, we employed a flow cytometric approach to distinguish recipient from donor-type macrophages in bronchoalveolar lavage fluids. Our data show that resident alveolar macrophages of untreated chimeric CD45.2 mice are very slowly replaced by constitutively immigrating CD45.1 positive monocytes, resulting in a replacement rate of approximately 40% by 1 yr. In contrast, more than 85% of the resident CD45.2 positive alveolar and lung homogenate macrophages were exchanged by donor CD45.1-expressing macrophages within 2 mo after treatment with Escherichia coli endotoxin (LPS). Importantly, fluorescence-activated cell sorter analysis of increased annexin V binding to both recipient and donor-type macrophages revealed increased apoptotic events to underlie this endotoxin-driven inflammatory macrophage turnover. Collectively, the data show that under baseline conditions the alveolar macrophage turnover exhibits very slow kinetics, whereas acute lung inflammation in response to treatment with LPS triggers a brisk acceleration of recruitment of monocytes that replace the resident alveolar macrophage population.     

Heat shock proteins are present in mallory bodies (cytokeratin aggresomes) in human liver biopsy specimens

Mallory bodies (MBs) are aggresomes, composed of cytokeratin and various other proteins, which form in diseased liver because of disruption in the ubiquitin-proteasome protein degradation pathway. Heat shock proteins (hsp's) are thought to be involved in this process because it was discovered that MB formation is induced by heat shock in drug-primed mice. It has been reported that ubiquitin and a mutant form of ubiquitin (UBB(+1)) are found in aggresomes formed in the neurons in Alzheimer's disease and in the liver MBs in various liver diseases. In addition, hsp 70 has been found in aggresomes in Alzheimer's and in MBs in drug-primed mice. Therefore, we hypothesized that hsp's might be involved in MB formation in human liver diseases. Liver biopsy sections were double-stained using ubiquitin and hsp 70 or 90b antibodies. Both hsps 70 and 90b were found in MBs in all liver diseases investigated including primary billiary cirrhosis, nonalcoholic steatohepatitis, hepatitis B and C, idiopathic cirrhosis, alcoholic hepatitis, and hepatocellular carcinoma. Ubiquitin and the hsp's colocalized in all MBs in the diseased liver sections. These results indicate that hsp involvement in MB formation is similar to that seen in aggresome formation in other conformational diseases.     

Mutations in the NHLRC1 gene are the common cause for Lafora disease in the Japanese population

Lafora disease (LD) is a rare autosomal recessive genetic disorder characterized by epilepsy, myoclonus, and progressive neurological deterioration. LD is caused by mutations in the EMP2A gene encoding a protein phosphatase. A second gene for LD, termed NHLRC1 and encoding a putative E3 ubiquitin ligase, was recently identified on chromosome 6p22. The LD is relatively common in southern Europe, the Middle East, and Southeast Asia. A few sporadic cases with typical LD phenotype have been reported from Japan; however, our earlier study failed to find EPM2A mutations in four Japanese families with LD. We recruited four new families from Japan and searched for mutations in EPM2A . All eight families were also screened for NHLRC1 mutations. We found five independent families having novel mutations in NHLRC1. Identified mutations include five missense mutations (p.I153M, p.C160R, p.W219R, p.D245N, and p.R253K) and a deletion mutation (c.897insA; p.S299fs13). We also found a family with a ten base pair deletion (c.822-832del10) in the coding region of EPM2A. In two families, no EPM2A or NHLRC1 mutation was found. Our study, in addition to documenting the genetic and molecular heterogeneity observed for LD, suggests that mutations in the NHLRC1 gene may be a common cause of LD in the Japanese population.     

M-30 and 4HNE are sequestered in different aggresomes in the same hepatocytes

M-30 and 4HNE adducts are two markers of active liver disease. M-30 is a serologic marker and 4HNE adducts are histologic markers. M-30 is a marker for apoptosis because it is a fragment of cytokeratin-18 left over from proteolysis by caspase 3. 4HNE is a marker of oxidative stress because it results from lipid peroxidation. Both markers are commonly found in nonalcoholic steatohepatitis and in alcoholic hepatitis. Liver biopsies from patients with steatohepatitis, 11 alcoholic and 11 non-alcoholics were stained for 4HNE and M-30. Almost all of the biopsies in both groups showed 4HNE- and M-30-positive aggresomes in hepatocytes. Mallory Denk bodies (MDB) stained variably positive for M-30, whereas 4HNE was present in aggresomes independent of MDBs. However, they were sometimes located in hepatocytes which also contained MDBs as shown by confocal microscopy of double stained biopsies. The results indicate that the formation of M-30 and 4HNE aggresomes occurs through different pathways of liver cell injury in both types of steatohepatitis.     

