Byron Kakulas

Two novel (M233T and R278T) presenilin-1 mutations in early-onset Alzheimer's disease pedigrees and preliminary evidence for association of presenilin-1 mutations with a novel phenotype

Eleven early-onset dementia families, all with affected individuals who have either presented clinical symptoms of early onset familial Alzheimers disease (EOFAD) or have been confirmed to have EOFAD by autopsy, and two early onset cases with biopsy-confirmed AD pathology, were screened for missense mutations in the entire coding region of presenilin-1 (PS-1) and -2 (PS-2) genes. Missense mutations were detected by direct sequence analysis of PCR products amplified from genomic DNA templates of affected individuals. Three pedigrees were attributable to known mutations in the PS-1 gene: P264L, E280A and the splice acceptor site (G to T) mutation, which results in the deletion of residues 290–319 of PS-1(PS-1 Δ290–319). In a fourth pedigree, a novel PS-1 mutation was identified in exon 7 (M233T), which is homologous to a pathogenic PS-2 mutation (M239V), and is characterized by a very early average age of onset (before the age of 35). In one early onset case, another novel PS-1 mutation was identified in exon 8 (R278T). Of the five remaining families and the other early onset case, none have missense mutations in the PS-1 or PS-2 genes, or in exon 16 and 17 of the APP gene. Moreover, two of the PS-1 mutations, PS-1 Δ290–319 and ρ278T, are associated with the co-presentation of familial spastic paraparesis (FSP) in some of the affected family members. Our data raise the possibility that the phenotypic spectrum associated with PS-1 mutations may extend beyond typical FAD to include FSP, a disease heretofore unsuspected to bear any relationship to FAD. In addition, our data suggest that other novel EOFAD loci, in addition to APP and the presenilin genes, are involved in the aetiology of up to 50% of EOFAD cases.

Neuropathology: the foundation for new treatments in spinal cord injury

The first step essential in the search for a cure of human spinal cord injury (SCI) is to appreciate the complexity of the disorder. In this regard, it is not only the loss of ambulation but the sensory and autonomic changes that are equally important in recovery. In addition, there are the serious social emotional psychological and lifestyle effects of SCI which should also be taken into account. It is also true that no two SCI lesions are alike as each is the result of a SCI unique to that individual. Clinically of utmost importance is the segmental level of injury and whether it is complete, incomplete or discomplete (loss of all neurological functions below the injury but with physiological or anatomical continuity of Central nervous system tracts across the lesion). We are not concerned here with primary and secondary prevention or methods designed to limit the severity of the lesion after the event, important as they are, but with the requirements for a cure. Clearly, the greater the number of nerve fibers that can be preserved in the acute stage, the better will be the end result. Our focus at present is on the end-stage lesion with the aim of showing that a cure for SCI will depend upon establishing functionally useful central axonal regeneration and reestablishing physiological reconnections. Existing experimental methods are based on stimulating axonal regeneration by neutralizing inhibitory factors, adding positive trophisms and creating a permissive environment. Better results are obtained by bridging the gap with grafts of peripheral nerves or transplants of Schwann cells and genetically engineered fibroblasts. Recently, the potential for stem cells to enhance this process has created great interest. This is because of the ability of pluripotential cells to differentiate into neural tissue. A cure based on the physiopathology of SCI requires pyramidal, extrapyramidal, sensory, cerebellar and autonomic pathways to be regenerated with their appropriate neurotransmitters restored and reflexes integrated physiologically and in synchrony. In human SCI, there is a very long distance anatomically for axonal regrowth to occur in order to reach their relevant nuclei. This is because of continuing Wallerian degeneration. It also presumes that the target neurons are intact and that there has been no transneuronal degeneration above or below the lesion. Alternatively, in place of regenerated long axons, a multisynaptic pathway may be constructed from stem cells that have developed into neurons. Whether such a pathway would restore useful neurological functions is unknown. At present, the transplant and grafting research teams are exploring these possibilities in experimental animals. Moderate success in gaining axonal regeneration has been reported; however, it must be appreciated that the human lesion differs considerably from that of the experimental animal. In order to be successful, the neuropathology and neurophysiology of human SCI must be taken into account. The purpose of this review is to place the requirements for a cure, using stem cells, within the context of the neuropathology of human SCI.

The applied neuropathology of human spinal cord injury.

The timeliness of a review of the neuropathology ofhuman spinal cord injury (SCI) centres on theincreasing interest in central axonal regeneration inthe search for a ‘cure’ of spinal paralysis. Neuropathol-ogy contributes to this end by describing the nature ofthe disorder to be cured. More broadly a knowledge ofthe neuropathology underlying SCI is essential also forthe clinician responsible for management of the patientas well as the neuroscientist working on SCI. There hasbeen spectacular progress in neurobiology in recentyears with CNS repair and regeneration now reportedby many centres. However, in order for thesediscoveries to be applied to human SCI an apprecia-tion of the neuropathology is required. The neurobiol-ogist must be aware of the nature of the condition tobe cured for the research to be relevant. Furthermore,before the experimental findings may be applied to thepatient it must be shown that the reactions to injuryare comparable in each. An outline of recent advancesin the neurobiology of experimental SCI is thereforeincluded in this review in order to bridge the gapbetween human SCI and the experimental models.Most importantly the insight provided by the complex-ity of the problem of finding a cure serves to informthose waiting for this to occur to be realistic in theirexpectations.

