hESC-derived striatal progenitors grafted into a Huntington's disease rat model support long-term functional motor recovery by differentiating, self-organizing and connecting into the lesioned striatum.

BACKGROUND: Huntington's disease (HD) is a motor and cognitive neurodegenerative disorder due to prominent loss of striatal medium spiny neurons (MSNs). Cell replacement using human embryonic stem cells (hESCs) derivatives may offer new therapeutic opportunities to replace degenerated neurons and repair damaged circuits. METHODS: With the aim to develop effective cell replacement for HD, we assessed the long-term therapeutic value of hESC-derived striatal progenitors by grafting the cells into the striatum of a preclinical model of HD [i.e., adult immunodeficient rats in which the striatum was lesioned by monolateral injection of quinolinic acid (QA)]. We examined the survival, maturation, self-organization and integration of the graft as well as its impact on lesion-dependent motor alterations up to 6 months post-graft. Moreover, we tested whether exposing a cohort of QA-lesioned animals to environmental enrichment (EE) could improve graft integration and function. RESULTS: Human striatal progenitors survived up to 6 months after transplantation and showed morphological and neurochemical features typical of human MSNs. Donor-derived interneurons were also detected. Grafts wired in both local and long-range striatal circuits, formed domains suggestive of distinct ganglionic eminence territories and displayed emerging striosome features. Moreover, over time grafts improved complex motor performances affected by QA. EE selectively increased cell differentiation into MSN phenotype and promoted host-to-graft connectivity. However, when combined to the graft, the EE paradigm used in this study was insufficient to produce an additive effect on task execution. CONCLUSIONS: The data support the long-term therapeutic potential of ESC-derived human striatal progenitor grafts for the replacement of degenerated striatal neurons in HD and suggest that EE can effectively accelerate the maturation and promote the integration of human striatal cells.

In vitro-derived medium spiny neurons recapitulate human striatal development and complexity at single-cell resolution.

Stem cell engineering of striatal medium spiny neurons (MSNs) is a promising strategy to understand diseases affecting the striatum and for cell-replacement therapies in different neurological diseases. Protocols to generate cells from human pluripotent stem cells (PSCs) are scarce and how well they recapitulate the endogenous fetal cells remains poorly understood. We have developed a protocol that modulates cell seeding density and exposure to specific morphogens that generates authentic and functional D1- and D2-MSNs with a high degree of reproducibility in 25 days of differentiation. Single-cell RNA sequencing (scRNA-seq) shows that our cells can mimic the cell-fate acquisition steps observed in vivo in terms of cell type composition, gene expression, and signaling pathways. Finally, by modulating the midkine pathway we show that we can increase the yield of MSNs. We expect that this protocol will help decode pathogenesis factors in striatal diseases and eventually facilitate cell-replacement therapies for Huntington's disease (HD).

The enhancement of activity rescues the establishment of Mecp2 null neuronal phenotypes.

MECP2 mutations cause Rett syndrome (RTT), a severe and progressive neurodevelopmental disorder mainly affecting females. Although RTT patients exhibit delayed onset of symptoms, several evidences demonstrate that MeCP2 deficiency alters early development of the brain. Indeed, during early maturation, Mecp2 null cortical neurons display widespread transcriptional changes, reduced activity, and defective morphology. It has been proposed that during brain development these elements are linked in a feed-forward cycle where neuronal activity drives transcriptional and morphological changes that further increase network maturity. We hypothesized that the enhancement of neuronal activity during early maturation might prevent the onset of RTT-typical molecular and cellular phenotypes. Accordingly, we show that the enhancement of excitability, obtained by adding to neuronal cultures Ampakine CX546, rescues transcription of several genes, neuronal morphology, and responsiveness to stimuli. Greater effects are achieved in response to earlier treatments. In vivo, short and early administration of CX546 to Mecp2 null mice prolongs lifespan, delays the disease progression, and rescues motor abilities and spatial memory, thus confirming the value for RTT of an early restoration of neuronal activity.

The antidepressant tianeptine reverts synaptic AMPA receptor defects caused by deficiency of CDKL5.