Five out of 16 plasma signaling proteins are enhanced in plasma of patients with mild cognitive impairment and Alzheimer's disease

Alzheimer's disease (AD) is a progressive neurodegenerative disorder with characteristic neuropathological changes of the brain. Great efforts have been undertaken to determine the progression of the disease and to monitor therapeutic interventions. Especially, the analysis of blood plasma had yielded incongruent results. Recently, Ray et al. (Nat. Med. 13, 2007, 1359f) identified changes of 18 signaling proteins leading to an accuracy of 90% in the diagnosis of AD. The aim of the present study was to examine 16 of these signaling proteins by quantitative Searchlight multiplex ELISA in order to determine their sensitivity and specificity in our plasma samples from AD, mild cognitive impairment (MCI), depression with and without cognitive impairment and healthy subjects. Quantitative analysis revealed an increased concentration in Biocoll isolated plasma of 5 out of these 16 proteins in MCI and AD patients compared to healthy subjects: EGF, GDNF and MIP1δ (in AD), MIP4 (in MCI) and RANTES (in MCI and AD). ROC analysis predicted a sensitivity of 65-75% and a specificity of 52-63% when comparing healthy controls versus MCI or AD. Depression without any significant cognitive deficits did not cause any significant changes. Depressed patients with significant cognitive impairment were not different from MCI patients. In conclusion, we detected a number of altered proteins that may be related to a disease specific pathophysiology. However, the overall expression pattern of plasma proteins could not be established as a biomarker to differentiate MCI from AD or from depression.     

Rab GTPases are recruited to chlamydial inclusions in both a species-dependent and species-independent manner

Chlamydiae are obligate intracellular bacteria that replicate within an inclusion that is trafficked to the peri-Golgi region where it fuses with exocytic vesicles. The host and chlamydial proteins that regulate the trafficking of the inclusion have not been identified. Since Rab GTPases are key regulators of membrane trafficking, we examined the intracellular localization of several green fluorescent protein (GFP)-tagged Rab GTPases in chlamydia-infected HeLa cells. GFP-Rab4 and GFP-Rab11, which function in receptor recycling, and GFP-Rab1, which functions in endoplasmic reticulum (ER)-to-Golgi trafficking, are recruited to Chlamydia trachomatis, Chlamydia muridarum, and Chlamydia pneumoniae inclusions, whereas GFP-Rab5, GFP-Rab7, and GFP-Rab9, markers of early and late endosomes, are not. In contrast, GFP-Rab6, which functions in Golgi-to-ER and endosome-to-Golgi trafficking, is associated with C. trachomatis inclusions but not with C. pneumoniae or C. muridarum inclusions, while the opposite was observed for the Golgi-localized GFP-Rab10. Colocalization studies between transferrin and GFP-Rab11 demonstrate that a portion of GFP-Rab11 that localizes to inclusions does not colocalize with transferrin, which suggests that GFP-Rab11's association with the inclusion is not mediated solely through Rab11's association with transferrin-containing recycling endosomes. Finally, GFP-Rab GTPases remain associated with the inclusion even after disassembly of microtubules, which disperses recycling endosomes and the Golgi apparatus within the cytoplasm, suggesting a specific interaction with the inclusion membrane. Consistent with this, GFP-Rab11 colocalizes with C. trachomatis IncG at the inclusion membrane. Therefore, chlamydiae recruit key regulators of membrane trafficking to the inclusion, which may function to regulate the trafficking or fusogenic properties of the inclusion.     

Changes in sleep theta rhythm are related to episodic memory impairment in early Alzheimer's disease

Impairments have been reported both in sleep structure and episodic memory in Alzheimer's disease [AD]. Our objective was to investigate the relationships between episodic memory deficits and electro-encephalography [EEG] abnormalities occurring during sleep in patients with early AD. Postlearning sleep was recorded in 14 patients with mild to moderate AD, and 14 healthy elderly controls after they performed an episodic memory task derived from the Grober and Buschke's procedure. For each sleep stage, the relative power and mean frequency in each band were analyzed. Relative to agematched controls, AD patients presented faster mean theta frequency in both REM sleep and slow wave sleep [SWS]. In AD patients, a correlative analysis revealed that faster theta frequency during SWS was associated with better delayed episodic recall. We assume that increased theta activity reflects changes in neuronal activity to maintain memory performance, indicating that compensatory mechanisms already described at the waking state could also be engaged during SWS.     

