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Molecular pathology of neurodegenerative diseases: principles and practice
  1. Gabor G Kovacs
  1. Laboratory Medicine Program, University Health Network, Toronto, Ontario, Canada
  1. Correspondence to Gabor G Kovacs, Laboratory Medicine Program, University Health Network, Toronto, ON M5G 2C4, Canada; Gabor.kovacs{at}uhnresearch.ca

Abstract

Neurodegenerative diseases are characterised by selective dysfunction and progressive loss of synapses and neurons associated with pathologically altered proteins that deposit primarily in the human brain and spinal cord. Recent discoveries have identified a spectrum of distinct immunohistochemically and biochemically detectable proteins, which serve as a basis for protein-based disease classification. Diagnostic criteria have been updated and disease staging procedures have been proposed. These are based on novel concepts which recognise that (1) most of these proteins follow a sequential distribution pattern in the brain suggesting a seeding mechanism and cell-to-cell propagation; (2) some of the neurodegeneration-associated proteins can be detected in peripheral organs; and (3) concomitant presence of neurodegeneration-associated proteins is more the rule than the exception. These concepts, together with the fact that the clinical symptoms do not unequivocally reflect the molecular pathological background, place the neuropathological examination at the centre of requirements for an accurate diagnosis. The need for quality control in biomarker development, clinical and neuroimaging studies, and evaluation of therapy trials, as well as an increasing demand for the general public to better understand human brain disorders, underlines the importance for a renaissance of postmortem neuropathological studies at this time. This review summarises recent advances in neuropathological diagnosis and reports novel aspects of relevance for general pathological practice.

  • amyloid-beta
  • alpha-synuclein, neurodegeneration
  • neuropathology
  • prion protein
  • proteinoopathy
  • tau
  • TDP-43
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Definition and classification of neurodegenerative diseases

Neurodegenerative diseases (NDDs) are characterised by progressive dysfunction of synapses, neurons, glial cells and their networks. A crucial component of NDDs is the deposition of physicochemically altered variants of physiological proteins in the nervous system. Importantly, not only neurons but glial cells also accumulate these pathological proteins.1

The classification of NDDs is based on the clinical presentation, anatomical regions and cell types affected, conformationally altered proteins involved in the pathogenetic process, and aetiology if known (ie, genetic variations or acquired pathways, for example, in prion diseases).2 3 Importantly, (1) the clinical symptoms are determined by the anatomical region showing neuronal dysfunction and not necessarily by the distribution of the altered protein; and (2) NDD-associated proteins show a wide spectrum of biochemical modifications and can accumulate in neurons or glial cells (intracellular), or deposit in extracellular locations such as plaques, including those showing amyloid characteristics. Accordingly, the best approach to NDDs is to define anatomical, cellular and protein vulnerability patterns.1 4

Clinical manifestations begin either as (1) cognitive decline, dementia and alterations in high-order brain functions (ie, involvement of the hippocampus, entorhinal cortex, limbic system and neocortical areas); (2) movement disorders, including hyperkinetic, hypokinetic, cerebellar, or upper and lower motor neuron dysfunction (ie, involvement of the basal ganglia, thalamus, brainstem nuclei, cerebellar cortex and nuclei, motor cortical areas, and lower motor neurons of the spinal cord); or (3) early combinations of these.2 A subset of dementia is called frontotemporal dementia, which is associated with the degeneration of the frontal and temporal lobes (frontotemporal lobar degeneration, FTLD). Affected areas show atrophy or altered metabolic activity in neuroimaging, and atrophy at postmortem macroscopical and neuronal loss and reactive astrogliosis at microscopical inspection.

The molecular pathological classification focuses on the distinction of synaptic, intracellular and extracellular protein accumulations.1 The subcellular location of the intracellular deposits (eg, nuclear, cytoplasmic or cell process) is also important. Many new antibodies have been developed for immunohistochemistry which describe novel immunostaining patterns. For the diagnostic classification, however, not all protein immunoreactive morphologies are considered. Although for subtyping of diseases morphological criteria are used predominantly, biochemistry and genetic analysis are often required as a complementary examination to immunohistochemistry. It should be mentioned that there are some forms of NDDs, exemplified by hereditary spastic paraplegia or some variants of spinocerebellar ataxia, where no specific protein inclusions are detected with currently available methods.

