Intravitreal gene therapy protects against retinal dysfunction and degeneration in sheep with CLN5 Batten disease.

Neuronal ceroid lipofuscinoses (NCL; Batten disease) are a group of inherited neurodegenerative diseases primarily affecting children. A common feature across most NCLs is the progressive loss of vision. We performed intravitreal injections of self-complementary AAV9 vectors packaged with either ovine CLN5 or CLN6 into one eye of 3-month-old CLN5-/- or CLN6-/- animals, respectively. Electroretinography (ERG) was performed every month following treatment, and retinal histology was assessed post-mortem in the treated compared to untreated eye. In CLN5-/- animals, ERG amplitudes were normalised in the treated eye whilst the untreated eye declined in a similar manner to CLN5 affected controls. In CLN6-/- animals, ERG amplitudes in both eyes declined over time although the treated eye showed a slower decline. Post-mortem examination revealed significant attenuation of retinal atrophy and lysosomal storage body accumulation in the treated eye compared with the untreated eye in CLN5-/- animals. This proof-of-concept study provides the first observation of efficacious intravitreal gene therapy in a large animal model of NCL. In particular, the single administration of AAV9-mediated intravitreal gene therapy can successfully ameliorate retinal deficits in CLN5-/- sheep. Combining ocular gene therapy with brain-directed therapy presents a promising treatment strategy to be used in future sheep trials aiming to halt neurological and retinal disease in CLN5 Batten disease.

Autosomal dominant neuronal ceroid lipofuscinosis: Clinical features and molecular basis.

The neuronal ceroid lipofuscinoses (NCLs) are at least 13 distinct progressive neurodegenerative disorders unified by the accumulation of lysosomal auto-fluorescent material called lipofuscin. The only form that occurs via autosomal-dominant inheritance exhibits adult onset and is sometimes referred to as Parry type NCL. The manifestations may include behavioral symptoms followed by seizures, ataxia, dementia, and early death. Mutations in the gene DNAJC5 that codes for the presynaptic co-chaperone cysteine string protein-α (CSPα) were recently reported in sporadic adult-onset cases and in families with dominant inheritance. The mutant CSPα protein may lead to disease progression by both loss and gain of function mechanisms. Iron chelation therapy may be considered as a possible pharmaceutical intervention based on our recent mechanism-based proposal of CSPα oligomerization via ectopic Fe-S cluster-binding, summarized in this review.

Interrater agreement of classification of photoparoxysmal electroencephalographic response.

Our goal was to assess the interrater agreement (IRA) of photoparoxysmal response (PPR) using the classification proposed by a task force of the International League Against Epilepsy (ILAE), and a simplified classification system proposed by our group. In addition, we evaluated IRA of epileptiform discharges (EDs) and the diagnostic significance of the electroencephalographic (EEG) abnormalities. We used EEG recordings from the European Reference Network (EpiCARE) and Standardized Computer-based Organized Reporting of EEG (SCORE). Six raters independently scored EEG recordings from 30 patients. We calculated the agreement coefficient (AC) for each feature. IRA of PPR using the classification proposed by the ILAE task force was only fair (AC = 0.38). This improved to a moderate agreement by using the simplified classification (AC = 0.56; P = .004). IRA of EDs was almost perfect (AC = 0.98), and IRA of scoring the diagnostic significance was moderate (AC = 0.51). Our results suggest that the simplified classification of the PPR is suitable for implementation in clinical practice.

Functional characterization of novel MFSD8 pathogenic variants anticipates neurological involvement in juvenile isolated maculopathy.

