Overview and recent advances in neuropathology. Part 2: Neurodegeneration

Pathology (February 2011) 43(2), pp. 93–102

INVITED REVIEW

Overview and recent advances in neuropathology. Part 2: Neurodegeneration COLIN L. MASTERS*{, JILLIAN J. KRIL*ô, GLENDA M. HALLIDAY*{{, ROGER PAMPHLETT***, STEVEN COLLINS*{z, ANDREW F. HILL*§ AND CATRIONA MCLEAN*jj *All authors contributed equally and should be considered equal first author; {Mental Health Research Institute, zDepartment of Pathology, §Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, jjFaculty of Medicine, Monash University, Melbourne, and Department of Anatomical Pathology, The Alfred Hospital, Prahran, Victoria, ôDisciplines of Medicine and Pathology, Sydney Medical School, **The Stacey MND Laboratory, Department of Pathology, The University of Sydney, Sydney, and {{Neuroscience Research Australia and the University of New South Wales, Randwick, New South Wales, Australia

Summary The sections in the following review cover six main neurodegenerative diseases. The first article on Alzheimer’s disease (AD) outlines the major evidence available to date that links Abamyloid peptide as a proximal cause of AD. The article also highlights how an initial finding of the protein content of the amyloid plaque seen in the brains of patients with AD has led to many very significant findings in the neuroscience field. The next section outlines the many and recent advances that have occurred in the field of frontotemporal lobar degeneration (FTLD), including the most recent finding related to the fused sarcoma gene (FUS) and the newest nomenclature whereby the FTLD is subtyped according to the presence of specific proteins seen at a microscopic level. The section on Lewy bodies outlines the latest advances in the relationship between the anatomical distribution of Lewy bodies and disease phenotype. The following section includes an overview of current known genetic links with familial causes of motor neuron disease (MND) and an update on the areas being researched into the causes of sporadic MND. The presence of TDP-43 within inclusions and its new diagnostic role in MND are discussed. The final article on prion diseases gives an overview of human prion diseases, including the phenotypic spectrum, epidemiology and diagnostic investigations relevant to disease. Key words: a-synucleinAb amyloid peptide, Alzheimer’s disease, amyloid protein precursor, Creutzfeldt-Jakob disease, dementia, frontotemporal dementia, frontotemporal lobar degeneration, gene mutations, human prion diseases, Lewy bodies, motor neuron disease, Parkinson’s disease. Abbreviations: AD, Alzheimer’s disease; ALS, amyotrophic lateral sclerosis; CBD, corticobasal degeneration; CJD, Creutzfeldt-Jakob disease; CSF, cerebrospinal fluid; FTD, frontotemporal dementia; FTLD, frontotemporal lobar degeneration; MND, motor neuron disease; PNFA, progressive nonfluent aphasia; PSP, progressive supranuclear palsy; SemD, semantic dementia. Received 18 October, accepted 9 November 2010

INTRODUCTION Over the past two decades major advances have been made in the field of neurodegeneration. Over the years a common Print ISSN 0031-3025/Online ISSN 1465-3931 DOI: 10.1097/PAT.0b013e3283426eee

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theme has emerged whereby different clinical diseases such as Alzheimer’s and Parkinson’s disease are associated with the presence of intra- and extra-cellular aggregates of diseasespecific abnormal forms of common intracellular proteins. At a diagnostic level, the neuropathologist can now specifically subtype many neurodegenerative diseases on the basis of accumulating protein within specific areas of the brain. Associated research including the linkage of the abnormal protein to genes in inherited forms of disease and disease pathogenesis has advanced our knowledge of the disease process. The sequel of this advance in knowledge is the development, trialling and commencement of targeted therapeutic options.

ALZHEIMER’S DISEASE: MOLECULAR PATHOLOGY DEFINED BY OLIGOMERIC SPECIES OF Ab AMYLOID PROTEIN Overview It is now more than 25 years since we defined the N-terminus of the Ab-amyloid peptide, the molecular subunit which polymerises into the amyloid fibrils of the extracellular plaques, the pathognomonic feature of Alzheimer’s disease (AD).1 Subsequent studies showed that the Ab peptide was derived by proteolytic processing of the transmembrane region of the amyloid protein precursor (APP),2 with the characteristics of a multi-domain type 1 neuronal plasma membrane glycoprotein.3 These findings have underpinned the current Ab theory of AD, and the Ab peptide itself has been the prime therapeutic and diagnostic target for the last two decades. There is compelling evidence that Ab is the proximal cause of AD: 1. Ab is the major macromolecular constituent in the plaque and perivascular amyloid deposits (see Fig. 1). 2. Mutations in the APP gene and the genes encoding proteases which give rise to Ab by cleaving APP also cause early onset AD. 3. Triplication of the APP gene in Down’s syndrome results in AD. 4. There are validated correlations of Ab in cerebrospinal fluid (CSF) and brain (as detected by Ab amyloid ligands

