Developmental deficits and staging of dynamics of age associated Alzheimer's disease neurodegeneration and neuronal loss in subjects with Down syndrome.

The increased life expectancy of individuals with Down syndrome (DS) is associated with increased prevalence of trisomy 21-linked early-onset Alzheimer's disease (EOAD) and dementia. The aims of this study of 14 brain regions including the entorhinal cortex, hippocampus, basal ganglia, and cerebellum in 33 adults with DS 26-72 years of age were to identify the magnitude of brain region-specific developmental neuronal deficits contributing to intellectual deficits, to apply this baseline to identification of the topography and magnitude of neurodegeneration and neuronal and volume losses caused by EOAD, and to establish age-based staging of the pattern of genetically driven neuropathology in DS. Both DS subject age and stage of dementia, themselves very strongly correlated, were strong predictors of an AD-associated decrease of the number of neurons, considered a major contributor to dementia. The DS cohort was subclassified by age as pre-AD stage, with 26-41-year-old subjects with a full spectrum of developmental deficit but with very limited incipient AD pathology, and 43-49, 51-59, and 61-72-year-old groups with predominant prevalence of mild, moderately severe, and severe dementia respectively. This multiregional study revealed a 28.1% developmental neuronal deficit in DS subjects 26-41 years of age and 11.9% AD-associated neuronal loss in DS subjects 43-49 years of age; a 28.0% maximum neuronal loss at 51-59 years of age; and a 11.0% minimum neuronal loss at 61-72 years of age. A total developmental neuronal deficit of 40.8 million neurons and AD-associated neuronal loss of 41.6 million neurons reflect a comparable magnitude of developmental neuronal deficit contributing to intellectual deficits, and AD-associated neuronal loss contributing to dementia. This highly predictable pattern of pathology indicates that successful treatment of DS subjects in the fourth decade of life may prevent AD pathology and functional decline.

Longitudinal telomere shortening and early Alzheimer's disease progression in adults with down syndrome.

Telomere shortening was shown to parallel Alzheimer's disease (AD) associated dementia. By using a dual PNA Probe system we have developed a practical method for comparing telomere length in T-lymphocyte interphases from individuals with Down syndrome (DS) with and without "mild cognitive impairment" (MCI-DS) and demonstrated that telomere length can serve as a valid biomarker for the onset of MCI-DS in this high-risk population. To verify progressive cognitive decline we have now examined sequential changes in telomere length in 10 adults with DS (N = 4 Female, N = 6 Male) developing MCI-DS. Cases were selected blind to telomere length from a sample of adults with DS previously enrolled in a prospective longitudinal study at 18-month intervals with clinical and telomere assessments: (1) MCI-DS group data were collected approximately three years prior to development of MCI-DS; (2) 18 months later; (3) when MCI-DS was first observed. These telomere measures were compared to those from another 10 adults with DS matched by sex and approximate age but without indications of MCI-DS (Controls). PNA (peptide nucleic acid) probes for telomeres together with a chromosome two centromere probe were used. Findings indicated telomere shortening over time for both Cases and Controls. Group differences emerged by 18-months prior to recognition of MCI-DS onset and completely non-overlapping distributions of telomere measures were observed by the time of MCI-DS onset. This study adds to accumulating evidence of the value of telomere length, as an early biomarker of AD progression in adults with Down syndrome.

Multiregional Age-Associated Reduction of Brain Neuronal Reserve Without Association With Neurofibrillary Degeneration or ß-Amyloidosis.

Increase in human life expectancy has resulted in the rapid growth of the elderly population with minimal or no intellectual deterioration. The aim of this stereological study of 10 structures and 5 subdivisions with and without neurofibrillary degeneration in the brains of 28 individuals 25-102-years-old was to establish the pattern of age-associated neurodegeneration and neuronal loss in the brains of nondemented adults and elderly. The study revealed the absence of significant neuronal loss in 7 regions and topographically selective reduction of neuronal reserve over 77 years in 8 brain structures including the entorhinal cortex (EC) (-33.3%), the second layer of the EC (-54%), cornu Ammonis sector 1 (CA1) (-28.5%), amygdala, (-45.8%), thalamus (-40.5%), caudate nucleus (-35%), Purkinje cells (-48.3%), and neurons in the dentate nucleus (40.1%). A similar rate of neuronal loss in adults and elderly, without signs of accelerating neuronal loss in agers or super-agers, appears to indicate age-associated brain remodeling with significant reduction of neuronal reserve in 8 brain regions. Multivariate analysis demonstrates the absence of a significant association between neuronal loss and the severity of neurofibrillary degeneration and ß-amyloidosis, and a similar rate of age-associated neuronal loss in structures with and without neurofibrillary degeneration.

Telomere longitudinal shortening as a biomarker for dementia status of adults with Down syndrome.

