ABC transporters function

       Using bioinformatic tools, we have identified 14 ABC transporters that are expressed in the brain. It is apparent that there are no brain-specific ABC transporters: all ABC transporters that have been identified as being expressed in brain are also expressed in at least one other tissue, and several are expressed quite widely. While Table 10.1 provides data about general tissue distribution, further analyses will be required to determine whether these transporters are localized to neurons, glia, or even to cerebral vasculature. The latter point is important, as ABC transporters such as MDR1 and MRP1 represent key elements of the blood-brain barrier and are prominently expressed in cerebrovascular endothelial cells (Cordon- Cardo et al., 1989). We have put forth the hypothesis that ABC transporters are involved in the process of detachment of AP from cellular membranes (Lam et al., 2001).

      A(3 is unlikely to aggregate while attached to the membrane, as the hydrophobic amino acids in the peptide COOH tail would be shielded by their association with the lipid bilayer. On the other hand, the likelihood of Ap aggregation increases substantially following membrane detachment. Thus, detachment of Ap} from the membrane may represent a critical change in the biophysical properties of the peptide, and may very well be a prerequisite to the aggregation events that are thought to be at the core of the pathology of Alzheimer's disease. In summary, we have identified the suite of ABC transporters that are expressed in the brain. Such bioinformatic analysis is a prerequisite to the functional expression of each of these transporters in model systems.

    With such information in hand, we will be able to determine which brainexpressed ABC transporters function as (3-amyloid efflux pumps. Such proteins represent novel targets for the development of drugs that can regulate p-amyloid levels in the brain.

ABC transporter genes

         Allikmets, Gerrard, Hutchinson, and Dean (1996) characterized a large number of human ABC transporter genes by searching the human EST database using sequences derived from ABC (Walker A, ABC signature, and Walker B) of MDR1, as well as the entire sequence of cystic fibrosis transmembrane conductane regulator (CFTR). Because many of the ESTs are derived from brain libraries, the data suggested that many of these ABC transporters were expressed in brain. Because the number of ESTs deposited in public databases has increased substantially in the past 4 years, we recently repeated this analysis in order to ensure that the full complement of brain-expressed ABC transporters was represented in the database.

      Moreover, to increase the power of our search, we developed consensus amino acid sequences for the Walker A motif, as well as the ABC signature and Walker B motifs, by comparing the sequences of the following human ABC transporters.

      The BLAST programs (Altschul, Gish, Miller, Myers, & Lipman, 1990) were used to search the human database EST using these consensus sequences, as well as the complete amino acid sequences of the human ABC-transporter proteins identified above. Human clones with the highest scores were retrieved and primers were designed using the program Mac Vector (Oxford Molecular, UK). Reverse transcriptase-polymerase chain reaction (RT-PCR) was performed using brain fetal and adult total RNA (Invitrogen) in order to confirm human brain expression, and PCR products were sequenced to authenticate the ESTs. Based on both the cDNA library source used to generate these ESTs, as well as the expression profile of RNA as seen in Northern blots (Allikmets et al. 1996, and unpublished data), we identified the 14 ABC transporters listed in Table 10.1 as being brain-expressed.

Neurofibrillary tangles, senile plaques, and neuronal death

       Alzheimer's disease is characterized by neurofibrillary tangles, senile plaques, and neuronal death. The neurofibrillary tangles contain paired helical filaments composed of hyperphosphorylated tau, while the senile plaques are comprised of an array of proteins deposited around a core of insoluble Ab peptide.

       The cause of neuronal death remains unknown, but considerable evidence suggests that it is secondary to an increase in the brain Ab load. The molecular events involved in the constitutive production of Ap are increasingly being understood.

     The first step appears to be cleavage of the amyloid precursor protein (APP) by (3-secretase (Vassar et al., 1999; Lin et al., 2000), yielding an extracellular fragment known as sAPP(3, which is shed into the extracellular space (Mills & Reiner, 1999). Cleavage of C99 within the membrane by an enzyme known as y-secretase, which appears to be identical to the presenilins (Wolfe et al., 1999; Lin et al., 2000), liberates intact A{3. Both the 40 and 42 amino acid versions of A(3 are amphipathic, consisting of 28 charged amino acids and either 12 or 14 hydrophobic amino acids (for A(3t_40 and A(3142, respectively).

Discussion about neurobiologic and clinical events

        The neurobiologic and clinical events leading to the development of clear-cut AD are now being clarified. Emphasis in therapeutic trials is increasingly focused on strategies for preventing the development and progression of AD.

      Screening tools that can be used both in routine clinical practice and in research studies may play an important role in the development and effective use of these new therapies.

Materials and Methods

       The interview was designed to require no more than 5 minutes to administer and to be standardized so that a person with no clinical training could conduct the interview over the phone. The interview contains one item testing for the delayed recall of a list of three objects. The interview also contains some simple questions about cognitively demanding activities of daily living. Initially, the interview was given to a group of AD patients and agematched controls to determine its acceptability and whether there were obvious problems with telephonic administration and scoring.

      This pilot evaluation lead to rewording of some items and to clarifications in the administration procedure. We are now conducting a reliability and concurrent validity study. There are 90 participants in this study, all of whom have received a cognitive and diagnostic evaluation by a team of dementia experts in the past 6 months. Some of the participants have AD and others are elderly, nondemented controls.

      The telephone interviewers are blind to the participant's diagnosis and cognitive evaluation data. To assess reliability, each participant is being interviewed twice during a 1-month period. Concurrent validity will be assessed by determining the correspondence between scores on the screening interview and the participant's most recent clinical diagnosis.

      More detailed analyses will look at the ability of the screening instrument to identify AD patients with very mild disease. Although the sample size is too small to draw definitive conclusions about the possible influence of confounding variables, we will also examine the relationship of screening test scores to factors such as education, age, and medical comorbidities. These analyses will determine the reliability of the screening tool and provide a first look at its ability to identify cases of AD. Further validity studies will be conducted, subsequently, provided that the reliability and validity data in the current study are acceptable. One additional study will involve elderly patients enrolled in a managed care plan. None of the participants in this study will have received a full diagnostic evaluation for dementia. Participants will be screened over the telephone and determined to be cognitively normal or possibly impaired. Subsets from each group will then be examined clinically to determine the extent to which the scr

METHODS AND RESULTS

        Allikmets, Gerrard, Hutchinson, and Dean (1996) characterized a large number of human ABC transporter genes by searching the human EST database using sequences derived from ABC (Walker A, ABC signature, and Walker B) of MDR1, as well as the entire sequence of cystic fibrosis transmembrane conductane regulator (CFTR). Because many of the ESTs are derived from brain libraries, the data suggested that many of these ABC transporters were expressed in brain. Because the number of ESTs deposited in public databases has increased substantially in the past 4 years, we recently repeated this analysis in order to ensure that the full complement of brain-expressed ABC transporters was represented in the database. Moreover, to increase the power of our search, we developed consensus amino acid sequences for the Walker A motif, as well as the ABC signature and Walker B motifs, by comparing the sequences of the following human ABC transporters.  The consensus sequences that emerged from this analysis were (G(X)2G(X)GK(X)T(X)4L(X)2L(X)2PT(X)3G for the Walker A motif, and LSGG(X)4L(X)2A(X)AL(X)3PKV(X)2LDE(X)TS(X) for the ABC signature and Walker B motifs.

       The BLAST programs (Altschul, Gish, Miller, Myers, & Lipman, 1990) were used to search the human database EST using these consensus sequences, as well as the complete amino acid sequences of the human ABC-transporter proteins identified above. Human clones with the highest scores were retrieved and primers were designed using the program Mac Vector (Oxford Molecular, UK). Reverse transcriptase-polymerase chain reaction (RT-PCR) was performed using brain fetal and adult total RNA (Invitrogen) in order to confirm human brain expression, and PCR products were sequenced to authenticate the ESTs.

       Based on both the cDNA library source used to generate these ESTs, as well as the expression profile of RNA as seen in Northern blots (Allikmets et al. 1996, and unpublished data), we identified the 14 ABC transporters listed in Table 10.1 as being brain-expressed.

