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present study1, deactivation within the default network decreased with slower response times, and presumably reflected a switch from internally to externally focused attention. The association of slower responses with greater default deactivation suggests that slower responses are associated with task- irrelevant processing during lapses of attention, rather than merely inefficient activation of the attentional control network. Lapses of attention may therefore involve the failure to switch from a focus on internal feelings and thoughts to the external task at hand. Functional connectivity analyses could be used to bolster this finding, as a lapse of attention should lead to both decreased activation in the prefrontal-parietal control network and increased deactivation in the default network12,13. The current results, however,

point to the necessity of understanding how multiple neural networks may compete for the internal versus external focus of attention.

Patients with right-hemisphere lesions are impaired on tasks of vigilance, with slowed response times on even simple tasks. Consistent with these data, activations associated with pre-stimulus focus and reorientation after a lapse of attention in the present study1 were all right lateralized. This work moves forward our understanding of the specific neural mecha-nisms that modulate the waxing and waning of human attention to the external world. In addi-tion to helping understand how people vary from focus to distraction, these findings may be useful in understanding the brain basis of dis-orders of attention, including attention-deficit hyperactivity disorder (ADHD) and deficits of attention after stroke or during aging.

1. Weissman, D.H., Roberts, K.C., Visscher, K.M. & Woldorff, M.G. Nat. Neurosci. 9, 971–978 (2006).

2. Navon, D. Cognit. Psychol. 9, 353–383 (1977).3. Collette, F. & Van der Linden, M. Neurosci. Biobehav.

Rev. 26, 105–125 (2002).4. Derfuss, J., Brass, M. & von Cramon, D.Y. Neuroimage

23, 604–612 (2004).5. Hampshire, A. & Owen, A.M. Cereb. Cortex, published

online January 25 2006 (doi:10.1093/cercor/bhj116).6. Wager, T.D. & Smith, E.E. Cogn. Affect. Behav. Neurosci.

3, 255–274 (2003).7. Buchel, C. & Friston, K.J. Cereb. Cortex 7, 768–778

(1997).8. Gazzaley, A., Cooney, J.W., McEvoy, K., Knight, R.T.

& D’Esposito, M. J. Cogn. Neurosci. 17, 507–517 (2005).

9. Astafiev, S.V., Shulman, G.L. & Corbetta, M. Eur. J. Neurosci. 23, 591–596 (2006).

10. Raichle, M.E. et al. Proc. Natl. Acad. Sci. USA 98, 676–682 (2001).

11. Greicius, M.D., Krasnow, B., Reiss, A.L. & Menon, V. Proc. Natl. Acad. Sci. USA 100, 253–258 (2003).

12. Lee, L., Friston, K. & Horwitz, B. Neuroimage 30, 1243–1254 (2006).

13. Rissman, J., Gazzaley, A. & D’Esposito, M. Neuroimage 23, 752–763 (2004).

Nitro-PDI incites toxic waste accumulationMark P Mattson

In Parkinson disease and related disorders, nitric oxide may disable PDI, an enzyme critical for proper protein folding in the endoplasmic reticulum, resulting in the accumulation of damaged proteins and eventually neuronal death.

Imagine your local garbage company going on strike. Pretty soon, the city would be clogged with trash, which would disrupt daily life. The situ-ation is similar inside cells; misfolded proteins are degraded by specific machinery in the cell and the components recycled. When this ‘gar-bage’ removal and recycling process goes awry, the consequences can be deadly. Neurons that succumb in Parkinson disease and Alzheimer disease contain abnormal accumulations of misfolded and aggregated proteins: α- synuclein and synphilin-1 in Parkinson disease, and amyloid-β peptide and tau in Alzheimer disease1. The proteins are synthesized and folded in the endoplasmic reticulum (ER), and, when misfolded or oxidatively damaged, are normally tagged for degradation in the proteasome by the addition of a ubiquitin group.

A common dogma is that in Parkinson disease and Alzheimer disease, cellular stress (metabolic, oxidative or ionic) can inter-fere with the proper folding of proteins and increase their susceptibility to damage, and can also impair proteasome function2,3. Although perturbed cellular Ca2+ homeostasis and oxi-

dative stress may contribute to ER and protea-some dysfunction, the underlying molecular mechanism is unknown. Now Uehara and col-leagues4, in a recent paper in Nature, show that a nitric oxide (NO)-mediated modification of protein disulfide isomerase (PDI, a specific ER protein) is critical for proper protein folding and that this modification is pivotal to the pathogenesis of Parkinson disease.

