Gabor G. Kovacs MD PhDInstitute of Neurology

Medical University of Viennagabor.kovacs@meduniwien.ac.at

Summary of presentation

• Current concepts of neurodegeneration

• Brief update on classification of neurodegenerative diseases(NDDs)

• Diagnostic guideline:– Clinical and general autopsy, sampling protocols– Essential stainings– Evaluation of stainings– Comprehensive evaluation of lesions: setting up the

diagnosis: Which regions to evaluate? Which criteria touse? When to suspect atypical or genetic forms?

GG Kovacs ed. Neuropathology of neurodegenerative diseases. Cambridge University Press 2014

INTRACELLULAR

Cytoplasmic/Cell processor Intranuclear

EXTRACELLULAR

Not only neuronsbut glial cells

GG Kovacs ed. Neuropathology of neurodegenerative diseases. Cambridge University Press 2014

GG Kovacs ed. Neuropathology of neurodegenerative diseases. Cambridge University Press 2014

GG Kovacs ed. Neuropathology of neurodegenerative diseases. Cambridge University Press 2014

Amyl

oid-

βPr

ion

prot

ein

Ph-T

DP43

a

-syn

ucle

inph

osph

o-Ta

u

Important for the diagnostic practice

“Maturation“ of protein deposits

Tau TauNon-argyrophilic ArgyrophilicNon-ubiquitinated Ubiquitinated

Hierachical spreading Stages and Phases

Concomitant pathologies are more the rule than the exception

1

2

3

Braak&Braak staging

EuroCNS-Neurodegenerative disease course-Gabor G. Kovacs-2014EuroCNS-Neurodegenerative disease course-Gabor G. Kovacs-201412

Phases of A-beta deposition

EuroCNS-Neurodegenerative disease course-Gabor G. Kovacs-2014EuroCNS-Neurodegenerative disease course-Gabor G. Kovacs-2014

Phase 1: frontal, parietal, temporal, or occipital neocortex. These depositsappear focally in small groups of diffuse plaques in layers II, III, IV, and V.

Phase 2: Abeta appears in the entorhinal region, CA1, and in the insularcortex. In 33–50% of the cases, single Abeta deposits occur in the amygdala,the cingulate gyrus, the presubicular region, the molecular layer of the fasciadentata.

Phase 3: subcortical regions: caudate nucleus, putamen, claustrum,basal forebrain nuclei, substantia innominata, thalamus, hypothalamus,molecular layer of the fascia dentata. Variable: central gray in the midbrain, thecolliculi superiores and inferiores, CA4, red nucleus, subthalamic nucleus.

Phase 4: further brainstem regions: inferior olivary nucleus, the reticularformation of the medulla oblongata, and the substantia nigra.

Phase 5: further brainstem regions and cerebellum: the reticularformation of the pons, the pontine nuclei, the central and dorsal raphe nuclei,the locus coeruleus, the parabrachial nuclei, the dorsal tegmental nucleus(Gudden), the reticulotegmental nucleus of the pons (Bechterew).

13

M. Parkinson: Verlauf und „Staging“ nach Braak

What does it mean : spreading?Example: alpha-Synuclein

According to the theory of Heiko Braak the dorsal vagus nucleus is the first site in thecentral nervous system

M. Parkinson: Verlauf und „Staging“ nach Braak

1

32

Stages of alpha-Synuclein deposition

M. Parkinson: Verlauf und „Staging“ nach Braak

4 4

5 6

Stages of alpha-Synuclein deposition

Michell, A. W. et al. Brain 2004 127:1693-1705; The role of biomarkers in the diagnosis of Parkinson's disease

Stage IIISubstantia nigra

Stages I-II:Premotor phase

Symptom Affected brain region (Braak St.)Disorder of olfatcion Bulbus und Tractus olfactorius (I)

Obstipation Dorsal Vagus nucleus (I)

REM-sleep disorder Raphe Nuclei (II)

Depression Locus coeruleus, Raphe Nuklei (II)

Parkinsonism Substantia nigra (III)

Cognitive decline Amygdala, Cortex (IV-VI)

Parkinson`s disease: a sytemicdisorder?