The following proteins are associated with the majority of sporadic and genetic adult-onset NDDs5: (1) amyloid-beta (Aβ), which is cleaved from the transmembrane amyloid precursor protein (APP), a 770-aa protein—the APP gene has been mapped to chromosome 21q21.3; (2) α-synuclein, a 140-aa protein encoded by a gene (SNCA) on chromosome 4; (3) prion protein (PrP), which is a 253-aa protein encoded by the gene of PrP (PRNP) located on chromosome 20; (4) the microtubule-associated protein tau is represented by different isoforms and encoded by a single gene (MAPT) on chromosome 17q21; (5) transactive response DNA-binding protein 43 (TDP-43), a highly conserved nuclear 414-aa protein encoded by the TARDBP gene on chromosome 1; and (6) FET (abbreviation of the following three proteins) proteins, which include the fused in sarcoma (FUS), Ewing’s sarcoma RNA-binding protein 1 (EWSR1) and TATA-binding protein-associated factor 15 (TAF15).6 There are further proteins associated with hereditary disorders such as neurological trinucleotide repeat disorders, neuroserpin, ferritin-related NDDs and familial cerebral amyloidosis.1

Accordingly, we distinguish tauopathies, α-synucleinopathies, TDP-43 proteinopathies, FUS/FET proteinopathies, prion diseases, trinucleotide repeat diseases, neuroserpinopathy, ferritinopathy and cerebral amyloidosis. Aβ, as one of the most frequently detected NDD-associated proteins, accumulates in Alzheimer’s disease (AD) together with tau. Immunoreactive morphologies of different proteinopathies and their distribution are shown in figures 1 and 2.

Figure 1

Protein immunoreactivities in neurodegenerative diseases. Amyloid-beta immunoreactive cored plaque (A1) and cerebral amyloid angiopathy (A2). Patchy/perivacuolar (B1) and kuru-plaque (B2) type immunoreactivity for the prion protein. Tau-positive neurofibrillary tangle (C1), pretangle (C2), 3-repeat tau isoform positive Pick body (C3), 4-repeat tau isoform positive spherical inclusion (C4), tau-positive neuropil threads in axons (C5) and grains in dendrites (C6). Tau-positive tufted astrocyte (C7), astrocytic plaque (C8), ramified astrocyte (C9), globular astroglial inclusion (C10), thorn-shaped astrocyte (C11), granular/fuzzy astrocyte (C12), oligodendroglial coiled body (C13) and globular oligodendroglial inclusion (C14). α-Synuclein-positive brainstem-type Lewy body (D1), cortical-type Lewy body (D2), Lewy neurite (D3), thin neurites (D4), astrocyte (D5), oligodendrocyte (D6) in Parkinson’s disease and neuronal cytoplasmic and nuclear inclusions (D7), and oligodendroglial cytoplasmic Papp-Lantos body (D8) in multiple system atrophy. TDP-43 immunoreactive granular (E1), compact (E2 and E3) and skein-like (E4) deposits in neurons; thin (E5) and thick (E6) neurites in the grey matter; and thin threads (E7) and oligodendroglial inclusion (E8) in the white matter. FUS immunoreactive compact cytoplasmic (F1; right side of image, compared with the physiological nuclear immunostaining of the neuron on the left side of the image) and vermiform nuclear (F2) neuronal inclusion in the granular cells of the dentate gyrus; compact (F3) and tangle-like (F4) inclusions in lower motor neurons; and white matter threads (F5) and oligodendroglial inclusion (F6). FUS, fused in sarcoma; TDP-43, transactive response DNA-binding protein 43.

Figure 2

Distribution of protein immunoreactivity in various neurodegenerative conditions. Amyloid-beta immunoreactivity in the frontal cortex in Alzheimer’s disease (A). The arrow indicates cerebral amyloid angiopathy. Diffuse/synaptic immunoreactivity for disease-associated prion protein in the frontal cortex in Creutzfeldt-Jakob disease (B). Tau-positive neurofibrillary tangles in Alzheimer’s disease (C) and Pick bodies (D) in the hippocampus; tufted astrocytes (arrow) and neuronal tau immunoreactivity (arrowhead) in progressive supranuclear palsy (E) and astrocytic plaques and threads in corticobasal degeneration (F) in the frontal cortex; pretangles and grains in argyrophilic grain disease (G) and globular oligodendroglial inclusions in the white matter (WM) contrasting the grey matter (GM) in globular glial tauopathy (H) in the hippocampus. α-Synuclein-positive intraneuronal Lewy bodies and Lewy neurites in Parkinson’s disease in the substantia nigra (I) and cortical Lewy bodies (one indicated by an arrowhead) together with neurites and immunoreactive astrocytes in dementia with Lewy bodies (J). Phosphorylated TDP-43 immunoreactive neuronal cytoplasmic and neuritic immunoreactive in frontotemporal lobar degeneration with TDP-43 pathology in the frontal cortex (K). FUS-positive neuronal cytoplasmic inclusion (arrowhead) contrasting the physiological nuclear staining (arrow) in the spinal cord in familial amyotrophic lateral sclerosis with FUS mutation. The bar in A represents 400 µm for A, B, D and F; 200 µm for E, G, H and J; 100 µm for I and K; and 40 µm for L. FUS, fused in sarcoma; TDP-43, transactive response DNA-binding protein 43.