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Fig. 1 Immediately after the N-terminal sequence of Ab was determined (in the Glenner laboratory for vescular amyloid and in the Beyreuther laboratory for plaque amyloid), rabbit antibodies were raised against the synthetic Ab peptide sequence in the Masters laboratory in Perth. These images are among the first (in 1985) to record the visualisation of Ab in the Alzheimer’s disease (AD) brain, revealing for the first time the vast extent of Ab deposition in AD.

5.

6.

7. 8.

using positron emission tomography; see Fig. 2) with the natural history of AD. The interactions of metal ions (Cuþþ, Znþþ) with Ab provide an explanation for the selective topographic localisation of amyloid deposition in regions where these ions are enriched around the glutamatergic synapse. The major genetic risk factor for AD, the polymorphic APOE gene, may play a role in Ab clearance from the interstitial fluid spaces of the brain; this clearance pathway has recently been shown to be compromised in sporadic cases of AD. There is preliminary evidence that therapeutic targeting of Ab is effective.4,5 Alternative theories of AD causation have not yet been developed.

While Ab is the proximal cause of AD, there are many events which occur upstream and downstream of its pathogenic actions. One such downstream event is the formation of neurofibrillary tangles and neurites. The tangles and neurites contain tau, a microtubule associated protein (MAP). The molecular basis by which Ab on the external surface of the neuron drives the aggregation of tau into tangles and neurites is an area of intense research. A consensus is emerging that Ab oligomers interact with the NR2B subunit of the NMDA receptor, which allows calcium dys-homeostasis in the dendritic (post-synaptic) spines. This in turn invokes a pathway which involves fyn (a kinase) and calcineurin (a phosphatase) ultimately leading to the accumulation of the tau MAP.6,7 It remains to be determined how these phosphorylation/dephosphorylation events might play into the cascade that leads to

Fig. 2 Approximately 20 years after the first visualisation of Ab by immunocytochemistry in the human brain (Fig. 1), the new technology of Ab-ligands for positron emission tomography (PET) emerged with the development of Pittsburgh compound B (PiB). PiB is an analog of thioflavin T which, together with Congo Red, had been known for many decades as histological markers for all types of amyloid. The 11C-PiB PET shows clearly the retention of signal in the live Alzheimer’s disease (AD) brain compared to a healthy control (HC) subject. (Figures kindly provided by V. Villemagne and C. Rowe, PET Centre, Austin Health.)

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NEURODEGENERATION

synaptic degeneration (the cause of cognitive impairment in AD). Networking The Australian Imaging, Biomarker and Lifestyle study (AIBL) has brought together a network which has provided a longitudinal cohort of subjects who are helping to define the role of Ab oligomers in the natural history of AD.8–13 Based on these studies, and equivalent investigations around the world, we are now laying down the infrastructure for the pre-symptomatic ascertainment of AD. From this, studies will emerge on diseasemodifying therapeutic strategies which aim to delay the onset of AD from a mean peak age of 80 years to 85 years. If successful, this will halve the impending epidemic of AD, and hold it at its current prevalence ratios. Disease-modifying therapeutics are on the horizon, but the difficulty in bringing these therapeutics to market are immense. Nevertheless, the upstream and downstream molecular pathways leading to AD have now been defined, which gives us a real chance to intervene.

FRONTOTEMPORAL LOBAR DEGENERATION Overview Frontotemporal dementia (FTD) shows a spectrum of clinical phenotypes broadly divided into behavioural and language variants of FTD (bv-FTD and lv-FTD, respectively) and associated disorders with prominent movement abnormalities, namely progressive supranuclear palsy (PSP), corticobasal degeneration (CBD) and motor neuron disease (FTDMND).14 Not only is there overlap with the classical parkinsonian movement disorders, but these disorders also show marked overlap in the cognitive disorders they manifest. Indeed the cognitive deficits are not exclusive and most patients show a combination of behavioural and language deficits together with movement abnormalities, with the diagnostic category reflecting the earliest or most marked clinical feature(s). Language variant FTD cases can be further subdivided into those with a progressive non-fluent aphasia (PNFA) and those with semanTable 1

