Neuropathology of CADASIL and other small artery diseases of the brain (SADB)

Hannu Kalimo
Turku and Helsinki Finland

 Hereditary small artery diseases of brain

CADASIL (cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy ) CAA (cerebral amyloid angiopathy) Swe-hMID (Swedish hereditary multi-infarct dementia) CARASIL (cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy) PADMAL (pontine autosomal dominant microangiopathy and leukoencephalopathy ) Retinal vasculopathy with cerebral leukodystrophy (RVCL) COL4A1 associated SADB Fabry disease Varia

Sporadic small artery diseases of brain

CAA (cerebral amyloid angiopathy) Arteriolosclerosis (Binswangers disease)

Notch3 undergoes 3 cleavages (S1-S3). NICD enters nucleus

and regulates transcription

Hes/Hey

MAML1

CoR HDAc

In CADASIL over 200 different mutations have been reported worldwide in EGF repeats (1-32 ) of the extracellular domain of Notch3 (N3ECD). Most often the mutation results in an uneven number of cysteines (either gain or loss of an uneven number of cysteines).

RBPJ

RBPJ

Signaling cell

Receiving cell (vascular smooth muscle cell)

CoR HDAc HAc

Control, cerebral White matter

CADASIL,cerebal white matter >

H&E

H&E

PAS

a- smooth muscle
actin

Notch3 extracellular domain (N3ECD, mab 1E4)

Collagen I

Cerebral Blood Flow

Positron emission tomography

Patient M/34 (strokes)

Control M/31
Tuominen et al, Stroke 2004;35:1063-67

Arterial Changes in CADASIL in Relation to Pathogenesis of Diffuse Myelin Loss of Cerebral White Matter: Examination of Cerebral Medullary
Arteries by Reconstruction of Serial Sections of an Autopsy Case..
Okeda et al. (Stroke 2002;33:2565-9)

Red zone: continuous loss of VSMCs

Red dotted

zone: discontinuous loss of VSMCs

Green zone: intimal fibrous thickening

Blue zone: adventitial fibrosis Although intimal fibrosis or hyalinosis was present, luminal occlusion was scarce.

Histogram of internal diameter of arterioles between CADASIL and control group in W M

30
Percent %
H&E

CADASIL (n=929) c ontrol (n=218)

20 10 0
5 20 35 50 65

80

Internal diameter of arterioles in mm

PAS

Miao et al. Brain Pathol 2004;14:358-364.

95

Cortex White matter

Stenosis

Occlusion and infarcted tissue In CADASIL occlusion of a penetrating cerebral artery results in a lacunar infarct (<), because penetrating arteries are terminal without collateral flow from the neighboring arteries.

< Lacunar infarct

Cortical vs. white matter arteries are metabolically different

CADASIL: Multiple small (lacunar) infarcts in WM and basal ganglia, whereas cerebral cortex is relatively spared

>

< <

<
<

<
CADASIL does not protect from classic risk factors: A CADASIL patient with p.R133C mutation had also severe medium-sized and small vessel atherosclerosis including complete occlusion of an insular branch of middle cerebral artery ( ) and a cortical parieto-occipital infarct ( ). CADASIL infarcts in the white matter (<)

CADASIL WM: arterioles (N3ECD)

CADASIL cerebral cortex: small arterioles and a capillary ( ) (N3ECD) nerve

CADASIL WM: capillary (N3ECD)

*
*

In CADASIL brain Notch3 extracellular domain (N3ECD/GOM) accumulates not only on WM arterioles, but also on WM capillaries (pericytes), as well as = arteries on these vessels in cerebral cortex, Since arteriopathy in CADASIL is gealthough cortical arterioles are not neralised, accumulation of N3ECD equally thickened. can also be shown by immunostaining a diagnostic skin biopsy, but...

VSMC

*
VSMC

E
Lumen

VSMC

* *
VSMC

VSMC

Electron micrograph of a small dermal artery: widened subendothelial

space ( ), irregularity of vascular smooth muscle cells (VSMCs) and granular osmiophilic material (GOM, arrows). E = endothelium

*
0.4 mm

1 mm

Specific to CADASIL is electron microscopic finding of granular osmiophilic material (GOM *) in indentations (notches) of and next to degenerating vascular smooth muscle cells. GOM has been demonstrated in all CADASIL cases with a Notch3 mutation resulting in an uneven number of cysteines in Notch3 extracellular domain. (N = nucleus). Tikka et al. Brain 2009; 132:933-939

Deposition of Notch3 extracellular domain (N3ECD) can also be shown with immunostaining, but EM demonstration of GOM is more reliable, esp. at the beginning of the disease with only small amounts of N3ECD/GOM. 19-year-old male CADASIL patient, electron microscopy of a dermal artery

N3ECD immunostaining Skin biopsy: N3ECD = arteries immunostaining *

*
Nerve

nerve

< <
Tikka et al. Brain 2009; 132:933-939

CADASIL and true GOM

Elastin fragments

Variable cell debris

Granular debris of unknown origin

Variable cell debris

Tikka et al. Brain 2009; 132:933-939

GOM has been demonstrated in all CADASIL cases with a Notch3 mutation resulting in an uneven number of cysteines in N3ECD. How about non-cys cases?

