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# Endoplasmic reticulum dysfunction in neurological disease

Benoit D. Roussel, Antonina J Kruppa, Elena Miranda, Damian C Crowther,

David A Lomas, Stefan J Marciniak

► To cite this version:

Benoit D. Roussel, Antonina J Kruppa, Elena Miranda, Damian C Crowther, David A Lomas, et al.. Endoplasmic reticulum dysfunction in neurological disease. The Lancet Neurology, 2013, 12 (1), pp.105 - 118. 10.1016/S1474-4422(12)70238-7 . inserm-01296824

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Manuscript Number: THELANCETNEUROLOGY-D-12-00012R3

# IMPLICATIONS OF ENDOPLASMIC RETICULUM DYSFUNCTION IN NEUROLOGICAL DISEASE

Article Type: Review

Corresponding Author: Dr Benoit D Roussel, Ph.D.

Corresponding Author's Institution: INSERM U919

First Author: Benoit D Roussel, Ph.D.

Order of Authors: Benoit D Roussel, Ph.D.; Antonina J Kruppa, Ph.D.; Elena Miranda, Ph.D.; Damian C Crowther, Ph.D.; David A Lomas, Ph.D.; Stefan J Marciniak, Ph.D.

**Abstract:** Endoplasmic reticulum (ER) dysfunction is important in the pathogenesis of many neurological diseases. In this review, we examine the evidence for ER dysfunction in a range of neurological conditions including cerebral ischaemia, sleep apnoea, Alzheimer's disease, multiple sclerosis, amyotrophic lateral sclerosis, the prion diseases and Familial Encephalopathy with Neuroserpin Inclusion Bodies (FENIB). Protein misfolding in the endoplasmic reticulum initiates a well-studied 'Unfolded Protein Response' in energy-starved neurones during stroke that is relevant to the toxicity of reperfusion. The toxic peptide Aβ induces 'ER stress' in Alzheimer's disease leading to activation of similar pathways, while the accumulation of polymeric neuroserpin in the neuronal ER triggers a poorly understood 'ER overload response'. In other neurological disorders such as Parkinson's and Huntington's diseases ER dysfunction is well recognised but the mechanisms for this are less clear. By targeting components of these signalling responses, it may prove possible to ameliorate their toxic effects and treat a range of neurodegenerative conditions.
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IMPLICATIONS OF ENDOPLASMIC RETICULUM DYSFUNCTION IN
NEUROLOGICAL DISEASE

Benoit D. Roussel<fnref n="1" />,<fnref n="4" /> PhD, Antonina J. Kruppa<fnref n="1" /> PhD, Elena Miranda<fnref n="2" /> PhD, Damian C. Crowther<fnref n="3" /> PhD, David A. Lomas<fnref n="1" /> PhD and Stefan J. Marciniak<fnref n="1" /> PhD

Correspondence should be addressed to:

Dr Benoit D Roussel & Dr Stefan J Marciniak

Cambridge Institute for Medical Research,

Wellcome Trust/MRC Building,

Hills Road, Cambridge CB2 0XY, United Kingdom

Email: [broussel@cyceron.fr](mailto:broussel@cyceron.fr), [sjm20@cam.ac.uk](mailto:sjm20@cam.ac.uk)

<footnote n="1">¹Department of Medicine, University of Cambridge, Cambridge Institute for Medical Research (CIMR), Wellcome Trust/MRC Building, Hills Road, Cambridge CB2 0XY, UK</footnote>

<footnote n="2">²Department of Biology and Biotechnology 'Charles Darwin' and Pasteur Institute-Cenci Bolognetti Foundation, University of Rome 'La Sapienza', piazzale Aldo Moro 5, 00185 Rome, Italy</footnote>

<footnote n="3">³Department of Genetics, University of Cambridge, Downing Site, Cambridge, CB2 3EH, UK</footnote>

<footnote n="4">⁴INSERM U919, University Caen Basse Normandie, Serine Proteases and Pathophysiology of the Neurovascular Unit, Public Interest Group CYCERON, F-14074 Caen, France (*actual address)</footnote>
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Key words: ER stress, Neurodegeneration, Cerebral ischaemia, Alzheimer's disease, Parkinson's disease, FENIB

Running title: ER dysfunction in the CNS

Word count: 5,988 (excluding references and legends)
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Endoplasmic reticulum (ER) dysfunction is important in the pathogenesis of many neurological diseases. In this review, we examine the evidence for ER dysfunction in a range of neurological conditions including cerebral ischaemia, sleep apnoea, Alzheimer's disease, multiple sclerosis, amyotrophic lateral sclerosis, the prion diseases and Familial Encephalopathy with Neuroserpin Inclusion Bodies (FENIB). Protein misfolding in the endoplasmic reticulum initiates a well-studied 'Unfolded Protein Response' in energy-starved neurones during stroke that is relevant to the toxicity of reperfusion. The toxic peptide Aβ induces 'ER stress' in Alzheimer's disease leading to activation of similar pathways, while the accumulation of polymeric neuroserpin in the neuronal ER triggers a poorly understood 'ER overload response'. In other neurological disorders such as Parkinson's and Huntington's diseases ER dysfunction is well recognised but the mechanisms for this are less clear. By targeting components of these signalling responses, it may prove possible to ameliorate their toxic effects and treat a range of neurodegenerative conditions.

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## Introduction

It can be argued that most neurodegenerative disorders arise from defective protein folding. This is clearly true for those rare disorders caused by the misfolding of mutated neuronal proteins such as Huntington's disease, but it is also the case for common disorders such as cerebral ischaemia where the lack of energy can impair normal protein folding ¹ and sporadic Alzheimer's disease where protein aggregation causes cellular stress ²⁻⁴. Even some infectious neurodegenerative disorders such as the prionopathies arise from abnormal protein folding ⁵⁻⁹. Consequently, the processes for disposing of misfolded proteins are the focus of much research. Although the subcellular location of protein misfolding may differ between these disorders, it appears that the interdependence of protein folding throughout the cell results in ER dysfunction being the final common pathway for many, if not all, of these diseases (Table 1).

## ER Dysfunction

The endoplasmic reticulum (ER) is required for folding of all secreted and membrane proteins and insults that impair its function induce a pathological state termed 'ER stress' ¹⁰. This triggers an adaptive programme called the Unfolded Protein Response (UPR), which combines the early inhibition of protein synthesis with a later upregulation of genes that promote protein folding or disposal ¹⁰. Both the translational and transcriptional components of the UPR protect the neurone from becoming overwhelmed by misfolded ER proteins; however, when the insult is too great, apoptotic cell death frequently ensues. While many cells
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are relatively protected from accumulating misfolded protein through continued
dilution of their ER by cell replication, this process is unavailable to post-mitotic
neurones, which depend exclusively on the UPR. Even glial cells, which can
replicate, are susceptible to ER stress owing to their highly developed secretory
pathway.

It is important that the UPR be distinguished from the less well-understood ER
Overload Response (EOR) ¹¹. The former is triggered by protein misfolding
within the ER lumen, while the latter occurs when well-folded or misfolded
proteins accumulate and distend the ER. In this review, we will discuss the
relevance of these forms of ER dysfunction to neurological disease, since a
better understanding of each is likely to identify novel therapeutic strategies
(Panel 1). We will focus on ischaemic stroke, Alzheimer's disease and
Parkinson's disease to discuss ER stress, while using Familial Encephalopathy
with Neuroserpin Inclusion Bodies (FENIB) to illustrate ER overload, since these
are the diseases for which the most evidence currently exists. In addition, we will
discuss the evidence for ER dysfunction in multiple sclerosis, amyotrophic lateral
sclerosis and the prion disorders. Some neurological disorders provoke ER
stress signalling despite being caused by the accumulation of protein within the
cytoplasm, for example Huntington's disease ¹². Such disorders emphasise the
interdependence of protein folding networks in the neurone and so shall be
discussed as examples of disordered protein homeostasis or 'proteostasis'.
## ER stress signalling

The signalling pathways triggered by ER stress comprise the UPR<sup>10</sup> (Figure 1). Although it may seem tiresome to dwell on individual molecular components, this is unavoidable when studying a phenomenon as complex as ER stress. Not least, because many of the measures commonly used to detect and quantify ER stress are themselves components of the UPR. Some of these are activated transiently and so their absence cannot be used as evidence for a lack of ER stress<sup>10, 13</sup>; conversely, certain components can be activated by stresses other than ER protein misfolding and so need to be interpreted with caution. We shall therefore begin with a review of the major mediators of ER stress signalling with an emphasis on their role in neurones before moving on to considering specific examples of neuropathology mediated by ER dysfunction.

### PERK

Whenever misfolded protein accumulates in the ER lumen, new protein synthesis is rapidly inhibited<sup>14</sup>. This occurs because ER stress activates the PKR-like Endoplasmic Reticulum eIF2α Kinase (PERK), also known as Pancreatic eIF2α kinase (Pek) because of the tissue in which it was first discovered<sup>14</sup>. This kinase is ubiquitously expressed but is most important in highly secretory tissues such as the brain where it functions to match protein synthesis with the efficiency of protein folding in the ER<sup>15</sup>. When protein folding is progressing well PERK is inactive, but when misfolded ER proteins accumulate the kinase is activated and phosphorylates eukaryotic translation initiation factor 2α (eIF2α) (Figure 1)<sup>16</sup>.
This substrate is a subunit of the heterotrimeric GTP-binding complex eIF2 that regulates the initiation of protein synthesis <sup>14</sup>. EIF2 recruits the methionyl-tRNA to the ribosome at the onset of protein translation, but when its eIF2α subunit is phosphorylated this activity is lost and translation ceases. The load of proteins entering the ER is thus reduced following PERK activation preventing the accumulation of more misfolded proteins.

## ATF4

Despite the inhibition of protein translation that accompanies phosphorylation of eIF2α, a small subset of mRNAs is translated more efficiently under these conditions <sup>17</sup>. The best characterised of these is Activating Transcription Factor 4 (ATF4), which triggers a transcriptional programme of stress-responsive genes <sup>17</sup>. Other members of the eIF2α kinase family can inhibit translation and activate these genes; for example PKR responds to viral infection, HRI to iron deficiency and GCN2 to amino acid starvation <sup>18</sup>. This ability of eIF2α phosphorylation to mediate a response to multiple stresses led to this pathway being named the Integrated Stress Response <sup>19</sup>. Surprisingly, within the brain this can provoke specific behavioural responses. A lack of essential dietary amino acids triggers the phosphorylation of eIF2α in the anterior piriform cortex by GCN2, which alters feeding behaviour in rodents <sup>20, 21</sup>. Thus, phosphorylation of eIF2α is not synonymous with ER stress and multiple lines of evidence should always be sought when attributing phosphorylation of eIF2α to ER dysfunction.
Phosphorylation of eIF2α is particularly important for the maintenance of axonal myelination in the human central nervous system (Table 1); indeed mutations that disrupt the regulation of eIF2α result in white matter hypomyelination disorders such as Childhood Ataxia with Central nervous system Hypomyelination (CACH syndrome) and Vanishing White Matter (VWM) <sup>22</sup>. This family of autosomal recessive conditions constitutes a spectrum of severity from congenital to adult-onset, involving the progressive loss of mental and motor function due to a loss of brain white matter. In all cases, the causative mutations are found within subunits of eIF2B, a guanine nucleotide exchange factor (GEF) responsible for recharging the eIF2 complex with GTP <sup>23, 24</sup> (Figure 1). These mutations interfere with the GEF activity of eIF2B and enhance the synthesis of ATF4. This deregulates many target genes including components of a negative feedback system, including CHOP and GADD34, that normally recover protein translation following its initial inhibition by PERK <sup>13, 25</sup>.

## • CHOP

The transcription factor CHOP is induced by ATF4 and has widely been thought to be pro-apoptotic during ER stress, since *Chop-/-* animals are resistant to ER stress-induced cell death <sup>13</sup>. However, this is inconsistent with observations made in neurological diseases. For example, CHOP expression is clearly anti-apoptotic in mouse models of the X-linked disorder Pelizaeus-Merzbacher leukodystrophy caused by abnormalities of the proteolipid protein 1 (PLP1) gene <sup>26</sup>. Indeed, identification of the target genes of CHOP failed to reveal a link with
apoptosis but instead identified genes that promote protein secretion<sup>13</sup>. This led us to suggest that in models of severe ER stress the promotion of protein secretion by CHOP might increase the intensity of stress and thus induce cell death <sup>13</sup>. By contrast, during conditions of milder ER stress, for example in Pelizaeus-Merzbacher leukodystrophy, it is likely that CHOP does not enhance ER stress sufficiently to trigger cell death <sup>26</sup>. These observations highlight the need to model not only the nature of the stress, but also its *intensity* and *duration* since differences in either can significantly affect the outcome and thus lead to beneficial or deleterious effects depending upon the context. There may also be cell type specific consequences of expressing CHOP. For example, while deletion of *Chop* worsens the central hypomyelination seen in Pelizaeus-Merzbacher leukodystrophy <sup>26</sup>, *Chop/-* animals are protected against the peripheral hypomyelination caused by mutant myelin protein zero (P0) in mouse models of Charcot Marie Tooth type 1B <sup>27</sup>.