NO is a highly diffusible gas that acts as an intra- and intercellular messenger. It is implicated in a wide range of adaptive structural and functional changes in the nervous system, including synaptic plasticity5. NO is produced in response to Ca2+ influx or release from intracellular stores; Ca2+ binds to calmodulin, which then activates NO synthase (NOS; Fig. 1). Transient moderate levels of NO production regulate a variety of physiological processes in neurons by directly or indirectly inducing post-translational modifications of proteins. NO can interact with sulfhydryl groups in a reaction called nitrosylation; such a modification of NMDA glutamate receptors provides a feedback mechanism to reduce Ca2+ influx through the NMDA receptor channel6. Alternatively, NO activates soluble guanylate cyclase to produce cyclic guanosine monophosphate (cGMP), which then activates a kinase (cGMP-dependent protein kinase) that phosphorylates various

protein substrates, including those involved in synaptic plasticity and cell survival. However, prolonged production of large amounts of NO can wreak havoc on cellular macromolecules, a process believed to contribute to the death of neurons in various disorders, including stroke and Parkinson disease7. NO damages cells by interacting with a superoxide anion radical to produce the nitrogen radical peroxynitrite. Peroxynitrite promotes membrane lipid peroxidation and can also damage proteins by nitrating tyrosine residues. Increasing evidence also points to a role for NO in protein garbage accumulation and in the degeneration of dopaminergic neurons in Parkinson disease; for example, parkin, an E3 ubiquitin ligase, which is associated with early-onset Parkinson disease, is S-nitrosylated in animal models of the disease, which then causes the accumulation of parkin substrates such as α-synuclein8.

To determine the consequences of S- nitrosylation of PDI in neurons, Uehara et al. exposed cultured cerebrocortical neu-rons to a cytotoxic concentration of NMDA to induce Ca2+ influx and NO production. Under these conditions, PDI was S-nitrosylated in a NO-dependent manner, and there was a large increase in the amount of polyubiquitinated proteins in the neurons, consistent with increased accumulation of misfolded proteins. PDI is normally upregulated in response

The author is in the Laboratory of Neurosciences,

National Institute on Aging Intramural Research

Program, 5600 Nathan Shock Drive, Baltimore,

Maryland 21224, USA.

e-mail: mattsonm@grc.nia.nih.gov

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to various cellular stressors and can protect neural cells against ischemic injury9.

Uehera et al. found that overexpression of wild-type PDI reduced the amounts of polyubiquitinated proteins in the neurons and protected them from being killed by NMDA. Native PDI, but not S- nitrosylated PDI, also protected cells against death induced by the ER stressors tunicamycin (a glycosylation inhibitor) and thapsigargin (an inhibitor of the ER Ca2+-ATPase). They also found that PDI was excessively S- nitrosylated in brain tissue samples from patients with Parkinson disease and Alzheimer disease compared to age-matched control subjects. Moreover, exposure of cultured neurons to the dopaminergic neurotoxin rotenone induced S-nitrosylation of PDI. The authors then identified the cysteine residues in PDI that were nitrosylated and found that one of the cysteine residues is further oxidized to sulphinic acid, a chemical modification that is likely to irreversibly impair the function of PDI.

The authors went on to show that NO- mediated impairment of PDI is a critical event in the formation of Lewy bodies, the intracellular accumulations of misfolded ubiquitinated proteins, such as α-synuclein and synphilin-1, that are characteristic of Parkinson disease. They found that PDI can prevent intracellular aggregates of polyubiquitinated synphilin-1 from forming in human neural cells and that NO compromises this beneficial function of PDI. Collectively, these findings suggest a specific molecular mechanism by which NO may cause the accumulation of damaged proteins and neuronal degeneration in Parkinson disease.