Previous studies have suggested that Alzheimer's disease (AD) causes an accelerated shortening of telomeres, the ends of chromosomes consisting of highly conserved TTAGGG repeats that, because of unidirectional 5'-3' DNA synthesis, lose end point material with each cell division. Our own previous work suggested that telomere length of T-lymphocytes might be a remarkably accurate biomarker for "mild cognitive impairment" in adults with Down syndrome (MCI-DS), a population at dramatically high risk for AD. To verify that the progression of cognitive and functional losses due to AD produced this observed telomere shortening, we have now examined sequential changes in telomere length in five individuals with Down syndrome (3F, 2M) as they transitioned from preclinical AD to MCI-DS (N = 4) or dementia (N = 1). As in our previous studies, we used PNA (peptide nucleic acid) probes for telomeres and the chromosome 2 centromere (as an "internal standard" expected to be unaffected by aging or dementia status), with samples from the same individuals now collected prior to and following development of MCI-DS or dementia. Consistent shortening of telomere length was observed over time. Further comparisons with our previous cross-sectional findings indicated that telomere lengths prior to clinical decline were similar to those of other adults with Down syndrome (DS) who have not experienced clinical decline while telomere lengths following transition to MCI-DS or dementia in the current study were comparable to those of other adults with DS who have developed MCI-DS or dementia. Taken together, findings indicate that telomere length has significant promise as a biomarker of clinical progression of AD for adults with DS, and further longitudinal studies of a larger sample of individuals with DS are clearly warranted to validate these findings and determine if and how factors affecting AD risk also influence these measures of telomere length.

Intellectual Disability, Mild Cognitive Impairment, and Risk for Dementia.

People with intellectual disability (ID) are living longer than ever before, raising concerns about old-age associated disorders. Dementia is among the most serious of these disorders, and theories relating cognitive reserve to risk predict that older adults with ID should be particularly vulnerable. Previous estimates of relative risk for dementia associated with ID have been inconsistent, and the present analyses examined the possible influence of variation in diagnostic criteria on findings. As expected, relaxation in the stringency of case definition for adults with ID increased relative risk, underscoring the importance of developing valid criteria for defining mild cognitive impairment, early dementia, and distinguishing between the two in adults with ID. Once available, these standards will contribute to more effective evidence-based planning.

Mild cognitive impairment identified in older individuals with Down syndrome by reduced telomere signal numbers and shorter telomeres measured in microns.

Previously, we established that short-term T lymphocyte cultures from people with Down syndrome (DS) and dementia (Alzheimer's disease) had shorter telomeres than did those from age- and sex-matched people with DS only, quantified as significantly reduced numbers of signals of peptide nucleic acid (PNA) telomere probes in whole metaphases [Jenkins et al. (2008); Neurosci Lett 440:340-343] as well as reduced telomere probe light intensity values in interphases [Jenkins et al. (2010); Neurobiol Aging 31:765-771]. We now describe shorter telomere length in adults with DS and mild cognitive impairment (MCI) compared to age- and sex-matched individuals with DS without MCI. Telomere length is quantified by reduced telomere signal numbers and shorter chromosome 1 telomeres measured in micrometers (microns). These findings were in agreement with quantitative light intensity measurements of chromosome 1 and chromosome 21 PNA telomere probes with and without the use of a "normalizing ratio" involving the fluorescence exhibited by a PNA probe for centromere 2, and with the use of light intensity measurements of interphase preparations. Most importantly, the distributions of chromosome 1 telomere lengths (in microns) were completely non-overlapping for adults with and without MCI, indicating that this measure has great promise as a biomarker for MCI as well as dementia in this population.

Does the cryogenic freezing process cause shorter telomeres?

We have observed evidence of increased telomere shortening in short-term T-lymphocyte cultures following freezing and thawing of the original inoculum obtained by ficoll-paque gradient centrifugation, compared to T-lymphocytes that were cultured immediately without freezing and thawing from the same blood sample from 3 female and 3 male adults. Because freezing may have similar effects on other cell types, and because telomere shortening may only manifest its effects after many years or decades, we suggest there is a pressing need for evaluation of the effects of freezing on any cells envisioned for clinical applications, including embryo implantation.

Link between DYRK1A overexpression and several-fold enhancement of neurofibrillary degeneration with 3-repeat tau protein in Down syndrome.

