Spinocerebellar ataxia type 4 is caused by a GGC expansion in the ZFHX3 gene and is associated with prominent dysautonomia and motor neuron signs.

BACKGROUND: Spinocerebellar ataxia 4 (SCA4), characterized in 1996, features adult-onset ataxia, polyneuropathy, and linkage to chromosome 16q22.1; its underlying mutation has remained elusive. OBJECTIVE: To explore the radiological and neuropathological abnormalities in the entire neuroaxis in SCA4 and search for its mutation. METHODS: Three Swedish families with undiagnosed ataxia went through clinical, neurophysiological, and neuroimaging tests, including PET studies and genetic investigations. In four cases, neuropathological assessments of the neuroaxis were performed. Genetic testing included short read whole genome sequencing, short tandem repeat analysis with ExpansionHunter de novo, and long read sequencing. RESULTS: Novel features for SCA4 include dysautonomia, motor neuron affection, and abnormal eye movements. We found evidence of anticipation; neuroimaging demonstrated atrophy in the cerebellum, brainstem, and spinal cord. [18F]FDG-PET demonstrated brain hypometabolism and [11C]Flumazenil-PET reduced binding in several brain lobes, insula, thalamus, hypothalamus, and cerebellum. Moderate to severe loss of Purkinje cells in the cerebellum and of motor neurons in the anterior horns of the spinal cord along with pronounced degeneration of posterior tracts was also found. Intranuclear, mainly neuronal, inclusions positive for p62 and ubiquitin were sparse but widespread in the CNS. This finding prompted assessment for nucleotide expansions. A polyglycine stretch encoding GGC expansions in the last exon of the zink finger homeobox 3 gene was identified segregating with disease and not found in 1000 controls. CONCLUSIONS: SCA4 is a neurodegenerative disease caused by a novel GGC expansion in the coding region of ZFHX3, and its spectrum is expanded to include dysautonomia and neuromuscular manifestations.

Hippocampal Expression of Cytochrome P450 1B1 in Penetrating Traumatic Brain Injury.

Hippocampal dysfunction contributes to multiple traumatic brain injury sequala. Female rodents' outcome is superior to male which has been ascribed the neuroprotective sex hormones 17ß-estradiol and progesterone. Cytochrome P450 1B1 (CYP1B1) is an oxidative enzyme influencing the neuroinflammatory response by creating inflammatory mediators and metabolizing neuroprotective 17ß-estradiol and progesterone. In this study, we aimed to describe hippocampal CYP1B1 mRNA expression, protein presence of CYP1B1 and its key redox partner Cytochrome P450 reductase (CPR) in both sexes, as well as the effect of penetrating traumatic brain injury (pTBI). A total 64 adult Sprague Dawley rats divided by sex received pTBI or sham-surgery and were assigned survival times of 1-, 3-, 5- or 7 days. CYP1B1 mRNA was quantified using in-situ hybridization and immunohistochemistry performed to verify protein colocalization. CYP1B1 mRNA expression was present in all subregions but greatest in CA2 irrespective of sex, survival time or intervention. At 3-, 5- and 7 days post-injury, expression in CA2 was reduced in male rats subjected to pTBI compared to sham-surgery. Females subjected to pTBI instead exhibited increased expression in all CA subregions 3 days post-injury, the only time point expression in CA2 was greater in females than in males. Immunohistochemical analysis confirmed neuronal CYP1B1 protein in all hippocampal subregions, while CPR was limited to CA1 and CA2. CYP1B1 mRNA is constitutively expressed in both sexes. In response to pTBI, females displayed a more urgent but brief regulatory response than males. This indicates there may be sex-dependent differences in CYP1B1 activity, possibly influencing inflammation and neuroprotection in pTBI.

Neuronal RARß Signaling Modulates PTEN Activity Directly in Neurons and via Exosome Transfer in Astrocytes to Prevent Glial Scar Formation and Induce Spinal Cord Regeneration.

