Jesus J. Garcia

The E-protein Tcf4 interacts with Math1 to regulate differentiation of a specific subset of neuronal progenitors

Proneural factors represent <10 transcriptional regulators required for specifying all of the different neurons of the mammalian nervous system. The mechanisms by which such a small number of factors creates this diversity are still unknown. We propose that proteins interacting with proneural factors confer such specificity. To test this hypothesis we isolated proteins that interact with Math1, a proneural transcription factor essential for the establishment of a neural progenitor population (rhombic lip) that gives rise to multiple hindbrain structures and identified the E-protein Tcf4. Interestingly, haploinsufficiency of TCF4 causes the Pitt–Hopkins mental retardation syndrome, underscoring the important role for this protein in neural development. To investigate the functional relevance of the Math1/Tcf4 interaction in vivo, we studied Tcf4−/− mice and found that they have disrupted pontine nucleus development. Surprisingly, this selective deficit occurs without affecting other rhombic lip-derived nuclei, despite expression of Math1 and Tcf4 throughout the rhombic lip. Importantly, deletion of any of the other E-protein-encoding genes does not have detectable effects on Math1-dependent neurons, suggesting a specialized role for Tcf4 in distinct neural progenitors. Our findings provide the first in vivo evidence for an exclusive function of dimers formed between a proneural basic helix–loop–helix factor and a specific E-protein, offering insight about the mechanisms underlying transcriptional programs that regulate development of the mammalian nervous system.

Mapmodulin/Leucine-rich Acidic Nuclear Protein Binds the Light Chain of Microtubule-associated Protein 1B and Modulates Neuritogenesis

We had previously described the leucine-rich acidic nuclear protein (LANP) as a candidate mediator of toxicity in the polyglutamine disease, spinocerebellar ataxia type 1 (SCA1). This was based on the observation that LANP binds ataxin-1, the protein involved in this disease, in a glutamine repeat-dependent manner. Furthermore, LANP is expressed abundantly in purkinje cells, the primary site of ataxin-1 pathology. Here we focused our efforts on understanding the neuronal properties of LANP. In undifferentiated neuronal cells LANP is predominantly a nuclear protein, requiring a bona fide nuclear localization signal to be imported into the nucleus. LANP translocates from the nucleus to the cytoplasm during the process of neuritogenesis, interacts with the light chain of the microtubule-associated protein 1B (MAP1B), and modulates the effects of MAP1B on neurite extension. LANP thus could play a key role in neuronal development and/or neurodegeneration by its interactions with microtubule associated proteins.

Calcium current and charge movement of mammalian muscle: action of amyotrophic lateral sclerosis immunoglobulins.

1. The Vaseline‐gap voltage clamp technique was used to record dihydropyridine (DHP)‐sensitive Ca2+ currents (ICa) and charge movement in single cut fibres from the rat extensor digitorum longus (EDL) muscle. Amyotrophic lateral sclerosis (ALS) immunoglobulin G (IgG) action on ICa and charge movement has been characterized. 2. ALS IgG reduced ICa amplitude. The peak ICa of EDL fibres (mean +/‐ S.E.M.) at 0 mV, expressed as amperes per membrane capacitance, was ‐4.79 +/‐ 0.029 A F‐1, while after 30 min incubation in ALS IgG it was ‐2.52 +/‐ 0.04 A F‐1. IgG from healthy patients, and from patients with other diseases (familial ALS, myasthenia gravis, chronic relapsing inflammatory polyneuritis, multiple sclerosis and one sample from Lambert‐Eaton syndrome, LES) did not affect ICa, while IgG from patients with Guillain‐Barré syndrome and one other sample from a patient with LES affected the ICa in a similar way as ALS IgG. 3. The time constant of ICa activation (alpha m) at 0 mV was 44.8 +/‐ 1.4 ms in control, and 36.6 +/‐ 1.5 ms after an incubation of 30 min in ALS IgG. The steady‐state activation curve (m infinity) was shifted to more positive potentials by ALS IgG. 4. The rate constants of activation (range ‐20 to 30 mV) were altered by ALS IgG: alpha m decreased while beta m increased. These data suggest that ALS IgG favours the permanence of the Ca2+ channels in the closed state. 5. The time constant of Ca2+ channels deactivation at ‐90 mV with a pre‐pulse to 0 mV was 4.4 +/‐ 0.5 ms in control and 4.1 +/‐ 0.6 ms in ALS IgG. The relationship between the deactivation time constant and membrane potential was not significantly modified by ALS IgG. 6. ICa inactivation was not affected by ALS IgG. The potentials of half‐inactivation were ‐32.1 and ‐36.6 mV in control and ALS IgG, respectively. Similarly, the rate constants of inactivation (alpha h and beta h) remained unaltered by ALS IgG. 7. We successfully blocked ICa with 100 microM‐TMB‐8 (3,4,5‐trimethoxybenzoic acid 8‐(diethylamino)octyl ester hydrochloride), without major effects on charge movement. We adopted this procedure to study charge movement. ALS IgG reduced charge movement without significant effects on the effective valence and voltage dependence. Qon and Qoff, the charges during and after the pulse, were similarly affected by ALS IgG. 8. The actions of ALS IgG on DHP‐sensitive Ca2+ current and charge movement suggest an interaction between ALS IgG and some component of the DHP‐receptor complex.

