Faculty of Sciences and Technology of University of Coimbra Biochemistry Master Metabolism 2011/2012

Compartmentation of glial and neural metabolism in Alzheimer’s Disease

Daniela Rodrigues José Pedro Mendes Lídia Grilo Filipa Moreira

3 years of work: Separate the individuals

Quantitive analysis of the metabolites present in hipocamppus

Differences of glial and neuronal metabolism

• Cognitive/behavior tests; • MRI, (FDG)-PET; • CFS expression of protein tau and βamyloid.

• 1H-MRS (single-voxel) • Glutamate/Glutamine; • Lactate; • GABA; • N-Acetyl-Aspartate

• Administration of [U13C] glucose and [2-13C] acetate • Metabolite fluxes • Steady state

early

Controls

vs

AD Patients

moderate

advanced 25 participants in each group – 100 in total

Aims Develop a protocol to discriminate AD patients in three states of the disease development, using several criteria’s: brain morphology, cognitive behavior, biochemical metabolism and proteins levels;

Characterization and quantification of the hippocampus metabolism in patients with AD in every state (early, moderate and advanced) using H-MRS technique.

Compartmentation of glial and neuronal metabolism

Alzheimer’ Disease (AD)

• Degenerative Disease • No cure • Treatment of the symptoms

worldwide scenario (estimated): 2010 – 35.6 million people 2030 – 65.7 million people 2050 – 115.4 million people.

Decreased schooling

Normally, appears in individual > 65 years

Major risk to have AD

Each patient suffers from Alzheimer's disease unique way, but there are commonalities

Memory

Fig.1 – Brain imaging of a healthy individual (left) and Alzheimer’ patient (right).

Cell morphology characteristics:  Neural loss

 Loss of connections between neurons  Senil Plaques  Neurofibrillary Tangle

Fig. 2 – Neural alterations in Alzheimer Disease

Stages / Symptoms • • • • • • •

1st Stage

Lost short term memory; Loss of attention; Apathy; Some disorientation of time and space; Only performs simple tasks; Difficulty in recognizing and identifying objects (agnosia) Difficulty in execution of movements (apraxia).

2nd Stage

• • • • • •

Difficulty of independence; Missing remembering vocabulary; Emotional instability; Irritability; Aggressiveness; Illusions

3rd Stage

• • • • • •

Completely dependent on people to perform tasks; Loss of speech; Aggressiveness; Apathy extreme; Tiredness; Loss of mobility.

Task 1- Diagnosis and prognosis of AD 1.1 AD diagnosis using Cognitive – behavior tests We will perform 3 types of tests:

• Mini–mental state examination (MMSE) • Alzheimer's disease Assessment Scale – Cognitive Subscale (ADAS-Cog) • Clinical Dementia Rating Scale Sum of Boxes (CDR-SB) • Caucasians • 60-80 years of age; • same level of education (less than 8 years) • Mediterranean diet during their life • Absence of genetical factors that leads to AD

Task 1- Diagnosis and prognosis of AD 1.1.1 Mini–mental state examination • Questionnaire test used to screen dementia and cognitive impairment • Simple questions and problems in a number of areas:     

the time and place of the test repeating lists of words arithmetic such as the serial sevens language use and comprehension basic motor skills

• It can be used to follow the course of cognitive changes in an individual over time.

decline per year: - less than 2 points - slow progressors, - 2 to 4 points - intermediate progressors - more than or equal to 5 points - rapid progressors

Task 1- Diagnosis and prognosis of AD 1.1.2. Alzheimer's disease Assessment Scale - Cognitive Subscale (ADAS-Cog)

•

Specifically designed to evaluate the severity of cognitive and non-cognitive behavioral dysfunctions characteristic of persons with AD

 more thorough than MMSE  considered the best brief exam for the study of language and memory skills

ADAS Cognitive domains evaluated: Cognitive

(ADAS-Cog)

NonCognitive

   

memory (50%) language (28%) praxis (14%) command understanding (8%)

The ADAS-Cog scores clearly distinguish clinically diagnosed AD patients from non-dementia control subjects! [1].

Task 1- Diagnosis and prognosis of AD 1.1.3. Clinical Dementia Rating Scale Sum of Boxes (CDR-SB)

•

Allow as to asess global performance of the participant  patient interview  mental status examination  an interview of a collateral source

Six cognitive domains or boxes:

CDR is credited with being able to discern very mild impairments.

     

memory orientation judgment/ problem solving community affairs home and hobbies personal care

Table I - Score ranges to MMSE, ADAS-Cog and CDR-SB test corresponding to normal participants and AD patients in an early, moderate and advanced stage.

Test / Group MMSE ADAS-Cog CDR-SB

Controls 25-30 ≤24 0-3

early 21-24 25-49 4-8

moderate 10-20 50-59 9-13

advanced ≤9 60-70 14-18

Task 1- Diagnosis and prognosis of AD 1.2. MRI and fluorodeoxyglucose (FDG)-PET brain analysis AD patients exhibit:  Regional cerebral atrophy - MRI  Hypometabolism - (FDG)-PET MRI morphometry: - hippocampal volume - entorhinal and retrospenial cortical thickness.

FDG-PET: - entorhinal, retrosplenial and lateral orbitofrontal metabolism.

MRI can be used to quantify regional atrophy distinguishing early and later preclinical stages of AD.