Mizuno et al. Inter Med 47: 2067-72, 2008 Brass et al. NEJM 2009; 360:1656-65

Scheid et al. Neurology 71:774-6, 2008

The NOTCH 3 protein

a-helix b-turn

b-sheet

Dimitrios Vlachakis and Sophia Kossida Biomedical Research Foundation, Academy of Athens

Arg

Trp
LOSS OF sheet STRUCTURE

T2* MRI

Microbleeds in CADASIL

MRI: Lesnik Oberstein et al.

Dichgans et al. (Stroke 2002;33:67-71):

Lesnik Oberstein et al. (Neurology 2001; 57: 10661070):

MBs found in 69% of 16 CADASIL patients and in no control subjects (p= 0.001) MBs found in 31% of 32 sympto Assoc. with age (p= 0.002) matic CADASIL patients. Located in cortical-subcortical Assoc. with age (p< 0.008) regions (38%), white matter (20%), Assoc. with antiplatelet use (p< thalamus (13%), brainstem (14%) 0.025) 82% located outside areas Located in thalamus (61%), subcorappearing hyperintense on T2w tical WM (26%), cerebellum (6%) images

WM: N3ECD

Cortex: N3ECD

CADASIL targets specifically white matter arterioles, whereas grey matter arterioles are better preserved and the thinner walls of grey matter arterioles are more prone to leak (> microbleeds).

Cortex: microbleed

CADASIL WM: fibrinogen

In CADASIL the BBB of the thickened arterioles appears to be relatively intact: extravasation of plasma proteins is minimal.
Breakdown of BBB in hypertension: fibrinogen

Microbleed in CADASIL cortex: fibrinogen

Control

Epilepsy

CADASIL VK

CADASIL Mk

Fibrinogen extravasated during breakdown of BBB diffuses to CSF and from it is taken up by cerebellar Purkinje cells (PC). In CADASIL PCs remain negative.

 Hereditary small artery diseases of brain

CADASIL (cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy ) Swe-hMID (Swedish hereditary multi-infarct dementia) CAA (cerebral amyloid angiopathy) CARASIL (cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy) PADMAL (pontine autosomal dominant microangiopathy and leukoencephalopathy ) Retinal vasculopathy with cerebral leukodystrophy (RVCL) COL4A1 associated SADB Fabry disease Varia

Sporadic small artery diseases of brain

CAA (cerebral amyloid angiopathy) Arteriolosclerosis (Binswangers disease)

1.

2. 3

A
Sourander and Wlinder original article Hereditary multi-infarct dementia (Acta Neuropathol 39:247-254;1977). A. Small infarcts in: 1. right frontal white matter , 2. right capsula interna and 3. left pallidum + small lacunes in the basal ganglia. a and b: Cystic infarcts in pons (two patients).

Swe-hMID T2w MRI Swedish hereditary multiinfarct dementia (Swe-hMID)

Swe-hMID T2w MRI

The family of Sourander and Wlinder (Hereditary multi-infarct dementia. Acta

Neuropathol 39:247-254;1977)

was long considered the first publ. CADASIL family.

Swe-hMID T2w MRI CADASIL p.R141C T2w MRI

(Low et al. Brain 130: 357367; 2007)

Feature

Swe-hMID

CADASIL (Stevens p.R141C)

No. affected

4
48 3

Mean age at onset ( SD) 34.6 3.4

Onset range
Mean age at death

29 - 38
44.3 4.3

39-57
60 7

Duration (years)
No. of stroke episodes

11 2.1
>4

14.6 4.5
>6
Low et al. Brain 2007; 130, 357367

Swe-hMID, N3ECD

CADASIL, N3ECD

The absence Notch3 ECD (C) and GOM (E) in Swe-hMID and their presence in CADASIL (D and F).