Failure of axonal regeneration in the central nervous system (CNS) is mainly attributed to a lack of intrinsic neuronal growth programs and an inhibitory environment from a glial scar. Phosphatase and tensin homolog (PTEN) is a major negative regulator of neuronal regeneration and, as such, inhibiting its activity has been considered a therapeutic target for spinal cord (SC) injuries (SCIs). Using a novel model of rat cervical avulsion, we show that treatment with a retinoic acid receptor ß (RARß) agonist results in locomotor and sensory recovery. Axonal regeneration from the severed roots into the SC could be seen by biotinylated dextran amine labeling. Light micrographs of the dorsal root entry zone show the peripheral nervous system (PNS)-CNS transition of regrown axons. RARß agonist treatment also resulted in the absence of scar formation. Mechanism studies revealed that, in RARß-agonist-treated neurons, PTEN activity is decreased by cytoplasmic phosphorylation and increased secretion in exosomes. These are taken up by astrocytes, resulting in hampered proliferation and causing them to arrange in a normal-appearing scaffold around the regenerating axons. Attribution of the glial modulation to neuronal PTEN in exosomes was demonstrated by the use of an exosome inhibitor in vivo and PTEN siRNA in vitro assays. The dual effect of RARß signaling, both neuronal and neuronal-glial, results in axonal regeneration into the SC after dorsal root neurotmesis. Targeting this pathway may open new avenues for the treatment of SCIs. SIGNIFICANCE STATEMENT: Spinal cord injuries (SCIs) often result in permanent damage in the adult due to the very limited capacity of axonal regeneration. Intrinsic neuronal programs and the formation of a glial scar are the main obstacles. Here, we identify a single target, neuronal retinoic acid receptor ß (RARß), which modulates these two aspects of the postinjury physiological response. Activation of RARß in the neuron inactivates phosphatase and tensin homolog and induces its transfer into the astrocytes in small vesicles, where it prevents scar formation. This may open new therapeutic avenues for SCIs.

COX-2 regulation and TUNEL-positive cell death differ between genders in the secondary inflammatory response following experimental penetrating focal brain injury in rats.

INTRODUCTION: Traumatic brain injury is followed by secondary neuronal degeneration, largely dependent on an inflammatory response. This response is probably gender specific, since females are better protected than males in experimental models. The reasons are not fully known. We examined aspects of the inflammatory response following experimental TBI in male and female rats to explore possible gender differences at 24 h and 72 h after trauma, times of peak histological inflammation and neuronal degeneration. METHODS: A penetrating brain injury model was used to produce penetrating focal TBI in 20 Sprague-Dawley rats, 5 males and 5 females for each time point. After 24 and 72 h the brains were removed and subjected to in situ hybridization and immunohistochemical analyses for COX-2, iNOS, osteopontin, glial fibrillary acidic protein, 3-nitrotyrosine, TUNEL and Fluoro-Jade. RESULTS: COX-2 mRNA and protein levels were increased in the perilesional area compared to the uninjured contralateral side and significantly higher in males at 24 h and 72 h (p < 0.05). iNOS mRNA was significantly increased in females at 24 h (p < 0.05) although protein was not. TUNEL was increased in male rats after 24 h (p < 0.05). Glial fibrillary acidic protein, osteopontin, 3-nitrotyrosine and Fluoro-Jade stained degenerating neurons were increased in the perilesional area, showing no difference between genders. CONCLUSIONS: COX-2 regulation differed between genders after TBI. The increased COX-2 expression in male rats correlated with increased apoptotic cell death detected by increased TUNEL staining at 24 h, but not with neuronal necrosis measured by Flouro-Jade. Astrogliosis and microgliosis did not differ, confirming a comparable level of trauma. The gender-specific trait of the secondary inflammatory response may be connected to prostaglandin regulation, which may partially explain gender variances in outcome after TBI.

Brain tissue saving effects by single-dose intralesional administration of Neuroprotectin D1 on experimental focal penetrating brain injury in rats.

