Matthew D. Howell

Canonical transient receptor channel 5 (TRPC5) and TRPC1/4 contribute to seizure and excitotoxicity by distinct cellular mechanisms.

Seizures are the manifestation of highly synchronized burst firing of a large population of cortical neurons. Epileptiform bursts with an underlying plateau potential in neurons are a cellular correlate of seizures. Emerging evidence suggests that the plateau potential is mediated by neuronal canonical transient receptor potential (TRPC) channels composed of members of the TRPC1/4/5 subgroup. We previously showed that TRPC1/4 double-knockout (DKO) mice lack epileptiform bursting in lateral septal neurons and exhibit reduced seizure-induced neuronal cell death, but surprisingly have unaltered pilocarpine-induced seizures. Here, we report that TRPC5 knockout (KO) mice exhibit both significantly reduced seizures and minimal seizure-induced neuronal cell death in the hippocampus. Interestingly, epileptiform bursting induced by agonists for metabotropic glutamate receptors in the hippocampal CA1 area is unaltered in TRPC5 KO mice, but is abolished in TRPC1 KO and TRPC1/4 DKO mice. In contrast, long-term potentiation is greatly reduced in TRPC5 KO mice, but is normal in TRPC1 KO and TRPC1/4 DKO mice. The distinct changes from these knockouts suggest that TRPC5 and TRPC1/4 contribute to seizure and excitotoxicity by distinct cellular mechanisms. Furthermore, the reduced seizure and excitotoxicity and normal spatial learning exhibited in TRPC5 KO mice suggest that TRPC5 is a promising novel molecular target for new therapy.

Spinal muscular atrophy: an update on therapeutic progress.

Humans have two nearly identical copies of survival motor neuron gene: SMN1 and SMN2. Deletion or mutation of SMN1 combined with the inability of SMN2 to compensate for the loss of SMN1 results in spinal muscular atrophy (SMA), a leading genetic cause of infant mortality. SMA affects 1 in ~6000 live births, a frequency much higher than in several genetic diseases. The major known defect of SMN2 is the predominant exon 7 skipping that leads to production of a truncated protein (SMNΔ7), which is unstable. Therefore, SMA has emerged as a model genetic disorder in which almost the entire disease population could be linked to the aberrant splicing of a single exon (i.e. SMN2 exon 7). Diverse treatment strategies aimed at improving the function of SMN2 have been envisioned. These strategies include, but are not limited to, manipulation of transcription, correction of aberrant splicing and stabilization of mRNA, SMN and SMNΔ7. This review summarizes up to date progress and promise of various in vivo studies reported for the treatment of SMA.

Diverse role of survival motor neuron protein

The multifunctional Survival Motor Neuron (SMN) protein is required for the survival of all organisms of the animal kingdom. SMN impacts various aspects of RNA metabolism through the formation and/or interaction with ribonucleoprotein (RNP) complexes. SMN regulates biogenesis of small nuclear RNPs, small nucleolar RNPs, small Cajal body-associated RNPs, signal recognition particles and telomerase. SMN also plays an important role in DNA repair, transcription, pre-mRNA splicing, histone mRNA processing, translation, selenoprotein synthesis, macromolecular trafficking, stress granule formation, cell signaling and cytoskeleton maintenance. The tissue-specific requirement of SMN is dictated by the variety and the abundance of its interacting partners. Reduced expression of SMN causes spinal muscular atrophy (SMA), a leading genetic cause of infant mortality. SMA displays a broad spectrum ranging from embryonic lethality to an adult onset. Aberrant expression and/or localization of SMN has also been associated with male infertility, inclusion body myositis, amyotrophic lateral sclerosis and osteoarthritis. This review provides a summary of various SMN functions with implications to a better understanding of SMA and other pathological conditions.

