The relationship between somatic mtDNA rearrangements, human heart disease and aging

The lifetime accumulation of low-abundance, somatic mtDNA re-arrangements (sublimons) has been proposed as a potential contributor to aging, and also to diseases such as cardiomyopathy or coronary heart disease. Tissue-specific sublimons, varying in abundance by three orders of magnitude between individuals, have recently been observed in myocardium of control subjects. To study the relationship between myocardial sublimon levels and various types of cardiac disease and aging, we applied a semi-quantitative fluorescent PCR assay on cellular DNA extracted from left ventricle in a series of 300 well characterized male victims of sudden death up to age 70 (Helsinki Sudden Death Study). The most prevalent classes of sublimons were present at <0.1 to 91 copies per cell, but their abundance did not correlate with any cardiac disease phenotype. In multiple regression analyses age (beta = 0.43, P < 0.0001) and smoking (bet = 0.25, P = 0.012) were the only independent factors found to correlate with sublimon levels. Thus, sublimons are inferred to accumulate with age in myocardium of a subset of individuals, but to levels where they do not appear to have any phenotypic effects during a typical life span. We propose that, instead of being a causal factor in cardiac aging, sublimons co-exist with wild-type mtDNA in an equilibrium which is regulated by as yet unknown mechanisms.     

Histological heterogeneity and somatic mtDNA mutations in gastric intraepithelial neoplasia.

Somatic mutations of mitochondrial DNA (mtDNA) are associated with various types of human cancer. To elucidate their role in gastric carcinogenesis, we analyzed mutations in the displacement loop region of mtDNA in 24 paraffin-embedded gastric intraepithelial neoplasias (formerly dysplasia) from a high gastric cancer risk area in northern Italy. Helicobacter pylori infection was assessed by histological examination (Giemsa staining). Gastritis was classified according to the guidelines of the Updated Sydney System. The mtDNA displacement loop region was amplified and sequenced from gastric intraepithelial neoplasia samples and adjacent non-neoplastic gastric mucosa. The gastric intraepithelial neoplasias were divided into two groups by their association with H. pylori gastritis. Group A with lesions arising on a background of H. pylori-positive gastritis contained 7 patients, and group B with lesions associated with H. pylori-negative gastritis contained 17 patients. Group A had a larger proportion of high-grade lesions than group B and showed a foveolar phenotype (type II dysplasia). Group B had a larger proportion of cases with mtDNA displacement loop region mutations than group A (P=0.004, Fisher's exact test) and exhibited an intestinal phenotype. No evidence of heteroplasmic variants in the mtDNA displacement loop, suggestive of mutations, was detected in gastric biopsies from 25 H. pylori-negative subjects and 60 cancer-unaffected H. pylori-positive patients. These results provide further evidence for the morphologic and mtDNA biomolecular differences of gastric intraepithelial neoplasias, and suggest the existence of two distinct pathways to gastric cancer--corpus-dominant H. pylori gastritis and the atrophy-metaplasia pathway.     

Convergent cholinergic activities in aging and Alzheimer's disease.

Choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) activities have been examined postmortem in a series of 66 individuals with no evidence of CNS disease, ranging in age from 24 gestational weeks to 95 years and in 33 cases of Alzheimer's disease (AD) aged 57-89 years. In the normal human hippocampus a striking and highly significant age-related decline in ChAT occurred from middle to old age (between 40 and 100 years); a trend apparent at a later stage and to a lesser extent in the hippocampal gyrus. In both areas enzyme activity in AD was inversely related to age at death; reductions compared with the normal were on average 70-80% in the 60-70 year old groups compared with 30-40% in the 80-90 year old group. A similar trend was apparent with respect to acetylcholinesterase (AchE) histochemical activity associated with fibers and terminals (predominantly cholinergic and concentrated in CA3 and 4 of the hippocampus) but not with reactive perikarya (considered to be noncholinergic) present in both hippocampus and cortex. These data indicate that the normal aging human hippocampus may constitute a useful model for investigating the dysfunction or degeneration of basal forebrain cholinergic neurons in AD.     