Identification of Brain-Expressed ABC Transporters That May Mediate Detachment of p-Amyloid From Biological Membranes

         Alzheimer's disease is characterized by neurofibrillary tangles, senile plaques, and neuronal death. The neurofibrillary tangles contain paired helical filaments composed of hyperphosphorylated tau, while the senile plaques are comprised of an array of proteins deposited around a core of insoluble A(3 peptide).

        The cause of neuronal death remains unknown, but considerable evidence suggests that it is secondary to an increase in the brain A(3 load. It has been known for nearly 10 years that the A|3 peptides are rapidly released from cells (Haass et al., 1992; Seubert et al., 1992; Shoji et al., 1992; Busciglio, Gabuzda, Matsudaira, & Yankner, 1993), but the hydrophobic amino acids at the COOH-terminus make it likely that the peptide will remain associated with the membrane following y-secretase cleavage. Thus, we hypothesized that an active process was required in order for A(3 to detach from the membrane. Selected members of the ATP-binding cassette (ABC) superfamily of transporters are responsible for the energydependent efflux of a variety of lipophilic and amphipathic molecules from cells, and the process bears a striking similarity to that which occurs with the AP peptide.
      ABC transporter known as MDR1 is an Ap efflux pump.

     We have identified a single ABC transporter, MDR1, as an AP efflux pump. Cells throughout the body constitutively produce and release Ap, yet the MDR1 protein is only expressed in a limited number of tissues, and is essentially undetectable in neurons

Discussion

       The neurobiologic and clinical events leading to the development of clear-cut AD are now being clarified.

     Emphasis in therapeutic trials is increasingly focused on strategies for preventing the development and progression of AD.
     Screening tools that can be used both in routine clinical practice and in research studies may play an important role in the development and effective use of these new therapies.

Materials and Methods

         The interview was designed to require no more than 5 minutes to administer and to be standardized so that a person with no clinical training could conduct the interview over the phone. The interview contains one item testing for the delayed recall of a list of three objects. 

    The interview also contains some simple questions about cognitively demanding activities of daily living.                                                                                                                                     

     Initially, the interview was given to a group of AD patients and agematched controls to determine its acceptability and whether there were obvious problems with telephonic administration and scoring. This pilot evaluation lead to rewording of some items and to clarifications in the administration procedure. We are now conducting a reliability and concurrent validity study. There are 90 participants in this study, all of whom have received a cognitive and diagnostic evaluation by a team of dementia experts in the past 6 months. 

    Some of the participants have AD and others are elderly, nondemented controls. The telephone interviewers are blind to the participant's diagnosis and cognitive evaluation data. To assess reliability, each participant is being interviewed twice during a 1-month period. 

     

      Concurrent validity will be assessed by determining the correspondence between scores on the screening interview and the participant's most recent clinical diagnosis. More detailed analyses will look at the ability of the screening instrument to identify AD patients with very mild disease. Although the sample size is too small to draw definitive conclusions about the possible influence of confounding variables, we will also examine the relationship of screening test scores to factors such as education, age, and medical comorbidities. 

     These analyses will determine the reliability of the screening tool and provide a first look at its ability to identify cases of AD. Further validity studies will be conducted, subsequently, provided that the reliability and validity data in the current study are acceptable. One additional study will involve elderly patients enrolled in a managed care plan. None of the participants in this study will have received a full diagnostic evaluation for dementia. Participants will be screened over the telephone and determined to be cognitively normal or possibly impaired. Subsets from each group will then be examined clinically to determine the extent to which the screening classification agrees with the results of a diagnostic evaluation.

TELEPHONE SCREENING FOR ALZHEIMER'S DISEASE

     A complete diagnostic evaluation for AD is time-consuming, expensive, and requires substantial expertise on the part of the examining physician.
    In clinical settings and in research studies, there is a need for brief screening tools that could help to identify those individuals who should receive a full diagnostic evaluation. Effective screening tools could help to ensure that scarce diagnostic services are focused on those individuals who are most likely to have dementia. Screening can be conducted in a variety of settings and, potentially, using a variety of different tools. As examples, screening could be done in clinics, doctors' offices, door-to-door surveys, by mail, or by telephone. Several mental-status interviews have been developed or adapted for use over the telephone (e.g., Brandt, Spencer, & Folstein, 1988; Roccaforte, Burke, Bayer, & Wengel, 1992). Most of these instruments are designed as telephonic versions of brief mental-status examinations, such as the MMSE and, therefore, have enough questions to measure the severity, as well as detect the presence of dementia.

    Recently, we have initiated a study to test the reliability and validity of a very brief telephonic-screening interview that could be given by nonprofessionals in a few minutes. The interview is not designed to enable a
specific dementia diagnosis or to evaluate the severity of dementia, but simply to identify persons with a high probability of being demented.

NEUROBIOLOGY OF EARLY ALZHEIMER'S DISEASE

     Details of the studies on early AD conducted at our center have been published. The population for these studies are the residents of the Jewish Home and Hospital (JHH), a long-term care facility affiliated with the Mount Sinai School of Medicine in New York.

    The two main campuses of the JHH have approximately 1,600 residents, with an average age of over 85. Cognitive screening is a routine part of clinical care in the JHH, and Mini-Mental State Examination (MMSE)
scores are available for nearly all residents. For the early AD study, all consenting residents with MMSE scores of 15 or greater are given a thorough diagnostic evaluation and are assigned a score on the Clinical
Dementia Rating (CDR; Morris et al., 1993). Those with CDR scores of 0 (no dementia), 0.5 (questionable dementia), or 1.0 (mild dementia) are administered a battery of neuropsychological tests.

     The JHH routinely requests autopsy permission when a resident dies and, over the period of the early AD project, more than 50 autopsies have been obtained from residents who died with CDR scores of 0 to 1.0, and evidenced either no significant neuropathology or had neuropathologic lesions associated with AD only. As might be expected with cases from a long-term care facility, many more autopsies have been obtained from residents who died with more severe dementia or with comorbid neuropathologic lesions.

     Neuropathologic studies of cases dying with no dementia, questionable dementia, or mild dementia have shown that senile plaques are more abundant in most areas of the neocortex in patients with CDR 0.5 than in persons dying without any evidence of dementia (CDR 0) (Haroutunian et al., 1998). Examination of specific amyloid fragments has shown that the Afi peptides, especially the A^ 42 peptide, are markedly elevated even in the CDR 0.5 cases, compared with the CDR 0 cases (Naslund et al., 2000). By contrast, neurofibrillary tangles were evident in the entorhinal cortex and hippocampus of virtually all brains from
persons over age 80, even those from nondemented individuals; extensive neurofibrillary tangles in the neocortex were evident only in patients with CDR 2.0 (moderate dementia) or greater (Haroutunian et al., 1999). These data support the view that overproduction and accumulation of amyloid protein, particularly the ^ 42 fragment, is a very early manifestation of AD. Neurofibrillary tangles in entorhinal cortex and hippocampus are age-related phenomena that are further influenced by AD as dementia progresses
from severe to terminal, while the development of tangles in the neocortex is associated with progression to moderate and severe dementia.

     Other biologic manifestations of AD have also been examined in this series. Cholinergic markers, including choline acetyltransferase (ChAT) and acetylcholinesterase, were not diminished in early AD cases, but were reduced substantially in those patients dying with moderate-to-severe dementia (Davis et al., 1999a).

NEUROPSYCHOLOGY OF EARLY ALZHEIMER'S DISEASE

      Cross-sectional studies comparing patients with very mild AD to agematched,  nondemented control subjects have demonstrated that a deficit in memory is the earliest and most prominent neuropsychological deficit in patients with diagnosed AD (Welsh, Butters, Hughes, Mohs, & Heyman,1991). This deficit is most pronounced on tasks for which the patients are asked to recall previously learned information (such as a short list of words) after a brief delay during which the patient engages in other cognitive activity.

