Kenneth L. Moya

Cellular prion protein localization in rodent and primate brain

The presence of an abnormal, protease‐resistant form of the prion protein (PrP) is the hallmark of various forms of transmissible spongiform encephalopathies (TSE) which can affect a number of mammalian species, including humans. The normal, cellular form of this protein, PrPc, while abundant in brain is also present in many tissues and a number of species. In order to address the unresolved question of the precise localization of normal cerebral PrPc, we used a free‐floating immunohistochemistry procedure to localize the protein at both the light and the electron microscopic levels in the brain of three TSE‐sensitive species: hamster, macaque and humans. This method shows that PrPc is abundant in synaptic terminal fields in olfactory bulb, limbic‐associated structures and in the striato‐nigral complex, whereas many other regions of the hamster brain are essentially devoid of immunoreactivity. With the striking exception of the olfactory nerve, in which axons are continually growing throughout life, PrPc is not abundant in fibre pathways. PrPc distribution in the primate hippocampus and cortex is very similar to the distribution observed in hamster. PrPc was present at synaptic profiles as shown by immunoelectron microscopy, but was not detectable in neuronal perikaryon either by light or electron microscopy. Our results show that PrPc is abundant in a number of brain structures known for ongoing plasticity, and are consistent with the hypothesis that the protein also plays a role in synaptic function.

High expression and anterograde axonal transport of aminoterminal sonic hedgehog in the adult hamster brain

Sonic hedgehog (SHH) is considered to play an important role in tissue induction and patterning during development, particularly in determining neuronal cell fate in the ventral neural tube and in the embryonic forebrain. SHH precursor is autoproteolytically cleaved to an aminoterminal fragment (SHHN) which retains all known SHH biological activities. Here, we demonstrate the expression of a 22‐kDa SHHN immunoreactive peptide in developing and adult hamster brain regions using a rabbit antiserum directed against a mouse SHHN fragment. Interestingly, SHHN was developmentally regulated with the highest expression observed in the adult brain, was resistant to Triton X‐100 solubilization at 4 °C and partitioned with the raft component ganglioside GM1 during density gradient centrifugation. In rat brain, Shh transcripts were identified by double in situ hybridization in GABAergic neurons located in various basal forebrain nuclei including globus pallidus, ventral pallidum, medial septum‐diagonal band complex, magnocellular preoptic nucleus and in cerebellar Purkinje cells as well as in motoneurons of several cranial nerve nuclei and of the spinal cord. We show that radiolabelled SHHN peptides are synthesized in the adult hamster retina and are transported axonally along the optic nerve to the superior colliculus in vivo. Our data indicate that SHHN is associated with cholesterol rich raft‐like microdomains and anterogradely transported in the adult brain, and suggest that the roles of this extracellular protein are more diverse than originally thought.

Engrailed protects mouse midbrain dopaminergic neurons against mitochondrial complex I insults

Mice heterozygous for the homeobox gene Engrailed-1 (En1) display progressive loss of mesencephalic dopaminergic (mDA) neurons. We report that exogenous Engrailed-1 and Engrailed-2 (collectively Engrailed) protect mDA neurons from 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a mitochondrial complex I toxin used to model Parkinsons disease in animals. Engrailed enhances the translation of nuclearly encoded mRNAs for two key complex I subunits, Ndufs1 and Ndufs3, and increases complex I activity. Accordingly, in vivo protection against MPTP by Engrailed is antagonized by Ndufs1 small interfering RNA. An association between Engrailed and complex I is further confirmed by the reduced expression of Ndufs1 and Ndufs3 in the substantia nigra pars compacta of En1 heterozygous mice. Engrailed also confers in vivo protection against 6-hydroxydopamine and α-synuclein-A30P. Finally, the unilateral infusion of Engrailed into the midbrain increases striatal dopamine content, resulting in contralateral amphetamine-induced turning. Therefore, Engrailed is both a survival factor for adult mDA neurons and a regulator of their physiological activity.