Aspects of disease pathogenesis with implications for the diagnostic procedure

Traditionally accepted pathogenic aspects of NDDs comprise molecular damage, dysregulation of energetic and ion homeostasis, and metabolic changes.7 The proteinopathy concept emphasises the role of protein processing systems and highlights important aspects of pathogenesis, such as the unfolded protein response,8 and protein elimination pathways, such as the ubiquitin-proteasome system and the autophagy-lysosome pathway.9 A novel concept, referred to as prion-like spreading, suggests that the proteins associated with NDDs propagate in the nervous system and involve anatomical areas in a sequential/hierarchical fashion. This concept stems from prion disease research and proposes template-directed protein misfolding and cell-to-cell propagation of pathological NDD-associated proteins.10 Exposure of susceptible hosts to cognate molecular templates has been proposed for cerebral neurodegenerative conditions and for systemic amyloidosis, also described as ‘inducible proteopathies’.11 Indeed, for most of the above-mentioned proteins, evidence from cell culture and animal experimental models has revealed that cells can take up pathological proteins and propagate pathology to surrounding cells.12 Cell-to-cell prion-like propagation should not, however, be confused with human-to-human transmissibility as seen in prion diseases. Therefore, the public health relevance is different for prion diseases and non-prion NDDs, such as AD and/or Parkinson’s disease (PD). The conceptual levels of protein propagation include basically four levels, reflected by the experimental approach used to demonstrate the spreading, such as molecular, tissue, systemic and infectious propagons.13 Infectious propagons have been defined as ‘proteins that transmit pathological conformation between individuals’.13 However, under very unusual situations, seeds of Aβ have been shown to propagate from human grafted dura mater containing pathological Aβ.14 Importantly, only in prion diseases the whole clinicopathological phenotype (‘phenotype propagon’),14 together with the pathological conformer of the PrP, can be effectively and rapidly transmitted from human to human. Although the current trend is to interpret the progressive neuronal damage as a consequence of cell-to-cell propagation, the classical selective vulnerability hypothesis suggests that protein aggregation is initiated in a subset of neurons that are vulnerable to certain harmful conditions.15

The property of self-propagation was exploited to establish real-time quaking-induced conversion assays (RT-QuIC) for the detection of disease-associated PrP in human Creutzfeldt-Jakob disease (CJD).16 Based on the concept of amyloid seeding, proteins capable of misfolding will seed the assembly of the recombinant protein into amyloid fibrils that generate fluorescence of thioflavin-T.17 Recent studies have shown that this method also works in biofluid samples, such as cerebrospinal fluid, for the detection of PrP, and for α-synuclein and different isoforms of tau protein.17

The recognition of sequential involvement of anatomical regions, supported by the recent cell-to-cell propagation theory, led to the development of stages and phases of pathological protein deposits (see below and in tables 1 and 2). Accordingly, examination of a single anatomical area (eg, neurosurgical biopsy) can be only suggestive of the presence of a certain disease. The propagation of proteins along nerves has implications for general pathologists also. Indeed, deposits of NDD-associated proteins can be detected in peripheral organs associated with neural structures.18 19 This has been studied and demonstrated most extensively for α-synuclein.20 Several studies suggest that α-synuclein has the ability to spread from the gastrointestinal tract to the brain and vice versa, which has led to the hypothesis that disorders of the gastrointestinal tract (ie, inflammation or dysbiosis of the gut microbiota) may trigger α-synuclein aggregation as an early step in the pathogenesis of α-synucleinopathies.21 Interestingly, a recent study described disease-associated PrP deposits in the vagus nerve in sporadic and genetic CJD, arguing for diverse transport mechanisms and direction of transport.22 The concept of proteinopathies, and particularly the idea on propagation, has led to the development of novel therapeutic strategies. The aim of these is either to interact with the processing or biochemical modification of a specific protein (eg, Aβ metabolism or tau modifications),23–27 or to target pathological protein forms with therapeutic antibodies (eg, Aβ, α-synuclein and tau).23–28 It must be noted, however, that for example for Aβ the therapeutic trials were not clearly effective,25–27 either due to the incorrectly defined target, too late initiation of the therapy (ie, patients already showing clinical symptoms), or due to comorbidities and multimorbidities (see below).

