PTEN deletion in spinal pathways via retrograde transduction with AAV-RG enhances forelimb motor recovery after cervical spinal cord injury; Sex differences and late-onset pathophysiologies.

Spinal cord injuries (SCI) cause permanent functional impairments due to interruption of motor and sensory pathways. Regeneration of axons does not occur due to lack of intrinsic growth capacity of adult neurons and extrinsic inhibitory factors, especially at the injury site. However, some regeneration can be achieved via deletion of the phosphatase and tensin homolog (PTEN) in cells of origin of spinal pathways. Here, we deployed an AAV variant that is retrogradely transported (AAV-rg) to deliver gene modifying cargos to the cells of origin of multiple pathways interrupted by SCI, testing whether this promoted recovery of motor function. PTENf/f;RosatdTomato mice and control RosatdTomato mice received injections of different doses (number of genome copies, GCs) of AAV-rg/Cre into the cervical spinal cord at the time of a C5 dorsal hemisection injury. Forelimb grip strength was tested over time using a grip strength meter. PTENf/f;RosatdTomato mice with AAV-rg/Cre (PTEN-deleted) exhibited substantial improvements in forelimb gripping ability in comparison to controls. Of note, there were major sex differences in the extent of recovery, with male mice exhibiting greater recovery than females. However, at around 5-7 weeks post-injury/injection, many mice with SCI and AAV-rg-mediated PTEN deletion began to exhibit pathophysiologies involving excessive scratching of the ears and back of the neck and rigid forward extension of the hindlimbs. These pathophysiologies increased in incidence and severity over time. Our results reveal that although intra-spinal injections of AAV-rg/Cre in PTENf/f;RosatdTomato mice can enhance forelimb motor recovery after SCI, late-developing functional abnormalities occur with the experimental conditions used here. Mechanisms underlying late-developing pathophysiologies remain to be defined.

Elevation of NAD+ by nicotinamide riboside spares spinal cord tissue from injury and promotes locomotor recovery.

Spinal cord injury (SCI)-induced tissue damage spreads to neighboring spared cells in the hours, days, and weeks following injury, leading to exacerbation of tissue damage and functional deficits. Among the biochemical changes is the rapid reduction of cellular nicotinamide adenine dinucleotide (NAD+), an essential coenzyme for energy metabolism and an essential cofactor for non-redox NAD+-dependent enzymes with critical functions in sensing and repairing damaged tissue. NAD+ depletion propagates tissue damage. Augmenting NAD+ by exogenous application of NAD+, its synthesizing enzymes, or its cellular precursors mitigates tissue damage. Nicotinamide riboside (NR) is considered to be one of the most promising NAD+ precursors for clinical application due to its ability to safely and effectively boost cellular NAD+ synthesis in rats and humans. Moreover, various preclinical studies have demonstrated that NR can provide tissue protection. Despite these promising findings, little is known about the potential benefits of NR in the context of SCI. In the current study, we tested whether NR administration could effectively increase NAD+ levels in the injured spinal cord and whether this augmentation of NAD+ would promote spinal cord tissue protection and ultimately lead to improvements in locomotor function. Our findings indicate that administering NR (500 mg/kg) intraperitoneally from four days before to two weeks after a mid-thoracic contusion-SCI injury, effectively doubles NAD+ levels in the spinal cord of Long-Evans rats. Moreover, NR administration plays a protective role in preserving spinal cord tissue post-injury, particularly in neurons and axons, as evident from the observed gray and white matter sparing. Additionally, it enhances motor function, as evaluated through the BBB subscore and missteps on the horizontal ladderwalk. Collectively, these findings demonstrate that administering NR, a precursor of NAD+, increases NAD+ within the injured spinal cord and effectively mitigates the tissue damage and functional decline that occurs following SCI.

PTEN deletion in spinal pathways via retrograde transduction with AAV-rg enhances forelimb motor recovery after cervical spinal cord injury; sex differences and late-onset pathophysiologies.

Spinal cord injuries (SCI) cause permanent functional impairments due to interruption of motor and sensory pathways. Regeneration of axons does not occur due to lack of intrinsic growth capacity of adult neurons and extrinsic inhibitory factors, especially at the injury site. However, some regeneration can be achieved via deletion of the phosphatase and tensin homolog (PTEN) in cells of origin of spinal pathways. Here, we deployed an AAV variant that is retrogradely transported (AAV-rg) to deliver gene modifying cargos to the cells of origin of multiple pathways interrupted by SCI, testing whether this promoted recovery of motor function. PTEN f/f ;Rosa tdTomato mice and control Rosa tdTomato mice received injections of different doses (number of genome copies, GCs) of AAV-rg/Cre into the cervical spinal cord at the time of a C5 dorsal hemisection injury. Forelimb grip strength was tested over time using a grip strength meter. PTEN f/f ;Rosa tdTomato mice with AAV-rg/Cre (PTEN-deleted) exhibited substantial improvements in forelimb gripping ability in comparison to controls. Of note, there were major sex differences in the extent of recovery, with male mice exhibiting greater recovery than females. However, at around 5-7 weeks post-injury/injection, many mice with SCI and AAV-rg-mediated PTEN deletion began to exhibit pathophysiologies involving excessive scratching of the ears and back of the neck and rigid forward extension of the hindlimbs. These pathophysiologies increased in incidence and severity over time. Our results reveal that although intra-spinal injections of AAV-rg/Cre in PTEN f/f ;Rosa tdTomato mice can enhance forelimb motor recovery after SCI, late-developing functional abnormalities occur with the experimental conditions used here. Mechanisms underlying late-developing pathophysiologies remain to be defined.

