Caroline Rouaux

Critical loss of CBP/p300 histone acetylase activity by caspase-6 during neurodegeneration.

By altering chromatin structure, histone acetyltransferases (HATs) act as transcriptional regulators. We observed in a model of primary neurons that histone acetylation levels decreased at the onset of apoptosis. The CREB‐binding protein (CBP) is a HAT of particular interest because it also acts as a co‐activator controlling, among others, CREB‐dependent transcriptional activity. It has been demonstrated that CREB exerts neuroprotective functions, but the fate of CBP during neuronal apoptosis remained unclear till now. This work provided evidence that CBP is specifically targeted by caspases and calpains at the onset of neuronal apoptosis, and CBP was futher identified as a new caspase‐6 substrate. This ultimately impinged on the CBP/p300 HAT activity that decreased with time during apoptosis entry, whereas total cellularHAT activity remained unchanged. Interestingly, CBP loss and histone deacetylation were observed in two different pathological contexts: amyloid precursor protein‐dependent signaling and amyotrophic lateralsclerosis model mice, indicating that these modifications are likely to contribute to neurodegenerative diseases. In terms of function, we demonstrated that fine‐tuning of CBP HAT activity is necessary to ensure neuroprotection.

The pRb/E2F cell-cycle pathway mediates cell death in Parkinson's disease.

The mechanisms leading to degeneration of dopaminergic neurons (DNs) in the substantia nigra of patients with Parkinsons disease (PD) are not completely understood. Here, we show, in the postmortem human tissue, that these neurons aberrantly express mitosis-associated proteins, including the E2F-1 transcription factor, and appear to duplicate their nuclear DNA. We further demonstrate that the dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine injected into mice and application of its active metabolite 1-methyl-4-phenylpyridinium to mesencephalic cultures activate the retinoblastoma–E2F pathway in postmitotic DNs. We also find that cell death rather than mitotic division followed the toxin-induced replication of DNA, as determined by BrdU incorporation in DNs. In addition, blocking E2F-1 transcription protected cultured DNs against 1-methyl-4-phenylpyridinium toxicity. Finally, E2F-1-deficient mice were significantly more resistant to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced dopaminergic cell death than their wild-type littermates. Altogether, BrdU incorporation in mature neurons and lack of evidence for newborn neurons argue against neuronal turnover in normal conditions or during pathological states in the substantia nigra. Instead, our results demonstrate that mitosis-like signals are activated in mature DNs in patients with PD and mediate neuronal death in experimental models of the disease. Inhibition of mitosis-like signals may therefore provide strategies for neuroprotection in PD.

Sodium Valproate Exerts Neuroprotective Effects In Vivo through CREB-Binding Protein-Dependent Mechanisms But Does Not Improve Survival in an Amyotrophic Lateral Sclerosis Mouse Model

Amyotrophic lateral sclerosis (ALS) is characterized by motoneuron (MN) degeneration, generalized weakness, and muscle atrophy. The premature death of MNs is thought to be a determinant in the onset of this disease. In a transgenic mouse model of ALS expressing the G86R mutant superoxide dismutase 1 (mSOD1), we demonstrated previously that CREB (cAMP response element-binding protein)-binding protein (CBP) and histone acetylation levels were specifically decreased in nuclei of degenerating MNs. We show here that oxidative stress and mSOD1 overexpression can both impinge on CBP levels by transcriptional repression, in an MN-derived cell line. Histone deacetylase inhibitor (HDACi) treatment was able to reset proper acetylation levels and displayed an efficient neuroprotective capacity against oxidative stress in vitro. Interestingly, HDACi also upregulated CBP transcriptional expression in MNs. Moreover, when injected to G86R mice in vivo, the HDACi sodium valproate (VPA) maintained normal acetylation levels in the spinal cord, efficiently restored CBP levels in MNs, and significantly prevented MN death in these animals. However, despite neuroprotection, mean survival of treated animals was not significantly improved (<5%), and they died presenting the classical ALS symptoms. VPA was not able to prevent disruption of neuromuscular junctions, although it slightly delayed the onset of motor decline and retarded muscular atrophy to some extent. Together, these data show that neuroprotection can improve disease onset, but clearly provide evidence that one can uncouple MN survival from whole-animal survival and point to the neuromuscular junction perturbation as a primary event of ALS onset.

Direct lineage reprogramming of post-mitotic callosal neurons into corticofugal neurons in vivo.

Once programmed to acquire a specific identity and function, cells rarely change in vivo. Neurons of the mammalian central nervous system (CNS) in particular are a classic example of a stable, terminally differentiated cell type. With the exception of the adult neurogenic niches, where a limited set of neuronal subtypes continue to be generated throughout life, CNS neurons are born only during embryonic and early postnatal development. Once generated, neurons become permanently post-mitotic and do not change their identity for the lifespan of the organism. Here, we have investigated whether excitatory neurons of the neocortex can be instructed to directly reprogram their identity post-mitotically from one subtype into another, in vivo. We show that embryonic and early postnatal callosal projection neurons of layer II/III can be post-mitotically lineage reprogrammed into layer-V/VI corticofugal projection neurons following expression of the transcription factor encoded by Fezf2. Reprogrammed callosal neurons acquire molecular properties of corticofugal projection neurons and change their axonal connectivity from interhemispheric, intracortical projections to corticofugal projections directed below the cortex. The data indicate that during a window of post-mitotic development neurons can change their identity, acquiring critical features of alternative neuronal lineages.