Table 1

Stages and phases reported for various tau pathologies and amyloid-beta deposition

Table 2

Stages, phases and patterns reported for various α-synuclein and TDP-43 pathologies

The pathological process of intracellular protein aggregation also undergoes a process of maturation. Preaggregates, detectable only by specific antibodies, will later become fibrillar and ubiquitinated, and can then be visualised by antiubiquitin, anti-p62 immunohistochemistry and various silver stainings.29–31 Thus, the protein-based classification and the frequent co-occurence of deposits composed of various proteins require the application of a wide spectrum of immunostainings for a diagnosis. Additional stainings useful for the characterisation of pathologies include silver stainings (Bielschowsky, Bodian, Campbell-Switzer and Gallyas)32 and thioflavin staining. Although silver techniques have greatly contributed to the understanding of NDDs, their use has been hampered by the complexity of the techniques and lack of standardised protocols, leading to interlaboratory variability.33 More recently, in diagnostic practice, ubiquitin and p62/sequestome-1 immunohistochemistry have replaced silver staining for the detection of various abnormal protein deposits.34

Finally, for diagnostic practice it is crucial to understand the concept of concomitant pathologies. This concept describes the frequent observation that, in addition to the hallmark lesions of a specific NDD, further pathological alterations can be observed in the same brain.35 36 While the threshold of clinical impairment may be reached by a sufficient amount of a single proteinopathy, it could alternatively be reached by the combined and additive presence of various disorders that in isolation are not sufficient to cause clinical symptoms.2 4 The concurrent presence of brain (ie, neurodegenerative disorder) and systemic (ie, metabolic or vascular) disorders with different pathogenic aspects has been defined as multimorbidity to distinguish from those with overlapping pathogenic aspects termed as comorbidity, such as combinations of neurodegenerative proteinopathies.37 Importantly, proteinopathy comorbidities are seen associated with several gene mutations.37 In the ageing brain particularly, a large variety of comorbidities and multimorbidities can be detected,4 which hampers the translation of neuropathological disease subtyping into clinically easily interpretable biomarkers and emphasises the critical role of postmortem examination, which should include the examination of peripheral organs as well.

Overview of diagnostic molecular neuropathology of NDDs

Alzheimer’s disease

AD is characterised by the extracellular deposition of Aβ fibrils and by the intraneuronal accumulation of abnormally phosphorylated tau protein. Aβ and tau pathology in AD often follows a stereotypical pattern. This is described as phases for Aβ deposition,38 based on the progressive involvement of isocortical areas (phase 1), hippocampus and entorhinal cortex,2 basal ganglia and diencephalon,3 brainstem,4 and cerebellum.5 Aβ cerebral amyloid angiopathy (CAA) frequently but not necessarily associates with the parenchymal deposits and is distinguished as capillary (type 1) or non-capillary type (type 2), also showing three stages.39 40 The Consortium to Establish a Registry for Alzheimer’s Disease (CERAD)41 criteria were based on the detection of argyrophilic neuritic plaques, which represent only a subset of Aβ immunomorphologies. Detecting Aβ deposits in the brain without tau pathology is not sufficient for the diagnosis of AD. However, this is now discussed as preclinical AD in vivo based on the presence of biomarkers,42 43 or postmortem by the presence of AD-type neuropathological alterations despite no signs of cognitive decline during life. Regarding tau pathology, the sequence of tau deposition starts in the transentorhinal cortex (stage I), followed by the entorhinal cortex (II), inferior (III) and middle temporal gyri (IV), followed by neocortical areas such as the occipital cortex (peristriate for stage V and striate cortex for stage VI).44–46 Recent studies have shown that subcortical nuclei also show early tau pathology,47 48 which are now discussed also as early subcortical stages a, b and c,49 50 or as early precortical phase.51 This also fits with the concept of preclinical AD; thus, protein deposition starts decades before the first clinical symptoms. Furthermore, based on the assessment of the distribution of neurofibrillary tangles (NFTs), clinicopathological subtypes of AD, for example, hippocampal sparing, typical or limbic-predominant, have been proposed,52 arguing for further subclassification of AD. In summary, for the current neuropathological diagnosis of AD, based on the recommendation of the National Institute on Aging-Alzheimer’s Association, an ‘ABC’ score for the description of AD neuropathological change incorporates the phases of Aβ deposition (score A for ‘amyloid’), stages of tau-positive neurofibrillary degeneration (score B, for ‘Braak’) and appearance of argyrophilic neuritic plaques (score C for ‘CERAD’), which have to be documented and interpreted together by transforming the ABC scores into one of four levels of AD neuropathological change: not, low, intermediate or high.53

