The role of lysosomes and autophagosomes in frontotemporal lobar degeneration.

INTRODUCTION: Cell biological and genetic evidence implicate failures in degrading aggregating proteins, such as tau and TDP-43, through the autophagy or lysosomal pathways in the pathogenesis of frontotemporal lobar degeneration (FTLD). METHODS: We investigated changes in the degradative pathways in 60 patients with different pathological or genetic forms of FTLD employing immunohistochemistry for marker proteins such as lysosomal-associated membrane proteins 1 (LAMP-1) and 2 (LAMP-2), cathepsin D (CTSD) and microtubule-associated protein 1 light chain 3 alpha (LC3A). Immunostained sections were qualitatively and semi-quantitatively assessed for the appearance, distribution and intensity of staining in neurones of the dentate gyrus (DG) and CA4 region of the hippocampus, and the temporal cortex (Tcx). RESULTS: Lower levels of neuronal LAMP-1 immunostaining were present in the DG and Tcx in FTLD-tau compared to FTLD-TDP. There was less LAMP-1 immunostaining in FTLD-tau with MAPT mutations, and FTLD-tau with Pick bodies, compared to FTLD-TDP types A and B, and less LAMP-1 immunostaining in FTLD-TDP type C than in FTLD-TDP types A and B. There was greater LAMP-1 immunostaining in GRN mutation which may reflect the underlying type A histology rather than mutation. There were no differences in neuronal LAMP-2, CTSD, EEA-1 or LC3A immunostaining between any of the five FTLD histological or four genetic groups, nor between FTLD-TDP and FTLD-tau. CONCLUSIONS: The underlying pathological mechanism in FTLD-tau may lie with a relative deficiency of lysosomes, or defective vesicular transport, whereas the failure to clear TDP-43 aggregates may lie with lysosomal dysfunction rather than a lack of available lysosomes or degradative enzymes.

Neurodegeneration in frontotemporal lobar degeneration and motor neurone disease associated with expansions in C9orf72 is linked to TDP-43 pathology and not associated with aggregated forms of dipeptide repeat proteins.

AIMS: A hexanucleotide expansion in C9orf72 is the major genetic cause of inherited behavioural variant Frontotemporal dementia (bvFTD) and motor neurone disease (MND), although the pathological mechanism(s) underlying disease remains uncertain. METHODS: Using antibodies to poly-GA, poly-GP, poly-GR, poly-AP and poly-PR proteins, we examined sections of cerebral cortex, hippocampus, thalamus, cerebellum and spinal cord, from 20 patients with bvFTD and/or MND bearing an expansion in C9orf72 for aggregated deposits of dipeptide repeat proteins (DPR). RESULTS: Antibodies to poly-GA, poly-GP and poly-GR detected numerous rounded cytoplasmic inclusions (NCI) within granule cells of hippocampal dentate gyrus and those of the cerebellum, as well as 'star-burst' shaped NCI in pyramidal neurones of CA3/4 region of hippocampus. NCI were uncommon in Purkinje cells, and only very rarely seen in anterior horn cells. Poly-PA antibody detected occasional NCI within CA3/4 neurones alone, whereas poly-PR antibody did not identify any NCI but immunostained the nucleus of anterior horn cells, CA3/4 neurones and Purkinje cells, in patients with or without expansion in C9orf72, as well as in normal controls. Poly-GA antibody generally detected more DPR than poly-GP, which in turn was greater than poly-GR. All patients with bvFTD + MND or MND showed plentiful p62/TDP-43 positive inclusions in remaining anterior horn cells. CONCLUSION: Degeneration and loss of anterior horn cells associated with expansions in C9orf72 occurs in the absence of DPR, and implies that changes involving loss of nuclear staining for and a cytoplasmic aggregation of TDP-43 are more likely to be the cause of this.

Review: Recent progress in frontotemporal lobar degeneration.

Frontotemporal lobar degeneration (FTLD) is a highly familial condition and is increasingly being recognized as an important form of dementia. The literature published on this disease is often difficult to collate due to the wide range in nomenclature used. Thankfully, consensus recommendations have now been published to address this issue and hopefully the community will adopt these as intended. Much progress has been made in our understanding of the clinical, pathological and genetic understanding of FTLD in recent years. Progranulin and TDP-43 have recently been identified as new important proteins involved in the pathophysiology of FTLD and this latter protein may have potential as a biomarker of this disease. However, much remains before we have a full picture of the genes that cause FTLD and the biological pathways in which they function. The purpose of this review is to summarize the current concepts and recent advances in our knowledge of this disease.

