Microangiopathy in temporal lobe epilepsy with diffusion MRI alterations and cognitive decline.

White matter microvascular alterations in temporal lobe epilepsy (TLE) may be relevant to acquired neurodegenerative processes and cognitive impairments associated with this condition. We quantified microvascular changes, myelin, axonal, glial and extracellular-matrix labelling in the gyral core and deep temporal lobe white matter regions in surgical resections from 44 TLE patients with or without hippocampal sclerosis. We compared this pathology data with in vivo pre-operative MRI diffusion measurements in co-registered regions and neuropsychological measures of cognitive impairment and decline. In resections, increased arteriolosclerosis was observed in TLE compared to non-epilepsy controls (greater sclerotic index, p < 0.001), independent of age. Microvascular changes included increased vascular densities in some regions but uniformly reduced mean vascular size (quantified with collagen-4, p < 0.05-0.0001), and increased pericyte coverage of small vessels and capillaries particularly in deep white matter (quantified with platelet-derived growth factor receptorß and smooth muscle actin, p < 0.01) which was more marked the longer the duration of epilepsy (p < 0.05). We noted increased glial numbers (Olig2, Iba1) but reduced myelin (MAG, PLP) in TLE compared to controls, particularly prominent in deep white matter. Gene expression analysis showed a greater reduction of myelination genes in HS than non-HS cases and with age and correlation with diffusion MRI alterations. Glial densities and vascular size were increased with increased MRI diffusivity and vascular density with white matter abnormality quantified using fixel-based analysis. Increased perivascular space was associated with reduced fractional anisotropy as well as age-accelerated cognitive decline prior to surgery (p < 0.05). In summary, likely acquired microangiopathic changes in TLE, including vascular sclerosis, increased pericyte coverage and reduced small vessel size, may indicate a functional alteration in contractility of small vessels and haemodynamics that could impact on tissue perfusion. These morphological features correlate with white matter diffusion MRI alterations and might explain cognitive decline in TLE.

Quantitative cellular pathology of the amygdala in temporal lobe epilepsy and correlation with magnetic resonance imaging volumetry, tissue microstructure, and sudden unexpected death in epilepsy risk factors.

OBJECTIVE: Amygdala enlargement can occur in temporal lobe epilepsy, and increased amygdala volume is also reported in sudden unexpected death in epilepsy (SUDEP). Apnea can be induced by amygdala stimulation, and postconvulsive central apnea (PCCA) and generalized seizures are both known SUDEP risk factors. Neurite orientation dispersion and density imaging (NODDI) has recently provided additional information on altered amygdala microstructure in SUDEP. In a series of 24 surgical temporal lobe epilepsy cases, our aim was to quantify amygdala cellular pathology parameters that could predict enlargement, NODDI changes, and ictal respiratory dysfunction. METHODS: Using whole slide scanning automated quantitative image analysis methods, parallel evaluation of myelin, axons, dendrites, oligodendroglia, microglia, astroglia, neurons, serotonergic networks, mTOR-pathway activation (pS6) and phosphorylated tau (pTau; AT8, AT100, PHF) in amygdala, periamygdala cortex, and white matter regions of interest were compared with preoperative magnetic resonance imaging data on amygdala size, and in 13 cases with NODDI and evidence of ictal-associated apnea. RESULTS: We observed significantly higher glial labeling (Iba1, glial fibrillary acidic protein, Olig2) in amygdala regions compared to cortex and a strong positive correlation between Olig2 and Iba1 in the amygdala. Larger amygdala volumes correlated with lower microtubule-associated protein (MAP2), whereas higher NODDI orientation dispersion index correlated with lower Olig2 cell densities. In the three cases with recorded PCCA, higher MAP2 and pS6-235 expression was noted than in those without. pTau did not correlate with SUDEP risk factors, including seizure frequency. SIGNIFICANCE: Histological quantitation of amygdala microstructure can shed light on enlargement and diffusion imaging alterations in epilepsy to explore possible mechanisms of amygdala dysfunction, including mTOR pathway activation, that in turn may increase the risk for SUDEP.