Prion diseases

Prion diseases are classified based on the aetiology as idiopathic/sporadic, acquired and genetic forms. A clinicopathological grouping is based on historical descriptions, which define phenotypes as Creutzfeldt-Jakob disease (CJD: spongiform encephalopathy; sporadic, iatrogenic, variant or genetic), kuru, Gerstmann-Sträussler-Scheinker disease (PrP-amyloidosis), and familial or sporadic fatal insomnia (selective thalamic degeneration without prominent spongiform change).54 Immunohistochemistry or western blotting for disease-associated PrP is important for the definitive diagnosis of prion diseases. Based on immunostaining for disease-associated PrP, biochemical examination of the size of the protease-resistant core of the abnormal PrP (ie, type 1 migrating at 21 kDa and type 2 at 19 kDa), combined with the genotype at the polymorphic codon 129 (methionine, M, or valine, V) of the PrP gene (PRNP), at least six major molecular subtypes and their combinations are recognised for sporadic CJD.55–58 The combination of these molecular features distinguishes the bovine spongiform encephalopathy-related acquired form variant CJD, iatrogenic CJD, the recently described disorder termed variably protease-sensitive prionopathy (VPSPr), and certain genetic forms of disease. In VPSPr, typical protease-resistant PrP was undetectable with standard diagnostic procedures, and abnormal PrP was detected only after enrichment at concentrations much lower than common prion diseases showing a distinct ladderlike electrophoretic profile.59

Immunohistochemistry for disease-associated PrP reveals a wide range of morphologies; however, it is critical to recognise florid plaque-like structures, which are a characteristic sign of variant CJD, although it has been described in iatrogenic CJD as well.60 Peripheral organs also accumulate disease-associated PrP in humans depending on the aetiological subtype.61–67 Most importantly, the lymphoreticular tissue shows PrP immunoreactivity in variant CJD.68 In certain genetic prion diseases, CAA has been described,69 while vessel wall deposition without amyloid characteristics has been described in variant and sporadic CJD.70 In summary, differentiation of acquired or genetic forms requires data on the aetiology (ie, to exclude acquired forms) and sequencing of the PRNP, together with morphology and immunoblotting of the disease-associated PrP.

α-Synucleinopathies

For the classification of α-synucleinopathies, currently only clinical and morphological aspects are considered. Two major groups are distinguished: the neuron-predominant α-synucleinopathies showing Lewy body pathology, and multiple system atrophy (MSA) which is dominated by glial cytoplasmic inclusions (Papp-Lantos bodies). Diseases with Lewy bodies are further stratified based on the clinical data (movement disorder or cognitive decline as early symptom) as dementia with Lewy bodies (DLB), PD, and PD with dementia. Lewy bodies may be detected incidentally without prominent clinical symptoms (ie, incidental Lewy body disease). One neuropathological approach to Lewy body disorders is the staging of α-synuclein pathology according to Braak and colleagues,71 which includes the sequential involvement of the medulla oblongata (stage 1), pons (2) mesencephalon (3; eg, the substantia nigra), limbic areas (4) and neocortical areas (5 and 6).72 However, this approach has been criticised since some cases do not strictly follow these stages, suggesting that there are disease forms with Lewy bodies where α-synuclein pathology is generated ‘spontaneously’ in a specific region.71 Another procedure is applied in the diagnostic criteria for DLB (ie, brainstem, limbic and neocortical types),73 which originates from the classification by Kosaka et al.74 Since the olfactory bulb is a region affected early, and there are disease forms where Lewy bodies are restricted to the amygdala,75 76 there are further suggestions for classification. The unified staging system for Lewy body disorders classifies Lewy body disorders as olfactory bulb only, brainstem-predominant, limbic-predominant, brainstem and limbic, and neocortical.77