## GADD34

A critical target gene of CHOP encodes Growth Arrest and DNA Damage-inducible protein 34 (GADD34), which is a regulatory subunit of protein phosphatase 1 (PP1) <sup>13</sup>. This dephosphorylates eIF2α and thus brings about the recovery of protein translation after its initial inhibition. It has been shown in mice that inactivation of the *Gadd34* gene can protect animals from tissue damage during ER stress by reducing the accumulation of misfolded proteins <sup>13</sup>. Subsequently, a small molecule called salubrinal was identified that increases
the level of eIF2α phosphorylation and promotes survival of ER-stressed cells²⁸.

It has been suggested that salubrinal may be an inhibitor of the eIF2α phosphatases ²⁸, but direct evidence for this is lacking. Recently, however, a selective inhibitor of GADD34, guanabenz, has been identified confirming the beneficial effects of GADD34 inhibition during ER stress at least in cultured cells ²⁹. It is therefore likely that future therapies involving the inhibition of GADD34 will be able to modulate ER stress.

## ATF6

In addition to the Integrated Stress Response genes regulated by PERK and ATF4, many additional genes are induced during the UPR by two further ER stress sensors, Inositol-Requiring Enzyme 1 (IRE1) and Activating Transcription Factor 6 (ATF6)¹⁰ (Figure 1). Like PERK, both are ER membrane proteins that are held inactive under normal conditions by the binding of the ER chaperone Binding Immunoglobulin Protein (BiP; also known as GRP78). During stress, misfolded proteins sequester BiP, freeing ATF6 to traffic to the Golgi apparatus where it is cleaved to release a soluble transcription factor. Of note, astrocytes express an ATF6-related protein called OASIS (Old Astrocyte Specifically Induced Substance) whose functions appear to overlap partially with those of ATF6 ³⁰. This might enable astrocytes to respond to ER stress in a tissue-specific manner.
While cleavage of ATF6 is strong evidence of ER stress, this is difficult to detect. Consequently, many studies fail to measure this arm of the UPR, but the use of sensitive reporters that measure the binding of ATF6 to specific DNA sequences can circumvent this problem *in vitro* <sup>11</sup>.

## IRE1

The activation of IRE1 closely resembles that of PERK but, unlike PERK, IRE1 triggers splicing of the mRNA encoding the transcription factor X-box Binding Protein 1 (XBP1) <sup>31</sup>. This induces a frame shift that generates the active form of this protein. Like phosphorylation of eIF2α, the splicing of XBP1 mRNA is transient <sup>13</sup>. Following the alleviation of ER stress by the induction of UPR target genes, spliced XBP1 mRNA is rapidly lost from the cell to be replaced by the unspliced form <sup>13</sup>. In laboratory studies, supra-physiological levels of ER stress can cause complete splicing of XBP1 mRNA, but *in vivo* stresses are frequently subtler and so it is rare to observe complete splicing and careful quantification is therefore necessary <sup>32</sup>.

If proteins fail to fold in the ER, they are prevented from progressing along the secretory pathway and eventually are targeted for degradation <sup>33</sup>. This ‘ER-associated protein degradation’ (or ERAD) returns terminally misfolded proteins to the cytosol where they are ubiquitinated by ER-associated ubiquitin ligases for degradation by the proteasome <sup>34</sup>. Many components of the ERAD machinery
are target genes of XBP1 and so the capacity for ERAD is increased by
activation of IRE1 <sup>35</sup>.

ER dysfunction in neurological disease

Cerebral hypoxia

In industrialized countries stroke is the third biggest killer and the leading cause of disability in adults. Acute ischaemia is the main cause of neuronal loss and, although its pathology is complex, appears to involve ER stress <sup>1, 15, 36-38</sup>. During cerebral ischaemia, neuronal depolarization due to energy depletion causes the uncontrolled release of glutamate. The consequent activation of NMDA receptors on nearby neurones generates further glutamate release and causes an ischaemic depolarization wave to spread outward from the initial site of damage leading to widespread disturbance of calcium homeostasis <sup>39</sup>. Unsurprisingly, many studies implicate ER calcium store release in the resulting excitotoxic death (reviewed in reference <sup>39</sup>).

The ER is the main site for calcium storage within the cell and so its chaperones
have evolved to function efficiently in this high calcium environment; indeed
many require high calcium to function <sup>40</sup>. Calcium is pumped into the ER by the
sarcoendoplasmic reticular calcium-ATPase (SERCA) and released back into the
cytosol by the inositol trisphosphate receptor (IP3R) in response to extracellular
signals and by the ryanodine receptor during calcium-induced calcium release <sup>39</sup>.
Inhibition of the SERCA pump by thapsigargin induces apoptosis in many cell
types including neuroblastoma cells and is often used to induce ER stress in the
experimental setting <sup>41</sup>. During cerebral ischaemia, energy depletion leads to
failure of the SERCA pump and thus redistribution of ER calcium into the cytosol.
This delivers a double blow with combined toxicity from uncontrolled cytosolic
calcium and ER stress due to chaperone dysfunction (Figure 2) <sup>39</sup>.

The UPR is activated in many rodent models of cerebral ischaemia ¹,³⁶. In these
models, protein synthesis is rapidly inhibited and accompanies the
phosphorylation of eIF2α. Although the inhibition of translation has been
attributed to aggregation of components of the translation machinery ⁴², the
primary mechanism appears to be through phosphorylation of eIF2α ⁴³, which
can remain phosphorylated for up to twelve hours following ischaemia ⁴⁴. PERK
is the sole eIF2α kinase activated by cerebral ischaemia and predictably Perk-/-
mice fail to show eIF2α phosphorylation following transient cerebral ischaemia
and fail to reduce protein translation during the reperfusion period ¹⁵. Even with
the far milder recurrent cerebral hypoxia seen in obstructive sleep apnoea,
activation of PERK has been described and has been suggested to play a role in
the loss of upper airway motor neurones in this condition ⁴⁵.

IRE1 is also activated during cerebral ischaemia leading to the induction of UPR
target genes ³⁷, ³⁸. It appears to promote activation of pro-apoptotic caspase-12
in cultured rodent cells and this has been detected in ischaemic rodent brains ⁴⁶.
However, caspase-12 is non-functional in the majority of humans, except those
from sub-Saharan Africa, owing to a common truncating mutation <sup>47</sup>. For this
reason, it has been proposed that during ER stress in humans, caspase-4 might
serve an analogous function to that of caspase-12 in rodents <sup>48</sup>.

Due to the problems of detection referred to earlier, the role of ATF6 during
ischaemia is unclear. ATF6 gene expression is elevated following transient
middle cerebral artery occlusion in rats <sup>36</sup>, but its activation has not been
detected in rat brain following cardiac arrest and resuscitation <sup>49</sup>. OASIS is also
upregulated and appears to play a neuroprotective role in response to kainate
excitotoxicity <sup>50</sup>.

CHOP is induced following forebrain ischaemia in a variety of rodent models
including bilateral common carotid <sup>51, 52</sup> or middle cerebral artery occlusion in
mice <sup>44</sup> and global cerebral ischaemia in rats <sup>53</sup>. The location within the brain of
CHOP induction appears to correlate well with subsequent cell death <sup>53, 54</sup> and
deletion of the *Chop* gene protects mice during bilateral common carotid artery
occlusion <sup>52</sup>. Similarly, depletion of CHOP using RNA interference partially
prevents the death of astrocyte cultures stressed by oxygen-glucose depletion <sup>55</sup>.
As discussed, some of the toxicity attributable to CHOP reflects increased protein
translation mediated by GADD34 <sup>13</sup>. Indeed, GADD34 induction is well-
recognised to accompany cerebral ischaemia <sup>56</sup> being detected at the peri-infarct
penumbra within two to twenty-four hours <sup>57</sup> and accounts for the transient nature
of eIF2α phosphorylation during stroke <sup>15</sup>. Attenuation of translation protects the
cell from accumulating misfolded proteins, thus treatment with salubrinal (a drug that enhances eIF2α phosphorylation) reduces ER stress and promotes cellular survival in kainate-induced neurotoxicity⁵⁸. Indeed, salubrinal has been shown to limit infarct size in a murine model of stroke ¹.

In addition to attenuating protein translation, the UPR leads to upregulation of ER
molecular chaperones, the most abundant being BiP ¹⁰. Following transient
forebrain ischaemia, BiP expression in the hippocampus and cortex peaks
between twelve to seventy-two hours after reperfusion ⁵⁴, ⁵⁹, but can remain
elevated for up to two weeks ³⁶. This is protective, since suppression of BiP
enhances apoptosis in hippocampal neurones exposed to excitotoxic and
oxidative insults ⁶⁰, while its overexpression in primary astrocyte cultures is
protective against oxygen-glucose deprivation ⁶¹. Indeed, higher levels of BiP
induction correlate with neuronal protection ³⁷, ⁶².

Induction of the ER chaperone ORP150 (150-kDa oxygen-regulated protein) is also seen in ischaemic neurones, and over-expression of ORP150 protects mice from cerebral ischaemia ⁶³. ORP150 modulates the activity of BiP, which exists in either an ATP- or ADP-bound form, each with different substrate-binding affinity. ORP150 functions as a nucleotide exchange factor for BiP to replace its bound ADP with ATP. Interesting, another BiP nucleotide exchange factor, SIL1, is mutated in the Marinesco-Sjogren syndrome of cerebellar ataxia in which homozygous loss of SIL1 leads to ER stress and selective loss of cerebellar
Purkinje cells<sup>64</sup>. When ATP-BiP binds its substrate protein, a co-chaperone of the DnaJ family is required to stimulate BiP's ATPase activity and so promote high affinity substrate binding. The relative levels of ADP-BiP and ATP-BiP appear to be important for neuronal survival, since the toxicity of SIL1 loss can be ameliorated by the deletion of an ER luminal DnaJ co-chaperone called p58<sup>IPK</sup><sup>65</sup>. While loss of SIL1 would be predicted to reduce the level of ATP-BiP through impaired nucleotide exchange, loss of p58<sup>IPK</sup> is likely to restore balance by lessening BiP's ATPase activity.

When taken together, these observations suggest that ER stress is induced by
cerebral ischaemia and that the UPR limits cerebral infarct size. There is good
evidence to suggest that manipulation of ER stress signalling by increasing
phosphorylation of eIF2α may have important therapeutic effects as an acute
intervention for cerebral ischaemia.

Alzheimer's disease

Often presenting with failing memory, Alzheimer's disease (AD) is a relentlessly
progressive neurodegenerative disorder and the most common cause of
dementia in adults. The classic histopathological features consist of extracellular
plaques of the β-amyloid (Aβ) peptide and intracellular neurofibrillary tangles of
hyperphosphorylated aggregates of the microtubule-associated protein tau.
Studies of familial AD demonstrate that the disorder is caused by overproduction
of the more aggregation-prone Aβ<sub>1-42</sub> peptide <sup>66</sup> (Figure 3). Despite decades of
study, the molecular pathology of this disease remains unclear. However,
increasing evidence suggests that ER stress may represent a new paradigm for
understanding this condition.

Studies using post-mortem tissue from the brains of individuals with AD have
provided evidence for ER dysfunction in this disease. But when looking at a
single marker of ER stress, such studies can generate confusing results. For
example, when BiP is examined in isolation it can show no change ³, a decrease
⁶⁷ or even an increase ², ⁶⁸, although BiP expression does seem to be higher in
brains with histologically more advanced disease ⁶⁸, ⁶⁹. However, whilst often a
useful indicator, the level of BiP expression is inadequate alone to assess the
activation of UPR signalling, especially in a chronic disorder in which histological
features evolve over time. It has therefore proved more useful to include
additional markers of the UPR in such studies. For example, protein disulphide
isomerase, another target of the UPR, is elevated in the temporal lobes of the
brains of individuals with AD ³, associated with neurofibrillary tangles in the
hippocampus and frontal lobes and in dystrophic neurites of senile plaques ⁴.
When one looks for more proximal markers of UPR signalling, there is activation
of PERK in the hippocampus of AD brains, which appears to co-localise with
staining for phosphorylated tau and correlates with histological staging ⁷⁰, ⁷¹. Yet
phosphorylated PERK is barely detectable in tangle-bearing neurones
themselves, suggesting that PERK is activated at the pre-tangle stage in
neurones of the hippocampus ⁷¹. In addition, there are elevated levels of spliced
XBP1 mRNA in the temporal cortex of AD patients when compared to age-
matched controls ³. Congruently, a marked increase in the number of neurones
immunoreactive for phosphorylated (active) IRE1α has been detected in the
hippocampus of individuals with AD ⁷¹.

There is good evidence for activation of PERK in laboratory models of AD.
Increased BiP expression observed upon application of exogenous Aβ to primary
cortical neurones suggests that accumulation of the peptide may directly activate
ER stress signalling ⁷², ⁷³. Moreover, exogenous Aβ has been found to increase
the phosphorylation of PERK and eIF2α in cultured neuronal cells ⁷³, ⁷⁴, while
silencing of PERK by siRNA in Aβ₄₂-treated cells limits eIF2α phosphorylation
and enhances cell death ⁷³. In addition, Aβ₄₂ treatment induces CHOP
expression both in cultured cells and in rabbit hippocampus ⁷⁵, ⁷⁶, while prior
treatment of cells with CHOP anti-sense RNA improves survival following
exposure to the Aβ peptide, suggesting a role for CHOP in Aβ-mediated death ⁷⁷.
In *Drosophila* neurones or mammalian PC-12 cells, exposure to Aβ induces
XBP1 mRNA splicing and in both models overexpression of spliced XBP1 is
protective, while knockdown of XBP1 exacerbates Aβ toxicity ⁷⁸.

