Lesson 5 Aging Cell Research Article

Applied Molecular Cellular Biology

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🧠 Aging, DNA Repair & BDNF — What’s the Connection?

🔎 Background

Aging & the brain: As we age, our brains accumulate DNA damage, especially from reactive oxygen species (ROS) ⚡️ produced by mitochondria during energy production.
DNA repair hero: The base excision repair (BER) pathway is the main system to fix oxidative DNA damage in both nuclear and mitochondrial DNA.
Problem: Neurons can’t divide (post-mitotic) and have low antioxidant defenses, so they really depend on BER. But how BER changes with age in humans wasn’t fully known.
Possible regulator: BDNF (Brain-Derived Neurotrophic Factor) 🧩 supports neurons and may control DNA repair via CREB (a transcription factor). Its activity declines with age.

🧪 Methods

Human brains: Samples from 57 people (20–99 years) across 4 regions:
- Hippocampus (HC) 🧩
- Entorhinal cortex (EC) 🔄
- Superior frontal gyrus (SFG) 🧠
- Postcentral gyrus (PCG) 🖐 → Microarrays measured gene expression.
In silico: Predicted CREB binding sites in BER gene promoters.
Lab experiments:
- Mouse hippocampal neurons treated with BDNF 🐭
- Electrophoretic mobility shift assays (EMSA) tested CREB binding
- Bdnf+/− mice used to mimic reduced BDNF.
- Western blots & activity assays checked DNA repair enzymes.

📊 Results

3.1 Aging reduces BER gene expression

Most BER genes went down with age in all 4 brain regions.
Key drops: POLB, APE1, NEIL2, LIG3, FEN1, NTHL1.
Only UNG went up (~1.5×).
NER pathway genes also changed, but less than BER → meaning BER is especially vulnerable in aging brains.

3.2 BER genes correlate with BDNF

BDNF levels also declined with age (↓ ~60% in EC, SFG, PCG).
Many BER genes (POLB, APE1, NEIL2, PNKP, FEN1, etc.) showed a positive correlation with BDNF.
Some (like UNG) showed negative correlation.

3.3 CREB binding to BER promoters

Most BER promoters had CRE sites (binding spots for CREB).
EMSA showed CREB binds to ~82% of tested sites.
Binding was lost if sites were methylated ➡️ suggests aging methylation might reduce repair.

3.4 BDNF boosts BER in neurons

Treating mouse hippocampal neurons with BDNF:
- ↑ Phosphorylation of Akt and CREB 🚦
- ↑ Protein levels of APE1, POLB, NEIL2
- ↑ Enzyme activities (incision & incorporation) → BDNF directly stimulates DNA repair!

3.5 Reduced BDNF impairs repair

Bdnf+/− mice (50% less BDNF):
- Lower POLB & APE1 in hippocampus, not cortex.
- BER activity slightly reduced (not always significant).
- Trend toward more DNA lesions in nuclear & mitochondrial DNA. → Suggests regional vulnerability.

💡 Discussion & Model

Main idea: Aging brains lose BER function, partly because BDNF declines.
BDNF → TrkB → CREB (and others like NFκB, ATF4, NRF2) → activates BER genes → DNA repair.
Less BDNF = less repair = more oxidative DNA damage → contributes to aging and neurodegeneration.
Therapeutic hint: Boosting BDNF (e.g., exercise 🏃‍♀️, drugs) might help maintain DNA repair in neurons.

🧩 Key Takeaways

Aging brains = less DNA repair (BER genes go down).
BDNF levels drop with age, and this tracks with BER decline.
CREB binds to many BER gene promoters.
BDNF treatment stimulates BER proteins & activity.
Mice with less BDNF show weaker DNA repair.
Conclusion: BDNF is a master regulator of DNA repair in the brain.

Quiz

Score: 0/30 (0%)

Q0. What is the main DNA repair pathway responsible for fixing oxidative DNA damage in neurons?