Antisense oligonucleotide mediated therapy of spinal muscular atrophy

Spinal muscular atrophy (SMA) is the leading genetic cause of infant mortality. SMA results from deletions or mutations of survival motor neuron 1 (SMN1), an essential gene. SMN2, a nearly identical copy, can compensate for SMN1 loss if SMN2 exon 7 skipping is prevented. Among the many cis-elements involved in the splicing regulation of SMN exon 7, intronic splicing silencer N1 (ISS-N1) has emerged as the most effective target for an antisense oligonucleotide (ASO)-mediated splicing correction of SMN2 exon 7. Blocking of ISS-N1 by an ASO has been shown to fully restore SMN2 exon 7 inclusion in SMA patient cells as well as in vivo. Here we review how ISS-N1 targeting ASOs that use different chemistries respond differently in the various SMA mouse models. We also compare other ASO-based strategies for therapeutic splicing correction in SMA. Given that substantial progress on ASO-based strategies to promote SMN2 exon 7 inclusion in SMA has been made, and that similar approaches in a growing number of genetic diseases are possible, this report has wide implications.

A Short Antisense Oligonucleotide Ameliorates Symptoms of Severe Mouse Models of Spinal Muscular Atrophy

Recent reports underscore the unparalleled potential of antisense-oligonucleotide (ASO)-based approaches to ameliorate various pathological conditions. However, in vivo studies validating the effectiveness of a short ASO (<10-mer) in the context of a human disease have not been performed. One disease with proven amenability to ASO-based therapy is spinal muscular atrophy (SMA). SMA is a neuromuscular disease caused by loss-of-function mutations in the survival motor neuron 1 (SMN1) gene. Correction of aberrant splicing of the remaining paralog, SMN2, can rescue mouse models of SMA. Here, we report the therapeutic efficacy of an 8-mer ASO (3UP8i) in two severe models of SMA. While 3UP8i modestly improved survival and function in the more severe Taiwanese SMA model, it dramatically increased survival, improved neuromuscular junction pathology, and tempered cardiac deficits in a new, less severe model of SMA. Our results expand the repertoire of ASO-based compounds for SMA therapy, and for the first time, demonstrate the in vivo efficacy of a short ASO in the context of a human disease.

Advances in therapeutic development for spinal muscular atrophy

Spinal muscular atrophy (SMA) is a leading genetic cause of infant mortality. The disease originates from low levels of SMN protein due to deletion and/or mutations of SMN1 coupled with the inability of SMN2 to compensate for the loss of SMN1. While SMN1 and SMN2 are nearly identical, SMN2 predominantly generates a truncated protein (SMNΔ7) due to skipping of exon 7, the last coding exon. Several avenues for SMA therapy are being explored, including means to enhance SMN2 transcription, correct SMN2 exon 7 splicing, stabilize SMN/SMNΔ7 protein, manipulate SMN-regulated pathways and SMN1 gene delivery by viral vectors. This review focuses on the aspects of target discovery, validations and outcome measures for a promising therapy of SMA.

Severe impairment of male reproductive organ development in a low SMN expressing mouse model of spinal muscular atrophy

Spinal muscular atrophy (SMA) is caused by low levels of survival motor neuron (SMN), a multifunctional protein essential for higher eukaryotes. While SMN is one of the most scrutinized proteins associated with neurodegeneration, its gender-specific role in vertebrates remains unknown. We utilized a mild SMA model (C/C model) to examine the impact of low SMN on growth and development of mammalian sex organs. We show impaired testis development, degenerated seminiferous tubules, reduced sperm count and low fertility in C/C males, but no overt sex organ phenotype in C/C females. Underscoring an increased requirement for SMN expression, wild type testis showed extremely high levels of SMN protein compared to other tissues. Our results revealed severe perturbations in pathways critical to C/C male reproductive organ development and function, including steroid biosynthesis, apoptosis, and spermatogenesis. Consistent with enhanced apoptosis in seminiferous tubules of C/C testes, we recorded a drastic increase in cells with DNA fragmentation. SMN was expressed at high levels in adult C/C testis due to an adult-specific splicing switch, but could not compensate for low levels during early testicular development. Our findings uncover novel hallmarks of SMA disease progression and link SMN to general male infertility.