      Longitudinal studies of nondemented persons who are at risk for dementia have examined the question of whether there are neuropsychological deficits that are measurable before patients are impaired enough to warrant a diagnosis of AD. In these studies, baseline neuropsychological data are used to compare performance of patients who subsequently were diagnosed with AD with those who remain dementia free.

     Results of these studies indicate that there are measurable deficits in memory and, to a lesser extent, in language and cognitive-processing speed at least 1 year before patients meet diagnostic criteria for dementia  Our own results from the JHH study confirm that patients with a CDR score of 0.0 who convert to CDR 0.5 1 year later have baseline memory scores that are poorer than the baseline memory scores of patients who remain CDR 0.0 on follow-up. Thus, data from a variety of sources indicates that poor scores on a test of memory, particularly delayed-recall memory, are a sensitive indicator of early dementia.

Early Clinical and Biological Manifestations of Alzheimer's Disease: Implications for Screening and Treatment

        Alzheimer's disease (AD) is a slowly progressive disease with an average of more than 10 years from first manifestations to death. Both the underlying biology of AD and its clinical manifestations change substantially over the course of the illness. This chapter reviews briefly some recent studies investigating the neurobiologic changes in the brains of AD patients over the course of the illness, with an emphasis on the earliest changes.

     Cognitive deficits also change over the course of illness and an understanding of the earliest cognitive changes may enable the development of screening instruments for AD. An ongoing set of studies designed to determine the reliability and validity of telephonic screening instruments is described. An instrument of this type could be useful in clinical trials of agents for the primary prevention of AD, as well as in health service-delivery organizations where there is a need to identify cases of AD that have not yet been diagnosed.

Positron Emission Tomography Imaging of Amyloid Senile Plaques and Neurofibrillary Tangles

      Several groups have been developing new, small-molecule probes to image the amyloid NPs and NFTs.        
     Current methods for measuring brain amyloid, such as histochemical stains, require tissue fixation on postmortem or biopsy material. Available in vivo methods for measuring NPs or NFTs are indirect. Studies that may lead to direct in vivo, human AP imaging include various radiolabeled probes using small organic and organometallic molecules capable of detecting differences in amyloid-fibril structure or amyloid-protein sequences. Investigators also have used chrysamine-G, a carboxylic acid analogue of Congo red, an amyloid-staining histologic dye, serum amyloid-P component, a normal plasma  glycoprotein that binds to amyloid-deposit fibrils (Lovat, O'Brien, Armstrong, et al., 1998), or monoclonal antibodies (Majocha et al, 1992).
      Methodological difficulties that hinder progress with these techniques include poor blood-brain barrier crossing and limited specificity and sensitivity. In addition, most approaches do not measure both NPs and NFTs. Recently, Barrio et al. (1999) reported using a hydrophobic, radiofluorinated derivative of l,l-dicyano-2-[6-(dimethylamino)naphthalen-2-yllpropene (FDDNP) (Jacobson, Petric, Hogenkamp, Sinur, & Barrio,1996) with PET to measure the cerebral localization and load of NFTs and SPs in AD patients. The probe showed visualization of NFTs, NPs, and diffuse amyloid in AD brain specimens using in vitro fluorescence microscropy, which matched results using conventional stains (e.g., thioflavin S) in the same tissue specimens. Such approaches may ultimately aid in the early detection of AD and brain-function monitoring during antidementia treatment trials, particularly those designed to interrupt accumulation of NPs and NFTs.

Preclinical Detection: Benefits and Strategies

      Although no cure exists for AD, preclinical disease detection has several benefits. When early detection assessments are negative, people with mild memory complaints can be reassured that their forgetfulness reflects a normal age-related change that probably will not progress.

     In addition, many people would like to know about a poor prognosis while still in a mildly impaired state in order to plan their futures while mental faculties remain. Perhaps the most compelling argument for preclinical detection strategies is to identify candidates for novel antidementia treatments before the dementing process causes extensive neuronal death, since new antidementia treatments are more likely to delay the dementing process than to reverse neuronal death.
      Although current cholinergic treatments have been shown to result in symptomatic, rather than disease-altering or structural effects, it would certainly be of interest to initiate treatments very early when searching for a disease-modifying effect. Moreover, both the expense and potential risks of treatment make it reasonable to reserve treatment only for those people who are at the greatest risk for developing the disease.

Brain Activation During Memory Tasks in People at Genetic Risk

      To determine the relationship between brain responses to memory tasks and genetic risk for AD, Bookheimer et al. (2000) performed apo E genotyping and functional MRI while cognitively intact older persons performed memory tasks. The study included 30 participants, aged 47 to 82 years, with mild memory complaints but normal memory performance, of whom 16 were apo E-e4 carriers and 14 were not. The age and prior educational achievement in the two groups were similar. Brain-activation patterns were determined from functional MRI scanning while participants memorized and recalled unrelated word pairs. Memory performance was reassessed on 14 participants 2 years later.

     The magnitude and spatial extent of brain activation during memory performance in regions affected by AD, including left hippocarnpal, parietal, and prefrontal regions, was greater in the participants with apo E-e4 alleles, as compared with those with no apo E-e4 alleles.

     During memory performance tasks, the apo E-e4 carriers demonstrated a greater percentage increase in hippocarnpal MRIsignal intensity and a greater number of activated regions throughout the brain than did participants without apo E-e4. Longitudinal assessment after 2 years indicated that greater baseline brain activation correlated with verbal-memory decline. These results indicate that brain-activation patterns during memory tasks differ according to genetic risk for AD and may provide information that eventually predicts future cognitive decline.

Cerebral Metabolic and Cognitive Decline in Apo E-e4 Carriers

     To determine cognitive and metabolic decline patterns according to genetic risk, Small et al. (2000) investigated cerebral metabolic rates using PET in middle-aged and older nondemented persons with normal memory performance.
      Participants were right-handed and in the 50- to 84-year-age range. Of the 54 participants with mild memory complaints, 27 were apo E-e4 carriers and 27 were noncarriers. A single copy of the apo E-e4 allele was associated with lowered inferior parietal, lateral temporal, and posterior cingulate metabolism, which predicted cognitive decline after 2 years of longitudinal follow-up. For the 20 nondemented participants followed longitudinally, memory-performance scores did not decline significantly,  but cortical metabolic rates did. In apo E-e4 carriers, a 4% left posterior cingulate metabolic decline was observed, and inferior parietal and lateral temporal regions demonstrated the greatest magnitude (5%) of metabolic decline after 2 years. These results have practical implications for clinical trials of dementiaprevention treatments. The right lateral temporal metabolism for apo E-e4 carriers at baseline and 2-year follow-up yielded an estimated power under the most conservative scenario (i.e., assuming that the points are connected exactly in reverse order) of 0.9, to detect a 1-unit decline from baseline to follow-up using a one-tailed test. A sample size of only 20 participants, therefore, would be needed in each treatment arm (i.e., active drug or placebo) to detect a drug-effect size of 0.8 (a = 0.05, power = 0.8).

     Thus, a clinical trial of a novel intervention to prevent cerebral metabolic decline would require only 40 participants over a 2-year treatment period. Such findings are consistent with previous PET studies showing stable and replicable results (Andreasen, Arndt, Cizadlo, et al., 1996), and suggest that combining PET and AD genetic-risk measures will allow investigators to use relatively small sample sizes when testing antidementia treatments in preclinical AD stages.

     These results indicate that the combination of cerebral metabolic rates and genetic-risk factors provides a means for preclinical AD detection that will assist in response monitoring during experimental treatments.

Brain Imaging Surrogate Markers for Detection and Prevention of Cognitive Aging and Alzheimer's Disease

       With increasing knowledge of the neuropathological and cognitive changes preceding Alzheimer's disease (AD), clinical trials have begun to focus on preventive treatments aimed at slowing age-related cognitive decline and delaying onset of AD.

      Studying participants with minimal deficits leads to diagnostic heterogeneity and a need for larger samples in order to detect active drug effects. This chapter summarizes results of several studies designed to address such issues. Middle-aged and older adults with mild memory complaints were studied using brain imaging and measures of the major known genetic risk for AD, the apolipoprotein E-4 (apo E-e4) allele.