Cortical neuronal hypertrophy and mTOR pathway activation in CAN regions in SUDEP.

OBJECTIVES: Dysfunctional connectivity and preexisting structural abnormalities of central autonomic network (CAN) regions have been shown on magnetic resonance imaging (MRI) in sudden unexpected death in epilepsy (SUDEP) and may be mechanistically relevant. In a previous postmortem study we reported increased microglia in CAN regions, including the superior temporal gyrus (STG) in SUDEP. In this current study we investigated mammalian target of rapamycin (mTOR) pathway activation and neuronal c-Fos activation in CAN regions in SUDEP compared to control groups. METHODS: In a series of 59 postmortem cases (SUDEP, n = 26; epilepsy controls [EPCs], n = 14; and nonepilepsy controls [NECs], n = 19), we quantified pS6-240/4, pS6-235/6 (markers of mTOR activation) and c-Fos neuronal densities and labeling index in the STG, anterior cingulate, insula, frontobasal, and pulvinar regions using immunohistochemistry with whole-slide automated image analysis. RESULTS: Significantly more pS6-positive neurons were present in the STG in cases with a history of recent seizures prior to death and also in SUDEP compared to other cause of death groups. No differences were noted for c-Fos neuronal labeling in any region between cause of death groups. Cortical neuronal hypertrophy in the STG was observed in some SUDEP cases and associated with pS6-240/4 expression. pS6-235/6 highlighted neuronal intranuclear inclusions, mainly in SUDEP cases and in the STG region. SIGNIFICANCE: Neuronal labeling for pS6 in the STG correlated with both seizure activity in the period prior to death and SUDEP. Further investigations are required to explore the significance of this region in terms of autonomic network dysfunction that may increase the vulnerability for SUDEP.

Serotonin transporter in the temporal lobe, hippocampus and amygdala in SUDEP.

Several lines of evidence link deficient serotonin function and SUDEP. Chronic treatment with serotonin reuptake inhibitors (SRIs) reduces ictal central apnoea, a risk factor for SUDEP. Reduced medullary serotonergic neurones, modulators of respiration in response to hypercapnia, were reported in a SUDEP post-mortem series. The amygdala and hippocampus have high serotonergic innervation and are functionally implicated in seizure-related respiratory dysregulation. We explored serotonergic networks in mesial temporal lobe structures in a surgical and post-mortem epilepsy series in relation to SUDEP risk. We stratified 75 temporal lobe epilepsy patients with hippocampal sclerosis (TLE/HS) into high (N = 16), medium (N = 11) and low risk (N = 48) groups for SUDEP based on generalised seizure frequency. We also included the amygdala in 35 post-mortem cases, including SUDEP (N = 17), epilepsy controls (N = 10) and non-epilepsy controls (N = 8). The immunohistochemistry labelling index (LI) and axonal length (AL) of serotonin transporter (SERT)-positive axons were quantified in 13 regions of interest with image analysis. SERT LI was highest in amygdala and subiculum regions. In the surgical series, higher SERT LI was observed in high risk than low risk cases in the dentate gyrus, CA1 and subiculum (p < 0.05). In the post-mortem cases higher SERT LI and AL was observed in the basal and accessory basal nuclei of the amygdala and peri-amygdala cortex in SUDEP compared to epilepsy controls (p < 0.05). Patients on SRI showed higher SERT in the dentate gyrus (p < 0.005) and CA4 (p < 0.05) but there was no difference in patients with or without a psychiatric history. Higher SERT in hippocampal subfields in TLE/HS cases with SUDEP risk factors and higher amygdala SERT in post-mortem SUDEP cases than epilepsy controls supports a role for altered serotonergic networks involving limbic regions in SUDEP. This may be of functional relevance through reduced 5-HT availability.

Progressive hemispheric atrophy in HIV: A Rasmussen's-like variant of CD8 encephalitis?