However, although NO-mediated impairment of PDI may contribute to neuronal degeneration in Parkinson disease, many questions remain unanswered. When in the course of the disease process does S- nitrosylation of PDI reach a criti-cal threshold level that results in the accumula-tion of damaged proteins? Moreover, although S-nitrosylation can impair PDI function, this may be a reversible process under normal condi-tions. Indeed, PDI itself possesses denitrosylation activity involving a nitroxyl disulfide interme-diate, which, after undergoing a one-electron oxidation, decomposes to yield NO plus dithiyl radical10. Dopaminergic neurons may be able to maintain PDI activity early in the disease pro-cess by increasing their expression of PDI and by reversing the S-nitrosylation process. However, when NO levels become excessive and PDI and parkin functions are impaired, misfolded and oxidatively damaged proteins accumulate to a point that overwhelms the proteasome, and the neurons succumb. Perhaps denitrosylation mechanisms themselves are compromised by oxidative stress, contributing to excessive pro-

tein nitrosylation. What is the cellular source of NO that is responsible for S-nitrosylation of PDI and parkin in dopaminergic neurons? Not all neurons in the brain express NOS and, indeed, the dopaminergic neurons that are vulnerable in Parkinson disease may produce little NO rela-tive to their postsynaptic target neurons in the striatum and to interneurons in the substantia nigra11. It is therefore possible that the NO that impairs PDI activity in dopaminergic neurons is produced by neighboring neurons (Fig. 1).

Another interesting question is why the various NO-mediated mechanisms that pro-mote neuronal survival fail to protect neurons against the toxic actions of NO. For example, brain-derived neurotrophic factor (BDNF) may protect neurons against Alzheimer disease and Parkinson disease, in part by initiating a NO-dependent signaling pathway that leads to S-nitrosylation of nuclear proteins that

associate with cyclic AMP response element binding protein target genes12. Apparently, when neurons are faced with progressive age-related increases in oxidative and metabolic stress and elevated levels of Ca2+, NO and pro-tein nitrosylation reach levels that irreversibly compromise ER functions.

It seems likely that NO-mediated accumula-tion of damaged proteins occurs most promi-nently in synaptic terminals because of the high levels of Ca2+ influx and oxyradical production at those sites. However, the subcellular local-ization of the various organellar and protein players in the scheme proposed by Uehara et al. complicates the placement of this new mechanism within the current view of where and how the neurodegenerative process begins in dopaminergic neurons in Parkinson disease. Evidence suggests that the neurodegenerative process in Parkinson disease begins in the pre-

O2–

Oxidative stress

Mitochondria

Endoplasmicreticulum

Misfolded proteins(e.g., SP1, αSN)

Ca2+

Ca2+

Ca2+

Ca2+

CM NOS

NO

PDI

OONO

Parkin

Proteasome Toxic protein accumulationSynaptic dysfunctionCell death

CM

GlutamateNMDAR

Oxidative stress

Jess

ica

Iann

uzzi

Figure 1 Involvement of nitric oxide (NO)-mediated modification of protein disulfide isomerase (PDI) in the accumulation of neurotoxic proteins in Parkinson disease. Age-related increases in membrane-associated oxidative stress and mitochondrial dysfunction foster excessive Ca2+ influx through NMDA receptors in the dendrites of dopaminergic neurons. Ca2+ binds to calmodulin CM, which in turn activates nitric oxide synthase (NOS), resulting in the production of NO. NO S-nitrosylates PDI in neurons, impairing the ability of PDI to properly regulate the folding of proteins in the endoplasmic reticulum. Abnormally folded proteins are normally targeted for degradation in the proteasome. In Parkinson disease, both the targeting of damaged protein by ubiquitin E3 ligases, such as parkin, and the function of proteasomes may be impaired by NO-mediated mechanisms. In addition to S-nitrosylation, NO may damage proteins by interacting with superoxide anion radical (O2

–) to generate the free radical peroxynitrite (OONO).

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synaptic terminals of dopaminergic neurons, where there are high concentrations of dopa-mine, parkin and α-synuclein, and where mito-chondrial alterations are believed to be pivotal to the neurodegenerative process13,14. However, the protein synthetic ER (and presumably PDI) is present in much higher amounts in the den-drites and cell body as compared to the axon terminals15. Similarly, NMDA receptors and NOS are concentrated in dendrites, suggesting that nitrosylation of PDI occurs mainly in den-drites. The findings of Uehara et al. therefore provide new evidence for dendritic abnormali-ties in the pathogenesis of Parkinson disease.