High frequency of mitochondrial complex I mutations in Parkinson's disease and aging

Idiopathic Parkinson’s disease (PD) involves a systemic loss of activity of complex I of the mitochondrial electron transport chain. This biochemical lesion plays a key pathogenic role. Transfer of PD mitochondrial DNA recapitulates this loss of activity and several other pathogenic features of PD suggesting that this lesion may arise, at least in part, from mitochondrial DNA. We investigated this possibility by an extensive clonal sequencing of the seven mitochondrial genes encoding complex I subunits in PD and age-matched control frontal cortex. Each gene was completely sequenced an average of 94.4 times for each subject. Aminoacid-changing mutations were found at the frequency of 59.3 per million bases in both PD and controls, corresponding to approximately 32% of the mitochondrial genomes in the average sample having at least one mutation in a complex I gene. Individual low frequency mutations had an abundance of 1–10%. Significant interindividual variation in mutation frequency was observed. Several aminoacid-changing mutations were identified and multiple PD brains but not in controls. Genetic algorithm analysis detected areas in ND genes with a higher mutation frequency in PD that allowed differentiation of PD from controls. Total mutational burden due to low-abundance heteroplasmy is high and may play a role in human disease.     

Presenilin mutations in Alzheimer's disease

The presenilins (PS-1 and PS-2) are 2 members of a novel family of genes encoding integral membrane proteins recently implicated in Alzheimer's disease (AD) pathology. To date, 43 mutations have been identified in PS-1 and 2 in PS-2 that lead to familial presenile AD (onset before age 65 years). The normal and pathological functions of the PS proteins (ps-1 and ps-2) are unknown, but their high degree of homology predicts similar biological activities. Homologies with ps from other species suggest that they may play a role in intracellular protein sorting and trafficking, in intercellular cell signaling, or in cell death. Since to date only missense mutations and in-frame deletions were identified, it is believed that mutated ps act through either a gain of (dys-)function or a dominant negative effect. In vivo and in vitro studies have linked PS mutations to amyloid deposition, an early pathological event in AD brains. Hum Mutat 11:183–190, 1998. © 1998 Wiley-Liss, Inc.     

Immunization reverses memory deficits without reducing brain Abeta burden in Alzheimer's disease model

We have previously shown that chronic treatment with the monoclonal antibody m266, which is specific for amyloid beta-peptide (Abeta), increases plasma concentrations of Abeta and reduces Abeta burden in the PDAPP transgenic mouse model of Alzheimer's disease (AD). We now report that administration of m266 to PDAPP mice can rapidly reverse memory deficits in both an object recognition task and a holeboard learning and memory task, but without altering brain Abeta burden. We also found that an Abeta/antibody complex was present in both the plasma and the cerebrospinal fluid of m266-treated mice. Our data indicate that passive immunization with this anti-Abeta monoclonal antibody can very rapidly reverse memory impairment in certain learning and memory tasks in the PDAPP mouse model of AD, owing perhaps to enhanced peripheral clearance and (or) sequestration of a soluble brain Abeta species.     

Implications of presenilin 1 mutations in Alzheimer's disease.

Mutations in presenilin 1 (PS1) and presenilin 2 (PS2) are the most common genetic factors underlying the development of early-onset familial Alzheimer's disease (FAD). To investigate the pathogenic mechanism of PS1 mutations linked to FAD, we established inducible mouse neuroblastoma (Neuro 2a) cell lines expressing the human wild-type (wt) or mutated PS1(M146L or deltaexon 10) under the control of the Lac repressor. Using this inducible PS1 system, the influence of PS1 mutations on the generation of endogenous murine Abeta species was assessed using a highly sensitive immunoblotting technique. The induction of mutated PS1 resulted in an increase in the extra- and intracellular levels of two distinct Abeta species ending at residue 42, Abeta1-42 and its N-terminally truncated variant(s), Abetax-42. In addition, the intracellular generation of these Abeta42 species was completely blocked by brefeldin A. In contrast, it exhibited differential sensitivities to monensin such that there was an increased accumulation of intracellular Abetax-42 but an inhibition of intracellular Abeta1-42 generation. These data strongly suggest that Abetax-42 is generated in a proximal Golgi compartment, whereas Abeta1-42 is generated in a distal Golgi and/or a post-Golgi compartment. Thus, it appears that PS1 mutations enhance the degree of 42-specific gamma-secretase cleavage which occurs (i) in the ER or the early Golgi apparatus prior to gamma-secretase cleavage, or (ii) in the distinct sites where Abetax-42 and Abeta1-42 are generated. To date, the site of Abeta42 generation has not been firmly established. Our data provide new information regarding the site of Abeta42 generation mediated by the FAD-linked mutant PS1.     

Fragile sites, Alzheimer's disease, and aging.

