Albert Martínez

Cerebellar GABAergic progenitors adopt an external granule cell-like phenotype in the absence of Ptf1a transcription factor expression.

We report in this study that, in the cerebellum, the pancreatic transcription factor Ptf1a is required for the specific generation of Purkinje cells (PCs) and interneurons. Moreover, granule cell progenitors in the external GCL (EGL) appear to be unaffected by deletion of Ptf1a. Cell lineage analysis in Ptf1aCre/Cre mice was used to establish that, in the absence of Ptf1a expression, ventricular zone progenitors, normally fated to produce PCs and interneurons, aberrantly migrate to the EGL and express typical markers of these cells, such as Math1, Reelin, and Zic1/2. Furthermore, these cells have a fine structure typical of EGL progenitors, indicating that they adopt an EGL-like cell phenotype. These findings indicate that Ptf1a is necessary for the specification and normal production of PCs and cerebellar interneurons. Moreover, our results suggest that Ptf1a is also required for the suppression of the granule cell specification program in cerebellar ventricular zone precursors.

Reelin Regulates Postnatal Neurogenesis and Enhances Spine Hypertrophy and Long-Term Potentiation

Reelin, an extracellular protein essential for neural migration and lamination, is also expressed in the adult brain. To unravel the function of this protein in the adult forebrain, we generated transgenic mice that overexpress Reelin under the control of the CaMKIIα promoter. Overexpression of Reelin increased adult neurogenesis and impaired the migration and positioning of adult-generated neurons. In the hippocampus, the overexpression of Reelin resulted in an increase in synaptic contacts and hypertrophy of dendritic spines. Induction of long-term potentiation (LTP) in alert-behaving mice showed that Reelin overexpression evokes a dramatic increase in LTP responses. Hippocampal field EPSP during a classical conditioning paradigm was also increased in these mice. Our results indicate that Reelin levels in the adult brain regulate neurogenesis and migration, as well as the structural and functional properties of synapses. These observations suggest that Reelin controls developmental processes that remain active in the adult brain.

A TrkB/EphrinA Interaction Controls Retinal Axon Branching and Synaptogenesis

Toward understanding topographically specific branching of retinal axons in their target area, we have studied the interaction between neurotrophin receptors and members of the Eph family. TrkB and its ligand BDNF are uniformly expressed in the retina and tectum, respectively, and exert a branch-promoting activity, whereas EphAs and ephrinAs are expressed in gradients in retina and tectum and can mediate a suppression of axonal branching. We have identified a novel cis interaction between ephrinA5 and TrkB on retinal ganglion cell axons. TrkB interacts with ephrinA5 via its second cysteine-rich domain (CC2), which is necessary and sufficient for binding to ephrinA5. Their functional interaction is twofold: ephrinA5 augments BDNF-promoted retinal axon branching in the absence of its activator EphA7–Fc, whereas EphA7–Fc application abolishes branching in a local and concentration-dependent manner. The importance of TrkB in this process is shown by the fact that overexpression of an isolated TrkB–CC2 domain interfering with the ephrinA/TrkB interaction abolishes this regulatory interplay, whereas knockdown of TrkB via RNA interference diminishes the ephrinA5-evoked increase in branching. The ephrinA/Trk interaction is neurotrophin induced and specifically augments the PI-3 kinase/Akt pathway generally known to be involved in the promotion of branching. In addition, ephrinAs/TrkB modulate axon branching and also synapse formation of hippocampal neurons. Our findings uncover molecular mechanisms of how spatially restricted axon branching can be achieved by linking globally expressed branch-promoting with differentially expressed branch-suppressing activities. In addition, our data suggest that growth factors and the EphA–ephrinA system interact in a way that affects axon branching and synapse development.

The Reelin Pathway Modulates the Structure and Function of Retinal Synaptic Circuitry

The formation of synaptic connections requires the coordination of specific guidance molecules and spontaneous neuronal activity. The visual system has provided a useful model for understanding the role of these cues in shaping the precise connections from the neural retina to the brain. Here, we demonstrate that two essential genes in the Reelin signaling pathway function during the patterning of synaptic connectivity in the retina. Physiological studies of mice deficient in either reelin or disabled-1 reveal an attenuation of rod-driven retinal responses. This defect is associated with a decrease in rod bipolar cell density and an abnormal distribution of processes in the inner plexiform layer. These results imply that, in addition to its essential role during neuronal migration, the Reelin pathway contributes to the formation of neuronal circuits in the central nervous system.