Two checkpoint complexes are independently recruited to sites of DNA damage in vivo

The Ddc1/Rad17/Mec3 complex and Rad24 are DNA damage checkpoint components with limited homology to replication factors PCNA and RF-C, respectively, suggesting that these factors promote checkpoint activation by "sensing" DNA damage directly. Mec1 kinase, however, phosphorylates the checkpoint protein Ddc2 in response to damage in the absence of all other known checkpoint proteins, suggesting instead that Mec1 and/or Ddc2 may act as the initial sensors of DNA damage. In this paper, we show that Ddc1 or Ddc2 fused to GFP localizes to a single subnuclear focus following an endonucleolytic break. Other forms of damage result in a greater number of Ddc1-GFP or Ddc2-GFP foci, in correlation with the number of damage sites generated, indicating that Ddc1 and Ddc2 are both recruited to sites of DNA damage. Interestingly, Ddc2 localization is severely abrogated in mec1 cells but requires no other known checkpoint genes, whereas Ddc1 localization requires Rad17, Mec3, and Rad24, but not Mec1. Therefore, Ddc1 and Ddc2 recognize DNA damage by independent mechanisms. These data support a model in which assembly of multiple checkpoint complexes at DNA damage sites stimulates checkpoint activation. Further, we show that although Ddc1 remains strongly localized following checkpoint adaptation, many nuclei contain only dim foci of Ddc2-GFP, suggesting that Ddc2 localization may be down-regulated during resumption of cell division. Lastly, visualization of checkpoint proteins localized to damage sites serves as a useful tool for analysis of DNA damage in living cells.     

Spatial and object working memory deficits in Parkinson's disease are due to impairment in different underlying processes

Working memory maintenance processes for visual-spatial and visual-object information were evaluated in patients with Parkinson's disease (PD). PD patients and controls performed a working memory task with two conditions that differed only in the aspect of the stimuli that the participant was instructed to remember: their locations or shapes. Maintenance processes were investigated by measuring accuracy over 1-s, 5-s, and 10-s delays. Results indicated that patients were impaired in maintaining object information over the delay. In contrast, the patients showed impairment on the spatial condition only when the to-be-remembered stimulus was highly similar in location to the probe, but this impairment was equivalent across the delays, suggesting that this deficit was not due to maintenance impairment. These results suggest that deficits in working memory for spatial and object information are mediated by distinct cognitive processes in nondemented patients with PD and may differ in their pathophysiological basis.     

Plasma antioxidants are similarly depleted in mild cognitive impairment and in Alzheimer's disease

In order to assess peripheral levels and activities of a broad spectrum of non-enzymatic and enzymatic antioxidants in elderly subjects with mild cognitive impairment (MCI) and Alzheimer's disease (AD), plasma levels of water-soluble (Vitamin C and uric acid) and of lipophilic (Vitamin A, Vitamin E and carotenoids including lutein, zeaxanthin, beta-cryptoxanthin, lycopene, alpha- and beta-carotene) antioxidant micronutrients as well as activities of plasma and red blood cell (RBC) superoxide dismutase (SOD) and of plasma glutathione peroxidase (GPx) were measured in 25 patients with MCI, 63 AD patients and 53 controls. Peripheral levels and activities of antioxidants were similarly lower in MCI and AD patients as compared to controls. As MCI may represent a prodromal stage of AD, and oxidative damage appears to occur as one of the earliest pathophysiological events in AD, an increased intake of antioxidants in patients with MCI could be helpful in lowering the risk of conversion to dementia.     

Laforin, a dual specificity phosphatase involved in Lafora disease, regulates insulin response and whole-body energy balance in mice

Laforin is a dual specificity protein phosphatase involved in Lafora disease (LD), a fatal form of progressive myoclonus epilepsy characterized by neurodegeneration and the presence of intracellular polyglucosan inclusions (Lafora bodies) in different tissues. In this work, we describe that mice lacking laforin (epm2a-/-) have enhanced insulin response leading to altered whole-body energy balance. This enhanced insulin response overactivates the Akt pathway which increases glucose uptake in the heart, resulting in increased glycogen levels and the formation of polyglucosan inclusions. In addition, enhanced insulin response resulted in increased liver lipid biosynthesis, resulting in hepatic steatosis. On the contrary, overexpression in rat hepatoma FTO2B cells of native laforin but not of a form lacking phosphatase activity (C266S) resulted in attenuation of insulin signaling. These results define laforin as a new regulator of insulin sensitivity, which provides novel insights into LD pathogenesis and identifies this phosphatase as a potential novel component of the insulin signaling cascade.     

