Katy Chalmers

APOEɛ4 influences the pathological phenotype of Alzheimer's disease by favouring cerebrovascular over parenchymal accumulation of Aβ protein

The relative amounts of amyloid β‐protein (Aβ) in cerebral blood vessels and parenchyma vary considerably amongst patients with Alzheimers disease (AD). Although several mechanisms have been proposed to explain this variability, the underlying genetic and environmental determinants are still unclear, as are the functional consequences. Polymorphisms in APOE, the gene for apolipoprotein E (ApoE), influence the risk of developing AD and of deposition of Aβ within the brain. We examined the relationship between the APOE genotype and the relative extent of accumulation of Aβ as plaques within the cerebral parenchyma and in cortical blood vessels in the form of cerebral amyloid angiopathy (CAA), in autopsy brain tissue from 125 AD cases and from 53 elderly, neurologically normal controls of which 19 had CAA without other neuropathological features of AD. In the AD cases, we also assessed whether the severity of CAA was related to the age of onset and duration of dementia, risk factors for atherosclerotic vascular disease, and histologically demonstrable cerebral in‐farcts or foci of haemorrhage. The APOE genotype was determined by a standard polymerase chain reaction‐based method. Paraffin sections of frontal, temporal and parietal lobes were immunolabelled for Aβ and the parenchymal Aβ load (total Aβ minus vessel‐associated Aβ) was quantified by computer‐assisted image analysis. CAA severity was scored for cortical and leptomeningeal vessels. The relevant clinical data were obtained from the database of the South West Brain Bank. In AD, we found the severity of CAA to be strongly associated with the number of ɛ4 alleles (P < 0.0001) but the parenchymal Aβ load to be independent of APOE genotype. Cases with severe CAA had a lower parenchymal Aβ load than had those with moderate CAA (P = 0.003). Neither the severity of CAA nor the parenchymal Aβ load correlated with age of onset, duration of disease or age at death, and the severity of CAA also did not correlate with the presence of cerebral infarcts or foci of haemorrhage. These findings indicate that possession of the APOEɛ4 allele favours vascular over parenchymal accumulation of Aβ in AD. This may influence the pathogenesis of neurodegeneration in ɛ4‐associated AD.

Decreased expression and activity of neprilysin in Alzheimer disease are associated with cerebral amyloid angiopathy

Neprilysin (NEP) degrades amyloid-&bgr; (A&bgr;) and is thought to contribute to its clearance from the brain. In Alzheimer disease (AD), downregulation of NEP has been suggested to contribute to the development of cerebral amyloid angiopathy (CAA). We examined the relationship among NEP, CAA, and APOE status in AD and elderly control cases. NEP was most abundant in the tunica media of cerebrocortical blood vessels and in pyramidal neurons. In homogenates of the frontal cortex, NEP protein levels were reduced in AD but not significantly; NEP enzymatic activity was significantly reduced in AD. Immunohistochemistry revealed a reduction of both vascular and parenchymal NEP. The loss of vessel-associated NEP in AD was inversely related to the severity of CAA, and analysis of cases with severe CAA showed that levels of vascular NEP were reduced to the same extent in A&bgr;-free and A&bgr;-laden vessels, strongly suggesting that the reduction in NEP is not simply secondary to CAA. Possession of APOE &egr;4 was associated with significantly lower levels of both parenchymal and vascular NEP. Colinearity of &egr;4 with the presence of moderate to severe CAA precluded assessment of the independence of this association from NEP levels. However, logistic regression analysis showed low NEP levels to be a significant independent predictor of moderate to severe CAA.

Angiotensin‐converting enzyme (ACE) levels and activity in Alzheimer's disease, and relationship of perivascular ACE‐1 to cerebral amyloid angiopathy