PC-12 cells overexpressing mutant forms of presenilin 1 (PS1), a component of
the γ-secretase, that has been linked to familial AD, show increased levels of
phosphorylated eIF2α, as do hippocampal homogenates from PS1 mutant
knock-in mice ⁷⁹. PC-12 cells expressing mutant PS1 have elevated levels of
CHOP, as do knock-in mice expressing mutant PS1⁷⁹. The nature of the interaction between PS1 and ER stress has yet to be fully elucidated, although it is known that PS1 and the SERCA calcium pump physically interact with one another⁸⁰. As discussed, disordered calcium homeostasis is a potent cause of ER stress and much evidence suggests that calcium signalling is perturbed in Alzheimer's disease⁸¹ (reviewed in reference ⁸²). Interestingly, ER stress induced by tunicamycin, a glycosylation inhibitor frequently used to induce ER stress in vitro, increases PS1 expression in multiple cell lines including neuroblastoma⁸⁰. Recent evidence suggests that phosphorylation of eIF2α by PERK during ER stress can increase the level of β-site APP cleaving enzyme-1 (BACE) and thereby promoting amyloid formation ⁸³. This suggests that the interaction between AD and ER stress may prove to be bi-directional.

ER stress has been shown to alter APP localization, processing and degradation by the ERAD machinery ⁸⁴ (Figure 4). During ER stress, immature forms of APP bind to BiP and when BiP is overexpressed Aβ generation is reduced as APP is retained in earlier compartments of the secretory pathway ⁸⁴. The ERAD ubiquitin ligase HRD1 promotes APP ubiquitination and degradation in HEK293 cells reducing the generation of both Aβ₄₀ and Aβ₄₂ ². In addition, an intronic polymorphism (IVS3-88A>G) of SEL1L, another component of the ERAD system, has been associated with AD in an Italian population ⁸⁵. These hints that ERAD is important in the pathogenesis of AD still require further validation. But there is accumulating evidence for elevated UPR signalling in AD and, combined with the
support from model systems, provides a potential mechanism by which the Aβ
peptide and mutants of PS1 might induce ER stress. This offers the potential to
identify new therapeutic targets in an otherwise incurable disease.

Parkinson's disease

With age, 2% of the population suffer the loss of dopaminergic neurones in their nigrostriatal pathway and so develop Parkinson's disease (PD) characterised by hypokinesia, rigidity and tremor. Post-mortem studies identify characteristic Lewy body inclusions in many affected brains, suggesting aberrant protein disposal as a possible cause, while toxins that cause Parkinsonism, such as MPTP, rotenone and 6OHDA, impair mitochondrial function and promote ER stress<sup>86-90</sup>.

Multiple lines of evidence point towards a role for ER stress in the pathogenesis
of PD. For example, excess levels of dopamine are toxic to PC-12 cells, which
correlates with their induction of ER chaperones <sup>91</sup>. The brains of patients
suffering from sporadic PD, but not controls, have elevated levels of
phosphorylated PERK and eIF2α <sup>92</sup>. Perk/- sympathetic neurones are
hypersensitive to 6OHDA <sup>92</sup>, while overexpression of spliced XBP1 is protective
both in cultured cells and animals treated with MMP+/MPTP <sup>87</sup>. CHOP
expression is increased both by 6OHDA and MPTP, and knockout of the Chop
gene is protective in mice treated with 6OHDA <sup>93</sup>.
In Japanese and European families with the juvenile onset autosomal recessive variant of PD (ARPD), classical genetic mapping identified the disease-associated gene *PARK2* <sup>94</sup>. This encodes an ubiquitin ligase Parkin, whose activity is lost with disease-associated mutations <sup>95</sup>. Parkin was suggested to localize to the ER and to be upregulated during the UPR <sup>96</sup>. A major function of Parkin appears to involve targeting defective mitochondria for destruction by autophagy <sup>97</sup>. It is possible that impaired mitochondrial function in patients with *PARK2* mutations manifests as ER stress owing to defects in energy supply during ER protein folding. However, another component of Lewy bodies, α-synuclein, can be found throughout the cell including the ER lumen and may also be a target of Parkin <sup>98</sup>. Mutations of α-synuclein cause an autosomal dominant form of PD <sup>99</sup>, while the glycosylated ER-form of α-synuclein has been shown to be ubiquitinated by Parkin and to accumulate in a non-ubiquitinated form in the brains of individuals with Parkin deficiency <sup>98</sup>. Overexpression of α-synuclein induces ER stress <sup>100</sup>, perhaps through binding to BiP <sup>101</sup>, while PDI appears to impair α-synuclein fibril formation <sup>100</sup>. Recently, oligomers of mutant α-synuclein were shown to accumulate within the ER in an animal model and to trigger the UPR, albeit with relatively little PERK activation <sup>102</sup>. When eIF2α phosphorylation was augmented with salubrinal, the accumulation of α-synuclein oligomers was ameliorated and disease onset was delayed. There is further evidence that modulating the levels of ER stress may be beneficial in PD, since 4-PBA, which has been shown to possess 'chemical chaperone activity' that can protect cells from ER stress, rescues cells from the neurotoxicity of rotenone <sup>103</sup> and 'pre-
conditioning' neuroblastoma cells with ER stress to up-regulate chaperone levels can protect the cells from subsequent challenge with 6OHDA <sup>104</sup>.

## Huntington disease

Huntington disease (HD) is an inherited autosomal dominant disorder characterised by motor dysfunction, psychiatric disturbances and intellectual decline. It is caused by expansions of CAG repeats within the *HTT* gene encoding huntingtin and so represents an archetypal poly-Q disease. In health, the normal *HTT* gene contains 6 to 35 repeats, while in patients affected by HD there may be more than 40, which generates a protein prone to aggregation. Although this aggregation takes place in the cytosol this has been shown to provoke ER stress <sup>105</sup>.

It is now appreciated that perturbations of protein folding within one organelle can be propagated to another. This concept of cell-wide protein homeostasis, or 'proteostasis', may be especially relevant to HD and other poly-Q disorders wherein cytoplasmic aggregates trigger the UPR <sup>106</sup>. The precise mechanism linking aggregation in the cytosol with malfunction of the ER is poorly understood; however, expression of poly-Q proteins and mutant huntingtin in cells has been shown to bind components of the ERAD machinery and impair their function <sup>12, 107</sup>. It is possible that huntingtin is a target of the ERAD ubiquitin ligase HRD1, since HRD1 can ameliorate the toxicity of the poly-Q protein <sup>107</sup>. By sequestering HRD1 and other components, huntingtin might reduce the capacity of a neurone
to cope with misfolded proteins by ERAD, thereby rendering them more
vulnerable to ER stress <sup>12</sup>. In addition, while chronic ER stress is known to
induce cell death in some circumstances, it might also impair intracellular
signalling in viable cells. Recent data suggest that ER calcium release via the
IP3R1 channel is impaired by ER stress in HD owing to the loss of a stimulatory
interaction between BiP and this channel <sup>108</sup>.

Amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) involves the progressive loss of motor neurones causing an irreversible decline of muscle power and death usually within five years. The aetiology remains largely unknown, although 5-10% of cases display a dominant pattern of inheritance suggestive of a toxic-gain-of-function, so-called Familial ALS (FALS). Of these, a fifth are caused by mutations in the copper-zinc superoxide dismutase 1 (SOD1) that accumulates as fibrils in Lewy Body-like hyaline inclusions, the histological hallmark of FALS.

Post-mortem brains from ALS patients show elevated levels of markers of ER stress <sup>109</sup>. Furthermore, disease models of FALS implicate ER stress in its pathogenesis. Although SOD1 was thought to be an abundant cytosolic protein, it is also secreted by multiple cell types and co-localises with ER markers <sup>110</sup>. When expressed in cultured cells or in animal neuronal tissue, mutant SOD1 accumulates within the ER and upregulates targets of the UPR <sup>111-113</sup>.
Activation of PERK can be detected in animals prior to the development of overt disease<sup>114</sup> and it has been shown in three models of FALS that a subset of motor neurones are more sensitive to the toxic effects of mutant SOD1 and show evidence of ER stress at a younger age<sup>115</sup>. When one allele of *Perk* is deleted in mice expressing a mutant form of SOD1, onset of disease is accelerated, suggesting that phosphorylation of eIF2α is protective in this disorder<sup>116</sup>. This is supported by the protection of vulnerable neurones in animals treated with salubrinal<sup>115</sup>.

Surprisingly, deletion of neuronal XBP1 has been shown to delay the onset of disease extending the lives of FALS mice<sup>117</sup>. In cultured neurones depletion of XBP1 reduces SOD1 aggregates and toxicity through enhanced autophagy, which might plausibly explain this apparent neuroprotection through increased clearance of aggregated protein. Similarly, enhanced degradation of SOD1 by overexpression of ERAD protein derlin1 ameliorates SOD1-induced cell death<sup>118</sup>. Though mutant SOD1 has been shown to interact with derlin1 and so impair ERAD to promote ER stress<sup>119</sup>.

## Multiple sclerosis

Regulated phosphorylation of eIF2α is important for the health of myelinated neurones within the central nervous system as illustrated by the CACH/VWD syndrome and Pelizaeus-Merzbacher leukodystrophy as mentioned previously. This holds true in multiple sclerosis (MS), although the timing of eIF2α
phosphorylation relative to the onset of disease determines its consequences. In the experimental autoimmune encephalitis (EAE) model of MS, ER stress can hasten or delay disease progression if induced after of before disease onset respectively ¹²⁰, ¹²¹; a phenomenon previously seen with interferon treatment. Indeed, both EAE and interferon cause phosphorylation of eIF2α by PERK. Only when animals are exposed to interferon prior to EAE is a protective 'pre-conditioning' seen and, importantly, the protection is lost if one allele of *Perk* is deleted ¹²⁰. In contrast, interferon treatment of animals with established EAE exacerbates their ER stress and worsens demyelination through impaired oligodendrocyte survival.

## FENIB

Ten years ago, the first cases of a new familial encephalopathy with inclusion bodies (FENIB) were described in two families living in the USA ¹²². The primary symptom of this autosomal dominant neurodegenerative disease was pre-senile dementia. In each family, eosinophilic neuronal inclusions were observed in the deeper layers of the cerebral cortex and in the *substantia nigra* (Figure 5). After isolation and sequencing of these inclusions, it was demonstrated that they were composed of a single protein, neuroserpin. When expressed in neurones, the mutants of neuroserpin accumulate to form acid-Schiff-positive diastase-resistant inclusions in the ER, also called Collins' bodies ¹²².
Neuroserpin is a member of the serine protease inhibitor (serpin) superfamily. Serpins are capable of polymerisation under certain circumstances and many examples of naturally occurring mutations exist that promote polymerisation leading to disease ¹²³, ¹²⁴. The retention of polymerised neuroserpin within the ER causes neurotoxicity through a toxic gain-of-function, while the lack of secretion causes the activation of proteolysis and hence disease through a loss-of-function ¹²³.

Despite being retained in the ER, the polymerised mutants of neuroserpin fail to activate the UPR ¹¹. Instead, these polymers induce NFκB signalling in a calcium-dependent manner ¹¹. This is reminiscent of the ER overload response (EOR), which is activated by the accumulation of well-folded protein as happens, for example, in some viral infections ¹²⁵. The degradation of mutant neuroserpin involves the co-ordinated activity of both the ERAD and autophagic pathways ¹²⁴. Since these pathways have successfully been targeted in preclinical studies of a related serpinopathy using the drug carbamazepine ¹²⁶, there is hope for pharmacological treatment of this and other ER overload disorders. Indeed, it is likely that similar strategies will prove beneficial in other diseases of protein accumulation, such as those described in the sections concerning ER stress.

## Prion diseases

The nature of the ER dysfunction observed in prion diseases remains less clear. While ER stress pathways were activated in a cell model of Creutzfeldt-Jakob
disease (CJD), UPR signalling was not detected in a transgenic mouse model for
prion disease <sup>5</sup>. Nevertheless, both primary neuronal cultures and PC-12 cells
expressing PrP mutants develop dramatic ER swelling suggesting that ER
homeostasis is perturbed <sup>6</sup>. The failure to detect splicing of XBP1 mRNA or
differences in BiP levels in prion disease models suggests that prion proteins
might not elicit classical ER stress <sup>5</sup>. However, pharmacological induction of ER
stress in neuronal cell lines expressing mutant PrP can decrease PrP levels <sup>7</sup>.
Recently it was shown that prion replication causes prolonged repression of
protein translation leading to synaptic failure of neuronal loss <sup>9</sup>. In contrast to
many ER stress-related disorders in which enhanced phosphorylation of eIF2α is
protective, prion-induced neurodegeneration is enhanced by salubrinal while
overexpression of GADD34 is protective. The relationship between the
prionopathies and ER dysfunction therefore requires further examination.

Concluding remarks and therapeutic potential

The evidence for activation of ER stress responsive pathways in a range of
neurological conditions is strong. What remains to be determined is how
successful strategies that target these responses will be in treating these
diseases. The UPR signalling field has matured to a point at which small
molecular inhibitors of its components are now being developed.