Triplication of chromosome 21 in Down syndrome (DS) results in overexpression of the minibrain kinase/dual-specificity tyrosine phosphorylated and regulated kinase 1A gene (DYRK1A). DYRK1A phosphorylates cytoplasmic tau protein and appears in intraneuronal neurofibrillary tangles (NFTs). We have previously shown significantly more DYRK1A-positive NFTs in DS brains than in sporadic Alzheimer disease (AD) brains. This study demonstrates a gene dosage-proportional increase in the level of DYRK1A in DS in the cytoplasm and the cell nucleus, and enhanced cytoplasmic and nuclear immunoreactivity of DYRK1A in DS. The results suggest that overexpressed DYRK1A may alter both phosphorylation of tau and alternative splicing factor (ASF). Two-dimensional electrophoresis revealed modification of ASF phosphorylation in DS/AD and AD in comparison to controls. Altered phosphorylation of ASF by overexpressed nuclear DYRK1A may contribute to the alternative splicing of the tau gene and an increase by 2.68 × of the 3R/4R ratio in DS/AD, and a several-fold increase in the number of 3R tau-positive NFTs in DS/AD subjects compared with that in sporadic AD subjects. These data support the hypothesis that phosphorylation of ASF by overexpressed DYRK1A may contribute to alternative splicing of exon 10, increased expression of 3R tau, and early onset of neurofibrillary degeneration in DS.

Shorter telomeres may indicate dementia status in older individuals with Down syndrome.

We have recently reported reduced telomere length in T lymphocytes of individuals with Down syndrome (DS) and dementia due to Alzheimer's disease (AD). We have now replicated and extended that study by finding that people with DS and mild cognitive impairment (MCI-DS) also have shorter telomeres than people with DS without MCI-DS. Additional new findings demonstrated that light intensity measurements from chromosome 21 alone, or in concert with chromosomes 1, 2, and 16, exhibited shorter telomeres in adults with DS and with either dementia or MCI-DS compared to aging per se. Chromosome 21 measurements appeared to be especially promising for use as a biomarker because there was no overlap in the distribution of light intensity measurement scores between demented or MCI-DS and non-demented participants. Given that early clinical symptoms of AD can be very difficult to recognize in this population of adults due to their pre-existing cognitive impairments, a valid biomarker would be of great value. Early detection is especially important because it would allow treatments to begin before significant damage to the central nervous system has occurred. Our findings suggest that it may be feasible to use telomere shortening as a biomarker for accurately inferring dementia status.

The role of overexpressed DYRK1A protein in the early onset of neurofibrillary degeneration in Down syndrome.

The gene encoding the minibrain kinase/dual-specificity tyrosine phosphorylated and regulated kinase 1A (DYRK1A) is located in the Down syndrome (DS) critical region of chromosome 21. The third copy of DYRK1A is believed to contribute to abnormal brain development in patients with DS. In vitro studies showing that DYRK1A phosphorylates tau protein suggest that this kinase is also involved in tau protein phosphorylation in the human brain and contributes to neurofibrillary degeneration, and that this contribution might be enhanced in patients with DS. To explore this hypothesis, the brain tissue from 57 subjects including 16 control subjects, 21 patients with DS, and 20 patients with sporadic Alzheimer's disease (AD) was examined with two antibodies to the amino-terminus of DYRK1A (7F3 and G-19), as well as two polyclonal antibodies to its carboxy-terminus (X1079 and 324446). Western blots demonstrated higher levels of full-length DYRK1A in the brains of patients with DS when compared to control brains. Immunocytochemistry revealed that DYRK1A accumulates in neurofibrillary tangles (NFTs) in subjects with sporadic AD and in subjects with DS/AD. Overexpression of DYRK1A in patients with DS was associated with an increase in DYRK1A-positive NFTs in a gene dosage-dependent manner. Results support the hypothesis that overexpressed DYRK1A contributes to neurofibrillary degeneration in DS more significantly than in subjects with two copies of the DYRK1A gene and sporadic AD. Immunoreactivity with antibodies against DYRK1A not only in NFTs but also in granules in granulovacuolar degeneration and in corpora amylacea suggests that DYRK1A is involved in all three forms of degeneration and that overexpression of this kinase may contribute to the early onset of these pathologies in DS.

Increased "absence" of telomeres may indicate Alzheimer's disease/dementia status in older individuals with Down syndrome.

We have reported previously that telomeres (ends of chromosomes consisting of highly conserved TTAGGG repeats) were shorter in metaphase and interphase preparations in T lymphocytes from short-term whole blood cultures of women with Down syndrome (DS) and dementia compared to age-matched women with DS but without dementia [E.C. Jenkins, M.T. Velinov, L. Ye, H. Gu, S. Li, E.C. Jenkins Jr., S.S. Brooks, D. Pang, D.A. Devenny, W.B. Zigman, N. Schupf, W.P. Silverman, Telomere shortening in T lymphocytes of older individuals with Down syndrome and dementia, Neurobiol. Aging 27 (2006) 41-45]. Our previous study was carried out by measuring changes in fluorescence intensity [using an FITC-labeled peptide nucleic acid (PNA) probe (Applied Biosystems; DAKO) and Applied Imaging software], and we now report on a substantially simpler metric, counts of signals at the ends of chromosomes. Nine adults with DS and dementia plus four who are exhibiting declines in cognition analogous to mild cognitive impairment in the general population (MCI-DS) were compared to their pair-matched peers with DS but without dementia or MCI-DS. Results indicated that the number of chromosome ends that failed to exhibit fluorescent signal from the PNA telomere probe was higher for people with dementia or mild cognitive impairment (MCI-DS). Thus, a simple count of chromosome ends for the "presence/absence" of fluorescence may provide a valid biomarker of dementia status. If this is the case, then after additional research for validation to assure high specificity and sensitivity, the test may be used to identify and ultimately guide treatment for people at increased risk for developing mild cognitive impairment and/or dementia.

