Lingling Kong

Impaired Synaptic Vesicle Release and Immaturity of Neuromuscular Junctions in Spinal Muscular Atrophy Mice

The motor neuron disease spinal muscular atrophy (SMA) causes profound muscle weakness that most often leads to early death. At autopsy, SMA is characterized by loss of motor neurons and muscle atrophy, but the initial cellular events that precipitate motor unit dysfunction and loss remain poorly characterized. Here, we examined the function and corresponding structure of neuromuscular junction (NMJ) synapses in a mouse model of severe SMA (hSMN2/delta7SMN/mSmn−/−). Surprisingly, most SMA NMJs remained innervated even late in the disease course; however they showed abnormal synaptic transmission. There was a two-fold reduction in the amplitudes of the evoked endplate currents (EPCs), but normal spontaneous miniature EPC (MEPC) amplitudes. These features in combination indicate reduced quantal content. SMA NMJs also demonstrated increased facilitation suggesting a reduced probability of vesicle release. By electron microscopy, we found a decreased density of synaptic vesicles that is likely to contribute to the reduced release probability. In addition to presynaptic defects, there were postsynaptic abnormalities. EPC and MEPC decay time constants were prolonged because of a slowed switch from the fetal acetylcholine receptor (AChR) γ-subunit to the adult ε-subunit. There was also reduced size of AChR clusters and small myofibers, which expressed an immature pattern of myosin heavy chains. Together these results indicate that impaired synaptic vesicle release at NMJs in severe SMA is likely to contribute to failed postnatal maturation of motor units and muscle weakness.

Mutations in TRPV4 cause Charcot-Marie-Tooth disease type 2C

Charcot-Marie-Tooth disease type 2C (CMT2C) is an autosomal dominant neuropathy characterized by limb, diaphragm and laryngeal muscle weakness. Two unrelated families with CMT2C showed significant linkage to chromosome 12q24.11. We sequenced all genes in this region and identified two heterozygous missense mutations in the TRPV4 gene, C805T and G806A, resulting in the amino acid substitutions R269C and R269H. TRPV4 is a well-known member of the TRP superfamily of cation channels. In TRPV4-transfected cells, the CMT2C mutations caused marked cellular toxicity and increased constitutive and activated channel currents. Mutations in TRPV4 were previously associated with skeletal dysplasias. Our findings indicate that TRPV4 mutations can also cause a degenerative disorder of the peripheral nerves. The CMT2C-associated mutations lie in a distinct region of the TRPV4 ankyrin repeats, suggesting that this phenotypic variability may be due to differential effects on regulatory protein-protein interactions.

Early Functional Impairment of Sensory-Motor Connectivity in a Mouse Model of Spinal Muscular Atrophy

To define alterations of neuronal connectivity that occur during motor neuron degeneration, we characterized the function and structure of spinal circuitry in spinal muscular atrophy (SMA) model mice. SMA motor neurons show reduced proprioceptive reflexes that correlate with decreased number and function of synapses on motor neuron somata and proximal dendrites. These abnormalities occur at an early stage of disease in motor neurons innervating proximal hindlimb muscles and medial motor neurons innervating axial muscles, but only at end-stage disease in motor neurons innervating distal hindlimb muscles. Motor neuron loss follows afferent synapse loss with the same temporal and topographical pattern. Trichostatin A, which improves motor behavior and survival of SMA mice, partially restores spinal reflexes, illustrating the reversibility of these synaptic defects. Deafferentation of motor neurons is an early event in SMA and may be a primary cause of motor dysfunction that is amenable to therapeutic intervention.

Overexpression of IGF-1 in Muscle Attenuates Disease in a Mouse Model of Spinal and Bulbar Muscular Atrophy

Expansion of a polyglutamine tract in the androgen receptor (AR) causes spinal and bulbar muscular atrophy (SBMA). We previously showed that Akt-mediated phosphorylation of AR reduces ligand binding and attenuates the mutant AR toxicity. Here, we show that in culture insulin-like growth factor 1 (IGF-1) reduces AR aggregation and increases AR clearance via the ubiquitin-proteasome system through phosphorylation of AR by Akt. In vivo, SBMA transgenic mice overexpressing a muscle-specific isoform of IGF-1 selectively in skeletal muscle show evidence of increased Akt activation and AR phosphorylation and decreased AR aggregation. Augmentation of IGF-1/Akt signaling rescues behavioral and histopathological abnormalities, extends the life span, and reduces both muscle and spinal cord pathology of SBMA mice. This study establishes IGF-1/Akt-mediated inactivation of mutant AR as a strategy to counteract disease in vivo and demonstrates that skeletal muscle is a viable target tissue for therapeutic intervention in SBMA.

