Epidural cerebellar stimulation drives widespread neural synchrony in the intact and stroke perilesional cortex.

BACKGROUND: Cerebellar electrical stimulation has shown promise in improving motor recovery post-stroke in both rodent and human studies. Past studies have used motor evoked potentials (MEPs) to evaluate how cerebellar stimulation modulates ongoing activity in the cortex, but the underlying mechanisms are incompletely understood. Here we used invasive electrophysiological recordings from the intact and stroke-injured rodent primary motor cortex (M1) to assess how epidural cerebellar stimulation modulates neural dynamics at the level of single neurons as well as at the level of mesoscale dynamics. METHODS: We recorded single unit spiking and local field potentials (LFPs) in both the intact and acutely stroke-injured M1 contralateral to the stimulated cerebellum in adult Long-Evans rats under anesthesia. We analyzed changes in the firing rates of single units, the extent of synchronous spiking and power spectral density (PSD) changes in LFPs during and post-stimulation. RESULTS: Our results show that post-stimulation, the firing rates of a majority of M1 neurons changed significantly with respect to their baseline rates. These firing rate changes were diverse in character, as the firing rate of some neurons increased while others decreased. Additionally, these changes started to set in during stimulation. Furthermore, cross-correlation analysis showed a significant increase in coincident firing amongst neuronal pairs. Interestingly, this increase in synchrony was unrelated to the direction of firing rate change. We also found that neuronal ensembles derived through principal component analysis were more active post-stimulation. Lastly, these changes occurred without a significant change in the overall spectral power of LFPs post-stimulation. CONCLUSIONS: Our results show that cerebellar stimulation caused significant, long-lasting changes in the activity patterns of M1 neurons by altering firing rates, boosting neural synchrony and increasing neuronal assemblies' activation strength. Our study provides evidence that cerebellar stimulation can directly modulate cortical dynamics. Since these results are present in the perilesional cortex, our data might also help explain the facilitatory effects of cerebellar stimulation post-stroke.

C9orf72 in myeloid cells suppresses STING-induced inflammation.

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are neurodegenerative disorders that overlap in their clinical presentation, pathology and genetic origin. Autoimmune disorders are also overrepresented in both ALS and FTD, but this remains an unexplained epidemiologic observation1-3. Expansions of a hexanucleotide repeat (GGGGCC) in the C9orf72 gene are the most common cause of familial ALS and FTD (C9-ALS/FTD), and lead to both repeat-containing RNA and dipeptide accumulation, coupled with decreased C9orf72 protein expression in brain and peripheral blood cells4-6. Here we show in mice that loss of C9orf72 from myeloid cells alone is sufficient to recapitulate the age-dependent lymphoid hypertrophy and autoinflammation seen in animals with a complete knockout of C9orf72. Dendritic cells isolated from C9orf72-/- mice show marked early activation of the type I interferon response, and C9orf72-/- myeloid cells are selectively hyperresponsive to activators of the stimulator of interferon genes (STING) protein-a key regulator of the innate immune response to cytosolic DNA. Degradation of STING through the autolysosomal pathway is diminished in C9orf72-/- myeloid cells, and blocking STING suppresses hyperactive type I interferon responses in C9orf72-/- immune cells as well as splenomegaly and inflammation in C9orf72-/- mice. Moreover, mice lacking one or both copies of C9orf72 are more susceptible to experimental autoimmune encephalitis, mirroring the susceptibility to autoimmune diseases seen in people with C9-ALS/FTD. Finally, blood-derived macrophages, whole blood and brain tissue from patients with C9-ALS/FTD all show an elevated type I interferon signature compared with samples from people with sporadic ALS/FTD; this increased interferon response can be suppressed with a STING inhibitor. Collectively, our results suggest that patients with C9-ALS/FTD have an altered immunophenotype because their reduced levels of C9orf72 cannot suppress the inflammation mediated by the induction of type I interferons by STING.

Restoring mitofusin balance prevents axonal degeneration in a Charcot-Marie-Tooth type 2A model.