Biallelic MFSD8 variants are an established cause of severe late-infantile subtype of neuronal ceroid lipofuscinosis (v-LINCL), a severe lysosomal storage disorder, but have also been associated with nonsyndromic adult-onset maculopathy. Here, we functionally characterized two novel MFSD8 variants found in a child with juvenile isolated maculopathy, in order to establish a refined prognosis. ABCA4 locus resequencing was followed by the analysis of other inherited retinal disease genes by whole exome sequencing (WES). Minigene assays and cDNA sequencing were used to assess the effect of a novel MFSD8 splice variant. MFSD8 expression was quantified with qPCR and overexpression studies were analyzed by immunoblotting. Transmission electron microscopy (TEM) was performed on a skin biopsy and ophthalmological and neurological re-examinations were conducted. WES revealed two novel MFSD8 variants: c.[590del];[439+3A>C] p.[Gly197Valfs*2];[Ile67Glufs*3]. Characterization of the c.439+3A>C variant via splice assays showed exon-skipping (p.Ile67Glufs*3), while overexpression studies of the corresponding protein indicated expression of a truncated polypeptide. In addition, a significantly reduced MFSD8 RNA expression was noted in patient's lymphocytes. TEM of a skin biopsy revealed typical v-LINCL lipopigment inclusions while neurological imaging of the proband displayed subtle cerebellar atrophy. Functional characterization demonstrated the pathogenicity of two novel MFSD8 variants, found in a child with an initial diagnosis of juvenile isolated maculopathy but likely evolving to v-LINCL with a protracted disease course. Our study allowed a refined neurological prognosis in the proband and expands the natural history of MFSD8-associated disease.

Loss of CLN7 results in depletion of soluble lysosomal proteins and impaired mTOR reactivation.

Defects in the MFSD8 gene encoding the lysosomal membrane protein CLN7 lead to CLN7 disease, a neurodegenerative lysosomal storage disorder belonging to the group of neuronal ceroid lipofuscinoses. Here, we have performed a SILAC-based quantitative analysis of the lysosomal proteome using Cln7-deficient mouse embryonic fibroblasts (MEFs) from a Cln7 knockout (ko) mouse model. From 3335 different proteins identified, we detected 56 soluble lysosomal proteins and 29 highly abundant lysosomal membrane proteins. Quantification revealed that the amounts of 12 different soluble lysosomal proteins were significantly reduced in Cln7 ko MEFs compared with wild-type controls. One of the most significantly depleted lysosomal proteins was Cln5 protein that underlies another distinct neuronal ceroid lipofuscinosis disorder. Expression analyses showed that the mRNA expression, biosynthesis, intracellular sorting and proteolytic processing of Cln5 were not affected, whereas the depletion of mature Cln5 protein was due to increased proteolytic degradation by cysteine proteases in Cln7 ko lysosomes. Considering the similar phenotypes of CLN5 and CLN7 patients, our data suggest that depletion of CLN5 may play an important part in the pathogenesis of CLN7 disease. In addition, we found a defect in the ability of Cln7 ko MEFs to adapt to starvation conditions as shown by impaired mammalian target of rapamycin complex 1 reactivation, reduced autolysosome tubulation and increased perinuclear accumulation of autolysosomes compared with controls. In summary, depletion of multiple soluble lysosomal proteins suggest a critical role of CLN7 for lysosomal function, which may contribute to the pathogenesis and progression of CLN7 disease.

Novel compound heterozygous mutations causing Kufs disease type B.

BACKGROUND: Kufs disease type B (also termed CLN13), an adult-onset form of neuronal ceroid lipofuscinosis (NCL), is genetically heterogeneous and challenging to diagnose. Recently, mutations in cathepsin-F have been identified as the causative gene for autosomal recessive Kufs disease type B. RESULTS: Here, we report a sporadic case of Kufs disease type B with novel compound heterozygous mutations, a novel missense mutation c.977G>T (p.C326F) and a novel nonsense mutation c.416C>A (p.S139X), in the cathepsin-F gene. The magnetic resonance imaging findings were consistent with those demonstrated in adult neuronal ceroid lipofuscinosis: diffuse cortical atrophy, mild hyperintensity and reduction of the deep white matter on T2-weighted images. A skin biopsy was negative for abnormalities. CONCLUSIONS: Altogether, our findings broaden the mutation database in relation to the neuronal ceroid lipofuscinosis, and the clinical diagnosis of Kufs disease type B was confirmed.

Glial cells are functionally impaired in juvenile neuronal ceroid lipofuscinosis and detrimental to neurons.