Sustained improvement of spinal muscular atrophy mice treated with trichostatin a plus nutrition

Early treatment with the histone deacetylase inhibitor, trichostatin A, plus nutritional support extended median survival of spinal muscular atrophy mice by 170%. Treated mice continued to gain weight, maintained stable motor function, and retained intact neuromuscular junctions long after trichostatin A was discontinued. In many cases, ultimate decline of mice appeared to result from vascular necrosis, raising the possibility that vascular dysfunction is part of the clinical spectrum of severe spinal muscular atrophy. Early spinal muscular atrophy disease detection and treatment initiation combined with aggressive ancillary care may be integral to the optimization of histone deacetylase inhibitor treatment in human patients. Ann Neurol 2008; 64:465–470

Survival Motor Neuron Protein in Motor Neurons Determines Synaptic Integrity in Spinal Muscular Atrophy

The inherited motor neuron disease spinal muscular atrophy (SMA) is caused by deficient expression of survival motor neuron (SMN) protein and results in severe muscle weakness. In SMA mice, synaptic dysfunction of both neuromuscular junctions (NMJs) and central sensorimotor synapses precedes motor neuron cell death. To address whether this synaptic dysfunction is due to SMN deficiency in motor neurons, muscle, or both, we generated three lines of conditional SMA mice with tissue-specific increases in SMN expression. All three lines of mice showed increased survival, weights, and improved motor behavior. While increased SMN expression in motor neurons prevented synaptic dysfunction at the NMJ and restored motor neuron somal synapses, increased SMN expression in muscle did not affect synaptic function although it did improve myofiber size. Together these data indicate that both peripheral and central synaptic integrity are dependent on motor neurons in SMA, but SMN may have variable roles in the maintenance of these different synapses. At the NMJ, it functions at the presynaptic terminal in a cell-autonomous fashion, but may be necessary for retrograde trophic signaling to presynaptic inputs onto motor neurons. Importantly, SMN also appears to function in muscle growth and/or maintenance independent of motor neurons. Our data suggest that SMN plays distinct roles in muscle, NMJs, and motor neuron somal synapses and that restored function of SMN at all three sites will be necessary for full recovery of muscle power.

Increased IGF-1 in muscle modulates the phenotype of severe SMA mice

Spinal muscular atrophy (SMA) is an inherited motor neuron disease caused by the mutation of the survival motor neuron 1 (SMN1) gene and deficiency of the SMN protein. Severe SMA mice have abnormal motor function and small, immature myofibers early in development suggesting that SMN protein deficiency results in retarded muscle growth. Insulin-like growth factor 1 (IGF-1) stimulates myoblast proliferation, induces myogenic differentiation and generates myocyte hypertrophy in vitro and in vivo. We hypothesized that increased expression of IGF-1 specifically in skeletal muscle would attenuate disease features of SMAΔ7 mice. SMAΔ7 mice overexpressing a local isoform of IGF-1 (mIGF-1) in muscle showed enlarged myofibers and a 40% increase in median survival compared with mIGF-1-negative SMA littermates (median survival = 14 versus 10 days, respectively, log-rank P = 0.025). Surprisingly, this was not associated with a significant improvement in motor behavior. Treatment of both mIGF-1(NEG) and mIGF-1(POS) SMA mice with the histone deacetylase inhibitor, trichostatin A (TSA), resulted in a further extension of survival and improved motor behavior, but the combination of mIGF-1 and TSA treatment was not synergistic. These results show that increased mIGF-1 expression restricted to muscle can modulate the phenotype of SMA mice indicating that therapeutics targeted to muscle alone should not be discounted as potential disease-modifying therapies in SMA. IGF-1 may warrant further investigation in mild SMA animal models and perhaps SMA patients.