Mitofusin-2 (MFN2) is a mitochondrial outer-membrane protein that plays a pivotal role in mitochondrial dynamics in most tissues, yet mutations in MFN2, which cause Charcot-Marie-Tooth disease type 2A (CMT2A), primarily affect the nervous system. We generated a transgenic mouse model of CMT2A that developed severe early onset vision loss and neurological deficits, axonal degeneration without cell body loss, and cytoplasmic and axonal accumulations of fragmented mitochondria. While mitochondrial aggregates were labeled for mitophagy, mutant MFN2 did not inhibit Parkin-mediated degradation, but instead had a dominant negative effect on mitochondrial fusion only when MFN1 was at low levels, as occurs in neurons. Finally, using a transgenic approach, we found that augmenting the level of MFN1 in the nervous system in vivo rescued all phenotypes in mutant MFN2R94Q-expressing mice. These data demonstrate that the MFN1/MFN2 ratio is a key determinant of tissue specificity in CMT2A and indicate that augmentation of MFN1 in the nervous system is a viable therapeutic strategy for the disease.

Cell transplantation strategies for acquired and inherited disorders of peripheral myelin.

Objective: To investigate transplantation of rat Schwann cells or human iPSC-derived neural crest cells and derivatives into models of acquired and inherited peripheral myelin damage. Methods: Primary cultured rat Schwann cells labeled with a fluorescent protein for monitoring at various times after transplantation. Human-induced pluripotent stem cells (iPSCs) were differentiated into neural crest stem cells, and subsequently toward a Schwann cell lineage via two different protocols. Cell types were characterized using flow cytometry, immunocytochemistry, and transcriptomics. Rat Schwann cells and human iPSC derivatives were transplanted into (1) nude rats pretreated with lysolecithin to induce demyelination or (2) a transgenic rat model of dysmyelination due to PMP22 overexpression. Results: Rat Schwann cells transplanted into sciatic nerves with either toxic demyelination or genetic dysmyelination engrafted successfully, and migrated longitudinally for relatively long distances, with more limited axial migration. Transplanted Schwann cells engaged existing axons and displaced dysfunctional Schwann cells to form normal-appearing myelin. Human iPSC-derived neural crest stem cells and their derivatives shared similar engraftment and migration characteristics to rat Schwann cells after transplantation, but did not further differentiate into Schwann cells or form myelin. Interpretation: These results indicate that cultured Schwann cells surgically delivered to peripheral nerve can engraft and form myelin in either acquired or inherited myelin injury, as proof of concept for pursuing cell therapy for diseases of peripheral nerve. However, lack of reliable technology for generating human iPSC-derived Schwann cells for transplantation therapy remains a barrier in the field.

C9orf72 is required for proper macrophage and microglial function in mice.

Expansions of a hexanucleotide repeat (GGGGCC) in the noncoding region of the C9orf72 gene are the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. Decreased expression of C9orf72 is seen in expansion carriers, suggesting that loss of function may play a role in disease. We found that two independent mouse lines lacking the C9orf72 ortholog (3110043O21Rik) in all tissues developed normally and aged without motor neuron disease. Instead, C9orf72 null mice developed progressive splenomegaly and lymphadenopathy with accumulation of engorged macrophage-like cells. C9orf72 expression was highest in myeloid cells, and the loss of C9orf72 led to lysosomal accumulation and altered immune responses in macrophages and microglia, with age-related neuroinflammation similar to C9orf72 ALS but not sporadic ALS human patient tissue. Thus, C9orf72 is required for the normal function of myeloid cells, and altered microglial function may contribute to neurodegeneration in C9orf72 expansion carriers.

C9orf72 BAC Transgenic Mice Display Typical Pathologic Features of ALS/FTD.

Noncoding expansions of a hexanucleotide repeat (GGGGCC) in the C9orf72 gene are the most common cause of familial amyotrophic lateral sclerosis and frontotemporal dementia. Here we report transgenic mice carrying a bacterial artificial chromosome (BAC) containing the full human C9orf72 gene with either a normal allele (15 repeats) or disease-associated expansion (∼100-1,000 repeats; C9-BACexp). C9-BACexp mice displayed pathologic features seen in C9orf72 expansion patients, including widespread RNA foci and repeat-associated non-ATG (RAN) translated dipeptides, which were suppressed by antisense oligonucleotides targeting human C9orf72. Nucleolin distribution was altered, supporting that either C9orf72 transcripts or RAN dipeptides promote nucleolar dysfunction. Despite early and widespread production of RNA foci and RAN dipeptides in C9-BACexp mice, behavioral abnormalities and neurodegeneration were not observed even at advanced ages, supporting the hypothesis that RNA foci and RAN dipeptides occur presymptomatically and are not sufficient to drive neurodegeneration in mice at levels seen in patients.

