Gonzalo Blanco

Mapping of the human and murine X11-like genes (APBA2 and Apba2), the murine Fe65 gene (Apbb1), and the human Fe65-like gene (APBB2): genes encoding phosphotyrosine-binding domain proteins that interact with the Alzheimer's disease amyloid precursor protein

Abnormal processing of the membrane-spanning amyloid precursor protein (APP), resulting in the production of increased amounts of fibrillogenic b-amyloid peptide (Ab), is considered to be one of the key metabolic events underlying Alzheimer’s disease (AD; Selkoe 1994). The function of APP is not fully understood, and the precise cellular mechanisms that lead to A b production are not clearly defined. However, one pathway for A b production involves the re-internalization of membrane-bound APP into lysosomes where fragments of APP containing intact A b are generated (Selkoe 1994). In common with a number of cell surface receptors, the carboxy terminal cytoplasmic domain of APP contains an AsnPro-Thr-Tyr (NPTY) motif which mediates re-internalization via clathrin-coated pits (Chen et al. 1990). This motif has also been demonstrated to be a consensus sequence for binding to phosphotyrosine binding/interacting domain (PTB)-bearing proteins (van der Geer and Pawson 1995). We and others have recently reported that the cytoplasmic domain of APP binds to four human PTB proteins: X11, X11-like, Fe65, and Fe65-like (Borg et al. 1996; Bressler et al. 1996; Fiore et al. 1995; Gue ́nette et al. 1996; McLoughlin and Miller 1996). It has been confirmed that the YENPTY sequence in the cytoplasmic domain of APP is responsible for mediating the interactions between the PTB domain in X11 and the second of two PTB domains in Fe65 (Borg et al. 1996; Fiore et al. 1995). PTB domain proteins are believed to be involved in signal transduction processes (van der Geer and Pawson 1995), and the interaction of APP with X11, X11-like, Fe65, and Fe65-like suggests a role for APP in such signal transduction mechanisms. Furthermore, as they interact with the YENPTY motif in APP, these PTB proteins may modulate processing of APP and hence formation of A b. Therefore, mapping of the genes coding for these proteins is important as they represent new candidate susceptibility genes for AD. The approved gene symbols for the members of these APP binding protein (APB) families are presented in Table 1. The gene for human X11 (APBA1) is already known to be on Chromosome (Chr) 9 close to marker D9S411E (Duclos et al. 1993), and the gene for human Fe65 (APBB1) has been localized to Chr 11 at 11p15 (Bressler et al. 1996). The existence of murine X11 and murine Fe65-like has not yet been reported. Here we report the chromosomal assignment of human APBA2 and APBB2 plus the chromosomal mapping of the murine homologs of X11-like (Apba2) and Fe65 ( Apbb1). In order to map the human APBA2 and APBB2 genes, we selected PCR primers from the previously identified cDNA clones (McLoughlin and Miller 1996) and overlapping sequences deposited in the databases (accession numbers R89683, R13010, R18654, and T16098 for APBA2 and accession number HSU62325 for APBB2). For APBA2 the following primer pair: forward, 58-TTACAAGTCGTGTCCTGGGAG-38, and reverse, 58-GACGTCTGGGGTCCTGTG-3 8, generated a small PCR product of 103 bp. For APBB2 the following primer pair: forward, 58-CACAGAGAAGAGTCTGGCCC-38 and reverse, 5 8-AGGTTGCTTGTGACAGGTCC-38, generated a PCR product of 114 bp. These PCR products were sequenced to confirm they originated from the correct genes. Both human APBA2 and APBB2 genes were mapped using the Genebridge 4 radiation hybrid panel (HGMP Resource Centre, Cambridge, UK) consisting of 94 hamster-derived cell lines. PCR amplification of human DNA with PCR primers designed for these genes resulted in products of the expected size, while no amplification products were obtained from the hamster DNA control sample. Scores for individual cell lines were submitted at the WICGR mapping service at http:// www.genome.wi.mit.edu. APBA2 was assigned to human Chr 15 between the markers WI-5590 (10.31 cR) and D15S144 (21.7 cR). APBB2 was assigned to human Chr 4 between the markers D4S405 (4.6 cR) and D4S496 (10.1 cR). To map theApba2andApbb1loci in the mouse, we used the EUCIB resource which comprises 982 interspecific backcross progeny for high-resolution genetic mapping across the mouse genome (Breen et al. 1994). It is clear from sequence alignments that the mouse sequence L34676 available in the Genbank database corresponds to the mouse homolog of APBA2 ( pba2) rather than to the mouse homolog of APBA1 (McLoughlin and Miller 1996). The following primer pair was selected for mouse Apba2 PCR amplifications: forward, 5 8-GCGCTCTGATCTCAATGG38; reverse, 58-GGAAATGATGCCACCTTC-38. This generated an approximately 1000-bp PCR product. Primers for mouse Apbb1 were designed from the published rat sequence (accession number X60468). The following primer pair was designed for mouse Apbb1 PCR amplifications: forward, 5 8-CTGGCACATCCCAACAGG-38; reverse, 58-AGCAAAGCCAGTCCAGGT-38. The PCR product was 202 bp. Both of these murine PCR products were sequenced to confirmed their origin. The mouseApba2andApbb1PCR products did not show any allelic size difference between C57BL/6 and Mus spretus, the two parental strains of the EUCIB interspecific backcross. However, in both cases, SSCP analysis (Chang et al. 1993) did show a clear polymorphism between C57BL/6 and Mus spretus.In the case of Apba2the large 1-kb PCR product was Sau3AI digested prior to loading on the SSCP gel. 92 random samples from the EUCIB backcross were analyzed for the segregation of C57BL/6 and Mus Correspondence to: D.M. McLoughlin at Dept. of Neuroscience Mammalian Genome 9, 473–475 (1998).