Progranulin gene mutations associated with frontotemporal dementia and progressive non-fluent aphasia.

Frontotemporal lobar degeneration (FTLD) refers to a focal, non-Alzheimer form of cerebral degeneration that encompasses the distinct clinical syndromes of frontotemporal dementia (FTD), progressive non-fluent aphasia (PNFA) and semantic dementia. Some patients show tau-based pathological changes and in familial cases mutations have been identified in the microtubule-associated protein tau gene (MAPT) on chromosome 17q21. However, many cases are tau-negative, showing instead ubiquitin-immunoreactive (UBQ-ir) neuronal cytoplasmic inclusions and neurites, and in some familial cases UBQ-ir neuronal intranuclear inclusions of a lentiform appearance. Very recently, mutations have been identified in familial cases in the progranulin (PGRN) gene, also on chromosome 17q21. Clinical, pathological and molecular diversity within FTLD highlights the importance of careful examination of clinical-pathological-genetic relationships. This paper reports, for the first time, a clinico-pathological investigation of two FTLD families with PGRN mutations, and compares the clinical characteristics with those of patients studied in the department with MAPT mutations. The clinical profile associated with PGRN mutations constituted, in some patients, a prototypical picture of FTD and in others one of PNFA, both profiles occurring within the same family. Patients with PGRN mutations exhibited phonological deficits, whereas in patients with MAPT mutations language abnormalities, when present in addition to the prominent behavioural disorder, take the form of semantic disturbance. The findings provide compelling evidence for the link between FTD and PNFA, while raising the possibility of identifiable clinical differences between FTLD patients with MAPT and PGRN mutations.

Corticobasal degeneration and progressive supranuclear palsy share a common tau haplotype.

OBJECTIVE: To analyze the association of polymorphisms in the tau gene with pathologically confirmed corticobasal degeneration (CBD). BACKGROUND: The authors previously described an extended tau haplotype (H1) that covers the human tau gene and is associated with the development of progressive supranuclear palsy (PSP). The authors now extend this analysis to CBD, a neurodegenerative condition with clinical and neuropathologic similarities to PSP. Like PSP, CBD is associated with accumulation of aggregates containing the 4-repeat isoforms of tau. Because of difficulty in diagnosis of CBD, the authors only analyzed cases with pathologically confirmed CBD. METHODS: The authors collected 57 unrelated, neuropathologically confirmed cases of CBD. Tau sequencing in these cases failed to show the presence of pathogenic mutations. Polymorphisms that spanned the tau gene were analyzed in all CBD cases and controls. RESULTS: Analyzing tau polymorphisms in CBD cases showed that the frequency of H1 and H1/H1 was significantly increased when analyzing all cases and when separating by country of origin. H1 frequency in all CBD cases was 0.921, compared with a control frequency of 0.766 (X(2) = 9.1, p = 0.00255 [1df], OR 3.56 [8.43 > CI 95% > 1.53]). The H1/H1 frequency was also significantly higher at 0.842 compared with 0.596 in age-matched controls (X(2) = 17.42, p = 0.00016, 2df), OR 3.61 [7.05 > CI 95% > 1.85]). CONCLUSIONS: The CBD tau association described here suggests that PSP and CBD share a similar cause, although the pathogenic mechanism behind the two diseases leads to a different clinical and pathologic phenotype.

The abundance of mRNA for dopamine D2 receptor isoforms in brain tissue from controls and schizophrenics.

The possibility that schizophrenia is associated with a differential expression, in the brain, of the short and long isoforms of the dopamine D2 receptor has been investigated by assessing the abundance of mRNA for each of the isoforms. Using a quantitative RNA-PCR technique, increased mRNA for both isoforms of the D2 receptor were observed in some brain regions, with no differential distribution between the isoforms in the schizophrenics compared to controls.

Detection of the interleukin 18 family in rat brain by RT-PCR.

Interleukin 18, an inflammatory cytokine, mediates its effects by interaction with its receptor complex, consisting of the IL-18 receptor (IL-18R) and receptor accessory protein (AcPL). A functional inhibitor of IL-18, the IL-18 binding protein (IL-18BP), has been identified recently. This study reports the detection of IL-18, IL-18R, AcPL and IL-18BP mRNA expression in the brain of normal adult rats using RT-PCR.