The muscle relaxant dantrolene targets IP3R channels of the ER to prevent the
release of calcium. This provides neuroprotection in models of HD <sup>127</sup> and of
cerebral hypoxia-reperfusion injury, where its action is associated with reduced levels of ER stress <sup>128</sup>. Prolonging the phosphorylation of eIF2α and thus delaying the recovery of protein translation with agents such as salubrinal or guanabenz is another promising approach <sup>28, 29</sup>. Salubrinal has already been shown to reduce the accumulation of α-synuclein in models of ALS <sup>102, 129</sup>, reduce the toxicity of Aβ to primary neurones <sup>73</sup>, and lessen the ER stress associated with cerebral ischemia/reperfusion and models of HD <sup>1, 130</sup>. More recently, agents have been developed that inhibit IRE1, including quinotrierixin <sup>131</sup> and 4μ8C <sup>132</sup>. Both selectively inhibit the splicing of XBP1 mRNA and the latter prevents expansion of the ER without obvious toxicity, which might have therapeutic utility to impede the formation of protein inclusions in neurones.

The list of neurological disorders with impaired ER protein folding is already long and appears set to grow further for some time to come (Table 1). It will therefore aid the neurologist and neuroscientist alike to appreciate the signalling pathways that communicate this failure of ER function to the rest of the cell, since it is likely these insights will inform research, diagnosis and ultimately therapy.

## Acknowledgements

Our labs are funded by grants from the MRC and ART and by Papworth NHS Trust. AJK is a Wellcome Trust PhD student and SJM is an MRC Senior Clinical Fellow (MRC Ref G1002610). DCC is an Alzheimer's Research UK Senior Research Fellow and is supported by the MRC and EPSRC (MRC Ref
G0700990). EM is funded by the Pasteur Institute-Cenci Bolognetti Foundation and by the Telethon Foundation.

**Contributions and conflicts of interest**

BDR and SJM planned and wrote the review. All authors commented on the final manuscript. BDR performed the literature search for and wrote the stroke, Parkinson's and Huntington's disease, multiple sclerosis and prionopathy sections and prepared figure 1. AJK performed the literature search for Alzheimer's disease, and contributed to drafting the Alzheimer's disease section and figure. EM helped draft the FENIB section. DCC helped draft the Alzheimer's section. DAL helped draft the FENIB section. SJM performed the literature search for ER stress and supervised the review project.
# Panel 1

## Search Strategy

We set out by searching the PubMed database with the terms "(endoplasmic reticulum) AND (brain OR neurological) AND disease" including only articles in English. These titles and abstracts were screened and relevant papers were obtained for detailed assessment. Their bibliographies were also screened for additional sources. Papers published prior to September 2012 are included. Where possible, emphasis is given to articles published within the last 5 years.

## Figure legends

<caption>**Figure 1. Endoplasmic reticulum stress signalling**</caption>

In health, newly synthesised secretory proteins are translocated into the ER lumen co-translationally. They fold with the aid of chaperones, for example BiP. When recognised as correctly folded they exit the ER in COPII-coated vesicles and traffic along the secretory pathway. During ER stress, misfolded proteins accumulate in the ER lumen and sequester BiP from the sensor molecules PERK, IRE1 and ATF6. PERK phosphorylates eIF2α, which binds and inhibits its guanine nucleotide exchange factor eIF2B. This blocks new secretory protein synthesis and triggers the integrated stress response (ISR) through increased levels of ATF4. Activation of IRE1 leads to generation of active XBP1, while
cleavage of ATF6 generates ATF6c. These transcription factors co-operate to
induce target genes of the unfolded protein response (UPR).

**Figure 2. ER stress during cerebral ischaemia**

During an ischaemic stroke, the lack of glucose and oxygen supply in neurones
induces a dramatic fall in ATP production. Without energy, BiP binds to unfolded
proteins less efficiently resulting in the accumulation of misfolded ER client
proteins and subsequent activation of the UPR. In parallel, failure of the SERCA
pump depletes ER calcium and worsens chaperone function still further.
Extracellular tPA (tissue-type plasminogen activator) cleaves the NMDA receptor
allowing calcium influx into the cytosol. The resultant elevation of cytosolic
calcium triggers calcium-induced calcium release via ryanodine receptors located
in the ER membrane leading to calcium depletion from the ER. In severely
affected neurones, elevated cytosolic calcium ultimately induces cell death. In
surviving neurones, induction of the eIF2α phosphatase GADD34 by ATF4 in a
CHOP-dependent manner enables the eventual recovery of protein translation.
This can contribute to reperfusion injury through increasing the level of ER
protein load.

**Figure 3. Alzheimer's disease and ER stress**

(A) In health, amyloid precursor protein (APP) is cleaved sequentially by β-secretase followed by γ-secretase in the TGN and early endosomes mainly
generating non-toxic Aβ₄₀. APP is recycled via the endosomal pathway. (B) In Alzheimer's disease, toxic Aβ₄₂ is generated and secreted. Exogenous Aβ₄₂ causes ER stress via a poorly understood mechanism. In some cases of familial Alzheimer's disease, mutations in PS1 impair trafficking of APP, thereby enhancing Aβ₄₂ secretion. Mutated PS1 may also lead to APP processing within the ER.

**Figure 4: ERAD of amyloid precursor protein**

Newly synthesised proteins, for example amyloid precursor protein (APP), interact with ER chaperones during multiple folding cycles until they reach their native folded state. Overexpression of BiP can increase retention of APP in the ER. If protein folding fails, the terminally misfolded client is targeted for degradation via ERAD. The interaction of glycoprotein substrates with both the folding machinery and the ERAD machinery is governed by cycles of de-mannosylation and re-mannosylation. Eventually, low mannose species can interact with EDEM and be targeted for dislocation from the ER lumen into the cytosol. This process is incompletely understood and displays substrate specific differences, but for APP involves a complex containing Hrd1 and Sel1L. Once in the cytosol, the ERAD substrate is ubiquitinated and eventually degraded by the proteasome.
**Figure 5: FENIB and the ER overload response**

(A) Eosin staining of cerebral cortex from individuals with wild-type neuroserpin (WT) and the S52R or G392E mutations. Mutant protein is retained as polymers within the ER to form Collins bodies (red). The rate of polymer formation correlates with the age at which symptoms develop and the amount of Collins' bodies that can be detected. (B) Monomeric serpins, for example neuroserpin, present a reactive centre loop (red) that functions as a pseudosubstrate for their target protease. However, point mutations in the shutter domain (purple circle) open β-sheet A (green) to allow the insertion of the reactive centre loop belonging to a second serpin molecule. If allowed to propagate, this loop-sheet linkage generates large stable pathological polymers that are retained in the ER to form Collins' bodies. (C) These polymers are formed of folded neuroserpin and therefore fail to activate the UPR. Instead they cause the release of ER calcium into the cytosol, which causes NFκB activation and target gene induction.
<table><thead><tr><td>Disease</td><td>Neurological phenotype</td><td>Cause</td><td>Proposed mechanism for ER dysfunction</td><td>Selected references</td></tr></thead><tbody><tr><td>CACH/VWM leukodystrophy</td><td>Cerebellar ataxia<br>Central hypomyelination<br>Autosomal recessive</td><td>eIF2B mutations</td><td>Enhanced ATF4 synthesis</td><td>22-24</td></tr><tr><td>Pelizaeus-Merzbacher leukodystrophy</td><td>Cerebellar ataxia<br>Central hypomyelination<br>X-linked</td><td>PLP1 gene duplication</td><td>Accumulation of PLP1 within the ER</td><td>26</td></tr><tr><td>Charcot-Marie tooth type 1A</td><td>Peripheral sensory neuropathy<br>Peripheral demyelination<br>Autosomal dominant</td><td>PMP22 mutation or duplication</td><td>Accumulation of myelin protein PMP22 within the ER</td><td>133</td></tr><tr><td>Charcot-Marie tooth type 1B</td><td>Peripheral sensory neuropathy<br>Peripheral demyelination<br>Autosomal dominant</td><td>MPZ mutation</td><td>Presumed accumulation of myelin protein P0 within the ER</td><td>27</td></tr><tr><td>Multiple sclerosis</td><td>Progressive motor and cognitive impairment<br>Patchy central demyelination<br>Sporadic</td><td>Inflammatory loss of myelination</td><td>Inflammation-driven ER stress</td><td>120, 121</td></tr><tr><td>Marinesco-Sjögren syndrome</td><td>Cerebellar ataxia<br>Purkinje cell loss<br>Autosomal recessive</td><td>SIL1 gene mutation</td><td>BiP dysfunction due to impaired SIL1 nucleotide exchange activity</td><td>64, 65</td></tr><tr><td>Alzheimer disease</td><td>Loss of cortical neurones<br>Progressive cognitive impairment<br>Sporadic &amp;</td><td>Poorly understood β-amyloid accumulation hyperphosphorylation of tau</td><td>Impaired ER calcium homeostasis<br>Direct Aβ toxicity</td><td>2-4, 71-73, 75, 80, 81, 83</td></tr></tbody></table>
<table><tr><td></td><td>Autosomal dominant</td><td></td><td></td><td></td></tr><tr><td rowspan="2">Parkinson disease</td><td>Progressive motor and cognitive dysfunction</td><td>Poorly understood loss of dopaminergic neurones from the substantia nigra</td><td>Impaired mitochondrial ATP generation causing secondary ER failure</td><td rowspan="2">87, 91, 93, 96, 101-104</td></tr><tr><td>Sporadic<br>Autosomal Recessive<br>Autosomal Dominant</td><td>Familial forms due to mutations in PARK2, SNCA and PINK1</td><td>ER accumulation of aggregated synuclein</td></tr><tr><td>Huntington disease</td><td>Progressive motor and cognitive dysfunction<br>Autosomal Dominant</td><td>Expansion of CAG repeats in the HTT gene leading to selective neuronal loss in caudate and putamen</td><td>Failure of proteostasis likely due to cytosolic aggregates of HTT impairing proteasome function</td><td>12, 106-108</td></tr><tr><td rowspan="2">Amyotrophic lateral sclerosis</td><td>Progressive motor impairment</td><td>Poorly understood loss of motor neurones</td><td rowspan="2">Accumulation of mutant SOD1 within the ER</td><td rowspan="2">109, 111-116, 119</td></tr><tr><td>Sporadic<br>&<br>Autosomal Dominant</td><td>Some familiar forms due to mutations in SOD1 gene</td></tr><tr><td>Prion diseases</td><td>Spongiform encephalopathy<br>Infective<br>&<br>Autosomal Dominant</td><td>Propagation of infective prion conformation of PrP protein</td><td>Impaired cellular proteostasis<br>Possible direct ER toxicity</td><td>5-7, 9</td></tr><tr><td>FENIB</td><td>Progressive dementia and epilepsy<br>Autosomal Dominant</td><td>Mutations of SERPINI1</td><td>Polymerised neuroserpin accumulates within the ER activating the ER overload response</td><td>11, 124</td></tr><tr><td>Tuberous sclerosis complex</td><td>Central hamartomas cause epilepsy often with cognitive and behavioural defects</td><td>TSC1 and TSC2 mutations</td><td>mTORC-mediated impairment of ER stress signalling</td><td>134</td></tr></table>
<table><tr><td></td><td>Autosomal Dominant</td><td></td><td></td><td></td></tr><tr><td>Epilepsy</td><td>Progressive neuronal loss<br>Sporadic</td><td>Repeated seizure activity</td><td>Incompletely understood<br>Likely involves disturbed<br>ER calcium homeostasis</td><td>135</td></tr><tr><td>Wolfram syndrome 1</td><td>Diabetes insipidus, optic<br>atrophy, deafness<br>Autosomal Recessive</td><td>Mutations of WFS1<br>gene.</td><td>WFS1 encodes an ER ion<br>channel whose dysfunction<br>induces ER stress</td><td>136</td></tr><tr><td>Torsion dystonia 1</td><td>Involuntary sustained muscle<br>contraction<br>Autosomal Dominant</td><td>Mutations in TOR1A<br>gene</td><td>Impairment of Torsin A, an<br>AAA ATPase chaperone in<br>the ER</td><td>137</td></tr><tr><td>Lipid storage<br>disorders (e.g.<br>Gaucher disease)</td><td>Progressive ataxia, mental<br>and motor deficiency<br>Autosomal Recessive</td><td>Mutations in GBA</td><td>Disturbed ER calcium<br>homeostasis and<br>accumulation of mutated<br>protein within the ER</td><td>138</td></tr><tr><td>Cerebral<br>ischaemia</td><td>Neuronal death<br>Sporadic</td><td>Ischaemia causing<br>impaired protein folding</td><td>Energy deficiency causing<br>ER chaperone and enzyme<br>dysfunction</td><td>1, 51, 54, 59, 61, 62</td></tr><tr><td>Sleep apnoea</td><td>Neuronal death<br>Sporadic.</td><td>Ischaemia causing<br>impaired protein folding</td><td>Energy deficiency causing<br>ER chaperone and enzyme<br>dysfunction</td><td>45</td></tr><tr><td>Trauma</td><td>Neuronal death<br>Sporadic</td><td>Neuronal trauma and<br>impaired perfusion</td><td>Incompletely understood<br>Likely involves disturbed<br>energy metabolism and ER<br>calcium homeostasis</td><td>139</td></tr></table>
**Table1: Neurodegenerative diseases and ER stress: an overview.**

