(E-174) Cell Site Specific Laser Induced Damage Signaling Response in Huntington’s Disease Astrocytes of Mouse Brain Models

Introduction::

Huntington’s disease (HD) is a rare neurodegenerative disease that is autosomal dominant and is caused by an excess of CAG nucleotide repeats in the HTT gene. It affects roughly 2.7 out of every 100,000 people worldwide.1 Symptoms often appear in the afflicted’s thirties and forties causing a breakdown of voluntary and involuntary muscle movements as well as general cognitive issues.2 This occurs because of the production of toxic mutant huntingtin protein that interferes with many cellular functions3. Some studies have implicated damage to astrocytes in the progression of HD, where expression of the HTT gene in astrocytes heightens associated damage.4 

In human brains, astrocytes are the most abundant cell type.5 Astrocyte function is complex and dynamic, including the upkeep of homeostasis throughout the central nervous system. Astrocyte homeostatic functions include assisting with glutamate transport, control of synaptic transmission, and general structural support. One way that astrocytes interact with other cells in the central nervous system is through gliotransmitters.6 Astrocytes propagate information through calcium oscillations in the brain. A proposed mechanism for dysfunction associated with HD is that some pathways mediated by astrocytes are under or over-activated, including signaling through molecules such as Kir4.1, Glt1, and calcium.7,8 A specific consequence of this can be reactive astrocytes with unregulated calcium levels that result in excitotoxicity and neurodegeneration.9 From this, we design our study to measure calcium levels in astrocytes near cells that were damaged by laser in a specified location.

Materials and Methods::

We first cultured cells as described in Wakida et al. 2022.10 We then used a rapidly progressing R6-2 HD model, compared to a non-transgenic control. On DIV 7 we used lipofectamine and optimem to transfect in the Salsa6f fusion protein which detects calcium activity. 

Transfected cells were incubated for 24 to 72 hours prior to laser damage. Our imaging and laser set up (figure 1) consists of a Zeiss inverted 200M microscope paired with a 800 nm Coherent Chameleon Ti:Sapphire femtosecond laser, working at a power of approximately 3.4x108 W/m2. We have the capacity to minimize cell damage with a laser diameter of 0.5 μm to 0.8 μm, or increase laser settings to cause immediate cell death by photolysis. Laser exposure is controlled through a Uniblitz electromechanical shutter to release pulses of laser in bursts as low as 10 ms. These pulses will be controlled by Robolase, which is a custom coded program written in LabView specifically for our lasers. A Hamamatsu Orca R2 CCD camera is attached to the microscope to capture images. Zeiss software Zen will be used to control light and fluorescence settings, and capture images. Zen will also allow for the acquisition of time series to capture images every 2-5 seconds, as determined at the time of use. The cells are held at stable conditions of a 5% ratio of CO2, a temperature 37°C, and a humidity of 50% through an Ibidi stage incubation system.11

Results, Conclusions, and Discussions::

Figure 2 demonstrates calcium fluorescence of astrocytes responding to laser damage. Both GCaMP6 and tdTomato signals are relatively stable before laser damage. Following laser damage, there is a spike in fluorescence that then fades as the targeted cell dies. We see a form of astrocyte response in figure 3 where the signal intensity of an attached cell in a cortical HD model shows minimal fluctuation in calcium leading up to the induced damage with images at -10:00 and -5:00. Following laser exposure at time 0 there is an increase in calcium signal fluctuations with amplitude changes in peaks.

One metric used to measure astrocyte response was dF/F as seen in figure 4. dF/F was calculated as the relative change in fluorescence before and after the laser was fired. Significance is shown above corresponding cell types, (p< 0.05) where we observe a significant increase across all categories of astrocytes compared to control cells. No difference in trends were observed between the HD and NT models. In figure 5 we observe significant increases in frequency of oscillation from the pre-injury to post-injury periods. Frequency was calculated by taking the number of peaks divided by the time elapsed. We observe a significant increase in network HD astrocytes, potentially implicating the disease affects networked astrocyte communication. In the HD attached striatum model, the increase had a p-value of 0.0561, further supporting this change within HD models.

We saw greater levels of relative increase in dF/F compared to their respective controls in our cortical model. This could point to astrocyte dysfunction specifically corresponding to damage to the cortex and therefore being associated with the cognitive issues that HD causes. Further, significant increases in frequency were observed in the post-injury period of HD networked astrocytes, suggesting a change in calcium communication in astrocyte networks for HD astrocytes. This difference could point to HD affecting specifically the frequency aspect of astrocyte communication. These conclusions could help treat HD as drug research could look into what factors influence astrocyte calcium frequency and work to decrease it to minimize damage from overreactivity. 

Acknowledgements (Optional): : This work was supported by the following NIH grants: R01 NS089076, R01 NS090390 and R35 NS116872(to LMT), 1F30AG060704-01A1, T32GM008620, Lorna Carlin Fellowship and T32AG000096 (to GMF), and the Hereditary Disease Foundation (to JSS). This work is also supported by the Air Force Office of Scientific Research under award number FA9550-20-1-0052 and ongoing support from the Beckman Laser Institute Foundation (to MWB). This work is also partially funded by UROP UCI.

References (Optional): :

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11. Wakida, N. M., Lau, A. L., Nguyen, J., Cruz, G. M., Fote, G. M., Steffan, J. S., Thompson,
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