      In a study of positron emission tomography during mental rest, glucose metabolic rates were significantly lower in apo E-e4 carriers in brain regions affected by AD. Another study using functional magnetic resonance imaging showed increased brain activation during memory tasks in apo E-e4 carriers in similar brain regions. Longitudinal follow-up after 2 years indicated the potential utility of such brain-imaging measures, combined with genetic-risk information, as surrogate markers in prevention-treatment trials for cognitive aging and AD.

      Additional development of novel approaches using positron emission tomography to directly measure the neuritic plaques and neurofibrillary tangles of AD offers promise of more specific measures of disease progression in future clinical trials.

The fourth method

     The fourth method produced population-based, average 3-D hippocampal surface maps for both the control and MCI groups, derived from the manual outlines produced across centers. The methodology for constructing a surface map was described and was initially applied to document the variability of the elderly control, MCI, and AD hippocampus in the Talairach atlas
space, arguing for a refinement of this commonly used space in the functional imaging assessment of the elderly and demented population. Hippocampal
contours were traced using a track ball on each relevant slice. The points that made up a traced contour, after smoothing the effects of irregular hand movements, were connected across slices to create regularly ordered 3-D meshes corresponding to the hippocampal surface. To quantify an individual patient's anatomy an average hippocampal 3-D model was resolved by averaging the vectorial displacements, on a point-by-point basis from the parametric mesh, for each patient's hippocampus.

    Hippocampal variability, expressed as a 3-D distance in the common coordinate space, was computed by taking the mean of the square roots of a 3-D displacement vector necessary to align each node along a patient's mesh onto the average representation of the population's hippocampus.

A linear measure of medial temporal lobe

   The third method was a linear measure of medial temporal lobe width that used the centimeter-distance scale of images that have the midpoint of the rostral caudal extent of the hippocampal formation at each side.

    This linear measure of the narrowest point of the medial temporal lobes adjacent to the brain stem used the procedure described by Jobst et al. (1998,1992). Image was accessed via the Internet, and measures were entered electronically over the web.

Assessment methods.

       Four assessment techniques were implemented at each center. The first was a traditional volumetric assessment technique with manual outline of left and right hippocampi according to the methods of Jack et al. (1990,1989). Contours were made using a Java-based outlining package distributed to all centers over the Internet, which can be compiled on all current personal computers, laptop computers, or unixbased workstations. The digital output of these contours were electronically sent to UCLA for processing.

    The second procedure was to reformat the scans axially, parallel to the long axis of the hippocampus, according to the methods of de Leon et al. (1993) and to provide an individual slice through each volume to obtain qualitative assessments of medial temporal atrophy using a 4-point scale. The images of each slice were accessed via the Internet, and scores were entered electronically over the web.

Statistical analysis.

     Volumetric assessment included normalization for interpatient variation of head size by dividing hippocampal volume by the total intracranial volume of that particular patient. Percentiles for normalized hippocampal volume had been previously reported. Percentile scores were then converted to a W score, which is a value from a standard normal distribution corresponding to the observed percentile (in a standard normal distribution, the 50th, 5th, and 2.5th percentiles are given by W scores of 0, -1.645, and -1.96, respectively).

       A W score of less than or equal to -2.5 would be associated with the 0.6th percentile of hippocampal volume. Sixty-seven of 80 MCI patients in a recent study (Jack et al., 1999) had W scores less than 0 (13 had W scores less than or equal to -2.5, which is 16.25% of the population of MCI patients studied). Assuming outliner variability as previously reported across studies of errors less than 1.9% coefficient of variation, then the number of MCI patients needed to produce sufficient power to find a significant effect would be 30.
       This would provide 80% power to detect a difference of 16% as a deviation from .5, with alpha equal to .01. If 10.5% of the 30 MCI patients fall below a W score of -2.5, rather than the expected 16.25%, we would have 80% power to detect an alpha at .01 (h = .45 at 0.6th percentile of the control's standard normal distribution) (Cohen, 1988). Sensitivity (true positives / (true positives + false negatives) and specificity (true negatives/ (false positives + true negatives) calculations were also computed for each method's ability to correctly identify incipient AD, MCI, and controls.

Techniques for identification of patients with MCI compared with controls

    The four techniques for evaluation were chosen based upon their demonstrated usefulness in evaluating medial temporal atrophy. These include:
1. The volumetric assessment technique of the Mayo group (Jack et al.,1990,1992).
2. The qualitative assessment of medial temporal lobe atrophy performed by the NYU group (de Leon et al., 1993,1989,1997).
3. The linear assessment technique of Jobst et al. (1998,1992).
4. The three-dimensional (3-D) mapping technique employed at UCLA (Thomspon & Toga, 1996; Thompson et al., 1996). The centers participating in this multisite protocol who share both common MCI definitions and MRI acquisition sequences are listed in Table6.1, along with the number of MCIs and controls to be studied.

Mild cognitive impairment (MCI) may have incipient Alzheimer's disease

    Elderly individuals with mild cognitive impairment (MCI) may have incipient Alzheimer's disease (AD) and are the most likely targets for therapies directed at disease modification or slowing progression to the
endpoint of overt dementia. Identification of those individuals who will eventually develop AD is assuming urgent importance in many centers.

    With the potential advent of disease-modifying drugs, biomarkers with good reliability, sensitivity, and specificity are needed. One promising biomarker for patients with incipient AD is hippocampal atrophy
assessed by magnetic resonance imaging (MRI).

Magnetic resonance

      Magnetic resonance imaging has been used routinely in the past to ensure diagnostic accuracy by ruling out stroke and other structural causes of cognitive impairment. Present technology, however, allows volumetric measurements of brain structures to be performed during the course of a trial that may serve additional purposes. Volumetric measurements obtained at the inception of a trial provide indices of whole brain and regional atrophy that relate to the fundamental pathology of the disease process. They offer an alternative approach to cognitive and clinical assessments for staging patients. They may be helpful in predicting disease progression and serve as potentially important explanatory covariates in efficacy analyses of therapeutic agents.

    Serial MRI volumetric measurements performed over the course of a trial permit investigators to directly monitor volume loss resulting from ongoing pathology, and may help distinguish whether a therapeutic agent has symptomatic or diseasemodifying effects.

     The relationships found so far between the hippocampal volume and the clinical assessments obtained in participants upon entering the MCI trial further point to the utility of MRI volumetric analysis as a valuable indicator of disease severity. They suggest that even within the relatively narrow continuum of MCI participants, MRI volumetric analysis can discriminate between participants on the basis of their performance on neuropsychological and clinical evaluations. These encouraging results support the contention that baseline MRI volumetric analysis may predict subsequent conversion to AD and be useful for measuring the effects of treatment.

Volumetric analyses have been performed on MRI

     Volumetric analyses have been performed on MRI scans obtained at the time of study entry from the first 77 participants enrolled in the MRI portion of the MCI protocol. Fifty-eight percent of these participants are men and 42% are women. They have a mean age of 73 years and a mean education of 15 years. They have a mean score of 28 on the Mini-Mental Status Examination (MMSE) (Folstein, Folstein, & McHugh, 1975), a mean score of 11 on the ADAS-COG, and a mean score of 1.8 on the CDR Sum of Boxes.