CACH: Childhood ataxia with central hypomyelination; VWM: vanishing white matter; eIF2: eukaryotic translation initiation factor 2; ATF4: activating transcription factor 4; PLP1: proteolipid protein 1; CHOP: CCAAT/enhancer binding protein; IRE1: Inositol-requiring enzyme-1; Bip: Binding immunoglobulin protein; PMP22: peripheral myelin protein 22; PS1: presenilin 1; ERAD: ER-associated protein degradation; SOD1: Cu/Zn superoxide dismutase enzyme; SERCA: sarco/endoplasmic reticulum Ca2+--ATPase; PLP1: proteolipid protein 1; MPZ: myelin protein zero
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33-44.
IMPLICATIONS OF ENDOPLASMIC RETICULUM DYSFUNCTION IN
NEUROLOGICAL DISEASE

Benoit D. Roussel<fnref n="1" />,<fnref n="4" /> PhD, Antonina J. Kruppa<fnref n="1" /> PhD, Elena Miranda<fnref n="2" /> PhD, Damian C. Crowther<fnref n="3" /> PhD, David A. Lomas<fnref n="1" /> PhD and Stefan J. Marciniak<fnref n="1" /> PhD

Correspondence should be addressed to:

Dr Benoit D Roussel & Dr Stefan J Marciniak

Cambridge Institute for Medical Research,

Wellcome Trust/MRC Building,

Hills Road, Cambridge CB2 0XY, United Kingdom

Email: [broussel@cyceron.fr](mailto:broussel@cyceron.fr), [sjm20@cam.ac.uk](mailto:sjm20@cam.ac.uk)

<footnote n="1">¹Department of Medicine, University of Cambridge, Cambridge Institute for Medical Research (CIMR), Wellcome Trust/MRC Building, Hills Road, Cambridge CB2 0XY, UK</footnote>

<footnote n="2">²Department of Biology and Biotechnology 'Charles Darwin' and Pasteur Institute-Cenci Bolognetti Foundation, University of Rome 'La Sapienza', piazzale Aldo Moro 5, 00185 Rome, Italy</footnote>

<footnote n="3">³Department of Genetics, University of Cambridge, Downing Site, Cambridge, CB2 3EH, UK</footnote>

<footnote n="4">⁴INSERM U919, University Caen Basse Normandie, Serine Proteases and Pathophysiology of the Neurovascular Unit, Public Interest Group CYCERON, F-14074 Caen, France (*actual address)</footnote>
Key words: ER stress, Neurodegeneration, Cerebral ischaemia, Alzheimer's disease, Parkinson's disease, FENIB

Running title: ER dysfunction in the CNS

Word count: 5,988 (excluding references and legends)
Endoplasmic reticulum (ER) dysfunction is important in the pathogenesis of many neurological diseases. In this review, we examine the evidence for ER dysfunction in a range of neurological conditions including cerebral ischaemia, sleep apnoea, Alzheimer's disease, multiple sclerosis, amyotrophic lateral sclerosis, the prion diseases and Familial Encephalopathy with Neuroserpin Inclusion Bodies (FENIB). Protein misfolding in the endoplasmic reticulum initiates a well-studied 'Unfolded Protein Response' in energy-starved neurones during stroke that is relevant to the toxicity of reperfusion. The toxic peptide Aβ induces 'ER stress' in Alzheimer's disease leading to activation of similar pathways, while the accumulation of polymeric neuroserpin in the neuronal ER triggers a poorly understood 'ER overload response'. In other neurological disorders such as Parkinson's and Huntington's diseases ER dysfunction is well recognised but the mechanisms for this are less clear. By targeting components of these signalling responses, it may prove possible to ameliorate their toxic effects and treat a range of neurodegenerative conditions.

Word count: 157
## Introduction

It can be argued that most neurodegenerative disorders arise from defective protein folding. This is clearly true for those rare disorders caused by the misfolding of mutated neuronal proteins such as Huntington's disease, but it is also the case for common disorders such as cerebral ischaemia where the lack of energy can impair normal protein folding ¹ and sporadic Alzheimer's disease where protein aggregation causes cellular stress ²⁻⁴. Even some infectious neurodegenerative disorders such as the prionopathies arise from abnormal protein folding ⁵⁻⁹. Consequently, the processes for disposing of misfolded proteins are the focus of much research. Although the subcellular location of protein misfolding may differ between these disorders, it appears that the interdependence of protein folding throughout the cell results in ER dysfunction being the final common pathway for many, if not all, of these diseases (Table 1).

## ER Dysfunction

The endoplasmic reticulum (ER) is required for folding of all secreted and membrane proteins and insults that impair its function induce a pathological state termed 'ER stress' ¹⁰. This triggers an adaptive programme called the Unfolded Protein Response (UPR), which combines the early inhibition of protein synthesis with a later upregulation of genes that promote protein folding or disposal ¹⁰. Both the translational and transcriptional components of the UPR protect the neurone from becoming overwhelmed by misfolded ER proteins; however, when the insult is too great, apoptotic cell death frequently ensues. While many cells
are relatively protected from accumulating misfolded protein through continued
dilution of their ER by cell replication, this process is unavailable to post-mitotic
neurones, which depend exclusively on the UPR. Even glial cells, which can
replicate, are susceptible to ER stress owing to their highly developed secretory
pathway.

It is important that the UPR be distinguished from the less well-understood ER
Overload Response (EOR) ¹¹. The former is triggered by protein misfolding
within the ER lumen, while the latter occurs when well-folded or misfolded
proteins accumulate and distend the ER. In this review, we will discuss the
relevance of these forms of ER dysfunction to neurological disease, since a
better understanding of each is likely to identify novel therapeutic strategies
*(Panel 1)*. We will focus on ischaemic stroke, Alzheimer's disease and
Parkinson's disease to discuss ER stress, while using Familial Encephalopathy
with Neuroserpin Inclusion Bodies (FENIB) to illustrate ER overload, since these
are the diseases for which the most evidence currently exists. In addition, we will
discuss the evidence for ER dysfunction in multiple sclerosis, amyotrophic lateral
sclerosis and the prion disorders. Some neurological disorders provoke ER
stress signalling despite being caused by the accumulation of protein within the
cytoplasm, for example Huntington's disease ¹². Such disorders emphasise the
interdependence of protein folding networks in the neurone and so shall be
discussed as examples of disordered protein homeostasis or 'proteostasis'.
## ER stress signalling

The signalling pathways triggered by ER stress comprise the UPR<sup>10</sup> (Figure 1). Although it may seem tiresome to dwell on individual molecular components, this is unavoidable when studying a phenomenon as complex as ER stress. Not least, because many of the measures commonly used to detect and quantify ER stress are themselves components of the UPR. Some of these are activated transiently and so their absence cannot be used as evidence for a lack of ER stress<sup>10, 13</sup>; conversely, certain components can be activated by stresses other than ER protein misfolding and so need to be interpreted with caution. We shall therefore begin with a review of the major mediators of ER stress signalling with an emphasis on their role in neurones before moving on to considering specific examples of neuropathology mediated by ER dysfunction.

### PERK

Whenever misfolded protein accumulates in the ER lumen, new protein synthesis is rapidly inhibited<sup>14</sup>. This occurs because ER stress activates the PKR-like Endoplasmic Reticulum eIF2α Kinase (PERK), also known as Pancreatic eIF2α kinase (Pek) because of the tissue in which it was first discovered<sup>14</sup>. This kinase is ubiquitously expressed but is most important in highly secretory tissues such as the brain where it functions to match protein synthesis with the efficiency of protein folding in the ER<sup>15</sup>. When protein folding is progressing well PERK is inactive, but when misfolded ER proteins accumulate the kinase is activated and phosphorylates eukaryotic translation initiation factor 2α (eIF2α) (Figure 1)<sup>16</sup>.
This substrate is a subunit of the heterotrimeric GTP-binding complex eIF2 that regulates the initiation of protein synthesis <sup>14</sup>. EIF2 recruits the methionyl-tRNA to the ribosome at the onset of protein translation, but when its eIF2α subunit is phosphorylated this activity is lost and translation ceases. The load of proteins entering the ER is thus reduced following PERK activation preventing the accumulation of more misfolded proteins.

## ATF4

Despite the inhibition of protein translation that accompanies phosphorylation of eIF2α, a small subset of mRNAs is translated more efficiently under these conditions <sup>17</sup>. The best characterised of these is Activating Transcription Factor 4 (ATF4), which triggers a transcriptional programme of stress-responsive genes <sup>17</sup>. Other members of the eIF2α kinase family can inhibit translation and activate these genes; for example PKR responds to viral infection, HRI to iron deficiency and GCN2 to amino acid starvation <sup>18</sup>. This ability of eIF2α phosphorylation to mediate a response to multiple stresses led to this pathway being named the Integrated Stress Response <sup>19</sup>. Surprisingly, within the brain this can provoke specific behavioural responses. A lack of essential dietary amino acids triggers the phosphorylation of eIF2α in the anterior piriform cortex by GCN2, which alters feeding behaviour in rodents <sup>20, 21</sup>. Thus, phosphorylation of eIF2α is not synonymous with ER stress and multiple lines of evidence should always be sought when attributing phosphorylation of eIF2α to ER dysfunction.
Phosphorylation of eIF2α is particularly important for the maintenance of axonal myelination in the human central nervous system (Table 1); indeed mutations that disrupt the regulation of eIF2α result in white matter hypomyelination disorders such as Childhood Ataxia with Central nervous system Hypomyelination (CACH syndrome) and Vanishing White Matter (VWM) <sup>22</sup>. This family of autosomal recessive conditions constitutes a spectrum of severity from congenital to adult-onset, involving the progressive loss of mental and motor function due to a loss of brain white matter. In all cases, the causative mutations are found within subunits of eIF2B, a guanine nucleotide exchange factor (GEF) responsible for recharging the eIF2 complex with GTP <sup>23, 24</sup> (Figure 1). These mutations interfere with the GEF activity of eIF2B and enhance the synthesis of ATF4. This deregulates many target genes including components of a negative feedback system, including CHOP and GADD34, that normally recover protein translation following its initial inhibition by PERK <sup>13, 25</sup>.

## • CHOP

The transcription factor CHOP is induced by ATF4 and has widely been thought to be pro-apoptotic during ER stress, since *Chop-/-* animals are resistant to ER stress-induced cell death <sup>13</sup>. However, this is inconsistent with observations made in neurological diseases. For example, CHOP expression is clearly anti-apoptotic in mouse models of the X-linked disorder Pelizaeus-Merzbacher leukodystrophy caused by abnormalities of the proteolipid protein 1 (PLP1) gene <sup>26</sup>. Indeed, identification of the target genes of CHOP failed to reveal a link with
apoptosis but instead identified genes that promote protein secretion<sup>13</sup>. This led us to suggest that in models of severe ER stress the promotion of protein secretion by CHOP might increase the intensity of stress and thus induce cell death <sup>13</sup>. By contrast, during conditions of milder ER stress, for example in Pelizaeus-Merzbacher leukodystrophy, it is likely that CHOP does not enhance ER stress sufficiently to trigger cell death <sup>26</sup>. These observations highlight the need to model not only the nature of the stress, but also its *intensity* and *duration* since differences in either can significantly affect the outcome and thus lead to beneficial or deleterious effects depending upon the context. There may also be cell type specific consequences of expressing CHOP. For example, while deletion of *Chop* worsens the central hypomyelination seen in Pelizaeus-Merzbacher leukodystrophy <sup>26</sup>, *Chop/-* animals are protected against the peripheral hypomyelination caused by mutant myelin protein zero (P0) in mouse models of Charcot Marie Tooth type 1B <sup>27</sup>.

## GADD34

A critical target gene of CHOP encodes Growth Arrest and DNA Damage-inducible protein 34 (GADD34), which is a regulatory subunit of protein phosphatase 1 (PP1) <sup>13</sup>. This dephosphorylates eIF2α and thus brings about the recovery of protein translation after its initial inhibition. It has been shown in mice that inactivation of the *Gadd34* gene can protect animals from tissue damage during ER stress by reducing the accumulation of misfolded proteins <sup>13</sup>. Subsequently, a small molecule called salubrinal was identified that increases
the level of eIF2α phosphorylation and promotes survival of ER-stressed cells²⁸.

It has been suggested that salubrinal may be an inhibitor of the eIF2α phosphatases ²⁸, but direct evidence for this is lacking. Recently, however, a selective inhibitor of GADD34, guanabenz, has been identified confirming the beneficial effects of GADD34 inhibition during ER stress at least in cultured cells ²⁹. It is therefore likely that future therapies involving the inhibition of GADD34 will be able to modulate ER stress.

## ATF6

In addition to the Integrated Stress Response genes regulated by PERK and ATF4, many additional genes are induced during the UPR by two further ER stress sensors, Inositol-Requiring Enzyme 1 (IRE1) and Activating Transcription Factor 6 (ATF6) ¹⁰ (Figure 1). Like PERK, both are ER membrane proteins that are held inactive under normal conditions by the binding of the ER chaperone Binding Immunoglobulin Protein (BiP; also known as GRP78). During stress, misfolded proteins sequester BiP, freeing ATF6 to traffic to the Golgi apparatus where it is cleaved to release a soluble transcription factor. Of note, astrocytes express an ATF6-related protein called OASIS (Old Astrocyte Specifically Induced Substance) whose functions appear to overlap partially with those of ATF6 ³⁰. This might enable astrocytes to respond to ER stress in a tissue-specific manner.
While cleavage of ATF6 is strong evidence of ER stress, this is difficult to detect. Consequently, many studies fail to measure this arm of the UPR, but the use of sensitive reporters that measure the binding of ATF6 to specific DNA sequences can circumvent this problem *in vitro* <sup>11</sup>.