SympathicGanglion cells

Gastrointestinalsystem

Cardialsympathicnerves

Adrenal medullaSubcutan

Wide spectrumof alterations in the brain

The possiblenumber ofcombinationsis very high

1 3

Acta Neuropathol (2013) 126:365–384DOI 10.1007/s00401-013-1157-y

ORIGINAL PAPER

Non-Alzheimer neurodegenerative pathologies and theircombinations are more frequent than commonly believedin the elderly brain: a community-based autopsy series

Gabor G. Kovacs· Ivan Milenkovic ·Adelheid Wöhrer · Romana Höftberger · Ellen Gelpi ·Christine Haberler · Selma Hönigschnabl ·Angelika Reiner-Concin · Harald Heinzl ·Susanne Jungwirth · Wolfgang Krampla · Peter Fischer · Herbert Budka

Received: 2 May 2013 / Revised: 15 July 2013 / Accepted: 18 July 2013 / Published online: 31 July 2013© Springer-Verlag Berlin Heidelberg 2013

novel forms), TDP-43 proteinopathy (13.3 %), vascularlesions (48.9 %), and others (15.1 %; inflammation, meta-bolic encephalopathy, and tumours). TDP-43 proteinopa-thy correlated with hippocampal sclerosis (p < 0.001) andAlzheimer-related pathology (CERAD score and Braakand Braak stages, p = 0.001). The presence of one specificvariable (cerebral amyloid angiopathy, Aβ parenchymaldeposits, TDP-43 proteinopathy, α-synucleinopathy, vascu-lar lesions, non-Alzheimer type tauopathy) did not increasethe probability of the co-occurrence of others (p = 0.24).The number of observed pathologies correlated with AD-neuropathologic change (p < 0.0001). In addition to AD-neuropathologic change, tauopathies associated well withdementia, while TDP-43 pathology and α-synucleinopathyshowed strong effects but lost significance when evaluatedtogether with AD-neuropathologic change. Non-AD neuro-degenerative pathologies and their combinations have beenunderestimated, but are frequent in reality as demonstrated

Abstract Neurodegenerative diseases are characterisedby neuronal loss and cerebral deposition of proteins withaltered physicochemical properties. The major proteins areamyloid-β (Aβ), tau, α-synuclein, and TDP-43. Althoughneuropathological studies on elderly individuals haveemphasised the importance of mixed pathologies, therehave been few observations on the full spectrum of pro-teinopathies in the ageing brain. During a community-basedstudy we performed comprehensive mapping of neurode-generation-related proteins and vascular pathology in thebrains of 233 individuals (age at death 77–87; 73 examinedclinically in detail). While all brains (from individuals withand without dementia) showed some degree of neurofibril-lary degeneration, Aβ deposits were observed only in 160(68.7 %). Further pathologies included α-synucleinopathies(24.9 %), non-Alzheimer tauopathies (23.2 %; including

Electronic supplementary material The online version of thisarticle (doi:10.1007/s00401-013-1157-y ) contains supplementarymaterial, which is available to authorizedusers.

G. G. Kovacs (* ) · I. Milenkovic ·A. Wöhrer · R. Höftberger ·E. Gelpi · C. Haberler · H. BudkaInstitute of Neurology, Medical University Vienna, AKH 4J,Währinger Gürtel 18-20, 1097 Vienna, Austriae-mail: gabor.kovacs@meduniwien.ac.at

Present Address:E. GelpiNeurological Tissue Bank of the Biobanc-Hospital Clinic-Institutd’Investigacions Biomediques August Pi i Sunyer (IDIBAPS),Barcelona, Spain