Swe-hMID, electron microscopy

CADASIL, electron microscopy

Differential diagnosis CADASIL vs. Swe-hMID

Migraine episodes before onset of strokes common in CADASIL, not recorded in Swe-hMID MRI: Lesions in temporal lobes and capsula externa common in CADASIL, unusual in Swe-hMID Skin biopsy: GOM regularly found in CADASIL, not in SwehMID In 4 members of the Swedish family no pathogenic mutations were found in the entire 8091 bp reading frame of NOTCH3 nor clear evidence for NOTCH3 gene linkage. In Stevens family p.R141C mutation confirmed CADASIL dg.
Low et al. Brain 2007; 130, 357367

Figures from Low et al. Brain 2007; 130, 357367

Sclerotic indices in Swe-hMID vs. CADASIL: Frontal white matter:

Swe-hMID, H&E CADASIL, H&E

Swe-hMID: mean 0.30 % with 0.50 4.4% CADASIL: mean 0.39 % with 0.50 7.4%

Basal ganglia:
Swe-hMID, a-SMA CADASIL, a-SMA

Swe-hMID: mean 0.33 % with 0.50 8.3% CADASIL: mean 0.36 % with 0.50 19.9%
Craggs et al. Brain Pathol 2013;23:547-557

Swe-hMID, medin

CADASIL, medin

 Hereditary small artery diseases of brain

CADASIL (cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy ) Swe-hMID (Swedish hereditary multi-infarct dementia) CAA (cerebral amyloid angiopathy) CARASIL (cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy) PADMAL (pontine autosomal dominant microangiopathy and leukoencephalopathy ) Retinal vasculopathy with cerebral leukodystrophy (RVCL) COL4A1 associated SADB Fabry disease Varia

Sporadic small artery diseases of brain

Arteriolosclerosis (Binswangers disease) CAA (cerebral amyloid angiopathy)

Herovici D

Smooth muscle actin E

Collagen I F

H&E H&E Herovici

Figs A-C from Kalimo et al. Greenfields Neuropathology 8th ed. 2008; Fig. D courtesy of Dr. H Vinters; Fig. E from Alafuzoff et al. Exp Gerontol 47 (2012) 825-33

C Small vessel disease A: Granular cortical atrophy B and C: small infarcts

Atherosclerosis in small arteries is in principle similar as in larger vessels. In brain arteries it creeps down to smaller branches and may cause stenosis or occlusion. Emboli detached from plaques of larger vessels can lodge in small arteries and cause occlusion.

 Hereditary small artery diseases of brain

CADASIL (cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy ) Swe-hMID (Swedish hereditary multi-infarct dementia) CAA (cerebral amyloid angiopathy) CARASIL (cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy) PADMAL (pontine autosomal dominant microangiopathy and leukoencephalopathy ) Retinal vasculopathy with cerebral leukodystrophy (RVCL) COL4A1 associated SADB Fabry disease Varia

Sporadic small artery diseases of brain

Arteriolosclerosis (Binswangers disease) CAA (cerebral amyloid angiopathy)

Italian recess (p.A673V)

Swedish (p.KM670-671NL)

b-secretase
Flemish (p.A692G) a-secretase

g-secretase

Arctic (p.E693G), Dutch (p.E693Q), Italian (p.E693K) Iowa (p.D694N)

Outer layer

V > F (Indiana), V > G or > I (London)

Florida (p.I716V)

If the mutation is in Ab, amylayer loid is mutated or Inner mut+wt. If the mutation is in molecules regulating Ab processing, amyloid is of wild type (wt)

Congo red

Thioflavin

Occipital cortex

+ birefringence

H&E

double barreling

Ab1-40

Ab1-40 Cerebral amyloid angiopathy (CAA) affects vessels of all size including small arteries down to capillaries.

Electron microscopy: Ab amyloid fibrils

(Arctic AD due to p.E693G mutation in APP)

CADASIL WM: arterioles (N3ECD)

(here the external diameters are the same

H&E

CADASIL

CAA ICH of lobar type

Congo red

< <

CADASIL CADASIL

CAA

Occipital cortex, Arctic AD due to p.E693G mutation in APP

Occipital cortex in sporadic AD

A.

Ab1-40

In CAA Ab often accumulates also in capillaries, in severe cases it causes capillary occlusion and CBF disturbances, but rarely overt infarcts. Capillary CAA may contribute to the degeneration of neurons in Alzheimers disease by altering/impairing CBF.

Fig. A: Kalimo et al. Acta Neuropath Comm 2013 in print, Figs B and C: Thal et al. Neurobiol Aging 30 (2009) 1936-48

Sclerotic index (SI) 1 (internal diameter/external diameter) i.e. the higher the value, the thicker is the wall, and in an obliterated artery SI = 1
SCLEROTIC INDEX (SI) ANALYSIS** SI STDEV CADASIL WM (n=929) CORTEX (n=250) CONTROLS WM (n=218) CORTEX (n=258) cwAD PS1D9 AD WM (n=232) 0,43 0,13 CORTEX (n=416) 0,48 0,13 **: blood vessels with external diameter less than 30 mm are excluded. 0,45 0,53 0,16 0,16 0,73 0,56 0,14 0,13 P<0.01 P=0.03