A developmental approach to diversifying neuroscience through effective mentorship practices: perspectives on cross-identity mentorship and a critical call to action.

Many early-career neuroscientists with diverse identities may not have mentors who are more advanced in the neuroscience pipeline and have a congruent identity due to historic biases, laws, and policies impacting access to education. Cross-identity mentoring relationships pose challenges and power imbalances that impact the retention of diverse early career neuroscientists, but also hold the potential for a mutually enriching and collaborative relationship that fosters the mentee's success. Additionally, the barriers faced by diverse mentees and their mentorship needs may evolve with career progression and require developmental considerations. This article provides perspectives on factors that impact cross-identity mentorship from individuals participating in Diversifying the Community of Neuroscience (CNS)-a longitudinal, National Institute of Neurological Disorders and Stroke (NINDS) R25 neuroscience mentorship program developed to increase diversity in the neurosciences. Participants in Diversifying CNS were comprised of 14 graduate students, postdoctoral fellows, and early career faculty who completed an online qualitative survey on cross-identity mentorship practices that impact their experience in neuroscience fields. Qualitative survey data were analyzed using inductive thematic analysis and resulted in four themes across career levels: (1) approach to mentorship and interpersonal dynamics, (2) allyship and management of power imbalance, (3) academic sponsorship, and (4) institutional barriers impacting navigation of academia. These themes, along with identified mentorship needs by developmental stage, provide insights mentors can use to better support the success of their mentees with diverse intersectional identities. As highlighted in our discussion, a mentor's awareness of systemic barriers along with active allyship are foundational for their role.

Temporary induction of hypoxic adaptations by preconditioning fails to enhance Schwann cell transplant survival after spinal cord injury.

Hypoxic preconditioning is protective in multiple models of injury and disease, but whether it is beneficial for cells transplanted into sites of spinal cord injury (SCI) is largely unexplored. In this study, we analyzed whether hypoxia-related preconditioning protected Schwann cells (SCs) transplanted into the contused thoracic rat spinal cord. Hypoxic preconditioning was induced in SCs prior to transplantation by exposure to either low oxygen (1% O2 ) or pharmacological agents (deferoxamine or adaptaquin). All preconditioning approaches induced hypoxic adaptations, including increased expression of HIF-1α and its target genes. These adaptations, however, were transient and resolved within 24 h of transplantation. Pharmacological preconditioning attenuated spinal cord oxidative stress and enhanced transplant vascularization, but it did not improve either transplanted cell survival or recovery of sensory or motor function. Together, these experiments show that hypoxia-related preconditioning is ineffective at augmenting either cell survival or the functional outcomes of SC-SCI transplants. They also reveal that the benefits of hypoxia-related adaptations induced by preconditioning for cell transplant therapies are not universal.

Harnessing rAAV-retro for gene manipulations in multiple pathways that are interrupted after spinal cord injury.

This paper explores the potential of rAAV2-retro to deliver gene modifying cargoes to the cells of origin of multiple pathways that are interrupted by spinal cord injury (SCI), summarizing data from previous studies and new data from additional experiments. rAAV-retro exhibits uniquely robust and reliable long-distance retrograde transport from pre-terminal axons and synapses back to neuronal bodies. Previous studies have documented that various AAV-based genetic modifications can enable axon regeneration after SCI, but these have targeted the cells of origin of one pathway at a time. In contrast, rAAV-retro can simultaneously transduce large numbers of neurons of origin of multiple spinal pathways with single injections into the spinal cord. Our initial studies use RosatdTomato and double transgenic PTENf/f; RosatdTomato mice in which transfection with rAAV-retro/Cre deletes PTEN and activates tdT expression in the same neurons. Injections of rAAV-retro/Cre into the cervical, thoracic and lumbar spinal cord led to topographically specific retrograde transduction in cortical motoneurons and neurons in subcortical regions that give rise to different spinal pathways. Our results confirm and extend previous studies indicating selective transduction of neurons that terminate at the level of the injection with minimal retrograde transduction of axons in transit to lower levels. We document feasibility of using rAAV-retro expressing shRNA against PTEN along with a GFP reporter (rAAV-retro-shPTEN/GFP) to effectively knock down PTEN in multiple populations of neurons, which can be used in any species. Some limitations and caveats of currently available rAAV-retros are discussed. Together, our results support the potential applications of rAAV-retro for AAV-based gene-modifications for SCI.