Developmental expression of the cellular prion protein in elongating axons

PrPc, a sialoglycoprotein present in the normal adult hamster brain, is particularly abundant in plastic brain regions but little is known about the level of expression and the localization of the protein during development. Western blot analysis of whole brain homogenates with mab3F4 show very low levels of the three main molecular weight forms of the protein at birth, in contrast to the strong and wide expression of mRNA transcripts. The PrPc levels increase sharply through P14 and are diminished somewhat in the adult. Regional analysis showed that in structures with ongoing growth or plasticity such as the olfactory bulb and hippocampus, PrPc remains high in the adult, while in areas where structural and functional relationships stabilize during development, such as the cortex and the thalamus, PrPc levels decline after the third postnatal week. In the neonate brain PrPc was prominent along fiber tracts similar to markers of axon elongation and in vitro experiments showed that the protein was present on the surface of elongating axons. PrPc is then localized to the synaptic neuropil in close spatio‐temporal association with synapse formation. The localization of PrPc on elongating axons suggests a role for the protein in axon growth. In addition, the relative abundance of the protein in developing axon pathways and during synaptogenesis may provide a basis for the age‐dependent susceptibility to transmissible spongiform encephalopathies.

Immunolocalization of the cellular prion protein in normal brain.

We examined the localization of PrPc in normal brain using free‐floating section immunohistochemistry and monclonal antibody 3F4. In the mature hamster and baboon brain, PrPc is localized to the neuropil with a synaptic distribution and the PrPc immunoreactivity is denser in regions known for ongoing plasticity. Cell bodies and major fiber tracts have little or no PrPc immunoreactivity. At the electron microscopic level, PrPc immunoreactivity decorates synaptic profiles, both pre‐ and postsynaptically. Results obtained with two additional antibodies, 3B5 and Pri‐304, showed similar patterns of PrPc bands on Western blots, although Pri‐304 was less sensitive. On sections through the adult hamster hippocampus, 3B5 and Pri‐304 both stained the synaptic neuropil while cell bodies in the pyramidal and dentate granule cell layers were not immunoreactive. Pri‐304 differentiated between synaptic layers in the hippocampus and closely resembled the pattern of staining obtained with 3F4. Preliminary results of developing brain showed that PrPc is initially localized along fiber tracts in the neonate brain. These results show that PrPc has a synaptic distribution in the adult brain and suggest that there are important changes in its distribution during brain development. These results also characterize two additional reagents for studies of PrPc localization. Microsc. Res. Tech. 50:58–65, 2000.

Enhanced detection and retrograde axonal transport of PrPc in peripheral nerve

Neuroinvasion of the CNS during orally acquired transmissible spongiform encephalopathies (TSEs) may involve the transport of the infectious agent from the periphery to the CNS via the peripheral nerves. If this occurs within axons, the mechanism of axonal transport may be fundamental to the process. In studies of peripheral nerve we observed that the cellular prion protein (PrPc) is highly resistant to detergent extraction. The implication of this is an underestimation of the abundance of PrPc in peripheral nerve. We have developed nerve extraction conditions that enhance the quantification of the protein in nerve 16‐fold. Application of these conditions to evaluate the accumulation of PrPc distal to a cut nerve now reveals that PrPc is retrogradely transported from the axon ending. These results provide a potential cellular mechanism for TSE infectivity to gain entry to the CNS from the periphery.

The potential role of tau protein O-glycosylation in Alzheimer's disease

Single O-linked N-acetylglucosamine (O-GlcNAc) sugar residues can compete with phosphate groups to occupy specific sites on certain nuclear and cytosolic proteins. Here we show that inhibiting cellular kinase activities resulted in changes in protein O-glycosylation levels in heat-stable cytoskeletal protein fractions derived from primary neuronal cells. As increased phosphorylation of the microtubule-associated protein tau is one of the pathological hallmarks of Alzheimers disease and glycosylation may play an influential role in this process. We observed a significant decrease in the protein O-GlcNAc glycosylation of a tau-enriched cytoskeletal fraction generated from AD post-mortem brain samples as compared with control, suggesting an inverse relationship between the two post-translational modifications. Finally, cells transfected with the cDNA coding for O-GlcNAc transferase (OGT) displayed altered tau phosphorylation patterns as compared with control cells, suggesting that changes in tau glycosylation may influence its phosphorylation state. The specificity of the changes in the phosphorylation of individual amino acid residues provides evidence for a targeted O-glycosylation of tau.

Engrailed homeoprotein recruits the adenosine A1 receptor to potentiate ephrin A5 function in retinal growth cones.

