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1.  TDP-43 in the olfactory bulb in Alzheimer’s disease 
doi:10.1111/nan.12309
PMCID: PMC5154242  PMID: 26810591
2.  Neuronal DNA damage response‐associated dysregulation of signalling pathways and cholesterol metabolism at the earliest stages of Alzheimer‐type pathology 
Aims
Oxidative damage and an associated DNA damage response (DDR) are evident in mild cognitive impairment and early Alzheimer's disease, suggesting that neuronal dysfunction resulting from oxidative DNA damage may account for some of the cognitive impairment not fully explained by Alzheimer‐type pathology.
Methods
Frontal cortex (Braak stage 0–II) was obtained from the Medical Research Council's Cognitive Function and Ageing Study cohort. Neurones were isolated from eight cases (four high and four low DDR) by laser capture microdissection and changes in the transcriptome identified by microarray analysis.
Results
Two thousand three hundred seventy‐eight genes were significantly differentially expressed (1690 up‐regulated, 688 down‐regulated, P < 0.001) in cases with a high neuronal DDR. Functional grouping identified dysregulation of cholesterol biosynthesis, insulin and Wnt signalling, and up‐regulation of glycogen synthase kinase 3β. Candidate genes were validated by quantitative real‐time polymerase chain reaction. Cerebrospinal fluid levels of 24(S)‐hydroxycholesterol associated with neuronal DDR across all Braak stages (r s = 0.30, P = 0.03).
Conclusions
A persistent neuronal DDR may result in increased cholesterol biosynthesis, impaired insulin and Wnt signalling, and increased GSK3β, thereby contributing to neuronal dysfunction independent of Alzheimer‐type pathology in the ageing brain.
doi:10.1111/nan.12252
PMCID: PMC5102584  PMID: 26095650
Ageing brain; DNA damage response; neurones; microarray; dementia; Alzheimer's
3.  Immunolocalization of platelet‐derived growth factor receptor‐β (PDGFR‐β) and pericytes in cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) 
Aims
Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is identified by aggregates of NOTCH3 extracellular domain (N3ECD) along capillaries and the deposition of granular osmiophilic material (GOM). We assessed the pattern of distribution of pericytes in relation to N3ECD deposits in cerebral microvessels of CADASIL subjects.
Methods
We assessed post mortem brains from (n = 50) subjects with CADASIL, cerebral small vessel disease, and similar‐age cognitively normal and older controls. Immunohistochemical and immunofluorescent staining methods were used to study the distribution and quantify immunoreactivities of the platelet‐derived growth factor receptor‐β (PDGFR‐β) (for pericytes) and microvascular markers in the frontal cortex and white matter.
Results
PDGFR‐β antibody stained cells typical of pericytes in capillaries and small arterioles in both the grey and white matter. PDGFR‐β reactive pericytes adopted ‘crescent’ morphology wrapped closely around capillary walls readily evident in cross‐sections. We noted considerable overlap between PDGFR‐β and N3ECD imunoreactivities in capillaries. Quantitative analysis of PDGFR‐β immunoreactivity revealed significant differences in PDGFR‐β %A in CADASIL compared with young controls (P < 0.05). PDGFR‐β %A was further positively correlated with the basement membrane marker collagen IV (r = 0.529, P = 0.009), but was not associated with GLUT‐1, the marker for endothelial cells.
Conclusions
Our results suggest increased expression of PDGFR‐β immunoreactive pericytes in cerebral microvessels in CADASIL compared with similar age controls. While we cannot confirm whether PDGFR‐β‐expressing pericytes produce N3ECD and hence GOM, our findings demonstrate that up‐regulation of pericyte‐like cells is associated with microvascular changes, including loss of vascular smooth muscle cells in CADASIL.
doi:10.1111/nan.12188
PMCID: PMC5098250  PMID: 25303037
CADASIL; capillaries; dementia; pericyte; platelet‐derived growth factor receptor; vascular smooth muscle cell; vascular dementia
4.  Bringing CLARITY to the human brain: visualization of Lewy pathology in three dimensions 
Aims
CLARITY is a novel technique which enables three‐dimensional visualization of immunostained tissue for the study of circuitry and spatial interactions between cells and molecules in the brain. In this study, we aimed to compare methodological differences in the application of CLARITY between rodent and large human post mortem brain samples. In addition, we aimed to investigate if this technique could be used to visualize Lewy pathology in a post mortem Parkinson's brain.