The relative abundance of dopamine D4 receptor mRNA in post mortem brains of schizophrenics and controls.

An increase in dopamine D4 receptors has recently been reported in post mortem brain samples from schizophrenics. We have attempted to complement this finding by assessing the levels of the specific messenger RNA (mRNA) for the D4 receptor, using the technique of quantitative RNA-PCR. No significant differences in the levels of expression of mRNA for the D4 receptor were found in the brains from schizophrenics compared to controls. The relationship between D4 receptors and schizophrenia, therefore, remains unclear.

ApoE2 allele, Down's syndrome, and dementia.

All individuals with Down's syndrome (trisomy 21-DS) develop the pathogenic hallmarks of Alzheimer's disease in old age (+40 years). The extent of pathology is variable, but it has been shown that the amount of beta-amyloid pathology is variable and related to age and the degree of dementia. Thus, in DS, growing old is associated with a progressive pathological process which results in cognitive decline. However, neuropsychological studies of older DS subjects have identified a clinical dementia in only a proportion of cases. These contradictory observations could be reconciled if some factor existed which modulated the rate and amount of beta-amyloid pathology. Recent studies demonstrate an association between the apolipoprotein E4 (ApoE4) allele and the earlier age of onset in both sporadic and familial AD. Increased amounts of beta-amyloid pathology can also be related to the E4 allele. However, at present there are no data documenting the effects of ApoE genotype on the expression or degree of clinical symptoms of the disease. We have examined the ApoE genotype in a cohort of clinically evaluated elderly patients with DS in order to examine the effects of ApoE genotype on the clinical symptoms of dementia. We report here that, despite the presence of an active disease process, the ApoE E2 allele is associated with longevity and preservation of cognitive functioning.

Apolipoprotein E epsilon 2 allele promotes longevity and protects patients with Down's syndrome from dementia.

Although individuals with Down's syndrome nearly always develop the clinical and pathological features of Alzheimer's disease, some clearly do not become demented despite living into their sixth and seventh decades. Genetic variation at the apolipoprotein E locus has recently been shown to be an important determinant of Alzheimer's disease, with the epsilon 4 allele having been shown to be associated with the disease and, at least in some cases, the epsilon 2 allele being negatively associated with the disease. Here we show, in a series of clinically assessed individuals with Down's syndrome, that the epsilon 2 allele of ApoE is associated with both longevity and the absence of clinical evidence of dementia. These data show that the clinical phenotype of Down's syndrome can be modulated by genes on chromosomes other than chromosome 21. The importance of this observation to the pathogenesis of Alzheimer's disease, both in Down's syndrome and in general, is discussed.

Apolipoprotein E allelic frequencies in patients with lobar atrophy.

Apolipoprotein E (ApoE) genotype was determined by polymerase chain reaction amplification and restriction digestion of DNA extracted from brain tissues of 26 patients with clinically diagnosed and pathologically verified lobar atrophy (LA) and from blood of a further 22 patients with clinical LA. Brain tissue from 50 patients with pathologically confirmed Alzheimer's disease (AD) was also examined. As has been previously reported, the ApoE E4 allele frequency was significantly increased in AD. However, no change in the frequency of ApoE alleles was found in two of the clinical and pathological forms of LA (dementia of frontal type and dementia of frontal type with motor neurone disease) although the ApoE E4 allele frequency was elevated in cases of non-fluent progressive aphasia in accordance with the presence of coincidental Alzheimer-type pathological changes.

Debrisoquine hydroxylase gene polymorphism frequencies in patients with amyotrophic lateral sclerosis.

Using PCR and restriction digest analysis, the frequencies of the variant cytochrome P450 debrisoquine hydroxylase CYP2D6 alleles CYP2D6(A) and CYP2D6(B) were investigated in 50 patients with amyotrophic lateral sclerosis (ALS) and 13 patients with ALS and frontotemporal dementia (FTD) and compared to those frequencies in patients with FTD alone and Alzheimer's disease (AD). The CYP2D6(T) allelic frequency was also assessed in ALS and ALS + FTD. Although the frequency of a poor metabolizer genotype was not increased in any disease group, there was a significant increase in the frequency of the CYP2D6(B) allele in the ALS patient group. This suggests that possession of a CYP2D6(B) allele may be a risk factor for the development of ALS, possibly conferring a 'gain of function' imposed by the mutation or reflecting linkage disequilibrium to a nearby susceptibility gene.