Control of neuronal branching by the death receptor CD95 (Fas/Apo-1)

The CD95 (Apo-1/Fas)/CD95 ligand (CD95L) system is best characterized as a trigger of apoptosis. Nevertheless, despite broad expression of CD95L and CD95 in the developing brain, absence of functional CD95 (lpr mice) or CD95L (gld mice) does not alter neuronal numbers. Here, we report that in embryonic hippocampal and cortical neurons in vivo and in vitro CD95L does not induce apoptosis. Triggering of CD95 in cultured immature neurons substantially increases neurite branches by promoting their formation. The branching increase occurs in a caspase-independent and death domain-dependent manner and is paralleled by an increase in the nonphosphorylated form of Tau. Most importantly, lpr and gld mutants exhibit a reduced number of dendritic branches in vivo at the time when synapse formation takes place. These data reveal a novel function for the CD95 system and add to the picture of guidance molecules in the developing brain.

Phosphorylation of tau and α-synuclein in synaptic-enriched fractions of the frontal cortex in Alzheimer’s disease, and in Parkinson’s disease and related α-synucleinopathies

Phosphorylation of tau and phosphorylation of alpha-synuclein are crucial abnormalities in Alzheimers disease (AD) and alpha-synucleinopathies (Parkinsons disease: PD, and dementia with Lewy bodies: DLB), respectively. The presence and distribution of phospho-tau were examined by sub-fractionation, gel electrophoresis and Western blotting in the frontal cortex of cases with AD at different stages of disease progression, PD, DLB pure form and common form, and in age-matched controls. Phospho-tauSer396 has been found in synaptic-enriched fractions in AD frontal cortex at entorhinal/transentorhinal, limbic and neocortical stages, thus indicating early tau phosphorylation at the synapses in AD before the occurrence of neurofibrillary tangles in the frontal cortex. Phospho-tauSer396 is also found in synaptic-enriched fractions in the frontal cortex in PD and DLB pure and common forms, thus indicating increased tau phosphorylation at the synapses in these alpha-synucleinopathies. Densitometric studies show between 20% and 40% phospho-tauSer396, in relation with tau-13, in synaptic-enriched fractions of the frontal cortex in AD stages I-III, and in PD and DLB. The percentage reaches about 95% in AD stage V and DLB common form. Yet tau phosphorylation characteristic of neurofibrillary tangles, as revealed with the AT8 antibody, is found in the synaptic fractions of the frontal cortex only at advanced stages of AD. Increased phosphorylated alpha-synucleinSer129 levels are observed in the synaptic-enriched fractions of the frontal cortex in PD and DLB pure and common forms, and in advanced stages of AD. Since tau-hyperphosphorylation has implications in microtubule assembly, and phosphorylation of alpha-synuclein at Ser129 favors alpha-synuclein aggregation, it can be suggested that synapses are targets of abnormal tau and alpha-synuclein phosphorylation in both groups of diseases. Tau phosphorylation at Ser396 has also been found in synaptic-enriched fractions in 12-month-old transgenic mice bearing the A53T alpha-synuclein mutation.

Mutant GlialCAM Causes Megalencephalic Leukoencephalopathy with Subcortical Cysts, Benign Familial Macrocephaly, and Macrocephaly with Retardation and Autism

Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a leukodystrophy characterized by early-onset macrocephaly and delayed-onset neurological deterioration. Recessive MLC1 mutations are observed in 75% of patients with MLC. Genetic-linkage studies failed to identify another gene. We recently showed that some patients without MLC1 mutations display the classical phenotype; others improve or become normal but retain macrocephaly. To find another MLC-related gene, we used quantitative proteomic analysis of affinity-purified MLC1 as an alternative approach and found that GlialCAM, an IgG-like cell adhesion molecule that is also called HepaCAM and is encoded by HEPACAM, is a direct MLC1-binding partner. Analysis of 40 MLC patients without MLC1 mutations revealed multiple different HEPACAM mutations. Ten patients with the classical, deteriorating phenotype had two mutations, and 18 patients with the improving phenotype had one mutation. Most parents with a single mutation had macrocephaly, indicating dominant inheritance. In some families with dominant HEPACAM mutations, the clinical picture and magnetic resonance imaging normalized, indicating that HEPACAM mutations can cause benign familial macrocephaly. In other families with dominant HEPACAM mutations, patients had macrocephaly and mental retardation with or without autism. Further experiments demonstrated that GlialCAM and MLC1 both localize in axons and colocalize in junctions between astrocytes. GlialCAM is additionally located in myelin. Mutant GlialCAM disrupts the localization of MLC1-GlialCAM complexes in astrocytic junctions in a manner reflecting the mode of inheritance. In conclusion, GlialCAM is required for proper localization of MLC1. HEPACAM is the second gene found to be mutated in MLC. Dominant HEPACAM mutations can cause either macrocephaly and mental retardation with or without autism or benign familial macrocephaly.