## IRE1

The activation of IRE1 closely resembles that of PERK but, unlike PERK, IRE1 triggers splicing of the mRNA encoding the transcription factor X-box Binding Protein 1 (XBP1) <sup>31</sup>. This induces a frame shift that generates the active form of this protein. Like phosphorylation of eIF2α, the splicing of XBP1 mRNA is transient <sup>13</sup>. Following the alleviation of ER stress by the induction of UPR target genes, spliced XBP1 mRNA is rapidly lost from the cell to be replaced by the unspliced form <sup>13</sup>. In laboratory studies, supra-physiological levels of ER stress can cause complete splicing of XBP1 mRNA, but *in vivo* stresses are frequently subtler and so it is rare to observe complete splicing and careful quantification is therefore necessary <sup>32</sup>.

If proteins fail to fold in the ER, they are prevented from progressing along the secretory pathway and eventually are targeted for degradation <sup>33</sup>. This 'ER-associated protein degradation' (or ERAD) returns terminally misfolded proteins to the cytosol where they are ubiquitinated by ER-associated ubiquitin ligases for degradation by the proteasome <sup>34</sup>. Many components of the ERAD machinery
are target genes of XBP1 and so the capacity for ERAD is increased by
activation of IRE1 <sup>35</sup>.

ER dysfunction in neurological disease

Cerebral hypoxia

In industrialized countries stroke is the third biggest killer and the leading cause of disability in adults. Acute ischaemia is the main cause of neuronal loss and, although its pathology is complex, appears to involve ER stress <sup>1, 15, 36-38</sup>. During cerebral ischaemia, neuronal depolarization due to energy depletion causes the uncontrolled release of glutamate. The consequent activation of NMDA receptors on nearby neurones generates further glutamate release and causes an ischaemic depolarization wave to spread outward from the initial site of damage leading to widespread disturbance of calcium homeostasis <sup>39</sup>. Unsurprisingly, many studies implicate ER calcium store release in the resulting excitotoxic death (reviewed in reference <sup>39</sup>).

The ER is the main site for calcium storage within the cell and so its chaperones
have evolved to function efficiently in this high calcium environment; indeed
many require high calcium to function <sup>40</sup>. Calcium is pumped into the ER by the
sarcoendoplasmic reticular calcium-ATPase (SERCA) and released back into the
cytosol by the inositol trisphosphate receptor (IP3R) in response to extracellular
signals and by the ryanodine receptor during calcium-induced calcium release <sup>39</sup>.
Inhibition of the SERCA pump by thapsigargin induces apoptosis in many cell
types including neuroblastoma cells and is often used to induce ER stress in the
experimental setting <sup>41</sup>. During cerebral ischaemia, energy depletion leads to
failure of the SERCA pump and thus redistribution of ER calcium into the cytosol.
This delivers a double blow with combined toxicity from uncontrolled cytosolic
calcium and ER stress due to chaperone dysfunction (Figure 2) <sup>39</sup>.

The UPR is activated in many rodent models of cerebral ischaemia ¹,³⁶. In these
models, protein synthesis is rapidly inhibited and accompanies the
phosphorylation of eIF2α. Although the inhibition of translation has been
attributed to aggregation of components of the translation machinery ⁴², the
primary mechanism appears to be through phosphorylation of eIF2α ⁴³, which
can remain phosphorylated for up to twelve hours following ischaemia ⁴⁴. PERK
is the sole eIF2α kinase activated by cerebral ischaemia and predictably Perk-/-
mice fail to show eIF2α phosphorylation following transient cerebral ischaemia
and fail to reduce protein translation during the reperfusion period ¹⁵. Even with
the far milder recurrent cerebral hypoxia seen in obstructive sleep apnoea,
activation of PERK has been described and has been suggested to play a role in
the loss of upper airway motor neurones in this condition ⁴⁵.

IRE1 is also activated during cerebral ischaemia leading to the induction of UPR
target genes ³⁷, ³⁸. It appears to promote activation of pro-apoptotic caspase-12
in cultured rodent cells and this has been detected in ischaemic rodent brains ⁴⁶.
However, caspase-12 is non-functional in the majority of humans, except those
from sub-Saharan Africa, owing to a common truncating mutation ⁴⁷. For this
reason, it has been proposed that during ER stress in humans, caspase-4 might
serve an analogous function to that of caspase-12 in rodents ⁴⁸.

Due to the problems of detection referred to earlier, the role of ATF6 during
ischaemia is unclear. ATF6 gene expression is elevated following transient
middle cerebral artery occlusion in rats ³⁶, but its activation has not been
detected in rat brain following cardiac arrest and resuscitation ⁴⁹. OASIS is also
upregulated and appears to play a neuroprotective role in response to kainate
excitotoxicity ⁵⁰.

CHOP is induced following forebrain ischaemia in a variety of rodent models
including bilateral common carotid ⁵¹, ⁵² or middle cerebral artery occlusion in
mice ⁴⁴ and global cerebral ischaemia in rats ⁵³. The location within the brain of
CHOP induction appears to correlate well with subsequent cell death ⁵³, ⁵⁴ and
deletion of the *Chop* gene protects mice during bilateral common carotid artery
occlusion ⁵². Similarly, depletion of CHOP using RNA interference partially
prevents the death of astrocyte cultures stressed by oxygen-glucose depletion ⁵⁵.
As discussed, some of the toxicity attributable to CHOP reflects increased protein
translation mediated by GADD34 ¹³. Indeed, GADD34 induction is well-
recognised to accompany cerebral ischaemia ⁵⁶ being detected at the peri-infarct
penumbra within two to twenty-four hours ⁵⁷ and accounts for the transient nature
of eIF2α phosphorylation during stroke ¹⁵. Attenuation of translation protects the
cell from accumulating misfolded proteins, thus treatment with salubrinal (a drug that enhances eIF2α phosphorylation) reduces ER stress and promotes cellular survival in kainate-induced neurotoxicity⁵⁸. Indeed, salubrinal has been shown to limit infarct size in a murine model of stroke ¹.

In addition to attenuating protein translation, the UPR leads to upregulation of ER molecular chaperones, the most abundant being BiP ¹⁰. Following transient forebrain ischaemia, BiP expression in the hippocampus and cortex peaks between twelve to seventy-two hours after reperfusion ⁵⁴, ⁵⁹, but can remain elevated for up to two weeks ³⁶. This is protective, since suppression of BiP enhances apoptosis in hippocampal neurones exposed to excitotoxic and oxidative insults ⁶⁰, while its overexpression in primary astrocyte cultures is protective against oxygen-glucose deprivation ⁶¹. Indeed, higher levels of BiP induction correlate with neuronal protection ³⁷, ⁶².

Induction of the ER chaperone ORP150 (150-kDa oxygen-regulated protein) is also seen in ischaemic neurones, and over-expression of ORP150 protects mice from cerebral ischaemia ⁶³. ORP150 modulates the activity of BiP, which exists in either an ATP- or ADP-bound form, each with different substrate-binding affinity. ORP150 functions as a nucleotide exchange factor for BiP to replace its bound ADP with ATP. Interesting, another BiP nucleotide exchange factor, SIL1, is mutated in the Marinesco-Sjogren syndrome of cerebellar ataxia in which homozygous loss of SIL1 leads to ER stress and selective loss of cerebellar
Purkinje cells<sup>64</sup>. When ATP-BiP binds its substrate protein, a co-chaperone of the DnaJ family is required to stimulate BiP's ATPase activity and so promote high affinity substrate binding. The relative levels of ADP-BiP and ATP-BiP appear to be important for neuronal survival, since the toxicity of SIL1 loss can be ameliorated by the deletion of an ER luminal DnaJ co-chaperone called p58<sup>IPK</sup><sup>65</sup>. While loss of SIL1 would be predicted to reduce the level of ATP-BiP through impaired nucleotide exchange, loss of p58<sup>IPK</sup> is likely to restore balance by lessening BiP's ATPase activity.

When taken together, these observations suggest that ER stress is induced by
cerebral ischaemia and that the UPR limits cerebral infarct size. There is good
evidence to suggest that manipulation of ER stress signalling by increasing
phosphorylation of eIF2α may have important therapeutic effects as an acute
intervention for cerebral ischaemia.

Alzheimer's disease

Often presenting with failing memory, Alzheimer's disease (AD) is a relentlessly
progressive neurodegenerative disorder and the most common cause of
dementia in adults. The classic histopathological features consist of extracellular
plaques of the β-amyloid (Aβ) peptide and intracellular neurofibrillary tangles of
hyperphosphorylated aggregates of the microtubule-associated protein tau.
Studies of familial AD demonstrate that the disorder is caused by overproduction
of the more aggregation-prone Aβ<sub>1-42</sub> peptide <sup>66</sup> (Figure 3). Despite decades of
study, the molecular pathology of this disease remains unclear. However,
increasing evidence suggests that ER stress may represent a new paradigm for
understanding this condition.

Studies using post-mortem tissue from the brains of individuals with AD have
provided evidence for ER dysfunction in this disease. But when looking at a
single marker of ER stress, such studies can generate confusing results. For
example, when BiP is examined in isolation it can show no change ³, a decrease
⁶⁷ or even an increase ², ⁶⁸, although BiP expression does seem to be higher in
brains with histologically more advanced disease ⁶⁸, ⁶⁹. However, whilst often a
useful indicator, the level of BiP expression is inadequate alone to assess the
activation of UPR signalling, especially in a chronic disorder in which histological
features evolve over time. It has therefore proved more useful to include
additional markers of the UPR in such studies. For example, protein disulphide
isomerase, another target of the UPR, is elevated in the temporal lobes of the
brains of individuals with AD ³, associated with neurofibrillary tangles in the
hippocampus and frontal lobes and in dystrophic neurites of senile plaques ⁴.
When one looks for more proximal markers of UPR signalling, there is activation
of PERK in the hippocampus of AD brains, which appears to co-localise with
staining for phosphorylated tau and correlates with histological staging ⁷⁰, ⁷¹. Yet
phosphorylated PERK is barely detectable in tangle-bearing neurones
themselves, suggesting that PERK is activated at the pre-tangle stage in
neurones of the hippocampus ⁷¹. In addition, there are elevated levels of spliced
XBP1 mRNA in the temporal cortex of AD patients when compared to age-
matched controls ³. Congruently, a marked increase in the number of neurones
immunoreactive for phosphorylated (active) IRE1α has been detected in the
hippocampus of individuals with AD ⁷¹.

There is good evidence for activation of PERK in laboratory models of AD.
Increased BiP expression observed upon application of exogenous Aβ to primary
cortical neurones suggests that accumulation of the peptide may directly activate
ER stress signalling ⁷², ⁷³. Moreover, exogenous Aβ has been found to increase
the phosphorylation of PERK and eIF2α in cultured neuronal cells ⁷³, ⁷⁴, while
silencing of PERK by siRNA in Aβ₄₂-treated cells limits eIF2α phosphorylation
and enhances cell death ⁷³. In addition, Aβ₄₂ treatment induces CHOP
expression both in cultured cells and in rabbit hippocampus ⁷⁵, ⁷⁶, while prior
treatment of cells with CHOP anti-sense RNA improves survival following
exposure to the Aβ peptide, suggesting a role for CHOP in Aβ-mediated death ⁷⁷.
In *Drosophila* neurones or mammalian PC-12 cells, exposure to Aβ induces
XBP1 mRNA splicing and in both models overexpression of spliced XBP1 is
protective, while knockdown of XBP1 exacerbates Aβ toxicity ⁷⁸.

PC-12 cells overexpressing mutant forms of presenilin 1 (PS1), a component of
the γ-secretase, that has been linked to familial AD, show increased levels of
phosphorylated eIF2α, as do hippocampal homogenates from PS1 mutant
knock-in mice ⁷⁹. PC-12 cells expressing mutant PS1 have elevated levels of
CHOP, as do knock-in mice expressing mutant PS1⁷⁹. The nature of the interaction between PS1 and ER stress has yet to be fully elucidated, although it is known that PS1 and the SERCA calcium pump physically interact with one another⁸⁰. As discussed, disordered calcium homeostasis is a potent cause of ER stress and much evidence suggests that calcium signalling is perturbed in Alzheimer's disease⁸¹ (reviewed in reference ⁸²). Interestingly, ER stress induced by tunicamycin, a glycosylation inhibitor frequently used to induce ER stress in vitro, increases PS1 expression in multiple cell lines including neuroblastoma⁸⁰. Recent evidence suggests that phosphorylation of eIF2α by PERK during ER stress can increase the level of β-site APP cleaving enzyme-1 (BACE) and thereby promoting amyloid formation ⁸³. This suggests that the interaction between AD and ER stress may prove to be bi-directional.

ER stress has been shown to alter APP localization, processing and degradation by the ERAD machinery ⁸⁴ (Figure 4). During ER stress, immature forms of APP bind to BiP and when BiP is overexpressed Aβ generation is reduced as APP is retained in earlier compartments of the secretory pathway ⁸⁴. The ERAD ubiquitin ligase HRD1 promotes APP ubiquitination and degradation in HEK293 cells reducing the generation of both Aβ₄₀ and Aβ₄₂ ². In addition, an intronic polymorphism (IVS3-88A>G) of SEL1L, another component of the ERAD system, has been associated with AD in an Italian population ⁸⁵. These hints that ERAD is important in the pathogenesis of AD still require further validation. But there is accumulating evidence for elevated UPR signalling in AD and, combined with the
support from model systems, provides a potential mechanism by which the Aβ
peptide and mutants of PS1 might induce ER stress. This offers the potential to
identify new therapeutic targets in an otherwise incurable disease.

