Alessia Maddalena

Antibodies against β-Amyloid Slow Cognitive Decline in Alzheimer's Disease

To test whether antibodies against beta-amyloid are effective in slowing progression of Alzheimers disease, we assessed cognitive functions in 30 patients who received a prime and a booster immunization of aggregated Abeta(42) over a 1 year period in a placebo-controlled, randomized trial. Twenty patients generated antibodies against beta-amyloid, as determined by tissue amyloid plaque immunoreactivity assay. Patients who generated such antibodies showed significantly slower rates of decline of cognitive functions and activities of daily living, as indicated by the Mini Mental State Examination, the Disability Assessment for Dementia, and the Visual Paired Associates Test of delayed recall from the Wechsler Memory Scale, as compared to patients without such antibodies. These beneficial clinical effects were also present in two of three patients who had experienced transient episodes of immunization-related aseptic meningoencephalitis. Our results establish that antibodies against beta-amyloid plaques can slow cognitive decline in patients with Alzheimers disease.

Treatment with the selective muscarinic ml agonist talsaclidine decreases cerebrospinal fluid levels of Aβ42 in patients with Alzheimer's disease

The amyloid p-peptides Aβ40 and Aβ42 are highly amyloidogenic constituents of brain Pamyloid plaques in Alzheimers disease (AD). Lowering their formation may be achieved by modulating the activities of proteases that cleave the amyloid precursor protein (AβPP), including α-β-, and γ-secretases. Talsaclidine is a functionally selective muscarinic ml agonist that stimulates non-amyloidogenic a-secretase processing in vitro. We compared cerebrospinal fluid (CSF) levels of Aβ40 and Aβ42 measured by ELISA before and at the end of 4 weeks of treatment with talsaclidine. The medication was administered in a double- blind, placebo-controlled, and randomized clinical study to 40 patients with AD. Talsaclidine (n=34) decreased CSF levels of Aβ42 by a median of 19% (p < 0.001) as compared to baseline. The mean diflerence between CSF levels of Aβ42 before and after treatment with talsaclidine (n=34) was -46.73 (SD) pg/ml as compared to 0.8 (SD)pg/ml with placebo (n=6) (p < 0.05). CSF levels of Aβ40 increased during treatement with placebo (n=6) while they remained stable during treatment with talsaclidine (n=31) (1.118±1.710 ng/ ml, and-0.170A0.967 ng/ml, respectively; p < 0.05). These data show that treatment with the ml agonist talsaclidine reduced Aβ peptides, and particularly Aβ42, in AD patients, suggesting it as a potential amyloid lowering therapy of AD.

Treatment with the Selective Muscarinic Agonist Talsaclidine Decreases Cerebrospinal Fluid Levels of Total Amyloid β‐Peptide in Patients with Alzheimer's Disease

Abstract: Brain amyloid load in Alzheimers disease (AD) is, at least in genetic forms, associated with overproduction of amyloid β‐peptides (Aβ). Thus, lowering Aβ production is a central therapeutic target in AD and may be achieved by modulating such key enzymes of amyloid precursor protein (APP) processing as β‐, γ‐, and α‐secretase activities. Talsaclidine is a selective muscarinic M1 agonist that stimulates the nonamyloidogenic α‐secretase pathway in model systems. Talsaclidine was administered double‐blind, placebo‐controlled, and randomized to 24 AD patients and cerebrospinal fluid (CSF) levels of total Aβ were quantitated before and after 4 weeks of drug treatment. We observed that talsaclidine decreases CSF levels of Aβ significantly over time within the treatment group (n=20) by a median of 16% as well as compared to placebo (n=4) by a median of 27%. We conclude that treatment with selective M1 agonists may reduce Aβ production and may thus be further evaluated as a potential amyloid‐lowering therapy of AD.

Cerebrospinal Fluid Profile of Amyloid β Peptides in Patients with Alzheimer’s Disease Determined by Protein Biochip Technology