Mutations in the X-linked cyclin-dependent kinase-like 5 (CDKL5) gene cause a complex neurological disorder, characterized by infantile seizures, impairment of cognitive and motor skills and autistic features. Loss of Cdkl5 in mice affects dendritic spine maturation and dynamics but the underlying molecular mechanisms are still far from fully understood. Here we show that Cdkl5 deficiency in primary hippocampal neurons leads to deranged expression of the alpha-amino-3-hydroxy-5-methyl-4-iso-xazole propionic acid receptors (AMPA-R). In particular, a dramatic reduction of expression of the GluA2 subunit occurs concomitantly with its hyper-phosphorylation on Serine 880 and increased ubiquitination. Consequently, Cdkl5 silencing skews the composition of membrane-inserted AMPA-Rs towards the GluA2-lacking calcium-permeable form. Such derangement is likely to contribute, at least in part, to the altered synaptic functions and cognitive impairment linked to loss of Cdkl5. Importantly, we find that tianeptine, a cognitive enhancer and antidepressant drug, known to recruit and stabilise AMPA-Rs at the synaptic sites, can normalise the expression of membrane inserted AMPA-Rs as well as the number of PSD-95 clusters, suggesting its therapeutic potential for patients with mutations in CDKL5.

Lack of Methyl-CpG Binding Protein 2 (MeCP2) Affects Cell Fate Refinement During Embryonic Cortical Development.

During differentiation, neurons progressively restrict their fate repressing the expression of specific genes. Here we describe the involvement in such developmental steps of the methyl-CpG binding protein 2 (MeCP2), an epigenetic factor that participates to chromatin folding and transcriptional regulation. We previously reported that, due to transcriptional impairments, the maturation of Mecp2 null neurons is delayed. To evaluate whether this could stem from altered progenitors proliferation and differentiation, we investigated whether lack of Mecp2 affects these features both in vitro and in vivo. We show that in Mecp2 null embryonic cortexes the expression of genes defining the identity of proliferating neuroprogenitors is enriched and that their permanence in the G1 phase is prolonged. Moreover, the number of cells transitioning from a stage of maturation to a more mature one is increased in Mecp2 null embryonic cortices, in line with the central role of G1 for cell identity refinement. We thus suggest that, possibly due to the lack of proper transcriptional control normally exerted by Mecp2, fate refinement is impaired in developing null cells. We propose that the maturation delay affecting the developing Mecp2 null cortex originates, at least in part, from deranged mechanisms of cell fate refinement.

MeCP2 Related Studies Benefit from the Use of CD1 as Genetic Background.

MECP2 mutations cause a number of neurological disorders of which Rett syndrome (RTT) represents the most thoroughly analysed condition. Many Mecp2 mouse models have been generated through the years; their validity is demonstrated by the presence of a broad spectrum of phenotypes largely mimicking those manifested by RTT patients. These mouse models, between which the C57BL/6 Mecp2tm1.1Bird strain probably represents the most used, enabled to disclose much of the roles of Mecp2. However, small litters with little viability and poor maternal care hamper the maintenance of the colony, thus limiting research on such animals. For this reason, past studies often used Mecp2 mouse models on mixed genetic backgrounds, thus opening questions on whether modifier genes could be responsible for at least part of the described effects. To verify this possibility, and facilitate the maintenance of the Mecp2 colony, we transferred the Mecp2tm1.1Bird allele on the stronger CD1 background. The CD1 strain is easier to maintain and largely recapitulates the phenotypes already described in Mecp2-null mice. We believe that this mouse model will foster the research on RTT.

Defects During Mecp2 Null Embryonic Cortex Development Precede the Onset of Overt Neurological Symptoms.

MeCP2 is associated with several neurological disorders; of which, Rett syndrome undoubtedly represents the most frequent. Its molecular roles, however, are still unclear, and data from animal models often describe adult, symptomatic stages, while MeCP2 functions during embryonic development remain elusive. We describe the pattern and timing of Mecp2 expression in the embryonic neocortex highlighting its low but consistent expression in virtually all cells and show the unexpected occurrence of transcriptional defects in the Mecp2 null samples at a stage largely preceding the onset of overt symptoms. Through the deregulated expression of ionic channels and glutamatergic receptors, the lack of Mecp2 during early neuronal maturation leads to the reduction in the neuronal responsiveness to stimuli. We suggest that such features concur to morphological alterations that begin affecting Mecp2 null neurons around the perinatal age and become evident later in adulthood. We indicate MeCP2 as a key modulator of the transcriptional mechanisms regulating cerebral cortex development. Neurological phenotypes of MECP2 patients could thus be the cumulative result of different adverse events that are already present at stages when no obvious signs of the pathology are evident and are worsened by later impairments affecting the central nervous system during maturation and maintenance of its functionality.