Stathmin-2 loss leads to neurofilament-dependent axonal collapse driving motor and sensory denervation.

The mRNA transcript of the human STMN2 gene, encoding for stathmin-2 protein (also called SCG10), is profoundly impacted by TAR DNA-binding protein 43 (TDP-43) loss of function. The latter is a hallmark of several neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). Using a combination of approaches, including transient antisense oligonucleotide-mediated suppression, sustained shRNA-induced depletion in aging mice, and germline deletion, we show that stathmin-2 has an important role in the establishment and maintenance of neurofilament-dependent axoplasmic organization that is critical for preserving the caliber and conduction velocity of myelinated large-diameter axons. Persistent stathmin-2 loss in adult mice results in pathologies found in ALS, including reduced interneurofilament spacing, axonal caliber collapse that drives tearing within outer myelin layers, diminished conduction velocity, progressive motor and sensory deficits, and muscle denervation. These findings reinforce restoration of stathmin-2 as an attractive therapeutic approach for ALS and other TDP-43-dependent neurodegenerative diseases.

Expandable Sendai-Virus-Reprogrammed Human iPSC-Neuronal Precursors: In Vivo Post-Grafting Safety Characterization in Rats and Adult Pig.

One of the challenges in clinical translation of cell-replacement therapies is the definition of optimal cell generation and storage/recovery protocols which would permit a rapid preparation of cell-treatment products for patient administration. Besides, the availability of injection devices that are simple to use is critical for potential future dissemination of any spinally targeted cell-replacement therapy into general medical practice. Here, we compared the engraftment properties of established human-induced pluripotent stem cells (hiPSCs)-derived neural precursor cell (NPCs) line once cells were harvested fresh from the cell culture or previously frozen and then grafted into striata or spinal cord of the immunodeficient rat. A newly developed human spinal injection device equipped with a spinal cord pulsation-cancelation magnetic needle was also tested for its safety in an adult immunosuppressed pig. Previously frozen NPCs showed similar post-grafting survival and differentiation profile as was seen for freshly harvested cells. Testing of human injection device showed acceptable safety with no detectable surgical procedure or spinal NPCs injection-related side effects.

Derivation of Sendai-Virus-Reprogrammed Human iPSCs-Neuronal Precursors: In Vitro and In Vivo Post-grafting Safety Characterization.

The critical requirements in developing clinical-grade human-induced pluripotent stem cells-derived neural precursors (hiPSCs-NPCs) are defined by expandability, genetic stability, predictable in vivo post-grafting differentiation, and acceptable safety profile. Here, we report on the use of manual-selection protocol for generating expandable and stable human NPCs from induced pluripotent stem cells. The hiPSCs were generated by the reprogramming of peripheral blood mononuclear cells with Sendai-virus (SeV) vector encoding Yamanaka factors. After induction of neural rosettes, morphologically defined NPC colonies were manually harvested, re-plated, and expanded for up to 20 passages. Established NPCs showed normal karyotype, expression of typical NPCs markers at the proliferative stage, and ability to generate functional, calcium oscillating GABAergic or glutamatergic neurons after in vitro differentiation. Grafted NPCs into the striatum or spinal cord of immunodeficient rats showed progressive maturation and expression of early and late human-specific neuronal and glial markers at 2 or 6 months post-grafting. No tumor formation was seen in NPCs-grafted brain or spinal cord samples. These data demonstrate the effective use of in vitro manual-selection protocol to generate safe and expandable NPCs from hiPSCs cells. This protocol has the potential to be used to generate GMP (Good Manufacturing Practice)-grade NPCs from hiPSCs for future clinical use.

Mechanism of STMN2 cryptic splice-polyadenylation and its correction for TDP-43 proteinopathies.