The neuronal ceroid lipofuscinoses (NCLs or Batten disease) are a group of inherited, fatal neurodegenerative disorders of childhood. In these disorders, glial (microglial and astrocyte) activation typically occurs early in disease progression and predicts where neuron loss subsequently occurs. We have found that in the most common juvenile form of NCL (CLN3 disease or JNCL) this glial response is less pronounced in both mouse models and human autopsy material, with the morphological transformation of both astrocytes and microglia severely attenuated or delayed. To investigate their properties, we isolated glia and neurons from Cln3-deficient mice and studied their basic biology in culture. Upon stimulation, both Cln3-deficient astrocytes and microglia also showed an attenuated ability to transform morphologically, and an altered protein secretion profile. These defects were more pronounced in astrocytes, including the reduced secretion of a range of neuroprotective factors, mitogens, chemokines and cytokines, in addition to impaired calcium signalling and glutamate clearance. Cln3-deficient neurons also displayed an abnormal organization of their neurites. Most importantly, using a co-culture system, Cln3-deficient astrocytes and microglia had a negative impact on the survival and morphology of both Cln3-deficient and wildtype neurons, but these effects were largely reversed by growing mutant neurons with healthy glia. These data provide evidence that CLN3 disease astrocytes are functionally compromised. Together with microglia, they may play an active role in neuron loss in this disorder and can be considered as potential targets for therapeutic interventions.

Retinal function in patients with the neuronal ceroid lipofuscinosis phenotype / Estudo da função retiniana em pacientes com o fenótipo da lipofuscinose ceróide neuronal

ABSTRACT Purpose: To analyze the clinical features, visual acuity, and full-field electroretinogram (ERG) findings of 15 patients with the neuronal ceroid lipofuscinosis (NCL) phenotype and to establish the role of ERG testing in NCL diagnosis. Methods: The medical records of five patients with infantile NCL, five with Jansky-Bielschowsky disease, and five with juvenile NCL who underwent full-field ERG testing were retrospectively analyzed. Results: Progressive vision loss was the initial symptom in 66.7% of patients and was isolated or associated with ataxia, epilepsy, and neurodevelopmental involution. Epilepsy was present in 93.3% of patients, of whom 86.6% presented with neurodevelopmental involution. Fundus findings ranged from normal to pigmentary/atrophic abnormalities. Cone-rod, rod-cone, and both types of dysfunction were observed in six, one, and eight patients, respectively. Conclusion: In our study, all patients with the NCL phenotype had abnormal ERG findings, and the majority exhibited both cone-rod and rod-cone dysfunction. We conclude that ERG is a valuable tool for the characterization of visual dysfunction in patients with the NCL phenotype and is useful for diagnosis.

RESUMO Objetivo: Analisar o quadro clínico, a acuidade visual e o eletrorretinograma de campo total (ERG) de 15 pacientes com o fenótipo da lipofuscinose ceróide neuronal (LCN), estabelecendo o papel do eletrorretinograma no seu diagnóstico. Métodos: Eletrorretinograma foi realizado em 5 pacientes com lipofuscinose ceróide neuronal infantil, 5 com doença de Jansky-Bielschowsky e 5 com lipofuscinose ceróide neuronal juvenil sendo feita uma análise retrospectiva dos registros médicos. Resultados: A perda progressiva da acuidade visual foi o sintoma inicial em 66,7%; isolada ou associada à ataxia, epilepsia e involução do desenvolvimento neuropsico motor. Epilepsia foi o sintoma inicial em 93,3% e 86,6% apresentaram involução do desenvolvimento neuropsicomotor. Achados fundoscópicos variaram de normal a alterações pigmentares/atróficas. Disfunção de cone-bastonete foi constatada em 6 pacientes, bastonete-cone em 1 e em 8 pacientes observou-se disfunção proporcional de ambos os sistemas. Conclusão: O eletrorretinograma foi alterado em todos os pacientes, e o achado mais frequente foi o comprometimento de cones e bastonetes. O eletrorretinograma constitui, portanto, uma ferramenta valiosa para caracterizar a disfunção visual em pacientes com o fenótipo da lipofuscinose ceróide neuronal, contribuindo para seu diagnóstico.

CLN2 Disease (Classic Late Infantile Neuronal Ceroid Lipofuscinosis).