Parkinson's disease

With age, 2% of the population suffer the loss of dopaminergic neurones in their nigrostriatal pathway and so develop Parkinson's disease (PD) characterised by hypokinesia, rigidity and tremor. Post-mortem studies identify characteristic Lewy body inclusions in many affected brains, suggesting aberrant protein disposal as a possible cause, while toxins that cause Parkinsonism, such as MPTP, rotenone and 6OHDA, impair mitochondrial function and promote ER stress <sup>86-90</sup>.

Multiple lines of evidence point towards a role for ER stress in the pathogenesis
of PD. For example, excess levels of dopamine are toxic to PC-12 cells, which
correlates with their induction of ER chaperones <sup>91</sup>. The brains of patients
suffering from sporadic PD, but not controls, have elevated levels of
phosphorylated PERK and eIF2α <sup>92</sup>. Perk/- sympathetic neurones are
hypersensitive to 6OHDA <sup>92</sup>, while overexpression of spliced XBP1 is protective
both in cultured cells and animals treated with MMP+/MPTP <sup>87</sup>. CHOP
expression is increased both by 6OHDA and MPTP, and knockout of the Chop
gene is protective in mice treated with 6OHDA <sup>93</sup>.
In Japanese and European families with the juvenile onset autosomal recessive variant of PD (ARPD), classical genetic mapping identified the disease-associated gene *PARK2* <sup>94</sup>. This encodes an ubiquitin ligase Parkin, whose activity is lost with disease-associated mutations <sup>95</sup>. Parkin was suggested to localize to the ER and to be upregulated during the UPR <sup>96</sup>. A major function of Parkin appears to involve targeting defective mitochondria for destruction by autophagy <sup>97</sup>. It is possible that impaired mitochondrial function in patients with *PARK2* mutations manifests as ER stress owing to defects in energy supply during ER protein folding. However, another component of Lewy bodies, α-synuclein, can be found throughout the cell including the ER lumen and may also be a target of Parkin <sup>98</sup>. Mutations of α-synuclein cause an autosomal dominant form of PD <sup>99</sup>, while the glycosylated ER-form of α-synuclein has been shown to be ubiquitinated by Parkin and to accumulate in a non-ubiquitinated form in the brains of individuals with Parkin deficiency <sup>98</sup>. Overexpression of α-synuclein induces ER stress <sup>100</sup>, perhaps through binding to BiP <sup>101</sup>, while PDI appears to impair α-synuclein fibril formation <sup>100</sup>. Recently, oligomers of mutant α-synuclein were shown to accumulate within the ER in an animal model and to trigger the UPR, albeit with relatively little PERK activation <sup>102</sup>. When eIF2α phosphorylation was augmented with salubrinal, the accumulation of α-synuclein oligomers was ameliorated and disease onset was delayed. There is further evidence that modulating the levels of ER stress may be beneficial in PD, since 4-PBA, which has been shown to possess 'chemical chaperone activity' that can protect cells from ER stress, rescues cells from the neurotoxicity of rotenone <sup>103</sup> and 'pre-
conditioning' neuroblastoma cells with ER stress to up-regulate chaperone levels
can protect the cells from subsequent challenge with 6OHDA <sup>104</sup>.

Huntington disease

Huntington disease (HD) is an inherited autosomal dominant disorder characterised by motor dysfunction, psychiatric disturbances and intellectual decline. It is caused by expansions of CAG repeats within the *HTT* gene encoding huntingtin and so represents an archetypal poly-Q disease. In health, the normal *HTT* gene contains 6 to 35 repeats, while in patients affected by HD there may be more than 40, which generates a protein prone to aggregation. Although this aggregation takes place in the cytosol this has been shown to provoke ER stress <sup>105</sup>.

It is now appreciated that perturbations of protein folding within one organelle can
be propagated to another. This concept of cell-wide protein homeostasis, or
'proteostasis', may be especially relevant to HD and other poly-Q disorders
wherein cytoplasmic aggregates trigger the UPR <sup>106</sup>. The precise mechanism
linking aggregation in the cytosol with malfunction of the ER is poorly understood;
however, expression of poly-Q proteins and mutant huntingtin in cells has been
shown to bind components of the ERAD machinery and impair their function <sup>12,107</sup>.
It is possible that huntingtin is a target of the ERAD ubiquitin ligase HRD1,
since HRD1 can ameliorate the toxicity of the poly-Q protein <sup>107</sup>. By sequestering
HRD1 and other components, huntingtin might reduce the capacity of a neurone
to cope with misfolded proteins by ERAD, thereby rendering them more
vulnerable to ER stress <sup>12</sup>. In addition, while chronic ER stress is known to
induce cell death in some circumstances, it might also impair intracellular
signalling in viable cells. Recent data suggest that ER calcium release via the
IP3R1 channel is impaired by ER stress in HD owing to the loss of a stimulatory
interaction between BiP and this channel <sup>108</sup>.

Amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) involves the progressive loss of motor neurones causing an irreversible decline of muscle power and death usually within five years. The aetiology remains largely unknown, although 5-10% of cases display a dominant pattern of inheritance suggestive of a toxic-gain-of-function, so-called Familial ALS (FALS). Of these, a fifth are caused by mutations in the copper-zinc superoxide dismutase 1 (SOD1) that accumulates as fibrils in Lewy Body-like hyaline inclusions, the histological hallmark of FALS.

Post-mortem brains from ALS patients show elevated levels of markers of ER stress <sup>109</sup>. Furthermore, disease models of FALS implicate ER stress in its pathogenesis. Although SOD1 was thought to be an abundant cytosolic protein, it is also secreted by multiple cell types and co-localises with ER markers <sup>110</sup>. When expressed in cultured cells or in animal neuronal tissue, mutant SOD1 accumulates within the ER and upregulates targets of the UPR <sup>111-113</sup>.
Activation of PERK can be detected in animals prior to the development of overt disease<sup>114</sup> and it has been shown in three models of FALS that a subset of motor neurones are more sensitive to the toxic effects of mutant SOD1 and show evidence of ER stress at a younger age<sup>115</sup>. When one allele of *Perk* is deleted in mice expressing a mutant form of SOD1, onset of disease is accelerated, suggesting that phosphorylation of eIF2α is protective in this disorder<sup>116</sup>. This is supported by the protection of vulnerable neurones in animals treated with salubrinal<sup>115</sup>.

Surprisingly, deletion of neuronal XBP1 has been shown to delay the onset of disease extending the lives of FALS mice <sup>117</sup>. In cultured neurones depletion of XBP1 reduces SOD1 aggregates and toxicity through enhanced autophagy, which might plausibly explain this apparent neuroprotection through increased clearance of aggregated protein. Similarly, enhanced degradation of SOD1 by overexpression of ERAD protein derlin1 ameliorates SOD1-induced cell death <sup>118</sup>. Though mutant SOD1 has been shown to interact with derlin1 and so impair ERAD to promote ER stress <sup>119</sup>.

**Multiple sclerosis**

Regulated phosphorylation of eIF2α is important for the health of myelinated neurones within the central nervous system as illustrated by the CACH/VWD syndrome and Pelizaeus-Merzbacher leukodystrophy as mentioned previously.
This holds true in multiple sclerosis (MS), although the timing of eIF2α
phosphorylation relative to the onset of disease determines its consequences. In the experimental autoimmune encephalitis (EAE) model of MS, ER stress can hasten or delay disease progression if induced after of before disease onset respectively ¹²⁰, ¹²¹; a phenomenon previously seen with interferon treatment. Indeed, both EAE and interferon cause phosphorylation of eIF2α by PERK. Only when animals are exposed to interferon prior to EAE is a protective 'pre-conditioning' seen and, importantly, the protection is lost if one allele of *Perk* is deleted ¹²⁰. In contrast, interferon treatment of animals with established EAE exacerbates their ER stress and worsens demyelination through impaired oligodendrocyte survival.

## FENIB

Ten years ago, the first cases of a new familial encephalopathy with inclusion bodies (FENIB) were described in two families living in the USA ¹²². The primary symptom of this autosomal dominant neurodegenerative disease was pre-senile dementia. In each family, eosinophilic neuronal inclusions were observed in the deeper layers of the cerebral cortex and in the *substantia nigra* (Figure 5). After isolation and sequencing of these inclusions, it was demonstrated that they were composed of a single protein, neuroserpin. When expressed in neurones, the mutants of neuroserpin accumulate to form acid-Schiff-positive diastase-resistant inclusions in the ER, also called Collins' bodies ¹²².
Neuroserpin is a member of the serine protease inhibitor (serpin) superfamily. Serpins are capable of polymerisation under certain circumstances and many examples of naturally occurring mutations exist that promote polymerisation leading to disease ¹²³, ¹²⁴. The retention of polymerised neuroserpin within the ER causes neurotoxicity through a toxic gain-of-function, while the lack of secretion causes the activation of proteolysis and hence disease through a loss-of-function ¹²³.

Despite being retained in the ER, the polymerised mutants of neuroserpin fail to activate the UPR ¹¹. Instead, these polymers induce NFκB signalling in a calcium-dependent manner ¹¹. This is reminiscent of the ER overload response (EOR), which is activated by the accumulation of well-folded protein as happens, for example, in some viral infections ¹²⁵. The degradation of mutant neuroserpin involves the co-ordinated activity of both the ERAD and autophagic pathways ¹²⁴. Since these pathways have successfully been targeted in preclinical studies of a related serpinopathy using the drug carbamazepine ¹²⁶, there is hope for pharmacological treatment of this and other ER overload disorders. Indeed, it is likely that similar strategies will prove beneficial in other diseases of protein accumulation, such as those described in the sections concerning ER stress.

## Prion diseases

The nature of the ER dysfunction observed in prion diseases remains less clear. While ER stress pathways were activated in a cell model of Creutzfeldt-Jakob
disease (CJD), UPR signalling was not detected in a transgenic mouse model for
prion disease <sup>5</sup>. Nevertheless, both primary neuronal cultures and PC-12 cells
expressing PrP mutants develop dramatic ER swelling suggesting that ER
homeostasis is perturbed <sup>6</sup>. The failure to detect splicing of XBP1 mRNA or
differences in BiP levels in prion disease models suggests that prion proteins
might not elicit classical ER stress <sup>5</sup>. However, pharmacological induction of ER
stress in neuronal cell lines expressing mutant PrP can decrease PrP levels <sup>7</sup>.
Recently it was shown that prion replication causes prolonged repression of
protein translation leading to synaptic failure of neuronal loss <sup>9</sup>. In contrast to
many ER stress-related disorders in which enhanced phosphorylation of eIF2α is
protective, prion-induced neurodegeneration is enhanced by salubrinal while
overexpression of GADD34 is protective. The relationship between the
prionopathies and ER dysfunction therefore requires further examination.

**Concluding remarks and therapeutic potential**

The evidence for activation of ER stress responsive pathways in a range of
neurological conditions is strong. What remains to be determined is how
successful strategies that target these responses will be in treating these
diseases. The UPR signalling field has matured to a point at which small
molecular inhibitors of its components are now being developed.

The muscle relaxant dantrolene targets IP3R channels of the ER to prevent the
release of calcium. This provides neuroprotection in models of HD <sup>127</sup> and of
cerebral hypoxia-reperfusion injury, where its action is associated with reduced levels of ER stress <sup>128</sup>. Prolonging the phosphorylation of eIF2α and thus delaying the recovery of protein translation with agents such as salubrinal or guanabenz is another promising approach <sup>28, 29</sup>. Salubrinal has already been shown to reduce the accumulation of α-synuclein in models of ALS <sup>102, 129</sup>, reduce the toxicity of Aβ to primary neurones <sup>73</sup>, and lessen the ER stress associated with cerebral ischemia/reperfusion and models of HD <sup>1, 130</sup>. More recently, agents have been developed that inhibit IRE1, including quinotrierixin <sup>131</sup> and 4μ8C <sup>132</sup>. Both selectively inhibit the splicing of XBP1 mRNA and the latter prevents expansion of the ER without obvious toxicity, which might have therapeutic utility to impede the formation of protein inclusions in neurones.

The list of neurological disorders with impaired ER protein folding is already long and appears set to grow further for some time to come (Table 1). It will therefore aid the neurologist and neuroscientist alike to appreciate the signalling pathways that communicate this failure of ER function to the rest of the cell, since it is likely these insights will inform research, diagnosis and ultimately therapy.

## Acknowledgements

Our labs are funded by grants from the MRC and ART and by Papworth NHS Trust. AJK is a Wellcome Trust PhD student and SJM is an MRC Senior Clinical Fellow (MRC Ref G1002610). DCC is an Alzheimer's Research UK Senior Research Fellow and is supported by the MRC and EPSRC (MRC Ref
G0700990). EM is funded by the Pasteur Institute-Cenci Bolognetti Foundation and by the Telethon Foundation.

