Donald R. McLachlan

Intramuscular desferrioxamine in patients with Alzheimer's disease

Although epidemiological and biochemical evidence suggests that aluminium may be associated with Alzheimers disease (AD), there is no convincing proof of a causal link for aluminium in disease progression. We have completed a two year, single-blind study to investigate whether the progression of dementia could be slowed by the trivalent ion chelator, desferrioxamine. 48 patients with probable AD were randomly assigned to receive desferrioxamine (125 mg intramuscularly twice daily, 5 days per week, for 24 months), oral placebo (lecithin), or no treatment. No significant differences in baseline measures of intelligence, memory, or speech ability existed between groups. Activities of daily living were assessed and videorecorded at 6, 12, 18, and 24 month intervals. There were no differences in the rate of deterioration of patients receiving either placebo or no treatment. Desferrioxamine treatment led to significant reduction in the rate of decline of daily living skills as assessed by both group means (p = 0.03) and variances (p less than 0.04). The mean rate of decline was twice as rapid for the no-treatment group. Appetite (n = 4) and weight (n = 1) loss were the only reported side-effects. We conclude that sustained administration of desferrioxamine may slow the clinical progression of the dementia associated with AD.

Priming of semantic autobiographical knowledge: A case study of retrograde amnesia

The case of a 36-year-old man who suffers dense retrograde and anterograde amnesia as a result of closed-head injury that caused extensive damage to his left frontal-parietal and right parieto-occipital lobes is described. Patient K.C. has normal intelligence and relatively well-preserved perceptual, linguistic, short-term memory, and reasoning abilities. He possesses some fragmentary general knowledge about his autobiographical past, but he does not remember a single personal event or happening from any time of his life. He has some preserved expert knowledge related to the work he did for 3 years before the onset of amnesia, although he has no personal recollections from that period. Some features of K.C.s retrograde amnesia can be interpreted in terms of the distinction between episodic and semantic memory, and in terms of the distinction between episodic and semantic autobiographical knowledge. K.C.s semantic knowledge, but not his episodic knowledge, showed progressive improvement, or priming, in the course of the investigation.

Reduction of vitamin D hormone receptor mRNA levels in Alzheimer as compared to Huntington hippocampus: correlation with calbindin-28k mRNA levels

Receptors for vitamin D hormone (VDR) and the calcium binding protein, calbindin-28k, have been localized in many tissues, including brain. In brain, VDR and calbindin-28k were reported to colocalize in hippocampal CA1 cells. We have shown that mRNA pool size for calbindin-28k was reduced, on average, by 35% in Alzheimer hippocampal CA1 cells, as compared to Huntington control (manuscript in preparation). In the present study, in situ hybridization with tritiated antisense RNA probes was used to examine VDR expression in paired Alzheimer and Huntington brain tissue. Message levels for VDR were reduced, on average, by 34% and 31%, respectively, in Alzheimer hippocampal CA1 and CA2 pyramidal cells, as compared to Huntington control. However, VDR message levels were not significantly different from control in Alzheimer temporal cortex or cerebellum. There was no correlation between VDR message levels and brain weight, autopsy interval, patient age or the extent of neurofibrillary degeneration. Instead, VDR mRNA pool size in hippocampal CA1 cells correlated significantly with calbindin-28k message levels (r = 0.52, P less than 0.001). Decreased message levels for VDR and calbindin-28k in these cells were due to an increased percentage of cells expressing lower message levels for these proteins. These results show that in Alzheimer hippocampal CA1 cells, VDR mRNA pool size is downregulated and that this downregulation may play a role in the reduction of calbindin-28k expression.

CAN THE CONTROVERSY OF THE ROLE OF ALUMINUM IN ALZHEIMER'S DISEASE BE RESOLVED? WHAT ARE THE SUGGESTED APPROACHES TO THIS CONTROVERSY AND METHODOLOGICAL ISSUES TO BE CONSIDERED?