CLN2 disease is an inherited metabolic storage disorder caused by the deficiency of the lysosomal enzyme tripeptidyl peptidase 1 (TPP1). The disease affects mainly the brain and the retina and is characterized by progressive dysfunction of the central nervous system, leading to dementia, epilepsy, loss of motor function and blindness. The classical late infantile type begins at around three years of age with epilepsy and/or a standstill of psychomotor development, followed by a rapid loss of all abilities and death in childhood. A late onset form in a small proportion of patients starts at the age of 4 to 10 years, but also leads to severe neurological deterioration. The deficiency of TPP1 causes the lysosomal accumulation of a material called ceroid lipofuscin. The natural substrate of TPP1 is not known, nor is the connection between storage process and neurodegeneration, which is characterized by loss of neurons. Among various experimental approaches to treatment, enzyme replacement therapy (ERT) and gene therapy have developed remarkably. Enzyme delivery through the cerebrospinal fluid led to wide distribution of enzyme activity in the brain and to attenuated neuropathology and disease progression in a TPP1-deficient mouse model as well as in a natural TPP1-deficient dog model. Safety of the intrathecal delivery, pharmacokinetics, and tissue distribution of the administered enzyme studied in non-human primates were encouraging, and a phase I/II clinical trial for intraventricular ERT in CLN2 patients is ongoing. A second approach uses intracerebral injection of viral vectors containing normal coding segments of the CLN2 gene. In a CLN2 mouse model, this procedure resulted in cerebral enzyme expression, reduced brain pathology and increased survival. A small number of patients have been treated the same way using an AAV2-vector for gene transfer to the brain. Although there were no serious adverse events unequivocally attributable to the vector used, there were some serious adverse effects, and a clinical benefit was not clearly evident under the conditions of the experiment. A phase I/phase II study using a AAVrh10 vector is presently recruiting patients.

Lysosomal cathepsins and their regulation in aging and neurodegeneration.

Lysosomes and lysosomal hydrolases, including the cathepsins, have been shown to change their properties with aging brain a long time ago, although their function was not really understood. The first biochemical and clinical studies were followed by a major expansion in the last 20 years with the development of animal disease models and new approaches leading to a major advancement of understanding of the role of physiological and degenerative processes in the brain at the molecular level. This includes the understanding of the major role of autophagy and the cathepsins in a number of diseases, including its critical role in the neuronal ceroid lipofuscinosis. Similarly, cathepsins and some other lysosomal proteases were shown to have important roles in processing and/or degradation of several important neuronal proteins, thereby having either neuroprotective or harmful roles. In this review, we discuss lysosomal cathepsins and their regulation with the focus on cysteine cathepsins and their endogenous inhibitors, as well as their role in several neurodegenerative diseases.

Rett-like onset in late-infantile neuronal ceroid lipofuscinosis (CLN7) caused by compound heterozygous mutation in the MFSD8 gene and review of the literature data on clinical onset signs.

BACKGROUND: We present clinical and molecular findings of a patient with ceroid-lipofuscinosis CLN7, with a compound heterozygous mutation of the MFSD8 gene, with Rett syndrome clinical signs onset and a later development of full picture of vLINCL. CASE PRESENTATION: A 7 years-old female patient with normal development until the age 12 months, developed Rett like clinical picture (psychomotor regression, microcephaly, stereotypic hands movements in the midline, hyperventilation episodes) present at the onset of her condition (age 18 months), features still present at the initial evaluation in our clinic at age 5 years. RESULTS: MECP2 (methyl CpG binding protein 2) gene mutation was negative. At age 6 years she was readmitted for severe ataxia and blindness, seizures, and severe developmental regression leading to NCL (neuronal ceroid lipofuscinosis) suspicion. EEG showed slow background with IRDA (intermittent rhythmic delta activity). A conjunctive biopsy showed abnormal curvilinear and fingerprint lysosomal deposits, and genetic analysis revealed two heterozygous mutations of MFSD8 gene (c.881C > A p.Thr294Lys and c.754 + 2T > A) each inherited from carrier parents and a heterozygous variant (c.470A>C p.Asp157Ala) of CLN5 gene. CONCLUSION: NCL should be suspected and MFSD8 genetic testing should also be considered in patients with Rett like phenotype at onset and negative MECP2 mutation. Such cases should be carefully and frequently re-evaluated in order to avoid delayed diagnosis and offer proper genetic advice to the family. In our knowledge, this might be the first case of CLN7 disease with Rett like onset described in the literature, which developed typical vLINCL clinical phenotype after age 5.5 years. A short review of the literature showing NCL onset modalities is presented.

