Immune proteins C1q and CD47 may contribute to aberrant microglia-mediated synapse loss in the aging monkey brain that is associated with cognitive impairment.

Cognitive impairment in learning, memory, and executive function occurs in normal aging even in the absence of Alzheimer's disease (AD). While neurons do not degenerate in humans or monkeys free of AD, there are structural changes including synapse loss and dendritic atrophy, especially in the dorsolateral prefrontal cortex (dlPFC), and these correlate with cognitive age-related impairment. Developmental studies revealed activity-dependent neuronal properties that lead to synapse remodeling by microglia. Microglia-mediated phagocytosis that may eliminate synapses is regulated by immune "eat me" and "don't eat me" signaling proteins in an activity-dependent manner, so that less active synapses are eliminated. Whether this process contributes to age-related synapse loss remains unknown. The present study used a rhesus monkey model of normal aging to investigate the balance between the "eat me" signal, complement component C1q, and the "don't eat me" signal, transmembrane glycoprotein CD47, relative to age-related synapse loss in dlPFC Area 46. Results showed an age-related elevation of C1q and reduction of CD47 at PSD95+ synapses that is associated with cognitive impairment. Additionally, reduced neuronal CD47 RNA expression was found, indicating that aged neurons were less able to produce the protective signal CD47. Interestingly, microglia do not show the hypertrophic morphology indicative of phagocytic activity. These findings suggest that in the aging brain, changes in the balance of immunologic proteins give microglia instructions favoring synapse elimination of less active synapses, but this may occur by a process other than classic phagocytosis such as trogocytosis.

Study of Alzheimer's disease- and frontotemporal dementia-associated genes in the Cretan Aging Cohort.

Using exome sequencing, we analyzed 196 participants of the Cretan Aging Cohort (CAC; 95 with Alzheimer's disease [AD], 20 with mild cognitive impairment [MCI], and 81 cognitively normal controls). The APOE ε4 allele was more common in AD patients (23.2%) than in controls (7.4%; p < 0.01) and the PSEN2 p.Arg29His and p.Cys391Arg variants were found in 3 AD and 1 MCI patient, respectively. Also, we found the frontotemporal dementia (FTD)-associated TARDBP gene p.Ile383Val variant in 2 elderly patients diagnosed with AD and in 2 patients, non CAC members, with the amyotrophic lateral sclerosis/FTD phenotype. Furthermore, the p.Ser498Ala variant in the positively selected GLUD2 gene was less frequent in AD patients (2.11%) than in controls (16%; p < 0.01), suggesting a possible protective effect. While the same trend was found in another local replication cohort (n = 406) and in section of the ADNI cohort (n = 808), this finding did not reach statistical significance and therefore it should be considered preliminary. Our results attest to the value of genetic testing to study aged adults with AD phenotype.

Aging compromises oligodendrocyte precursor cell maturation and efficient remyelination in the monkey brain.

Age-associated cognitive decline is common among otherwise healthy elderly people, even in the absence of Alzheimer's disease and neuron loss. Instead, white matter loss and myelin damage are strongly associated with cognitive decline. Myelin is subject to lifelong oxidative stress that damages the myelin sheath, which is repaired by cells of the oligodendrocyte lineage. This process is mediated by oligodendrocyte precursor cells (OPCs) that sense the damage and respond by proliferating locally and migrating to the region, where they differentiate into mature myelinating oligodendrocytes. In aging, extensive myelin damage, in combination with inefficient remyelination, leads to chronically damaged myelin and loss of efficient neuronal conduction. This study used the rhesus monkey model of normal aging to examine how myelin regeneration capacity is affected by age. Results show that older subjects have reduced numbers of new BCAS1 + myelinating oligodendrocytes, which are newly formed cells, and that this reduction is associated with poorer cognitive performance. Interestingly, this does not result from limited proliferation of progenitor OPCs. Instead, the transcription factor NKX2.2, which regulates OPCs differentiation, is significantly decreased in aged OPCs. This suggests that these OPCs have a diminished potential for differentiation into mature oligodendrocytes. In addition, mature oligodendrocytes have reduced RNA expression of two essential myelin protein markers, MBP and PLP. These data collectively suggest that in the normal aging brain, there is a reduction in regenerative OPCs as well as myelin production that impairs the capacity for remyelination.

Crystal structure of glutamate dehydrogenase 2, a positively selected novel human enzyme involved in brain biology and cancer pathophysiology.

