The elevated neuronal MHCI expression in NSE-Db
mice is associated with lower levels of synaptic markers and fewer neurons in some regions of their hippocampus, as well as a smaller area of contralateral retina projections in their dLGN 
. The current studies were aimed at further understanding how elevated neuronal MHCI levels affect neuronal connections using an in vitro
embryonic retina-thalamic co-culture system. In wildtype thalami, MHCI expression is up-regulated postnatally and is activity-dependent 
. We observed that embryonic retina explants that were cultured near to a embryonic wildtype thalamus extended neurites which formed connections the thalami, as expected. In contrast, there was a conspicuous lack of neurite outgrowth from retina explant regions that were proximal to thalamic explants from NSE-Db
mice, suggesting that this thalamic tissue released a factor that inhibited nearby neurite outgrowth. Since our previous studies showed that recombinant MHCI monomers (but not heavy chain alone or β2M alone) inhibited neurite outgrowth in vitro 
, we surmised that the NSE-Db
thalami were producing sMHCI which inhibited neurite outgrowth from neighboring neurons. Indeed, in other tissues, upregulated MHCI expression leads to increased release of sMHCI 
. However, it was also possible that the transgene in some way reduced thalamic secretion of neurotrophic factors or induced the expression of a soluble inhibitory factor(s) other than sMHCI. We observed that neurite outgrowth could be rescued in large part by adding a conformation-dependent anti-Db
mAb to these cultures, pointing to diffusible conformationally correct Db
sMHCI as the inhibitory factor released from NSE-Db
We also examined how retina neurites interacted with monkey COS cells that were programmed to express a full-length Db cDNA. FACS analysis with a conformation-dependent mAb confirmed the presence of Db-MHCI on their cell surface. While retina neurites made contacts with a nearby COS cell aggregates transfected with a control plasmid, they had little neurite outgrowth from regions near to Db-expressing COS cell aggregates and made few contacts with these cells. These studies show that transfected cells that differ in their expression of a full-length mouse MHCI heavy chain cDNA differentially affect neurite outgrowth from nearby neurons.
Our studies with COS cell-produced sMHCI(1–298) showed that sMHCI(1–298) inhibited neurite outgrowth at 50–100 pM, a level similar to that of recombinant Db
-MHCI monomers loaded with a particular mouse self-peptide 
. The Db
-sMHCI released from the transfected COS cells present a vast array of different peptides from monkey cell proteins. This provides another line of evidence indicating that RGC MHCI receptors are not sensitive to the peptide presented by MHCI. Because of this lack of specificity for the peptide presented, any conformationally correct sMHCI is a potential ligand, which may be why neurons are sensitive to such low levels of sMHCI. Notably, little or no sMHCI is detected in CSF of healthy individuals 
indicating that the blood brain barrier effectively blocks sMHCI in peripheral blood from entering the CNS. Consequently, the level of MHCI expression in a particular brain region may be the major regulator of local sMHCI levels.
In the immune system, T cells that engage soluble MHCI, or membrane-bound MHCI without sufficient co-stimulatory signals, enter a state of inactivity (anergy) or can apoptose 
. T cell inactivation by MHCI can be prevented by blocking the T cell receptor's signaling cascade with an antagonist (Rp-cAMPS) of cAMP-dependent PKA 
. Similarly, sMHCI down-regulates NK cell function or induces NK cell apoptosis 
, and compounds that elevate cAMP suppress both NK and dendritic cell function 
. We found that Rp-cAMPS itself had no discernable effect on retinal neurite outgrowth in vitro.
However, its inclusion in cultures containing sMHCI(1–298) prevented the inhibitory effects of sMHCI(1–298) on neurite outgrowth. Evidently, sMHCI can inhibit cellular functions by pathways that involve PKA in both the nervous and immune systems. Conceivably, the neuroinhibitory activity of sMHCI that we have observed may be a neurological counterpart of its inhibitory action on NK cells or T cells that engage sMHCI or membrane-bound MHCI in the absence of co-stimulatory signals.