Loss of nuclear TDP-43 is a hallmark of neurodegeneration in TDP-43 proteinopathies, including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). TDP-43 mislocalization results in cryptic splicing and polyadenylation of pre-messenger RNAs (pre-mRNAs) encoding stathmin-2 (also known as SCG10), a protein that is required for axonal regeneration. We found that TDP-43 binding to a GU-rich region sterically blocked recognition of the cryptic 3' splice site in STMN2 pre-mRNA. Targeting dCasRx or antisense oligonucleotides (ASOs) suppressed cryptic splicing, which restored axonal regeneration and stathmin-2-dependent lysosome trafficking in TDP-43-deficient human motor neurons. In mice that were gene-edited to contain human STMN2 cryptic splice-polyadenylation sequences, ASO injection into cerebral spinal fluid successfully corrected Stmn2 pre-mRNA misprocessing and restored stathmin-2 expression levels independently of TDP-43 binding.

Precision spinal gene delivery-induced functional switch in nociceptive neurons reverses neuropathic pain.

Second-order spinal cord excitatory neurons play a key role in spinal processing and transmission of pain signals to the brain. Exogenously induced change in developmentally imprinted excitatory neurotransmitter phenotypes of these neurons to inhibitory has not yet been achieved. Here, we use a subpial dorsal horn-targeted delivery of AAV (adeno-associated virus) vector(s) encoding GABA (gamma-aminobutyric acid) synthesizing-releasing inhibitory machinery in mice with neuropathic pain. Treated animals showed a progressive and complete reversal of neuropathic pain (tactile and brush-evoked pain behavior) that persisted for a minimum of 2.5 months post-treatment. The mechanism of this treatment effect results from the switch of excitatory to preferential inhibitory neurotransmitter phenotype in dorsal horn nociceptive neurons and a resulting increase in inhibitory activity in regional spinal circuitry after peripheral nociceptive stimulation. No detectable side effects (e.g., sedation, motor weakness, loss of normal sensation) were seen between 2 and 13 months post-treatment in naive adult mice, pigs, and non-human primates. The use of this treatment approach may represent a potent and safe treatment modality in patients suffering from spinal cord or peripheral nerve injury-induced neuropathic pain.

The role of spinal cord extrasynaptic α5 GABAA receptors in chronic pain.

Chronic pain is an incapacitating condition that affects a large population worldwide. Until now, there is no drug treatment to relieve it. The impairment of GABAergic inhibition mediated by GABAA receptors (GABAA R) is considered a relevant factor in mediating chronic pain. Even though both synaptic and extrasynaptic GABAA inhibition are present in neurons that process nociceptive information, the latter is not considered relevant as a target for the development of pain treatments. In particular, the extrasynaptic α5 GABAA Rs are expressed in laminae I-II of the spinal cord neurons, sensory neurons, and motoneurons. In this review, we discuss evidence showing that blockade of the extrasynaptic α5 GABAA Rs reduces mechanical allodynia in various models of chronic pain and restores the associated loss of rate-dependent depression of the Hoffmann reflex. Furthermore, in healthy animals, extrasynaptic α5 GABAA R blockade induces both allodynia and hyperalgesia. These results indicate that this receptor may have an antinociceptive and pronociceptive role in healthy and chronic pain-affected animals, respectively. We propose a hypothesis to explain the relevant role of the extrasynaptic α5 GABAA Rs in the processing of nociceptive information. The data discussed here strongly suggest that this receptor could be a valid pharmacological target to treat chronic pain states.

Spinal subpial delivery of AAV9 enables widespread gene silencing and blocks motoneuron degeneration in ALS.

Gene silencing with virally delivered shRNA represents a promising approach for treatment of inherited neurodegenerative disorders. In the present study we develop a subpial technique, which we show in adult animals successfully delivers adeno-associated virus (AAV) throughout the cervical, thoracic and lumbar spinal cord, as well as brain motor centers. One-time injection at cervical and lumbar levels just before disease onset in mice expressing a familial amyotrophic lateral sclerosis (ALS)-causing mutant SOD1 produces long-term suppression of motoneuron disease, including near-complete preservation of spinal α-motoneurons and muscle innervation. Treatment after disease onset potently blocks progression of disease and further α-motoneuron degeneration. A single subpial AAV9 injection in adult pigs or non-human primates using a newly designed device produces homogeneous delivery throughout the cervical spinal cord white and gray matter and brain motor centers. Thus, spinal subpial delivery in adult animals is highly effective for AAV-mediated gene delivery throughout the spinal cord and supraspinal motor centers.