     We performed correlational analyses between the MRI hippocampal volumes at study entry and the participants' concurrent performance on (ADAS-COG) and functional (CDR Sum of Boxes) measures. The results indicate that hippocampal volume in these participants predicted performance on the ADAS-COG (r = -.31, P < .01) and the CDR Sum of Boxes (r = -.33, P < .01). These correlations remained significant after controlling for age and gender. The relationships are shown graphically in Figures 5.1 and 5.2. For illustration purposes, the MRI hippocampal volumes are divided into tertiles from smallest to highest.

intrarater test-retest coefficient of variation of hippocarnpal volumetric measurements is 1.9%

     Validation studies demonstrate that the intrarater test-retest coefficient of variation of hippocarnpal volumetric measurements is 1.9% with this method (Jack, Bentley, Twomey, & Zinsmeister, 1990). The 3D-image data set of each patient is realigned into an orientation perpendicular to the principal axis of the left hippocarnpal formation. The imaging data are then interpolated in-plane to the equivalent of a 512 x 512 matrix and magnified two times. The voxel size of the fully processed image data is 0.316 mm3. The borders of the hippocampi are manually traced on the workstation screen for each image slice sequentially from posterior to anterior.
     Typically, 40 to 50 imaging slices are measured for each hippocampus. In-plane hippocampal anatomic boundaries are defined to include the CA1 to CA4 sectors of the hippocampus proper, the dentate gyrus, and the subiculum (Jack et al., 1997). The posterior boundary of the hippocampus is determined by the oblique coronal anatomic section, in which the crura of the fornices are defined in full profile.

     Pearson correlation coefficients were calculated between the hippocampal volume measured from MRIs obtained at study entry, as well as the participants' baseline Alzheimer's Disease Assessment Scale-Cognitive portion (ADAS-COG) (Rosen, Mohs, & Davis, 1984) and Clinical Dementia Rating (CDR) Sum of Boxes (Morris, 1997), in order to determine the relationship between hippocampal volume and cognitive and functional performance at baseline.

treatment with vitamin E, donepezil, or placebo

         Approximately 720 participants with MCI are currently being randomized to treatment with vitamin E, donepezil, or placebo for 3 years, with roughly an equal number of participants (240) in each treatment group.
      From previous data, it is known that individuals who meet criteria for MCI convert to AD at a rate of approximately 12% to 15% per year (Petersen, Smith, Ivnik, et al, 1995; Grundman, Petersen, Morris, et al., 1996b; Petersen et al., 1999). Additional data suggest that this conversion rate may be even higher (up to approximately 20% per year over the first 3 years) in MCI participants who are screened with a memory test assessing delayed recall. Based on these figures, it is expected that at the end of the 3 years, 45% to 60% of placebo-treated participants may develop AD.

     The trial is designed to evaluate whether the two treatment interventions are effective at reducing the rate of conversion to AD by approximately one third over the course of the 3 years. The MRI component of the protocol stipulates that MRI scans are obtained on participants at entrance into the trial, upon completion of the trial 3 years later, and at the time of diagnostic crossover to AD. In all participants the MRI study is performed according to a defined set of imaging sequences that are sufficiently generic so that they can be executed across a variety of MRI vendors. The cornerstone of the planned volumetric analyses includes a 3-dimensional (3D) volumetric imaging sequence that provides optimal spatial resolution in all three anatomic dimensions.

     Guidelines for the storage and archival procedures are provided to each of the individual participating sites. Magnetic resonance imaging data cassettes from participating sites are sent to the central data repository at the Mayo Clinic in Rochester, Minnesota, where imaging data are downloaded, checked for compliance with the prescribed imaging sequences, and then catalogued. Catalogued images are cross-checked with case report forms forwarded from participating clinical sites to the ADCS coordinating
center in San Diego, California. After the images are analyzed, the data are merged with other clinical data on each participant residing in the ADCS central database.

    The proposed analyses focus primarily on the medial temporal lobe, particularly the hippocampal formation. Nevertheless, with the imaging sequences outlined in the study protocol, the capability exists to measure serial whole brain, lobar, sublobar, and ventricular volumes, as well as gray and white matter volumes, and leukoaraiosis. Based on prior studies, we anticipate that the hippocampal volume or other baseline measurements on the baseline MRI scans will predict which participants are most likely to develop AD. Analyses of the serial images obtained over the course of the study will examine whether treatment with either vitamin E or donepezil alters the rate of atrophy in the whole brain, lobar and sublobar-medial temporal lobe, entorhinal cortex, as well as the gray and white matter. Similar analyses will examine whether there are treatmen differences in the rate of ventricular enlargement.

The earliest pathological changes in the brains of AD

    The earliest pathological changes in the brains of AD patients occur in the medial temporal lobe. The entorhinal cortex and its perforant pathway projections to the hippocampus are among the earliest brain regions to exhibit the neuropathological hallmarks of AD (Braak & Braak, 1996). The resulting loss of neurons and synapses in this region is manifested at a macroscopic level by medial temporal lobe atrophy, which can be observed by visual inspection of the brain at autopsy or by MRI in living patients.

    Paralleling the neuropathological changes, clinical symptoms of AD often involve cognitive impairments and fluctuations in body weight that are closely linked to medial temporal lobe atrophy (Grundman, Corey-Bloom, Jernigan, Archibald, & Thai, 1996a). In recent years, considerable effort has been devoted to imaging these structures to see if they serve as a useful adjunct in diagnosing and following patients with early AD.
    Previous work demonstrates that MRI-based quantitative measurements of the hippocampal formation are useful in differentiating patients with AD from normal control participants (Jack, Petersen, Xu, et al., 1997).
They found that the best discriminator between AD patients and normal controls was the hippocampal volume when different medial temporal lobe structures were compared. Participants with mild AD had mean hippocampal volumes that were approximately two standard deviations below the control mean.

   Volumetric measurements of the hippocampal formation may also be useful for predicting future conversion to AD in MCI participants. Jack, Petersen, Xu, et al. (1999) measured MRI-based hippocampal volume in
80 consecutive patients who met criteria for the diagnosis of MCI, and followed them longitudinally with approximately annual clinical and cognitive assessments. The primary endpoint was the crossover of individual MCI patients to a clinical diagnosis of AD during follow-up.

    During the period of observation, which averaged 33 months, 27 of 80 MCI patients developed dementia. Hippocampal atrophy at baseline was significantly associated with subsequent conversion from MCI to AD. The risk of AD was 50% within 3 years in MCI patients with moderate hippocampal atrophy; however, it was only 26% in patients with mild hippocampal atrophy (approximately 50% reduction in risk of conversion based solely on differences
in hippocampal volume). In MCI patients with normal hippocampal volume, the risk of developing AD dropped even further, with only 9% crossing over to AD within 3 years.

Orientation-Memory-Concentration test

    Approximately 830 individuals over age 75 years were on the computer roster of a large outpatient clinic affiliated with a managed care organization in Minneapolis, Minnesota. We selected those individuals from this roster without diagnoses of dementia, Alzheimer's disease (AD), and their synonymous diagnostic codes in any patient encounter in the prior 2 years. Of the 611 individuals who met these criteria, we sent an introductory letter to 447 potential participants, informing them that they would be receiving a phone call as part of this study.
    Upon reaching a participant on the telephone, the examiner (an RN experienced in geriatrics and formally trained to administer the mental status examinations) informed the individual of who she was and the
nature of the study. For those who agreed to participate, they were given either the Orientation-Memory-Concentration (OMC) test (Katzman, Brown, Fuld, et al., 1983) or the Minnesota Cognitive Acuity Screen (MCAS) (Knopman, Knudson, Yoes, & Weiss, 2000). Administration of the two generally alternated, except in a few circumstances in which a participant would agree only to the shorter of the two tests.

     After completion of the mental status examination, the participants were questioned on four issues. The first question asked how they thought they performed on the mental status examination ("How do you
think that you did on the tests we just finished?"), and the responses were good, fair, poor, or very poor. The second question was, "Apart from this interview, in the past few months, have you felt that you have had memory problems?", with the rating scheme as follows: "my memory is excellent," "my memory is as good as anyone else my age," "my memory isn't as good as it used to be, but it doesn't cause me any problems," "my memory has definitely deteriorated in the past few years and interferes with my daily activities," or "my memory is severely impaired." The third question was whether they would follow-up with their physician if the  were told that they had done poorly ("Suppose that the present interview showed that you had memory problems, would you be willing to have a medical assessment for memory problems at your clinic?", with yes, no, or possibly as the responses.
      The final question was open-ended, asking what they thought of the process of telephonic examinations. The study was approved by the HealthPartners Institutional Review Board. Oral consent was obtained to conduct the phone interview.

