The study of circadian cycles is TIMELESS

Circadian rhythmicity is an essential biological process that regulates the sleep-wake cycle through both internal processes located primarily within the suprachiasmatic nucleus, as well as external environmental cues like light. Many circadian proteins work to maintain this rhythmicity, with one being TIMELESS which is within a feedback loop with other proteins such as BMAL1/CLOCK to promote or repress clock-dependent genes. TIMELESS has a clear role in maintaining our “biological clock” but its role in other processes such as synaptic plasticity is less apparent. In order to examine the role TIMELESS plays in memory and learning, Barrio-Alonso et al. utilized a combination of conditional region specific knockouts of TIMELESS done using CRE-Lox and subsequent behavioral and quantification experiments.

To obtain behavioral data, the mice were placed in a Y-maze and underwent a contextual fear conditioning (CFC) test. In the Y-maze the mice were given 10 min to interact with two arms of the maze while a third arm was blocked off. After 1 hr, the mice would be reintroduced to the maze with the third arm “novel arm” now available. The preference for the novel arm served as an index of short-term working memory. The conditional knockout (CKO) mice had a substantially lower preference for the novel arm compared to wild type mice (Figure 1A). The decreased preference indicated the CKO mice had impaired working memory. However, the mice explored the maze at similar distances, indicating no changes in exploration between the CTRL and CKO mice (Figure 1B).  In the CFC test the mice were subjected to paired stimuli, they were played a neutral tone while simultaneously they received a mild foot shock. This would result in the mice instinctively freezing due to fear. The neutral tone was the conditioned stimuli (CS) and the foot shock was the unconditioned stimulus (US). Over the course of three days the mice were subjected to different stimuli.  

Day 1: CS+US 

Day 2: CS only with the same context. 

Day 3: CS only with different context.   

On each day the freezing behavior was scored but had different implications for each day. On Day 2 freezing served as a measure of contextual fear while Day 3 freezing served as a measure of cued fear memory. On the first day, the CKO and control group had similar results indicating memory acquisition was intact as mice in both groups associated the neutral tone with the foot shock (Figure 1C). However, on day 2, the CKO group had reduced freezing behavior when in the contextual setting without the CS, indicating impaired memory (Figure 1D). On Day 3, the CKO group exhibited reduced freezing behavior in the presence of the conditioned stimulus, as well (Figure 1E). They repeated the setup of days 1-3 but instead tested after 60 min post training. With this, no difference between the CKO and control groups were observed. With these results, they concluded Timeless must contribute to long term memory. They repeated this experiment with mice at ZT10 and found no long term fear memory deficits as seen with the ZT12 mice. This implied the effects of TIMELESS ablation in a circadian context were most prominent at ZT12. 

Kathryn Casey and Brianna Tsakh

Figure 1: TIMELESS deletion impairs short-term spatial memory and retrieval of long-term memory. (A) Quantification of the preference for the “novel” arm compared to the “familiar arm” between CTRL and CKO mice. (B) Distance explored by CTRL mice vs. CKO mice in the Y-Maze. (C) Measurement of the ability of the CTRL vs. CKO mice to learn and associate a conditioned stimulus (neutral tone) with an unconditioned stimulus (toe shock). (D) Measurement of the ability of the CTRL vs. CKO mice to freeze in response to being in the same context without adding the conditioned stimulus. (E)  Measurement of the ability of the CTRL vs. CKO mice to freeze in response to the addition of the conditioned stimulus.

Reference: Barrio-Alonso E, Lituma PJ, Notaras MJ, Albero R, Bouchekioua Y, Wayland N, Stankovic IN, Jain T, Gao S, Calderon DP, Castillo PE, Colak D. Circadian protein TIMELESS regulates synaptic function and memory by modulating cAMP signaling. Cell Rep. 2023 Apr 25;42(4):112375. doi: 10.1016/j.celrep.2023.112375. Epub 2023 Apr 11. PMID: 37043347; PMCID: PMC10564971.

TDP-43 depletion disrupts blood brain barrier in neurodegeneration

Capillary epithelial cells within the blood brain barrier (BBB) breakdown in neurodegenerative disorders. A loss of a DNA binding protein called TDP-43 is thought to be the reason for blood-brain barrier breakdown. ECs and microglia are usually underrepresented in single nuclei analysis of the brain, so ERG transcription factor was stained to enrich them and sort them based on ERG subtype. Three clusters of ECs were identified: aging, degenerative and healthy. The degenerative group showed downregulation of β-catenin and upregulation of TNF/NF-kB with a loss of TDP-43. Capillary ECs were further separated into five clusters based on the top genes expressed within a cluster. Reactive clusters (REV1) were enriched degenerative ECs and homeostatic clusters (HC) were enriched normal ECs. REV1 had increased TNF/NF-kB signaling and decreased Wnt/ β-catenin compared to the HCs.Researchers also saw capillary ECs switching to a proinflammatory subtype during disease, which is thought to contribute to the breakdown of the BBB seen in neurodegenerative diseases. They used Uniform Manifold Approximation and Projection (UMAP) visualization which showed a clear increase in nuclear β-catenin in the HC cluster. Additionally, β-catenin transcriptional targets, such as TCF/LEF1, ABCG2, and APCDD1, were significantly upregulated in the HC cluster compared to REV1. These findings suggest that the REV1 population exhibits reduced nuclear β-catenin, lower TDP-43 levels, and diminished expression of Wnt signaling genes. NF-kB and Wnt function to maintain the BBB in the presence of TDP-43 and so it is theorized that a loss of TDP-43 shifts NF-kB to its proinflammatory functions causing the BBB to breakdown.