The mere presence of oligodendroglial inclusions (Papp-Lantos bodies) is sufficient for the diagnosis of MSA.78 Clinical subtypes such as MSA-P (parkinsonism dominant) and MSA-C (cerebellar symptom-predominant) are defined for MSA where the distribution of glial inclusions might follow striatonigral or olivopontocerebellar predominance.79 These differences cannot be clearly translated into biochemical or morphological differences. Recently, a further type has been described with FTLD.80 81

Further underappreciated α-synuclein immunoreactivities in Lewy body diseases comprise those in astrocytes and oligodendrocytes.82 83 α-Synuclein immunoreactive deposits can be observed in the ependyma, perivascular cells, cranial nerves, retina, gastrointestinal tract, peripheral organs and skin.84–97 α-Synuclein immunoreactivity in MSA has been described in subpial and periventricular astrocytes98 and in Schwann cells.99 Furthermore, α-synuclein immunoreactivity in skin biopsy tissues in MSA shows differences from that in patients with PD.94 97

Tauopathies

Tauopathies are classified as primary (when tau pathology is the driving force in the pathogenesis) or secondary. Primary tauopathies are grouped based on the ratio of 3 repeat (R)-tau and 4R-tau isoforms and two or three major bands (60, 64 and 68 kDa) in western blot of sarkosyl-insoluble fractions, furthermore, based on the distinct involvement of anatomical areas and cell types, and ultrastructural features of tau filaments.100 Mutations in the microtubule-associated tau (MAPT) gene show a wide spectrum of tau pathologies but typically recapitulate features of sporadic tauopathies.101 102

The 3R tauopathy Pick’s disease is characterised by neuronal spherical Pick bodies, ramified astrocytes and less frequently, small oligodendroglial globular inclusions.103 104 4R tauopathies comprise progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), globular glial tauopathies (GGT) and argyrophilic grain disease (AGD). PSP is characterised by NFTs predominating the brainstem-subcortical areas associated with variable amounts of tufted astrocytes and coiled bodies. CBD is characterised by spherical neuronal inclusions (which are 4R-tau immunoreactive contrasting the 3R immunoreactive Pick bodies) and diffuse cytoplasmic tau immunoreactivity of neurons. These are seen together with astrocytic plaques, threads and coiled bodies of oligodendrocytes predominating cortical and subcortical (eg, basal ganglia) and less the brainstem areas.105 The distinguishing features of GGTs are the abundant astroglial and/or oligodendroglial inclusions with globular morphology.106 Comparably less neuronal inclusions comprising 4R immunoreactive spherical inclusions, globular NFT-like structures and diffuse cytoplasmic tau immunoreactivity are seen. AGD is characterised by neuronal dendritic grains and diffuse cytoplasmic tau immunoreactivity with additional granular/fuzzy astrocytes and oligodendroglial coiled bodies in a limbic distribution.107 The mixed 3R and 4R tauopathy affecting the medial temporal lobe, termed also as NFT-predominant dementia, represents the severe end of primary age-related tauopathy (PART).108 The concept of PART has been developed to recognise an almost universally detectable pathology at autopsy among elderly individuals clinically either lacking symptoms or showing amnestic cognitive changes. PART can be interpreted as part of early AD pathology but can represent also an independent process eventually progressing to NFT-predominant dementia.108 A further frequent finding in the ageing brain is ageing-related tau astrogliopathy (ARTAG), which describes tau-positive astrocytes in subpial, subependymal and perivascular locations, and white and grey matter.109 Finally, due to its high societal impact, chronic traumatic encephalopathy (CTE) has to be mentioned; this neuropathological term includes tau pathology of neuronal and astroglial tau morphologies following mild repetitive head injury that shows a peculiar distribution (eg, depth of cortical sulci).110 Although astroglial tau pathology shows overlap with ARTAG, strict criteria emphasise that the diagnosis of CTE requires the combined presence of neuronal and astroglial pathologies.110

TDP-43 proteinopathies

TDP-43 is a major component of the ubiquitin-positive inclusions that characterise amyotrophic lateral sclerosis (ALS) and a common form of FTLD. Importantly, a high number of cases (up to 40%) show genetic alterations, such as mutations in the C9orf72 (most frequent), granulin (GRN), valosin-containing protein (VCP), TARDBP (the gene encoding TDP-43), SQSTM1 (sequestome), DCTN1 (dynactin), TBK1 (TANK-binding kinase) and OPTN (optineurin) genes.111–121 Importantly, C9orf72 expansion repeat mutation cases contain additional p62-positive, TDP-43-negative neuronal cytoplasmic inclusions composed of dipeptide repeat proteins, which are translated from the C9orf72 expansion repeats.114