INTRODUCTION: Mammalian glutamate dehydrogenase (hGDH1 in human cells) interconverts glutamate to α-ketoglutarate and ammonia while reducing NAD(P) to NAD(P)H. During primate evolution, humans and great apes have acquired hGDH2, an isoenzyme that underwent rapid evolutionary adaptation concomitantly with brain expansion, thereby acquiring unique catalytic and regulatory properties that permitted its function under conditions inhibitory to its ancestor hGDH1. Although the 3D-structures of GDHs, including hGDH1, have been determined, attempts to determine the hGDH2 structure were until recently unsuccessful. Comparison of the hGDH1/hGDH2 structures would enable a detailed understanding of their evolutionary differences. This work aimed at the determination of the hGDH2 crystal structure and the analysis of its functional implications. Recombinant hGDH2 was produced in the Spodoptera frugiperda ovarian cell line Sf21, using the Baculovirus expression system. Purification was achieved via a two-step chromatography procedure. hGDH2 was crystallized, X-ray diffraction data were collected using synchrotron radiation and the structure was determined by molecular replacement. The hGDH2 structure is reported at a resolution of 2.9 Å. The enzyme adopts a novel semi-closed conformation, which is an intermediate between known open and closed GDH1 conformations, differing from both. The structure enabled us to dissect previously reported biochemical findings and to structurally interpret the effects of evolutionary amino acid substitutions, including Arg470His, on ADP affinity. In conclusion, our data provide insights into the structural basis of hGDH2 properties, the functional evolution of hGDH isoenzymes, and open new prospects for drug design, especially for cancer therapeutics.

PS1 FAD mutants decrease ephrinB2-regulated angiogenic functions, ischemia-induced brain neovascularization and neuronal survival.

Microvascular pathology and ischemic lesions contribute substantially to neuronal dysfunction and loss that lead to Alzheimer disease (AD). To facilitate recovery, the brain stimulates neovascularization of damaged tissue via sprouting angiogenesis, a process regulated by endothelial cell (EC) sprouting and the EphB4/ephrinB2 system. Here, we show that in cultures of brain ECs, EphB4 stimulates the VE-cadherin/Rok-α angiogenic complexes known to mediate sprouting angiogenesis. Importantly, brain EC cultures expressing PS1 FAD mutants decrease the EphB4-stimulated γ-secretase cleavage of ephrinB2 and reduce production of the angiogenic peptide ephrinB2/CTF2, the VE-cadherin angiogenic complexes and EC sprouting and tube formation. These data suggest that FAD mutants may attenuate ischemia-induced brain angiogenesis. Supporting this hypothesis, ischemia-induced VE-cadherin angiogenic complexes, levels of neoangiogenesis marker Endoglin, vascular density, and cerebral blood flow recovery, are all decreased in brains of mouse models expressing PS1 FAD mutants. Ischemia-induced brain neuronal death and cognitive deficits also increase in these mice. Furthermore, a small peptide comprising the C-terminal sequence of peptide ephrinB2/CTF2 rescues angiogenic functions of brain ECs expressing PS1 FAD mutants. Together, our data show that PS1 FAD mutations impede the EphB4/ephrinB2-mediated angiogenic functions of ECs and impair brain neovascularization, neuronal survival and cognitive recovery following ischemia. Furthermore, our data reveal a novel brain angiogenic mechanism targeted by PS1 FAD mutants and a potential therapeutic target for ischemia-induced neurodegeneration. Importantly, FAD mutant effects occur in absence of neuropathological hallmarks of AD, supporting that such hallmarks may form downstream of mutant effects on neoangiogenesis and neuronal survival.

Apolipoprotein E ɛ4 (APOE ɛ4) Allele is Associated with Long Sleep Duration Among Elderly with Cognitive Impairment.