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tic dementia (SemD) based on the nature of their language impairment.15,16 Pathologically, there is also marked variation in the severity and distribution of neurodegeneration, and the location and type of abnormal protein deposits (Table 1). Collectively these entities are referred to as frontotemporal lobar degeneration (FTLD), reflecting the dominant pathology that is degeneration of the frontal and/or temporal lobes. The protein deposits that identify the individual subclasses of pathology are then indicated by the nomenclature FTLD-(protein), for example FTLD with tau positive pathology is termed FTLD-tau17,18 (Table 1). The degree of atrophy varies with FTLD subtype and disease duration, but can be grouped using a simple staging scheme.19 Briefly, examination of coronal sections (i) of the frontal lobe including the temporal pole and caudate nucleus, and (ii) at the lateral geniculate nucleus reveals four grades of atrophy (stage 1–4) of the frontal and temporal cortices, hippocampus, basal ganglia and white matter. Using this scheme, atrophy in bvFTD, lv-FTD and CBD has been shown to progressively increase with disease duration and by post-mortem most cases are stage 4.19–21 In contrast, the majority of PSP cases show more mild atrophy and are predominantly stages 1 and 2.21 In addition to its application to post-mortem sections, this scheme has also been successfully applied with minor modification to assess atrophy and disease progression in coronal magnetic resonance imaging (MRI) scans.16 Quantification of atrophy in bv-FTD reveals that, although the frontal and temporal lobes are most severely damaged, the remaining lobes are not spared.22,23 While by stage 4 the volume of the frontal and temporal cortices are reduced to approximately 50% of control values, there is also significant atrophy of parietal (49–77%, average 64%) and occipital (73%) cortices. Notably, the hippocampus, an area selectively damaged in AD, also shows marked atrophy by stage 4 (46%). Neuronal loss and gliosis in FTLD reflects the anatomical location and severity of atrophy. Early in the disease there is loss of approximately 25% of neurons from layers III and V of

Pathological subtypes of frontotemporal lobar degeneration (FTLD) and the location and staining characteristics of the pathology

FTLD-tau

Subtypes

Histopathology

Location

Pick’s disease Corticobasal degeneration Progressive supranuclear palsy Argyrophilic grain disease18 Tauopathy, NOS

Argyrophilic, intracytoplasmic inclusions

Dentate gyrus; hippocampal and cortical neurons

Hyperphosphorylated tau accumulation in neurons and/or glia See17,18 for descriptions Relocation of TDP from nucleus to cytoplasm and neurites

Any

FTLD-TDP

Other rare disorders (see17) Subtypes 1–485

FTLD-FUS

None described

FTLD-UPS

None described

FTLD-ni

None described

Rare subtypes

Neuronal intermediate filament inclusion disease Basophilic inclusion body disease

Tau and TDP-43 negative, FUS and ubiquitin positive intracytoplasmic and intranuclear inclusions, and neurites Tau, TDP-43 and FUS negative, ubiquitin positive intracytoplasmic inclusions Neuronal loss and gliosis consistent with FTLD in absence of recognisable inclusions See17,18 for descriptions

See17,18 for descriptions Dentate gyrus; frontal and temporal cortex (subtyping based on density and proportion of cytoplasmic inclusions and neuritis) Dentate gyrus; frontal and temporal cortex

Dentate gyrus

See17,18 for descriptions

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the cortex and 20% of neurons from the CA1 sector of the hippocampus.24 As the disease progresses this loss becomes more marked and involves all cortical laminae. It is also accompanied by a 3–5-fold increase in the density of astrocytes.24,25 In keeping with the prominent atrophy of the hippocampus, there is a loss of approximately 60% of CA1 neurons in late stage FTLD.25 Routine neuropathological examination also reveals prominent neuronal loss and gliosis in the basal ganglia, especially the caudate nucleus, and substantia nigra. Recent advances The abnormal protein accumulation in FLTD can be broadly divided into tau positive and tau negative17,18 (Table 1). Tau deposits can be within neurons (e.g., Pick bodies in Pick’s disease, neurofibrillary tangles in PSP or accumulations of hyperphosphorylated tau which are not fibrillar or argyrophilic seen in patients with mutations in the tau gene or sporadic tauopathies, nos), within glia (e.g., thorny or tufted astrocytes in PSP, coiled bodies in CBD), or extracellular (e.g., astrocytic plaques in CBD). Tau negative inclusions were previously described as ubiquitin positive inclusions (FTLD-U), but most contain the TAR DNA binding protein-43 (TDP-43; FTLDTDP). These inclusions are seen in the cytoplasm of neurons in the dentate gyrus, and to a lesser extent, the CA1 region of the hippocampus, frontal and temporal cortices and inferior olivary