The fragile site expression under conditions of folate deprivation was compared in the chromosomes from 5 Alzheimer's disease (AD) female patients, 5 healthy elderly females and 5 healthy young females. Although different fragile sites were observed in the three groups, nevertheless, more similarities were found between the AD patients and elderly normal donors. The only fragile site common to all groups was 3p14. This site was the most frequent in the young donors group. In both AD and elderly control groups we observed a higher frequency of fragility in 6p21, but not in the young controls. Other interesting fragility points observed in these two groups were: 6q21 and 14q24 (in the AD patients) and 9q13, 14q24 and 17q21 (in the healthy aged). 6p21 and 17q21 have been proposed as 'new' fragile sites. We confirm the existence of these fragile sites and comment that in these bands the genes MTBT2 and MTBT1, which are microtubule (beta) associated protein tau-like and tau 1, respectively, are mapped. The tau protein is a component of paired helical filaments which accumulate in degenerating neurons in the brain of patients with AD and with less intensity of normal elderly individuals.     

Neurohypophyseal peptides in aging and Alzheimer's disease

The neurohypophyseal hormones arginine-vasopressin (AVP) and oxytocin (OT) are produced in the neurons of the hypothalamic supraoptic (SON) and paraventricular (PVN) nucleus and in the much smaller cells of the suprachiasmatic (SCN) nucleus. The SON is the main source of plasma AVP. Part of the AVP and OT neurons of the PVN join the hypothalamo-neurohypophyseal tract, whereas others send projections to the median eminence or various brain areas, where AVP and OT are involved in a number of central functions as neurotransmitters/neuromodulators. AVP and OT from the PVN can also regulate via the autonomous innervation endocrine glands and fat tissue. OT is produced for a major part in the PVN but some OT neurons are present in the SON. Moreover, both AVP and OT containing neurons are observed in the "accessory nuclei", i.e. islands situated between the SON and PVN. The SCN is the biological clock, and the number of AVP expressing neurons in the SCN shows both diurnal and seasonal rhythms. In addition to these hypothalamic areas, AVP and OT may be found to a lesser extent in some other brain areas, such as the bed nucleus of the stria terminalis, diagonal band of Broca, nucleus basalis of Meynert, lateral septal nucleus, globus pallidus and the anterior amygdaloid nucleus, as well as in the peripheral tissues. The AVP and OT containing neurons should not be considered as one system. Prominent functional differences exist between the different nuclei. The heterogeneity also becomes clear from the marked differences in the neurohypophyseal peptides containing neurons of the SON, PVN and SCN during aging, and in the most prevalent age-related neurodegenerative diseases, i.e. Alzheimer's disease (AD). For those reasons, we will discuss the SON, PVN and SCN separately.     

Processing of semantic relations in normal aging and Alzheimer's disease.

The decline in semantic memory observed in Alzheimer's disease is presumed to result from progressive loss of the attributes underlying category representation. Here, we explored the possibility that semantic deterioration would affect attributes differently, depending on the type of semantic relationship connecting the subject and the object of the attribution. We compared the performance of 50 patients with probable Alzheimer's disease (APs) to that of 30 elderly controls in two semantic tasks: a verbal sentence verification task and a visual test of analogical relations, both including several types of semantic relations. On the sentence verification task, the performance of APs was comparable to that of elderly controls when statements were true, but deteriorated significantly when statements were false. This result was interpreted as a failure of controlled processes to successfully search semantic space when statements were incongruent or false. In addition, all participants found some semantic relations more difficult to process than others, with relative difficulty being consistent across tasks. Taxonomic semantic relations were the most difficult, while part/whole relations were the easiest, but also the ones to deteriorate most rapidly. In contrast, functional attributes were comparatively preserved as the disease progressed. These results emphasize the role of attention and semantic context in jointly determining access to relevant attributes and categories. Furthermore, they suggest that semantic memory impairments in Alzheimer's are affected by the type of processing and semantic relationship required by the task.     

Noradrenaline deficiency in brain increases beta-amyloid plaque burden in an animal model of Alzheimer's disease

Loss of Locus coeruleus (LC) noradrenergic (NA) neurons occurs in several neurodegenerative conditions including Alzheimer's disease (AD). In vitro and in vivo studies have shown that NA influences several features of AD disease including inflammation, neurodegeneration, and cognitive function. In the current study we tested if LC loss influenced beta amyloid (Abeta) plaque deposition. LC neuronal degeneration was induced in transgenic mice expressing mutant V717F human amyloid precursor protein (APP) by treatment with the selective neurotoxin N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine DSP4 (5mg/kg every 2 weeks beginning at age 3 months). At 9 months of age, when control mice show low amyloid load, DSP4-treated mice showed an approximately 5-fold increase in the average number of Abeta plaques. This was accompanied by an increase in the levels of APP C-terminal cleavage fragments. DSP4-treatment increased both microglial and astroglial activation. In vivo, DSP4-treatment decreased expression and activity of the Abeta degrading enzyme neprilysin, while in vitro NA increased phagocytosis of Abeta1-42 by microglia. These findings suggest that noradrenergic innervation from LC are needed to maintain adequate Abeta clearance, and therefore that LC degeneration could contribute to AD pathogenesis.     