AXONAL AND NEUROMUSCULAR SYNAPTIC PHENOTYPES IN Wld S , SOD1 G93A AND ostes MUTANT MICE IDENTIFIED BY FIBER-OPTIC CONFOCAL MICROENDOSCOPY

We used live imaging by fiber-optic confocal microendoscopy (CME) of yellow fluorescent protein (YFP) expression in motor neurons to observe and monitor axonal and neuromuscular synaptic phenotypes in mutant mice. First, we visualized slow degeneration of axons and motor nerve terminals at neuromuscular junctions following sciatic nerve injury in Wld(S) mice with slow Wallerian degeneration. Protection of axotomized motor nerve terminals was much weaker in Wld(S) heterozygotes than in homozygotes. We then induced covert modifiers of axonal and synaptic degeneration in heterozygous Wld(S) mice, by N-ethyl-N-nitrosourea (ENU) mutagenesis, and used CME to identify candidate mutants that either enhanced or suppressed axonal or synaptic degeneration. From 219 of the F1 progeny of ENU-mutagenized BALB/c mice and thy1.2-YFP16/Wld(S) mice, CME revealed six phenodeviants with suppression of synaptic degeneration. Inheritance of synaptic protection was confirmed in three of these founders, with evidence of Mendelian inheritance of a dominant mutation in one of them (designated CEMOP_S5). We next applied CME repeatedly to living Wld(S) mice and to SOD1(G93A) mice, an animal model of motor neuron disease, and observed degeneration of identified neuromuscular synapses over a 1-4day period in both of these mutant lines. Finally, we used CME to observe slow axonal regeneration in the ENU-mutant ostes mouse strain. The data show that CME can be used to monitor covert axonal and neuromuscular synaptic pathology and, when combined with mutagenesis, to identify genetic modifiers of its progression in vivo.

Candidate testis‐determining gene, Maestro (Mro), encodes a novel HEAT repeat protein

Mammalian sex determination depends on the presence or absence of SRY transcripts in the embryonic gonad. Expression of SRY initiates a pathway of gene expression resulting in testis development. Here, we describe a novel gene potentially functioning in this pathway using a cDNA microarray screen for genes exhibiting sexually dimorphic expression during murine gonad development. Maestro (Mro) transcripts are first detected in the developing male gonad before overt testis differentiation. By 12.5 days postcoitus (dpc), Mro transcription is restricted to the developing testis cords and its expression is not germ cell‐dependent. No expression is observed in female gonads between 10.5 and 14.5 dpc. Maestro encodes a protein containing HEAT‐like repeats that localizes to the nucleolus in cell transfection assays. Maestro maps to a region of mouse chromosome 18 containing a genetic modifier of XX sex reversal. We discuss the possible function of Maestro in light of these data. Developmental Dynamics 227:600–607, 2003.