Prevalence of amyloid-β deposition in the cerebral cortex in Parkinson's disease

The pathological basis for the dementia which occurs in 20 to 40% of patients with idiopathic Parkinsons disease (PD) remains uncertain. In the present postmortem study, we compared the prevalence and severity of parenchymal and vascular amyloid‐β (Aβ) deposition in the cerebral cortex in a group of 57 PD brains, including 13 cases with dementia, and in 100 control brains. A higher proportion of PD brains had vascular Aβ deposition, whereas the proportions and severity of parenchymal Aβ were similar in the PD and control groups. There was a poor correlation between Aβ deposition and neurofibrillary tangles which were present in only small numbers in a minority of cases. Cortical Aβ deposition was present in only 6 of the 13 cases with dementia and only 3 fulfilled the Consortium to Establish a Registry for Alzheimers Disease (CERAD) criteria for definite Alzheimers disease. The present findings confirm that dementia in PD is only infrequently due to fully established Alzheimers disease. However, vascular and parenchymal Aβ deposition could still contribute to dementia and cognitive decline when combined with other changes such as α‐synuclein deposition in the cerebral cortex and cortical Lewy bodies.

Regeneration of muscle in Duchenne muscular dystrophy: an electron microscope study.

Electron-microscopic observations have been made on skeletal muscle from cases of Duchenne muscular dystrophy and particular attention has been paid to the regenerative changes. The degenerative changes encountered were similar to those which have been found by previous workers. The most basic change was a segmental one and consisted of supercontraction of myofibrils which appeared to precede their fusion and subsequent breakdown. It is suggested that the supercontraction may represent a state of contracture or failure of relaxation due to a relative deficiency of ATP in the muscle fibre. Regenerative changes were found in considerable numbers of muscle fibres particularly in the younger cases. In fibres showing advanced degenerative changes, “satellite cells” were commonly found to be enlarged and to have a more elaborate organelle content than that of the resting “satellite cell”. Thin filaments were found in the cytoplasm of some “satellite cells” confirming their ability to differentiate into myoblasts. Ribosomal aggregates were found within the degenerate sarcoplasm of some fibres but were not associated with new filament formation. Regenerating fibres were recognized by the presence of polyribosomes in the sarcoplasm and large nuclei with dispersed chromatin and prominent nucleoli. Although in some fibres the pattern of myofibrillar development appeared to be quite normal, in many regenerating fibres the myofibrils were poorly aligned and at times completely disorganized and often showed abnormalities such as streaming of the Z-band or other focal deficiencies in isolated sarcomeres. Mitochondria were sparse in the vicinity of developing myofibrils. In some small fibres which ended blindly, regenerative changes appeared to have become arrested. The present observations indicate that although dystrophic muscle is capable of regeneration, the pattern of morphological development of the contractile elements in regenerating fibres is often abnormal. Moreover, there is evidence of metabolic abnormalities in such fibres. The observed abnormalities in the regeneration of dystrophic muscle correlate with the known abnormal growth pattern of dystrophic muscle in tissue culture. The possible reasons for these abnormalities are considered.

Use of the dog model for Duchenne muscular dystrophy in gene therapy trials

Golden retriever muscular dystrophy (GRMD) is an excellent model for the study of the efficacy of gene therapy in dystrophin deficient myopathies for there are many similarities between affected dogs and Duchenne muscular dystrophy (DMD) in boys. GRMD is not caused by deletion mutation but results from a point mutation in the consensus splice acceptor in intron 6 of the canine dystrophin gene. As a result exon 7 is skipped during processing of the GRMD dystrophin messenger RNA. We have developed a rapid test which makes direct use of exon 7 specific genomic PCR products. We have undertaken preliminary experiments on gene therapy using the mini-gene and the full length gene alone and in combination with lipofectin and/or the bacterial beta-galactosidase reporter gene Lac Z. Following direct injection of the Lac Z plasmid, either alone or with lipofectin, about 50% of the sites showed expression when biopsied some 14 days later. The beta-galactosidase activity was present in muscle and granulation tissue but was never abundant. Pups injected intraperitoneally with Lac Z were found to have positive material in their mesenteric lymph nodes, liver and spleen. Those injected with Lac Z and lipofectin also had positive material in the diaphragm, intercostal muscles and abdominal muscles, but again only a small amount of positive material was present at any of the sites. In animals directly injected into the muscle with the dystrophin mini-gene, half had positive staining for dystrophin in biopsies taken 14 days later. Of the 6 sites in the muscles of animals given the mini-gene and lipofectin only one had fibres positive for dystrophin when examined 14 days later. Six pups were injected directly with full-length gene construct and when biopsies were taken 10 days later two of the animals had strongly stained peripheries to a small number of fibres.

