The Interaction of Genetic Mutations in PARK2 and FA2H Causes a Novel Phenotype in a Case of Childhood-Onset Movement Disorder

Mutations in the PARK2 gene have been implicated in the pathogenesis of early-onset Parkinson's disease. We present a case of movement disorder in a 4-year-old child from consanguineous parents and with a family history of Dopamine responsive dystonia, who was diagnosed with early-onset Parkinson's disease based on initial identification of a pathogenic PARK2 mutation. However, the evolution of the child's clinical picture was unusually rapid, with a preponderance of pyramidal rather than extrapyramidal symptoms, leading to re-investigation of the case with further imaging and genetic sequencing. Interestingly, a second homozygous mutation in the FA2H gene, implicated in Hereditary spastic paraplegia, was revealed, appearing to have contributed to the novel phenotype observed, and highlighting a potential interaction between the two mutated genes

Cyclin-dependent-like kinase 5 is required for pain signaling in human sensory neurons and mouse models

Cyclin-dependent-like kinase 5 (Cdkl5) gene mutations lead to an X-linked disorder that is characterized by infantile epileptic encephalopathy, developmental delay and hypotonia. However, we found that a substantial percentage of these patients also report a previously unrecognised anamnestic deficiency in pain perception. Consistent with a role in nociception, we discovered that Cdkl5 is expressed selectively in nociceptive dorsal root ganglia (DRG) neurons in mice and in iPS-derived human nociceptors. CDKL5 deficient mice display defective epidermal innervation and conditional deletion of Cdkl5 in DRG sensory neurons impairs nociception, phenocopying CDKL5 deficiency disorder in patients. Mechanistically, Cdkl5 interacts with CaMKIIα to control outgrowth as well as TRPV1-dependent signaling, which are disrupted in both Cdkl5 mutant murine DRG and human iPS-derived nociceptors. Together, these findings unveil a previously unrecognized role for Cdkl5 in nociception, proposing an original regulatory mechanism for pain perception with implications for future therapeutics in CDKL5 deficiency disorder

Dose-dependent neuroprotection of VEGF165 in Huntington's disease striatum

Huntington's disease (HD) is a devastating neurodegenerative disorder caused by abnormal polyglutamine expansion in the huntingtin protein (Exp-Htt). Currently, there are no effective treatments for HD. We used bidirectional lentiviral transfer vectors to generate in vitro and in vivo models of HD and to test the therapeutic potential of vascular endothelial growth factor 165 (VEGF165). Lentiviral-mediated expression of Exp-Htt caused cell death and aggregate formation in human neuroblastoma SH-SY5Y and rat primary striatal cultures. Lentiviral-mediated VEGF165 expression was found to be neuroprotective in both of these models. Unilateral stereotaxic vector delivery of Exp-Htt vector in adult rat striatum led to progressive inclusion formation and striatal neuron loss at 10 weeks post-transduction. Coinjection of a lower dose VEGF165 significantly attenuated DARPP-32 + neuronal loss, enhanced NeuN staining and reduced Exp-Htt aggregation. A tenfold higher dose VEGF165 led to overt neuronal toxicity marked by tissue damage, neovascularization, extensive astrogliosis, vascular leakage, chronic inflammation and distal neuronal loss. No overt behavioral phenotype was observed in these animals. Expression of VEGF165 at this higher dose in the brain of wild-type rats led to early mortality with global neuronal loss. This report raises important safety concerns about unregulated VEGF 165 CNS applications. © The American Society of Gene & Cell Therapy

Not Just Loss-of-Function Variations: Identification of a Hypermorphic Variant in a Patient With a CDKL5 Missense Substitution