T-TEST

Weller et al. Brain Pathology 2008; 18: 253266

CARASIL
(cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy)

First reported by Maeda et al. in three sibling patients from a

Japanese family preliminarily in 1965 and in greater detail 1976 with S. Maeda as the first authors, hence been also called Maeda syndrome. About 50 cases diagnosed, mainly in Japan, but also in China and 1 Caucasian patient in Spain. Onset during patients twenties to early thirties as a vasculopathic subcortical encephalopathy without hypertension. Neurologic symptoms: severe psychomotor deterioration caused by subcortical encephalopathy: e.g. dysarthria, dysphagia, emotional instability, and spastic gait. Non-neurologic symptoms are e.g: alopecia, vertebral disc herniation, and spondylosis deformans.

Yamamoto et al. Neuropathology and Applied Neurobiology 37, 94113

CARASIL, WM artery

CARASIL, meningeal artery

CARASIL, WM artery Control, meningeal artery

CARASIL: fibrous intimal proliferation, splitting of the internal elastic lamina, and occasional hyaline degeneration, but results on luminal stenosis discrepant. Sclerotic index: (n = 2) Controls: 0.17; 0.28 CARASIL WM: 0.25; 0.39 CADASIL WM: 0.61
Oide et al. Neuropathol, 2008; 28:132142

CADASIL, mening. artery CARASIL, mening. artery

T2w MRI

T1w MRI

T2*-GE MRI

First Caucasian (Spanish) CARASIL patient : T2- and T1-weighted MRI changes: Diffuse leukoencephalopathy involving periventricular and deep WM, including anterior temporal lobes and external capsules. Multiple lacunar infarcts deep WM and GM and brainstem (C and D). Multiple micro-bleeds in pons, basal ganglia, and hemispheric subcortical WM (E).
Mendioroz et al. Neurology 2012; 75: 2033-37

 Hereditary small artery diseases of brain

CADASIL (cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy ) Swe-hMID (Swedish hereditary multi-infarct dementia) CAA (cerebral amyloid angiopathy) CARASIL (cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy) PADMAL (pontine autosomal dominant microangiopathy and leukoencephalopathy ) Retinal vasculopathy with cerebral leukodystrophy (RVCL) COL4A1 associated SADB Fabry disease Varia

Sporadic small artery diseases of brain

Arteriolosclerosis (Binswangers disease) CAA (cerebral amyloid angiopathy)

PADMAL

Ding et al J Neuroimaging 2010; 20:134-140

PADMAL, van Gieson-elastica

PADMAL, collagen IV

PADMAL, a-SMA

PADMAL, CD68 microgia PADMAL, van Gieson-elastica

PADMAL
PADMAL

Hagel et al. Acta Neuropath 2004; 108:231-40

PADMAL, collagen IV

PADMAL, EM

 Hereditary small artery diseases of brain

CADASIL (cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy ) Swe-hMID (Swedish hereditary multi-infarct dementia) CAA (cerebral amyloid angiopathy) CARASIL (cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy) PADMAL (pontine autosomal dominant microangiopathy and leukoencephalopathy ) Retinal vasculopathy with cerebral leukodystrophy (RVCL) COL4A1 associated SADB Fabry disease Varia

Sporadic small artery diseases of brain

Arteriolosclerosis (Binswangers disease) CAA (cerebral amyloid angiopathy)

Retinal vasculopathy with cerebral leukodystrophy (RVCL), of type HERNS (hereditary endotheliopathy, retinopathy, nephropathy, with stroke-like episodes), is caused by mutations in TREX1 (three prime repair exonuclease 1)

CD68 for microglia

Neurofilament

Figures courtesy of Dr. H Vinters

H&E

Nordic CADASIL research group

Clinical studies Matti Viitanen U of T, Karol Inst Susanna Roine U of T Auli Verkkoniemi U of H Pathology Hannu Kalimo U of H Liisa Myllykangas U of H Qing Miao U of T Genetics Proteomics and molecular biology
Marc Baumann U of H Saara Tikka U of H Maciej Lalowski U of H Urban Lendahl and team Karol Inst

Imaging and PET studies Juha Rinne U of T Susanna Roine U of T Ophthalmology Tero Kivel U of H Paula Summanen U of H
U of H = Univ of Helsinki, Finland U of T = Univ of Turku, Finland Karol Inst = Karolinska Inst, Sweden

Minna Pyhnen U of H Maija Siitonen (nee Junna)* U of T Petra Pasanen* U of T

Vaasa Joensuu

Pori

R133C

Turku

Helsinki

R182C

CADASIL: T2w MRI with confluent hyperintensities

Turku coat of arms