Lectican proteoglycans, their cleaving metalloproteinases, and plasticity in the central nervous system extracellular microenvironment.

The extracellular matrix (ECM) in the central nervous system actively orchestrates and modulates changes in neural structure and function in response to experience, after injury, during disease, and with changes in neuronal activity. A component of the multi-protein, ECM aggregate in brain, the chondroitin sulfate (CS)-bearing proteoglycans (PGs) known as lecticans, inhibit neurite outgrowth, alter dendritic spine shape, elicit closure of critical period plasticity, and block target reinnervation and functional recovery after injury as the major component of a glial scar. While removal of the CS chains from lecticans with chondroitinase ABC improves plasticity, proteolytic cleavage of the lectican core protein may change the conformation of the matrix aggregate and also modulate neural plasticity. This review centers on the roles of the lecticans and the endogenous metalloproteinase families that proteolytically cleave lectican core proteins, the matrix metalloproteinases (MMPs) and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTSs), in neural plasticity. These extracellular metalloproteinases modulate structural neural plasticity-including changes in neurite outgrowth and dendritic spine remodeling-and synaptic plasticity. Some of these actions have been demonstrated to occur via cleavage of the PG core protein. Other actions of the proteases include cleavage of non-matrix substrate proteins, whereas still other actions may occur directly at the cell surface without proteolytic cleavage. The data convincingly demonstrate that metalloproteinases modulate physiological and pathophysiological neural plasticity.

How the discovery of ISS-N1 led to the first medical therapy for spinal muscular atrophy

Spinal muscular atrophy (SMA), a prominent genetic disease of infant mortality, is caused by low levels of survival motor neuron (SMN) protein owing to deletions or mutations of the SMN1 gene. SMN2, a nearly identical copy of SMN1 present in humans, cannot compensate for the loss of SMN1 because of predominant skipping of exon 7 during pre-mRNA splicing. With the recent US Food and Drug Administration approval of nusinersen (Spinraza), the potential for correction of SMN2 exon 7 splicing as an SMA therapy has been affirmed. Nusinersen is an antisense oligonucleotide that targets intronic splicing silencer N1 (ISS-N1) discovered in 2004 at the University of Massachusetts Medical School. ISS-N1 has emerged as the model target for testing the therapeutic efficacy of antisense oligonucleotides using different chemistries as well as different mouse models of SMA. Here, we provide a historical account of events that led to the discovery of ISS-N1 and describe the impact of independent validations that raised the profile of ISS-N1 as one of the most potent antisense targets for the treatment of a genetic disease. Recent approval of nusinersen provides a much-needed boost for antisense technology that is just beginning to realize its potential. Beyond treating SMA, the ISS-N1 target offers myriad potentials for perfecting various aspects of the nucleic-acid-based technology for the amelioration of the countless number of pathological conditions.

ADAMTS expression and function in central nervous system injury and disorders.

The components of the adult extracellular matrix in the central nervous system form a lattice-like structure that is deposited as perineuronal nets, around axon initial segments and as synapse-associated matrix. An abundant component of this matrix is the lecticans, chondroitin sulfate-bearing proteoglycans that are the major substrate for several members of the ADAMTSs (a disintegrin and metalloproteinase with thrombospondin motifs) family. Since lecticans are key regulators of neural plasticity, ADAMTS cleavage of lecticans would likely also contribute to neuroplasticity. Indeed, many studies have examined the neuroplastic contribution of the ADAMTSs to damage and recovery after injury and in central nervous system disease. Much of this data supports a role for the ADAMTSs in recovery and repair following spinal cord injury by stimulating axonal outgrowth after degradation of a glial scar and improving synaptic plasticity following seizure-induced neural damage in the brain. The action of the ADAMTSs in chronic diseases of the central nervous system appears to be more complex and less well-defined. Increasing evidence indicates that lecticans participate in synaptic plasticity in neurodegenerative disease states. It will be interesting to examine how ADAMTS expression and action would affect the progression of these diseases.