AAV vectors accumulate in the pineal gland after injections into the brain or spinal cord.

AAV vectors are being used extensively for gene-modifying therapies for neurological disorders. Here, we report the surprising discovery that injections of different AAVs into the brain, spinal cord, or cerebrospinal fluid (CSF) lead to robust transduction of cells in the pineal gland. We document transduction of cells in the pineal gland following focal injections of AAV2/9-shPTEN-zsGreen into the sensorimotor or hippocampus of rats and injections of AAV2/Cre into the spinal cord of transgenic mice with a stop-flox tdT reporter. Pineal transduction was evident even when AAV2/Cre injections were made into the lumbar spinal cord many millimeters distant from the pineal gland. Immunostaining with antibodies for cell types in the pineal gland revealed that pinealocytes were transduced. Pineal transduction was also observed with intracerebroventricular (i.c.v.) injections of AAV2/9-shPTEN-zsGreen, suggesting that pineal transduction following focal injections of AAV into CNS parenchyma may be caused by diffusion of the vector from the injection sites into the CSF and then accumulation in the pineal gland. Together, these findings suggest the need for vigilance for functional consequences and possible adverse effects of off-target accumulation of therapeutic AAVs in the pineal gland and AAV-driven expression of therapeutic cargos in pinealocytes.

Rostro-Caudal Specificity of Corticospinal Tract Projections in Mice.

Rostro-caudal specificity of corticospinal tract (CST) projections from different areas of the cortex was assessed by retrograde labeling with fluorogold and retrograde transfection following retro-AAV/Cre injection into the spinal cord of tdT reporter mice. Injections at C5 led to retrograde labeling of neurons throughout forelimb area of the sensorimotor cortex and a region in the dorsolateral cortex near the barrel field (S2). Injections at L2 led to retrograde labeling of neurons in the posterior sensorimotor cortex (hindlimb area) but not the dorsolateral cortex. With injections of biotinylated dextran amine (BDA) into the main sensorimotor cortex (forelimb region), labeled axons terminated selectively at cervical levels. With BDA injections into caudal sensorimotor cortex (hindlimb region), labeled axons passed through cervical levels without sending collaterals into the gray matter and then elaborated terminal arbors at thoracic sacral levels. With BDA injections into the dorsolateral cortex near the barrel field, labeled axons terminated at high cervical levels. Axons from medial sensorimotor cortex terminated primarily in intermediate laminae and axons from lateral sensorimotor cortex terminated primarily in laminae III-V of the dorsal horn. One of the descending pathways seen in rats (the ventral CST) was not observed in most mice.

AAVshRNA-mediated PTEN knockdown in adult neurons attenuates activity-dependent immediate early gene induction.

Genetic deletion or knockdown of PTEN enables regeneration of CNS axons, enhances sprouting of intact axons after injury, and induces de novo growth of uninjured adult neurons. It is unknown, however how PTEN deletion in mature neurons alters neuronal physiology. As a first step to address this question, we used immunocytochemistry for activity-dependent markers to assess consequences of PTEN knockdown in cortical neurons and granule cells of the dentate gyrus. In adult rats that received unilateral intra-cortical injections of AAV expressing shRNA against PTEN, immunostaining for c-fos under resting conditions (home cage, HC) and after 1 h of exploration of a novel enriched environment (EE) revealed no hot spots of c-fos expression that would suggest abnormal activity. Counts revealed similar numbers of c-fos positive neurons in the area of PTEN deletion vs. homologous areas in the contralateral cortex in the HC and similar induction of c-fos with EE. However, IEG induction in response to high frequency stimulation (HFS) of the cortex was attenuated in areas of PTEN deletion. In rats with AAVshRNA-mediated PTEN deletion in the dentate gyrus, induction of the IEGs c-fos and Arc with HFS of the perforant path was abrogated in areas of PTEN deletion. Immunostaining using phosphospecific antibodies for phospho-S6 (a downstream marker for mTOR activation) and phospho-ERK1/2 revealed abrogation of S6 phosphorylation in PTEN-deleted areas but preserved activation of phosphorylation of ERK1/2. SIGNIFICANCE STATEMENT: Deletion or knockdown of the tumor suppressor gene PTEN enables regenerative growth of adult CNS axons after injury, which is accompanied by enhanced recovery of function. Consequently, PTEN represents a potential target for therapeutic interventions to enhance recovery after CNS injury. Here we show that activity-dependent IEG induction is attenuated in PTEN-depleted neurons. These findings raise the intriguing possibility that functional recovery due to regenerative growth may be limited by the disruption of plasticity-related signaling pathways, and that recovery might be enhanced by restoring PTEN expression after regenerative growth has been achieved.