Low frequency of mtDNA point mutations in patients with PEO associated with POLG1 mutations

Mitochondrial myopathy in progressive external ophthalmoplegia (PEO) has been associated with POLG1 mutations. POLG1 encodes the catalytic alpha subunit of polymerase gamma and is the only polymerase known to be involved in mtDNA replication. It has two functionally different domains, one polymerase domain and one exonuclease domain with proofreading activity. In this study we have investigated whether mtDNA point mutations are involved, directly or indirectly, in the pathogenesis of PEO. Muscle biopsy specimens from patients with POLG1 mutations, affecting either the exonuclease or the polymerase domain, were investigated. Single cytochrome c oxidase (COX)-deficient muscle fibers were dissected and screened for clonally expanded mtDNA point mutations using a sensitive denaturing gradient gel electrophoresis analysis, in which three different regions of mtDNA, including five different tRNA genes, were investigated. To screen for randomly distributed mtDNA point mutations in muscle, two regions of mtDNA including deletion breakpoints were investigated by high-fidelity PCR, followed by cloning and sequencing. Long-range PCR revealed multiple mtDNA deletions in all the patients but not the controls. No point mutations were identified in single COX-deficient muscle fibers. Cloning and sequencing of muscle homogenate identified randomly distributed point mutations at very low frequency in patients and controls (<1:50 000). We conclude that mtDNA point mutations do not appear to be directly or indirectly involved in the pathogenesis of mitochondrial disease in patients with different POLG1 mutations.     

Lipoprotein lipase mutations and Alzheimer's disease

Lipoprotein lipase (LPL) helps transfer lipids from lipoprotein particles to cells. In the brain, LPL is present in Alzheimer's disease (AD) amyloid plaques. LPL binds apolipoprotein E (ApoE) lipoprotein particles and low-density lipoprotein receptor-related protein (LRP), an ApoE receptor. Since polymorphisms in both ApoE and LRP influence AD risk, we sought to determine whether LPL mutations also affect AD risk. In a case-control study, the frequencies of two of the most common known LPL mutations were measured in European-Americans either clinically diagnosed or pathologically confirmed as AD or normal control (N) subjects. In clinically diagnosed subjects, the Ser447Ter mutation comprised 9.8% (62/630) of alleles in N and 3.8% (9/238) in AD, a significant difference (P = 0.0057), while the Asn291Ser mutation comprised 1.1% (5/460) of alleles in N and 5.1% (8/158) in AD, also a significant difference (P = 0.0073), though in pathologically confirmed subjects the allele frequencies for AD did not significantly differ from N for either mutation. In clinically diagnosed subjects, LPL mutations were associated with altered AD risk, suggesting a potential role for LPL in the causation of AD. Further studies in different populations should help clarify the questions raised by these results.     

Microglia show altered morphology and reduced arborization in human brain during aging and Alzheimer's disease

Changes in microglia function are involved in Alzheimer's disease (AD) for which ageing is the major risk factor. We evaluated microglial cell process morphologies and their gray matter coverage (arborized area) during ageing and in the presence and absence of AD pathology in autopsied human neocortex. Microglial cell processes were reduced in length, showed less branching and reduced arborized area with aging (case range 52–98 years). This occurred during normal ageing and without microglia dystrophy or changes in cell density. There was a larger reduction in process length and arborized area in AD compared to aged‐matched control microglia. In AD cases, on average, 49%–64% of microglia had discontinuous and/or punctate Iba1 labeled processes instead of continuous Iba1 distribution. Up to 16% of aged‐matched control microglia displayed discontinuous or punctate features. There was no change in the density of microglial cell bodies in gray matter during ageing or AD. This demonstrates that human microglia show progressive cell process retraction without cell loss during ageing. Additional changes in microglia occur with AD including Iba1 protein puncta and discontinuity. We suggest that reduced microglial arborized area may be an aging‐related correlate of AD in humans. These variations in microglial cells during ageing and in AD could reflect changes in neural‐glial interactions which are emerging as key to mechanisms involved in ageing and neurodegenerative disease.     