Methods
Rodent and human brain samples were clarified and immunostained using the passive version of the CLARITY technique. Samples were then immersed in different refractive index matching media before mounting and visualizing under a confocal microscope.
Results
We found that tissue clearing speed using passive CLARITY differs according to species (human vs. rodents), brain region and degree of fixation (fresh vs. formalin‐fixed tissues). Furthermore, there were advantages to using specific refractive index matching media. We have applied this technique and have successfully visualized Lewy body inclusions in three dimensions within the nucleus basalis of Meynert, and the spatial relationship between monoaminergic fibres and Lewy pathologies among nigrostriatal fibres in the midbrain without the need for physical serial sectioning of brain tissue.
Conclusions
The effective use of CLARITY on large samples of human tissue opens up many potential avenues for detailed pathological and morphological studies.
doi:10.1111/nan.12293
PMCID: PMC5053282  PMID: 26526972
CLARITY; human post mortem brain; Lewy body pathology; three‐dimensional visualization; tissue clearing
5.  Review: Modulating the unfolded protein response to prevent neurodegeneration and enhance memory 
Recent evidence has placed the unfolded protein response (UPR) at the centre of pathological processes leading to neurodegenerative disease. The translational repression caused by UPR activation starves neurons of the essential proteins they need to function and survive. Restoration of protein synthesis, via genetic or pharmacological means, is neuroprotective in animal models, prolonging survival. This is of great interest due to the observation of UPR activation in the post mortem brains of patients with Alzheimer's, Parkinson's, tauopathies and prion diseases. Protein synthesis is also an essential step in the formation of new memories. Restoring translation in disease or increasing protein synthesis from basal levels has been shown to improve memory in numerous models. As neurodegenerative diseases often present with memory impairments, targeting the UPR to both provide neuroprotection and enhance memory provides an extremely exciting novel therapeutic target.
doi:10.1111/nan.12211
PMCID: PMC5053297  PMID: 25556298
memory; neurodegeneration; neurodegenerative diseases; therapeutics; unfolded protein response
6.  Audit of practice in sudden unexpected death in epilepsy (SUDEP) post mortems and neuropathological findings 
Aims
Sudden unexpected death in epilepsy (SUDEP) is one of the leading causes of death in people with epilepsy. For classification of definite SUDEP, a post mortem (PM), including anatomical and toxicological examination, is mandatory to exclude other causes of death. We audited PM practice as well as the value of brain examination in SUDEP.
Methods
We reviewed 145 PM reports in SUDEP cases from four UK neuropathology centres. Data were extracted for clinical epilepsy details, circumstances of death and neuropathological findings.
Results
Macroscopic brain abnormalities were identified in 52% of cases. Mild brain swelling was present in 28%, and microscopic pathologies relevant to cause or effect of seizures were seen in 89%. Examination based on whole fixed brains (76.6% of all PMs), and systematic regional sampling was associated with higher detection rates of underlying pathology (P < 0.01). Information was more frequently recorded regarding circumstances of death and body position/location than clinical epilepsy history and investigations.
Conclusion
Our findings support the contribution of examination of the whole fixed brain in SUDEP, with high rates of detection of relevant pathology. Availability of full clinical epilepsy‐related information at the time of PM could potentially further improve detection through targeted tissue sampling. Apart from confirmation of SUDEP, complete neuropathological examination contributes to evaluation of risk factors as well as helping to direct future research into underlying causes.
doi:10.1111/nan.12265
PMCID: PMC4864133  PMID: 26300477
audit; neuropathology; post mortem; Sudden unexpected death in epilepsy; SUDEP
7.  Neuroprotective effects of enriched environment housing after transient global cerebral ischaemia are associated with the upregulation of insulin‐like growth factor‐1 signalling 
Aims
Use of enriched environment (EE) housing has been shown to promote recovery from cerebral ischaemic injury but the underlying mechanisms of their beneficial effects remains unclear. Here we examined whether the beneficial effects of EE housing on ischaemia‐induced neurodegeneration and cognitive impairment are associated with increased insulin‐like growth factor‐1 (IGF‐1) signalling in the hippocampus.
Methods
Forty‐two adult male Wistar rats were included in the study and received either ischaemia or sham surgery. Rats in each group were further randomized to either: EE or standard laboratory cage housing (control). Rats were placed in their assigned housing condition immediately after recovery from anaesthesia. Behavioural testing in the cued learning and discrimination learning tasks were conducted 2 weeks after ischaemia. Rats were euthanized after behavioural testing and the hippocampus was analysed for IGF‐1 level, IGF‐1 receptor (IGF‐1R) activation, protein kinase B (Akt) pathway activation, neurone loss and caspase 3 expression.