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nucleus. TDP-43 positive dystrophic neurites are also seen in the cortex and hippocampus. A minor proportion FTLD-U cases do not contain TDP-43, and instead stain for the fused in sarcoma gene product (FUS; FTLD-FUS). Clinicopathological correlations have revealed a number of associations between clinical and pathological subtypes in FTD.26,27 The most clear of these is FTD-MND where all patients show FTLD-TDP inclusions in the hippocampus, cortex and brainstem and spinal cord motor neurons. In other subtypes the associations are less than complete, but nevertheless some patterns have emerged. In general, patients with the CBD clinical syndrome have CBD pathology, however the converse is not true as a number of patients with bv-FTD also have CBD pathology.26 The majority of SemD have FTLDTPD while PNFA are usually tau positive, including a proportion with AD pathology. However, for the largest clinical subset of FTD, bv-FTD, the pathology is not able to be predicted, with slightly more than half having FLTD-TDP and the remainder FTLD-tau. While research clinics linked to prospective brain donor programs such as the Australian Brain Bank Network have significantly advanced our understanding of the clinicopathological correlations of FTD, further research is required to clarify the association between clinical and pathological phenotypes, and to establish valid biomarkers for these disorders (Fig. 3).

Fig. 3 Pathological subtypes of frontotemporal lobar degeneration (FTLD). (A) Pick’s disease: arygrophilic intracytoplasmic inclusions are seen in neurons of the dentate gyrus (arrows). (B) Astrocytic plaque in corticobasal degeneration stained for hyperphosphorylated tau (AT8). (C) Coiled body in the white matter of corticobasal degeneration stained for hyperphosphorylated tau (AT8). (D) Neurofibrillary tangles in the subthalamic nucleus of progressive supranuclear palsy identified using silver impregnation (modified Bielschowsky method). (E) Ubiquitin-positive intracytoplasmic inclusion in the dentate gyrus. (F) Dentate gyrus inclusions stained for FUS (arrows). (G) Normal nuclear staining of TDP-43. (H) TDP-43 positive intracytoplasmic inclusions in the dentate gyrus (small arrows). Note the loss of normal nuclear staining (large arrow) in inclusion bearing neurons. (I) TDP-43 positive neuritis in the frontal cortex. Scale bar ¼ 20 mm. Scale in (F), (G), (H) and (I) same as (A); scale in (C), (D), and (E) same as (B).

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Fig. 4 (A) Transverse section through the midbrain showing the dark pigmented region of the substantia nigra in a control without Parkinson’s disease at left, and a patient with Parkinson’s disease at right. The box at right indicates the region of higher magnification in B. (B,C) Sections stained immunohistochemically for a-synuclein and counterstained with cresyl violet for Nissl substance. (B) Some remaining pigmented dopamine neurons are seen in the substantia nigra of patients with Parkinson’s disease, with many small phagocytic microglia containing neuronal remnants. Inset: a higher magnification of an a-synuclein-immunoreactive haloed Lewy body within a pigmented dopamine neuron. (C) Considerably more a-synuclein-immunoreactive Lewy bodies and Lewy neurites are observed in limbic and cortical structures in dementia with Lewy bodies. Inset: A typical a-synuclein-immunoreactive Lewy body in a cortical neuron. (D) The rate of Lewy body accumulation in the brain differs substantially between patients with Parkinson’s disease and dementia with Lewy bodies, although at end-stage the pathological accumulation and distribution can look identical.

In parallel with other neurodegenerative disorders, monogenic inherited forms of FTLD, although rare, offer valuable insights into the pathogenesis of the disorder. Interestingly two genes in close proximity on chromosome 17 (17p21) have been shown to cause FTD (http://www.molgen.ua.ac.be/ADMutations/). The first mutations to be identified were in the microtubule associated protein tau gene (MAPT). These patients show the clinical manifestations of FTD, often with associated parkinsonism, and neuronal and glial accumulation of hyperphosphorylated tau. Subsequently, mutations in the progranulin gene (PRGN) were also identified. The link between these mutations and FTD is less clear although granulins, which are cleaved from progranulin, are involved in a range of functions including the mediation of inflammation.28 The pathology in PRGN mutations is FTLD-TDP, including, rarely, intranuclear TDP inclusions. In addition to the MAPT and PRGN mutation that have been reported in large numbers of families worldwide, small numbers of cases with mutations in the valosincontaining protein (VCP) or chromatin-binding protein 2B (CHMP2b) have been reported (http://www.molgen.ua.ac.be/ ADMutations/). Interestingly, while TDP-43 and FUS mutations have been reported in a number of families with MND, there are only rare instances of these mutations occurring in FTD.29

