Heterogeneous splicing patterns resulting from KIF5A variants associated with amyotrophic lateral sclerosis.

Single-nucleotide variants (SNVs) in the gene encoding Kinesin Family Member 5A (KIF5A), a neuronal motor protein involved in anterograde transport along microtubules, have been associated with amyotrophic lateral sclerosis (ALS). ALS is a rapidly progressive and fatal neurodegenerative disease that primarily affects the motor neurons. Numerous ALS-associated KIF5A SNVs are clustered near the splice-site junctions of the penultimate exon 27 and are predicted to alter the carboxy-terminal (C-term) cargo-binding domain of KIF5A. Mis-splicing of exon 27, resulting in exon exclusion, is proposed to be the mechanism by which these SNVs cause ALS. Whether all SNVs proximal to exon 27 result in exon exclusion is unclear. To address this question, we designed an in vitro minigene splicing assay in human embryonic kidney 293 cells, which revealed heterogeneous site-specific effects on splicing: only 5' splice-site (5'ss) SNVs resulted in exon skipping. We also quantified splicing in select clustered, regularly interspaced, short palindromic repeats-edited human stem cells, differentiated to motor neurons, and in neuronal tissues from a 5'ss SNV knock-in mouse, which showed the same result. Moreover, the survival of representative 3' splice site, 5'ss, and truncated C-term variant KIF5A (v-KIF5A) motor neurons was severely reduced compared with wild-type motor neurons, and overt morphological changes were apparent. While the total KIF5A mRNA levels were comparable across the cell lines, the total KIF5A protein levels were decreased for v-KIF5A lines, suggesting an impairment of protein synthesis or stability. Thus, despite the heterogeneous effect on ribonucleic acid splicing, KIF5A SNVs similarly reduce the availability of the KIF5A protein, leading to axonal transport defects and motor neuron pathology.

Impaired motor unit recovery and maintenance in a knock-in mouse model of ALS-associated Kif5a variant.

Kinesin family member 5A (KIF5A) is an essential, neuron-specific microtubule-associated motor protein responsible for the anterograde axonal transport of various cellular cargos. Loss of function variants in the N-terminal, microtubule-binding domain are associated with hereditary spastic paraplegia and hereditary motor neuropathy. These variants result in a loss of the ability of the mutant protein to process along microtubules. Contrastingly, gain of function splice-site variants in the C-terminal, cargo-binding domain of KIF5A are associated with amyotrophic lateral sclerosis (ALS), a neurodegenerative disease involving death of upper and lower motor neurons, ultimately leading to degradation of the motor unit (MU; an alpha motor neuron and all the myofibers it innervates) and death. These ALS-associated variants result in loss of autoinhibition, increased procession of the mutant protein along microtubules, and altered cargo binding. To study the molecular and cellular consequences of ALS-associated variants in vivo, we introduced the murine homolog of an ALS-associated KIF5A variant into C57BL/6 mice using CRISPR-Cas9 gene editing which produced mutant Kif5a mRNA and protein in neuronal tissues of heterozygous (Kif5a+/c.3005+1G>A; HET) and homozygous (Kif5ac.3005+1G>A/c.3005+1G>A; HOM) mice. HET and HOM mice appeared normal in behavioral and electrophysiological (compound muscle action potential [CMAP] and MU number estimation [MUNE]) outcome measures at one year of age. When subjected to sciatic nerve injury, HET and HOM mice have delayed and incomplete recovery of the MUNE compared to wildtype (WT) mice suggesting an impairment in MU repair. Moreover, aged mutant Kif5a mice (aged two years) had reduced MUNE independent of injury, and exacerbation of the delayed and incomplete recovery after injury compared to aged WT mice. These data suggest that ALS-associated variants may result in an impairment of the MU to respond to biological challenges such as injury and aging, leading to a failure of MU repair and maintenance. In this report, we present the behavioral, electrophysiological and pathological characterization of mice harboring an ALS-associated Kif5a variant to understand the functional consequences of KIF5A C-terminal variants in vivo.

Continual cerebrospinal fluid sampling in the neonatal domestic piglet for biomarker and discovery studies.

BACKGROUND: Longitudinal access to cerebrospinal fluid (CSF) is useful for biomarker discovery in neurological disorders or diseases affecting CSF composition. Here, we aim to test a new method for insertion of a permanent intrathecal catheter, facilitating longitudinal collection of CSF. NEW METHOD: We surgically placed a permanent intrathecal catheter into the cisterna magna of anesthetized neonatal piglets. The thecal sac was accessed at the L5-S1 spinal level and a radiopaque catheter was inserted under fluoroscopic x-ray guidance to position the tip at the cisterna magna. A titanium access port was connected to the catheter and anchored subcutaneously. Immediately after surgery, we confirmed CSF flow through the catheter and port via needle aspiration. Catheter patency over a two-month study period was determined through periodic CSF collection from the port. RESULTS: Frequent (up to 3 times weekly), longitudinal sampling of CSF was achievable in neonatal piglets up to 60 days after implantation. CSF was readily accessible through the port without major adverse events. Catheterized piglets demonstrated slower, but normal, weight gain compared to control piglets. Post-operative complications were managed with standard access precautions and medications. There were no complications involving the implanted hardware. COMPARISON WITH EXISTING METHOD(S): This method fills a critical gap in the existing methods for longitudinal CSF sampling through an implanted intrathecal catheter system in neonatal piglets. CONCLUSIONS: This novel method is both safe and effective for longitudinal CSF access in the domestic piglet. Catheter patency and access to CSF is maintained over multiple months without major adverse events.

