Prevention of age-associated neuronal hyperexcitability with improved learning and attention upon knockout or antagonism of LPAR2.

Recent studies suggest that synaptic lysophosphatidic acids (LPAs) augment glutamate-dependent cortical excitability and sensory information processing in mice and humans via presynaptic LPAR2 activation. Here, we studied the consequences of LPAR2 deletion or antagonism on various aspects of cognition using a set of behavioral and electrophysiological analyses. Hippocampal neuronal network activity was decreased in middle-aged LPAR2-/- mice, whereas hippocampal long-term potentiation (LTP) was increased suggesting cognitive advantages of LPAR2-/- mice. In line with the lower excitability, RNAseq studies revealed reduced transcription of neuronal activity markers in the dentate gyrus of the hippocampus in naïve LPAR2-/- mice, including ARC, FOS, FOSB, NR4A, NPAS4 and EGR2. LPAR2-/- mice behaved similarly to wild-type controls in maze tests of spatial or social learning and memory but showed faster and accurate responses in a 5-choice serial reaction touchscreen task requiring high attention and fast spatial discrimination. In IntelliCage learning experiments, LPAR2-/- were less active during daytime but normally active at night, and showed higher accuracy and attention to LED cues during active times. Overall, they maintained equal or superior licking success with fewer trials. Pharmacological block of the LPAR2 receptor recapitulated the LPAR2-/- phenotype, which was characterized by economic corner usage, stronger daytime resting behavior and higher proportions of correct trials. We conclude that LPAR2 stabilizes neuronal network excitability upon aging and allows for more efficient use of resting periods, better memory consolidation and better  performance in tasks requiring high selective attention. Therapeutic LPAR2 antagonism may alleviate aging-associated cognitive dysfunctions.

Growth-Promoting Treatment Screening for Corticospinal Neurons in Mouse and Man.

Neurons of the central nervous system (CNS) that project long axons into the spinal cord have a poor axon regenerative capacity compared to neurons of the peripheral nervous system. The corticospinal tract (CST) is particularly notorious for its poor regeneration. Because of this, traumatic spinal cord injury (SCI) is a devastating condition that remains as yet uncured. Based on our recent observations that direct neuronal interleukin-4 (IL-4) signaling leads to repair of axonal swellings and beneficial effects in neuroinflammation, we hypothesized that IL-4 acts directly on the CST. Here, we developed a tissue culture model for CST regeneration and found that IL-4 promoted new growth cone formation after axon transection. Most importantly, IL-4 directly increased the regenerative capacity of both murine and human CST axons, which corroborates its regenerative effects in CNS damage. Overall, these findings serve as proof-of-concept that our CST regeneration model is suitable for fast screening of new treatments for SCI.

A predominantly glial origin of axonal ribosomes after nerve injury.

Axonal mRNA transport and local protein synthesis are crucial for peripheral axon regeneration. To date, it remains unclear how ribosomes localize to axons. They may be co-transported with mRNAs or, as suggested by recent studies, transferred from Schwann cells (SC). Here, we generated transgenic "RiboTracker" mice expressing tdTomato-tagged ribosomal protein L4 in specific cell types when crossed with Cre lines. Two neuronal RiboTracker-Cre lines displayed extremely low levels of axonal L4-tdTomato-positive ribosomes. In contrast, two glial RiboTracker-Cre lines revealed tagged ribosomes in sciatic nerve (SN) axons with increasing amounts after injury. Furthermore, non-RiboTracker dorsal root ganglia co-cultured with L4-tdTomato-expressing SCs displayed tagged ribosomes in axons. These data provide unequivocal evidence that SN axons receive ribosomes from SCs upon injury and indicate that glial cells are the main source of axonal ribosomes.

Alpha9 integrin promotes neurite outgrowth on tenascin-C and enhances sensory axon regeneration.