Amyloid-β peptides (Aβ) are major components of amyloid plaques in the Alzheimer’s disease (AD) brain and have been proposed as diagnostic markers in cerebrospinal fluid (CSF). Aβ derived from brain may be processed into fragments before emerging in CSF. Therefore, we determined mass profiles of Aβ peptides in CSF of patients with AD and age-matched healthy control subjects (CTR) by using protein biochip technology. Aβ peptides were captured on the chip surfaces (spots) by the specific monoclonal antibody 6E10 and were then analyzed by integrated surface-enhanced laser desorption and ionization time-of-flight mass spectrometry (SELDI-TOF-MS). We found Aβ species with mean molecular masses at 1,583.3 Da (corresponding to Aβ_2–14), 2,068.5 Da (Aβ_1–17), 2,166.4 Da (Aβ_1–18), 3,676.6 Da (Aβ_1–33), 3,789.4 Da (Aβ_1–34), 4,076.9 Da (Aβ_1–37), 4,134.0 Da (Aβ_1–38), 4,233.3 Da (Aβ_1–39), 4,332.4 Da (Aβ_1–40) and 4,516.8 Da (Aβ_1–42) in both AD (n = 24) and CTR (n = 24) subjects. Aβ_1–38 appeared to be a major Aβ species in human CSF along with Aβ_1–40. Quantitation revealed that CSF levels of Aβ_1–38 were significantly decreased in AD as compared to CTR subjects. The CSF profile of Aβ peptides may be used for diagnostic and therapeutic purposes in clinical studies.

Early Induction of Angiogenetic Signals in Gliomas of GFAP-v-src Transgenic Mice

Angiogenesis is a prerequisite for solid tumor growth. Glioblastoma multiforme, the most common malignant brain tumor, is characterized by extensive vascular proliferation. We previously showed that transgenic mice expressing a GFAP-v-src fusion gene in astrocytes develop low-grade astrocytomas that progressively evolve into hypervascularized glioblastomas. Here, we examined whether tumor progression triggers angiogenetic signals. We found abundant transcription of vascular endothelial growth factor (VEGF) in neoplastic astrocytes at surprisingly early stages of tumorigenesis. VEGF and v-src expression patterns were not identical, suggesting that VEGF activation was not only dependent on v-src. Late-stage gliomas showed perinecrotic VEGF up-regulation similarly to human glioblastoma. Expression patterns of the endothelial angiogenic receptors flt-1, flk-1, tie-1, and tie-2 were similar to those described in human gliomas, but flt-1 was expressed also in neoplastic astrocytes, suggesting an autocrine role in tumor growth. In crossbreeding experiments, hemizygous ablation of the tumor suppressor genes Rb and p53 had no significant effect on the expression of VEGF, flt-1, flk-1, tie-1, and tie-2. Therefore, expression of angiogenic signals is an early event during progression of GFAP-v-src tumors and precedes hypervascularization. Given the close similarities in the progression pattern between GFAP-v-src and human gliomas, the present results suggest that these mice may provide a useful tool for antiangiogenic therapy research.

Cholesterol 25-Hydroxylase on Chromosome 10q Is a Susceptibility Gene for Sporadic Alzheimer’s Disease

Alzheimer’s disease (AD) is the most common cause of dementia. It is characterized by β-amyloid (Aβ) plaques, neurofibrillary tangles and the degeneration of specifically vulnerable brain neurons. We observed high expression of the cholesterol 25-hydroxylase (CH25H) gene in specifically vulnerable brain regions of AD patients. CH25H maps to a region within 10q23 that has been previously linked to sporadic AD. Sequencing of the 5′ region of CH25H revealed three common haplotypes, CH25Hχ2, CH25Hχ3 and CH25Hχ4; CSF levels of the cholesterol precursor lathosterol were higher in carriers of the CH25Hχ4 haplotype. In 1,282 patients with AD and 1,312 healthy control subjects from five independent populations, a common variation in the vicinity of CH25H was significantly associated with the risk for sporadic AD (p = 0.006). Quantitative neuropathology of brains from elderly non-demented subjects showed brain Aβ deposits in carriers of CH25Hχ4 and CH25Hχ3 haplotypes, whereas no Aβ deposits were present in CH25Hχ2 carriers. Together, these results are compatible with a role of CH25Hχ4 as a putative susceptibility factor for sporadic AD; they may explain part of the linkage of chromosome 10 markers with sporadic AD, and they suggest the possibility that CH25H polymorphisms are associated with different rates of brain Aβ deposition.

No Complementation Between TP53 or RB‐1 and v‐src in Astrocytomas of GFAP‐v‐src Transgenic Mice