Lentiviral-induced high-grade gliomas in rats: the effects of PDGFB, HRAS-G12V, AKT, and IDH1-R132H.

In human gliomas, the RTK/RAS/PI(3)K signaling pathway is nearly always altered. We present a model of experimental gliomagenesis that elucidates the contributions of genes involved in this pathway (PDGF-B ligand, HRAS-G12V, and AKT). We also examine the effect on gliomagenesis by the potential modifier gene, IDH1-R132H. Injections of lentiviral-encoded oncogenes induce de novo gliomas of varying penetrance, tumor progression, and histological grade depending on the specific oncogenes used. Our model mimics hallmark histological structures of high-grade glioma, such as pseudopalisades, glomeruloid microvascular proliferation, and diffuse tumor invasion. We use our model of gliomagenesis to test the efficacy of an experimental brain tumor gene therapy. Our model allowed us to test the contributions of oncogenes in the RTK/RAS/PI(3)K pathway, and their potential modification by over-expression of mutated IDH1, in glioma development and progression in rats. Our model constitutes a clinically relevant system to study gliomagenesis, the effects of modifier genes, and the efficacy of experimental therapeutics.

Targeting RNA foci in iPSC-derived motor neurons from ALS patients with a C9ORF72 repeat expansion.

Amyotrophic lateral sclerosis (ALS) is a severe neurodegenerative condition characterized by loss of motor neurons in the brain and spinal cord. Expansions of a hexanucleotide repeat (GGGGCC) in the noncoding region of the C9ORF72 gene are the most common cause of the familial form of ALS (C9-ALS), as well as frontotemporal lobar degeneration and other neurological diseases. How the repeat expansion causes disease remains unclear, with both loss of function (haploinsufficiency) and gain of function (either toxic RNA or protein products) proposed. We report a cellular model of C9-ALS with motor neurons differentiated from induced pluripotent stem cells (iPSCs) derived from ALS patients carrying the C9ORF72 repeat expansion. No significant loss of C9ORF72 expression was observed, and knockdown of the transcript was not toxic to cultured human motor neurons. Transcription of the repeat was increased, leading to accumulation of GGGGCC repeat-containing RNA foci selectively in C9-ALS iPSC-derived motor neurons. Repeat-containing RNA foci colocalized with hnRNPA1 and Pur-α, suggesting that they may be able to alter RNA metabolism. C9-ALS motor neurons showed altered expression of genes involved in membrane excitability including DPP6, and demonstrated a diminished capacity to fire continuous spikes upon depolarization compared to control motor neurons. Antisense oligonucleotides targeting the C9ORF72 transcript suppressed RNA foci formation and reversed gene expression alterations in C9-ALS motor neurons. These data show that patient-derived motor neurons can be used to delineate pathogenic events in ALS.

B cells are critical to T-cell-mediated antitumor immunity induced by a combined immune-stimulatory/conditionally cytotoxic therapy for glioblastoma.

We have demonstrated that modifying the tumor microenvironment through intratumoral administration of adenoviral vectors (Ad) encoding the conditional cytotoxic molecule, i.e., HSV1-TK and the immune-stimulatory cytokine, i.e., fms-like tyrosine kinase 3 ligand (Flt3L) leads to T-cell-dependent tumor regression in rodent models of glioblastoma. We investigated the role of B cells during immune-mediated glioblastoma multiforme regression. Although treatment with Ad-TK+Ad-Flt3L induced tumor regression in 60% of wild-type (WT) mice, it completely failed in B-cell-deficient Igh6(-/-) mice. Tumor-specific T-cell precursors were detected in Ad-TK+Ad-Flt3L-treated WT mice but not in Igh6(-/-) mice. The treatment also failed in WT mice depleted of total B cells or marginal zone B cells. Because we could not detect circulating antibodies against tumor cells and the treatment was equally efficient in WT mice and in mice with B-cell-specific deletion of Prdm 1 (encoding Blimp-1), in which B cells are present but unable to fully differentiate into antibody-secreting plasma cells, tumor regression in this model is not dependent on B cells' production of tumor antigen-specific immunoglobulins. Instead, B cells seem to play a role as antigen-presenting cells (APCs). Treatment with Ad-TK+Ad-Flt3L led to an increase in the number of B cells in the cervical lymph nodes, which stimulated the proliferation of syngeneic T cells and induced clonal expansion of antitumor T cells. Our data show that B cells act as APCs, playing a critical role in clonal expansion of tumor antigen-specific T cells and brain tumor regression.

