Fred Baskin

Platelet amyloid precursor protein processing: A bio-marker for Alzheimer's disease

The amyloid precursor protein (APP) in brain is processed either by an amyloidogenic pathway by beta-secretase and gamma-secretase to yield Abeta (beta-amyloid 4 kDa) peptide or by alpha-secretase within the beta-amyloid domain to yield non-amyloidogenic products. We have studied blood platelet levels of a 22-kDa fragment containing the Abeta (beta-amyloid 4 kDa) peptide, beta-secretase (BACE1), alpha-secretase (ADAM10), and APP isoform ratios of the 120-130 kDa to 110 kDa peptides from 31 Alzheimers disease (AD) patients and 10 age-matched healthy control subjects. We found increased levels of Abeta4, increased activation of beta-secretase (BACE1), decreased activation of alpha-secretase (ADAM10) and decreased APP ratios in AD patients compared to normal control subjects. These observations indicate that the blood platelet APP is processed by the same amyloidogenic and non-amyloidogenic pathways as utilized in brain and that APP processing in AD patients is altered compared to control subjects and may be a useful bio-marker for the diagnosis of AD, the progression of disease and for monitoring drug responses in clinical trials.

Platelet APP isoform ratios correlate with declining cognition in AD

Background: Platelets and neurons both contain large quantities of two carboxyl-truncated 120 to 130 and 110 kDa Alzheimer amyloid precursor proteins (APPs). Platelets taken from patients with AD have been reported to contain a reduced ratio of these APPs. Objective: To further study the AD specificity of reduced platelet APP ratios and to determine whether, after 3 years, cognitive losses in AD are accompanied by similarly reduced platelet APP ratios. Methods: To test the AD specificity of reduced platelet APP ratios, we quantitated these APPs in eight patients with PD and six patients with hemorrhagic stroke (HS). To determine whether further cognitive losses correlate with platelet APP ratio reductions in patients with AD, the authors re-examined platelet APPs and Mini-Mental State Examination (MMSE) scores of 10 patients with AD and 11 controls, who were tested 3 years ago. APP ratios were determined by the average of six assays using Western blotting with m22C11 monoclonal antibody, enhanced chemoluminescence, and digital scanning of autoradiographs. Results: APP ratios were normal in the patients with PD and HS, further supporting the AD specificity of this assay. After 3 years, the MMSE scores and APP ratios of our control subjects changed by <4%. However, the average MMSE scores of our patients with AD declined from 16.4 to 8.3, and their average 120 to 130/110 kDa APP ratios declined from 5.8 to 3.6. The difference between AD and control APP ratios, with no overlap, is significant and the correlation between the 3-year decline in AD MMSE scores and reduced APP ratios (r = 0.69) was significant. Conclusions: Although the number of subjects analyzed was limited, reduced platelet APP ratios appear to be a specific biological marker of AD and a biological index of the severity of cognitive loss in AD.

Cholinergic Neuron‐Specific Expression of the Human Choline Acetyltransferase Gene Is Controlled by Silencer Elements

Abstract: Choline acetyltransferase (ChAT) is specifically expressed in Cholinergic neurons. To identify control mechanisms regulating the cell‐specific expression of the gene encoding ChAT, transient expression of the luciferase gene driven by human ChAT gene 5’ flanking sequences was compared in cholinergic and noncholinergic cell lines. Analysis of the gene indicated the presence of two regulatory elements with selective silencing activity. These elements, located between nucleotides −2043 to −3347 and nucleotides −3347 to −6550, act cooperatively to repress promoter activity > 10‐fold in a human adrenergic neuroblastoma cell line, SHSY5Y, and a human osteosarcoma cell line, 143 TK, while exhibiting less than a two‐fold effect in Cholinergic cell lines. Deletion of either nucleotides −2043 to −3347 or nucleotides −3348 to −6550 reduced cell‐specific repression by approximately half. Such differential repression appears to be responsible for the selective expression of the ChAT component of the Cholinergic phenotype.

Altered apolipoprotein E secretion in cytokine treated human astrocyte cultures

Apolipoprotein E (ApoE), postulated to be a major lipid carrier protein in brain, is synthesized and secreted primarily by astrocytes and is involved in brain development and repair. We have analyzed its secretion in primary cultures of older (high passage) slowly dividing and younger (lower passage) rapidly dividing fetal human astrocytes exposed to various inflammatory and anti-inflammatory cytokines, alone and in combination. ApoE secretion was reduced in high passage astrocytes when compared to lower passage astrocytes. A further reduction in ApoE secretion in high passage cells was consistently produced by the combination of cytokines interleukin 1 (IL-1) alpha and beta and interferon (IFN-gamma) cytokines or by the basic fibroblast growth factor (basic-FGF) alone. Epidermal growth factor (EGF) increased ApoE secretion. The combination of these cytokine effects in chronically degenerating brain regions of Alzheimers disease and other neurodegenerative diseases could reduce the amount of ApoE available for neuronal regeneration. EGF, or agents inducing EGF, could ameliorate these ApoE deficiencies.

Joseph disease and Huntington disease Protein patterns in fibroblasts and brain

Article abstract-Proteins were separated on two-dimensional acrylamide gels obtained from brain samples of patients with Joseph disease, Huntington disease (HD) and multiple sclerosis. Similar protein separations were made from cultured skin fibroblasts of Joseph disease patients. Two major classes of proteins, one with a MW of 50,000 probably representing the glial filamentous acidic protein, or another class with a MW of 40,000 (proteins Jc, Jd, L1 and L2) were increased in the cerebellum of six Joseph disease patients. The same protein species were abnormally increased in HD brains, mainly in the basal ganglia and frontal cortex. These identical classes of protein changes were present in two nosologically separate autosomal dominant neurological disorders, Joseph disease (a spinocerebellar degeneration) and HD (a basal ganglia and cerebral cortical degeneration) and may reflect a biochemical correlation of gliosis and neuronal disease. However, these changes may be evidence that the two diseases are allelic mutations of the same gene. The dominantly inherited spinocerebellar degenerations may result from a primary defecit of glial-neuronal interaction, resulting in neuronal loss but with a compensatory increase in the number of glial cells attempting to provide additional trophic-metabolic support.