LEWY BODIES Overview Lewy bodies are named after Dr Friedrich Heinrich Lewy (1885–1950), a German-born neurologist, who first discovered the ‘spherical . . . neuronal inclusions’ in the brain of a deceased patient with Parkinson’s disease in 1912.30 The major protein constituent in Lewy bodies was described in 1997 as a-synuclein, which is a synaptic protein that abnormally aggregates into the filaments that form the spherical Lewy body mass displacing other neuronal organelles (insets Fig. 4).31 Genetic studies show that increasing the cellular concentration of a-synuclein changes this soluble protein into a protein that self-aggregates.32 For most cases the mechanism/s underlying Lewy body production remain speculative. Lewy bodies are diagnostic for both Parkinson’s disease33 and dementia with Lewy bodies,34 but are also found in lower concentrations in the aged and a variety of neurodegenerative disorders.35 Depending on the syndrome, they can occur in the central, peripheral and autonomic nervous system. In patients with Parkinson’s disease, Lewy bodies concentrate in brainstem areas as well as in the periphery and autonomic nervous systems; however, it is the characteristic cell loss in

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the substantia nigra that is associated with the motor symptoms of this disorder (Fig. 4A,B).33 This contrasts with the distribution of Lewy bodies in dementia with Lewy bodies, where Lewy bodies concentrate in higher cortical regions contributing to the clinical features of this disorder (Fig. 4C).33,34 Recent advances in Parkinson’s disease Despite the dominance of motor impairment in Parkinson’s disease, it has recently been highlighted that there is a much more widespread deposition of Lewy bodies in the brains of patients with end-stage Parkinson’s disease.36 In fact the distribution of Lewy body pathology in patients with endstage Parkinson’s disease is not different from that observed in dementia with Lewy bodies, although the time to develop the pathology is markedly different (a few years for dementia with Lewy bodies but decades for Parkinson’s disease, Fig. 4D).37 The concept that in Parkinson’s disease Lewy bodies take considerable time to develop and infiltrate different regions of the nervous system over time has led to the idea that Lewy pathology occurs in restricted regions prior to the onset of clinical disease.36 Confirmation of a slow infiltration of Lewy pathology in Parkinson’s disease has been shown in therapeutic fetal transplants, where grafted replacement substantia nigra neurons start to demonstrate Lewy pathology only after 10 years of transplantation.38 These findings have stimulated new research and ideas on how Lewy bodies slowly invade the different vulnerable sets of neurons in patients with Parkinson’s disease, and suggests that the propagation and/or disease mechanism may differ between Lewy body disorders. Recent advances in dementia with Lewy bodies The clinical syndrome of dementia with Lewy bodies requires Lewy body pathology in association with a dominant dementia syndrome.34 Additional clinical features are fluctuations in consciousness, well-formed visual hallucinations, and/or parkinsonism. In most instances, however, such patients with dementia also have significant amounts of neuritic Alzheimer-type pathology that correlates better with the severity of dementia.39 – 41 This makes it difficult to separate the clinical syndrome of dementia with Lewy bodies from Alzheimer’s disease, especially as many patients with Alzheimer’s disease have some Lewy body pathology in limbic brain regions.42 In patients with both neuropathologies, there is a more rapid disease progression.43 The differences in the rate of disease progression between patients with Parkinson’s disease and those with dementia with Lewy bodies (Fig. 4D) are likely to relate to the accelerated protein deposition and cognitive impairment due to interactions between a-synuclein, tau and Ab that enhances the aggregation of each other, as recently shown in animal models.44 That many patients with Alzheimer’s disease will also have Lewy bodies has implications for assuming that all incidental Lewy body disease cases are preclinical cases of Parkinson’s disease, especially as a relatively large number of clinically normal elderly have some cortical rather than subcortical Lewy body pathology.45 Future research to determine whether there are similar or different preclinical changes in incidental Lewy body patients who may develop a dominant motor phenotype compared with a dominant dementia phenotype is now required.