Oral cysteamine bitartrate and N-acetylcysteine for patients with infantile neuronal ceroid lipofuscinosis: a pilot study.

BACKGROUND: Infantile neuronal ceroid lipofuscinosis is a devastating neurodegenerative lysosomal storage disease caused by mutations in the gene (CLN1 or PPT1) encoding palmitoyl-protein thioesterase-1 (PPT1). We have previously reported that phosphocysteamine and N-acetylcysteine mediate ceroid depletion in cultured cells from patients with this disease. We aimed to assess whether combination of oral cysteamine bitartrate and N-acetylcysteine is beneficial for patients with neuronal ceroid lipofuscinosis. METHODS: Children between 6 months and 3 years of age with infantile neuronal ceroid lipofuscinosis with any two of the seven most lethal PPT1 mutations were eligible for inclusion in this pilot study. All patients were recruited from physician referrals. Patients received oral cysteamine bitartrate (60 mg/kg per day) and N-acetylcysteine (60 mg/kg per day) and were assessed every 6-12 months until they had an isoelectric electroencephalogram (EEG, attesting to a vegetative state) or were too ill to travel. Patients were also assessed by electroretinography, brain MRI and magnetic resonance spectroscopy (MRS), and electron microscopic analyses of leukocytes for granular osmiophilic deposits (GRODs). Children also underwent physical and neurodevelopmental assessments on the Denver scale. Outcomes were compared with the reported natural history of infantile neuronal ceroid lipofuscinosis and that of affected older siblings. This trial is registered with ClinicalTrials.gov, number NCT00028262. FINDINGS: Between March 14, 2001, and June 30, 2012, we recruited ten children with infantile neuronal ceroid lipofuscinosis; one child was lost to follow-up after the first visit and nine patients (five girls and four boys) were followed up for 8 to 75 months. MRI showed abnormalities similar to those in previous reports; brain volume and N-acetyl aspartic acid (NAA) decreased steadily, but no published quantitative MRI or MRS studies were available for comparison. None of the children acquired new developmental skills, and their retinal function decreased progressively. Average time to isoelectric EEG (52 months, SD 13) was longer than reported previously (36 months). At the first follow-up visit, peripheral leukocytes in all nine patients showed virtually complete depletion of GRODs. Parents and physicians reported less irritability, improved alertness, or both in seven patients. No treatment-related adverse events occurred apart from mild gastrointestinal discomfort in two patients, which disappeared when liquid cysteamine bitartrate was replaced with capsules. INTERPRETATION: Our findings suggest that combination therapy with cysteamine bitartrate and N-acetylcysteine is associated with delay of isoelectric EEG, depletion of GRODs, and subjective benefits as reported by parents and physicians. Our systematic and quantitative report of the natural history of patients with infantile neuronal ceroid lipofuscinosis provides a guide for future assessment of experimental therapies. FUNDING: National Institutes of Health.

Recurrent generalized seizures, visual loss, and palinopsia as phenotypic features of neuronal ceroid lipofuscinosis due to progranulin gene mutation.

We detail the phenotype of a novel form of neuronal ceroid lipofuscinosis due to a homozygous progranulin gene mutation (c.813_816del; CLN11 MIM #614706). The symptoms appeared in two young adult siblings, and included progressive retinopathy, recurrent generalized seizures, moderate ataxia, and subtle cognitive dysfunction. Long-lasting episodes of palinopsia were a recurring symptom and associated with polyphasic visual-evoked potential waveform that suggested hyperexcitability of the occipital cortex. Electroencephalography showed rare spike-wave paroxysms, and magnetic resonance imaging revealed selective cerebellar atrophy. Skin biopsy revealed fingerprint storage and the absence of progranulin protein. Electron microscopy of peripheral blood leukocytes showed fingerprint profiles in 1/100 lymphocytes. These findings define a novel phenotype and provide clues for better understanding of progranulin function. A PowerPoint slide summarizing this article is available for download in the Supporting Information section here.