Damaged CNS axons are prevented from regenerating by an environment containing many inhibitory factors. They also lack an integrin that interacts with tenascin-C, the main extracellular matrix glycoprotein of the CNS, which is upregulated after injury. The alpha9beta1 integrin heterodimer is a receptor for the nonalternatively spliced region of tenascin-C, but the alpha9 subunit is absent in adult neurons. In this study, we show that PC12 cells and adult rat dorsal root ganglion (DRG) neurons do not extend neurites on tenascin-C. However, after forced expression of alpha9 integrin, extensive neurite outgrowth from PC12 cells and adult rat DRG neurons occurs. Moreover, both DRG neurons and PC12 cells secrete tenascin-C, enabling alpha9-transfected cells to grow axons on tissue culture plastic. Using adeno-associated viruses to express alpha9 integrin in vivo in DRGs, we examined axonal regeneration after cervical dorsal rhizotomy or dorsal column crush in the adult rat. After rhizotomy, significantly more dorsal root axons regrew into the dorsal root entry zone at 6 weeks after injury in alpha9 integrin-expressing animals than in green fluorescent protein (GFP) controls. Similarly, after a dorsal column crush injury, there was significantly more axonal growth into the lesion site compared with GFP controls at 6 weeks after injury. Behavioral analysis after spinal cord injury revealed that both experimental and control groups had an increased withdrawal latency in response to mechanical stimulation when compared with sham controls; however, in response to heat stimulation, normal withdrawal latencies returned after alpha9 integrin treatment but remained elevated in control groups.

Axonal mRNAs: characterisation and role in the growth and regeneration of dorsal root ganglion axons and growth cones.

We have developed a compartmentalised culture model for the purification of axonal mRNA from embryonic, neonatal and adult rat dorsal root ganglia. This mRNA was used un-amplified for RT-qPCR. We assayed for the presence of axonal mRNAs encoding molecules known to be involved in axon growth and guidance. mRNAs for beta-actin, beta-tubulin, and several molecules involved in the control of actin dynamics and signalling during axon growth were found, but mRNAs for microtubule-associated proteins, integrins and cell surface adhesion molecules were absent. Quantification of beta-actin mRNA by means of qPCR showed that the transcript is present at the same level in embryonic, newborn and adult axons. Using the photoconvertible reporter Kaede we showed that there is local translation of beta-actin in axons, the rate being increased by axotomy. Knock down of beta-actin mRNA by RNAi inhibited the regeneration of new axon growth cones after in vitro axotomy, indicating that local translation of actin-related molecules is important for successful axon regeneration.

Fast direct neuronal signaling via the IL-4 receptor as therapeutic target in neuroinflammation.

Ongoing axonal degeneration is thought to underlie disability in chronic neuroinflammation, such as multiple sclerosis (MS), especially during its progressive phase. Upon inflammatory attack, axons undergo pathological swelling, which can be reversible. Because we had evidence for beneficial effects of T helper 2 lymphocytes in experimental neurotrauma and discovered interleukin-4 receptor (IL-4R) expressed on axons in MS lesions, we aimed at unraveling the effects of IL-4 on neuroinflammatory axon injury. We demonstrate that intrathecal IL-4 treatment during the chronic phase of several experimental autoimmune encephalomyelitis models reversed disease progression without affecting inflammation. Amelioration of disability was abrogated upon neuronal deletion of IL-4R. We discovered direct neuronal signaling via the IRS1-PI3K-PKC pathway underlying cytoskeletal remodeling and axonal repair. Nasal IL-4 application, suitable for clinical translation, was equally effective in improving clinical outcome. Targeting neuronal IL-4 signaling may offer new therapeutic strategies to halt disability progression in MS and possibly also neurodegenerative conditions.

Sciatic nerve regeneration in mice and rats: recovery of sensory innervation is followed by a slowly retreating neuropathic pain-like syndrome.