MOTOR NEURON DISEASE Overview Amyotrophic lateral sclerosis (ALS) is the most common form of motor neuron disease (MND). In ALS, both frontal cortex Betz cells (upper motor neurons) and spinal anterior horn cells (lower motor neurons) are progressively lost. ALS usually leads to death in 2–5 years from muscle weakness. The disease affects about 1300 Australians at any one time, and one person in Australia a day dies of ALS. Mutations in SOD1 account for about 10% of the patients where ALS runs in families (about one-sixth of all ALS cases are familial). Smaller numbers of patients with familial ALS have mutations in TDP-43 or FUS. In the more common sporadic ALS, no other family members are affected, and the cause of this form of the disease remains unknown. A central question in ALS research is whether the sporadic form of the disease is due to an environmental agent, a genetic variant, or a combination of the two. Because familial and sporadic cases are virtually identical both clinically and pathologically, an increasingly popular view is that genetic mechanisms underlie sporadic as well as familial ALS. An intensive search for genetic abnormalities in sporadic ALS has been largely fruitless, however, with mutations in the above genes being found in only a few percent of patients. One widespread idea is that sporadic ALS is a complex disease, involving interactions between different genes, or between genes and the environment. However, no genetic susceptibility has so far been found that convincingly explains the great majority of sporadic ALS patients, and most positive case-control genetic association studies have not been replicated. Furthermore, the polymorphisms found have differed in each study, and the odds ratios have been minimal. A number of researchers are now looking for new models to explain how a genetic mechanism could be responsible for an apparently sporadic disease such as sporadic ALS. Epigenetic changes46 and somatic mutations47 have been put forward as possible mechanisms that can explain the sporadic nature of this disease. Recent advances For the pathologist, one of the most relevant advances has been the widespread use of TDP-43 immunostaining to aid in the post-mortem diagnosis of ALS. TDP-43 inclusions are seen in the motor neuron cytoplasm of all sporadic, and most familial cases of ALS (mutant SOD1 ALS is an exception).48,49 In normal motor neurons, TDP-43 is confined to the nucleus, but in ALS a number of neurons will have absent nuclear staining, together with finely granular, filamentous or compact cytoplasmic TDP-43 inclusions (Fig. 5). A phosphorylated TDP-43 antibody has recently been developed which stains only the inclusions (which are made up of hyperphosphorylated TDP43);50 this can aid the detection of scanty inclusions. Even with a total loss of motor neurons, TDP-43 staining can help make the diagnosis, since inclusions can also be seen in the cytoplasm of ALS glial cells. The post-mortem diagnosis of ALS is usually straight-forward, based on a loss of spinal motor neurons and corticospinal tract degeneration. Difficulties in diagnosis can arise if only the brain has been removed, since assessment of Betz cell loss in the frontal motor strip is complicated, unless the brain has been sectioned horizontally.51 The only brain stem lower motor neurons that are commonly affected by the disease are in the

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ALS; in peripheral neuropathies, myofibres within these small groups tend to be of one myofibre type.56 This can be a valuable feature to help distinguish between these two diagnoses. National networking The Australian MND DNA Bank collects blood DNA samples and epidemiological data from patients with MNDs and makes these available to approved researchers. The Australian Brain Bank Network co-ordinates the collection of brain and spinal cord samples from people with MND who pre-donate tissue to state-based tissue resource centres. This tissue is also made available to approved researchers.

HUMAN PRION DISEASES Overview Fig. 5 In the anterior horn of the spinal cord of a patient with ALS are four motor neurons showing: (A) a compact cytoplasmic TDP-43 inclusion and a pale nucleus; (B) filamentous TDP-43 inclusions and a pale nucleus; (C) a normal neuron with a TDP-43 stained nucleus; (D) a pale nucleus with fine cytoplasmic TDP-43 speckling (pre-inclusions). Proteintech TDP-43 immunostaining with anti-rabbit TARDBP and haematoxylin counterstain.

hypoglossal nucleus, but detecting mild losses of these neurons is tricky. However, finding TDP-43 inclusions in hypoglossal neurons will usually allow a confident diagnosis of MND to be made. The detection of TDP-43 inclusions in spinal motor neurons is also useful when only a small segment of spinal cord is available for examination, since spinal motor neuron loss may be patchy in ALS. The pathogenetic role of TDP-43 in sporadic ALS remains unclear, and it is possible that a number of different pathogenetic pathways lead to the formation of TDP-43 inclusions. The distribution of TDP-43 inclusions appears to be the same in ALS patients who have ALS with or without TDP-43 mutations,52 indicating that the abnormal protein is likely to play a major role in the disease. It has been suggested that the inclusions seen in a number of neurodegenerative diseases are protective, rather than harmful, in nature. This does not seem to be the case with TDP-43, since motor neurons containing TDP43 inclusions are readily destroyed in MND.53 In progressive muscular atrophy, which comprises up to 20% of MND, only lower motor neuron signs are found. Again, TDP-43 immunostaining can help post-mortem diagnosis, since TDP-43 inclusions are seen in surviving motor neurons in progressive muscular atrophy as well as in ALS.54 It is also worthwhile immunostaining the spinal cord of progressive muscular atrophy cases with a macrophage marker such as CD68, since in about 50% of cases this will demonstrate corticospinal tract degeneration that is missed on myelin stains.55 It seems increasingly likely that ALS and progressive muscular atrophy will turn out to be the same underlying disease. Muscle biopsies are sometimes performed on people suspected to have ALS to make sure the weakness is not due to muscle or peripheral nerve disease. Many of the usual histological features of neurogenic atrophy may be seen in the biopsied muscle, though myofibre type grouping (the hallmark of chronic neurogenic atrophy) may be slight in ALS. Separating ALS from peripheral neuropathies can be clinically difficult, so it is useful to note that the presence of mixed type I and II myofibres within small groups of atrophic myofibres may be distinctive for spinal motor neuron disorders such as