Aims: Several observations point to the involvement of angiotensin‐converting enzyme‐1 (ACE‐1) in Alzheimers disease (AD): ACE‐1 cleaves amyloid‐β peptide (Aβ) in vitro, the level and activity of ACE‐1 are reportedly increased in AD, and variations in the ACE‐1 gene are associated with AD. We analysed ACE‐1 activity and expression in AD and control brains, particularly in relation to Aβ load and cerebral amyloid angiopathy (CAA). Methods: ACE‐1 activity was measured in the frontal cortex from 58 control and 114 AD cases of known Aβ load and CAA severity. The distribution of ACE‐1 was examined immunohistochemically. In five AD cases with absent or mild CAA, five with moderate to severe CAA and five controls with absent or mild CAA, levels of vascular ACE‐1 were assessed by quantitative immunofluorescence. Results: ACE‐1 activity was increased in AD (P < 0.001) and correlated directly with parenchymal Aβ load (P = 0.05). Immunohistochemistry revealed ACE‐1 in neurones and cortical blood vessels – in the intima but most abundant perivascularly. Cases with moderate to severe CAA had significantly more vessel‐associated ACE‐1 than did those with little or no CAA. Perivascular ACE‐1 did not colocalize with Aβ, smooth muscle actin, glial fibrillary acidic protein, collagen IV, vimentin or laminin, but was similarly distributed to extracellular matrix (ECM) proteins fibronectin and decorin. Conclusions: Our findings indicate that ACE‐1 activity is increased in AD, in direct relationship to parenchymal Aβ load. Increased ACE‐1, probably of neuronal origin, accumulates perivascularly in severe CAA and colocalizes with vascular ECM. The possible relationship of ACE‐1 to the deposition of perivascular ECM remains to be determined.

Insights into the pathogenesis and pathogenicity of cerebral amyloid angiopathy.

Amyloid-beta (Abeta) cerebral amyloid angiopathy (CAA) affects most Alzheimers disease (AD) patients and ~30% of otherwise-normal elderly people. APOE epsilon 4 is a major risk factor for CAA in AD. Neurons are probably the source of the vascular Abeta. CAA develops when Abeta is deposited in the vessel walls along or across which it normally passes into the CSF and bloodstream. Vascular deposition is facilitated by factors that increase Abeta40:Abeta42, impede perivascular passage of Abeta or raise its concentration. The levels of some Abeta-degrading enzymes are reduced in AD patients with CAA. However, angiotensin-converting enzyme activity is increased and may act via angiotensin II to increase transforming growth factor beta1, a potent inducer of ECM synthesis. CAA is a cause of intracerebral haemorrhage and cerebral ischaemic damage. In AD, neuritic degeneration is accentuated around Abeta-laden vessels. Rarely, CAA is associated with angiitis. The balance between parenchymal and cerebrovascular degradation of Abeta, and regulation of perivascular extracellular matrix production, are likely to be key determinants of Abeta distribution and pathogenicity within the brain.

Neurofibrillary tangles may interfere with Smad 2/3 signaling in neurons

Transforming growth factor (TGF)-&bgr; is a multifunctional cytokine with anti-inflammatory, reparative and neuroprotective functions. Increased levels of TGF&bgr; in Alzheimer disease (AD) are associated with perivascular deposition of extracellular matrix, which may impair clearance of &bgr;-amyloid and contribute to the development of cerebral amyloid angiopathy. TGF&bgr; signaling is transduced by Smad proteins: on TGF&bgr; receptor activation, Smads 2 and 3 are released from sequestration by microtubules, phosphorylated (forming pSmad2/3), and, together with Smad 4, translocated to the nucleus, where they initiate the transcription of multiple genes. Neuronal microtubule assembly is disturbed in AD when tau, a microtubule-stabilizing protein, is hyperphosphorylated and forms neurofibrillary tangles. We have investigated the relationship between Ser202 phospho-tau and pSmads 2 and 3 in the temporal lobe in AD. Within neurons in control brains, pSmads 2 and 3 were almost exclusively intranuclear. In AD, pSmad 3 bound to phospho-tau (mostly insoluble tau) and accumulated in the cytoplasm of tangle-bearing neurons; this was accompanied by a marked decrease in nuclear pSmad3. pSmads 2 and 3 were also present in neuronal granulovacuolar inclusions. Our findings suggest that neurofibrillary tangles sequester pSmad3, preventing its translocation into the nucleus and the induction of gene transcription. Interference with the Smad signaling may adversely affect survival of tangle-bearing neurons in AD.

Contributors to white matter damage in the frontal lobe in Alzheimer's disease

Abnormalities of cerebral white matter are present in a majority of patients with Alzheimers disease (AD) and probably contribute to motor dysfunction and cognitive impairment. The white matter abnormalities are usually attributed to degenerative vascular disease and cerebral amyloid angiopathy (CAA) but the evidence is scanty or inconclusive. In the present study we examined sections of frontal lobe from 125 autopsy‐confirmed cases of AD and assessed the relationship of degenerative large and small vessel disease, CAA, parenchymal Aβ load and APOE genotype, to several objective measures of white matter damage: extent of immunolabelling for glial fibrillary acidic protein (GFAP), axonal accumulation of amyloid precursor protein (APP), axon density in superficial and deep white matter, and intensity of staining for myelin. We found no association between atherosclerosis, arteriolosclerosis, CAA or APOE genotype and white matter damage. However, labelling of white matter for GFAP correlated strongly with the parenchymal Aβ load (P = 0.0003) and with APP accumulation (P = 0.008). Our findings suggest that severity of frontal white matter damage in AD is closely related to parenchymal Aβ load and that in most cases the contribution of degenerative vascular disease, CAA and APOE is relatively minor.