We report two cases of progressive lateralising encephalopathy in adult patients with treated HIV in the absence of opportunistic infection or vasculitis. One case was characterised by CD8 cortical infiltrates and was steroid responsive and may represent a variant of CD8 encephalitis. The other case presented with focal seizures and episodes of status epilepticus and pathology showed severe cortical atrophy with features reminiscent of the chronic phase of Rasmussen's encephalitis.

Review: Neuropathology findings in autonomic brain regions in SUDEP and future research directions.

Autonomic dysfunction is implicated from clinical, neuroimaging and experimental studies in sudden and unexpected death in epilepsy (SUDEP). Neuropathological analysis in SUDEP series enable exploration of acquired, seizure-related cellular adaptations in autonomic and brainstem autonomic centres of relevance to dysfunction in the peri-ictal period. Alterations in SUDEP compared to control groups have been identified in the ventrolateral medulla, amygdala, hippocampus and central autonomic regions. These involve neuropeptidergic, serotonergic and adenosine systems, as well as specific regional astroglial and microglial populations, as potential neuronal modulators, orchestrating autonomic dysfunction. Future research studies need to extend to clinically and genetically characterized epilepsies, to explore if common or distinct pathways of autonomic dysfunction mediate SUDEP. The ultimate objective of SUDEP research is the identification of disease biomarkers for at risk patients, to improve post-mortem recognition and disease categorisation, but ultimately, for exposing potential treatment targets of pharmacologically modifiable and reversible cellular alterations.

Regional microglial populations in central autonomic brain regions in SUDEP.

OBJECTIVE: Sudden unexpected death in epilepsy (SUDEP) may arise as a result of autonomic dysfunction during a seizure. The central autonomic networks (CANs) modulate brainstem cardiorespiratory regulation. Recent magnetic resonance imaging (MRI) studies in SUDEP have shown cortical and subcortical volume changes and altered connectivity between CAN regions, but the pathological correlate is unknown. Because neuroinflammation is both a cause and a consequence of seizures and may relate to regional brain pathology, our aim was to evaluate microglial populations in CANs in SUDEP. METHODS: In 55 postmortem cases, including SUDEP, epilepsy controls without SUDEP and nonepilepsy controls, we quantified Iba1-expressing microglia in 14 cortical and thalamic areas that included known CAN regions. RESULTS: Mean Iba1 labeling across all brain regions was significantly higher in SUDEP cases compared to epilepsy and nonepilepsy controls. There was significant regional variation in Iba1 labeling in SUDEP cases only, with highest labeling in the medial thalamus. Significantly higher labeling in SUDEP cases than epilepsy and nonepilepsy controls was consistently noted in the superior temporal gyrus. In cases with documented seizures up to 10 days prior to death, significantly higher mean Iba1 labeling was observed in SUDEP compared to epilepsy controls. SIGNIFICANCE: Our findings support microglial activation in SUDEP, including cortical and subcortical regions with known autonomic functions such as the thalamus and superior temporal gyrus. This may be relevant to cellular pathomechanisms underlying cardioregulatory failure during a seizure.

Polyglucosan bodies in medullary catecholaminergic neurones in SUDEP.

Polyglucosan bodies have been reported in the context of hypoxic-ischaemic perinatal brain injury, mainly in the pallidum but with rare reports in brainstem neurons. We report a case of a five-year-old boy with cerebral palsy and complex neurological features including epilepsy who experienced sudden nocturnal death. At post-mortem long-standing bilateral necrosis of basal ganglia and hippocampal atrophy was identified in keeping with hypoxic-ischaemic perinatal injury. In addition numerous polyglucosan bodies, which were PAS, p62 and ubiquitin positive, were noted in brainstem neurones and dendrites, primarily involving the ventrolateral and dorsomedial medulla. Immunohistochemistry confirmed relative preservation of medullary neuronal populations in the reticular formation, including catecholaminergic (tyrosine hydroxylase, TH), serotonergic (tryptophan hydroxylase) and neurokinin1 receptor/somatostatin positive neurones. The polyglucosan bodies predominated in catecholaminergic neurones which could indicate their selective vulnerability and a functional deficiency, which during a critical peri-ictal period contributed to the sudden unexpected death in epilepsy.