Human prion diseases are a diverse group of rare neurodegenerative disorders, of which Creutzfeldt-Jakob disease (CJD) is the most common phenotype.57 Transmissibility and the neuropathology are distinguishing hallmarks from other neurodegenerative disorders, with pathogenesis and transmission linked to misfolded conformers (PrPSc) of the constitutively expressed prion protein (PrPC), with PrPSc typically accumulating as extracellular deposits within the central nervous system of afflicted individuals. Although the precise biophysical composition remains to be determined, considerable evidence supports the infectious unit (‘prion’) to be composed predominantly by PrPSc (the ‘protein only’ hypothesis).58-61 Phenotypic spectrum and epidemiology Human prion disease, exemplified by CJD, can be sporadic, genetic or acquired. Most CJD cases (85–90%) are sporadic, occurring at a rate of 1–2 per million people per year, with a similar rate worldwide.62,63 Sporadic CJD occurs most usually in individuals between 65 and 75 years of age. Approximately 10–14% of cases are due to a mutation (genetic CJD) in the gene encoding PrPC (PRNP), with mis-sense and non-sense mutations, as well as octapeptide repeat insertions and deletions recognised.64,65 In contrast to Alzheimer’s and Parkinson’s diseases, CJD due to additional copies of the PRNP gene has not been reported.66 Rapidly progressive dementia is the usual presentation of sporadic CJD, with the phenotype of genetic CJD more variable but often indistinguishable (particularly those due to the E200K and V210I mutations) requiring PRNP genotyping to confidently differentiate the two forms. Across the sporadic CJD spectrum, six clinico-pathological subtypes were originally delineated, appearing to be determined by the combination of codon 129 polymorphism (either methionine or valine) and the mobility of the unglycosylated band of PrPSc (type 1 or 2) on Western blots following proteinase K digestion67 (Fig. 6). Since this seminal observation, greater complexity has ensued due to a number of developments, such as recognition that these six apparent molecular subtypes may really only correspond to four actual sporadic CJD strains,68 as well as the frequent recognition of more than one PrPSc type in the brain, perhaps causing merged phenotypes,69,70 and the recent recognition of ‘protease-sensitive prionopathies’ with reduced amounts of PrPSc and more complex Western blot profiles following proteinase treatment.71 Other principal human prion diseases include GerstmannStra¨ussler-Scheinker syndrome; fatal familial insomnia; and

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Fig. 6 Western immunoblot developed using anti-PrP monoclonal antibody 3F4 (Covance, USA) of brain tissue homogenate from a case of variant CJD showing the presence of immunoreactive bands following treatment of the homogenate with proteinase K (PK). PK digestion removes an amino-terminal fragment of PrPSc, resulting in the altered mobility on the Western blot. Lane 1, pre-PK; Lane 2, post-PK.

kuru.72 Fatal familial insomnia is associated with the D178N mutation in PRNP, while multifarious changes underlie Gerstmann-Stra¨ussler-Scheinker syndrome.65 Fatal familial insomnia usually presents with insomnia and dysautonomia, with neuropathological changes emphasised in the thalami.73 The third aetiologic subgroup is acquired human prion disease. Kuru is prototypical, with iatrogenic and variant CJD also included in this category. Iatrogenic CJD has been reported in relation to dura mater grafts, corneal transplants, human cadaveric pituitary (especially growth) hormone treatment and neurosurgical instrument cross-contamination.74 The phenotype of iatrogenic CJD may show variation from sporadic CJD, with cerebellar ataxia often initially observed in persons acquiring disease through inadvertent peripheral inoculation of prions. Kuru, an endemic disease predominantly affecting the Fore linguistic group of the Eastern Highlands of Papua-New Guinea was first reported in the 1950s and was eventually shown to be acquired through cannabalistic mourning rituals of deceased relatives. Although almost eradicated, the incubation period for kuru is now recognised to extend beyond 50 years,75 while for iatrogenic CJD in relation to pituitary hormones it can be over 30 years. Most recently and sensationally added to the subgroup of acquired human prion disease is the zoonosis, variant CJD (vCJD). First reported in 1996,76 it has predominantly occurred in the United Kingdom, where the greatest numbers of bovine spongiform encephalopathy (‘mad cow disease’) also arose and entered the human food chain. Despite waning numbers of vCJD, the altered tropism of PrPSc in vCJD, additionally found in lymphoreticular organs such as spleen and lymph nodes,77 with the proven efficient human transmission through blood products,78 has perpetuated concerns regarding the possible ongoing occurrence of vCJD through secondary transmissions, especially when it remains uncertain how many individuals may harbour long-term subclinical disease.79