Cholinesterase inhibitors reduce cortical Aβ in dementia with Lewy bodies

Cholinesterase inhibitors (ChEIs) are effective symptomatic treatments in dementia with Lewy bodies (DLB), although effects on pathologic mechanisms are unknown. In the first human autopsy study examining the impact of ChEI treatment on brain pathology, we compared treated patients with DLB with matched untreated patients for cortical β-amyloid (Aβ) and tau pathologies. Treated patients with DLB had significantly less parenchymal Aβ deposition, which is relevant to disease management and treatment of dementia patients using ChEI.

Cholinesterase inhibitors reduce cortical Abeta in dementia with Lewy bodies.

Cholinesterase inhibitors (ChEIs) are effective symptomatic treatments in dementia with Lewy bodies (DLB), although effects on pathologic mechanisms are unknown. In the first human autopsy study examining the impact of ChEI treatment on brain pathology, we compared treated patients with DLB with matched untreated patients for cortical β-amyloid (Aβ) and tau pathologies. Treated patients with DLB had significantly less parenchymal Aβ deposition, which is relevant to disease management and treatment of dementia patients using ChEI.

Distribution of the branched chain aminotransferase proteins in the human brain and their role in glutamate regulation

The branched chain aminotransferase enzymes (BCAT) serve as nitrogen donors for the production of 30% of de novo glutamate synthesis in rat brain. Despite the importance of this major metabolite and excitatory neurotransmitter, the distribution of BCAT proteins in the human brain (hBCAT) remains unreported. We have studied this and report, for the first time, that the mitochondrial isoform, hBCATm is largely confined to vascular endothelial cells, whereas the cytosolic hBCATc is restricted to neurons. The majority of hBCATc‐labelled neurons were either GABA‐ergic or glutamatergic showing both cell body and axonal staining indicating a role for hBCATc in both glutamate production and glutamate release during excitation. Strong staining in hormone secreting cells suggests a further role for the transaminases in hormone regulation potentially similar to that proposed for insulin secretion. Expression of hBCATm in the endothelial cells of the vasculature demonstrates for the first time that glutamate could be metabolized by aminotranferases in these cells. This has important implications given that the dysregulation of glutamate metabolism, leading to glutamate excitotoxicity, is an important contributor to the pathogenesis of several neurodegenerative conditions, where the role of hBCATm in metabolizing excess glutamate may factor more prominently.

Relationship of neurofibrillary pathology to cerebral amyloid angiopathy in Alzheimer's disease.

Over 90% of patients with Alzheimers disease (AD) develop cerebral amyloid angiopathy (CAA). Severe dyshoric CAA, in which amyloid extends into the surrounding brain parenchyma, may be associated with adjacent clustering of tau‐immunopositive neurites but the relationship of CAA to neurofibrillary pathology has not been systematically investigated. In the present study this relationship was examined in sections of frontal, temporal and parietal cortex from 25 AD patients with moderate to severe CAA and 26 with mild or absent CAA. We measured immunolabelling of abnormally phosphorylated tau adjacent to Aβ‐laden and non‐Aβ‐laden arteries and arterioles, and in cortex away from arteries and arterioles. We also analysed the possible influence of APOE genotype on these measurements. There were no significant differences between the lobes in measurements of tau labelling, either around blood vessels or elsewhere in the cortex. However, tau labelling around Aβ‐laden arteries and arterioles significantly exceeded that around non‐Aβ‐laden blood vessels (P < 0.001) and this, in turn was greater than the labelling of cortex away from blood vessels (P < 0.001). There was no association between APOE ɛ4 and the immunolabelling density for tau, whether around amyloid‐ or non‐amyloid‐laden arteries and arterioles, or in the cerebral cortex away from these. We propose that both CAA and peri‐vascular accumulation of hyperphosphorylated tau may be a consequence of elevated levels of soluble Aβ around cortical arteries and arterioles.