GlialCAM, a Protein Defective in a Leukodystrophy, Serves as a ClC-2 Cl− Channel Auxiliary Subunit

Summary Ion fluxes mediated by glial cells are required for several physiological processes such as fluid homeostasis or the maintenance of low extracellular potassium during high neuronal activity. In mice, the disruption of the Cl− channel ClC-2 causes fluid accumulation leading to myelin vacuolation. A similar vacuolation phenotype is detected in humans affected with megalencephalic leukoencephalopathy with subcortical cysts (MLC), a leukodystrophy which is caused by mutations in MLC1 or GLIALCAM. We here identify GlialCAM as a ClC-2 binding partner. GlialCAM and ClC-2 colocalize in Bergmann glia, in astrocyte-astrocyte junctions at astrocytic endfeet around blood vessels, and in myelinated fiber tracts. GlialCAM targets ClC-2 to cell junctions, increases ClC-2 mediated currents, and changes its functional properties. Disease-causing GLIALCAM mutations abolish the targeting of the channel to cell junctions. This work describes the first auxiliary subunit of ClC-2 and suggests that ClC-2 may play a role in the pathology of MLC disease. Video Abstract

Cell-autonomous inactivation of the reelin pathway impairs adult neurogenesis in the hippocampus.

Adult hippocampal neurogenesis is thought to be essential for learning and memory, and has been implicated in the pathogenesis of several disorders. Although recent studies have identified key factors regulating neuroprogenitor proliferation in the adult hippocampus, the mechanisms that control the migration and integration of adult-born neurons into circuits are largely unknown. Reelin is an extracellular matrix protein that is vital for neuronal development. Activation of the Reelin cascade leads to phosphorylation of Disabled-1, an adaptor protein required for Reelin signaling. Here we used transgenic mouse and retroviral reporters along with Reelin signaling gain-of-function and loss-of-function studies to show that the Reelin pathway regulates migration and dendritic development of adult-generated hippocampal neurons. Whereas overexpression of Reelin accelerated dendritic maturation, inactivation of the Reelin signaling pathway specifically in adult neuroprogenitor cells resulted in aberrant migration, decreased dendrite development, formation of ectopic dendrites in the hilus, and the establishment of aberrant circuits. Our findings support a cell-autonomous and critical role for the Reelin pathway in regulating dendritic development and the integration of adult-generated granule cells and point to this pathway as a key regulator of adult neurogenesis. Moreover, our data reveal a novel role of the Reelin cascade in adult brain function with potential implications for the pathogenesis of several neurological and psychiatric disorders.

Molecular pathogenesis of megalencephalic leukoencephalopathy with subcortical cysts: mutations in MLC1 cause folding defects

Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a rare type of leukodystrophy, most often caused by mutations in the MLC1 gene. MLC1 is an oligomeric plasma membrane (PM) protein of unknown function expressed mainly in glial cells and neurons. Most disease-causing missense mutations dramatically reduced the total and PM MLC1 expression levels in Xenopus oocytes and mammalian cells. The impaired expression of the mutants was verified in primary cultures of rat astrocytes, as well as human monocytes, cell types that endogenously express MLC1, demonstrating the relevance of the tissue culture models. Using a combination of biochemical, pharmacological and imaging methods, we also demonstrated that increased endoplasmatic reticulum-associated degradation and endo-lysosomal-associated degradation can contribute to the cell surface expression defect of the mutants. Based on these results, we suggest that MLC1 mutations reduce protein levels in vivo. Since the expression defect of the mutants could be rescued by exposing the mutant-protein expressing cells to low temperature and glycerol, a chemical chaperone, we propose that MLC belongs to the class of conformational diseases. Therefore, we suggest the use of pharmacological strategies that improve MLC1 expression to treat MLC patients.