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Diagnostic investigations Pre-mortem diagnostic capability for sporadic CJD has improved considerably over the last 15 years, in part due to the success of coordinated international surveillance consortia. Validation of the utility of 14-3-3 protein detection in the CSF as a confirmatory diagnostic investigation,80 with sensitivities of around 85% in large unselected patient series,70 was the first major breakthrough, with distillation of those MRI brain features sufficiently typical and discriminatory of sporadic CJD to allow incorporation in classification criteria the most recent advance.81 Currently, only very few patients, probably in the order of <5%, do not evince typical features on one of either electroencephalogram (EEG), CSF or MRI examination, with the ‘proteinase-sensitive prionopathy’ subgroup exemplary of this uncommon but recognised difficulty.71 The major acknowledged limitation of these surrogate biomarkers, in particular 14-3-3 protein detection, is their non-specificity, a difficulty which is being successfully addressed by harnessing techniques based on the protein misfolding cyclic amplification assay, which can directly amplify minute amounts of PrPSc.82 Post-mortem neuropathology remains the gold standard for diagnosis. There is no recommended role for diagnostic brain biopsy during life. The key neuropathological diagnostic criteria for CJD include a classic triad; neuronal loss, gliosis and spongiform encephalopathy (Fig. 7). These changes are seen throughout all areas of grey matter: cortex, basal ganglia and cerebellar cortex. The extent of pathology varies depending upon the length of illness. Early stage disease may show patchy changes within the lower laminae of cortex only. Prion protein immunoperoxidase studies using PrP antibodies (3F4 Signet and/or 12F10 Cayman) support the diagnosis with positivity seen in various patterns; synaptic, perineuronal and plaque (Fig. 8). Paraffin embedded tissue (PET) blot technique, although not routine, can also be used in equivocal cases.83 Variations in the extent and distribution of pathology may be seen in fatal familial insomnia, genetic CJD, and acquired CJD. National surveillance systems and epidemiological research Public health concerns directly relating to the potential zoonotic transmission of bovine spongiform encephalopathy (BSE), led to the almost simultaneous initiation of numerous national surveillance centres across Europe and elsewhere in the 1990s.63 The Australian National Creutzfeldt-Jakob Disease Registry (ANCJDR) commenced activities in 1993

Fig. 7 Classical triad of changes seen in Creutzfeldt-Jakob disease: spongiform degeneration, gliosis, and neuronal loss. A central plaque is also seen (H&E).

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NEURODEGENERATION

Fig. 8 Perineuronal PrP immunoreactivity. A background synaptic pattern is seen (12F10 immunoperoxidase).

to monitor the occurrence of further cases of iatrogenic CJD related to pituitary hormone therapy but in 1996 broadened activities by joining an international consortium of national surveillance centres (EUROCJD).62 In addition to the specific surveillance outcomes afforded to each participant country, this multi-national alliance, through prospective harmonised data collection and standardised case definitions, has been very successful at providing a wealth of descriptive epidemiological data such as delineating the phenotypic spectrum of all forms of human prion disease, including the determinants of survival,83 as well as offering rigorous assessment and validation of routine diagnostic investigations.70,81 Further, such collaborations continue to facilitate relative risk comparisons, which can be used to inform important public health decisions for a participant country.84 Acknowledgements: Colin Masters was the author for the section on Alzheimer’s disease; Jillian Kril was the author for the section on frontotemporal lobar degeneration; Glenda Halliday was the author for the section on Lewy bodies; Roger Pamphlett was the author for the section on motor neuron disease; and Andrew Hill, Steven Collins and Catriona McLean were the authors for the section on human prion diseases. Address for correspondence: Professor C. McLean, Department of Anatomical Pathology, The Alfred Hospital, Prahran, Vic 3181, Australia. E-mail: [email protected]

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