Noam Y. Harel

Can regenerating axons recapitulate developmental guidance during recovery from spinal cord injury

The precise wiring of the adult mammalian CNS originates during a period of stunning growth, guidance and plasticity that occurs during and shortly after development. When injured in adults, this intricate system fails to regenerate. Even when the obstacles to regeneration are cleared, growing adult CNS fibres usually remain misdirected and fail to reform functional connections. Here, we attempt to fill an important niche related to the topics of nervous system development and regeneration. We specifically contrast the difficulties faced by growing fibres within the adult context to the precise circuit-forming capabilities of developing fibres. In addition to focusing on methods to stimulate growth in the adult, we also expand on approaches to recapitulate development itself.

Subcutaneous Nogo Receptor Removes Brain Amyloid-β and Improves Spatial Memory in Alzheimer's Transgenic Mice

The production and aggregation of cerebral amyloid-β (Aβ) peptide are thought to play a causal role in Alzheimers disease (AD). Previously, we found that the Nogo-66 receptor (NgR) interacts physically with both Aβ and the amyloid precursor protein (APP). The inverse correlation of Aβ levels with NgR levels within the brain may reflect regulation of Aβ production and/or Aβ clearance. Here, we assess the potential therapeutic benefit of peripheral NgR-mediated Aβ clearance in APPswe/PSEN-1ΔE9 transgenic mice. Through site-directed mutagenesis, we demonstrate that the central 15–28 aa of Aβ associate with specific surface-accessible patches on the leucine-rich repeat concave side of the solenoid structure of NgR. In transgenic mice, subcutaneous NgR(310)ecto-Fc treatment reduces brain Aβ plaque load while increasing the relative levels of serum Aβ. These changes in Aβ are correlated with improved spatial memory in the radial arm water maze. The benefits of peripheral NgR administration are evident when therapy is initiated after disease onset. Thus, the peripheral association of NgR(310)ecto-Fc with central Aβ residues provides an effective therapeutic approach for AD.

Reticulon-4A (Nogo-A) Redistributes Protein Disulfide Isomerase to Protect Mice from SOD1-Dependent Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron disease inherited in a small subset of patients. The SOD1(G93A) transgenic mouse models this subset of patients, and studies of this strain have suggested that endoplasmic reticulum (ER) stress and deficits in ER chaperone function are contributors to ALS pathophysiology. Here, we demonstrate that the reticulon family of proteins is a novel regulator of the ER chaperone protein disulfide isomerase (PDI), and that through PDI, reticulon-4A (Nogo-A) can protect mice against the neurodegeneration that characterizes ALS. We show that overexpressing reticulon protein induces a punctate redistribution of PDI intracellularly, both in vitro and in vivo. Conversely, reduction of endogenous NogoA expression causes a more homogeneous expression pattern in vivo. These effects occur without induction of the unfolded protein response. To examine the effect of PDI redistribution on ALS disease progression, we conducted survival and behavior studies of SOD1(G93A) mice. Deletion of a single copy of the NogoA,B gene accelerates disease onset and progression, while deletion of both copies further worsens disease. We conclude that NogoA contributes to the proper function of the ER resident chaperone PDI, and is protective against ALS-like neurodegeneration. Our results provide a novel intracellular role for reticulon proteins and support the hypothesis that modulation of PDI function is a potential therapeutic approach to ALS.

LGI1-associated epilepsy through altered ADAM23-dependent neuronal morphology.

Most epilepsy genes encode ion channels, but the LGI1 gene responsible for autosomal dominant partial epilepsy with auditory features produces a secreted protein. LGI1 is suggested to regulate PSD-95 via ADAM22. However, no unbiased screen of LGI1 action has been conducted. Here, we searched for brain genes supporting high affinity LGI-1 binding. ADAM23 was the only LGI1 interactor identified. The related proteins, ADAM22 and ADAM11, but not ADAM12, bind LGI1. Neither ADAM23 nor ADAM11, nor some forms of ADAM22, contain PDZ-interacting sequences, suggesting PSD-95-independent mechanisms in ADPEAF. Because ADAMs modulate integrins, we examined LGI1 effect on neurite outgrowth. LGI1 increases outgrowth from wild-type but not ADAM23-/- neurons. Furthermore, CA1 pyramidal neurons of ADAM23-/- hippocampi have reduced dendritic arborization. ADAM23-/- mice exhibit spontaneous seizures, while ADAM23+/- mice have decreased seizure thresholds. Thus, LGI1 binding to ADAM23 is necessary to correctly pattern neuronal morphology and altered anatomical patterning contributes to ADPEAF.