Results
Our data showed that EE housing: (1) mitigated ischaemia‐induced neuronal loss; (2) attenuated ischaemia‐induced increase in caspase 3 immunoreactivity in the hippocampus; (3) ameliorated ischaemia‐induced cognitive impairments; and (4) increased IGF‐1R activation and signalling through the Akt pathway after ischaemic injury.
Conclusion
Ultimately, these findings suggest the possibility that IGF‐1 signalling may be one of the underlying mechanisms involved in the beneficial effects of EE in optimizing recovery following cerebral ischaemic injury.
doi:10.1111/nan.12146
PMCID: PMC4201886  PMID: 24750178
Akt signalling; caspase 3; complex environment; contextual learning; Fluoro‐Jade; water maze
8.  Investigating complex I deficiency in Purkinje cells and synapses in patients with mitochondrial disease 
Aims
Cerebellar ataxia is common in patients with mitochondrial disease, and despite previous neuropathological investigations demonstrating vulnerability of the olivocerebellar pathway in patients with mitochondrial disease, the exact neurodegenerative mechanisms are still not clear. We use quantitative quadruple immunofluorescence to enable precise quantification of mitochondrial respiratory chain protein expression in Purkinje cell bodies and their synaptic terminals in the dentate nucleus.
Methods
We investigated NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 13 protein expression in 12 clinically and genetically defined patients with mitochondrial disease and ataxia and 10 age‐matched controls. Molecular genetic analysis was performed to determine heteroplasmy levels of mutated mitochondrial DNA in Purkinje cell bodies and inhibitory synapses.
Results
Our data reveal that complex I deficiency is present in both Purkinje cell bodies and their inhibitory synapses which surround dentate nucleus neurons. Inhibitory synapses are fewer and enlarged in patients which could represent a compensatory mechanism. Mitochondrial DNA heteroplasmy demonstrated similarly high levels of mutated mitochondrial DNA in cell bodies and synapses.
Conclusions
This is the first study to use a validated quantitative immunofluorescence technique to determine complex I expression in neurons and presynaptic terminals, evaluating the distribution of respiratory chain deficiencies and assessing the degree of morphological abnormalities affecting synapses. Respiratory chain deficiencies detected in Purkinje cell bodies and their synapses and structural synaptic changes are likely to contribute to altered cerebellar circuitry and progression of ataxia.
doi:10.1111/nan.12282
PMCID: PMC4973693  PMID: 26337858
ataxia; mitochondrial disease; mitochondrial DNA; Purkinje cells; respiratory chain deficiency; synapses
10.  A neuronal DNA damage response is detected at the earliest stages of Alzheimer's neuropathology and correlates with cognitive impairment in the Medical Research Council's Cognitive Function and Ageing Study ageing brain cohort 
Aims
Population‐based studies have shown that approximately 20% of the ageing population (aged 65 years and over) with dementia have little or no classical Alzheimer‐type neuropathology. Cumulative DNA damage and a reduced capacity of DNA repair may result in neuronal dysfunction and contribute to cognitive impairment independent of Alzheimer‐type pathology in the ageing brain.
Methods
We investigated expression of the DNA damage response (DDR)‐associated molecules γH2AX and DNA‐PKcs using immunohistochemistry and western blotting, and senescence‐associated β‐galactosidase in the frontal association neocortex of cases with low levels of Alzheimer‐type pathology (Braak & Braak stage 0–II), and explored their relationship to cognitive impairment in a population‐representative sample from the Medical Research Council's Cognitive Function and Ageing Study cohort.
Results
Increases in both γH2AX + (r s = −0.36, P = 0.025) and DNA‐PKcs + (r s = −0.39, P = 0.01) neuronal counts were associated with a lower Mini‐Mental State Examination score. Increasing levels of senescence associated‐β‐gal+ pyramidal neurones were weakly associated with the total number of DNA‐PKcs+ neurones (P = 0.08), but not with traditional senescence‐associated signalling molecules, including p53 and p16.