Medullary tyrosine hydroxylase catecholaminergic neuronal populations in sudden unexpected death in epilepsy.

Sudden unexpected death in epilepsy (SUDEP) is mechanistically complex and one probable cause is seizure-related respiratory dysfunction. Medullary respiratory regulatory nuclei include the pre-Bötzinger complex (pre-BötC) in the ventrolateral medulla (VLM), the medullary raphé nuclei (MR) and nucleus of solitary tract in the dorsomedial medulla (DMM). The region of the VLM also contains intermingled tyrosine hydroxylase (TH) catecholaminergic neurones which directly project to the pre-BötC and regulate breathing under hypoxic conditions and our aim was to evaluate these neurones in SUDEP cases. In post-mortem cases from three groups [SUDEP (18), epilepsy controls (8) and non-epilepsy controls (16)] serial sections of medulla (obex + 2 to + 13 mm) were immunolabeled for TH. Three regions of interest (ROI) were outlined (VLM, DMM and MR) and TH-immunoreactive (TH-IR) neurones were evaluated using automated detection for overall labeling index (neurones and processes) and neuronal densities and compared between groups and relative to obex level. C-fos immunoreactivity was also semi-quantitatively evaluated in these regions. We found no significant difference in the density of TH-IR neurones or labeling index between the groups in all regions. Significantly more TH-IR neurones were present in the DMM region than VLM in non-epilepsy cases only (P < 0.01). Regional variations in TH-IR neurones with obex level were seen in all groups except SUDEP. We also identified occasional TH neurones in the MR region in all groups. There was significantly less c-fos labeling in the VLM and MR in SUDEP than non-epilepsy controls but no difference with epilepsy controls. In conclusion, in this series we found no evidence for alteration of total medullary TH-IR neuronal numbers in SUDEP but noted some differences in their relative distribution in the medulla and c-fos neurones compared to control groups which may be relevant to the mechanism of death.

Adenosine kinase and adenosine receptors A1 R and A2A R in temporal lobe epilepsy and hippocampal sclerosis and association with risk factors for SUDEP.

OBJECTIVE: The "adenosine hypothesis of SUDEP" (sudden unexpected death in epilepsy) predicts that a seizure-induced adenosine surge combined with impaired metabolic clearance can foster lethal apnea or cardiac arrest. Changes in adenosine receptor density and adenosine kinase (ADK) occur in surgical epilepsy patients. Our aim was to correlate the distribution of ADK and adenosine A2A and A1 receptors (A2A R and A1 R) in surgical tissue from patients with temporal lobe epilepsy and hippocampal sclerosis (TLE/HS) with SUDEP risk factors. METHODS: In 75 cases, patients were stratified into high-risk (n = 16), medium-risk (n = 11) and low-risk (n = 48) categories according to the frequency of generalized seizures before surgery. Using whole-slide scanning Definiens image analysis we quantified the labeling index (LI) for ADK, A2A R, and A1 R in seven regions of interest: temporal cortex, temporal lobe white matter, CA1, CA4, dentate gyrus, subiculum, and amygdala and relative to glial and neuronal densities with glial fibrillary acidic protein (GFAP) and neuronal nuclear antigen (NeuN). RESULTS: A1 R showed predominant neuronal, A2A R astroglial, and ADK nuclear labeling in all regions but with significant variation. Compared with the low-risk group, the high-risk group had significantly lower A2A R LI in the temporal cortex. In HS cases with severe neuronal cell loss and gliosis predominantly in the CA1 and CA4 regions, significantly higher A1 R was present in the amygdala in high-risk than in low-risk cases. There was no significant difference in neuronal loss or gliosis between the risk groups or differences for ADK labeling. SIGNIFICANCE: Reduced cortical A2A R suggests glial dysfunction and impaired adenosine modulation in response to seizures in patients at higher risk for SUDEP. Increased neuronal A1 R in the high-risk group could contribute to periictal amygdala dysfunction in SUDEP.