BACKGROUND: Apolipoprotein E gene (APOE) ɛ4 allele increases the risk for Alzheimer's disease (AD). Furthermore, among patients with cognitive impairment, longer sleep duration is associated with worse cognitive performance. To date, literature examining the associations between APOE ɛ4 allele and objective sleep duration is limited. OBJECTIVE: Our aim was to assess the association between APOE ɛ4 and objective sleep duration, among patients with mild cognitive impairment (MCI) and AD. A sub-sample of 89 patients with AD (n = 49) and MCI (n = 40) were recruited from a large, population-based cohort of 3,140 elders (>60 years) residing on Crete, Greece. METHODS: All participants underwent medical history/physical examination, extensive neuropsychiatric and neuropsychological evaluation, 3-day 24 h actigraphy and APOE ɛ4 allele genotyping. Comparisons of sleep duration variables between APOE ɛ4 allele carriers and non-carriers were assessed using ANCOVA, controlling for confounders. RESULTS: The sample included 18 APOE ɛ4 carriers and 71 non-carriers, aged 78.6±6.6 and 78.2±6.5 years, respectively. Comparisons between the APOE ɛ4 carriers and non-carriers revealed no significant differences in terms of demographic and clinical variables. In terms of objective sleep duration across the two groups, APOE ɛ4 carriers compared to non-carriers had significantly longer nighttime Total Sleep Time (nTST) (7.7±1.4 versus 7.2±1.3  h, respectively, p = 0.011), as well as 24 h TST (8.5±1.6 versus 7.8±1.5  h, respectively, p = 0.012). CONCLUSION: Among patients with MCI and AD, APOE ɛ4 carriers have longer objective nighttime and 24 h sleep duration compared to non-carriers. These findings further support that objective long sleep duration is a genetically-driven pre-clinical marker associated with worse prognosis in elderly with cognitive impairment.

Presenilin1 familial Alzheimer disease mutants inactivate EFNB1- and BDNF-dependent neuroprotection against excitotoxicity by affecting neuroprotective complexes of N-methyl-d-aspartate receptor.

Excitotoxicity is thought to play key roles in brain neurodegeneration and stroke. Here we show that neuroprotection against excitotoxicity by trophic factors EFNB1 and brain-derived neurotrophic factor (called here factors) requires de novo formation of 'survival complexes' which are factor-stimulated complexes of N-methyl-d-aspartate receptor with factor receptor and presenilin 1. Absence of presenilin 1 reduces the formation of survival complexes and abolishes neuroprotection. EPH receptor B2- and N-methyl-d-aspartate receptor-derived peptides designed to disrupt formation of survival complexes also decrease the factor-stimulated neuroprotection. Strikingly, factor-dependent neuroprotection and levels of the de novo factor-stimulated survival complexes decrease dramatically in neurons expressing presenilin 1 familial Alzheimer disease mutants. Mouse neurons and brains expressing presenilin 1 familial Alzheimer disease mutants contain increased amounts of constitutive presenilin 1-N-methyl-d-aspartate receptor complexes unresponsive to factors. Interestingly, the stability of the familial Alzheimer disease presenilin 1-N-methyl-d-aspartate receptor complexes differs from that of wild type complexes and neurons of mutant-expressing brains are more vulnerable to cerebral ischaemia than neurons of wild type brains. Furthermore, N-methyl-d-aspartate receptor-mediated excitatory post-synaptic currents at CA1 synapses are altered by presenilin 1 familial Alzheimer disease mutants. Importantly, high levels of presenilin 1-N-methyl-d-aspartate receptor complexes are also found in post-mortem brains of Alzheimer disease patients expressing presenilin 1 familial Alzheimer disease mutants. Together, our data identify a novel presenilin 1-dependent neuroprotective mechanism against excitotoxicity and indicate a pathway by which presenilin 1 familial Alzheimer disease mutants decrease factor-depended neuroprotection against excitotoxicity and ischaemia in the absence of Alzheimer disease neuropathological hallmarks which may form downstream of neuronal damage. These findings have implications for the pathogenic effects of familial Alzheimer disease mutants and therapeutic strategies.

Differential interaction of hGDH1 and hGDH2 with manganese: Implications for metabolism and toxicity.