Joseph disease Protein patterns in fibroblasts and brain

The separation of brain and fibroblast proteins was analyzed on two-dimensional acrylamide gels. Proteins were examined from skin fibroblast cultures and brain homogenates from the frontal cerebral cortex, putamen, and cerebellum. Protein species from skin fibroblast cultures of controls and patients with Joseph disease or Huntington disease were not significantly different. The proteins from homogenates of the cerebral cortex, putamen, and cerebellum from controls differed from those of one Joseph disease patient. Two major classes of proteins were increased in the patients putamen and cerebellum. Proteins of 40,000 and 50,000 daltons-including the glial filamentous acidic protein complex (molecular weight 50,000), and two proteins which migrated near actin-were increased in the cerebellum. The glial filamentous acidic protein complex increased 3.7-fold in the putamen of the patient. These protein changes probably represent gliosis, but may also be an expression of the primary genetic mutation.

Correlation of statin-increased platelet APP ratios and reduced blood lipids in AD patients.

Platelets, like neurons, contain 120- to 130- and 110-kd amyloid precursor proteins (APPs). Their ratio is reduced in AD, further reductions correlating with reduced Mini-Mental Status Examination scores [r(11) = 0.69, p < 0.05]. As statins alter APP processing, platelet APPs were analyzed in patients with AD given anticholesterol drugs for 6 weeks. APP ratios increased [t(37) = −3.888, p = 0.0004], proportionally with reduced cholesterol [r(36) = −0.45, p = 0.005]. Longer trials may reveal slowed cognitive loss, validating this index.

An AP-2 binding sequence within exon 1 of human and porcine choline acetyltransferase genes enhances transcription in neural cells

The gene for choline acetyltransferase, synthesizing acetylcholine, is induced by several neurotrophic factors. A role for AP-2 in enhancing this transcription and limiting it to neural cells is strongly suggested. Previous studies demonstrated that base pairs +465-727 within the untranslated exon 1 of the porcine gene enhanced the expression of a reporter gene transfected into PC-12 cells. Deletion and mutation experiments indicate that base pairs +465-472 (CCGCGGGG) in the porcine gene, or +307-314 (CCTCGGGG) in the human sequence, were necessary and sufficient for increased gene expression in cholinergic or adrenergic but not liver cells. Constructs containing active sequences, but not inactive mutated sequences, specifically bind nuclear proteins from neuroblastoma cells, but not liver cells, in gel shift experiments. The human and porcine sequences are in agreement with an AP-2 consensus binding sequence, a nuclear transcription factor expressed only in cells derived from the neural crest. Gel shift experiments using recombinant AP-2 confirm this identification. AP-2 antibody further retarded the mobility of these DNA-nuclear extract or DNA-AP-2 complexes. These results support the importance of this AP-2 binding sequence in enhancing and limiting choline acetyltransferase expression in neural cells.

Idiopathic polyneuropathy associated with cytotoxic anti-neuroblastoma serum. IgG and IgM immunoglobulin studies.

Serums from six patients with progressive idiopathic acute or chronic polyneuropathy possessed a cytolytic activity against transformed mouse cholinergic or noncholinergic neuroblasts but not against transformed rat astrocytes. This activity was not qualitatively nor quantitatively present in serums from normal controls or from patients with a variety of other motor system disorders and other neurologic disorders. Fluorescein conjugated goat antihuman IgG and IgM monospecific immunoglobulins were used to characterize further the cytotoxic activity from patient serums and these studies suggested the presence of immunoglobulin G (IgG) and immunoglobulin M (IgM) directed against a cell surface neuroblastoma antigen. Cold reactive immunoglobulins of the IgG and IgM type were present in the serums of all six patients. A bioassay is described that may be helpful in evaluating other patients with progressive idiopathic polyneuropathies.

Platelet APP isoform ratios in asymptomatic young adults expressing an AD-related presenilin-1 mutation

The Alzheimers disease (AD) related amyloid precursor protein (APP) is stored, cleaved and released similarly from neurons and from platelets. We have reported that the proportion of 120-130 to 110 kDa carboxyl-cleaved APP present in the platelets of AD patients is significantly lower than that of platelets of age-matched controls. This reduced APP isoform ratio, not seen in several other disease groups, is further reduced as the severity of AD increases. Since the neuropathology of AD is believed to begin many years before the onset of cognitive loss, we have also compared platelet APP ratios of four pre-symptomatic young adults carrying a presenilin-1 mutation to seven siblings homozygous for the normal PS-1 gene in an effort to determine whether reduced APP ratios are present before apparent cognitive loss in familial AD. Decreased platelet APP ratios were not seen in any of these subjects at this time. We will continue to monitor these subjects as they near the mean age of AD onset in these families. As the magnitude of the APP ratio reduction is proportional to the severity of cognitive loss in sporadic AD, these cognitively normal incipient AD subjects would not be expected to present significant reductions in this AD severity index at this time. Alternatively, the absence of platelet APP ratio reductions may result from a failure of platelets from familial PS-1 AD subjects to manifest altered APPs, as has been reported for PS-2 AD subjects, unlike those of sporadic AD patients. Continued monitoring of cognitive status in our sub-set of controls with AD-like low APP ratios may yet validate the ability of this assay to detect incipient sporadic AD.