Disscusion

       Under conditions that might be present in many clinical practices, the acceptance/completion rate for telephonic mental status assessment among participants 75 years and older was only 55%. Of those who underwent cognitive assessment, the yield of cognitively impaired participants was low. Only 3% scored in the range that was indicative of cognitive impairment.

      Among the interviewees, willingness to consult with a physician regarding memory problems detected during a telephonic screen was quite high (87%).

       The low number of individuals who scored in the impaired range suggests that the telephonic strategy did not serve to uncover undiagnosed cognitive impairment in this elderly cohort. We believe we should have
encountered 20 screen failures (roughly a 10% prevalence in this 75+ year only age group), in contrast to the seven we found.
       We suspect that the lower-than-expected number of cases occurred as a result of selective refusal by participants with cognitive difficulties to participate in the telephonic screening. Unfortunately, we have no way of determining the cognitive status of those individuals who refused to be interviewed. From other studies in which cognitive status was available from an earlier visit prior to a refusal to continue to participate, those who refused to participate in follow-up had lower performance at baseline. In the Canadian experience, of 147 participants whose cognitive screening was abnormal, but who refused to undergo a clinical examination, their mental status score was more than seven points lower on the Modified Mini-Mental State examination, compared with those who continued to participate in the study (81.3 vs. 88.8).
     The low overall rate of completion of interviews could be attributed to a basic flaw in the telephonic strategy. In current American culture, telephone marketing is widespread, and many people, upon receiving phone
calls from strangers, may resent the intrusion. An alternative strategy would have been for us to have engaged the primary physicians in this clinic and encouraged them to promote participation. Because patients might see their physicians only every 6 months or less, such an approach would work only if our project had had a longer time scale.
      The findings of the present study suggest to us that telephonic screening is a useful and efficient strategy to identify those who do not need face-to-face cognitive assessments. Assuming that telephonic screening
were to be identified to the health plan membership as "required" or "strongly recommended," as opposed to an optional activity tied to research as was necessarily the case with the present project, the completion rate
could be higher. The number of elders who would then need face-to-face examinations would be perhaps only a half or a third of all individuals over age 75 years.

Magnetic resonance imaging (MRI)

       Magnetic resonance imaging (MRI) volumetric analysis is a relatively new assessment tool in clinical trials for Alzheimer's disease (AD). It is a technique that is particularly well suited for clinical trials designed to prevent AD or slow its progression. The Alzheimer's Disease Cooperative Study, a multicenter consortium that performs AD-related clinical trials, is presently conducting a clinical trial with vitamin E and donepezil (Aricept®) in participants with mild cognitive impairment (MCI). The objective of the trial is to determine if either agent is capable of delaying a diagnosis of AD.

          Mild cognitive impairment participants are randomized to treatment with vitamin E, donepezil, or placebo, and followed longitudinally for 3 years. A subset of randomized participants is undergoing MRI of the brain prior to beginning treatment and during the course of the trial. The objectives of the MRI component of this trial are to determine (a) whether MRI volumes predict cognitive and functional performance in a well-defined cohort of participants with MCI; (b) whether hippocampal volume or other baseline volumetric measurements predict subsequent crossover to clinical AD; and (c) whether treatment with either vitamin E or donepezil alters the rate of brain atrophy over the course of treatment.

         Although the trial is still recruiting participants, analysis of the MRI scans collected thus far indicate that hippocampal volumes measured at the time of study entry predict baseline cognitive and functional performance. These results support the hypothesis that brain volumetric analysis may also be useful to predict future prognosis and for monitoring the effects of treatment on disease progression.

Autosomal recessive genes

    Consanguinity increases the prevalence in a population of autosomal recessive genes that may have undesirable characteristics. For example, an additional 1/16 of the variation of DNA is made homozygous by the inbreeding of a marriage of first-cousins. Conversely, the probability of identifying recessive or quasirecessive susceptibility factors is enhanced by inbreeding. Childhood mortality is increased in offspring of first-cousin marriages by a factor of 1.4 to 1.7, and children born to consanguineous unions have poorer health than the offspring of nonconsanguineous unions (including malignancies, congenital abnormalities, mental retardation, and physical handicap). Curiously, there are few studies of the effects of inbreeding on the health of adults. We have hypothesized that consanguinity in the Wadi Ara community has increased the prevalence of autosomal recessive genes that are responsible, in part, for the increased prevalence of the disease.
    It has been reported that 44% of all Arab marriages in Israel are consanguineous, with a mean inbreeding coefficient of .0192 (Jaber, Shohat, Rotter, & Shohat, 1997). The inbreeding rates for Israeli Arabs may be particularly high (as compared with Egyptians or Syrians) because mobility was reduced for centuries by the Turks. At times in Arab communities, it has been required for children to marry into their own family in part to avoid sharing resources with competing family groups.

All of the known Alzheimer-related genes (on chromosomes 21, 14, 1, and 19) are dominant (chromosomes 21,14,1) or codominant (chromosome 19). There are no known genes affecting the development of AD that are recessive, perhaps because there have been few studies of AD in populations with high levels of inbreeding. De Braekeleer et al. (1989) reported an association between inbreeding and the development of AD (inbreeding coefficient 9 times higher in AD cases than in controls) in a rural population in Quebec. Studies in the Old Order Amish have found a low prevalence of disease in this inbred community, also having a low frequency of the apo E-e4 allele (.037). Inbreeding could be linked to AD through a confounding effect of education, but the association of inbreeding and education is controversial (found for a Saudi population but not for Israeli Arabs). We expect that there are recessive factors for AD because of indirect evidence: genetic modeling studies of AD in apo E-e4 negative families are unable to reject a recessive model (Rao et al., 1996).
    However, it is difficult to evaluate recessive models in outbred populations. The high prevalence we have seen in our highly inbred Arab population in Israel might be due to the inculcation of recessive AD susceptibility alleles or to a dominant gene that became frequent by founder effect.

Alzheimer's disease from which the disease occurs

Brain - soft nerve tissue, it should be protected by destroyers and poisons: stress, alcohol, narcotics, psychotropic drugs. In Alzheimer's disease brain cells atrophy and die "bundles". I had to deal with the disease up close.

First, the disease goes unnoticed: chudit people sometimes forget what happened yesterday, but remembers well what happened in childhood. However, there comes a critical moment, as a rule on the background of a stressful situation when the problem becomes obvious: the person loses touch with the outside world ceases to recognize the closest, he was haunted by delusions. There are periods of exacerbation when patients become unbearable. It is terrible, irreparable, but will have to adapt.

What does it happen?

In Alzheimer's disease in the brain is the deposition of protein in the form of "senile plaques" and the formation of neurofibrillary tangles of so-called, consisting of damaged neurons - both of these processes lead to the destruction of nerve pathways of the brain.

While still not clear what exactly causes these changes, but in recent years, scientists were able to identify specific genes responsible for human predisposition to Alzheimer's disease.

And what is interesting: the disease is more common in poorly educated people with unskilled occupations. A person with high intelligence are less likely to face the manifestations of this disease for the reason that it has a greater number of connections between nerve cells. So when the death of some cells lost the functions can be transferred to others who have not previously been involved.

Genetic and Environmental Risk Factors for Alzheimer's Disease in Arabs Residing in Israel

There is relatively little known about the distribution of Alzheimer's disease (AD) around the world. Knowledge of high- or low-prevalence foci of disease may aid our understanding of disease mechanisms. Alzheimer's disease occurs less frequently in Asia than in Europe and North America, probably because of lower allele frequency of the apolipoprotein E (apo E) e4 allele. The disease appears to be also less common in India and Africa.

There are few studies of the disease in Arabs, and few in populations with a high level of consanguinity. We have found a remarkably high prevalence of the disease in Arabs residing in Israel. In a study in Tunisia, unspecified dementia was found to be 3 times less prevalent than in the United States. Treves, Chandra, and Korczyn (1993) found a lower prevalence of presenile AD in Sephardi, as compared with Ashkenazi Jews, but more late-onset disease in Sephardi than Ashkenazi, perhaps because of lower levels of education in Sephardi. The state of Israel is comprised of about 5 million Jews and 1 million non-Jews, mostly Moslem Arabs. This Arab population of Israel is valuable for medical research because of a high level of inbreeding, large family size, high level of smoking exposure, high level of medical care available through the Israeli Health System, excellent demographic databases available from the government, absence of alcohol use because of the religious proscription, and high participation rate (>98%).