Neuropeptide depletion in the amygdala in sudden unexpected death in epilepsy: A postmortem study.

OBJECTIVE: Sudden unexpected death in epilepsy (SUDEP) is typically unwitnessed but can be preceded by seizures in the period prior to death. Peri-ictal respiratory dysfunction is a likely mechanism for some SUDEP, and central apnea has been shown following amygdala stimulation. The amygdala is enriched in neuropeptides that modulate neuronal activity and can be transiently depleted following seizures. In a postmortem SUDEP series, we sought to investigate alterations of neuropeptidergic networks in the amygdala, including cases with recent poor seizure control. METHODS: In 15 SUDEP cases, 12 epilepsy controls, and 10 nonepilepsy controls, we quantified the labeling index (LI) for galanin, neuropeptide Y (NPY), and somatostatin (SST) in the lateral, basal, and accessory basal nuclei and periamygdala cortex with whole slide scanning image analysis. Within the SUDEP group, seven had recent generalized seizures with recovery 24 hours prior to death (SUDEP-R). RESULTS: Galanin, NPY, and SST LIs were significantly lower in all amygdala regions in SUDEP cases compared to epilepsy controls (P < .05 to P < .0005), and galanin LI was lower in the lateral nucleus compared to nonepilepsy controls (P < .05). There was no difference in the LI in the SUDEP-R group compared to other SUDEP. Higher LI was noted in epilepsy controls than nonepilepsy controls; this was significant for NPY in lateral and basal nuclei (P < .005 and P < .05). SIGNIFICANCE: A reduction in galanin in the lateral nucleus in SUDEP could represent acute depletion, relevant to postictal amygdala dysfunction. In addition, increased amygdala neuropeptides in epilepsy controls support their seizure-induced modulation, which is relatively deficient in SUDEP; this could represent a vulnerability factor for amygdala dysfunction in the postictal period.

Characterisation of medullary astrocytic populations in respiratory nuclei and alterations in sudden unexpected death in epilepsy.

Central failure of respiration during a seizure is one possible mechanism for sudden unexpected death in epilepsy (SUDEP). Neuroimaging studies indicate volume loss in the medulla in SUDEP and a post mortem study has shown reduction in neuromodulatory neuropeptidergic and monoaminergic neurones in medullary respiratory nuclear groups. Specialised glial cells identified in the medulla are considered essential for normal respiratory regulation including astrocytes with pacemaker properties in the pre-Botzinger complex and populations of subpial and perivascular astrocytes, sensitive to increased pCO2, that excite respiratory neurones. Our aim was to explore niches of medullary astrocytes in SUDEP cases compared to controls. In 48 brainstems from three groups, SUDEP (20), epilepsy controls (10) and non-epilepsy controls (18), sections through the medulla were labelled for GFAP, vimentin and functional markers, astrocytic gap junction protein connexin43 (Cx43) and adenosine A1 receptor (A1R). Regions including the ventro-lateral medulla (VLM; for the pre-Bötzinger complex), Median Raphe (MR) and lateral medullary subpial layer (MSPL) were quantified using image analysis for glial cell populations and compared between groups. Findings included morphologically and regionally distinct vimentin/Cx34-positive glial cells in the VLM and MR in close proximity to neurones. We noted a reduction of vimentin-positive glia in the VLM and MSPL and Cx43 glia in the MR in SUDEP cases compared to control groups (p < 0.05-0.005). In addition, we identified vimentin, Cx43 and A1R positive glial cells in the MSPL region which likely correspond to chemosensory glia identified experimentally. In conclusion, altered medullary glial cell populations could contribute to impaired respiratory regulatory capacity and vulnerability to SUDEP and warrant further investigation.

Hippocampal morphometry in sudden and unexpected death in epilepsy.