Effective intravenous therapy for neurodegenerative disease with a therapeutic enzyme and a peptide that mediates delivery to the brain.

The blood-brain barrier (BBB) presents a major challenge to effective treatment of neurological disorders, including lysosomal storage diseases (LSDs), which frequently present with life-shortening and untreatable neurodegeneration. There is considerable interest in methods for intravenous delivery of lysosomal proteins across the BBB but for the most part, levels achievable in the brain of mouse models are modest and increased lifespan remains to be demonstrated. In this study, we have investigated delivery across the BBB using a mouse model of late-infantile neuronal ceroid lipofuscinosis (LINCL), a neurodegenerative LSD caused by loss of tripeptidyl peptidase I (TPP1). We have achieved supraphysiological levels of TPP1 throughout the brain of LINCL mice by intravenous (IV) coadministration of recombinant TPP1 with a 36-residue peptide that contains polylysine and a low-density lipoprotein receptor binding sequence from apolipoprotein E. Importantly, IV administration of TPP1 with the peptide significantly reduces brain lysosomal storage, increases lifespan and improves neurological function. This simple "mix and inject" method is immediately applicable towards evaluation of enzyme replacement therapy to the brain in preclinical models and further exploration of its clinical potential is warranted.

In a model of Batten disease, palmitoyl protein thioesterase-1 deficiency is associated with brown adipose tissue and thermoregulation abnormalities.

Infantile neuronal ceroid lipofuscinosis (INCL) is a fatal neurodegenerative disorder caused by a deficiency of palmitoyl-protein thioesterase-1 (PPT1). We have previously shown that children with INCL have increased risk of hypothermia during anesthesia and that PPT1-deficiency in mice is associated with disruption of adaptive energy metabolism, downregulation of peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α), and mitochondrial dysfunction. Here we hypothesized that Ppt1-knockout mice, a well-studied model of INCL that shows many of the neurologic manifestations of the disease, would recapitulate the thermoregulation impairment observed in children with INCL. We also hypothesized that when exposed to cold, Ppt1-knockout mice would be unable to maintain body temperature as in mice thermogenesis requires upregulation of Pgc-1α and uncoupling protein 1 (Ucp-1) in brown adipose tissue. We found that the Ppt1-KO mice had lower basal body temperature as they aged and developed hypothermia during cold exposure. Surprisingly, this inability to maintain body temperature during cold exposure in Ppt1-KO mice was associated with an adequate upregulation of Pgc-1α and Ucp-1 but with lower levels of sympathetic neurotransmitters in brown adipose tissue. In addition, during baseline conditions, brown adipose tissue of Ppt1-KO mice had less vacuolization (lipid droplets) compared to wild-type animals. After cold stress, wild-type animals had significant decreases whereas Ppt1-KO had insignificant changes in lipid droplets compared with baseline measurements, thus suggesting that Ppt1-KO had less lipolysis in response to cold stress. These results uncover a previously unknown phenotype associated with PPT1 deficiency, that of altered thermoregulation, which is associated with impaired lipolysis and neurotransmitter release to brown adipose tissue during cold exposure. These findings suggest that INCL should be added to the list of neurodegenerative diseases that are linked to alterations in peripheral metabolic processes. In addition, extrapolating these findings clinically, impaired thermoregulation and hypothermia are potential risks in patients with INCL.

Synergistic effects of central nervous system-directed gene therapy and bone marrow transplantation in the murine model of infantile neuronal ceroid lipofuscinosis.