Traumatic brain injury (TBI) is followed by a secondary inflammation in the brain. Neuroprotectin D1 (NPD1) is synthesized from docosahexaenoic acid (DHA) and has anti-inflammatory and antiapoptotic effects in experimental models of neurodegenerative disease and brain ischemia-reperfusion. It is not known whether intralesional administration of NPD1 ameliorates inflammation and cell death after severe TBI. We therefore investigated the effects of NPD1 following a severe form of focal penetrating TBI. A total of 30 male Sprague-Dawley rats weighing between 350 and 450 g were exposed to focal penetrating TBI or sham surgery. The rats were randomized to NPD1 treatment (50 ng intralesionally, immediately following TBI) or no treatment. The rats were sacrificed at 24 or 72 h. All subgroups consisted of 5 rats. Brains were removed, fresh frozen, cut in 14-µm coronal sections and subjected to Fluoro-Jade, TUNEL, MnSOD, 3-NT, COX-2, Ox-42 and NF-κB immuno-staining and lesion size analyses. NPD1 decreased the lesion area at 72 h compared to no treatment with a mean change 42% (NPD1 14.1 mm2; no treatment 24.5 mm2) (p < 0.01). No difference was detected in markers for neuronal degeneration, apoptosis, anti-inflammatory or antioxidative enzymes, or immune cells. In conclusion, single-dose intralesional administration of NPD1 had brain tissue sparing effects after focal penetrating TBI, which may be beneficial in preventing brain tissue damage, making NPD1 a potential candidate for further clinical applications. Exact mechanisms of action could not be determined and it is possible that continuous or multiple administration regimens may increase efficacy in sequential preclinical studies.

COX-2 Inhibition by Diclofenac Is Associated With Decreased Apoptosis and Lesion Area After Experimental Focal Penetrating Traumatic Brain Injury in Rats.

Traumatic brain injury (TBI) is followed by a secondary inflammation in the brain. The inflammatory response includes prostanoid synthesis by the inducible enzyme cyclooxygenase-2 (COX-2). Inhibition of COX-2 is associated with improved functional outcome in experimental TBI models, although central nervous system-specific effects are not fully understood. Animal studies report better outcomes in females than males. The exact mechanisms for this gender dichotomy remain unknown. In an initial study we reported increased COX-2 expression in male rats, compared to female, following experimental TBI. It is possible that COX-2 induction is directly associated with increased cell death after TBI. Therefore, we designed a sequential study to investigate the blocking of COX-2 specifically, using the established COX-2 inhibitor diclofenac. Male Sprague-Dawley rats weighing between 250 and 350 g were exposed to focal penetrating TBI and randomly selected for diclofenac treatment (5 µg intralesionally, immediately following TBI) (n = 8), controls (n = 8), sham operation (n = 8), and normal (no manipulation) (n = 4). After 24 h, brains were removed, fresh frozen, cut into 14 µm coronal sections and subjected to COX-2 immunofluorescence, Fluoro Jade, TUNEL, and lesion area analyses. Diclofenac treatment decreased TUNEL staining indicative of apoptosis with a mean change of 54% (p < 0.05) and lesion area with a mean change of 55% (p < 0.005). Neuronal degeneration measured by Fluoro Jade and COX-2 protein expression levels were not affected. In conclusion, COX-2 inhibition by diclofenac was associated with decreased apoptosis and lesion area after focal penetrating TBI and may be of interest for further studies of clinical applications.

Structural and Functional Substitution of Deleted Primary Sensory Neurons by New Growth from Intrinsic Spinal Cord Nerve Cells: An Alternative Concept in Reconstruction of Spinal Cord Circuits.

In a recent clinical report, return of the tendon stretch reflex was demonstrated after spinal cord surgery in a case of total traumatic brachial plexus avulsion injury. Peripheral nerve grafts had been implanted into the spinal cord to reconnect to the peripheral nerves for motor and sensory function. The dorsal root ganglia (DRG) containing the primary sensory nerve cells had been surgically removed in order for secondary or spinal cord sensory neurons to extend into the periphery and replace the deleted DRG neurons. The present experimental study uses a rat injury model first to corroborate the clinical finding of a re-established spinal reflex arch, and second, to elucidate some of the potential mechanisms underlying these findings by means of morphological, immunohistochemical, and electrophysiological assessments. Our findings indicate that, after spinal cord surgery, the central nervous system sensory system could replace the traumatically detached original peripheral sensory connections through new neurite growth from dendrites.

Study of Autophagy and Microangiopathy in Sural Nerves of Patients with Chronic Idiopathic Axonal Polyneuropathy.