Our observations concerning sMHCI, together with the abnormalities observed in NSE-Db
mouse neurodevelopment 
, suggest several new possibilities concerning MHCI's neurobiological activities. Neuronal MHCI expression in the thalamus peaks during the period of activity-dependent remodeling 
. Studies with other tissues have shown that increased MHCI expression is accompanied by increased release of sMHCI 
. This could be due to increased shedding of intact MHCI, increased heavy chain transcription leading a concomitant increase in alternatively spliced transcripts and/or more substrate for the action of proteases. After appropriate connections are formed, elevated membrane-bound MHCI and sMHCI levels may help limit axon outgrowth into that region and additional synaptogenesis. Indeed, we have observed that the NSE-Db
mice have reduced synaptic markers in some hippocampal regions and are very deficient in compensatory neuronal sprouting responses after a hippocampal lesion 
. The sMHCI might bind to neuronal MHCI receptors in ways that activate receptor signaling, or conversely, prevent the MHCI receptor from binding to membrane-bound MHCI. Additionally, MHCI receptor interaction with sMHCI without the co-stimulatory signals that accompany MHCI receptor interaction with membrane-bound MHCI, may lead to different biological outcomes. Alternatively, like some other axon guidance molecules, MHCI and sMHCI may have bi-functional effects, exerting either negative or positive influences on axon growth depending on the context (e.g., their concentration, the levels of cyclic nucleotides, and the stage in development) 
Interestingly, mice deficient in complement protein C1q or C3 have impairments in retinogeniculate synapse elimination, similar to that seen in MHCI-deficient mice 
, suggesting that complement acts to tag supernumerary synapses for removal by phagocytotic mechanisms. Complement proteins can be synthesized by neurons and glia and their expression peaks during activity dependent remodeling 
. C1q combines with serine esterases C1r and C1s to form the C1 protein. Notably, C1 can associate with β2M and its serine esterase activity can cleave MHCI between its α2 and α3 domains, releasing a biologically active sMHCI consisting of the α1 and α2 domains associated with β2M 
. C3 also contributes to the formation of proteases. Both C1q expression and neuronal MHCI expression peak in the LGN during activity-dependent remodeling 
. The concurrent upregulation of both MHCI and complement protein expression could synergistically elevate sMHCI levels in the microenvironment which we have shown can act in trans
to inhibit neurite outgrowth (
and herein). Such complement cleavage would also reduce MHCI cis
-associations with other molecules on the same cell surface. Additionally, MHCI molecules can dissociate, leaving free heavy chains on the cell surface which can associate in cis
with other free heavy chains and receptors on the same cell surface and modulate cellular functions (reviewed in 
). Complement cleavage of free heavy chains could reduce these cis
-associations as well. Importantly, our recent studies suggest that the extent of MHCI cis
interactions on the neuronal cell surface modulate axon outgrowth and polarity (Tina Bilousova and DLK, manuscript in preparation). Cleavage also leaves behind MHCI's α3 domain which can interact with some classical MHCI receptors of the innate immune system 
. Further studies are needed to address these possibilities, but are hampered by the lack of nucleic acid probes and antibodies that can distinguish sMHCI and membrane-bound MHCI in tissue sections.
Neuronal MHCI and complement protein expression are upregulated in response to various insults (e.g. seizures, injury, cytokines and other inflammatory signals) 
. Our results in the current study, together with our previous observations of abnormalities in neuronal connections and neurorepair responses in the NSE-Db
mouse CNS suggest that such insults may increase local levels of MHCI and sMHCI which could affect neurodevelopment and neurorepair responses. It is of interest that the HLA region has been genetically associated with schizophrenia and autism in humans 
. If MHCI and/or sMHCI are found to have potential deleterious neurological effects, MHCI antibodies, soluble MHCI receptors or anti-inflammatory medications may useful to block these effects.