OBJECTIVE: Infantile neuronal ceroid lipofuscinosis (INCL) is an inherited childhood neurodegenerative disorder caused by the loss of palmitoyl protein thioesterase-1 (PPT1) activity. Affected children suffer from blindness, epilepsy, motor dysfunction, cognitive decline, and premature death. The Ppt1(-/-) mouse shares the histological and clinical features of INCL. Previous single-therapy approaches using small molecule drugs, gene therapy, or neuronal stem cells resulted in partial histological correction, with minimal improvements in motor function or lifespan. Here, we combined central nervous system (CNS)-directed adeno-associated virus (AAV)2/5-mediated gene therapy with bone marrow transplantation (BMT) in the INCL mouse. METHODS: At birth, Ppt1(-/-) and wild-type mice were given either intracranial injections of AAV2/5-PPT1 or bone marrow transplantation, separately as well as in combination. To assess function, we measured rotorod performance monthly as well as lifespan. At terminal time points, we evaluated the therapeutic effects on several INCL-specific parameters, such as cortical thickness, autofluorescent accumulation, and glial activation. Finally, we determined levels of PPT1 enzyme activity and bone marrow engraftment in treated mice. RESULTS: AAV2/5-mediated gene therapy alone resulted in significant histological correction, improved motor function, and increased lifespan. Interestingly, the addition of BMT further increased the lifespan of treated mice and led to dramatic, sustained improvements in motor function. These data are truly striking, given that BMT alone is ineffective, yet it synergizes with CNS-directed gene therapy to dramatically increase efficacy and lifespan. INTERPRETATION: AAV2/5-mediated gene therapy in combination with BMT provides an unprecedented increase in lifespan as well as dramatic improvement on functional and histological parameters.

Osmoregulation of ceroid neuronal lipofuscinosis type 3 in the renal medulla.

Recessive inheritance of mutations in ceroid neuronal lipofuscinosis type 3 (CLN3) results in juvenile neuronal ceroid lipofuscinosis (JNCL), a childhood neurodegenerative disease with symptoms including loss of vision, seizures, and motor and mental decline. CLN3p is a transmembrane protein with undefined function. Using a Cln3 reporter mouse harboring a nuclear-localized bacterial beta-galactosidase (beta-Gal) gene driven by the native Cln3 promoter, we detected beta-Gal most prominently in epithelial cells of skin, colon, lung, and kidney. In the kidney, beta-Gal-positive nuclei were predominant in medullary collecting duct principal cells, with increased expression along the medullary osmotic gradient. Quantification of Cln3 transcript levels from kidneys of wild-type (Cln3(+/+)) mice corroborated this expression gradient. Reporter mouse-derived renal epithelial cultures demonstrated a tonicity-dependent increase in beta-Gal expression. RT-quantitative PCR determination of Cln3 transcript levels further supported osmoregulation at the Cln3 locus. In vivo, osmoresponsiveness of Cln3 was demonstrated by reduction of medullary Cln3 transcript abundance after furosemide administration. Primary cultures of epithelial cells of the inner medulla from Cln3(lacZ/lacZ) (CLN3p-null) mice showed no defect in osmolyte accumulation or taurine flux, arguing against a requirement for CLN3p in osmolyte import or synthesis. CLN3p-deficient mice with free access to water showed a mild urine-concentrating defect but, upon water deprivation, were able to concentrate their urine normally. Unexpectedly, we found that CLN3p-deficient mice were hyperkalemic and had a low fractional excretion of K(+). Together, these findings suggest an osmoregulated role for CLN3p in renal control of water and K(+) balance.

Kufs' disease: diagnostic difficulties in the examination of extracerebral biopsies.

Kufs' disease or NCL4 (neuronal ceroid lipofuscinosis type 4) is a rare and poorly characterized, adult-onset form of NCL. The mutation in gene CLN, underlying Kufs' disease, still remains unknown. The diagnosis of this disease is difficult because it is based only on clinical and ultrastructural examinations. We report the case of a 45-year-old woman referred to the Neurological Department with suspicion of Creutzfeldt-Jakob disease (CJD). CJD as well as infectious, autoimmune and some lysosomal diseases were excluded. Since clinical symptoms, i.e. psychotic, auditory and visual hallucinations as well as behavioural disturbances, still suggested metabolic or neurodegenerative disease, a skin and muscle biopsy was performed. On ultrastructural examination the muscle biopsy revealed the subsarcolemmal accumulation of lipofuscin, lipofuscin-like and granular osmiophilic deposits (GRODs). The most unique fingerprint deposits (FP) and curvilinear profiles (CP) for diagnosis of Kufs' disease were located in vascular smooth muscle cells (VSMCs). In these cells lipofuscin-like deposits and GRODs were also visible. The fact that FP and CP were found exclusively in VSMCs jointly with clinical and laboratory data allows us to diagnose Kufs' disease in our patient.