Conclusion
The association between the neuronal DDR and cognitive impairment, independent of AD pathology in the ageing brain, may be suggestive of a causal link via neuronal dysfunction.
doi:10.1111/nan.12202
PMCID: PMC4861215  PMID: 25443110
cognitive impairment; DNA damage response; DNA‐PKcs; neurone; γH2AX
11.  Clusterin/Apolipoprotein J immunoreactivity is associated with white matter damage in cerebral small vessel diseases 
Aim
Brain clusterin is known to be associated with the amyloid‐β deposits in Alzheimer's disease (AD). We assessed the distribution of clusterin immunoreactivity in cerebrovascular disorders, particularly focusing on white matter changes in small vessel diseases.
Methods
Post‐mortem brain tissues from the frontal or temporal lobes of a total of 70 subjects with various disorders including cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), cerebral amyloid angiopathy (CAA) and AD were examined using immunohistochemistry and immunofluorescence. We further used immunogold electron microscopy to study clusterin immunoreactivity in extracellular deposits in CADASIL.
Results
Immunostaining with clusterin antibodies revealed strong localization in arterioles and capillaries, besides cortical neurones. We found that clusterin immunostaining was significantly increased in the frontal white matter of CADASIL and pontine autosomal dominant microangiopathy and leukoencephalopathy subjects. In addition, clusterin immunostaining correlated with white matter pathology severity scores. Immunostaining in axons ranged from fine punctate deposits in single axons to larger confluent areas with numerous swollen axon bulbs, similar to that observed with known axon damage markers such as non‐phosphorylated neurofilament H and the amyloid precursor protein. Immunofluorescence and immunogold electron microscopy experiments showed that whereas clusterin immunoreactivity was closely associated with vascular amyloid‐β in CAA, it was lacking within the granular osmiophilic material immunolabelled by NOTCH3 extracelluar domain aggregates found in CADASIL.
Conclusions
Our results suggest a wider role for clusterin associated with white matter damage in addition to its ability to chaperone proteins for clearance via the perivascular drainage pathways in several disease states.
doi:10.1111/nan.12248
PMCID: PMC4949672  PMID: 25940137
Alzheimer's disease; CADASIL; cerebral amyloid angiopathy; cerebrovascular disease; clusterin; granular osmiophilic material
12.  Identification of clinical target areas in the brainstem of prion‐infected mice 
Aims
While prion infection ultimately involves the entire brain, it has long been thought that the abrupt clinical onset and rapid neurological decline in laboratory rodents relates to involvement of specific critical neuroanatomical target areas. The severity and type of clinical signs, together with the rapid progression, suggest the brainstem as a candidate location for such critical areas. In this study we aimed to correlate prion pathology with clinical phenotype in order to identify clinical target areas.
Method
We conducted a comprehensive survey of brainstem pathology in mice infected with two distinct prion strains, which produce different patterns of pathology, in mice overexpressing prion protein (with accelerated clinical onset) and in mice in which neuronal expression was reduced by gene targeting (which greatly delays clinical onset).
Results
We identified specific brainstem areas that are affected by prion pathology during the progression of the disease. In the early phase of disease the locus coeruleus, the nucleus of the solitary tract, and the pre‐Bötzinger complex were affected by prion protein deposition. This was followed by involvement of the motor and autonomic centres of the brainstem.
Conclusions
Neurodegeneration in the locus coeruleus, the nucleus of the solitary tract and the pre‐Bötzinger complex predominated and corresponded to the manifestation of the clinical phenotype. Because of their fundamental role in controlling autonomic function and the overlap with clinical signs in sporadic Creutzfeldt–Jakob disease, we suggest that these nuclei represent key clinical target areas in prion diseases.
doi:10.1111/nan.12189
PMCID: PMC4949711  PMID: 25311251
brainstem; clinical target areas; cre‐lox system; locus coeruleus; neurodegeneration; prions
13.  Molecular and cellular pathogenesis of adamantinomatous craniopharyngioma 
Adamantinomatous craniopharyngiomas (ACPs) are the most common pituitary tumours in children. Although histologically benign, these are clinically aggressive tumours, difficult to manage and associated with poor quality of life for the patients. Several human and mouse studies have provided unequivocal evidence that the over‐activation of the WNT/β‐catenin signalling pathway underlies the molecular aetiology of these tumours. Recently, research using genetically modified mouse models of human ACP have revealed a critical and unexpected non‐cell autonomous role for pituitary stem cells in ACP tumourigenesis, which has expanded the cancer stem cell paradigm. As the result of this basic research, the pathogenesis of ACP is being unveiled, with promising implications for the development of novel treatments against these childhood neoplasms. These benign tumours may additionally represent a unique model to provide insights into the initial steps of oncogenesis.