Amyloid beta protein deposition in patients with frontotemporal lobar degeneration: relationship to age and apolipoprotein E genotype.

Amyloid beta protein (Abeta) deposition was investigated in the frontal cortex of 54 autopsy cases of frontotemporal lobar degeneration (FTLD) using methenamine silver staining, and immunohistochemistry employing the monoclonal end-specific antibodies BC05 and BA27 to visualize deposits containing Abeta(42(43)) and Abeta(40), respectively. Abeta was detected in 14 (26%) patients, nearly always in the form of diffuse Abeta(42(43)) containing plaques though some cored, neuritic plaques with trace amounts of Abeta(40) were occasionally seen. The 14 patients showing Abeta deposits were significantly older at onset of illness than those 40 patients without Abeta. It was only possible to genotype 46/54 cases, 16 of whom bore at least one copy of the Apolipoprotein E (APOE) epsilon4 allele, giving an allele frequency of 20%. Possession of APOE epsilon4 allele was significantly associated with deposition of Abeta such that 10/16 epsilon4 allele bearers had Abeta deposits. Eight of these ten patients showed only mild to moderate amounts of Abeta, but in two patients, one homozygous and one heterozygous for epsilon4 allele, there was extensive neuritic plaque and neurofibrillary tangle formation. In contrast, only few non-epsilon4 allele bearers (4/30) showed minor Abeta deposits. When stratifying for APOE epsilon4 allele, both bearers and non-bearers of epsilon4 allele with Abeta deposits had a significantly later age at onset than their respective groups without Abeta deposits. We conclude that the likelihood of Abeta deposition, as a secondary and coincidental feature unrelated to the primary pathological process, within the brains of individuals with FTLD will be high if patients have a sufficiently late onset of illness or happen to be a bearer of the APOE epsilon4 allele. Indeed 9/14 patients with Abeta deposits studied here had an onset of illness after 55 years of age and bore APOE epsilon4 allele.

Pathological relationships between microglial cell activity and tau and amyloid beta protein in patients with Alzheimer's disease.

The extent of microglial cell activation (microglial cell load) was estimated by image analysis of ferritin-immunostained sections of frontal cortex from 72 patients with pathologically confirmed Alzheimer's disease (AD), and correlated with the amount of pathological tau and amyloid beta protein (Abeta), as both Abeta(40) and Abeta(42) load, in adjacent sections of the same cases. Microglial cell load did not correlate with either Abeta(40) or Abeta(42) load but was significantly correlated with pathological tau load. Microglial cell load was unrelated to age at onset of disease or duration of illness. It is possible that because the presence of microglial cells predates that of pathological tau proteins within the cerebral cortex in AD, neurofibrillary damage to nerve cells may stem from the release of proinflammatory and other potentially neurotoxic molecules from microglial cells.

The extended haplotype of the microtubule associated protein tau gene is not associated with Pick's disease.

Pick's disease (PiD) is a rare neurodegenerative condition and is a member of a heterogeneous group of disorders known as tauopathies, so-called because of the predominantly neuronal aberrant tau accumulations found in these diseases. The tauopathy, familial frontotemporal dementia (FTD), is caused by mutations in the tau gene. Moreover, progressive supranuclear palsy (PSP) is associated with the tau H1 haplotype. In certain familial forms of FTD and in PSP the microtubule-binding four repeat tau isoform principally accumulates in neuropathological lesions. However, in PiD three repeat tau accumulations are found. We therefore investigated whether either the tau H1 or H2 haplotype was associated with PiD. Our results indicate a slight increase in H2H2 frequency in Pick's cases which is not statistically significant.

Allelic variations in apolipoprotein E and prion protein genotype related to plaque formation and age of onset in sporadic Creutzfeldt-Jakob disease.

Prion gene sequence is thought to affect the phenotypic expression of prion disease and the E2 variant of apolipoprotein E (Apo E) can be neuroprotective in dementia. We determined codon 129 of the prion gene and the Apo E variants in Creutzfeldt-Jakob disease (CJD) using PCR and restriction digest. We found a significant correlation between valine at codon 129 of the prion protein gene and the presence of plaque in CJD and a later age of onset in CJD cases possessing the Apo E2 allele. This study provides further evidence that sequence variations in the prion gene can modify disease pathology and the neuroprotection afforded by Apo E2 is not confined to Alzheimer's disease.

