Major histocompatibility complex class I molecules protect motor neurons from astrocyte-induced toxicity in amyotrophic lateral sclerosis.

Astrocytes isolated from individuals with amyotrophic lateral sclerosis (ALS) are toxic to motor neurons (MNs) and play a non-cell autonomous role in disease pathogenesis. The mechanisms underlying the susceptibility of MNs to cell death remain unclear. Here we report that astrocytes derived from either mice bearing mutations in genes associated with ALS or human subjects with ALS reduce the expression of major histocompatibility complex class I (MHCI) molecules on MNs; reduced MHCI expression makes these MNs susceptible to astrocyte-induced cell death. Increasing MHCI expression on MNs increases survival and motor performance in a mouse model of ALS and protects MNs against astrocyte toxicity. Overexpression of a single MHCI molecule, HLA-F, protects human MNs from ALS astrocyte-mediated toxicity, whereas knockdown of its receptor, the killer cell immunoglobulin-like receptor KIR3DL2, on human astrocytes results in enhanced MN death. Thus, our data indicate that, in ALS, loss of MHCI expression on MNs renders them more vulnerable to astrocyte-mediated toxicity.

Microglia induce motor neuron death via the classical NF-κB pathway in amyotrophic lateral sclerosis.

Neuroinflammation is one of the most striking hallmarks of amyotrophic lateral sclerosis (ALS). Nuclear factor-kappa B (NF-κB), a master regulator of inflammation, is upregulated in spinal cords of ALS patients and SOD1-G93A mice. In this study, we show that selective NF-κB inhibition in ALS astrocytes is not sufficient to rescue motor neuron (MN) death. However, the localization of NF-κB activity and subsequent deletion of NF-κB signaling in microglia rescued MNs from microglial-mediated death in vitro and extended survival in ALS mice by impairing proinflammatory microglial activation. Conversely, constitutive activation of NF-κB selectively in wild-type microglia induced gliosis and MN death in vitro and in vivo. Taken together, these data provide a mechanism by which microglia induce MN death in ALS and suggest a novel therapeutic target that can be modulated to slow the progression of ALS and possibly other neurodegenerative diseases by which microglial activation plays a role.

A single administration of morpholino antisense oligomer rescues spinal muscular atrophy in mouse.

Spinal muscular atrophy (SMA) is an autosomal-recessive disorder characterized by α-motor neuron loss in the spinal cord anterior horn. SMA results from deletion or mutation of the Survival Motor Neuron 1 gene (SMN1) and retention of SMN2. A single nucleotide difference between SMN1 and SMN2 results in exclusion of exon 7 from the majority of SMN2 transcripts, leading to decreased SMN protein levels and development of SMA. A series of splice enhancers and silencers regulate incorporation of SMN2 exon 7; these splice motifs can be blocked with antisense oligomers (ASOs) to alter SMN2 transcript splicing. We have evaluated a morpholino (MO) oligomer against ISS-N1 [HSMN2Ex7D(-10,-29)], and delivered this MO to postnatal day 0 (P0) SMA pups (Smn-/-, SMN2+/+, SMNΔ7+/+) by intracerebroventricular (ICV) injection. Survival was increased markedly from 15 days to >100 days. Delayed CNS MO injection has moderate efficacy, and delayed peripheral injection has mild survival advantage, suggesting that early CNS ASO administration is essential for SMA therapy consideration. ICV treatment increased full-length SMN2 transcript as well as SMN protein in neural tissue, but only minimally in peripheral tissue. Interval analysis shows a decrease in alternative splice modification over time. We suggest that CNS increases of SMN will have a major impact on SMA, and an early increase of the SMN level results in correction of motor phenotypes. Finally, the early introduction by intrathecal delivery of MO oligomers is a potential treatment for SMA patients.

Systemic gene delivery in large species for targeting spinal cord, brain, and peripheral tissues for pediatric disorders.

Adeno-associated virus type 9 (AAV9) is a powerful tool for delivering genes throughout the central nervous system (CNS) following intravenous injection. Preclinical results in pediatric models of spinal muscular atrophy (SMA) and lysosomal storage disorders provide a compelling case for advancing AAV9 to the clinic. An important translational step is to demonstrate efficient CNS targeting in large animals at various ages. In the present study, we tested systemically injected AAV9 in cynomolgus macaques, administered at birth through 3 years of age for targeting CNS and peripheral tissues. We show that AAV9 was efficient at crossing the blood-brain barrier (BBB) at all time points investigated. Transgene expression was detected primarily in glial cells throughout the brain, dorsal root ganglia neurons and motor neurons within the spinal cord, providing confidence for translation to SMA patients. Systemic injection also efficiently targeted skeletal muscle and peripheral organs. To specifically target the CNS, we explored AAV9 delivery to cerebrospinal fluid (CSF). CSF injection efficiently targeted motor neurons, and restricted gene expression to the CNS, providing an alternate delivery route and potentially lower manufacturing requirements for older, larger patients. Our findings support the use of AAV9 for gene transfer to the CNS for disorders in pediatric populations.

Early heart failure in the SMNDelta7 model of spinal muscular atrophy and correction by postnatal scAAV9-SMN delivery.

Proximal spinal muscular atrophy (SMA) is a debilitating neurological disease marked by isolated lower motor neuron death and subsequent atrophy of skeletal muscle. Historically, SMA pathology was thought to be limited to lower motor neurons and the skeletal muscles they control, yet there are several reports describing the coincidence of cardiovascular abnormalities in SMA patients. As new therapies for SMA emerge, it is necessary to determine whether these non-neuromuscular systems need to be targeted. Therefore, we have characterized left ventricular (LV) function of SMA mice (SMN2+/+; SMNΔ7+/+; Smn-/-) and compared it with that of their unaffected littermates at 7 and 14 days of age. Anatomical and physiological measurements made by electrocardiogram and echocardiography show that affected mouse pups have a dramatic decrease in cardiac function. At 14 days of age, SMA mice have bradycardia and develop a marked dilated cardiomyopathy with a concomitant decrease in contractility. Signs of decreased cardiac function are also apparent as early as 7 days of age in SMA animals. Delivery of a survival motor neuron-1 transgene using a self-complementary adeno-associated virus serotype 9 abolished the symptom of bradycardia and significantly decreased the severity of the heart defect. We conclude that severe SMA animals have compromised cardiac function resulting at least partially from early bradycardia, which is likely attributable to aberrant autonomic signaling. Further cardiographic studies of human SMA patients are needed to clarify the clinical relevance of these findings from this SMA mouse.

Rescue of the spinal muscular atrophy phenotype in a mouse model by early postnatal delivery of SMN.

Spinal muscular atrophy (SMA), the most common autosomal recessive neurodegenerative disease affecting children, results in impaired motor neuron function. Despite knowledge of the pathogenic role of decreased survival motor neuron (SMN) protein levels, efforts to increase SMN have not resulted in a treatment for patients. We recently demonstrated that self-complementary adeno-associated virus 9 (scAAV9) can infect approximately 60% of motor neurons when injected intravenously into neonatal mice. Here we use scAAV9-mediated postnatal day 1 vascular gene delivery to replace SMN in SMA pups and rescue motor function, neuromuscular physiology and life span. Treatment on postnatal day 5 results in partial correction, whereas postnatal day 10 treatment has little effect, suggesting a developmental period in which scAAV9 therapy has maximal benefit. Notably, we also show extensive scAAV9-mediated motor neuron transduction after injection into a newborn cynomolgus macaque. This demonstration that scAAV9 traverses the blood-brain barrier in a nonhuman primate emphasizes the clinical potential of scAAV9 gene therapy for SMA.