Peripheral nerve regeneration has been studied extensively in the sciatic nerve crush model, at the level of both function and gene expression. The crush injury allows full recovery of sensory and motor function in about 3 weeks as assessed by the foot reflex withdrawal test and De Medinacelli walking patterns. We used the recently developed CatWalk paradigm to study walking patterns in more detail in mice and rats. We found that, following the recovery of sensory function, the animals developed a state of mechanical allodynia, which retreated slowly over time. The motor function, although fully recovered with the conventional methods, was revealed to be still impaired because the animals did not put weight on their previously injured paw. The development of neuropathic pain following successful sensory recovery has not been described before in crush-lesioned animals and may provide an important new parameter to assess full sensory recovery.

Homeobox gene expression in adult dorsal root ganglia during sciatic nerve regeneration: is regeneration a recapitulation of development?

After damage of the sciatic nerve, a regeneration process is initiated. Neurons in the dorsal root ganglion regrow their axons and functional connections. The molecular mechanisms of this neuronal regenerative process have remained elusive, but a relationship with developmental processes has been conceived. This chapter discusses the applicability of the developmental hypothesis of regeneration to the dorsal root ganglion; this hypothesis states that regeneration of dorsal root ganglion neurons is a recapitulation of development. We present data on changes in gene expression upon sciatic nerve damage, and the expression and function of homeobox genes. This class of transcription factors plays a role in neuronal development. Based on these data, it is concluded that the hypothesis does not hold for dorsal root ganglion neurons, and that regeneration-specific mechanisms exist. Cytokines and the associated Jak/STAT (janus kinase/signal transducer and activator of transcription) signal transduction pathway emerge as constituents of a regeneration-specific mechanism. This mechanism may be the basis of pharmacological strategies to stimulate regeneration.

Cortical gene expression in spinal cord injury and repair: insight into the functional complexity of the neural regeneration program.

Traumatic spinal cord injury (SCI) results in the formation of a fibrous scar acting as a growth barrier for regenerating axons at the lesion site. We have previously shown (Klapka et al., 2005) that transient suppression of the inhibitory lesion scar in rat spinal cord leads to long distance axon regeneration, retrograde rescue of axotomized cortical motoneurons, and improvement of locomotor function. Here we applied a systemic approach to investigate for the first time specific and dynamic alterations in the cortical gene expression profile following both thoracic SCI and regeneration-promoting anti-scarring treatment (AST). In order to monitor cortical gene expression we carried out microarray analyses using total RNA isolated from layer V/VI of rat sensorimotor cortex at 1-60 days post-operation (dpo). We demonstrate that cortical neurons respond to injury by massive changes in gene expression, starting as early as 1 dpo. AST, in turn, results in profound modifications of the lesion-induced expression profile. The treatment attenuates SCI-triggered transcriptional changes of genes related to inhibition of axon growth and impairment of cell survival, while upregulating the expression of genes associated with axon outgrowth, cell protection, and neural development. Thus, AST not only modifies the local environment impeding spinal cord regeneration by reduction of fibrous scarring in the injured spinal cord, but, in addition, strikingly changes the intrinsic capacity of cortical pyramidal neurons toward enhanced cell maintenance and axonal regeneration.

CNS expression pattern of Lmx1b and coexpression with ptx genes suggest functional cooperativity in the development of forebrain motor control systems.

In the central nervous system, acquisition of regional specification is an important developmental process. The regional specification is reflected by restricted and overlapping expression of homeobox genes, which are regulators of this event. Here, we detail the expression pattern of Lmx1b during late embryonic brain development and show that this gene is expressed in multiple regions and diverse sets of neurons. Noteworthy, the Lmx1b expression domain is shared by Ptx2 in posterior hypothalamic regions and by Ptx3 in the dopaminergic neurons of the ventral midbrain. In addition, the mutual cofactor Ldb1 is expressed in these regions. The expression of these gene sets is maintained in the adult brain. The subthalamic nucleus, where Lmx1b is coexpressed with Ptx2, and the substantia nigra/ventral tegmental area, where Lmx1b is coexpressed with Ptx3, are both ancillary nuclei of the motor control circuitry, but use different neurotransmitters. These data point to a combinatorial gene network that allows Lmx1b to diversify its regulatory actions by cooperation with specific Ptx genes.