The importance of PDI impairment in the abnormal protein accumulations and neuro-nal death in Parkinson disease, relative to other mechanisms (mitochondrial impairment, oxidative stress, proteasome dysfunction

and others), remains to be established. Nevertheless, PDI is a newcomer to the list of potential targets for therapeutic intervention in Parkinson disease and other protein aggre-gation disorders. Several possible approaches can be envisioned, including drugs that induce the expression of PDI or enhance its enzymatic activity, agents that block S-nitrosylation of PDI, such as general scavengers of NO or selec-tive inhibitors of PDI nitrosylation, and agents that inhibit NO production. Because NO is believed to be important in other neurodegen-erative disorders, such as Alzheimer disease, Huntington disease, amyotrophic lateral scle-rosis and stroke, among others, it is important to determine whether NO-mediated impair-ment of PDI function is also critical for the abnormal accumulation of protein garbage and neuronal death in these disorders.

1. Forloni, G. et al. Neurobiol. Aging 23, 957–976 (2002).

2. Hashimoto, M. et al. Neuromolecular Med. 4, 21–36 (2003).

3. Forman, M.S. et al. Trends Neurosci. 26, 407–410 (2003).

4. Uehara, T. et al. Nature 441, 513–517 (2006).5. Ahern, G.P., Klyachko, V.A. & Jackson, M.B. Trends

Neurosci. 25, 510–517 (2002).6. Choi, Y.B. et al. Nat. Neurosci. 3, 15–21 (2000).7. Dawson, V.L. & Dawson, T.M. Prog. Brain Res. 118,

215–229 (1998).8. Chung, K.K. et al. Science 304, 1328–1331 (2004).9. Tanaka, S. et al. J. Biol. Chem. 275, 10388–10393

(2000).10. Sliskovic, I. et al. J. Biol. Chem. 280, 8733–8741

(2005).11. Downen, M. J. Neuropathol. Exp. Neurol. 58, 12–21

(1999).12. Riccio, A. et al. Mol. Cell 21, 283–294 (2006).13. Betarbet, R. et al. Nat. Neurosci. 3, 1301–1306

(2000).14. Abou-Sleiman, P.M. et al. Nat. Rev. Neurosci. 7,

207–219 (2006).15. Gardiol, A. et al. J. Neurosci. 19, 168–179 (1999).

Trailblazers of the cortex

On page 880 of this issue, there is a paper with the grand title “The first neurons of the human cerebral cortex.” In rare specimens of human embryos aborted in the fifth week of development, before the neural tube is even fully closed, Bystron and colleagues were able to detect cells that look convincingly like neurons and express the established neuron marker βIII- tubulin. The cells are found in the prospective cortex, in a layer just below the pial surface of the incipient telencephalon, before any neurogenesis takes place in the local telencephalic neuroepithelium. It appears as though these cells enter the telencephalic domain tangentially from underlying areas, though their exact origin remains unclear. The authors call these cells “predecessor neurons.” The image shows these neurons, stained golden for βIII-tubulin, on top of the as-yet undifferentiated telencephalic ventricular zone from a 35-day-old human embryo. The blue stain labels all nuclei.

The earliest cortical neurons have been widely assumed to be the Cajal-Retzius cells, which also spread across the telencephalic surface after tangentially migrating long distances from their birthplaces. Cajal-Retzius cells are most famous for their expression of reelin, which is crucial in guiding the development of the stratified neocortex. The predecessor neurons make no reelin, however, and are thus an entirely new neuron population. Furthermore, the first Cajal-Retzius cells in the emerging human cortex appear a week later than the predecessors. The authors show predecessor and Cajal-Retzius neurons coexisting in the primordial plexiform layer of 7-week-old embryos, with Cajal-Retzius cells coming to lie in the emerg-ing marginal zone above the predecessors.

The new predecessor neurons raise many, many questions. What is their origin, and what is their function? How long do they persist in the developing brain? Could the cortex develop without them? To tackle these issues, analogous early cortical neurons would need to be identified in experimentally amenable species. Unless, of course, the predecessor neurons turn out to be specific to higher primates, which would make them especially interesting. In any case, Bystron and colleagues have opened a new chapter in the developmental biology of the brain, and we eagerly await the sequel.

Annette Markus

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