S. Hönigschnabl ·A. Reiner-ConcinInstitute of Pathology, Danube Hospital, Vienna, Austria

H. HeinzlCenter for Medical Statistics, Informatics, and IntelligentSystems, Medical University Vienna, Vienna, Austria

S. JungwirthLudwig Boltzmann Institute of Ageing Research,Vienna, Austria

W. KramplaDepartment of Radiology, Danube Hospital,Vienna, Austria

P. FischerPsychiatric Department, Medical Research Society Vienna, D.C.,Danube Hospital, Vienna, Austria

Present Address:H. BudkaInstitute of Neuropathology, University Hospital Zurich, Zurich,Switzerland

Author'spersonal copy

Summary of presentation

• Current concepts of neurodegeneration

• Brief update on classification of neurodegenerative diseases(NDDs)

• Diagnostic guideline:– Clinical and general autopsy, sampling protocols– Essential stainings– Evaluation of stainings– Comprehensive evaluation of lesions: setting up the

diagnosis: Which regions to evaluate? Which criteria touse? When to suspect atypical or genetic forms?

Before starting....

• Critical evaluation of your aims and possibilities– Performed in neuropathology diagnostic service with or without

special focus on neurodegeneration,– It may be performed in the frame of brain banking primarily for

research purposes.– Also general pathologists may be required to indicate their first

opinion on a neuropathological condition and know further whenconsultation of specialised neuropathological service is essential.

All approaches should reach a common level of diagnostic quality!

Step-1

Clinical information and general autopsy

• Sex and age of the patient; duration of symptoms; presence of dementia,movement disorder or both; consumption of drugs/alcohol; evidence forconcomitant and systemic disease,

• Clinico-neuropathological correlation• Neuroradiology reports and images are also helpful (are vascular lesions

compatible with the clinical course?; distribution of brain atrophy?;hydrocephalus? etc.).

• Importantly, a suspicion of prion disease should be considered beforestart of the neuropathological work-up (result of CSF examination; 14-3-3performed or not; EEG report: periodic sharp wave complex, triphasicwaves mentioned or not; rapidly progressive disease course of severalmonths might raise the suspicion).

• Information from the general autopsy: Cause of death; state of thecardiovascular system; neoplastic, inflammatory or infectious disease, etc.

Step-2

Sampling

Sampling protocolssee homepage of BrainNet Europe: http://www.brainnet-europe.org/index.php?

option=com_content&view=article&id=99&Itemid=99).

Step-3

Essential stainings1) Hematoxylin and Eosin (HE) and luxol fast blue counterstained with nuclear

fast red (LFB/NFR) on all blocks;2) As silver stain, generally we use Bielschowsky, or in selected cases Gallyas.

3) For amyloid, Congo red or alternatively Thioflavine may be used;

4) For the detection of protein markers, we use antibodies against the six mostimportant proteins related to NDDs: anti-A (detecting both 40 and 42), anti-prion protein (PrP), anti--synuclein, anti-phospho-Tau, anti-phospho-TDP-43 (if not available, then anti-TDP-43 can be used as well), and anti-FUS.

5) Further antibodies to be eventually used are anti-ubiquitin, anti-p62,microglial/macrophage markers (HLA-DR, CD68; e.g. to detect tractdegeneration), glial fibrillar acidic protein (GFAP; e.g. to detect reactiveastrogliosis, or where there is a discrepancy between protein deposits andtissue pathology), or myelin basic protein and axonal markers (e.g. SMI-31).

6) Rarely we use anti-huntingtin immunostain, and other disorders withintranuclear protein deposits (e.g. polyglutamine repeat disorders) may requireadditional antibodies.

Step-4

Evaluation ofsections-1

Step-5

Pick bodies Cortical Lewy bodies Marinesco-body Lewy body Intranuclear inclusion body

Neurofilment inclusions Neurofibrillary tangles Neurofibrillary tangles Granulovacuolar degeneration

Amyloid plaque-1 Amyloid plaque and neurites Dystrophic neurites Amyloid plaque-2 + Torpedo

2

Neurofibrillary tangles Granulovacuolar degeneration Dystrophic neurites Plaques and neurites