40-Hz steady state response in Alzheimer's disease and mild cognitive impairment

The 40-Hz steady state response (SSR) reflects early sensory processing and can be measured with electroencephalography (EEG). The current study compared the 40-Hz SSR in groups consisting of mild Alzheimer's disease patients (AD) (n = 15), subjects with mild cognitive impairment (MCI) (n = 20) and healthy elderly control subjects (n = 20). All participants were naïve for psychoactive drugs. Auditory click trains at a frequency of 40-Hz evoked the 40-Hz SSR. To evaluate test-retest reliability (TRR), subjects underwent a similar assessment 1 week after the first. The results showed a high TRR and a significant increase of 40-Hz SSR power in the AD group compared to MCI and controls. Furthermore a moderate correlation between 40-Hz SSR power and cognitive performance as measured by ADAS-cog was shown.The results suggest that 40-Hz SSR might be an interesting candidate marker of disease progression.     

Serum MCP-1 levels are increased in mild cognitive impairment and mild Alzheimer's disease

Upregulation of a number of chemokines, including monocyte chemotactic protein-1 (MCP-1), is associated with Alzheimer's disease (AD) pathological changes. Emerging evidence suggests that inflammatory events precede the clinical development of AD, as cytokine disregulation has been observed also in patients with mild cognitive impairment (MCI).

MCP-1 levels were evaluated in serum samples from 48 subjects with MCI, 94 AD patients and 24 age-matched controls. Significantly increased MCP-1 levels were found in MCI and mild AD, but not in severe AD patients as compared with controls. mRNA levels in peripheral blood mononuclear cells (PBMC), evaluated by quantitative RT-PCR analysis, paralleled serum MCP-1 levels. Moreover, a progressive MCP-1 decrease was observed over a 1-year follow up in a subgroup of MCI subjects converted to AD. MCP-1 upregulation is likely to be a very early event in AD pathogenesis, by far preceding the clinical onset of the disease. Nevertheless, as MCP-1 is likely to play a role in several pathologies with an inflammatory component, a possible usfulness as an early AD biomarker would be possible only in combination with other molecules.     

Id helix-loop-helix proteins are differentially expressed in gestational trophoblastic disease

AIMS: To assess the expression of Id proteins in trophoblastic tissues and to correlate this with clinical parameters, proliferative and apoptotic indices as well as to related oncogene expression. METHODS AND RESULTS: Immunohistochemistry for Id1, Id2, Id3 and Id4 was performed on 83 trophoblastic tissues including 17 normal first-trimester placentas, seven term placentas, 47 hydatidiform moles (HM), and 12 spontaneous miscarriages. The four Id proteins were predominantly expressed in the villous and implantation site intermediate trophoblast. Expression of Id1 in HM was significantly higher than that in normal placenta (P = 0.0006) and spontaneous miscarriage (P = 0.0001) but did not correlate with subsequent development of gestational trophoblastic neoplasia (GTN). Id1 expression correlated with the proliferation index as assessed by MCM7 (P = 0.003) and Ki67 (P = 0.017) and with the apoptotic activity assessed by TUNEL (P = 0.001) and M30 CytoDeath antibody (P = 0.013). Moreover, the expression of Id1 correlated with the expression of p53 (P = 0.004), p21(WAF1) (/CIP1) (P = 0.003) but not with p16 (P = 0.107). CONCLUSIONS: Id proteins may play a role in the regulation of proliferative and apoptotic activity in trophoblastic tissue and are potentially useful in differentiating molar and non-molar gestation, but are not helpful in predicting GTN.     

CD28+ intraepithelial lymphocytes with long telomeres are recruited within the inflamed ileal mucosa in Crohn disease.

Crohn disease is a chronic inflammatory bowel disease that involves all the intestine but predominantly alters the ileum. The disease largely depends on T cells, but the biologic role of intestinal intraepithelial lymphocytes (IEL) in transmural inflammation remains poorly characterized. To address this issue, a comparison of IEL and lamina propria lymphocytes (LPL) isolated from the uninvolved and the inflamed ileal mucosa of Crohn disease patients was performed. More CD8+ IEL (26% versus 8%) from the inflamed ileal mucosa expressed the CD28 receptor and the CD11a integrin than IEL from the uninvolved ileal mucosa, which were mostly CD28-. IEL had longer telomeres in the inflamed than in the uninvolved areas and a TCR Vbeta repertoire more similar to circulating T cells, suggesting that the increased proportion of CD28+ TCRalphabeta+ IEL within the inflamed mucosa is more likely due to recruited lymphocytes from the periphery that populate the epithelial layer than to the acquisition of the CD28 molecule by activated resident lymphocytes. In the uninvolved ileal mucosa, IEL from Crohn disease patients had shorter telomeric lengths than IEL from control patients, suggesting that they have been chronically stimulated. Such perturbation of the IEL population within the ileal mucosa could contribute to the inflammation in Crohn disease.