Non-transgenic guinea pig strains exhibit divergent age-related changes in hippocampal mitochondrial respiration

Abstract

Aim: Alzheimer's disease (AD) is the most common form of dementia. However, while 150+ animal models of AD exist, drug translation from preclinical models to humans for treatment usually fails. One factor contributing to low translation is likely the absence of neurodegenerative models that also encompass the multi-morbidities of human aging. We previously demonstrated that, in comparison to the PigmEnTed (PET) guinea pig strain which models "typical" brain aging, the Hartley strain develops hallmarks of AD like aging humans. Hartleys also exhibit age-related impairments in cartilage and skeletal muscle. Impaired mitochondrial respiration is one driver of both cellular aging and AD. In humans with cognitive decline, diminished skeletal muscle and brain respiratory control occurs in parallel. We previously reported age-related declines in skeletal muscle mitochondrial respiration in Hartleys. It is unknown if there is concomitant mitochondrial dysfunction in the brain.

Methods: Therefore, we assessed hippocampal mitochondrial respiration in 5- and 12-month Hartley and PET guinea pigs using high-resolution respirometry.

Results: At 12 months, PETs had higher complex I supported mitochondrial respiration paralleling their increase in body mass compared to 5 months PETs. Hartleys were also heavier at 12 months compared to 5 months but did not have higher complex I respiration. Compared to 5 months Hartleys, 12 months Hartleys had lower complex I mitochondrial efficiency and compensatory increases in mitochondrial proteins collectively suggesting mitochondrial dysfunction with age.

Conclusions: Therefore, Hartleys might be a relevant model to test promising therapies targeting mitochondria to slow brain aging and AD progression.

Keywords: brain aging; guinea pig; hippocampus; mitochondrial respiration; non‐transgenic.

Figure 1.. Study Design and Anthropometrics.

Hippocampal samples from male PET and Hartley guinea pigs were collected at 5 and 12 mo of age (n = 12/age/strain; total N = 48 guinea pigs) to assess complex-specific mitochondrial oxygen consumption and protein content (A). Both PET and Hartley guinea pigs were heavier at 12 versus 5 mo of age (B), but there was no difference in whole brain weight (C). Interestingly, compared to their 5 mo counterparts 12 mo PET and Hartley guinea pigs had significantly lower relative brain weights (D). 5 mo PET n = 12; 5 mo Hartley n = 12; 12 mo PET n = 12; 12 mo Hartley n = 11 for all panels. One 12-month-old guinea pig died prior to final tissue collection due to reasons unrelated to the study; no gross pathology was observed on necropsy. † p<0.01, ‡ p<0.001

Figure 2.. PET but not Hartley guinea pigs have higher complex I supported maximal hippocampal mitochondrial respiration with age.

The SUIT 1 ADP titration assesses complex I supported (with glutamate, malate, and pyruvate) submaximal to maximal respiration to calculate the Vmax and Km values for maximal respiratory potential and ADP sensitivity, respectively. Panel A displays a raw tracing of the Oroboros O2k and supplemental table 1 reports the protocols in detail. Averages of the Michaelis Menten curves are shown in panel B for each of the 4 groups. There was a significant age by strain interaction for ADP Vmax (p = 0.0066) (C). Specifically, 5 mo Hartley guinea pigs had a higher ADP Vmax compared to 5 mo PETs (p = 0.032). PETs had a significantly higher ADP Vmax at 12 mo compared to 5 mo of age (p = 0.0438) (C). There was a similar finding with ADP sensitivity, the amount of ADP required to achieve ½ Vmax, with an age by strain significant interaction (p = 0.0474) (D). Specifically, 5 mo Hartley guinea pigs had a trend for lower ADP sensitivity versus 5 mo PETs (p = 0.0502). 12 mo Hartley guinea pigs also had a trend for higher ADP sensitivity compared to 5 mo of age (p=0.0502). 5 mo PET n = 12; 5 mo Hartley n = 11; 12 mo PET n = 12; 12 mo Hartley n = 10 for all panels. One 5 mo Hartley guinea pig sample was not preserved correctly, and one 12 mo Hartley guinea pig was excluded from analysis because of a cytochrome c response. See methods and Figure S1 to see how these cytochrome c thresholds were established in line with best practices. *p<0.05

Figure 3.. PET and Hartley guinea pigs do not have age-related differences in submaximal or maximal complex I and II supported hippocampal mitochondrial respiration.