Twenty-five percent of polyneuropathies are idiopathic. Microangiopathy has been suggested to be a possible pathogenic cause of chronic idiopathic axonal polyneuropathy (CIAP). Dysfunction of the autophagy pathway has been implicated as a marker of neurodegeneration in the central nervous system, but the autophagy process is not explored in the peripheral nervous system. In the current study, we examined the presence of microangiopathy and autophagy-related structures in sural nerve biopsies of 10 patients with CIAP, 11 controls with inflammatory neuropathy and 10 controls without sensory polyneuropathy. We did not find any significant difference in endoneurial microangiopathic markers in patients with CIAP compared to normal controls, though we did find a correlation between basal lamina area thickness and age. Unexpectedly, we found a significantly larger basal lamina area thickness in patients with vasculitic neuropathy. Furthermore, we found a significantly higher density of endoneurial autophagy-related structures, particularly in patients with CIAP but also in patients with inflammatory neuropathy, compared to normal controls. It is unclear if the alteration in the autophagy pathway is a consequence or a cause of the neuropathy. Our results do not support the hypothesis that CIAP is primarily caused by a microangiopathic process in endoneurial blood vessels in peripheral nerves. The significantly higher density of autophagy structures in sural nerves obtained from patients with CIAP and inflammatory neuropathy vs. controls indicates the involvement of this pathway in neuropathy, particularly in CIAP, since the increase in density of autophagy-related structures was more pronounced in patients with CIAP than those with inflammatory neuropathy. To our knowledge this is the first report investigating signs of autophagy process in peripheral nerves in patients with CIAP and inflammatory neuropathy.

On acute gene expression changes after ventral root replantation.

Replantation of avulsed spinal ventral roots has been show to enable significant and useful regrowth of motor axons in both experimental animals and in human clinical cases, making up an interesting exception to the rule of unsuccessful neuronal regeneration in central nervous system. Compared to avulsion without repair, ventral root replantation seems to rescue lesioned motoneurons from death. In this study we have analyzed the acute response to ventral root avulsion and replantation in adult rats with gene arrays combined with cluster analysis of gene ontology search terms. The data show significant differences between rats subjected to ventral replantation compared to avulsion only. Even though number of genes related to cell death is similar in the two models after 24 h, we observed a significantly larger number of genes related to neurite growth and development in the rats treated with ventral root replantation, possibly reflecting the neuroregenerative capacity in the replantation model. In addition, an acute inflammatory response was observed after avulsion, while effects on genes related to synaptic transmission were much more pronounced after replantation than after avulsion alone. These data indicate that the axonal regenerative response from replantation is initiated at an earlier stage than the possible differences in terms of neuron survival. We conclude that this type of analysis may facilitate the comparison of the acute response in two types of injury.

Observations at the CNS-PNS Border of Ventral Roots Connected to a Neuroma.

Previous studies have shown that numerous sprouts originating from a neuroma, after nerve injury in neonatal animals, can invade spinal nerve roots. However, no study with a focus on how such sprouts behave when they reach the border between the central and peripheral nervous system (CNS-PNS border) has been published. In this study we have in detail examined the CNS-PNS border of ventral roots in kittens with light and electron microscopy after early postnatal sciatic nerve resection. A transient ingrowth of substance P positive axons was observed into the CNS, but no spouts remained 6 weeks after the injury. Using serial sections and electron microscopy it was possible to identify small bundles of unmyelinated axons that penetrated from the root fascicles for a short distance into the CNS. These axons ended blindly, sometimes with a growth cone-like terminal swelling filled with vesicles. The axon bundles were accompanied by p75 positive cells in both the root fascicles and the pia mater, but not in the CNS. It may thus be suggested that neurotrophin presenting p75 positive cells could facilitate axonal growth into the pia mater and that the lack of such cells in the CNS compartment might contribute to the failure of growth into the CNS. A maldevelopment of myelin sheaths at the CNS-PNS border of motor axons was observed and it seems possible that this could have consequences for the propagation of action potential across this region after neonatal nerve injury. Thus, in this first detailed study on the behavior of recurrent sprouts at the CNS-PNS border.