Engineering the brain tumor microenvironment enhances the efficacy of dendritic cell vaccination: implications for clinical trial design.

PURPOSE: Glioblastoma multiforme (GBM) is a deadly primary brain tumor. Clinical trials for GBM using dendritic cell (DC) vaccination resulted in antitumor immune responses. Herein, we tested the hypothesis that combining in situ (intratumoral) Ad-Flt3L/Ad-TK-mediated gene therapy with DC vaccination would increase therapeutic efficacy and antitumor immunity. EXPERIMENTAL DESIGN: We first assessed the immunogenicity of tumor lysates generated by Ad-TK (+GCV), temozolomide (TMZ), or freeze/thawing cycles (FTC) in a syngeneic brain tumor model. We also assessed phenotypic markers, cytokine release, and phagocytosis of bone marrow-derived DCs generated by fms-like tyrosine kinase 3 ligand (Flt3L) + IL-6 or by granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin (IL) 4. Inhibition of tumor progression and production of anti-GBM antibodies was assessed following vaccination with (i) tumor cell lysates, (ii) DCs generated with either Flt3L/IL-6 or GM-CSF/IL-4 loaded with either Ad-TK/GCV-, TMZ-, or FTC-generated tumor lysates, or (iii) DCs in combination with in situ Ad-Flt3L/Ad-TK gene therapy. RESULTS: DCs loaded with tumor cell lysates generated with either Ad-TK/GCV or TMZ led to increased levels of phagocytosis, therapeutic efficacy, and humoral immune response. In situ immunogene therapy in combination with DC vaccination led to brain tumor regression and long-term survival in about 90% of animals, a significant increase when compared with either therapy alone. CONCLUSIONS: Our results indicate that modifying the tumor microenvironment using intratumoral Ad-Flt3L/Ad-TK-mediated gene therapy potentiates therapeutic efficacy and antitumor immunity induced by DC vaccination. These data support novel phase I clinical trials to assess the safety and efficacy of this combined approach.

Gene therapy-mediated delivery of targeted cytotoxins for glioma therapeutics.

Restricting the cytotoxicity of anticancer agents by targeting receptors exclusively expressed on tumor cells is critical when treating infiltrative brain tumors such as glioblastoma multiforme (GBM). GBMs express an IL-13 receptor (IL13Rα2) that differs from the physiological IL4R/IL13R receptor. We developed a regulatable adenoviral vector (Ad.mhIL-4.TRE.mhIL-13-PE) encoding a mutated human IL-13 fused to Pseudomonas exotoxin (mhIL-13-PE) that specifically binds to IL13Rα2 to provide sustained expression, effective anti-GBM cytotoxicity, and minimal neurotoxicity. The therapeutic Ad also encodes mutated human IL-4 that binds to the physiological IL4R/IL13R without interacting with IL13Rα2, thus inhibiting potential binding of mhIL-13-PE to normal brain cells. Using intracranial GBM xenografts and syngeneic mouse models, we tested the Ad.mhIL-4.TRE.mhIL-13-PE and two protein formulations, hIL-13-PE used in clinical trials (Cintredekin Besudotox) and a second-generation mhIL-13-PE. Cintredekin Besudotox doubled median survival without eliciting long-term survival and caused severe neurotoxicity; mhIL-13-PE led to ∼40% long-term survival, eliciting severe neurological toxicity at the high dose tested. In contrast, Ad-mediated delivery of mhIL-13-PE led to tumor regression and long-term survival in over 70% of the animals, without causing apparent neurotoxicity. Although Cintredekin Besudotox was originally developed to target GBM, when tested in a phase III trial it failed to achieve clinical endpoints and revealed neurotoxicity. Limitations of Cintredekin Besudotox include its short half-life, which demanded frequent or continued administration, and binding to IL4R/IL13R, present in normal brain cells. These shortcomings were overcome by our therapeutic Ad, thus representing a significant advance in the development of targeted therapeutics for GBM.

A novel bicistronic high-capacity gutless adenovirus vector that drives constitutive expression of herpes simplex virus type 1 thymidine kinase and tet-inducible expression of Flt3L for glioma therapeutics.