Recovery from Chronic Spinal Cord Contusion after Nogo Receptor Intervention

Several interventions promote axonal growth and functional recovery when initiated shortly after central nervous system injury, including blockade of myelin‐derived inhibitors with soluble Nogo receptor (NgR1, RTN4R) decoy protein. We examined the efficacy of this intervention in the much more prevalent and refractory condition of chronic spinal cord injury.

Nogo Receptor Deletion and Multimodal Exercise Improve Distinct Aspects of Recovery in Cervical Spinal Cord Injury

We tested the ability of two plasticity-promoting approaches to enhance recovery in a mouse model of incomplete spinal cord injury (SCI). Genetically, we reduced myelin-mediated inhibition of neural plasticity through Nogo66-receptor (NgR) gene deletion. Behaviorally, we utilized a novel multimodal exercise training paradigm. Adult mice of wild-type or NgR-null genotype were subjected to partial lateral hemisection (LHx) at C3-C4 with the intent of producing anatomically and functionally mild deficits. Exercise training or control treatment proceeded for 14 weeks. Behavioral outcomes were assessed prior to tract tracing and histological analysis. Genotype and training exerted differing effects on performance; training improved performance on a test related to the training regimen (task-specific benefit), whereas genotype also improved performance on more generalized behaviors (task-non-specific benefit). There were no significant histological differences across genotype or training assignment with regard to lesion size or axonal tract staining. Thus either NgR gene deletion or exercise training benefits mice with mild cervical spinal injury. In this lesion model, the effects of NgR deletion and training were not synergistic for the tasks assessed. Further work is required to optimize the interaction between pharmacological and physical interventions for SCI.

Functional MRI and other non-invasive imaging technologies: Providing visual biomarkers for spinal cord structure and function after injury

Substantial progress has been made towards understanding the molecular basis for limited endogenous central nervous system (CNS) axonal growth after injuries such as spinal cord trauma. Realization of the potential benefit of therapeutic interventions requires methods to assess axonal growth and functional reorganization over time after neurological damage. Here, we discuss the technical challenges of analyzing and interpreting the effects of various interventions on CNS repair, specifically in the context of spinal cord injury. Evolving technologies such as functional magnetic resonance imaging and other non-invasive imaging techniques will be reviewed. These technologies should revolutionize our ability to track changes in both CNS structure and function.

Multimodal exercises simultaneously stimulating cortical and brainstem pathways after unilateral corticospinal lesion.

In the context of injury to the corticospinal tract (CST), brainstem-origin circuits may provide an alternative system of descending motor influence. However, subcortical circuits are largely under subconscious control. To improve volitional control over spared fibers after CST injury, we hypothesized that a combination of physical exercises simultaneously stimulating cortical and brainstem pathways above the injury would strengthen corticobulbar connections through Hebbian-like mechanisms. We sought to test this hypothesis in mice with unilateral CST lesions. Ten days after pyramidotomy, mice were randomized to four training groups: (1) postural exercises designed to stimulate brainstem pathways (BS); (2) distal limb-grip exercises preferentially stimulating CST pathways (CST); (3) simultaneous multimodal exercises (BS+CST); or (4) no training (NT). Behavioral and anatomical outcomes were assessed after 20 training sessions over 4 weeks. Mice in the BS+CST training group showed a trend toward greater improvements in skilled limb performance than mice in the other groups. There were no consistent differences between training groups in gait kinematics. Anatomically, multimodal BS+CST training neither increased corticobulbar fiber density of the lesioned CST rostral to the lesion nor collateral sprouting of the unlesioned CST caudal to the lesion. Further studies should incorporate electrophysiological assessment to gauge changes in synaptic strength of direct and indirect pathways between the cortex and spinal cord in response to multimodal exercises.

Serum Nogo-A levels are not elevated in amyotrophic lateral sclerosis patients

Improved biomarkers would facilitate the diagnosis and treatment of amyotrophic lateral sclerosis (ALS). Muscle content of the neuritic outgrowth inhibitor Nogo-A is increased in patients with ALS and other denervating conditions. Seeking a less invasive diagnostic method, we sought to determine whether or not Nogo increases in the serum of ALS patients. We developed a dissociation-enhanced lanthanide fluorescent immunoassay (DELFIA) protocol to screen serum samples from 172 ALS patients and 172 healthy controls for Nogo-A immunoreactivity. Unexpectedly, there was a trend toward decreased levels of serum Nogo-A in ALS. Mean serum Nogo-A level in ALS patients was 0.71 nM (95% confidence interval (CI) 0.42–1.00), as opposed to 1.15 nM (95% CI 0.72–1.59) in healthy controls. A significantly larger percentage of healthy control sera (11.0% vs 4.7%) displayed markedly elevated levels of Nogo-A. Additional study is required to determine the factors that lead to elevated Nogo-A levels in a subset of both ALS patients and healthy controls.

Nogo-A marks motor neuron disease.