The role of LANP and ataxin 1 in E4F-mediated transcriptional repression

The leucine‐rich acidic nuclear protein (LANP) belongs to the INHAT family of corepressors that inhibits histone acetyltransferases. The mechanism by which LANP restricts its repression to specific genes is unknown. Here, we report that LANP forms a complex with transcriptional repressor E4F and modulates its activity. As LANP interacts with ataxin 1—a protein mutated in the neurodegenerative disease spinocerebellar ataxia type 1 (SCA1)—we tested whether ataxin 1 can alter the E4F–LANP interaction. We show that ataxin 1 relieves the transcriptional repression induced by the LANP–E4F complex by competing with E4F for LANP. These results provide the first functional link, to our knowledge, between LANP and ataxin 1, and indicate a potential mechanism for the transcriptional aberrations observed in SCA1.

Generation and characterization of LANP/pp32 null mice.

ABSTRACT The leucine-rich acidic nuclear protein (LANP) belongs to a family of evolutionarily conserved proteins that are characterized by an amino-terminal domain rich in leucine residues followed by a carboxy-terminal acidic tail. LANP has been implicated in the regulation of a variety of cellular processes including RNA transport, transcription, apoptosis, vesicular trafficking, and intracellular signaling. Abundantly expressed in the developing cerebellum, this protein has also been hypothesized to play a role in cerebellar morphogenesis. LANP has been implicated in disease biology as well, both as a mediator of toxicity in spinocerebellar ataxia type 1 and as a tumor suppressor in cancers of the breast and prostate. To better understand the function of this multifaceted protein, we have generated mice lacking LANP. Surprisingly, these mice are viable and fertile. In addition we could not discern any derangements in any of the major organ systems, including the nervous system, which we have studied in detail. Overall our results point to a functional redundancy of LANPs function, most likely provided by its closely related family members.

Ca2+ current and charge movement in adult single human skeletal muscle fibres.

1. The Vaseline‐gap technique was used to record calcium currents (ICa) and charge movement in single cut fibres from normal human muscle. Experiments were carried out in 2 or 10 mM‐extracellular Ca2+ concentration ([Ca2+]o) and at 17 or 27 degrees C. 2. The passive electrical properties of the fibres with this technique were: membrane resistance for unit length rm = 59.4 k omega cm; longitudinal resistance per unit length ri = 4.9 M omega/cm; longitudinal resistance per unit length under the Vaseline seals re = 438 M omega/cm; specific membrane resistance Rm = 1.176 k omega cm2; input capacitance = 5.53 nF; specific membrane capacitance = 8.9 microF/cm2. 3. The maximum amplitude of ICa at 17 degrees C was: in 2 mM [Ca2+]o, ‐0.42 microA/microF and in 10 mM [Ca2+]o, ‐1.44 microA/microF. At 27 degrees C and in 10 mM [Ca2+]o, it increased to ‐3.04 microA/microF. The calculated temperature coefficient (Q10) for the increase in amplitude from 17 to 27 degrees C was 2.1. 4. Ca2+ permeability (PCa) was calculated using the Goldman‐Katz relation; in 2 mM [Ca2+]o at 17 degrees C, PCa = 1.26 x 10(‐6) cm/s; in 10 mM [Ca2+]o at 17 degrees C, PCa = 2.23 x 10(‐6) cm/s; in 10 mM [Ca2+]o at 27 degrees C, PCa = 4.03 x 10(‐6) cm/s. 5. The activation curve calculated from the PCa was shifted by 10 mV to positive potentials when raising [Ca2+]o from 2 to 10 mM. Increasing the temperature did not change the curve. The mid‐point potentials (Va 1/2) and steepness (k) of the activation curves were: at 17 degrees C, in 2 mM [Ca2+]o, Va 1/2 = ‐1.53 mV and k = 6.7 mV; in 10 mM [Ca2+]o, Va 1/2 = 9.96 mV and k = 6.8 mV; at 27 degrees C and 10 mM [Ca2+]o, Va 1/2 = 11.3 mV and k = 7.7 mV. The activation time constant in 10 mM [Ca2+]o reached a plateau at potentials positive to 10 mV, with a value of 93.8 ms at 17 degrees C and 17.4 ms at 27 degrees C. The calculated Q10 was 4.5. 6. The deactivation of the current was studied from tail currents at different membrane potentials in 10 mM [Ca2+]o.(ABSTRACT TRUNCATED AT 400 WORDS)