Neurofilament Levels in CSF and Serum in an Adult SMA Cohort Treated with Nusinersen.

OBJECTIVE: To retrospectively evaluate the utility of serum and cerebrospinal fluid (CSF) levels of neurofilament light chain (NfL) and phosphorylated neurofilament heavy chain (pNfH) as biomarkers for spinal muscular atrophy (SMA) progression and response to nusinersen treatment. METHODS: NfL and pNfH levels were quantified using single molecular array (SIMOA) in CSF of 33 adult SMA patients (SMN copy number 3-5) before and in response to nusinersen treatment. In 11 of the patients, blood serum samples were also collected. CSF NfL and pNfH from patients were compared to CSF Nfs from age-matched controls without neurological disease (n = 6). For patients, pearson correlation coefficients (r) were calculated to investigate associations between Nf levels and other functional outcome measures. RESULTS: Nf levels were similar between SMA and control adults and showed no change in response to nusinersen treatment in CSF or serum. Cross-sectional analyses showed an increase in CSF NfL and pNfH with age in patients (NfL p = 0.0013; pNfH p = 0.0035) and an increase in CSF NfL in controls (p = 0.002). In non-ambulatory patients, baseline serum pNfH showed a negative correlation with multiple strength and functional assessment metrics including Revised Upper Limb Module (r = -0.822, p = 0.04), upper extremity strength (r = -0.828, p = 0.042), lower extremity strength (r = -0.860, p = 0.028), and total strength (r = -0.870, p = 0.024). CONCLUSIONS: Nf levels did not change in response to nusinersen in adults with SMA and were not different from controls. In patients and controls, we detected an age-related increase in baseline CSF NfL and pNfH levels. Though some associations were identified, our results suggest Nf levels are not preditive or prognostic biomarkers in this population.

Premature delivery in the domestic sow in response to in utero delivery of AAV9 to fetal piglets.

Numerous pediatric neurogenetic diseases may be optimally treated by in utero gene therapy (IUGT); but advancing such treatments requires animal models that recapitulate developmental physiology relevant to humans. One disease that could benefit from IUGT is the autosomal recessive motor neuron disease spinal muscular atrophy (SMA). Current SMA gene-targeting therapeutics are more efficacious when delivered shortly after birth, however postnatal treatment is rarely curative in severely affected patients. IUGT may provide benefit for SMA patients. In previous studies, we developed a large animal porcine model of SMA using AAV9 to deliver a short hairpin RNA (shRNA) directed at porcine survival motor neuron gene (Smn) mRNA on postnatal day 5. Here, we aimed to model developmental features of SMA in fetal piglets and to demonstrate the feasibility of prenatal gene therapy by delivering AAV9-shSmn in utero. Saline (sham), AAV9-GFP, or AAV9-shSmn was injected under direct ultrasound guidance between gestational ages 77-110 days. We developed an ultrasound-guided technique to deliver virus under direct visualization to mimic the clinic setting. Saline injection was tolerated and resulted in viable, healthy piglets. Litter rejection occurred within seven days of AAV9 injection for all other rounds. Our real-world experience of in utero viral delivery followed by AAV9-related fetal rejection suggests that the domestic sow may not be a viable model system for preclinical in utero AAV9 gene therapy studies.

Update on Biomarkers in Spinal Muscular Atrophy.

The availability of disease modifying therapies for spinal muscular atrophy (SMA) has created an urgent need to identify clinically meaningful biomarkers. Biomarkers present a means to measure and evaluate neurological disease across time. Changes in biomarkers provide insight into disease progression and may reveal biologic, physiologic, or pharmacologic phenomena occurring prior to clinical detection. Efforts to identify biomarkers for SMA, a genetic motor neuron disease characterized by motor neuron degeneration and weakness, have culminated in a number of putative molecular and physiologic markers that evaluate biological media (eg, blood and cerebrospinal fluid [CSF]) or nervous system function. Such biomarkers include SMN2 copy number, SMN mRNA and protein levels, neurofilament proteins (NFs), plasma protein analytes, creatine kinase (CK) and creatinine (Crn), and various electrophysiology and imaging measures. SMN2 copy number inversely correlates with disease severity and is the best predictor of clinical outcome in untreated individuals. SMN mRNA and protein are commonly measured in the blood or CSF of patients receiving SMA therapies, particularly those aimed at increasing SMN protein expression, and provide insight into current disease state. NFs have proven to be robust prognostic, disease progression, and pharmacodynamic markers for SMA infants undergoing treatment, but less so for adolescents and adults. Select plasma proteins are altered in SMA individuals and may track response to therapy. CK and Crn from blood correlate with motor function and disease severity status and are useful for predicting which individuals will respond to therapy. Electrophysiology measures comprise the most reliable means for monitoring motor function throughout disease course and are sensitive enough to detect neuromuscular changes before overt clinical manifestation, making them robust predictive and pharmacodynamic biomarkers. Finally, magnetic resonance imaging and muscle ultrasonography are non-invasive techniques for studying muscle structure and physiology and are useful diagnostic tools, but cannot reliably track disease progression. Importantly, biomarkers can provide information about the underlying mechanisms of disease as well as reveal subclinical disease progression, allowing for more appropriate timing and dosing of therapy for individuals with SMA. Recent therapeutic advancements in SMA have shown promising results, though there is still a great need to identify and understand the impact of biomarkers in modulating disease onset and progression.