doi:10.1111/nan.12226
PMCID: PMC4949713  PMID: 25611703
adamantinomatous craniopharyngioma; pituitary; Sox2; stem cells; WNT pathway; β‐catenin
14.  Accumulation of dipeptide repeat proteins predates that of TDP‐43 in frontotemporal lobar degeneration associated with hexanucleotide repeat expansions in C9ORF72 gene 
Aims
Frontotemporal lobar degeneration (FTLD) and motor neurone disease are linked by the possession of a hexanucleotide repeat expansion in C9ORF72, and both show neuronal cytoplasmic inclusions within cerebellar and hippocampal neurones which are TDP‐43 negative but immunoreactive for p62 and dipeptide repeat proteins (DPR), these being generated by a non‐ATG RAN translation of the expanded region of the gene.
Methods
Twenty‐two cases of FTLD from Newcastle were analysed for an expansion in C9ORF72 by repeat primed PCR and Southern blot. Detailed case note analysis was performed, and blinded retrospective clinical impressions were achieved by review of clinical histories. Sections from all major brain regions were immunostained for TDP‐43, p62 and DPR. The extent of TDP‐43 and DPR pathology in expansion bearers was compared with that in 13 other previously identified cases from the Manchester Brain Bank with established disease.
Results
Three Newcastle patients bearing an expansion in C9ORF72 were identified. These three patients died prematurely, two from bronchopneumonia within 10 months and 3 years of onset, and one from myocardial infarction 3 years after onset. In all three, DPR were plentiful throughout all cerebral cortical regions, hippocampus and cerebellum, but TDP‐43 pathological changes were sparse. The severity of DPR pathological changes in these three patients was similar to that in the Manchester series, although the extent of TDP‐43 pathology was significantly less.
Conclusion
Widespread accumulation of DPR within nerve cells may occur much earlier than that of TDP‐43 in patients with FTLD bearing expansion in C9ORF72.
doi:10.1111/nan.12178
PMCID: PMC4934135  PMID: 25185840
C9ORF72; dipeptide repeat proteins; frontotemporal lobar degeneration; hexanucleotide repeat expansion
15.  Changes to the lateral geniculate nucleus in Alzheimer's disease but not dementia with Lewy bodies 
Aims
Complex visual hallucinations occur in 70% of dementia with Lewy bodies (DLB) cases and significantly affect patient well‐being. Visuo‐cortical and retinal abnormalities have been recorded in DLB and may play a role in visual hallucinations. The present study aimed to investigate the lateral geniculate nucleus (LGN), a visual relay centre between the retina and visual cortex, to see if changes to this structure underlie visual hallucinations in DLB.
Methods
Fifty‐one [17 probable DLB, 19 control and 15 probable Alzheimer's disease (AD)] cases were recruited for a functional magnetic resonance imaging study, in which patients' response to a flashing checkerboard stimulus was detected and measured in the LGN, before comparison across experimental groups. Additionally, post mortem  LGN tissue was acquired for a cross‐sectional study using 20 (six DLB, seven control and seven AD) cases and analysed using stereology. α‐Synuclein, phosphorylated tau and amyloid‐β pathology was also assessed in all cases.
Results
DLB cases did not significantly differ from controls on neuroimaging, morphometry or pathology. However, a significant increase in amyloid‐β pathology, a reduction in number of parvocellular neurones and magnocellular gliosis was found in AD cases compared with control and DLB cases.
Conclusions
These findings suggest that the early visual system is relatively spared in DLB, which implies that upstream visual structures may be largely responsible for the generation of hallucinatory percepts. The significance of the degeneration of the LGN in AD cases is uncertain.
doi:10.1111/nan.12249
PMCID: PMC4913748  PMID: 25967384
dementia with Lewy bodies; fMRI; lateral geniculate nucleus; neuropathology; stereology
16.  Neuroprotective Effects of Enriched Environment Housing After Transient Global Cerebral Ischemia are Associated with the Upregulation of Insulin-like Growth Factor-1 Signaling 
Aims
Use of enriched environment (EE) housing has been shown to promote recovery from cerebral ischemic injury but the underlying mechanisms of their beneficial effects remains unclear. Here we examined whether the beneficial effects of EE housing on ischemia-induced neurodegeneration and cognitive impairment are associated with increased insulin-like growth factor-1 (IGF-1) signaling in the hippocampus.