Novel mutations in human and mouse SCN4A implicate AMPK in myotonia and periodic paralysis

Corrochano Sanchez et al. identify a novel mutation (I588V) in SCN4A, which encodes the Nav1.4 voltage-gated sodium channel, in a patient with myotonia and periodic paralysis. By generating and characterizing a mouse model (‘draggen’) carrying the equivalent point mutation (I582V), they uncover novel pathological and metabolic features of SCN4A channelopathies.

Myofibrillar myopathy caused by a mutation in the motor domain of mouse MyHC IIb

Ariel is a mouse mutant that suffers from skeletal muscle myofibrillar degeneration due to the rapid accumulation of large intracellular protein aggregates. This fulminant disease is caused by an ENU-induced recessive mutation resulting in an L342Q change within the motor domain of the skeletal muscle myosin protein MYH4 (MyHC IIb). Although normal at birth, homozygous mice develop hindlimb paralysis from Day 13, consistent with the timing of the switch from developmental to adult myosin isoforms in mice. The mutated myosin (MYH4(L342Q)) is an aggregate-prone protein. Notwithstanding the speed of the process, biochemical analysis of purified aggregates showed the presence of proteins typically found in human myofibrillar myopathies, suggesting that the genesis of ariel aggregates follows a pathogenic pathway shared with other conformational protein diseases of skeletal muscle. In contrast, heterozygous mice are overtly and histologically indistinguishable from control mice. MYH4(L342Q) is present in muscles from heterozygous mice at only 7% of the levels of the wild-type protein, resulting in a small but significant increase in force production in isolated single fibres and indicating that elimination of the mutant protein in heterozygotes prevents the pathological changes observed in homozygotes. Recapitulation of the L342Q change in the functional equivalent of mouse MYH4 in human muscles, MYH1, results in a more aggregate-prone protein.

Identification of a Z-band associated protein complex involving KY, FLNC and IGFN1

The KY protein underlies a form of muscular dystrophy in the mouse but its role in muscle remains elusive. Immunodetection of endogenous KY protein in C2C12-derived myotubes and expression of a recombinant form in neonatal cardiomyocytes indicated that KY is a Z-band associated protein. Moreover, characterization of a KY interacting protein fragment led to the identification of Igfn1 (Immunoglobulin-like and fibronectin type 3 domain containing 1). Igfn1 is a transcriptionally complex locus encoding many protein variants. A yeast two-hybrid screen identified the Z-band protein filamin C (FLNC) as an interacting partner. Consistent with this, expression of an IGFN1 recombinant fragment showed that the three N-terminal globular domains, common to at least five IGFN1 variants, are sufficient to provide Z-band targeting. Taken together, the yeast two-hybrid, biochemical and immunofluorescence data support the notion that KY, IGFN1 and FLNC are part of a Z-band associated protein complex likely to provide structural support to the skeletal muscle sarcomere.

Constitutive upregulations of titin-based signalling proteins in KY deficient muscles.

An increase in the expression of stretch/stress response elements in fast and slow muscles has been previously described in a transcriptional profiling of KY deficient muscles. Here, we have characterized the induction of this titin-based family of signalling proteins in ky/ky muscles at the protein level. Changes in expression of MLP, MARP2 and Xin have been related to the onset of dystrophic and adaptive changes that operate in ky/ky muscles. Our results indicate that induction of this set of genes is an early consequence of the interference caused by the absence of the KY protein. A search of muscle profiles of mouse models revealed such molecular hallmark only in muscles subjected to a single bout of eccentric contractions and specific titin mutants. Based on the role of this family as titin-based stress response molecules, it is suggested that titin structural/signalling instability is common to ky and titin mouse mutants and eccentric contractions.