Intraneuronal Abeta immunoreactivity is not a predictor of brain amyloidosis-beta or neurofibrillary degeneration.

Amyloid beta (Abeta) immunoreactivity in neurons was examined in brains of 32 control subjects, 31 people with Down syndrome, and 36 patients with sporadic Alzheimer's disease to determine if intraneuronal Abeta immunoreactivity is an early manifestation of Alzheimer-type pathology leading to fibrillar plaque formation and/or neurofibrillary degeneration. The appearance of Abeta immunoreactivity in neurons in infants and stable neuron-type specific Abeta immunoreactivity in a majority of brain structures during late childhood, adulthood, and normal aging does not support this hypothesis. The absence or detection of only traces of reaction with antibodies against 4-13 aa and 8-17 aa of Abeta in neurons indicated that intraneuronal Abeta was mainly a product of alpha- and gamma-secretases (Abeta(17-40/42)). The presence of N-terminally truncated Abeta(17-40) and Abeta(17-42) in the control brains was confirmed by Western blotting and the identity of Abeta(17-40) was confirmed by mass spectrometry. The prevalence of products of alpha- and gamma -secretases in neurons and beta- and gamma-secretases in plaques argues against major contribution of Abeta-immunopositive material detected in neuronal soma to amyloid deposit in plaques. The strongest intraneuronal Abeta(17-42) immunoreactivity was observed in structures with low susceptibility to fibrillar Abeta deposition, neurofibrillary degeneration, and neuronal loss compared to areas more vulnerable to Alzheimer-type pathology. These observations indicate that the intraneuronal Abeta immunoreactivity detected in this study is not a predictor of brain amyloidosis or neurofibrillary degeneration. The constant level of Abeta immunoreactivity in structures free from neuronal pathology during essentially the entire life span suggests that intraneuronal amino-terminally truncated Abeta represents a product of normal neuronal metabolism.

Telomere shortening in T lymphocytes of older individuals with Down syndrome and dementia.

Telomere shortening has been recently correlated with Alzheimer's disease status. Therefore, we hypothesized that a possible association might exist for adults with Down syndrome (DS). Using blind, quantitative telomere protein nucleic acid FISH analyses of metaphase and interphase preparations from 18 age-matched trisomy 21 female study participants with and without dementia, we have observed increased telomere shortening in adults with DS and dementia (p < .01). From this initial study, we conclude that telomere shortening is associated with dementia in this high-risk population and suggest that additional research may show that telomere shortening may be a biological marker of dementia status.

Cell type- and brain structure-specific patterns of distribution of minibrain kinase in human brain.

The minibrain kinase (Mnb/Dyrk1A) gene is localized in the Down syndrome (DS) critical region of chromosome 21. This gene encodes a proline-directed serine/threonine protein kinase (minibrain kinase-Mnb/Dyrk1A), which is required for the proliferation of distinct neuronal cell types during postembryonic neurogenesis. To study the distribution of Mnb/Dyrk1A during human brain development and aging, we raised Mnb/Dyrk1A-specific antibody (mAb 7F3) and examined 22 brains of normal subjects from 8 months to 90 years of age. We found that neurons were the only cells showing the presence of 7F3-positive product in both cell nucleus and cytoplasm. Nuclear localization supports the concept that Mnb/Dyrk1A may be involved in control of gene expression. Synaptic localization of Mnb/Dyrk1A also supports our previous studies suggesting that Mnb/Dyrk1A is a regulator of assembly of endocytic apparatus and appears to be involved in synaptic vesicle recycling and synaptic signal transmission. Accumulation of numerous 7F3-positive corpora amylacea in the memory and motor system subdivisions in subjects older than 33 years of age indicates that Mnb/Dyrk1A is colocalized with markers of astrocyte and neuron degeneration. Differences in the topography and the amount of Mnb/Dyrk1A in neurons, astrocytes, and ependymal and endothelial cells appear to reflect cell type- and brain structure-specific patterns in trafficking and utilization of Mnb/Dyrk1A.