 

 

Reference:

Omar, O.M.F., Kimble, A.L., Cheemala, A. et al. Endothelial TDP-43 depletion disrupts core blood–brain barrier pathways in neurodegeneration. Nat Neurosci (2025). 

A PCR approach to disease diagnosis

 

Hello Readers! 

We are part of the PCR team in the Scimemi Lab at SUNY UAlbany: Catherine Lienemann, Alaina Jeeson, and Dr. Phillip Albrecht, our lab manager.

The polymerase chain reaction (PCR) is a laboratory technique that is used to amplify DNA fragments. Amplifying DNA fragments is useful as it allows us to create large amounts or copies of a DNA sample that is required for analysis, using only one copy of DNA doesn’t give us enough information for any molecular or genetic analyses (Nhgri, 2019). In the lab, we use PCR to confirm the genotypes of mice in our mouse colony. We extract DNA from mice tail snips and make digestions as well as 1:1 dilutions of the DNA. We then set up reaction samples that include DNA polymerase, DNA primers, and DNA dilutions, which go into the thermocyclers. Lastly, we conduct gel electrophoresis which separates the contents of the samples and image them to analyze further. Components needed for PCR include double-stranded template DNA, DNA primer, and DNA polymerase. DNA primers are short DNA fragments that bind to specific complementary sequences in the template DNA strand, this prepares the DNA strand for amplification. DNA polymerase is an enzyme that connects nucleotides, makes DNA molecules that form PCR products, and initiates replication. The PCR process consists of three consecutive steps including: (i) denaturation; (ii) annealing; (iii) elongation. DNA denaturation happens at high temperatures (95°C in our case). During denaturation, the DNA double-strands are separated from each other. The annealing process allows primers to bind to the single-stranded DNA. Lastly, elongation takes place where the DNA polymerase initiates DNA replication and new DNA strands are synthesized in the 5’-3’ direction, making multiple copies of DNA. All of the previously mentioned steps are accomplished in a thermocycler which can control temperatures and incubation times, allowing for many PCR cycles to amplify DNA (Khehra et al., 2023).  

PCR is a tool not limited to the world of genotyping and research labs, it found itself center stage in labs around the globe as diagnostic technology during the COVID-19 Pandemic. A paper by Velavan, T. P et al. (2021) dives into the use of “real-time reverse transcriptase Polymerase-Chain Reactions (RT-PCR)” which detects SARS CoV-2 easily by converting the viral RNA into complementary DNA (cDNA) through reverse transcription, and then amplifying specific segments of the cDNA. The genetic sample is obtained using swab tests of the upper respiratory system such as the nose or mouth. There can be some hiccups in this process such as improper swabbing technique or False-negatives. These false-negatives can occur through the mutation of the reverse transcriptase-PCR primer and the probe target segments of the virus’s genome. The RNA is then extracted and diluted with the standard elements of a PCR  such as “forward and reverse primers, nuclease-free water, a fluorophore-quencher probe and a reaction mix (magnesium, transcriptase, nucleotides, polymerase, and additives)” (Velavan, T. P et al., 2021). 

A paper by Fallon et al. (2022), discusses the use of PCR in the diagnosis of uveitis, a disease that is identified by inflammation of the eye. The aqueous or vitreous fluid of the eye was used to run PCR to detect Herpes Simplex Virus, Varicella Zoster Virus, Cytomegalovirus, Toxoplasmosis gondii, Mycobacterium tuberculosis and Epstein-Barr Virus. In this study, data that was used included the pre-PCR test diagnosis and treatment and the post-PCR test diagnosis and treatment from 116 patients. Using this data Fallon et al. (2022) wanted to see the significance of PCR in diagnosing and treating uveitis. 49% of patients had a diagnosis change and 27% of patients had a treatment change based on the results of PCR testing.

The results of the study by Fallon et al. (2022) showed that PCR testing in patients allows for treatment changes and improved diagnosis of infectious uveitis. It was also noted that the PCR testing results had a higher impact on uveitis diagnosis in patients who didn’t have a pre-test diagnosis or had an unknown diagnosis. The CDC also performed a study using the Fulgent COVID-19 RT-PCR test to examine the testing’s specificity and accuracy. The results out of a total 2039 subjects was a testing sensitivity of 94.7% and a specificity of 100% making it one of the most accurate tests on the market for the diagnosis of COVID-19 (Velavan, T. P et al. (2021). These results show us that PCR testing is confirmatory in nature, and can greatly impact patient care treatments, diagnosis, and management even in cases where patients have been previously diagnosed using other laboratory methods. 

References

• Fallon, J., Narayan, S., Lin, J., Sassoon, J., & Llop, S. (2022). The impact of polymerase chain reaction (PCR) on diagnosis and management of infectious uveitis at a tertiary care facility. Journal of Ophthalmic Inflammation and Infection, 12(1).  
• Khehra, N., Padda, I. S., & Swift, C. J. (2023). Polymerase chain Reaction (PCR). StatPearls - NCBI Bookshelf.  
• Nhgri. (2019, March 9). Polymerase Chain Reaction (PCR) fact sheet. Genome.gov. 
• Velavan, T. P., & Meyer, C. G. (2021). COVID-19: A PCR-defined pandemic. International journal of infectious diseases : IJID : official publication of the International Society for Infectious Diseases, 103, 278–279.