Subpial AAV Delivery for Spinal Parenchymal Gene Regulation in Adult Mammals.

The use of adeno-associated virus (AAV) vectors has become an attractive method for treatment of a variety of neurodegenerative disorders by permitting targeted gene upregulation or silencing in the CNS. Systemic and intrathecal infusion, while preferable routes of vector delivery, have shown encouraging but variable efficacy due to the poor permeability of AAV into spinal cord and brain parenchyma in adult mammals. Recently we have developed a novel and relatively noninvasive technique of spinal subpial vector delivery. This technique confers widespread transgene expression throughout the spinal parenchyma, including both white and gray matter. We have demonstrated that this technique can be performed safely, with a high level of accuracy, and is effective in both small (mouse or rat) and large preclinical (adult pig or nonhuman primate) animal models. In this chapter we provide a comprehensive description of the subpial vector delivery technique in adult rodents (mouse and rat) and large preclinical animals (adult pig and nonhuman primates).

Predominant role of spinal P2Y1 receptors in the development of neuropathic pain in rats.

The role of P2X2/3, P2X3, P2X4 or P2X7 and P2Y2, P2Y6, and P2Y12 receptors in neuropathic pain has been widely studied. In contrast, the role of P2Y1 receptors is scarcely studied. In this study we assessed the role of P2Y1 receptors in several neuropathic pain models in the rat. Furthermore, we analyzed the expression of P2Y1 receptors in the ipsilateral dorsal root ganglia (DRG) and dorsal part of the spinal cord during the development and maintenance of neuropathic pain. We also determined the effect of the P2Y1 receptor antagonist on the expression of P2Y1 receptors. Chronic constriction injury (CCI), spared nerve injury (SNI) or spinal nerve ligation (SNL) produced tactile allodynia from 1 to 14 days after nerve injury. CCI, SNI and SNL enhanced expression of P2Y1 receptors in DRG but not in the dorsal part of the spinal cord at 1-3 days after injury. Intrathecal injection of the selective P2Y1 receptor antagonist MRS2500, but not vehicle, reduced tactile allodynia in rats 1-3 days after CCI, SNI, or SNL. Moreover, intrathecal injection of MRS2500 (at day 1 or 3) reduced neuropathy-induced up-regulation of P2Y1 receptors expression. Intrathecal injection of MRS2500 lost most of the antiallodynic effect when injected 14 days after injury. At this time, MRS2500 did not modify nerve-injury-induced P2Y1 receptors up-regulation. Our results suggest that P2Y1 receptors are localized in DRG, are up-regulated by nerve injury and play a pronociceptive role in development and, to a lesser extent, maintenance of neuropathic pain.

The α5 subunit containing GABAA receptors contribute to chronic pain.

It has been recently proposed that α5-subunit containing GABAA receptors (α5-GABAA receptors) that mediate tonic inhibition might be involved in pain. The purpose of this study was to investigate the contribution of α5-GABAA receptors in the loss of GABAergic inhibition and in formalin-induced, complete Freund's adjuvant (CFA)-induced and L5 and L6 spinal nerve ligation-induced long-lasting hypersensitivity. Formalin or CFA injection and L5 and L6 spinal nerve ligation produced long-lasting allodynia and hyperalgesia. Moreover, formalin injection impaired the rate-dependent depression of the Hofmann reflex. Peripheral and intrathecal pretreatment or post-treatment with the α5-GABAA receptor antagonist, L-655,708 (0.15-15 nmol), prevented and reversed, respectively, these long-lasting behaviors. Formalin injection increased α5-GABAA receptor mRNA expression in the spinal cord and dorsal root ganglia (DRG) mainly at 3 days. The α5-GABAA receptors were localized in the dorsal spinal cord and DRG colabeling with NeuN, CGRP, and IB4 which suggests their presence in peptidergic and nonpeptidergic neurons. These receptors were found mainly in small and medium sized neurons. Formalin injection enhanced α5-GABAA receptor fluorescence intensity in spinal cord and DRG at 3 and 6 days. Intrathecal administration of L-655,708 (15 nmol) prevented and reversed formalin-induced impairment of rate-dependent depression. These results suggest that α5-GABAA receptors play a role in the loss of GABAergic inhibition and contribute to long-lasting secondary allodynia and hyperalgesia.