Genetic and Environmental Risk Factors for Alzheimer's Disease in Arabs Residing in Israel

There is relatively little known about the distribution of Alzheimer's disease (AD) around the world. Knowledge of high- or low-prevalence foci of disease may aid our understanding of disease mechanisms. Alzheimer's disease occurs less frequently in Asia than in Europe and North America, probably because of lower allele frequency of the apolipoprotein E (apo E) e4 allele. The disease appears to be also less common in India and Africa.

There are few studies of the disease in Arabs, and few in populations with a high level of consanguinity. We have found a remarkably high prevalence of the disease in Arabs residing in Israel.

In a study in Tunisia, unspecified dementia was found to be 3 times less prevalent than in the United States. Treves, Chandra, and Korczyn (1993) found a lower prevalence of presenile AD in Sephardi, as compared with Ashkenazi Jews, but more late-onset disease in Sephardi than Ashkenazi, perhaps because of lower levels of education in Sephardi. The state of Israel is comprised of about 5 million Jews and 1 million non-Jews, mostly Moslem Arabs. This Arab population of Israel is valuable for medical research because of a high level of inbreeding, large family size, high level of smoking exposure, high level of medical care available through the Israeli Health System, excellent demographic databases available from the government, absence of alcohol use because of the religious proscription, and high participation rate (>98%).
Consanguinity increases the prevalence in a population of autosomal recessive genes that may have undesirable characteristics. For example, an additional 1/16 of the variation of DNA is made homozygous by the inbreeding of a marriage of first-cousins. Conversely, the probability of
identifying recessive or quasirecessive susceptibility factors is enhanced by inbreeding. Childhood mortality is increased in offspring of first-cousin marriages by a factor of 1.4 to 1.7, and children born to consanguineous unions have poorer health than the offspring of nonconsanguineous unions (including malignancies, congenital abnormalities, mental retardation,
and physical handicap). Curiously, there are few studies of the effects of inbreeding on the health of adults. We have hypothesized that consanguinity in the Wadi Ara community has increased the prevalence of autosomal recessive genes that are responsible, in part, for the
increased prevalence of the disease.

It has been reported that 44% of all Arab marriages in Israel are consanguineous, with a mean inbreeding coefficient of .0192 (Jaber, Shohat, Rotter, & Shohat, 1997). The inbreeding rates for Israeli Arabs may be particularly high (as compared with Egyptians or Syrians) because mobility
was reduced for centuries by the Turks. At times in Arab communities, it has been required for children to marry into their own family in part to avoid sharing resources with competing family groups.

Linkage studies by the Boston University group and others focused on inbred Arab families have helped to locate genes for several inherited, neurological diseases, including Wilson's disease, spinal muscular atrophy, sensorineural deafness, and autosomal recessive Duchenne-like
muscular dystrophy. Of critical relevance, successes in mapping genes for Wilson's disease and deafness using inbred Arab kindreds facilitated the cloning of these genes, which were subsequently shown to have diseasecausing mutations prevalent in outbred populations from other parts of the world, including the United States.

All of the known Alzheimer-related genes (on chromosomes 21, 14, 1, and 19) are dominant (chromosomes 21,14,1) or codominant (chromosome 19). There are no known genes affecting the development of AD that are recessive, perhaps because there have been few studies of AD in populations with high levels of inbreeding. De Braekeleer et al. (1989) reported an association between inbreeding and the development of AD (inbreeding coefficient 9 times higher in AD cases than in controls) in a rural population in Quebec. Studies in the Old Order Amish have found a low prevalence of disease in this inbred community, also having a low frequency
of the apo E-e4 allele (.037). Inbreeding could be linked to AD through a confounding effect of education, but the association of inbreeding and education is controversial (found for a Saudi population but not for Israeli Arabs). We expect that there are recessive factors for AD Genetic and Environmental Risk Factors 15 because of indirect evidence: genetic modeling studies of AD in apo E-e4 negative families are unable to reject a recessive model (Rao et al., 1996).

However, it is difficult to evaluate recessive models in outbred populations. The high prevalence we have seen in our highly inbred Arab population in Israel might be due to the inculcation of recessive AD susceptibility alleles or to a dominant gene that became frequent by founder effect.
In a population-based study of AD, we have screened all elderly residents of Wadi Ara, an Arab community in northern Israel, and observed an unusually high prevalence (20.5% of those >60 years, 60.5% of those >85 years). This prevalence is higher than that found in other populations in Israel, China, Europe, or the United States, even after adjustment for
age, education, and gender, and is not due to increased frequency of the apo E-e4 allele (Corder, Saunders, Strittmatter, et al., 1993; Saunders, Strittmatter, Schmechel, et al., 1993) which is actually reduced in this community (0.035 for nondemented elders) as compared with other Caucasians.

DNA samples were collected randomly from 256 participants of the Wadi Ara study, aged 75 ± 9, 118 male, and their apo E genotype was determined by a PCR-based method (Chapman, Estupinan, Asherov, & Goldfarb, 1996). Of the 256 cases examined, 22 carried an apo E-e4 allele
(all heterozygous), including 3 of 34 with AD (apo E-e4 allele frequency 0.04), 8 of 128 nondemented elderly participants (apo E-e4 allele frequency 0.03), 7 of 56 with age-associated memory impairment (AAMI; apo E-e4 allele frequency 0.06), and 4 of 38 with other types of dementia and pseudodementia (apo E-e4 allele frequency 0.05). These data suggest that the
apo E-e4 allele is relatively uncommon in Arabs in Wadi Ara. In fact, this is the lowest frequency of apo E-e4 ever recorded. Although the possibility that it is associated with dementia was not excluded, it cannot explain the high AD prevalence in this population.

Pedigree studies showed that more than one of three of the 168 prevalent cases were from one hamula (extended family) of the 14 found in Wadi Ara. A lOcM genome scan showed significant association with a site that has been narrowed to 1.6 cM. Evidence for linkage stemmed primarily from excess homozygosity of one of the alleles for a marker in this region in the total sample of cases (15%) compared with controls (3%). The crude odds ratio of AD associated with this genotype was 5.7 (95% CI = 1.5,21.7).

The odds increased to 10.0 (1.3-75.9) after adjustment for age, sex, and systolic blood pressure. The observation of significant but different patterns of association within multiple hamulas suggests the existence of multiple recombination between the marker and the AD susceptibility locus. This genetic location notably overlaps a region showing evidence for linkage to AD in a genome scan (Kehoe et al., 1999). This location includes several genes, including a gene strongly related to lipid metabolism. We are currently working on the precise identification of the linked gene. We are also investigating the possibility that the high prevalence of AD in Wadi Ara is related to environmental risk factors and medical illness, including a high-fat diet, altered lipid metabolism, thyroid disease, smoking, hypertension, heart disease, and stroke.

DEMENTIA PREVALENCE IN EXTREME OLD AGE

Numerous researchers have extrapolated from dementia and AD prevalence rates in younger people that all centenarians should have at least some degree of cognitive impairment. Yet our studies, as well as those of other centenarian studies, indicate that approximately 20% to 30% are cognitively intact.
Dr. Silver, our lead neuropsychologist, examined a series of 74 centenarians during a 3-year period. Some of their characteristics are noted in Table 2.1, and Table 2.2 lists the neuropsychological tests performed. We are collaborating with other centenarian studies to establish norms for components of the Mattis Dementia Rating Scale and other tests.

The neuropsychological examination, as well as other phenotyping, is performed where the person lives. Specific maneuvers are performed to accommodate for hearing and vision deficits. Multiple visits are performed to prevent fatigue from factoring into the testing, and family is often invited to participate in order to help language barriers.

Table 2.3 summarizes Dr. Silver's neuropsychological test results of 74 subjects. The neuropathological findings for 14 of these participants are summarized in Table 2.4.