The SUIT 2 protocol examined complex I (glutamate, malate, and pyruvate) and complex II (succinate) submaximal and maximal mitochondrial respiration prior to examining non-coupled respiratory capacity. Panel A displays a raw tracing of the Oroboros O2k and supplemental table 2 reports the protocols in detail. Changes in oxygen consumption for each titration step are displayed in panel B. There were no age or strain differences in submaximal complexes I through IV 0.5 mM ADP phosphorylating conditions (C). Further, there were no differences in maximal 1 mM ADP (D) or supramaximal 6 mM ADP phosphorylating conditions (E). When rotenone was added to inhibit complex I and examine complex II through IV maximal respiration there were also no differences (F). There were also no differences in non-coupled mitochondrial respiration when FCCP was added to uncouple the electrochemical gradient (G). Finally, when examining complex IV specific respiration there were also no differences between age or strain (H). 5 mo PET n = 8; 5 mo Hartley n = 7; 12 mo PET n = 6; 12 mo Hartley n = 7 for all panels. The n-value is lower due to the necessity for the same technician to respire all samples and decrease variability in line with best practices. Further, O2k samples must be freshly respired decreasing the number of assays that can be performed in each day.

Figure 4.. Hartley guinea pigs have lower hippocampal complex I mitochondrial efficiency with age.

To understand where these changes lie in the mitochondrial reticulum the respiratory control ratio (RCR), as maximal state 3 (CI_P or CI+CII_P) respiration divided by state 2 (Leak) as an indirect measure of mitochondrial coupling efficiency, where a higher ratio is indicative of greater mitochondrial efficiency of oxygen consumption coupled to ATP production. We calculated this ratio in both SUIT 1 and SUIT 2 protocols and under both complex I and complex I and II phosphorylating conditions. Panels A and B were calculated from SUIT 1 where the maximal bolus of ADP was 8 mM and Panel C was calculated from SUIT 2 where the maximal bolus of ADP was 6 mM ADP. Additionally, in SUIT 1 pyruvate, glutamate, and malate were added prior to 10 sequential titrations to achieve ADP kinetics and then succinate was added; however, in SUIT 2 pyruvate, glutamate, malate, and succinate were added prior to the addition of ADP. Given the primary outcome of this study was not to determine RCR we did not plan titrations around testing this hypothesis. There was a significant age by strain interaction (p = 0.0035) in complex I RCR where 5 mo PET guinea pigs had a lower RCR compared to 5 mo Hartleys (p =0.009); however, Hartleys had a lower RCR at 12 mo compared to 5 mo suggesting a decrease in complex I efficiency (A). There was no significant interaction upon addition of succinate for RCR in SUIT 1; however, there was an overall effect of strain (p = 0.0370) and at 5 mo Hartleys had a higher RCR than PETs (p = 0.0276) (B). Finally, the RCR calculated from the SUIT 2 resulted in no significant differences in age or stain when examining the combined complex I and complex II RCR (C). SUIT 1 (Panels A and B) - 5 mo PET n = 12; 5 mo Hartley n = 11; 12 mo PET n = 11; 12 mo Hartley n = 10 for all panels. There was one statistical outlier in the 12 mo PET group. One 5 mo Hartley guinea pig sample was not preserved correctly, and one 12 mo Hartley guinea pig was excluded from analysis because of a cytochrome c response. SUIT 2 (Panel C) - 5 mo PET n = 8; 5 mo Hartley n = 7; 12 mo PET n = 6; 12 mo Hartley n = 7 for the panel. The n-value is lower due to the necessity for the same technician to respire all samples and decrease variability in line with best practices. *p<0.05, † p<0.001

Figure 5.. Hartley but not PET guinea pigs have higher hippocampal mitochondrial protein content with age.

An OXPHOS western blot quantified subunits for each of the 5 mitochondrial protein complexes. There was an overall effect of age or trend in complexes I (A), II (B), IV (D), and V (E) in that mitochondrial protein content was higher at 12 mo compared to 5 mo of age. The planned multiple comparison revealed trends suggesting that this higher mitochondrial content in 12 mo compared to 5 mo hippocampal samples was only in Hartleys and for complex I (p = 0.0718; A) and complex II (p = 0.0792; B). Representative OXPHOS blot and total protein stain (F). VDAC is the most abundant protein in the outer mitochondrial membrane, and we observed that there was a significant age by strain interaction and a trend for an effect of age (p = 0.0472) (G). This effect of age was driven by PETs having lower VDAC at 12 mo compared to 5 mo (p = 0.0464). For TOM20, another mitochondrial protein, there was a significant age by strain interaction (p<0.001) (H). Specifically, at 5 mo PETs had higher TOM20 than Hartleys (p = 0.0023) whereas at 12 mo Hartleys had higher TOM20 than PETs (p = 0.0006). Further PETs had lower TOM20 at 12 mo compared to 5 mo (p = 0.0004), and Hartleys had higher TOM20 protein content at 12 mo compared to 5 mo (p = 0.0062), paralleling the OXPHOS results. Representative VDAC and TOM20 blot and total protein stains (I). 5 mo PET n = 6; 5 mo Hartley n = 6; 12 mo PET n = 6; 12 mo Hartley n = 6 for panels A, B, C, D, E, and H. Panel G had 5 mo PET n = 6; 5 mo Hartley n = 5, 12 mo PET n = 6; 12 mo Hartley n = 6 as one 5 mo Hartley sample was an outlier for the TOM20 blot. *p<0.05, † p<0.01, ‡ p<0.001