Thoracic 9 Spinal Transection-Induced Model of Muscle Spasticity in the Rat: A Systematic Electrophysiological and Histopathological Characterization.

The development of spinal hyper-reflexia as part of the spasticity syndrome represents one of the major complications associated with chronic spinal traumatic injury (SCI). The primary mechanism leading to progressive appearance of muscle spasticity is multimodal and may include loss of descending inhibitory tone, alteration of segmental interneuron-mediated inhibition and/or increased reflex activity to sensory input. Here, we characterized a chronic thoracic (Th 9) complete transection model of muscle spasticity in Sprague-Dawley (SD) rats. Isoflurane-anesthetized rats received a Th9 laminectomy and the spinal cord was transected using a scalpel blade. After the transection the presence of muscle spasticity quantified as stretch and cutaneous hyper-reflexia was identified and quantified as time-dependent changes in: i) ankle-rotation-evoked peripheral muscle resistance (PMR) and corresponding electromyography (EMG) activity, ii) Hoffmann reflex, and iii) EMG responses in gastrocnemius muscle after paw tactile stimulation for up to 8 months after injury. To validate the clinical relevance of this model, the treatment potency after systemic treatment with the clinically established anti-spastic agents baclofen (GABAB receptor agonist), tizanidine (α2-adrenergic agonist) and NGX424 (AMPA receptor antagonist) was also tested. During the first 3 months post spinal transection, a progressive increase in ankle rotation-evoked muscle resistance, Hoffmann reflex amplitude and increased EMG responses to peripherally applied tactile stimuli were consistently measured. These changes, indicative of the spasticity syndrome, then remained relatively stable for up to 8 months post injury. Systemic treatment with baclofen, tizanidine and NGX424 led to a significant but transient suppression of spinal hyper-reflexia. These data demonstrate that a chronic Th9 spinal transection model in adult SD rat represents a reliable experimental platform to be used in studying the pathophysiology of chronic spinal injury-induced spasticity. In addition a consistent anti-spastic effect measured after treatment with clinically effective anti-spastic agents indicate that this model can effectively be used in screening new anti-spasticity compounds or procedures aimed at modulating chronic spinal trauma-associated muscle spasticity.

Role of spinal P2Y6 and P2Y11 receptors in neuropathic pain in rats: possible involvement of glial cells.

BACKGROUND: The participation of spinal P2X receptors in neuropathic pain is well recognized. However, the role of P2Y receptors has been less studied. The purpose of this study was to investigate the contribution of spinal P2Y6,11 receptors following peripheral nerve damage induced by spinal nerve ligation. In addition, we determined the expression of P2Y6,11 receptors in the dorsal spinal cord in presence of the selective P2Y6,11 receptors antagonists. Furthermore, we evaluated the participation of spinal microglia and astrocytes in the pronociceptive role of P2Y6,11 receptors. RESULTS: Spinal administration of the selective P2Y6 (MRS2578, 10-100 µM) and P2Y11 (NF340, 0.3-30 µM) receptor antagonists reduced tactile allodynia in spinal nerve ligated rats. Nerve injury increased the expression of P2Y6,11 receptors at 7, 14 and 21 days after injury. Furthermore, intrathecal administration of MRS2578 (100 µM/day) and NF340 (30 µM/day) for 3 days significantly reduced spinal nerve injury-induced increase in P2Y6,11 receptors expression, respectively. Spinal treatment (on day 14 after injury) with minocycline (100 µg/day) or fluorocitrate (1 nmol/day) for 7 days reduced tactile allodynia and spinal nerve injury-induced up-regulation in Iba-1 and GFAP, respectively. In addition, minocycline reduced nerve injury-induced up-regulation in P2Y6,11 receptors whereas that fluorocitrate diminished P2Y11, but not P2Y6, receptors up-regulation. Intrathecal treatment (on day 21 after injury) with the selective P2Y6 (PSB0474, 3-30 µM) and P2Y11 (NF546, 1-10 µM) receptor agonists produced remarkable tactile allodynia in nerve ligated rats previously treated with minocycline or fluorocitrate for 7 days. CONCLUSIONS: Our data suggest that spinal P2Y6 is present in spinal microglia while P2Y11 receptors are present in both spinal microglia and astrocytes, and both receptors are up-regulated in rats subjected to spinal nerve injury. In addition, our data suggest that the spinal P2Y6 and P2Y11 receptors participate in the maintenance of neuropathic pain.