OBJECTIVE: To determine hippocampal morphometric measures, including granule cell dispersion and features of malrotation, as potential biomarkers for sudden unexpected death in epilepsy (SUDEP) from an archival postmortem series. METHODS: In a retrospective study of 187 archival postmortems from 3 groups, SUDEP (68; 14 with hippocampal sclerosis [HS]), non-SUDEP epilepsy controls (EP-C = 66; 25 with HS), and nonepilepsy controls (NEC = 53), Nissl/hematoxylin & eosin-stained sections from left and right hippocampus from 5 coronal levels were digitized. Image analysis was carried out for granule cell layer (GCL) thickness and measurements of hippocampal dimensions (HD) for shape (width [HD1], height [HD2]) and medial hippocampal positioning in relation to the parahippocampal gyrus (PHG) length (HD3). A qualitative evaluation of hippocampal malrotational (HMAL) features, dentate gyrus invaginations (DGI), and subicular/CA1 folds (SCF) was also made. RESULTS: GCL thickness was increased in HS more than those without (p < 0.001). In non-HS cases, increased GCL thickness was noted in EP-C compared to NEC (p < 0.05) but not between SUDEP and NEC. There was no difference in the frequency of DGI, SCF, measurements of hippocampal shape (HD1, HD2, or ratio), or medial positioning among SUDEP, EP-C, and NEC groups, when factoring in HS, coronal level, and age at death. Comparison between left and right sides within cases showed greater PHG lengths (HD3) on the right side in the SUDEP group only (p = 0.018). CONCLUSIONS: No hippocampal morphometric features were identified in support of either excessive granule cell dispersion or features of HMAL as definitive biomarkers for SUDEP. Asymmetries in PHG measurements in SUDEP warrant further investigation as they may indicate abnormal central autonomic networks.

The ventrolateral medulla and medullary raphe in sudden unexpected death in epilepsy.

Sudden unexpected death in epilepsy (SUDEP) is a leading cause of premature death in patients with epilepsy. One hypothesis proposes that sudden death is mediated by post-ictal central respiratory depression, which could relate to underlying pathology in key respiratory nuclei and/or their neuromodulators. Our aim was to investigate neuronal populations in the ventrolateral medulla (which includes the putative human pre-Bötzinger complex) and the medullary raphe. Forty brainstems were studied comprising four groups: 14 SUDEP, six epilepsy controls, seven Dravet syndrome cases and 13 non-epilepsy controls. Serial sections through the medulla (from obex 1 to 10 mm) were stained for Nissl, somatostatin, neurokinin 1 receptor (for pre-Bötzinger complex neurons) and galanin, tryptophan hydroxylase and serotonin transporter (neuromodulatory systems). Using stereology total neuronal number and densities, with respect to obex level, were measured. Whole slide scanning image analysis was used to quantify immunolabelling indices as well as co-localization between markers. Significant findings included reduction in somatostatin neurons and neurokinin 1 receptor labelling in the ventrolateral medulla in sudden death in epilepsy compared to controls (P < 0.05). Galanin and tryptophan hydroxylase labelling was also reduced in sudden death cases and more significantly in the ventrolateral medulla region than the raphe (P < 0.005 and P < 0.05). With serotonin transporter, reduction in labelling in cases of sudden death in epilepsy was noted only in the raphe (P ≤ 0.01); however, co-localization with tryptophan hydroxylase was significantly reduced in the ventrolateral medulla. Epilepsy controls and cases with Dravet syndrome showed less significant alterations with differences from non-epilepsy controls noted only for somatostatin in the ventrolateral medulla (P < 0.05). Variations in labelling with respect to obex level were noted of potential relevance to the rostro-caudal organization of respiratory nuclear groups, including tryptophan hydroxylase, where the greatest statistical difference noted between all epilepsy cases and controls was at obex 9-10 mm (P = 0.034), the putative level of the pre-Bötzinger complex. Furthermore, there was evidence for variation with duration of epilepsy for somatostatin and neurokinin 1 receptor. Our findings suggest alteration to neuronal populations in the medulla in SUDEP with evidence for greater reduction in neuromodulatory neuropeptidergic and mono-aminergic systems, including for galanin, and serotonin. Other nuclei need to be investigated to evaluate if this is part of more widespread brainstem pathology. Our findings could be a result of previous seizures and may represent a pathological risk factor for SUDEP through impaired respiratory homeostasis during a seizure.

c-Jun in Schwann cells promotes axonal regeneration and motoneuron survival via paracrine signaling.