Aluminum (Al) is unquestionably neurotoxic in both experimental animals and certain human diseases. Minute quantities injected intracerebrally into rabbits will induce severe neurological symptoms and neuropathological features of neurodegeneration. Hyper-aluminemia often develops in patients with renal failure being treated with intermittent hemodialysis on a chronic basis, and in severe cases results in an encephalopathy. Uremic adults and premature infants not on dialysis treatment also can develop encephalopathy due to Al toxicity, as is the case when large amounts of alum are used as a urinary bladder irrigant. There are many other examples of Al-induced neurotoxicity; however, the question as to whether Al presents a health hazard to humans as a contributing factor to Alzheimers disease is still the subject of debate. Several lines of evidence are presented that have formed the basis of the debate concerning the possible pathogenic role for Al in Alzheimers disease. Important evidence for an Al-Alzheimers causal relationship is the observation by laser microprobe mass analysis (LMMS) of the presence of Al in neurofibrillary tangles, although there are conflicting data on the extent of the Al deposition. The relatively poor sensitivity of some of the analytical instruments available for these challenging in situ microanalyses could explain the discrepant results, although LMMS and perhaps secondary ion mass spectrometry (SIMS) appear to be sufficiently sensitive. Harmonization of the techniques is an essential next step. There is new evidence that exposure to Al from drinking water might result in cognitive impairment and an increased incidence of Alzheimers disease. However, these epidemiological studies have inherent problems that must be scrutinized to determine if an association really does exist. An understanding of a possible enhanced bioavailability of Al in this type of exposure, versus other exposures such as antacid intake or industrial exposure, needs to be considered and explored. There has been one promising clinical trial of the treatment of Alzheimers disease patients with the Al chelator desferrioxamine (DFO). Further studies are needed, and if confirmation is forthcoming then such data could also support an Al-Alzheimers disease link as well as suggesting that DFO offers potential as a therapeutic agent. The possibility that iron might be the offending agent needs to be considered since DFO is a very strong iron chelator. The significance of Al-induced neurofibrillary degeneration in experimental animals should be assessed especially in light of new data showing that this model exhibits abnormally phosphorylated tau protein structures in the neuronal perikarya. Thus the key questions that must be answered before it can be asserted that Al possesses causal relationship to Alzheimers disease, are as follows and are addressed in this present discussion: (1) Are there elevations of the concentration of Al in the brains of Alzheimers disease patients? (2) Is there a relationship between environmental exposure to Al, particularly in drinking water, and an increased risk of Alzheimers disease? (3) Is treatment with DFO a potentially useful therapeutic approach and to what extent might beneficial effects of DFO implicate Al in the etiology of Alzheimers disease? (4) Are there similarities between the experimental animal studies and Alzheimers disease particularly in the development of abnormal forms of tau seen in neurofibrillary tangles? (5) Does Al promote the deposition of the A beta peptide in Alzheimers disease? (6) Does hyperaluminemia associated with long-term hemodialysis treatment induce neurofibrillary degeneration? If the answer to each of these six questions is yes, then does this assert that Al possesses a causal relationship to Alzheimers disease? On the other hand, must all six be met to be able to make this assertion?

α1-Antichymotrypsin Binding to Alzheimer Aβ Peptides Is Sequence Specific and Induces Fibril Disaggregation In Vitro

Abstract: The serine protease inhibitor α1‐antichymotrypsin (ACT) consistently colocalizes with amyloid deposits of Alzheimers disease (AD) and may contribute to the generation of amyloid proteins and/or physically affect fibril assembly. AD amyloid fibrils are composed primarily of Aβ, which is a proteolytic fragment of the larger β‐amyloid precursor protein. Using negative‐stain and immunochemical electron microscopy, we have investigated the binding of ACT to the fibrils formed by four synthetic Aβ analogues corresponding to the wild‐type human 1–40 sequence [HWt(1–40)], a 1–40 peptide [HDu(1–40)] containing the Glu22→ Gln mutation found in hereditary cerebral hemorrhage with amyloidosis of the Dutch type, the N‐terminal 1–28 residues [β(1–28)], and an internal fragment of Aβ containing residues 11–28 [β(11–28)]. Each of these peptide analogues assembled into 70–90‐Å‐diameter fibrils resembling native amyloid and, except for β(11–28), bound ACT, as indicated by the appearance of 80–100‐Å globular particles that adhered to preformed fibrils and that could be decorated with anti‐ACT antibodies. Under the conditions used, ACT binding destabilized the in vitro fibrils and produced a gradual dissolution of the macromolecular assemblies into constituent filaments and shorter fragments. The internal fragment (11–28) did not exhibit ACT binding or any structural changes. These results suggest that a specific sequence likely contained within the N‐terminal 10 residues of Aβ is responsible for the formation of the ACT‐amyloid complex. Although the observed fibril disassembly is surprising in view of the notion that ACT contributes directly to the physical process involved in amyloid fibril formation, the induced structural changes may expose new domains in Aβ for additional proteolysis or for interactions with cell‐surface receptors.

Controlled induction of paired helical filaments of the Alzheimer type in cultured human neurons, by glutamate and aspartate

One characteristic histopathological feature of brains of patients with Senile Dementia of the Alzheimer Type (SDAT) is a neuronal change known as neurofibrillary degeneration. Ultrastructurally, this degeneration presents as aggregates of Paired Helical Filaments (PHFs). We have reported (De Boni and Crapper 1978) that brain affected with SDAT contains a factor which induces PHFs similar to those occurring in Alzheimers disease, in human CNS neurons in vitro. We now report that PHFs may be induced in cultured, fetal human spinal cord lesions by factors other than extracts from brain affected by SDAT. Specifically, ultrastructural analyses have shown that PHFs closely similar to those found in SDAT are induced by the excitotoxic aminoacids, glutamate (Glu) and aspartate (Asp). PHFs were found with Glu and Asp in combination at 1.1 and 0.45 mM, respectively and by Glu alone at 2.2 mM, when added to the culture medium. Cultures grown and maintained in MEM Eagle medium without Glu and Asp were invariably composed of tightly packed neuropil with abundant synaptic contacts, were free of vacuolated somata or processes and did not exhibit the presence of PHFs. In contrast, cultures exposed to Glu and Asp invariably responded with the presence of vacuolated neuronal somata and degenerating neuronal processes containing intermediate filaments, frequently paired into PHFs. These induced PHFs are composed of intermediate filaments morphologically identical (diameter 10 +/- 0.9 nm, n = 55) to neurofilaments. While the induced PHFs exhibit a range of periodicities (50-540 nm) with an overall mean period of 140 +/- 68 nm (n = 245), approximately 4% are morphologically closely similar (mean period 76 +/- 10 nm, n = 10) to those in SDAT.