Methods
Forty-two adult male Wistar rats were included in the study and received either ischemia or sham surgery. Rats in each group were further randomized to either: EE or standard laboratory cage housing (control). Rats were placed in their assigned housing condition immediately after recovery from anesthesia. Behavioral testing in the cued learning and discrimination learning tasks were conducted 2 weeks after ischemia. Rats were euthanized after behavioral testing and the hippocampus was analyzed for IGF-1 level, IGF-1 receptor (IGF-1R) activation, protein kinase B (Akt) pathway activation, neuron loss, and caspase 3 expression.
Results
Our data showed that EE housing: (1) mitigated ischemia-induced neuronal loss, (2) attenuated ischemia-induced increase in caspase-3 immunoreactivity in the hippocampus, (3) ameliorated ischemia-induced cognitive impairments, and (4) increased IGF-1R activation and signaling through the Akt pathway after ischemic injury.
Conclusion
Ultimately, these findings suggest the possibility that IGF-1 signaling may be one of the underlying mechanisms involved in the beneficial effects of EE in optimizing recovery following cerebral ischemic injury.
doi:10.1111/nan.12146
PMCID: PMC4201886  PMID: 24750178
complex environment; contextual learning; water maze; Akt signaling; Fluoro-Jade; caspase 3
17.  Alterations in global DNA methylation and hydroxymethylation are not detected in Alzheimer's disease 
Aims
Genetic factors do not seem to account fully for Alzheimer disease (AD) pathogenesis. There is evidence for the contribution of environmental factors, whose effect may be mediated by epigenetic mechanisms. Epigenetics involves the regulation of gene expression independently of DNA sequence and these epigenetic changes are influenced by age and environmental factors, with DNA methylation being one of the best characterized epigenetic mechanisms. The human genome is predominantly methylated on CpG motifs, which results in gene silencing; however methylation within the body of the gene may mark active transcription. There is evidence suggesting an involvement of environmental factors in the pathogenesis of Alzheimer's disease (AD), which prompted our study examining DNA methylation in this disorder.
Methods
Using immunohistochemistry with 5‐methylcytosine/5‐hydroxymethylcytosine antibodies we studied, in comparison with age matched controls, DNA methylation in sporadic and familial AD cases in the entorhinal cortex that exhibits substantial pathology and the cerebellum, which is relatively spared.
Results
Neuronal nuclear labelling with 5‐methylcytosine (5mC) and 5‐hydroxymethylcytosine (5hmC) was evident in all cases studied. We did not detect any significant change in the levels of nuclear staining in the AD samples compared to neurologically normal controls. In the entorhinal cortex we also examined global DNA methylation and hydroxymethylation using an enzyme‐linked immunosorbent assay (ELISA).
Conclusion
No significant differences were found between AD and control cases in global levels of 5mC and 5hmC in the entorhinal cortex using immunohistochemistry and enzyme‐linked immunosorbent assays.
doi:10.1111/nan.12183
PMCID: PMC4879505  PMID: 25201696
Alzheimer's disease; epigenetics; hydroxymethylation; methylation
18.  A neuronal DNA damage response is detected at the earliest stages of Alzheimer’s neuropathology and correlates with cognitive impairment in the Medical Research Council’s Cognitive Function and Ageing Study ageing brain cohort 
Aims
Population-based studies have shown that approximately 20% of the ageing population (aged 65 years and over) with dementia have little or no classical Alzheimer-type neuropathology. Cumulative DNA damage and a reduced capacity of DNA repair may result in neuronal dysfunction and contribute to cognitive impairment independent of Alzheimer-type pathology in the ageing brain.
Methods
We investigated expression of the DNA damage response (DDR)-associated molecules γH2AX and DNA-PKcs using immunohistochemistry and western blotting, and senescence-associated β-galactosidase in the frontal association neocortex of cases with low levels of Alzheimer-type pathology (Braak & Braak stage 0–II), and explored their relationship to cognitive impairment in a population-representative sample from the Medical Research Council’s Cognitive Function and Ageing Study cohort.
Results
Increases in both γH2AX+ (rs = −0.36, P = 0.025) and DNA-PKcs+ (rs = −0.39, P = 0.01) neuronal counts were associated with a lower Mini-Mental State Examination score. Increasing levels of senescence associated-β-gal+ pyramidal neurones were weakly associated with the total number of DNA-PKcs+ neurones (P = 0.08), but not with traditional senescence-associated signalling molecules, including p53 and p16.