Prevalence of Stroke in Parkinson's Disease: A Postmortem Study

The results of previous epidemiological studies of the relationship between Parkinsons disease and stroke have been conflicting; some showing a reduced risk of ischaemic and haemorrhagic stroke during life, and others indicating an increased likelihood of stroke‐related death. We compared the frequency of cerebral infarcts and haemorrhages at postmortem in 100 cases of pathologically verified idiopathic Parkinsons disease and 100 age‐matched control brains. No significant differences were found in the numbers of infarcts or haemorrhages or stroke‐related deaths between the two groups. Our findings do not indicate either a protective effect against stroke, or a greater susceptibility to death from stroke, in the population studied.

Novel mutation in the myelin protein zero gene in a family with intermediate hereditary motor and sensory neuropathy

OBJECTIVES To determine the molecular basis for autosomal dominant intermediate hereditary motor and sensory neuropathy (HMSN) in a four generation family. The gene defects in families with intermediate HMSN are not known, but it has been suggested that most have X linked HMSN. METHODS All participating family members were examined clinically. Genomic DNA was obtained from 10 affected and seven unaffected members. Linkage analysis for the known HMSN loci was first performed. Mutations in the peripheral myelin protein zero gene (PMP0) were sought in two affected members, using one unaffected member for comparison, by amplification of the six exons of the gene followed by single strand conformation polymorphism (SSCP) analysis, dideoxy fingerprinting (ddF), and sequencing. Subsequently, the mutation was screened for in all affected and unaffected members in the family using Alu I digestion and in 100 unrelated control subjects using “snap back” SSCP analysis. Sequencing of cDNA from a sural nerve biopsy from an affected member was also performed. RESULTS The clinical phenotype was of variable severity, with motor nerve conduction velocities in the intermediate range. Linkage to PMP0 was demonstrated. Analysis of genomic DNA and cDNA for PMP0 identified a novel codon 35 GAC to TAC mutation. The mutation produces an inferred amino acid change of aspartate to tyrosine at codon six of the processed protein (Asp6Tyr) in the extracellular domain and was present in all affected family members but not in 100 unrelated controls. CONCLUSIONS The present findings further extend the range of phenotypes associated with PMP0 mutations and indicate that families with “intermediate” HMSN need not necessarily be X-linked as previously suggested.

The clinical neuropathology of spinal cord injury a guide to the future

The neuropathology of spinal cord injury (SCI) is reviewed in the light of clinical problems and as a guide to future research. The pathology of SCI in the acute stage suggests that the spinal cord may be partly preserved even in the most severe injuries. This finding emphasises the need for great care in roadside management. In the acute phase there are irreversible changes and possibly reversible changes which have not been adequately identified. Even a small percentage of nerve fibres escaping the initial injury would be of great benefit to the patient.In the subacute stage when transynaptic degeneration is proceeding there may also be associated functional changes leading to abnormal reflex activity. It is possible through an improved understanding of the neuropathology and neurophysiology of the isolated or partly isolated segments of the cord that new reflex connections may be stimulated to develop by artificial means. In the chronic stage there are well recognised complications such as osteoarthrosis with spinal stenosis, post-traumatic syringomyelia and traumatic nerve root neuroma formation, which may lead to clinical deterioration and which may be amenable to treatment.In a more theoretical sense it is possible that improved understanding of CNS plasticity and transplant neurobiology using recombinant DNA technology, grafting and ‘re-education’ of the regenerated tissues may be rewarding in the longterm future. Although this outcome is entirely hypothetical at this stage basic research deserves great emphasis.

TGF-β1 and TGF-β2 expression after traumatic human spinal cord injury

Study design:Immunohistochemical investigation in control and lesioned human spinal cords.Objectives:To assess the spatial and temporal expression patterns of transforming growth factor-β1 and -β2 (TGF-β1 and TGF-β2) in the human spinal cord after traumatic injury.Setting:Germany, Aachen, Aachen University Hospital.Methods:Sections from human spinal cords from 4 control patients and from 14 patients who died at different time points after traumatic spinal cord injury (SCI) were investigated immunohistochemically.Results:In control cases, TGF-β1 was confined to occasional blood vessels, intravascular monocytes and some motoneurons, whereas TGF-β2 was only found in intravascular monocytes. After traumatic SCI, TGF-β1 immunoreactivity was dramatically upregulated by 2 days after injury (the earliest survival time investigated) and was detected within neurons, astrocytes and invading macrophages. The staining was most intense over the first weeks after injury but gradually declined by 1 year. TGF-β2 immunoreactivity was first detected 24 days after injury. It was located in macrophages and astrocytes and remained elevated for up to 1 year. In white matter tracts undergoing Wallerian degeneration, there was no induction of either isoform.Conclusion:The early induction of TGF-β1 at the point of SCI suggests a role in the acute inflammatory response and formation of the glial scar, while the later induction of TGF-β2 may indicate a role in the maintenance of the scar. Neither of these TGF-β isoforms appears to contribute to the astrocytic scar formation in nerve fibre tracts undergoing Wallerian degeneration.