TDP-43 immunoreactive structures are observed as neuronal cytoplasmic inclusions, dystrophic neurites, neuronal intranuclear inclusions and glial cytoplasmic inclusions; furthermore, skein-like and spherical inclusions are described in ALS.122–127 A harmonised classification system distinguishes four subtypes (A–D) based on the predominance and layer, predominance of neuritic and neuronal cytoplasmic, or intranuclear inclusions.128 These patterns can also predict the genes involved in hereditary forms. Finally a further type (E) has been described in rapidly progressive forms (duration <3 years)129; this awaits further confirmation from larger cohorts.

TDP-43 pathology frequently associates with other disorders, including AD, DLB, hippocampal sclerosis of ageing (a disorder affecting about 10% of individuals over the age of 85 years)130 and CTE.131 Some of these associations are related to genetic or other risk factors.5 130 132–135 The cellular distribution of TDP-43 pathology may resemble that observed in FTLD-TDP, with only rare neuronal nuclear inclusions and variable presence of glial deposits. To recognise a stereotypical medial temporal lobe-predominant TDP-43 proteinopathy in older adults, with or without coexisting hippocampal sclerosis pathology that is associated with an amnestic dementia syndrome mimicking AD-type dementia, recently an entity termed limbic-predominant age-related TDP-43 encephalopathy (LATE) has been defined.136 LATE is supposed to describe the clinical disorder, while the neuropathological changes are termed LATE-NC.136

FET/FUS proteinopathies

Rare sporadic disorders associated with FTLD, such as basophilic inclusion body disease, atypical FTLD-U and neuronal intermediate filament inclusion disease, show neuronal (cytoplasmic/nuclear) and glial cytoplasmic inclusions immunoreactive for FET proteins.6 137–139 The inclusion types and their regional distribution in FTLD distinguish these forms in the majority of cases. In contrast, FUS gene mutation-related ALS cases are only immunoreactive for FUS and not for further FET proteins.6

Rare disease forms

Rare disease forms include mostly genetic NDDs with abnormal protein inclusions. One group of disorders is associated with the expansion of unstable trinucleotide repeats, such as inherited ataxias and Huntington’s disease. Further rare inherited disorders associated with proteins and genes include neuroserpin.140–144 In ferritin-related NDDs, the molecular genetic defect resides in the ferritin light polypeptide gene and leads to the accumulation of ferritin deposits in the nucleus and cytoplasm of neurons and glia, and also in endothelial cells, cells of the vascular adventitia, choroid plexus and leptomeningeal cells, and peripheral organs.145 Furthermore, there are hereditary cerebral amyloidoses where the deposited proteins include Aβ, transthyretin, gelsolin, cystatin, PrP and BRI2 protein.146 Some further disorders show inclusion bodies, which are immunoreactive only for the ubiquitin-proteasome system (ie, FTLD-UPS), or a variety of proteins as seen in intranuclear inclusion body disease. Finally, there are diseases where no specific inclusions have been described (eg, FTLD-ni; no inclusions),115 although histological signs of neurodegeneration can be observed. Further NDD conditions include those associated with brain iron accumulation, postencephalitic disorders, head trauma (eg, CTE) or others described in restricted geographical areas (eg, parkinsonism-dementia complex of Guam) that associate with various spectra of proteinopathy lesions.

The approach to comorbidities and multimorbidities

All of the above-mentioned disorders, even hereditary forms, show various degrees of comorbid proteinopathies.37 However, since pure forms of proteinopathies are still frequently observed,147 the notion of the current molecular classification is justified. This is of particular importance to elucidate which pathogenic pathways lead to neurodegeneration. Comorbid proteinopathies might show a different clinical course, which has implications for biomarker and therapeutic development, and therefore the neuropathological report has to include this information. The interpretation is complicated by the fact that abnormally deposited proteins exist in different phases or states of cell type, specific intracellular or extracellular aggregation or fibrillisation, and distinct protein depositions follow different phases or stages of sequential involvement of anatomical brain areas.10 The latter phenomenon has been described for NFTs,45 46 PSP-related tau pathology,148 astrocytic tau pathologies,30 Pick’s disease,149 AGD,107 150 TDP-43 protein-related FTLD and ALS,151 152 TDP-43 pathology accompanying AD or other disorders,153 154 α-synuclein in PD72 and in MSA,155 156 and Aβ deposition (tables 1 and 2).38 Hence comorbid cases might show an end stage or phase of one proteinopathy and an early stage or phase of another NDD. In addition, genetic variations, age and gender effects157 158 and systemic and vascular disorders interpreted as multimorbidities influence the phenotype, dynamics and course of the disease. This implies that (1) a minimum number of strategic blocks should be used for the delineation of regional involvement of different proteinopathies; (2) finding lesions characteristic of an NDD should not prompt termination of mapping for other NDD-associated proteins; and (3) documentation of non-NDD multimorbidities should be included.