Cerebral microvascular pathology in aging and Alzheimer's disease

The aging of the central nervous system and the development of incapacitating neurological diseases like Alzheimer's disease (AD) are generally associated with a wide range of histological and pathophysiological changes eventually leading to a compromised cognitive status. Although the diverse triggers of the neurodegenerative processes and their interactions are still the topic of extensive debate, the possible contribution of cerebrovascular deficiencies has been vigorously promoted in recent years. Various forms of cerebrovascular insufficiency such as reduced blood supply to the brain or disrupted microvascular integrity in cortical regions may occupy an initiating or intermediate position in the chain of events ending with cognitive failure. When, for example, vasoconstriction takes over a dominating role in the cerebral vessels, the perfusion rate of the brain can considerably decrease causing directly or through structural vascular damage a drop in cerebral glucose utilization. Consequently, cerebral metabolism can suffer a setback leading to neuronal damage and a concomitant suboptimal cognitive capacity. The present review focuses on the microvascular aspects of neurodegenerative processes in aging and AD with special attention to cerebral blood flow, neural metabolic changes and the abnormalities in microvascular ultrastructure. In this context, a few of the specific triggers leading to the prominent cerebrovascular pathology, as well as the potential neurological outcome of the compromised cerebral microvascular system are also going to be touched upon to a certain extent, without aiming at total comprehensiveness. Finally, a set of animal models are going to be presented that are frequently used to uncover the functional relationship between cerebrovascular factors and the damage to neural networks.     

Attentional networks in normal aging and Alzheimer's disease

By combining a flanker task and a cuing task into a single paradigm, the authors assessed the effects of orienting and alerting on conflict resolution and explored how normal aging and Alzheimer's disease (AD) modulate these attentional functions. Orienting failed to enhance conflict resolution; alerting was most beneficial for trials without conflict, as if acting on response criterion rather than on information processing. Alerting cues were most effective in the older groups--healthy aging and AD. Conflict resolution was impaired only in AD. Orienting remained unchanged across groups. These findings provide evidence of different life span developmental and clinical trajectories for each attentional network.     

Herpesviruses in brain and Alzheimer's disease

It has been established, using polymerase chain reaction (PCR), that herpes simplex virus type 1 (HSV1) is present in a high proportion of brains of elderly normal subjects and Alzheimer's disease (AD) patients. It was subsequently discovered that the virus confers a strong risk of AD when in brain of carriers of the type 4 allele of the apolipoprotein E gene (apoE-epsilon4). This study has now sought, using PCR, the presence of three other herpesviruses in brain: human herpesvirus 6 (HHV6)-types A and B, herpes simplex virus type 2 (HSV2) and cytomegalovirus (CMV). HHV6 is present in a much higher proportion of the AD than of age-matched normal brains (70% vs. 40%, p=0.003) and there is extensive overlap with the presence of HSV1 in AD brains, but HHV6, unlike HSV1, is not directly associated in AD with apoE-epsilon4. In 59% of the AD patients' brains harbouring HHV6, type B is present while 38% harbour both type A and type B, and 3% type A. HSV2 is present at relatively low frequency in brains of both AD patients and normals (13% and 20%), and CMV at rather higher frequencies in the two groups (36% and 35%); in neither case is the difference between the groups statistically significant. It is suggested that the striking difference in the proportion of elderly brains harbouring HSV1 and HSV2 might reflect the lower proportion of people infected with the latter, or the difference in susceptibility of the frontotemporal regions to the two viruses. In the case of HHV6, it is not possible to exclude its presence as an opportunist, but alternatively, it might enhance the damage caused by HSV1 and apoE-epsilon4 in AD; in some viral diseases it is associated with characteristic brain lesions and it also augments the damage caused by certain viruses in cell culture and in animals.     

Mitochondrial DNA rearrangements in aging human brain and in situ PCR of mtDNA

Deletions of the mitochondrial DNA (mtDNA) have been shown to accumulate with age in a variety of species regardless of mean or maximal life span. This implies that such mutations are either a molecular biomarker of senescence or that they are more causally linked to senescence itself. One assay that can be used to detect these mtDNA mutations is the long-extension polymerase chain reaction assay. This assay amplifies approximately 16 kb of the mtDNA in mammalian mitochondria and preferentially amplifies mtDNAs that are either deleted or duplicated. We have applied this assay to the aging human brain and found a heterogeneous array of rearranged mtDNAs. In addition, we have developed in situ polymerase chain reaction to detect mtDNA within individual cells of both the mouse and the human brain as a first step in identifying and enumerating cells containing mutant mtDNAs in situ.