No abstract

CA2 is an understudied subregion of the hippocampus that is critical for social memory. Previous studies identified multiple components of the mitochondrial calcium uniporter (MCU) complex as selectively enriched in CA2. The MCU complex regulates calcium entry into mitochondria, which in turn regulates mitochondrial transport and localization to active synapses. We found that MCU is strikingly enriched in CA2 distal apical dendrites, precisely where CA2 neurons receive entorhinal cortical input carrying social information. Furthermore, MCU-enriched mitochondria in CA2 distal dendrites are larger compared to mitochondria in CA2 proximal apical dendrites and neighboring CA1 apical dendrites, which was confirmed in CA2 with genetically labeled mitochondria and electron microscopy. MCU overexpression in neighboring CA1 led to a preferential localization of MCU in the proximal dendrites of CA1 compared to the distal dendrites, an effect not seen in CA2. Our findings demonstrate that mitochondria are molecularly and structurally diverse across hippocampal cell types and circuits, and suggest that MCU can be differentially localized within dendrites, possibly to meet local energy demands.

CA2 is an understudied subregion of the hippocampus that is critical for social memory. Previous studies identified multiple components of the mitochondrial calcium uniporter (MCU) complex as selectively enriched in CA2, however the functional significance of this enrichment remains unclear. The MCU complex regulates calcium entry into mitochondria, which in turn regulates mitochondrial transport and localization to active synapses. We found that MCU is strikingly enriched in CA2 distal apical dendrites, precisely where CA2 neurons receive entorhinal cortical input carrying social information. Further, MCU-enriched mitochondria in CA2 distal dendrites are larger compared to mitochondria in CA2 proximal apical dendrites and neighboring CA1 apical dendrites. Genetic knockdown of MCU in CA2 resulted in smaller mitochondria in CA2 distal dendrites, indicating that MCU expression plays a role in regulating mitochondrial mass in CA2. MCU overexpression in neighboring CA1 led to larger mitochondria preferentially in proximal dendrites compared to distal dendrites and GFP controls. Our findings demonstrate that mitochondria are molecularly and structurally diverse across hippocampal cell types and circuits, and that MCU expression cell-autonomously regulates mitochondrial mass, but layer-specific dendritic localization depends on cell type. Our data support the idea that CA2 mitochondria are functionally distinct from CA1 mitochondria, which may confer unique synaptic and circuit properties underlying CA2 function in social memory.

Protein expansion microscopy (proExM) is a powerful technique that crosslinks proteins to a swellable hydrogel to physically expand and optically clear biological samples. The resulting increased resolution (~70 nm) and physical separation of labeled proteins make it an attractive tool for studying the localization of subcellular organelles in densely packed tissues, such as the brain. However, the digestion and expansion process greatly reduces fluorescence signals making it necessary to optimize ExM conditions per sample for specific end goals. Here we describe a proExM workflow optimized for resolving subcellular organelles (mitochondria and the Golgi apparatus) and reporter-labeled spines in fixed mouse brain tissue. By directly comparing proExM staining and digestion protocols, we found that immunostaining before proExM and using a Proteinase K based digestion for 8 hours consistently resulted in the best fluorescence signal to resolve subcellular organelles while maintaining sufficient reporter labeling to visualize spines and trace individual neurons. With these methods, we more accurately quantified mitochondria size and number and better visualized Golgi ultrastructure in reconstructed CA2 neurons of the hippocampus.

Severe malaria occurs most in young children but is poorly understood due to the absence of a developmentally-equivalent rodent model to study the pathogenesis of the disease. Though functional and quantitative deficiencies in innate response and a biased T helper 1 (Th1) response are reported in newborn pups, there is little information available about this intermediate stage of the adaptive immune system in murine neonates. To fill this gap in knowledge, we have developed a mouse model of severe malaria in young mice using 15-day old mice (pups) infected with Plasmodium chabaudi. We observe similar parasite growth pattern in pups and adults, with a 60% mortality and a decrease in the growth rate of the surviving young mice. Using a battery of behavioral assays, we observed neurological symptoms in pups that do not occur in infected wildtype adults. CD4 + T cells were activated and differentiated to an effector T cell (Teff) phenotype in both adult and pups. However, there were relatively fewer and less terminally differentiated pup CD4 + Teff than adult Teff. Interestingly, despite less activation, the pup Teff expressed higher T-bet than adults' cells. These data suggest that Th1 cells are functional in pups during Plasmodium infection but develop slowly.