Practical aspects for general pathologists

Neuropathological examination is performed in neuropathology diagnostic services for clinical reasons or in the frame of brain banking primarily for research purposes. General pathologists may also be asked to indicate a first opinion on a neuropathological condition and recognise when consultation of a specialised neuropathological service is essential. To enhance the quality of the neuropathological report, clinical and neuroradiological information is essential. In particular any suspicion of prion disease should be considered before start of the neuropathological work-up. This requires studying the clinical data for any rapidly progressive neuropsychiatric disorder (<2 years’ duration), the result of cerebrospinal fluid examination (eg, protein 14-3-3 or RT-QuIC examination), brain MRI (high signal intensities in the basal ganglia, thalamus and/or cortical areas) and electroencephalography report (eg, periodic sharp wave complex and/or triphasic waves). In cases with a suspicion for prion disease, specific autopsy measures159 and country-specific regulations must be followed. The quality of the neuropathological report is greatly enhanced when the results of the general autopsy (cause of death; state of the cardiovascular system; inflammatory, infectious or neoplastic diseases; and so on) are considered. Sampling for the diagnostic procedure may be complemented by a series of additional blocks to be embedded in paraffin and stored. In particular, paraffin blocks are more suitable for future studies than resampling after prolonged formalin fixation.160 For brain banking purposes, blocks for freezing, preferably the immediate adjacent area for paraffin embedding, should be prepared. Finally, reports on peripheral organs and NDD-associated proteins should be followed closely, for example, when a gastrointestinal biopsy tissue will be requested to be evaluated, for example, for α-synuclein immunoreactivity.

In summary, the state-of-the-art neuropathological examination should include several anatomical regions and immunostainings to be able to report stages or phases of proteinopathies and their combinations, and should be complemented by the examination of peripheral organs for NDD-associated proteins and for multimorbidities affecting the clinical course of the disease.

Future perspectives

The number of autopsies performed is in decline worldwide, which has a major impact on the fate of neuropathology. However, novel therapies and biomarkers continue to appear on the market, which need strict quality control and definite diagnoses, including consideration of the spectrum of comorbidities and multimorbidities. As a result of the general public becoming more informed, in part due to the internet, the interest of the general public is increasing for transparent studies on the human brain. Frequently, by providing feedback on the disease, neuropathologists are able to impart a positive psychological effect on family members, helping them come to terms with the situation that their relative has deceased due to a yet unfathomable devastating disorder. Scientific examinations on the human brain have enormous benefits compared with the required costs, and these aspects should be recognised by health services and by the stakeholders of scientific developments. To emphasise the expert role of the neuropathologist, Edward B Lee161 recommends the integration of all the diagnostic modalities available that assist in the classification of disease in the form of a layered diagnosis (eg, integrated diagnosis; histological, clinical, molecular classification; and additional layers such as biochemistry).161 Thus, we should return back to the previously successful times of communication between neuropathologists, clinicians, neuroradiologists and general pathologists to better serve the needs of society with excellent and transparent quality control, and last but not least to increase the value and appreciation of the neuropathological diagnostic service.

Take home messages

  • Loss of neurons and accumulation of proteins characterise neurodegenerative diseases.

  • Most of these proteins follow a sequential distribution pattern in the brain.

  • Seeding mechanism and cell-to-cell propagation of proteins is suggested.

  • Some of the neurodegeneration-associated protein can be detected in peripheral organs.

  • Concomitant presence of neurodegeneration-associated proteins is frequent.

Acknowledgments

The author would like to acknowledge the Edmond J Safra Program in Parkinson's Disease and the Rossy Program for PSP research.

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View Abstract

Footnotes

  • Handling editor Runjan Chetty.

  • Funding The author has not declared a specific grant for this research from any funding agency in the public, commercial or not-for-profit sectors.

  • Competing interests None declared.

  • Patient consent for publication Not required.

  • Provenance and peer review Not commissioned; externally peer reviewed.

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