The AP-1 transcription factor c-Jun is a master regulator of the axonal response in neurons. c-Jun also functions as a negative regulator of myelination in Schwann cells (SCs) and is strongly reactivated in SCs upon axonal injury. We demonstrate here that, after injury, the absence of c-Jun specifically in SCs caused impaired axonal regeneration and severely increased neuronal cell death. c-Jun deficiency resulted in decreased expression of several neurotrophic factors, and GDNF and Artemin, both of which encode ligands for the Ret receptor tyrosine kinase, were identified as novel direct c-Jun target genes. Genetic inactivation of Ret specifically in neurons resulted in regeneration defects without affecting motoneuron survival and, conversely, administration of recombinant GDNF and Artemin protein substantially ameliorated impaired regeneration caused by c-Jun deficiency. These results reveal an unexpected function for c-Jun in SCs in response to axonal injury, and identify paracrine Ret signaling as an important mediator of c-Jun function in SCs during regeneration.

Downstream effector molecules in successful peripheral nerve regeneration.

The robust axon regeneration that occurs following peripheral nerve injury is driven by transcriptional activation of the regeneration program and by the expression of a wide range of downstream effector molecules from neuropeptides and neurotrophic factors to adhesion molecules and cytoskeletal adaptor proteins. These regeneration-associated effector molecules regulate the actin-tubulin machinery of growth-cones, integrate intracellular signalling and stimulatory and inhibitory signals from the local environment and translate them into axon elongation. In addition to the neuronally derived molecules, an important transcriptional component is found in locally activated Schwann cells and macrophages, which release a number of cytokines, growth factors and neurotrophins that support neuronal survival and axonal regeneration and that might provide directional guidance cues towards appropriate peripheral targets. This review aims to provide a comprehensive up-to-date account of the transcriptional regulation and functional role of these effector molecules and of the information that they can give us with regard to the organisation of the regeneration program.

Neuronal c-Jun is required for successful axonal regeneration, but the effects of phosphorylation of its N-terminus are moderate.

Although neural c-Jun is essential for successful peripheral nerve regeneration, the cellular basis of this effect and the impact of c-Jun activation are incompletely understood. In the current study, we explored the effects of neuron-selective c-Jun deletion, substitution of serine 63 and 73 phosphoacceptor sites with non-phosphorylatable alanine, and deletion of Jun N-terminal kinases 1, 2 and 3 in mouse facial nerve regeneration. Removal of the floxed c-jun gene in facial motoneurons using cre recombinase under control of a neuron-specific synapsin promoter (junΔS) abolished basal and injury-induced neuronal c-Jun immunoreactivity, as well as most of the molecular responses following facial axotomy. Absence of neuronal Jun reduced the speed of axonal regeneration following crush, and prevented most cut axons from reconnecting to their target, significantly reducing functional recovery. Despite blocking cell death, this was associated with a large number of shrunken neurons. Finally, junΔS mutants also had diminished astrocyte and microglial activation and T-cell influx, suggesting that these non-neuronal responses depend on the release of Jun-dependent signals from neighboring injured motoneurons. The effects of substituting serine 63 and 73 phosphoacceptor sites (junAA), or of global deletion of individual kinases responsible for N-terminal c-Jun phosphorylation were mild. junAA mutants showed decrease in neuronal cell size, a moderate reduction in post-axotomy CD44 levels and slightly increased astrogliosis. Deletion of Jun N-terminal kinase (JNK)1 or JNK3 showed delayed functional recovery; deletion of JNK3 also interfered with T-cell influx, and reduced CD44 levels. Deletion of JNK2 had no effect. Thus, neuronal c-Jun is needed in regeneration, but JNK phosphorylation of the N-terminus mostly appears to not be required for its function.