Malaria and other chronic infections generate effector and effector memory T cells, but the infection is not completely controlled. While malnutrition has long been suggested to be protective against malaria, recent studies have shown that malnourished children are more susceptible to malaria infections, with increased mortality rates. Current vaccination protocols result in immunity that decays, but the influence of malnutrition in the target population has not been explored. To better understand how malnutrition affects the development of protective T cells in this infection, we used a diet of moderate malnutrition to determine the activation of effector (Teff), central memory (Tcm) and effector memory (Tem) T cells. C57Bl/6 mice were fed on either protein sufficient (16%) or moderately malnourished diets (3% protein, and deficient in both Iron and Zinc) for 5 weeks. Upon infection, we observed a significant decrease in the number of effector cells at day 7 post-infection. This was accompanied by a significant drop in weight among the malnourished mice. Surprisingly, there was no significant deference in parasitemia by day 7p.i. At the memory timepoints (d60 p.i), we observed that the spleens of the malnourished mice were much smaller than the well-nourished control mice. There was a significant drop in the number of CD4 T cells and Tcm but the proportion of Tem increased in the malnourished mice and a lower cell number due to the few total CD4 T cells in the malnourished group. Our data indicate that the nutritional status of the host affect the pool of memory CD4 T cells generated in malaria infection. This could be one mechanism as to why T cell immunity decays in malaria patients as the disease is prevalent in resource-limited nations.

The immune system plays an essential role in the elimination of malaria parasites. CD4+ T cells are imperative in the immune response to the infection, but immune response to malaria especially in young children is poorly understood, due to a lack of a better young animal model to study the pathogenesis of the disease. We have developed a young mouse (pup) model using day 15 pups to help understand the pathogenesis, and development of protective CD4 T cells in malaria infection. Here, we determined the ability of pup cells to protect immunocompromised RAGKO mice from malaria infection. We infected day 15 old pups with Plasmodium chabudi at 1×105 iRBCs, and transferred splenocytes to RAGKO mice on day 8 post-infection. RAGKO mice were then infected. Mice that received pup cells looked healthier and active throughout the experiment, compared to their counterparts who received adult spleen cells. During the peak of infection, mice that received adult cells lost more weight than the pup cell recipients. On day 60 post-transfer, we observed same numbers of CD4 T cells, central memory and effector memory T cells. There were significantly lower proportions and numbers of IFNγ and TNFα positive cells from pups compared to adult cells. We next determined if there was any difference in the proliferation of CD4 T cells and all splenocytes in pup cells. We purified CD4+ T cells using EasySep kit and cultured them on αCD3/CD28 pre-coated plates, compared to all splenocytes. There was a significant growth in purified CD4 T cells from adult than pup cells. Interestingly, all splenocytes from pups proliferated better than adults. Therefore, our data suggest that pup cells protect RAGKO mice from death, but do not develop into functional memory due to poor proliferation.

The immune system plays an important role in the elimination of Plasmodium parasites that cause malaria, which affect children the most worldwide. Immunity to malaria, especially in young children is poorly understood due to the absence of a developmentally-equivalent rodent model to study the pathogenesis of disease. We have developed a mouse model using 15-day old mice (pups) of malaria infection in neonatal mice. Using C57BL/6 pups, we determined that P. chabaudi infection decreases the growth rate of young mice compared to controls, and results in 60% mortality, and neurological damage not present in adults, as indicated by a battery of behavioral assays. When all splenic cells were stimulated in vitro stimulation, cells from pups proliferated faster than adult cells, but purified CD4 T cells were slower. Upon infection with Plasmodium parasites, both adult and pup CD4+ T cells were activated and differentiated to an effector T cell (Teff) phenotype; however, pup CD4+ Teff were less differentiated than adult Teff. Pup CD4+ T cells also produced more IL-2 than cells from adult B6 mice, and TNF-a was increased in parasite-specific BALB/c pup T cells. Interestingly, there were more pup CD4+T-bethi Teff after infection suggestive of increased Th1 commitment, potentially contributing to cerebral symptoms.