The extent of amyloid deposition in brain in patients with Down's syndrome does not depend upon the apolipoprotein E genotype.

The extent of deposition of amyloid beta protein (A beta) was investigated in 20 elderly patients with Down's syndrome, using the end-specific monoclonal antibodies BC05 and BA27 to detect the presence of A beta 42(43) and A beta 40 (respectively), and related to apolipoprotein E (ApoE) genotype. No significant differences in the amount of A beta deposited in the brain, either as A beta 42(43) or A beta 40, were noted in patients possessing an ApoE E4 allele, compared to those without. Patients with an ApoE E4 allele in general died at an earlier age than those with only ApoE E3 alleles, the latter in turn being outlived by those with an ApoE E2 allele. In Down's syndrome therefore, ApoE may influence the timing of onset, or the rate of progression, of disease but without affecting the type or total amount of pathology accumulated.

Preferential deposition of amyloid beta protein (Abeta) in the form Abeta40 in Alzheimer's disease is associated with a gene dosage effect of the apolipoprotein E E4 allele.

The effect of apolipoprotein E (ApoE) genotype on the deposition of amyloid beta protein (Abeta) was examined in 54 patients with Alzheimer's disease. No difference in the amount of Abeta deposited as Abeta42(43) was seen between genotype groups with no, one or two E4 alleles. However, the amount of Abeta40 deposited increased according to the copy number of E4 alleles with patients possessing one E4 allele containing more than twice, and those with two E4 alleles, four times, the amount of Abeta40 in their brains compared to patients without an E4 allele. The increase in total Abeta deposited within the tissue (i.e. Abeta40 plus Abeta42(43) loads) in the presence of an E4 allele is therefore due entirely to an enhanced deposition of Abeta40. These data are consistent with the suggestion that the presence of E4 within pre-existing Abeta42(43) containing plaques may lower the threshold to fibrilization of Abeta40 thereby promoting its subsequent deposition. Thus, although the total amount of Abeta initially deposited in the brain as Abeta42(43) is not affected by the binding of any one particular ApoE isoform this does influence the subsequent maturation of plaques with a greater proportion transforming into Abeta40 containing cored plaques when the E4 isoform is present.

An intronic polymorphism in the presenilin-1 gene does not influence the amount or molecular form of the amyloid beta protein deposited in Alzheimer's disease.

The frequency of the allele-1 polymorphism in intron 8 of the presenilin-1 (PS-1) gene, and the proportion of individuals homozygous in this respect, was investigated in 57 patients with autopsy verified Alzheimer's disease (AD). In 33 of these patients the amount of amyloid beta protein (A beta) was compared across the three PS-1 genotype groups (1/1, 1/2, 2/2). No excess of the allele-1 was detected in these patients with confirmed AD and no variations in the extent of A beta deposition, as either A beta 40 or A beta 42, in terms of plaque number or percentage area of tissue occupied, were found. We conclude that this intronic PS-1 polymorphism does not influence the pathological phenotype of AD, at least as far as A beta deposition is concerned.

A polymorphism within intron 11 of the tau gene is not increased in frequency in patients with sporadic Alzheimer's disease, nor does it influence the extent of tau pathology in the brain.

There are numerous polymorphisms within the tau gene but these are in complete linkage disequilibrium and exist as two common extended haplotypes H1 and H2. We have investigated the frequency of these haplotypes in 83 cases of sporadic Alzheimer's disease (AD) using the +34 polymorphism in intron 11 of the tau gene as a marker of H1 and H2 haplotypes. The total amount of hyperphosphorylated tau protein (tau load), present as neurofibrillary tangles, neuropil threads or plaque neurites, was quantified in the frontal cortex of these patients and related to tau haplotype. We found no increase in H1H1 haplotype in this autopsy population of cases with AD compared to published control data. Stratification of cases for apolipoprotein E (APO E) genotype showed a slight, but not statistically significant, overrepresentation of epsilon 4 allele amongst bearers of H2 haplotype. There were no overall differences in tau load between haplotype groups though cases within each haplotype group bearing APO E epsilon 4 allele had a significantly higher tau load than those without epsilon 4 allele. Neither age at onset or duration of illness differed according to tau haplotype. We conclude that the frequency of tau gene H1 haplotype is not elevated in AD and possession of this has no impact upon the amount of tau pathology in AD.