Background and objectives: CDKL5 deficiency disorder (CDD) is a neurodevelopmental encephalopathy characterized by early-onset epilepsy and impaired psychomotor development. Variations in the X-linked CDKL5 gene coding for a kinase cause CDD. Molecular genetics has proved that almost all pathogenic missense substitutions localize in the N-terminal catalytic domain, therefore underlining the importance for brain development and functioning of the kinase activity. CDKL5 also features a long C-terminal domain that acts as negative regulator of the enzymatic activity and modulates its subcellular distribution. CDD is generally attributed to loss-of-function variations, whereas the clinical consequences of increased CDKL5 activity remain uncertain. We have identified a female patient characterized by mild epilepsy and neurologic symptoms, harboring a novel c.2873C>G nucleotide substitution, leading to the missense variant p.(Thr958Arg). To increase our comprehension of genetic variants in CDKL5-associated neurologic disorders, we have characterized the molecular consequences of the identified substitution. Methods: MRI and video EEG telemetry were used to describe brain activity and capture seizure. The Bayley III test was used to evaluate the patient development. Reverse transcriptase PCR was used to analyze whether the identified nucleotide variant affects messenger RNA stability and/or splicing. The X chromosome inactivation pattern was analyzed determining the DNA methylation status of the androgen receptor (AR) gene and by sequencing of expressed alleles. Western blotting was used to investigate whether the novel Thr958Arg substitution affects the stability and/or enzymatic activity of CDKL5. Immunofluorescence was used to define whether CDKL5 subcellular distribution is affected by the Thr958Arg substitution. Results: Our data suggested that the proband tends toward a skewed X chromosome inactivation pattern in favor of the novel variant. The molecular investigation revealed that the p.(Thr958Arg) substitution leads to a significant increase in the autophosphorylation of both the TEY motif and residue Tyr171 of CDKL5, as well as in the phosphorylation of the target protein MAP1S, indicating an hyperactivation of CDKL5. This occurs without evidently affecting the kinase subcellular distribution. Discussion: Our data provide a strong indication that the c.2873C>G nucleotide substitution represents an hypermorphic pathogenic variation of CDKL5, therefore highlighting the importance of a tight control of CDKL5 activity in the brain

Lentivector-mediated delivery of GDNF protects complex motor functions relevant to human Parkinsonism in a rat lesion model

Although viral vector-mediated delivery of glial cell-line derived neurotrophic factor (GDNF) to the brain has considerable potential as a neuroprotective strategy in Parkinson's disease (PD), its ability to protect complex motor functions relevant to the human condition has yet to be established. In this study, we used an operant task that assesses the selection, initiation and execution of lateralized nose-pokes in Lister Hooded rats to assess the efficacy with which complex behaviours are protected against neurotoxic lesions by prior injection of a lentiviral vector expressing GDNF. Unilateral injection of 6-hydroxydopamine (6-OHDA) into the medial forebrain bundle (MFB) caused rats to attempt fewer trials and to make more procedural errors. Lesioned rats also developed a pronounced ipsilateral bias, with a corresponding drop in contralateral accuracy. They were also slower to react to contralateral stimuli and to execute movements bilaterally. Rats that were pre-treated 4 weeks prior to lesion surgery with an equine infectious anaemia virus (EIAV) vector carrying GDNF [EIAV-GDNF, injected into the striatum and above the substantia nigra (SN)] performed significantly better on all of these parameters than control rats. In addition to the operant task, EIAV-GDNF successfully rescued contralateral impairments in the corridor, staircase, stepping and cylinder tasks, and prevented drug-induced rotational asymmetry. This study confirms that GDNF can protect against 6-OHDA-induced impairments in complex as well as simple behaviours, and reinforces the use of EIAV-based vectors for the treatment of PD

Lymphotoxin-alpha expression in the meninges causes lymphoid tissue formation and neurodegeneration

Organised meningeal immune cell infiltrates are suggested to play an important role in cortical grey matter pathology in the multiple sclerosis brain, but the mechanisms involved are as yet unresolved. Lymphotoxin-alpha plays a key role in lymphoid organ development and cellular cytotoxicity in the immune system and its expression is increased in the cerebrospinal fluid of naïve and progressive multiple sclerosis patients and post-mortem meningeal tissue. Here we show that persistently increased levels of lymphotoxin alpha in the cerebral meninges can give rise to lymphoid-like structures and underlying multiple sclerosis-like cortical pathology. Stereotaxic injections of recombinant lymphotoxin-alpha into the rat meninges led to acute meningeal inflammation and subpial demyelination that resolved after 28 days, with demyelination being dependent on prior sub-clinical immunisation with myelin oligodendrocyte glycoprotein. Injection of a lymphotoxin-alpha lentiviral vector into the cortical meningeal space, to produce chronic localised over-expression of the cytokine, induced extensive lymphoid-like immune cell aggregates, maintained over 3 months, including T-cell rich zones containing podoplanin+ fibroblastic reticular stromal cells and B-cell rich zones with a network of follicular dendritic cells, together with expression of lymphoid chemokines and their receptors. Extensive microglial and astroglial activation, subpial demyelination and marked neuronal loss occurred in the underlying cortical parenchyma. Whereas subpial demyelination was partially dependent on prior myelin oligodendrocyte glycoprotein immunisation, the neuronal loss was present irrespective of immunisation. Conditioned medium from LTα treated microglia was able to induce a reactive phenotype in astrocytes. Our results show that chronic lymphotoxin-alpha overexpression alone is sufficient to induce formation of meningeal lymphoid-like structures and subsequent neurodegeneration, similar to that seen in the progressive multiple sclerosis brain