Human low‐grade astrocytomas frequently recur and progress to states of higher malignancy. During tumor progression TP53 alterations are among the first genetic changes, while derangement of the p16/p14ARF/RB‐1 system occurs later. To probe the pathogenetic significance of TP53 and RB‐1 alterations, we introduced a v‐src transgene driven by glial fibrillary acidic protein (GFAP) regulatory elements (which causes preneoplastic astrocytic lesions and stochastically astrocytomas of varying degrees of malignancy) into TP53+/‐ or RB‐1+/‐ mice. Hemizygosity for TP53 or RB‐1 did not increase the incidence or shorten the latency of astrocytic tumors in GFAP‐v‐src mice over a period of up to 76 weeks. Single strand conformation analysis of exons 5 to 8 of non‐ablated TP53 alleles revealed altered migration patterns in only 3/16 tumors analyzed. Wild‐type RB‐1 alleles were retained in all RB‐1+/‐ GFAP‐v‐src mice‐derived astrocytic tumors analyzed, and pRb immunostaining revealed protein expression in all tumors. Conversely, the GFAP‐v‐src transgene did not influence the development of extraneural tumors related to TP53 or RB‐1 hemizygosity. Therefore, the present study indicates that neither loss of RB‐1 nor of TP53 confer a growth advantage in vivo to preneoplastic astrocytes expressing v‐src, and suggests that RB‐1 and TP53 belong to one single complementation group along with v‐src in this transgenic model of astrocytoma development. The stochastic development of astrocytic tumors in GFAP‐v‐src, TP53+/‐ GFAP‐v‐src, and RB‐1+/‐ GFAP‐v‐src transgenic mice indicates that additional hitherto unknown genetic lesions of astrocytes contribute to tumorigenesis, whose elucidation may prove important for our understanding of astrocytoma initiation and progression.

Genetic polymorphisms and cerebrospinal fluid levels of tissue inhibitor of metalloproteinases 1 in sporadic Alzheimer's disease

Tissue inhibitor of metalloproteinases 1 (TIMP-1) inhibits several proteinases including a disintegrin and metalloproteinase 10 (ADAM10), a major &agr;-secretase that cleaves the &bgr;-amyloid precursor protein within its amyloidogenic A&bgr; domain. The gene encoding TIMP-1 (TIMP1) maps to the short arm of the X chromosome, in a region previously suggested as conferring genetic susceptibility for Alzheimers disease (AD). To determine whether genetic variability of TIMP1 contributes to the pathogenesis of AD, we analysed one single nucleotide polymorphism within TIMP1 and one single nucleotide polymorphism in the 5′-untranslated region of TIMP1 in patients with AD and control subjects from two independent and ethnically different populations. We did not observe any association between TIMP1 genotypes and the diagnosis of AD in men or women. We also measured TIMP-1 protein levels in the cerebrospinal fluid of patients with AD, healthy control subjects, and patients with other neurological disorders. TIMP-1 levels were similar in all groups. In addition, no significant differences were observed after stratification for TIMP1 genotypes. Our data show that neither genetic variability nor protein levels of TIMP-1 are associated with AD.

Transgenic mice as research tools in neurocarcinogenesis

Transgenic animal models for neurocarcinogenesis have provided significant insights into the molecular mechanisms underlying carcinogenic processes, including those which affect the nervous system. In view of the very rapid pace of acquisition of knowledge, it is not possible to cover all transgenic mouse models for neural tumors. Instead, this article discusses some of the most important technical innovations for manipulation of the mammalian genome (notably the various methods for targeted genome modifications, as well as the technology for introducing large DNA fragments into the germ line of mice), and presents a selection of the transgenic mouse models which are proving most promising for furthering our understanding of the pathogenetic basis of cancer in the nervous system.

Cerebrospinal fluid biomarkers for the diagnosis of Alzheimer's disease

The antemortem diagnosis of probable Alzheimer’s disease (AD) requires time-consuming and costly procedures. Therefore, biomarkers that can direct the physician rapidly to the correct diagnosis are highly desirable. According to the consensus report of the working group on “Molecular and Biochemical Markers of Alzheimer’s Disease,” the ideal biomarker for AD should have a sensitivity of >80% for detecting AD and a specificity of >80% for distinguishing other dementias. Candidate diagnostic markers were identified by quantitating proteins associated with the characteristic histopathological hallmarks of AD: β-amyloid plaques and neurofibrillary tangles (NFT). Cerebrospinal fluid (CSF) levels of amyloid β-peptide (Aβ42) and total tau protein, as well as combinations of the two, were shown to corroborate the clinical diagnosis of AD; however, they did so without fully meeting the working group guidelines. Combined CSF measurements of phosphorylated tau protein (phospho-tau) and Aβ42 and calculation of the phospho-tau/Aβ42 ratio resulted in a further, slight improvement of diagnostic accuracy (Maddalena et al. 2003). Thus, measurement of the CSF phospho-tau/Aβ42 ratio meets the working group guidelines for sensitivity and comes close to meeting the guidelines for specificity. It is currently unclear how both sensitivity and specificity values can be set at levels higher than 90%. It is unlikely that this goal can be achieved by measurement of Aβ peptides and tau proteins alone, because the AD neuropathology is characterized by a variety of other lesions, such as infarcts, gliosis, argyrophilic grains and Lewy bodies. Moreover, the neuropathology of other dementing conditions, such as frontotemporal lobe dementias, displays partially AD histopathological features.