More than a century after Charcot’s description of amyotrophic lateral sclerosis (ALS), the diagnosis, monitoring, and treatment of ALS continue to be based almost entirely on clinical examination. Electromyography has become an essential supplementary test for confirming lower motor neuron (LMN) degeneration. Notwithstanding the evolving use of diffusion tensor magnetic resonance imaging and transcranial magnetic stimulation, there are currently no validated molecular markers for ALS or for upper motor neuron pathology in general. In this issue of Annals, Pradat and colleagues add to their growing body of work suggesting that muscle expression of the Nogo-A protein serves as a biomarker for ALS. Nogo-A was identified as a protein expressed on the surface of oligodendrocytes that inhibits axonal outgrowth. Myelin-derived Nogo-A, myelin-associated glycoprotein, and oligodendrocyte myelin glycoprotein all bind to an axonal Nogo-66 receptor and prevent axonal sprouting and regeneration in the adult central nervous system. It has been demonstrated that blocking this inhibitory pathway increases sprouting, plasticity, and regeneration in the brain and spinal cord, with documented efficacy in promoting recovery from spinal cord trauma and stroke in preclinical models. In contrast with the pattern in oligodendrocytes, the expression of Nogo-A by skeletal muscle and by neurons is substantial during development but much lower in the healthy adult. Previous findings by Loeffler and colleagues had shown that muscle Nogo-A expression increases in both humans and a widely used mouse model of ALS, and that Nogo-A muscle levels correlate with severity on the ALS-functional rating scale. In the current study, Pradat and colleagues investigate the utility of Nogo as a biomarker in an important clinical scenario: the prediction of which patients presenting solely with LMN symptoms will progress toward full-blown ALS as opposed to a more benign pathology. Pradat and colleagues examined muscle biopsies from 33 patients who presented with “lower motor neuron syndromes” (LMNSs) that did not meet El Escorial criteria for diagnosis of possible, probable, or definite ALS. Biopsies were probed by immunoblot for Nogo-A. Patients were then clinically followed for a median of 2 to 3 years (or death) by investigators blinded to Nogo-A status. Remarkably, of the 17 patients in whom muscle Nogo-A was detected, 15 progressed to a diagnosis of ALS, whereas only 1 of 16 Nogo-A–negative patients progressed to ALS. These results demonstrate 94% sensitivity and 88% specificity for the prediction of progression from LMNS to ALS. The potential benefits of a reliable biomarker for ALS are multiple. The first is earlier diagnosis. Musclespecific Nogo expression was detected as early as 3 months after LMN symptom onset. Earlier diagnosis allows earlier initiation of treatment (or treatment trials), potentially delaying or even preventing clinical onset of upper motor neuron symptoms. The second benefit is more accurate diagnosis. Together with the findings presented in Pradat and colleagues’ study, this group has previously shown that muscle-specific Nogo-A expression is not merely a consequence of denervation, because denervation under nonALS conditions did not result in increased muscle Nogo-A expression. The third benefit is more accurate tracking of disease progression. A previous article by this group suggested that absolute levels of muscle-specific Nogo-A expression correlate with severity on the ALS-functional rating scale. However, this study by necessity examined muscle biopsies at only one time point per patient, precluding the ability to make firm conclusions regarding Nogo levels and disease progression. Another benefit is insight into ALS pathogenesis. The greatest significance of the current study may eventually come from fresh insights into the pathophysiology of ALS. Nogo-A might play a role in motor neuron disease through one or more mechanisms. Its ability to inhibit axonal sprouting and regeneration in the context of spinal cord injury and stroke has been well-documented. Thus, if abnormally expressed in muscle, Nogo could conceivably limit the ability of surviving motor neurons to form compensatory sprouts to replace lost neighboring motor units. Such a mechanism would imply a role for Nogo-66 receptor in ALS. Indeed, previous work by this group showed that Nogo overexpression in soleus muscle caused retraction of nerve terminals from the neuromuscular junction. Alternatively, Nogo’s membership in the Reticulon family of proteins suggests a potential link to motor neuron disease. Though not as well understood, intracellular Reticulons are thought to play a role in endoplasmic reticulum structure and trafficking. This Reticulon function is cell autonomous and independent of Nogo-66 receptor. Mutations in the endoplasmic reticulum protein VAP-B, as well as the vesicletrafficking factor Alsin, have been associated with familial forms of ALS. Perhaps Nogo plays an asyet-unidentified role in endoplasmic reticulum metabolism that modifies motor neuron pathology. As the patients in the current study lacked a family history of ALS, Nogo may play more of a reactive rather than an initiating role in disease onset. Though Pradat and colleagues’ study establishes Nogo-A as an interesting candidate biomarker for ALS, EDITORIALS