Excitatory Projection Neuron Subtypes Control the Distribution of Local Inhibitory Interneurons in the Cerebral Cortex

In the mammalian cerebral cortex, the developmental events governing the integration of excitatory projection neurons and inhibitory interneurons into balanced local circuitry are poorly understood. We report that different subtypes of projection neurons uniquely and differentially determine the laminar distribution of cortical interneurons. We find that in Fezf2⁻/⁻ cortex, the exclusive absence of subcerebral projection neurons and their replacement by callosal projection neurons cause distinctly abnormal lamination of interneurons and altered GABAergic inhibition. In addition, experimental generation of either corticofugal neurons or callosal neurons below the cortex is sufficient to recruit cortical interneurons to these ectopic locations. Strikingly, the identity of the projection neurons generated, rather than strictly their birthdate, determines the specific types of interneurons recruited. These data demonstrate that in the neocortex individual populations of projection neurons cell-extrinsically control the laminar fate of interneurons and the assembly of local inhibitory circuitry.

Fezf2 directs the differentiation of corticofugal neurons from striatal progenitors in vivo

In the developing cerebral cortex, cell-extrinsic and cell-intrinsic signals govern the establishment of neuron subtype-specific identity. Here we show that, within the niche of the striatum, the expression of a single transcription factor, Fezf2, is sufficient to generate corticofugal neurons from progenitors fated to become medium spiny neurons. This demonstrates that a specific population of cortical projection neurons can be directed to differentiate outside of the cortex by cell-autonomous signaling.

Toxic gain of function from mutant FUS protein is crucial to trigger cell autonomous motor neuron loss

FUS is an RNA‐binding protein involved in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Cytoplasmic FUS‐containing aggregates are often associated with concomitant loss of nuclear FUS. Whether loss of nuclear FUS function, gain of a cytoplasmic function, or a combination of both lead to neurodegeneration remains elusive. To address this question, we generated knockin mice expressing mislocalized cytoplasmic FUS and complete FUS knockout mice. Both mouse models display similar perinatal lethality with respiratory insufficiency, reduced body weight and length, and largely similar alterations in gene expression and mRNA splicing patterns, indicating that mislocalized FUS results in loss of its normal function. However, FUS knockin mice, but not FUS knockout mice, display reduced motor neuron numbers at birth, associated with enhanced motor neuron apoptosis, which can be rescued by cell‐specific CRE‐mediated expression of wild‐type FUS within motor neurons. Together, our findings indicate that cytoplasmic FUS mislocalization not only leads to nuclear loss of function, but also triggers motor neuron death through a toxic gain of function within motor neurons.

HP1α guides neuronal fate by timing E2F-targeted genes silencing during terminal differentiation

A critical step of neuronal terminal differentiation is the permanent withdrawal from the cell cycle that requires the silencing of genes that drive mitosis. Here, we describe that the α isoform of the heterochromatin protein 1 (HP1) protein family exerts such silencing on several E2F‐targeted genes. Among the different isoforms, HP1α levels progressively increase throughout differentiation and take over HP1γ binding on E2F sites in mature neurons. When overexpressed, only HP1α is able to ensure a timed repression of E2F genes. Specific inhibition of HP1α expression drives neuronal progenitors either towards death or cell cycle progression, yet preventing the expression of the neuronal marker microtubule‐associated protein 2. Furthermore, we provide evidence that this mechanism occurs in cerebellar granule neurons in vivo, during the postnatal development of the cerebellum. Finally, our results suggest that E2F‐targeted genes are packaged into higher‐order chromatin structures in mature neurons relative to neuroblasts, likely reflecting a transition from a ‘repressed’ versus ‘silenced’ status of these genes. Together, these data present new epigenetic regulations orchestrated by HP1 isoforms, critical for permanent cell cycle exit during neuronal differentiation.

Nogo receptor antagonizes p75NTR-dependent motor neuron death.

The Nogo-66 receptor (NgR) plays a critical role in restricting axon regeneration in the central nervous system. This inhibitory action is in part mediated by a neuronal receptor complex containing p75NTR, a multifunctional receptor also well known to trigger cell death upon binding to neurotrophins such as NGF. In the present study, we show that Pep4 and NEP1–40, which are two peptides derived from the Nogo-66 sequence that modulate NgR-mediated neurite outgrowth inhibition, prevent NGF-stimulated p75NTR-dependent death of cultured embryonic motor neurons. They also confer protection on spinal cord motor neurons after neonatal sciatic nerve axotomy. These findings demonstrate an as-yet-unknown function of NgR in maintaining neuronal survival that may be relevant for motor neuron development and degeneration.

HDAC‐3 Participates in the Repression of e2f‐Dependent Gene Transcription in Primary Differentiated Neurons

Abstract: Activation of e2f‐1 gene expression is an event that has been now established in many models of neuronal apoptosis. Accumulated E2F‐1 protein has also been observed in post mortem brains obtained from patients suffering from different neurodegenerative diseases. We have previously shown in primary neuronal cultures that e2f‐1 gene transcription was actively repressed in neuroprotective conditions through HDAC‐dependent regulation on the E2F‐responsive elements (E2F‐REs) located in the e2f‐1 gene promoter. Here, we further investigated the protein complex bound to these sites by gel shift analysis. We found that the specific protein binding to E2F‐REs is altered in apoptotic conditions compared to neuroprotective conditions, suggesting that the proteic constituents of the complex are likely to be modified upon apoptosis onset. Indeed, Western blot analysis showed a time‐dependent degradation of the Rb/E2F binding protein HDAC‐3 during apoptosis, a degradation that is caspase‐dependent. Altogether, these data point to HDAC‐3 as a good candidate involved in the active e2f‐1 repression necessary for neuroprotection.