Glioblastoma multiforme (GBM) is a deadly primary brain tumor. Conditional cytotoxic/immune-stimulatory gene therapy (Ad-TK and Ad-Flt3L) elicits tumor regression and immunological memory in rodent GBM models. Since the majority of patients enrolled in clinical trials would exhibit adenovirus immunity, which could curtail transgene expression and therapeutic efficacy, we used high-capacity adenovirus vectors (HC-Ads) as a gene delivery platform. Herein, we describe for the first time a novel bicistronic HC-Ad driving constitutive expression of herpes simplex virus type 1 thymidine kinase (HSV1-TK) and inducible Tet-mediated expression of Flt3L within a single-vector platform. We achieved anti-GBM therapeutic efficacy with no overt toxicities using this bicistronic HC-Ad even in the presence of systemic Ad immunity. The bicistronic HC-Ad-TK/TetOn-Flt3L was delivered into intracranial gliomas in rats. Survival, vector biodistribution, neuropathology, systemic toxicity, and neurobehavioral deficits were assessed for up to 1 year posttreatment. Therapeutic efficacy was also assessed in animals preimmunized against Ads. We demonstrate therapeutic efficacy, with vector genomes being restricted to the brain injection site and an absence of overt toxicities. Importantly, antiadenoviral immunity did not inhibit therapeutic efficacy. These data represent the first report of a bicistronic vector platform driving the expression of two therapeutic transgenes, i.e., constitutive HSV1-TK and inducible Flt3L genes. Further, our data demonstrate no promoter interference and optimum gene delivery and expression from within this single-vector platform. Analysis of the efficacy, safety, and toxicity of this bicistronic HC-Ad vector in an animal model of GBM strongly supports further preclinical testing and downstream process development of HC-Ad-TK/TetOn-Flt3L for a future phase I clinical trial for GBM.

Study of the efficacy, biodistribution, and safety profile of therapeutic gutless adenovirus vectors as a prelude to a phase I clinical trial for glioblastoma.

Glioblastoma multiforme (GBM) is the most common and most aggressive primary brain tumor in humans. Systemic immunity against gene therapy vectors has been shown to hamper therapeutic efficacy; however, helper-dependent high-capacity adenovirus (HC-Ad) vectors elicit sustained transgene expression, even in the presence of systemic anti-adenoviral immunity. We engineered HC-Ads encoding the conditional cytotoxic herpes simplex type 1 thymidine kinase (TK) and the immunostimulatory cytokine fms-like tyrosine kinase ligand 3 (Flt3L). Flt3L expression is under the control of the regulatable Tet-ON system. In anticipation of a phase I clinical trial for GBM, we assessed the therapeutic efficacy, biodistribution, and clinical and neurotoxicity with escalating doses of HC-Ad-TetOn-Flt3L + HC-Ad-TK in rats. Intratumoral administration of these therapeutic HC-Ads in rats bearing large intracranial GBMs led to long-term survival in approximately 70% of the animals and development of antiglioma immunological memory without signs of neuropathology or systemic toxicity. Systemic anti-adenoviral immunity did not affect therapeutic efficacy. These data support the idea that it would be useful to develop HC-Ad vectors further as a therapeutic gene-delivery platform to implement GBM phase I clinical trials.

Gene therapy for brain cancer: combination therapies provide enhanced efficacy and safety.

Glioblastoma multiforme (GBM) is the most common primary brain cancer in adults. Despite significant advances in treatment and intensive research, the prognosis for patients with GBM remains poor. Therapeutic challenges for GBM include its invasive nature, the proximity of the tumor to vital brain structures often preventing total resection, and the resistance of recurrent GBM to conventional radiotherapy and chemotherapy. Gene therapy has been proposed as a useful adjuvant for GBM, to be used in conjunction with current treatment. Work from our laboratory has shown that combination of conditional cytotoxic with immunotherapeutic approaches for the treatment of GBM elicits regression of large intracranial tumor masses and anti-tumor immunological memory in syngeneic rodent models of GBM. In this review we examined the currently available animal models for GBM, including rodent transplantable models, endogenous rodent tumor models and spontaneous GBM in dogs. We discuss non-invasive surrogate end points to assess tumor progression and therapeutic efficacy, such as behavioral tests and circulating biomarkers. Growing preclinical and clinical data contradict the old dogma that cytotoxic anti-cancer therapy would lead to an immune-suppression that would impair the ability of the immune system to mount an anti-tumor response. The implications of the findings reviewed indicate that combination of cytotoxic therapy with immunotherapy will lead to synergistic antitumor efficacy with reduced neurotoxicity and supports the clinical implementation of combined cytotoxic-immunotherapeutic strategies for the treatment of patients with GBM.