Evidence for the participation of peripheral α5 subunit-containing GABAA receptors in GABAA agonists-induced nociception in rats.

The activation of GABAA receptor by γ-amino butyric acid (GABA) in primary afferent fibers produces depolarization. In normal conditions this depolarization causes a reduction in the release of neurotransmitters. Therefore, this depolarization remains inhibitory. However, previous studies have suggested that in inflammatory pain, GABA shifts its signaling from inhibition to excitation by an increased GABA-induced depolarization. The contribution of peripheral α5 subunit-containing GABAA receptors to the inflammatory pain is unknown. The purpose of this study was to investigate the possible pronociceptive role of peripheral α5 subunit-containing GABAA receptors in the formalin test. Formalin (0.5%) injection into the dorsum of the right hind paw produced flinching behavior in rats. Ipsilateral local peripheral pre-treatment (-10min) with exogenous GABA (0.003-0.03µg/paw) or common GABAA receptor agonists muscimol (0.003-0.03µg/paw), diazepam (0.017-0.056µg/paw) or phenobarbital (1-100µg/paw) significantly increased 0.5% formalin-induced nociceptive behavior. The pronociceptive effects of GABA (0.03µg/paw), muscimol (0.03µg/paw), diazepam (0.056µg/paw) and phenobarbital (100µg/paw) were prevented by either the GABAA receptor antagonist bicuculline (0.01-0.1µg/paw) or selective α5 subunit-containing GABAA receptor inverse agonist L-655,708 (0.017-0.17µg/paw). The α5 subunit-containing GABAA receptor protein was expressed in dorsal root ganglion (DRG) and dorsal spinal cord of naïve rats. The formalin injection did not modify α5 subunit-containing GABAA receptor expression. Overall, these results suggest that peripheral α5 subunit-containing GABAA receptors play a pronociceptive role in the rat formalin test.

Spinal 5-HT5A receptors mediate 5-HT-induced antinociception in several pain models in rats.

The antinociceptive role of spinal 5-HT5A receptors in rat models of pain along with their expression was evaluated in the spinal cord and dorsal root ganglion (DRG). Nociception was assessed in the formalin, capsaicin, and acetic acid writhing tests. The expression of 5-HT5A receptors was determined by Western blot analysis. Intrathecal treatment with serotonin (5-HT, 10-100 nmol) or 5-carboxamidotryptamine (5-CT, 0.03-0.3 nmol) dose-dependently prevented 1% formalin-induced nociception. Furthermore, 5-HT reduced capsaicin- and acetic acid-induced nociception. 5-HT- or 5-CT-induced antinociception in the formalin test was diminished by the selective 5-HT5A receptor antagonist N-[2-(dimethylamino)ethyl]-N-[[4'-[[(2-phenylethyl)amino] methyl][1,1'-biphenyl]-4-yl]methyl]cyclopentanepropanamide dihydrochloride (SB-699551; 3 and 10 nmol). In addition, 5-HT-induced spinal antinociception in the capsaicin and acetic acid tests was blocked by SB-699551 (10 nmol). Given alone, intrathecal injection of SB-699551 did not affect nociception induced by any irritant. 5-HT5A receptors were expressed in the dorsal spinal cord and DRG, even though formalin injection increased after 24h 5-HT5A receptor expression only in the spinal cord. Data suggest that 5-HT and 5-CT produce antinociception by activation of spinal 5-HT5A receptors in both the spinal cord and DRG. Furthermore, our results suggest that spinal 5-HT5A receptors play an antinociceptive role in several pain models in rats. 5-HT5A receptors may provide a therapeutic target to develop analgesic drugs.