Engrailed 1 and engrailed 2 homeoprotein transcription factors (collectively Engrailed) display graded expression in the chick optic tectum where they participate in retino-tectal patterning. In vitro, extracellular Engrailed guides retinal ganglion cell (RGC) axons and synergises with ephrin A5 to provoke the collapse of temporal growth cones. In vivo disruption of endogenous extracellular Engrailed leads to misrouting of RGC axons. Here we characterise the signalling pathway of extracellular Engrailed. Our results show that Engrailed/ephrin A5 synergy in growth cone collapse involves adenosine A1 receptor activation after Engrailed-dependent ATP synthesis, followed by ATP secretion and hydrolysis to adenosine. This is, to our knowledge, the first evidence for a role of the adenosine A1 receptor in axon guidance. Based on these results, together with higher expression of the adenosine A1 receptor in temporal than nasal growth cones, we propose a computational model that illustrates how the interaction between Engrailed, ephrin A5 and adenosine could increase the precision of the retinal projection map.

Inhibition of N-glycan processing alters axonal transport of synaptic glycoproteins in vivo.

The principal aim of this study was to examine the relative contributions from the neuronal and endothelial isoforms of nitric oxide synthase (nNOS and eNOS, respectively) in their capacity to modulate intra-ischemic cerebral blood flow (CBF) changes, in the ischemically vulnerable hippocampus and striatum. CBF changes were monitored, using laser-Doppler flowmetry, in rats subjected to 30 min of forebrain ischemia (right common carotid occlusion+hemorrhagic hypotension). Rats were pretreated with a selective nNOS inhibitor (ARR 17477), a NOS inhibitor that blocks both eNOS and nNOS (N(G)-nitro-L-arginine; L-NNA), or saline (control). In initial experiments, where ischemic MABP was targeted to exactly 30 mmHg, NOS inhibition reduced intra-ischemic cortical CBF from the control level of approximately 20% of baseline to 3% (L-NNA) or 6% (ARR 17477) of baseline. The statistically similar effects of the two NOS inhibitors confirmed that nNOS is the predominant NO source supporting intra-ischemic vasodilation in the cortex. In subsequent experiments, CBF was measured in the right hippocampus, and striatum, as well as the cortex, and, to reduce data variability, blood withdrawal was adjusted to achieve an intra-ischemic cortical CBF of 20% (controls) or 5% (NOS inhibited rats) of baseline. In those groups, mean ischemic MABP levels ranged from 28 to 32 mmHg. In controls, intra-ischemic CBF fell to 20%, 45%, and 47% of baseline in the cortex, hippocampus, and striatum, respectively. With nNOS inhibition, intra-ischemic CBF was further reduced to 5%, 15%, and 18% of baseline, respectively. However, with combined eNOS/nNOS inhibition, the CBF values were 5%, 37%, and 21%, respectively. These results suggest that the nNOS contribution to intra-ischemic vasodilation in vulnerable regions is substantially greater than eNOS. The significantly higher intra-ischemic CBF level in the hippocampus in combined eNOS/nNOS vs nNOS-inhibited rats may relate, in contrast to other regions, to a low eNOS influence on vascular function in that structure and CBF redistribution to the hippocampus when eNOS activity is blocked globally.&NA; Synaptic glycoproteins are synthesized and glycosylated in the neuronal cell body, and conveyed to terminals by fast axonal transport. We used the α‐mannosidase inhibitor, 2‐deoxyman‐nojirimycin (dMan), to investigate the effects of disrupting N‐glycan processing on the axonal trafficking of proteins in vivo. dMan significantly reduced rapid axonal transport in retinal ganglion cells to about 34% of control values 4 h after metabolic labeling; at 8 h post‐labeling the inhibition was reversed. 2‐D gel analysis showed that dMan completely inhibited the arrival of radiolabeled L1 and NCAM at axon terminals, and resulted in the appearance of two novel proteins of 230 kDa and 155 kDa. Our results show that disruption of the N‐glycosylation pathway has an immediate inhibitory effect on total axonal transport and longer lasting effects on the trafficking of specific glycoproteins to axon terminals in vivo.

A novel cellular prion protein isoform present in rapid anterograde axonal transport

We studied the axonal transport of PrP(C) in hamster retinal and sciatic nerve axons. Our results show that a novel 38kDa form is the predominant form in rapid anterograde axonal transport while the 36kDa and 33kDa PrP(C) forms, abundant in nerve and brain, appear to be either stationary or slowly transported. We did not detect any significant retrograde transport of PrP(C). These results show that 38kDa PrP(C) is the form exported from the cell body to the axonal compartment where it may represent the precursor to the more abundant PrP(C) forms after its modification in nerve fibres or terminals.