The genetics of spinal muscular atrophies.

PURPOSE OF REVIEW This article reviews clinical, genetic, and therapeutic advances in spinal muscular atrophies (SMAs), inherited disorders characterized by motor neuron loss and muscle weakness. RECENT FINDINGS There has been progress in defining the clinical and genetic features of at least 16 distinct forms of SMA. The genes associated with 14 of these disorders have been identified in the last decade, including four within the last year: TRPV4, ATP7A, VRK1, and HSPB3. Genetic testing is now available for many SMAs, providing important diagnostic and prognostic information. Cell and animal models of SMAs have been used to further understand how mutations in SMA-associated genes, which code for proteins involved in diverse functions such as transcriptional regulation, RNA processing, and cytoskeletal dynamics, lead to motor neuron dysfunction and loss. In the last year, there has also been remarkable progress in preclinical therapeutics development for proximal SMA using gene therapy, antisense oligonucleotides, and small molecules. SUMMARY The advances in the clinical and genetic characterization of different forms of SMAs have important implications for clinical evaluation and management of patients. The identification of multiple, novel SMA-causing genes will lead to an improved understanding of motor neuron disease biology and may provide novel targets for therapeutics development.

Improvement of neuromuscular synaptic phenotypes without enhanced survival and motor function in severe spinal muscular atrophy mice selectively rescued in motor neurons.

In the inherited childhood neuromuscular disease spinal muscular atrophy (SMA), lower motor neuron death and severe muscle weakness result from the reduction of the ubiquitously expressed protein survival of motor neuron (SMN). Although SMA mice recapitulate many features of the human disease, it has remained unclear if their short lifespan and motor weakness are primarily due to cell-autonomous defects in motor neurons. Using Hb9Cre as a driver, we selectively raised SMN expression in motor neurons in conditional SMAΔ7 mice. Unlike a previous study that used choline acetyltransferase (ChATCre+) as a driver on the same mice, and another report that used Hb9Cre as a driver on a different line of conditional SMA mice, we found no improvement in survival, weight, motor behavior and presynaptic neurofilament accumulation. However, like in ChATCre+ mice, we detected rescue of endplate size and mitigation of neuromuscular junction (NMJ) denervation status. The rescue of endplate size occurred in the absence of an increase in myofiber size, suggesting endplate size is determined by the motor neuron in these animals. Real time-PCR showed that the expression of spinal cord SMN transcript was sharply reduced in Hb9Cre+ SMA mice relative to ChATCre+ SMA mice. This suggests that our lack of overall phenotypic improvement is most likely due to an unexpectedly poor recombination efficiency driven by Hb9Cre. Nonetheless, the low levels of SMN were sufficient to rescue two NMJ structural parameters indicating that these motor neuron cell autonomous phenotypes are very sensitive to changes in motoneuronal SMN levels. Our results directly suggest that even those therapeutic interventions with very modest effects in raising SMN in motor neurons may provide mitigation of neuromuscular phenotypes in SMA patients.

The Antisense Transcript SMN-AS1 Regulates SMN Expression and Is a Novel Therapeutic Target for Spinal Muscular Atrophy

The neuromuscular disorder spinal muscular atrophy (SMA), the most common inherited killer of infants, is caused by insufficient expression of survival motor neuron (SMN) protein. SMA therapeutics development efforts have focused on identifying strategies to increase SMN expression. We identified a long non-coding RNA (lncRNA) that arises from the antisense strand of SMN, SMN-AS1, which is enriched in neurons and transcriptionally represses SMN expression by recruiting the epigenetic Polycomb repressive complex-2. Targeted degradation of SMN-AS1 with antisense oligonucleotides (ASOs) increases SMN expression in patient-derived cells, cultured neurons, and the mouse central nervous system. SMN-AS1 ASOs delivered together with SMN2 splice-switching oligonucleotides additively increase SMN expression and improve survival of severe SMA mice. This study is the first proof of concept that targeting a lncRNA to transcriptionally activate SMN2 can be combined with SMN2 splicing modification to ameliorate SMA and demonstrates the promise of combinatorial ASOs for the treatment of neurogenetic disorders.