Role of peripheral and spinal 5-HT2B receptors in formalin-induced nociception.

In this study we assessed the role of local peripheral and spinal serotonin 2B (5-HT(2B)) receptors in rats submitted to the formalin test. For this, local peripheral ipsilateral, but not contralateral, administration of the highly selective 5-HT(2B) receptor antagonist 2-amino-4-(4-fluoronaphth-1-yl)-6-isopropylpyridine (RS-127445, 0.01-1 nmol/paw) significantly prevented 1% formalin-induced flinching behavior. Moreover, local peripheral ipsilateral, but not contralateral, of the selective 5-HT(2) receptor agonist (±)-2,5-dimethoxy-4-iodoamphetamine hydrochloride (DOI, 1-10 nmol/paw) augmented 0.5% formalin-induced nociceptive behavior. The local pronociceptive effect of the 5-HT(2) receptor agonist DOI (10 nmol/paw) was significantly prevented by the local injection of RS-127445 (0.01 nmol/paw). Moreover, intrathecal injection of the selective 5-HT(2B) receptor antagonist RS-127445 (0.1-10 nmol/rat) also prevented 1% formalin-induced nociceptive behavior. In contrast, spinal injection of the 5-HT(2) receptor agonist DOI (1-10 nmol/rat) significantly increased flinching behavior induced by 0.5% formalin. The spinal pronociceptive effect of the 5-HT(2) receptor agonist DOI (10 nmol/rat) was prevented by the intrathecal injection of the 5-HT(2B) receptor antagonist RS-127445 (0.1 nmol/rat). Our results suggest that the 5-HT(2B) receptors play a pronociceptive role in peripheral as well as spinal sites in the rat formalin test. 5-HT(2B) receptors could be a target to develop analgesic drugs.

Role of peripheral and spinal 5-HT(3) receptors in development and maintenance of formalin-induced long-term secondary allodynia and hyperalgesia.

The role of peripheral and spinal 5-HT(3) receptors in formalin-induced secondary allodynia and hyperalgesia in rats was assessed. Formalin produced acute nociceptive behaviors (flinching and licking/lifting) followed by long-term secondary mechanical allodynia and hyperalgesia in both paws. In experiments where the test drug was anticipated to augment or antagonize the response, 0.5 or 1% formalin, respectively, was used for injection. Peripheral ipsilateral, but not contralateral, pre-treatment (-10 min) with serotonin (5-HT, 10-100 nmol/paw) and the selective 5-HT(3) receptor agonist 1-(m-chlorophenyl)-biguanide (m-CPBG, 10-300 nmol/paw) increased 0.5% formalin-induced secondary allodynia and hyperalgesia in both paws. Moreover, spinal pre-treatment with m-CPBG (10-300 nmol/rat) increased 0.5% formalin-induced secondary hyperalgesia but not allodynia in both paws. Accordingly, peripheral ipsilateral (30-300 nmol/paw), but not contralateral (300 nmol/paw), and spinal (10-100 nmol) pre-treatment with the selective 5-HT(3) receptor antagonist ondansetron prevented 1% formalin-induced secondary mechanical allodynia and hyperalgesia in both paws. The peripheral pronociceptive effects of 5-HT (100 nmol/paw) and m-CPBG (300 nmol/paw) as well as the spinal effect of m-CPBG (300 nmol/rat) were completely prevented by the peripheral (10 nmol/paw) and spinal (1 nmol/rat) injection, respectively, of ondansetron. At these doses, ondansetron did not modify per se formalin-induced nociceptive behaviors. Spinal (30-300 nmol/rat), but not peripheral (300 nmol/paw), post-treatment (on day 6) with ondansetron reversed established formalin-induced secondary mechanical allodynia and hyperalgesia in both paws. Results suggest that a barrage of afferent input induced by 5-HT at peripheral 5-HT(3) receptors participates in the development of formalin-induced long-term secondary allodynia and hyperalgesia in the rat. In addition, our data suggest that spinal 5-HT(3) receptors play an important role during development and maintenance of these evoked long-term behaviors.