**Contributions and conflicts of interest**

BDR and SJM planned and wrote the review. All authors commented on the final manuscript. BDR performed the literature search for and wrote the stroke, Parkinson's and Huntington's disease, multiple sclerosis and prionopathy sections and prepared figure 1. AJK performed the literature search for Alzheimer's disease, and contributed to drafting the Alzheimer's disease section and figure. EM helped draft the FENIB section. DCC helped draft the Alzheimer's section. DAL helped draft the FENIB section. SJM performed the literature search for ER stress and supervised the review project.
# Panel 1

## Search Strategy

We set out by searching the PubMed database with the terms "(endoplasmic reticulum) AND (brain OR neurological) AND disease" including only articles in English. These titles and abstracts were screened and relevant papers were obtained for detailed assessment. Their bibliographies were also screened for additional sources. Papers published prior to September 2012 are included. Where possible, emphasis is given to articles published within the last 5 years.

## Figure legends

<caption>**Figure 1. Endoplasmic reticulum stress signalling**</caption>

In health, newly synthesised secretory proteins are translocated into the ER lumen co-translationally. They fold with the aid of chaperones, for example BiP. When recognised as correctly folded they exit the ER in COPII-coated vesicles and traffic along the secretory pathway. During ER stress, misfolded proteins accumulate in the ER lumen and sequester BiP from the sensor molecules PERK, IRE1 and ATF6. PERK phosphorylates eIF2α, which binds and inhibits its guanine nucleotide exchange factor eIF2B. This blocks new secretory protein synthesis and triggers the integrated stress response (ISR) through increased levels of ATF4. Activation of IRE1 leads to generation of active XBP1, while
cleavage of ATF6 generates ATF6c. These transcription factors co-operate to
induce target genes of the unfolded protein response (UPR).

**Figure 2. ER stress during cerebral ischaemia**

During an ischaemic stroke, the lack of glucose and oxygen supply in neurones
induces a dramatic fall in ATP production. Without energy, BiP binds to unfolded
proteins less efficiently resulting in the accumulation of misfolded ER client
proteins and subsequent activation of the UPR. In parallel, failure of the SERCA
pump depletes ER calcium and worsens chaperone function still further.
Extracellular tPA (tissue-type plasminogen activator) cleaves the NMDA receptor
allowing calcium influx into the cytosol. The resultant elevation of cytosolic
calcium triggers calcium-induced calcium release via ryanodine receptors located
in the ER membrane leading to calcium depletion from the ER. In severely
affected neurones, elevated cytosolic calcium ultimately induces cell death. In
surviving neurones, induction of the eIF2α phosphatase GADD34 by ATF4 in a
CHOP-dependent manner enables the eventual recovery of protein translation.
This can contribute to reperfusion injury through increasing the level of ER
protein load.

**Figure 3. Alzheimer's disease and ER stress**

(A) In health, amyloid precursor protein (APP) is cleaved sequentially by β-secretase followed by γ-secretase in the TGN and early endosomes mainly
generating non-toxic Aβ₄₀. APP is recycled via the endosomal pathway. (B) In Alzheimer's disease, toxic Aβ₄₂ is generated and secreted. Exogenous Aβ₄₂ causes ER stress via a poorly understood mechanism. In some cases of familial Alzheimer's disease, mutations in PS1 impair trafficking of APP, thereby enhancing Aβ₄₂ secretion. Mutated PS1 may also lead to APP processing within the ER.

**Figure 4: ERAD of amyloid precursor protein**

Newly synthesised proteins, for example amyloid precursor protein (APP), interact with ER chaperones during multiple folding cycles until they reach their native folded state. Overexpression of BiP can increase retention of APP in the ER. If protein folding fails, the terminally misfolded client is targeted for degradation via ERAD. The interaction of glycoprotein substrates with both the folding machinery and the ERAD machinery is governed by cycles of de-mannosylation and re-mannosylation. Eventually, low mannose species can interact with EDEM and be targeted for dislocation from the ER lumen into the cytosol. This process is incompletely understood and displays substrate specific differences, but for APP involves a complex containing Hrd1 and Sel1L. Once in the cytosol, the ERAD substrate is ubiquitinated and eventually degraded by the proteasome.
**Figure 5: FENIB and the ER overload response**

(A) Eosin staining of cerebral cortex from individuals with wild-type neuroserpin (WT) and the S52R or G392E mutations. Mutant protein is retained as polymers within the ER to form Collins bodies (red). The rate of polymer formation correlates with the age at which symptoms develop and the amount of Collins' bodies that can be detected. (B) Monomeric serpins, for example neuroserpin, present a reactive centre loop (red) that functions as a pseudosubstrate for their target protease. However, point mutations in the shutter domain (purple circle) open β-sheet A (green) to allow the insertion of the reactive centre loop belonging to a second serpin molecule. If allowed to propagate, this loop-sheet linkage generates large stable pathological polymers that are retained in the ER to form Collins' bodies. (C) These polymers are formed of folded neuroserpin and therefore fail to activate the UPR. Instead they cause the release of ER calcium into the cytosol, which causes NFκB activation and target gene induction.
<table><thead><tr><td>Disease</td><td>Neurological phenotype</td><td>Cause</td><td>Proposed mechanism for ER dysfunction</td><td>Selected references</td></tr></thead><tbody><tr><td>CACH/VWM leukodystrophy</td><td>Cerebellar ataxia<br>Central hypomyelination<br>Autosomal recessive</td><td>eIF2B mutations</td><td>Enhanced ATF4 synthesis</td><td>22-24</td></tr><tr><td>Pelizaeus-Merzbacher leukodystrophy</td><td>Cerebellar ataxia<br>Central hypomyelination<br>X-linked</td><td>PLP1 gene duplication</td><td>Accumulation of PLP1 within the ER</td><td>26</td></tr><tr><td>Charcot-Marie tooth type 1A</td><td>Peripheral sensory neuropathy<br>Peripheral demyelination<br>Autosomal dominant</td><td>PMP22 mutation or duplication</td><td>Accumulation of myelin protein PMP22 within the ER</td><td>133</td></tr><tr><td>Charcot-Marie tooth type 1B</td><td>Peripheral sensory neuropathy<br>Peripheral demyelination<br>Autosomal dominant</td><td>MPZ mutation</td><td>Presumed accumulation of myelin protein P0 within the ER</td><td>27</td></tr><tr><td>Multiple sclerosis</td><td>Progressive motor and cognitive impairment<br>Patchy central demyelination<br>Sporadic</td><td>Inflammatory loss of myelination</td><td>Inflammation-driven ER stress</td><td>120, 121</td></tr><tr><td>Marinesco-Sjögren syndrome</td><td>Cerebellar ataxia<br>Purkinje cell loss<br>Autosomal recessive</td><td>SIL1 gene mutation</td><td>BiP dysfunction due to impaired SIL1 nucleotide exchange activity</td><td>64, 65</td></tr><tr><td>Alzheimer disease</td><td>Loss of cortical neurones<br>Progressive cognitive impairment<br>Sporadic &amp;</td><td>Poorly understood β-amyloid accumulation hyperphosphorylation of tau</td><td>Impaired ER calcium homeostasis<br>Direct Aβ toxicity</td><td>2-4, 71-73, 75, 80, 81, 83</td></tr></tbody></table>
<table><tr><td></td><td>Autosomal dominant</td><td></td><td></td><td></td></tr><tr><td rowspan="2">Parkinson disease</td><td>Progressive motor and cognitive dysfunction</td><td>Poorly understood loss of dopaminergic neurones from the substantia nigra</td><td>Impaired mitochondrial ATP generation causing secondary ER failure</td><td rowspan="2">87, 91, 93, 96, 101-104</td></tr><tr><td>Sporadic<br>Autosomal Recessive<br>Autosomal Dominant</td><td>Familial forms due to mutations in PARK2, SNCA and PINK1</td><td>ER accumulation of aggregated synuclein</td></tr><tr><td>Huntington disease</td><td>Progressive motor and cognitive dysfunction<br>Autosomal Dominant</td><td>Expansion of CAG repeats in the HTT gene leading to selective neuronal loss in caudate and putamen</td><td>Failure of proteostasis likely due to cytosolic aggregates of HTT impairing proteasome function</td><td>12, 106-108</td></tr><tr><td rowspan="2">Amyotrophic lateral sclerosis</td><td>Progressive motor impairment</td><td>Poorly understood loss of motor neurones</td><td rowspan="2">Accumulation of mutant SOD1 within the ER</td><td rowspan="2">109, 111-116, 119</td></tr><tr><td>Sporadic<br>&<br>Autosomal Dominant</td><td>Some familiar forms due to mutations in SOD1 gene</td></tr><tr><td>Prion diseases</td><td>Spongiform encephalopathy<br>Infective<br>&<br>Autosomal Dominant</td><td>Propagation of infective prion conformation of PrP protein</td><td>Impaired cellular proteostasis<br>Possible direct ER toxicity</td><td>5-7, 9</td></tr><tr><td>FENIB</td><td>Progressive dementia and epilepsy<br>Autosomal Dominant</td><td>Mutations of SERPINI1</td><td>Polymerised neuroserpin accumulates within the ER activating the ER overload response</td><td>11, 124</td></tr><tr><td>Tuberous sclerosis complex</td><td>Central hamartomas cause epilepsy often with cognitive and behavioural defects</td><td>TSC1 and TSC2 mutations</td><td>mTORC-mediated impairment of ER stress signalling</td><td>134</td></tr></table>
<table><tr><td></td><td>Autosomal Dominant</td><td></td><td></td><td></td></tr><tr><td>Epilepsy</td><td>Progressive neuronal loss<br>Sporadic</td><td>Repeated seizure activity</td><td>Incompletely understood<br>Likely involves disturbed<br>ER calcium homeostasis</td><td>135</td></tr><tr><td>Wolfram syndrome 1</td><td>Diabetes insipidus, optic atrophy, deafness<br>Autosomal Recessive</td><td>Mutations of WFS1 gene.</td><td>WFS1 encodes an ER ion channel whose dysfunction induces ER stress</td><td>136</td></tr><tr><td>Torsion dystonia 1</td><td>Involuntary sustained muscle contraction<br>Autosomal Dominant</td><td>Mutations in TOR1A gene</td><td>Impairment of Torsin A, an AAA ATPase chaperone in the ER</td><td>137</td></tr><tr><td>Lipid storage disorders (e.g. Gaucher disease)</td><td>Progressive ataxia, mental and motor deficiency<br>Autosomal Recessive</td><td>Mutations in GBA</td><td>Disturbed ER calcium homeostasis and accumulation of mutated protein within the ER</td><td>138</td></tr><tr><td>Cerebral ischaemia</td><td>Neuronal death<br>Sporadic</td><td>Ischaemia causing impaired protein folding</td><td>Energy deficiency causing ER chaperone and enzyme dysfunction</td><td>1, 51, 54, 59, 61, 62</td></tr><tr><td>Sleep apnoea</td><td>Neuronal death<br>Sporadic.</td><td>Ischaemia causing impaired protein folding</td><td>Energy deficiency causing ER chaperone and enzyme dysfunction</td><td>45</td></tr><tr><td>Trauma</td><td>Neuronal death<br>Sporadic</td><td>Neuronal trauma and impaired perfusion</td><td>Incompletely understood<br>Likely involves disturbed energy metabolism and ER calcium homeostasis</td><td>139</td></tr></table>
**Table1: Neurodegenerative diseases and ER stress: an overview.**

CACH: Childhood ataxia with central hypomyelination; VWM: vanishing white matter; eIF2: eukaryotic translation initiation factor 2; ATF4: activating transcription factor 4; PLP1: proteolipid protein 1; CHOP: CCAAT/enhancer binding protein; IRE1: Inositol-requiring enzyme-1; Bip: Binding immunoglobulin protein; PMP22: peripheral myelin protein 22; PS1: presenilin 1; ERAD: ER-associated protein degradation; SOD1: Cu/Zn superoxide dismutase enzyme; SERCA: sarco/endoplasmic reticulum Ca2+-ATPase; PLP1: proteolipid protein 1; MPZ: myelin protein zero
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33-44.
Benoit D. Roussel

PhD

Tel: +44 1223 762818 (secretary)

Fax: +44 1223 748178

E-mail: bdr24@cam.ac.uk

<figure><img src="image_3.png" /></figure>

UNIVERSITY OF
CAMBRIDGE

Cambridge Institute for Medical Research
Wellcome Trust/MRC Building
Hills Road
Cambridge CB2 0XY
United Kingdom

Dear Dr Craven

Thank you for offering us the possibility to publish our manuscript. Please find here our revised manuscript with the editorial changes you suggested: we have included a contributions and conflicts of interest section under the same heading, there is now a panel to detail our searches on Pubmed, and there is only one qualification for each author.

Best regards

Dr BD Roussel.
<figure><img src="image_4.png" /><figcaption>Figure 1</figcaption></figure>

Health

<figure><img src="image_5.png" /></figure>

ER stress

UPR gene transcription
<figure><img src="image_6.png" /><figcaption>Figure 2</figcaption></figure>
<caption>Figure 3</caption>

A

<figure><img src="image_7.png" /></figure>

Health

B

<figure><img src="image_8.png" /></figure>

Alzheimer's disease

<figure><img src="image_9.png" /></figure>
<figure><img src="image_10.png" /><figcaption>Figure 4</figcaption></figure>
<figure><img src="image_11.png" /><figcaption>Pathway to serpin polymerisation</figcaption></figure>

<figure><img src="image_12.png" /><figcaption>ER overload response</figcaption></figure>