Upregulation of PKD1L2 provokes a complex neuromuscular disease in the mouse

Following a screen for neuromuscular mouse mutants, we identified ostes, a novel N-ethyl N-nitrosourea-induced mouse mutant with muscle atrophy. Genetic and biochemical evidence shows that upregulation of the novel, uncharacterized transient receptor potential polycystic (TRPP) channel PKD1L2 (polycystic kidney disease gene 1-like 2) underlies this disease. Ostes mice suffer from chronic neuromuscular impairments including neuromuscular junction degeneration, polyneuronal innervation and myopathy. Ectopic expression of PKD1L2 in transgenic mice reproduced the ostes myopathic changes and, indeed, caused severe muscle atrophy in Tg(Pkd1l2)/Tg(Pkd1l2) mice. Moreover, double-heterozygous mice (ostes/+, Tg(Pkd1l2)/0) suffer from myopathic changes more profound than each heterozygote, indicating positive correlation between PKD1L2 levels and disease severity. We show that, in vivo, PKD1L2 primarily associates with endogenous fatty acid synthase in normal skeletal muscle, and these proteins co-localize to costameric regions of the muscle fibre. In diseased ostes/ostes muscle, both proteins are upregulated, and ostes/ostes mice show signs of abnormal lipid metabolism. This work shows the first role for a TRPP channel in neuromuscular integrity and disease.

A nonsense mutation in mouse tardbp affects TDP43 alternative splicing activity and causes limb-clasping and body tone defects.

Mutations in TARDBP, encoding Tar DNA binding protein-43 (TDP43), cause amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Attempts to model TDP43 dysfunction in mice have used knockouts or transgenic overexpressors, which have revealed the difficulties of manipulating TDP43, whose level is tightly controlled by auto-regulation. In a complementary approach, to create useful mouse models for the dissection of TDP43 function and pathology, we have identified a nonsense mutation in the endogenous mouse Tardbp gene through screening an N-ethyl-N-nitrosourea (ENU) mutant mouse archive. The mutation is predicted to cause a Q101X truncation in TDP43. We have characterised TardbpQ101X mice to investigate this mutation in perturbing TDP43 biology at endogenous expression levels. We found the TardbpQ101X mutation is homozygous embryonic lethal, highlighting the importance of TDP43 in early development. Heterozygotes (Tardbp+/Q101X) have abnormal levels of mutant transcript, but we find no evidence of the truncated protein and mice have similar full-length TDP43 protein levels as wildtype littermates. Nevertheless, Tardbp+/Q101X mice have abnormal alternative splicing of downstream gene targets, and limb-clasp and body tone phenotypes. Thus the nonsense mutation in Tardbp causes a mild loss-of-function phenotype and behavioural assessment suggests underlying neurological abnormalities. Due to the role of TDP43 in ALS, we investigated potential interactions with another known causative gene, mutant superoxide dismutase 1 (SOD1). Tardbp+/Q101X mice were crossed with the SOD1G93Adl transgenic mouse model of ALS. Behavioural and physiological assessment did not reveal modifying effects on the progression of ALS-like symptoms in the double mutant progeny from this cross. In summary, the TardbpQ101X mutant mice are a useful tool for the dissection of TDP43 protein regulation, effects on splicing, embryonic development and neuromuscular phenotypes. These mice are freely available to the community.

Molecular phenotyping of the mouse ky mutant reveals UCP1 upregulation at the neuromuscular junctions of dystrophic soleus muscle

The ky mutant mouse displays a muscular dystrophy that affects almost exclusively slow type muscles in which persistent muscle regeneration, neuromuscular junction instability and an absence of the hypertrophic response are prominent features. In order to gain insights into the pathogenesis of this muscular dystrophy we have undertaken RNA profiling of the extensor digitorum longus, a fast unaffected muscle, and the highly pathological soleus slow muscle, followed by further expression studies to validate the results. In dystrophic soleus, there is a coordinated change in the expression level of genes encoding energy transducing mitochondrial proteins and an increase in the expression of stretch response genes. Upregulation of uncoupling proteins 1 and 2 is a unique molecular signature of the ky muscular dystrophy and was further characterised at the protein level. Our results show a spatial and temporal association between disorganisation of acetylcholine receptor clusters and upregulation of uncoupling protein 1. There is also evidence of a breakdown of neuromuscular junction muscle-specific kinase-dependent signalling in adult mutant soleus. Sarcolemma-associated proteins implicated in muscular dystrophies revealed no differences on microarrays and were confirmed as normally distributed by immunofluorescence. Altogether, the data presented suggest that the ky muscular dystrophy develops by a distinctive pathogenic mechanism.