Nucleotide excision repair (NER)

Base excision repair (BER)

Mismatch repair (MMR)

Double-strand break repair (DSBR)

Q1. Which factor was found to positively correlate with BER gene expression across human brain regions?

BDNF

p53

NF-κB

AP-1

Q2. Which transcription factor is directly activated by BDNF through the TrkB receptor?

CREB

HIF1α

STAT3

p53

Q3. Which BER gene was significantly upregulated with aging instead of downregulated?

UNG

POLB

APE1

FEN1

Q4. What was the main experimental method used to measure gene expression changes across brain regions and ages?

Microarray analysis

RT-qPCR

RNA sequencing

Western blotting

Q5. In the study, which brain regions were analyzed for BER expression changes?

Frontal cortex, striatum, cerebellum, medulla

Entorhinal cortex, hippocampus, superior frontal gyrus, postcentral gyrus

Amygdala, cerebellum, pons, occipital cortex

Thalamus, medulla, hypothalamus, basal ganglia

Q6. What is the function of POLB in the BER pathway?

DNA ligation

Recognition of oxidized bases

Gap filling with new nucleotides

Cutting the DNA backbone

Q7. What was observed when primary mouse hippocampal neurons were treated with BDNF?

Decreased POLB protein level

Increased APE1, NEIL2, and POLB levels

Reduced CREB phosphorylation

No change in BER enzyme activity

Q8. Which experimental technique was used to confirm CREB binding to predicted promoter regions?

EMSA (Electrophoretic Mobility Shift Assay)

ChIP-seq

Co-immunoprecipitation

ELISA

Q9. What happens when CRE sites in BER promoters are methylated?

CREB binding is enhanced

CREB binding is abolished

CREB binding becomes unspecific

No effect

Q10. In Bdnf+/− mice, what was observed in the hippocampus compared to wild-type?

Increased APE1 and POLB levels

Reduced APE1 and POLB protein levels

No change in BER proteins

Complete loss of DNA repair

Q11. Which DNA lesion is primarily repaired by OGG1?

Uracil

8-oxoguanine

Thymine glycol

Abasic sites

Q12. What is the role of APE1 in BER?

Ligates DNA ends

Removes damaged bases

Cuts the DNA backbone at AP sites

Adds nucleotides

Q13. Why were protease inhibitors used during sample preparation?

To block DNA degradation

To prevent protein degradation

To enhance fluorescence

To inhibit RNAse activity

Q14. What was the significance of p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001 in the figures?

Levels of gene expression

Levels of statistical significance

Fold change of mRNA

Protein molecular weight

Q15. The BER pathway is the main mechanism for removing oxidative DNA damage.

True

False

Q16. NER is more affected by aging than BER in the human brain.

True

False

Q17. CREB binds only to full CRE sites, not half-sites.

True

False

Q18. BDNF levels decline with age in multiple cortical regions.

True

False

Q19. Microarray analysis allows detection of DNA mutations in single cells.

True

False

Q20. APE1 and POLB protein levels increased after BDNF treatment of neurons.

True

False

Q21. CREB phosphorylation leads to recruitment of transcriptional cofactors and activation of target genes.

True

False

Q22. Methylation of CRE sites enhances transcription factor binding.

True

False

Q23. Bdnf+/− mice have double the normal BDNF protein level.

True

False

Q24. Incorporation activity measures the gap-filling step of BER.

True

False

Q25. Reactive oxygen species damage DNA mainly by removing phosphate groups.

True

False

Q26. CREB binding was confirmed for 9 out of 11 predicted BER promoter sites.

True

False

Q27. Phosphorylation of NEIL2 by PKC activates its glycosylase activity.

True

False

Q28. Long-range PCR was used to assess DNA damage by measuring amplification efficiency of large fragments.

True

False

Q29. BDNF likely contributes to maintaining genomic stability during aging.

True

False