Fig .3- The regions of interest used: 1) hippocampus, 2) entorhinal, 3) parahippocampal, 4) retrosplenial, 5) precuneus, 6) inferior parietal, 7) supramarginal, 8) middle temporal, 9) lateral orbitofrontal and 10) medial orbitofrontal cortices [1].

Task 1- Diagnosis and prognosis of AD 1.2. MRI and fluorodeoxyglucose (FDG)-PET brain analysis Experimental protocol:  Subjects will be scanned after a 4 hour fast (water only).  Plasma glucose has to be ≤ 180 mg/dL for FDG to be injected.  Dosage ranges from 0.1 to 0.2 mCi per kg of body weight.  Imaging begins at 30 min post injection  The scan will be acquired as six 5-min frames. Fig. 4Typical positron emission tomography (PET) facility

The distribution of 18F-FDG is a good reflection of the distribution of glucose uptake

PET scan of the brain

2-Deoxy-2-fluoro-D-glucose Or Fluorodeoxyglucose (18F)

Task 1- Diagnosis and prognosis of AD 1.3. CFS expression of protein tau and β-amyloid Biochemical analysis of the CSF to establish the levels on the 3 stages of AD of:

• Aβ42 • t-tau • p-tau Immunoassay method - CSF samples will be obtained by lumbar puncture - ratio of t-tau/Aβ42 and p-tau /Aβ42 will be calculated

High CSF tau levels probably reflect axonal degeneration In AD, there are high levels of p-tau, consequence of the self-assembly of tangles of paired helical filaments and straight filaments.

Fig 5. Representation of amyloid plaques formation.

Controls

AD patients

Aβ42

200 pg/mL

140 pg/mL

T-tau

70 pg/mL

125 pg/mL

P-tau

25 pg/mL

45 pg/mL

T-tau/Aβ42

0,20

1

P-tau/Aβ42

0,15

0,36

Task 2- Quantitive analysis of brain metabolites 1H-MRS

single voxel

N-acetyl-aspartate compounds

Glutamate/Glutamine; GABA;

Lactate.

N-Acetyl-Aspartate: activation of NMDA (ionotropic receptor). Marker for mitochondrial functions and loss of neuronal cells;

Lactate: barley used by neurons and glial cells, increase levels in AD indicates; GABA- inhibitory neurotransmitter. GABAergic neurons synthesize GABA by the descarboxylation of glutamate via Glutamate descaboxylase

Task 2- Quantitive analysis of brain metabolites Deficiency in glutamate/glutamine cycle

Equal changes observed in AD

See glutamate levels by H-MRS

Fig. 6 - Conversion of glutamate to glutamine by GS in the synaptic cleft. The glutamine is exported to the neuron reconverted into glutamate

Task 3 – 13C MRS measure of glial and neural metabolism  Advanced technique that allows non-invasive in vivo evaluation of metabolite concentrations  13C MRS is feasible on a clinical 1.5 T MR scanner  Specific diagnostic information - value in neurological research Step 1 Step 2 Step 3 Step 4

• Intravenous administration of [U-13C]glucose • Intravenous administration of [2-13C]acetate • Determination of relative metabolic fluxes • Evaluation of Steady-State conditions

• Evaluation/Comparation of metabolites between controls and AD patients Step 5

Step 1- [U-13C]Glucose

Fig. 7 Metabolism of [U-13C] glucose administred to all individuals, where labeled pyruvate is converted in acetly-CoA by pyruvate dehydrogenase.

Fig. 8 Metabolism of [U-13C] glucose administred to all individuals, where labeled pyruvate is converted to oxaloacetate by pyruvate carboxylase. [5]

Step 2- [2-13C]acetate

Fig. 9 Pathway of [2-13C] acetate in glial and neural cells. [6]

Step3-Determination of relative metabolic fluxes Obtained 13C isotopomer data

Tca CAL Program

Fractional contributions to acetyl-CoA in SS

 Decreased requirement of astrocytes to replenish oxaloacetate in the TCA cycle  Lower flux through astrocytic PC

The clearance of GABA by astrocytes Conversion into succinate TCA cycle

Step4: Evaluation of Steady-State Condition [U-13C]glucose and [2-13C]acetate are oxidized

Glutamate C4 becomes enriched in the 1st turn of the TCA cycle

C3 and C2 isotopomers become enriched in the following TCA turns

The glutamate C3/C4 enrichment ratio used to evaluate metabolic Steady-State (7.5 h after infusion)

Possible results from 13C MRS In normal : Glucose carbons are transported to the neuron entering the TCA cycle: - Glu4 (round 1) - Glu2 (round 2) - CO2 (round 3). - Glutamate returns carbon to the glia after neurotransmission for conversion to glutamine (Gln4 and Gln2). In AD: - All neuronal processes are systemically reduced to half - Glial glucose enriches glutamine before glutamate - Excess glucose in AD is metabolized only as far as lactate.

Fig. 10 Traffic of glutamate between neurons and astrocytes in normal and AD individuals.

Budget Reagents [U- 13C] Glucose

29 000 €

[2-13C] acetate

20 300 €

MRI

36 000 €

PET

24 000 €

2-Deoxy-2-fluoro-Dglucose

500 €

1H-MRS

15 000 €

13C-MRS

15 000 €

Other material

40 000 €

TOTAL

179 800 €

[U- 13C] Glucose

145€/0,5g (Cambridge Isotope Lab)

[2-13C] acetate

203.50€ /g (SigmaAldrich)

2-Deoxy-2-fluoro-Dglucose

813.00/100mg (SigmaAldrich)

We expect to have the support of HUC people! (for free) 

References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.

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