Ligand specific effects on aluminum incorporation and toxicity in neurons and astrocytes.

Aluminum is present in many manufactured foods and medicines and is added to drinking water for purification purposes. It has been proposed that aluminum is a contributing factors to several neurodegenerative disorders such as Alzheimers disease. However, this remains controversial primarily due to the unusual properties of aluminum and a lack of information on its cellular sites of action. To resolve some of these questions, we have examined aluminum uptake in both neuronal and astroglial cells as well as the role of metal speciation. The relative accumulation of four aluminum salts, aluminum maltolate, aluminum lactate, aluminum chloride and aluminum fluoride, was investigated and correlated with cell viability and intracellular distribution as determined by morin staining. Significant differences in aluminum incorporation and toxicity were observed in both neuronal and glia cells with the largest effects exhibited by the maltol species. This was accompanied by a nuclear accumulation in the neuronal cell line that was contrasted by the perinuclear, vesicular distribution in astrocytes that partially co-localized with cathepsin D, a lysosomal marker. These findings demonstrate differences in aluminum species and highlights the importance of these factors in modulating the toxic effect of aluminum.

Sensitive detection of metallothioneins-1, -2 and -3 in tissue homogenates by immunoblotting: a method for enhanced membrane transfer and retention

Standard immunoblotting procedures were unable to detect metallothioneins-1 and 2 (MT-1, MT-2) and metallothionein-3 (MT-3)/growth inhibitory factor (GIF) in unfractionated brain homogenates. We have developed a novel process which involves the inclusion of 2 mM CaCl2 in electrophoretic transfer buffers and glutaraldehyde fixation following transfer to either nitrocellulose or polyvinylidene difluoride (PVDF) membranes. Using commercial MT antibodies and a specific MT-3 polyclonal antibody raised in our laboratory, we have been able to detect all three MTs on both membrane types with a detection limit of approx. 10 ng for MT-1 and MT-2. Nitrocellulose membrane pore size had no noticeable effect on detection sensitivity. These modifications enable more sensitive MT detection than previously described blotting methods. In addition, this technique eliminates the need for indirect monitoring approaches and simplifies quantification since sample fractionation or enrichment are not required.

Chromatin proteins share antigenic determinants with neurofilaments.

Antigenic determinants common to distinct proteins may be unambiguously identified by the use of monoclonal antibodies. Some monoclonal antibodies to mammalian neurofilaments have recently been shown to cross‐react with the neurofibrillary tangles found at high density in the brains of senile dements with Alzheimers disease (SDAT). Here, we show that these antibodies also cross‐react with chromatin proteins, including the linker histones H1 and H10. Elevated levels of histone H10 have also been reported in SDAT brains.

Run-on gene transcription in human neocortical nuclei. Inhibition by nanomolar aluminum and implications for neurodegenerative disease

The incorporation of [α-32P]-uridine triphosphate into DNA transcription products was examined in short post-mortem interval (PMI) human brain neocortical nuclei (n, 22; PMI, 0.5–24 h) using run-on gene transcription. Reverse Northern dot-blot hybridization of newly synthesized RNA against either total cDNA or Alu repetitive DNA indicated that human brain neocortical nuclei of up to 4-h PMI were efficient in incorporating radiolabel into new transcription products, after which there was a graded decline in de novo RNA biosynthetic capacity. To test the effects of 0–3000 nM concentrations of ambient aluminum on RNA polymerase I (RNAP I) and RNA polymerase II (RNAP II) transcription, dot blots containing 0.5, 1.0, 2.0, and 5.0 µg of DNA for (1) the human-specific Alu repetitive element (2) the neurofilament light (NFL) chain, and (3) glial fibrillary acidic protein (GFAP) were Northern hybridized against newly synthesized radiolabeled total RNA. These DNAs represent heterogeneous nuclear RNA (hnRNA), neuronal-, and glial-specific markers, respectively. We report here a dose-dependent repression in the biosynthetic capabilities of brain RNAP II in the range of 50–100 nM aluminum, deficits similar to those previously described using a rabbit neocortical nuclei transcription system and at concentrations that have been reported in Alzheimer’s disease (AD) euchromatin. Transcription from RNAP II and the neuron-specific NFL gene in the presence of aluminum was found to be particularly affected. These findings support the hypothesis that brain gene transcription in the presence of trace amounts of ambient aluminum impairs mammalian brain DNA to adequately read out genetic information.