Manganese (Mn) is an essential trace element that serves as co-factor for many important mammalian enzymes. In humans, the importance of this cation is highlighted by the fact that low levels of Mn cause developmental and metabolic abnormalities and, on the other hand, chronic exposure to excessive amounts of Mn is characterized by neurotoxicity, possibly mediated by perturbation of astrocytic mitochondrial energy metabolism. Here we sought to study the effect of Mn on the two human glutamate dehydrogenases (hGDH1 and hGDH2, respectively), key mitochondrial enzymes involved in numerous cellular processes, including mitochondrial metabolism, glutamate homeostasis and neurotransmission, and cell signaling. Our studies showed that, compared to magnesium (Mg) and calcium (Ca), Mn exerted a significant inhibitory effect on both human isoenzymes with hGDH2 being more sensitive than hGDH1, especially under conditions of low ADP levels. Specifically, in the presence of 0.25 mM ADP, the Mn IC50 was 1.14 ± 0.02 mM and 1.54 ± 0.08 mM for hGDH2 and for hGDH1, respectively (p = 0.0001). Increasing Mn levels potentiated this differential effect, with 3 mM Mn inhibiting hGDH2 by 96.5% and hGDH1 by 70.2%. At 1 mM ADP, the Mn IC50 was 1.84 ± 0.02 mM and 2.04 ± 0.07 mM (p = 0.01) for hGDH2 and hGDH1, respectively, with 3 mM Mn inhibiting hGDH2 by 93.6% and hGDH1 by 70.9%. These results were due to the sigmoidal inhibitory curve of Mn that was more pronounced for hGDH2 than for hGDH1. Indeed, at 0.25 mM, the Hill coefficient value was higher for hGDH2 (3.42 ± 0.20) than for hGDH1 (1.94 ± 0.25; p = 0.0002) indicating that interaction of Mn with hGDH2 was substantially more co-operative than for hGDH1. These findings, showing an enhanced sensitivity of the hGDH2 isoenzyme to Mn, especially at low ADP levels, might be of pathophysiological relevance under conditions of Mn neurotoxicity.

The odyssey of a young gene: structure-function studies in human glutamate dehydrogenases reveal evolutionary-acquired complex allosteric regulation mechanisms.

Mammalian glutamate dehydrogenase (GDH) catalyzes the reversible inter-conversion of glutamate to α-ketoglutarate and ammonia, interconnecting carbon skeleton and nitrogen metabolism. In addition, it functions as an energy switch by its ability to fuel the Krebs cycle depending on the energy status of the cell. As GDH lies at the intersection of several metabolic pathways, its activity is tightly regulated by several allosteric compounds that are metabolic intermediates. In contrast to other mammals that have a single GDH-encoding gene, humans and great apes possess two isoforms of GDH (hGDH1 and hGDH2, encoded by the GLUD1 and GLUD2 genes, respectively) with distinct regulation pattern, but remarkable sequence similarity (they differ, in their mature form, in only 15 of their 505 amino-acids). The GLUD2 gene is considered a very young gene, emerging from the GLUD1 gene through retro-position only recently (<23 million years ago). The new hGDH2 iso-enzyme, through random mutations and natural selection, is thought to have conferred an evolutionary advantage that helped its persistence through primate evolution. The properties of the two highly homologous human GDHs have been studied using purified recombinant hGDH1 and hGDH2 proteins obtained by expression of the corresponding cDNAs in Sf21 cells. According to these studies, in contrast to hGDH1 that maintains basal activity at 35-40 % of its maximal, hGDH2 displays low basal activity that is highly responsive to activation by rising levels of ADP and/or L-leucine which can also act synergistically. While hGDH1 is inhibited potently by GTP, hGDH2 shows remarkable GTP resistance. Furthermore, the two iso-enzymes are differentially inhibited by estrogens, polyamines and neuroleptics, and also differ in heat-lability. To elucidate the molecular mechanisms that underlie these different regulation patterns of the two iso-enzymes (and consequently the evolutionary adaptation of hGDH2 to a new functional role), we have performed mutagenesis at sites of difference in their amino acid sequence. Results showed that the low basal activity, heat-lability and estrogen sensitivity of hGDH2 could be, at least partially, ascribed to the Arg443Ser evolutionary change, whereas resistance to GTP inhibition has been attributed to the Gly456Ala change. Other amino acid substitutions studied thus far cannot explain all the remaining functional differences between the two iso-enzymes. Also, the Arg443Ser/Gly456Ala double mutation in hGDH1 approached the properties of wild-type hGDH2, without being identical to it. The insights into the structural mechanism of enzymatic regulation and the implications in cell biology provided by these findings are discussed.

Age at onset in Huntington's disease: replication study on the association of HAP1.

In recent years two association studies investigating the HAP1 T441M (rs4523977) polymorphism as a potential modifying factor of the age at onset (AAO) of Huntington's disease (HD), have been reported. Initially evidence for association was found between the M441 risk allele and the AAO. Subsequently, a second study, although failing to replicate these findings, found evidence for association between the same risk allele and AAO of motor symptoms (mAAO). In the present study, the role of the HAP1 T441M polymorphism as a modifier of the AAO in HD was investigated in a cohort of 298 Greek HD patients. In this cohort the CAG repeat number accounted for 55% of the variance in AAO. No association was found between the HAP1 T441M polymorphism and the AAO of HD.