EPO mediated enhancement in synaptic plasticity

Several reports have successfully demonstrated enhanced neurogenesis in the hippocampus due to various factors such as VEGF, NT-3, Notch ligand Delta-like 1, IL-1b, IL-6, FGF-2 (Barkho et al., 2006; Delgado et al., 2014; Faigle & Song, 2013; Kang &

Hebert, 2015; Kawaguchi et al., 2013). However, the critical question often raised in the field consists of doubting the extent of positive effect of a few newly generated neurons on functional consequences. Many have reported that a small number of newly generated neurons can influence behavior as demonstrated by stimulation of

DEBIA WAKHLOO 132 single pyramidal neurons in the rat motor cortex. These newly generated neurons could also induce whisker movements (Brecht et al., 2004; Shadlen et al., 1996).

Enhanced neurogenesis and synaptic integration together could contribute to positive effects observed at functional or behavioral level. Unstimulated neurogenesis is followed by an early survival phase, where the newly generated neurons show dramatic decline in their numbers within the first few days (Biebl et al., 2000; Kuhn et al., 2005). During this phase, majority of the cells are eliminated well before they could make functional connections in the target regions of the hippocampus (Kempermann et al., 2015). However, it is essential that the surviving neurons integrate into the existing architecture of the hippocampus by the formation of synaptic connections with the pre-existing neurons. The initiation of dendrite and spine development generally follows the process of axonal elongation (Sun et al., 2013). Once both these processes are underway, the newly generated neurons are said to be integrated and functional active. During normal neurogenesis in a healthy brain, a large proportion of neural progenitors undergo apoptosis within their first days. By rapidly removing cell debris, the scavenging properties of microglia could have an important regulatory role at early stages of neurogenesis (Sierra et al., 2010). However, it is not completely understood whether the phagocytosis is beneficial for the surrounding newborn neurons by reducing pro-inflammatory mediators, or whether they are detrimental by further inducing apoptotic neuronal death (Magnus et al., 2001; Neher et al., 2011).

Using transgenic mouse line (Thy1-YFP), which specifically labels dendrites and spines of pyramidal neurons, a significant increase in the density of dendrites as well as spines was observed. This result could serve as another mechanism of augmented network function and therefore strengthen the evidence of cognitive improvement upon EPO administration. This observation is in accordance with reports suggesting increase in dendritic length and complexity appears to be mediated by a cell autonomous, autocrine action of another neurotrophic factor BDNF (Wang et al., 2015). In our findings, based on their morphology, the increase in the density of spines is constituted by an increase in stubby spines, which are immature in nature. The increase in immature stubby spines supports the hypothesis that the newly generated neurons form new spines, which are yet to be mature. This maturation trend is similar to that

DEBIA WAKHLOO 133 observed with trajectory analysis in neurons, which demonstrated that these neurons are present in continuum, and are driven to maturity upon EPO administration. It is very likely, that upon appropriate cognitive stimulation, these immature stubby spines would mature into stable mushroom spines. However, evidence that this is the case, is currently the ongoing work.

Though likely, it is not established how finely tuned cross talk between microglia and adult born neurons would function and how it leads to any changes upon microglia activation. Previous work from our group also has hinted to the effect of EPO, where EPO treatment was shown to dampen microglial activity (Mitkovski et al., 2015). In our present results, along with neuronal and dendritic spine increase, a reduction in the number of microglia upon EPO treatment was observed. This decrease in microglia was also corroborated by single cell RNA-Sequencing. These results together point towards enhanced neurogenesis and maybe the newly differentiated neurons do not require excessive pruning as they begin to form synaptic connections. Our findings are in accordance with Paolicelli and colleagues, where they observed a transient decrease in microglia in the first to fourth postnatal week in CXCR1 knockout mice and a corresponding increase in the dendritic spine density (Paolicelli et al., 2011). An explanation could be attributed to the previously stated concept of ‘pro-neurogenic’

function of microglia. Disruption of the microglia expressed receptor CX3CR1 pathway in young rodents’ decreased survival and proliferation of hippocampal neural progenitor cells (Bachstetter et al., 2011). The other hypothetical reasoning could be that the microglia in the presence of EPO transdifferentiate and give rise to some newly generated neurons in the CA1. The microglial transdifferentiation as compared to the astrocytic transdifferentiation has not been extensively studied; however, some reports demonstrate that using 70% fetal bovine serum upregulates Sox2 in microglia, which transforms them into Map2 positive neurons (Nonaka et al., 2008; Zhang et al., 2013). Our current ongoing work will attempt to shed light on this matter. On a rodent behavioral level, the increase in the number of neurons and their synaptic connections might contribute to the brain function via alteration in the structural properties of the circuitry. Enhanced synaptic plasticity is also essential for mediating pattern separation in memory formation and cognition (Clelland et al., 2009; Nakashiba et al., 2012; Sahay

DEBIA WAKHLOO 134 et al., 2011). However, on an over-arching human level, these results may provide explanation of the MRI-documentable increase in the dimensions of certain brain areas (Hassouna et al., 2016) or the reduction of brain matter loss upon EPO treatment in mental illness (Miskowiak et al., 2015; Wustenberg et al., 2011).