Diffuse plaques Pick bodies Astrocytic plaque Tufted astrocyte

Coiled bodies Oligodendroglial deposits-1 Oligodendroglial deposits-2 Grains

Evaluation of sections-3Immunohistochemistry: Extracellular deposits

Step-6

Capillary deposition of A-beta

Diffuse/synaptic Perineuronal-I Perineuronal-IIPatchy/perivacuolar

Plaque-like PrP IR Kuru type Multicentricamyloid plaque amyloid plaque

Evaluation ofsections-3

Immunohistochemistry:Intracellular deposits

Step-6

I.a. Pretangle (more correct: diffuse cytoplasmic neuronal IR)Diffuse or dispersed fine granular staining of neuronal cytoplasm and, to a variableextent, the processes. Perinuclear enhancement may be noted.

Comment: By definition these are ubiquitin immunonegative and are notargyrophilic.

I.b. Neurofibrillary tangleDensely stained fibrillar, usually circumscribed,intracellular cytoplasmic structures showing flame or globoseshape.

Comment: These are ubiquitin immunopositive and are argyrophilic. TAU

I.c. Pick bodyWell demarcated usually single, rarely multiple cytoplasmic fibrillarspherical structures usually as big or bigger than the neuronal nucleus.

Comment: The full definition requires argyrophilia with Bielshowsky butnot with Gallyas method, furthermore ubiquitin/p62 and 3R tauimmunoreactivity.

I.d. Spherical inclusionThis term is used in some scientific reports to indicate usually single,round cytoplasmic structures that are smaller than theneuronal nucleus, when the anatomical distribution orstaining patterns are not fitting with current definitions ofPick body.

TAU

Perinuclear ringStrong perinuclear ring-formed tau immunoreactivity usually with a lack of tauimmunopositivity of other parts of the neuronal perikaryon.

TAU

Dystrophic neurites Neuropil threads Grains

Tau immunoreactivity

TAU

Tufted astrocyte Fine granular IR in astrocytic process Astrocytic plaque

Tufted A-Plaque Ramified Thorny Bushy Globular

TAU

Coiled bodies

Globular oligodendroglial inclusions

TAU

Brainstem and corticalLewy bodies

Granular cytoplasmic IRThick and thin neurites

A-Synuclein

„Maturation“ of Lewy bodies

A-Synuclein

Astrocytic and oligodendroglial coiled body-like inclusionsin Parkinson`s disease and Lewy body dementia

A-Synuclein

Neuronal cytoplasmic and nuclear and oligodendroglial cytoplasmicinclusions in multiple system atrophy

A-Synuclein

Compact round NCI Granular NCI Compact NCISkein-like NCI Skein-like NCI Compact and granular NCI

phTDP-43: NEURONAL

TDP-43

Use stageing and diagnostic criteriaStep-7

Suggest or characterizeStep-8

Suggest (indicatedwith black boxes)

Or

characterize(indicated with grayboxes)

Evaluating unusual forms or variants of sporadic disorders,and suspicion of genetic NDD

Further proteins…If none of the above steps was helpful, neuroserpinopathy and

ferritinopathy might be considered.It is also important to keep polyglutamine repeat diseases in

mind; here anti-ubiquitin immunostaining and carefulscreening for intranuclear inclusions may help.

In FTLD cases without TDP-43 and tau pathology, anti-FUS orfurther immunostaining for ubiquitin and p62 may be useful

Step-9

Atypical patterns and variants reported in the literature

or

And when you think you arefinished....

Step-10

Alpha-synuclein TDP-43

AD AD AD AD AD AD

Limbic

Limbic + Subcortical

Amygdala

Brainstem (+ other)

None

Concomitant other tauopathies

Evaluate concomitant pathologyStep-10

Screening with p62 can be sufficient in the regions indicated by a red box

Conclusions

• Widespread sampling and staining is needed toevaluate the spectrum of pathological alterations inthe neurodegenerating brain

• “Prion-like“ spreading: research should be followedwhether it has any implications for public health

• Involvement of peripheral tissues: a tool for in vivodiagnostic biopsies?

First when relevant therapy can be offered....