Hippocampal synaptic connectivity in phenylketonuria

Abstract

In humans, lack of phenylalanine hydroxylase (Pah) activity results in phenylketonuria (PKU), which is associated with the development of severe mental retardation after birth. The underlying mechanisms, however, are poorly understood. Mutations of the Pah gene in Pah(enu2)/c57bl6 mice result in elevated levels of phenylalanine in serum similar to those in humans suffering from PKU. In our study, long-term potentiation (LTP) and paired-pulse facilitation, measured at CA3-CA1 Schaffer collateral synapses, were impaired in acute hippocampal slices of Pah(enu2)/c57bl6 mice. In addition, we found reduced expression of presynaptic proteins, such as synaptophysin and the synaptosomal-associated protein 25 (SNAP-25), and enhanced expression of postsynaptic marker proteins, such as synaptopodin and spinophilin. Stereological counting of spine synapses at the ultrastructural level revealed higher synaptic density in the hippocampus, commencing at 3 weeks and persisting up to 12 weeks after birth. Consistent effects were seen in response to phenylalanine treatment in cultures of dissociated hippocampal neurones. Most importantly, in the hippocampus of Pah(enu2)/c57bl6 mice, we found a significant reduction in microglia activity. Reorganization of hippocampal circuitry after birth, namely synaptic pruning, relies on elimination of weak synapses by activated microglia in response to neuronal activity. Hence, our data strongly suggest that reduced microglial activity in response to impaired synaptic transmission affects physiological postnatal remodelling of synapses in the hippocampus and may trigger the development of mental retardation in PKU patients after birth.

Altered DNA methylation in PAH deficient phenylketonuria

“…Down-regulation of SLC6A9 and KCNA1 may impact ion balance in astrocytes. Several proteins either involved in synaptic function or known to reside in the synapse are downregulated including: VAMP8 [28], SNAP25 [29], SYT2 [35], SLC10A4 [27], and GABRA1 [36]. These findings are congruent with those of Horling et al [29] that recently described synaptic dysfunction in PKU.…”

“…Several proteins either involved in synaptic function or known to reside in the synapse are downregulated including: VAMP8 [28], SNAP25 [29], SYT2 [35], SLC10A4 [27], and GABRA1 [36]. These findings are congruent with those of Horling et al [29] that recently described synaptic dysfunction in PKU. Dysregulation of these synaptic genes along with GFAP in astrocytes has the potential to impact all members of the so-called tripartite synapse [37].…”

“…However, in the PHE enu2 mouse model, no additional data are available relative to functional significance of these findings. Overall data generated in this study along with those of Horling et al [29] suggest that synaptic dysfunction via neurons or glia participates in PKU neural dysfunction. Follow-up brain disassociation studies should prove useful in allowing methylation and expression studies in more homogeneous cell populations (oligodendrocytes, astrocytes, neurons).…”

“…SNAP 25 is downregulated (see Table 2B) but not differentially methylated. Down-regulation of SNAP25 was recently described in hippocampal synaptic connectivity in the PAH enu2 mouse [29]. SYT2 is a synaptotagmin and also part of the SNARE complex.…”

Rescue of Synaptic Phenotypes and Spatial Memory in Young Fragile X Mice

“…Although histochemical levels of postsynaptic density (PSD)-95 or synaptophysin have been found to correspond well with those detected at the biochemical levels (Glantz et al, 2007;Horling et al, 2015), Western blots measure the total protein levels in the tissue and may have results that are different from histochemistry at a single structure or cell level, owing to protein expression or transport to a specific structure (Hongpaisan et al, 2013). Our study, however, focused on the structure changes, and we thus used immunohistochemistry to confirm the morphologic studies using electron microscopy (see below) and confocal microscopy of DiI staining.…”