Conclusion
The association between the neuronal DDR and cognitive impairment, independent of AD pathology in the ageing brain, may be suggestive of a causal link via neuronal dysfunction.
doi:10.1111/nan.12202
PMCID: PMC4861215  PMID: 25443110
cognitive impairment; DNA damage response; DNA-PKcs; neurone; γH2AX
19.  Review: Insights into molecular mechanisms of disease in neurodegeneration with brain iron accumulation: unifying theories 
Neurodegeneration with brain iron accumulation (NBIA) is a group of disorders characterized by dystonia, parkinsonism and spasticity. Iron accumulates in the basal ganglia and may be accompanied by Lewy bodies, axonal swellings and hyperphosphorylated tau depending on NBIA subtype. Mutations in 10 genes have been associated with NBIA that include Ceruloplasmin (Cp) and ferritin light chain (FTL), both directly involved in iron homeostasis, as well as Pantothenate Kinase 2 (PANK2), Phospholipase A2 group 6 (PLA2G6), Fatty acid hydroxylase 2 (FA2H), Coenzyme A synthase (COASY), C 19orf12, WDR 45 and DCAF 17 (C 2orf37). These genes are involved in seemingly unrelated cellular pathways, such as lipid metabolism, Coenzyme A synthesis and autophagy. A greater understanding of the cellular pathways that link these genes and the disease mechanisms leading to iron dyshomeostasis is needed. Additionally, the major overlap seen between NBIA and more common neurodegenerative diseases may highlight conserved disease processes. In this review, we will discuss clinical and pathological findings for each NBIA‐related gene, discuss proposed disease mechanisms such as mitochondrial health, oxidative damage, autophagy/mitophagy and iron homeostasis, and speculate the potential overlap between NBIA subtypes.
doi:10.1111/nan.12242
PMCID: PMC4832581  PMID: 25870938
autophagy; mitochondria; NBIA; neurodegeneration; Tau; α‐synuclein
20.  Mitochondrial and inflammatory changes in sporadic inclusion body myositis 
Aims
Sporadic inclusion body myositis (sIBM) is the most common late onset muscle disease causing progressive weakness. In light of the lack of effective treatment, we investigated potential causes underlying muscle wasting. We hypothesized that accumulation of mitochondrial respiratory deficiency in muscle fibres may lead to fibre atrophy and degeneration, contributing to muscle mass reduction.
Methods
Histochemical and immunohistochemical analyses were performed on muscle biopsies from 16 sIBM patients to detect activity of mitochondrial enzymes and expression of mitochondrial respiratory chain proteins along with inflammatory markers respectively. Mitochondrial DNA mutations were assessed in single muscle fibres using real‐time PCR.
Results
We identified respiratory‐deficient fibres at different stages of mitochondrial dysfunction, with downregulated expression of complex I of mitochondrial respiratory chain being the initial feature. We detected mitochondrial DNA rearrangements in the majority of individual respiratory‐deficient muscle fibres. There was a strong correlation between number of T lymphocytes and macrophages residing in muscle tissue and the abundance of respiratory‐deficient fibres. Moreover, we found that respiratory‐deficient muscle fibres were more likely to be atrophic compared with respiratory‐normal counterparts.
Conclusions
Our findings suggest that mitochondrial dysfunction has a role in sIBM progression. A strong correlation between the severity of inflammation, degree of mitochondrial changes and atrophy implicated existence of a mechanistic link between these three parameters. We propose a role for inflammatory cells in the initiation of mitochondrial DNA damage, which when accumulated, causes respiratory dysfunction, fibre atrophy and ultimately degeneration of muscle fibres.
doi:10.1111/nan.12149
PMCID: PMC4833191  PMID: 24750247
atrophy; complex I; inflammation; mitochondria; mitochondrial deletions; sIBM
21.  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.
doi:10.1111/nan.12292
PMCID: PMC4832296  PMID: 26538301
C9orf72; dipeptide repeat proteins; frontotemporal lobar degeneration; motor neurone disease; TDP‐43
22.  Review: Multiple system atrophy: emerging targets for interventional therapies 
Multiple system atrophy (MSA) is a fatal orphan neurodegenerative disorder that manifests with rapidly progressive autonomic and motor dysfunction. The disease is characterized by the accumulation of α‐synuclein fibrils in oligodendrocytes that form glial cytoplasmic inclusions, a neuropathological hallmark and central player in the pathogenesis of MSA. Here, we summarize the current knowledge on the etiopathogenesis and neuropathology of MSA. We discuss the role of α‐synuclein pathology, microglial activation, oligodendroglial dysfunction and putative cell death mechanisms as candidate therapeutic targets in MSA.