It is noteworthy that evidence of microvascular disease was conspicuously absent in the 14 cases found in Table 2.4. We have noted the absence of diagnoses and blood pressure measurements for many of our participants, which would predispose to vascular disease.
We are now in the process of recruiting individuals who are willing to eventually proceed to postmortem autopsy, following them longitudinally with annual detailed neuropsychological examinations. Our aim is to recruit 33 centenarians per year, who live in close enough proximity to the Massachusetts ADRC to ensure that the brain can be obtained within 4 hours of death.

TABLE 2.1 Centenarians Who Have Undergone Neuropsychological Testing
Age range 100-110 years
Gender 86% women, 14% men
Living situation 7% live alone, 26% with family, 67% in nursing homes
Country of origin 50% foreign born

Most frequent birthplaces Italy, Ireland, and Canada Education Mean = 11 years
Range = 1-20 years


TABLE 2.2 Neuropsychological Test Battery

Mini-Mental State Exam
• Mattis Dementia Rating Scale
• Boston Naming Test (CERAD)
• Trail Making Tests
A and B
• Clock Drawing
• Drilled Word Span
• Cowboy Story
(Boston-Rochester)
• Presidents since FDR
Inventory: Self report and Observer report
• Spiers' Calculations


Geriatric Depression Scale
* Telephone Interview for Cognitive Status
• Test for Severe Impairment
• Tactile Naming
• Cognition and Health History (Informant)
• Psychiatry History (Informant)
• Clinical Dementia Rating Scale
• NEO-Five Factor Personality

Intrathecal corticosteroids might slow Alzheimer’s disease progression

Anti-inflammatory drugs for treatment and prevention of Alzheimer’s disease have to date proved disappointing, including a large study of low-dose prednisone, but higher dose steroids significantly reduced amyloid secretion in a small series of nondemented patients. In addition, there is a case report of a patient with amyloid angiopathy who had complete remission from two doses of dexamethasone, and very high dose steroids are already used for systemic amyloidosis. This paper presents the hypothesis that pulse-dosed intrathecal methylprednisolone or dexamethasone will produce detectable slowing of Alzheimer’s progression, additive to that obtained with cholinesterase inhibitors and memantine. A protocol based on treatment regimens for multiple sclerosis and central nervous system lupus is outlined, to serve as a basis for formulating clinical trials. Ultimately intrathecal corticosteroids might become part of a multi-agent regimen for Alzheimer’s disease and also have application for other neurodegenerative disorders.

Epidemiological evidence suggests that drugs which counteract inflammation might have efficacy for the prevention and treatment of Alzheimer’s disease, but up to now clinical trials have failed to show any clear-cut benefits for nonsteroidal anti-inflammatory drugs (NSAIDs), hydroxychloroquine, anti-leprosy agents or prednisone. In the case of prednisone, however, it may be that much higher doses might be effective where lower doses were not. In the largest clinical trial to date, patients were given prednisone starting at 20 mg per day for one month, followed by one year at 10 mg daily and then tapering off over another 4 months. By contrast, in a series of 16 nondemented patients aged 25 to 82 who were given prednisolone 30–60 mg per day for at least month for treatment of various conditions, there was significant reduction in cerebrospinal fluid amyloid beta peptides in 15 out of 16 patients, up to about a 50% decline for patients receiving 50 and 60 mg of prednisolone. If amyloid is indeed a cause of Alzheimer’s disease, the above data suggests high dose steroids could suppress Alzheimer’s disease by reducing amyloid.

If the above line of reasoning is on the right track, it is important to note that high dose corticosteroids are already used clinically for treatment of primary systemic amyloidosis, in amounts orders of magnitude greater than those used in the Alzheimer’s disease trial. For example, in one recently published study of a regimen combining dexamethasone and interferon, patients were given several days of dexamethasone in an amount of 40 mg per day, which would be equivalent to 400 mg of prednisone daily.

Finally, there is a case report of a 64-year-old man with multiple myeloma who had a temporary remission of Alzheimer’s disease while receiving chemotherapy with vincristine, carmustine, melphalan, cyclophosphamide, and prednisone.

Obviously megadose of anabolic steroids or cytotoxic chemotherapy as a treatment for Alzheimer’s disease would be hard to justify, but if one can knock down amyloid production, inflammation or both with corticosteroids alone, there should be at least some therapeutic effects. The key is delivering high enough doses of corticosteroids to have efficacy without devastating or killing patients with steroid-induced side effects in the process. Building on several case reports of patients with central nervous system lupus erythematosus and multiple sclerosis who responded to intrathecal prednisolone, dexamethasone, or triamcinalone after failing oral and intravenous steroids, this paper proposes that a similar therapeutic strategy could be pursued for patients with Alzheimer’s disease. Studies in rhesus monkeys and pigs indicate that that intrathecal steroids maximize biodistribution within the brain and minimize it within the rest of the body.

Genetic Correlates of Successful Cognitive Aging

IDENTIFYING GENES PREDISPOSING TO SUCCESSFUL COGNITIVE AGING

The hypothesis driving this study is that centenarians are a select group of people who have a history of aging relatively slowly and who have either markedly delayed or entirely escaped diseases normally associated with aging, such as Alzheimer's disease (AD), cancer, stroke, and heart disease.
The ultimate challenge in this area of research is to identify the genes that are associated with such a survival advantage and the ability to age so well for such a long time without cognitive impairment. We have very exciting preliminary results revealing that centenarians can be the
key to discovering these genes.
Primarily because of funding by the Institute for the Study of Aging, continue careful annual neuropsychological testing and eventual neuropathological study of the centenarian subjects.

Approximately 20% of our participants wish to be postmortem brain donors, but we anticipate
increasing this rate to 50%, again, in part, due to the generosity of the Institute. These neuropsychological-neuropathological correlations will help us better understand what disease-free aging of the brain means and what it looks like; if causes of dementia are different in the extremely old compared with younger individuals; and the type and quantity of changes in the brain that correlate with different levels of cognitive impairment.

At the same time, we are studying the centenarians for genes that may play pivotal roles in determining how centenarians markedly delay or, in some cases, escape cognitive impairment. We have obtained the largest collection of centenarian sibships in the world (N ~ 200). Genome-wide scans from these individuals were performed and the data was given to our statistics colleagues at the Whitehead Institute and Rutgers University for linkage analyses. Based upon scans of 308 individuals making up 137 families, currently we have noted statistically significant linkage to a 20 cM region on chromosome 4. A manuscript reporting these results is currently in the review process. Discovery of genes that powerfully affect processes as broad as rates of aging and/or susceptibility to diseases, such as AD and stroke, would have dramatic impact upon understanding the underpinnings of aging and could, ultimately, lead to promising targets for drug discovery.


In our attempts to locate and recruit sibships, we have enrolled five families with many members achieving extreme old age. Such clustering lends itself to linkage studies similar to those performed in families with clustering for rare diseases (such as Tay-Sachs, cystic fibrosis, and sickle-cell anemia).
In this instance, we are not looking for the cause of a disease but rather a fantastic advantage. Four of these families are described in a recent article published in the Journal of the American Geriatrics Society. Unfortunately, because there is likely to be more than one gene (or the lack of a specific mutation) increasing the probability of achieving exceptional old age, we believe that even 10 members of one family being included in a linkage study is unlikely to produce statistically significant results. However, as we include cousins and perhaps children, such studies might prove to be feasible.
In another approach to gene discovery, we have established a collaboration with gene - expression expert Steven Gullens, PhD, from the Brigham and Women's Hospital. Because we are able to obtain brain tissue at postmortem autopsy within 4 hours of death, we will provide Dr. Gullens with ideal brain tissue samples from regions of specific interest regarding AD, as well as regions that play critical roles in cognition (e.g., frontal lobe and its influence upon executive function) for differential gene expression studies. Determining which genes are active in cognitively intact participants versus those with various causes of cognitive impairment versus
other younger controls should be an efficient approach to discovering genes critical to AD pathogenesis.