doi:10.1111/nan.12304
PMCID: PMC4788141  PMID: 26785838
α‐synuclein; aetiology; clinical trial; multiple system atrophy; neuropathology; pathogenesis
23.  Extensive respiratory chain defects in inhibitory interneurones in patients with mitochondrial disease 
Aims
Mitochondrial disorders are among the most frequently inherited cause of neurological disease and arise due to mutations in mitochondrial or nuclear DNA. Currently, we do not understand the specific involvement of certain brain regions or selective neuronal vulnerability in mitochondrial disease. Recent studies suggest γ‐aminobutyric acid (GABA)‐ergic interneurones are particularly susceptible to respiratory chain dysfunction. In this neuropathological study, we assess the impact of mitochondrial DNA defects on inhibitory interneurones in patients with mitochondrial disease.
Methods
Histochemical, immunohistochemical and immunofluorescent assays were performed on post‐mortem brain tissue from 10 patients and 10 age‐matched control individuals. We applied a quantitative immunofluorescent method to interrogate complex I and IV protein expression in mitochondria within GABAergic interneurone populations in the frontal, temporal and occipital cortices. We also evaluated the density of inhibitory interneurones in serial sections to determine if cell loss was occurring.
Results
We observed significant, global reductions in complex I expression within GABAergic interneurones in frontal, temporal and occipital cortices in the majority of patients. While complex IV expression is more variable, there is reduced expression in patients harbouring m.8344A>G point mutations and POLG mutations. In addition to the severe respiratory chain deficiencies observed in remaining interneurones, quantification of GABAergic cell density showed a dramatic reduction in cell density suggesting interneurone loss.
Conclusions
We propose that the combined loss of interneurones and severe respiratory deficiency in remaining interneurones contributes to impaired neuronal network oscillations and could underlie development of neurological deficits, such as cognitive impairment and epilepsy, in mitochondrial disease.
doi:10.1111/nan.12238
PMCID: PMC4772453  PMID: 25786813
cognition; epilepsy; interneurones; mitochondrial DNA; respiratory chain deficiency
24.  At the centre of neuronal, synaptic and axonal pathology in murine prion disease: degeneration of neuroanatomically linked thalamic and brainstem nuclei 
Aims
The processes by which neurons degenerate in chronic neurodegenerative diseases remain unclear. Synaptic loss and axonal pathology frequently precede neuronal loss and protein aggregation demonstrably spreads along neuroanatomical pathways in many neurodegenerative diseases. The spread of neuronal pathology is less studied.
Methods
We previously demonstrated severe neurodegeneration in the posterior thalamus of multiple prion disease strains. Here we used the ME7 model of prion disease to examine the nature of this degeneration in the posterior thalamus and the major brainstem projections into this region.
Results
We objectively quantified neurological decline between 16 and 18 weeks post‐inoculation and observed thalamic subregion‐selective neuronal, synaptic and axonal pathology while demonstrating relatively uniform protease‐resistant prion protein (PrP) aggregation and microgliosis across the posterior thalamus. Novel amyloid precursor protein (APP) pathology was particularly prominent in the thalamic posterior (PO) and ventroposterior lateral (VPL) nuclei. The brainstem nuclei forming the major projections to these thalamic nuclei were examined. Massive neuronal loss in the PO was not matched by significant neuronal loss in the interpolaris (Sp5I), while massive synaptic loss in the ventral posteromedial nucleus (VPM) did correspond with significant neuronal loss in the principal trigeminal nucleus. Likewise, significant VPL synaptic loss was matched by significant neuronal loss in the gracile and cuneate nuclei.
Conclusion
These findings demonstrate significant spread of neuronal pathology from the thalamus to the brainstem in prion disease. The divergent neuropathological features in adjacent neuronal populations demonstrates that there are discrete pathways to neurodegeneration in different neuronal populations.
doi:10.1111/nan.12232
PMCID: PMC4744702  PMID: 25727649
axon; cathepsin D; chronic neurodegeneration; neuroanatomical spread; phagocytosis; synapse
25.  Corrigendum 
doi:10.1111/nan.12176
PMCID: PMC4738599

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